Nigarin Sultana,Shahidul Islam,Angela Juhasz,Wujun Ma*

State Agriculture Biotechnology Centre,Murdoch University,Perth,WA 6150,Australia

ABSTRACT Wheat leaf senescence is a developmental process that involves expressional changes in thousands of genes that ultimately impact grain protein content (GPC),grain yield (GY),and nitrogen use efficiency.The onset and rate of senescence are strongly influenced by plant hormones and environmental factors e.g.nitrogen availability.At maturity,decrease in nitrogen uptake could enhance N remobilization from leaves and stem to grain,eventually leading to leaf senescence.Early senescence is related to high GPC and somewhat low yield whereas late senescence is often related to high yield and somewhat low GPC.Early or late senescence is principally regulated by up and down-regulation of senescence associated genes.Integration of external and internal factors together with genotypic variation influence senescence associated genes in a developmental age dependent manner.Although regulation of genes involved in senescence has been studied in rice,Arabidopsis,maize,and currently in wheat,there are genotypespecific variations yet to explore.A major effort is needed to understand the interaction of positive and negative senescence regulators in determining the onset of senescence.In wheat,increasing attention has been paid to understand the role of positive senescence regulator,e.g.GPC-1,regulated gene network during early senescence time course.Recently,gene regulatory network involved early to late senescence time course revealed important senescence regulators.However,the known negative senescence regulator TaNAC-S gene has not been extensively studied in wheat and little is known about its value in breeding.Existing data on senescence-related transcriptome studies and gene regulatory network could effectively be used for functional study in developing nitrogen efficient wheat varieties.

Keywords:Wheat (Triticum aestivum L.)Senescence Nitrogen use efficiency NAC transcription factor Stay-green

1.Introduction

Wheat(Triticumspp.)is the world’s principal and commercially important food crop.It belongs to the family Poaceae (grasses),tribe Triticeae,genusTriticum[1,2].Its ability to self-pollinate greatly facilitated the selection of many distinct domesticated varieties.Global wheat consumption has increased in the past four decades to around 700 Mt annually and accounts for approximately 25% of worldwide protein supply [3].

Senescence is a highly coordinated developmental program in wheat with a significant impact on grain yield and protein content.It starts early after entering the reproductive stage,while after anthesis the whole plant undergoes the stage.Senescence process benefits plant aptness by confirming its best supply of nutrients to the sink organs to promote the adaptability of its offspring with their given environment.Upon senescence,leaf cells undergo dramatic physiological,biochemical,metabolic and transcriptomic changes in an orderly manner.During monocarpic senescence,up to 90% of the nitrogen is remobilized from the vegetative plant parts to the grain [4,5].Depending on environmental and genetic factors,the onset and rate of senescence determine the potential of nitrogen use efficiency (NUE) [6–8].Although the importance of leaf senescence has been reported for a long time,identification of major genes and their regulatory networks controlling leaf senescence is still lacking in the context of NUE.The regulation of leaf senescence is not straightforward,rather underlying a complex network of genes that are directly or indirectly associated with the senescence program.Among the different transcription factors involved in senescence regulation,NAC transcription factors have been studied extensively in barley,rice,and wheat.In this review,we will mainly focus on the regulation of senescence in wheat in the context of GPC,GY,and NUE.

2.Leaf Senescence

Senescence in plants occurs at various levels but most conspicuously at the final developmental stage of plant cells which represents a vital role in controlling degenerative physiological changes.There are two types of leaf senescence:mitotic or replicative senescence and postmitotic senescence [9].Mitotic senescence is the arrest of cell division or replication activity in apical meristematic tissue ensuing the retardation of growth of leaves and flowers.Postmitotic senescence occurs mainly in plant somatic organs such as leaves and floral petals.Once the cellular differentiation and maturation of these organs has completed,their senescence is followed by an active and programmed degenerative process [10].

Leaf senescence is a postmitotic senescence which involve an orderly disassembly of different macromolecules in response to various environmental signals and leaf age information under stringent genetic control [11–13].It is accompanied by a visible change in leaf color as a consequence of the breakdown of chlorophylls.During the development and growth stages,the leaf becomes photosynthetically competent and accumulate nutrients facilities,then enter the senescence phase immediately after the anthesis.Progression of senescence is followed by a decrease in rate of nutrient uptake and decline in photosynthesis activity while an increase in the degradation of RNA,protein,lipid and DNA degradation to be recycled into the sink organ which ultimately lead to the death of senescing leaf [14,15].About 80% of nitrogen contained in the grain is provided by the salvaging of chloroplast proteins during the senescence process[16,17].As leaf senescence is integrated with source/sink demands of the plant,a complex gene network with the coordinated action of plant hormone,nutrient availability,and stress response is involved in its regulation[18–21].

Senescence is significantly influenced by genetic and environmental factors.Abiotic(e.g.water and nutrient deficiency,extreme temperature,and day-length) and biotic environmental factors(e.g.pathogen infection)can influence the onset and rate of senescence which in turn can impact the nutrient remobilization from vegetative parts of the plant to the grain [10,22,23].For instance,the low N condition leads to accelerated senescence,while high N condition can delay senescence [24,25],because senescence depends on the balance between N availability and demand in the plant[26].Under optimal conditions,the interaction of genetic and environmental factors ensues senescence in an age-dependent way [27].Sequential senescence involves the degradation and transfer of nutrients from older leaves to younger leaves and other growing parts of the plant.Under abrupt change in environmental condition,plant can follow senescence of stressed leaf locally to acclimate with the environment for survival [10,28].

Under the control of external and internal factors,the gradual degradation of cells called programmed cell death (PCD) during leaf senescence starts from mesophyll and then propagates into the entire leaf area to remobilize the accumulated nutrients from leaf to seed [29].Primarily,PCD results in the structural changes of intracellular organelles such as chloroplast whereas the organelles required for gene expressions such as nucleus and mitochondria are degraded at the later stage of senescence for the proficient remobilization of nutrients until the end of a senescing leaf [30].Degradation and hydrolysis of chloroplast proteins such as ribulose biphosphate carboxylase (Rubisco) into amino acids is accompanied by the action of various endo-and exopeptidases metabolism of membrane lipid in the senescing leaves is escorted by enzymes like lipases,phosphatases,and hydrolases provide energy for the protein degradation process and remobilization of amino acids [31,32].A shreds of evidence showed that the continuous decrease in anabolic processes involved in nutrient assimilation while increase in catabolic processes involved in macromolecule degradation and nutrient remobilization along with the senescence progression [33].Degradation of chlorophyll content results in the decrease photosynthesis activity leading to the impaired carbon fixation and sugar transport to the developing seeds.In contrast,the enhanced degradation of protein facilitates its remobilization from leaf to seed ultimately balance the C/N ratio in seed.Increasing the length of photosynthetic activity during delaying the senescence process can result in an increased grain yield whereas increasing the rate of amino acid remobilization from degraded proteins during accelerated senescence can increase the GPC [34].Therefore,better performance of yield can negatively affect the GPC and vice versa[35,36].Hence,investigating senescence will provide valuable information about recycling of nutrients from vegetative parts to the grains,and may also lead to ways of manipulating senescence for improving grain yield,protein content,and NUE in wheat.

3.Leaf Senescence is a major determinant of efficient nitrogen remobilization

Monocarpic crop plant development starts with nitrogen uptake which stored temporarily in the vegetative parts as structural components or metabolites and ends up in seeds where nutrients are remobilized to in varying extents[37].The ability of plants to effectively remobilize N into the grains is of crucial importance for overall NUE.During leaf senescence,nitrogen along with potassium is remobilized more efficiently (drop by approximately 80%)compare to other elements such as Zn,Cu,Fe,S,Cr,P,and Mo(drop by over 40%)[4].Nitrogen is preferably remobilized in the form of amino acids and peptides which is also associated with carbon remobilization.Inorganic nitrogen molecules can be remobilized from the senescencing leaves to sinks in the form of nitrate,ammonium,and urea,induced by corresponding transporters[38–40].In general,the extent of nitrogen remobilized to the grain is believed to be controlled by the quantity of N available in the canopy,not by the grain need[41].Conversely,a study reported that wheat grainfilling is not source-limited under optimum conditions[42],and N remobilization was ceased once the developing ears were removed[43].Contribution ofNRT1.7andNRT2.5nitrate transporters are involved in remobilizing nitrate from the source leaves to the sink organs [44–46].DUR3urea transporter in senescencing Arabidopsis leaves contributes to the retranslocation of N from urea[38,39].Similarly,during the senescence progression,NRT1.6,NRT1.5,andAMT1.5transporters participate in the remobilization of nitrate and ammonium[47].Studies on proteolysis and nitrogen remobilization during leaf senescence revealed that the major remobilization processes involve the proteolysis of proteins like Rubisco into amino acid by proteases and their translocation to the sink organ.It is also known that,during the senescence process,genes associated with nitrogen transport and proteases showed up-regulated expression[40,48,49].Among the over 800 proteases identified in plant genomes,serine-proteases and Cysteineproteases (CysProt) have been reported as the most predominant enzymes associated with leaf senescence [49].However,due to the post-transcriptional regulation of some proteases [50] or inhibition of protease activity by endogenous inhibitors [51,52],detailed investigation is needed to identify the potential targets of proteases involved in nitrogen remobilization.In particular,members of the papain-like subfamily C1A CysProt are synthesized as inactive or little active precursors which are self-processed or processed by other enzymes to be activated.The pro-sequences play important modulatory role in the formation of mature enzymes.Although the interaction of plant protease-inhibitor in response to senescence has not been understood in detail yet,CysProt-cystanin interaction has been reported in barley [53],spinach[54],and some transgenic plants[55,56].Other protease inhibitors that mainly target serine proteases has also been reported to increase abiotic stress tolerance but still there is lack of information in senescence control.

The selection of plants with delayed or early senescence has appeared as an alternative approach to influence the length of nitrogen uptake and remobilization [57].During the progression of leaf senescence,the reduced photosynthetic activity and active protein degradation change the C/N ratio which ultimately affect the GPC and GY.The C/N ratio is influenced by start and end of senescence timing as well as by its duration[5,22,35,58].At maturity,how much N will be remobilized is predominantly determined by the amount of N taken up by the plant before anthesis[59],and this N uptake is the key factor determining nitrogen remobilization in wheat [60].Usually,decrease in N uptake during grain filling in turn could enhance N remobilization from leaves and stem,ultimately leading to leaf senescence.In wheat,NAM-B1,a close homolog of ArabidopsisAtNAPhas been reported to have a strong influence on precocious senescence and increased nitrogen remobilization and grain filling efficiency [61].Recently,NAC-Sgene in wheat has been reported to have a positive correlation with delayed senescence and GPC without a significant reduction in GY[62].Using the dataset(NCBI Sequence Read Archive(SRA)submission:SUB7918524)of our own transcriptome study[63],senescence related gene network analysis (unpublished results) of leaf samples collected at three stages of senescence (flowering time,10 and 20 days post anthesis (DPA)) from three wheat cultivars showed a clear clustering of nitrogen metabolism related genes(Table S1) along with major senescence related genes i.e.AtNAP,VRN2,ELF3,EIN3,NAMB1,stay green,etc.The highest number of nitrogen metabolism genes was found in the NAP cluster followed by the stay green gene cluster and VRN2 cluster.These gene clusters represent genes with molecular functions primarily related to the senescence initiation.Similarly,higher enrichment of nitrate transporters (High affinity nitrate transporters,NRT/PTR 5.5 and NPF 4.1 families) in these clusters confirms the relationship between early senescence stages and changes in nitrogen metabolism.Therefore,study the regulatory mechanism of the senescence-associated genes with an impact on nitrogen remobilization can be a potential approach for wheat improvement.

4.Stay-green phenotype and early senescence phenotype in relation to NUE

Improving NUE is a major challenge in modern biotechnological era which consists of with two key problems for crop yield and fertilizer application.The first is that traditional breeding strategies to improve crop plants may have reached a plateau.The second is that further increases in N application may not result in yield improvements but will lead to serious environmental problems[64–67].Global N fertilizer consumption is increased by 1% every year [68].It has been estimated that the crop plant can use only 30%–40%of the N supplied,while the remaining losses by evaporation and leaching [69].Thus,to increase crop production with less expense,it is important to develop cultivar with improved NUE.Many studies have identified the key traits to improve nitrogen uptake and utilization efficiency of wheat cultivars.Factors affecting nitrogen uptake efficiency include root architecture,expression of more proficient transporters (HATS and LATS belonging to the NRT2 and NRT1/NPF gene families),genes that play a role in regulating root architecture,better storage,and assimilation [70–72].Deep-rooted genotypes are able to take out water from deep soil thus showing ‘Stay green’ trait,making plants to have a delayed senescence allowing a longer period of photosynthesis [73–75]and increased N uptake ability from the soil during grain filling[76].On the other hand,NUE can be affected by a number of physiological traits such as the effect of N on carbohydrate partitioning,active carbon fixation during grain-filling,the storage of N,and the remobilization of N from senescent tissues [69,77,78].Among these,delaying the process of leaf senescence during grain-filling is probably the most promising one for NUE improvement as it would increase the amount of fixed carbon available for grainfilling whilst utilizing the same amount of nitrogen [62].

Stay-green is a term used to describe genotypes with extended foliar greenness during grain filling,associated with greater aboveground biomass,grain protein,and yield production.It has been suggested that stay-green genotypes with slower senescence would provide a longer grain filling period led to assimilate and uptake more carbon and nitrogen than early senescence genotypes.Improved the nutrients uptake and accumulation by stay-green phenotypes is a result of greater biomass accumulation during grain filling phase in response to increased sink demand.Hence,stay-green genotypes are capable of superior nutrient uptake and accumulation,which ultimately improve the NUE [79,80].Several studies have been conducted to investigate the genetic variation of stay-green phenotypes on NUE in Sorghum [81,82],Maize[83,84],barley [85],and wheat [75,86].The total photosynthesis across the whole plant cycle can be improved by extending the duration of active photosynthesis [87–89].The stay-green phenotype is classified into five types based on the onset and the rate of leaf senescence during the grain filling [90].In Type A staygreens,senescence starts at late but then progresses at a normal rate whereas in Type B stay-greens,senescence is initiated on time with a slower rate of senescence progression.Type C stay-green maintains the chlorophyll indefinitely resulting from lesions but lack photosynthetic capacity whereas Type D stay-green due to rapid leaf death by freezing,boiling or drying.In Type E,the leaves of the stay-green genotypes tend to be greener at maturity than those of the early genotype.In general,therefore,only types A and B could consider as functional stay-green due to maintaining photosynthetic capacity during the grain filling phase.Positive corelation between the stay-green phenotype and yield under stress and non-stress conditions has been reported in wheat[75,91–93].Negative effects of stay green phenotype on yield have also been reported[94].In winter wheat,a positive relationship of flag leaf stay-green duration and harvest index with water use efficiency has shown during grain development [95].The late-season retention of chlorophyll and decreased in photosynthetic capacity has been resulted in a longer grain filling period and delayed senescence with higher grain yield [96].In contrast,selecting wheat genotypes with early flowering or/and short-grain filling period could improve productivity due to the avoidance of some unfavorable seasonal conditions [97].Several lines of studies reported that the functionalNAM-B1gene can accelerate senescence and increases nutrient remobilization,resulting in a higher grain protein content [61,99,100].However,this gene was also reported to shortenthe duration of active photosynthetic and grain filling period,and thus reduce grain yield as a consequence of enhanced senescence [101].A recent study usedNAMRNAi plants with delayed senescence has found that starch accumulation and final grain yield is determined by grain filling capacity rather than the duration of photosynthesis [102].Taken together,we believe that identification of various senescence associated genes and their regulatory mechanism in wheat cultivars can facilitate wheat improvement.

5.Senescence is linked to GPC and GY in wheat

Inheritance of GPC and GY traits has been reported in several studies on monocarpic crop like wheat[103].Several pieces of evidence from different studies proposed alternative strategies to improve GY and GPC simultaneously [104,105].In wheat,an intense reliance of GPC on post-anthesis nitrogen uptake and nitrogen remobilization efficiency has been reported [94].Although for a long time,high GPC has been the main emphasis for breeding[98],the progress is not satisfactory [10].Firstly,GPC is not only controlled by genotype but also by the environment,in which the wheat is grown such as timing and dosage of nitrogen application,availability of water,macro-and micronutrients and temperature during plant development,in particular during the grain filling period [106,107].Environmental factors,especially the higher temperature and water stress during grain maturing may result in a relative increase in GPC together with a loss of starch accumulation.Thereby,the selection of progeny with high GPC from a heterogenous segregating population is challenging[108,109].Secondly,a negative correlation exists between GY and GPC [110,111].This relationship is a consequence of interactions between nitrogen and carbon metabolism,which finally affect GPC or GY respectively [77].In wheat,a large proportion of the final grain yield is derived from post-anthesis assimilation and translocation of metabolites,particularly C and N [112].Thirdly,GPC is largely dependent on the additional provision of nitrogen fertilizer,optimization of N management and selecting cultivars with high N uptake and utilization efficiency [79,113-115].Increase use of nitrogen fertilizer dramatically raises the cost as well as the use of N fertilizer from 12 to 104 Tg per year in the last 40 years with significant and undesirable impacts on the environment globally [66].Therefore,wheat breeders have been searching for approaches to enhance GPC in wheat grain,without using extra nitrogen input.This indicates that new solutions are needed to obtain potential yields while maintaining substantial GPC.Senescence starts immediately after anthesis and its progression is interrelated with duration and activeness of photosynthesis,thereby both grain yield and GPC are strongly influenced by senescence.Although progression of senescence in the whole plant is not uniform,it starts in the older and bottom leaf layers and then continue upwards and usually end up with the senescence of flag leaf [116].In wheat～18% of the kernel N per spike [117] and～50% of the assimilates for grain filling [118] are contributed by flag leaf.However,the overall physiological process of plant is also affected by adverse environmental conditions such as drought,heat or nitrogen stress which ultimately affect GPC and GY.A number of studies have been reported a positive relationship between stay-green phenotype or late senescence and yield [75,92,119]whereas a few reports has been demonstrated on negative influence of late senescence on yield [94,120].Late or early senescence in controlling GPC and GY has also been influenced by field condition.Alhabbar et al.[121] reported thatNAM-A1aallele in wheat accelerates senescence in the Mediterranean conditions and thus can improve grain yield through the efficient nitrogen utilization by reducing the grain filling period and grains start to ripe before the arrival of unsuitable seasonal conditions in Western Australia.Because the inadequate water during the dry season negatively affect nitrogen remobilization from vegetative part to grain.Further study with largescale phenotyping of stay-green and early senescence phenotypes under different field conditions could enhance our understanding of the influence of senescence mechanisms on grain yield and GPC.A number of quantitative trait loci(QTL)studies identified various loci which are related to GPC associated traits and thus provide effective tools for breeders to improve GPC in wheat[122–124].The allele for high GPC and accelerated senescence,GPC-B1orNAM-B1was first identified in wild emmer wheatTriticum turgidumssp.Dicoccoides[125].Functional characterization ofGPC-1genes in wheat revealed that silencing of all homoeologousGPC-1genes exibits a significant delay in senescence and a reduction in grain protein as well as zinc and iron content[61,125–127].Recently,overexpression of late senescencerelatedTaNAC-Sgene in transgenic bread wheat lines showed an increase in grain protein concentration without yield penalty[128].Therefore,genes that are primarily related to senescence can be explored to improve GPC and GY in wheat.

6.Regulation of leaf senescence in wheat

The survival strategy and death of monocarpic plants is correlated with their reproduction which is critically controlled by developmental programme[129].However,environmental signals have an influence on flowering time [130].Thereby,the onset of leaf senescence in monocarpic plants is ultimately determined by the integration of developmental programme and environmental signals.The transcriptomic studies of leaf senescence in Arabidopsis,wheat,rice,maize,barley,switchgrass,and many other plants have revealed that the sequential events occurring during senescence is finely regulated in a coordinated manner [131-134].During senescence,a large number of genes showed up–regulated expression are called the senescence associated genes (SAG) or positive senescence regulators.Positive senescence regulators must exist for senescence to proceed.Identification of SAGs in different crop plants and their expression analysis reveal that a complex regulatory mechanism is involved in the regulation of senescence [132,135,136].On the other hand,a number of genes works against the ageing process by down-regulating their expression and are called negative senescence regulator.The negative senescence regulators are also important to prevent senescence from occurring prematurely.Many of these regulatory elements may contribute imperceptibly in the senescence phenotype due to redundant functions in senescence.

In general,a basic senescence process is followed across the plant species,although there are differences between wheat and dicots in regard to secondary metabolite formation.In wheat,leaf senescence has been studied with regard to GPC,which is a major determinant of wheat quality and affects its demand in international market [137].The major quantitative trait locus (QTL) on chromosome 6B for grain protein content in tetraploid wheat also explored the association ofGpc-B1(encodes a NAC transcription factor) with senescence-related traits such as flag leaf chlorophyll degradation and change in peduncle color [36].In addition,the effect of genetic variation inNAM-A1andNAM-B1genes in Australian wheat varieties in relation to senescence and NUE has also been studied recently [121,138].Transcriptome study by Borrill et al.[8] showed that flag leaf and peduncle senescence is significantly delayed in NAM-A2/NAM-B2 double mutants and thus the genes that are closely interacted with NAM-A2 and NAM-B2 in gene network analysis can have significant regulatory role on senescence.In wheat,immediately after anthesis a large number of genes start to be down-regulated in wheat flag leaf which are mainly associated with photosynthesis,carbohydrate and amino acid metabolism,translation,ribosomal RNA processing,hormone biosynthesis and signalling (e.g.ABA biosynthesis,salicylic acid biosynthesis,ethylene signalling,JA signalling,response to cytokinin) [8].At about 10 DPA,a large number of genes start to be up-regulated,which are predominantly associated with remobilization of protein component(e.g.vesicle-mediated transport and proteasome) and transport process (e.g.nitrogen transmembrane transport,amino acid transport,phosphate transport).At the later stage of senescence,the number of down-regulated genes becomes decreased and the up-regulated genes increased.The up-regulated response of hormones like ethylene was observed at the later stages of senescence [8].However,the rate of senescence and its molecular nature is strongly influenced by various environmental(e.g.low and high temperature,nutrient availability,drought,pathogen attack,low nitrogen)and internal factors(e.g.hormones,reproductive development)which are integrated into developmental age-dependent senescence phases.A senescence regulation model (Fig.1) has been established based on the available literature,showing a complex regulatory network is maintained in determining the onset and rate of senescence through crosstalking of senescence-associated genes influenced by external and internal factors as well as genetic variation.These regulatory pathways up-or down-regulate distinct sets of senescenceassociated genes,such asNAC1andWRKY,to determine the onset of senescence [8,23,139].

6.1.Environmental regulation

The senescence process including senescence rate and molecular nature is intimately influenced by various environmental factors.The environmental cues that affect leaf senescence include stresses such as high or low temperature,drought,ozone,nutrient deficiency,pathogen infection,and shading,etc[23].Any environmental stress with a negative consequence of growth conditions may result in the induction or enhancement of plant senescence.However,little information is available about the regulatory pathways involved in mediating the environmental stress signals.Recent studies proposed that the complex regulatory networks involve hormones and genes in integrating environmental signals into developmental senescence [140,141].Still,it is not clear that to what extent the regulatory pathways like age-dependent,developmental senescence,and environmental stress-activated are kept distinct and where they converge for different senescence stimuli e.g.oxidative status,sugar or hormone action.It is likely that the convergence occurs upstream of the gene expression which is involved with the mechanisms of cellular and macromolecule breakdown leading to nutrient recycling.Plant internal systems or factors like different hormones might sense the environmental stress and translate them into molecular signal leading to the expression of relevant genes including the senescence associated genes(SAGs).In a study,the induction of senescence initiation process by developmental ageing or by different factors(hormones or stresses) showed divergence in gene expression profile of SAGs.However,at the later stage of senescence,a large number of SAGs showed convergence in their expression induced developmentally or by different treatments [142].Therefore,many transcriptional changes due to environmental signals overlapped with those observed during senescence [143,144].

Fig.1.A model for senescence regulation.Multiple factors can induce the expression of negative senescence regulators related to active photosynthesis and chlorophyll biosynthesis prior to the start of senescence.Co-induction of positive and negative senescence regulators at the onset of senescence and photosynthesis activity become declined.Dominance of positive regulator results in active aging and degradation program and decline in photosynthesis and chlorophyll biosynthesis.

A number of transcriptome studies showed that NAC is the major TF family that significantly up-regulated in integrating the abiotic stress signal [145].In Arabidopsis,a senescenceassociated regulatory network involvingANAC092/ORE1which induce aging-related cell-death by integrating various environmental signals (e.g.salt stress,drought,light/dark) via feed forwarded loop comprises Ethylene Insensitive2 (EIN2),ANAC092/ORE1and microRNA164(miRNA164) into the developmental program [146].The expression ofANAC092/ORE1is negatively regulated by at the post-transcriptional level.At the later stage of senescence,down-regulated expression ofmiRNA164induceANAC092/ORE1which directly activate chlorophyll catabolic genes(CCGs) and SAGs as well as by inhibiting the role ofGLK.1i.e.,GOLDEN2-LIKE1via interacting with its promoter[147].Under high salt concentration,the transcriptional repression activity ofVNI2is compromised but the transcriptional activation activity becomes induced,contributing plant fitness to an enhanced salt stress [8].VNI2gene is related to delaying senescence by inducing the activity ofcold-regulated (COR)and responsive to dehydration(RD)genes in an ABA-dependent manner.Transcriptome study under N stress condition by Sultana et al.[63] showed that different wheat cultivars can adapted to N stress by down-regulating the expression of photosystem II 10 kDa,chlorophylla–bbinding protein,catalase,cytochrome P450 family protein,gibberellin receptor GID1A,cytokinin oxidase/dehydrogenase,and methyltransferase predominantly.In contrast,the up-regulated genes associated with N stress adjustment involved RADIALIS-like transcription factor,glycosyltransferase,placenta specific 8 (PLAC8) family proteins,aminotransferase,plasma membrane ATPase and CRT-binding factor mainly.As discussed above,genes important for nitrogen stress tolerance can also be the potential candidate for the regulation nitrogen stress dependent senescence because of the convergence between stress induced and age dependent senescence.

6.2.Hormonal regulation

Phytohormones play a major role in senescence control through the integration and synchronization of environmental signals with plant developmental process [27].A considerable number of studies have revealed that various biotic and abiotic stress-responsive phytohormones,including auxin,ethylene,abscisic acid (ABA),cytokinin (CK),gibberellic acid (GA),salicylic acid (SA),brassinosteroids (BRs),strigolactones (SLs),and jasmonic acid (JA) paved their regulatory role all the way started from early to later stages of senescence.Senescence process is facilitated by ethylene,JA,ABA,SA,BRs,and SLs whereas auxin,GA,and cytokinins(CKs)contribute to retardation of senescence.

Ethylene hormone is known to contribute in regulation of a wide variety of plant developmental processes include cell division and elongation,senescence,and biotic and abiotic stress responses[148,149].Ethylene hormone is known as a positive regulator of leaf senescence which signals to its receptors to induce the senescence promoting genes while suppressing the senescence delaying genes[150–152].Senescence timing can be controlled by inserting mutation in ethylene-responsive genes.Ethylene insensitive2(EIN2) plays important role in crosstalk of other hormones with ABA and MeJA in controlling senescence [153].In Arabidopsis,increased expression ofEIN2induce directly the up-regulated expression of positive senescence regulatorANAC092/ORE1.AlthoughORE1expression is negatively regulated by MicroRNA(miR164),increased expression ofEIN2with the progression of senescence gradually down-regulate miR164 expression which leads to the up-regulatedORE1expression.Thus,EIN2also indirectly up-regulate ORE1 expression through the down-regulated expression ofmiR164[146].Similarly,during leaf aging,accumulation of ethylene results in the activation of Ethylene-Insensitive 3(EIN3),a positive regulator of leaf senescence [13].The increased expression ofEIN3can inhibitmiR164which in turn promotesORESARA 1(ORE1) expression.The increased expression ofORE1andEIN3can regulate the major senescence-associated genes likeNAC083,NAC102,andSAG29using the facility of motifs present in their promoter [146,154,155].Among the 1314 target genes ofEIN3reported,269 are SAGs.In response to environmental signals,ethylene receptors can transfer the signal toEIN3and its homologEIL3,which in turn activates a series of genes i.e.ERFs,EREBPthat contribute to the regulation of senescence [156].

Jasmonic acid is a lipid derived phytohormone which play important role in promoting leaf senescence[157,158].Exogenous application of MeJA can induce the expression ofSAGsleading to the prompt degradation of chlorophyll content and decreased photosynthesis activity [159].In rice,RNAi knock-down experiment showed thatOsDOSis a negative regulator of senescence which can integrate JA induced signal into the developmental senescence[160].Teosinte Branched/Cycloidea/PCF4(TCP)transcription factor play a bifunctional role in integrating JA signal into senescence.Senescence can be repressed by the decreased accumulation of JA as a consequence of the repressed biosynthesis of Lipoxygenase2(LOX2)by the activation oftcp9andtcp20[161].In contrast,senescence can be accelerated by the increased level of JA resulted from the activation ofLOX2biosynthesis bytcp4[162].Recently,in wheat,a novel WRKY-type promoter of leaf senescence,TaWRKY42-B,has been identified which accelerate leaf senescence initiation by promoting JA biosynthesis via interaction withAtLOX3and its orthologTaLOX3(TraesCS4B02G295200)[163].JA production showed down-regulation at the early stage of aging and later integrating the signals from external and internal factors eventually leading to the onset of senescence.Salicylic acid is(SA)is a phenolic phytohormone,which plays major role in regulating developmental leaf senescence as well as biotic and abiotic stress responses [164].In Arabidopsis,during the progression of senescence when the degradation of chlorophyll is predominantly higher,a significant increase in SA indicates the involvement of SA at later stages of senescence [131].Approximately 60% of SA biosynthesis and signalling genes were up-regulated in senescing leaves of Arabidopsis[165].Also,the exogenous SA treatment promotes expression of many SAGs,including WRKY transcription factors WRKY6 [166],WRKY53 [167],WRKY54 [168] and WRKY70[169].Leaf senescence is also regulated by SA by means of its influence in lipid metabolism[170],autophagy[171],and production of ROS[172].Besides senescence progression,SA also influence onset of senescence by consecutive induction of positive and negative senescence regulators [168].

Abscisic acid is a sesquiterpenoid (15-carbon) hormone which is involved in regulation of substantial number of plant growth and developmental processes comprising leaf senescence,regulation of shoot and root growth,seed germination,response to biotic and abiotic stress [173,174].Abscisic acid is known to have positive regulatory role during leaf senescence [136,175].Under the environmental stressed condition such as high salt concentration,drought,extreme temperature,expression ofSAGscan be induced by an increased level of ABA in leaf.In Arabidopsis,ABA inducible receptor-like kinase (RPK1) gene showed up-regulated expression with the senescence progression [23].A number of studies reported the underlying mechanism of ABA-induced accelerated senescence.Exogenous application of ABA can increase the accumulation of H2O2[176].ABA can also take part in senescence progression and completion in an age-dependent manner by inducing the expression of superoxide dismutase (SOD),ascorbate peroxidase (APOD),and catalase (CAT) like anti-oxidative enzymes[165].ABI5can positively regulate ORESARA1 (ORE1),NAC-like,activated by APETALA3/PISTILLATA (NAP) [155,177,178].NAPcan directly induce ABA biosynthesis gene abscisic aldehyde oxidase(AAO3)in accelerating the senescence process[177,178].Alongside up-regulating the expression of senescence-associated NAC TFs,ABA can promote activation ofMAPKKK18,MKK3,andMPK1/2/7.In Arabidopsis,the inactivation ofMAPKKK18showed delayed leaf senescence [179].ABI5can also control the expression of genes involved in the degradation of chlorophylli.e.non-yellow coloring1 (NYC1) and stay-green1 (SGR1) [180].In wheat,under water stressed condition,increased expression of ABA has been implicated in accelerated rate of carbon remobilization and grain filling[181].Taking together,ABA is crucial in regulating leaf senescence through integrating the environmental signals with developmental stages.

Brassinosteroidsare polyhydroxysteroids,well known to play a vital role in regulating plant growth and development,biotic and abiotic stress response and senescence [182].BRs are sensed by the brassinosteroid-insensitive 1 (BRI1),a member of the leucine rich repeat receptor kinase(LRR-RK)family[183].Brassinosteroids increase chlorophyll breakdown and thereby accelerate senescence[184]while BR-deficient.Mutants show delayed senescence[185].Moreover,effect of exogenous application of epibrassinolide (eBL)in excised wheat leaf segments has been reported.Application of high eBL doses accelerate leaf senescence through the increased chlorophyll degradation and peroxidase activity while lower doses are not able to accelerate the senescence process [184].The BRI1(BR insensitive 1) null mutants exhibit declined level of SAG transcripts and thus show extended lifetime [186] whereas the BRI1-EMS-suppressor 1 (BES1) shows hastened senescence as a result of up-regulation of BR response pathway [187].The degradation of the central BR signal regulators brassinazole-resistant 1 (BZR1)andBES1byORE9gene encoding an F-box protein results in precocious senescence [188].BZR1can positively regulates the expression ofWRKY57which can repressSAG4andSAG12genes result in delayed senescence [189].BES1andBZR1transcription factors also have been reported to bind to the promoters of BR-regulated target genes likeNAC002,NAC019,NAC055,andNAC072[188].BES1transcription factor can directly repress two related transcription factors,golden2-like 1 (GLK1) and golden2-like 2 (GLK2)transcription factors that are required for chloroplast development and induce expression of genes related to photosynthesis and lightharvesting [189–191].Transcription factorBZR1coordinates the BR and GA signalling through negative interaction withRGA(repressor of ga1-3) to control senescence [192].

Strigolactones are a group of terpenoid lactones which play role in regulation of seed germination,shoot branching,and senescence[193–196].SL biosynthesis is largely influenced by nutrient availability such as phosphate and nitrate [197].Strigolactone receptor DWARF 14 (D14)belongs to the alpha/beta hydrolase family and has a significant role in SL perception [193–195].The SL ligandbound D14 protein forms a signalling complex with an F box leucine-rich repeat-containing protein MAX2 (for More Axillary Branches2/ORE 9)and various target proteins leading to the transduction of the hormonal signal and to the polyubiquitination and proteasomal degradation of target proteins [198].The target proteins of this complex include D53 (DWARF 53),Suppressor of MAX 1-like genes(e.g.SMAXL6,SMAXL7,SMAXL8)which are ortholog of D53 in rice,and BES1.BES1is a positive regulator of BR signalling which is primarily suppressed byMAX2[199].Degradation of BES1 and suppressor of MAX 2-like can lead to retardation of shoot branching and delay in senescence [199,200].In wheat,a positive relationship of SLs with drought and its role in drought tolerance has been reported [201].Similar to drought stress,in our recent transcriptome study,a positive relationship of SLs with nitrogen stress has also been observed (https://researchrepository.murdoch.edu.au/id/eprint/58315/) (unpublished results).Thus,further research on strigolactone-brassinosteroid perception(Fig.2) can open a new door under nitrogen stress related senescence regulation.

Cytokinins,known as adenine-or phenylurea-based chemicals,are broadly involved in regulation of plant growth and developmental processes [202] as well as in various stress tolerance[203].Cytokinin hormone acts as a negative regulator of senescence by impeding the degradation of chloroplast [204].Rather than controlling senescence initiation,it controls the progression of senescence [205].Over-expression of CK biosynthesis gene isopentyl transferase (IPT) under the control ofSAG12promoter can delay the age-dependent senescence process[206].The underlying mechanism behind the CK induced senescence retardation involves induction of hexose uptake and transport.At the site of phloem loading,sucrose can be hydrolyzed by extracellular invertase into the hexose monomers and promote the transport of monomers to the sink tissue to increase sink-strength[207].In wheat,interaction of cytokinin and nitrogen metabolism has been revealed.In wheat cultivar with functional stay-green phenotype,application of cytokinin showed positive correlation with grain yield under normal nitrogen condition due to delayed senescence [181].

Gibberellic acid is a tetracyclic di-terpenoid compound widely known for its regulation on flowering,seed germination,cell elongation,senescence,environmental stress tolerance [208,209].GA biosynthesis gene GA Requiring1 (GA1) can negatively regulate the expression of DELLA proteins.In presence of GA,DELLA can be proteosomally degraded by GA receptor genes such asGibberellin Insensitive Dwarf1(GID1) which ultimately results in the down-regulation of known molecular marker of senescenceSAG12andSAG29.While in the absence of GA,DELLA repress the GA signalling pathway [210,211].

Fig.2.Strigolactone-brassinosteroid perception of senescence regulation.D14 binds with MAX2 in presence of SL.Binding of MAX2-D14-SL complex with target proteins like D53,BES1,and SMAXL6,7,8 can lead to the proteasomal degradation of target proteins.Degradation of target proteins related to accelerated senescence and shoot branching ultimately can result in repression of shoot branching and delay in senescence.

Auxin hormone has diverse regulatory role on plant growth and development and is known to negatively associated with senescence [23,212].Although during the senescence progression,its biosynthesis is temporarily up-regulated which activate genes i.e.tryptophan synthase (TSA1),IAAld oxidase (AO1),and nitrilases(NIT1-3) involved in Indole-3-acetic acid (IAA) [165].Expression ofWRKY 57can be repressed by auxin which in turn can inhibitSAG12expression leading to late senescence.

Although a considerable study has been done in understanding the leaf senescence regulation by different hormones,the complex cross-talking among the hormones involved in different signalling pathways need to be further explored.

6.3.Molecular regulation

Although the apparent symptoms of leaf senescence appear similar,the molecular nature of the senescence state influenced by environmental and internal factors might be distinctive [213].During the onset and progression of senescence,several thousand genes are differentially expressed [7,132].Senescence-associated gene-regulatory networks in Arabidopsis,rice,and wheat have uncovered the significant role of genes in the transcription factors family NAC and WRKY [8,28,214,215].In Arabidopsis thaliana,approximately 2500 genes transcribed during leaf senescence have been identified (～130 are transcription factors) [131].In Wheat approximately 52,905 high confidence genes are expressed(～2210 are TFs) during senescence,of them 9533 are identified as differentially expressed gene (DEGs) (～341 are TFs) [8].

Senescence regulatory network in wheat comprises the differentially expressed TFs mainly enriched in NAC (61),MYB-related(43),WRKY (27),and AP2/EREBP (16).During the senescence program,some genes’ expression is higher at an early stage whereas others at a later stage.Also,the expression of some genes decreases gradually whereas others increase gradually,and some genes expression is consistently high or low during the time course.During the senescence process in wheat,a set of genes are upregulated at the earlier stages of senescence involved RWP-RK,pseudo-ARR-B,and CCAAT_HAP2 (NF-YA) families whereas the genes up-regulated at the later stages involved CAMTA,GRAS,MADS_II,and NAC.Along with the senescence promoting TFs,a decent number of genes also work against the aging process showed down-regulated expression with senescence progression.TFs such as AS2/LOB,bHLH,TCP,and MADS_I were predominantly down-regulated at the earlier stages whereas C2C2 GATA,GARP G2-like,and MADS_II TFs down-regulated at the later stages of senescence.However,in the senescence regulatory transcriptome study,none of the differentially expressed TFs were significant for down-regulated expression over the early to late senescence time period [8].

Taken together,transcriptional networks involved different transcription factors that are directed by different plant hormones represent age-dependent mechanisms of senescence regulation.

6.4.Regulation of leaf senescence by WRKY transcription factors

TF family WRKY is known to have an important regulatory function in onset and progression of leaf senescence by inducing expression ofSAGs.In Arabidopsis,WRKY53is a positive regulator of senescence of which the expression is repressed byWHIRLY1.WHY1 protein can be accumulated as a consequence of chloroplast degradation during senescence progression [167].In contrast toWHY1,transcription factor REVOLUTA(REV)can induce expression ofWRKY53and knockdown ofREVcan result in delayed senescence[216].WRKY53also induceORE9gene which contribute to accelerated senescence through the disruption ofBES1andBZR1regulated signalling pathway.Recent transcriptome study by Borrill et al.[8]reported 27 differentially expressed WRKY TFs during senescence(Table S2).Among them,14 WRKY TFs showed up-regulated expression and 13 WRKY TFs showed down-regulated expression with the senescence progression [8].In wheat,TaWRKY42-Bhas been identified as a novel positive regulator of leaf senescence which is functionally conserved withAtWRKY53in the initiation of age-dependent leaf senescence.Overexpression ofTaWRKY42-Bin Arabidopsis and silencingTaWRKY42-Bin wheat has revealed the role ofTaWRKY42-Bin inducing the of developmental and dark-induced leaf senescence.Interaction ofTaWRKY42-BwithTaLOX3,a JA biosynthesis gene,lead to the accumulation of JA which ultimately results in premature leaf senescence in a JA dependent signalling pathway [163].Another WRKY TF gene known asWRKY6showed up-regulated expression during the senescence progression.WRKY6can regulate many senescenceassociated genes including senescence-induced receptor-like kinase (SIRK) and pathogenesis-related genes [217].In wheat,TaWRKY7shows up-regulation during the natural leaf senescence process.The ectopic over-expression ofTaWRKY7in Arabidopsis(Arabidopsis thaliana) drastically speed up early leaf senescence under darkness treatment [218].TaWRKY7expression is also induced under drought stress[219].During the progression of leaf senescence,expression ofTaWRKY40-D,a novel WRKY transcription factor,is increased.Virus-induced gene silencing ofTaWRKY40-Din wheat shows a stay-green phenotype,whereas its overexpression in Arabidopsis shows early leaf senescence by modifying the biosynthesis and signalling of genes involved in JA and ABA pathways [220].

6.5.Regulation of leaf senescence by NAC transcription factors

The NAC gene family name was derived from the names of three transcription factors:(i) NAM (no apical meristem,Petunia),(ii)ATAF1–2,and (iii) CUC2 (cup-shaped cotyledon,Arabidopsis)[221,222].NAC protein family members are highly conserved at the N-terminal NAC DNA binding domain consisting of approximately 160 amino acid residues and have a highly variable Cterminal domain which do not contain any known protein domains[222].This variable C-terminal domain of NAC proteins generally operates as a functional domain and acts as a transcriptional activator or repressor [223–225].The NAC transcription factors are multifunctional proteins with various roles in the plant life cycle,such as maintenance of the shoot apical meristem[224],cotyledon development [221],lateral root development [226],flower formation [227],hormone signalling [228],response to pathogen infection [222,229] plant organ senescence[132],embryo development [230],response to different abiotic stresses[223,229],seed development [231],and senescence [61].

NAC is the largest TF family that plays a crucial role in the regulation of leaf senescence [232,233].In Arabidopsis,about more than 100 NAC genes have been reported to have a changing pattern in expression along with senescence.Among those NAC transcription factors,>30 genes e.g.ANAC016,ANAC029/AtNAP,ANAC059/ORS1,andANAC092/ORE1exibit increased expression whereas relatively less number of genes likeANAC042/JUB1andANAC083/VNI2show decreased expression during the progression of senescence[234].Also,ANAC019,ANAC055,andRD26(ANAC072)were reported as senescence-associated NAC TFs due to their differential expression during senescence [131].

In rice,NAC transcription factors,[235],OsNAC5[231,236],OsNAC6[229],OsORE1[237],ONAC106[238],andOsNAP[239]are known to be associated with senescence program.Among them,OsNAPandONAC106can directly control the expression of SAGs,OsSGR,OsNYC1[239].OsNAPin rice is a homolog ofAtNAP,which positively regulates leaf senescence in an ABA-dependent manner.Studies showed thatOsNAPdirectly induces the expression of genes involved in chloroplast degradation and nutrient transport.Overexpression ofOsNAPin plants showed an earlysenescence phenotype and knockdown ofOsNAPresulted in a significant delay in leaf senescence complemented by a prolonged grain-filling phase and consequently an increased grain yield (of up to 10%) [239].In addition,in rice expression of the ABAdependent NAC gene,OsNAC5is related to Fe,Zn and amino acid remobilization from source to sink tissue and gradually increased during senescence [231].

Among the 168 NAC genes identified in wheat[240],NAM-B1is most closely related to the Arabidopsis NAC proteinsANAC025,NAM/NARS2(ANAC018) andNARS1/NAC2(ANAC056) showed increased expression during senescence and mediate remobilization of nutrients from flag leaves to ears during grain filling [61].Three allelic variants ofNAM-B1are known,including the wild,mutated and deletion type [241].The functional allele ofNAM-B1increases grain protein,zinc and iron content but reduces the grain filling period (accelerate senescence),resulting in a decreased grain weight in some genetic backgrounds [101].The wild emmer wheat(Triticum turgidumssp.Dicoccoides),which carry a functionalNAM-B1allele,is recognized as one of the rich sources of genetic variation in GPC.However,in most of the modern wheat cultivars,theNAM-B1gene is absent or non-functional(mutated),which has been reported to have influence in delaying senesce [99,242,243].Studies onNAM-B1gene using different growth environments and genotypes revealed a positive correlation of increased GPC with accelerated grain filling [36].However,wheat cultivars showed high GPC had a trivial yield penalty that might be due to a decreased level of carbon supply to the root to uptake N as the photosynthesis period became shortened [244].The level of this negative impact is depended on the genetic background and the environment [245–248].On the other hand,knock outNAM-B1is associated with delayed senescence and reduced GPC with no penalty in grain yield [61,126].As gene regulation is a complex process,early or late onset of senescence can be regulated by many genes instead of singleNAM-B1gene.Thus,deviation from the negative relation between GPC and Grain yield is becoming one of the major goals of current wheat improvement research.

A recent transcriptome study on senescence regulatory network revealed that Arabidopsis orthologANAC029,ANAC082,andANAC090showed up-regulation during the senescence progression.However,SAG12,ANAC092/ORE1,andJUB1were not differentially expressed[8].In Arabidopsis,ANAC092/ORE1is a key positive regulator in leaf senescence which can inhibit chlorophyll biosynthesis by interacting with Golden2-Like1 (GLK1) and Golden2-Like2 (GLK2) through its C-terminal region [249].In addition,induced by phytochrome-interacting TFsPIF4,PIF5,EIN3,andABI5orEEL,ORE1can accelerates chlorophyll lost by directly activating the transcription of Chlorophyll Catabolic Genes (CCGs) like Non-Yellowing Coloring1 (NYC1),and Pheophorbide A Oxygenase(PAO),as well as Bifunctional Nuclease1 (BFN1),Senescence-Associated Gene 29/Sugars Will Eventually be Exporter Transporters 15 (SAG29/SWEET15) and Seven in Absentia (SINA) to promote the degradation of nucleic acid,proteins and transport processes [249–251].AtNAP/ANAC029is known to be related to precocious senescence[252].NAC family genesANAC017,ANAC090andANAC082,named as ‘‘NACtroika”can regulate senescence in a positive or negative manner in Arabidopsis [253].JUNGBRUNNEN 1(NAC042)plays a central longevity regulatory role and the higher expression level of this gene in Arabidopsis contributes to the increased expression levels of reactive-oxygen species-responsive genes,such as glutathione reductase and some heat shock proteins[254].

Senescence related transcriptome study in wheat by Borril et al.[8]showed that among the 63 differentially expressed NAC TFs,61 showed up-regulation during the senescence time course(Table S3).Moreover,among the top 36 regulatory genes of senescence regulatory gene network,NAM-A1was represented as the topmost based on‘edge weight threshold’used in that study.Gene regulatory network also revealedNAM-D1,NAM-A2andNAM-D2as the targets ofNAM-A1gene.However,no down-regulated NAC TF was included in the network development because of no significant down-regulation of NAC TF.This may be due to the involvement of only one cultivar (Bobwhite) for the study [8].Thus,the role of negative regulators of senescence of NAC family genes in wheat senescence gene regulatory network is still obscure.

7.Predicted regulatory pathway of wheat senescence involving TaNAC-S

TaNAC-Sis a novel NAC transcription factor that acts as a potential negative regulator of leaf senescence.This gene was detected in a transcriptomics study(Affymetrix wheat1 gene chip:Ta.16423.1.S1_at) of six wheat varieties grown with varying levels of N[128].The full-length coding sequence containing 5′and 3′non-coding regions,was obtained from theHM037184sequence accession homologous toCK208366,and was namedTaNAC-S.This gene was considered as a candidate gene of senescence regulator because it showed a potent positive correlation with leaf N concentration and N remobilization during grain filling[255].Later study ofTaNAC-Srevealed that overexpression of this gene in transgenic wheat line led to an increased chlorophyll content and prolonged photosynthesis.More importantly,the increase in grain protein concentration did not lead to a yield penalty.Generally,it is believed that late senescence is associated with lower GPC while early senescence results in an increased GPC but low yield[61,256].This contradictory evidence unlocks the thought that unknown physiological mechanisms may exist in boosting grain N concentrations.

Due to the above-mentioned high potential of usingTaNAC-Sgene variation in simultaneously improving wheat GPC and yield,further research is needed to thoroughly characterize the gene in order to efficiently capture its gene effects in breeding.To exploreTaNAC-Sgene,we have carried out a large experiment and revealed its allelic variation,association with other genes and gene networks,genetic effects,and sensitivity to various environmental factors such as N concentration (https://researchrepository.murdoch.edu.au/id/eprint/58315/).Combining the genotyping,phenotyping,TaNAC-Spromoter,and gene network analysis it made clear that the up-or down-regulation ofTaNAC-Sis dependent on brassinosteroid-strigolactone perception.Under nitrogen stress conditions (Fig.3),the level of SL hormone decreased[257].The decreased SL level can repress its receptor D14/rsbQ to interact with Max2/ORE9,a known senescence accelerator[258] leading to an increased level of some target proteins of SLD14-ORE9 complex i.e.BES1.BES1 can directly repressGLKwhich is required for chloroplast development[189–191]and in turn can repress the expression of its target gene such asNAC-S.The reduced expression ofGLKcan promoteORE1,NAC019,andPIFexpression which can directly bindNAC-Spromoter to downregulate its expression.BES1 can also directly bind to the promoter of NAC-S to inhibit its expression.The decreased expression ofNAC-Scan result in an increased expression of genes related to chlorophyll catabolism and accelerated senescence such asSDQ1andCLH2.Oppositely,under high nitrogen conditions (Fig.4),the increased level of SL can trigger the binding of D14 with ORE9.SL-D14-ORE9 can lead to a degradation of their target protein BES1.The decreased level of BES1 can induce an increased expression ofNAC-Sby triggering its binding toGLK.The increasedGLKandNAC-Slevel also can repress the expression of senescence promoting genes and induce the chloroplast biosynthesis-related genes likeJUB1andVNI2ultimately making results in delayed senescence.Further study is needed to explore the SL regulated senescence under nitrogen stress condition in wheat.

Fig.3.Model for NAC-S in regulating senescence under nitrogen stressed condition.

Fig.4.Model for NAC-S in regulating senescence under high nitrogen conditions.

8.Conclusions

Recent advances in transcriptome study on senescence regulatory gene network in wheat facilitated the identification of key senescence regulators.Nevertheless,a large part of its gene regulatory network is still unclear and needs to be further explored.It is important to identify the interacting partners of the regulatory genes together with functional study so that the regulatory mechanism can be clearer.Variations of senescence types and their associated regulatory networks or genes are also worth further study,transcriptome study should be further expanded to more cultivars with contrasting maturity.Uncover varying mechanisms for different types of delayed senescence will ultimately lead to the identification of key senescence delaying genes in wheat and their role in controlling senescence.Finally,as senescence is interrelated with NUE and influenced by nitrogen levels,study the relationship between N metabolism and senescence gene network will potentially unleash the senescence-related gene effects on wheat NUE improvement.

CRediT authorship contribution statement

Nigarin Sultana:carried out the literature review and manuscript writing.Shahidul Islam:participated in manuscript writing and revising.Angela Juhasz:helped in concept formation and manuscript revising.Wujun Ma:supervised the project and finalised the writing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work is financially supported by Australia Grain Research&Development Corporation Project (UMU00048) and Murdoch University International Postgraduate Research Scholarship.

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2021.01.004.