Stay-green Phenotype Research Articles

Delayed degradation of chlorophylls and photosynthetic proteins in Arabidopsis autophagy mutants during stress-induced leaf yellowing

Plant autophagy, one of the essential proteolysis systems, balances proteome and nutrient levels in cells of the whole plant. Autophagy has been studied by analysing Arabidopsis thaliana autophagy-defective atg mutants, but the relationship between autophagy and chlorophyll (Chl) breakdown during stress-induced leaf yellowing remains unclear. During natural senescence or under abiotic-stress conditions, extensive cell death and early yellowing occurs in the leaves of atg mutants. A new finding is revealed that atg5 and atg7 mutants exhibit a functional stay-green phenotype under mild abiotic-stress conditions, but leaf yellowing proceeds normally in wild-type leaves under these conditions. Under mild salt stress, atg5 leaves retained high levels of Chls and all photosystem proteins and maintained a normal chloroplast structure. Furthermore, a double mutant of atg5 and non-functional stay-green nonyellowing1-1 (atg5 nye1-1) showed a much stronger stay-green phenotype than either single mutant. Taking these results together, it is proposed that autophagy functions in the non-selective catabolism of Chls and photosynthetic proteins during stress-induced leaf yellowing, in addition to the selective degradation of Chl-apoprotein complexes in the chloroplasts through the senescence-induced STAY-GREEN1/NYE1 and Chl catabolic enzymes.

Arabidopsis Thylakoid Formation 1 Is a Critical Regulator for Dynamics of PSII–LHCII Complexes in Leaf Senescence and Excess Light

In higher plants, photosystem II (PSII) is a large pigment-protein supramolecular complex composed of the PSII core complex and the plant-specific peripheral light-harvesting complexes (LHCII). PSII-LHCII complexes are highly dynamic in their quantity and macro-organization to various environmental conditions. In this study, we reported a critical factor, the Arabidopsis Thylakoid Formation 1 (THF1) protein, which controls PSII-LHCII dynamics during dark-induced senescence and light acclimation. Loss-of-function mutations in THF1 lead to a stay-green phenotype in pathogen-infected and senescent leaves. Both LHCII and PSII core subunits are retained in dark-induced senescent leaves of thf1, indicative of the presence of PSII-LHCII complexes. Blue native (BN)-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis showed that, in dark- and high-light-treated thf1 leaves, a type of PSII-LHCII megacomplex is selectively retained while the stability of PSII-LHCII supercomplexes significantly decreased, suggesting a dual role of THF1 in dynamics of PSII-LHCII complexes. We showed further that THF1 interacts with Lhcb proteins in a pH-dependent manner and that the stay-green phenotype of thf1 relies on the presence of LHCII complexes. Taken together, the data suggest that THF1 is required for dynamics of PSII-LHCII supramolecular organization in higher plants.

Stay-green trait-antioxidant status interrelationship in durum wheat (Triticum durum) flag leaf during post-flowering

Three independent durum wheat mutant lines that show delayed leaf senescence or stay-green (SG) phenotype, SG196, SG310 and SG504, were compared to the parental genotype, cv. Trinakria, with respect to the photosynthetic parameters and the cellular redox state of the flag leaf in the period from flowering to senescence. The SG mutants maintained their chlorophyll content and net photosynthetic rate for longer than Trinakria, thus revealing a functional SG phenotype. They also showed a better redox state as demonstrated by: (1) a lower rate of superoxide anion production due to generally higher activity of the antioxidant enzymes superoxide dismutase and catalase in all of the SG mutants and also of the total peroxidase in SG196; (2) a higher thiol content that can be ascribed to a higher activity of the NADPH-providing enzyme glucose-6-phosphate dehydrogenase in all of the SG mutants and also of the NADP(+)-dependent malic enzyme in SG196; (3) a lower pro-oxidant activity of lipoxygenase that characterises SG196 and SG504 mutants close to leaf senescence. Overall, these results show a general relationship in durum wheat between the SG phenotype and a better redox state. This relationship differs across the different SG mutants, probably as a consequence of the different set of altered genes underlying the SG trait in these independent mutant lines.

The Single-Stranded DNA-Binding Protein WHIRLY1 Represses WRKY53 Expression and Delays Leaf Senescence in a Developmental Stage-Dependent Manner in Arabidopsis

Leaf senescence in plants involves both positive and negative transcriptional regulation. In this work, we show evidence for the single-stranded DNA-binding protein WHIRLY1 (WHY1) that functions as an upstream suppressor of WRKY53 in a developmental stage-dependent manner during leaf senescence in Arabidopsis (Arabidopsis thaliana). The why1 mutant displayed an early-senescence phenotype. In this background, the expression levels of both WRKY53 and the senescence-associated protease gene SAG12 increased. WHY1 bound to the sequence region that contains an elicitor response element motif-like sequence, GNNNAAATT, plus an AT-rich telomeric repeat-like sequence in the WRKY53 promoter in in vivo and in vitro mutagenesis assays as well as in a chromatin immunoprecipitation assay. This binding to the promoter of WRKY53 was regulated in a developmental stage-dependent manner, as verified by chromatin immunoprecipitation-polymerase chain reaction assay. This direct interaction was further determined by a transient expression assay in which WHY1 repressed β-GLUCURONIDASE gene expression driven by the WRKY53 promoter. Genetic analysis of double mutant transgenic plants revealed that WHY1 overexpression in the wrky53 mutant (oeWHY1wrky53) had no effect on the stay-green phenotype of the wrky53 mutant, while a WHY1 knockout mutant in the wrky53 mutant background (why1wrky53) generated subtle change in the leaf yellow/green phenotype. These results suggest that WHY1 was an upstream regulator of WRKY53 during leaf senescence.

Overexpression of a pea DNA helicase (PDH45) in peanut (Arachis hypogaea L.) confers improvement of cellular level tolerance and productivity under drought stress.

Peanut, a major edible oil seed crop globally is predominantly grown under rainfed conditions and suffers yield losses due to drought. Development of drought-tolerant varieties through transgenic technology is a valid approach. Besides superior water relation traits like water mining, intrinsic cellular level tolerance mechanisms are important to sustain the growth under stress. To achieve this objective, the focus of this study was to pyramid drought adaptive traits by overexpressing a stress responsive helicase, PDH45 in the background of a genotype with superior water relations. PCR, Southern, and RT-PCR analyses confirmed stable integration and expression of the PDH45 gene in peanut transgenics. At the end of T₃ generation, eight transgenic events were identified as promising based on stress tolerance and improvement in productivity. Several transgenic lines showed stay-green phenotype and increased chlorophyll stability under stress and reduced chlorophyll retardation under etherel-induced simulated stress conditions. Stress-induced root growth was also substantially higher in the case of transformants. This was reflected in increased WUE (low Δ¹³C) and improved growth rates and productivity. The transgenics showed 17.2 and 26.75 % increase in yield under non-stress and stress conditions over wild type ascertaining the feasibility of trait pyramiding strategy for the development of drought-tolerant peanut.

Identification of an AtCRN1-like chloroplast protein BeCRN1 and its distinctive role in chlorophyll breakdown during leaf senescence in bamboo (Bambusa emeiensis ‘Viridiflavus’)

CRN1/PPH/NYC3 is one of the key genes responsible for chlorophyll degradation during senescence in both Arabidopsis and rice. In this study, BeCRN1 and its promoter, BeCRN1p, were isolated from, Bambusa emeiensis ‘Viridiflavus’, a bamboo variety, for the first time. BeCRN1 consists of 1,646 bp, with an open reading frame of 1,473 bp, encoding a predicted polypeptide of 490 amino acids. Besides, BeCRN1 contained 5 exons and 4 introns, and harbored a distinctive microsatellite in the fourth intron. Differences in BeCRN1p are observed primarily in the AtCRN1 promoter, while sharing similar cis element composition with the rice CRN1 promoter. BeCRN1 is localized within the chloroplast, and it is strongly induced by senescence signals. Although constitutive overexpression of BeCRN1 in crn1 has reversed the stay-green phenotype of crn1 to the wild type phenotype, its promoter has failed to do so with AtCRN1 following dark treatment. The efficiency of BeCRN1p is only about one-third of that of the AtCRN1 promoter.

Plant senescence and crop productivity

Senescence is a developmental process which in annual crop plants overlaps with the reproductive phase. Senescence might reduce crop yield when it is induced prematurely under adverse environmental conditions. This review covers the role of senescence for the productivity of crop plants. With the aim to enhance productivity, a number of functional stay-green cultivars have been selected by conventional breeding, in particular of sorghum and maize. In many cases, a positive correlation between leaf area duration and yield has been observed, although in a number of other cases, stay-green cultivars do not display significant effects with regards to productivity. In several crops, the stay-green phenotype is observed to be associated with a higher drought resistance and a better performance under low nitrogen conditions. Among the approaches used to achieve stay-green phenotypes in transgenic plants, the expression of the IPT gene under control of senescence-associated promoters has been the most successful. The promoters employed for senescence-regulated expression contain cis-elements for binding of WRKY transcription factors and factors controlled by abscisic acid. In most crops transformed with such constructs the stay-green character has led to increased biomass, but only in few cases to increased seed yield. A coincidence of drought stress resistance and stay-green trait is observed in many transgenic plants.

Molecular cloning and characterization of a chlorophyll degradation regulatory gene from bamboo

Leaf senescence constituted the final stage of leaf development and it is always accompanied by the leaf yellowing. The non-yellowing gene (NYE1), initially identified from Arabidopsis in our laboratory, is a key regulatory gene responsible for chlorophyll degradation during senescence. In this study, an orthologue of AtNYE1 was isolated from the bamboo (Bambusa emeiensis cv. Viridiflavus) and tentatively named BeNYE1. The full length sequence of 1 386 bp contains an open reading frame of 801 bp. The protein encoded by BeNYE1 consists of 266 amino acids. Sequence analysis revealed that BeNYE1 had high similarity with other NYE/SGR proteins from various monocotyledon species. BeNYE1 was strongly induced by natural senescence and dark-induced senescence in bamboo. Driven by a 1.5 kb upstream fragment of AtNYE1, BeNYE1 could rescue the stay-green phenotype of nye1-1. The constitutive over-expression of BeNYE1 could accelerate the chlorophyll degradation. These results indicated that BeNYE1 might play an important role in the regulation of chlorophyll degradation during leaf senescence in bamboo.

Improved drought resistance in a wheat stay-green mutant tasg1 under field conditions

We investigated the drought resistance of a wheat (Triticum aestivum L.) stay-green mutant tasg1 and its wild-type (WT) in field experiments conducted for two years. Drought stress was imposed by controlling irrigation and sheltering the plants from rain. Compared with the WT, tasg1 exhibited a distinct delayed senescence under both normal and drought stress conditions, as indicated by slower degradation of chlorophyll and decrease in net photosynthetic rate than in WT. At the same time, tasg1 mutants maintained more integrated chloroplasts and thylakoid ultrastructure than did WT plants under drought stress. Lower malondialdehyde content and higher antioxidative enzyme activities in tasg1, compared to WT, may be involved in the stay-green phenotype and drought resistance of tasg1.

The antioxidative defense system is involved in the delayed senescence in a wheat mutant tasg1

Wheat, which is the most important food crop worldwide, is a cereal that presents considerable potential for increased yield. A new wheat (Triticum aestivum L.) mutant tasg1 with delayed leaf senescence was constructed using ethyl methane sulfonate as a mutagen. Natural senescence in tasg1 was distinctly delayed in the field, as indicated by the slower progression of chlorophyll degradation, net photosynthetic rate than its wild type. Further, the malondialdehyde and the hydrogen peroxide content was lower and antioxidative enzyme activity higher in tasg1 than those in its wild type during both natural senescence and methyl viologen-induced oxidative stress. The results suggest that tasg1 is a functional stay-green wheat mutant with the Type B (in which senescence initiates on schedule, but progresses at a rate lower than that in the respective WTs) or Type A (in which senescence initiates late but proceeds at a normal rate) and B combination and that the competence of the antioxidant defense system is one of the most important mechanisms underlying the expression of the stay-green phenotype.

Isolation and characterization of a chlorophyll degradation regulatory gene from tall fescue

The non-yellowing gene (NYE1), initially identified from Arabidopsis, is a key regulatory gene responsible for chlorophyll degradation during senescence. Here, FaNYE1, an orthologue of AtNYE1, was further identified from a major type of cool-season turf grass, tall fescue (Festuca arundinacea Schreb.), by RACE-PCR. It consists of 1,441bp, with an open reading frame of 834bp, encoding a predicted polypeptide of 278 amino acids. Sequence similarity as well as exon and intron characteristics clearly suggested that FaNYE1 encoded an AtNYE1-like chloroplast protein. FaNYE1 could be strongly induced by dark treatment and natural senescence. FaNYE1, driven by a 1.5kb upstream fragment of AtNYE1, could rescue the stay-green phenotype of nye1-1. Constitutive overexpression of FaNYE1 in Arabidopsis resulted in the whole spectrum of leaf yellowing phenotypes, the severity of which correlates with its transcript level. These results collectively indicate that FaNYE1 might play an important regulatory role in chlorophyll degradation during senescence in tall fescue, and therefore is a valuable gene for improving the green period or lawn color of turf grasses by genetic engineering.

Silencing of the LeSGR1 gene in tomato inhibits chlorophyll degradation and exhibits a stay-green phenotype

The full-length cDNA of LeSGR1 was cloned from tomato by RT-PCR and RACE. The cDNA encoded a protein of 272 amino acid residues and was deposited in GenBank (accession No. DQ100158). Northern analysis suggests that LeSGR1 gene specifically expresses in senescent leaves and mature fruits of tomatoes. Desiccation and flooding induce the expression of LeSGR1 in tomato leaves and stems. Both in ethylene-insensitive mutants (Nr) and ripening inhibitor mutants (rin), the expression of LeSGR1 is markedly decreased compared with that in the wild type. Alignment of the nucleotide sequence of SGR1 cloned from the tomato green flesh (gf) mutant with that from the wild type tomato shows a single nucleotide change leading to an amino acid substitution in gf mutant. Furthermore, LeSGR1 gene silencing by RNA interference results in inhibited chlorophyll degradation similar to the phenotype in gf mutant. Thus, we conclude that LeSGR1 is crucial to chlorophyll degradation and the mutation of SGR1 protein might be responsible for gf tomato properties.

Stay-green quantitative trait loci's effects on water extraction, transpiration efficiency and seed yield depend on recipient parent background

A stay-green phenotype enhances the adaptation of sorghum (Sorghum bicolor (L.) Moench) to terminal drought, although the mechanisms leading to its expression remain unclear. Differences in tillering and leaf area at anthesis, transpiration efficiency (TE), water extraction, harvest index (HI) and yield under terminal drought and fully irrigated conditions were assessed in 29 introgression lines (IL) targeting stay-green quantitative trait loci (QTLs) Stg1, Stg2, Stg3, Stg4, StgA and StgB in an S35 background, and 16 IL targeting Stg1, Stg3, Stg4 and StgB in an R16 background. TE was increased by StgB in the R16 background, whereas there was no effect in the S35 background. Water extraction was increased by Stg1 in the S35 background but not in R16. StgB modified the proportion of water extracted before and after anthesis in the S35 background. While tillering and leaf area at anthesis were decreased by Stg1 and Stg3 in S35, there was no effect in R16. Yield data under fully irrigated conditions showed higher tiller grain yield in Stg1, Stg2 and Stg3 ILs. Although yield differences were mostly explained by HI variation, the yield variation unexplained by HI was closely related to TE in S35 (R2=0.29) and R16 (R2=0.72), and was closely related to total water extracted in S35 (R2=0.41) but not in R16. These data indicate the potential for several stay-green QTLs to affect traits related to plant water use. However, these effects depend on the interaction between the genetic background and individual QTLs.

Genetic variability of transpiration response to vapor pressure deficit among sorghum genotypes

Simulation studies have demonstrated that limited maximum transpiration rate (TR) at high air vapor pressure deficit (VPD) in water-limited environments could result in significant increases in sorghum yield. However, such a restriction on TR at high VPD has not been documented in sorghum. The objective of this study was to search within sorghum germplasm for the possibility of restricted TR at high VPD. Twenty six genotypes were selected for measurement of VPD response based on field observations including yield, leaf temperature, and the stay-green phenotype. These genotypes were grown in a greenhouse for about 24-d growth, and then placed into individual chambers in which VPD was varied and TR measured. The results of this study showed marked variation among sorghum genotypes in TR response to VPD. Seventeen genotypes were identified as exhibiting a breakpoint in their VPD response in the range from 1.6 to 2.7 kPa, above which there was little or no further increase in TR. Therefore, these genotypes with a breakpoint have the possibility of soil water conservation when VPD during the midday cycle exceeds the breakpoint VPD. This trait would be desirable in less humid environments for increasing yields in water-deficit seasons. The observed range in the value of the BP among genotypes offers the possibility of developing genotypes with BP appropriate for specific environments.

Identification and Application of a Rice Senescence-Associated Promoter

SAG39 is a rice (Oryza sativa) gene that encodes a cysteine protease. SAG39 shares 55% homology with the Arabidopsis (Arabidopsis thaliana) senescence-associated protein SAG12. The promoter for SAG39 (P(SAG39)) was isolated, and SAG39 expression was determined to be relatively low in mature leaves, while not expressed in the endosperm. SAG39 mRNA levels increased as senescence progressed, with maximum accumulation of transcripts at late senescence stages. Gel retardation assays indicated that two cis-acting elements in P(SAG39), HBOXCONSENSUSPVCHS and WRKY71OS, responded to leaf senescence. To test if P(SAG39) could be useful for increasing rice yields by increasing cytokinin content and delaying senescence, homozygous transgenic plants were obtained by linking P(SAG39) to the ipt gene and introducing it into Zhonghua 11. The chlorophyll level of the flag leaf was used to monitor senescence, confirming the stay-green phenotype in P(SAG39):ipt transgenic rice versus wild-type plants. Changes in the cytokinin content led to early flowering and a greater number of emerged panicles 70 d after germination in the transgenic rice. Measurements of sugar and nitrogen contents in flag leaves demonstrated a transition in the source-sink relationship in transgenic plants triggered at the onset of leaf senescence, with the nitrogen content decreasing more slowly, while sugars were removed more rapidly than in wild-type plants. The importance of these changes to rice physiology, yield, and early maturation will be discussed.

Nuclear targeted AtS40 modulates senescence associated gene expression in Arabidopsis thaliana during natural development and in darkness

The Arabidopsis thaliana AtS40-3 gene belongs to a group of genes sharing the conserved DUF548 sequence motif with up to now unknown function. One member of this group, the barley HvS40, was shown before to play a role in regulation of leaf senescence. Similar as the barley gene, AtS40-3 is induced during senescence and is also regulated in response to dark treatment, ABA, salicylic acid and pathogen attack. By localization of the GUS fusion protein, the AtS40-3 gene was shown to encode a nucleus targeted protein. The s40-3a mutant with a T-DNA insertion in the promoter region of the gene was observed to have a staygreen phenotype. By quantitative real-time PCR analyses expression of the AtS40-3 gene in this mutant was observed to be constitutive and not induced during senescence. This coincided with WRKY53 gene expression in nonsenescent leaves and lowered expression levels of WRKY53 and SAG12 at later stages of development. While in the wildtype expression of AtS40-3 was activated by darkness, in the s40-3a mutant the expression of AtS40-3 stayed at a low level. This coincided with lower expression of the SEN1 and SAG12 genes. In another promoter mutant with a T-DNA insertion further upstream of the coding sequence the levels of AtS40-3 and SAG12 transcripts increased in parallel both in a natural light-dark regime and in darkness. The data on gene expression in promoter T-DNA insertion mutants of the s40-3 gene indicate that AtS40 regulates senescence either by modulation of WRKY53 or by activation of SAG12 independent of WRKY53.

EFFECT OF NITROGEN FERTILIZATION ON STAY-GREEN AND SENESCENT SORGHUM HYBRIDS IN SAND CULTURE

Nitrogen (N) is vital in the production of N containing compounds in cereal crops. Stay-greenness in sorghum [Sorghum bicolor (L.) Moench] is characterized by the plant's ability to retain water and chlorophyll content at maturity. A greenhouse experiment was conducted to investigate the effect of the genotypic variability between stay-green (BT × 643) and senescent (RT × 7000) hybrids for leaf senescence, dry matter partitioning and N partitioning under three N levels (0.45, 0.90, 1.80 g plant−1). The stay-green hybrid had a higher percentage green leaf area at 120 DAE with a higher residual N in leaves. The chlorophyll meter (SPAD-502) values for the stay-green hybrid during anthesis (75 and 85 DAE) were higher compared to its senescent counterpart. The results indicated that the expression of stay-green phenotype in sorghum was contingent on a minimum N supply of 1.35 g plant−1 and associated with greater N acquisition. Nitrogen use and uptake in the stay-green hybrid were higher.

Correction

The Plant JournalVolume 60, Issue 6 p. 1110-1110 Free Access Correction First published: 08 December 2009 https://doi.org/10.1111/j.1365-313X.2009.04019.xCitations: 1AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat The authors would like to report that there was an error in figure 1 of their paper (Morita et al., 2009). Parts c and d were incorrect. The correct figure is as follows: The authors apologize for this error. Reference Morita, R., Sato, Y., Masuda, Y., Nishimura, M. and Kusaba, M. (2009) Defect in non-yellow coloring 3, an α/β hydrolase-fold family protein, causes a stay-green phenotype during leaf senescence in rice. Plant J. 59, 940– 952. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Citing Literature Volume60, Issue6December 2009Pages 1110-1110 ReferencesRelatedInformation

A survey of cultivated heirloom tomato varieties identifies four new mutant alleles at the green-flesh locus

The process of crop domestication occurs through the selection and subsequent propagation of novel alleles that improve traits of interest. Cultivated tomato (Solanum lycopersicum), particularly heirloom varieties, exhibit a wide range of variation in fruit size, shape and color. The green-flesh mutant of tomato possesses a stay-green phenotype resulting in fruits that ripen to a red-brown color, due to the retention of chlorophyll and the simultaneous accumulation of lycopene. The recent identification of the GREEN-FLESH gene provides a molecular tool with which to investigate the origin of a subset of cultivated tomato varieties that resemble the green-flesh mutant. Sequence analysis of the GF locus from 26 varieties revealed the existence of four previously unidentified null alleles. This study illustrates the potential of cultivated tomato varieties, including heritage cultivars, heirlooms, and land races, for uncovering new alleles in genes of interest.

Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis.

During leaf senescence, chlorophyll is removed from thylakoid membranes and converted in a multistep pathway to colorless breakdown products that are stored in vacuoles. Dephytylation, an early step of this pathway, increases water solubility of the breakdown products. It is widely accepted that chlorophyll is converted into pheophorbide via chlorophyllide. However, chlorophyllase, which converts chlorophyll to chlorophyllide, was found not to be essential for dephytylation in Arabidopsis thaliana. Here, we identify pheophytinase (PPH), a chloroplast-located and senescence-induced hydrolase widely distributed in algae and land plants. In vitro, Arabidopsis PPH specifically dephytylates the Mg-free chlorophyll pigment, pheophytin (phein), yielding pheophorbide. An Arabidopsis mutant deficient in PPH (pph-1) is unable to degrade chlorophyll during senescence and therefore exhibits a stay-green phenotype. Furthermore, pph-1 accumulates phein during senescence. Therefore, PPH is an important component of the chlorophyll breakdown machinery of senescent leaves, and we propose that the sequence of early chlorophyll catabolic reactions be revised. Removal of Mg most likely precedes dephytylation, resulting in the following order of early breakdown intermediates: chlorophyll --> pheophytin --> pheophorbide. Chlorophyllide, the last precursor of chlorophyll biosynthesis, is most likely not an intermediate of breakdown. Thus, chlorophyll anabolic and catabolic reactions are metabolically separated.