Journal of Experimental Botany, Vol. 53, No. 368, pp. 473-481,
March 1, 2002
© 2002 Oxford University Press
Original Papers |
Leaf initiation and development in soybean under phosphorus stress
1 Department of Botany, North Carolina State University, Raleigh, NC 27695-7612, USA
2 Department of Crop Science, North Carolina State University, Raleigh, NC 27695-7620, USA
Received 15 May 2001; Accepted 28 September 2001
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Abstract |
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Experiments investigated changes in leaf development in young soybean plants progressing into P stress. The apical meristem and leaf structure were examined anatomically to evaluate the involvement of cell division and cell expansion in the restriction of leaf number and individual leaf size. Seedlings were deprived of P for 32 d following germination. Leaf initiation rates declined noticeably after about 2 weeks, even though the apical dome was of similar size and had a similar number of cells as controls. Primordia appeared morphologically similar also. Expansion of primary and the first three trifoliolate leaves of -P plants was severely reduced, and expansion of each leaf ceased, uniformly, when an area of about 40 cm2 was obtained. Leaf epidermal cell size in the lateral plane was unaffected. The results indicate that expansion of leaves under P stress was limited by the number of cell divisions, which would imply control of cell division by a common regulatory factor within the leaf canopy.
Key words: Cell division, cell expansion, growth, leaf development, phosphorus, shoot apex.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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One of the earliest and most pronounced responses to P-deficiency is a decrease in shoot growth, specifically in leaf number and leaf size (Lynch et al., 1991
). Little is known, however, about the physiological events responsible for the growth restriction. Shoot development requires the iterative production of leaves by the shoot apical meristem and their subsequent expansion. A decreased number of leaves with P-deficiency implies changes in leaf initiation rates and activity of the shoot apical meristem. Decreased size of individual leaves can be related to changes in either cell division or cell expansion, or both.
Few studies have focused on factors controlling leaf development under stress conditions. Maksymowych, in a study with Xanthium leaves, characterized the timing of cell division and expansion events (Maksymowych, 1963). Since then, other experiments have implicated epidermal cell development in the regulation of leaf and shoot expansion (Masuda and Yamamoto, 1972; Waldron and Terry, 1987). It was shown that the P concentration in leaf epidermal cells was lower when P nutrition was limited (Treeby et al., 1987), suggesting that the inhibition of leaf expansion might be a direct effect of P deprivation and the restriction of leaf epidermal cell expansion (Fredeen et al., 1989). In studies with cotton, it was found that the inhibition of leaf expansion was associated with smaller leaf epidermal cells (Radin and Eidenbock, 1984). The authors proposed that epidermal cell expansion was inhibited by reduced water flow to shoots, resulting from decreased hydraulic conductance in roots. The response pattern for leaf and cell expansion under P-deficiency was similar to that occurring under nitrogen stress in sunflower plants (Radin and Boyer, 1982) and, thus, could be viewed as a common response associated with nutritional stresses.
In this study, the factors controlling altered leaf development in young soybean plants progressing into P stress are examined. The approach involved the general characterization of adjustments in leaf initiation and expansion, accompanied by anatomical examination of the apical meristem and leaf structure. The primary intent was to assess the relative involvement of cell division and cell expansion in the reduction of leaf size. In particular, it was intended to evaluate critically whether decreases in leaf size resulted from restricted epidermal cell expansion.
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Materials and methods |
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Plant growth conditions
Soybean (Glycine max [L.] Merr. cv. Ransom) seeds were germinated in paper towel rolls placed in deionized water in a dark chamber at 25 °C for 3 d. Seedlings with radicles 8–12 cm in length were transferred to two 84 l continuous-flow hydroponic systems in a growth chamber located in the North Carolina State University Phytotron (Thomas and Downs, 1991
). Each hydroponic system initially contained 48 seedlings. Seedlings in one system received a -P nutrient solution containing 800 µM CaSO4, 300 µM KNO3 and MgSO4, and a micronutrient solution at one-sixteenth Hoagland's strength with iron supplied as Fe-DTPA. Seedlings in the other hydroponic system received the same nutrient solution supplemented with 150 µM KH2PO4. The pH of the solutions was automatically monitored and adjusted to 5.8 throughout the experiments by additions of 0.1 M H2SO4. The growth chamber was maintained at a day/night temperature of 24/22 °C and a humidity level of 50±10%. A combination of cool white fluorescent and incandescent lamps supplied a PPFD (400–700 nm) of approximately 500 µmol m-2 s-1 at plant level during the 9 h photoperiod. Incandescent lamps provided a 3 h low intensity interruption at 20 µmol m-2 s-1 during the middle of the 15 h dark period to repress flowering.
Growth measurements
Beginning on the fourth day after transfer to treatment solutions, three plants were harvested from each treatment every 4 d. At each harvest, the shoot, root, and individual leaves were separated and individual leaf areas measured with a Li-Cor 3100 leaf area meter (Li-Cor Instruments, Lincoln, NE). Total leaf area was calculated as the sum of all measurable (>5.0 cm2) attached individual leaves. Abscised leaves were not included. The tissues were placed in a convection oven at 60 °C for 24 h and dry weights of shoot, root, and individual leaves were measured. The experiment was repeated and the results represent a data set with six replicates for each mean. The means and the standard errors were calculated and plotted to establish general growth curves and the timing of diverging growth events.
Additional plants were harvested for P analysis on the day the -P treatment began, and 12, 24 and 32 d later. Plants were separated into shoot and root, tissues were freeze-dried and ground, and the P concentration was determined by inductively coupled plasma atomic emission spectrometry.
Tissue preparation and microscopy
At each harvest, three separate plants were sampled from each solution to obtain shoot apical meristems. The apices were fixed in FAA, (70% ethanol, formalin and glacial acetic acid, 90:5:5, by vol.) and later dissected. The number of leaf primordia was combined with the number of macroscopic leaves to obtain total leaf number. Leaf number was cumulative, so it included abscised leaves. Leaf initiation per 4 d sample interval was estimated by subtracting cumulative leaf number at the beginning of the interval from that at the end.
Fixed apices from 4, 16, and 28 d after transfer to treatment solutions were dehydrated through a graded alcohol series from 50% to 95% and then infiltrated with JB4 embedding medium (Polysciences, Inc., Warrington, PA). The embedded tissues were sectioned with a MT-2 ‘Porter-Blum’ ultra-microtome (Ivan Sorvall, Inc., Norwalk, CN) and stained with toluidine blue. The sections were imaged using a Spot 2 digital camera and software (Diagnostic Instruments, Inc., Sterling Heights, MI). The apical dome was defined as the area above the points of insertion of the two youngest distinguishable leaf primordia. The area of the apical dome and the number of cells were measured from five different imaged sections from the median portion of three apices from each treatment using Image Pro Plus ver. 2.0 computer software (Media Cybernetics, Silver Spring, MD). Cell size was calculated by dividing the mean total apical area by the mean number of cells for each apex.
Cell number in the third trifoliolate leaf was determined by sampling whole leaves at early stages of development or by removing a 1.0 cm2 punch of leaf tissue from the widest laminal area adjacent to the midrib when leaves were larger. The tissues were fixed in FAA, embedded as described above, and sectioned. Then randomly selected sections were placed on slides and examined microscopically. The section selections were from areas of the leaf that were spatially separated at least 200 µm to avoid examination of adjacent cells. Three sections from each of three replicate leaves were imaged and measured as with the apices above. Data for transverse cell expansion were derived from measures of total thickness and individual cell layers (adaxial and abaxial epidermal, two palisade layers, paraveinal, and spongy mesophyll). Lateral cell expansion was estimated from the number of epidermal cells and cells in the first palisade layer intersecting a 100 µm line.
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Results |
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The experiments were designed to follow key morphological events as young soybean plants progressed through different stages of P stress. The -P plants were never exposed to P in nutrient media, so they were entirely dependent on P supplied from the seed. Visually, the P-deprived plants were similar to controls until about day 8, when they became darker green. This is often noticed with plants experiencing mild P stress (Hecht-Bucholz, 1967
). As the P stress condition progressed, the primary leaves developed necrotic spots and eventually abscised. A similar visual pattern of senescence was repeated sequentially with the trifoliolate leaves.
Whole plant growth and P content
Whole plant dry weight of the -P plants began to deviate from that of the controls at 16 d after transfer to the solution culture (Fig. 1a). While the control plants were able to enter an exponential growth phase, growth of the -P plants remained linear and their dry weights were 19% of the controls at the end of the experiment. Shoot dry weight was affected more than root dry weight in the -P treatment, with the result that shoot:root dry weight ratios were lower than the control throughout most of the experiment (Fig. 1b). Abscised lower leaves in the -P treatment were not included in dry weight calculations. It should be mentioned that development of axillary branches was completely suppressed in the -P plants. Therefore, axillary branches from control plants were not included in any of the dry weight or area measurements reported.
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Analysis of the tissues confirmed that total P remained relatively constant in -P plants, while control plants accumulated P exponentially in a pattern similar to whole plant growth (Fig. 1a
). In control plants, the P concentration of the shoot steadily increased from germination through much of the experiment, from about 0.6% to 1.0%, while the shoot P concentration in -P plants decreased and tended to stabilize just under 0.2% after day 12 (data not shown).
Canopy leaf area
As implied by the dry weight effects, an alteration in shoot development was the most striking effect of phosphorus deprivation. With control plants, main shoot leaf area entered an exponential phase, while leaf area for the -P plants remained relatively unchanged after about 16 d and was only 10% of that for control plants after 32 d (Fig. 2a).
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Leaf initiation
A leaf was considered initiated when a buldge was observed on the flanks of the apex under a dissecting microscope (after Lyndon, 1998; Fig. 4). For the purposes of this manuscript, this is considered to be a primordium. For control plants, the cumulative number of main shoot leaves steadily increased, and 19 leaves per plant had been initiated by the end of the study (Fig. 2b). In contrast, with the -P treatment the cumulative number of main shoot leaves diverged from that of the control plants at 16 d, and only 15 leaves per plant were initiated by 32 d (Fig. 2b).
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A plot of leaf initiation per 4 d sample interval illustrates when the treatment differences became apparent (Fig. 3
). Leaf initiation of control plants was maintained at about 2.5 leaves per 4 d interval from day 12 to day 20 and then decreased to about 1.0. For -P plants, leaf initiation declined to 1.0 on day 12, remained there until day 20, and then temporarily increased to a level similar to the control.
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Shoot apical meristem
Examination of the shoot apical meristem on days 4, 16 and 28 of the experiment indicated few histogenetical or morphological differences between the control and -P plants (Fig. 4). Morphological features were similar to those observed for Glycine max (Sun, 1957). Even after 28 d, when the -P plants were experiencing extreme P stress, the integrity of the apical meristem appeared to be intact. A two-layered tunica and corpus structure was evident as in the control plants. Quantitative examination of the apical dome indicated that the dome area, cell number, and cell size were somewhat smaller in -P plants, but differences were not statistically different from the controls (Table 1).
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Individual leaf expansion
With control plants, areas of each of the first four leaves at full expansion were greater at successively higher positions on the main stem (Fig. 5a). The area of the primary leaves reached a plateau at approximately 50 cm2 on day 12, the first trifoliolate leaf at 75 cm2 on day 16, and the second and third trifoliolate leaves reached their full-expansion plateaus at 90 cm2 and 140 cm2 on days 20 and 28, respectively. With -P plants, each successive leaf reached full expansion at about the same time as its respective control, but their areas were less (Fig. 5b). In fact, expansion of each leaf stopped when it reached an area of about 40 cm2. Leaf abscission was responsible for declines in mean leaf area after full expansion. When expressed as a percentage of the control, the restriction of leaf expansion in the -P plants resulted in greater degrees of inhibition at higher leaf positions because successive leaves in the +P treatment were larger. At full expansion, leaf area of the -P primary leaves was 80%, the first trifoliolate leaf 53%, the second trifoliolate leaf 44%, and the third trifoliolate leaf 28% of the respective controls.
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Since the duration of expansion for each leaf in the -P treatment remained similar to the control and the final leaf area was very different, the rates of leaf expansion were affected. This can be seen by comparing the slope for each leaf position of -P plants with that of the respective control during the expansion phase (Fig. 5a
, b). Leaf abscission had no impact on measured areas during expansion; it became involved only after full expansion was reached.
The third trifoliolate leaf
Microscopic examinations of the apical meristem indicated that the third trifoliolate leaf was initiated early in seedling development, less than 4 d into the experiment, when the control and -P plants appeared morphologically similar. Yet, as previously mentioned, expansion of the third trifoliolate leaf was severely affected by the -P treatment by the end of the experimental period. A separate plot of third trifoliolate leaf area data is provided in Fig. 6a. The third trifoliolate leaf was selected to assess changes in cell division and cell expansion in detail.
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Lateral expansion
As leaves develop, they expand in a lateral plane (as measured by leaf area) and in a transverse plane (as measured by leaf thickness in cross-section). The rates of lateral expansion of the third trifoliolate leaf differed greatly between treatments from day 20 to day 28 (Fig. 6a). Because expansion of each -P leaflet was restricted similarly; the terminal leaflet was selected for detailed examination. Length and width measurements of the terminal leaflets were plotted at various times (day 16 to day 32) to evaluate whether proportionate lateral expansion remained similar to the controls. A linear relationship was maintained for both treatments (Fig. 6b), indicating that the restriction of growth processes was exerted uniformly in the lateral plane.
Changes in lateral expansion of leaves could result from changes in cell division or cell expansion. The effect that P availability had on cell expansion on the lateral plane was investigated by measuring cell number per 100 µm in the adaxial and abaxial epidermal layers of the third trifoliolate terminal leaflet. Measurements were made during the period between day 16 and day 32, when rapid leaf development was occurring (cf. Fig. 6a). Leaf cell number of -P plants appeared to be slightly greater early on, as -P terminal leaflets had 14±0.6 cells and the control plants had 12±1.6 epidermal cells per 100 µm at 16 d (Fig. 7a, b). In the later stages of expansion, however, cell numbers were virtually the same. At day 32, when the leaf was fully expanded, the adaxial and abaxial epidermal layers had 5–6 cells per 100 µm in both treatments. This indicates that epidermal cells were expanding at about the same rate and that final epidermal cell sizes on the upper and lower leaf surfaces were not different in the two treatments.
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Transverse expansion and differentiation
Histologically, the terminal leaflet of the third trifoliolate leaf of -P plants was similar to the control, with recognizable epidermal, palisade, paraveinal, and spongy mesophyll cell layers (Fig. 8). Nonetheless, there was a delay in the rate of development and thinner leaves at full expansion in the -P treatment. At day 16, for example, the cells in the paraveinal layer were noticeably smaller and vacuoles were less developed compared to the controls. Thinner leaves under -P were evident on day 24 and day 28. The latter was a time when leaves in both treatments had reached full lateral expansion (Fig. 9). Effects on all six layers of the leaf mesophyll contributed to thinner leaves (Table 2), however, the spongy mesophyll layer appeared to be affected the most, with a 32% size reduction compared to controls.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Leaf initiation
These experiments with soybean are the first to examine the apical dome under P stress and relate its structural characteristics to leaf initiation. Microscopic examination of the apical dome showed that it was similar in size to controls throughout the experiment. The apical dome area and cell number were only slightly decreased and leaf primordia appeared to be about the same size as those initiated by the well-nourished control plants. This suggests that the structural integrity of the shoot apex was maintained even as the whole plant was experiencing severe P stress. Nonetheless, the functional activity of the apex changed as the P stress progressed. The delay in the rate of appearance of new leaves indicates slowing of leaf initiation. Quantifying the extent to which initiation was slowed was difficult, because leaf initiation in -P plants was erratic. But, during the day 12 to day 20 intervals, leaf initiation slowed by about 66%.
Slower leaf initiation implies down-regulation of the rate of cell division at the meristem. Such regulatory adjustments are not unexpected. Cell suspension culture studies have shown that mineral nutrient supply affects the rate of cell division (Hoefnagel et al., 1993). In particular, studies with BY-2 cell suspension cultures have shown that total P deprivation results in cells entering a ‘static state’ for a relatively long period of time, and that upon re-supply of phosphorus the cell cycle is re-established (Sano et al., 1999). In a whole plant system such as used in this experiment, the supply of P is not entirely shut off, as P is remobilized from storage pools and senescing tissue. The specific mechanisms co-ordinating P supply and cell division rate are entirely obscure at this time. General possibilities include regulatory mechanisms that respond to P concentrations at the meristem, or perhaps simply the rate of P arriving at the meristem from redistributions within the plant, which could limit nucleic acid synthesis directly or through decreased energy (ATP) availability.
Attempts have been made previously to relate size of the apical dome with leaf size at full expansion (Allsopp, 1954; Wardlaw, 1952). Results of this study do not indicate a relationship between the size of the dome, or leaf primordia, and the extent of leaf expansion. The third trifoliolate leaf primordium, for example, was initiated early in the experiment from shoot apices that appeared similar and with roughly equivalent cell numbers in both control and -P plants (Table 1, day 4). Leaf expansion occurred over similar time periods (Fig. 6a), but the final size of the leaves differed greatly. Thus, the effects of cell division or expansion that occurred after initiation were primarily responsible for changes in leaf size.
Cell expansion
One of the most surprising observations in this study was the lack of effect of P stress on leaf epidermal cell expansion. Anatomical measurement of both epidermal (Fig. 7) and palisade mesophyll (not shown) cell layers revealed no differences in lateral cell expansion rate in comparison with the controls. In addition, despite the differences in final leaf size, leaves of control and -P plants completed their expansion within the same time period, so there was no indication of restricted epidermal cell expansion. Indeed, the decreased rate of leaf expansion (Fig. 5) can be explained simply by fewer cells expanding at the same rate. These observations were true for leaves expanding early in the stress period and others expanding later on when the plant was experiencing severe P stress. The anatomical measurements were done on the third trifoliolate leaf, which would be expected to be the most severely affected by the lack of P availability (Fig. 6).
The absence of a P stress effect on epidermal cell size in the lateral plane is at odds with the observations with cotton (Radin and Eindenbock, 1984). In their experiments, plants were grown in sand and in solution culture with up to six levels of P (0–0.5 mM). Leaf epidermal cell size was calculated by counting the number of cells in a known area, and those values were plotted against estimated leaf area. They found a linear relationship and concluded that P nutrition limits the ‘degree to which the cells could expand’. They did find that somewhat fewer cells were present per unit of leaf area with decreasing leaf size, but cell size was primarily responsible for the smaller leaves. There is not an obvious explanation for the inconsistency in the results from the two studies. Although not stated in their paper, one would assume that their measurements of cell number were done on fully expanded leaves. If that is true, one is left with the inadequate explanation that cotton responds differently to P stress than soybean or that unidentified treatment differences were present in the experiments.
While no evidence of a P stress effect on cell size could be found in the lateral plane, a different result was evident in cell size in transverse sections. Cell layers were slower to develop and cells were smaller, which resulted in leaves that were 
30% thinner under P stress than the controls. The response pattern could have implications for the increase in green colour observed in the early days of P stress. Decreased transverse expansion of the palisade mesophyll increased transverse cell density in the leaf, which would, presumably, increase the concentration of chlorophyll in the vertical plane. It has been suggested before that darker green leaves were the consequence of inhibited growth and continued production of chlorophyll (Hecht-Buchholz, 1967).
Cell division as a determinant of leaf size
Without major changes in epidermal cell size, leaf expansion in this study must have been related to changes in cell division. A causal relationship between cell division and the size of individual leaves, of course, involves more than the events at the apical meristem after leaf initiation. The number of cells in a leaf primordium is very small compared to the final number of cells in a fully expanded leaf. It can be estimated from visual counts, for example, that primordia contain only about 0.05–0.1% of the cells present in the fully expanded first trifoliolate leaf and even less in the expanded third trifoliolate leaf of soybean.
An interesting observation in these experiments is that individual leaf areas were similar at all leaf positions in the P stress treatment. The final area for each successive leaf was about 40 cm2 (Fig. 6b). Although the data were not reported here, epidermal cell size did not differ greatly in leaves of different node number, thus, a similar number of cell divisions occurred for each leaf. The uniformity in leaf area occurred even though the leaves were initiated and developed over different time intervals and presumably under different and increasing degrees of P stress. It is as if a regulatory mechanism were engaged during the early stages of P stress and limited the number of cell divisions that could occur. At present there is no way of knowing what the sensing mechanism might be. The same amount of P was present in the plants throughout the experiment, so regulation could have been triggered by the dilution and lowered P concentrations early in the stress period.
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Notes |
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3 To whom correspondence should be addressed. Fax: +19195155315. E-mail: tom_rufty{at}ncsu.edu
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References |
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