Download Growth of Zea mays L. plants with their seminal

Journal of Experimental Botany, Vol. 48, No. 311, pp. 1229-1239, June 1997
Journal of
Experimental
Botany
Growth of Zea mays L. plants with their seminal roots
only. Effects on plant development, xylem transport,
mineral nutrition and the flow and distribution of abscisic
acid (ABA) as a possible shoot to root signal
W. Dieter Jeschke1'3, Margita Holobrada2 and Wolfram Hartung1
1
Julius-von-Sachs-lnstitut fOr Biowissenschaften, Lehrstuhl fiir Botanik I, Universita't Wurzburg, Mittlerer
Dallenbergweg 64, D-97082 WQrzburg, Germany
2
Institute of Botany, Slovak Academy of Sciences, Dubravska' cesta 14, SK-84223
Bratislava, Slovak Republic
Received 1 November 1996; Accepted 20 February 1997
Abstract
Maize l£ea mays L.) was grown in quartz sand culture
either with a normal root system (controls) or with
seminal roots only ('single-rooted'). Development of
adventitious roots was prevented by using plants with
an etiolated mesocotyl and the stem base was positioned 5 - 8 cm above the sand. Even though the roots
of the single-rooted plants were sufficiently supplied
with water and nutrients, the leaves experienced water
deficits and showed decreased transpiration as transpirational water flow was restricted by the constant
number of xylem vessels present in the mesocotyl. As
a consequence of this restriction, transpirational water
flow velocities in the metaxylem vessels reached mean
values of 270 m h " 1 and phloem transport velocities
of 5.2 m h " ' . Despite limited xylem transport mineral
nutrient concentrations in leaf tissues were not
decreased in single-rooted plants, but shoot and particularly stem development was somewhat inhibited.
Due to the lack of adventitious roots the shoot:root
ratio was strongly increased in the single-rooted
plants, but the seminal roots showed compensatory
growth compared to those in control plants. Consistent
with decreased leaf conductance, ABA concentrations
in leaves of single-rooted plants were elevated up to
10-fold, but xylem sap ABA concentrations in these
plants were lower than in controls, in good agreement
with the well-watered conditions experienced by the
seminal roots. Surprisingly, however, ABA concentrations in tissues of the seminal roots of the singleJ
To whom correspondence should be addressed: Fax: +49 931 8886156.
Oxford University Press 1997
rooted plants were clearly increased compared to the
controls, presumably due to increased ABA import via
phloem from the water-stressed leaves. The results
are discussed in relation to the role of ABA as a shoot
to root signal.
Key words: Zea mays, seminal roots, plant development,
xylem transport, mineral nutrition, ABA, shoot-to-root
signal.
Introduction
Growth and development of the shoot of a whole plant
is strongly dependent on concomitant and unrestricted
development of the root system, as evident from the
effects of root pruning (Jesko, 1972a, b; Carmi and Koller,
1978; Milligan and Dale, 1988) and the restriction of the
rooting volume Carmi et ah, 1983; Robbins and Pharr,
1988; Creswell and Causton, 1988; Peterson et al, 1991a).
In both of these cases the capacity of a major assimilate
sink in the plant was decreased by restricting the size of
the root and this led to a commensurate decrease in shoot
growth and, usually, in the rate of leaf photosynthesis.
In the case of monocotyledons which lack secondary
thickening the size of the root system can be severely
restricted if adventitious nodal roots are excised (Jesko,
1972a; Jeschke, 1984) or their development is prevented
(Passioura, 1972; Passioura and Ashford, 1974) and the
root system is then confined to the primary root. Passioura
used single-rooted wheat plants, in which the hydraulic
1230
Jeschke et al.
resistance to water flow was greatly increased, as a means
to decrease water consumption by the whole plant. In
this way he was able substantially to increase the grain
yield in comparison with plants having the full root
system, if both the single-rooted and the control plants
were dependent on a limited water resource (Passioura,
1972).
While effects of a restricted root size on shoot and leaf
development and photosynthesis have been widely studied
(Carmi et al., 1983; Robbins and Pharr, 1988; Cresswell
and Causton, 1988; Peterson et al., 1991a), any possible
water deficit and restriction of the mineral nutrition of
the shoot, which may also arise, have attracted somewhat
less attention (Richards and Rowe, 1977; Hameed et al.,
1987). In preliminary experiments with single-rooted
maize {Zea mays L.) plants continually supplied with
water and nutrients not only was decreased water uptake
(see Passioura, 1972) observed, but also signs of severe
water deficits. The aim of the present paper is firstly to
study the effects of a severely restricted root system,
consisting only of the primary and adventitious seminal
roots, on the water relations, the xylem sap composition
and the mineral supply of maize plants. Secondly, it is
intended to reveal possible root to shoot or shoot to root
signals acting in maintaining economical water consumption in the whole plant with an inadequately small root
system which is, nevertheless, exposed to adequate soil
moisture. In this case, neither biosynthesis of abscisic acid
(ABA), which seems to play a well established role as a
root-to-shoot stress signal (Davies and Zhang, 1991),
should be increased in the root nor its concentration in
the xylem sap elevated, because roots are well supplied
with water and nutrients and because root volume is not
restricted. Restricted root volume was shown by Liu and
Latimer (1995) and Ternesi et al. (1994) to increase ABA
concentrations in the xylem. One could expect therefore
an increased ABA formation in the leaves. Part of this
ABA could be translocated in the phloem back to the roots.
Materials and methods
Plant culture
Zea mays L., cv. Garant, FAO 240 hybrid maize seeds were
soaked and germinated in 10 1 pots in quartz sand moistened
with 1/16 Hoagland nutrient solution at different light conditions. Control plants (control) were allowed to germinate in
the greenhouse in the light; nodal roots developed in due course
at the base of the stem. In order to produce plants having their
seminal roots only, some of the pots were kept in the dark and
these seedlings developed a strongly etiolated, elongated mesocotyl. The coleoptile node was about 5-8 cm above-ground.
Throughout cultivation the shoot of these plants was supported
so that it remained above-ground and though nodal roots were
initiated, they were unable to reach the quartz sand substrate
(Plate 1). In the text these plants will be termed 'single-rooted
plants'. Starting from 6d afteT sowing (DAS) all plants were
initially cultivated in the greenhouse with 16 h d " 'supplemental
Plate 1. Basal part of the shoot of a single rooted maize plant. The
arrow denotes the etiolated mesocotyl; note the undeveloped initials of
adventitious roots. Age of the plant: 11 weeks.
illumination (Osram HQL 400, 300-500 ^onol photon m 2 s '),
15-25 CC and 45-70% relative humidity and later both variants
were grown outside the greenhouse under ambient light,
temperature and humidity conditions.
Plants were grown in quartz sand culture and watered daily
with nutrient solution. The concentration was gradually
increased from an initial 1/16 to a final 1/2 strength of Hoagland
solution (2.5 mM Ca(NO3)2, 2.5 mM KNO3) 1 mM MgSO4,
1 mM KH2PO4, 0.5 mM H3BO3) with additional micronutrients
and Fe-citrate.
Measurement of total plant transpiration and leaf conductance
Total plant transpiration was measured gravimetrically by
determining the daily or sometimes 2 h water loss and correcting
for the loss from the substrate by weighing a pot without a
plant. Leaf conductance of all leaves of control and singlerooted plants was measured with a LiCor 1600 steady-state
porometer under ambient conditions. Measurements were done
at the same time of the day (1-3 p.m.), but only under sunny
conditions.
Harvesting of plants and collection of root pressure xylem exudate
Harvests were made in experiment 1 at 77 DAS at early fruiting
and in experiment 2 at 68 DAS at the time of tasseUing. Plants
were harvested and separated into roots, stem and individual
Maize plant growth
leaves. Leaf laminae were further subdivided into basal (b) and
apical (a) parts. Roots were carefully washed to remove quartz
sand particles. Samples of all plant parts were weighed, freezedried and dry weight was determined. After fine grinding,
sample aliquots of dry matter were used for chemical analyses.
Xylem sap was collected at the day of harvesting. After
excision of the shoots silicon rubber tubing was connected to
the tops of the mesocotyl of the single-rooted plants or to the
stump of the stem base of control plants. The xylem sap
collected during the first few min was discarded to avoid
contamination resulting from cut tissues (Else et al., 1994).
Collected xylem sap volumes were recorded and samples were
frozen before use for further analyses.
1231
mesocotyl plus root was obtained from the sum of all individual
conductivities and the pressure difference Ap needed for driving
the measured volume flow (total water loss from the plant) was
obtained from (2). Using (1), the volume flow [cm 3 h"'] in
each of the vessels at the prevailing pressure difference Ap was
then estimated and from this volume and the cross-sectional
vessel area [cm2] theflowvelocity [cm h - 1 ] or [m h" 1 ] occurring
in each individual vessel was calculated.
Flow velocities in the phloem were estimated on the basis of
the measured sieve tube area, the quantities of carbon needed
for root growth and respiration and an assumed sucrose
concentration of 20% in the phloem sap, for details see below
in the Discussion.
Chemical analyses
Cations in tissues and in the xylem sap were determined using
atomic absorption spectrometry (FMD 3, Carl Zeiss,
Oberkochen), anions by anion chromatography (Anionlenchromatograph, Biotronik Co., Maintal, Germany). Amino
acids were determined using an amino acid analyser (Biotronik Co., Maintal, Germany).
Analysis of free and conjugated ABA
Freeze-dried tissue samples were homogenized and extracted in
80% methanol. Extracts were passed through a Sep Pak C18cartridge. Methanol was removed under reduced pressure and
the aqueous residue partitioned three times against ethyl acetate
at pH 3.0. The ethyl acetate of the combined organic fractions
was removed under reduced pressure. The residue was taken
up in TBS-buffer (TRIS-buffered saline; 150mmol I" 1 NaCl
lmmol I" 1 MgCl2> and 50mmol I" 1 TRIS; pH 7.8) and
subjected to an immunological ABA assay (ELISA) as described
earlier (Peuke et al., 1994; Mertens et al. 1985). For xylem saps
the Sep-Pak C18 purification step was omitted. The aqueous
phase after partitioning against ethyl acetate was hydrolysed
for 1 h at room temperature with 1 M NaOH. This fraction
was acidified with concentrated hydrochloric acid to pH 3, and
partitioned three times against ethyl acetate. ABA which had
been released from conjugates (predominantly ABA-glucose
ester) was analysed by ELISA in the organic phase. The
accuracy of the ELISA was verified for Zea in earlier
investigations (Hartung et al., 1994). Recoveries of ABA during
purification procedures were checked routinely using radioactive
ABA and found to be more than 95%. The immunochemicals
were generously supplied by Professor Weiler, Ruhr Universitfit
Bochum (Germany).
Evaluation of volume flow conductivities and flow velocities of the
xylem vessels
Using transverse sections of the mesocotyl (Plate 2) or the basal
stem of control plants the radii of xylem vessels r, were
measured. According to Hagen-Poiseuilles law (Stuart and
Klages, 1988; Nobel, 1991) the volume flow J, [cm3 s"1]
through a capillary vessel with the radius r, is
Jv = A V/At = - (
(1)
and for a series of parallel vessels
(2)
with A V the volume flowing in the interval A t under the
pressure difference Ap, I the length of the vessel and TJ the
viscosity of the solution (for water 77=0.01002 dynes cm" 2 ).
For each vessel the individual volume flow conductivity Arf/S-q
I was estimated using 30 cm for the length / from the soil to the
upper end of the mesocotyl. The total conductivity of the
Results
Plant growth and development
The single-rooted plants developed an etiolated 8-15 cm
long and 2-3 mm wide mesocotyl. The anatomical structure of the etiolated mesocotyl in its mature state was
distinguished from other stem parts by a tertiary and in
parts double endodermis with strongly thickened walls
(Plate 2). The central cylinder showed the arrangement
of xylem and phloem as in the root but contained further
mechanical elements in the outer part of the central pith.
Interestingly one or two lacunae were also present
(Plate 2) which resembled the protoxylem lacunae in the
stem bundles. Amongst the vessels (Table 1) there were
7-11 larger late metaxylem vessels on average 59 ^m wide
which were responsible for 89% of the water transport,
compared to c. 450 vessels 87 ±3 fun wide in the basal
stem above the uppermost nodal roots of the control
plants (not counting the protoxylem lacunae).
Until 45 DAS, plant height was not affected, but later
on it was decreased by about 10% in the single-rooted
plants. Similarly, leaf area expansion in the uppermost
leaves, which completed expansion after 52 DAS, was
depressed in response to the reduced root system (Fig. 1A,
leaves younger than leaf 8). During the 68 or 77 d of
growth in the two experiments the primary seminal root
plus the adventitious seminal roots (Jesko, 1991) grew
profusely and branched remarkably to reach final dry
weights of 3.4 or 4.0 g. Nevertheless, at harvest, root
biomass was the most strongly decreased compared to
that of the control, followed by that of the stem (Table 2),
while leaf dry matter production was least inhibited in
single-rooted plants (93% or 78% of the controls in
experiments one and two respectively). The severe restriction of root development, caused by the lack of nodal
root growth, led to strongly elevated shoot-to-root ratios
of 15.7 or 11.9 compared to 3.5 or 6.1 in controls in
experiments one and two, respectively. Inflorescence
development was delayed and probably decreased, since
it occurred during and after the first signs of water deficits
were visible in the single-rooted plants (see Fig. IB). In
the first experiment the fruiting inflorescences of single-
1232
Jeschke et al.
Plate 2. Transverse cross-section of the etiolated mesocotyl of Zea mays L. at the age of 68 days after sowing (DAS). (A) Inner part of the cortex
(Co) and stele. (B) Detail of stele and inner part of cortex. En = endodermis, ph = phloem, MX = metaxylem vessel, Sc = sclerenchyma, Pi = pith,
Lac=>lacunae. The length of the bar corresponds to 250 ^m in (A) and to 100 (im in (B). Fixing: ethanol-fonnaldehyde-acetic acid, 6-8 fun thick
sections (HN34 microtome, Jung, Germany). Staining has been performed by R Wacker, University of WOrzburg, Biozentrum, Physiological
Chemistry, D-97218 Gerbrunn.
rooted plants weighed only 13% of those of the control,
but this was also due to a delay in flowering. Plants were
harvested too early to judge effects of single-rootedness
on yield (Passioura, 1972).
Whilst specific signs of severe mineral deficiency and
leaf necroses were not visible, rolling of the leaves of
single-rooted plants indicated incipient water deficits and
with time slightly chlorotic bands developed along the
leaves.
Water relations and leaf photosynthesis of control and
single-rooted plants
As was to be expected, the dependence of maize plants
on one single root had the most substantial effects on
their water relations (Table 3, items i to vii). As a direct
consequence of the much smaller root biomass, the water
uptake and hence loss per plant (i) was severely decreased
(by 72% or 41% in experiments one and two, respectively).
Since leaf area was somewhat smaller, water loss relative
to leaf area (ii) was less depressed (by 42% or 27%), but
leaf conductance was substantially lower in single-rooted
than in control plants (Fig. 2; iii in Table 3).
Remarkably, however, these differences between singlerooted and control plants disappeared or were even
inverted, if the transpirational water loss was related to
the root size and the water uptake per root weight was
calculated (neglecting the small amounts of water used
for growth). Relative to root fresh weight, water uptake
Maize plant growth
means stable, but it decreased with time as shoot growth
and size progressively exceeded that of the root.
CO2 assimilation by leaves of single-rooted plants was
decreased (viii in Table 3) as has similarly been found in
plants with a restricted rooting volume (Robbins and
Pharr, 1988), but not in sorghum after excision of nodal
roots (Jesko, 1972a). The difference most probably was
due to the much shorter (39 d) duration of the experiments with sorghum.
500
control
400E
300-
e
200-
u
100-
L6
L7
tuOy expanded at: 4 2
47
L8
L9
leaf number
52
57
L10
82
Mineral element concentrations in root and leaf tissues of
control and single-rooted plants
L11
66 DAS
100
B
75
I
o
1
20
r——
watf loss
led area
50
15
10
o
25
*>a'
0
50
60
As seen in Fig. 3, for no element, except for Zn in roots,
was the concentration in root or leaf tissues decreased
significantly in single-rooted compared to control plants.
On the contrary, even in leaf tissues, which would have
been more likely to become inadequately supplied as a
consequence of the restricted root size, the concentrations
of all major and minor nutrient elements more or less
exceeded those in leaves of the control plants. This was
also true for Mg, which might have been deficient, as
suggested by slightly chlorotic bands along the leaves.
5
pressure gradient
o—""'
40
1233
70
days after sowing (D A S)
Fig. 1. (A) Final leaf area of leaves No. 6 to 11 of control (—D—)
and single-rooted ( V ) plants of Zea mays L. The numbers below
the x-axis show the time in days after sowing when expansion of each
leaf was completed and indicate that the x-axis also is a time-axis,
which is almost synchronous with that of (B). (B) Time-dependence of
the water loss per leaf area of single-rooted maize plants, given as % of
the controls (—•—) and time-dependence of the pressure gradient
needed to drive xylem sap at the prevailing rate through the mesocotyl
of single-rooted plants of Zea mays L. ( — D — ) (right hand scale).
Bars indicate SE of the mean.
in single-rooted plants even slightly exceeded that in the
controls (iv in Table 3). Interestingly, the same result was
seen in the volume flows of root pressure exudates (vii)
which, though much slower than transpirational water
flow, were 14% higher in single-rooted than control plants.
As seen in Fig. 3B, in relation to the control plants the
water supply available from the single root was by no
Concentration of free and conjugated ABA in root, tassel
and leaf tissues of control and single-rooted plants
In Fig. 4 concentrations of abscisic acid in root, tassel
and leaf tissues of the apical half of control and singlerooted plants are shown. Whereas almost constant ABA
levels of 0.05 to O.lnmolg" 1 FW were found in all
control leaves, ABA concentrations were twice as high in
young leaves of single-rooted plants and increased with
leafage to reach in older leaves 12-fold higher levels than
in the controls. Tassel ABA concentration was increased
by 60% and roots contained an 8-fold elevated ABA level
(Fig. 4). Root ABA concentrations are in a range similar
to that found by Zhang and Davies (1989).
Concentrations of conjugated ABA, which consists
mainly of ABA-glucose ester (ABA-GE) were also determined and slightly lower concentrations than those of free
ABA were found. Similar to free ABA, young leaves of
single-rooted plants contained 2-fold higher levels of conjugated ABA than controls and concentrations increased
with leaf age to reach 4-fold higher levels compared to the
Table 1. Number, diameter and volume flow conductivity of the xylem vessels in the etiolated mesocotyl of single-rooted maize plants
Type of vessels
Late metaxylem vessels
Smaller vessels*
Experiment
Number of
vessels
Mean diameter
Range
(fun)
Conductivity*
11
9 ±0.4
112
36 + 3
59 ±4.2
55.8 ±4.2
21.9± 1.7
23.6 ±1.2
41-85
35-93
9-37
10-45
1.45x10
0.2x10
' Mostly early metaxylem vessels.
* Estimated with Hagen-Poiseuille's law as -n^/i^l for all individual vessels. The length / of vessels was assumed to be 30 cm from the top of the
mesocotyl down to within the primary root. The choice of / has no effect on the relative contribution of the differently wide vessels to total
conductivity.
1234
Jeschke et al.
Table 2. Dry matter content of control and single-rooted Zea mays plants recorded at the day of harvesting, Le. 68 d after sowing
The results are presented as mean values ±SE of 4 control and 10 single-rooted plants in experiment 2.
Organs
Experiment 2
Experiment 1*
Control plants
(gDW plant" 1 )
Leaves
Tassel
Cob
Stem
Shoot
Root
Shoot/root
Single-rooted plants
% of Control
(gDW plant" 1 )
13.1 ±0.2
3.4 ±0.1
12.1 ±0.4
2.6±0.2
40.3 ±3.9
56.8 ±4.0
9.3±1.1
6.10
25.3 ±0.9
40.1 ±2.3
3.4±0.3
11.90
% of Control
93
78
—
63
71
36
78
—
13
58
56
16
* For comparison, data for the single-rooted plants in experiment 1 are given as % of the respective control. Since the harvest was later (at
77 DAS) in this experiment, dry weights of all organs in the single-rooted plants were more depressed than in experiment 2 and a cob was formed.
controls in the oldest leaves. Conjugated ABA was also
higher in roots (8-fold) and the tassel (2-fold) of singlerooted plants (Fig. 5). The increased leaf ABA concentrations of single-rooted plants were well related to the
observed decreases in leaf conductances (Fig. 2).
ing from the fully developed root system was two times
faster than from the single primary root (vi in Table 3).
If volume flows were related to the root fresh weight,
however, single-rooted plants bled 14% more rapidly than
the controls, a quite remarkable result (vii in Table 3)
which agrees, however, with the high water uptake capacity of maize seminal roots observed by Navara et al.
(1994).
In full agreement with this somewhat higher volume
flow and owing to the much higher hydraulic resistance
Xylem sap flow and composition in control and singlerooted plants
Both control and single-rooted maize plants produced
root pressure exudate after shoot excision, although bleed-
Table 3. Water loss and transport parameters and leaf photosynthesis in control and single-rooted plants o/Zea mays L.
Item
Experiment 1*
Control
Experiment 2
Single-rooted' %of
Control
Control
Single-rooted'
%of
Control
Water relations
Mean water loss per plant
[gh" 1 plant" 1 ]
Water loss per leaf area
Leaf conductance g
Water uptake per root fresh weight
vi
vii
Flow velocity in large metaxylem vessels
[mh-'Htcms- 1 ])
Xylem sap flow (root pressure) per plant
[cm'h" 1 plant" 1 ]
Xylem sap flow (root pressure)
[cm 3 gFW" 1 h" 1 ]
29.2±6
8.2 ±2.4
28
14.2±3.0
8.4 ±1.0
59
1460±31
840±70
58
1290±27
940± 11
73
n.d.'
1.3±0.2
(0.3)
—
n.d'
0.21 ±0.02
1.4 ±0.4
107
e
2400
270±35
(7.5)
0.1 ±0.009
50
0.4 ±0.1
0.44 ±0.05
110
3.2±0.2
1.6±0.2
50
0.028 ±0.006
0.032 ±0.003
114
53.2±3.0
45.5 ±2.4
86
Photosynthesis
CO2 assimilation
[/Imol m- 2 s~ 1 ]
n.d.'
n.d.'
n.d.'
5.2'
Phloem transport
Flow velocity in the sieve tubes
[rnh" 1 ]
* Measurements of water loss and transport in experiment 1 are means over the period 40-60 DAS.
* Measurements of water loss in experiment 2 were made over the period 36-67 DAS.
' Single-rooted maize plants with etiolated mesocotyl.
' n.d. = Not determined.
' Mean flow velocity estimated for the mean water loss (i) = 8.2, the range was 193-374 m h " ' depending on the size of the vessels. This mean
flow velocity increased with the size of the transpiring leaf area and reached, e.g. 510 m h" 1 or 14 cms" 1 at 63 DAS.
1
Mean flow velocity was estimated on the basis of the measured sieve tube area, the carbon use in the root and a sucrose concentration of 20%.
Maize plant growth
1235
0.6-
O) 0 4 -
o
<
;
0.2-1
V
0
CD
8
increasing leaf age
Leaf
9
10
1 1
5x
root
number
Fig. 2. Leaf conductance g as measured in leaves of control (—•—)
and single-rooted ( V ) plants of Zea mays L. as a function of leaf
number (and age).
4+5
leaf number
7
8
9
10 11 + 1 2tassel
Fig. 4. Concentration of abscisic acid (ABA) in tissues of the apical
leaf half, roots and tassel of control (—•—) and single-rooted
( V ) plants of Zea mays L.
-0.25-
T
o
E0.15<
CD
<
0.1-
220.05^
V
3
o
D
0—Q5x
root
4+5
leal number
7
8
9
10 11 + 12tassel
Fig. 5. Concentration of abscisic acid conjugates (ABA-GE) in tissues
of the apical leaf half, roots and tassel of control (—•—) and singlerooted ( V ) plants of Zea mays L.
Fig. 3. Element concentrations in ^mol g ' FW in root and leaf tissues
of control (ED) and single-rooted plants (Q) of Zea mays L. Note that
major elements are given at a 20- or 10-fold reduced scale and minor
elements at a 5- or 10-fold enlarged scale and that the y-axis scale for
the root is 2-fold enlarged.
to flow (see below) the concentrations of some solutes,
notably K + , NOf, Mg2 + , SO*" and amino acids, in the
exudate from single-rooted plants were substantially
higher than in that from control plants (Fig. 6). Owing
to the slightly increased volume flow (Table 3), solute
flows of nutrients were even more increased than were
their concentrations. Levels of other ions such as Ca2 + ,
H2POJ and malate were the same in exudates from the
two types of plants (Fig. 6). Consistent with results of
Fig. 3, however, none of the nutrient ions showed lower
levels in xylem sap of single-rooted than control plants.
In addition to an overall higher concentration, the
amino acid composition in exudates from single-rooted
plants was also altered compared to that of the controls
(Table 4). The concentrations of basic amino acids and
in particular of amides were increased while those of
acidic amino acids were lower.
Concentrations of ABA in xylem exudates from the
mesocotyl of single-rooted plants were on average 7 times
lower than in those from the control stem base.
Concentrations of conjugated ABA, however, were
2.5-fold higher in single-rooted than control plants
(Table 5). The changes in xylem sap ABA and ABA-GE
concentrations clearly were not due to the slightly higher
flow rates (see Else et al., 1994) in single-rooted plants,
since (a) ABA and ABA-GE levels changed in opposing
1236
Jeschke et al.
40-,
control
single-rooted
8
n
20
10
O
O>
<3
CO
O
Fig. 6. Concentrations of ions and of total amino acids in the root
pressure xylem exudate collected from control (ED) and single-rooted
plants (Of) of Zea mays L. Solute flow rates may be obtained from
concentrations and the volume flow rates given in Table 3.
Table 4. N-compounds in xylem sap of control and single-rooted
plants of Zea. mays L.; data are means ±SEM
Acidic amino acids
Basic amino acids
Neutral amino acids
NH 3
Amides
NO3
Control plants
(mmol m" 3 )
Single-rooted plants
(mmol m~3)
0.18 ±0.02
0.76 ±0.23
3.13±1.45
0.67 ±0.13
4.68± 1.15
8.11 ±3.79
0.15 ±0.02
1.02±0.11
4.10±0.22
1.52±0.13
16.47 ±1.29
34.09 ±2.77
direction and since (b) root pressure exudates and not
pressure-induced sap was used here. Similar differences
were observed when solute flows of ABA and ABA-GE
relative to the root weight were calculated using the
volume flows in Table 3.
Discussion
Roof growth and water transport
Restriction of root development to that of the seminal
roots eventually led to a reduction in shoot growth but
it also favoured growth of these roots. Their dry matter
(3.4 or 4 g at 68 or 7 DAS, respectively) was clearly larger
than in normally grown maize plants in which the sum
of the primary and adventitious seminal roots had a dry
weight of 2.3 g (64 DAS) and 2.8 g (79 DAS) (Jesko,
personal communication), indicating a compensatory
though limited additional growth when nodal roots failed
to develop. This corresponds with pronounced compensatory growth particularly in the seminal roots of Zea
mays in response to soil water deficits (Jesko et al, 1996).
The root treatment used in the present experiments,
i.e. limiting the root system to the seminal root(s) in a
monocotyledon is principally distinguished (A) from the
restriction of the rooting volume (Carmi et al, 1983;
Robbins and Pharr, 1988; Cresswell and Causton, 1988;
Peterson et al., 1991a) and also (B) from root pruning in
a dicotyledon (Carmi and Koller, 1978; Milligan and
Dale, 1988). In contrast to (A), the primary and adventitious seminal roots were allowed to grow in a fairly
unrestricted volume (the only restriction being rooting
depth) and had free access to water, minerals and oxygen
(for effects of root constriction see Peterson et al, 19916)
while its development was limited by internal factors,
such as the lack of secondary thickening and possibly an
insufficient assimilate supply. In contrast to (B), the
primary root was fully intact. As opposed to both (A)
and (B) where vascular bundles between root and shoot
can develop freely and do not excessively hinder xylem
transport, in the single-rooted maize plants, similar to
wheat (Passioura, 1972), all of the water transport is
largely confined to the few major vessels in the stele of
the mesocotyl (Plate 2), so that the hydraulic resistance
is greatly increased in comparison to the controls having
numerous and larger vessels in their nodal roots and the
adjacent stem interaodes. This results in enormous mean
flow velocities of 270 mh" 1 (7.5 cms" 1 ) which by far
exceed those in the controls (11 mh" 1 ), those reported
for herbaceous plants (10-60mh" 1 ) or even in lianas
(150mb" 1 , Strasburger, 1991). The pressure gradients
required for these flow rates were estimated on the basis
of vessel diameters (Nobel, 1991) and were 5 kPa cm" 1
for the flow through the mesocotyl of single-rooted plants
compared to O.HkPacm" 1 for the controls or
0.09 kPa cm" 1 for trees (Nobel, 1991). Assuming a length
of 30 cm from the top of the mesocotyl through the
primary root, a pressure of about 150 kPa or 1.5 bar is
required for water transport in addition to that needed
for other hydraulic resistances. Moreover, since the
number of vessels in the primary root and the mesocotyl
was constant whilst the size of the supplied shoot and its
water loss increased, flow rates (Table 3) and the required
pressure gradients were substantially elevated with time
as seen in Fig. IB.
Phloem transport
A corresponding constriction as for xylem flow exists for
the descending phloem transport with a restricted total
phloem area in the mesocotyl of about 4.7 x 10"5cm2.
Table 5. Concentration of free and conjugated ABA in the root
pressure xylem sap collected from control and single-rooted 7JZ2L
mays L. plants
Plants
ABA [nM]
Conjugated ABA [nM]
Controls
Single-rooted plants
30.9 ±6.1
4.1 ±0.4
0.40 ±0.08
1.0±0.2
Maize plant growth
Assuming a relative growth rate of the single root of
0.022 d ~' and using the measured C concentration of
42% in root dry matter, the daily carbon increment of
the root was 31.9 mg C. When assuming further that root
respiration amounted to 70% of the C increment (Jeschke
et ai, 1996), then the root required 54.2 mg C or 128.7 mg
sucrose or 0.6 ml of a phloem sap containing 20% sucrose
per day. Together with the sieve tube area this required
a flow velocity in the sieve tubes of 5.2 mh" 1 . These data
correspond to a sieve tube area of 1.35 x 10~5cm2 and
transport velocities o f 6 m h " ' reported for single-rooted
wheat (Passioura and Ashford, 1974) and are much
higher than usual (0.5-1 mh" 1 , Strasburger, 1991).
Whereas in wheat the single root reached 60% of the
control dry weight and root growth was therefore not
likely to be limited by assimilate transport (Passioura and
Ashford, 1974), the stronger inhibition down to 16% in
the present experiments with maize suggests that resistance to assimilate transport in the mesocotyl restricted
root development and led to the high shoot to root ratio
of 11.9 or 15.7 as found in the two experiments with
maize. Indirect indications for a build-up of assimilates
above the mesocotyl as a bottle neck were seen from the
appearance of highly thickened but non-growing initials
for nodal roots (Fig. 1). A build-up of assimilates in the
shoot also occurred in plants with restricted rooting
volume Robbins and Pharr, 1988).
Mineral nutrition
Given the small primary root and the high shoot-to-root
ratio, the capacity of the root to take up nutrient ions
might have limited shoot growth. In this case, the concentration in shoot tissues of limiting nutrients would be
expected to lower than of those in the controls. However,
all nutrient concentrations in leaves of the single-rooted
maize were equal to or exceeded those in the controls,
indicating that ion uptake by the branched seminal root
was adequate. Similar conclusions were reached for bean
plants grown with a restricted root volume (Carmi and
Heuer, 1981) and for similarly treated peach (Richards
and Rowe, 1977), although in this latter case root restriction led to somewhat decreased N, P and Ca levels
in leaves.
Adequate nutrient ion uptake follows also from the
composition of the root pressure xylem exudates. The
composition of the control xylem sap was well comparable
with published data (Engels and Marschner, 1993), but
it was altered distinctly in single-rooted plants. In essence,
the concentrations of three solutes, K + , NO3~, and amino
acids were substantially elevated, while Mg 2+ , Cl" and
SO2," were somewhat increased, which altogether led to
a doubling of the osmotic concentration of the exudate
from 50 (control) to 98 mM (single-rooted). This increase
was obviously needed to enable xylem exudation despite
1237
the higher hydraulic resistance due to much lower root
surface area and to the restricted number of vessels. On
the basis of the flow rates of 1.6 ml h" 1 in single-rooted
and 3.2 ml h" 1 in control shoot stumps, an additional
pressure of 27 kPa was needed to drive the exudate
through the mesocotyl compared to only 1.8 kPa in the
controls. The measured water potential differences
between external medium and exudate were —190 and
— 78 kPa, respectively, for single-rooted and control
plants and hence well adequate to overcome the radial
and longitudinal resistance within the root up to the base
of the shoot.
Characteristically the concentrations of NO3~ and K +
were elevated (Fig. 6), both of which are the basic ions
for xylem transport (Triplett et ai, 1980; Rufty et ai,
1981). When NO3~~ was provided as the major anion,
xylem sap volume flow was twice as high as with SO2."
(Triplett et ai, 1980) and also under saline conditions
K + and NO3" contributed decisively to the generation of
a positive root pressure (Jeschke et ai, 1995). Besides
these two ions, amino acids showed substantially higher
xylem sap concentrations as similarly found for
Leptochloa fusca under saline conditions (Jeschke et ai,
1995), indicating that synthesis of amino acids in the root
and their release into the xylem vessels contribute to
generating root pressure. Based on the ratio of reduced
to total N in the xylem sap (Pate, 1973), the single root
contributed somewhat less (62%) to nitrate reduction in
the plant than the control roots (82%). Although these
ratios were somewhat higher than found for maize by
Rufty et ai (1981), the apparently depressed nitrate
reduction in the single compared to control roots may be
attributed to lower assimilate import through the
mesocotyl.
Whereas single-rooted wheat produced higher grain
yield than controls with a fully developed root system
(Passioura, 1972) single-rooted maize showed decreased
shoot growth, smaller leaves and decreased photosynthetic rate. The decisive difference was that control and
single-rooted wheat plants grew on a limited water supply
and the single-rooted ones conserved water during vegetative growth and had larger resources during the later
stages (Passioura, 1972). In the present case with continuous water supply, however, the control plants had no
disadvantage in the later stages of growth, whereas in the
single-rooted maize plants the much smaller root led to
a reduction in photosynthesis, presumably due to a sinkinduced limitation (Herold, 1980; Plaut et ai, 1987) and
in later stages to a progressively severe reduction in the
water supply to the shoot (Fig. 1).
ABA and a possible shoot-to-root signal
Roots that are exposed to drying soil seem to be able to
measure the soil water content and as a response to
1238
Jeschke et al.
synthesize and release a chemical stress signal to the
xylem and hence to the shoot. Here this signal regulates
leaf conductance and leaf growth. It has been shown that
ABA is that stress signal in maize plants (Davies and
Zhang, 1991; Zhang and Davies, 1991; Hartung et al.,
1994). In the present experiments, the shoots of the singlerooted maize plants were experiencing water shortage
although their roots were exposed to conditions of optimal
water and nutrient supply and of sufficient rooting
volume, conditions which are likely to result in low ABA
biosynthesis rates within the root. Indeed ABA was not
increased, but rather significantly reduced in the xylem
sap from seminal roots of single-rooted plants. Lebreton
et al. (1995) using QTL (quantitative trait loci) analysis
have shown that in Zea a significant portion of xylem
sap ABA derives from nodal rather than the seminal
roots. This agrees with the findings. Under these present
conditions the leaves experienced water deficits and were
hence the site to trigger and perform synthesis of additional ABA as a regulator of stomatal movement and
this led to clearly increased ABA levels in young and
particularly in adult leaves and concomitantly reduced
leaf conductances.
Surprisingly in this context, however, ABA in root
tissues of single-rooted plants was substantially increased
compared to the controls. Since, as stated above, an
increase of ABA biosynthesis in the well-watered seminal
roots seems very unlikely, their elevated ABA levels must
have resulted from increased ABA phloem import from
the shoot. In contrast to other plants where shoot-derived
ABA was in part recirculated back to the shoot (Peuke
et al. 1994), in the single-rooted maize plants ABA
apparently was retained in root tissues and not released
to the xylem vessels. Thesefindingsagree with conclusions
of Slovik et al. (1995) drawn from computer simulations.
They predicted that a large proportion of ABA synthesized in leaves is loaded to the phloem and transported
towards the root as a hormonal shoot to root signal. At
present it is only possible to speculate about a stress
physiological role of this signal. Two options can be
envisaged: (1) ABA could be responsible for the observed
enhancement of growth and branching of the seminal
roots. It has been shown earlier that ABA can enhance
the formation of root hairs and lateral roots (Biddington
and Dearman, 1982; Hartung and Heilmeier, 1992) and
hence increase the uptake area of a root system. (2) ABA
might be instrumental in stimulating water and solute
flow through the root into the xylem as is the case of the
single-rooted plants (Table 3). There are many reports in
the literature of ABA-stimulated water and volume flow
through root systems of a wide range of species
(Karmoker and van Steveninck, 1978; Ludewig et al.,
1988; Bassirirad and Radin, 1992; Zhang et al. 1995, and
literature cited therein).
Conjugated ABA, which consists mainly of ABA glu-
cose ester (ABA-GE), was relatively high in leaves of
control plants (the ABA/ABA-GE ratio was (2). Its
concentration was slightly elevated in single-rooted plants,
although a large difference between treatments was
observed in the root (approximately 8-fold increase in
single-rooted plants). In contrast to free ABA, the concentration of conjugated ABA was doubled in the xylem of
single-rooted plants. It seems that a small portion of extra
ABA which had been deposited in roots after phloem
import was released to the xylem in the form of ABA-GE
and transported to the leaves. After hydrolysis this compound to a certain degree may contribute to the elevated
ABA levels in the leaves. The physiological significance
of this sequence of events is unclear, although a function
of ABA-GE as a hormonal stress signal has been postulated recently by Bano et al. (1993).
Acknowledgements
This work was supported by SFB 251 of the Deutsche
Forschungsgemeinschaft. M Holobrada especially would like to
thank the Deutsche Forschungsgemeinschaft for a travel grant
and the Julius-von-Sachs Institute fur Biowissenschaften der
Universitat Wurzburg, Lehrstuhl Botanik I, Germany for
enabling the experimental work during the visit. Thanks are
extended to Mrs Andrea Hilpert and Barbara Dierich for skilled
technical assistance, to Mrs Marion Reinhardt, Elfriede Reisberg
and Eva Wirth for analyses and to Mr Robin Wacker,
Wurzburg, for help with microtomy and staining of mesoctyl
sections.
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