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Relationships between flag leaf carbon isotope discrimination and several morpho-physiological traits in durum wheat genotypes under Mediterranean conditions
Environmental and Experimental Botany 45 (2001) 63 – 71 www.elsevier.com/locate/envexpbot
Relationships between flag leaf carbon isotope discrimination and several morpho-physiological traits in durum wheat genotypes under Mediterranean conditions Othmane Merah a,b,*, P. Monneveux b, E. Dele´ens a a
Institut de Biotechnologie des Plantes, UMR 8618, Bat 630, Uni6ersite´ de Paris-Sud, Centre d’Orsay, 91405 Orsay Cedex, France b UFR de Ge´ne´tique et Ame´lioration des Plantes, ENSA-INRA, 2 Place Viala, 34060 Montpellier Cedex, France Received 23 June 2000; received in revised form 9 October 2000; accepted 10 October 2000
Abstract Relationships between flag leaf carbon isotope discrimination (D), water status parameters, residual transpiration (RT) and stomatal density (SD) were examined on a collection of 144 durum wheat accessions. Associations between D, grain yield (GY) and harvest index (HI) were also studied. The field trial was conducted under Mediterranean conditions. The crop cycle was characterised by a period of drought from February until maturity. A broad range of values we obtained for D (16.5–19.9‰) and other physiological traits. Flag leaf D was positively and significantly correlated with both HI and GY. D was better correlated with HI than with GY, which suggests that higher D values indicate higher efficiency of carbon partitioning to the kernel, leading to higher GY. D was found positively related with RT and negatively related with SD. This relationship may indicate a possible SD component of RT due to the association between conductance and SD. Strong positive correlations were found between D and water status parameters, suggesting that D may provide a good indication of plant water status in durum wheat under rainfed Mediterranean conditions. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Drought tolerance; Harvest index; Residual transpiration; Stomatal density; Triticum durum; Water status
1. Introduction Durum wheat is probably one of the oldest cultivated plants in the world. This species is mainly grown in the Mediterranean region under
* Corresponding author. E-mail address: [email protected] (O. Merah).
rainfed conditions without irrigation (Baldy, 1986). In these areas, where the annual range of precipitation varies between 200 and 900 mm, plants are often exposed to periods of water deficit that have a negative impact on leaf gas exchange, growth and yield (Baldy, 1986; Araus et al. 1997). Therefore, drought tolerance improvement of durum wheat cultivars is a major objective for all breeders in Mediterranean countries. Several morpho-physiological traits have been
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proposed as screening criteria for drought tolerance, such as relative water content (RWC) and osmotic potential (OP) (Gummurulu et al., 1989) and stomatal parameters (Venora and Calcagno, 1991). Transpiration efficiency (TE) (dry matter produced/water transpired) is an interesting attribute for growth in dry areas. The use of carbon isotope discrimination (D) as a related criterion affords an easy way of screening for TE. The negative relationship between D and TE was established firstly by Farquhar and Richards (1984) and confirmed for numerous species by several authors, who then proposed D as an indirect selection criterion for TE (Condon et al., 1987; Ehdaie and Waines, 1993; Acevedo, 1993). However, selection for high TE may favour genotypes with low production under water deficit, since high D was found frequently associated with high grain yield (GY) (Acevedo, 1993; Ehdaie and Waines, 1993; Morgan et al., 1993; Sayre et al., 1995; Araus et al., 1997, 1998; Merah et al., 1999a). Few studies have approached the relationship between flag leaf D and harvest index (HI). Otherwise, there are limited insights into relationships between D and morpho-physiological traits under water stress, especially under field conditions. The realised studies mostly focused on the effect of stomatal conductance or leaf structure on D (Condon et al., 1987; Morgan et al., 1993; Poorter and Farquhar, 1994; Araus et al., 1997; Geber and Dawson, 1997; Fischer et al., 1998). Conversely, relationships between D, plant water status, residual transpiration (RT) and stomatal parameters are undocumented in cereals. The aims of this study were then (1) to describe the variation of flag leaf D depicting the species variability, using a collection of 144 durum wheat accessions (2) to analyse and discuss the relationships between D and several flag leaf morphophysiological traits, and (3) to examine relationships between D, GY and HI under water limited field conditions. The present study was designed to examine the dependence of D variation on water status differences under field Mediterranean conditions. We hypothesised that D could be a good predictor of plant water status. We also attempted to relate D to other leaf characters, such as stomatal density (SD), RT and
specific leaf dry weight (SLDW). These relationships, which were scarcely documented in cereals, could clarify the interdependence between these traits. Associations between D and both SD and RT may exist through relationship between D and stomatal conductance.
2. Materials and methods
2.1. Plant material About 144 wheat accessions (Triticum durum Desf.) originating from 18 countries and constituting the Durum Wheat Core Collection (DWCC) of International Centre for Agricultural Research in Dry Areas were (ICARDA) used. This collection included landraces (66), improved varieties (53) and ICARDA/ Centro Internacional para Mejoramiento de Maiz Y Trigo (CIMMYT) advanced lines (25).
2.2. Site and crop management The trial was carried out in Montpellier (South of France) in 1994/1995. The soil is sandy-loam with a depth of about 0.6 m and a low water retention capacity. A randomised complete block design was used with two replicates per accession. In each plot, seeds were sown in four rows — 1.50 m long with a row space of 25 cm apart and inter-plant space of 3 cm. The sowing was done on 4th November, 1994, anthesis took place between mid April and the first week of May 1995 and maturity occurred at the end of June 1995.
2.3. Water a6ailability during the plant growth cycle Cumulative rainfall (R) during the plant growth cycle (November –June) was 285 mm, which was quite low for this region. The Penman evapotranspiration (PET) was 486 mm during the same period, i.e. 170% higher than rainfall, suggesting that water stress occurred during this plant growth cycle. Moreover, the rainfall was unequally distributed along the plant cycle. More than 65% of the total rainfall occurred during the
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first 3 months of the cropping cycle with a R/PET ratio of 5.06, which exceeds the crop demand during this period. The plant growth cycle was characterised by a drought period from February until the end of the plant cycle (R/PET decreased strongly to 0.26). A strong terminal water stress occurred during grain filling stage. Indeed, only 31 mm of rainfall were registered in May and June, whereas PET attained 266 mm (R/PET = 0.13). Monthly detailed informations on rainfall, PET, temperatures, radiation and air relative humidity are reported elsewhere by Merah et al. (1999b).
2.4. Measurements 2.4.1. Flag leaf traits The RT was measured according to Clarke et al. (1991). At anthesis (at pollen ripeness for the majority of flowers), four flag leaves per accession were excised and immediately brought to the laboratory. Then, they remained for half an hour under ambient room conditions. They were weighed (W1, in g) after this period and again 180 min later (W2, in g); the flag leaf area (LA) (in cm2) was determined using an area meter (LI3000, Li-Cor, Lambda Instruments Co., USA). RT (g H2O cm − 2 min − 1 ×10 − 5) was determined as: RT =
W1 − W2 . LA ×180
RWC(%) =
then transferred at − 20°C. OP of the cell sap (expressed in MPa), extracted by pressing the total leaf tissue in a syringe, was measured with a micro-osmometer (Ro¨ebling 16R.1, Berlin, Germany). OP at full turgor (i.e. 100% RWC) was calculated according to Wilson et al. (1979) as: OP100 = OP ×
FW − DW ×100. TW − DW
The OP was determined on four other excised flag leaves, immediately frozen in liquid nitrogen,
RWC − B , 100− B
where B (the apoplasmic water content) was considered as a constant (15% for durum wheat) as reported by Gaudillie`re and Barcelo (1990). The SD was evaluated using flag leaf prints made in the middle part of the adaxial surface by using a Formvar-chloroform mixture. After drying at room temperature (22°C), the film was peeled off and mounted on a slide by means of an adhesive film. For each accession, photographs of four microscopic fields were used to determine the numbers of stomata per unit area (SD). The flag leaf D and carbon content (CC) were measured on 20 flag leaves randomly detached at anthesis for each accession and immediately ovendried for 48 h at 80°C. The dry leaf samples were ground to a fine powder. CC and carbon isotope composition (d13C‰) were determined with an isotope mass spectrometer Optima (Micromass, Villeurbane, France) coupled to the elemental analyser (Carlo-Erba, Thermoquest, Courtaboeuf, France), as: l 13C(%) =
The flag leaf DW was obtained after oven drying at 80°C during 48 h. The SLDW was calculated as SLDW=DW/LA. The RWC was measured on four other flag leaves. The fresh weight (FW) was measured immediately after excision, the full turgid weight (TW) after the rehydration of the leaves by placing them in a test tube containing distilled water for 24 h at 4°C in darkness, and the DW after oven drying at 80°C during 48 h. The RWC was calculated from the following equation:
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n
R sample − 1 × 1000, R reference
R being 13C/12C ratio. The discrimination (D) was then calculated using the following formulae (Farquhar and Richards, 1984), D=
la− lp 1+ lp
where lp is the d13C of the leaves and la, the d13C of the atmospheric CO2, − 8‰.
2.4.2. Plant phenology and agronomic traits The number of days from sowing to heading (HD) was recorded, when 50% of the plants (for a given genotype) were at this stage. At maturity, GY per plant and HI were also recorded by weighing the bulked harvested seeds.
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2.5. Statistical analysis All the data were subjected to an analysis of variance using the GLM procedure of SAS (SAS Institute, 1987, Cary, NC, USA). Correlation analysis was performed to determine the relationships between the traits using the SAS CORR procedure. Power of the regression functions and the 95% limits were also calculated. Adjusted means (subtracting of HD effect) were also calculated by using the number of days from sowing to HD as a covariate.
3. Results The analysis of variance has revealed significant differences among genotypes for all the measured traits (Table 1). The difference between extreme genotypes for D was 3.4‰. The leaf CC also exhibited a large range of variation. SLDW and LA varied three-fold, whereas SD and RT varied two-fold (Table 1). Flag leaf RWC ranged from 72 to 91%, OP from −3.0 to −1.5 MPa, whereas OP at full turgor (OP100) ranged from − 2.7 to − 1.3 MPa. GY varied from 0.76 to 4.13 g per
plant. HI also showed a large difference within the studied collection. In this study, the number of days from sowing to HD differed by more than 2 weeks between extreme genotypes (Trinakria, Cannizara and Sicilia Lutri three Italian landraces are the earliest genotypes, whereas Barba de Lobo, Santa Marta from Portugal and Rescio Raspinegro from Spain are the latest ones). These differences may result in D and GY variations. An analysis of variance, where HD was used as covariate, was performed in order to test, if HD could be at the origin of D variation. No significant HD effect on D was noted. Even when HD effect was significant on SLDW and GY, differences between genotypes remained highly significant (Table 1). Adjusted means were generated by fixing the HD effect. Therefore, it appears that the genotypic variability in durum wheat for D and the measured traits is not attributable only to difference in phenology. A significant negative relationship was found between D and leaf CC (Table 2). This correlation improved (r= − 0.35, P B 0.001) after subtracting the effect of HD on the two traits. No significant correlation was noted between D and either SLDW or LA (Table 2). Significant positive rela-
Table 1 Minimum, maximum, mean and standard error (S.E.) values of all parameters determined for flag leaves and whole plants measured on 144 grown-field durum wheat accessions in water limited rainfed conditions at Montpellier during the growing season of 1994/1995a Traits
D (‰) CC (mg g−1 DW) SD (number mm−2) RT (g H2O min−1 cm−2×10−5) RWC (%) OP (MPa) OP100 (Mpa) SLDW (g m−2) Flag LA (cm2) Days to HD (d) HI GY (g per plant) a
Minimum
16.5 380 66.9 2.11 71.9 −3.05 −2.46 38.5 7.5 121 0.14 0.76
Maximum
19.9 457 117.7 5.83 91.0 −1.50 −1.32 99.5 24.2 141 0.47 4.13
Mean
18.2 431 90.0 3.65 84.0 −2.21 −1.78 63.8 15.0 129 0.28 1.92
S.E
0.7 12.8 9.7 0.58 3.3 0.32 0.22 13.1 3.1 5.8 0.05 0.64
Source Genotype (d.f. = 143)
HD (d.f. = 1)
*** ** *** *** *** *** *** *** *** *** *** ***
ns ns ns ns ns ns ns ** ns -ns *
Asterisks indicate the results of variance analysis, where the genotype effect is significant, ** P50.01; *** P50.001. Number of days from sowing to HD was taken as a covariate. Asterisks indicate where HD effect is significant *PB0.05, ** P50.01, and ns indicate a not significant HD effect. The degrees of freedom (d.f.) of genotype and HD effects are also displayed.
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Table 2 Correlation between flag leaf D and some flag leaf morphophysiological traitsa
Flag leaf D
CC
SD
RWC
OP
OP100
SLDW
LA
−0.27**
-0.38***
0.31***
0.52***
0.49***
0.16
0.15
a All traits were measured on flag leaf (with four replicates) of a collection of 144 grown-field durum wheat genotypes cultivated under rainfed condition at Montpellier during the growing season of 1994/95. CC, carbon content; SD, stomatal density; RWC, relative water content; OP, osmotic potential; OP100, osmotic potential at full turgor; SLDW, specific leaf dry weight; LA leaf area. Asterisks indicate the significance of the correlation, ** P50.01; *** P50.001.
tions were noticed between D and both RWC and OP. Plants with higher OP100 showed lower TE according to the positive correlation found between D and OP100 (Table 2). D and SD were negatively related (Fig. 1), suggesting that leaves having the highest stomatal limitation at anthesis (low D) presented a high SD and conversely. A significant positive correlation was found between RT and D (Fig. 2). Leaf D and both HI and GY were positively related (Fig. 3).
photosynthetic capacity (Condon et al. 1987). Otherwise, SLDW is currently considered as an indicator of leaf thickness and photosynthesis capacity (Araus et al., 1997). The no significant relationship found between D and SLDW under our conditions allows to postulate that D variation results, as previously postulated by Morgan et al. (1993), Geber and Dawson (1997) and Fischer et al. (1998), from differences in stomatal conductance, rather than from photosynthetic capacity differences.
4. Discussion
4.1. Relationship between Z, CC, LA and SLDW A negative relationship was found between D and CC (Table 2). This result, which is in accordance with those of Barthes et al. (1994), Araus et al. (1998) and Merah et al. (1999a), express that, when a plant is able to accumulate more dry matter per water supply unit (low D), the mineral accumulation is low and CC consequently high. However, the biological relevance of this relation is low according to the coefficient of determination (r 2 = 0.123). No significant correlation was noted between D and either SLDW and LA (Table 2). Similarly, neither LA nor SLDW were significantly related to water use efficiency (WUE) in durum wheat cultivated under controlled conditions (Gummurulu et al., 1989). A weak or no correlation was found between D and SLDW by Poorter and Farquhar (1994) and Araus et al. (1997) under stressed conditions. The variation of D depends on differences in stomatal conductance and/or
Fig. 1. Relationship between flag leaf D and SD after subtracting the flag LA effect on SD in a collection of 144 durum wheat genotypes grown at Montpellier. Solid line is the fitted linear regression line. Dashed lines represent the 95% confidence limits.
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carbon isotope composition and water availability in large plant communities and consider, thus, the 13 C natural abundance as a biological integrator of plant water status. Similarly, D may provide a useful tool for the plant water status characterisation in durum wheat under rainfed Mediterranean conditions according to the positive correlation observed between this trait and water status parameters.
Fig. 2. Relationship between flag leaf D and RT in a collection of 144 durum wheat genotypes grown at Montpellier. Solid line is the fitted linear regression line. Dashed lines represent the 95% confidence limits.
4.2. Relationship between Z and water status The broad range of variation of water status observed in our study is comparable with those found by Gummurulu et al. (1989) and Venora and Calcagno (1991) in durum wheat varieties grown under controlled conditions. Significant positive relations were noticed between D and RWC, OP and OP100 (Table 2). Differences in water status parameters may result from differences in leaf structure. Indeed, Rascio et al. (1990) reported a correlation between leaf structure and OP in durum wheat. They hypothesised that an increase in cell wall thickness and a reduction of OP contribute to turgor maintenance, when the leaf water potential decreases. In our study, however, no significant correlations were noted between SLDW and water status parameters, suggesting that these traits are not interdependant. Moreover, the positive correlation registered between OP100 and RWC (r= 0.435, PB 0.001) values could be the consequence of tissue dehydration, which agrees with the positive relation between D and RWC (Table 2). Stewart et al. (1995) found correlations between
Fig. 3. Relationships between flag leaf D and either GY (a) and HI (b) after correcting for the number of days from sowing to HD effect on GY in a collection of 144 durum wheat genotypes grown at Montpellier. Solid lines are the fitted linear regression line. Dashed lines represent the 95% confidence limits.
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4.3. Relationship between Z, SD and RT A negative correlation was found between D and SD (Table 2). The variation of SD may be influenced by leaf size, as suggested by the significant correlation noted between LA and SD (r= − 0.18, P B 0.05). However, the correlation between D and SD remained significant after subtracting LA effect on SD (Fig. 1). This result was unexpected, since the relationship between D and SD was scarcely documented. Morgan et al. (1993) did not find a significant correlation between SD and D in bread wheat, but the range of genotypic variability for these traits in their experiment was low. Stomatal regulation, rather than stomata distribution, could probably be responsible for the gas exchange under water limitation and, thus, could influence WUE and D (Venora and Calcagno, 1991; El Hafid et al., 1998). These authors, however, did not examine in details the relationships between SD and either D or stomatal conductance. The negative correlation observed in our experiment between SD and D needs, then, to be further investigated precisely, if a causal relationship exists between these traits. A significant positive correlation was found between RT and D (Fig. 2), which is in agreement with the negative correlation between WUE and RT noted by Clarke et al. (1991) under water stressed conditions. Moreover, leaves with numerous stomata tended to have lower RT than leaves with low SD (r = − 0.25, P B0.01). This association may indicate a possible SD component of RT due to the association between stomatal conductance and SD (Miskin et al. 1972). Our data do not allow to determine which, from the stomatal or the cuticular components of the RT, are responsible for the negative relationship between SD and RT. The correlation observed between leaf D (which reflects the stomatal conductance until anthesis) and RT may suggest that stomatal transpiration could constitute an important component of RT.
4.4. Relationships between Z, GY and HI Positive correlations were found between D and both HI and GY (Fig. 3). In cereals, it is known
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that higher TE (and thus lower D) is frequently associated with a low GY (Condon et al., 1987; Acevedo, 1993; Morgan et al., 1993; Sayre et al., 1995; Araus et al., 1997; Merah et al., 1999a, 2000). Moreover, D correlated better with HI than with GY, suggesting that higher D values also reflect higher efficiency of carbon partitioning to the kernel, which results in higher GY (Ehdaie and Waines, 1993). The positive correlation between flag leaf D and both HI and GY emphasises the dependence of these three traits on stomatal conductance (Morgan et al., 1993; Fischer et al., 1998). Merah et al. (1999a) reported that plants that transpire more per unit of dry matter produced (high D) have higher ash concentrations in leaf dry matter and higher GY resulting from greater carbon partitioning efficiency to the kernel.
5. Conclusion Previous field and glasshouse studies showed a large D variation in cereals (Condon et al., 1987; Acevedo, 1993; Ehdaie and Waines, 1993; Morgan et al., 1993; Sayre et al., 1995; Araus et al., 1997, 1998; Merah et al., 1999a; Watanabe et al., 2000). Our work also has shown a wide genetic variability for flag leaf D existed within the large durum wheat collection studied. The flag leaf D was positively correlated with both GY and HI; higher D value results in higher efficiency of carbon partitioning to the kernel, which leads to higher yield. This result confirms the potential interest of D for selecting for GY under Mediterranean conditions. The analysis of relationships between D and the studied morpho-physiological traits showed some intriguing correlations. Unexpectedly, D was correlated negatively with SD and positively with RT. Further studies are, however, needed to find precisely which from the stomatal or cuticular component of the RT is mainly responsible for this association. The positive correlation observed between D and RWC, OP and OP100 within the studied collection of durum wheat emphasises the dependence of D on water status parameters. Thus, D may represent a good indicator of plant water status. These results,
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which need to be confirmed in other experiments, could lead to a better understanding of physiological functioning under drought conditions.
Acknowledgements Merah O. was supported by a French-Algerian fellowship. Financial support for this study was provided by the French Ministry of Foreign Affairs through the ENSAM-INRA/UPS/ICARDA joint program ‘Biotechnology and Durum Wheat Breeding’. Thanks to I. Souyris (INRA, Montpellier), O. Roche and R. Boyer (IBP, Universite´ de Paris-Sud) for technical help.
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Relationships between flag leaf carbon isotope discrimination and several morpho-physiological traits in durum wheat genotypes under Mediterranean conditions Othmane Merah a,b,*, P. Monneveux b, E. Dele´ens a a
Institut de Biotechnologie des Plantes, UMR 8618, Bat 630, Uni6ersite´ de Paris-Sud, Centre d’Orsay, 91405 Orsay Cedex, France b UFR de Ge´ne´tique et Ame´lioration des Plantes, ENSA-INRA, 2 Place Viala, 34060 Montpellier Cedex, France Received 23 June 2000; received in revised form 9 October 2000; accepted 10 October 2000
Abstract Relationships between flag leaf carbon isotope discrimination (D), water status parameters, residual transpiration (RT) and stomatal density (SD) were examined on a collection of 144 durum wheat accessions. Associations between D, grain yield (GY) and harvest index (HI) were also studied. The field trial was conducted under Mediterranean conditions. The crop cycle was characterised by a period of drought from February until maturity. A broad range of values we obtained for D (16.5–19.9‰) and other physiological traits. Flag leaf D was positively and significantly correlated with both HI and GY. D was better correlated with HI than with GY, which suggests that higher D values indicate higher efficiency of carbon partitioning to the kernel, leading to higher GY. D was found positively related with RT and negatively related with SD. This relationship may indicate a possible SD component of RT due to the association between conductance and SD. Strong positive correlations were found between D and water status parameters, suggesting that D may provide a good indication of plant water status in durum wheat under rainfed Mediterranean conditions. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Drought tolerance; Harvest index; Residual transpiration; Stomatal density; Triticum durum; Water status
1. Introduction Durum wheat is probably one of the oldest cultivated plants in the world. This species is mainly grown in the Mediterranean region under
* Corresponding author. E-mail address: [email protected] (O. Merah).
rainfed conditions without irrigation (Baldy, 1986). In these areas, where the annual range of precipitation varies between 200 and 900 mm, plants are often exposed to periods of water deficit that have a negative impact on leaf gas exchange, growth and yield (Baldy, 1986; Araus et al. 1997). Therefore, drought tolerance improvement of durum wheat cultivars is a major objective for all breeders in Mediterranean countries. Several morpho-physiological traits have been
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proposed as screening criteria for drought tolerance, such as relative water content (RWC) and osmotic potential (OP) (Gummurulu et al., 1989) and stomatal parameters (Venora and Calcagno, 1991). Transpiration efficiency (TE) (dry matter produced/water transpired) is an interesting attribute for growth in dry areas. The use of carbon isotope discrimination (D) as a related criterion affords an easy way of screening for TE. The negative relationship between D and TE was established firstly by Farquhar and Richards (1984) and confirmed for numerous species by several authors, who then proposed D as an indirect selection criterion for TE (Condon et al., 1987; Ehdaie and Waines, 1993; Acevedo, 1993). However, selection for high TE may favour genotypes with low production under water deficit, since high D was found frequently associated with high grain yield (GY) (Acevedo, 1993; Ehdaie and Waines, 1993; Morgan et al., 1993; Sayre et al., 1995; Araus et al., 1997, 1998; Merah et al., 1999a). Few studies have approached the relationship between flag leaf D and harvest index (HI). Otherwise, there are limited insights into relationships between D and morpho-physiological traits under water stress, especially under field conditions. The realised studies mostly focused on the effect of stomatal conductance or leaf structure on D (Condon et al., 1987; Morgan et al., 1993; Poorter and Farquhar, 1994; Araus et al., 1997; Geber and Dawson, 1997; Fischer et al., 1998). Conversely, relationships between D, plant water status, residual transpiration (RT) and stomatal parameters are undocumented in cereals. The aims of this study were then (1) to describe the variation of flag leaf D depicting the species variability, using a collection of 144 durum wheat accessions (2) to analyse and discuss the relationships between D and several flag leaf morphophysiological traits, and (3) to examine relationships between D, GY and HI under water limited field conditions. The present study was designed to examine the dependence of D variation on water status differences under field Mediterranean conditions. We hypothesised that D could be a good predictor of plant water status. We also attempted to relate D to other leaf characters, such as stomatal density (SD), RT and
specific leaf dry weight (SLDW). These relationships, which were scarcely documented in cereals, could clarify the interdependence between these traits. Associations between D and both SD and RT may exist through relationship between D and stomatal conductance.
2. Materials and methods
2.1. Plant material About 144 wheat accessions (Triticum durum Desf.) originating from 18 countries and constituting the Durum Wheat Core Collection (DWCC) of International Centre for Agricultural Research in Dry Areas were (ICARDA) used. This collection included landraces (66), improved varieties (53) and ICARDA/ Centro Internacional para Mejoramiento de Maiz Y Trigo (CIMMYT) advanced lines (25).
2.2. Site and crop management The trial was carried out in Montpellier (South of France) in 1994/1995. The soil is sandy-loam with a depth of about 0.6 m and a low water retention capacity. A randomised complete block design was used with two replicates per accession. In each plot, seeds were sown in four rows — 1.50 m long with a row space of 25 cm apart and inter-plant space of 3 cm. The sowing was done on 4th November, 1994, anthesis took place between mid April and the first week of May 1995 and maturity occurred at the end of June 1995.
2.3. Water a6ailability during the plant growth cycle Cumulative rainfall (R) during the plant growth cycle (November –June) was 285 mm, which was quite low for this region. The Penman evapotranspiration (PET) was 486 mm during the same period, i.e. 170% higher than rainfall, suggesting that water stress occurred during this plant growth cycle. Moreover, the rainfall was unequally distributed along the plant cycle. More than 65% of the total rainfall occurred during the
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first 3 months of the cropping cycle with a R/PET ratio of 5.06, which exceeds the crop demand during this period. The plant growth cycle was characterised by a drought period from February until the end of the plant cycle (R/PET decreased strongly to 0.26). A strong terminal water stress occurred during grain filling stage. Indeed, only 31 mm of rainfall were registered in May and June, whereas PET attained 266 mm (R/PET = 0.13). Monthly detailed informations on rainfall, PET, temperatures, radiation and air relative humidity are reported elsewhere by Merah et al. (1999b).
2.4. Measurements 2.4.1. Flag leaf traits The RT was measured according to Clarke et al. (1991). At anthesis (at pollen ripeness for the majority of flowers), four flag leaves per accession were excised and immediately brought to the laboratory. Then, they remained for half an hour under ambient room conditions. They were weighed (W1, in g) after this period and again 180 min later (W2, in g); the flag leaf area (LA) (in cm2) was determined using an area meter (LI3000, Li-Cor, Lambda Instruments Co., USA). RT (g H2O cm − 2 min − 1 ×10 − 5) was determined as: RT =
W1 − W2 . LA ×180
RWC(%) =
then transferred at − 20°C. OP of the cell sap (expressed in MPa), extracted by pressing the total leaf tissue in a syringe, was measured with a micro-osmometer (Ro¨ebling 16R.1, Berlin, Germany). OP at full turgor (i.e. 100% RWC) was calculated according to Wilson et al. (1979) as: OP100 = OP ×
FW − DW ×100. TW − DW
The OP was determined on four other excised flag leaves, immediately frozen in liquid nitrogen,
RWC − B , 100− B
where B (the apoplasmic water content) was considered as a constant (15% for durum wheat) as reported by Gaudillie`re and Barcelo (1990). The SD was evaluated using flag leaf prints made in the middle part of the adaxial surface by using a Formvar-chloroform mixture. After drying at room temperature (22°C), the film was peeled off and mounted on a slide by means of an adhesive film. For each accession, photographs of four microscopic fields were used to determine the numbers of stomata per unit area (SD). The flag leaf D and carbon content (CC) were measured on 20 flag leaves randomly detached at anthesis for each accession and immediately ovendried for 48 h at 80°C. The dry leaf samples were ground to a fine powder. CC and carbon isotope composition (d13C‰) were determined with an isotope mass spectrometer Optima (Micromass, Villeurbane, France) coupled to the elemental analyser (Carlo-Erba, Thermoquest, Courtaboeuf, France), as: l 13C(%) =
The flag leaf DW was obtained after oven drying at 80°C during 48 h. The SLDW was calculated as SLDW=DW/LA. The RWC was measured on four other flag leaves. The fresh weight (FW) was measured immediately after excision, the full turgid weight (TW) after the rehydration of the leaves by placing them in a test tube containing distilled water for 24 h at 4°C in darkness, and the DW after oven drying at 80°C during 48 h. The RWC was calculated from the following equation:
65
n
R sample − 1 × 1000, R reference
R being 13C/12C ratio. The discrimination (D) was then calculated using the following formulae (Farquhar and Richards, 1984), D=
la− lp 1+ lp
where lp is the d13C of the leaves and la, the d13C of the atmospheric CO2, − 8‰.
2.4.2. Plant phenology and agronomic traits The number of days from sowing to heading (HD) was recorded, when 50% of the plants (for a given genotype) were at this stage. At maturity, GY per plant and HI were also recorded by weighing the bulked harvested seeds.
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2.5. Statistical analysis All the data were subjected to an analysis of variance using the GLM procedure of SAS (SAS Institute, 1987, Cary, NC, USA). Correlation analysis was performed to determine the relationships between the traits using the SAS CORR procedure. Power of the regression functions and the 95% limits were also calculated. Adjusted means (subtracting of HD effect) were also calculated by using the number of days from sowing to HD as a covariate.
3. Results The analysis of variance has revealed significant differences among genotypes for all the measured traits (Table 1). The difference between extreme genotypes for D was 3.4‰. The leaf CC also exhibited a large range of variation. SLDW and LA varied three-fold, whereas SD and RT varied two-fold (Table 1). Flag leaf RWC ranged from 72 to 91%, OP from −3.0 to −1.5 MPa, whereas OP at full turgor (OP100) ranged from − 2.7 to − 1.3 MPa. GY varied from 0.76 to 4.13 g per
plant. HI also showed a large difference within the studied collection. In this study, the number of days from sowing to HD differed by more than 2 weeks between extreme genotypes (Trinakria, Cannizara and Sicilia Lutri three Italian landraces are the earliest genotypes, whereas Barba de Lobo, Santa Marta from Portugal and Rescio Raspinegro from Spain are the latest ones). These differences may result in D and GY variations. An analysis of variance, where HD was used as covariate, was performed in order to test, if HD could be at the origin of D variation. No significant HD effect on D was noted. Even when HD effect was significant on SLDW and GY, differences between genotypes remained highly significant (Table 1). Adjusted means were generated by fixing the HD effect. Therefore, it appears that the genotypic variability in durum wheat for D and the measured traits is not attributable only to difference in phenology. A significant negative relationship was found between D and leaf CC (Table 2). This correlation improved (r= − 0.35, P B 0.001) after subtracting the effect of HD on the two traits. No significant correlation was noted between D and either SLDW or LA (Table 2). Significant positive rela-
Table 1 Minimum, maximum, mean and standard error (S.E.) values of all parameters determined for flag leaves and whole plants measured on 144 grown-field durum wheat accessions in water limited rainfed conditions at Montpellier during the growing season of 1994/1995a Traits
D (‰) CC (mg g−1 DW) SD (number mm−2) RT (g H2O min−1 cm−2×10−5) RWC (%) OP (MPa) OP100 (Mpa) SLDW (g m−2) Flag LA (cm2) Days to HD (d) HI GY (g per plant) a
Minimum
16.5 380 66.9 2.11 71.9 −3.05 −2.46 38.5 7.5 121 0.14 0.76
Maximum
19.9 457 117.7 5.83 91.0 −1.50 −1.32 99.5 24.2 141 0.47 4.13
Mean
18.2 431 90.0 3.65 84.0 −2.21 −1.78 63.8 15.0 129 0.28 1.92
S.E
0.7 12.8 9.7 0.58 3.3 0.32 0.22 13.1 3.1 5.8 0.05 0.64
Source Genotype (d.f. = 143)
HD (d.f. = 1)
*** ** *** *** *** *** *** *** *** *** *** ***
ns ns ns ns ns ns ns ** ns -ns *
Asterisks indicate the results of variance analysis, where the genotype effect is significant, ** P50.01; *** P50.001. Number of days from sowing to HD was taken as a covariate. Asterisks indicate where HD effect is significant *PB0.05, ** P50.01, and ns indicate a not significant HD effect. The degrees of freedom (d.f.) of genotype and HD effects are also displayed.
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Table 2 Correlation between flag leaf D and some flag leaf morphophysiological traitsa
Flag leaf D
CC
SD
RWC
OP
OP100
SLDW
LA
−0.27**
-0.38***
0.31***
0.52***
0.49***
0.16
0.15
a All traits were measured on flag leaf (with four replicates) of a collection of 144 grown-field durum wheat genotypes cultivated under rainfed condition at Montpellier during the growing season of 1994/95. CC, carbon content; SD, stomatal density; RWC, relative water content; OP, osmotic potential; OP100, osmotic potential at full turgor; SLDW, specific leaf dry weight; LA leaf area. Asterisks indicate the significance of the correlation, ** P50.01; *** P50.001.
tions were noticed between D and both RWC and OP. Plants with higher OP100 showed lower TE according to the positive correlation found between D and OP100 (Table 2). D and SD were negatively related (Fig. 1), suggesting that leaves having the highest stomatal limitation at anthesis (low D) presented a high SD and conversely. A significant positive correlation was found between RT and D (Fig. 2). Leaf D and both HI and GY were positively related (Fig. 3).
photosynthetic capacity (Condon et al. 1987). Otherwise, SLDW is currently considered as an indicator of leaf thickness and photosynthesis capacity (Araus et al., 1997). The no significant relationship found between D and SLDW under our conditions allows to postulate that D variation results, as previously postulated by Morgan et al. (1993), Geber and Dawson (1997) and Fischer et al. (1998), from differences in stomatal conductance, rather than from photosynthetic capacity differences.
4. Discussion
4.1. Relationship between Z, CC, LA and SLDW A negative relationship was found between D and CC (Table 2). This result, which is in accordance with those of Barthes et al. (1994), Araus et al. (1998) and Merah et al. (1999a), express that, when a plant is able to accumulate more dry matter per water supply unit (low D), the mineral accumulation is low and CC consequently high. However, the biological relevance of this relation is low according to the coefficient of determination (r 2 = 0.123). No significant correlation was noted between D and either SLDW and LA (Table 2). Similarly, neither LA nor SLDW were significantly related to water use efficiency (WUE) in durum wheat cultivated under controlled conditions (Gummurulu et al., 1989). A weak or no correlation was found between D and SLDW by Poorter and Farquhar (1994) and Araus et al. (1997) under stressed conditions. The variation of D depends on differences in stomatal conductance and/or
Fig. 1. Relationship between flag leaf D and SD after subtracting the flag LA effect on SD in a collection of 144 durum wheat genotypes grown at Montpellier. Solid line is the fitted linear regression line. Dashed lines represent the 95% confidence limits.
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carbon isotope composition and water availability in large plant communities and consider, thus, the 13 C natural abundance as a biological integrator of plant water status. Similarly, D may provide a useful tool for the plant water status characterisation in durum wheat under rainfed Mediterranean conditions according to the positive correlation observed between this trait and water status parameters.
Fig. 2. Relationship between flag leaf D and RT in a collection of 144 durum wheat genotypes grown at Montpellier. Solid line is the fitted linear regression line. Dashed lines represent the 95% confidence limits.
4.2. Relationship between Z and water status The broad range of variation of water status observed in our study is comparable with those found by Gummurulu et al. (1989) and Venora and Calcagno (1991) in durum wheat varieties grown under controlled conditions. Significant positive relations were noticed between D and RWC, OP and OP100 (Table 2). Differences in water status parameters may result from differences in leaf structure. Indeed, Rascio et al. (1990) reported a correlation between leaf structure and OP in durum wheat. They hypothesised that an increase in cell wall thickness and a reduction of OP contribute to turgor maintenance, when the leaf water potential decreases. In our study, however, no significant correlations were noted between SLDW and water status parameters, suggesting that these traits are not interdependant. Moreover, the positive correlation registered between OP100 and RWC (r= 0.435, PB 0.001) values could be the consequence of tissue dehydration, which agrees with the positive relation between D and RWC (Table 2). Stewart et al. (1995) found correlations between
Fig. 3. Relationships between flag leaf D and either GY (a) and HI (b) after correcting for the number of days from sowing to HD effect on GY in a collection of 144 durum wheat genotypes grown at Montpellier. Solid lines are the fitted linear regression line. Dashed lines represent the 95% confidence limits.
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4.3. Relationship between Z, SD and RT A negative correlation was found between D and SD (Table 2). The variation of SD may be influenced by leaf size, as suggested by the significant correlation noted between LA and SD (r= − 0.18, P B 0.05). However, the correlation between D and SD remained significant after subtracting LA effect on SD (Fig. 1). This result was unexpected, since the relationship between D and SD was scarcely documented. Morgan et al. (1993) did not find a significant correlation between SD and D in bread wheat, but the range of genotypic variability for these traits in their experiment was low. Stomatal regulation, rather than stomata distribution, could probably be responsible for the gas exchange under water limitation and, thus, could influence WUE and D (Venora and Calcagno, 1991; El Hafid et al., 1998). These authors, however, did not examine in details the relationships between SD and either D or stomatal conductance. The negative correlation observed in our experiment between SD and D needs, then, to be further investigated precisely, if a causal relationship exists between these traits. A significant positive correlation was found between RT and D (Fig. 2), which is in agreement with the negative correlation between WUE and RT noted by Clarke et al. (1991) under water stressed conditions. Moreover, leaves with numerous stomata tended to have lower RT than leaves with low SD (r = − 0.25, P B0.01). This association may indicate a possible SD component of RT due to the association between stomatal conductance and SD (Miskin et al. 1972). Our data do not allow to determine which, from the stomatal or the cuticular components of the RT, are responsible for the negative relationship between SD and RT. The correlation observed between leaf D (which reflects the stomatal conductance until anthesis) and RT may suggest that stomatal transpiration could constitute an important component of RT.
4.4. Relationships between Z, GY and HI Positive correlations were found between D and both HI and GY (Fig. 3). In cereals, it is known
69
that higher TE (and thus lower D) is frequently associated with a low GY (Condon et al., 1987; Acevedo, 1993; Morgan et al., 1993; Sayre et al., 1995; Araus et al., 1997; Merah et al., 1999a, 2000). Moreover, D correlated better with HI than with GY, suggesting that higher D values also reflect higher efficiency of carbon partitioning to the kernel, which results in higher GY (Ehdaie and Waines, 1993). The positive correlation between flag leaf D and both HI and GY emphasises the dependence of these three traits on stomatal conductance (Morgan et al., 1993; Fischer et al., 1998). Merah et al. (1999a) reported that plants that transpire more per unit of dry matter produced (high D) have higher ash concentrations in leaf dry matter and higher GY resulting from greater carbon partitioning efficiency to the kernel.
5. Conclusion Previous field and glasshouse studies showed a large D variation in cereals (Condon et al., 1987; Acevedo, 1993; Ehdaie and Waines, 1993; Morgan et al., 1993; Sayre et al., 1995; Araus et al., 1997, 1998; Merah et al., 1999a; Watanabe et al., 2000). Our work also has shown a wide genetic variability for flag leaf D existed within the large durum wheat collection studied. The flag leaf D was positively correlated with both GY and HI; higher D value results in higher efficiency of carbon partitioning to the kernel, which leads to higher yield. This result confirms the potential interest of D for selecting for GY under Mediterranean conditions. The analysis of relationships between D and the studied morpho-physiological traits showed some intriguing correlations. Unexpectedly, D was correlated negatively with SD and positively with RT. Further studies are, however, needed to find precisely which from the stomatal or cuticular component of the RT is mainly responsible for this association. The positive correlation observed between D and RWC, OP and OP100 within the studied collection of durum wheat emphasises the dependence of D on water status parameters. Thus, D may represent a good indicator of plant water status. These results,
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which need to be confirmed in other experiments, could lead to a better understanding of physiological functioning under drought conditions.
Acknowledgements Merah O. was supported by a French-Algerian fellowship. Financial support for this study was provided by the French Ministry of Foreign Affairs through the ENSAM-INRA/UPS/ICARDA joint program ‘Biotechnology and Durum Wheat Breeding’. Thanks to I. Souyris (INRA, Montpellier), O. Roche and R. Boyer (IBP, Universite´ de Paris-Sud) for technical help.
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