Stay‐green: A consequence of the balance between supply and demand for nitrogen during grain filling?

Retention of green leaf area in grain sorghum under post‐anthesis drought, known as stay‐green, is associated with greater biomass production, lodging resistance and yield. The stay‐green phenomenon can be examined at a cell, leaf, or whole plant level. At a cell level, the retention of chloroplast proteins such as LHCP2, OEC33 and Rubisco until late in senescence has been reported in sorghum containing the KS19 source of stay‐green, indicating that photosynthesis may be maintained for longer during senescence in these genotypes. At a leaf level, longevity of photosynthetic apparatus is intimately related to nitrogen (N) status. At a whole plant level, stay‐green can be viewed as a consequence of the balance between N demand by the grain and N supply during grain filling. To examine some of these concepts, nine hybrids varying in the B35 and KS19 sources of stay‐green were grown under a post‐anthesis water deficit. Genotypic variation in delayed onset and reduced rate of leaf senescence were explained by differences in specific leaf nitrogen (SLN) and N uptake during grain filling. Matching N supply from age‐related senescence and N uptake during grain filling with grain N demand found that the shortfall in N supply for grain filling was greater in the senescent than stay‐green hybrids, resulting in more accelerated leaf senescence in the former. We hypothesise that increased N uptake by stay‐green hybrids is a result of greater biomass accumulation during grain filling in response to increased sink demand (higher grain numbers) which, in turn, is the result of increased radiation use efficiency and transpiration efficiency due to higher SLN. Delayed leaf senescence resulting from higher SLN should, in turn, allow more carbon and nitrogen to be allocated to the roots of stay‐green hybrids during grain filling, thereby maintaining a greater capacity to extract N from the soil compared with senescent hybrids.     

硫氮配施对持绿型小麦氮素运转及叶片衰老的影响

以持绿型小麦品种‘豫麦66号’、‘潍麦8号’及非持绿型品种‘小偃6号’为材料,采用以氮肥为主区硫肥为副区的田间裂区试验,研究了2个施氮水平[纯氮120kg/hm2(N120)、220kg/hm2(N220)]和3个施硫水平[纯硫0kg/hm2(S0)、20kg/hm2(S20)、60kg/hm2(S60)]下植株各部位的含氮量、叶片干鲜重及叶片叶绿素含量的变化,探讨硫氮配施对不同类型小麦氮吸收及衰老的影响。结果表明:在相同处理下,持绿型小麦植株的总含氮量、氮素转运量、叶绿素含量、叶片含水量及穗粒数和千粒重均高于非持绿型小麦。N120处理条件下,不同硫肥处理时持绿型小麦与非持绿型小麦变化趋势相同,开花期茎和叶含氮量及叶绿素含量在S60处理下均低于其他2个硫肥处理,生育后期叶片含水量下降幅度也明显高于其他处理;在N220处理条件下,3个品种开花期叶含氮量、收获期总氮累积量、氮收获指数、叶绿素含量及叶片含水量在S60处理下均高于其他2个处理,其中非持绿型小麦在高硫处理条件下灌浆期的叶绿素含量的增长率明显高于持绿型小麦,而灌浆中后期叶片含水量的下降幅度则明显低于持绿型小麦。研究发现,施用硫肥在氮肥不足时会对小麦植株氮素的吸收利用及叶片衰老等方面产生负面影响,但在氮肥充足时却在氮素的吸收利用、延缓叶片衰老及最终籽粒产量和总生物量等方面表现出正面效应;本实验条件下,220kg/hm2左右施氮量和60kg/hm2左右施硫量有利于各品种小麦生长发育和产量提高;高N和高S水平对于延缓小麦的衰老而言,非持绿性小麦比持绿型小麦更明显。     

Nutrient partitioning and grain yield of TaNAM-RNAi wheat under abiotic stress

Aims

Decreased expression of TaNAM genes by RNAi results in delayed senescence and decreased grain protein, iron, and zinc concentrations. Here, we determined whether NAM expression level alters onset of senescence under stress conditions, whether delayed senescence in the TaNAM-RNAi line resulted in improved tolerance to post-anthesis abiotic stress, and determined the effects of post-anthesis abiotic stress on N and mineral remobilization and partitioning to grain.

Methods

Greenhouse-grown WT and TaNAM-RNAi wheat were characterized in two studies:three levels of N fertility or water limitation during grain fill. Studies were conducted under both optimal and heat stress temperatures. Senescence onset was determined by monitoring flag leaf chlorophyll.

Results

Under optimal tempertures, TaNAM-RNAi plants had a yield advantage at lower N. TaNAM-RNAi plants had delayed senescence relative to the WT and lower grain protein and mineral concentrations, N remobilization efficiency, and partitioning of N and most minerals to grain.

Conclusions

Nutritional quality of TaNAM-RNAi grain was consistently lower than WT. Delayed senescence of TaNAM-RNAi plants provided a yield advantage under optimal temperatures but not under water or heat stress. Discovery of specific NAM protein targets may allow separation of the delayed senescence and nutrient partitioning traits, which could be used for improvement of wheat.     

Wheat Grain Filling Is Limited by Grain Filling Capacity rather than the Duration of Flag Leaf Photosynthesis: A Case Study Using NAM RNAi Plants

It has been proposed that delayed leaf senescence can extend grain filling duration and thus increase yields in cereal crops. We found that wheat (Triticum aestivum) NAM RNAi plants with delayed senescence carried out 40% more flag leaf photosynthesis after anthesis than control plants, but had the same rate and duration of starch accumulation during grain filling and the same final grain weight. The additional photosynthate available in NAM RNAi plants was in part stored as fructans in the stems, whereas stem fructans were remobilised during grain filling in control plants. In both genotypes, activity of starch synthase was limiting for starch synthesis in the later stages of grain filling. We suggest that in order to realise the potential yield gains offered by delayed leaf senescence, this trait should be combined with increased grain filling capacity.     

Increased cytokinin levels in transgenic PSAG12–IPT tobacco plants have large direct and indirect effects on leaf senescence, photosynthesis and N partitioning

We studied the impact of delayed leaf senescence on the functioning of plants growing under conditions of nitrogen remobilization. Interactions between cytokinin metabolism, Rubisco and protein levels, photosynthesis and plant nitrogen partitioning were studied in transgenic tobacco (Nicotiana tabacum L.) plants showing delayed leaf senescence through a novel type of enhanced cytokinin syn‐thesis, i.e. targeted to senescing leaves and negatively auto‐regulated (P_SAG12–IPT), thus preventing developmental abnormalities. Plants were grown with growth‐limiting nitrogen supply. Compared to the wild‐type, endogenous levels of free zeatin (Z)‐ and Z riboside (ZR)‐type cytokinins were increased up to 15‐fold (total ZR up to 100‐fold) in senescing leaves, and twofold in younger leaves of P_SAG12–IPT. In these plants, the senescence‐associated declines in N, protein and Rubisco levels and photosynthesis rates were delayed. Senescing leaves accumulated more (¹⁵N‐labelled) N than younger leaves, associated with reduced shoot N accumulation (–60%) and a partially inverted canopy N profile in P_SAG12–IPT plants. While root N accumulation was not affected, N translocation to non‐senescing leaves was progressively reduced. We discuss potential consequences of these modified sink–source relations, associated with delayed leaf senescence, for plant productivity and the efficiency of utilization of light and minerals.     

The effect of fertilizer‐N on the inter‐specific competition among three wheat aphids under elevated CO2

Increasing atmospheric carbon dioxide (ab. CO₂) and fertilizer‐nitrogen (ab. N) applications may have marked direct effects on the plant growth of agricultural crops, and in turn affect the higher trophic level of insect herbivores. In this study, the effects of elevated CO₂ (i.e., 650 µl/L vs. ambient 400 µl/L) and fertilizer‐N (0, 50, 100, 200 kg/ha) on the population abundances and the inter‐specific competition among three co‐occurring species of wheat aphids, Sitobion avenae, Rhopalosiphum padi and Schizaphis graminum, were studied. The grain weight per ear and the 1,000‐grain weight were generally increased when grown under elevated CO₂ and showed a significant effect at the 100 kg/ha (grain weight per ear) and 0, 50 and 100 kg/ha (1,000‐grain weight) N. These two yield indexes increased with increasing fertilizer‐N levels within reasonable limits and reached a maximum at 100 kg/ha. Elevated CO₂ combined with fertilizer‐N levels formed complex indirect effects on the three wheat aphids through the wheat crops they fed on. Elevated CO₂ significantly decreased the niche overlap index (ab. NOI) between S. avenae and R. padi under 0 and 100 kg/ha and that between R. padi and S. graminum under 0 kg/ha, while significantly increased the three NOIs under 50 kg/ha and that between R. padi and S. graminum under 100 and 200 kg/ha. S. avenae and R. padi had the larger population and stronger competition in low‐N condition (0 and 50 kg/ha), which was harmful to wheat yield and quality when combined with its own poor nutrition. Overall, the 100 kg/ha N level was the best option based on the aphid population, competition and wheat yields. Therefore, the balanced relationship formed among fertilizers, plants and insects under 100 kg/ha N was vital for the interactive ecosystem.     

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.     

Physiological characterization of 'stay green' mutants in durum wheat

Four mutants with delayed leaf senescence were selected from seed of durum wheat mutagenized with ethylmethane sulphonate. Changes in net photosynthetic rate, efficiency of photosystem II and chlorophyll concentration during the maturation and senescence of the flag leaves of both mutant and parental plants were determined under glasshouse conditions. The four mutant lines maintained photosynthetic competence for longer than the parental line and are therefore functionally 'stay green'. The mutant lines also had higher seed weights and grain yields per plant than the parental line.     

Increasing yield potential through manipulating of an ARE1 ortholog related to nitrogen use efficiency in wheat by CRISPR/Cas9

Wheat (Triticum aestivum L.) is a staple food crop consumed by more than 30% of world population. Nitrogen (N) fertilizer has been applied broadly in agriculture practice to improve wheat yield to meet the growing demands for food production. However, undue N fertilizer application and the low N use efficiency (NUE) of modern wheat varieties are aggravating environmental pollution and ecological deterioration. Under nitrogen-limiting conditions, the rice (Oryza sativa) abnormal cytokinin response1 repressor1 (are1) mutant exhibits increased NUE, delayed senescence and consequently, increased grain yield. However, the function of ARE1 ortholog in wheat remains unknown. Here, we isolated and characterized three TaARE1 homoeologs from the elite Chinese winter wheat cultivar ZhengMai 7698. We then used CRISPR/Cas9-mediated targeted mutagenesis to generate a series of transgene-free mutant lines either with partial or triple-null taare1 alleles. All transgene-free mutant lines showed enhanced tolerance to N starvation, and showed delayed senescence and increased grain yield in field conditions. In particular, the AABBdd and aabbDD mutant lines exhibited delayed senescence and significantly increased grain yield without growth defects compared to the wild-type control. Together, our results underscore the potential to manipulate ARE1 orthologs through gene editing for breeding of high-yield wheat as well as other cereal crops with improved NUE.     

Influence of reproductive organs on plant senescence in rice and wheat

The chlorophyll and protein contents of the flag, second and third leaves gradually decreased during the reproductive development of rice (Oryza sativa L. cv. Rasi) and wheat (Triticum aestivum L. cv. Sonalika) plants, whereas proline accumulation increased up to the grain maturation stage and slightly decreased thereafter. In rice plant, the rate of decrease in chlorophyll and protein and increase in proline level were higher in the flag leaf than in the second leaf. It was opposite in wheat plant. The export of [³²P]-phosphate from leaves to grains gradually increased reaching a maximal stage at the grain development stage, and then declined. The export of this radioisotope was greater in rice than in wheat. Removal of panicle at the anthesis and grainfilling stages delayed leaf senescence of rice plant, while in wheat the ponicle removal at any stage did not have a marked effect on delaying leaf senescence. The contents of chlorophyll and protein of glumes were higher in wheat than in rice. The variation of such source-sink relationship might be one of the possible reasons for the above effect on leaf senescence.     

Mutation of Oryza sativa CORONATINE INSENSITIVE 1b (OsCOI1b) delays leaf senescence

Jasmonic acid (JA) functions in plant development, including senescence and immunity. Arabidopsis thaliana CORONATINE INSENSITIVE 1 encodes a JA receptor and functions in the JA‐responsive signaling pathway. The Arabidopsis genome harbors a single COI gene, but the rice (Oryza sativa) genome harbors three COI homologs, OsCOI1a, OsCOI1b, and OsCOI2. Thus, it remains unclear whether each OsCOI has distinct, additive, synergistic, or redundant functions in development. Here, we use the oscoi1b‐1 knockout mutants to show that OsCOI1b mainly affects leaf senescence under senescence‐promoting conditions. oscoi1b‐1 mutants stayed green during dark‐induced and natural senescence, with substantial retention of chlorophylls and photosynthetic capacity. Furthermore, several senescence‐associated genes were downregulated in oscoi1b‐1 mutants, including homologs of Arabidopsis thaliana ETHYLENE INSENSITIVE 3 and ORESARA 1, important regulators of leaf senescence. These results suggest that crosstalk between JA signaling and ethylene signaling affects leaf senescence. The Arabidopsis coi1‐1 plants containing 35S:OsCOI1a or 35S:OsCOI1b rescued the delayed leaf senescence during dark incubation, suggesting that both OsCOI1a and OsCOI1b are required for promoting leaf senescence in rice. oscoi1b‐1 mutants showed significant decreases in spikelet fertility and grain weight, leading to severe reduction of grain yield, indicating that OsCOI1‐mediated JA signaling affects spikelet fertility and grain filling.