Download Symplasmic networks in secondary vascular tissues

Journal of Experimental Botany, Vol. 65, No. 7, pp. 1829–1848, 2014
doi:10.1093/jxb/ert459 Advance Access publication 22 January, 2014
Review paper
Symplasmic networks in secondary vascular tissues:
parenchyma distribution and activity supporting
long-distance transport
Rachel Spicer
Department of Botany, Connecticut College, New London, CT 06320, USA
To whom correspondence should be addressed. E-mail: [email protected]
Received 1 October 2013; Revised 25 November 2013; Accepted 26 November 2013
Abstract
Stems that develop secondary vascular tissue (i.e. xylem and phloem derived from the vascular cambium) have unique
demands on transport owing to their mass and longevity. Transport of water and assimilates must occur over long
distances, while the increasing physical separation of xylem and phloem requires radial transport. Developing secondary tissue is itself a strong sink positioned between xylem and phloem along the entire length of the stem, and
the integrity of these transport tissues must be maintained and protected for years if not decades. Parenchyma cells
form an interconnected three-dimensional lattice throughout secondary xylem and phloem and perform critical roles
in all of these tasks, yet our understanding of their physiology, the nature of their symplasmic connections, and their
activity at the symplast–apoplast interface is very limited. This review highlights key historical work as well as current
research on the structure and function of parenchyma in secondary vascular tissue in the hopes of spurring renewed
interest in this area, which has important implications for whole-plant transport processes and resource partitioning.
Key words: Cambium, contact cells, parenchyma, phloem, rays, xylem.
Introduction
Xylem and phloem transport are often presented in isolation, or worse, in distinct contrast: xylem transport of water
and mineral nutrients occurs through dead cells in which the
water is under tension (negative pressure) and is an entirely
passive process; transport of photoassimilates in the phloem,
on the other hand, occurs through living cells under positive
hydrostatic pressure and often (although not always) requires
an input of energy. The focus is traditionally on structure/
function relationships of the conducting elements: the vessels and tracheids of xylem and the sieve tubes and sieve
cells of phloem. In fact, the two transport processes are
highly interdependent and the activity of parenchyma cells
provides a critical metabolic and energetic link. Highly specialized parenchyma cells are distributed throughout xylem
and phloem and take on particular importance in secondary
vascular tissues where the physical separation of water and
assimilate streams, long transport distances, and perennial
lifestyle place new and at times extreme demands on transport. Some parenchyma cells act as storage sites for carbohydrates and thus may function as either sources or sinks,
despite being heterotrophic by definition. Other parenchyma
cells serve as an intermediary between transport and storage tissues and function in protecting and maintaining the
integrity of the transport system. The primary goal of this
review is to draw attention to the diverse and active roles of
parenchyma in supporting transport in stems with secondary
growth. By integrating knowledge from traditionally distinct
fields, taking a comparative approach to highlight the broad
themes that emerge above natural variation, and identifying
key areas of both accelerated progress and woeful neglect, it is
hoped that this review will stimulate new avenues of research
in this important but understudied field.
© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
For permissions, please email: [email protected]
1830 | Spicer
The activity of parenchyma cells in secondary vasculature is
particularly relevant in light of the increasing recognition that
phloem source–sink relationships in trees are complex (van Bel,
1996; van Bel and Ehlers, 2000; Hafke et al., 2005; Thompson,
2006; Turgeon, 2010; Fu et al., 2011). The canonical source
leaves and terminal sinks (root, fruit, or seed) are separated by
a great distance, while a strong axial sink, the vascular cambium and its rapidly expanding derivatives, is just several cells
away from the phloem along the entire length of the stem.
Parenchyma storing carbohydrates in both xylem and phloem
may function as diffuse, local sources—even of water—and contribute to phloem turgor gradients (Sevanto et al., 2011, 2013).
The fact that trees do not have large pressure gradients in their
phloem (Gamalei, 1989; Rennie and Turgeon, 2009; Turgeon,
2010; Fu et al., 2011) further suggests that activity along the
entire length of the stem—activity that relies on energy and carbohydrates from neighbouring parenchyma cells—needs to be
incorporated into models of long-distance phloem transport.
In plants with secondary growth, the stem must no longer be
viewed as a simple transport route linking above- and belowground organs, but instead as a distribution manifold. A great
deal more work needs to be done in this area.
It is impossible to discuss the role of parenchyma in stems
undergoing secondary growth without frequent reference to
anatomical features and a clear image of the three-dimensional arrangement of cells. To anyone not well versed in
plant anatomy, the terminology can be daunting. A primer on
relevant anatomy is thus included here in order to make the
work accessible to newcomers from different fields, as well as
to highlight classical work, some of which is quite old but still
important and at risk of being forgotten. Rather than a thorough review, the anatomy covered here is meant to provide
an overview of variation found in nature with a focus on the
aspects that are most relevant to long- and short-distance vascular transport. Although all seed plants forming secondary
vascular tissues are technically considered, the information
on nonconiferous gymnosperms (cycads, Gnetales, Ginkgo)
is so sparse and/or the anatomy so derived that the discussion
is effectively limited to conifers and angiosperms. In addition, although some extreme cases of variation due to atypical cambial activity (e.g. successive cambia) are mentioned,
the emphasis is on common patterns rather than a thorough
coverage of existing variation. Lastly, although the focus of
this review is the activity of parenchyma in secondary vascular tissues, evidence will at times be drawn from work on
primary tissues when detailed physiological roles have only
been studied in the latter and where there is good reason to
believe the processes are similar.
Three-dimensional arrangement and
specialization of parenchyma in secondary
vascular tissue
Parenchyma cells in secondary vascular tissue are oriented
either radially or axially, and, with both orientations typically
present, they form a highly interconnected network—effectively a three-dimensional lattice—of living cells throughout
the xylem and phloem (Fig. 1). A subset of both axial and
radial parenchyma are in physical contact with the conducting elements of the xylem and phloem, either directly via pits
in the cell wall or indirectly via the apoplast, and are therefore
able to modify the composition of the transpiration stream
and phloem sap, respectively. The reverse is also true, such
that parenchyma cells are supplied with materials and signalling molecules travelling in both transport streams. A great
deal more is known about the comparative anatomy of
parenchyma in xylem than in phloem, in part due to the commercial importance of wood, diagnostic use of parenchyma
characteristics in species identification, and greater longevity
of the tissue. However, both xylem and phloem parenchyma
show complex spatial patterns of functional significance that
are often taxa specific. In general, conifers have far less radial
and axial parenchyma in xylem than angiosperms, with lianas and stem succulents representing some of the most
parenchyma-rich stems in the plant world, both xylem and
phloem included (Table 1). Although comparable values for
parenchyma in phloem are not widely reported, there is often
a correlation in patterns between the two tissues. Lastly, it
should be noted that while cellular structure and arrangement is described here, a brief discussion of cellular contents
has been incorporated into several sections on parenchyma
function.
Rays are complex structures with specialized cell types
Radially oriented parenchyma cells are grouped together in
rays that are continuous through the cambium such that a
single ray is at once both a xylem ray and a phloem ray. This
is a function of the fact that secondary xylem and phloem
are produced by a bifacial vascular cambium, such that all
cells within a single radial file are derived from the same cambial initial (i.e. a single initial will produce both xylem and
phloem derivatives; Larson, 1994). This presents a unique
situation whereby materials to be exchanged between xylem
and phloem must pass through meristematic tissue, including the true cambial initials and differentiating cells on either
side. The structure and composition of mature xylem rays has
received a great deal of attention, that of phloem rays less
so, and limited work examines the nature of symplasmic connections both among parenchyma and across the cambium
linking the two tissues.
In the central portion of both xylem- and phloem-side rays
are typically radially elongate cells (radial/longitudinal length
ratio 1.5–40:1) termed ‘procumbent’ cells (Fig. 2). Based on
their shape, a high density of plasmodesmata linking cells
radially (Sauter and Kloth, 1986; van der Schoot and van Bel,
1989a; Chaffey and Barlow, 2001), and evidence of symplasmic
continuity through both fluorescent tracer studies and membrane potential measurements (van der Schoot and van Bel,
1990; Sokolowska and Zagorska-Marek, 2012), these cells are
thought to be specialized for radial transport. In conifers these
cells are stacked to form narrow plates just one cell wide (i.e.
the ray is ‘uniseriate’) or less frequently two cells wide (‘biseriate’). Angiosperm rays are typically ‘multiseriate’ (1–15+ cells
wide) and may form two or more discrete size classes. There are
Symplasmic networks in secondary xylem and phloem | 1831
Fig. 1. Cross-section of a stem with secondary vascular tissue. The cambial zone is sink tissue during active growth and is never a
source, such that secondary phloem is adjacent to a strong sink along the entire length of a woody stem. A subset of axial and radial
parenchyma function in carbohydrate and/or nitrogen storage, and therefore may act as sinks or sources depending on phenology.
Radial parenchyma provide a route of symplasmic transport between xylem and phloem that must pass through the undifferentiated
cambial initials and young derivatives. Symplasmic connections exist between ray and axial parenchyma, and subsets of both cell types
are in direct contact with the transpiration stream, whereas contact with the assimilate transport stream is likely indirect. Both axial and
radial parenchyma remain alive for many years after secondary xylem and phloem have ceased to conduct water and photosynthate,
respectively. Xylem parenchyma die upon heartwood formation, prior to which they synthesize secondary metabolites and cell wall
material (i.e. tyloses) and may further lignify. Subsets of phloem parenchyma expand, develop heavily lignified cell walls, and often
proliferate via renewed mitotic divisions in nonconducting phloem, but they do not die until periderm formation. Note that the distribution
of axial parenchyma shown here (short tangential bands) is generic and that many specialized configurations exist (this figure is available
in colour at JXB online).
important functional implications of ray width in that only the
cells on the outer margins will be in contact with conducting
elements. Given this, every parenchyma cell in a conifer ray will
be in some contact with a tracheid or sieve cell, whereas cells in
the centre of wide angiosperm rays will be isolated from vessels
and sieve tubes. The terms ‘contact’ and ‘isolation’ cells have
long been in use to make this distinction (Braun, 1970, 1984;
Sauter and Kloth, 1986), but it is important to note that there
is a great deal of variation in the configuration of conducting
element–parenchyma contacts (Fig. 2).
Cells at the longitudinal tips of rays (i.e. the top and bottom when viewed in tangential section) are often structurally
quite different from procumbent cells and suggest functional
specialization, but work in this area is almost exclusively based
on anatomy. When two or more distinct cell types are present
in a single ray it is described as ‘heterogeneous’, but the cell
types present differ between xylem and phloem and between
conifers and angiosperms such that this term is nonspecific.
In conifers, xylem-side rays are either composed exclusively
of procumbent cells (i.e. the ray is ‘homogenous’) or have ray
tracheids at their tips. Ray tracheids are nearly identical to
longitudinal tracheids in structure and hence dead at maturity, and although a function in radial transport of water has
long been presumed, their importance in this capacity has
recently been challenged (Barnard et al., 2013). In contrast,
‘erect’ parenchyma cells, which range from square to vertically elongate (radial–longitudinal length ratio 1:1–10), are
often located at the tips of phloem-side rays in conifers and
of both phloem- and xylem-side rays in angiosperms (Fig. 2).
In conifers, Strasburger cells (also called ‘albuminous’ cells)
are interspersed among erect cells and so considered part of
the ray (Grillos and Smith, 1959; Alfieri and Kemp, 1983;
Alfieri and Mottola, 1983; Allen and Hiatt, 1994). Found in
all gymnosperms, Strasburger cells are thought to be functionally equivalent to companion cells in angiosperms and
hence function in phloem loading (Sauter et al., 1976; Sauter,
1980). Plasmodesmatal connections between Strasburger cells
and sieve cells are extensive and provide a direct link between
radial and axial symplasmic transport, further highlighting the
fact that short-distance transfer between diffuse local sources
and sinks is occurring along the length of the long-distance
transport pathway in secondary phloem. Companion cells are
consistent features of the transport phloem of angiosperms,
but with few exceptions they are not part of the radial system.
It has been suggested that erect (also called ‘upright’) cells
in angiosperm xylem-side rays are specialized to function in
exchange of material with vessels (Höll, 1975; Sauter and
Kloth, 1986), as in some taxa they alone are connected to vessels via pits in the cell wall (e.g. Populus and Salix). However,
there is tremendous variation in patterns of ray parenchyma
1832 | Spicer
Table 1. Volume proportion of secondary xylem occupied by
radial and axial parenchyma
Conifers have narrow rays (1 or 2 cells wide) and minimal axial
parenchyma. Angiosperm rays are often much wider (commonly 3–6
cells, but up to 15 or more cells wide). Axial parenchyma ranges from
minimal to abundant, especially in lianas and trees described as stem
succulents. aLianas (woody vine); bPachycauls (bottle-tree growth
form). Sources: DeSmidt, (1922), Myer (1922), Panshin and de Zeeuw
(1980), Chapotin et al. (2006b), Brandes and Barros (2008), and
Hearn (2009a).
Angiosperms
Acer
Populus
Betula
Ulmus
Fraxinus
Carya
Juglans
Quercus
Piptadeniaa
Dalbergiaa
Senegaliaa
Adansoniab
Adeniab
Conifers
Pinus
Picea
Larix
Pseudotsuga
Abies
Cupressus
Juniperus
Radial parenchyma
(% of volume)
Axial parenchyma
(% of volume)
12–18
10–14
10–16
10–18
12–16
17–26
10–17
15–40
15
12
8–14
70–80 + (total parenchyma)
40–95 + (total parenchyma)
<0.1
<0.2
2
2–8
3–10
8–12
9–14
8–24
25
33
32–43
4–8
5–7
6–10
7
5–7
6
6–8
–
–
–
–
–
<0.2
<0.2
vessel pitting and in many cases both procumbent and erect
cells are highly pitted in walls shared with vessels such that
both cell types are capable of exchange (Fig. 2). This would
serve to maximize the symplast/apoplast contact area, which
is important given that internal parenchyma cells are isolated.
Similarly, the size and frequency of pits and/or plasmodesmatal connections linking parenchyma longitudinally and
tangentially within a single ray have clear implications for
the efficiency of transport within a ray, but these aspects are
not well documented. It is striking that the erect cells typically form uniseriate portions of the ray (Fig. 2). Although the
functional implications of this arrangement are not known,
these radial cell files could link axial elements (either vessels or
axial parenchyma) on opposite sides of a ray in a way that a
given radial file in the multiseriate portion of the ray cannot.
Axial parenchyma distribution also suggests functional
specialization
Axial parenchyma forms an intermediary between radial
parenchyma and the axial conducting elements in secondary vascular tissue and is often arranged in distinct
taxa-specific spatial patterns. In general these patterns
are well documented in xylem where they are preserved in
time (see Carlquist, 2001a for an excellent review), but far
less so in phloem where parenchyma is difficult to distinguish from neighbouring sieve element and companion or
Strasburger cells, and cellular organization is lost due to
sieve element collapse and ray dilatation (see discussion
on longevity). Individual cells are usually short in the axial
direction and longitudinally aligned in strands (sometimes
called ‘strand’ parenchyma, derived from a fusiform cambial initial that rapidly subdivides into several shorter cells;
Larson, 1994), but in plants forming only small amounts
of secondary growth (i.e. the unfortunately named ‘woody
herbs’) there may be an additional type that is long and
tapered, apparently derived from a fusiform initial without
any further transverse divisions (Eames and MacDaniels,
1947; Zahur, 1959). Both types are present surrounding a
vessel in Solanum lycopersicum in Fig. 3A (van der Schoot
and van Bel, 1989a, b).
In conifers, axial parenchyma constitutes a far lower proportion of secondary xylem than in angiosperms (Table 1)
and may be lacking entirely, present as single strands scattered throughout the xylem, form short (2–6 cells wide)
tangential bands, or be concentrated at the growth ring
boundary (Panshin and de Zeeuw, 1980; Braun, 1984). In
contrast, it is common for axial parenchyma in angiosperms
to occupy anywhere from under 1% to over 25% of total
xylem volume. It often surrounds vessels in partial or complete sheaths (Figs 3 and 4A) or forms wide tangential bands
in which smaller vessels are nested (Carlquist, 2001a). This
configuration may underscore the role of axial parenchyma
in maintaining water transport in angiosperm vessels, several
aspects of which are discussed later in the review. Where less
abundant, axial parenchyma forms narrow tangential bands
(Fig. 4B) or a one- to several-cell-thick band at the growth
ring boundary (Fig. 4). This arrangement, which is also
found in many conifers, is likely important during reinitiation of cambial activity in seasonal climates. Interestingly, in
temperate regions these cells often overwinter in the cambial
zone as partially differentiated cells (Jane, 1934; Chowdhury,
1936; Fuchs et al., 2010b). Together with partially differentiated phloem elements (including parenchyma; Grillos and
Smith, 1959), these are the first cells of the growing season to
complete maturation such that the newly activated cambium
is located between two layers of parenchyma. The activities
of these cells in relation to supporting new growth deserves
further attention.
Axial parenchyma in secondary phloem is often more
abundant proportionally than in secondary xylem of both
conifers and angiosperms, although it is more difficult to
distinguish from other living elements. Strasburger and companion cells are in essence highly specialized parenchyma,
but there is a rich body of literature on the structure and
physiology of these cells and they are only included here
in the context of symplasmic continuity with parenchyma.
As in xylem, the volume of axial parenchyma in secondary
phloem is lower and more consistent in conifers (sieve cells
dominate) and typically exists as scattered strands, small
Symplasmic networks in secondary xylem and phloem | 1833
Fig. 2. Ray parenchyma arrangement in angiosperm secondary xylem shown in radial (left) and tangential (right) sections. Vertical
dashed lines indicate regions in contact with a vessel. Rays may be uniseriate (A–C), multiseriate (D, E, H, I), or contain both uniseriate
and multiseriate regions (F, G, J–L). Erect parenchyma cells are either square or vertically elongate and occur at the upper and lower
margins of rays (A, B, E, F, I, J) or in vertical bands that alternate with procumbent cells (D, G, K, L). Pits in parenchyma cell wall
regions shared with vessels (delineated by a dashed outline) may occur in both types of ray parenchyma (A, D–G, I, K, L) or be absent
in some (C, H) or all (B, J) procumbent cells. The degree of variation in ray structure suggests that similar variation exists in functional
specialization. In some species, tile cells form via sequential radial divisions (i.e. formation of tangential walls) of developing procumbent
cells (G). It is interesting to speculate on the function role of this massive proliferation of cell wall for radial transport. Rhomboid crystals,
typically composed of calcium oxalate, are shown in the erect parenchyma cells of a few species (D, I, J). Species shown are (A)
Guilfoylia monostylis, (B) Salix caerulea, (C) Eucalypus regnans, (D) Hopea nutans, (E) Panax elegans, (F) Endospermum peltatum,
(G) Durio oxleyanas, (H) Acer pseudoplatanus, (I) Canarium mehenbethene, (J) Cyclostemon sp., (K) Madhuca utilis, (L) Palaquium
galatoxylum. Figures adapted from Chattaway M. 1951. Morphological and functional variations in the rays of pored timbers. Australian
Journal of Biological Sciences 4, 12–27, with permission from the publisher, CSIRO Publishing (http://www.publish.csiro.au/nid/280/
paper/BI9510012.htm).
1834 | Spicer
Fig. 3. Vessel contacts and symplasmic connections among living elements in the secondary vascular cylinder of Solanum
lycopersicum. (A) Elongate axial parenchyma (AP), parenchyma strands (PS), and radial contact cells (C) all form a symplast–apoplast
interface at pits (not shown) in the walls shared with vessels (V) and are thus capable of exchange with the transpiration stream. (B)
Intracellular injections with Lucifer Yellow CH demonstrate extensive symplasmic connections among living elements, where an asterisk
indicates the cell that was impaled and arrows indicate intercellular dye transfer. The cambial zone is shown on the right as three
adjacent files of four cells each, all of which have distinctly narrow radial widths. Three files of phloem cells are thus shown at the far
right; everything to the left of the cambial zone is xylem. Axial parenchyma in contact with a vessel were strongly coupled (a) as were
living fibres (e). Dye injected into xylem ray parenchyma moved to like-cells in the same radial file (c, d, i) as well as into adjacent living
fibres (f). Dye injected into phloem ray parenchyma moved through the cambial zone into the xylem (j). Cambial cells showed axial dye
transfer (h). Figures adapted from (A) van der Schoot C, van Bel AJE. 1989. Architecture of the internodal xylem of tomato (Solanum
lycopersicum) with reference to longitudinal and lateral transfer. American Journal of Botany 76, 487–503, permission of the publisher,
Botanical Society of America, and (B) van der Schoot C, van Bel AJE. 1990. Mapping membrane-potential differences and dye-coupling
in internodal tissues of tomato (Solanum lycopersicon L.). Planta 182, 9–21, with permission of the publisher, Springer.
clusters of strands, or short tangential bands that alternate
with sieve and Strasburger cells, and fibres if present (Grillos
and Smith, 1959; Alfieri and Evert, 1968; Alfieri and Kemp,
1983; Alfieri and Mottola, 1983; Castro et al., 2005, 2006).
Axial parenchyma in angiosperm secondary phloem is more
variable and although attempts have been made to categorize
major pattern types, it is best described as a continuum, as
with most anatomical characteristics. When production by
the cambium is examined closely however, specific repeating
patterns of alternating cell types are observed (Evert, 1963b;
Alfieri and Kemp, 1983; Barlow and Lueck, 2006). When
sparse, axial parenchyma is found in thin tangential bands;
when abundant it forms the majority of the phloem in which
clusters of sieve elements and companion cells are nested
(+/–fibres; Zahur, 1959; den Outer, 1983, 1993). This latter
condition is especially common in lianas (den Outer, 1993).
Axial parenchyma in phloem appears to be symplasmically
isolated from the conducting elements and associated companion and Strasburger cells (van Bel and Kempers, 1991;
van Bel, 1996; van Bel and Ehlers, 2000; Evert, 2006), but
prominent clusters of pits are found in the walls in contact
with ray parenchyma.
The nature of contacts among parenchyma
and axial conducting elements
Connections are formed where radial and axial parenchyma
are in direct contact and where both types of parenchyma abut
conducting elements of the xylem and phloem. Nearly all of
these connections occur at pits, which are effectively holes in the
cell wall. In the absence of pits, exchange of important materials may occur via the apoplast. When one considers the diversity of plants with secondary growth and the multitude of cell
types present, the number of specific types of contacts is quite
large. Only a few of these types have been studied in detail, but
it is clear that in order to understand intercellular symplasmic
transfer as well as exchange across the apoplast–symplast interface, the physical nature of these contacts should be studied in
detail and with a comparative approach wherever possible.
The apoplast–symplast interface in xylem occurs at
cell wall pits occupied by a unique cell wall layer
The site of exchange between the transpiration stream and
either radial or axial parenchyma occurs at a unique interface
Symplasmic networks in secondary xylem and phloem | 1835
Fig. 4. Axial parenchyma highlighted in black with image processing software (A, B) or left unstained and appearing white (C) in
cross-sections of Fraxinus americana (A), Quercus rubra (B), and Adansonia za (C). Axial parenchyma forms a band at the growth ring
boundary in F. americana and often completely surrounds latewood vessels (A; a large earlywood vessel is shown in the lower left, with
small latewood vessels above), whereas in Q. rubra latewood, it forms thin tangential bands connecting latewood vessels surrounded by
tracheids and limited parenchyma. In A. za, the proportion of parenchyma is extremely high (C), shown here with the cell walls of lignified
elements heavily stained but the background matrix of both axial and ray parenchyma unstained and appearing white. * indicates a
uniseriate ray; ** indicates a multiseriate ray. Bars, 50 µm (A, B) and 1 mm (C). Figures adapted from (A, B) Spicer R, Holbrook NM.
2007. Parenchyma cell respiration and survival in secondary xylem: does metabolic activity decline with cell age? Plant, Cell and
Environment 30, 934–943, with permission of the publisher, Wiley, and (C) Chapotin SM, Razanameharizaka JH, Holbrook NM. 2006.
A biomechanical perspective on the role of large stem volume and high water content in baobab trees (Adansonia spp.; Bombacaceae).
American Journal of Botany 93, 1251–1264, with permission of the publisher, Botanical Society of America.
within the cell wall pits. In conifers, all ray parenchyma cells
have pitting in the wall shared with tracheids and show taxaspecific variation in pit size and shape (Panshin and de Zeeuw,
1980). In fact, cross-field pitting, where ‘cross-field’ refers to
the rectangular region of cell wall shared between ray parenchyma and tracheids, is one of the most useful characters for
identifying conifer woods. Pits range in shape from single
broad ovals or quadrangles that nearly fill the cross-field to
up to six small, round pits with prominent arched borders in
the tracheid wall (Phillips, 1941). Although parenchyma-vessel pitting in angiosperms has received less attention in terms
of comparative anatomy, there is greater variation in the
types of parenchyma present in xylem-side rays and pitting
ranges from closely spaced pits that are small and round to
larger, oblong pits (Fig. 2). As already noted, these pits may
occur along the entire length of an angiosperm ray where it
contacts a vessel, or in isolated regions.
A unique cell wall layer—referred to here as the amorphous layer—is found in most parenchyma in secondary
xylem, and its chemical composition and function deserve
a great deal more attention. Most radial and axial parenchyma cells in xylem have some form of secondary cell wall,
although it typically lacks the complex laminar structure of
tracheary elements and may be more comparable to a thickened primary wall (Panshin and de Zeeuw, 1980). Once secondary cell wall formation is complete, an additional layer of
loosely woven and irregularly aligned microfibrils is deposited with hemicelluloses in a matrix rich in pectin but with
little-to-no lignin in the space between the plasma membrane
and newly deposited cell wall (Schmid and Machado, 1968;
Chafe, 1974; Schaffer and Wisniewski, 1989; Barnett et al.,
1993). This amorphous layer is thickest near pits that face
a vessel or tracheid such that it fills the parenchyma-side pit
cavity (Fig. 5), but it has consistently been shown to surround
the entire parenchyma protoplast in a thin layer (Schaffer
and Wisniewski, 1989; Murakami et al., 1999). Various functions have been ascribed to the amorphous layer over the past
50 years but the simple truth is that we have very little idea of
its role(s), largely because work is limited to ultrastructural
studies. It appears to be involved in tylosis formation (Foster,
1967; Meyer and Cote, 1968; Rioux et al., 1988; Bonsen and
Kucera, 1990; Rudelle et al., 2005) but it is also present in
species in which tyloses do not form, including conifers. It
has been hypothesized to protect the parenchyma protoplast
from changes in hydrostatic pressure in the adjacent transpiration stream (van Bel and van der Schoot, 1988), but it also
surrounds the protoplast on all sides. The amorphous layer
has also been suggested to form an important barrier that
prevents damaging ice crystal formation and allows parenchyma cells to supercool in freezing conditions (Wisniewski
and Davis, 1989; Wisniewski et al., 1991). Here, pectin in the
amorphous layer is thought to function as a hydrogel and
protect against freeze-induced desiccation. Perhaps the most
compelling argument however is that this loose layer forms an
extension of the apoplast in cells where the cell wall may be
quite impermeable due to a dense laminate structure and lignification (Barnett et al., 1993), thereby greatly expanding the
surface area available for exchange across the plasma membrane, much like transfer cell wall invaginations (Pate and
Gunning, 1972; Offler et al., 2003). This would be in keeping
with the observation that the amorphous layer also fills cavities of ‘blind pits’ (van der Schoot and van Bel, 1989a), pits
1836 | Spicer
Fig. 5. Axial parenchyma (AP) cell in Cercocarpus betuloides
shown adjacent to a vessel (V) and two tracheids (T). Arrowheads
point to the loosely fibrillar amorphous layer filling the two halfbordered pit cavities facing the vessel and the cavity of a simple
pit facing a tracheid. Bar, 2.5 µm. Figure adapted from Dute
R, Patel J, Jansen S. 2010. Torus-bearing pit membranes in
Cercocarpus. IAWA Journal 31, 53–66, with permission of the
publisher, Brill.
in parenchyma that lead simply to vessel or tracheid walls.
Usually interpreted as developmental mishaps and effectively dead ends, they can be quite abundant and could also
serve to increase the area of the parenchyma plasma membrane exposed to the apoplast. Most likely the amorphous
layer serves many of these functions and shows taxonomic
variation in its specialization. Lastly, it should be noted that
many names have been used to describe this and related
loosely fibrillar layers, including the ‘protective layer’ and
‘isotropic layer’. Although the protective layer has been used
most widely, the amorphous layer is preferred here because it
describes the structure without implying any function.
Symplasmic continuity among secondary vascular
parenchyma is extensive and dynamic
Extensive symplasmic connections exist among the various
types of parenchyma cells in secondary vascular tissue, typically in the form of simple pits (i.e. pits without arched borders) crossed by multiple plasmodesmata. A crude—yet still
highly labour intensive—assessment of potential symplasmic
continuity can be made through quantitative ultrastructural
studies, but a complete picture of symplasmic fields requires
tests to determine the size exclusion limit of plasmodesmatal
connections and in vivo imaging following symplasmic dye
injection. Given the thick lignified walls that are characteristic of secondary xylem elements, phloem fibres, and even
many parenchyma cells, this is naturally extremely challenging work. Combine these technical difficulties with the
diversity of cell types and distribution patterns and it is easy
to appreciate why so little progress has been made in this area.
Nevertheless, a few systems are well characterized (S. lycopersicum and Populus spp.) and suggest that symplasmic continuity shifts rapidly during cell differentiation, and that once
mature there are symplasmic fields of cells that are partially
or wholly isolated from each other. There is also evidence
for taxonomic variation in these patterns, as well as seasonal
shifts in symplasmic continuity in the temperate zone.
As already noted, the central ray cells are often radially
elongate with high plasmodesmatal densities in their tangential walls and thus appear specialized to transport material radially (Sauter and Kloth, 1986; van der Schoot and
van Bel, 1989a; Chaffey and Barlow, 2001). Although dyecoupling experiments are scarce, there is some evidence for
continuity through xylem-side and phloem-side rays (van der
Schoot and van Bel, 1990; Sokolowska and Zagorska-Marek,
2012), and circumstantial evidence as well as immunolabelling suggests that carbohydrates and nitrogenous compounds
(Chaffey and Barlow, 2001; Fuchs et al., 2010a) are trafficked
through the rays via plasmodesmata. Little is known about
symplasmic continuity within rays in the longitudinal and
radial directions, but pitting in radial and transverse walls is
a diagnostic feature (Panshin and de Zeeuw, 1980) and the
potential for continuity can be inferred from anatomy in a
few cases. Pain-staking studies of symplasmic continuity in
S. lycopersicum internodes in which dye coupling, metabolic
activity, and membrane potential were mapped onto a threedimensional reconstruction that includes living fibres, xylemand phloem-side rays cells, and cambial derivatives illustrate
both the complexity and taxon-specific nature of these symplasmic networks (Fig. 3).
An improved understanding of symplasmic continuity in
secondary growth is particularly important in the context of
the vascular cambium, a large and critically important sink
that must not only radially import all materials needed for
cell division, expansion and differentiation, but also relies on
(presumably) positional signals from neighbours to determine
cell fate. Recent work shows that the number of plasmodesmatal connections between cells in the cambial zone changes
during differentiation (Ehlers and van Bel, 2010; Fuchs et al.,
2010b, 2011). During active growth in Populus, cambial initials are linked via plasmodesmata in their tangential walls
to the immediate derivatives on either side, but there is then
a dramatic but transient increase in plasmodesmatal frequency in the same wall between mother and daughter cells
(i.e. radial continuity increases). In later stages of maturation,
tangential wall plasmodesmatal numbers decrease, and they
decrease more rapidly on the xylem side. Cambial initials
appear to have very few plasmodesmata in their radial walls
(i.e. tangential continuity is low), but these also increase in
number in their immediate derivatives. During dormancy, the
number of plasmodesmatal connections is greatly reduced in
all directions but increases upon cambial reactivation in the
spring. Dye coupling in the cambial zone in S. lycopersicum
supported roughly analogous changes in plasmodesmatal
number, with injections into immediate cambium derivatives showing movement through one or more cells in the
Symplasmic networks in secondary xylem and phloem | 1837
directions of both xylem and phloem, whereas older phloemside derivatives appeared isolated and longitudinal transfer of
dye was extremely rare (Ehlers and van Bel, 2010). Given the
potential for regulation of plasmodesmatal aperture and role
of phloem- and/or neighbour-derived signalling molecules,
it is interesting to speculate on the effects of transient isolation during xylem and phloem differentiation. Unfortunately,
the major technical barriers to working with woody tissue
are likely to continue to impede progress in this area in the
absence of a major advance in live cell imaging technology.
Symplasmic connections among axial and radial parenchyma in both xylem and phloem are perhaps the least
characterized. Axial parenchyma surrounding vessels in S.
lycopersicum show ready axial transfer of dye (Fig. 3B), as do
clusters of living fibres, and dye moves freely from an injected
ray cell into a fibre cluster (van der Schoot and van Bel,
1990). Similarly, injections into mature xylem and phloem
parenchyma result in dye movement into like cell types both
radially and longitudinally (Ehlers and van Bel, 2010). Of
particular interest is the degree of symplasmic isolation of
sieve element/companion cell and sieve cell/Strasburger cell
complexes (each pair of course is in high symplasmic continuity) from the adjacent axial and radial parenchyma in the
phloem. Substantial evidence exists for complete isolation
in a number of herbaceous and woody species (van Bel and
Kempers, 1991; van Bel and van Rijen, 1994; Hafke et al.,
2005; Evert, 2006; with particularly good discussions in van
Bel, 1996; van Bel and Ehlers, 2000). If phloem conducting
elements and their respective companion/Strasburger cells
are symplasmically isolated from adjacent parenchyma, any
exchange of materials would require release into the apoplast and subsequent transfer across a plasma membrane.
Cell-specific localization studies of transport proteins in the
plasma membrane of parenchyma in both xylem and phloem
are welcome additions to this field, particularly those able to
resolve asymmetric localization with respect to orientation of
neighbouring cells. Work in this area is minimal and almost
exclusively focused on vessel-associated xylem parenchyma.
Relevant findings are included in the following sections on
storage and embolism repair functions.
Functions of parenchyma in secondary
vascular tissue
Parenchyma cells function in many different and overlapping
capacities to facilitate and maintain transport in secondary
vascular tissue. Although a traditional breakdown of the
most obvious functions includes carbohydrate storage, radial
transport, and xylem–phloem exchange, these categories are
too broad to be useful. For example, some axial and radial
parenchyma store substantial amounts of carbohydrates in
the form of starch, but the timescale over which that starch
is broken down and mobilized (e.g. seasonally versus diurnally), its final point of use (e.g. spring leaf expansion, cambial growth, embolism repair, freezing tolerance), and the
means by which it travels (e.g. symplasmically through rays or
release into the apoplast) all vary. While much of the work on
parenchyma storage and implied transport is descriptive (e.g.
a fair body of work documents temporal changes in the cellular contents of parenchyma), it is rarely paired with experimental work to establish the fates of those contents. Similarly,
experimental work in plant physiology may implicate parenchyma but rarely includes cell-type-specific observations. An
attempt is made here to discuss specific parenchyma functions
in the context of whole-plant physiology. Some of the most
promising research areas are those that combine physiological manipulations with cellular level gene expression, protein
localization, and quantitative measures of cell composition.
Secondary vascular parenchyma serve as transfer
stations for carbohydrates, nitrogenous compounds,
and water
The function of secondary vascular parenchyma in seasonal
storage and short- and long-term redistribution of organic
and inorganic compounds is one of the most important and
arguably most studied. Many observations are descriptive,
based on macroscopic tissue samples, and specific to the species and environment studied and therefore difficult to generalize. However, it is clear that subsets of parenchyma in
secondary xylem and phloem store carbohydrates as soluble
sugars, starch, and/or lipids (Höll, 2000) as well as nitrogen in
the form of vegetation storage proteins (Stepien et al., 1994).
Excellent reviews in this area include Höll (1975, 2000),
Pate (1975) van Bel (1990, 1995), Pate and Jeschke (1995),
and Hoch et al. (2003) and only a brief summary is included
here. There is also increasing recognition that parenchyma in
secondary xylem function in water storage and recirculation
between xylem and phloem (Chapotin et al., 2006b; Holtta
et al., 2006; Sevanto et al., 2011; Hearn et al., 2013). The relationships are complex, but these studies are some of the most
informative regarding the role of parenchyma in xylem and
phloem exchange.
Despite climate- and taxa-specific variation, there are several patterns of seasonal carbohydrate storage in secondary tissues with respect to growth. Following leaf flush and
expansion in temperate deciduous trees, starch stores increase
in xylem parenchyma throughout the period of active growth
(Sauter and Kloth, 1987; Kozlowski, 1992; Sauter and
Neumann, 1994; Sauter and van Cleve, 1994; Sauter and
Wellenkamp, 1998; Barbaroux and Breda, 2002; Hoch et al.,
2003; Landhausser and Lieffers, 2003; Wong et al., 2003; Bazot
et al., 2013). Where present, stored lipids start to increase as
leaves begin to senesce (Sauter and Neumann, 1994; Hoch
et al., 2003). Levels of stored starch gradually decrease
throughout dormancy and then decrease significantly at the
onset of bud break and cambial activity, usually corresponding to a transient increase in sugar levels in both xylem and
phloem sap. There are also reports of soluble sugar stores
increasing during dormancy in cold climates and in response
to chilling, suggesting a role in freezing tolerance (Sauter
and Kloth, 1987; Sauter and Neumann, 1994; Wong et al.,
2003). Similar seasonal changes in starch and soluble sugars
have been reported in the phloem (Landhausser and Lieffers,
2003; Bazot et al., 2013). Although rarely documented, lipid
1838 | Spicer
stores show far less seasonal variation than starch (Hoch
et al., 2002, 2003). In the seasonally dry tropics, starch storage increases prior to the onset of the dry season and there is
evidence of mobilization to support leaf flush (Mooney et al.,
1992; Latt et al., 2001; Newell et al., 2002; Wurth et al., 2005).
In contrast, in conifers the previous-year needles are thought
to provide new growth with the needed assimilates and a large
decrease in stored carbohydrates at budbreak is lacking (Höll,
1985; Fischer and Höll, 1991, 1992). Overlaid onto seasonal
patterns of storage are differences in tissue age and there is
evidence that starch stored in parenchyma closer to the cambium (e.g. in younger cells) or the foliage (e.g. in small twigs)
show the largest fluctuations (Bullock, 1992; Mooney et al.,
1992; Barbaroux and Breda, 2002; Landhausser and Lieffers,
2003; Wong et al., 2003; Chapotin et al., 2006b).
Nitrogen is stored in both xylem and phloem parenchyma as vegetative storage proteins localized to the vacuole (Sauter et al., 1988; Sauter and van Cleve, 1989; Sauter
et al., 1989; Wetzel and Greenwood, 1991; Harms and Sauter,
1992; Stepien et al., 1994). Protein bodies increase in abundance at the end of the growing season as leaves senesce and
then decrease in the spring (Harms and Sauter, 1991, 1992;
Langheinrich and Tischner, 1991; Lawrence et al., 1997;
Sauter and Wellenkamp, 1998; Bazot et al., 2013), concomitant with a transient appearance in xylem sap (van Cleve
et al., 1991). Xylem parenchyma are active in retrieving
amino acids from the transpiration stream and transferring
them to the phloem, as well as effectively concentrating them
in xylem streams destined for young leaves (van Bel et al.,
1979; van Bel and van der Schoot, 1979; Dickson et al., 1985;
Vogelmann et al., 1985; Pate and Jeschke, 1995).
The extent to which secondary vascular parenchyma function in water storage and/or recirculation between the xylem
and phloem is gaining increasing attention. Parenchyma in
secondary xylem are thought to store and release water, most
notably in stems with high parenchyma volumes (Mooney
et al., 1992; Chapotin et al., 2006a,b,c; Hearn, 2009b; Hearn
et al., 2013). However, carbon stores are utilized for many
competing processes (e.g. supporting new growth; Mooney
et al., 1992; Sauter and van Cleve, 1994; Sauter, 2000; Latt
et al., 2001; Barbaroux and Breda, 2002; Newell et al., 2002;
Hoch et al., 2003; Wong et al., 2003; Wurth et al., 2005),
freezing tolerance (Sauter et al., 1996; Hoch et al., 2002;
Kuroda et al., 2003; Decourteix et al., 2006; Kasuga et al.,
2007; Takata et al., 2007), recovery from episodic defoliation
(Gregory and Wargo, 1986; Landhausser and Lieffers, 2012),
defence (Imaji and Seiwa, 2010; Goodsman et al., 2013; Lahr
and Krokene, 2013), and transpiration stream maintenance
and repair (discussed below), all of which represent important tradeoffs. A renewed interest in the effects of drought
stress on forest trees highlights the roles of carbon storage
and radial exchange of water between secondary xylem and
phloem (Holtta et al., 2006; Sala et al., 2010; Sevanto et al.,
2011; Mencuccini et al., 2013; Sevanto et al., 2013). Field
measurements, carbon supply manipulations, and models of
stem diameter changes in trees demonstrate the importance
of radial exchange of water between xylem and phloem and
the fact that axial transport of water and assimilates are
linked (Holtta et al., 2006; De Schepper and Steppe, 2010; De
Schepper et al., 2010; Sevanto et al., 2011; Mencuccini et al.,
2013). For instance, increasing tension in the xylem under
drought requires an increase in phloem osmotic pressure to
sustain assimilate transport. If soluble sugars released from
stored carbon are used as osmoticum, internal stores of carbon—stores that will be needed in prolonged drought if stomata close—will be depleted. Although parenchyma play key
roles in this process, little is known about the path of radial
water movement or ultimate source of osmoticum. This key
area of research illustrates the importance of xylem–phloem
exchange in whole-plant resource partitioning in trees, where
mechanistic studies have largely been restricted to smaller
plants of agricultural importance (Pate, 1975; van Bel, 1990,
1995; Pate and Jeschke, 1995).
The mechanism of transport within the parenchyma
symplast is still poorly understood
One of the most striking gaps in our understanding of
parenchyma function in storage is the lack of a proposed
mechanism for transport through the various parenchyma
symplasmic connections (i.e. how materials are physically
moved once they have been signalled to be mobilized). Early
work focused on the high activity of acid phosphatases in
ray parenchyma, which is highest during active growth and
localized more heavily to the tangential cell walls facing
the cambium in both xylem and phloem (Höll, 1975; Fink,
1982). However, this would imply transmembrane transport
whereas most evidence suggests that cell-cell transport is
symplasmic, including plasmodesmatal counts, dye coupling,
and cytoskeleton immunolocalization (Chaffey and Barlow,
2001). Particularly convincing is the immunolocalization of
protein particles to within plasmodesmata during budbreak.
More focus has been placed on the mechanism of symplast–
apoplast exchange, where both H+-ATPases (Fromard et al.,
1995; Alves et al., 2001, 2004, 2007; Arend et al., 2002, 2004)
and sucrose and hexose transporters (Decourteix et al., 2006,
2008) have been localized to the plasma membranes of parenchyma cells associated directly with vessels (‘vessel-associated
cells’ or ‘contact cells’). Interestingly however, both types of
sugar transporters were also localized to radial parenchyma.
Given that radial transport can occur both centripetally and
centrifugally, it is interesting to ask whether this can occur
at the same time in a given tissue (e.g. in separate rays) or
whether a single transport direction dominates. A great deal
more work needs to be done in this area.
Parenchyma function in xylem embolism repair
Due to the strong cohesive forces between water molecules,
liquid water is actually able to sustain a negative hydrostatic
pressure, or tension (Apfel, 1972). Indeed, it is this critical
property of water coupled with its tendency to adhere to
the hydrophilic components of the cell wall (namely, cellulose microfibrils) that provides the foundation for the
cohesion-tension theory of xylem transport. Although a
positive hydrostatic pressure in the xylem can occur, during
Symplasmic networks in secondary xylem and phloem | 1839
active transpiration the xylem water column is under varying
degrees of tension and hence considered ‘metastable’, and in
this state is at risk of cavitating due the expansion of small gas
bubbles (Sperry and Tyree, 1988; Tyree and Sperry, 1989). If
these gas bubbles expand to block liquid flow through a conduit, an ‘embolism’ is said to have formed, and the hydraulic
capacity of the shoot in which it forms is correspondingly
reduced. The field of cavitation research is large, active, and
well beyond the scope of this review, but for one fact: there is
a body of evidence suggesting that parenchyma in secondary
xylem play a role in refilling embolized conduits with water.
Embolisms may form either under increasing tension in
the xylem (e.g. drought-induced embolism; Sperry and Tyree,
1988) or following freezing when gas bubbles trapped during
ice formation expand upon thawing (freeze-induced embolism; Mayr and Sperry, 2010). There is variation in the time
course of both embolism formation and reversal, with freezeinduced embolism repair generally thought to occur seasonally. In contrast, drought-induced embolism in angiosperms
has increasingly been viewed as highly dynamic process, with
some evidence for diurnal cycles of cavitation and refilling
(Zwieniecki and Holbrook, 1998; Zwieniecki et al., 2000; Bucci
et al., 2003), leaving puzzling questions as to how water under
positive pressure might be driven into empty conduits adjacent to conduits filled with water under tension (Holbrook and
Zwieniecki, 1999; Zwieniecki and Holbrook, 2009). Far less
is known about refilling conifer tracheids (but see McCulloh
et al., 2011). It is especially important to note that at the time
of writing this review, a recent discovery that evidence for
diurnal cycles of cavitation repair in several angiosperm species is the result of an experimental artefact (Wheeler et al.,
2013) may call into question a great many experimental results
in the area of xylem refilling (Sperry, 2013). However, there is
still compelling evidence for refilling of embolized conduits
in xylem over longer time scales and/or under minimal tension (e.g. in the absence of sufficient root-generated pressure),
and several lines of research suggest the importance of parenchyma in this process. Most recently, in vivo imaging with high
resolution X-ray computed tomography shows water droplets
forming, expanding, and coalescing during vessel refilling in
grapevine (Brodersen et al., 2010). These exciting images confirm those of prior cryo-SEM studies (Canny, 1997; Utsumi
et al., 1998; Canny et al., 2007) and show droplets specifically
forming in the proximity of contacts with ray parenchyma.
Both techniques are thus capable of discerning the contributions of specific parenchyma cell types.
Many models of refilling dynamics have been proposed
and all rely on the activity of parenchyma cells in contact
with xylem vessels to draw water into the embolized conduit (Brodersen and McElrone, 2013). In most cases, this is
thought to be achieved by the deposition of solutes in the
form of sugars, inorganic cations, or a combination of both.
One notable exception is a model in which a high osmotic
pressure in isolated cells creates a high hydrostatic pressure
surrounding the parenchyma adjacent to embolized vessels
such that water is physically forced into the xylem via reverse
osmosis (Canny, 1997), but this model and its parent, the
compensating pressure theory of sap ascent (Canny, 1995,
1998), have received little to no acceptance (Comstock, 1999;
Stiller and Sperry, 1999). There is considerable evidence that
partially or fully girdling the phloem (i.e. reducing phloem
transport) reduces or prevents xylem refilling (Salleo et al.,
1996, 2004, 2006; Zwieniecki et al., 2000; Bucci et al., 2003)
and that starch levels in parenchyma adjacent to xylem vessels decline on a timescale coincident with apparent refilling
(Bucci et al., 2003; Sakr et al., 2003; Salleo et al., 2004, 2006,
2009; Secchi and Zwieniecki, 2011). There is also evidence
that inhibiting the activity of several H+-ATPases in xylem
parenchyma prevents refilling, while stimulating the activity of H+-ATPase promotes it (Salleo et al., 2004), suggesting that proton-coupled membrane transport is involved.
Particularly exciting is a report using an elegant technique
to collect xylem sap from functional and nonfunctional (i.e.
embolized) vessels separately in water stressed trees. Sap from
nonfunctional vessels—which was substantial in volume—
had a significantly lower pH and up to 5-times the osmotic
potential as functional vessels, where inorganic ions made
up only half of the osmoticum, the rest coming from soluble
sugars (Secchi and Zwieniecki, 2012).
Gene expression studies provide further evidence for a role
of parenchyma in osmotically driven water movement into
embolized vessels, with parenchyma in the secondary xylem
of Populus showing an upregulation of genes encoding aquaporins (primarily PtPIP2s), sucrose transporters (PtSUC2.1
and PtSUT2b, albeit transient), di- and tri-valent cation
tansporters, and α- and β-amylases in response to artificially
induced embolism (Secchi and Zwieniecki, 2010, 2001; Secchi
et al., 2011). Although not strictly in secondary vascular tissue, similar changes in gene expression were found in petioles
during diurnal cycles of water stress and recovery in grape
(Perrone et al., 2012). As key candidate genes and transport
proteins are identified, reporter constructs and immunolabelling studies should allow for cell-specific resolution of parenchyma activity in xylem refilling.
Parenchyma in secondary xylem may directly affect
hydraulic conductivity through ion exchange
Both radial and axial parenchyma in secondary xylem have
direct access to the transpiration stream where the plasma
membrane (and amorphous layer) abut a vessel or tracheid
pit, and some evidence suggests that their activity could have
rapid and reversible effects on hydraulic conductivity. Flow
rates through vessels have been shown to increase by up to 2.5
times in response to low (i.e. physiologically relevant) concentrations of disassociating solutes (KCl, NaCl, KNO3, CaCl2)
in the xylem sap solution (Zimmermann, 1978; van Ieperen
et al., 2000; Zwieniecki et al., 2001; Gasco et al., 2006). This
effect is independent of the presence of living parenchyma
cells and thus thought to be localized to the connections
between vessels (i.e. flow through a single vessel was unaffected, but see van Ieperen et al., 2000 for contrasting results),
namely the intervessel pit membranes, which are composed of
partially hydrolysed primary cell wall material (Choat et al.,
2008). Intervessel pit ‘membranes’ consist largely of loosely
woven cellulose microfibrils and bear no relationship to true
1840 | Spicer
plasma membranes. One hypothesis is that residual pectins in
the pit membrane function as a hydrogel and shrink and swell
in response to ionic interactions, effectively changing the size
of the pores through which water flows (Zwieniecki et al.,
2001; Gasco et al., 2006; van Ieperen, 2007). However, there
is some evidence that pectins are effectively removed from
the pit membrane during enzymatic hydrolysis of the primary wall/middle lamella complex in angiosperms (O’Brien
and Thimann, 1967; O’Brien, 1970; Butterfield and Meylan,
1982; Plavcova and Hacke, 2011; van Doorn et al., 2011; Kim
and Daniel, 2013). Alternative mechanisms include (a) other
polymers like lignin and hemicelluloses shrinking and swelling via ionic interactions (van Doorn et al., 2011) and (b) the
viscoelectric effect, in which charge interactions effectively
alter the viscosity of a fluid, causing an apparent change in
conductivity (Santiago et al., 2013).
Regardless of the mechanism, it is thought that xylem
parenchyma could significantly alter flow by changing the
ionic composition and/or pH of the transpiration stream.
This would allow plants to spatially regulate xylem resistance and potentially compensate for lost hydraulic capacity
due to embolism, as well as favour transport to or from certain xylem sectors (Zwieniecki et al., 2003). Although the in
planta significance of this effect remains unclear (van Ieperen,
2007), there is evidence that phloem-derived ions added to
the transpiration stream can increase hydraulic conductivity
of the xylem (Zwieniecki et al., 2004). Potassium ions (K+)
could be loaded into the xylem via recycling from the phloem
(Zwieniecki et al., 2001, 2004), and H+-ATPases in xylem
parenchyma membranes could modify pH (Fromard et al.,
1995). However, there is also evidence that the inclusion of
physiologically realistic Ca2+ concentrations greatly decreases
the ion-mediated effect on hydraulic conductivity (van
Ieperen and van Gelder, 2006), calling into question the biological significance of the effect (van Ieperen, 2007). It is not
clear how widespread this phenomenon is however, as some
woody species maintain a significant effect of KCl even with
the inclusion of Ca2+ (Nardini et al., 2007). Lastly, it has been
shown that an inverse relationship exists across all vascular
plants between the degree of primary wall lignification and
the magnitude of ion-mediated hydraulic response (Boyce
et al., 2004), suggesting large-scale evolutionary implications
of this effect. Interestingly, the authors attribute the relationship to an effective shielding of pectins by lignin deposition,
but it could also easily support the alternative hypothesis
proposed by van Doorn (2011). Clearly more work is needed
to understand the true in planta capacity of parenchyma to
regulate xylem resistance.
Parenchyma exhibit extreme longevity, remain
pluripotent, and serve to protect the integrity
of the stem
Most axial and radial parenchyma in secondary vascular tissue remain alive after the conducting elements cease
to function, underscoring that their roles are complex and
may change with age. In senescing secondary xylem, parenchyma help defend tissue against microorganisms by rapidly
synthesizing phenolic compounds (Chattaway, 1952; Bamber
and Fukazawa, 1985; Magel, 2000) and by plugging both
vessels and resin canals with newly produced cell wall material (i.e. tyloses and tylosoids, respectively), gums, and resins (Chattaway, 1949; Czaninski, 1977; Bonsen and Kucera,
1990). These activities occur shortly before parenchyma cells
die and characterize heartwood formation, in which a nonconducting cylinder devoid of all living cells is compartmentalized in the centre of the stem (Chattaway, 1952; Spicer,
2005). In angiosperms in which only the outermost xylem
is conductive, parenchyma may live for 5–25+ years after
water transport ceases (Spicer and Holbrook, 2005, 2007;
Chapotin et al., 2006b), continuing to function in storage
and wound response until the onset of heartwood formation
or tree death. Interestingly however, there is evidence that
axial parenchyma surrounding vessels die much earlier than
ray parenchyma in the highly parenchymatous Adansonia
(Chapotin et al., 2006b). Parenchyma in secondary phloem
also remain alive well after the tissue has ceased conducting
photosynthate, which is usually at the end of the first growing season (or ‘flush’ of growth in nontemperate regions) and
rarely more than two (Esau, 1948, 1950, 1964; Schneider,
1955; Evert, 1961, 1963a; Alfieri and Evert, 1968; Alfieri and
Kemp, 1983). Although the Strasburger and companion cells
die when their partnered conducting elements form definitive
callose and lose their protoplast, many ray and axial parenchyma remain alive, in some cases for 20+ years. In nonconducting phloem, axial parenchyma cells expand in diameter,
contributing to the collapse of sieve elements. Some axial
parenchyma develop thick lignified walls at this point, effectively differentiating into sclereids late in life (Esau, 1950),
functioning in mechanical support and defence. An equally
conspicuous change is in the radial parenchyma, which
expand in tangential diameter and often undergo renewed
divisions in a process called ‘ray dilatation’ (Schneider, 1955).
This dramatically increases the tangential diameter of a subset of the rays, thereby allowing the phloem to keep pace with
the expanding circumference of the stem and remain intact.
The ability of both ray and axial parenchyma cells to divide
is retained throughout their life, such that these cells also
function in wound response through the formation of callus
tissue (Wargo, 1977; Shigo, 1984). Highly parenchymatous
stems like pachycauls and lianas (discussed below; Table 1)
appear to have particularly active parenchyma in this regard,
which may be an adaptation to having mechanically weak,
easily injured stems (Dobbins and Fisher, 1986; Fisher, 1981;
Fisher and Ewers, 1989, 1991).
Repeated evolution of parenchymatous secondary
tissue underscores the importance of parenchyma
in whole-plant physiology
A very different approach to understanding the physiological significance of parenchyma at the whole-plant level is to
consider its role in evolutionary diversification. Although it
is beyond the scope of this review to cover these patterns in
any detail, there are several growth forms in angiosperms in
which an enormous amount of parenchyma is produced by the
Symplasmic networks in secondary xylem and phloem | 1841
vascular cambium, including lianas, pachycauls (swollen stems,
or bottle-trees), and a subset of stem or root tubers (Table 1).
The diverse and often overlapping functions of parenchyma
are encapsulated within these unique growth forms: parenchyma in lianas allows for increased stem flexibility (Gartner,
1991; Putz and Holbrook, 1991) and recovery from injury
(Fisher and Ewers, 1991), pachycaulous stems store both carbohydrates and water as part of a complex suite of adaptations
to arid environments (Borchert, 1994; Chapotin et al., 2006b;
Hearn, 2009b; Hearn et al., 2013), and subterranean tubers
store carbohydrates for survival through cold or dry periods
(Pate and Dixon, 1981; Hearn, 2009b). Within each of these
growth forms are multiple developmental paths to producing
large amounts of parenchyma, some of which occur together
in the same organ, while some singularly characterize whole
species or genera. Each of these developmental paths has
evolved independently multiple times (Hearn, 2009b; Hearn
et al., 2013) and most result in wide bands of parenchyma that
alternate with vascular tissue, underscoring the importance of
a close association of the two tissues. This evolution of development approach also highlights the inextricable link between
anatomy and physiology, where selective pressures act on both
to produce adaptations to particular environments.
Highly parenchymatous secondary growth may be a product of a vascular cambium functioning ‘normally’ but simply
producing large amounts of parenchyma, but more typically
results from cambial activity that is unusual when compared
to the majority of trees and shrubs. The production of successive or ‘supernumery’ cambia is common in lianas, pachycauls, and tubers (e.g. Beta spp.; Carlquist, 2001b). Here, a
normal cambium forms and produces secondary xylem and
phloem in the expected positions, but later a new cambium
dedifferentiates from parenchyma, first in the cortex and then
later in the secondary phloem (Fahn and Zimmermann, 1982;
Carlquist, 2001b, 2007; Rajput et al., 2008, 2012a,b, c; Robert
et al., 2011). Each new cambium produces a disproportionate
amount of parenchyma (usually axial xylem parenchyma but
variation exists and standard definitions are difficult to apply)
as well as conducting elements, the end result being a highly
parenchymatous organ with repeating layers of both xylem
and phloem. A second means to build up large volumes of
parenchyma is to allow parenchyma to proliferate (renew
mitotic divisions) at some distance from the cambium. This
occurs in secondary phloem normally during ray dilatation
but is common in the xylem (less commonly the phloem) of
pachycauls (Carlquist, 1998; Chapotin et al., 2006b; Hearn,
2009a,b). A related phenomenon occurs when alternating
tangential segments of the cambium produce significantly
different ratios of xylem/phloem such that the cambium
becomes furrowed and/or disjunct (i.e. the outward expansion
of alternating segments occurs at different rates). This produces a stem with wedges or strips of phloem running deep
into the xylem and is usually associated with proliferation of
parenchyma as well (Carlquist, 2001b; Pace et al., 2009, 2011;
Angyalossy et al., 2012). Lastly, many lianas achieve high
proportions of parenchyma by having exceptionally wide,
tall, unlignified rays, some of which may undergo postcambial proliferation (Carlquist, 1991; Angyalossy et al., 2012).
Conclusion
It is difficult to overstate the importance of parenchyma in
supporting transport in secondary vascular tissues. Although
our understanding of the physicochemical mechanisms driving xylem and phloem transport have improved dramatically,
we still lack a fundamental understanding of the mechanism
behind radial transport. Studies focused on the apoplast–
symplast interface and plasma membrane transporters have
provided a good foundation for xylem/parenchyma exchange,
but the role of the amorphous layer filling pit cavities is still
poorly understood. Although studies of transporter gene
expression and protein quantification are welcome additions,
emphasis should be placed on cell-specific techniques (in situ
hybridization, immunolocalization, and reporter constructs)
and applied to secondary tissues of different ages and physical positions within the stem. Most importantly, improved
model systems for in vivo dye and radiolabelled tracer studies as well as techniques for measuring parenchyma cell pressures and internal compositions need to be developed or
more widely employed. Given the increasing recognition that
diffuse sources and sinks are located in close proximity to
the phloem in woody stems, as well as increased interaction
among biologists studying xylem and phloem, there is good
reason to expect continued progress in this area.
Acknowledgements
The author would like to thank Aart van Bel and two anonymous reviewers for insightful comments on a previous version
of this review.
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