A high-grain protein content locus on barley (Hordeum vulgare) chromosome 6 is associated with increased flag leaf proteolysis and nitrogen remobilization

Leaf senescence and nitrogen remobilization from senescing tissues are two important factors determining grain protein content (GPC) in cereals. We compared near-isogenic barley ( Hordeum vulgare L.) germplasm varying in the allelic state of a major GPC quantitative trait locus on chromosome 6, delineated by molecular markers HVM74 and ABG458 and explaining approximately 46% of the variability in this trait. High GPC was consistently associated with earlier whole-plant senescence. SDS–PAGE and immunoblot analysis of flag leaf proteins indicated earlier leaf protein [including ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)] degradation in high-GPC germplasm. This was accompanied by enhanced availability of ammonium and glutamine in developing kernels, suggesting increased phloem retranslocation of nitrogen. Based on previous microarray analysis, we performed a detailed expression study of six leaf genes, tentatively involved in plastidial proteolysis, vacuolar proteolysis, intermediary N metabolism and N transport. All of these were upregulated in high-GPC barley, mostly around 21 to 28 days past anthesis, prior to or around the time demonstrating maximal differences in leaf protein (including Rubisco) levels. Therefore, these genes represent potential targets to manipulate grain protein accumulation. It appears likely that their functional analysis will enhance our understanding of whole-plant N recycling. Additionally, earlier leaf (photosynthetic) protein degradation may lead to reduced N carbon assimilation in high-GPC germplasm, explaining past studies demonstrating a negative correlation between GPC and yield.     

Control of barley (Hordeum vulgare L.) development and senescence by the interaction between a chromosome six grain protein content locus, day length, and vernalization

Regulatory processes controlling traits such as anthesis timing and whole-plant senescence are of primary importance for reproductive success and for crop quality and yield. It has previously been demonstrated that the presence of alleles associated with high grain protein content (GPC) at a locus on barley chromosome six leads to accelerated leaf senescence, and to strong (>10-fold) up-regulation of several genes which may be involved in senescence control. One of these genes (coding for a glycine-rich RNA-binding protein termed HvGR-RBP1) exhibits a high degree of similarity to Arabidopsis glycine-rich RNA-binding protein 7 (AtGRP7), which has been demonstrated to accelerate flowering under both long-day (LD) and short-day (SD) conditions, but not after vernalization. Development of near-isogenic barley lines, differing in the allelic state of the GPC locus, was compared from the seedling stage to maturity under both SD and LD and after vernalization under LD. Intriguingly, pre-anthesis plant development [measured by leaf emergence timing and pre-anthesis (sequential) leaf senescence] was enhanced in high-GPC germplasm. Differences were more pronounced under SD than under LD, but were eliminated by vernalization, associating observed effects with floral induction pathways. By contrast, differences in post-anthesis flag leaf and whole-plant senescence between low- and high-GPC germplasm persisted under all tested conditions, indicating that the GPC locus, possibly through HvGR-RBP1, impacts on both developmental stages. Detailed molecular characterization of this experimental system may allow the dissection of cross-talk between signalling pathways controlling early plant and floral development on one side, and leaf/whole-plant senescence on the other side.     

Anthesis date mainly explained correlations between post-anthesis leaf senescence, grain yield, and grain protein concentration in a winter wheat population segregating for flowering time QTLs

The genetic variability of the duration of leaf senescence during grain filling has been shown to affect both carbon and nitrogen acquisition. In particular, maintaining green leaves during grain filling possibly leads to increased grain yield, but its associated effect on grain protein concentration has not been studied. The aim of this study was to dissect the genetic factors contributing to correlations observed at the phenotypic level between leaf senescence during grain filling, grain protein concentration, and grain yield in winter wheat. With this aim in view, an analysis of quantitative trait locus (QTL) co-locations for these traits was carried out on a doubled haploid mapping population grown in a large multienvironment trial network. Pleiotropic QTLs affecting leaf senescence and grain yield and/or grain protein concentration were identified on chromosomes 2D, 2A, and 7D. These were associated with QTLs for anthesis date, showing that the phenotypic correlations with leaf senescence were mainly explained by flowering time in this wheat population. Study of the allelic effects of these pleiotropic QTLs showed that delaying leaf senescence was associated with increased grain yield or grain protein concentration depending on the environments considered. It is proposed that this differential effect of delaying leaf senescence on grain yield and grain protein concentration might be related to the nitrogen availability during the post-anthesis period. It is concluded that the benefit of using leaf senescence as a selection criterion to improve grain protein concentration in wheat cultivars may be limited and would largely depend on the targeted environments, particularly on their nitrogen availability during the post-anthesis period.     

Enhanced CO2 alters the relationship between photosynthesis and defence in cyanogenic Eucalyptus cladocalyx F. Muell.

The effect of elevated CO₂ and different levels of nitrogen on the partitioning of nitrogen between photosynthesis and a constitutive nitrogen-based secondary metabolite (the cyanogenic glycoside prunasin) was examined in Eucalyptus cladocalyx . Our hypothesis was that the expected increase in photosynthetic nitrogen-use efficiency of plants grown at elevated CO₂ concentrations would lead to an effective reallocation of available nitrogen from photosynthesis to prunasin. Seedlings were grown at two concentrations of CO₂ and nitrogen, and the proportion of leaf nitrogen allocated to photosynthesis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), protein and prunasin compared. Up to 20% of leaf nitrogen was allocated to the cyanogenic glycoside, although this proportion varied with leaf age, position and growth conditions. Leaf prunasin concentration was strongly affected by nitrogen supply, but did not increase, on a dry weight basis, in the leaves from the elevated CO₂ treatments. However, the proportion of nitrogen allocated to prunasin increased significantly, in spite of a decreasing pool of leaf nitrogen, in the plants grown at elevated concentrations of CO₂. There was less protein in leaves of plants grown at elevated CO₂ in both nitrogen treatments, while the concentration of active sites of Rubisco only decreased in plants from the low-nitrogen treatment. These changes in leaf chemistry may have significant implications in terms of the palatability of foliage and defence against herbivores.     

Effect of two contrasted N fertilisations on rapeseed growth and nitrate metabolism

Winter oilseed rape was grown under two nitrogen fertilisation conditions. The N1-plants and N5-plants were respectively supplied with 4.5 g N per plant (N-limiting condition) and 22.5 g N per plant (non-N-limiting condition). Growth parameters and nitrate reducing capacity were monitored at five sampling stages interspersed with ammonium nitrate applications. N5-plants showed a higher growth rate producing more leaves and stems, early flower and silique formation and delayed leaf senescence. They also contained more nitrate and a higher nitrate reductase activity (NRA) especially in leaves which represented the main site of nitrate reduction before flowering. However, stems and siliques contributed to NRA especially in nitrogen-limited plants that lost their leaves early. This present study outlines the importance of siliques as individual sinks reducing nitrate essentially in the pod walls. The soluble protein content decreased in senescing leaves which was indicative of the reallocation of proteinic nitrogen towards stems and siliques. In non-limiting conditions, other nitrogen compounds of leaves may account for such a reallocation. Hence, the timing of leaf fall could contribute to the low nitrogen recovery in rapeseed.     

Autumnal changes in total nitrogen, salt-extractable proteins and amino acids in leaves and adjacent bark of black alder, eastern cottonwood and white basswood

Autumnal changes in total nitrogen, salt-extractable protein and amino acid concentrations in leaves and adjacent bark of black alder [ Ainus glutinosa (L.) Gaertn.], eastern cottonwood ( Populus deltoides Bartr. ex Marsh.) and white basswood ( Tilia heterophylla Vent.) were determined for trees growing on minespoils and a prairiederived loamy soil in central Illinois. The composition of free amino acids in foliage was also determined at peak concentration for each tree species during late senescence. Total nitrogen concentration in the leaves decreased slowly throughout most of the fall for all species. In the final stages of senescence, total leaf nitrogen concentrations were about halved in eastern cottonwood and white basswood but continued to decrease slowly in black alder. The concentration of salt-extractable proteins in leaves of all species peaked early in the fall and then declined prior to leaf abscission. This decline coincided with an increase in the concentration of free amino acids in the leaves. The increase stabilized in both eastern poplar and white basswood but continued in black alder. Glutamine in black alder and eastern cottonwood, and asparagine in white basswood were the most abundant free amino acids at the time of peak concentration of total free amino acids in senescent leaves. Bark of trees of all species had higher nitrogen concentrations and higher proportions of salt-extractable proteins to estimated total proteins after leaf senescence than during the preceding summer. Results indicate that autumnal fluxes in leaf and bark nitrogen fractions of alder can differ substantially from fluxes in other broadleaved winter-deciduous trees in a way which suggests that alder does not effectively conserve leaf nitrogen through retranslocation to bark tissue.     

Supply of nitrogen can reverse senescence processes and affect expression of genes coding for plastidic glutamine synthetase and lysine-ketoglutarate reductase/saccharopine dehydrogenase

Nitrogen availability has a strong influence on developmental processes in plants. We show that the time of nitrogen supply regulates the course of leaf senescence in flag leaves of Hordeum vulgare . The senescence-specific decrease in chlorophyll content and photosystem II efficiency is clearly delayed when plants are fertilised with nitrate at the onset of leaf senescence. Concurrently, the additional supply of nitrate affects expression patterns of two marker genes of nitrogen metabolism. As shown by quantitative RT-PCR analyses, senescence-specific downregulation of plastidic glutamine synthetase ( GS2 ) and senescence-specific upregulation of lysine-ketoglutarate reductase/saccharopine dehydrogenase ( LKR/SDH ) are both clearly retarded. Depletion of nitrogen in experiments using hydroponic growth systems results in premature primary leaf senescence. The already started senescence processes can be even reversed by later nitrogen addition, as proved by a further increase in photosystem II efficiency and chlorophyll content, returning to the high values of controls which had not been deprived of nitrogen. Although both addition of nitrate or ammonium effectively reversed nitrogen depletion-induced primary leaf senescence, addition of urea did not. Additionally, effects of nitrogen supply on the course of leaf senescence were analysed in the model plant Arabidopsis thaliana. Leaves of A. thaliana show the same reversion of senescence processes after receiving additional nitrogen supply, indicating that the nitrogen response of leaf development is conserved in different plant species.     

Varietal effects of eight paired lines of transgenic Bt maize and near-isogenic non-Bt maize on soil microbial and nematode community structure

A glasshouse experiment was undertaken to provide baseline data on the variation between conventional maize (Zea mays L.) varieties and genetically modified maize plants expressing the insecticidal Bacillus thuringiensis protein (Bt, Cry1Ab). The objective was to determine whether the variation in soil parameters under a range of conventional maize cultivars exceeded the differences between Bt and non-Bt maize cultivars. Variations in plant growth parameters (shoot and root biomass, percentage carbon, percentage nitrogen), Bt protein concentration in shoots, roots and soil, soil nematode abundance and soil microbial community structure were determined. Eight paired varieties (i.e. varieties genetically modified to express Bt protein and their near-isogenic control varieties) were investigated, together with a Bt variety for which no near-isogenic control was available (NX3622, a combined transformant expressing both Bt and herbicide tolerance) and a conventional barley (Hordeum vulgare L.) variety which was included as a positive control. The only plant parameter which showed a difference between Bt varieties and near-isogenic counterparts was the shoot carbon to nitrogen ratio; this was observed for only two of the eight varieties, and so was not attributable to the Bt trait. There were no detectable differences in the concentration of Bt protein in plant or soil with any of the Bt-expressing varieties. There were significant differences in the abundance of soil nematodes, but this was not related to the Bt trait. Differences in previously published soil nematode studies under Bt maize were smaller than these varietal effects. Soil microbial community structure, as determined by phospholipid fatty acid (PLFA) analysis, was strongly affected by plant growth stage but not by the Bt trait. The experimental addition of purified Cry1Ab protein to soil confirmed that, at ecologically relevant concentrations, there were no measurable effects on microbial community structure.     

Cytoplasmic male sterility in barley

The maternal male sterile barley msm1 with or without a dominant gene, Rfmla, which restores male fertility, was studied. Determined with SDS-PAGE, the polypeptide pattern in the anthers of unrestored msm1 plants remains juvenile in the middle of anther development, two major zones being absent or weak. At the stage when anther development stops in msm1 plants, the anther proteins appear to be hydrolyzed to short-chain peptides. Restored plants, heterozygous for the restorer gene, Rfmla, behaved like the near-isogenic normal barley, cv. Adorra. The total leaf protein pattern of young leaf tissue and the chloroplastidic membrane protein pattern are normal in msm1 cytoplasm when studied with this technique. Chlorophyll b is unnecessary for restoration by Rfmla, though the restored plants have a lower chlorophyll a/b ratio than an unrestored plant in the mature stem leaf. Mature stem leaf pieces of unrestored msm1 plants were induced to senesce with 20 mM NaCl solution. This senescence was inhibited by exogenous kinetin. Leaf pieces of restored msm1 plants or those of near-isogenic normal barley behaved in the same way in the NaCl solution as in distilled water. Many features of the physiology of restored plants can be explained as the functions of cytokinins. Kernels of male sterile plants have a more rapid root elongation at germination than near-isogenic normal barley.     

CRISPR/Cas9 gene editing and natural variation analysis demonstrate the potential for HvARE1 in improvement of nitrogen use efficiency in barley

Nitrogen is a major determinant of grain yield and quality. As excessive use of nitrogen fertilizer leads to environmental pollution and high production costs, improving nitrogen use efficiency (NUE) is fundamental for a sustainable agriculture. Here, we dissected the role of the barley abnormal cytokinin response1 repressor 1 (HvARE1) gene, a candidate for involvement in NUE previously identified in a genome-wide association study, through natural variation analysis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing. HvARE1 was predominantly expressed in leaves and shoots, with very low expression in roots under low nitrogen conditions. Agrobacterium-mediated genetic transformation of immature embryos (cv. Golden Promise) with single guide RNAs targeting HvARE1 generated 22 T₀ plants, from which four T₁ lines harbored missense and/or frameshift mutations based on genotyping. Mutant are1 lines exhibited an increase in plant height, tiller number, grain protein content, and yield. Moreover, we observed a 1.5- to 2.8-fold increase in total chlorophyll content in the flag leaf at the grain filling stage. Delayed senescence by 10–14 d was also observed in mutant lines. Barley are1 mutants had high nitrogen content in shoots under low nitrogen conditions. These findings demonstrate the potential of ARE1 in NUE improvement in barley.     

Roles for redox regulation in leaf senescence of pea plants grown on different sources of nitrogen nutrition

Leaf senescence and associated changes in redox components were monitored in commercial pea (Pisum sativum L. cv. Phoenix) plants grown under different nitrogen regimes for 12 weeks until both nodules and leaves had fully senesced. One group of plants was inoculated with Rhizobium leguminosarum and grown with nutrient solution without nitrogen. A second group was not inoculated and these were grown on complete nutrient solution containing nitrogen. Leaf senescence was evident at 11 weeks in both sets of plants as determined by decreases in leaf chlorophyll and protein. However, a marked decrease in photosynthesis was observed in nodulated plants at 9 weeks. Losses in the leaf ascorbate pool preceded leaf senescence, but leaf glutathione decreased only during the senescence phase. Large decreases in dehydroascorbate reductase and catalase activities were observed after 9 weeks, but the activities of other antioxidant enzymes remained high even at 11 weeks. The extent of lipid peroxidation, the number of protein carbonyl groups and the level of H(2)O(2) in the leaves of both nitrate-fed and nodulated plants were highest at the later stages of senescence. At 12 weeks, the leaves of nodulated plants had more protein carbonyl groups and greater lipid peroxidation than the nitrate-fed controls. These results demonstrate that the leaves of nodulated plants undergo an earlier inhibition of photosynthesis and suffer enhanced oxidation during the senescence phase than those from nitrate-fed plants.     

Leaf yellowing and anthocyanin accumulation are two genetically independent strategies in response to nitrogen limitation in Arabidopsis thaliana

For the first time in Arabidopsis thaliana, this work proposes the identification of quantitative trait loci (QTLs) associated with leaf senescence and stress response symptoms such as yellowing and anthocyanin-associated redness. When Arabidopsis plants were cultivated under low nitrogen conditions, we observed that both yellowing of the old leaves of the rosette and whole rosette redness were promoted. Leaf yellowing is a senescence symptom related to chlorophyll breakdown. Redness is a symptom of anthocyanin accumulation related to whole plant ageing and nutrient limitation. In this work, Arabidopsis is used as a model system to dissect the genetic variation of these parameters by QTL mapping in the 415 recombinant inbred lines of the Bay-0xShahdara population. Fifteen new QTLs and two epistatic interactions were described in this study. The yellowing of the rosette, estimated by visual notation and image processing, was controlled by four and five QTLs, respectively. The visual estimation of redness allowed us to detect six QTLs among which the major one explained 33% of the total variation. Two main QTLs were confirmed in near-isogenic lines (heterogenous inbred family; HIF), thus confirming the relevance of the visual notation of these traits. Co-localizations between QTLs for leaf yellowing, redness and nitrogen use efficiency described in a previous publication indicate complex interconnected pathways involved in both nitrogen management and senescence- and stress-related processes. No co-localization between QTLs for leaf yellowing and redness has been found, suggesting that the two characters are genetically independent.     

Leaf senescence in Brassica napus: expression of genes encoding pathogenesis-related proteins

Genes that are expressed during leaf senescence in Brassica napus were identified by the isolation of representative cDNA clones. DNA sequence and deduced protein sequence from two senescence-related cDNAs, LSC94 and LSC222, representing genes that are expressed early in leaf senescence before any yellowing of the leaves is visible, showed similarities to genes for pathogenesis-related (PR) proteins: a PR-1a-like protein and a class IV chitinase, respectively. The LSC94 and LSC222 genes showed differential regulation with respect to each other; an increase in expression was detected at different times during development of healthy leaves. Expression of both genes was induced by salicylic acid treatment. These findings suggest that some PR genes, as well as being induced by pathogen infection, may have alternative functions during plant development, for example in the process of leaf senescence.