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Developmental Plant Anatomy
CEJ Botha 2005
Contents
1. TOWARDS THE SUPRACELLULAR ORGANISM ............................................................................ 4
Control of development ........................................................................................................................................... 4
Communication & domains ..................................................................................................................................... 4
Sources and sinks ..................................................................................................................................................... 6
2. GUARANTEEING A SUPPLY OF CELLS -THE VASCULAR CAMBIUM..................................... 7
The cell division process.............................................................................................................................. 8
Structure of the cambium............................................................................................................................. 9
Perennials ............................................................................................................................................................... 10
Wood Structure ...................................................................................................................................................... 10
Distribution of Parenchyma ................................................................................................................................... 11
Secondary Growth in Monocotyledons. ................................................................................................................. 11
Anomalous secondary structures ............................................................................................................................ 12
3. LEAF STRUCTURE-FUNCTION RELATIONSHIPS ....................................................................... 14
INTRODUCTION ............................................................................................................................................... 14
LEAF STRUCTURE ........................................................................................................................................... 15
LEAF DEVELOPMENT ...................................................................................................................................... 16
Major vein differentiation .......................................................................................................................... 17
The minor veins ......................................................................................................................................... 17
Structural specialisation of companion cells. ............................................................................................ 18
LEAF ARCHITECTURE: CLASSIFICATION OF MAJOR AND MINOR VEINS ............................................................. 18
THE MONOCOTYLEDONOUS FOLIAGE LEAF ..................................................................................................... 19
Leaf blade bundle anatomy in grasses ...................................................................................................... 20
Leaf Anatomy in the Cyperaceae ............................................................................................................... 22
4. PHLOEM TRANSLOCATION ............................................................................................................. 25
STRUCTURE-FUNCTION RELATIONSHIPS .......................................................................................................... 25
The symplasm-apoplasm concept .............................................................................................................. 25
Structure related to function ...................................................................................................................... 26
Comparison of Characteristics of Xylem and Phloem............................................................................................ 27
The Pathway of Translocation: ................................................................................................................. 27
1. Bark incisions..................................................................................................................................................... 28
2. Exudation from aphid stylets.............................................................................................................................. 28
3. Analysis of honeydew ........................................................................................................................................ 28
The source .............................................................................................................................................................. 29
Function, movement and regulation within the phloem and allied parenchymatous tissues .................................. 29
5. LEAVES AND TRANSPORT ................................................................................................................ 32
Is metabolic energy required? ................................................................................................................................ 33
MECHANISMS OF PHLOEM LOADING ............................................................................................................... 34
Transfer cells: .................................................................................................................................................... 34
Phloem loading.......................................................................................................................................... 35
Sieve-element loading and phloem loading ........................................................................................................... 37
Ultrastructural features of the cells in the loading zone ......................................................................................... 38
Phloem loading zone structure ............................................................................................................................... 38
PLASMODESMATAL FREQUENCY AND DISTRIBUTION ...................................................................................... 38
Diagnostic value of plasmodesmograms ................................................................................................... 40
The loading and transport sections of the phloem..................................................................................... 41
Potential phloem loading pathways........................................................................................................................ 42
The Phloem loading pathway and composition of the phloem sap ........................................................................ 43
Leaf disks ............................................................................................................................................................... 45
Intracellular injection ............................................................................................................................................. 46
New evidence for a symplasmic loading pathway in grasses .................................................................... 46
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Developmental Plant Anatomy
cross veins – implication in transport, loading and unloading ........................................................................... 46
PHYSIOLOGICAL EVIDENCE FOR DIFFERENT MODES OF PHLOEM LOADING....................................................... 47
Companion cell-sieve tube connectivity and phloem loading ................................................................................ 47
mechanism of symplasmic phloem loading................................................................................................ 47
Mechanism of apoplasmic phloem loading ............................................................................................... 49
1. release from the mesophyll symplast .................................................................................................................. 50
2. Transport in the mesophyll symplast .................................................................................................................. 50
3. The apoplasmic passage compartment ............................................................................................................... 51
4. Sugar uptake by the SE-CC-complex ................................................................................................................. 52
5. Kinetic studies .................................................................................................................................................... 52
6. Evidence for sucrose/proton symports ............................................................................................................... 52
Phloem loading -- system mode efficiency................................................................................................. 53
The humble potato – a seasonal source to sink conversion ....................................................................... 54
Ecophysiological concept of phloem loading ............................................................................................ 55
The monocot leaf – a case for thin and thick-walled sieve tubes .......................................................................... 58
Virus trafficking................................................................................................................................................. 63
In summation ......................................................................................................................................................... 65
ACKNOWLEDGEMENTS ........................................................................................................................ 66
Developmental Plant Anatomy. Core Notes. Page
3
1. towards the supracellular organism
Control of development
How do cells differentiate? How does a leaf form? How do transport cells such as
sieve elements differentiate? What controls short and long distance intercellular
transport? These are some of the questions that are central to understanding plant
structure-function relationships. The subject becomes more fascinating as research
reveals more information concerning the control of development of specific
structures, such as those differentiating from the shoot or root apex, developing
leaves, or even specialised cells such as the companion cell-sieve element complex.
Much attention is focussed on the processes involved, from the molecular, through
the ultrastructural and developmental to the whole plant level.
When we observe living cells under the microscope, or when we observe living or
fixed material with the light and or electron microscope, several things should
become clear immediately.
1. The multitude of different cell types involved in and associated with particular
tissue systems and the distribution of tissues in all parts of the plant.
2. The almost infinite variation in cell structure and substructural organelle
composition.
3. That the variations appear to be controlled by cellular function, and that function
(and hence, the appearance) may differ with season which will be manifest by
changes in growth patterns within the plant under investigation. All of these must be
influenced by the physiological state of the organelles, the cells, and the tissues that
make up the plant parts themselves.
Communication & domains
The living cells of the plant constitute the avenue for intercellular communication.
In higher plants, communication systems have evolved into two separate, distinct,
yet inter-associated physiological transport systems. The symplast, is defined as that
part of the plant where interconnection of organelles, and of adjacent cell cytoplasm,
through minute structures called plasmodesmata form a continuum which may
extend across a few cells, or may involve many thousands or hundreds of thousands
of cells. In contrast, the cell walls, intercellular spaces and the non-living xylem,
constitutes what is physiologically termed the apoplast. The apoplast is thus a
continuum, which extends from the finest roots, through hairs on the aerial parts of
the plant. It is the major pathway of (energy-free) travel and communication within
plants. In addition, the apoplast is the pathway followed by dissolved ions and
molecules from one plant part to another. The apoplast is often termed the “free
space” in the literature.
Several distinct domains may exist in any one plant. For example, the uptake
(loading) of sucrose from the mesophyll and its subsequent transport to the
companion cell-sieve tube complex, may involve domain crossing. Sugars have to
cross an apoplasmic barrier during loading, in recognised apoplasmic loaders..
Apoplasmic loading is thought to be an evolutionary step, which has occurred in
many of the taxonomically advanced families, and that this ‘phloem loading
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Developmental Plant Anatomy
ecophysiology’ has led to recognition of several types of loading in higher plants.
The exact ecophysiological advantages of a mixed or apoplasmic loading pathways
are not entirely clear1.
Longevity is assured by the primary and secondary meristems, which together
produce new cells to add to the body of the primary plant, throughout its life. The
plant may be viewed as an expanding system, composed of hundreds, thousands,
millions or sometimes, billions of cells - each with specific roles and functions in a
co-ordinated structural entity, some less, some more specialised than others.
The apical meristem is one of the
simplest-looking structures in the higher
plant, yet, the processes controlling its
differentiation sequencing is not yet fully
understood.
We recognize that changes have to be
effected in the way in which neighbour
cells
communicate
(or
stop
communicating) prior to, during and
after a cell division event in this
structure. This topic explores the
concept of domain control in higher
plants, specifically in the shoot apex.
A
M
outer zone
domain 2
=
plasmodesma
closed
inner
zone
domain 1
1
Turgeon, Robert; Medville, Richard, and Nixon, Kevin C.2001.he evolution of minor vein phloem and
phloem loading. Am. J. Bot. 88:1331-1339.
Developmental Plant Anatomy. Core Notes. Page
5
Sources and sinks
Biochemically, higher plants function as sources and sinks - sources of essential
carbon skeletons, and sinks where these carbon skeletons are either accumulated, or
metabolised and converted into other more useful secondary plant metabolites
associated either with growth or further development of the plant.
As plant evolution advanced plants became exposed to more varying and variable
environments, adaptation to the environmental variables became necessary, if
survival was to occur. For example, plants which occupy habitats where water is not
in short supply and where temperature extremes do not apply, have evolved an
almost exclusive, symplasmic phloem loading pathway. In contrast, plants which
occupy more xeric environments, with broader temperature extremes, have adopted
an increasingly mixed to apoplasmic phloem loading pathway.
Plant evolution has thus come a long way from the simple single-celled organism
that manufactured its own carbon skeletons, to the multicellular, where a degree of
division of labour became the norm, to what we term today, the ‘supracellular
organism’ which has arisen due to the specific adaptive needs of the plant to
changing environment. This resulted in the evolution of two disparate transport
pathways and a plethora of mechanisms which individually regulate, control and
enhances transport and cell-to-cell communication phenomena.
Hopefully, these core notes will be of some use- I believe they fill a gap that exists
in the literature, which makes this material highly relevant.
CEJ BOTHA, 6/28/2017
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Developmental Plant Anatomy
2. Guaranteeing a supply of cells -the vascular cambium
The shoot apical meristem has an intrinsic capacity for self regulation and the
positional specification of its cells implies the existence of an elaborate and versatile
communication network. Van der Schoot and Rinne2 proposed a model that pictures
this network as a system of overlapping signal circuits, which support local tasks as
well as coordinating indeterminate shoot development.
The vascular cambium is unlike the primary meristems of the plant (root and shoot
apex) in that it produces new cells and tissues which add to the axial system (i.e. the
conducting system) as well as to the radial system (i.e. the lateral transport
pathway). The apical meristems of the shoot and root on the other hand add only to
the axial system. Thus the cells of the vascular cambium do not fit the regular
concept of meristematic cells (i.e. small dense, with large nuclei, and of isodiametric
shape). However, it is clear that partial (symplasmic) isolation events are necessary
to ensure that controlled planar (domain related) cell divisions can take place.
However, it is possible that during division, that they function in much the same
way as described above for meristematic cells within the shoot and root apex.
Cambial cells are usually highly vacuolate and occur in two forms, namely fusiform
cells and ray cells. The term fusiform implies that the cell is shaped like a spindle,
but it is approximately prismatic and wedge-shaped at both ends. Ray cells on the
other hand, are short squat cells. Tangentially, both cell types are wider than they
appear in radial section or view. The diagram below should assist in orientating you,
with respect to the planes of cell division and of observation needed to study the
cambium.
AXIAL
AXIAL: Longitudinal
translocation, Xylem
and Phloem elements.
RADIAL: Lateral translocation.
Carbohydrate from phloem to
central regions, water from xylem
to phloem.
Fig. 1 sectional planes of reference
These two different cell types (fusiform and ray cells) have unique functions.
Fusiform cells usually only produce cells associated with the axial system - that is
they produce either new elements of the xylem, or elements of the phloem., and thus
add to the AXIAL conducting system. Ray cells on the other hand, produce under
2
van der Schoot, C, Rinne, P 1999. Networks for shoot design Trends in plant science 4, 31-37
Developmental Plant Anatomy. Core Notes. Page
7
normal circumstances, ONLY ray cells and thus add to the RADIAL system of the
plant.
The cell division process.
The division of fusiform and ray cells follows a rather set pattern, with
PERICLINAL division of the fusiform cells being (under normal circumstances)
responsible for the production of either xylem or phloem elements.
Endarch
Tangential face
Exarch
TANGENTIAL face
RADIAL face
Plane of cell division
Fig. 2 Fusiform cambial cell faces
Periclinal cell division is thus best described as being parallel to the two tangential
faces.
Periclinal cell division thus produces a NEW cambial initial and a mother cell:endarch = xylem; exarch = phloem.
The ray cells behave in exactly the same way as fusiform cells, with the one
exception being that ray cells may divide periclinally, along the tangential plane, to
add to the radial (ray) system, or they may divide anticlinally (Radial, periclinal) to
add to the overall dimension of the cambium itself.
Endarch
Tangential face
Exarch
Tangential
face
Radial face
Plane of cell division
Fig. 3 Planes of cell division, fusiform cells
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Developmental Plant Anatomy
inner
tangential face
Radial
face
Outer tangential face
This division adds to the ray (xylem side)
or to the phloem side.
Fig. 4 & 5(below) Planes of cell division, in ray initials
inner
tangential face
Radial
face
Outer tangential face
This plane of cell division adds to the circumference
of the vascular cambium itself.
Recently, Crawford and Zambryski3 Suggested that plasmodesmata can alter their
transport capacity temporally during development and spatially in different regions
of the plant. Plasmodesmata are, they suggest, major gatekeepers of signalling
molecules that essentially facilitate or regulate developmental programs, maintain
physiological status, and respond to pathogens. Cell division then, depends upon
temporary closure of plasmodesmata, to allow local re-alignment of cell function
and differentiation.
Structure of the cambium
The vascular cambium usually forms a thin cylinder of cells, exarch to the primary
xylem in dicotyledonous and gymnospermous stems. In roots, the cambium appears
first between the metaxylem and the metaphloem, and like the stem, fills in laterally,
until a complete cylinder of cambial cells and their derivatives are formed.
Cambium which is initiated within fascicles or vascular bundles, is termed
fascicular cambium, whist that formed between bundles, it termed the
interfascicular cambium. The cylinder, once complete, then divides predominantly
periclinally, forming a cylinder of secondary xylem endarch to, and a ring of
3 Crawford, K, Zambryski, P 2000. Plasmodesmata: Gatekeepers for cell-to-cell transport of developmental
signals in plants. Annu. Rev. Cell Dev. Biol. 16 393-421
Developmental Plant Anatomy. Core Notes. Page
9
secondary phloem exarch to the cambial zone. In general, the cambium produces
more xylem than phloem (about 4:1), with little effect evident which may be
ascribed directly to the environment.
The walls of cambial cells are thin, and the cells are highly vacuolate, unlike most
other meristematic cells. Cambial cells contain large populations of mitochondria,
and dictyosomes, all embedded within a ribosome-rich cytoplasm.
The two kinds of cells within the cambium (fusiform and ray initials) form, in some
species, a stratified and in others, a non-stratified structure. If the cambium is
stratified, it will produce a stratified or storied wood; if it is not, then it will produce
a non-stratified or non-storied wood.
The fusiform cells of the cambium do not follow the general pattern of cell division
seen in other parts of the plant - instead, they normally divide vertically, in the
longitudinal plane (contrary to the “normal” situation whereby a cell will divide by a
wall of minimal area, i.e., horizontally would be the wall of minimal area here).
However, horizontal divisions do, occur infrequently, when the fusiform cells divide
to produce new ray initials (from declining fusiform cells). The normal plane of
division (longitudinal and periclinal) may be interrupted from time to time, and
longitudinal anticlinal divisions take place, to enable expansion of the cambium,
accommodating the increased diameter of the stem. During spring, it is common to
see a fairly wide band of undifferentiated cells, termed the cambial zone, but only
one layer of true cambial initials is present. Ray initials, produce or add to the
parenchymatous vascular rays, which traverse the xylem, and usually terminate
within the primary or secondary phloem (thus termed primary or secondary rays).
Whilst many studies have been undertaken on cambial cells and their derivatives,
little if any significant difference exists between the two cell types. Clearly, the
products formed, must be under nuclear control.
Perennials
In perennial plants, cambial activity is a seasonal phenomenon, occurring only
during the season of active growth, beginning in the spring. In more temperate
climates, the seasonal alteration between flushes of cambial activity, and inactivity,
results in the formation of annual or growth rings, of secondary xylem and
secondary phloem. The age of such trees may be estimated by counting the number
of growth rings. The phenomenon of seasonality is connected with longevity - in
some Sequoiadendron specimens are reported to be 3 - to 4000 years old. Clearly,
this illustrates that the cambial initials are not only capable of intermittent activity,
but also considerable activity more or less for an indefinitely - thus, one could
describe cambial cells as (almost) immortal!
Wood Structure
Commercial woods consist almost entirely of secondary xylem, and are therefore a
product of the cambium. They are usually classified as softwoods - the wood of
gymnosperms, principally conifers, and hardwoods, - the wood of angiosperms,
mainly dicotyledons. Softwoods contain mostly tracheids, and hardwoods contain
vessels and tracheids.
It is necessary to examine the structure of wood in three planes of section in order to
understand and comprehend the structure. Thus, transverse, radial longitudinal and
10
Developmental Plant Anatomy
tangential longitudinal sections must be examined. In this way, a thorough
understanding of the relationship to each other, of the various kinds of cells making
up the wood, may be gained. Particular differences may be seen when examining the
horizontally-orientated components - the rays. With the exception of the Gnetales,
gymnosperm wood lacks vessels, and, in transection, the wood is composed of
uniform tracheids. In contrast, the wood of angiosperms, contains both vessels and
tracheids, thus presenting a less uniform appearance at first glance than that in
gymnosperm wood.
As mentioned, woody species from temperate climates, exhibit seasonal cambial
activity, which leads to the formation of growth rings. These growth rings, may, in
some genera (Acer, Betula) have vessels which are approximately even-sized. As
such the wood can be described as diffuse porous, whilst in genera such as Quercus,
and Fraxinus, the vessels which are formed in the early part of the season, are of
much larger diameter than those which are produced towards the end of the growing
season. So, growth rings contain early (spring) wood which have wide vessels, and
late (summer, autumn) wood, which contains distinctly-narrower-diameter vessels.
This type of wood is described as ring porous. Diffuse and ring porous wood is
more easily recognised in transections of the specimen.
Distribution of Parenchyma
Xylem parenchyma within wood is variable both in amount, as well as in its
distribution. Timber may be classified according to the distribution of the
parenchyma within the wood. Again, classification is easier when viewed in
transection. In the broad sense, paratracheal parenchyma is topographically
associated with vessels, whilst apotracheal parenchyma is not associated with the
tracheary elements. Additionally, we recognise the banding in xylem parenchyma.
Apotracheal parenchyma may occur as isolated strands of axial parenchyma
(diffuse), or in bands which are tangentially-arranged (metatracheal). Paratracheal
parenchyma is classified further. For example, if it encircles the tracheary elements,
then it is described as vasicentric; or it may form wings, on either side of the
tracheary elements aliform; or it may form fairly extensive bands, and is then termed
banded. Wood anatomy is very important in terms of taxonomy - if you get a
chance, look at The Anatomy of the Dicotyledons by Metcalfe and Chalk. This
definitive text, written at Kew Gardens, is an example in which anatomy’s central
role in plant taxonomy comes to the fore.
Secondary Growth in Monocotyledons.
Whilst it is generally true that monocotyledons lack real secondary growth, there are
nonetheless, some examples where a more or less “woody” stems are formed in the
mature stem. As an example, Yucca and the palms. In such species, the vascular
tissue, as is the case in all monocotyledons, is entirely primary, much of the growth
can be described as a type of secondary growth, related to and produced by what we
term a primary thickening meristem. The primary thickening meristem originates
through periclinal cell divisions, below the regions of attachment of young leaf
primordia. This meristem contributes to the diameter of the young stem and
subsequently, also to its height. Some authorities consider this to be an end result of
primary growth in these species.
Developmental Plant Anatomy. Core Notes. Page 11
In some species, a type of vascular cambium may be evident. This cambium is
apparently continuous with the primary thickening meristem, is present in older
regions of the stem, and contributes to its diameter. It may be considered to be the
end result of the juvenile stage of growth. The primary thickening meristem may be
seen in longitudinal sections, as a several layers of elongate to rectangular cells,
which are orientated parallel to the surface of the stem. In some monocotyledons, a
type of vascular cambium, which is apparently continuous with the primary
thickening meristem may be seen. This cambium does not function like that in
dicotyledons, but rather gives rise to entire vascular bundles, and looks like an
amphivasal arrangement of vascular tissues, all embedded in parenchymatous
tissues, which are referred to as conjunctive tissues. Conjunctive tissue is often
lignified in old stems. Cordyline spp. and Dracaena. In the latter example, great
height and girth are possible, in very old (5-6000 years) plants.
Anomalous secondary structures
Many dicotyledons, especially those that are climbers, show interesting variations of
structural modifications to the vascular system in the secondary plant body. Because
they differ in structure and arrangement of the vascular tissues, such plants are
referred to as demonstrating anomalous growth. This changed pattern may be due to
a normal cambium, but giving rise to unusual arrangements of the secondary xylem
and phloem, or, a cambium which is itself is unusual or abnormally situated, thus
giving rise to abnormal distribution of secondary vascular tissues. Another option is
that the additional or accessory cambia may be formed.
In climbers such as Vitis and Clematis, the interfascicular cambium produces only
parenchymatic elements. In Aristolochia and its relatives, the encircling
sclerenchymatous perivascular fibre sheath becomes ruptured, and parenchymatous
intrusions occur in the ruptured zones. Mature Aristolochia stems have a fluted
appearance. In some stems, the cambium ceases to produce new cells, except in two
opposing arcs, thus producing a flattened stem. In others, wedges of phloem tissue
occur within the secondary xylem. This is common in Bignonia and Doxantha
species.
Figure 10.1 in Esau’s Anatomy of Seed Plants illustrates the structure of the vascular
cambium in Fig. 10.1 relation to derivative tissues. In this Figure, (A,B) show
xylem and phloem (both primary) surrounded by the pericycle. Note that the
elements between the metaphloem (in B) and the metaxylem, are parenchymatic. In
Fig. 10.1 C and D, the beginnings of the formation of the vascular cambium between
these two tissue types is illustrated. The cambium is shown as the lightly stippled
layer in these diagrams. Division of the fusiform and ray initials is not uniform, with
more secondary xylem produced opposite the metaxylem than the protoxylem. The
net effect is the rounding out of the cambium itself (E and F). The radial lines in
these line drawings (depicted in diagram E) represent medullary as well as
secondary rays leading from the xylem to the secondary phloem. Note the loss of
cortical layers, evident in E and F to some extent.
The development of secondary tissues in roots follows roughly similar stages as it
does in stems. Esau Anatomy of Seed Plants Fig. 15.1 illustrates different stages of
12
Developmental Plant Anatomy
development of the secondary plant body in Alfalfa roots. After several seasons, the
cambium has produced considerable secondary vascular tissue, and the cortex is
long gone -- replaced by a periderm after several seasons of growth.
Developmental Plant Anatomy. Core Notes. Page 13
3. Leaf structure-function relationships
Introduction
Leaves are the major lateral organs of the stem and form an integral part of the
aerial axis of the plant. Leaves are typically, organs of determinate growth and of
dorsiventral symmetry. The generally flattened shape being ideal for maximising
exposure to sunlight for photosynthesis. Leaves may be classified as microphylls or
macrophylls . In phylogenetic terms, a macrophyll is a modified branch system and
is therefore cauline in origin. In contrast, the smaller microphylls are generally
enations or outgrowths of the axis, which is not associated with leaf gaps. The
vascular system in microphylls is rudimentary and not extensively connected to that
of the axis. Both types of leaves originate from a primordium at the shoot apex.
Obviously, one may argue that the small size of microphylls represents a failure to
undergo the extensive growth and elaboration usually associated with macrophylls.
Microphyllous leaves occur in the Psilotales, the club mosses and some
pteridophytes.
Leaves serve several vital functions in the day-to-day life of higher plants and it is
of interest that we look at their development, (ontogeny) their structure as well as
their numerous functions. There are three major functions of leaves - each of which
is inter-related and interdependent to some degree, upon the functioning status of the
others.
1. Photosynthesis
2. Translocation
3. Transpiration.
Each of these is either initiated or takes place directly in the mesophyll of leaves.
That leaves have vastly differing internal structure, is demonstrated by even the
lowly mesophyll cells, which are arranged in different patterns and locations, which
may be ascribed directly to the functional processes of the photosynthetic cycle
occurring within the leaf. Leaves of Dicotyledonous plants differ greatly to those of
Monocotyledonous plants and to those of Gymnosperms. Of course, there is some
degree of intergradation, but generally, it is possible to separate these leaves, using
some basic diagnostic criteria. Dicotyledons generally have a mesophyll which is
composed of two differing photosynthetic cell types - palisade and spongy
mesophyll cells. Leaves may be isolateral, isobilateral, dorsiventral or even needlelike in cross-section. Whatever the leaf shape, chloroplasts are concentrated within
the cytoplasmic matrix of these cells and, for the most part, the majority of the
chloroplasts are to be found in the upper palisade mesophyll cells. Mitochondrial
populations in these obviously-photosynthetic cells may be high as well. The xylem
is responsible for the major proportion of apoplasmic transport in vascular plants.
Apoplasmic transport is not limited totally to water transport, but in addition, the
transport of various macro and micronutrients, amino acids and other important
inorganic substances, from the roots to the stem and ultimately, the leaf via the
apoplasmic continuum.
The phloem is responsible for the transport of the major proportion of soluble
carbohydrate as well as other essential products. The phloem forms the major long-
14
Developmental Plant Anatomy
distance symplasmic transport pathway in all vascular plants. Translocation usually
takes place from a site of synthesis of assimilated material (called a source) to a site
or sites of utilisation (called sinks). The assimilated material is translocated in a
water-based medium, which emphasises the essential inter-relationship between the
xylem and phloem, more particularly so in the leaf where most of the phloem
loading takes place in mature plants.
It can be argued that transpiration is the driving force which facilitates the
movement of solutes through the xylem. Transpiration requires that water entry is
facilitated via the roots and that it occurs in a conducting system, and that the water
is translocated to other regions of the plant where it is utilised in numerous
biochemical and growth-related reactions. The xylem is also the principal pathway
through which water is translocated from point of entry, to a point of exit, which in
higher plants are the stomata. This process is termed transpiration. Transpiration
itself facilitates leaf cooling by evapotranspirational heat loss to the atmosphere.
Leaf structure
There are several examples of plants where the leaf changes form during maturation
- this is termed heteroblastic development, whereas in many species leaves do not
undergo changes in form during the plants development from juvenile to adult. In
such cases, development is termed homoblastic.
In general terms, all leaves have the similar features -- an epidermis, mesophyll,
vascular tissue and stomata. However, the arrangement of these four components is,
to a large extent, dictated by the physical environment - water availability, light
intensity and ecological niche. Thus it is the interplay of these environmental
parameters, which serve to modify leaf structure. The epidermis may, for example,
be simple or compound, there may be either a thick or a thin culticular covering,
there may be a hypodermis associated with the epidermis, stomatal distribution may
be amphistomatous (stomata on both sides of the leaf) or hypostomatous (stomata on
one side of the leaf only) and they may be raised above the general leaf surface,
flush with the leaf surface, or in some cases, sunken into crypts. The ground tissue
(mesophyll) may be specialized or unspecialized. If specialized, a palisade or
spongy layer may exist and in some leaves, palisade tissue may exist on both sides
of the leaf (isobilateral) as is the case in many succulents. The mesophyll may be
compact, with few intercellular spaces as in xerophytes, or may contain a large
intercellular space volume, as in some mesophytes and hydrophytes. Some
Developmental Plant Anatomy. Core Notes. Page 15
H2O
CO2
O2
Epidermis
WATER (Xylem)
ASSIMILATES (Phloem)
The leaf: simple mechanics
Fig. 6. Illustrates the general mechanical requirements of a typical leaf. Adequate gas
exchange, and functional transport pathways are essential.
Dicotyledonous foliage leaves contain a specialized, longitudinally-orientated
mesophyll, called the paraveinal mesophyll, which separates the upper palisade from
the lower spongy mesophyll. In most monocotyledonous plants, the mesophyll is not
differentiated into spongy and palisade layers. The vascular bundles are surrounded
by an initially-parenchymatous bundle sheath, which may undergo lignification as
the cells mature. There may be a specialized, concentric arrangement of the
photosynthetic mesophyll surrounding the bundle sheath cells as in C4 plants. The
mechanics of a typical leaf is illustrated in Fig. 1.
Leaf development
All leaves develop from a foliar buttress, which, in simple terms, is a meristematic
projection above the general surface of the protoderm. Foliar buttresses are initiated
near the apex, in regular sequence and lead to the formation of mature leaves. The
ontogenetic sequence for a typical dorsiventral dicotyledonous foliage leaf is
illustrated in Fig. 2 below.
Leaf Development: Ontogenetic Relationships
adaxial epidermis
palisade tissue
MI
SI
PC ->
procambium -> vascular bundles
middle (spongy) mesophyll
lower spongy mesophyll
abaxial epidermis
Fig. 7. Ontogenetic relationship between the marginal and submarginal initials during leaf
development.
16
Developmental Plant Anatomy
The marginal initials (MI) give rise to the adaxial and abaxial epidermis, whilst the
submarginal initials (SI) give rise to all internal leaf tissues, including the
procambium, from which all vascular tissues are differentiated. In dicotyledonous
plants the transition from photoassimilate sink to source status begins shortly after
the leaf has begun to unfold, at which point, the major morphogenetic events that
determine leaf shape are to all intents and purposes over.
Differentiation of Major Veins
ACROPETAL
Major veins
Mid vein
Fig. 8. Illustrates the acropetal differentiation of the major vein network in a typical
dicotyledonous leaf.
Maturation of the phloem and xylem in the midrib and the higher-order veins, which
occurs in an acropetal directions is largely complete before the transition begins .
During leaf unfolding, the functional maturation of the minor veins begins in a
basipetal direction. There is thus a degree of maturation of the leaf from the base to
the tip o the lamina during the sink to source transition. The minor venation
network, forms the distribution network of the leaf which provides first an importing
and then an exporting network as the leaves continue to expand.
Major vein differentiation
The sequence of events which takes place within the dicotyledonous foliage leaf
may be summarised as follows. Once the blade or lamina starts to expand, due to
anticlinal and periclinal cell division within the marginal and sub-marginal initials,
the procambial strands begin to form -- first of these to become evident, is the
midrib or main vein. This vein is blocked out acropetally, and vascular tissues
differentiate in regular sequence (protophloem followed by protoxylem then
metaphloem, followed by metaxylem) towards the tip of the still-immature
expanding leaf. The major veins of the lamina follow suite, initiating at the base of
the leaf, they too, differentiate acropetally. Thus the first-formed of the major
lamina veins matures first, and the last-formed (apical) major veins, differentiate
and mature last.
The minor veins
In dicotyledonous foliage leaves, the minor veins differentiate basipetally, from the
apex and the leaf margin, back towards the major vein network. Thus, it is quite
feasible for the tip of the developing leaf to mature with respect to transport, before
the base of the leaf. The apex could therefore conceivable, export photo-assimilated
material to the still immature basal part of the leaf, during the overall maturation and
Developmental Plant Anatomy. Core Notes. Page 17
development process. According to Haritatos et al., (2000)4, the definition of
"minor" veins in leaves is arbitrary and of uncertain biological significance.
Generally, the term refers to the smallest vein classes in the leaf, believed to
function in phloem loading. They found that a galactinol synthase promoter, cloned
from melon (Cucumis melo), directs expression of the gusA gene to the smallest
veins of mature Arabidopsis and cultivated tobacco (Nicotiana tabacum) leaves.
This expression pattern is apparently consistent with the role of galactinol synthase
in sugar synthesis and phloem loading in cucurbits. The expression pattern in
tobacco is especially noteworthy since galactinol is not synthesized in the leaves of
this plant. Also, they unexpectedly found that expression in tobacco is limited to
two of three companion cells in class-V veins, which are the most extensive in the
leaf. Thus, the "minor" vein system is defined and regulated at the genetic level, and
there is heterogeneity of response to this system by different companion cells of the
same vein, according to Haritatos et al., (2000).
Structural specialisation of companion cells.
The term companion cell is used to describe that cell, or group of cells which are
derived from the same mother (procambial) cell as the sieve tube member. However,
the identification of the 'companion cell' may be problematic is some species, more
especially so in monocotyledons. In contrast, transfer cells are relatively easy to
identify as they always have prominent wall ingrowths that are assumed to enhance
cell uptake either from associated symplasmic continuia, or directly from the
apoplast. This is achieved through the increased surface area of the
plasmamembrane, associated with the wall ingrowths. In contrast, the term
intermediary cell is applied to large parenchymatic cells, with dense cytoplasm,
which loosely abut the parenchymatous bundle sheath in several dicotyledonous
species. Such intermediary cells have been identified in Cucurbita pepo, Cucumis
melo and Coleus blumei. Thus, companion cells , transfer cells, and intermediary
cells all have similar functions in the plant -- involvement to some degree in the
loading of sugars in source leaf veins. Several attempts have been made to correlate
companion cell type with plasmodesmatal frequency, or the type of sugar or sugar
alcohol transported. However, the data for this is scanty and incomplete.
Leaf architecture: Classification of major and minor veins
All leaves have two components to their networks - the so-called major and minor
vein system. What makes them different? Simply, the major veins in dicotyledonous
foliage leaves, occupy much of the cross-sectional area of the leaf, and are often
associated with hypodermal collenchymatous or sclerenchymatous strands. Viewed
in cross section, they may even show signs of a cambial zone. However, this
cambial zone displays only limited secondary growth, which is more evident nearer
the base of the leaf and (if present) down in the petiole, where the vasculature of the
main vascular supply to the leaf, assumes a more cauline appearance (that is, it is
more stem-like). In contrast, minor veins lack associated mechanical supporting
tissue. Unlike the major network veins, the minor veins are usually embedded within
the interface between the palisade and spongy mesophyll layers. As mentioned,
4 Haritatos, E; Ayre, B G; Turgeon, R 2000. Identification of phloem involved in assimilate loading in leaves
by the activity of the galactinol synthase promoter Plant Physiol. 123, 929-937.
18
Developmental Plant Anatomy
minor veins are embedded within a horizontally orientated mesophyll, termed the
paraveinal mesophyll in some dicotyledons, which, judging by the relatively high
plasmodesmatal frequencies recorded between adjacent cells, is the principal
symplasmic solute conduction pathway from the palisade and spongy layers, into the
surrounding parenchymatous bundle-sheath cells, terminating at the sieve tubes
within major and minor veins.
Major and MInor Veins
Hypodermal
support tissue
Parenchymatous
bundle-sheath
Fig. 9. Diagram illustrating the differences between major and minor veins in a generalised
dicotyledonous foliage leaf.
The monocotyledonous foliage leaf
A commonly-mentioned anatomical feature of C4 plants is the orderly arrangement
of mesophyll cells with reference to the bundle sheath cells, forming concentric
layers around the vascular bundle as seen in transection. Bundle sheath cells of C4
plants have few if any intercellular air spaces between them, which is in direct
contrast to the sometimes large intercellular space volume between mesophyll cells
in C3 plants. Observations of the concentric arrangement of mesophyll and bundle
sheath cells of certain grasses and sedges, prompted Halberland to compare the
mesophyll layer to a Kranz (wreath-like) structure. The structure of
monocotyledonous foliage leaves depends to a large extent on the type of
photosynthesis (i.e. C3; C4) and on the environmental conditions that the plants grow
in (i.e. xerophytic, mesophytic, or hydrophytic). All monocotyledonous foliage
leaves are basically parallel-veined, but large numbers of cross-veins serve to
interconnect the parallel vein system. Parallel veins in monocotyledons are
classified as either large, intermediate or small.
Classification of leaf-blade vein order size is based upon the following criteria:
(a) Large bundles. These bundles are characterized by the presence of large
metaxylem vessels on either side of the protoxylem, which is often represented by a
lacuna. Obliterated protophloem is evident on the abaxial side of the bundle.
Conspicuous girders (either collenchyma or sclerenchyma) extend from the bundle
to either both ad- and abaxial leaf surfaces., or the abaxial surface only.
(b) Intermediate bundles. These bundles lack large metaxylem vessels and
protoxylem lacunae. Hypodermal strands or girders occur on both ad- and abaxial,
or on the abaxial surface only.
(c) Small bundles. In addition to lacking large metaxylem vessels and protoxylem
lacunae, these bundles are not associated with either hypodermal girders or strands.
Developmental Plant Anatomy. Core Notes. Page 19
Girder
Strand
X
PBS
P
X
P
Fig. 10. Illustrates the difference between a hypodermal strand (left) and a girder (right) in a
generalised monocotyledonous leaf.
Girders are defined as structures which interrupt the parenchymatous bundle sheath,
whist strands do not interrupt the bundle sheath.
Leaf blade bundle anatomy in grasses
Amongst the grasses, two anatomical variations are noteworthy - that is, the
Panicoid (Fig. 6) and Pooid (Fig. 7) groups.
Figs. 11 and 12. Line drawings based on electron micrographs of typical Panicoid (Fig.11)
and Pooid (Fig. 12) leaf blade bundle anatomy. PS=parenchymatous (Kranz) sheath,
BS=parenchymatous bundle sheath; MS=mestome sheath; VP=vascular parenchyma cell.
In the Panicoid grasses, the mesophyll is radially-arranged and surrounds a
parenchymatous bundle sheath. Panicoid grasses contain dimorphic chloroplasts,
with granal chloroplasts within the radiating (Kranz) mesophyll and generally agranal chloroplasts within the parenchymatous bundle sheath cells. Bundle sheath
chloroplasts are much larger than the Kranz chloroplasts and lack Rubisco - the
Calvin cycle is thus not supported within Kranz mesophyll cells.
Instead, these cells are associated with the initial incorporation of CO2 aspartate,
which is transported to the bundle sheath cells, via numerous plasmodesmata, where
malate or aspartate is decarboxylated, and the liberated CO2 is immediately
incorporated via Rubisco, into the Calvin cycle. Panicoid grasses are thus C4
photosynthetic species. In many Panicoid species, an additional cell layer exists
between the bundle sheath and the vascular tissues below. This layer, which consists
of thick-walled lignified cells, is termed the mestome sheath. Ontogenetically, the
20
Developmental Plant Anatomy
mestome sheath is derived from the procambium. Mestome sheath cells may either
completely surround the vascular tissue, or surround the phloem tissue only within
the vascular bundles. The middle lamella between the bundle sheath cells and, in
some species that between the mestome sheath cells, contains a suberized layer,
termed the suberin lamella. The compound middle lamella has been shown to
restrict the movement of solutes, forcing transport (i.e. photoassimilate inwards and
water outwards) to take a symplasmic route, via plasmodesmata. The suberin
lamella may have important ecological consequences, preventing the excessive
movement of water from the apoplast, under conditions of water stress. Pooid
grasses, like the Panicoid species, may be associated with a mestome sheath (Fig. 7).
Unlike the Panicoid grasses however, the Pooid species do not exhibit chloroplast
polymorphism, do not have compartmentalised Rubisco activity and all follow the
C3 photosynthetic pathway.
Fig. 13. Electron micrograph, showing a small transverse vein of Saccharum officinarum in
transverse view.
This vein is surrounded by a suberized bundle sheath and consists of a solitary tracheary
element (above,) a companion cell (lower right) one sieve tube (centre) and a
parenchymatous element (lower right). Note hydrolyses tracheary element cell wall bordering
the vascular parenchyma cell.
Whilst monocotyledonous foliage leaves are described as being parallel-veined,
large numbers of transverse veins exist within the leaf blade. Fig. 8 is an electron
micrograph of a transverse vein in such sites, the wall of the parenchymatic element
(reported here in Saccharum officinarum) may contain a well-developed suberin
lamella, which may have a regulatory role in solute loading and water loss from the
xylem to the mesophyll. Transverse veins such as that illustrated here, join adjacent
parallel veins, forming effective, interconnected transpiration and translocation
pathways.
Each transverse vein usually contains a single tracheary element and a solitary sieve
tube, with their associated parenchymatic elements. Although there are some reports
in the literature that transverse veins may lack functional sieve tubes, most, as
illustrated by S. Officinarum, do. The single file of sieve tubes are in direct contact
with the tracheary elements. Several files of parenchymatic elements are common,
and again, these are in direct contact with the tracheary and phloem elements in
these cross veins. Examination of the vein in Fig. 8, reveals that portions of the
Developmental Plant Anatomy. Core Notes. Page 21
walls of the tracheary element contiguous to the parenchymatic elements, lacks
secondary wall thickening. At such sites, the walls of tracheary and parenchymatic
elements appear swollen and loosely-fibrillar. In Zea mays, the distance between
transverse veins varies greatly (0.06-1.9mm) but is much greater than the
corresponding distance between the longitudinal veins. The presence of a bundle
sheath, which contains a suberized compound middle lamella, ensures that these
cross veins retain water and, like the parallel veins, that apoplasmic transport is
limited, forcing water and solutes to take a symplasmic route to the surrounding
mesophyll. Occasionally however, the transverse veins are discontinuous, and
mesophyll or intercellular spaces may be in direct contact with a parenchymatic
element of the vein. At such sites, the wall of the parenchymatic element (illustrated
here for S. officinarum) may contain a suberin lamella, which as stated, may have a
regulatory role in solute loading and controlling influence with respect to water loss
from the xylem to the mesophyll.
Leaf Anatomy in the Cyperaceae
Fig. 14. Line drawings showing the basic anatomical features of leaf blade bundle structure in
the Cyperaceae. The variation of cell thickness is most notable in the cell walls of the
endodermis. Note the distribution of chloroplasts in the border parenchyma and the presence
of large chloroplasts (agranal in some species) in the border parenchyma. Examples are:
Left: C. fastigiatus; C. esculentus; Mariscus congestus; centre: C. sexangularis; C. pulcher
and C. accutiformis; right: C. albostriatus; C. textilis and C. papyrus.
The accompanying line drawings (Fig. 14 above) illustrate the variation that has
been found amongst local Cyperaceae to date. Like the Poaceae, the Cyperaceae are
photosynthetically either C3 or C4. Like the Poaceae, the phloem within the leaf
blade vascular bundles in the Cyperaceae contain two types of sieve tube -- early,
thin-walled sieve tubes and late-formed thick-walled metaphloem, the latter
generally in close spatial association with the metaxylem, and lacking obvious
companion cells. Several anatomical variations are evident when Cyperaceae leaf
blade bundles are examined at the electron microscope level. The major differences
22
Developmental Plant Anatomy
in Cyperaceae compared to Poaceae, lies in the distribution of the chloroplastcontaining parenchyma and in the shape of and thickness of the wall of what has
been equated to the mestome sheath of grasses. The leaf blade bundle of C.
albostriatus is surrounded by a thickened and lignified mestome sheath, which is
devoid of obvious organelles and generally, this layer lacks chloroplasts. The
surrounding parenchymatous sheath too, contains few chloroplasts. Suberin lamellae
may be present in either outer tangential and/or inner radial tangential walls of this
mestome sheath layer. Closer examination of the phloem endarch to the mestome
sheath, reveals the presence of chloroplast-containing parenchymatous cells. This
layer has been referred to in the literature as 'border parenchyma' (BP, Fig. 10). In
contrast the C4 species contain a similar ring of chloroplast-containing parenchyma
cells endarch to the thickened, lignified ring (again referred to in the literature as the
‘mestome sheath’). However, these chloroplasts are large and obviously agranal.
Chloroplast dimorphism, lack of grana in these primary carbon reduction (PCR)
cells are indicative of the C4 syndrome. Experimental evidence exists for the positive
localisation of Rubisco in these large agranal chloroplasts only, which is additive
support for the plants being C4..
A possible explanation of the origin of the C4 syndrome in Cyperaceae is illustrated
in Fig.10. Fig.10a shows the typical C3 anatomy of many Cyperaceae species, in
which the parenchymatous bundle sheath encloses a lignified mestome sheath layer,
which, in turn, surrounds the xylem and phloem.
The border parenchyma zone (Fig.10b) commonly encircles either both xylem and
phloem, or only the phloem. Possibly, the C4 syndrome evolved in Cyperaceae with
leaf blade anatomies similar to that depicted in Fig.10b, which would have required
the loss of Rubisco activity in the mesophyll (PCA) cells, concentration of Rubisco
activity within the parenchymatous bundle sheath (PBS), loss of grana from border
parenchyma cells and hence, division of the photosynthetic process into primary
carbon assimilation (PCA, producing malate or aspartate) the export of malate or
aspartate through the mestome sheath-like cell layer, to the internally located
parenchymatous sheath. Anatomically, there are distinct similarities between the C4
Cyperaceae (Fig.10c) and C4 Panicoid (Fig.10d) Poaceae.
One major problem which still requires resolution is the terminology used to
describe the layers of lignified cells separating the PCA from the PCR cells - do we
call this layer a mestome sheath or not? Its position between PCA and PCR cells
seems to dictate that the layer should not be termed a 'mestome' sheath - rather an
endodermoid sheath, as it completely encases the underlying PCR and vascular
tissue. However, this will not be resolved without a detailed ontogenetic study of
leaf blade development.
Developmental Plant Anatomy. Core Notes. Page 23
A
D
C
D
Fig. 15. Diagrams showing possible evolutionary pathways, leading to the development of the
unique arrangement of the C4 subtypes in the Cyperaceae.
In C3 Cyperaceae, the vascular bundles are surrounded mesophyll and a
Cyperaceae, the vascular bundles are surrounded mesophyll and a parenchymatous
bundle sheath, beneath which, is a lignified mestome sheath. Anatomically, C3
Cyperaceae are similar to C3 Poaceae. Many of the Cyperaceae have an additional
parenchymatous layer beneath the mestome sheath, which contains granal
chloroplasts, forming border parenchyma., thus separating the mestome sheath from
the underlying vascular tissues. C4 The evolutionary step to Cyperaceae from the C3,
border-parenchyma type, may simply have required the loss of grana, coupled with
the compartmentalisation of Rubisco in these now agranal chloroplasts. The question
which remains unanswered, is , is the lignified layer a mestome sheath or is it an
endodermoid sheath? Typical C4 Poaceae bundle anatomy is illustrated above right
for comparative purposes.
24
Developmental Plant Anatomy
4. Phloem translocation
It is now more than one hundred and fifty years since Hartig first recognised and
associated phloem transport with sieve-elements through which all the cellular
foodstuffs are transported throughout the plant. Since then, much effort has been
expended in the elucidation of their structure and their mode of function in the plant.
The evidence is clear that the companion cell has an important role in the transport
system. In dicotyledonous plants the phloem and its associated companion cells can
be easily recognised. In monocotyledonous plants on the other hand, companion
cells are not so easily recognised. It is clear that the companion cell, and its
counterpart in the Gymnosperms, must be involved in the supply of energy to
maintain the metabolic integrity of the sieve tube and sieve cell. The recent findings
based on plasmolytic and plasmodesmatal frequency studies, lend support to several
mechanisms which may be involved in the two main phases of phloem transport,
namely:- Short-distance (from the mesophyll to the sieve tube) and long-distance
(from source sieve tubes to the various sinks) translocation. It is apparent that
osmotic forces have an important role in this transport system, which does not
preclude a requirement for considerable metabolic energy to maintain membrane
integrity and to assist with the loading process itself.
Structure-function relationships
The growth of plants and their normal functioning requires the integrated activity of
a variety of processes. It is clear that the absorption of water and of nutrients and
mineral elements by roots, as well as the biochemical processes of photosynthesis
and respiration is elementary to the normal functioning of plants. Add to this other
factors such as water stress, and a new gamut of potential controlling or regulating
factors emerge.
The transport of soluble carbonate from a region of synthesis (source) to a region of
biochemical utilisation (sink) is thus an essential process which all plants must
complete successfully if they are to increase their biomass significantly. The
transport pathway and its function will be dealt with in detail later.
The symplasm-apoplasm concept
The terms symplasmic and apoplasmic provide a useful means of discussing the
process whereby substances move through plants. According to this concept, the
term symplasmic refers to the total mass of the living cells in a plant in which the
individual protoplasts are interconnected via plasmodesmata. The plasmalemma
traverses the plasmodesmata, thus all protoplasts of living cells are interconnected,
forming a continuum. The symplast thus constitutes the sum of the total living
protoplast. The tonoplast is the limiting membrane which separates the protoplast
from the vacuole. Vacuoles are thus individual, and do not form part of the
cytoplasmic continuum directly. For example, ions taken up by the roots will be
taken up through the plasmalemma, and may migrate from cell to cell without
having to cross a permeability barrier. Movement from cell to cell in this simple
example is diffusive, and can be accelerated by protoplasmic streaming.
Developmental Plant Anatomy. Core Notes. Page 25
In contrast, the apoplast is the sum of all the non-living cell wall and its associated
intercellular spaces, forming a continuum of the plant, through which substances
such as water and solutes may move freely.
It has been proposed that the sieve tube constitutes a highly specialised phase of the
symplasmic transport pathway in plants. The principal features of their structural
and functional specialisation are the loss of nuclei at maturity, lack of the tonoplast,
parentally arranged cytoplasm and their continuity. The xylem is considered to be a
specialised phase of the apoplast, allowing rapid, long-distance transport of water
and dissolved inorganic and organic solutes, from roots to shoots. These conduits
have lost their protoplasts during differentiation and allow virtually unimpeded
flow.
It is important to remember the close spatial relationship between the xylem and
phloem throughout the of the living plant. One should also bear in mind the fact that
the functioning of the phloem is dependent on an adequate supply of water, as water
affects the osmotic potential of the contents of the sieve tubes, driving up the
pressure, and thus forcing the accumulated solutes to move away, carried by the
solvent.
Structure related to function
The xylem and phloem form the principal conduits for apoplasmic and symplasmic
transport in plants. Each function in their own unique way to ensure that the
translocation processes take place.
In the Table over the page, the principle anatomical and physiological differences
between xylem and phloem are compared. These systems, are the basis of the
apoplasmic (xylem) and symplasmic (phloem) transport systems within all vascular
plants. Other differences will manifest themselves, according to whether the plant is
a Gymnosperm, an Angiosperm (dicotyledonous or monocotyledonous) or any other
vascular plant.
26
Developmental Plant Anatomy
Comparison of Characteristics of Xylem and Phloem
XYLEM
PHLOEM
Principal conduit: vessel + tracheid
Sieve tubes
Composed of: elements
Sieve tube members Sieve areas at ends of
members and sometimes have lateral sieve
areas
Have open ends.
Long and narrow, to short and wide
Primary and secondary wall which is rigid
withstands
negative
(suction)
pressure
withstands
positive pressures -- extensible.
nacreously thickened primary wall
have
PHYSIOLOGICALLY
Dead
at
maturity
(no
Permeable to solute and solvent
cytoplasm)
Low sap conc. -5 to -15 atm Negative turgor
Semi-permeable. Alive at maturity contains
cytoplasm and include may not contain a
nucleus at maturity.(M + C).
High sap conc. (-15 to -34 atm)
High turgor when plants turgid
Partially collapsed when functioning. Absorb
water or air when cut
Exude sap when cut. Distended by pressure
when functioning
Translocate both solute and solvent. Rate up to
4,500 cm/hr
Translocate solutes - from 1.5cm/hr Salix
sp. to 7,200cm/hr as in Glycine max.
The Pathway of Translocation:
Early work on phloem transport was largely concerned with the mechanism
responsible for solute movement. Mason and Maskell's important early research
showed however, that regardless of the mechanism of transport, that movement of
assimilated material takes place from assimilating green tissues, to non-green,
growing, and actively respiring tissues in the plant. Munch (1930) presented a
simple hypothesis which illustrated his concept of phloem transport in plants, in
which osmotic forces alone, could account for the movement from source to sink.
Phloem exudate has been examined from many economically important plants, a
large number of ornamentals and weed species. The average dry matter content of
phloem exudate is roughly 10-25%. Of this, about 90% appears to be sugar. Amino
acids may make up about 0,5% of the exudate in the summer months, and
considerably more in the autumn months. According to Crafts and Crisp, there is a
general paucity of enzymes, with the exception of the alkaline and acid
phosphatases. These enzymes may have a role in the translocation process, perhaps
by assisting in membrane maintenance, or perhaps with the transport process itself.
Evidence for phosphatases is not circumstantial, in that they have been detected for
the past 15-20 years at the electron microscope level.
Developmental Plant Anatomy. Core Notes. Page 27
The work of Mason and Maskell (1928a, b): Annals of Botany 42: 189-253; 571636 demonstrated that regardless of mechanism, movement of assimilates takes
place from green assimilating tissue to non-green, growing and actively respiring
tissue. In short movement was from a source to a sink. Despite the green leaves of
plants representing the major sources of organic foods and hence serving as the
major activating organs for translocation it has been shown that storage tissues (i.e.
corms and tubers) may act as local sources in a temporary capacity, at the onset of
growth from such organs.
There are three experimental procedures by which the content of the sieve tubes and
their rates of translocation can be studied:
1. Exudation from bark or stem incisions
2. Exudation from cut stylet tips, and
3. Analysis of honeydew droplets from aphids.
1. Bark incisions
All the early work on phloem exudation and composition was done by simple
incisions - such exudate although contaminated with the contents of cut parenchyma
cells nevertheless gives a reasonably accurate picture of the contents of a
functioning sieve tube, and thus, the assimilate stream.
2. Exudation from aphid stylets
Kennedy and Mittler (1953) first reported (Nature 17: 528) on the stylet technique
as a satisfactory means for the collection of phloem exudate. These type of
experiments have yielded the data on substances translocated in sieve tubes
mentioned earlier.
Zimmerman (1961, 1963) was the first to illustrate the location of an aphid stylet tip
in an individual sieve tube - (I shall show this picture later on) - proof that the cut
stylet technique of analysis yields only phloem exudate.
3. Analysis of honeydew
It is preferable, when conducting experiments on translocation via phloem to disturb
the system as little as possible. However, analysis of honeydew droplets will not
yield as accurate a picture of the composition of the sieve tube sap as will the
excised stylet method, but the disturbance caused by severing the stylets of a feeding
aphid with the resultant release of sap via the feeding canal of the stylet groups, is
much greater than if the aphid colony is just left to feed on happily in its own time.
The advantage of utilising a colony of feeders rather than severed stylets is that one
may determine the rate of translocation via the phloem more accurately than by
severing the stylets - which would result in a rapid loss of pressure within the
systems.
This is particularly advantageous if long-term experiments involving the
translocation of radioisotopes is envisaged.
Distribution patterns:
Here we can generalise and state that:-
28
Developmental Plant Anatomy
(1) The lower leaves of a plant export primarily to the roots
(2) Upper leaves export to the apex
(3) Intermediate leaves export in both directions
(4) Mature leaves only export
(5) Young leaves only import
It is important to realise however, that the above serves only a generalisation and
that translocation may vary with the size, shape and general organization of the plant
and is also dependent on the presence or absence of reproductive organs and fruits.
The source
It is commonly accepted that the leaves of a plant constitute the source, and that the
main axis, root system, meristems and fruits constitute the sinks. However, the work
of Weatherley and associates 1959 (J. Exp. Bot. 10: 1-16) dispels this simple
picture. Using severed aphid mouthparts, they come to the conclusion that the whole
phloem makes up an integrated system of sources and sinks. The sucrose potential
being the factor most intimately involved in determining the direction in which this
material will be transported at any one time and under any [one] set of conditions.
Most work on source sink relationships are complex, subject to the influence of
light, temperature, water, growth storage capacity, senescence, disease and many
other factors. With this complexity in mind then, let us look more closely at the
source itself.
Obviously, photosynthesis is the primary process that provides the green portions of
a plant with the solutes responsible, through the process of osmosis, with driving
force of pressure flow.
What about the anatomical and physiological factors which enable the green tissues,
leaves, stems, roots, shoots and fruits to activate the translocation process? In order
to understand this we need to examine the role of leaves in transport.
Function, movement and regulation within the phloem and allied
parenchymatous tissues
The phloem is a conduit through which many substances can travel, carried for the
most part, by tha assimilate stream. As such, it presents a point of entry for
undesirable elements, such a viruses. Over time, plant phloem has developed means
to contain, to regulate or even to block the movement of potentially harmful entities.
The discourse that follows, emphasises just a few examples of how regulation or
prevention is carried out.
The relationships between the sieve element and the companion cell are central to
the function of the phloem. The companion cell-sieve element complex has been
described as a ‘traffic control centre’5 For example, recent evidence for the
localization of sucrose synthase, and its apparent companion-cell specificity in
phloem unloading zones of citrus fruit, as well as in intermediated and small veins
5
Oparka, K.J., Turgeon, R. 1999. Sieve Elements and Companion Cells—Traffic Control
Centers of the Phloem. Plant Cell, 11: 39-750.
Developmental Plant Anatomy. Core Notes. Page 29
in Zea mays and Citrus paradisi where high activity of sucrose synthase has been
demonstrated in vascular bundles during periods of rapid import. Curiously, sucrose
synthase protein was not associated with adjacent cells surrounding the vascular
strands in this tissue. The companion-cell specificity of sucrose synthase in phloem
of both importing and exporting structures of these diverse species, implies that this
may be a widespread association and underscores its potential importance to the
physiology of vascular bundles and of the intimately bound functions of the
companion cell-sieve element complex. Molecular biology studies using
Arabidopsis, direct the way forward to a clearer understanding of the unique
relationship that exists between these cells. Arabidopsis plants6 have been used to
monitor the movement these green fluorescent proteins (GFP-fusion proteins) from
companion cells into the sieve elements, and the subsequent translocation in the
phloem and unloading at distant sinks was monitored using various techniques,
including confocal microscopy. From studies like these, it is now apparent that the
pore plasmodesma connecting sieve elements to their companion cells, have a
molecular size exclusion limit (SEL) of more than 67 kDa in loading phloem. In
contrast, unloading in the roots is restricted to a narrow band of parenchymatous
cells associated with the companion cell sieve element complexes, forming a
restrictive domain, which controls the movement of large proteins into the root tip,
by limiting the SEL to 27-36 KDa.
Viroids are highly structured plant pathogenic RNAs that do not code for any
protein, and thus, their long-distance movement within the plant must be mediated
possibly through some direct interaction with cellular factors, which at this time
remain to be described. In addition to viroid RNAs, there is convincing recent
evidence which indicates that endogenous RNAs may move through the phloem,
and in so doing, that they acting as macromolecular signals which may be involved
in plant defence and development. The form in which these RNA molecules are
transported to distal parts of the plant remains, as yet, unclear. Gomez and Pallas
(2004) argue that viroids have the potential to be excellent model system which
could be used to try to identify translocatable proteins that could assist the vascular
movement of RNA molecules7. These authors have demonstrated that the phloem
protein 2 from cucumber (CsPP2) is able to interact in vivo with viroid RNA and
that the translocated viroid is symptomatic, and that the CsPP2 protein may assist in
the long-distance movement of the viroid RNA within the plant.
A protein, CmPP16 from Cucurbita maxima has been cloned8 and which has been
shown to have properties similar to those of viral movement proteins. The
messenger RNA (mRNA) for CmPP16 is present in phloem tissue, whereas the
protein itself, appears to be confined to sieve elements (SE). Microinjection and
6
Stadler R, Wright KM, Lauterbach C, Amon G, Gahrtz M, Feuerstein A, Oparka KJ, Sauer
N. 2005. Expression of GFP-fusions in Arabidopsis companion cells reveals non-specific
protein trafficking into sieve elements and identifies a novel post-phloem domain in roots.
Plant J. 41:319-31.
7
Gomez G, Pallas V 2004. A long-distance translocatable phloem protein from cucumber
forms a ribonucleoprotein complex in vivo with Hop stunt viroid RNA. J Virol. 78:10104-10.
8 Xoconostle-Cazares B, Xiang Y, Ruiz-Medrano R, Wang HL, Monzer J, Yoo BC, McFarland
KC, Franceschi VR, Lucas WJ. 1999. Plant paralog to viral movement protein that potentiates
transport of mRNA into the phloem. Science. 283:94-8.
30
Developmental Plant Anatomy
grafting studies revealed that CmPP16 moves from cell to cell, mediates the
transport of sense and antisense RNA, and moves together with its mRNA into the
SE of scion tissue. CmPP16 possesses the characteristics that are likely required to
mediate RNA delivery into the long-distance translocation stream. Thus, RNA may
move within the phloem as a component of a plant information superhighway.
Recent research has shown that phloem-mobile endogenous RNA is trafficked
selectively into the shoot apex of N. benthamiana. In contrast, most viruses and
long-distance post-transcriptional gene silencing (PTGS) signals are excluded from
the shoot apex. These observations suggest the operation of an underlying regulatory
mechanism. A potexvirus movement protein, known to modify cell-to-cell
trafficking and PTGS, was expressed ectopically in transgenic plants in order to
examine this possibility further. Plants in which the potexvirus movement protein
was expressed, were found to be compromised in their capacity to exclude both viral
RNA and silencing signals from the shoot apex. This resulted in the transgenic
plants also displaying various degrees of abnormal leaf polarity, which was
dependent on the level of transgene expression. Normal patterns of organ
development were restored by either virus- or Agrobacterium tumefaciens–mediated
induction of PTGS. The experiments by Foster et al. (2002)9 thus demonstrated that
an RNA signal surveillance system, which can act to allow the selective entry of
RNA into the shoot apex exists. The authors proposed that this surveillance system
regulates signalling and protects the shoot apex, in particular the cells that give rise
to reproductive structures, from viral invasion.
9
Toshi M. Foster, Tony J. Lough, Sarah J. Emerson, Robyn H. Lee, John L. Bowman, Richard L. S. Forster
and William J. Lucas 2002. A Surveillance System Regulates Selective Entry of RNA into the Shoot Apex.
The Plant Cell, 14:1497-1508.
Developmental Plant Anatomy. Core Notes. Page 31
5. Leaves and transport
Of prime importance is the pattern of venation. Most obvious here is the close
relationship between the mesophyll tissue and the vascular tissue.
For example, measurements of 6 species of Dicots revealed that the total length of
veins averaged 102 cm/cm.. of leaf blade. This thorough distribution of veins is
further reflected in the value of 430m for the average distance between veins. Esau
further points out a correlation between vein distribution and the structure of the
mesophyll. The larger the volume of non-conducting tissue, e.g. palisade, the closer
together are the vascular bundles.
Thus sun leaves having a strongly developed palisade layer, will generally speaking
have a better developed venation pattern and have greater lengths of vein tissue than
shade plants.
2. Physiologically: Despite the thorough distribution of vein tissue in leaves,
diffusion alone could not account for the rapid export of labelled assimilates from
leaves reported.
Protoplasmic streaming undoubtedly accelerates the movement of molecules from
chloroplasts to the phloem. Cyclosis is an active process which carries the plastids
around the cytoplasm, in such a way as to "stir" up the cytoplasm ensuring that the
cytoplasm is never very far from a pit area through which export may take place.
Many plants do not exhibit such movement but the strands of cytoplasm show a
streaming type of movement of varying intensity.
Observation of structure of major and minor veins shows very clearly the close
relationship between the tracheids in these veins and the sieve tubes. Often these
vascular elements are in direct contact, a fact which tends to serve as an illustration
of the semi-permeable nature of the sieve tube plasmalemma. Where the sieve tube
plasmalemma not impervious to assimilate molecules the pressure flow mechanism
would fail to operate.
Esau also describes the layer of parenchyma cells which completely surround the
small and minor veins of a leaf. This bundle sheath, or as the cells are more
correctly known in dicotyledonous plants as the border parenchyma, may contain
chloroplasts, but there are usually less chloroplasts here than in the palisade and
spongy mesophyll.
The bundle sheath must function in the passage of molecules between the palisade
and the sieve tubes by symplasmic movement. Their protoplasts are also bathed by
transpiration water and are therefore supplied with the inorganic nutrients adsorbed
and absorbed by the roots.
In terminal bundles the difference in size and contents between the border
parenchyma and companion cells may be minor or lacking.
Transfer cells of minor veins are in the same category. Note that companion cells,
border parenchyma and so-called transfer cells are in all instances (in minor veins at
least) larger in cross-sectional area than their corresponding sieve tubes.
32
Developmental Plant Anatomy
Is metabolic energy required?
Many of the workers active in this field insist that metabolic energy is required for
the movement of assimilates from border parenchyma into the sieve tubes. However,
I must point out that my far the majority of the research dealing with this topic is of
the mass analysis type, which affords no measure of the actual concentrations of
sugars present in the symplast.
It is very probable that sugars move along this route in phosphorylated forms.
However, there is no stoichiometric relationship between sugars and phosphorus in
the phloem sap, i.e. the K+ must be recycled to the palisade cells, possibly via the
transpiration stream after removal(?) from phloem sieve tubes by companion cells
for example.
It is often very difficult to determine whether a given cell in a vein ending is a
phloem parenchyma cell or a companion cell. Some of these nucleated cells stain
more densely and have more granular contents than others, there is no sharp
demarcation between the different nucleate cells. This may well reflect a similarity
of function of nucleate cells in this position. The association of sieve tubes,
companion cells and phloem parenchyma cells in the minor veins suggests a cell
system concerned with transfer of sugars from the mesophyll to the phloem
conduits.
To return for a moment to the nucleate cells surrounding the sieve-tubes, early
workers referred to these as ubergangzellen (transitional cells). The use of the term
ubergangzellen.) still persisting in much of modern-day literature. Pate Gunning and
Briarty (1968) decided to name these cells transfer cells, because by implication,
they were involved in the transfer of materials from one physiological cell type to
another. In this context two types – First, those that transfer to solutes from xylem to
mesophyll and sieve tubes (xylem transfer cells), second, those associated with
sieve tubes and companion cells (phloem transfer cells). Note also here that in
leaves especially in minor veins, companion cells occupy greater cross-sectional
area than do the sieve tubes.
Esau (1967) showed the intimate relationship between sieve tubes and tracheids.
She said that in minor veins, the primary wall of mature tracheids may be largely
disintegrated, so that the walls of the adjacent parenchyma cells and sieve tubes are
virtually in contact with the contents of the tracheary elements - thus the apoplast
could well serve as a rapid exchange route for solutes moving in the transpiration
stream. Thus overall there is an effective exchange mechanism in existence between
the xylem and phloem and photosynthetic cells.
Accepting the common concept that the movement of sugars out of the
photosynthetic and into the phloem depends on active metabolic energy transfer that
must involve enzyme function, she pointed out the strong phosphatase activity of
phloem parenchyma, and the abundance of ribosomes shown in these cells by the
electron microscope.
Thus it may seem that the nucleate cells of the phloem may have an active carrier
system bringing about the (active) transfer of sugars across membranes by a
succession of phosphorylations and dephosphorylations, resulting in their secretion
into the sieve tubes.
Developmental Plant Anatomy. Core Notes. Page 33
Turgor may therefore be easily established in the sieve tubes due to closeness of
sieve tubes to tracheary elements. The sensitivity of transport rates in the phloem to
water deficits reflects this association.
However, the physiological proof remains to be shown for the theory of
phosphorylation in translocation
Mechanisms of phloem loading
In higher plants, sugars and sugar alcohols form the bulk of the material, apart from
water which is transported from the leaves. Sucrose is the most commonly
transported sugar. The presence of only non-reducing sugars in the phloem sap is a
reflection of the selectivity of phloem which is an important factor in the control of
export of assimilates from the leaf.
The loading of sugars into the companion cell-sieve tube complex from the apoplast
is an active, energy-dependent process. Until recently, little or no information was
available about this process at the cell membrane level. Within the past 2-3 years,
several workers have provided evidence which indicates that the driving force for
solute accumulation may be provided by a proton pump mechanism, which is
energised by ATP, at the plasmalemma, and involving a sucrose proton co-transport
system.
The cytochemical localisation of considerable ATP-ase activity at the plasmalemma
provides concrete evidence for the involvement of this enzyme in phloem loading.
ATPase occurs in phloem parenchyma cells, transfer cells, and in companion cells,
lending support to the idea of an ATP-driven electrogenic proton co-transport
system at that site
How does this mechanism operate?
Results obtained from work by Malek and Baker using Ricinus indicated that a
coupled H+ efflux K+ influx pump is envisaged which establishes a proton gradient,
down which protons move from the free space into the sieve element, cotransporting sugar inwards. (N.B. mention was made earlier that only non-reducing
sugars were selectively taken up into the phloem). Such a scheme is consistent with
the composition of phloem sap, its high pH, High [K+], and with the action of
inhibitors on the loading and long-distance transport. Inhibitors are presumed to
inhibit the function of the H+ K+ ionic pump mechanism.
In addition to controlling the composition of the organic substances transported
from the leaf, phloem loading serves as the source of the driving force for the mass
flow of the assimilate stream. With an increase in solute concentration in the
companion cell-sieve tube complex resulting from active loading, a hydrostatic
pressure gradient is set up across the plasmalemma. Water, supplied by the xylem,
then enters the sieve tube and initiates a flow of solution down the translocation
pathway.
Transfer cells:
Parenchymatic cells of the phloem in the minor veins of some herbaceous
dicotyledons develop wall ingrowths, that result in a greatly increased plasmalemma
surface. Such parenchymatic cells are called transfer cells and are of two types, viz.
A and B. A-type transfer cells are companion cells with typically dense protoplasts
34
Developmental Plant Anatomy
and wall ingrowths on all walls, but often less abundant on the wall where
plasmodesmata connect the transfer cell with the sieve tube. B-type transfer cells are
phloem parenchyma cells with cell ingrowths best developed opposite the sieve
elements or their companion cells.
Two further categories of transfer cell exist - "C" and "D" - modified xylem
parenchyma and bundle sheath cells respectively.
Two main roles have been proposed for phloem transfer cells.
1. to collect and pass on photosynthates and ;
2 to retrieve and recycle solutes that enter the leaf apoplast in the transpiration
stream. Several workers regard the "cell wall membrane apparatus" of the transfer
cell as an adaptation facilitating apoplast-symplast exchanges of solutes across the
plasmalemma.
Transfer cells are never present in monocotyledons . However, Evert and his coworkers have recently observed tubular extensions of the plasmalemma on vascular
parenchyma cells and also in other cell types in Zea mays. It has been suggested that
the absence of transfer cells and the presence of tubular extensions may result in the
same process being carried out by these tubular extensions.
It is important however to note that all parenchymatic cells, with or without wall
ingrowths, or tubules associated with the walls of sieve tube members may be
concerned with the collection and or retrieval of photosynthates and other solutes
and their transfer to the sieve tubes.
Phloem loading
Turgeon (2000) 10 recently stated that “Photoassimilates and other substances
accumulate against concentration gradients in the phloem, a process known as
loading. In mature leaves, the sieve element-companion cell complexes (SE-CCCs)
of minor veins, where loading occurs, are connected to surrounding cells by
plasmodesmata. These pores appear to participate in loading in plants that
translocate raffinose and stachyose, but in sucrose- and polyol-translocating species
their function is less certain. Indeed, large numbers of plasmodesmata between the
SE-CCCs and surrounding cells should cause a dissolution of the concentration
gradient unless the size exclusion limit of the pores is small enough to retain
accumulated solute species. In leaves of willow, Salix babylonica L., a sucrosetranslocating plant with a high degree of symplastic connectivity into the minor vein
phloem, the sucrose concentration gradient is absent between mesophyll and
phloem, leading to the conclusion that phloem loading does not occur. Once inside
the SE-CCC, solute may be able to pass freely between sieve elements and
companion cells since they are also symplastically connected. However, due to net
flux into the sieve tubes in source leaves, this should cause a continual drain of
metabolic intermediates out of companion cells. It is proposed that this transport
step is regulated in minor veins to prevent continual loss of needed solute molecules
to the translocation stream.”
10
Turgeon, R. 2000. Plasmodesmata and solute exchange in the phloem. Australian Journal of Plant
Physiol. 27: 21-529.
Developmental Plant Anatomy. Core Notes. Page 35
In Arabidopsis thaliana 11 Phloem parenchyma cells appear to be specialized for
delivery of photoassimilate from the bundle sheath to sieve element-companion cell
complexes: they make numerous contacts with the bundle sheath and with
companion cells and they have transfer cell wall ingrowths where they are in contact
with sieve elements. Furthermore, these authors state that plasmodesmatal
frequencies are high at interfaces involving phloem parenchyma cells. The
plasmodesmata between phloem parenchyma cells and companion cells are
structurally distinct in that there are several branches on the phloem parenchyma
cell side of the wall and only one branch on the companion cell side. Most of the
translocated sugar in A. thaliana is sucrose, but raffinose is also transported. Based
on structural evidence, Haritatos et al (2000) state that the most likely route of
sucrose transport is from bundle sheath to phloem parenchyma cells through
plasmodesmata, followed by efflux into the apoplasm across wall ingrowths and
carrier-mediated uptake into the sieve element-companion cell complex.
Phloem loading and phloem translocation are thus important determinants of plant
growth and are topics, which have been reviewed frequently over the past years.
Our understanding of phloem loading mechanisms has been tempered by the quest
for a unifying concept of phloem loading. The (thermodynamic) elegance of the
apoplasmic phloem-loading concept conveniently met the demand for such a
universal loading mechanism. Investigators envisaged the existence of a sort of
‘Krebs Cycle’ of phloem loading, an expectation that made different forms of
phloem loading (apoplasmic vs. symplasmic) seem incompatible. Not unlike the
argument for or against particular transport mechanisms, arguments in favour of the
one concept were taken as arguments against the other. In recent years the most
notable change in the conceptual thinking about phloem loading has been the
growing tendency to see it as a process that may differ among the various plant
families. This review presents the ultrastructural and physiological arguments
leading to a more flexible concept of phloem loading. The emerging picture is of
higher plants using various strategies of phloem loading to achieve the export of
photosynthate from the source leaf. The different strategies are argued to have
ecophysiological significance, as they may well be associated with specific environmental conditions.
11
Haritatos E.; Medville R.; Turgeon R 2000. Minor vein structure and sugar transport in Arabidopsis
thaliana Planta 211: 105-111
36
Developmental Plant Anatomy
Sieve-element loading and phloem loading
pms
pm
st
bs
st
cc se
pvm pvm
bs
smc
sm
production
collection
transport
& export
Fig. 16 The diagram above (redrawn and modified after the diagram kindly provided by
Professor AJE van Bel University of Giessen Germany) highlights current thinking about the
processes involved in phloem loading. Here, three phases are recognised – a production
compartment (the mesophyll) a collecting compartment (the companion cell-sieve element
complex (and an transport & export compartment ,made up of the total transport phloem in
the leaf, connected to that in the stem.
The intricacy of potential transport pathways leading to the sieve elementcompanion cell complex (SE/CC-complex) has given rise to considerable ambiguity
as to the precise nature of the term "phloem loading." Following the proposal by
Oparka and van Bel, a distinction must be made here between the terms phloem
loading and sieve element loading. These authors have suggested that phloem
loading refers to the entire pathway that photosynthates follow from the mesophyll
to the SE/CC-complex. Sieve element loading on the other hand, strictly describes
the entrance of photosynthate into the SE/CC-complex. Sieve element loading is
thus described as symplasmic, provided that demonstrated functional symplasmic
continuity exists between the SE/CC-complex and the adjoining cells.
When solutes enter the SE/CC-complex via the plasmamembrane, but from the free
space surrounding the SE/CC complex (i.e., the cell wall) then sieve element
loading is termed apoplasmic. The term symplasmic phloem loading is employed
when the entire functional pathway, to and including the SE/CC-complex, is
symplasmic (in other words, assumedly functional plasmodesmata must occur in
reasonable numbers, along the entire path). Phloem loading is termed apoplasmic if
plasmodesmata are (virtually) absent at some point in the pathway irrespective of
the site of symplasmic discontinuity. According to these definitions, phloem loading
in a species can be termed apoplasmic, while the sieve element loading is
symplasmic.
Sieve element loading, phloem loading, and the spatial distribution of phloem
loading activity over the vein network are determined by three partly overlapping
Developmental Plant Anatomy. Core Notes. Page 37
levels of structural complexity. Knowledge of this structural framework is vital to
our understanding the phloem loading mechanism.
Ultrastructural features of the cells in the loading zone
Several features need to be discussed - in particular, the distribution of
plasmodesmata and their relative frequencies; the structure of the cells in the
loading pathway; the functional relationships between mesophyll and the cells along
the pathway to the vascular tissue.
Phloem loading zone structure
Identification of the minor vein configuration alone is not sufficient to assess the
nature of the phloem loading pathway, as sieve element-loading is only the last step
prior to phloem loading. Because the phloem loading pathway extends from the
mesophyll to the sieve elements, symplasmic connections between the SE-CC
complex and other vascular and extra-vascular cells may be equally important in
determining the pathway and mechanism of phloem loading.
Plasmodesmatal frequency and distribution
The plasmodesmatal frequency between cells in the phloem loading pathway may
have a strong influence on the mode and rate of phloem loading. Plasmodesmatal
connectivity between cells, in general, is presumed to be an important determinant
of intercellular transport The assumption is that the greater the number of
plasmodesmata at a given interface, the greater is the potential for symplasmic
transfer across that interface. The plasmodesmatal frequency between the
SE/CC-complex and the adjacent cells in various species shows a vast range of
symplasmic continuity.
Gamalei has distinguished several gradations (types) of plasmodesmatal
connectivity between the SE/CC-complex and adjacent cells in dicotyledonous
families: [Type 1] exhibits abundant plasmodesmatal contacts, [Type 1-2a] exhibits
moderate contacts, [Type 2a] sporadic, and [Type 2b] virtually no plasmodesmatal
contacts. The types differ in frequency by about a factor of 10, so that the span in
plasmodesmatal frequency between Type 1 and Type 2b is in the order of 103.
The quantitative diversity in symplasmic contacts may correlate with differences in
the architecture of the minor veins and the ultrastructure of the companion cells. The
sieve elements in the minor veins are accompanied by cells with a diverse
ultrastructure.
Evidence has been presented for a cristae-rich mitochondrial network in the
companion cells of many species, that occupies approximately 20% of the cytosol,
in some dicotyledons (e.g. Cucurbitaceae). Together with the density of the
cytoplasmic matrix an expanded mitochondrial network indicates high metabolic
activity. In Type-1 minor veins, the companion cells (intermediary cells in
Cucurbitaceae) may contain extensive endoplasmic reticulum labyrinths.
Chloroplasts are absent and other plastids may be small and scarce. In Type-2 minor
veins, the companion cells are smaller. Here, companion cells may contain several
small vacuoles and mitochondria are embedded in an exceptionally dense matrix. In
Type-2b minor veins, specialized companion cells (transfer cells) often develop
numerous cell wall invaginations.
38
Developmental Plant Anatomy
The companion-cell ultrastructure, the degree of symplasmic connectivity of the
SE/CC-complex, and the minor vein architecture has been collectively defined as
the minor vein configuration (see Figure 12). Occasionally, combined minor vein
configurations have been encountered, i.e. individual sieve elements in one minor
vein are symplasmically connected with neighbouring cells in a dissimilar way.
Combinations of Type 1 + Type 2a and Type 1 + Type 2b have been identified with
different ultrastructural properties of the companion cells in the same minor vein.
The minor vein configuration tends to stay the same within taxonomic families.
Only 12 (the polytypical families), out of 116 dicotyledonous families studied thus
far, possess more than one type of minor vein configuration. The obvious
supposition is that the minor vein configurations are associated with specific modes
of phloem loading. Type-l minor veins may perform symplasmic phloem loading,
whereas Type-2 minor veins may carry out apoplasmic phloem loading.
Fig. 17. Relationship between vein loading type and temperature and water availability.
Diagram provided by Prof Y Gamalei, Komarov Institute St Petersburg, Russia.
Tabulated data do not allow immediate appreciation of the spatial distribution of
symplasmic continuity, therefore, plasmodesmatal frequencies have been depicted in
several more imaginative ways. Possibly the quickest interpretation of the
plasmodesmatal patterns is obtained with plasmodesmograms. In these diagrams, the
striping density between the individual cell types has been used to represent the
plasmodesmatal frequencies. The striping visualises the absolute or relative number
of plasmodesmata per µm or µm2 of interface or per mm of vascular tissue. Later,
more quantitative methods have been evolved which suggest an assortment of
potential phloem loading pathways, in the dicotyledons and monocotyledons.
Developmental Plant Anatomy. Core Notes. Page 39
Diagnostic value of plasmodesmograms
Fig. 18. The plasmodesmograms show the % frequency of plasmodesmata, calculated on a
% average plasmodesma/m cell wall and a % per m cell wall interface. However, as stated
in Botha and van Bel 12
Obviously plasmodesmograms must be used with caution for a number of reasons:
1. A prominent disadvantage of the plasmodesmograms is that they reveal only
potential symplasmic pathways. Mapping of symplasmic continuity does not offer
any guarantee whether the plasmodesmata are operative or not, i.e. occlusion of, or
temporary closure of plasmodesmata are not visualised. Moreover, the permeability
of the plasmodesmata may be radically altered by endogenous changes, or changes
in the cellular. For instance, plasmodesmata of Chara are plugged with osmiophilic
substances or break at some stage of their development.
2. Even given the diverse structure of plasmodesmata it is unlikely that they differ
greatly in the mode of function. C4 grasses may have up to four different types of
plasmodesmata in the phloem loading pathway in leaves.
3. There may be large differences in the functional diameters of plasmodesmata.
Plasmodesmata of the bundle-sheath cells of C4-plants are wider in diameter than
those of other cells .
4. Plasmodesmata are aggregated in fields, which results in lack of homogeneity and
correspondingly large standard deviations in counting.
5. The plasmodesmograms merely provide information on the highest-order veins.
Lower-order veins with a disparate plasmodesmatal connectivity may also perform
phloem loading.
6. The first plasmodesmograms published were based on percent plasmodesmatal
frequencies in order to enable comparison between species. It has been argued that
the percentage distribution greatly underestimates the absolute number of
12
Botha, C. E J and van Bel, A. J E. 1992. Quantification of symplastic continuity as visualised by
plasmodesmograms: diagnostic value for phloem-loading pathways. Planta. 187: 359-366
40
Developmental Plant Anatomy
plasmodesmata at the mesophyll-bundle sheath interface, and plasmodesmograms
may be inadequate to allow prediction of intercellular assimilate fluxes.
Coincidentally, the efficacy of precise computations on flux rates on the basis of
plasmodesmatal frequencies is questionable. Symplasmic fluxes usually have been
calculated on the basis of percent interface coverage by plasmodesmata and the
diameter of individual plasmodesmata (20 - 130 nm) in EM-pictures. However,
because the operational diameter within plasmodesmata is about 2-3nm, the
symplasmic fluxes must be about 50 [252/.12)/g] times lower than those based on
the optical diameter provided that nine sub channels are available for transport
through the plasmodesma There is some correlation between the number of
plasmodesmata at the mesophyll-bundle sheath interface and the net assimilation
rate in grasses. The universal applicability of this observation to phloem loading
studies is doubtful, because the above-mentioned multiplicity of plasmodesmatal
structures and diameters renders the calculation of photosynthate fluxes virtually
impossible.
Thus it must be the diameter of the narrowest section in the symplasmic pathway,
which must be a determinant of the rate of photosynthate transfer.
Plasmodesmograms can only assist in the identification of the symplasmic
bottlenecks in the phloem loading pathway and may in addition, provide a useful
means to enable us to predict the symplasmic and apoplasmic stages in the phloem
loading process within a given species. Only if substantial differences in
plasmodesmatal frequencies occur between species, may quantitative comparisons
between species be justified.
The loading and transport sections of the phloem
Intuitively one presumes that phloem loading is located close to the vein endings in
the highest-order veins. However, the vein endings that protrude in the areole
formed by higher-order veins, often lack sieve elements in dicotyledons or have
sieve tubes ending at various intermediate points. The variation in sieve tube
arrangement in Rudbeckia is equal to that reported collectively for all angiosperms :
about a third of the vein endings are devoid of sieve tubes, only 10% have sieve
tubes extending to the tip and most vein endings (about 60%) have sieve tubes
terminating at some intermediate point. This enormous variability is likely to be
common among dicotyledons.
In dicotyledons, the most likely candidates for the major part of phloem loading are
the veins that encompass the areole, which is often demarcated by the two
highest-order veins. That these vein orders show a qualitative and quantitative
difference in phloem anatomy and introduces new difficulties to the assessment of
the phloem loading pathway. In Solanum, only abaxial sieve elements occur in the
7th-order veins, whereas in the 6th and lower-order veins adaxial sieve elements
also are present. In Cananga, the variability of the highest-order veins is considerable. To complicate the situation further, several dicotyledonous species (e.g. from
Cucurbitaceae, Lamiaceae, Solanaceae) possess two types of phloem, the sieve
elements of which are symplasmically connected with the mesophyll symplast in a
disparate fashion.
A high degree of anatomical diversity has been observed in the transverse veins of
grasses. The vein system consists of transverse veins the smallest branches of the
Developmental Plant Anatomy. Core Notes. Page 41
vein system in monocotyledons and small, intermediate, and large longitudinal
bundles. The transverse veins may be involved as storage units for co-ordinated
translocation in the longitudinal veins. It is doubtful, that transverse veins play any
major role in phloem loading.
In monocotyledonous families like Gramineae and Commelinaceae, the veins
contain early metaphloem (thin-walled) sieve elements and late-metaphloem
(thick-walled) sieve elements. These sieve elements have different symplasmic
continuities with the mesophyll. The function of the thin- and thick-walled sieve
tubes in photosynthate loading and export may be quite diverse. The thick-walled
sieve elements almost disappear in the leaf sheath of Saccharum, do not occur in the
large bundles of Zea, but contribute equally to the cross-sectional area in all vein
orders in other grasses. Thick-walled sieve elements do not seem to transport over
long distances in wheat, and may retrieve minerals and other solutes from the xylem
in maize.
It should be noted that the extremities of the minor veins in EM-sections are
sometimes difficult to locate with certainty. The minor veins may be spatially
unevenly distributed, especially in thicker leaves, and ranking of the vein order in a
cross-section thus may present some problems.
Potential phloem loading pathways
Linkage of vein ultrastructure and phloem loading physiology may be risky.
Nevertheless, it may be worthwhile to sketch the broad correlative outlines. The
central idea is that the degree of symplasmic connectivity in the phloem zone, which
may correspond to the mode of phloem loading. An abundance of plasmodesmata, in
particular at the interface between mesophyll and SE/CC-complex, may indicate a
continuous symplasmic pathway from the mesophyll to the sieve elements;
symplasmic phloem loading thus requires only one symplasmic domain. Absence or
paucity of plasmodesmata at some point in the phloem-loading pathway is presumed
to reflect a symplasmic constriction that necessitates apoplasmic transfer of
photosynthate. Apoplasmic phloem loading demands at least two symplasmic
domains: the mesophyll symplast (from which photosynthate is released) and the
SE/CC-complex symplast by which photosynthate is accumulated. Published
plasmodesmograms of the sieve elements loading zone suggest the existence of the
following modes of phloem loading
1. Symplasmic phloem loading in species with the Type-l minor vein configuration
commonly present in families with many trees and shrubs.
2. Apoplasmic phloem loading commonly present in families with the Type 2a or
Type-2b configurations. In these families herbaceous plants predominate, among
them important crop families of the temperate zone such as Asteraceae,
Brassicaceae, Chenopodiaceae, Fabaceae, and Solanaceae. Constrictions of the
symplasmic pathway at sites other than the SE/CC-complex indicate phloem loading
with successive apoplasmic steps. The plasmodesmograms of Vicia faba, for
instance, suggest a three-step mode of apoplasmic loading. Low densities of
plasmodesmata between mesophyll cells of Kalanchoe, sugar beet, and spinach
could also hint at extra veinal release of photosynthate and an inherent multi-step
apoplasmic phloem loading process.
42
Developmental Plant Anatomy
3. Combined symplasmic and apoplasmic phloem loading in families with the
Type-2a configuration. Here the potential for symplasmic sieve element-loading is
complemented by a potential apoplasmic sieve element-loading in the same sieve
element.
4. Parallel symplasmic and apoplasmic phloem loading in minor veins with sieve
elements differently connected to the mesophyll symplast. These "mixed"
configurations may fall in different categories: (a) Type-l (with intermediary cell)
and Type-2 (with common companion cell or transfer cell) sieve elements both in
the collateral minor vein, as in Coleus and Acanthus; (b) Type-l and Type-2 sieve
elements in parallel, respectively, at the abaxial and ad axial side, as in the
bicollateral minor vein of Cucurbita and Cucumis; (c) abaxial and ad axial Type-2a
sieve elements, with the abaxial SE/CC-complex slightly more symplasmically
isolated, as in the bicollateral minor vein of Solanum and (d) the thin- and
thick-walled sieve elements in the transverse or small veins in leaves of some
monocotyledons such as Zea, Themeda, Panicum, Saccharum and may other grasses
and Cyperaceae in general, as well as in Commelina for example.
The significance of the two mesophyll-to-sieve element conduits in some dicotyledons is obscure. Are they both acting in phloem loading? It has been put forward
that only one of the sieve element types is involved in phloem loading in cucurbits this may be true for potato as well. Initial export of soluble carbohydrates from the
Cucurbita leaf coincides with the structural maturation of the abaxial sieve
elements. Histochemical techniques and microautoradiography of the
l4
C-photosynthate export pathways corroborate the view that the abaxial phloem is
mainly involved in the export of photosynthesis products. It was speculated that the
adaxial phloem retrieved sugars from the free space. The two potential
mesophyll-sieve element pathways in monocotyledons raise the same question with
regard to their significance for phloem loading. The thick-walled sieve elements in
maize where shown to transfer materials from the xylem to the thin-walled sieve
elements in Zea leaves and do not seem to be involved in phloem loading of
photosynthate.
5. Symplasmic and apoplasmic phloem loading may occur in veins of different
orders. Different vein orders may provide disparate structural facilities for phloem
loading, for example, apoplasmic phloem loading in the highest-order veins and
symplasmic loading in the lower-order ones have been proposed for rice leaves. The
behaviour of fluorescent dyes injected into the mesophyll suggests the reverse in
tomato leaves: a potential symplasmic route for phloem loading in the 5th-order
veins, but an apoplasmic one in the 4th-order veins.
The importance of combined phloem loading in different-order veins may increase
with the antiphonal distance, which varies between 50 and 500 m.
Leaves with combined phloem loading potentials are expected to exhibit identical
responses to physiological challenges. It is hypothesised that Type-1-2a leaves and
leaves with mixed loading configurations are able to switch the mode of phloem
loading in response to environmental changes (135).
The Phloem loading pathway and composition of the phloem sap
The phloem exudate collected from petioles or stems contains an assortment of
sugars. This mixture of mono- (glucose, fructose), di- (sucrose), oligosaccharides
Developmental Plant Anatomy. Core Notes. Page 43
(e.g. raffinose, stachyose, verbascose), sugar alcohols (sorbitol, mannitol), and other
components is highly variable from species to species. The sugar composition of the
phloem exudates that were collected at some distance from the site of phloem
loading appears to correlate with the minor vein configuration. Families with a
Type-l configuration translocate 20-80% of the sugars in the form of
raffinose-related compounds, whereas Type-2 families translocate almost
exclusively sucrose.
It is an attractive option to link variations in sugar composition and minor vein
ultrastructure. In species with a mixed loading configuration, oligosaccharides may
load via the symplasmic pathway and sucrose via the apoplasmic pathway with
correspondingly different mechanisms of photosynthate transfer. In minor veins
with mixed configurations, different sugars may be loaded in parallel in different
sieve elements. The flexibility of the mixed loading configuration may be reflected
by the fact that substantial changes in the sugar composition correlate with the
weather conditions and the season.
The limitations of experimental approaches in determining phloem loading
mechanisms
As mentioned phloem loading is carried out by an array of several highly specialized
cells. These cells are not readily accessible to experimental approaches intended to
identify their individual contribution to the loading mechanism. Separation of the
cells is virtually impossible because of their strong coherence and their minimal
size. Disruption of the intercellular connections disturbs the closely-knit metabolic
processes. To maintain the metabolic network that extends over the entire phloem
loading pathway, phloem physiologists are limited to the use of integrated systems
such as (stripped) leaf segments and intact leaves. In these systems, the structural
and metabolic complexity and the intimate and variable intercellular connections are
major handicaps to acquiring unequivocal information on the phloem loading
mechanism. The obvious drawbacks often invalidate the interpretation of the
experiments. A short summary of the objections may suffice to cast a critical light
on the present physiological evidence for any mechanism of phloem loading
1. In general, the mode of phloem loading (apoplasmic or symplasmic) is difficult to
assess in stripped leaf discs, because minor veins are capable of both collection
(uptake of photosynthate arriving from the mesophyll into the sieve elements) and
retrieval (resorption back into the sieve elements of photosynthate leaking away).
Even minor veins of species loading via the symplast are able to accumulate sucrose
from a medium and show uptake characteristics similar to those of "apoplasmic
loaders."
2. Conclusions about the mode of phloem loading using mesophyll cells are
hazardous. The rationale of these experiments is that photosynthate is released from
the mesophyll symplast prior to accumulation by the symplast of the
SE/CC-complex. However, the plasmodesmatal connectivity in most Type-2 species
reveals a symplasmic disjunction the minor vein between vascular parenchyma and
the SE/CC-complex. This indicates that photosynthate must be released from
vascular cells into the apoplasmic space close to the ST-CC-complex. Consequently,
one has to consider the possibility of leakage from mesophyll cells or protoplasts,
even though this is not directly pertinent to the mechanism of phloem loading,
except in species where extra veinal apoplasmic transport would take place. In most
44
Developmental Plant Anatomy
species, therefore, release (and resorption) of photosynthate by isolated mesophyll
cells only reflects the photosynthate retention capacity in the symplasmic section of
the phloem loading pathway. Further, the techniques used to isolate the cells
inevitably alter them in an unknown fashion. It appears that experiments with
isolated mesophyll cells provide no argument whatsoever for the mode of phloem
loading.
3. Experiments with (stripped) leaf discs, or segments, provide a more true-to-life
situation for phloem loading, at least for "apoplasmic loaders." But the disc systems
still fail to determine the exact site and precise mechanisms of release from the
mesophyll symplast and uptake by the SE/CC-complex.
Leakage from discs probably includes several processes: loss from the mesophyll,
leakage from the cells normally engaged in the photosynthate release from the
mesophyll symplast, lateral release from the transport phloem and, one cannot
exclude leakage from the cut veins and cells. The involvement of several leakage
processes may explain the inconsistent effect of parachloromercuribenzenesulfonic
acid (PCMBS) on photosynthate release from discs. The release is inhibited by
PCMBS in Fabaceae, but was promoted in Nicotiana and Commelina. The action of
the sulfhydryl-blocker PCMBS is not entirely clear--it may bind to the sucrose
carrier or to the membrane-bound ATPase or to both. Nonetheless, it effectively
inhibits the plasmamembrane transport of sucrose.
Precise analysis of the uptake of l4C-labelled solutes by stripped leaf tissue suffers
from contributions in unknown proportions of mesophyll, bundle sheath, and vein
cells to the collective uptake. Autoradiographs of leaf discs probably provide
optically misleading information regarding the location of uptake. The apparently
dominant accumulation of organic substrates by the vein is possibly the sum of
direct uptake by the veins and rapid "by-pass" transfer via the mesophyll. This
supposition is supported by elegant experiments with Ricinus cotyledons. An
estimate of 50% uptake by the mesophyll cells of Ricinus cotyledons has been
reported. Comparative experiments with isolated veins and mesophyll cells support
the observation that uptake by veins and mesophyll hardly differs in absolute terms.
But it should be remembered that the uptake parameters may have been affected in
these experiments by the isolation procedure.
Leaf disks
The use of leaf discs may introduce another source of error. Usually, leaf discs are
taken from plants grown under normal day night regimes and minor veins in these
discs immediately accumulate l4C-sucrose administered via an aqueous medium. By
contrast, radiolabeling of the veins in discs does not occur when 14C is supplied in
the form of 14CO2. This suggests differential photosynthate compartmentation
dependent on the mode of 14C-supply, with strong implications for the degree of
vein loading.
Last but not least, vein loading has been assumed--(but not determined) to be
grossly identical with sieve element loading. As an example, after a 20 minute
incubation of Ricinus cotyledons in l4C-labelled sucrose, 10-15% of the label was in
the vein tissues and only 2-3% in the sieve tubes.
The notion that cells other than minor vein sieve elements contribute significantly to
uptake, and that sucrose applied via a medium hardly reaches the sieve elements, has
Developmental Plant Anatomy. Core Notes. Page 45
radical consequences for the interpretation of the results and making positive
statements about the mode of phloem loading and the functioning of the sieve
elements becomes a risky undertaking. If the experimental procedures suffer from
the above flaws, results obtained with leaf discs must be treated with extreme
caution. The massive and selective uptake of sucrose, the specific conversion of
sugars, the kinetics and other features of sugar transport (e.g. proton-driven
transport) may be due to the metabolic activity of cells other than sieve elements.
Intracellular injection
Intracellular injection of membrane-impermeable fluorescent dyes has been
employed to provide support for the existence of symplasmic phloem loading in a
number of species. The aim is usually to show symplasmic continuity between the
mesophyll and the sieve elements. In some instances, dye was transferred to the
vein, but the arrival of dye in the sieve elements could not be distinguished. Fore
example, dye moved to the minor vein phloem in leaves of Commelina but the
uninterrupted movement from mesophyll to sieve element has not been clearly
demonstrated. In Cucurbita, dye injected into the intermediary cell was never
observed as a longitudinal strand with a rapidly moving front, which would have
indicated movement in the sieve element. Lucifer Yellow injected into the mesophyll and mestome sheath of the barley leaf arrived in the mestome sheath and the
sieve element. Direct dye transfer from mesophyll to sieve element was however
never observed. Poor cell-cell conductivity in leaf tissues may result from closure of
the plasmodesmata in response to stripping, maceration, or injection procedures.
Even so, one has to realise that the conductivity that is observed through
dye-coupling, only reveals potential cell cell transfer and is not fully conclusive of
eventual symplasmic phloem loading.
New evidence for a symplasmic loading pathway in grasses
Recent evidence which is based on experiments in which attached leaves have been
‘fed’ with a fluorophore 5,6-carboxyfluorescine (5,6-CF) have revealed that there is
indeed a slow symplasmic transport system available, in which the 5,6-CF is taken
up from mesophyll cells and is transferred to the thin-walled (early metaphloem)
sieve tubes. Evidence for this comes from experiments in which the trafficking of
5,6-CF is closely monitored in leaf material. Our experiments show that the small
(collecting) veins are involved in uptake, and once the 5,6-CF is within the phloem
in these small veins, that it is rapidly transferred to the intermediate and then large
(transport veins) via numerous interconnected cross veins.
cross veins – implication in transport, loading and unloading
These cross veins have a generally simple structure, and consist of a single sieve
tube and associated companion cell, a vascular parenchyma cell, and a single xylem
strand. IN many instances, there is no bundle sheath surrounding this vein, which
could therefore be described as ‘naked’. Yet, surprisingly, there is no evidence of
leakage out of the cross vein sieve tube, into the surrounding mesophyll in source
leaves. However, the situation in sink leaves is different. In sink leaves, there is
evidence that 5,6-CF is offloaded into the mesophyll from the small veins, and that
the cross veins are implicated in this unloading process.
46
Developmental Plant Anatomy
Physiological evidence for different modes of phloem loading
The structural variety and physiological complexity of the experimental systems
present a formidable challenge to unravelling the phloem loading mechanism.
Diverse minor vein typology lends credibility to the concept of multi programmed
phloem loading. This concept predicts the existence of several modes of phloem
loading. Some physiological proof has been obtained for a symplasmic mode of
phloem loading. Good evidence for symplasmic phloem loading was acquired with
micro-autoradiography of minor veins of Cucumis leaves. After feeding intact leaves
with 14CO2 labelled photosynthate showed up in those sieve elements
symplasmically connected with the mesophyll symplast. Furthermore, PCMBS was
unable to block vein loading in Ipomea leaf discs and in leaf discs or intact leaves of
Coleus, thus evidence for a symplasmic loading pathway in these species at least.
Companion cell-sieve tube connectivity and phloem loading
Understanding the ultrastructure of the companion cell (intermediary cell, common
companion cell, or transfer cell) and its role in metabolic functions within itself, and
the potential regulation of the sieve element, in terms of its transport capacity and its
own metabolic requirements seems to be the key to the mechanism of loading.
Symplasmic connectivity is, possibly an indispensable accessory, but not the
absolute key factor for the mode of phloem loading. For example, the plasmodesmatal frequency between the sieve element/transfer cell-complex and the
adjacent cells is relatively high in Sonchus, a representative of the Type-2b family
Asteraceae. But, here the discontinuity in the symplasmic domain lies elsewhere.
The apparently tight relationship between the vein configuration and the reaction to
PCMBS indicates that Type-l species do not load apoplasmically, but most likely via
a symplasmic pathway. Given the variations in the plasmodesmatal connectivity in
the phloem loading zone, it is surprising that the reaction to PCMBS follows so
exactly the demarcation between the Type-1 and Type-2b configuration.
Plasmodesmograms may reveal potential two-way loading paths in many species,
although there is some uncertainty as to whether the plasmodesmograms always
represent the highest-order veins or whether both paths are in use for phloem
loading. Further, the plasmodesmograms may predict an occasional potential for
apoplasmic passage outside the vein. Despite the structural variation, the lack of
effect of PCMBS in Type-l leaves rules out any significant loading via a 'concealed"
apoplasmic pathway.
Some evidence exists for enhanced metabolism within the companion cells as the
crucial factor in phloem loading and is supported by the morphological response to
photosynthate accumulation and concurrent osmotic changes.
There is evidence that chilling caused discernible ultrastructural changes in some
plants. These changes have been linked to the mode of uptake by companion (or
intermediary) cells. However, there is as yet, not enough evidence to substantiate the
chilling effect.
mechanism of symplasmic phloem loading
Although there is good physiological evidence for symplasmic phloem loading, its
mechanism remains a source of controversy. The principle obstacle to accepting the
Developmental Plant Anatomy. Core Notes. Page 47
symplasmic mode of phloem loading is that sugar accumulation by the
SE/CC-complex must occur against a concentration gradient. Phloem loading is
undoubtedly against the over-all osmotic gradient in most cases. The question is
whether osmotic gradient and photosynthate concentration are synonymous. Neither
the osmotic substances nor their compartmentation have been identified. There is,
however, some evidence for the direction of the photosynthate gradient in Type-l
species. The observed absence of an osmotic gradient between mesophyll and sieve
element was presumed to indicate loading down the gradient in Cucurbita. By
contrast, plasmolysis studies showed that the osmotic values in the sieve element
and intermediary cells of Cucurbita and Coleus were much higher than those of the
mesophyll cells. Histochemical studies also evidenced a steep upward sugar gradient
between mesophyll and sieve element intermediary cell.
The first serious attempt to explain symplasmic transfer against the gradient was via
a mechanism in which precompartmentation of photosynthate in the mesophyll has
to occur. After pre-loading of a small compartment in the mesophyll, photosynthate
would be able to diffuse via plasmodesmata to the SE/CC-complex. In the
apoplasmic counterpart to symplasmic loading concept, photosynthate transfer in the
mesophyll is thought to occur down a concentration gradient and that at some point,
assimilate must cross the cell wall (possibly at the vascular parenchyma-companion
cell complex) using sucrose transporters.
One of the major issues related to symplasmic sucrose loading, surfaced in those
species in which the intermediary cells (modified companion cells) have high
plasmodesmal frequencies. Fig 15 illustrates the accepted loading mechanism, now
called the "polymerisation trap mechanism" first introduced by Turgeon (1989) 13.
The hypothesis is that sucrose and galactinol produced by the mesophyll diffuse
along their concentration gradient to the intermediary cells. There, they are
biosynthetically converted to raffinose, or eventually stachyose or verbascose. The
diameter of the raffinose molecules exceeds the “space” available within
plasmodesmata, and it exceeds the molecular exclusion limit of the plasmodesmata
between mesophyll and intermediary cells, thereby preventing back-flow to the
mesophyll. The diameter of the branched plasmodesmata between intermediary cell
and sieve element may be large enough to allow passage of the sucrosyl galactosides
into the sieve element. The special construction of the plasmodesmata between
mesophyll and sieve element is a hint that they may perform a specific task in
phloem loading. In this context, it is noteworthy that the plasmodesmata are formed
by branching and splitting in the direction of the mesophyll during the import stage
and by continued branching in the opposite direction at the beginning of the export
stage.
13
Turgeon, R 1989. The sink-source transition in leaves
138.
48
Annu. Rev Plant Physiol. & Mol. Biol. 40, 119-
Developmental Plant Anatomy
Fig. 19. The Turgeon model for the conversion of sucrose in the intermediary cells to larger
molecules a, to raffinose, or eventually stachyose or verbascose. The diameter of the
raffinose molecules exceeds the “space” available within plasmodesmata, and it exceeds the
molecular exclusion limit of the plasmodesmata between mesophyll and intermediary cells,
thereby preventing back-flow to the mesophyll.
In many dicotyledons Intermediary cells (IC) and phloem parenchyma cells (PP)
define the abaxial border of the minor vein. Intermediary cells may be quite dense in
comparison to phloem parenchyma cells. Sieve elements may be associated with an
intermediary cell or an "ordinary" companion cell (CC). Ordinary companion cells
are normally not in direct contact with the bundle sheath and do not have the fields
of plasmodesmata characteristic of intermediary cells.
It is important to note that high quantities of sucrose in the phloem sap of
presumptive symplasmic loaders appears to conflict with the hypothesis. The
discrepancy may be explained by the fact that phloem sap collected at some distance
from the point of phloem loading does not account for the sieve tube content at
the actual loading site. In other words, it may become biochemically altered again,
after loading, in the transport phloem. There are other options, including the
possibility that only the minor vein types accumulate raffinose or stachyose, whilst
the larger veins accumulate sucrose using active sucrose transporters, via a a parallel
apoplasmic sucrose-loading pathway
Mechanism of apoplasmic phloem loading
That apoplasmic phloem loading occurs in many species seems beyond doubt in
view of the ultrastructural framework of the loading zone and the thermodynamic
elegance of the concept. The question is to what extent is the concept substantiated
by experimental data? This reserve was originally expressed by Giaquinta, and since
then uncertainties have continued to cloud the issue. Experiments with leaf discs
inevitably introduce ambiguity with regard to the interpretation. At present,
therefore, knowledge of the mechanism of apoplasmic phloem loading may be
characterised as fragmentary at best.
To begin with, a few examples may illustrate the existing uncertainties.
First, the location and mechanism of photosynthate release from the mesophyll
symplast is a matter of debate. The low plasmodesmatal frequency between SE/CC
complexes and other vascular elements in numerous species is supportive of
photosynthate release close to the SE/CC-complex. In stripped discs, however,
Developmental Plant Anatomy. Core Notes. Page 49
different cells types may contribute to the photosynthate release, which could
amplify the variability of the release.
Second, assertions with regard to the proton motive force as the energy source for
uptake by the sieve elements likely and as active as they may be, are founded on the
properties of transport phloem and the composition of transport phloem exudate.
Local measurements of membrane potential and pH of the sieve elements in the
minor veins have never been performed.
Third, the contribution of substances other than sugars (e.g. amino acids, potassium)
to the osmotic potential of the sieve tube is high. Nevertheless, the differences in
osmotic potential between the mesophyll and the SE/CC-complexes are commonly
discussed in terms of sugar concentrations only.
Several areas need to be addressed, and evidence examined systematically, in order
to trace serious gaps in the evidence for the mechanism(s) of apoplasmic phloem
loading:
1.
2.
3.
4.
5.
6.
release from the mesophyll symplast,
transport in the mesophyll symplast,
the apoplasmic passage,
sucrose uptake by the SE/CC-complex symplast.
kinetic studies and,
evidence for sucrose/proton symports.
1. release from the mesophyll symplast
The availability of photosynthesis products for release into the apoplast depends on
a number of sequential and parallel processes such as the production and
partitioning of photosynthate and the intra- and intercellular compartmentation of
assimilates. It is possible that a limited number of cells are specialized to function in
the release process, and that the photosynthate is channelled to these cells by the
other units of the mesophyll symplast. For instance, the plasmodesmatal connectivity and position of the paraveinal mesophyll cells in soybean and poplar, may
dictate that all photosynthate produced in the palisade and spongy mesophyll must
pass these cells en route to the phloem.
Data on the site and mechanism of release from the apoplast are scarce and often
ambiguous. One fact appears to be undisputed: sugars occur in the apoplast and
show day/night oscillations. However the values reported for sugar concentrations in
the apoplast vary strongly.
Leakage results are related to the entire apoplast volume. These considerations fit
the view that the release is situated in a very small compartment of the leaf apoplast
in the vicinity of the sieve elements, in order to minimise the influence of
transpiration and thus increase the efficiency of phloem loading. Exceptions to this
rule, though, are not excluded (See the earlier discussion on unloading of 5,6-CF on
page 46).
2. Transport in the mesophyll symplast
The current view is that transport within the mesophyll symplast is diffusional. This
demands a photosynthate gradient throughout the mesophyll symplast, and an
implicitly higher photosynthate concentration (that remains to be demonstrated) in
50
Developmental Plant Anatomy
the cytosol of the proximal cells of the mesophyll symplast. Effective channelling of
photosynthate from the mesophyll to the SE/CC-complex, requires constant retrieval
by the mesophyll, with the exception of the terminal cells of the mesophyll
symplast, where massive release would focus the photosynthate transfer into the
apoplast. The leakage mechanisms from the passage cells and terminal cells of the
mesophyll symplast may therefore possess entirely different kinetics. The net
release from the passage mesophyll is generally low and seems to be controlled by
proton motive force-driven retrieval of photosynthate. The general principles
involved are outlined in the Figure below.
palisade
PVM (paraveinal
mesophyll)
Diffusional gradient
along ‘paraveinal’ mesophyll
BS
VP
CC
SE
Proximal
(loading end)
mesophyll
Release
retrieval
Distal
(unloading
end)
mesophyll
VP
CC
SE
Disjunction
(apoplasmic release)
Fig. 20 Diagram of the mechanism of flow of assimilate from the proximal through distal
mesophyll, and loading in the phloem. ‘Paraveinal mesophyll forms a loading ‘passage’ from
site of assimilation/accumulation, to the bundle sheath. Loading of the CC/SE complex can
be entirely symplasmic, or could be associated with an apoplasmic disjunction.
Light increases the release of photosynthate from leaf tissues, apparently as a result
of current photosynthesis. However, it has been determined that light stimulates
release even in the absence of CO2. Light-induced release of sucrose, amino acids,
and several minerals have been observed in Commelina benghalensis leaf discs,
which suggests that light may also affect the release at the membrane level 14.
3. The apoplasmic passage compartment
If sucrose is released in the vicinity of the SE/CC-complex, the local apoplast is
expected to display a range of adaptations to the massive transfer of solutes. A
pectin layer of nonfibrillar material, adjacent to the sieve elements in the minor
veins of Beta, Cucurbita, Gossypium, Phaseolus, and high pectinisation of the walls
of sieve tubes in Zea, Themeda and other grasses may be an important adaptation.
The high degree of hydration of pectins may allow the rapid accumulation of
sucrose and other solutes. The walls of sieve tubes may be penetrated by numerous
microvilli-like evaginations of the plasma membrane (as in Barley) giving the cell
wall plasmamembrane interface the appearance of a brush border. The apparent
14 Van Bel, A J E, Koops, A, Dueck, T. 1986 Does light promoted export from Commelina benghalensis
leaves result from differential light-sensitivity of the cells in the mesophyll-to-sieve tube path ? Physioll Plant
67: 227-234.
Developmental Plant Anatomy. Core Notes. Page 51
transverse arrangement of microfibrils in the wall may indicate some involvement in
transport.
4. Sugar uptake by the SE-CC-complex
Pioneering experiments on the concentration-dependence of sucrose uptake
provided evidence of a dual isotherm for sucrose uptake. That sucrose uptake does
not obey simple Michaelis-Menten kinetics was also deduced from disc
experiments. There are a few potential reasons for the complex kinetics, the most
important of which may be the differential contribution of mesophyll and veins to
the uptake. Data analysis pointed to a high-affinity saturatable and a low-affinity
linear uptake component. There may be a shift in the proportional contribution of
high-affinity low-capacity and low-affinity high-capacity systems in the successive
cells of the phloem loading system. High-affinity systems seem to prevail in
mesophyll cells, and low-affinity systems in the veins. This distribution of
different-affinity systems thus appears to function in the efficient channelling of
photosynthate to the sieve tubes--retrieval by high-affinity systems in the mesophyll
of the few sugars leaking away and massive uptake by low-affinity systems of
sugars in the SE-CC-complexes.
5. Kinetic studies
It is difficult to discriminate in kinetic studies between the involvement of two
carriers or that of one allosteric carrier with binding sites of different affinities. The
nature and ingestion of the high-affinity component is hardly disputed as being a
carrier-mediated proton motive force driven uptake mechanism. But one of the
arguments in favour of proton motive for given sucrose/proton co-transport remains
questionable: ATPase activity at the plasma membrane of the SE/CC-complex has
been invoked to support apoplasmic phloem loading.
The stoicheiometry of sucrose/proton uptake may be 2:1 at concentrations lower
than 5 mol m-3 , but shifts to 6:1 between 5 and 15 mol m-3 sucrose. This is
consistent with a carrier transiently operative in a protonated and an unprotonated
configuration. Nevertheless, the nature of the low-affinity component, which may be
operative at the ambient concentration in the apoplast in the phloem loading zone, is
not clear. Even saturation of the linear component at concentrations above 200 mol
m-3 can be explained by saturation of unprotonated carriers. So, is ATP needed?
6. Evidence for sucrose/proton symports
The preparation of right-side-out plasma membrane has made possible more precise
observations of the sucrose uptake mechanism. Controlling the sucrose composition
at both sides of the vesicle membrane and uncoupling the transmembrane transport
from cellular metabolism enable exact assessment of the kinetics and energetics of
the uptake system. The vesicles were shown to be capable of a proton-coupled
sucrose influx occurring against an appreciable concentration gradient. The flux is
carrier-mediated: it is sensitive to inhibitors like PCMBS and diethylpyrocarbonate
and showed little competition with other sugars. The apparent Km for sucrose entry
is approximately 1 mol m-3 and the stoicheiometry of the sucrose/proton symport
was 1:1. Manipulation of the pH by changing the medium pH and imposing  by
the use of K+ and valinomycin showed that both contribute to sucrose/proton
symport. The relative contribution of pH and  is difficult to estimate, as the
52
Developmental Plant Anatomy
proton motive force,  in particular, is rapidly dissipated in vesicles. As result,
sucrose accumulation rate is lower than in protoplasts.
Phloem loading -- system mode efficiency
Phloem loading rates may be lower in symplasmic than in apoplasmic loaders and
mass transfer rates in apoplasmic loaders generally exceed those in symplasmic
loaders. At least under controlled temperate conditions, symplasmic loaders appear
to generate less pressure flow. Reasons for that may be a smaller build-up of
osmotic potential in the sieve elements, a lower osmotic potential difference
between source and sink, or a higher resistance along the path than in apoplasmic
loaders. As a result, symplasmic loaders may be less efficient in quantitative terms-expressed as C-equivalents expressed per g of leaf weight, mesophyll cells, or
chloroplasts. In addition, symplasmic continuity may require conversion of sugars,
prior to loading.
The restricted export capacity in symplasmic loaders may originate from altered
metabolism within the loading (intermediary) cells in transport veins. The
ultrastructure of the intermediary cells indicates a limited capacity for the
production and transfer of sugars. The number of ER-vesicles in the intermediary
cells has been shown to increase with increased quantum flux density of light until it
reaches a threshold above which starch accumulates in the mesophyll (van Bel and
Gamalei, 1990 15Gamalei et al 2000 16). The functioning of the enzymes engaged in
the formation of sucrosyl-galactosides may impose limitations on oligosaccharide
production. The high end-product inhibition of stachyose synthase could prevent a
strong accumulation of sugars in the intermediary cell and sieve element. Evidence
on this point is confusing. For example, no osmotic gradient between mesophyll and
the intermediary cell was observed in Cucurbita).
Plasmolysis experiments have however, revealed a much higher osmotic potential in
the intermediary cell and sieve element than in the adjoining mesophyll in Cucurbita
and Coleus.
Deficient transport in the sieve tubes is another possibility for limited transport
capacity. Mass transfer of C-skeletons in the sieve tubes seems to be related to the
sugar concentration and the viscosity of the sieve tube sap. Maximised mass flow of
photosynthate is attained at 700 mol m-3 sucrose, whereas a raffinose solution
becomes saturated (at about 250 mol m-3) before such a level of transport is reached.
The high viscosity constitutes an endogenous resistance in mass flow processes. It
seems likely, therefore, that sucrosyl-oligosaccharides may build up an osmotic
potential sufficient to generate a substantial pressure flow. Auxiliary non-sugar
osmotic equivalents may account for the observed high osmotic potential of the
sieve element/intermediary cell-complex.
15 Van Bel, A. J E and Gamalei, Y. V. Multiprogrammed phloem loading. In: JL Bonneman, S. Delrot, WJ
Lucas and J Dainty eds. Recent advances in phloem transport and assimilate compartmentation. 1990;
128-140.
16 Gamalei, YV; Pakhomova, MV; Syutkina, AV, and Voitsekhovskaja, OV. 2000. Compartmentation of
assimilate fluxes in leaves. Plant Biol. 2:98-106.
Developmental Plant Anatomy. Core Notes. Page 53
The humble potato – a seasonal source to sink conversion
During its development the potato tuber starts out as a strong sink for assimilates,
and, later on during sprouting, will be a strong source of carbohydrate for the
developing vegetative plant.
Sieve element unloading in the potato tuber may occur during the rapid (mature)
growth phase of the vegetative plant. Sucrose is converted to starch, which may
account for up to 70% of the final tuber dry matter. (Oparka et al, 1992 17 thus the
tuber acts as a strong sink under these conditions.. After the shoots cease growth, a
period of dormancy ensues, and under appropriate conditions, sprouting may occur.
Prior to sprouting anatomical evidence suggests that starch mobilization first occurs
round existing phloem regions in the perimedulla – the same tissue in which phloem
unloading occurs, when the tuber is acting as a strong sink. Interestingly, sucrose
mobilization is directed towards the internal phloem strands. Sucrose may move
symplasmically, as the storage cells remain symplasmically coupled. Oparka et al
(1992) reported that sucrose uptake into the storage cells is essentially diffusional
and is unaffected by p-chloromercuribenzine sulphonic acid (PCMBS), which
inhibits the sucrose carrier or by the protonophore carbonyl cyanide –chlorophenyl
hydrazone (CCCP). Within these cells, sensitivity to cell turgor is lost, which
,means that sucrose is essentially able to diffuse to the apoplast, since the retrieval
mechanisms are either lost or inactivated at this time. Another interesting point is
that phloem loading , but not phloem unloading, of sucrose is sensitive to PCMBS –
in the presence of PCMBS, phloem loading was for all intents and purposes,
eliminated, confirming that loading is carrier-mediated. A question which arises
from this research is how can the anatomy of the transport pathway be compatible
with both the above transport mechanisms? One possibility raised by Oparka et al
(1992) is that different SE are involved in SE unloading (plasmodesmata present)
and SE loading (plasmodesmata absent). A second, and perhaps more acceptable
solution would be that plasmodesmata are present in both types of phloem, but that
during the loading phase, these plasmodesmata are blocked, perhaps due to a high
turgor differential between the SE-CC complex and surrounding cells generated
during SE loading.
Indeed, the humble potato begins to offer some explanation for the role of
plasmodesmata in regulating cell to cell transport and brings me back to the starting
point. The data presented by van der Schoot and Rinne 18and others, suggesting
regulated transient closure of plasmodesmata in developing meristematic tissues,
simply shows that plasmodesma may be the key to many things – all related to
regulation of trafficking within the supracellular organism.
17 Oparka, KJ, Viola, R, Wright, KM, and DAM Prior. 1992. Sugar transport in the potato tuber. In: C.
Pollock, J Farrar and T. Gordon eds. Carbon partitioning within and between organisms. Bios Scientific
Publishers, Oxford.
18 van der Schoot, C, Rinne, P 1999. Networks for shoot design Trends in Plant Science 4: 31-37
54
Developmental Plant Anatomy
UNLOADING
(sink)
LOADING
(source)
Sieve element
Companion
cell
complex
Fig. 21 Unloading in the sink tissue occurs vial plasmodesmata followed by sucrose storage
in the vacuole and starch synthesis in amyloplasts. The active, turgor-sensitive sucrose
transport system on the plasmamembrane functions a retrieval system for sucrose escaping
to the apoplast. SE loading in the source tissue is apoplasmic, (across the plasmalemma of
the SE-CC complex for example). Inactivation of the retrieval system of the storage cells
allows diffusion to the apoplast. Circles in walls represent membrane transport events.
Adapted and redrawn from Oparka, et al., 199219. See also Viola et al (2001)20
Ecophysiological concept of phloem loading
Gamalei and van Bel have stated that disparities in the mode of phloem loading may
have an ecophysiological significance. Certain minor vein configurations appear to
show a preference for specific climatic zones. Provided that the coincidence
between minor vein configuration and the mode of phloem loading is universal,
apoplasmic loading prevails in the temperate and arid zones, whereas symplasmic
phloem loading predominates in the tropical rainforest.
19
Oparka, KJ, Viola, R, Wright, KM, and DAM Prior. 1992. Sugar transport in the potato tuber. In: C.
Pollock, J Farrar and T. Gordon eds. Carbon partitioning within and between organisms. Bios Scientific
Publishers, Oxford.
20 Roberto Viola, Alison G. Roberts, Sophie Haupt, Silvia Gazzani, Robert D. Hancock, Nelson Marmiroli,
Gordon C. Machray, and Karl J. Oparka 2001. Tuberization in Potato Involves a Switch from Apoplastic to
Symplastic Phloem Unloading. Plant Cell. 13: 385–398.
Developmental Plant Anatomy. Core Notes. Page 55
Projection of the minor vein configuration on the Takhtajan system for higher-plant
evolution shows that ancient families possess the Type-1 configuration. The minor
vein configuration turns out to be layered in evolutionary zones throughout the
system. With progressive evolution, Types 1-2a, 2a, and 2b emerged in this order.
Symplasmic phloem loading would then be the most ancient mode of photosynthate
transfer to the phloem.
The present geographical distribution of the families raises speculations on the
selective pressures on the evolutionary changes in minor vein configuration and the
corresponding mode of phloem loading. The transformation from the symplasmic to
the apoplasmic mode of phloem loading may have been provoked by temperature or
water stress. Likely shortcomings of the symplasmic mode of phloem loading under
temperatures below 10° are: (a) plasmodesmal dysfunctioning, probably induced by
increased Ca++ levels of cytosolic calcium, which could provoke the formation of
the callose-synthesising enzyme 1, 3-D-glucansynthase. Deposition of callose
around the orifices of the plasmodesmata would be consistent with the observed
blockage of intracellular contact by an elevated level of intracellular calcium. (b)
Metabolic activity and the implicit capacity to produce oligosacchacharides by
intermediary cells may be drastically reduced at temperatures below 10oC.
None the less, there is merit in pursuing this approach- the ecophysiological
significance of phloem loading as based on vein typology, remains one of the most
important and exiting areas of the broad research field which we call phloem
loading and phloem transport.
Fig. 22. Options for symplasmic and apoplasmic phloem loading. The top diagram indicated
an entirely symplasmic pathway, the middle one, where high plasmodesmal frequencies exist
between the vascular parenchyma and the intermediary cells, (see Type 1, page 38) and the
lower one, where an apoplasmic disjunction exists between the vascular parenchyma and the
companion cell(see type 2a-2b, page 38). Reproduced with the permission of Professor AJE
van Bel, Giessen University, Germany.
56
Developmental Plant Anatomy
Fig. 23. Diagram of symplasmic (left) and apoplasmic (right) phloem loading processes
reproduced with the permission of Professor AJE van Bel, Giessen University, Germany.
Recent work has demonstrated that the two modes of phloem loading operate in
many different species, and that these may well be related to a number of
physiological issues, including availability (or lack thereof) of water, temperature
and climatic considerations. Phylogenetic analysis carried out by Turgeon et al
(2001)21 provides a rational basis for comparative studies of phloem structure and
phloem loading. These authors state that although several types of minor vein
companion cellError! Bookmark not defined. have been identified, and progress
has been made in correlating structural features of these cells with loading
mechanisms, little is known about the phylogenetic relationships of the different
types. To add to the available data on companion cells, they analyzed the
ultrastructure of minor veins in Euonymus fortunei and Celastrus orbiculatis
(Celastraceae) leaves and determined that in these species they are specialized as
intermediary cells. This cell type has been implicated in symplastic phloem loading.
The data were added to published data sets on minor vein phloem characteristics,
which were then mapped to a well-supported molecular tree.
The analysis indicates that extensive plasmodesmatal continuity between minor vein
phloem and surrounding cells in the angiosperms is an ancestral trait. Reduction in
plasmodesmatal frequency at this interface is a general evolutionary trend,
punctuated by instances of the reverse. This is especially true in the case of
intermediary cells that have many plasmodesmata, but other distinguishing
characteristics as well, and have arisen independently at least four, and probably six,
times in derived lineages.
Turgeon et al (2001) state that the character of highly reduced plasmodesmatal
frequency in minor vein phloem, common in crop plants, has several points of origin
21
Turgeon, Robert; Medville, Richard, and Nixon, Kevin C. 2001. The evolution of minor vein phloem and
phloem loading. Am. J. Bot. 88:1331-1339.
Developmental Plant Anatomy. Core Notes. Page 57
in the tree. Thus, caution should be exercised in generalizing results on apoplastic
phloem loading obtained from model species. Turgeon et al (2001) also caution that
transfer cells have many independent points of origin, not always from lineages with
reduced plasmodesmatal frequency.
The monocot leaf – a case for thin and thick-walled sieve tubes
The phloem within the longitudinal veins in leaf blades of grasses, with their two
distinct sieve tube types, poses interesting questions relative to the role that we
know the companion cell plays in the dicotyledonous foliage leaf. Whilst we
recognise that companion cells have an important role to play in the functionality of
the phloem -- Oparka and Turgeon (1999)22 have likened the companion cells to the
‘traffic control centers' of the phloem, this cell is, as mentioned, absent in the lateformed metaphloem in all grass leaves examined thus far. If traffic control is
absent, then are these TWST non-functional, or are the adjacent large vascular
parenchyma cells functioning in a companion-cell like manner?
It seems obvious that any research on phloem loading in grass leaves should be
focused on the small and intermediate longitudinal veins and that this requires that
the studies involve an integration of structural and physiological data. Based upon
plasmodesmal frequencies, the thick-walled sieve tubes tend to be isolated from the
thin-walled ones, and the thick-walled sieve tubes lack companion cells. The whole
concept of the SE-CC complex, nut, therefore, be reviewed and re-appraised to take
the differences in these sieve tube types into account. The model presented here is
based upon ongoing research and represents current thinking about the pathway
from the mesophyll to the sieve tubes in grasses.
22
Oparka K, Turgeon R 1999. Sieve elements and companion cells - traffic control centers of the
phloem. The Plant Cell 11: 739-750
58
Developmental Plant Anatomy
Fig. 24. Shows the relationship of thick- (TWST) to thin-walled (ST) sieve tubes and their
associated companion cells (CC) in a typical grass leaf. TWST lack companion cells, and are
thus associated with phloem parenchyma cells. TWST are usually isolated from the ST. Solid
bars connecting cells represent plasmodesma, which are frequent between the bundle
sheath (BS) and mestome sheath (MS) decreasing to the phloem vascular parenchyma
(PVP). An apoplasmic barrier is assumed between PVP and the CC-ST complex. An
alternative pathway to the ST and TWST which have low plasmodesmal frequencies, is
suggested from studies using 5,6-CF, as well as from solute retrieval studies, in which 5,6CFDA was allowed to be taken up in the transpiration stream, illustrated in Fig. 20.
The diagram shows the complexity of the conduits for assimilate transport within
the phloem loading pathway in a typical grass leaf. Assimilates move from the
mesophyll via plasmodesmata, which occur at all interfaces from the mesophyll,
down to the vascular parenchyma (VP) cells. Frequency studies (Table 1) generally
suggest that there are fewer plasmodesma between VP and the CC-SE complex in
species examined. Where plasmodesma are scarce between VP and the CC-SE
complex, apoplasmic transport is assumed to take place. Direct connection between
the VP and sieve tubes is possible for both thin-and thick-walled SE. However, with
the exception of Zea mays (source and sink leaf) and Hordeum vulgare Black
Hullless, plasmodesmal frequencies tend to be low, suggesting an apoplasmic
loading step. Our evidence an suggests that symplasmic loading of the thin-walled
sieve tube can thus follow two paths: First, the BS-.VP->CC->ST route with loading
of the thin-walled sieve tubes achieved via pore-plasmodesmal units (PPU’s) and
Developmental Plant Anatomy. Core Notes. Page 59
alternatively the BS->MS->VP>ST route, with loading of the thin-walled sieve
tubes achieved via plasmodesma. Both pathways have been presented in previous
papers (see Botha and Cross, 1997, and literature cited23). Plasmodesmal
frequencies between the VP and thick-walled sieve tubes are variable (see Table 1)
but, given their presence, suggests that a single potential symplasmic loading
pathway may exist along the BS->MS->VP->TWSE route, with loading of the thickwalled sieve tube via plasmodesma on the phloem loading pathway and, hence on
the phloem loading mechanism, in combination with studies on the uptake of the
fluorochrome 5,6 carboxyfluorescein diacetate (5,6-CFDA) and its transfer after
cleavage of the acetate moiety into the phloem.
Based upon plasmodesmal frequencies, it is clear that an apoplasmic loading step
must exist between the VP and the CC-SE complex, and that this appears common
to many of the grasses studied to date. Additionally, the lack of a CC association
with the TWST (even though this is reported elsewhere, see Haupt et al., 2001)24,
suggests either a non-functional role for the TWST in phloem loading processes, or
if loaded, that it could be loaded apoplasmically. If the TWST are non-functional,
then apoplasmic loading of the ST seems the most likely route to be taken by
loading assimilates. Recently, Matsiliza and Botha25 demonstrated that aphids fed
preferentially from the thin-walled sieve tubes in barley, which suggests that either
there is not enough assimilate trafficking in the thick-walled sieve tubes to be
attractive (i.e., that they are non-functional in assimilate transport) or that there
simply was more assimilate in the thin-walled sieve tubes, with concomitantly, a
higher osmotic potential, from which the aphids fed by choice.
Table 1 summarizes information available on the connectivity of the sieve tubes in
monocotyledonous leaves which have been studied to date. The data presented in
the table suggests that the thick-walled sieve tubes are for the most part, isolated
from the companion cell-thin-walled sieve tube complex. With the possible
exception of Zea mays, low plasmodesmal frequencies to the companion cell sieve
tube (thin walled) complex, does not support a major symplasmic loading pathway
to either sieve tube type in grasses. Given the results from aphid feeding
experiments, the role of the thin-walled sieve tubes seems clear. However, the case
for having two types of sieve tube is not, with much research needed to establish
why they have developed. Perhaps the thick-walled sieve tube is a remnant system
that was functional in the past, but has now lost its primary function in plants that
have both thick- and thin-walled sieve tubes. hat is intriguing though, is that studies
conducted using the known phloem-mobile fluorophore, 5,6-carboxyfluorescine
(5,6-CF) have shown that when its non-fluorescent apoplasmically-transported
diacetate form (5,6-carboxyfluorescine diacetate, 5,6-CFDA) is offered to leaves,
that 5,6-CFDA is rapidly taken up by living cells, where the diacetate is cleaved and
utilised in respiration. The resultant fluorescent molecule, 5,6-CF is taken up by
23
Botha CEJ and RHM Cross (1997) Plasmodesmatal frequency in relation to short-distance transport and
phloem loading in leaves of barley (Hordeum vulgare). Phloem is not loaded directly from the symplast.
Physiol. Plantarum 99: 355-362
24 Haupt S, Duncan GH, Holzberg S, Oparka KJ (2001) Evidence for symplastic phloem unloading in sink
leaves of barley. Plant Physiol. 125: 209-218
25 Matsiliza, B and CEJ Botha, (2002) Aphid (Sitobion yakini) investigation suggests thin-walled sieve tubes
in barley (Hordeum vulgare) to be more functional than thick-walled sieve tubes. Physiol. Plantarum 115:
137-143.
60
Developmental Plant Anatomy
symplasmically-connected cells, and end up in the phloem tissue including sieve
tubes (see Virus trafficking, page 63).
Developmental Plant Anatomy. Core Notes. Page 61
Table 1: Summary of plasmodesmal frequencies between bundle sheath/mestome sheath cells,
vascular parenchyma (VP), companion cells (CC), thin-walled sieve tubes (ST) and thickwalled sieve tubes (TWST) in source leaves26.
Species
Cell-Cell Interface
Bundle Sheath/Mestome Sheath
1
Zea mays
Themeda triandra 2
Hordeum vulgare cv Morex3
Hordeum vulgare cv Dyan4
Eragrostis plana 5
Panicum maximum 5
Bromus unioloides 5
Zea mays 1
Themeda triandra 2
Hordeum vulgare cv Morex3
Hordeum vulgare cv Dyan4
Eragrostis plana 5
Panicum maximum 5
Bromus unioloides 5
Zea mays 1
Themeda triandra 2
Hordeum vulgare cv Morex3
Hordeum vulgare cv Dyan4
Eragrostis plana 5
Panicum maximum 5
Bromus unioloides 5
VP
CC
ST
TWST




























VP
***
****
****
**
**
****
Vascular Parenchyma
CC
ST














Companion Cells
CC
ST
***



**









TWST
***






TWST







Key:
a=
Bundle sheath or vascular parenchyma, concomitant with vascular parenchyma
*****
= very high
****
= plentiful/high
***
= abundant
**
= low

= scarce

= rare
 = absent
= not structurally possible
26
Botha, C.E.J. Interaction of phloem and xylem during phloem loading – Functional symplasmic roles for
thin- and thick-walled sieve tubes in monocotyledons? Chapter 6. pp 115-130. In: Vascular Transport in
Plants Ed Noel M. Holbrook, Biology Department , Harvard University. Elsevier Publishers (early expected
release late April 2005).
62
Developmental Plant Anatomy
1 = Evert et al., 1977, aggregated data for small and intermediate vascular bundles; 2 = Botha and Evert
1988; 3 = Evert et al., 1996a; 4 = Botha and Cross, 1997; 5 = Botha, 1992.
Virus trafficking
Intriguingly, many of the grasses are host to a range of systemic viruses which
suggests that the phloem is not symplasmically isolated in all cases (see Jackson et
al., 1991;27 Schenk et al., 199528). Virus transmission and movement must,
therefore, implicate functional plasmodesma along the route. If plasmodesma are
present, are their sufficient to support a symplasmic loading process or not? If there
are then where and how is loading carried out? The loading strategy in the grass leaf
is possibly equitable to a mixed-mode system (which is, remember, also not
uncommon in dicotyledons). The significant difference is though, that the grass
leaves have two distinct types of sieve tube, which seem to be isolated from each
other, have some differences in their ontogeny and must have different roles to play
in assimilate uptake and unloading in source and sink leaves respectively. A major
question that needs further detailed study is if these sieve tubes load differently,
how is this achieved? Symplasmically, apoplasmically, in using a mixed-mode
loading mechanism? In order to try to answer this suite of questions, we have
recently started to examine the loading and retrieval systems in grass leaves, using
the xenobiotic, 5,6-CFDA. Our studies suggest that 5,6-CFDA enters the phloem
loading pathway where the acetate molecules are cleaved, exposing
carboxyfluorescein a polar molecule, which cannot cross membranes. In other
words, once the acetate is cleaved, the xenobiotic has to traffic via plasmodesma
thus entering the parenchymatic elements associated with the phloem as shown in
Fig. 25.
Using sophisticated microscopic techniques, including laser scanning confocal
microscopy (LSCM) it is possible to obtain thing optical slices through living tissue
and including images through something as thick as a leaf. LSCM makes use of a
laser beam, which is focussed to an incredibly narrow diameter, and photons of laser
light must pass through a spinning disk, penetrate the specimen of interest, (a
scanned beam of photons is employed to do this) after which the excited, reflected
energy (which returns at lower energy levels) is reflected back through a series if
filters, through the spinning disk and the information in the returning photons is
amplified and recorded. These individual bits of information are imaged producing a
micrograph, rather like the longitudinal section showing 5,6-CF transport in the
phloem of a wheat leaf, shown in Fig. 26.
27
Jackson AO, Petty IDT, Jones RW, Edwards, MC, French R (1991) Molecular genetic-analysis of barley
stripe mosaic-virus pathogenicity determinants. Can J Plant Pathol 13: 163-177
28 Schenk PM, Antoniw JF, Batista MD, Jacobi V, Adams MJ, Stebniss HH(1995) Movement of barley mild
mosaic and barley yellow mosaic-viruses in leaves and roots of barley. Ann Appl Biol 126: 291-305
Developmental Plant Anatomy. Core Notes. Page 63
Fig. 25. Colocalization epifluorescence micrograph, showing a large transport vein in a source Zea mays
leaf blade. Exposure of the cut base of the leaf for 60min to 5,6-CFDA was followed by a 30 min chase
using Texas Red. Cell walls (apoplast) fluoresce orange-red. 5,6-CFDA was offloaded into the xylem
parenchyma associated with the large metaxylem vessels, and appears in this section, to have moved in
both directions – adaxially through the xylem parenchyma, and abaxially into the phloem tissue, where all
parenchymatic elements appear to contain some fluorochrome. Under these conditions, the bundle sheath
cells (BS) contained little evidence of 5,6-CF.
Fig. 26. Laser scanning confocal micrograph showing transport of the fluorophore, 5,6-CF in
the sieve tubes of a cross vein (left, and in the lower left micrograph) as well as within sieve
tubes of a longitudinal vein (right). The fluorophore was loaded via mesophyll cells, and whole
leaves were examined using the confocal microscope 2 – 3 h later. Imaged in the laboratory
of Prof AJE van Bel, at the University of Giessen,
64
Developmental Plant Anatomy
It is experiments like that illustrated in Figs. 25 and 26 above that hold the key to
unravelling the loading pathways in grasses. Clearly, whilst it has been long-known
that this system functions in grass leaf blades (see Evert 1996)29 retrieval of solutes
from the xylem involved transport across the wall structure between mature xylem
vessels and associated xylem vascular parenchyma cells. It is only once the 5,6CFDA has trafficked across the margo, that cleavage can occur, and the 5,6-CF is
expressed. Once in the xylem vascular parenchyma cells, the 5,6-CF traffics
symplasmically, via plasmodesma, towards the sieve tubes in loading as well as
unloading systems. Thee data represent new and exciting ways of demonstrating in
situ transport in whole plants.
However exciting this may be, there are many related questions which are still
waiting to be answered.
In summation
Intercellular transport and intercellular trafficking in plants remains a vibrant field
of study, world-wide. A number of laboratories are perusing the questions that I
have alluded to in this document. There are many possibilities, and many
techniques, using biomolecular fluorescence, transmission and light microscopy,
genetic, gene sequencing, calcium blockading technologies and many others are
employed.
Whatever happens, it is clear that plasmodesma are the key to the evolution of the
supracellular organism, and understanding plasmodesma, will allow us to more
clearly understand trafficking intercellular transport and issues such as virus
transport.
29
Evert RF (1996) Phloem loading in maize. In: Regulation of carbon and nitrogen reduction and
utilization in maize JC Shannon DP Knievel and CD Bayer eds. American Society of Plant Physiologists
pp 67-81.
Developmental Plant Anatomy. Core Notes. Page 65
Acknowledgements
Much of the material that is presented in these core notes, is extracted from the
Book Manuscript entitled “Plant Anatomy – an Applied Approach” by Cutler, Botha
and Stevenson. This manuscript will appear as a Blackwell’s text book, carrying the
same title in 2005.
Other sources of information are credited where cited.
CEJB 28 June 2017
66
Developmental Plant Anatomy
5,6-carboxyfluorescine, 46, 50, 60
abaxial, epidermal, 17, 19, 41, 43, 49
agranal chloroplasts, 20, 22, 23, 24
amphistomatous stomata, 15
amphivasal, 12
angiosperms, 10, 11, 41, 57
anomalous, 12
apical meristem, 7
apoplast (-ic, domain), 4, 18, 21, 26, 33, 34, 35, 50,
51, 52
apoplastic continuum, 14
apoplastic transfer, 42
apoplastic transport, 14
apotracheal parenchyma, 11
Assimilates, 59
Barley, 51
Beta, 51
bicollateral vascular bundle, 43
Bignonia, 12
bundle sheath, 16, 18, 19, 20, 21, 22, 23, 24, 32, 35,
36, 41, 45, 46, 49
C3 photosynthetic plant, 19, 21, 22, 23, 24
C4 photosynthetic plant, 19, 20, 22, 23, 24, 40
callose, 56
cambium, 7, 8, 9, 10, 12, 13
Cauline (stem-like), 14, 18
Chenopodiaceae, 42
Coleus, 18, 43, 47, 48, 53
collateral vascular bundle, 43
companion cell, 4, 18, 21, 22, 25, 32, 33, 34, 35, 36,
37, 38, 39, 43, 46, 47, 48, 49, 58
companion cell - companion cell - see phloem, 18, 21,
22, 25, 32, 33
companion cell - see phloem, 33, 34, 37, 38, 39, 43,
47
companion cell (companion cell - companion cell - see
phloem), 18, 21, 22, 25, 32, 33
companion cell (companion cell - see phloem), 4, 33,
34, 37, 38, 43, 47
companion cells, 18, 22, 25, 32, 33, 34, 35, 36, 38, 39,
47, 48, 49, 57, 58
confocal, 63, 64
Cordyline, 12
cotyledons, 45
cross vein, monocotyledonous leaf, 21, 46
Cucurbita, 18, 43, 46, 48, 51, 53
Cucurbitaceae, 38, 41
Cyperaceae, 22, 23, 24, 43
cytosolic calcium, 56
dicotyledonous leaf, 17
dicotyledons, 10, 12, 19, 34, 38, 39, 41, 43, 63
domain - see symplastic, apoplastic, 42, 47
domain see symplastic, apoplastic, 42
dorsiventral dicotyledonous leaf, 14, 16
Doxantha, 12
early metaphloem, 42, 46
enations (leaf/leaf-like), 14
endodermis, 22
endodermoid, 23, 24
epidermis, 15, 17
fascicular, 9
foliar leaf buttress, 16
fusiform cell, 7, 8, 10
Gossypium (cotton), 51
Gramineae, 42
Gymnosperms, 14, 25
heteroblastic, 15
homoblastic, 15
hypostomatous stomata, 15
interfascicular, 9, 12
Intermediary cell, 18, 38, 43, 46, 47, 48, 53, 56
Intermediary cell (see companion cell), 18, 38, 43, 46,
47, 48, 53, 56
intermediate, photosynthetic, 19, 41, 42, 46, 58
isobilateral, 14, 15
Isolateral leaf, 14
lacuna, 19
lacunae, 19
leaf, 4, 11, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 32,
34, 35, 36, 37, 42, 43, 44, 45, 46, 47, 49, 50, 51,
53, 58, 59, 63
Leaf mesophyll, 4, 14, 15, 16, 18, 19, 20, 21, 22, 23,
24, 25, 32, 33, 35, 37, 38, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 58, 59
leaves, 4, 14, 15, 16, 17, 18, 19, 21, 28, 29, 32, 33, 34,
35, 40, 42, 43, 44, 46, 47, 48, 53, 57, 58, 60, 63
mass flow (transport), 34, 53
mesophytes, 15
mestome sheath, 20, 23, 24, 46
metaphloem, 9, 12, 17, 22, 42, 58
metatracheal parenchyma, 11
metaxylem, 9, 12, 17, 19, 22
monocotyledons, 11, 12, 18, 19, 35, 39, 42, 43
mother cell, 8
Nicotiana, 18, 45
palisade mesophyll, 14, 15, 16, 18, 32, 33, 50
Panicoid, 20, 23
paratracheal parenchyma, 11
paraveinal mesophyll (leaf), 16, 19, 50
parenchymatous cells, 23
passage cell (see endodermis), 51
passage cells (see endodermis), 51
pectin, 51
pericycle, 12
Phaseolus, 51
phloem, 5, 6, 7, 8, 10, 12, 14, 17, 18, 21, 22, 23, 25,
26, 27, 28, 29, 32, 33, 34, 35, 36, 37, 38, 40, 41,
43, 46, 49, 50, 51, 53, 54, 57, 58, 60
Phloem, 25, 27, 35, 36, 37, 38, 48
phloem loading, 5, 6, 15, 18, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 50, 53, 57
phloem loading (mechanism), 5, 15, 34, 36, 37, 38,
39, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50, 52, 53,
55, 56
phloem, sieve tube, 4, 18, 19, 21, 22, 25, 26, 28, 32,
33, 34, 35, 41, 42, 45, 46, 47, 49, 50, 51, 52, 53,
58, 59, 63
plasmodesma, 40, 41, 54
Developmental Plant Anatomy. Core Notes. Page 67
plasmodesmata, 4, 9, 20, 25, 35, 36, 37, 38, 39, 40,
41, 42, 46, 48, 49, 54, 55, 56, 57, 59
Pooid, 20, 21
potato, (see Solanaceae), 43, 54
pressure flow (transport), 29, 32, 53
primary xylem, 9
procambial, 17, 18
procambium, 17, 21
protoderm, 16
protophloem, 17, 19
protoxylem, 12, 17, 19
Quercus, 11
raffinose, 44, 48, 49, 53
ray cell, 7, 8
ray initial, 10, 12
Rubisco, 20, 23, 24
Saccharum officinarum, 21, 42, 43
secondary growth, 11, 18
Sedges, 19
see phloem, 4, 18, 21, 22, 25, 32, 33, 34, 35, 36, 37,
38, 39, 43, 46, 47, 48, 49, 58
sieve tube, 4, 18, 19, 21, 22, 25, 26, 28, 32, 33, 34,
35, 41, 42, 45, 46, 47, 49, 50, 51, 53, 58, 59, 63
Solanaceae, 41, 42
spongy mesophyll, 14, 15, 16, 18, 32, 50
stachyose, 44, 48, 49, 53
starch, 53, 54, 55
stems, 9, 11, 12, 29, 43
stomata, 15
stomatal crypt, 15
68
suberin lamella, 21, 22
supracellular, 4, 6, 54, 65
symplast (-ic, domain), 4, 25, 33, 35, 41, 42, 43, 44,
45, 47, 49, 50
symplastic transfer, 38, 48
Themeda, 43, 51
Thick-walled sieve tube, 58, 60
thick-walled sieve tube (see metaphloem,
monocotyledons), 42, 43, 58, 60
Thin-walled sieve tube, 44, 59
thin-walled sieve tube (see metaphloem,
monocotyledons), 22, 43, 59, 63
tracheid, 27
transfer cell, 18, 32, 33, 34, 35, 38, 43, 47
transfer cells, 18, 32, 33, 34, 35, 38, 58
Transfer cells, 32, 34, 35
transpiration, 15, 21, 32, 33, 35, 50
vascular bundles, 9, 12, 16, 21, 22, 24, 32
vascular tissue, 11, 12, 13, 15, 17, 20, 23, 24, 32, 38,
39
verbascose, 44, 48, 49
vessel, 27
Vitis, 12
xerophytic - see xerophytic plants, 19
xylem, 4, 7, 8, 9, 10, 11, 12, 14, 15, 17, 21, 22, 23, 26,
33, 34, 35, 42, 43, 46
xylem, tracheid, 27
xylem, vessel, 27
Zea mays, 22, 35, 59
Developmental Plant Anatomy