Journal of Experimental Botany, Vol. 53, No. 368, pp. 489-503,
March 1, 2002
© 2002 Oxford University Press
Original Papers |
Growth-induced water potentials and the growth of maize leaves
College of Marine Studies and College of Agriculture and Natural Resources, University of Delaware, Lewes, DE 19958, USA
Received 5 June 2001; Accepted 28 September 2001
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Abstract |
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Profiles of water potential (
w) were measured from the soil through the plant to the tip of growing leaves of fully established maize (Zea mays L.). The profiles revealed gradients in transpiration-induced w extending upward along the transpiration path, and growth-induced w extending radially between the veins in the elongating region of the leaf base. Water moving upward required a small gradient while that moving radially required a much larger gradient primarily because the protoxylem vessels were encased in many small, undifferentiated cells that were likely to act as a barrier to radial flow. Upon maturation, these small cells enlarged and some began to conduct water, probably decreasing the barrier. In the mature leaf, the growth-induced w were absent but the transpiration-induced w remained. When leaves were growing, the growth-induced w moved water into the elongating cells during the day and night, and it shifted with changes in transpiration-induced w. The shift involved solutes accumulating in the growing region. When water was withheld, the growth-induced w disappeared and leaf elongation ceased even though turgor pressure was at its highest. Turgor was maintained by osmotic adjustment that doubled the osmotic potential of the elongating cells. If elongation resumed at night or with rewatering, the growth-induced w reappeared. If pressure was applied to the soil/root system to cause guttation and re-establish the growth-induced w, elongation resumed immediately. These findings support the hypothesis that the primary control of growth is the disappearance and reappearance of the growth-induced w because the potential changed in the xylem and nearby cells, blocking or permitting radial water movement and thus blocking or permitting growth. Key words: Gradients, leaf elongation, osmotic adjustment, osmotic potential, turgor, Zea mays L.
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Introduction |
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This study was undertaken to determine how plant growth is affected by the water status of growing tissues in fully established plants. The question arises not only because water is frequently in short supply but also because water is required to generate turgor pressure (
p) that expands the cell walls. As the walls yield, p is prevented from becoming as high as it otherwise would thus creating a w below that of the water supply, a growth-induced w (Boyer, 2001
). This low w causes water to move into the growing cells. It is too small to detect in large-celled algae (Zhu and Boyer, 1992) but is as large as 0.5 MPa in multicellular plants (Westgate and Boyer, 1984, 1985). It can disappear when water is in short supply (Westgate and Boyer, 1984, 1985; Nonami and Boyer, 1990a; Nonami et al., 1997).
The linkage between growth and growth-induced w was studied mostly in seedlings during germination (Boyer, 1985, 1988). Dark-grown soybean seedlings produce a hypocotyl (later indistinguishable from stem) with an apical meristem and elongating region above the basal mature tissue. In the elongating region, a growth-induced gradient in w exists and causes water to move radially from the xylem into the surrounding tissues. Hypocotyl growth depends on this water (Matyssek et al., 1991b) and decreased immediately when the seedlings were transplanted to vermiculite having a low water content, i.e. a low w (Nonami and Boyer, 1990a; Nonami et al., 1997). The low w in the vermiculite caused the xylem potential to decrease, rapidly inverting the gradient in growth-induced w near the xylem and blocking most water movement into the growing cells (Nonami et al., 1997). The remaining growth relied on water from nearby mature tissues having a high w (Matyssek et al., 1991a, b). The p was unchanged in most of the growing cells (Nonami and Boyer, 1989). Afterward, abscisic acid levels increased (Bensen et al., 1988), gene expression for certain cell wall proteins increased (Bozarth et al., 1987; Creelman et al., 1990; Mason et al., 1988a; Surowy and Boyer, 1991), cell wall properties changed (Nonami and Boyer, 1990a, b), and the cells adjusted in osmotic potential as solute import exceeded use for a few hours (Meyer and Boyer, 1972, 1981). While these metabolic events occurred, the w of the whole growing region gradually decreased and allowed the inverted gradient to recover (Nonami and Boyer, 1990a; Nonami et al., 1997). Growth increased to an intermediate level probably governed by the changes in metabolism that had occurred (Nonami and Boyer, 1990a; Nonami et al., 1997).
In order to determine whether similar principles apply to fully established plants carrying on photosynthesis and transpiration, maize plants were grown and analysed for the factors controlling leaf growth. The leaves contain a meristem at the base, an adjacent elongating region, and mature tissue immediately above, i.e. a developmental pattern similar to that of soybean hypocotyls and differing only in orientation. The similarity in pattern defined the paths for water flow and allowed the same kinds of tests of growth that had been made in hypocotyls.
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Materials and methods |
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Plant material
For most of the experiments, maize plants (Zea mays L. cv. B73xMo17) were grown in a 1:1:1 mixture of soil:peat moss:perlite (pH 6.5) in a controlled environment chamber (Environmental Growth Chambers, Ohio, USA) with day/night temperature of 25/20 °C and humidity of 60/90%. Photosynthetically active radiation of 700–800 µmol photons m-2 s-1 was provided for 14 h d-1 from cool white fluorescent bulbs. Seeds were germinated directly in the soil mix in PVC pots consisting of pipe (15 cm inside diameter and 18 cm height) and a bottom plate (17 cm square) held together by two rubber cords. After germination, seedlings were thinned to five plants per pot. Nutrients were supplied as a modified Hoagland solution consisting of 6 mM Ca(NO3)2, 4 mM KNO3, 2 mM KH2PO4, 2 mM MgSO4, 25 µM H3BO3, 10 µM MnSO4, 2 µM ZnSO4, 0.5 µM CuSO4, 0.5 µM H2MoO4, and 50 µM Fe-citrate. Nutrient solution was added until excess drained from the pot.
Water conducting xylem
Transpiration was determined in the controlled environment chamber on 16-d-old plants by weighing the pot/soil/plant system. The water movement for growth was determined from the volume increase of the elongating region of leaf 5 (counted from the base of the shoot) measured with a caliper and a ruler. Both rates were expressed as fluxes based on the projected exposed area for the blade of leaf 5.
The xylem path for these flows was determined in separate plants. Seeds were germinated between two sheets of germinating papers wetted with 0.1 mM CaCl2 in a seedling incubator having a saturated atmosphere, darkness, and temperature of 29 °C for 3 d. One seedling was transplanted to the soil mix in a pot (Plexiglas tube 23 cm long and 6 cm inside diameter having a Plexiglas disc taped to the bottom) and covered with aluminium foil in the incubator. The seedling remained in the dark for another day after which the mesocotyl was inserted through a seal in a metal plate on top of the pot. All further shoot development occurred above the seal, which served as a barrier to prevent nodal roots originating from the shoot from entering the soil. This ensured that all the water passed through the mesocotyl and was distributed to all of the leaves.
The vascular paths for water movement in leaf 5 (from the base of the plant) were identified in 16-d-old plants by supplying a lignin stain (safranin solution, 0.07% w/v) to the roots after removing the lower half of the soil in the pot and cutting the exposed roots in the solution, as in Fig. 1. After supplying the dye to the roots for 1 h, free-hand cross-sections of fresh leaves were made and viewed with a stereo microscope in white light or with a Zeiss 510 LSM inverted confocal microscope (10x Plan-Neo objective with N.A. 0.3) using the helium neon laser (543 nm excitation) with a 560 nm long pass emission filter.
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Growth-induced w in intact plants
Seeds were germinated in vermiculite for 3 d in the seedling incubator. Two of them were gently removed and placed in a guillotine psychrometer (Boyer et al., 1985) with the roots extending outside into 0.1 mM CaCl2. The growing region or mature basal region of the mesocotyl was sealed in the vapour chamber. The seedlings were intact, but only the growing or mature region was sealed in the vapour chamber so that the w of each region could be determined independently of the w in the rest of the plant. After the w was measured isopiestically (Boyer, 1995), the tissue in the vapour chamber was excised with a cylindrical cutter fitted outside the vapour chamber. The excision allowed the w of the intact tissue to be compared with that of the same tissue when excised and did not disturb the system other than to remove the other parts of the seedling. In a different seedling in the vermiculite, the mesocotyl and coleoptile were marked with Higgins' Black India Ink at 0.5–1 cm intervals and the elongation profile was determined 3 h later.
Profiles in hydrated plants
The total leaf elongation was monitored by clamping the leaf base to a rigid bar and attaching a Kevlar thread to the leaf tip using double-sided mounting tabs (Ace Hardware Corp., Illinois, USA). The thread was wrapped around a wheel on an optical incremental encoder (25G, Sequential Information Systems Inc., New York, USA) that recorded motion by rotating as the leaf grew. This encoder had the advantage of being unaffected by temperature or supply voltage. At 2 s intervals, the data were sent to a datalogger (CR7, Campbell Scientific, Inc. Utah, USA) which stored them as a 1 min average of total accumulated counts during the minute and converted the counts to the elongation rate. The rate was downloaded to a computer for plotting.
The profile of leaf elongation was measured in leaf 5 by pushing a pin through the shoot at 1 cm intervals from the leaf base up to about 15 cm, then at 5–10 cm intervals to the leaf tip. After a few hours, the outer leaves were removed and the distance between consecutive holes was measured with a ruler. After pinning, elongation occurred at 70–80% of the unaltered rate. The profile of root elongation was measured by removing the lower half of the pot and marking exposed nodal roots with Higgins' Black India Ink at 5 cm intervals from the root/shoot transition toward the tip. The length between consecutive marks was measured at the same time leaf elongation was measured. From this profile, the mature region of the root was identified and used to sample for root w.
The corresponding profile of w was measured in leaf 5 of another plant in the same pot by first excising the shoot inside the controlled environment chamber and immediately transferring it to a humidity-saturated glove box. Three to four 2 cm leaf segments were excised from a position on the leaf and transferred into a psychrometer cup coated with petrolatum. The cup was immediately attached to the heat sink, sealed, and placed in an isopiestic psychrometer system (Isopiestics Co., Delaware, USA). After sampling each leaf position, the pot was moved to a humidity-saturated room, the lower half of the pot was removed and 10–15 cm of mature nodal root was sampled after removing the root tips. The soil was sampled by inserting a cork borer deeply into the bulk and covering the psychrometer chamber at least 2 mm deep with soil from inside the borer. After measuring w of each sample isopiestically (Boyer, 1995), the leaf and root tissues were frozen and thawed, and the s was measured by the same technique. The p was calculated from w-s.
Profiles in dehydrated plants
The leaf elongation and profiles of w were measured as above except nutrient solution was supplied for 12 d and subsequently withheld for 10 d, then resupplied. In order to keep the soil w uniform, 2 cm of dry Perlite was layered on top to minimize water loss from the soil surface. The whole pot was placed on four rubber stoppers in a larger container (28 cm diameter). The edge of the larger container was sealed to the edge of the plant pot with plastic sheet. The sealed space outside the pot prevented water condensation on the inner wall of the pot caused by day/night temperature changes. Elongation was measured with the optical encoder and profiles of w, s, and p were determined as in the hydrated plants. The profiles were measured every other day, alternating between two pots grown simultaneously. This gave 4 d between sampling for the plants remaining in the pots to recover from any soil disturbance. The profiles were measured at the end of the light or dark period to ensure that steady conditions had been achieved in the plants.
Root pressurization
A pot containing a plant with the mesocotyl passing through the top of a pressure chamber was sealed inside the pressure chamber (7.6 cm inside diameter, 26.7 cm depth). Compressed air was applied to the soil/root medium while leaf length was continuously monitored with the encoder as above in the controlled environment. A controller (DP25-E Process Meter, Omega Engineering Inc., Connecticut, USA) connected to a pressure transducer (PG856–250, Statham Laboratories Inc., Puerto Rico) controlled the pressure applied to the root medium. Guttation, if any, was collected and its s measured with the isopiestic psychrometer (Boyer, 1995). After 2–3 h of pressurizing, the shoot was excised and the cut surface was rinsed and blotted dry. The initial 10 µl of root exudate was collected and its s was measured with the isopiestic psychrometer.
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Results |
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Water conducting xylem
By day 16 in the controlled environment, leaf 5 was losing about 1.6 mmol m-2 s-1 of water as transpiration. About 2% of that amount was consumed to grow leaf 5 (Table 1
). At night, transpiration was less (0.094 mmol m-2 s-1) but leaf 5 continued to grow and the growth accounted for about 23% of the water flux. Because the transpiration stream passed upward through the leaf bases, a large upward flux continually moved through the growing region of leaf 5 while water was withdrawn radially for growth.
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The vascular path for the transpiration stream involved nearly every vascular bundle in the elongating region (Fig. 1A
) and moved upward in the same vascular bundles to the mature blade (Fig. 1C). The bundles were about 0.7–1.2 mm apart in the elongating region and 1–2 mm apart in the mature region because the leaf widened after it elongated (Westgate and Boyer, 1984). In controls, no bundles were stained when no dye was supplied to the water around the cut roots (Fig. 1B, D).
In each vascular bundle, one or two protoxylem vessels were conducting in the elongating region (Fig. 2A). Many densely-packed small cells encased the protoxylem and showed little or no dye (Fig. 2A, B). As elongation slowed in the upper part of the elongating region, dye was detected in a few cells around the protoxylem but the encasing cells remained small except for immature metaxylem (Fig. 2C, D). Immediately above the elongating region (Fig. 2E, F), some of the small cells had increased in diameter and become tracheids (Esau, 1953). Water passed from the protoxylem to the tracheids to the newly differentiating metaxylem (Fig. 2E, F). New vascular bundles were beginning to appear in the mesophyll but were non-conducting. Above this region, the two metaxylem vessels became major conducting vessels (Fig. 2G, H). After the leaf widened, the protoxylem became less distinct (Fig. 2J, K). The phloem was mostly unstained throughout the length of the vascular bundle.
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), 15 d (
) and 22 d (
) after planting. (C, E, G) Profiles of Growth-induced
w in intact plantsThe elongating regions of the young leaves were covered by older leaves and thus not readily accessible. It was necessary to excise the leaves in order to measure their
w. To test whether excision affected w, maize mesocotyls were used because they have a large exposed elongating region (4 cm, Fig. 3A) that is continuous with the coleoptile and leaf growing region. When they were placed in a guillotine psychrometer for measuring w in intact plants (Boyer et al., 1985), the elongating region of the mesocotyl had w of -0.38 MPa in the intact seedlings and -0.41 MPa after excision in the psychrometer, on average (Fig. 3B and inset table). The mature tissue had w of -0.11 MPa in the intact seedlings and it did not change after excision (Fig. 3C and inset table). As a result, growth-induced w were (-0.38)–(-0.11) =-0.27 MPa in the intact plant and -0.30 MPa after excision.
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This excision effect was small enough to ignore and justified the use of excised tissue in the subsequent experiments. It is worth noting that the isopiestic psychrometer used in the present work is the only psychrometer capable of measuring
w close to zero, where growth occurs (Boyer, 1995). The w, s, and p measured with this method are similar to those measured with the pressure chamber (Nonami and Boyer, 1987) and the pressure probe (Nonami et al., 1987; Nonami and Boyer, 1989, 1993) in growing tissues and were shown to be highly accurate (Boyer, 1966).
Profiles in hydrated plants
In plants 18-d-old, leaf 5 elongated in the region between 1 cm below and 8 cm above the soil surface (Fig. 4A), and the corresponding potential profile showed w of -0.07 to -0.1 MPa in the soil (Fig. 4B, S), -0.15 MPa in the mature root tissue (Fig. 4B, R), -0.52 to -0.61 MPa in the elongating region of the leaf (Fig. 4B, G), and -0.13 to -0.17 MPa in the mature leaf tissue (Fig. 4B, Ma, Mb, Mc). Because the w of the mature tissues was the same below and above the elongating region, the xylem running through the region had a w of about -0.13 to -0.17 MPa (Fig. 4B, X). Accordingly, the growth-induced w was located outside of the xylem. The growth-induced w was approximately -0.44 MPa during the day and -0.38 MPa at the end of the night (G–X in Fig. 4B). Throughout the shoot tissues, the w were more negative at the end of the day than at the end of the night, particularly in large plants, indicating that transpiration-induced w also were present during the day.
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Whenever leaf 5 increased in length (until day 20 in Fig. 5A
) or thickness and width (until day 27 in Fig. 5A), a growth-induced w of about 0.4 MPa could be detected in the w profile measured from the soil to the tip of the leaf (Fig. 5B). No growth-induced w was present after day 29 when leaf 5 had fully matured (Fig. 5C). Transpiration-induced w were always present during the day (difference between day and night w in Fig. 5B, C).
The s was -0.7 to -1 MPa at night throughout the plant but during the day, the s became more negative in the exposed part of the leaf probably reflecting solute produced by the activity of photosynthesis (Fig. 5D, E). The s also was slightly more negative in the growing region during the day (Fig. 5D), indicating that some solute had accumulated. The p was nearly unchanged between day and night, remaining 0.7–1.0 MPa throughout the leaf except in the elongating basal region, where it was only about 0.4 MPa (Fig. 5F). As leaf 5 matured, the p in the basal region rose to about 0.8 MPa (Fig. 5G).
The w gradient for the transpiration path was about -0.1 MPa/0.60 m=-0.17 MPa m-1 calculated from the leaf tip to the leaf base (Table 1). By contrast, the w gradient for the growth path was -0.44 MPa/ 0.00044 m=-1000 MPa m-1 calculated from the growth-induced w and the half distance between protoxylem of adjacent vascular bundles (Table 1).
Profiles in dehydrated plants
After water was withheld from the soil, elongation in leaf 5 ceased on the fifth day (Fig. 6A). When water was supplied on the tenth day, elongation resumed. Soil w was about -0.1 MPa before water was withheld (Fig. 6B), decreased to -1.1 to -1.5 MPa by the eighth day (Fig. 6C), and returned to -0.1 MPa when water was resupplied (Fig. 6D). The w profiles in the plant were affected by these soil w, becoming more negative as the soil dehydrated, but recovering when water was resupplied. The w decreased more in the mature tissues than in the elongating region as the soil dehydrated. In effect, the w of the mature tissues overtook the w in the elongating tissues, and the growth-induced w disappeared (Fig. 6C). The growth-induced w reformed when water was resupplied (Fig. 6D). By contrast, transpiration-induced w were present regardless of the water supply (Fig. 6B–D).
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The
s showed a similar pattern, becoming more negative as the soil dehydrated and less negative after water was resupplied (Fig. 6E–G). The s throughout the leaf was always more negative during the day than at night while the leaf grew. The p decreased markedly in the mature tissues, but only slightly in the elongating region when water was withheld (Fig. 6H, J). On the eighth day, the mature tissue was visibly wilted and rolled. After water was resupplied, the p recovered in the mature tissues and slightly in the elongating tissues (Fig. 6K).
Nocturnal recovery
The disappearance and reappearance of growth-induced w seemed to be correlated with leaf elongation during the dehydration experiment. Close scrutiny showed that when leaf elongation was rapid prior to the inhibition, growth-induced w were present during the day and the night (Fig. 7A1, A2). When leaf elongation ceased on the sixth day, growth-induced w were absent (Fig. 7B1). However, elongation resumed spontaneously the following night, and the growth-induced w reappeared (Fig. 7B2). By the tenth day, no elongation occurred during the day or night, and the growth-induced w were always absent (Fig. 7C1, C2). After water was resupplied, elongation resumed and the growth-induced w reappeared during the day and night (Fig. 7D1, D2).
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Figure 8
summarizes this sequence for the entire experiment and indicates that the growth-induced w disappeared when elongation ceased during the day and reappeared when elongation resumed at night (Fig. 8A, B, shaded areas). The growth-induced w also reappeared after rewatering. Until day 18, the p was fully maintained (Fig. 8C) because solute was accumulating rapidly in the elongating region (Fig. 8D). The s decreased from -0.8 MPa to -1.5 MPa during this time, i.e. the solute concentration nearly doubled. By day 18, p had become higher than at any other time even though elongation had ceased. The high p indicated that the osmotic adjustment was complete. After night 18, however, p decreased (Fig. 8C) because osmotic adjustment was unable to keep up as dehydration became more severe.
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Root pressurization
Passioura showed that pressure applied to the soil/root system could raise the pressure in leaf xylem until it equalled the atmospheric pressure (Passioura, 1988). When pressure was applied to the soil/root system of the intact maize plants, guttation appeared at the leaf margins and indicated that the potential X (Fig. 4) had been raised, confirming the observations of Passioura (Passioura, 1988). Therefore, this pressurization technique was used to raise X and cause the growth-induced w to reform in the dehydrated plants.
At the end of the fourth day after water was withheld, leaf elongation was about half its maximum rate because of the water deficit (Fig. 7A, 9A) and X had decreased to about -0.45 MPa (Fig. 7A1). Applying pressure of 0.46 MPa to the soil/root system (Fig. 9A) caused guttation to occur around the leaf margins (Table 2). The guttate was nearly pure water. The xylem solution at the base of the leaf was nearly as dilute (Table 2), measured as exudate from the roots after the shoot was removed. Therefore, when guttation appeared, X was nearly zero even though no new water was supplied to the soil. Under these conditions, leaf elongation recovered to the maximum steady rate (Fig. 9A). The initial peak in the elongation rate was ignored because it was caused mostly by the deformation of the pressure seal around the base of the plant (spike at 1.5 h in Fig. 9A).
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By contrast, plants at the end of the sixth day had X of -1.1 MPa (Fig. 7B1
) and raising the pressure to 1.5 MPa around the soil/root system did not cause guttation (Fig. 9B). The lack of guttation indicates that X did not recover to near-zero upon pressurization. When elongation was measured, the initial deformation of the pressure seal was observed, but elongation was almost absent. Upon removing the shoot, only a few microliters of exudate could be obtained. The exudate had a s of -0.11 MPa (Table 2).
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Discussion |
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Water potential gradients and growth
There was a strong relationship between growth and gradients in the growth-induced
w in leaves of fully established maize plants. In every instance of varying elongation, the growth-induced w were present when elongation was rapid and disappeared as the leaves ceased to elongate. The presence or absence of the growth-induced w depended on the xylem potential X, which changed more than in the surrounding tissues. As X decreased in water-deficient plants, it overtook the w of the surrounding tissues and caused the disappearance of the growth-induced w. With spontaneous recovery of X at night, or rewatering and recovery of X, the growth-induced w reappeared. In moderately water-deficient plants, pressurizing the roots rehydrated the xylem, increasing X and re-establishing the growth-induced w and elongation. In severely water-deficient plants, pressurizing the roots did not recover X and elongation did not recover. Therefore, the changes in X had a major effect on the gradient moving water into the surrounding elongating tissue. It should be noted that pressurizing the soil/root system altered X without changing any other factor in the shoot or the water supply to the soil, which allowed a strong test of whether the collapse of the growth-induced w caused the inhibition of leaf elongation. The pressure results show that the collapse of the growth-induced w was a primary cause of the early growth inhibition.
All of the water moving upward for transpiration had to pass first through the growing region in the protoxylem of each vascular bundle. The potential of this water was higher than in the surrounding growing tissues, creating the radial gradient of growth-induced w in the growing region. The radial gradient extended downward from the protoxylem and because distances were small between the vessels, the gradient was especially steep (-1000 MPa m-1). The steepness probably resulted from the densely-packed small cells encasing the protoxylem. These cells separated the protoxylem from the bulk of the growing tissues and formed many plasmalemma/cell wall barriers through which water had to pass before entering the surrounding tissues. Nonami et al. observed similar structures in the stems of soybean seedlings (Nonami, 1997), and they reported a low diffusivity for water in these cells that required a large gradient in potential to move water radially from the protoxylem (Nonami and Boyer, 1993). Extending this concept to maize, Fig. 10 shows the generalized anatomy of the elongating region of the maize leaf with the small cells in the vascular bundle separating the protoxylem vessels from most of the growing cells. The proposed gradient is steepest within the bundle, and the bulk of the growing tissue would have a low w. The psychrometer probably measured mostly this bulk and thus accounts for the low w of the growing region.
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This is in contrast to the mature leaf where the vessels had elongated, widened, and then differentiated to form metaxylem and tracheids. The metaxylem directly contacted the mature bundle sheath cells, and the flow path led directly to the site where water was being used in photosynthetic metabolism. The small cells characteristic of the elongating region were absent and radial flow should have occurred more readily than in the elongating region. This is supported by the small gradient in
w for the transpiration path (-0.17 MPa m-1). It extended to the evapourating surface of the mature leaf (the psychrometer measured the vapour pressure of the evapourating surface after equilibration with the water in the leaf interior). As a result, the enlargement and differentiation of the small cells around the xylem markedly increased the ability of water to move through the mature tissues.
The transpiration gradients coexist with the gradients moving water radially for growth as shown in Fig. 11A during the day. At night, the transpiration-induced gradient is small and the growth-induced gradient predominates. On the other hand, after the leaves mature, only the transpiration-induced gradient is observed and the growth-induced gradient is absent (Fig. 11B). In the elongating region, the growing cells compete with transpiration for the water in the protoxylem. The growth-induced gradient shifted between the day and night as X responded to transpiration. Westgate and Boyer also observed this shift and reported that solutes accumulated in the growing region during the day, making it possible for the w of the growing cells to shift to lower w and thus maintain radial water uptake for growth (Westgate and Boyer, 1984, 1985). In the present work, the shift continued when water was withheld from the soil, indicating that osmotic adjustment is a dynamic process occurring whenever X decreased. Its capability was large because the solute concentration doubled in the elongating tissues in the early phases of the water deficit, which completely maintained p. The complete maintenance of p indicated that there was no dehydration in most of the elongating cells, and osmotic adjustment had completely compensated for the lower w of most of the cells.
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Because the solutes for osmotic adjustment were derived mostly from photosynthetic products (Acevedo et al., 1979
; Michelena and Boyer, 1982), the lack of leaf elongation at this time could not be attributed to a lack of photosynthetic substrates or p. Instead, the growth-induced w disappeared, preventing water uptake because X had decreased more than the w of the surrounding tissues in the elongating region. It is likely that the decrease in X occurred first and osmotic adjustment occurred afterward. Meyer and Boyer showed that growth was inhibited before solutes accumulated in soybean hypocotyls subjected to low w (Meyer and Boyer, 1981). The solutes accumulated because they were unused in the growth process. Nonami and Boyer detected an immediate change in X, and solute accumulation was detected about 1 h later in similar hypocotyls (Nonami and Boyer, 1990a).
Signal transduction
This sequence of events suggests that, when soil or atmospheric conditions change, there is a transduction path signalling the change to the leaf. The signal starts with X, which changes rapidly. The gradient of growth-induced w is altered in a few cells next to the xylem, which alters the water flow to the outlying cells, and elongation responds rapidly (Nonami et al., 1997). As elongation changes, the demand for solute changes and the solute subsequently accumulates or is depleted, resulting in osmotic adjustment (Meyer and Boyer, 1981). The adjustment typically maintains p in the cells except for the few next to the protoxylem where the growth-induced w is altered (Nonami and Boyer, 1989). As shown in soybean hypocotyls, metabolic events probably follow that result in changed wall properties (Bozarth et al., 1987; Nonami et al., 1990a), altered gene expression for certain wall proteins (Mason et al., 1988b; Surowy and Boyer, 1991), and altered availability of plant growth regulators (Bensen et al., 1988; Creelman et al., 1990).
Because this signal path consists of the series X–w gradient–elongation–osmotic adjustment, it can be controlled by the action of growth regulators on wall yielding and the w gradient. Boyer showed that growth-induced w develop because the cell wall yields as enlargement occurs, preventing p from becoming as high as it otherwise would (Boyer, 2001). The low p results in a low w in the cells that is transmitted as a tension to the apoplast connecting to the xylem. The tension can be measured (Nonami and Boyer, 1987) and extracts water from the xylem for the growth process. In the absence of a yielding wall, growth-induced w are absent and no growth occurs.
When Maruyama and Boyer removed the auxin supply to hypocotyls of dark-grown soybean seedlings (Maruyama and Boyer, 1994), the growth (wall yielding) ceased and the growth-induced w disappeared. Ikeda et al. likewise found growth responses closely correlated with growth-induced w when the auxin/cytokinin supply was altered in tissue-cultured embryos (Ikeda et al., 1999). There is evidence that roots supply more ABA to shoots at low w (Davies and Zhang, 1991), and there was less shoot growth (Dodd and Davies, 1996; Zhang and Davies, 1990). In maize seedlings at low w, continued root elongation depended on increased ABA (Munns and Sharp, 1993; Saab et al., 1990; Sharp et al., 1994). These regulators appear to be synthesized in or near the growing cells, but also in roots and are transported to the shoot (Davies and Zhang, 1991). Because they are present in or transported to the growing tissues and are likely to act on wall yielding, they can alter the signal path at an important control point.
It is noteworthy that p acts largely through its local effect on the gradient in growth-induced w. Growth responds immediately to changes in p in single cells surrounded by water (Proseus et al., 1999, 2000) and it may at first seem that a similar response would occur in multicellular plants. However, in the present experiments, p in most of the cells was at its highest as elongation was being completely inhibited by low soil w. This opposite response indicates that growth was controlled by some other factor. In soybean hypocotyls, Nonami and Boyer suggested that a local change in p next to the xylem could disrupt the w gradient for growth, thus preventing water uptake for all the outlying cells (Nonami and Boyer, 1989). Subsequent work identified this disruption as a primary cause of the growth inhibition (Nonami and Boyer, 1990a; Nonami et al., 1997). In maize leaves, the data show that a similar mechanism was at work. This mechanism does not include the effects of p in single cells surrounded by water because they are masked by the local effects of p on the growth-induced w. As a result, the direct response of cell enlargement to p is not apparent in multicellular growing regions even though it may be occurring in the background.
Gradients in different flow paths
The transpiration-induced gradients were steepest in the root/shoot transition region but shallow in the leaf (Fig. 11B). The steepness in the transition region probably reflected complexity in the vasculature connecting the root to the stem. Above the transition, the vasculature consisted of simple linear xylem vessels similar to those found in tall fescue (Martre et al., 2000), and the gradient of -0.17 MPa m-1 was similar to one of about -0.20 MPa m-1 reported previously (Martre et al., 2001). By comparison, in the elongating region, the radial gradient of -1000 MPa m-1 was nearly 6000 times steeper than the upward one for transpiration despite the slow flow and small distances for growth. Nonami and Boyer found a radial gradient of about -300 MPa m-1 in the growing region of soybean hypocotyls (Nonami and Boyer, 1993), and a similar one is described in growing regions of tall fescue leaves (Martre et al., 2001).
In these stems and leaves with intercalary meristems, the upward transpiration stream clearly bypassed most of the enlarging cells. The paths were defined by the anatomy, but in leaves of dicotyledonous plants such as sunflower the transpiration stream also bypassed most of the mesophyll cells and evapouration appeared to occur from deep in the leaf (Boyer, 1974). Isotopically-labelled water was supplied to roots of sunflower, tobacco, and cotton and it was found that a large fraction bypassed the leaf mesophyll cells (Raney and Vaadia, 1965; Yakir et al., 1990). From a detailed study of leaf anatomy, Rayan and Matsuda also concluded that the transpiration stream largely bypassed the growing cells in barley leaves (Rayan and Matsuda, 1988). The result was thus the same despite the marked differences in anatomy between these species.
Rapidity of growth response
In the present work, most of the potentials were measured at the end of the day or night to ensure stable conditions, but the root pressurization experiment illustrates that changes in X occur rapidly with equally rapid effects on leaf elongation. The local reversal of the growth-induced w also occurs rapidly (Nonami et al., 1997), which is consistent with rapid changes in leaf elongation. Rapid changes in growth are frequently observed in plant organs (Acevedo et al., 1971; Hsiao et al., 1998; Kitano and Eguchi, 1992; Matyssek et al., 1991b; Milligan and Dale, 1988; Munns et al., 2000a, b; Nonami and Boyer, 1990a; Passioura and Munns, 2000). Since the first observations of growth-induced w (Boyer, 1968), these w have been reported in leaves of gramineae (Barlow, 1986; Fricke et al., 1997; Fricke and Flowers, 1998; Martre et al., 1999; Westgate and Boyer, 1984), stems of soybean (Boyer et al., 1985; Cavalieri and Boyer, 1982; Molz and Boyer, 1978; Nonami et al., 1997), and all of the growing tissues of maize (Westgate and Boyer, 1985). It seems safe to conclude that growth-induced w are of near-universal occurrence in actively growing regions of multicellular plants, and local changes in growth-induced w gradients could be important for rapid changes in growth as well as the steady ones reported here.
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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In the maize used in the present work, the plants were fully established and there was active photosynthesis and transpiration in contrast to the extensively-studied, dark-grown soybean seedlings where photosynthesis did not occur and transpiration was negligible. In the seedlings, the sequence of events controlling hypocotyl growth at low
w involved the growth-induced w in a primary, early way (Nonami and Boyer, 1990a; Nonami et al., 1997) followed by many changes in metabolism and growth regulators. Despite the differences between the experimental systems, the growth-induced w played a similar, central role in the fully-established maize and the following events were similar to those in the hypocotyls. As a result, leaf growth was more closely controlled by the growth-induced w than by p, and the growth-induced w occupied a central position in the signal pathway controlling the rate of leaf growth.
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Acknowledgments |
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This study was supported by DOE grant DE-FG02-87ER13776 to JSB. We thank Dr Mark E Westgate for helpful suggestions.
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Notes |
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1 To whom correspondence should be addressed. Fax: +13026454007. E-mail: boyer{at}udel.edu
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Abbreviations |
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X, water potential in lumen of xylem;
w, water potential; s, osmotic potential; p, turgor pressure. |
References |
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