Effect of leaf structure and water status on carbon isotope discrimination in field-grown durum wheat

The relationships between leaf and kernel carbon isotope discriminations (Δ) and several leaf structural parameters that are indicators of photosynthetic capacity were studied in durum wheat grown in the field under three water regimens. A set of 144 genotypes were cultivated in two rain-fed trials, and 125 of these were grown under supplementary irrigation before heading. Total chlorophyll and nitrogen (N) contents, the dry mass per unit leaf area (LDM, the reciprocal of specific leaf area) and carbon isotope discrimination (Δ) were measured in penultimate leaves and Δ of mature kernels was also analysed. Both LDM and N per unit area showed significant (P≤ 0.001) negative correlation (r=–0.60 and r=–0.36, respectively) with leaf Δ in the wettest trial. Little or no correlation was found for any structural parameter and leaf Δ in the rain-fed trials. In contrast, in the two rain-fed trials LDM was the parameter with the strongest positive correlation (P≤ 0.001) with kernel Δ (r= 0.47 and 0.30) and grain yield (r= 0.43 and 0.29), whereas no correlation was found in the irrigation trial. These correlations, rather than representing a causal link between the amount of photosynthetic tissue and Δ, were probably indirect associations caused by a parallel effect of water status and phenology on leaf structure, grain Δ and yield. Correlations across trials (i.e. environments) between leaf structure and either Δ and yield were very high, although also spurious. Our results suggest that LDM should be used to cull segregating population differences in leaf Δ based on the internal photosynthetic capacity only in the absence of drought. Selecting for kernel Δ and grain yield on the basis of LDM is worthwhile for rain-fed trials.     

Contribution of the ear and the fl ag leaf to grain fi lling in durum wheat inferred from the carbon isotope signature: Genotypic and growing conditions eff ects FA

The ear, together with the flag leaf, is believed to play a major role as a source of assimilates during grain filling in C3cereals. However, the intrusive nature of most of the available methodologies prevents reaching conclusive results in this regard. This study compares the carbon isotope composition（d13C） in its natural abundance in the water‐soluble fractions of the flag leaf blade and the ear with the d13C of mature kernels to assess the relative contribution of both organs to grain filling in durum wheat（Triticum turgidum L. var.durum）. The relative contribution of the ear was higher in landraces compared to modern cultivars, as well as in response to nitrogen fertilization and water stress. Such genotypic and environmentally driven differences were associated with changes in harvest index（HI）, with the relative contribution of the ear being negatively associated with HI. In the case of the genotypic differences, the lower relative contribution of the ear in modern cultivars compared with landraces is probably associated with the appearance in the former of a certain amount of source limitation driven by a higher HI. In fact, the relative contribution of the ear was far more responsive to changes in HI in modern cultivars compared with landraces.     

Contribution of the ear and the flag leaf to grainfilling in durum wheat inferred from the carbon isotope signature: Genotypic and growing conditions effects

The ear, together with the flag leaf, is believed to play a major role as a source of assimilates during grain filling in C3cereals. However, the intrusive nature of most of the available methodologies prevents reaching conclusive results in this regard. This study compares the carbon isotope composition(d13C) in its natural abundance in the water‐soluble fractions of the flag leaf blade and the ear with the d13C of mature kernels to assess the relative contribution of both organs to grain filling in durum wheat(Triticum turgidum L. var.durum). The relative contribution of the ear was higher in landraces compared to modern cultivars, as well as in response to nitrogen fertilization and water stress. Such genotypic and environmentally driven differences were associated with changes in harvest index(HI), with the relative contribution of the ear being negatively associated with HI. In the case of the genotypic differences, the lower relative contribution of the ear in modern cultivars compared with landraces is probably associated with the appearance in the former of a certain amount of source limitation driven by a higher HI. In fact, the relative contribution of the ear was far more responsive to changes in HI in modern cultivars compared with landraces.     

The combined effect of constant water deficit and nitrogen supply on WUE, NUE and Δ13C in durum wheat potted plants

Water scarcity and nitrogen shortage are the main constraints on durum wheat productivity. This paper examines the combined effects of a constant water deficit and nitrogen supply (NS) on growth, photosynthesis, stomatal conductance (g_s) and transpiration, instantaneous and time‐integrated water use efficiency (WUE) and nitrogen use efficiency (NUE) and carbon isotope discrimination (Δ¹³C) in durum wheat genotypes grown in pots under greenhouse conditions. Three water levels (40%, 70% and 100% container capacity), two nitrogen doses (high and low N) and four genotypes were assayed in a total of 24 experimental treatments. Water and nitrogen treatments were imposed 2 weeks after plant emergence. The growth, nitrogen content and Δ¹³C of the shoot and the gas exchange in the flag leaf were determined about 2 weeks after anthesis. As expected, both water and NS had a strong positive effect on growth. However, a reduction in water supply had low effect decreasing photosynthesis and transpiration, Δ¹³C and NUE and increasing WUE. On the contrary, increasing the level of nitrogen supplied had a significant negative effect on g_s, which decreased significantly the ratio of intercellular to ambient CO₂ concentrations and Δ¹³C, and increased both instantaneous and time‐integrated WUE. In addition, a higher N level also negatively affected the instantaneous and time‐integrated NUE. The Δ¹³C of shoots correlated significantly and negatively with either instantaneous or time‐integrated measurements of WUE. Moreover, within each NS, Δ¹³C also correlated negatively with the integrated NUE. We concluded that under our experimental conditions, Δ¹³C gives information about the efficiency with which not just water but also nitrogen are used by the plant. In addition, this study illustrates that a steady water limitation may strongly affect biomass without consistent changes in WUE. The lack of effect of the different water regimes on gas exchange, WUE and Δ¹³C illustrate the importance of how stress is imposed during growth.     

Epidermal conductance in different parts of durum wheat grown under Mediterranean conditions: the role of epicuticular waxes and stomata

Abstract. Epidermal (non-stomatally-controlled) conductance from the fourth leaf, first node leaf, flag leaf and ear of durum wheat (Triticum turgidum var durum L.) grown under Mediterranean field conditions has been measured, along with leaf stomatal frequency and the amount and distribution of epicuticular waxes. Measurements were carried out on varieties and land-races from the Middle East, North Africa, ‘Institut National de la Recherche Agricole’ (INRA) and ‘Centra Internacional de Mejora de Maiz y Trigo’ (CIMMYT). Significant differences were observed among genotypes in the epidermal conductances (g_e) of the four organs. For each of the four organs tested, genotypes from the Middle East and CIMMYT showed higher g_e. values than those from North Africa and INRA. Ears showed epidermal conductances that were more than four times higher than those of leaves when g_e. values were expressed per unit dry weight. The amount of epicuticular waxes was higher in the fourth leaves, intermediate in the first node and flag leaves and lower in the ears. For each organ, g_e differences among genotypes were unrelated with the amount of epicuticular waxes. Removal of epicuticular waxes by dipping the organs into chloroform significantly increased the epidermal conductance for the fourth and first node leaves and the ear. However, this did not occur for the flag leaf. For the fourth leaf, g_e of intact leaves and g_e of leaves in which epicuticular waxes were removed were unrelated (r = -0.265). The regression coefficient of this relation for the first node and flag leaves showed values of 0.666 and 0.650 (P > 0.05), respectively, and values were even higher in the ear (r > m 0.892, P > 0.01). Scanning electron microscope analysis showed that wax bloom decreased from the fourth leaf to the flag leaf, whereas the extent of amorphous wax increased. Wax bloom in leaves consisted mainly of deposits of thin wax plates. In the ears and the adaxial surface of flag leaves, fibrillar waxes predominated. In the first node and flag leaves, the wax deposits on the adaxial side cover the surface of the leaf more densely and uniformly than those on the abaxial side. There was no significant correlation between g_e and total stomatal density, or between g_e and either adaxial or abaxial stomatal density for any sample of the three different leaves. The contribution of epicuticular waxes plus total stomatal frequency only explained 42.4, 11.8, 28.3 and 16% of g_e (per unit leaf area) variations for the fourth leaf, first node leaf, flag leaf and the combined variation of the three leaves together, respectively. From these results, it is concluded that complex interrelationship between different morphophysiological characteristics probably control g_e differences among genotypes and that these interrelationships differ for each different plant part.     

Relationships between carbon isotope discrimination, dry matter production, and harvest index in durum wheat

Carbon isotope discrimination has been proposed as a criterion for the indirect selection to improve transpiration efficiency and grain yield in bread wheat and barley. Less attention has been devoted to durumwheat (Triticum durumDesf.) despite its economic importance in the Mediterranean basin. The Δ genetic variation and its relationships to dry matter production and harvest index in durum wheat were investigated in this study. For this purpose, field experiments were conducted on 144 durum wheat accessions under Mediterranean conditions (South of France) during three consecutive years with contrasting climatic conditions. Grain yield, above-ground biomass, harvest index, and carbon isotope discrimination of flag leaf and kernel were measured. Differences between years, noted for both leaf and kernel carbon isotope discrimination, were probably related to the variation in water availability from year to year. A large genotypic variation was also noticed for both leaf and kernel carbon isotope discrimination. The two traits were found to be positively correlated with grain yield within and across years, which confirms the interest in carbon isotope discrimination for selection for grain yield improvement under Mediterranean conditions. Both kernel and leaf carbon isotope discrimination correlated better with harvest index than with grain yield, suggesting that carbon isotope discrimination could reflect the efficiency of carbon partitioning to the kernel. The lack of correlation between leaf carbon isotope discrimination and both harvest index and grain yield in favourable water conditions (1996) was probably due to the difference in water availability between the period until flag leaves sampling (favourable conditions) and the strong water stress which accompanied the grain filling. Kernel carbon isotope discrimination correlated better with both harvest index and grain yield than did leaf carbon isotope discrimination. Moreover, a higher broad-sense heritability was obtained for kernel carbon isotope discrimination than for leaf carbon isotope discrimination. As a result, kernel carbon isotope discrimination appeared to be a better predictive criterion for efficiency of the carbon partitioning to the kernel (harvest index), and hence for grain yield, than did flag leaf carbon isotope discrimination.     

The effect of growth at elevated CO2 concentrations on photosynthesis in wheat

Rising levels of atmospheric CO₂ will have profound, direct effects on plant carbon metabolism. In this study we used gas exchange measurements, models describing the instantaneous response of leaf net CO₂ assimilation rate (A) to intercellular CO₂ partial pressure (C_i), in vitro enzyme activity assay, and carbohydrate assay in order to investigate the photosynthetic responses of wheat (Triticum aestivum L., cv. Wembley) to growth under elevated partial pressures of atmospheric CO₂ (C_a). At flag leaf ligule emergence, the modelled, in vivo, maximum carboxylation velocity for RuBisCO was significantly lower in plants grown at elevated C_a than in plants grown at ambient C_a (70 Pa compared with 40 Pa). By 12 d after ligule emergence, no significant difference in this parameter was detectable. At ligule emergence, plants grown at elevated C_a exhibited reduced in vitro initial activities and activation states of RuBisCO. At their respective growth C_i values, the photosynthesis of 40-Pa-grown plants was sensitive to p(O₂) and to p(CO₂) whereas that of 70-Pa-grown plants was insensitive. Both sucrose and starch accumulated more rapidly in the leaves of plants grown at 70 Pa. At flag leaf ligule emergence, modelled non-photorespiratory respiration in the light (R_d) was significantly higher in 70-Pa-grown plants than in 40-Pa-grown plants. By 12 d after ligule emergence no significant differences in R_d were detectable.     

The utilization of pre-anthesis reserves in grain filling of wheat. Assessment by steady-state 13CO2/12CO2 labelling

δ, C isotope composition relative to Pee Dee Belemnite
WSC, water-soluble carbohydrates
N, nitrogen
C, carbon
cv, cultivar
ME, efficiency of mobilized pre-anthesis C utilization in grain filling (g C g^–1C)

Significant mobilization of protein and carbohydrates in vegetative plant parts of wheat regularly occurs during grain filling. While this suggests a contribution of reserves to grain filling, the actual efficiency of mobilized assimilate conversion into grain mass (ME) is unknown. In the present study the contribution of pre-anthesis C (C fixed prior to anthesis) to grain filling in main stem ears of two spring wheat (Triticum aestivum L.) cultivars was determined by ¹³C/¹²C steady-state labelling. Mobilization of pre-anthesis C in vegetative plant parts between anthesis and maturity, and the contributions of water-soluble carbohydrates (WSC) and protein to pre-anthesis C mobilization were also assessed. Experiments were performed with two levels of N fertilizer supply in each of 2 years. Pre-anthesis reserves contributed 11–29% to the total mass of C in grains at maturity. Pre-anthesis C accumulation in grains was dependent on both the mass of pre-anthesis C mobilized in above-ground vegetative plant parts (r² = 0·87) and ME (defined as g pre-anthesis C deposited in grains per g pre-anthesis C mobilized in above-ground vegetative plant parts; r² = 0·40). ME varied between 0·48 and 0·75. The effects of years, N fertilizer treatments and cultivars on ME were all related to differences in the fractional contribution of WSC to pre-anthesis C mobilization. Multiple regression analysis indicated that C from mobilized pre-anthesis WSC may be used more efficiently in grain filling than C present in proteins at anthesis and mobilized during grain filling. Possible causes for variability of ME are discussed.     

Water use efficiency in C3 cereals under Mediterranean conditions: a review of physiological aspects

In this review, we will discuss physiological traits of C₃ cereals related to water use efficiency (WUE) in Mediterranean environments, from leaf (WUE_{instantaneous}) to crop level (WUE_yield or ‘water productivity’). First, we analyse the WUE_{instantaneous} and the possible trade‐off between improving this parameter and growth/yield performance. Ways to ameliorate WUE without penalties are discussed. We also analyse in what cases breeding by high or low WUE_{instantaneous} is a suitable criterion to maintain grain yield under drought (Mediterranean) conditions. This question is approached in the framework of carbon isotope discrimination, (Δ¹³C), the main indirect parameter used to integrate (at time and space scale) the WUE_{instantaneous} in C₃ plants. A negative correlation between these two parameters has been confirmed by several studies. The relationship between Δ¹³C and grain yield, however, is more complex, and may differ from one environment to another. In Mediterranean conditions with moderate or no water stress, a positive correlation between Δ¹³C and grain yield is found in barley and wheat, whereas in ‘stored‐water’ crops (such as in some regions of Australia), lower Δ¹³C (i.e. higher WUE_{instantaneous}) is associated with higher grain yield, particularly in more stressful conditions. These apparent inconsistencies and their possible implications for plant breeding are discussed. One physiological trait that has received minor attention in attempts to improve WUE_{instantaneous} is the role of ear photosynthesis. Ears of barley and durum wheat have a higher WUE_{instantaneous} than the flag leaf, both in well‐watered and in drought conditions. The underlying causes of the higher WUE_{instantaneous} of ears are not fully understood, but their refixation capacity (i.e. the capacity to re‐assimilate respired carbon dioxide) could be important. Although the genotypic variability of this trait has not been extensively studied, some data support the idea that variation in refixation capacity may be attributable to genetic factors. At the crop level, decreasing soil evaporation is a crucial factor in efforts to improve the WUE_yield in Mediterranean conditions, and fast initial growth of the crop (i.e. early vigour) seems to be relevant. In wheat, modern varieties with dwarfing genes (giberellic acid – insensitive) have higher yields but, concomitantly, they have lower initial growth performance. Recently, semi‐dwarf cultivars (giberellic acid – sensitive) with high grain yield and simultaneously high early vigour were found, opening new avenues to increase WUE_yield in wheat. The negative effects of futile water loss by cuticular and nocturnal transpiration are also commented. Finally, we discuss some agronomic practices (in particular, ‘deficit irrigation’ systems) linked to physiological traits that confer higher WUE_yield,, in particular, in the cases of Mediterranean regions.     

Gas exchange and carbon isotope composition of Ananas comosus in response to elevated CO2 and temperature

Ananas comosus L. (Merr.) (pineapple) was grown at three day/night temperatures and 350 (ambient) and 700 (elevated) μ mol mol^–1 CO₂ to examine the interactive effects of these factors on leaf gas exchange and stable carbon isotope discrimination ( Δ ,‰). All data were collected on the youngest mature leaf for 24 h every 6 weeks. CO₂ uptake (mmol m^–2 d^–1) at ambient and elevated CO₂, respectively, were 306 and 352 at 30/20 °C, 175 and 346 at 30/25 °C and 187 and 343 at 35/25 °C. CO₂ enrichment enhanced CO₂ uptake substantially in the day in all environments. Uptake at night at elevated CO₂, relative to that at ambient CO₂, was unchanged at 30/20 °C, but was 80% higher at 30/25 °C and 44% higher at 35/25 °C suggesting that phosphoenolpyruvate carboxylase was not CO₂-saturated at ambient CO₂ levels and a 25 °C night temperature. Photosynthetic water use efficiency (WUE) was higher at elevated than at ambient CO₂. Leaf Δ -values were higher at elevated than at ambient CO₂ due to relatively higher assimilation in the light. Leaf Δ was significantly and linearly related to the fraction of total CO₂ assimilated at night. The data suggest that a simultaneous increase in CO₂ level and temperature associated with global warming would enhance carbon assimilation, increase WUE, and reduce the temperature dependence of CO₂ uptake by A. comosus .     

Elevated concentrations of atmospheric CO2 protect against and compensate for O3 damage to photosynthetic tissues of field-grown wheat

The effects of elevated concentrations of atmospheric carbon dioxide and ozone on diurnal patterns of photosynthesis have been investigated in field-grown spring wheat ( Triticum aestivum ). Plants cultivated under realistic agronomic conditions, in open-top chambers, were exposed from emergence to harvest to reciprocal combinations of two carbon dioxide and two ozone treatments: [CO₂] at ambient (380 μmol mol⁻¹, seasonal mean) or elevated (692 μmol mol⁻¹) levels, [O₃] at ambient (27 nmol mol⁻¹, 7 hr seasonal mean) or elevated (61 nmol mol⁻¹) levels. After anthesis, diurnal measurements were made of flag-leaf gas-exchange and in vitro Rubisco activity and content. Elevated [CO₂] resulted in an increase in photoassimilation rate and a loss of excess Rubisco activity. Elevated [O₃] caused a loss of Rubisco and a decline in photoassimilation rate late in flag-leaf development. Elevated [CO₂] ameliorated O₃ damage. The mechanisms of amelioration included a protective stomatal restriction of O₃ flux to the mesophyll, and a compensatory effect of increased substrate on photoassimilation and photosynthetic control. However, the degree of protection and compensation appeared to be affected by the natural seasonal and diurnal variations in light, temperature and water status.     

Changes in leaf Δ13C of herbarium plant species during the last 3 centuries of CO2 increase

Δ¹³C were determined for herbarium specimens of 12 C₃ plants (trees, shrubs and herbs) collected during the last 240 years in Catalonia, an area with a Mediterranean climate. Values were 19.91 (S.E. = 0.32, n= 21) for 1750–1760, 19–86 (S.E. = 0.21, n= 49) for 1850–1890 and 19.95 (S.E. = 0.29, n= 25) for 1925–1950, and decreased significantly to 18.87 (S.E. = 0.31, n= 29) for 1982–1988. More irregular temporal changes were found in Δ¹³C of two C₄ species, but they also suggest a decrease in discrimination in recent decades. These results suggest that either carbon assimilation rates have increased or stomatal conductance has decreased, and therefore, that there has been an increase in water use efficiency over the last few decades.     

CO2 transfer conductance, leaf structure and carbon isotope composition of Polygonum cuspidatum leaves from low and high altitudes

Anatomy and some physiological characteristics of the leaves in Polygonum cuspidatum Sieb. et Zucc., a dioecious clonal herb, were compared between two populations, one from a lowland in Shizuoka City (10 m above sea level), and another from a highland on Mt. Fuji (2500 m above sea level). Leaf mass per area (LMA) of the highland plants was about twice that of the lowland plants. The greater leaf thickness, thicker mesophyll cell walls and higher mesophyll cell density in the highland leaves contributed to the larger LMA. Although mesophyll area exposed to intercellular airspaces was greater in the highland leaves than in the lowland leaves by 30%, the surface area of chloroplasts facing intercellular airspaces was similar between these leaves. CO₂ transfer conductance inside the leaf (g_i) of the highland leaves (0·75 μmol m^?2 s^?1 Pa^?1) is the lowest recorded for herbaceous plants and was only 40% of that in the lowland leaves. On the other hand, the difference in stomatal conductance was small. δ¹³C values in the leaf dry matter were greater in the highland leaves by 4‰. These data and the estimation of CO₂ partial pressures in the intercellular air spaces and in the chloroplast suggested that the greater dry matter δ¹³C in the highland leaves, indicative of lower long‐term ratio of the chloroplast stroma to the ambient CO₂ partial pressures, would be mainly attributed to their lower g_i.