Functional Stay-green Research Articles

Suppressing ASPARTIC PROTEASE 1 prolongs photosynthesis and increases wheat grain weight.

The elongation of photosynthesis, or functional staygreen, represents a feasible strategy to propel metabolite flux towards cereal kernels. However, achieving this goal remains a challenge in food crops. Here we report the cloning of wheat CO2 assimilation and kernel enhanced 2 (cake2), the mechanism underlying the photosynthesis advantages and natural alleles amenable to breeding elite varieties. A premature stop mutation in the A-genome copy of the ASPARTIC PROTEASE 1 (APP-A1) gene increased the photosynthesis rate and yield. APP1 bound and degraded PsbO, the protective extrinsic member of photosystem II critical for increasing photosynthesis and yield. Furthermore, a natural polymorphism of the APP-A1 gene in common wheat reduced APP-A1's activity and promoted photosynthesis and grain size and weight. This work demonstrates that the modification of APP1 increases photosynthesis, grain size and yield potentials. The genetic resources could propel photosynthesis and high-yield potentials in elite varieties of tetraploid and hexaploid wheat.

Genetic modification of PIN genes induces causal mechanisms of stay-green drought adaptation phenotype.

The stay-green trait is recognized as a key drought adaptation mechanism in cereals worldwide. Stay-green sorghum plants exhibit delayed senescence of leaves and stems, leading to prolonged growth, a reduced risk of lodging, and higher grain yield under end-of-season drought stress. More than 45 quantitative trait loci (QTL) associated with stay-green have been identified, including two major QTL (Stg1 and Stg2). However, the contributing genes that regulate functional stay-green are not known. Here we show that the PIN FORMED family of auxin efflux carrier genes induce some of the causal mechanisms driving the stay-green phenotype in sorghum, with SbPIN4 and SbPIN2 located in Stg1 and Stg2, respectively. We found that nine of 11 sorghum PIN genes aligned with known stay-green QTL. In transgenic studies, we demonstrated that PIN genes located within the Stg1 (SbPIN4), Stg2 (SbPIN2), and Stg3b (SbPIN1) QTL regions acted pleiotropically to modulate canopy development, root architecture, and panicle growth in sorghum, with SbPIN1, SbPIN2, and SbPIN4 differentially expressed in various organs relative to the non-stay-green control. The emergent consequence of such modifications in canopy and root architecture is a stay-green phenotype. Crop simulation modelling shows that the SbPIN2 phenotype can increase grain yield under drought.

Temporal trends in canopy temperature and greenness are potential indicators of late-season drought avoidance and functional stay-green in wheat

The ability to avoid dehydration is a drought resistance mechanism becoming increasingly more important even in temperate regions. In wheat, dehydration avoidance can be associated with a maintained canopy cooling during dry periods. However, in an average year under temperate conditions, drought periods are rather short which makes it difficult to routinely screen for drought avoidance using canopy temperature (CT). Furthermore, confounding factors such as differences in height, shoot biomass, canopy structure and phenology complicate the interpretation of differences in CT. We aimed to use temporal trends in CT and canopy greenness during short-term drought and heat events in the early grain filling phase to circumvent these problems. During this phase, evaporative demand is high and phenology-driven senescence has yet little effect on CT. Diverse sets of 354 and 71 wheat genotypes where grown in the field phenotyping platform of ETH Zurich in 2018 and 2019, respectively. CT was repeatedly measured during early grain filling by means of drone-based imaging. The temporal trends in CT during early grain filling showed a moderate to high within-year heritability (h2 = 0.35 and h2 = 0.88 in 2018 and 2019, respectively). These trends were largely independent of confounding factors when compared to single time point measurements and likely represent genotype-specific reactions to decreasing water availability more directly than absolute CT. CT trends were also largely independent of the temporal trends in stay-green indices. Therefore, we used a combination of time-resolved CT and stay-green trends to identify genotypes combining both traits. Significant differences were observed in the combined time-resolved index among three replicated check varieties. We therefore propose to use the combined time-resolved index to identify genotypes with improved drought avoidance and functional stay green.

NOL-mediated functional stay-green traits in perennial ryegrass (Lolium perenne L.) involving multifaceted molecular factors and metabolic pathways regulating leaf senescence.

Loss of chlorophyll (Chl) is a hallmark of leaf senescence, which may be regulated by Chl catabolic genes, including NON-YELLOW COLORING 1 (NYC1)-like (NOL). The objective of this study was to determine molecular factors and metabolic pathways underlying NOL regulation of leaf senescence in perennial grass species. LpNOL was cloned from perennial ryegrass (Lolium perenne L.) and found to be highly expressed in senescent leaves. Transient overexpression of LpNOL accelerated leaf senescence and Chl b degradation in Nicotiana benthamiana. LpNOL RNA interference (NOLi) in perennial ryegrass not only significantly blocked Chl degradation in senescent leaves, but also delayed initiation and progression of leaf senescence. This study found that NOL, in addition to functioning as a Chl b reductase, could enact the functional stay-green phenotype in perennial grass species, as manifested by increased photosynthetic activities in NOLi plants. Comparative transcriptomic analysis revealed that NOL-mediated functional stay-green in perennial ryegrass was mainly achieved through the modulation of Chl catabolism, light harvesting for photosynthesis, photorespiration, cytochrome respiration, carbohydrate catabolism, oxidative detoxification, and abscisic acid biosynthesis and signaling pathways.

Deciphering the Role of Stay-Green Trait to Mitigate Terminal Heat Stress in Bread Wheat

The present study aimed to reveal the impact of the stay-green trait in bread wheat under terminal heat stress. Field experiments (early and late sowing; for two consecutive years) were conducted to investigate the influence of terminal heat stress on the morpho-physiological traits in different stay-green types i.e., non-stay-green, moderately non-stay-green, moderately stay-green, and stay-green. In addition, the greenhouse experiment was performed to dissect the stay-green trait in functional stay-green, non-functional stay-green, and non-stay-green genotypes. The results of the field experiments confirmed that genotypes exhibiting the stay-green trait have a significantly high chlorophyll content, normalized difference vegetative index, grain yield, biological yield, kernel weight, and low canopy temperature under control and heat stress conditions. In the greenhouse experiment, functional stay-green and non-functional stay-green genotypes showed a high chlorophyll content and photochemical efficiency, whereas biological yield and grain yield showed a significant relation with the functional stay-green genotype under control and terminal heat stress treatments. The sequencing and expression analysis of chlorophyllide a oxygenase (CaO), light-harvesting complex (Cab), stay-green (SGR), and red chlorophyll catabolite reductase (RCCR) in functional stay-green, non-functional stay-green, and non-stay-green genotypes revealed variations in the exons of CaO and RCCR; and significant difference in the regulation of CaO and Cab at 7 days after anthesis under terminal heat stress. This study confirms that genotypes displaying the stay-green trait can aid wheat breeders to cope with increasing temperature in the impending decades.

Interactions between cytokinin and nitrogen contribute to grain mass in wheat cultivars by regulating the flag leaf senescence process

Premature senescence after anthesis reduces crop yields. Delaying leaf senescence could maintain photosynthetic activity for a longer period and lead to a higher photosynthetic rate. Recent studies have provided some insights into the interaction between cytokinin and nitrogen (N) in the regulation of plant development. In the present study, foliar application of exogenous 6-benzylaminopurine (6-BA) and lovastatin, an inhibitor of cytokinin synthesis, was combined with three N rates [0 kg ha−1 (low nitrogen, LN), 240 kg ha−1 (normal nitrogen, NN), and 360 kg ha−1 (high nitrogen, HN)] in two wheat cultivars, Wennong 6 (with a staygreen phenotype) and Jimai 20 (with a non-staygreen phenotype). Flag leaf senescence was assessed using a Gompertz growth curve. Grain mass, dry matter accumulation and distribution, total N of flag leaf, and concentrations of zeatin riboside (ZR) and abscisic acid (ABA) were also used to evaluate the functional characteristics of flag leaves. Grain mass was negatively correlated with initial senescence rate (r0) and duration of rapid chlorophyll loss (Chlloss), whereas it was positively correlated with maximum senescence rate (rmax), average senescence rate (raver), persistence phase (Chlper), total duration of flag leaf (Chltotal) and inflection point cumulative temperature (M). Compared to Jimai 20, Wennong 6 had larger raver, Chlper, and Chltotal. The concentration of ZR was highest under the 6-BA × NN treatment, followed by the 6-BA × HN and 6-BA × LN treatments. However, the concentration of ABA showed the opposite trend. It was concluded that the staygreen phenotype Wennong 6 was associated with greater grain mass and altered cytokinin metabolism and could be classified as a functional staygreen type. Foliar application of 6-BA interacting with N at the NN level (240 kg ha−1) may be a beneficial strategy for improving grain yield of wheat by regulating endogenous hormones and the flag leaf senescence process. Increasing endogenous cytokinin promoted the transport of dry matter to grain.

Contribution of Nitrogen Uptake and Retranslocation during Reproductive Growth to the Nitrogen Efficiency of Winter Oilseed-Rape Cultivars (Brassica napus L.) Differing in Leaf Senescence

Genotypic variation in N efficiency defined as high grain yield under limited nitrogen (N) supply of winter oilseed-rape line-cultivars has been predominantly attributed to N uptake efficiency (NUPT) through maintained N uptake during reproductive growth related to functional stay-green. For investigating the role of stay-green, N retranslocation and N uptake during the reproductive phase for grain yield formation, two line cultivars differing in N starvation-induced leaf senescence were grown in a field experiment without mineral N (N0) and with 160 kg N·ha−1 (N160). Through frequent harvests from full flowering until maturity N uptake, N utilization and apparent N remobilization from vegetative plant parts to the pods could be calculated. NUPT proved being more important than N utilization efficiency (NUE) for grain yield formation under N-limiting (N0) conditions. For cultivar differences in N efficiency, particularly N uptake during flowering (NUPT) and biomass allocation efficiency (HI) to the grains, were decisive. Both crop traits were related to delayed senescence of the older leaves. Remobilization of N particularly from stems and leaves was more important for pod N accumulation than N uptake after full flowering. Pod walls (high N concentrations) and stems (high biomass) mainly contributed to the crop-residue N at maturity. Decreasing the crop-inherent high N budget surplus of winter oilseed-rape requires increasing the low N remobilization efficiency particularly of pod-wall N to the grains. Addressing this conclusion, multi-year and -location field experiments with an extended range of cultivars including hybrids are desirable.

Genetic Analysis of Staygreen Trait and its Association with Morpho-physiological Traits under Water Deficit Stress in Wheat

The present study was conducted to assess the relation of staygreen trait with yield attributes and physiological traits under water deficit condition in wheat and to identify genetically diverse and agronomically desirable functional staygreen parents for incorporation in the breeding programmes for the development of drought tolerant genotypes. Heritability estimates along with genetic advance are high for leaf senescence rate (LSR). Present study also demonstrated significant positive association of staygreen trait LSR with RWC, photosynthetic rate, ear weight, thousand kernel weight and grain yield. Grouping genotypes for drought tolerance and staygreen trait suggested, CHIRYA7 as the most drought tolerant (DSI = 0.58) staygreen genotype (LSR=0.13) and CBW38 as drought susceptible (DSI = 1.12 ) fast senescing (0.48) genotype. D2-clustering grouped the genotypes into six clusters on the basis of similarity in the traits studied. Cluster III genotypes display staygreen character because it exhibited maximum cluster mean for photosynthetic rate, grain yield, test weight, SCMR, ear weight, biological yield, RWC and minimum cluster mean for DSI and LSR. There is a maximum intercluster distance between Cluster III and cluster VI hence staygreen genotypes from cluster III viz. CHIRYA7, HW2041 can be selected and crossed with cluster VI genotypes viz. HW2063 for incorporation of functional staygreen trait for development of drought tolerant genotypes. The clustering of genotypes will help to identify divergent genetic material for obtaining highly heterotic functional staygreen wheat hybrid for drought condition.

Quantitative Trait Locus Mapping and Candidate Gene Analysis for Functional Stay-Green Trait in Rice

Functional stay-green (FSG) delays leaf yellowing, maintaining photosynthetic competence, whereas nonfunctional stay-green (NFSG) retains only leaf greenness without sustaining photosynthetic activity. Retention of chlorophylls and photosynthetic capacity is important for increasing crop yield. We determined the main-effect quantitative trait loci (QTLs) for FSG traits in the japonica rice SNU-SG1 and isolated candidate genes. To identify QTLs influencing FSG, we analyzed eight traits: (1) 1 day after heading-degree of chlorophyll content of flag leaf, (2) 1 day after heading-degree of chlorophyll content of second leaf, (3) 1 day after heading-degree of chlorophyll content of flag and second leaves, (4) 50 day after heading-degree of chlorophyll content of flag leaf, (5) 50 day after heading-degree of chlorophyll content of second leaf, (6) 50 day after heading-degree of chlorophyll content of flag and second leaves, (7) relative decline degree of chlorophyll content of flag and second leaves, and (8) flowering time. We carried out QTL analysis with F7 RIL from a cross of japonica rice ‘SNU-SG1’ and indica rice ‘Milyang23 (M23)’. Using 131 molecular markers, we identified 18 QTLs for the eight traits with a threshold LOD value > 2.8. Sequence analysis identified 16 candidate genes for 10 main-effect QTLs. Of these, we have chosen seven strong candidate genes for the 10 main-effect QTLs. These genetic resources will be useful for breeding high-yielding rice cultivars.

Differences between winter oilseed rape (Brassica napus L.) cultivars in nitrogen starvation-induced leaf senescence are governed by leaf-inherent rather than root-derived signals.

Nitrogen (N) efficiency of winter oilseed rape (Brassica napus L.) line-cultivars (cvs.), defined as high grain yield under N limitation, has been primarily attributed to maintained N uptake during reproductive growth (N uptake efficiency) in combination with delayed senescence of the older leaves accompanied with maintained photosynthetic capacity (functional stay-green). However, it is not clear whether genotypic variation in N starvation-induced leaf senescence is due to leaf-inherent factors and/or governed by root-mediated signals. Therefore, the N-efficient and stay-green cvs. NPZ-1 and Apex were reciprocally grafted with the N-inefficient and early-senescing cvs. NPZ-2 and Capitol, respectively and grown in hydroponics. The senescence status of older leaves after 12 days of N starvation assessed by SPAD, photosynthesis and the expression of the senescence-specific cysteine protease gene SAG12-1 revealed that the stay-green phenotype of the cvs. NPZ-1 and Apex under N starvation was primarily under the control of leaf-inherent factors. The same four cultivars were submitted to N starvation for up to 12 days in a time-course experiment. The specific leaf contents of biologically active and inactive cytokinins (CKs) and the expression of genes involved in CK homeostasis revealed that under N starvation leaves of early-senescing cultivars were characterized by inactivation of biologically active CKs, whereas in stay-green cultivars synthesis, activation, binding of and response to biologically active CKs were favoured. These results suggest that the homeostasis of biologically active CKs was the predominant leaf-inherent factor for cultivar differences in N starvation-induced leaf senescence and thus N efficiency.

The stay-green trait

Stay-green (sometimes staygreen) refers to the heritable delayed foliar senescence character in model and crop plant species. In a cosmetic stay-green, a lesion interferes with an early step in chlorophyll catabolism. The possible contribution of synthesis to chlorophyll turnover in cosmetic stay-greens is considered. In functional stay-greens, the transition from the carbon capture period to the nitrogen mobilization (senescence) phase of canopy development is delayed, and/or the senescence syndrome proceeds slowly. Yield and composition in high-carbon (C) crops such as cereals, and in high-nitrogen (N) species such as legumes, reflect the source-sink relationship with canopy C capture and N remobilization. Quantitative trait loci studies show that functional stay-green is a valuable trait for improving crop stress tolerance, and is associated with the domestication syndrome in cereals. Stay-green variants reveal how autumnal senescence and dormancy are coordinated in trees. The stay-green phenotype can be the result of alterations in hormone metabolism and signalling, particularly affecting networks involving cytokinins and ethylene. Members of the WRKY and NAC families, and an ever-expanding cast of additional senescence-associated transcription factors, are identifiable by mutations that result in stay-green. Empirical selection for functional stay-green has contributed to increasing crop yields, particularly where it is part of a strategy that also targets other traits such as sink capacity and environmental sensitivity and is associated with appropriate crop management methodology. The onset and progress of senescence are phenological metrics that show climate change sensitivity, indicating that understanding stay-green can contribute to the design of appropriate crop types for future environments.

Physiological characteristics of a functional stay-green rice “SNU-SG1” during grain-filling period

Functional stay-green has been regarded as a promising characteristic to be introduced for improving rice yield potential. A functional stay-green rice “SNU-SG1” that was identified from japonica rice collections was compared with two regular high-yielding rice cultivars (HYVs) for the temporal change of leaf chlorophyll, soluble protein, and root activity, and nitrogen accumulation and remobilization during the grain-filling period. SNU-SG1 had slower decreasing rate and maintained higher concentration of chlorophyll and soluble protein in upper four leaves during the grain-filling period than HYVs “Suweon490” and “Andabyeo”, revealing a typical stay-green characteristic. Even though SNU-SG1 remobilized almost the same proportion of N accumulated before heading as HYVs to grain, it maintained much higher leaf N concentration due to the significantly higher N accumulation that is ascribable to the higher root activity sustenance during grain-filling period. The functional stay-green trait of SNU-SG1 seems to stem not only from the genetic control preventing chlorophyll degradation but also from the higher capacity to absorb N from soil due to the sustained strong root activity during grain-filling period. SNU-SG1 exhibited higher crop growth rate during late grain-filling period than HYVs, resulting in higher grain-filling percentage and non-structural carbohydrate re-accumulation in the stem at the final stage of grain filling. It is concluded that SNU-SG1 has a promising trait “functional stay-green” contributable to rice yield potential improvement through the improved grain filling.

Improving drought tolerance in maize: a view from industry

Significant yield losses in maize ( Zea mays L.) from drought are expected to increase with global climate change as temperatures rise and rainfall distribution changes in key traditional production areas. The success of conventional crop improvement over the past 50 years for drought tolerance forms a baseline against which new genetic methods must be compared. Selection based on performance in multi-environment trials (MET) has increased grain yield under drought through increased yield potential and kernel set, rapid silk exertion, and reduced barrenness, though at a lower rate than under optimal conditions. Knowledge of the physiology of drought tolerance has been used to dissect the trait into a series of key processes. This has been complemented by genetic dissection through the identification of QTL associated with these same traits. Both have been used to identify suitable organ- and temporal-specific promoters and structural genes. Phenotyping capacity has not kept pace with the exponential increase in genotypic knowledge, and large-scale managed stress environments (MSE) are now considered essential to further progress. These environments provide ideal settings for conducting massively parallel transcript profiling studies, and for validating candidate regions and genes. Genetic and crop physiological models of key processes are now being used to confirm the value of traits for target environments, and to suggest efficient breeding strategies. Studies of gene to phenotype relationships suggest that most putative drought tolerance QTL identified thus far are likely to have limited utility for applied breeding because of their dependency on genetic background or their sensitivity to the environment, coupled with a general lack of understanding of the biophysical bases of these context dependencies. Furthermore, the sample of weather conditions encountered during progeny selection within the multi environment testing of conventional breeding programs can profoundly affect allele frequency in breeding populations and the stress tolerance of elite commercial products. We conclude that while gains in kernels per plant can be made by exploiting native genetic variation among elite breeding lines, improvements in functional stay-green or in root distribution and function may require additional genetic variation from outside the species. Genomic tools and the use of model plants are considered indispensable tools in this search for new ways of optimizing maize yield under stress.