Abstract: Glycine max (L.) Merril. cultivars TGX 536-02D cv. A) and TGX 923-2E (cv. B) were subjected to water stress for 7 days at the vegetative stage (T2), flowering/fruiting stage (T3), seed development stage (T4) and the control (T1) with water potential (ψw) values of about -1.5, -2.3, -2.7 and -0.3 MPa, respectively. Stressed T3 and T4 plants of both cultivars had very low tolerance (20-46%). Trichomes were present in the abaxial epidermis of T3 and T4 plants with higher occurrence in cv.B and stomata were sunken. Generally, abaxial and adaxial stomatal aperture of stressed cultivars decreased in the order T2>T3>T4 stages and there was a corresponding increase in stomatal index in stressed plants. The low tolerance of T3 and T4 plants coupled with significant reduction in stomatal aperture with increase in stomatal index may have caused the drastic reduction in number of pods and seed yield of these stressed plants. While yield of T2 plants of cv. B was also significantly reduced (p<0.05), 100 seed weight of cv. A was significantly enhanced. Both cultivars are capable of osmotic adjustments as shown by the accumulation of sugars and the reduction of lipids and starch in seeds of stressed plants with greater accumulation of sugar in cv. B. Hence the reproductive stage is critical in the growth of both cultivars but cv. B shows greater osmotic and structural adjustments than cv. A.
INTRODUCTION
Plants have evolved various means of tolerating or avoiding adverse effects of unfavourable environmental factors. Such tolerance or resistance may change as a plant grows and develops. A plant may be susceptible to stress induced injury at one stage of development but at another stage, it may be completely resistant. The stage at which water stress imposes very drastic effects on the plant is referred to as the crucial/critical stage (Forbes and Watson, 1992). Generally, water stress at the vegetative stage of growth has little effect on growth and seed yield but deficit at reproductive stage, reduces yield (Ma et al., 2006). Severe water stress levels of about -3.3 MPa did not cause desiccation injuries in leaf tissues of several sorghum varieties prior to anthesis but at post anthesis stage these plants were badly affected (Sullivan, 1972). Water stress at the vegetative stage even caused a two-fold enhancement in growth and yield in Spigelia anthelmia (Umebese and Iloba, 2001).
The stomatal complex which consists of the stoma, together with its bordering guard cells and subsidiary cells controls leaf gas exchange and water loss. Plants respond to water deficits by closing their stomata. Thus, stomatal aperture reduces, stomatal resistance increases and stomatal conductance is decreased (Steudle, 2000). However, the drawback of the stomatal closure for plants is that their carbon gain is lowered and growth is impaired (Kramer and Boyer, 1995).
Plants adapt to stresses by different mechanisms, including changes in morphological patterns as well as physiological and biochemical processes (Bohnert et al., 1995). These include metabolic adjustments that lead to accumulation of organic solutes such as sugars, proline, starches, lipids and proteins (Gill et al., 2003).
This study compares the stomatal response, tolerance, adaptive features, yield and osmotic adjustments of two cultivars of Glycine max subjected to water stress at various stages of growth.
MATERIALS AND METHODS
Plant Material and Growth Experiment
Seeds of two cultivars of Glycine max (TGX536-02D and TGX 923-2E
designated cv.A and cv.B, respectively) were collected from the International
Institute for Agriculture (IITA), Ibadan, Nigeria.
Plants were raised in a nursery bed for 2 week. Thereafter, plants of the same height were transplanted to plastic pots each filled with 4 kg garden soil. The two cultivars were planted together in each pot to ensure inter-mixing of the root system subjected to similar moisture availability. The first batch of 75 pots was watered daily (control-T1). Three batches: T2, T3 and T4 were subjected to 7 days water stress at the vegetative (23 days after planting, DAP), flowering/fruit set (65 DAP) and seed development (75 DAP) stages, respectively. The plants were arranged in a randomized complete block design with 5 replications. The pots were kept at the greenhouse of the Botanic garden of the University of Lagos with 12 h photoperiod and a relative humidity of 60% during the day and 68% at night. Temperature range was 29.3±0.3 to 32.3±0.3°C during the day and 21.6±0.5 to 23.4±0.5°C at night.
Measurement of Leaf Water Potential
The degree of stress in the plant was measured by the determination
of the leaf water potential (ψw), using a pressure bomb
chamber (Model 600L, Chas. W. Cook and Sons, England) by the method of
Boyer (1967). A pressure as low as -0.3 MPa was regarded as low deficit
(high ψw) while a pressure as high as -2.0 MPa was regarded
as relatively high deficit (low øw). Measurements were
taken in three replicates at the end of each period of water stress, from
4-6 pm daily.
Measurement of Stress Tolerance
After 7 days water stress at different stages of growth the stress
was released by re-watering the plants. The plants were observed for recovery.
Stress tolerance was measured by calculating the percentage number of
plants that recovered from the treatment (Sullivan and Eastin, 1974).
Anatomical Investigations of Changes in the Stomatal Complex
Abaxial and adaxial epidermal strips were prepared by the techniques
of Weyers and Travis (1981) and observed under a light microscope (x400,
x1000 magnifications and 0.35, 0.26 μ resolutions, respectively).
Features observed and studied include, stomatal frequency (observed in
5 replicates and expressed as the number of stomata per mm2
leaf surface), trichomes, nature of stomata and epidermal cells. The Stomatal
Index (SI) which provides a more stable estimate of the stomatal distribution,
was calculated from the stomatal frequency (Sfreq) and expressed as: (Salisbury,
1928)
Measurement of Stomatal Aperture
Whole leaves were immersed in Formalin-Acetic Acid-Alcohol (FAA) for
48 h. Thereafter, they were dehydrated, fixed, impregnated and embedded
(Sass, 1958). Transverse sections of 10 μm in thickness were cut
through the midrib using a rotating microtome (422 Erma, Tokyo) and these
were spread in water at 40°C before placing on microscope slides smeared
with egg albumen and the slides were placed on an electric warmer at 55°C
for 25 h. Thereafter, slides were placed in xylene for 5 min, hydrated
in graded alcohol for 3 min each, rinsed rapidly and then stained with
safranin, dehydrated in 90% and absolute alcohol for 1 min before dipping
in xylene for 3 min. Sections were mounted in Canada balsam and observed
under a light microscope at x400 and x1000 magnifications. Stomatal measurements
were made with a micrometer screw guage inserted in the eye piece of the
microscope.
Pod and Seed Yield
Final harvest was done at pod maturity, when pods were yellow and
rattled when shaken (Gbikpi and Crookson, 1981). Number of pods, mean
seed yield (dry weight/plant) and 100 seed weight produced by each plant,
were recorded.
Chemical Composition of Harvested Seeds
Harvested seeds were dried at 80°C for 3 days. Powdered samples
were analyzed for total ethanol soluble sugar, starch, lipid, protein
and crude fibre contents. Ethanol soluble sugar was determined by the
phenol sulphuric acid method of Dubois et al. (1956). Starch content
was determined according to the Anthrone reagent method described by Southgate
(1969), while the lipid content was determined as described by AOAC (1980).
Crude protein content was determined according to Lowry et al. (1951),
while the crude fibre content was estimated by the method of Diamond and
Denman (1973).
Statistical Analyses
Analyses of Variance (ANOVA) was applied and tests of significance
between treatments at p<0.05 and p<0.01 were performed using Duncan`s
multiple range test.
RESULTS
Water stress was moderate at the T2 stage with a leaf ψw of -1.53 to -1.57 MPa, while it was severe at the reproductive stages (ψw: -2.23 to -2.67 MPa). Plants had a high rate of recovery (96.7 and 86.7% for cv.A and cv.B, respectively) when stress was given at the T2 stage (Table 1). Corresponding to the drastic reduction in ψw at the T3 and T4 stages, there was a drastic reduction in the stress tolerance of both cultivars. However, at these stages, cv.B had higher percentage recovery (36.7-46.7%) than cv.A (20.0-36.7%).
Stress treatment induced the production of trichomes at the abaxial epidermis of T3 and T4 plants with higher occurrence in cv.B (Table 2). T3 and T4 plants had sunken stomata. Furthermore, epidermal cells shrank, resulting in increased number of small cells at these stages. In the course of water stress T3 and T4 plants of cv.B shed older leaves and produced new leaves with smaller leaf area.
| Table 1: | Leaf water potential (ψw) and stress tolerance (% No. of plants that recovered and continued growth) of soybean under water stress |
| *Difference between stressed and control ψw is significant at p<0.01 | |
| Table 2: | Observed adaptive features of soybean cv. A and cv. B plants subjected to water stress at different stages of growth |
| *Present, **Abundant | |
| Fig. 1: | Abaxial and adaxial area of stomatal aperture of soybean cv.A and cv.B subjected to water stress at different stages of growth. Vertical bars with similar letters are not significantly different (p<0.05) |
Generally, both cultivars had higher area of stomatal aperture in the abaxial than the adaxial epidermis (Fig. 1). Almost all stress treatments caused significant reductions (p<0.05) in the area of stomatal aperture of both abaxial and adaxial epidermis. Reduction in aperture in both abaxial and adaxial epidermis was more marked in T3 and T4 plants than the T2 plants. Stomatal index, (expressed from stomatal frequency) increased significantly (p<0.05) in the abaxial epidermis of all stressed plants and in the adaxial epidermis of T4 and T3 plants of cv.A and cv.B respectively (Fig. 2). The reduction in aperture size corresponded mostly with the increase in stomatal index.
| Fig. 2: | Abaxial and adaxial stomatal index of soybean cv.A and cv.B subjected to water stress at different stages of growth. Vertical bars with similar letters are not significantly different (p<0.05) |
| Table 3: | Yield of soybean cultivars subjected to water stress at different growth stages |
| Treatment means with similar letter(s) on the horizontal axis are not significantly different at p<0.05 | |
Water stress caused significant reductions (p<0.05) in pod number, seed yield and 100 seed weight of both cultivars at the T3 and T4 stages (Table 3). Cv.B was also affected at the T2 stage but cv.A showed significant enhancement in 100 seed weight. The marked reduction in aperture size of T3 and T4 plants of both cultivars and increased stomatal index corresponded with the drastic reduction in yield.
| Table 4: | Chemical composition of harvested seeds of soybean plants subjected to water stress at different growth stages |
| Treatment means with similar letter(s) on the horizontal axis are not significantly different at p<0.05 | |
Stress treatment induced various changes in the chemical composition of the seeds (Table 4). Sugar content was significantly increased in stressed plants (p<0.05). However, starch and protein contents followed different patterns in both cultivars. In cv.A, starch was significantly reduced from early vegetative to seed development (T2-T4 stages) and protein was significantly increased while in cv.B, there was a significant increase in starch content and a significant decrease in protein content from T2-T4. stages. Lipid content and crude fibre were reduced in both cultivars from T2-T4 and all but the crude fibre of cv.B plants were significantly different (p<0.05) from the control (T1).
DISCUSSION
Severe water deficit affects many of the physiological processes associated with growth and death of plants may result (Belaygue, 1996). A leaf water potential of -2.23 to -2.65 MPa at the reproductive stage of soybean, cultivars TGX 536-02D: cv.A and TGX 923: cv.B, is severe since it caused very low recovery rate in both cultivars. Death of plants may result when there is uncontrolled destruction of the photosynthetic apparatus due to the accumulation of Reactive Oxygen Species (ROS). These are toxic forms of oxygen formed in plants under environmental stress conditions like drought, which limit CO2 fixation (Krause, 1994; Hopkins and Hüner, 2004). The effects of water deficit vary with the degree, duration of water deficit, variety and growth stage of the plant (Belaygue, 1996; Adejare and Umebese, 1998). Recovery rate of cv.A was higher than cv.B when stress was given at the vegetative stage but lower with stress at the reproductive stage.
Under water stress, the anatomy of root tissue changes because stress induces the development of apoplastic barriers to water and ion flow (Steudle, 2000). Similarly, the leaf tissues of soybean showed remarkable changes in anatomy by the presence of trichomes in T3 and T4 plants which hinders the outward loss of water by transpiration and contributes to the reduction of incident radiation on the plant while sunken stomata give a low stomatal conductance. The shedding of older leaves and production of new leaves with smaller leaf area in cv.B is referred to as leaf area adjustment; another mechanism for reducing transpiration during limited water availability (Hopkins and Hüner, 2004). Similar decreases in leaf area expansion during water deficit were observed in cassava and this arrested growth assists plants in attaining high productivity in environments with cycles of intermittent drought followed by re-watering (Alves and Setter, 2004a, b). Cv.B developed greater changes in anatomy and is thus, more adapted to water stress than cv.A at the reproductive stages (T3 and T4).
A method of altering the water status of the plant is to decrease stomatal conductance (Steudle, 2000). When water status in a leaf falls below a threshold value, stomata respond by closing which buffers the effects of water stress (Bradford and Hsiao, 1982). Water stress reduced the stomatal aperture of the abaxial and adaxial epidermis of both soybean cultivars with greater reduction in T3 and T4 plants of cv.B than those of cv.A. There was also a general increase in stomatal index in the abaxial and adaxial epidermis. The reduction of stomatal aperture reduces leaf transpiration and prevents the development of excessive water deficits in the tissues (Kramer and Boyer, 1995). Stomatal closure during water stress is also associated with the maintenance of xylem integrity controlling the risk of embolism (Jones and Sutherland, 1991). It has been shown to maintain the water pressure in the leaf rachis xylem preventing extensive development of cavitation (Cochard et al., 2002). Cavitation renders xylem conduits non-conductive (Pickard, 1981). Cavitation avoidance is a physiological function associated with stomatal regulation during water stress (Cochard et al., 2002).
Stomatal closure may prevent excessive water loss in plants but it reduces carbon gain and at certain stages of plant development, this closure reduces growth and yield. When carbon fixation is limited there is a reduction in NADP+ regeneration by the Calvin cycle. This leads to severe reduction in photosynthetic electron transport chain, forming ROS in the chloroplasts, which is destructive (Krause, 1994). In many plants growth and yield are not affected when stress is given at the vegetative stage (Ma et al., 2006) and some plants even show enhancement in growth and yield (Umebese and Iloba, 2001). Cv.A showed slight enhancement in yield when it was subjected to stress at the vegetative stage. Water deficit reduces biomass production in these soybean cultivars and the effect is more pronounced when water deficit is given at the reproductive stage (Adejare and Umebese, 1998). This result from the observed significant reduction in net assimilation rate and relative growth rate of the two cultivars stressed at the reproductive stage. Severe reduction in yield can occur when plants are subjected to water stress at the reproductive stage. A single drought event at the juvenile stage had little effect on seed yield while stress at anthesis or seed-fill stage caused reduction in seed yield (Ma et al., 2006). This is evident in the significant reduction in pod number in both cultivars stressed at the reproductive stage.
The chemical composition of seeds of stressed plants showed that the seeds of both cultivars have large capacities for osmotic adjustments when subjected to water stress. This is indicated by their ability to accumulate solutes under stress. Seeds of cv.B accumulated more sugars than cv.A at the reproductive stage. The increased accumulation of sugars is due to the increase in hydrolytic enzymes such as ribonuclease and amylase under water stress (Todd, 1972). Accumulation of sugars, a characteristic of mature seeds appears to be very important for the development of desiccation tolerance (Hoekstra et al., 2001). Enzymes produced under stress conditions also convert the oils and crude fibres of oil seeds to carbohydrates, which can be used for metabolic activities (Cholan, 1978). This was evident in the decrease of lipids and crude fibres of seeds of both cultivars under stress at all stages of growth.
The reproductive stage (T3 and T4) of soybean cvs. A and B is the critical stage in which water stress affects yield drastically. Application of water at this stage will greatly benefit the plant. Cv. A tolerates water stress better than cv. B at the vegetative stage of growth but at the reproductive stage cv. B is more resistant to water stress. The adaptive features of cv. B include the prompt response of stomata to loss of turgor, shedding of old leaves and the production of small leaves with numerous trichomes. Cv.B is also more osmotically adjusted to water stress than cv.A in having higher sugar and starch contents under stress.