The genetic analysis of stomatal frequency and size, stomatal conductance, photosynthetic rate and yield in wheat (Triticum aestivum L.) using substitution lines series

Roghaye Aminian, Shahram Mohammadi, Sadolla A. Hoshmand and Mahmood Khodambashi

Department of Agronomy and Plant Breeding, Faculty of Agriculture and Environmental Sciences, Shahrekord University, Shahrekord, Iran, P.O. Box 115.

Corresponding Author: Shahram Mohammadi

E-mail: mohammadyshahram@yahoo.com

Abstract

This study was aimed to determine the effects of stomatal size and frequency on stomatal conductance (gs), photosynthetic rate (Pr) and yield and to locate the genes controlling these traits. Therefore, substitution lines series of 'Timstein' (Tim) into genetic background of 'Chinese Spring' (CS) were tested in a completely randomized block design with three replications under two normal and water stress conditions. Water-stress condition was started at stem elongation stage and irrigation periods were done based on pre-determined 'Growth degree day' (GDD) in the length of stress period. Significant differences were found among substitution lines in terms of stomatal frequency, stomatal size, stomatal conductance, photosynthetic rate and yield. Although stomatal characteristics were found not to be strongly correlated with yield, photosynthetic rate and stomatal conductance, the significant correlation was found between the yield with stomatal size and stomatal frequency in stress and non-stress condition, respectively. In addition, there were significant correlations between yield with photosynthetic rate and stomatal conductance both in stress and non-stress conditions. For yield, 1A, 2B, 3B, 7B and 5D substitution lines showed significant differences from their recipient parents both in stress and non-stress conditions. However, the role of group B chromosomes was more prominent than the other two groups.

Key Words: Stomatal frequency, Stomatal size, Stomatal conductance, Photosynthetic rate

Introduction 

Water stress is a crucial climatic variable in wheat growing areas of the world under such conditions. Plant features, which increase their capabilities to sustain their physiological processes, may increase their growth and productivity (Gales 1983). Maintaining gas exchange properties in normal rates is among those features, which may increase plant growth and yield. Stomata are the major gates for gas exchange of leaves (Maghsoudi and Maghsoudi moud 2008).

Stomata play an important role in the control of water evaporation and gas exchange in plant leaves. Transpiration and photosynthesis are regulated by changing the size of stomatal pores. In addition, the operation of the stomatal apparatus is influenced by plant environment (Heichel 1971) and, therefore, stomatal regulation of transpiration is exerted by both internal and external factors. To cope better with dry condition, stomata must be open to allow CO² uptake and close during drought condition to minimize water loss by leaves (Elizabeth and Alistair 2007). Stomata can adjust stomatal conductance under environmental conditions to optimize CO² uptake and transpiration rates (Hyeon-Hye et al. 2004). Stomata are responsive to environmental factors such as light, relative humidity, CO² concentration and plant water status (Jarvis 1976). However, different cultivars of crop plants may have different gas exchange capabilities because they have various numbers of stomata per unit of leaf area and various stomatal sizes (Ciha and Brown 1975; Farquhar et al. 2002). Significant differences have been found among plant species in the responses of their stomata to changing environment (Raschke 1975). During the past decades, stomatal size and frequency have been used as an indicator of water loss by many investigators (Venora and Calcagno 1991; Wang and Clarke 1993a; Singh and Sethi 1995) but based on our knowledge no published work was found to use stomatal pore width as a trait, which determines the capacity of stomata to reduce water loss.

Selection and variation for stomatal characteristics has been reported in bread wheat (Bkagwat and Bhatia 1993). Mohammady (2002) found significant differences for stomatal length on adaxial and abaxial surfaces of bread wheat. He also reported that stomatal length is more effective than stomatal width on water transpiration. It has been suggested that wheat cultivars having wider somatal aperture produce higher yields without consuming more water (Shimshi and Ephrat 1975). Miskin et al. (1972) found a 25% decrease in frequency of stomata reduced transpiration rates by about 24%. They found that stomatal frequency did not influence the rate of photosynthesis. Wang and Clarke (1993b) reported that stomatal frequency positively correlated with the rate of water loss. This indicates breeding for smaller and fewer stomata may lead to reduction in water loss. Gaskell and Pearce (1983) found that stomatal density negatively correlated with grain yield and with stomatal size. Stomatal frequency in wheat was shown to be greater on the adaxial than on the abaxial surface. Mean ratios (abaxial/adaxial) were reported 0.748 for the first leaf and 0.728 for the second leaf (Teare et al. 1971).

Stomatal frequency and size have also been used as morphological markers for identifying ploidy level in many plant species (Beck et al. 2003; Beck et al. 2005, Kharazian 2007; Khazaei et al. 2009). Aryavand et al. (2003) and Khazaei et al. (2009) reported that significant variation was found in stomatal frequency between the ploidy levels for flag leaves in Aegilops neglecta and Triticum, respectively. However, it was shown that stomatal frequency and size high negatively correlated. Tanzarella and Blanco (1979) found that stomatal frequency of the flag leaf negatively correlated with stomatal length in durum wheat. It was postulated by Zhang et al. (2007) that stomatal frequency is closely linked to water use efficiency through its influence on stomatal conductance. Higher stomatal frequency has been suggested by Hardy et al. (1995) to be associated, together with photosynthetic pathways, to higher water use efficiency in C₄ plants, compared to C₃ plants. Due to the complexity of drought tolerance, efforts to increase drought tolerance could be achieved through selection for yield, which integrates all the unknown factors important for improving drought tolerance. Accordingly, many yield-based parameters such as stomatal characteristic, stomatal conductance, photosynthesis rate, transpiration, water use efficiency and so on were suggested to evaluate drought tolerance.

Substitution lines are promising genetic stocks for studying the role of chromosomes in controlling quantitative traits including drought tolerance related traits and they are widely used for studying inheritance of quantitative traits in wheat (Law et al. 1978 a, Law et al. 1978b). The aims of this study were to analyze the relationship between stomatal characteristics, yield, photosynthetic rate and stomatal conductance, and to locate the genes controlling those traits.

Materials and Methods

The present study involved chromosome substitution lines series of 'Timstein' (donor) into the genetic background of 'Chinese Spring' (recipient) developed at Ibaraki University (the University of Japan) by Dr. Watanabe.

This experiment was conducted in the field of agricultural research station Shahrekord University (latitude, 50^o 49', longitude; 32^o 21' altitude). According to Koupen method, Shahrekord climate is classified as moderately cold (average annual precipitation 420 mm). The soil was silty loam (pH = 7.8). The experiment had a randomized complete block design with three replications. Needed amount of nitrogen and phosphorous fertilizers were calculated based on soil test and were added to the soil before planting. Each plot consisted of four rows, which were 200 cm in size and distanced 20 cm from each other. After planting all plots were irrigated to assure a full crop establishment.

In non-stress condition, plants were irrigated after seven days and in water stress condition irrigation was withheld before stem elongation at Zadoks' scale 29 ( Zadoks et al. 1974) to simulate what would happen under drought stress condition. Stress period length was measured in preliminary experiment based on growth degree-day (GDD). Plants were irrigated when receiving this GDD.

The number of days from sowing to Zadoks' scale 58 (Zadoks et al. 1974) was noted for each substitution line. At this stage, the fully developed flag leaf of the main tillers was randomly selected from five plants of each plot for determination of stomatal characteristics.

Stomatal frequency (SF) and stomatal length (SL) and stomatal width (SW) were measured in the middle part of the adaxial and abaxial surfaces by impression method (Wang and Clarke 1993a). There is less deviation among sub samples taken at the center of the leaf than among sub samples taken at either the tip or the base (Teare et al. 1971). The number of stomata was counted from ten different microscopic fields of view at 160-x magnification. To find the stomatal frequency, the number of stomata per field of view was converted to the number of stomata per one mm² of leaf using a standard scale. Stomatal length and stomatal width were measured on the both surfaces from the impressions using a scaled eyepiece of microscope and then stomatal size was converted to μm. Stomatal area was estimated as the product of stomatal length and width. The measurements were made randomly on ten stomata in each impression and the mean values of the ten measurements were used for statistical analyses. Gas exchange measurements were determined in the middle part of the abaxial and adaxial sides of fully expanded flag leaves using a portable gas exchange system (LI-COR 6400; LI-COR Inc., Lincoln, NE, USA). Leaf temperatures were set at 25ºC for all measurements. The leaf cuvette was held in the horizontal position and caution was used not to shade any portion of the leaf. One leaf per row from each of four different rows (repeated measures) was randomly selected in the area of each plot. Reported measurements were the mean of four measurements recorded over 10 min. Full mature plants were harvested from the middle parts of the rows and grain yield was measured based on the mean of the grain yield of 10 plants per plot for each repeat.

Data were analyzed using SAS version 9.2 (SAS Institute Inc Cary, NC, USA). Analyses of variance were preformed using the GLM procedure. Correlation analysis was performed to determine the relationship between the traits using the CORR procedure and comparisons between means were made using LSD test at (P<0.05).

Results and Discussion

Analysis of variance indicated that significant differences existed among the substitution lines for stomatal frequency (SF) both in adaxial and abaxial surfaces of flag leaves in stress and non-stress conditions (Table1). Significant correlation was found between SF on adaxial flag leaf surface and yield in non-stress condition, while significant negative correlation was found between SF on abaxial flag leaf surface and yield in stress condition. Gaskell and Pearce (1983) showed that stomatal density was negatively correlated with grain yield. Mohammady (2002) found higher stomatal frequency and smaller stomata in the bread wheat cultivar 'Falchetto' (drought tolerant), compared to the cultivar 'Oxley' (drought susceptible). Conversely, Merah et al. (2001) and Heichel (1971) reported a negative relationship between stomatal frequency and drought tolerance in a collection of 144 durum wheat accessions and some varieties of barley, respectively. The inconsistency of this relationship was likely due to the influence of other characteristics than stomata in transpiration, water loss and yield. In water limiting condition stomatal size had more effects on yield than SF. On the other hand, low SF would appear to be a logical means of developing drought tolerant varieties. Presumably, cultivars with low SF would have greatest advantage when soil moisture is in short supply, a condition common to much of the world's agriculture. The means of characteristics across the genotypes are also presented in Table 2 for the both conditions. As can be seen from the table, there is no significant differences between the two conditions for the traits. This implies that some of the lines have performed better in the water-stress experiment compared to normal conditions.

There was significant and positive correlation between SF and stomatal conductance (gs) in non-stress condition, while there was no relationship between these traits in stress condition (Table 3 and Table 4). This observation also agrees with those from Heichel (1971) who observed that lines having low stomatal frequencies had lower stomatal conductance and transpired less water than lines with more stomata. Pr was significantly correlated with SF on adaxial surface in non-stress condition but the negative relationship was observed between Pr and SF in stress condition (Table 3 and Table 4). Miskin et al. (1972) found that stomatal frequency did not influence the rate of photosynthesis. A similar situation existed in varieties of bean and less dense stomata had faster net photosynthesis (Izhar and Wallace 1967), suggesting that stomatal pore area, transpiration or other factors are of equal or greater importance than stomatal frequency in regulating net photosynthesis in stress condition. Wang and Clarke (1993a) reported a positive correlation between SF and the rate of water loss. It was postulated by Zhang et al. (2007) that SF is closely linked to water-use efficiency through its influence on stomatal conductance. For all substitution lines, the adaxial surface has been higher SF than the abaxial surface. Besides, significant differences were found between the two sides. Other studies have reported significant differences between adaxial and abaxial surfaces in wheat (Teare et al. 1971; Rajendra et al. 1978; Singh and Sethi 1995; Mohammady 2002; Khazaei et al. 2009). The ratio of SF on abaxial to adaxial surface was 0.78 and 0.81 over all studied substitution lines in stress and non-stress conditions, respectively. The ratio of 0.78 and 0.81 observed in wheat substitution lines was closed to the ratio of 0.748 reported by Teare et al. (1971) and the ratio of 0.81 reported by Khazaei et al. (2009) in the same species. Mohammady (2002) obtained this ratio 0.69 for T. aestivum, and suggested that the ratio is possibly more stable that the absolute number of stomata in either surfaces of flag leaf.

A significant negative correlation was observed between SF and stomatal size (Table 3 and Table 4). The negative relationship found between stomatal frequency and size is consistent with findings of Miskin et al. (1972), Venora and Calcagno (1991), Singh and Sethi (1995), Aryavand et al. (2003) and Khazaei et al. (2009). However, Teare et al. (1971) reported a non-significant correlation between the two traits. The negative association between stomatal frequency and size appears to be a compensatory relationship in such a way that the total pore area is approximately equal (Miskin et al. 1972). Hence, lower stomatal density was compensated for by stomatal aperture and increased stomatal density by reducing stomatal aperture.

As figure 1A shows, there were significant differences regarding SF on adaxial surface between substitution lines and their recipient parent in both stress and non-stress conditions. As for SF on adaxial surface both in stress and non-stress conditions 3B, 1D and 7D lines were significantly different from recipient parent (Table 5). Quantitative trait loci analysis in rice showed that stomatal density was controlled by several QTLs having relatively small effects. Significant regions affecting stomatal density were identified on chromosomes 1, 2, 3, 4, and 6 (Ma et al. 2010). As figure 1A shows, the chromosomes of the group 'D' had the most stomatal frequency among all in both stress and non-stress conditions, and 7D line had maximum stomatal frequency in stress and non-stress conditions. The mean number of stomata per mm² was increased in half of the substitution lines in stress condition. Water stress significantly increased the number of stomata on both the adaxial and abaxial surfaces of soybean (Ciha and Brown 1975). The increase in stomatal frequency with water stress suggests that restricted leaf expansion caused this increase in these substitution lines, although there was also a decrease in the formation of stomata, possibly by inhibition of stomatal mother cell differentiation.

The significant differences were found between substitution lines in terms of stomatal length on both the adaxial and abaxial surface of flag leaf in stress and non-stress conditions implied on sufficient variation between lines (Table 1). According to the data shown in Table 4, there was significant correlation between stomatal length and yield in stress condition, while a significant negative relationship was found between stomatal frequency and stomatal length among substitution lines on both the adaxial and abaxial surface. Such a negative correlation has been also reported in sugar beet, soybean and barley (Ciha and Brown 1975). The significant positive correlation was observed between stomatal length and photosynthetic rate in non-stress condition, but there was negative relationship between stomatal length and photosynthetic rate in stress condition (Table 3 and Table 4). The mean of stomatal length in 1B and 3A substitution lines for abaxial surface and in the all substitution lines for adaxial surface were significantly different from recipient parent in non-stress and stress conditions, respectively (Table5).

Significant differences were found for stomatal width (Table 1). Therefore, there was suitable genetic variation between substitution lines. There was significant correlation between stomatal width with yield and photosynthetic rate in stress condition (Table 4), while there was no significant correlation between those traits in non-stress condition. It was suggested by Singh and Sethi (1995) that stomatal frequency and size influence water loss. In durum wheat grown under non-limiting conditions, Venora and Calcagno (1991) found a negative association between water loss and stomatal size. This indicates that breeding for bigger and fewer stomata may lead to reduction in water loss and an increase of yield in water limitation condition.

In water-stress conditions, 1A, 2A, 6A, 5B, and 6D lines on adaxial surfaces and 3A as well as 2B lines on abaxial surface were found significantly different from recipient parent for stomatal width, while in non-stress condition 5A, 6A, 1B, 2B, 4B, 1D, and 7D lines on adaxial surfaces and 4B, 5B, 6B, 3D and 5D lines on abaxial surfaces were found significantly different from recipient parent (Table 5). Hence, the role of the chromosomes of 'A' genome was the most prominent of all in stress condition, but the role of the chromosomes of 'B and D' genomes was more prominent than 'A' genome in non-stress condition in controlling stomatal width.

The results of analysis of variance showed that significant differences existed among the substitution lines for stomatal area on both the adaxial and abaxial surfaces of flag leaf in stress and non-stress conditions (Table 1). There was positive correlation between Pr and gs with stomatal area on both adaxial and abaxial surfaces of flag leaf in stress condition (Table 4). The influence of differences in stomatal frequency and size on gs, Pr and yield under field conditions could be obscured by transpiration, the degree of opening or length of time the stomata are open, environmental factors such as light and temperature, genotypic factors like photosynthetic capacity, and enzyme activity. As figure 1B shows, the chromosomes of the group 'B' had the fewest stomatal area among all in both stress and non-stress conditions. It is assumed that the 'B' genome of 'Timstein' cultivar have the genes that reduce the stomatal area. All the substitution lines except 2B, 3D, and 5D on adaxial surface, 3A, 2B, and 3D on abaxial surface in stress condition, 5A, 4B, and 1D on adaxial surface as well as 3A, 4B, 5B, 6B, 3D, 5D, and 7D on abaxial surface in non stress condition were significantly different from 'Chinese Spring' (recipient plant) (Table5).

To our knowledge, no report has been made on the chromosomes or loci associated with stomatal frequency and size in Triticum aestivum. In this study, we identified chromosomes in substitution lines, which were significantly different from those chromosomes in 'Chinese Spring' (recipient plant) for under studied traits. Because each chromosomal substitution line is exactly similar to recipient plant except in one chromosome, therefore the role of that chromosome in controlling the special trait could be found by comparing the substitution line to recipient plant.

As figure 2A shows, there were significant differences among substitution lines for yield in both stress and non-stress conditions. The chromosomes of 'A' and 'B' genome had the lowest and the highest yield as compared to the other two groups both in stress and non-stress conditions, respectively. Probably the genome 'B' of ‘Timstein' cultivar had drought tolerance genes. Therefore, the role of group 'B' in increasing yield was more prominent than the other two groups. Significant differences were found among substitution lines regarding Pr and gs in stress condition (see Table 1). The chromosomes of group 'A' had fewer Pr and gs than two other groups both in stress and non-stress conditions (Figure 2B and C). Therefore, the role of group 'B and D' in increasing those traits was more prominent than 'A' genome. There was not strong but significant correlation between yield with Pr and gs both in stress and non-stress condition. Maghsoudi and Maghsoudi moud (2008) found those stomatal characteristics are poorly correlated with grain yield and transpiration in some drought tolerant wheat cultivars. 1A, 5A, 2B, 3B, 4B, 7B, and 5D substitution lines as well as 1A, 3A, 2B, 3B, 6B, 7B, 1D, 5D, and 6D lines in terms of seed yield were significantly different from recipient parent in stress and non-stress condition, respectively. 3A, 2B, 3B, 4B, and 3D in stress condition for photosynthetic rate, 3A, 2B, 3B, 4B, and 1D lines, 1A, 3B, 1D, 6D, and 7D lines regarding gs were significantly different from 'Chinese Spring' in stress and non-stress conditions, respectively. Most of the substitution lines except 1A, 3A, 7A, 2B, 3B, 1D, and 5D had lower gs in stress condition. These differences in gs in stress and non-stress condition may be due to the degree of opening or length of time that the stomata are open and also to transpiration rate. Generally, substitution lines of 3A, 2B, 3B, 4B and 6D were the most prominent lines of all for most of the studied traits. Therefore, they must have yield effective genes that by changing stomatal size and frequency affect yield, Pr, and gs. The results of this experiment showed that the role of the chromosomes of group 'B' was more prominent than the other two groups as for stomatal characteristics. However, these results show that stomatal characteristics are not strongly correlated with grain yield, Pr and gs as with many other morpho-physiological characteristics. Stomatal features are not in close relation with yield. This may be due to polygenic nature of yield efforts.

During the past decades, efforts have been focused on finding plant characteristics which are closely correlated to yield particularly under environmental stresses such as drought. This would mean that changing those features makes it possible to change the yield. However, because genetic basis of yield is extremely complex, breeding improved genotypes for drought stressed conditions was not successful. Finding mechanisms which somehow affect yield has been the focus of many researches works (Maghsoudi and Maghsoudi moud 2008).

Acknowledgement

The authors offer grateful thanks to Shahrekord University for financial assistance. Thanks are also due to Dr. Watanabe for helpful information and technical assistance for development of the substitution lines series at Ibaraki University, Japan.

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