Maria Dolores Serret

Combined use of δ13C, δ18O and δ15N tracks nitrogen metabolism and genotypic adaptation of durum wheat to salinity and water deficit

• Accurate phenotyping remains a bottleneck in breeding for salinity and drought resistance. Here the combined use of stable isotope compositions of carbon (δ¹³C), oxygen (δ¹⁸O) and nitrogen (δ¹⁵N) in dry matter is aimed at assessing genotypic responses of durum wheat under different combinations of these stresses. • Two tolerant and two susceptible genotypes to salinity were grown under five combinations of salinity and irrigation regimes. Plant biomass, δ¹³C, δ¹⁸O and δ¹⁵N, gas-exchange parameters, ion and N concentrations, and nitrate reductase (NR) and glutamine synthetase (GS) activities were measured. • Stresses significantly affected all traits studied. However, only δ¹³C, δ¹⁸O, δ¹⁵N, GS and NR activities, and N concentration allowed for clear differentiation between tolerant and susceptible genotypes. Further, a conceptual model explaining differences in biomass based on such traits was developed for each growing condition. • Differences in acclimation responses among durum wheat genotypes under different stress treatments were associated with δ¹³C. However, except for the most severe stress, δ¹³C did not have a direct (negative) relationship to biomass, being mediated through factors affecting δ¹⁸O or N metabolism. Based upon these results, the key role of N metabolism in durum wheat adaptation to salinity and water stress is highlighted.

Comparative performance of δ13C, δ18O and δ15N for phenotyping durum wheat adaptation to a dryland environment

Grain yield and the natural abundance of the stable isotope compositions of carbon (δ13C), oxygen (δ18O) and nitrogen (δ15N) of mature kernels were measured during 3 consecutive years in 10 durum wheat genotypes (five landraces and five modern cultivars) subjected to different water and N availabilities in a Mediterranean location and encompassing a total of 12 trials. Water limitation was the main environmental factor affecting yield, δ13C and δ18O, whereas N fertilisation had a major effect on δ15N. The genotypic effect was significant for yield, yield components, δ13C, δ18O and δ15N. Landraces exhibited a higher δ13C and δ15N than cultivars. Phenotypic correlations of δ13C and δ18O with grain yield were negative, suggesting that genotypes able to sustain a higher water use and stomatal conductance were the most productive and best adapted; δ15N was also negatively correlated with grain yield regardless of the growing conditions. δ13C was the best isotopic trait in terms of genetic correlation with yield and heritability, whereas δ18O was the worst of the three isotopic abundances. The physiological basis for the different performance of the three isotopes explaining the genotypic variability in yield is discussed.

Shoot δ15N gives a better indication than ion concentration or Δ13C of genotypic differences in the response of durum wheat to salinity

We compared the performance of different physiological traits that reveal genotypic variations in tolerance to salinity in durum wheat. A set of 114 genotypes was grown in hydroponics for over 3 months. Three conditions: control, moderate (12 dS m-1) and severe (17 dS m-1) salinity, were maintained for nearly 8 weeks before harvest. The genotype biomass in control conditions correlated with the biomass at the two salinity levels. Subsequently, two subsets of 10 genotypes each were selected on the basis of extreme differences in biomass at the two salinity levels while showing relatively similar biomass in control conditions. Carbon isotope discrimination (Δ13C), nitrogen isotope composition (δ15N), and the concentration of nitrogen, phosphorus and several ions (K+, Na+, Ca2+, Mg2+) were analysed in the two subsets for the three treatments. At 12 dS m-1, K+ concentration, K+/Na+ ratio, Δ13C and δ15N correlated positively and Na+ correlated negatively with shoot biomass. Under control conditions and at 17 dS m-1 no correlation was observed. However, the trait that correlated best with genotypic differences in biomass was δ15N at 12 dS m-1. This trait was the first variable chosen at each of the two salinity levels in a stepwise analysis. We consider the possible mechanisms relating δ15N to biomass and the use of this isotopic signature as a selection trait.

Effect of salinity and water stress during the reproductive stage on growth, ion concentrations, Δ13C, and δ15N of durum wheat and related amphiploids

The physiological performance of durum wheat and two related amphiploids was studied during the reproductive stage under different combinations of salinity and irrigation. One triticale, one tritordeum, and four durum wheat genotypes were grown in pots in the absence of stress until heading, when six different treatments were imposed progressively. Treatments resulted from the combination of two irrigation regimes (100% and 35% of container water capacity) with three levels of water salinity (1.8, 12, and 17 dS m(-1)), and were maintained for nearly 3 weeks. Gas exchange and chlorophyll fluorescence and content were measured prior to harvest; afterwards shoot biomass and height were recorded, and Delta(13)C, delta(15)N, and the concentration of nitrogen (N), phosphorus, and several ions (K(+), Na(+), Ca(2+), Mg(2+)) were analysed in shoot material. Compared with control conditions (full irrigation with Hoagland normal) all other treatments inhibited photosynthesis through stomatal closure, accelerated senescence, and decreased biomass. Full irrigation with 12 dS m(-1) outperformed other stress treatments in terms of biomass production and physiological performance. Biomass correlated positively with N and delta(15)N, and negatively with Na(+) across genotypes and fully irrigated treatments, while relationships across deficit irrigation conditions were weaker or absent. Delta(13)C did not correlate with biomass across treatments, but it was the best trait correlating with phenotypic differences in biomass within treatments. Tritordeum produced more biomass than durum wheat in all treatments. Its low Delta(13)C and high K(+)/Na(+) ratio, together with a high potential growth, may underlie this finding. Mechanisms relating delta(15)N and Delta(13)C to biomass are discussed.

Comparative response of δ13C, δ18O and δ15N in durum wheat exposed to salinity at the vegetative and reproductive stages.

This study compared the performance of the stable isotope composition of carbon (δ(13) C), oxygen (δ(18) O) and nitrogen (δ(15) N) by tracking plant response and genotypic variability of durum wheat to different salinity conditions. To that end, δ(13) C, δ(18) O and δ(15) N were analysed in dry matter (dm) and the water-soluble fraction (wsf) of leaves from plants exposed to salinity, either soon after plant emergence or at anthesis. The δ(13) C and δ(18) O of the wsf recorded the recent growing conditions, including changes in evaporative conditions. Regardless of the plant part (dm or wsf), δ(13) C and δ(18) O increased and δ(15) N decreased in response to stress. When the stress conditions were established just after emergence, δ(15) N and δ(13) C correlated positively with genotypic differences in biomass, whereas δ(18) O correlated negatively in the most severe treatment. When the stress conditions were imposed at anthesis, relationships between the three isotope signatures and biomass were only significant and positive within the most severe treatments. The results show that nitrogen metabolism, together with stomatal limitation, is involved in the genotypic response to salinity, with the relative importance of each factor depending on the severity and duration of the stress as well as the phenological stage that the stress occurs.

Root traits and δ13C and δ18O of durum wheat under different water regimes

Plant growth, root characteristics and the stable carbon (δ13C) and oxygen (δ18O) composition were studied in durum wheat. Four recombinant inbred lines with good agronomic adaptation were grown under well watered (WW) and water stress (WS) conditions until mid-grain filling in lysimeters. Gas exchange was measured in the flag leaf just before harvest and then the aerial dry matter (Aerial DM), root weight density (RWD) and root length density (RLD) and the specific root length (SRL) were evaluated and the δ13C and δ18O of the roots, the flag leaf blade and the spike were analysed. Water stress decreased stomatal conductance, plant accumulated transpiration and Aerial DM, whereas δ13C and δ18O increased. Genotypic differences were found for all gas-exchange and root traits and isotope signatures. Aerial DM was positively correlated with RLD, regardless of the water regime, whereas it was negatively correlated with δ13C and δ18O, but only under WW conditions. Moreover, RWD and RLD were negatively related to both δ13C and δ18O under the WW regime, but no clear pattern existed under WS. Our study supports the use of δ13C and δ18O as proxies for selecting root traits associated with higher growth in the absence of water stress.

Gene expression and physiological responses to salinity and water stress of contrasting durum wheat genotypes

Elucidating the relationships between gene expression and the physiological mechanisms remains a bottleneck in breeding for resistance to salinity and drought. This study related the expression of key target genes with the physiological performance of durum wheat under different combinations of salinity and irrigation. The candidate genes assayed included two encoding for the DREB (dehydration responsive element binding) transcription factors TaDREB1A and TaDREB2B, another two for the cytosolic and plastidic glutamine synthetase (TaGS1 and TaGS2), and one for the specific Na(+) /H(+) vacuolar antiporter (TaNHX1). Expression of these genes was related to growth and different trait indicators of nitrogen metabolism (nitrogen content, stable nitrogen isotope composition, and glutamine synthetase and nitrate reductase activities), photosynthetic carbon metabolism (stable carbon isotope composition and different gas exchange traits) and ion accumulation. Significant interaction between genotype and growing conditions occurred for growth, nitrogen content, and the expression of most genes. In general terms, higher expression of TaGS1, TaGS2, TaDREB2B, and to a lesser extent of TaNHX1 were associated with a better genotypic performance in growth, nitrogen, and carbon photosynthetic metabolism under salinity and water stress. However, TaDREB1A was increased in expression under stress compared with control conditions, with tolerant genotypes exhibiting lower expression than susceptible ones.

Wheat ear carbon assimilation and nitrogen remobilization contribute significantly to grain yield

The role of wheat ears as a source of nitrogen (N) and carbon (C) in the grain filling process has barely been studied. To resolve this question, five wheat genotypes were labeled with 15 N-enriched nutrient solution. N remobilization and absorption were estimated via the nitrogen isotope composition of total organic matter and Rubisco. Gas exchange analyses showed that ear photosynthesis contributed substantially to grain filling in spite of the great loss of C due to respiration. Of the total kernel N, 64.7% was derived from the N acquired between sowing and anthesis, while the remaining 35.3% was derived from the N acquired between anthesis and maturity. In addition, 1.87 times more N was remobilized to the developing kernel from the ear than from the flag leaf. The higher yielding genotypes showed an increased N remobilization to the kernel compared to the lower yielding genotypes. In addition, the higher yielding genotypes remobilized more N from the ears to the kernel than the lower yielding genotypes, while the lower yielding genotypes remobilized more N from the flag leaf to the kernel. Therefore, the ears contribute significantly toward fulfilling C and N demands during grain filling.

Relative Contribution of Nitrogen Absorption, Remobilization, and Partitioning to the Ear During Grain Filling in Chinese Winter Wheat

Knowledge of the function of the ear as a key organ in the uptake, remobilization and partitioning of nitrogen is essential for understanding its contribution to grain filling and thus guiding future breeding strategies. In this work, four Chinese winter wheat genotypes were grown on a 15N-enriched nutrient solution. N absorption and further remobilization to the flag leaf, the ear and the mature grains were calculated via the 15N atom % excess. The results indicated that the high yields of the Chinese wheat genotype were determined by higher grain numbers per ear, with greater plant height and a larger ear size, while the thousand-grain weight did not affect grain yield. In the mature grains, 66.7% of total N was remobilized from the pre-anthesis accumulation in the biomass, while the remaining 33.3% was derived from the N taken up during post-anthesis. From anthesis to 2 weeks after the anthesis stage, the flag leaf remobilized 3.67 mg of N outwards and the ear remobilized 3.87 mg of N inwards from the pre-anthesis accumulation in each plant. The positive correlation between ear Nrem and grain Nrem indicated that the ear was an important organ for providing N to the grain, whereas the remobilized N stream from the leaves was not correlated with grain Nrem, thus indicating that flag leaf N was not translocated directly to the grain. The grain Nrem was negatively correlated with the ear N concentration throughout grain filling, which suggested that higher-yielding genotypes had better sink activity in the ear, while Rubisco played a critical role in N deposition. Therefore, to improve yield potential in wheat, the N accumulation in the ear and the subsequent remobilization of that stored N to the grains should be considered. N accumulation and remobilization in the ear may at least be valuable for Chinese breeding programs that aim at optimizing the sink/source balance to improve grain filling.