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.     

Partitioning of reserve and newly assimilated carbon in roots and leaf tissues of Lolium perenne during regrowth after defoliation: assessment by 13C steady-state labelling and carbohydrate analysis

The relative significance of the use of stored or currently absorbed C for the growth of leaves or roots of Lolium perenne L. after defoliation was assessed by steady-state labelling of atmospheric CO₂. Leaf growth for the first two days after defoliation was to a large extent dependent on the use of C reserves. The basal part of the elongating leaves was mainly new tissue and 91% of the C in this part of the leaf was derived from reserves assimilated prior to defoliation. However, half of the sucrose in the growth zone was produced from photosynthesis by the emerged leaves. Fructans that were initially present in elongating leaf bases were hydrolysed (loss of 93 to 100%) and the resulting fructose was found in the new leaf bases, suggesting that this pool may be used to support cell division and elongation. Despite a negative C balance at the whole-plant level, fructans were synthesized from sucrose that was translocated to the new leaf bases. After a regrowth period of 28 d, 45% of the C fixed before defoliation was still present in the root and leaf tissue and only 1% was incorporated in entirely new tissue.     

Kinetics and relative significance of remobilized and current C and N incorporation in leaf and root growth zones of Lolium perenne after defoliation: assessment by 13C and 15N steady-state labelling

The contribution of pre-defoliation reserves and current assimilates to leaf and root growth was examined in Lolium perenne L. during regrowth after defoliation. Differential steady-state labelling with ¹³C (CO₂ with δ¹³C = -0.0281 and -0.0088) and ¹⁵N (NO₃^? with 1.0 and 0.368 atom percentage, i.e. δ¹⁵N = 1.742 and 0.0052, respectively) was applied for 2 weeks after defoliation. Rapidly growing tissues were isolated, i.e. the basal elongation and maturation zones of the most rapidly expanding leaves and young root tips, with a biomass turnover rate > 1 d^?1. C and N weights of the elongation zone showed a transient decline. The dry matter and C concentration in fresh biomass of leaf growth zones transiently decreased by up to 25% 2 d after defoliation, while the N concentration remained constant. This ‘dilution’ of growth zone C indicates a decreased net influx of carbohydrates relative to growth-related influx of water and N in expanding cells, immediately after defoliation. Recovery of the total C and N weights of the leaf elongation zone coincided with net incorporation of currently absorbed C and N, as shown by the kinetics of δ¹³C and atom percentage ¹⁵N in the growth zones after defoliation. C isotope discrimination (Δ¹³C) in leaf growth zones was about 23‰, 1–2‰ higher than the Δ in root tips. Δ¹⁵N in the leaf and root growth zones was 10±3‰. The leaf elongation zones (at 0–0.03 m from the tiller base) and the distant root tips (about 0.2 m from the base) exhibited similar kinetics of current C and N incorporation. The amount of pre-defoliation C and N in the growth zones, expressed as a fraction of total C and N, decreased from 1.0 to 0.5 at 3 (C) and 5 (N) d after defoliation, and to 0.1 at 5 (C) and 14 (N) d after defoliation. Thus, the dependence of growth zones on current assimilate supply was significant, and stronger for C than for N. The important roles of current assimilates (as compared to pre-defoliation reserves) and ‘dilution’ of dry matter in regrowth after defoliation are discussed in relation to the method of labelling and the functional and morphological heterogeneity of shoot tissues.     

Genetic parameters and QTL analysis of δ13C and ring width in maritime pine

Classical quantitative genetics and quantitative trait dissection analysis (QTL) approaches were used in order to investigate the genetic determinism of wood cellulose carbon isotope composition (δ¹³C, a time integrated estimate of water use efficiency) and of diameter growth and their relationship on adult trees (15 years) of a forest tree species (maritime pine). A half diallel experimental set‐up was used to (1) estimate heritabilities for δ¹³C and ring width and (2) to decompose the phenotypic δ¹³C/growth correlation into its genetic and environmental components. Considerable variation was found for δ¹³C (range of over 3‰) and for ring width (range of over 5 mm) and significant heritabilities (narrow sense 0·17/0·19 for δ¹³C and ring width, respectively, 100% additivity). The significant phenotypic correlation between δ¹³C and ring width was not determined by the genetic component, but was attributable to environmental components. Using a genetic linkage map of a full‐sib family, four significant and four suggestive QTLs were detected for δ¹³C, the first for δ¹³C in a forest tree species, as far as known to the authors. Two significant and four suggestive QTLs were found for ring width. No co‐location of QTLs was found between δ¹³C and growth.     

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.     

Incomplete 13C labelling of α-pinene content of Quercus ilex leaves and appearance of unlabelled C in α-pinene emission in the dark

α ‐Pinene is formed in and emitted by Quercus ilex leaves. The carbon emitted as α ‐pinene is rapidly and totally labelled by ¹³C in CO₂ in air, but α ‐pinene contained in the leaf shows a fraction of completely unlabelled carbon even after long exposures to air containing only ¹³CO₂. When the labelled leaf is darkened, α ‐pinene emission drops but is still measurable for about 10 h, and carbon becomes partially unlabelled. After an 11 h darkening the α ‐pinene content is still as high as in the light but the carbon is mostly unlabelled. If the leaf is re‐illuminated but photosynthesis is inhibited by removing CO₂ and lowering O₂, a burst of emission occurs and the content of α ‐pinene is depleted. Our experiments suggest that a pool of α ‐pinene which is not directly generated by photosynthesis intermediates exists. Since this pool does not contribute relevantly to the emission in the light, we hypothesize that it is inhibited in the light and spatially located in a different compartment from chloroplasts. As we discuss, glycolysis in the cytoplasm and leucine catabolism in the mitochondria are both possible extra‐chloroplasts sources of carbon for isoprenoids.     

Fructan contents and dry matter deposition in different tissues of the wheat grain during development

The role of fructan metabolism in the assimilate relations of the grain of wheat (Triticum aestivum L.) was investigated by determination of the dry matter and fructan content of grain components at short intervals during grain filling. During the initial phase of rapid expansion, most of the assimilates entering the grain were partitioned to the outer pericarp. A large fraction of these assimilates were used for the synthesis of fructan. Dry matter deposition and fructan synthesis in the outer pericarp ceased at about 5d after anthesis. At the same time, the endosperm and the inner pericarp and testa started to accumulate dry matter at a fast rate. This was also associated with significant fructan synthesis in the latter tissues. The outer pericarp lost about 45% of its former maximum dry weight between 9 and 19 d after anthesis. This loss was due almost entirely to the near complete disappearance of water-soluble carbohydrates, most of which was fructan. The inner pericarp and testa accumulated dry matter until about mid-grain filling. The fructan contents of the inner pericarp and testa and the endosperm decreased slowly towards the end of grain filling. Most of the fructans in the inner pericarp and testa and the endosperm had a low molecular weight, whereas higher molecular weight fructans predominated in the outer pericarp. The embryo did not contain fructan. The presence of low molecular weight fructans in the endosperm cavity at mid-grain filling was confirmed. It is suggested that fructan synthesis is closely linked to growth-related water deposition in the different tissues of the wheat grain and serves to sequester the surplus of imported sucrose.     

Ear photosynthesis, carbon isotope discrimination and the contribution of respiratory CO2 to differences in grain mass in durum wheat

The role of ear photosynthesis in grain filling was studied in a number of durum wheat (Triticum turgidum var durum L.) landraces and varieties from the Middle East, North Africa, and from the collections of ‘Institut National de la Recherche Agronomique’ (INRA, France) and ‘Centro International de Mejora de Maiz y Trigo’ (CIMMYT, Mexico). Plants were grown in the field in a Mediterranean climate. Flag leaves (blade plus sheath) and ears were kept in the dark from 1 week after anthesis to maturity which reduced grain weight by 22.4% and 59.0%, respectively. In a further experiment, the carbon isotope discrimination ratio (Δ) of ear bracts, awns and flag leaves was measured on samples taken at anthesis and on mature kernels. The mean value of Δ for the water soluble fraction of bracts (17.0‰) and awns (17.7‰) were lower than those of leaves (19.5‰) and fairly similar to those of kernels (17.4‰) averaged across all genotypes. Data indicate that most of the photosynthates in the grain come from ear parts and not from flag leaves. In addition, a higher water use efficiency (WUE) of ear parts than of the flag leaf is suggested by their lower Δ values. Gas exchange in ears and flag leaves was measured during grain filling. Averaged over all genotypes, CO₂ diffusive conductance was about five times higher in the flag leaf than in the spike (with distal portions of awns outside the photosynthetic chamber) 2 weeks after anthesis. In absolute terms, the dark respiration rate (R_d) was greater than the net photosynthesis rate (P_n) by a factor of 1.74 in the spike, whereas R_d was much smaller, only 22.1, 65.7 and 24.8% of P_n in blade, sheath and awns, respectively. Data indicate that photosynthesis, and hence the water use efficiency (photosynthesis/transpiration), is greatly underestimated in ears because of the high rates of respiration which diminish the measured rates of net CO₂ exchange. Results of ¹³C discrimination and gas exchange show that genotypes from North Africa have higher WUE than those from the Middle East. The high R_d values of ears as well as their low diffusive conductance suggest that CO₂ from respiration may be used as source of carbon for ear photosynthesis. In the same way, the anatomy of glumes, for example, supports the role of bracts using internal CO₂ as source of photosynthesis. In the first experiment, the Δ in mature grains from culms with darkened ears compared with control culms provided further evidence in support of this hypothesis. Thus, the Δ from kernels of control plants was 0.40 higher than that from ear-darkened plants, probably because of some degree of refixation (recycling) of respired CO₂ in the grains.     

Regulation of growth, water use efficiency and δ13C by the nitrogen source in Casuarina equisetifolia Forst. & Forst.

Carbon isotope composition (δ¹³C) was measured in a glasshouse experiment with N₂-fixing and NO₃^–- or NH₄⁺-fed Casuarina equisetifolia Forst. & Forst plants, both under well-watered and drought conditions. The abundance of ¹³C was higher (more positive δ¹³C) for NH₄⁺- than for NO₃^– -grown plants and was lowest for N₂-fixing plants. NH₄⁺-fed plants had more leaf area and dry weight and higher water use efficiency (on a biomass basis) than N₂- and NO₃^–-grown plants and had lower water consumption than plants supplied with NO₃^–, either with high or low water supply. Specific leaf areas and leaf area ratios were higher with NH₄⁺ than with NO₃^– or N₂ as the N source. The difference observed in δ¹³C between plants grown with different N sources was higher than that predicted by theory and was not in the right direction (NH₄⁺-grown plants with a more negative δ¹³C) to be explained by differences in plant composition and engagement of the various carboxylation reactions. The more positive δ¹³C in NH₄⁺- than in NO₃^–-grown plants is probably due to a decreased ratio of stomatal to carboxylation conductances, which accounts for the lower water cost of C assimilation in NH₄⁺-grown plants.