Physiological characters associated with water-stress tolerance under pre anthesis water-stress conditions in wheat
Shahram Mohammady-D
Faculty of Agriculture, University of Shahrekord, Iran
Corresponding author: Shahram Mohammady-D
E-mail: shfaza@hotmail.com
Abstract
This study was conducted to evaluate previously identified water-stress tolerance and water-stress susceptible varieties, including Falchetto and Chinese spring (CS) (water-stress tolerant) and Oxley and Flat (water-stress susceptible), for some physiological characters affecting the performance of varieties under water-stress conditions. The results indicated that water-stress tolerant varieties had higher Stomatal Resistance (SR), higher Leaf Relative Water Content (LRWC), higher Vegetative Water Use Efficiency (WUEveg) and higher Vegetative Evapotranspiration Efficiency (ETEveg) than water- stress susceptible varieties. Varieties with higher LRWC generally had higher SR. This indicates that higher LRWC is, at least in part, due to higher SR. The variety Oxley (water-stress susceptible) showed no significant differences from Falchetto (water-stress tolerance) for stomatal area on both surfaces of the flag leaf but Falchetto had higher number of stomata per unit area and smaller stomata than Oxley. This implies that low frequency of stomata is compensated by high stomatal size. This also indicates that differences in SR between these two varieties are possibly due to differences in stomatal response to water-stress or due to differences in cuticle resistance. The variety Falchetto (water-stress tolerant) had lower carbon isotope discrimination (Δ) than the water-stress susceptible variety Oxley under both water-stress and normal conditions. Δ indicated a significant negative relationship with WUEveg, ETEveg and DM, but no significant relationship was observed between Δ and other characters. The absence of a significant relationship between Δ and SR under the conditions of this experiment revealed that variation in Δ is due to differences in photosynthetic capacity rather than differences in stomatal resistance.
Introduction
Assessment of wheat varieties for physiological components of water-stress tolerance has been used as a quick technique for screening water-stress tolerant varieties (Adjei and Kirkham 1980; Blum et al. 1981; Chaves 1991; Dhanda and Sethi 1998; Ehdaie 1995; Golestani Araghi and Assad 1998; Gumuluru et al. 1989; Winter et al. 1988). Physiological characters of plants, particularly those related to plant water status, have a great deal of importance in growth and development of plants during water-stress conditions. Leaf relative water content (LRWC), stomatal resistance (SR) water use efficiency (WUE) and its components, stomatal characteristics and carbon isotope discrimination (Δ) have been used as examples of the characters which determine water-stress tolerance of wheat genotypes.
LRWC is the relationship between fully turgid water content and actual water content of plant tissues when they are subjected to water-stress. Therefore, LRWC indicates the ability of plants to keep their water status at a reasonable level when they experience water-stress. Chaves (1991) pointed out that LRWC represents the water status of plants, while other parameters like water potential can be affected by soil, plant and atmospheric water status. Therefore, LRWC may be a more appropriate indicator of plant water status. LRWC is also closely related to cell volume and reflects the balance between water supply and transpiration (Schonfeld et al. 1988). In addition, genotypic variation for LRWC has been reported by various investigators in wheat (Blum and Johnson 1993; Dhanda and Sethi 1998; Mentewab and Sarrafi 1998; Teulat et al. 1997). This variation helps plant breeders to select for high LRWC in wheat cultivars and landraces.
Water use efficiency indicates the amount of assimilate produced for the amount of water used. Ehdaie and Waines (1993) defined water use efficiency (WUE) as the ratio of grain yield to total water used (GY/TWU). Total water used includes both soil evaporation and plant transpiration. In some papers, water use efficiency has been defined as the ratio of photosynthesis (net CO2 assimilation) to total water transpired (Fischer and Turner 1978). Water use efficiency can be defined in different ways according to the point of view of different scientists (Prof H Griffiths, personal communication). Agronomists define WUE as a ratio of yield to total water evaporated from the soil and transpired from the plant (g yield/kg water) and physiologists define WUE, based on gas exchange, as the ratio of CO2 assimilation to transpiration over growth cycle (μ mol CO2 m-2 s-1 /μ mol H2O m-2 s-1). WUE, in fact, indicates how efficiently the varieties use water supply.
Stomatal resistance (s cm-1) is a character leading to water regulation of plants. This character has been used as a criteria to screen water-stress tolerant varieties by several researchers (Adjei and Kirkham 1980; Blum et al. 1981; Golestani Araghi and Assad 1998; Gumuluru et al. 1989; Jones 1987; Shimshi and Ephrat 1975). Golestani Araghi and Assad (1998) working on Iranian varieties reported that stomatal resistance was recognised as a beneficial water-stress resistance indicator. Since most of the water escapes through the stomata (Wang and Clarke 1993b), stomatal size and frequency are among factors which influence stomatal resistance.
Carbon isotope discrimination (Δ) is a character which indicates the amount of 13C which is depleted by photosynthesis mechanisms. This character is related to water-stress tolerance indicators such as stomatal resistance and water use efficiency (Farquhar and Richards 1984; Griffiths 1993; Taiz and Zeiger 1998). Furthermore, this character has indicated negative association with grain yield under some water-stress conditions (Richards et al. 1998).
The above mentioned physiological characters are likely to indicate differences between tolerant and susceptible varieties. Identifying differences between the varieties differing in water-stress response for the above characters, therefore, provides considerable information about physiological characters associated with water-stress tolerance. In the previous experiments, the varieties Falchetto and Chinese Spring showed water-stress tolerance and varieties Oxley and Falat showed susceptibility to water-stress imposed at the growth stages around meiosis. The aims of the experiments presented in this paper were to investigate the effect of pre-anthesis water-stress on some physiological characters of both tolerant and susceptible varieties, and to identify characters associated with water-stress tolerance. To achieve the aims, three experiments were conducted. In the first experiment, WUEveg, ETEveg, SR and LRWC of the four varieties were compared and carbon isotope discrimination was studied only in varieties Oxley and Falchetto using samples derived from dried flag leaves. In the second experiment, stomatal frequency and size were studied in Oxley and Falchetto.
Material and methods
Experiment 1
This experiment was carried out in a greenhouse cubicle at the Close House Field Station,UK. This experiment was started in the middle of July when natural light and day length was adequate for the plants. The temperature ranged from 17 to 31 °C. The relative humidity fluctuated from 45-80%.
Four varieties Falchetto, Falat, CS and Oxley, were included in this experiment. Falchetto is an early flowering semi-winter variety bred in Italy and grown in the Sudan which possesses the Akakomugi semi-dwarf gene (Rht8) (Yassin 1980). This variety is tolerant to water-stress (Haider 1997) and has hairy glumes and compact spikes. Falat is reported to be susceptible by Golestani Araghi and Assad (1998). This variety has shown tolerance to water-stress and a gene for osmoregulation has been identified on chromosome 7A (Morgan 1991). Oxley is a semi-dwarf, semi-winter variety from Australia with the Rht-B1b gene and shows susceptibility to heat and water- stresses (Stone and Nicolas 1995).
Seeds of the varieties were germinated and then vernalised for 4 weeks in a cold cabinet at 2-7°C and an 8 hour photoperiod. 20 seedlings of each variety were transplanted in 10-cm pots containing John Innes No. 2 compost. The pots were divided into two blocks so that plants in each block were similar in vigour and size. The pots were arranged randomly within each block.
At growth stage 37-39 of Zadok's scale (Zadoks et al. 1974) (flag leaf emerging), plants in one of the blocks were subjected to water-stress and the plants in the other block remained under full irrigation conditions. In the full irrigation treatment, the compost was maintained at optimum water content. Several pots of each variety were weighed every three days and rewatered according to the amount of water lost. The water-stress treatment was initiated by bringing all the pots to optimum water content in the morning of the first day of treatment. The treated plants were subjected to water-stress by withholding water for 6 days.
Stomatal resistance in both full irrigation and water-stress treatments was measured at mid-day (11am-2pm) on the adaxial surface of flag leaf using a Delta-T Diffusion porometer model AP4. This equipment measures stomatal resistance based on the water vapor coming out through the leaf tissues in a unit of seconds per centimeter (s cm-1). Because of the effects of light intensity and temperature on the stomatal resistance values, artificial light was used when the sky was cloudy. Stomatal resistance was measured 3 days after starting water-stress for all of the varieties.
The leaf relative water content was determined by using the last fully expanded leaf (flag leaf) from each plant at the end of water-stress period. LRWC was calculated using the following equation:
LRWC=[(Fw-Dw)/(Tw-Dw)] x100
Where Fw = fresh weight, Dw = dry weight, Tw = turgid weight.
The fresh weights of leaf samples were obtained immediately after excision. Then the leaves were put into test tubes containing distilled water. After 24 hours, both adaxial and abaxial surfaces of leaves were dried with paper towel and turgid weights were obtained. Dry weights were obtained after oven drying the flag leaves at 80°C for 48 hours.
Vegetative Evapotranspiration Efficiency (ETEveg) and Vegetative Water Use Efficiency (WUEveg)
ETEveg and WUEveg were calculated based on vegetative dry matter production as follows:
ETEveg= total dry matter/total water evaporated and transpired from the plant surface excluding the soil
WUEveg= total dry matter/ total water evaporated and transpired from both the plant and soil surfaces (total water used).
At the end of the water-stress period, the plants were harvested at the surface of the soil, and weighed after oven drying at 80°C for 48 hours. Total water used was obtained by subtracting the weight of pots after harvesting the plants from the weight of pots at the transplant time and adding the amount of water added during growth period. Total water evaporated and transpired only from the plant surfaces was obtained by subtracting the total water evaporated from the unplanted pots from the total water used.
Carbon isotope discrimination (Δ)
This character was measured in 5 plants of varieties Oxley and Falchetto under both control and water-stress conditions. To prepare plant samples, flag leaf fresh tissues were oven-dried at 80 °C for 48 hours. Since water-stress was applied to the plants when flag leaves were half way emerged, the leaves were divided into two parts in order to identify whether there are differences between the tip and the base of leaves for Δ. Leaves were finely ground to a powder to ensure homogeneity and to achieve greater accuracy in determination of carbon ratio (Boutton 1991). Only a small amount of plant material is required for the majority of combustion systems (Griffiths 1993), therefore samples were weighed in amounts of 1±0.05 mg. The carbon composition (δ‰) of samples was determined using an elemental analyser isotope ratio mass spectrometer known as ANCA-SL (Automated Nitrogen Carbon Analysis unit for Solids and Liquids), PDZ Europe 20/20 mass spectrometer. Δ was calculated using the equation Δ (‰)=(δa-δp)/(1+ δp) assuming δa=-7.6‰ (Farquhar and Richards 1984).
Experiment 2
This experiment was conducted to study stomatal frequency and stomatal size in varieties Oxley and Falchetto. Plants from the two varieties were grown in completely randomized design with 5 replications in a greenhouse. The plants were irrigated evenly by putting them on a wet mat which was irrigated by an automatic water supply. When flag leaves of main stems were just visible the plants were transferred into a growth chamber where day/night temperatures were 20-23/15-17 °C, photoperiod was 16 hours and light intensity varied from 180 to 210 μ m m-2 s-1 on the plant surface. In this experiment, when flag leaves were fully developed, stomatal frequency and size were measured on the adaxial and abaxial surfaces of flag leaves by the impression method (Wang and Clarke 1993a). Impressions were taken from the middle of both adaxial and abaxial surfaces of flag leaves.
Stomata counts were made on the impressions from 3 randomly selected microscopic fields of view (1.37 mm2) of each impression for both adaxial and abaxial surfaces, and length (SL) and width (SW) of stomatal guard cells were measured on both surfaces from the impressions using a scaled eyepiece of a light microscope. These measurements were made randomly on 25 stomata in each impression. Stomatal area per unit leaf area (SA) was calculated using a modified method of Wang and Clarke (1993a). They calculated SA as the product of SF and SL. In order to bring stomatal width into account, SA was calculated as a product of SFxSLxSW in this experiment.
Statistical methods
In Exp1, analysis of variance (ANOVA) was performed to determine whether there was any difference between varieties under water-stress conditions. Comparison of mean values between varieties was performed by Tukey-Test (Cochran and Cox 1992). Student t-test was performed to compare the treatments within each variety. In the case of carbon isotope discrimination and stomatal characteristics in which only two varieties were assessed, student t-test was used to compare varieties or treatments. LRWC was calculated as a percentage. Therefore, the values for this character were transformed into Arc Sin values before the analysis of variance.
Results and Discussion
Leaf Relative Water Content (LRWC) and Stomatal Resistance (SR)
For all varieties, the LRWC differences between water-stress and well water conditions were significant (Fig 1) indicating the effect of water-stress on leaf water status. Falchetto and CS had higher LRWC than varieties Falat and Oxley under water-stress conditions. SR of all four varieties increased with water-stress. Falchetto showed the most and Falat showed the least amount of SR. Falchetto exhibited significant differences from Falat and Oxley for LRWC and SR but the differences between Oxley and CS were not significant for these characters. CS showed an intermediate response to water-stress and showed no significant differences from other varieties (Table1).
LRWC of plants after a period of water-stress represents the ability of plants to retain the water in their tissues. The leaf water retention, when subjected to water-stress, has been reported to increase yield in wheat (Sharma and Sethi 1998). Wheat varieties have shown variation for LRWC under both well water and water-stress treatments. A possible explanation of variation between varieties for LRWC under well water conditions is that this variation might be due to differences between varieties leaf thickness and stomatal conductance. Maintenance of LRWC, under water-stress conditions, can be due to osmotic adjustment or reduction in transpiration (Teulat et al. 1997).
In the present experiment, varieties with high LRWC in the water-stress treatment had generally high stomatal resistance, which is in agreement with Teulat et al. (1997) obtained from a study on barley. This result can lead to a conclusion that high LRWC is, at least in part, due to high stomatal resistance and consequently decrease in water loss. Blum and Johnson (1993) have also reported that under drying top soil, LRWC and leaf water potential increased in cultivars with high SR indicating that stomatal activity was controlling leaf water status. Chandrasekar et al. (2000) studied LRWC and ABA accumulation in two hexaploid varieties C306 (water-stress tolerant) and Hira (water-stress susceptible) under pot conditions. They reported that the water-stress imposed for a uniform period caused a decline in LRWC and increased accumulation of ABA in the two genotypes. They found that little reduction in LRWC was accompanied with high ABA accumulation under water-stress conditions. They also found that the water-stress tolerant variety (C306) showed less reduction in LRWC and higher ABA accumulation than the susceptible variety (Hira), but proline increased in both tolerant and susceptible varieties indicating ineffective role of proline accumulation on water-stress tolerance. Since ABA accumulation has been reported to stimulate stomatal closure in wheat (Mittelheucer and Van Steveninck 1969; Quarrie and Jones 1976), again it can be concluded that in this experiment lower reduction in LRWC is partly due to increases in stomatal resistance.
Theoretically, it is expected that plants with low stomatal resistance have more dry matter production due to more gas exchange. In water-stress conditions, the effect of stomatal resistance depends on intensity and type of water-stress. When the amount of water is limited in respect to the duration of water-stress, any factor that promotes transpiration can bring the plants to a lethal level of leaf water content at the end of the period of water-stress. In this situation, plants lose a considerable amount of their vegetative growth (late tillers and old leaves) which could contribute to assimilation after stress. In this situation low SR does not contribute to plant production particularly when the duration of water-stress is long. Conversely, when there is a considerable amount of water in the soil so that water supply is adequate in respect to water-stress duration, low SR provides a situation for the plants that they can take up more water from the soil and also contribute to higher water content of tissues by the end of the stress period.
With regard to stomatal resistance, another aspect to be considered is the duration of stomatal closure. Stomatal closure for a long period negatively affects potential crop yield (Venora and Calcagno 1991). Therefore it seems that partial closure of stomata particularly at mid day when temperature is high and opening of stomata when temperature is not high are beneficial to plant yield. Under water-stress conditions, SR mainly plays its positive role through water conservation and consequently by reducing water loss. In this situation, plants endure water-stress without severe damage.
Teulat et al. (1997) reported that LRWC is negatively correlated to total number of leaves in barley. In the present experiment, such a result was not observed. Falat, despite having less number of tillers than Falchetto and Chinese Spring, showed less LRWC than both of them. These results indicated that amount of water loss is not only dependent on vegetative growth but also regulating response processes such as stomatal behaviour are of great importance, in particular under serious water-stress conditions. An important point in comparing varieties with different amounts of vegetative growth in pot experiments is that the duration of withholding water from the pots must be enough to impose water-stress on the varieties. If the water-stress period is not long enough, varieties with low vegetative growth might not be exposed to water-stress.
Lilley and Ludlow (1996) reported that LRWC affects the ability of plants to recover from the stress and consequently influences yield and yield stability. Similar results were found in this experiment. Varieties CS and Falchetto with high LRWC showed a better recovery after water-stress than Oxley and Falat. Data from the previous experiment (Mohammady, 2002) indicated that varieties Falat and Oxley lost their late tillers because of water-stress, while Falchetto kept their late tillers and eventually produced more fertile tillers. Another consequence of high LRWC is the delaying of leaf senescence (Turner 1997). In this experiment, varieties with high LRWC postponed leaf senescence and leaf wilting even in old leaves. This character helps the plants to attain good recovery after stress and maintenance of assimilates transfer to the ear after water-stress.
In addition to LRWC, researchers have identified some other characters to indicate water-status of leaf under water-stress. Golestani Araghi and Assad (1998) calculated Relative Water Loss (RWL) as the ratio of water lost from the excised leaf during interval periods of time to the total water content of leaf. Sharma and Sethi (1998) calculated Excised-leaves Water Retention (ELWR) as another character which indicates water status of leaves and used this character as a selection criterion for water-stress tolerance. They expressed ELWR as a percentage of water remaining in an excised-leaf after 48 hours of air-drying. Dhanda and Sethi (1998) also used excised leaf water lost (ELWL) to compare water status of different genotypes. They calculated ELWL as the ratio of (Fresh weight-Weight 6 hours after excision) to (Fresh weight ?Dry weight). ELWL and ELWR have shown less stable values than LRWC (Dhanda and Sethi 1998). ELWL and ELWR also seem to be more affected by the temperature and relative humidity where the excised leaves are stored so that the amount of reduction in leaf weight after 6 hours might not be significantly different from the fresh weight. In addition, none of these characters are calculated using turgid weight of leaf except LRWC. In this experiment, LRWC was used to compare the varieties for water status since it seemed a better indicator of reduction in turgor pressure and consequently cell volume as a result of water-stress.
WUEveg and ETEveg
Varieties were assessed for WUE and ETE based on the amount of dry matter production at the vegetative stage. Fig. 1 (b, c) indicates the effect of water-stress on WUEveg and ETEveg. A small effect of water-stress was observed on these characters. A possible explanation for this result is that the duration of water stress in respect to whole growth period was too short to affect significantly the characters. In other words, assimilates stored in the leaves and water lost during the water-stress period are small in comparison with total biomass production and transpiration. It also could be due to a balanced reduction in both growth and water used during the water stress period.
Comparison between varieties indicated that CS had the highest WUEveg and ETEveg under water-stress conditions (Table 2), but Oxley had the lowest values for these characters. Differences between Falat and Oxley and between Falchetto and CS were not significant (Table 2). Since the effect of water-stress on WUEveg and ETEveg was not significant (Fig. 1), it can be concluded that differences between the varieties are due to differences in dry matter production for the amount of water used during the whole period of growth. It also can be concluded that water-stress imposed by withholding water for a short period does not produce any significant effect on WUEveg and ETEveg calculated immediately after the water-stress period. The effect of this kind of water-stress generally appears in final dry matter production of plants due to the effect of water-stress on recovery of leaves and tillers after water-stress. Such a result was observed in previous experiments (Mohammady, 2002) where water-stress caused a significant reduction in economic water-use efficiency in some varieties.
WUE and ETE are generally affected by dry matter production and water consumption in a reverse direction. Sometimes reduction in dry matter production is compensated by reduction in the amount of water used during the growth season. In this situation, WUE and ETE remain insensitive to water-stress even when the plants are exposed to continuous water-stress in the field. This kind of insensitivity has been reported by Fischer and Turner (1978) in the field experiments. This may seem surprising since it is well documented that water-stress causes stomatal closure and, in theory, temporary closure of stomata when evaporative demand is high should improve water use efficiency. Ludlow and Muchow (1990) suggested that closure of stomata, caused by an increase in temperature, might reduce transpiration efficiency due to a reduction in photosynthesis.
Where reduction in assimilates is not compensated by reduction in water use, WUE and ETE are mainly affected by the amount of water loss. The amount of water loss itself depends on the amount of water evaporated from the soil and this, in turn, depends on the early growth and the ability of plants to uptake water from the soil (Ehdaie and Waines 1993).
According to the literature, different methods have been used to define WUE and ETE. These characters were determined by Ehdaie and Waines (1996) as total dry matter/total water used and grain yield/ total water used, respectively. These definitions are different to those used in this experiment (see Material and Methods). In fact, the difference between ETE and WUE should reflect the ways by which water is consumed in the experiment and not the kind of dry matter production. WUE can be calculated as the amount of leaf dry matter, total dry matter and grain yield produced for the total amount of water used (water evaporated from the soil and evapotranspired from the plant surface). ETE can also be calculated as the amount of leaf dry matter, total dry matter and grain yield for the amount of water evapotranspired from the plant surface only. In fact ETE can be used as a modified method of water use efficiency by which the effect of evaporation from the soil on WUE of the plants is omitted. In this situation, uptake efficiency can be evaluated in the experiment. If a variety shows higher ETE but lower WUE in comparison with other varieties, it can be concluded that this variety has a low uptake efficiency since a large portion of the available water is evaporated at the soil surface instead of being absorbed by the plants. In this regard, Gorny (1999) defined two types of WUE based on how dry matter was used. He calculated WUEeco by dividing grain weight by water transpired and WUEveg by dividing dry matter of vegetative plant parts by units of transpired water. In this calculation, in fact, water use efficiency is calculated with respect to partitioning of dry matter in different plant parts.
Carbon isotope discrimination (Δ‰)
A significantly higher carbon isotope discrimination (Δ) was consistently observed in Oxley than in Falchetto for both treatments (Fig. 2 b). Δ was significantly lower (P<0.01) in plants subjected to water-stress than in well watered plants except for the base of the flag leaf of Oxley that was significant only at 5% (Fig. 2 a). Merah et al. (2001a) reported a similar reduction in Δ due to water stress in 6 species of durum wheat grown in the field for two subsequent years. According to their results, Δ was lower in all of the species in the first year where the severe early water-stress was noted. Farquhar and Richards (1984) suggested that the reduction in Δ under water-stress was due to an increase in stomatal resistance and consequently a reduction in the ratio of Pi/Pa under stress treatments.
The slightly higher values for Δ in the tip of the flag leaf compared to the base were not significantly different except in the case of the Oxley control plants (Fig. 2 c). This implies that carbon isotope composition in the tip and the base of flag leaf is not significantly different. Therefore measurement of Δ in samples prepared from the whole leaf can lead to a saving in time and expenditure and can sufficiently identify possible differences between varieties and treatments. In other words, from the present data, there was no firm evidence to support the hypothesis about the existence of a significant difference in 13C accumulation between the base and the tip of flag leaf. However, in both treatments and both varieties, Δ of the base was somewhat lower than the tip. Senescence and wilting is more severe in the leaf tip when water-stress is imposed on the plants. Nonetheless, this did not result in a significant reduction in carbon discrimination compared to the base. Since the increase in discrimination in the tip compared with the base is similar in the well watered and water-stressed treatment, the difference probably existed before the water-stress was imposed. The difference could result from a reduction in photosynthesis in the tip due to ageing effects.
Comparison between varieties based on pooled data (irrespective of leaf section) indicated significant differences between the treatments and between the two varieties (Fig 3). The overall reduction in Δ due to water-stress for Oxley was 0.84±0.25 which was less than the reduction for Falchetto (1.06±0.15). This indicates that discrimination against 13C decreases to a greater extent in the variety Falchetto as a result of water-stress. Since Δ is negatively correlated with WUE (Griffiths, 1993), the high WUEveg of Falchetto in comparison with Oxley is probably partly due to the lower value for Δ in Falchetto. Despite wide use of Δ as an indicator of water use efficiency, Condon et al. (2002) believe that measuring Δ as an indicator of water use efficiency has several shortcomings. For example, they have reported that measuring Δ provides no information on the magnitude of either assimilates or transpiration or whether variation in Δ is due to variation in photosynthesis capacity or stomatal conductance.
The variation in Δ depends on differences in either stomatal conductance or photosynthetic capacity (Condon et al. 1987). Since in the present experiment, there was a significant difference in Δ between the two varieties under both water-stress and well water conditions and the difference for stomatal resistance was significant only under water-stress conditions, it can be postulated that variation of Δ between the two varieties is due to both stomatal conductance and photosynthetic capacity. In addition, it seems that stomatal resistance and photosynthetic capacity interact positively in the reduction of Δ under water-stress conditions. This is a possible explanation for higher differences between the varieties under water-stress conditions than under normal conditions. So, under normal conditions, where there is no significant difference between the two varieties for stomatal resistance, lower Δ in the variety Falchetto can be accounted for by the higher photosynthetic capacity of this variety. But, under water-stress conditions, the lower Δ of Falchetto is partly due to higher stomatal resistance and partly due to higher photosynthetic capacity in this variety.
Relationship between Δ and other physiological characters under water-stress conditions
Carbon isotope data from the tip and base were pooled for 5 plants of Falchetto and Oxley and the relationship between the data and some physiological traits was investigated for these plants (n=10). As can be seen from Fig 4, significant relationships were found between Δ and ETEveg (R2=0.53, df=8, P<0.01), WUEveg (R2=0.51, df=8, P<0.01) and DM (R2=0.42, df=8, P<0.05). On the other hand, no significant relationship was found between Δ and either LRWC or SR.
The absence of a significant relationship between Δ and SR under the conditions of this experiment allows us to postulate that variation in Δ is due to differences in photosynthetic capacity rather than differences in stomatal resistance. However, Merah et al. (2001b) did not find a significant correlation between specific leaf dry weight and Δ in 144 accessions of durum wheat grown under rain fed field conditions. They concluded that variation in Δ results from differences in stomatal resistance rather than from photosynthetic capacity. They also reported a positive significant correlation between LRWC and Δ. In the present study such a relationship was not observed. Environmental differences and duration of water-stress period are two possible reasons behind these inconsistent results. However differences between the varieties and temperature could also affect these relationships.
Soil water is exhausted due to water-stress under pot conditions. Thus stomatal resistance in this situation can lead to higher leaf relative water contents and to a lower Δ. If so, a negative relationship was to be expected between Δ and either LRWC or SR. Unexpectedly, these relationships were not significant. This is due to the fact that SR is difficult to measure reliably (K Moore and S A Quarrie, personal communications) and it is not the only character which influences Δ but the potential capacity of photosynthesis is also involved. On the other hand, the relationship between Δ and ETE was significant. This was possibly due to a co-operation between SR and photosynthetic capacity which both increase WUE and ETE of the plants.
A negative relationship between WUE and Δ has been reported by several workers in wheat (Al Hakimi et al. 1996; Codon et al. 1990; Farquhar and Richards 1984; Griffiths 1993; Richards et al 1998; Quarrie et al. 1999) and other plants (Martin et al. 1999). Condon et al. (1993) studied the relationship between WUE and Δ in wheat in a dry land experiment. They reported a positive relationship between total dry matter (DM) and Δ when there was little limitation of water supply. In this situation, the relationship between WUE and Δ was also positive but non-significant. They also pointed out that when the crops encountered water-stress after anthesis, the relationship between Δ and DM tended to be negative. Farquhar and Richards (1984) measuring WUE and Δ in different water-stress regimes, reported higher WUE and lower Δ in severer water-stress stress treatments. Quarrie et al. (1999) suggested Δ is an integral measure of WUE and selecting for this character will result in higher yield under water-stress conditions.
Stomatal characteristics
Means and standard errors for SF, SL and SW are presented in Table 3 for varieties Oxley and Falchetto. Falchetto had a significantly higher number of stomata per unit area of flag leaf than Oxley on the adaxial surface (P<0.01), but the difference was not significant on the abaxial surface of the flag leaf (Table 3). However, Oxley had longer stomata than Falchetto on both surfaces of the flag leaf. This result was expected since it is now well documented that stomatal frequency is commonly inversely related to the size of guard cells (Singh and Sethi 1995; Venora and Calcagno 1991; Wang and Clarke 1993a). In the present experiment, however, the stomatal width in Oxley was not significantly different from that in Falchetto. This indicates that stomatal length is more effective than stomatal width to compensate for the loss of stomatal area due to lower SF. Stomatal length has been used as an indicator of stomatal size by many investigators (Singh and Sethi 1995; Venora and Calcagno 1991; Wang and Clarke 1993a) but no published work was found to use stomatal pore width as a trait which determines the capacity of stomata to reduce water loss. This is possibly due to difficulties which appear during the measurement of stomatal pores especially under unfavourable conditions. Venora and Calcagno (1991) measured and used stomatal width as an indicator of stomatal aperture. This application seems to explain differences between the varieties for water loss only under non-limiting conditions provided varieties with larger size of stomata have larger stomatal pores. In unfavourable conditions, particularly under water-stress conditions, stomatal aperture is not the main determination of water lost because it is extremely variable as a result of the influence of atmospheric factors (Wang and Clarke 1993b).
With regard to differences between leaf surfaces, data analysis indicated that there were significant differences (P<0.01) between adaxial and abaxial surfaces in both varieties for SF. A highly significant difference was also observed between adaxial and abaxial surfaces for SL in the variety Oxley. This difference was significant only at the P<0.05 level in Falchetto. SW did not differ significantly between the leaf surfaces in both varieties.
Higher number of stomata on the adaxial surface has also been reported by other workers (Malone et al. 1993; Singh and Sethi 1995; Teare et al. 1971). Teare et al. (1971) studied SF in different species of wheat. Their results indicated that the number of stomata was significantly greater on the adaxial than on the abaxial surface of the leaves of Triticum. This was the case for all species studied in the genus. The ratio of SF on abaxial to adaxial surface was 0.6902 over 4 varieties of common wheat. In the present study, this ratio was 0.6887 and 0.6909 for Oxley and Falchetto, respectively. These results are very close to those obtained by Teare et al, (1971). From these results, it can be suggested that the ratio of SF on the abaxial to that on the adaxial surfaces is possibly more stable than the absolute number of stomata in either surfaces of flag leaves in common wheat.
It is theoretically expected that varieties with higher number of stomata per unit area and greater length and width of stomata lose more water during the growth period. This happens if stomata remain open during the water-stress period. Any response of stomata to water-stress can be discussed in relation to stomatal resistance and LRWC. Reduction in water loss from leaf surfaces during periods of severe water-stress is an important water-stress tolerance indicator. Low rate of cuticle transpiration, therefore, may reduce leaf dehydration and promote leaf survival (Wang and Clarke 1993b). When water-stress develops, the response of stomata to water-stress seems to be of great importance in reducing water loss. Stomatal frequency of Falchetto was significantly higher than Oxley, but Falchetto had smaller stomata. On the other hand, our results from experiment 1 revealed that Falchetto had a higher LRWC and SR than Oxley. These results indicated that SF is not always correlated with plant water status. This is because stomatal size, response of stomata to environmental stress and even cuticle resistance are also involved in determining plant water status particularly under water-stress conditions. In the case of cuticle resistance, Gupta et al. (2001) using diurnal observations, reported that a water-stress tolerant variety (C-306) had higher leaf diffusive resistance than a water-stress sensitive variety (Kalyansona). Since stomata are mostly closed at night, it can be concluded that differences in diffusive resistance are mostly due to differences in cuticle resistance.
The results of other workers concerning the relationship between stomatal characteristics and plant water status are inconsistent. McCaig and Romagosa (1989) found no consistent differences in SF between two durum genotypes with different water-retention capabilities. Wang and Clarke (1993b) reported that SF was not correlated with relative water loss and LRWC in field experiments. However, their results indicated that SF was positively correlated with the rate of water loss but not with LRWC under growth room experiments. The inconsistency of this relationship is possibly due to the influence of other characteristics of stomata rather than SF and due to negative relationships between stomatal size and frequency. In addition to SF and stomatal size, the stomatal responses to water-stress and cuticle resistance are other factors which influence water status of plants under water-stress conditions. Thus, the results presented in this section and those explained above indicate that stomatal characteristics are affecting water status of plants as a complex, and every component of this complex should be studied in relation to other components and with other factors which influence water status of plants.
The increase or decrease in transpiring area under stress conditions may not be achieved by selecting for high or low SF due to the negative correlation between SF and stomatal size (Venora and Calcagno 1991). For this reason, it seems that SA as a combination of SF, SL and SW is a better determination of water status in plants. As can be seen from Table 4, there were no significant differences between the two varieties for SA on both surfaces. However, Oxley had a slightly higher SA on both adaxial and abaxial surfaces than Falchetto. These results indicate that higher SR of Falchetto on the adaxial surface is not due to SA but is possibly due to either differences in stomatal response to water-stress or differences in cuticle resistance. Higher leaf relative water content of this variety also seems to be due to either more extensive stomatal closure on the adaxial surface or to lower stomatal area on the abaxial surface. Identification of ABA accumulation in these two varieties might produce further evidence about the contribution of stomatal closure to water-stress tolerance under pre anthesis water-stress conditions.
To sum up, the results of the current study indicated that physiological characters such as SR, LRWC, WUE and Δ are among the characters that can confer tolerance to pre-anthesis water-stress to wheat varieties. Since much of the genetic variation for improving stress tolerance has been lost during selection and modern breeding (Araus et al., 2002), other genetic materials such as landraces rather than modern varieties should be used to obtain a large improvement in stress tolerance. Selected landraces can contribute to the enhancement of wheat production in dry regions by direct use for cultivation or by using in various methods of plant breeding in order to improve high yielding but drought susceptible varieties so that they can tolerate drought. The varieties Oxley and Falchetto can be used as controls for screening the landraces for water use efficiency, Δ, SR and LRWC.
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