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Plants maintain their internal temperature under environments with fluctuating temperatures by adjusting their morphology and architecture, an adaptive process termed thermomorphogenesis. Notably, the rhythmic patterns of plant thermomorphogenesis are governed by day-length information. However, it remains elusive how thermomorphogenic rhythms are regulated by photoperiod. Here, we show that warm temperatures enhance the accumulation of the chaperone GIGANTEA (GI), which thermostabilizes the DELLA protein, REPRESSOR OF ga1-3 (RGA), under long days, thereby attenuating PHYTOCHROME INTERACTING FACTOR 4 (PIF4)-mediated thermomorphogenesis. In contrast, under short days, when GI accumulation is reduced, RGA is readily degraded through the gibberellic acid-mediated ubiquitination-proteasome pathway, promoting thermomorphogenic growth. These data indicate that the GI-RGA-PIF4 signaling module enables plant thermomorphogenic responses to occur in a day-length-dependent manner. We propose that the GI-mediated integration of photoperiodic and temperature information shapes thermomorphogenic rhythms, which enable plants to adapt to diel fluctuations in day length and temperature during seasonal transitions.

MYB-type transcription factors (TFs) play important roles in plant growth and development, and in the rapid responses to unfavorable environmental conditions. We recently reported the isolation and characterization of a rice (Oryza sativa) MYB TF, OsMYB102, which is involved in the regulation of leaf senescence by downregulating abscisic acid (ABA) biosynthesis and the downstream signaling response. Based on the similarities of their sequences and expression patterns, OsMYB102 appears to be a homolog of the Arabidopsis thaliana AtMYB44 TF. Since AtMYB44 is a key regulator of leaf senescence and abiotic stress responses, it is important to examine whether AtMYB44 homologs in other plants also act similarly. Here, we generated transgenic Arabidopsis plants expressing OsMYB102 (OsMYB102-OX). The OsMYB102-OX plants showed a delayed senescence phenotype during dark incubation and were more susceptible to salt and drought stresses, considerably similar to Arabidopsis plants overexpressing AtMYB44. Real-time quantitative PCR (RT-qPCR) revealed that, in addition to known senescence-associated genes, genes encoding the ABA catabolic enzymes AtCYP707A3 and AtCYP707A4 were also significantly upregulated in OsMYB102- OX, leading to a significant decrease in ABA accumulation. Furthermore, protoplast transient expression and chromatin immunoprecipitation assays revealed that OsMYB102 directly activated AtCYP707A3 expression. Based on our findings, it is probable that the regulatory functions of AtMYB44 homologs in plants are highly conserved and they have vital roles in leaf senescence and the abiotic stress responses. [BMB Reports 2019; 52(11): 653-658].

The stay-green mutant of Arabidopsis thaliana, ore10 forms stable light-harvesting complex II (LHCII) aggregates during dark-induced senescence, which showed a single base deletion (G1351) in the coding region of the pheophytinase (PPH) gene. PPH specifically dephytylates the Mg-free chlorophyll (Chl) pigment pheophytin, yielding pheophorbide. In both ore10 and pph-1 mutants, pheophytin a accumulated due to the deficiency of PPH gene, but the amount was relatively smaller than that of degraded Chl, and most of the pheophytin a was bound to the stable LHCII forming aggregates with some other Chl-protein (CP) complexes. Comparison of Chl a/b ratios in thylakoids, aggregates, and LHCII indicated that the suppression of Chl b to Chl a conversion was stronger when Chl b reductase was missing and weak when PPH is missing in the large Chl catabolic complex, which allowed the partial degradation of Chl b. These results suggest that the PPH-dependent pathway is not specific for LHCII, but common for all CP complexes, including LHCII. In PPH-deficient mutants, the degradation of LHCII was suppressed by the formation of aggregates, and some of the remaining CP complexes and pheophytin a were included in the aggregates. Non-included CP complexes were degraded via an unknown mechanism.

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), a multifunctional E3 ligase protein with many target proteins, is involved in diverse developmental processes throughout the plant's lifecycle, including seed germination, the regulation of circadian rhythms, photomorphogenesis, and the control of flowering time. To function, COP1 must form multimeric complexes with SUPPRESSOR OF PHYA1 (SPA1), i.e., [(COP1)2(SPA1)2] tetramers. We recently reported that the blue-light receptor FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX1) represses COP1 activity by inhibiting its homodimerization, but it is not yet clear whether FKF1 affects the formation of COP1-containing multimeric complexes. To explore this issue, we performed size exclusion chromatography (SEC) of Arabidopsis thaliana proteins and found that the levels and composition of COP1-containing multimeric complexes varied throughout a 24-h period. The levels of 440–669 kDa complexes were dramatically reduced in the late afternoon compared to the morning and at night in wild-type plants. During the daytime, the levels of these complexes were reduced in FKF1-overexpressing plants but not in fkf1-t, a loss-of-function mutant of FKF1, suggesting that FKF1 is closely associated with the destabilization of COP1 multimeric protein complexes in a light-dependent manner. We also analyzed the SEC patterns of COP1 multimeric complexes in transgenic plants overexpressing mutant COP1 variants, including COP1L105A (which forms homodimers) and COP1L170A (which cannot form homodimers), and found that COP1 multimeric complexes were scarce in plants overexpressing COP1L170A. These results indicate that COP1 homodimers serve as basic building blocks that assemble into COP1 multimeric complexes with diverse target proteins. We propose that light-activated FKF1 inhibits COP1 homodimerization, mainly by destabilizing 440–669 kDa COP1 complexes, resulting in the repression of CONSTANS-degrading COP1 activity in the late afternoon in long days, but not in short days, thereby regulating photoperiodic flowering in Arabidopsis.

Arabidopsis EARLY FLOWERING3 (ELF3) functions in modulating light input to the circadian clock, as a component of ELF3-ELF4-LUX ARRHYTHMO (LUX) evening complex. However, the role of ELF3 in stress responses remains largely unknown. In this study, we show that ELF3 enhances plants' resilience to salt stress: ELF3-overexpressing (ELF3-OX) plants are salt-tolerant, while elf3 mutants are more sensitive to salt stress. The expressions of many salt stress- and senescence-associated genes are altered in elf3-1 and ELF3-OX plants compared with wild-type. During salt stress, ELF3 suppresses factors that promote salt stress response pathways, mainly GIGANTEA (GI), at the post-translational level, and PHYTOCHROME INTERACTING FACTOR4 (PIF4), at the transcriptional level. To enhance the salt stress response, PIF4 directly downregulates the transcription of JUNGBRUNNEN1 (JUB1/ANAC042), encoding a transcription factor that upregulates the expression of stress tolerance genes, DREB2A and DELLA. Furthermore, PIF4 directly upregulates the transcription of ORESARA1 (ORE1/ANAC092) and SAG29, positive regulators of salt stress response pathways. Based on our results, we propose that ELF3 modulates key regulatory components in salt stress response pathways at the transcriptional and post-translational levels.

Summary Drought is one of the major abiotic stresses that directly implicate plant growth and crop productivity. Although many genes in response to drought stress have been identified, genetic improvement to drought resistance especially in food crops is showing relatively slow progress worldwide. Here, we reported the isolation of abscisic acid, stress and ripening (ASR) genes from upland rice variety, IRAT109 (Oryza sativa L. ssp. japonica), and demonstrated that overexpression of OsASR5 enhanced osmotic tolerance in Escherichia coli and drought tolerance in Arabidopsis and rice by regulating leaf water status under drought stress conditions. Moreover, overexpression of OsASR5 in rice increased endogenous ABA level and showed hypersensitive to exogenous ABA treatment at both germination and postgermination stages. The production of H2O2, a second messenger for the induction of stomatal closure in response to ABA, was activated in overexpression plants under drought stress conditions, consequently, increased stomatal closure and decreased stomatal conductance. In contrast, the loss‐of‐function mutant, osasr5, showed sensitivity to drought stress with lower relative water content under drought stress conditions. Further studies demonstrated that OsASR5 functioned as chaperone‐like protein and interacted with stress‐related HSP40 and 2OG‐Fe (II) oxygenase domain containing proteins in yeast and plants. Taken together, we suggest that OsASR5 plays multiple roles in response to drought stress by regulating ABA biosynthesis, promoting stomatal closure, as well as acting as chaperone‐like protein that possibly prevents drought stress‐related proteins from inactivation.

NAM/ATAF1/ATAF2/CUC2 (NAC) is a plant-specific transcription factor (TF) family, and NACs participate in many diverse processes during the plant life cycle. Several Arabidopsis thaliana NACs have important roles in positively or negatively regulating leaf senescence, but in other plant species, including rice, the senescence-associated NACs (senNACs) remain largely unknown. Here we show that the rice senNAC TF ONAC106 negatively regulates leaf senescence. Leaves of onac106-1D (insertion of the 35S enhancer in the promoter region of the ONAC106 gene) mutants retained their green color under natural senescence and dark-induced senescence conditions. Genome-wide transcriptome analysis revealed that key senescence-associated genes (SGR, NYC1, OsNAC5, OsNAP, OsEIN3 and OsS3H) were differentially expressed in onac106-1D during dark-induced senescence. In addition to delayed senescence, onac106-1D also showed a salt stress-tolerant phenotype; key genes that down-regulate salt response signaling (OsNAC5, OsDREB2A, OsLEA3 and OsbZIP23) were rapidly up-regulated in onac106-1D under salt stress. Interestingly, onac106-1D also exhibited a wide tiller angle phenotype throughout development, and the tiller angle-related gene LPA1 was down-regulated in onac106-1D. Using yeast one-hybrid assays, we found that ONAC106 binds to the promoter regions of SGR, NYC1, OsNAC5 and LPA1. Taking these results together, we propose that ONAC106 functions in leaf senescence, salt stress tolerance and plant architecture by modulating the expression of its target genes that function in each signaling pathway.

In the facultative long-day (LD) plant Arabidopsis thaliana, FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) is activated by blue light and promotes flowering through the transcriptional and post-translational regulation of CONSTANS under inductive LD conditions. By contrast, the facultative short day (SD) plant rice (Oryza sativa) flowers early under inductive SD and late under non-inductive LD conditions; the regulatory function of OsFKF1 remains elusive. Here we show that osfkf1 mutants flower late under SD, LD and natural LD conditions. Transcriptional analysis revealed that OsFKF1 up-regulates the expression of the floral activator Ehd2 and down-regulates the expression of the floral repressor Ghd7; these regulators up- and down-regulate Ehd1 expression, respectively. Moreover, OsFKF1 can up-regulate Ehd1 expression under blue light treatment, without affecting the expression of Ehd2 and Ghd7. In contrast to the LD-specific floral activator Arabidopsis FKF1, OsFKF1 likely acts as an autonomous floral activator because it promotes flowering independent of photoperiod, probably via its distinct roles in controlling the expression of rice-specific genes including Ehd2, Ghd7 and Ehd1. Like Arabidopsis FKF1, which interacts with GI and CDF1, OsFKF1 also interacts with OsGI and OsCDF1 (also termed OsDOF12). Thus, we have identified similar and distinct roles of FKF1 in Arabidopsis and rice.

Degradation of chlorophyll (Chl) by Chl catabolic enzymes (CCEs) causes the loss of green color that typically occurs during senescence of leaves. In addition to CCEs, staygreen1 (SGR1) functions as a key regulator of Chl degradation. Although sgr1 mutants in many plant species exhibit a stay-green phenotype, the biochemical function of the SGR1 protein remains elusive. Many recent studies have examined the physiological and molecular roles of SGR1 and its homologs (SGR2 and SGR-LIKE) in Chl metabolism, finding that these proteins have different roles in different species. In this review, we summarize the recent studies on SGR and discuss the most likely functions of SGR homologs.

Background Rice zebra mutants are leaf variegation mutants that exhibit transverse sectors of green/yellow or green/white in developing or mature leaves. In most cases, leaf variegation is caused by defects in chloroplast biogenesis pathways, leading to an accumulation of reactive oxygen species in a transverse pattern in the leaves. Here, we examine a new type of leaf variegation mutant in rice, zebra3 (z3), which exhibits transverse dark-green/green sectors in mature leaves and lacks the typical yellow or white sectors. Results Map-based cloning revealed that the Z3 locus encodes a putative citrate transporter that belongs to the citrate-metal hydrogen symport (CitMHS) family. CitMHS family members have been extensively studied in bacteria and function as secondary transporters that can transport metal-citrate complexes, but whether CitMHS family transporters exist in eukaryotes remains unknown. To investigate whether Z3 acts as a citrate transporter in rice, we measured citrate levels in wild-type leaves and in the dark-green and green sectors of the leaves of z3 mutants. The results showed that citrates accumulated to high levels in the dark-green sectors of z3 mutant leaves, but not in the green sectors as compared with the wild-type leaves. Conclusions These results suggest that leaf variegation in the z3 mutant is caused by an unbalanced accumulation of citrate in a transverse pattern in the leaves. Taking these results together, we propose that Z3 plays an important role in citrate transport and distribution during leaf development and is a possible candidate for a CitMHS family member in plants. Electronic supplementary material The online version of this article (10.1186/s12284-017-0196-8) contains supplementary material, which is available to authorized users.

High salinity, one of the most severe abiotic stresses encountered by land plants, often results from water deficit and also induces whole-plant senescence. Thus, salt treatment provides a useful technique for stress-mediated induction of senescence in plants. In this chapter, we describe the procedures to induce senescence in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), using NaCl or KCl. Furthermore, we present experimental approaches to measure salt stress-induced leaf senescence.

In Arabidopsis thaliana, CONSTANS (CO) plays an essential role in the regulation of photoperiodic flowering under long-day conditions. CO protein is stable only in the afternoon of long days, when it induces the expression of FLOWERING LOCUS T (FT), which promotes flowering. The blue-light photoreceptor FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (FKF1) interacts with CO and stabilizes it by an unknown mechanism. Here, we provide genetic and biochemical evidence that FKF1 inhibits CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1)-dependent CO degradation. Light-activated FKF1 has no apparent effect on COP1 stability but can interact with and negatively regulate COP1. We show that FKF1 can inhibit COP1 homo-dimerization. Mutation of the coiled-coil domain in COP1, which prevents dimer formation, impairs COP1 function in coordinating flowering time. Based on these results, we propose a model whereby the light- and day length-dependent interaction between FKF1 and COP1 controls CO stability to regulate flowering time., CONSTANS promotes flowering under long-day conditions in Arabidopsis but is rapidly degraded in short-day conditions. Here the authors show that the blue-light photoreceptor FKF1 can interact with the E3 ligase COP1 in a light-dependent manner and prevent degradation of CO in long-day conditions.

Rice (Oryza sativa) Phytochrome-Interacting Factor-Like1 (OsPIL1), a basic helix-loop-helix transcription factor, plays an important role in the elongation of internode cells. Recently, we found that OsPIL1 participates in chlorophyll biosynthesis by directly upregulating several genes encoding components of the photosystem apparatus. Here, we show that OsPIL1 negatively regulates leaf senescence in rice. During dark-induced senescence (DIS), ospil1 mutants senesced earlier than wild type; this is opposite to mutants of Arabidopsis PIF4 and PIF5, the closest homologs of OsPIL1. Microarray analysis revealed that during DIS, several senescence-associated genes were upregulated and OsGLKs, negative regulators of leaf senescence, were strongly repressed in ospil1 mutants. Transgenic Arabidopsis plants overexpressing OsPIL1 showed an early senescing phenotype during DIS. In addition, OsPIL1 expressed in Arabidopsis upregulates the transcription of ORESARA1, a major senescence-inducing NAC transcription factor and one of the downstream genes of Arabidopsis PIF4, by directly binding the promoter region. These results indicate that OsPIL1 and Arabidopsis PIF4 have similar functions, but their downstream regulatory cascades have opposite effects.

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

During leaf senescence in Arabidopsis, STAYGREEN 1 (SGR1) and SGR2 regulate chlorophyll degradation positively and negatively, respectively. SGR-LIKE (SGRL) is also expressed in pre-senescing leaves, but its function remains largely unknown. Here we show that under abiotic stress, Arabidopsis plants overexpressing SGRL exhibit early leaf yellowing and sgrl-1 mutants exhibit persistent green color of leaves. Under salt stress, SGR1 and SGRL act synergistically for rapid Chl degradation prior to senescence. Furthermore, SGRL forms homo- and heterodimers with SGR1 and SGR2 in vivo, and interacts with LHCII and chlorophyll catabolic enzymes. The role of SGRL under abiotic stress is discussed.

Chlorophyll (Chl) degradation causes leaf yellowing during senescence or under stress conditions. For Chl breakdown, STAY-GREEN1 (SGR1) interacts with Chl catabolic enzymes (CCEs) and light-harvesting complex II (LHCII) at the thylakoid membrane, possibly to allow metabolic channeling of potentially phototoxic Chl breakdown intermediates. Among these Chl catabolic components, SGR1 acts as a key regulator of leaf yellowing. In addition to SGR1 (At4g22920), the Arabidopsis thaliana genome contains an additional homolog, SGR2 (At4g11910), whose biological function remains elusive. Under senescence-inducing conditions, SGR2 expression is highly up-regulated, similarly to SGR1 expression. Here we show that SGR2 function counteracts SGR1 activity in leaf Chl degradation; SGR2-overexpressing plants stayed green and the sgr2-1 knockout mutant exhibited early leaf yellowing under age-, dark-, and stress-induced senescence conditions. Like SGR1, SGR2 interacted with LHCII but, in contrast to SGR1, SGR2 interactions with CCEs were very limited. Furthermore, SGR1 and SGR2 formed homo- or heterodimers, strongly suggesting a role for SGR2 in negatively regulating Chl degradation by possibly interfering with the proposed CCE-recruiting function of SGR1. Our data indicate an antagonistic evolution of the functions of SGR1 and SGR2 in Arabidopsis to balance Chl catabolism in chloroplasts with the dismantling and remobilizing of other cellular components in senescing leaf cells.

Loss of chlorophyll is the visible symptom of leaf senescence and staygreen refers to the delayed leaf senescence in plants. The staygreen gene (SGR) in rice (Oryza sativa L.) has been identified as its mutation maintains greenness during leaf senescence, and encodes a chloroplast protein required for the initiation of chlorophyll breakdown in plants. In this study, we isolated a rice SGR-homologous gene in creeping bentgrass (Agrostis stolonifera L.), and transgenic creeping bentgrass plants were obtained by introducing pCAMBIA3301 vector harboring antisense SGR gene under control of the senescence-specific SAG12 promoter. Transgenic plants were selected by herbicide resistance assays and genomic integration of the transgenes was confirmed by PCR analysis. Subsequent analyses demonstrated the staygreen phenotype of the transgenic creeping bentgrass plants with decreased chlorophyll loss during leaf senescence. These results suggest that the antisense SGR expression in creeping bentgrass delays leaf senescence, which provides a way to develop genetically engineered turfgrass varieties with the commercially useful staygreen trait. Staygreen refers to the heritable delayed foliar senescence character in plants, usually due to impaired or delayed chlorophyll catabolism.

Plant breeders have focused on improving plant architecture as an effective means to increase crop yield. Here, we identify the main-effect quantitative trait loci (QTLs) for plant shape-related traits in rice (Oryza sativa) and find candidate genes by applying whole genome re-sequencing of two parental cultivars using next-generation sequencing. To identify QTLs influencing plant shape, we analyzed six traits: plant height, tiller number, panicle diameter, panicle length, flag leaf length, and flag leaf width. We performed QTL analysis with 178 F7 recombinant in-bred lines (RILs) from a cross of japonica rice line ‘SNUSG1’ and indica rice line ‘Milyang23’. Using 131 molecular markers, including 28 insertion/deletion markers, we identified 11 main- and 16 minor-effect QTLs for the six traits with a threshold LOD value > 2.8. Our sequence analysis identified fifty-four candidate genes for the main-effect QTLs. By further comparison of coding sequences and meta-expression profiles between japonica and indica rice varieties, we finally chose 15 strong candidate genes for the 11 main-effect QTLs. Our study shows that the whole-genome sequence data substantially enhanced the efficiency of polymorphic marker development for QTL fine-mapping and the identification of possible candidate genes. This yields useful genetic resources for breeding high-yielding rice cultivars with improved plant architecture.

CONSTANS (CO) induces the expression of FLOWERING LOCUS T (FT) in the photoperiodic pathway, and thereby regulates the seasonal timing of flowering. CO expression is induced and CO protein is stabilized by FLAVIN-BINDING KELCH REPEAT F-BOX PROTEIN 1 (FKF1) in the late afternoon, while CO is degraded by CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) during the night. These regulatory cascades were thought to act independently. In our study, we investigated the relationship between FKF1 and COP1 in the regulation of CO stability in response to ambient light conditions. A genetic analysis revealed that FKF1 acts as a direct upstream negative regulator of COP1, in which cop1 mutation is epistatic to fkf1 mutation in the photoperiodic regulation of flowering. COP1 activity requires the formation of a hetero-tetramer with SUPPRESSOR OF PHYA-105 (SPA1), [(COP1)2(SPA1)2]. Light-activated FKF1 has an increased binding capacity for COP1, forming a FKF1-COP1 hetero-dimer, and inhibiting COP1 homo-dimerization at its coiled-coil (CC) domain. Mutations in the CC domain result in poor COP1 dimerization and misregulation of photoperiodic floral induction. We propose that FKF1 represses COP1 activity by inhibiting COP1 dimerization in the late afternoon under long-day conditions, resulting in early flowering. [BMB Reports 2018; 51(4): 163-164].

Heading date and photoperiod sensitivity are fundamental traits that determine rice adaptation to a wide range of geographic environments. By quantitative trait locus (QTL) mapping and candidate gene analysis using whole-genome re-sequencing, we found that Oryza sativa Pseudo-Response Regulator37 (OsPRR37; hereafter PRR37) is responsible for the Early heading7-2 (EH7-2)/Heading date2 (Hd2) QTL which was identified from a cross of late-heading rice 'Milyang23 (M23)' and early-heading rice 'H143'. H143 contains a missense mutation of an invariantly conserved amino acid in the CCT (CONSTANS, CO-like, and TOC1) domain of PRR37 protein. In the world rice collection, different types of nonfunctional PRR37 alleles were found in many European and Asian rice cultivars. Notably, the japonica varieties harboring nonfunctional alleles of both Ghd7/Hd4 and PRR37/Hd2 flower extremely early under natural long-day conditions, and are adapted to the northernmost regions of rice cultivation, up to 53° N latitude. Genetic analysis revealed that the effects of PRR37 and Ghd7 alleles on heading date are additive, and PRR37 down-regulates Hd3a expression to suppress flowering under long-day conditions. Our results demonstrate that natural variations in PRR37/Hd2 and Ghd7/Hd4 have contributed to the expansion of rice cultivation to temperate and cooler regions.

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in chlorophyll (Chl) synthesis, and is required for prolamellar body (PLB) formation in etioplasts. Rice faded green leaf (fgl) mutants develop yellow/white leaf variegation and necrotic lesions during leaf elongation in field-grown plants. Map-based cloning revealed that FGL encodes OsPORB, one of two rice POR isoforms. In fgl, etiolated seedlings contained smaller PLBs in etioplasts, and lower levels of total and photoactive Pchlide. Under constant or high light (HL) conditions, newly emerging green leaves rapidly turned yellow and formed lesions. Increased levels of non-photoactive Pchlide, which acts as a photosensitizer, may cause reactive oxygen accumulation and lesion formation. OsPORA expression is repressed by light and OsPORB expression is regulated in a circadian rhythm in short-day conditions. OsPORA was expressed at high levels in developing leaves and decreased dramatically in fully mature leaves, whereas OsPORB expression was relatively constant throughout leaf development, similar to expression patterns of AtPORA and AtPORB in Arabidopsis. However, OsPORB expression is rapidly upregulated by HL treatment, similar to the fluence rate-dependent regulation of AtPORC. This suggests that OsPORB function is equivalent to both AtPORB and AtPORC functions. Our results demonstrate that OsPORB is essential for maintaining light-dependent Chl synthesis throughout leaf development, especially under HL conditions, whereas OsPORA mainly functions in the early stages of leaf development. Developmentally and physiologically distinct roles of monocot OsPORs are discussed by comparing with those of dicot AtPORs.

The loss of green coloration via chlorophyll (Chl) degradation typically occurs during leaf senescence. To date, many Chl catabolic enzymes have been identified and shown to interact with light harvesting complex II to form a Chl degradation complex in senescing chloroplasts; this complex might metabolically channel phototoxic Chl catabolic intermediates to prevent oxidative damage to cells. The Chl catabolic enzyme 7-hydroxymethyl Chl a reductase (HCAR) converts 7-hydroxymethyl Chl a (7-HMC a) to Chl a. The rice (Oryza sativa) genome contains a single HCAR homolog (OsHCAR), but its exact role remains unknown. Here, we show that an oshcar knockout mutant exhibits persistent green leaves during both dark-induced and natural senescence, and accumulates 7-HMC a and pheophorbide a (Pheo a) in green leaf blades. Interestingly, both rice and Arabidopsis hcar mutants exhibit severe cell death at the vegetative stage; this cell death largely occurs in a light intensity-dependent manner. In addition, 7-HMC a treatment led to the generation of singlet oxygen (1O2) in Arabidopsis and rice protoplasts in the light. Under herbicide-induced oxidative stress conditions, leaf necrosis was more severe in hcar plants than in wild type, and HCAR-overexpressing plants were more tolerant to reactive oxygen species than wild type. Therefore, in addition to functioning in the conversion of 7-HMC a to Chl a in senescent leaves, HCAR may play a critical role in protecting plants from high light-induced damage by preventing the accumulation of 7-HMC a and Pheo a in developing and mature leaves at the vegetative stage.

In Arabidopsis, EARLY FLOWERING3 (ELF3) has pivotal roles in controlling circadian rhythm and photoperiodic flowering. In addition, ELF3 negatively regulates leaf senescence by repressing the transcription of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PHYTOCHROME-INTERACTING FACTOR5 (PIF5); elf3 mutants senesce earlier and ELF3-overexpressing (ELF3-OX) plants senesce later than wild type (WT). Here, we show that in contrast to Arabidopsis ELF3, which represses senescence, the rice homolog OsELF3.1 promotes leaf senescence; oself3.1 mutants showed delayed senescence and OsELF3.1-OX plants senesced earlier under both dark-induced and natural senescence conditions. Microarray analysis revealed that in the senescing leaves, a number of senescence-associated genes, phytohormone-related genes, and NAC and WRKY family genes (OsNAP, ONAC106, and OsWRKY42) were differentially expressed in oself3.1 mutants compared with WT. Interestingly, we found that Arabidopsis plants overexpressing OsELF3.1 show delayed leaf senescence, produce short petioles, and flower late in long days, just like Arabidopsis ELF3-OX plants. This demonstrates that the regulatory functions of ELF3 and OsELF3.1 are conserved between Arabidopsis and rice, but the downstream regulatory cascades have opposite effects.

Xanthomonas oryzae pv. oryzae causing bacterial leaf blight disease in rice produces and secretes Hpa1 protein that belongs to harpin protein family. Previously it was reported that Hpa1 induced defense responses when it was produced in tobacco. In this study, we expressed hpa1 gene in rice and Arabidopsis to examine the effects of Hpa1 expression on disease resistance to both fungal and bacterial pathogens. Expression of hpa1 gene in rice enhanced disease resistance to both X. oryzae pv. oryzae and Magnaporthe grisea. Interestingly, individual transgenic rice plants could be divided into four groups, depending on responses to both pathogens. hpa1 expression in Arabidopsis also enhanced disease resistance to both Botrytis cineria and Xanthomonas campestris pv. campestris. To examine genes that are upregulated in the transgenic rice plants after inoculation with X. oryzae pv. oryzae, known defense-related genes were assessed, and also microarray analysis with the Rice 5 K DNA chip was performed. Interestingly, expression of OsACS1 gene, which was found as the gene that showed the highest induction, was induced earlier and stronger than that in the wild type plant. These results indicate that hpa1 expression in the diverse plant species, including monocot and dicot, can enhance disease resistance to both fungal and bacterial plant pathogens.

Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most serious diseases in wheat (Triticum aestivum) and barley (Hordeum vulgare). Dahongmil is an elite Korean wheat cultivar with relatively high resistance to FHB. To identify differentially expressed genes in the resistant cultivar Dahongmil and the susceptible cultivar Urimil after inoculation of F. graminearum, we used the Affymetrix GeneChip® Wheat Genome Array to identify 328 ESTs that were differentially expressed in inoculated seedling tissues of the two cultivars. From these, we selected 16 induced genes and found that they have defense functions, such as genes encoding pathogen resistance proteins, oxidative stress-related proteins, metabolism, and proteins involved in defense mechanisms. To verify the DNA microarray results, we tested seven of these genes by semiquantitative RT-PCR and confirmed that these defense- and stress-related genes were expressed at much higher levels in the resistant Dahongmil cultivar. We next developed a hypothetical functional gene network and identified 89 interaction pairs mediated by four of the differentially expressed genes in the hypothetical network. We further refined the network by identifying nine genes showing significant up- or down-regulation after FHB challenge in the resistant cultivar and two genes having multiple interactions with queried proteins. We hope that the set of induced genes identified in this study can be used for development of new wheat and barley cultivars with improved resistance to FHB.

The Arabidopsis transcriptional factor NAC016 directly activates chlorophyll degradation during leaf senescence by binding to the promoter of SGR1 and upregulating its transcription. During leaf senescence or abiotic stress in Arabidopsis thaliana, STAYGREEN1 (SGR1) promotes chlorophyll (Chl) degradation, acting with Chl catabolic enzymes, but the mechanism regulating SGR1 transcription remains largely unknown. Here, we show that the Arabidopsis senescence-associated NAC transcription factor NAC016 directly activates SGR1 transcription. Under senescence-promoting conditions, the expression of SGR1 was downregulated in nac016-1 mutants and upregulated in NAC016-overexpressing (NAC016-OX) plants. By yeast one-hybrid and chromatin immunoprecipitation assays, we found that NAC016 directly binds to the SGR1 promoter, which contains the NAC016-specific binding motif (termed the NAC016BM). Furthermore, nac016-1 SGR1-OX plants showed an early leaf yellowing phenotype, similar to SGR1-OX plants, confirming that NAC016 directly activates SGR1 expression in the leaf senescence regulatory cascade. Although we found that NAC016 activates SGR1 expression in senescing leaves, this transcriptional regulation is considerably weaker in maturing seeds; the seeds of sgr1-1 mutants (also known as nonyellowing1-1, nye1-1) stayed green, while the seeds of nac016-1 mutants turned from green to yellow normally. We also found that the abscisic acid (ABA) signaling-related transcription factor genes ABI5 and EEL and the ABA biosynthesis gene AAO3, which activate SGR1 expression directly or indirectly, were significantly downregulated in nac016-1 mutants and upregulated in NAC016-OX plants. However, the NAC016BM does not exist in their promoter regions, indicating that NAC016 may indirectly activate these ABA signaling and biosynthesis genes, probably by directly activating transcriptional cascades regulated by the NAC transcription factor NAP. The NAC016-mediated regulatory cascades of SGR1 and other Chl degradation-related genes are discussed.

Suppression of Flowering Locus C (FLC) is critical for the transition from the vegetative to reproductive phase in Arabidopsis. FLC represses the expression of several genes involved in floral induction and its transcription is positively or negatively regulated by FRIGIDA or by vernalization, respectively. However, the regulatory mechanisms for the level and stability of FLC protein throughout development are unknown. Here, we show that SINAT5 colocalizes with FLC in the nuclear bodies and that its zinc finger motif interacts directly with the MADS-box domain of FLC. In addition, it is shown that SINAT5 has an E3 ubiquitin ligase activity towards FLC as a substrate, suggesting that SINAT5 participates in the regulation of flowering time through the ubiquitin-mediated proteolysis of FLC.

Floral transition is influenced by environmental factors such as light and temperature. Plants are capable of integrating photoperiod and ambient temperature signaling into their developmental program. Despite extensive investigations on individual genetic pathways, little is known about the molecular components that integrate both pathways. Here, we demonstrate that the RING finger–containing E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) acts as an integrator of photoperiod and ambient temperature signaling. In addition to the role in photoperiodic destabilization of CONSTANS (CO), COP1 also regulates temperature sensitivity by controlling the degradation of GIGANTEA (GI). COP1-impaired mutants showed reduced sensitivity to low ambient temperature. Notably, COP1 is more stabilized at low temperature and accelerates GI turnover in a 26S proteasome-dependent manner. The direct association of GI with the promoter of FLOWERING LOCUS T (FT) was reduced because of its ambient temperature-dependent protein stability control and thus COP1-triggered GI turnover delays flowering at low temperatures via a CO-independent pathway. Taken together, our findings indicate that environmental conditions regulate the stability of COP1 and conditional specificity of its target selection stimulates proper developmental responses and ensures reproductive success.

Light regulates leaf senescence and light deprivation causes large-scale transcriptional reprogramming to dismantle cellular components and remobilize nutrients to sink organs, such as seeds and storage tissue. We recently reported that in Arabidopsis (Arabidopsis thaliana), Phytochrome-Interacting Factor4 (PIF4) and PIF5 promote dark-induced senescence and natural senescence by directly activating the expression of typical senescence-associated genes (SAGs), including ORESARA1 (ORE1) and ETHYLENE INSENSITIVE3 (EIN3). In contrast, phytochrome B (PhyB) inhibits leaf senescence by repressing PIF4 and PIF5 at the post-translational level. Although we found how red light signaling represses leaf senescence in Arabidopsis, it remains unknown whether PhyB and/or PhyA are involved in leaf senescence in rice (Oryza sativa). Here we show that rice phyB knockout mutants (osphyB-1, -2, and -3) exhibited an early senescence phenotype during dark-induced senescence, but an osphyA knockout mutant (osphyA-3) senesced normally. The RT-qPCR analysis revealed that several senescence-associated genes, including OsORE1 and OsEIN3, were significantly up-regulated in osphyB-2 mutants, indicating that OsPhyB also inhibits leaf senescence, like Arabidopsis PhyB. We also found that leaf segments of osphyB-2 senesced faster even under light conditions. Supplementation with nitrogen compounds, such as KNO₃ and NH₄NO₃, rescued the early senescence phenotype of osphyB-2, indicating that starvation is one of the major signaling factors in the OsPhyB-dependent leaf senescence pathway.

Background Gibberellic acid (GA; or gibberellin) affects the development of floral organs, especially anthers and pollen, and perturbation of development of male floral organs can cause sterility. Many studies of GA signaling have concentrated on anther development, but the effect of GA on grain production remains to be examined. Results Using a cross of ‘Milyang23 (M23)’, which has a functional allele of Early flowering1 (EL1), and ‘H143’, which has a nonfunctional el1 allele, we generated heterogeneous inbred family-near isogenic lines (HNILs) that are homozygous for EL1 [HNIL(M23)] or el1 [HNIL(H143)]. Here, we found that HNIL(H143) exhibited anther deformities and low pollen viability. The expression of GAMYB, a major activator of GA signaling, and its downstream genes CYP703A3 and KAR, mainly involved in pollen formation, increased abnormally during spikelet development; this activation of GA signaling may cause the sterility. To confirm the negative effect of the el1 mutation on spikelet fertility, we examined a line carrying a T-DNA insertion el1 mutant [hereafter ZH11(el1)] and its parental cultivar ‘Zhonghua11 (ZH11)’. ZH11(el1) showed nearly identical defects in anther development and pollen viability as HNIL(H143), leading to decreased seed setting rate. However, the elite japonica cultivar Koshihikari, which has a nonfunctional el1 allele for early flowering in long days, produces fertile spikelets and normal grain yields, like other elite japonica cultivars. This indicates that as-yet-unknown regulator(s) that can overcome the male sterile phenotype of the el1 mutation must have been introduced into Koshihikari. Conclusions The el1 mutation contributes to early flowering in japonica rice under long days but fails to limit GA signaling, thus negatively affecting spikelet fertility, which results in a loss of grain yield. Thus, EL1 is essential for photoperiod sensitivity in flowering as well as spikelet fertility in grain production.

Arabidopsis flowers early under long days (LD) and late under short days (SD).The repressor of photomorphogenesis DE-ETIOLATED1 (DET1) delaysflowering; det1-1 mutants flower early, especially under SD, but themolecular mechanism of DET1 regulation remains unknown. Here we examine theregulatory function of DET1 in repression of flowering. Under SD, the det1-1mutation causes daytime expression of FKF1 and CO; however, theiraltered expression has only a small effect on early flowering in det1-1mutants. Notably, DET1 interacts with GI and binding of GI to the FT promoterincreases in det1-1 mutants, suggesting that DET1 mainly restricts GIfunction, directly promoting FT expression independent of COexpression. Moreover, DET1 interacts with MSI4/FVE, which epigenetically inhibitsFLC expression, indicating that the lack of FLC expression indet1-1 mutants likely involves altered histone modifications at theFLC locus. These data demonstrate that DET1 acts in both photoperiod andautonomous pathways to inhibit expression of FT and SOC1. Consistentwith this, the early flowering of det1-1 mutants disappears completely in theft-1 soc1-2 double mutant background. Thus, we propose that DET1 is astrong repressor of flowering and has a pivotal role in maintaining photoperiodsensitivity in the regulation of flowering time.

Drought and other abiotic stresses negatively affect plant growth and development and thus reduce productivity. The plant-specific NAM/ATAF1/2/CUC2 (NAC) transcription factors have important roles in abiotic stress-responsive signaling. Here, we show that Arabidopsis thaliana NAC016 is involved in drought stress responses; nac016 mutants have high drought tolerance, and NAC016 -overexpressing ( NAC016 -OX) plants have low drought tolerance. Using genome-wide gene expression microarray analysis and MEME motif searches, we identified the NAC016-specific binding motif (NAC16BM), GATTGGAT[AT]CA, in the promoters of genes downregulated in nac016-1 mutants. The NAC16BM sequence does not contain the core NAC binding motif CACG (or its reverse complement CGTG). NAC016 directly binds to the NAC16BM in the promoter of ABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN1 ( AREB1 ), which encodes a central transcription factor in the stress-responsive abscisic acid signaling pathway and represses AREB1 transcription. We found that knockout mutants of the NAC016 target gene NAC-LIKE, ACTIVATED BY AP3/PI ( NAP ) also exhibited strong drought tolerance; moreover, NAP binds to the AREB1 promoter and suppresses AREB1 transcription. Taking these results together, we propose that a trifurcate feed-forward pathway involving NAC016 , NAP , and AREB1 functions in the drought stress response, in addition to affecting leaf senescence in Arabidopsis.

The identification of molecular markers and marker-aided selection are essential to the efficient breeding of drought-tolerant plants. However, because that characteristic is controlled by many quantitative trait loci, such markers that can screen and trace desirable barley genotypes in a segregating population or germplasm have not yet been determined. Relative water content has been used to estimate drought tolerance in plants because it is highly correlated with the drought index of yield. To develop reliable gene-specific markers for identifying tolerant versus susceptible genotypes, we performed suppression subtractive hybridization to identify candidate genes. We used two domestic barley cultivars, one having the highest RWC (drought-tolerant ‘Chalbori’) and the other having the lowest (drought-susceptible ‘Daebaekbori’). In response to dehydration at the early seedling stage, rapid upregulation ofDehydrin3 (Dhn3) andDhn4 occurred in the drought-tolerant genotypes, but not in the susceptible ones. Similar results were obtained with mature plants growing under frequent drought stress in the greenhouse. In addition,Dhn3 andDhn4 conferred higher drought tolerance when they were over-expressed in transgenicArabidopsis. Thus, in addition to using assessments of RWC, we propose thatDhn3 andDhn4 expressions can serve as drought-induced gene-specific markers to determine drought-tolerant barley genotypes at the seedling stage.

The floral transition in Arabidopsis is regulated by at least four flowering pathways: the long-day, autonomous, vernalization, and gibberellin (GA)-dependent pathways. Previously, we reported that the MADS-box transcription factor SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) integrates the long-day and vernalization/autonomous pathways. Here, we present evidences that SOC1 also integrates signaling from the GA-dependent pathway, a major flowering pathway under non-inductive short days. Under short days, the flowering time of GA-biosynthetic and -signaling mutants was well correlated with the level of SOC1 expression; overexpression of SOC1 rescued the non-flowering phenotype of ga1-3, and the soc1 null mutant showed reduced sensitivity to GA for flowering. In addition, we show that vernalization-induced repression of FLOWERING LOCUS C (FLC), an upstream negative regulator of SOC1, is not sufficient to activate SOC1; positive factors are also required. Under short days, the GA pathway provides a positive factor for SOC1 activation. In contrast to SOC1, the GA pathway does not regulate expression of other flowering integrators FLC and FT. Our results explain why the GA pathway has a strong effect on flowering under short days and how vernalization and GA interact at the molecular level.

The germination of rice (Oryza sativa L.) seed has been known to be unaffected by light or darkness, but a photoblastic rice (PBR) whose germination was favored by light was discovered in weedy rice. The effects of light, temperature, and soil burial depth on the seed dormancy and germination of PBR were examined. At 30°C, the seeds germinated 100% under white or red (R) light and below 1% in darkness. They showed 67 and 20% of germination under continuous and a brief pulse of far-red (FR) light, respectively, and photoreversible germination under alternating irradiation of R and FR light, indicating that the induction of germination occurs through very-low-fluence response and low-fluence response of phytochromes. The prompt and delayed induction of germination by a pulse of R and FR light, respectively, following dark imbibition suggests that phytochrome B exists in dormant seeds and phytochrome A is synthesized during dark imbibition. Dark imbibition for longer than 3 d induced secondary dormancy. In darkness, the germination frequency was about 28% at 15 to 20°C and below 1% at 25 to 40°C. At 12 h-diurnal fluctuations of 20 and 10°C and 25 and 15°C in darkness, the germination frequencies were 77 and 27%, respectively. When the seeds were sown in the soil, emergence frequency decreased as burial depth increased, and 12-h diurnal fluctuation of 20 and 10°C induced more seedling emergence than constant 15°C. Conclusively, PBR seeds are capable of germinating by sensing light or proximity to the soil surface during seasonal fluctuations in diurnal temperatures.

Plants initiate senescence to shed photosynthetically inefficient leaves. Light deprivation induces leaf senescence, which involves massive transcriptional reprogramming to dismantle cellular components and remobilize nutrients. In darkness, intermittent pulses of red light can inhibit senescence, likely via phytochromes. However, the precise molecular mechanisms transducing the signals from light perception to the inhibition of senescence remain elusive. Here, we show that in Arabidopsis, dark-induced senescence requires phytochrome-interacting transcription factors PIF4 and PIF5 (PIF4/PIF5). ELF3 and phytochrome B inhibit senescence by repressing PIF4/PIF5 at the transcriptional and post-translational levels, respectively. PIF4/PIF5 act in the signalling pathways of two senescence-promoting hormones, ethylene and abscisic acid, by directly activating expression of EIN3, ABI5 and EEL. In turn, PIF4, PIF5, EIN3, ABI5 and EEL directly activate the expression of the major senescence-promoting NAC transcription factor ORESARA1, thus forming multiple, coherent feed-forward loops. Our results reveal how classical light signalling connects to senescence in Arabidopsis.

The highly ordered process of senescence forms the final stage of leaf development; a large set of senescence-associated genes (SAGs) execute this orderly dismantling of the photosynthetic apparatus and remobilization of cellular components. A number of transcription factors (TFs) modulate SAG expression to promote or delay senescence. Here we show that NAC016, the previously uncharacterized senescence-associated NAM/ATAF1/2/CUC2 (senNAC) TF in Arabidopsis thaliana, promotes senescence. Leaves of nac016 mutants remained green under senescence-inducing conditions, and leaves of NAC016-overexpressing (NAC016-OX) plants senesced early. Under dark-induced senescence (DIS) conditions, nac016 mutants had low ion leakage, and retained the proper balance of photosystem proteins and normal grana thylakoid shape much longer than wild-type plants, suggesting that nac016 acts as a functional stay-green type senescence mutant. Under DIS conditions, SAGs (NYC1, PPH, SGR1/NYE1 and WRKY22), including senNACs (JUB1, NAP, ORE1, ORS1 and VNI2), were down-regulated in nac016 mutants and up-regulated in NAC016-OX plants. In addition to its role in senescence, NAC016 also affects abiotic stress. Under salt and oxidative stress conditions, NAC016 expression rapidly increased in developing leaves, possibly to promote senescence. Indeed, under the stress conditions, nac016 mutants stayed green and NAC016-OX plants senesced rapidly. To identify direct targets of the NAC016 TF in the regulation of leaf senescence, we conducted yeast one-hybrid assays, which strongly suggested that NAC016 binds to the promoters of NAP and ORS1. Based on these results, we propose that NAC016 regulatory mechanisms promoting leaf senescence exhibit cross-talk with the salt and oxidative stress-responsive signaling pathways.

The phytohormone gibberellic acid (GA) has essential signaling functions in multiple processes during plant development. In the "Green Revolution", breeders developed high-yield rice cultivars that exhibited both semi-dwarfism and altered GA responses, thus improving grain production. Most studies of GA have concentrated on germination and cell elongation, but GA also has a pivotal role in floral organ development, particularly in stamen/anther formation. In rice, GA signaling plays an important role in spikelet fertility; however, the molecular genetic and biochemical mechanisms of GA in male fertility remain largely unknown. Here, we review recent progress in understanding the network of GA signaling and its connection with spikelet fertility, which is tightly associated with grain productivity in cereal crops.

A temperature-sensitive mutant of Capsicum chinense, sy-2, shows a normal developmental phenotype when grown above 24°C. However, when grown at 20°C, sy-2 exhibits developmental defects, such as chlorophyll deficiency and shrunken leaves. To understand the underlying mechanism of this temperature-dependent response, phenotypic characterization and genetic analysis were performed. The results revealed abnormal chloroplast structures and cell collapse in leaves of the sy-2 plants grown at 20°C. Moreover, an excessive accumulation of reactive oxygen species (ROS) resulting in cell death was detected in the chlorophyll-deficient sectors of the leaves. However, the expression profile of the ROS scavenging genes did not alter in sy-2 plants grown at 20°C. A further analysis of fatty acid content in the leaves showed the impaired pathway of linoleic acid (18:2) to linolenic acid (18:3). Additionally, the Cafad7 gene was downregulated in sy-2 plants. This change may lead to dramatic physiological disorder and alteration of leaf morphology in sy-2 plants by losing low-temperature tolerance. Genetic analysis of an F(2) population from a cross between C. chinense 'sy-2' and wild-type C. chinense 'No. 3341' showed that the sy-2 phenotype is controlled by a single recessive gene. Molecular mapping revealed that the sy-2 gene is located at a genomic region of the pepper linkage group 1, corresponding to the 300 kb region of the Ch1_scaffold 00106 in tomato chromosome 1. Candidate genes in this region will reveal the identity of sy-2 and the underlying mechanism of the temperature-dependent plant response.

Seasonal changes in day length are perceived by plant photoreceptors and transmitted to the circadian clock to modulate developmental responses, such as flowering time. Blue light-sensing cryptochromes, the E3 ubiquitin-ligase COP1, and clock-associated proteins ELF3 and GI, regulate this process, although the regulatory link between them is unclear. Here, we present data showing that COP1 acts with ELF3 to mediate day length signaling from CRY2 to GI within the photoperiod flowering pathway. We found that COP1 and ELF3 interact in vivo and show that ELF3 allows COP1 to interact with GI in vivo, leading to GI degradation in planta. Accordingly, mutation of COP1 or ELF3 disturbs the pattern of GI cyclic accumulation. We propose a model in which ELF3 acts as a substrate adaptor, enabling COP1 to modulate light input signal to the circadian clock through targeted destabilization of GI.

Loss of green color in leaves results from chlorophyll (Chl) degradation in chloroplasts, but little is known about how Chl catabolism is regulated throughout leaf development. Using the staygreen (sgr) mutant in rice (Oryza sativa), which maintains greenness during leaf senescence, we identified Sgr, a senescence-associated gene encoding a novel chloroplast protein. Transgenic rice overexpressing Sgr produces yellowish-brown leaves, and Arabidopsis thaliana pheophorbide a oxygenase–impaired mutants exhibiting a stay-green phenotype during dark-induced senescence have reduced expression of Sgr homologs, indicating that Sgr regulates Chl degradation at the transcriptional level. We show that the leaf stay-greenness of the sgr mutant is associated with a failure in the destabilization of the light-harvesting chlorophyll binding protein (LHCP) complexes of the thylakoid membranes, which is a prerequisite event for the degradation of Chls and LHCPs during senescence. Transient overexpression of Sgr in Nicotiana benthamiana and an in vivo pull-down assay show that Sgr interacts with LHCPII, indicating that the Sgr-LHCPII complexes are formed in the thylakoid membranes. Thus, we propose that in senescing leaves, Sgr regulates Chl degradation by inducing LHCPII disassembly through direct interaction, leading to the degradation of Chls and Chl-free LHCPII by catabolic enzymes and proteases, respectively.

Light is an important environmental signal that governs plant growth and development. One important light-signalling component involved in plant light responses is COP1. The pleiotropic phenotypes of the cop1 mutant suggest that COP1 regulates not only photomorphogenesis, but also other developmental processes. We investigated the role of COP1 by identifying genes that are regulated by COP1. We report that AtMYB21, a gene encoding a flower-specific transcription factor, is ectopically expressed in the cop1 mutant. Analysis shows that dark-grown transgenic seedlings expressing AtMYB21-GR fusion protein display some features of the cop1 mutant, including decreased hypocotyl cell expansion, open cotyledons in the dark, and seedling lethality in the presence of dexamethasone. Light-grown adult transgenic plants expressing AtMYB21 have shorter stems, smaller and narrower leaves, narrower petals, and malformed carpels. In addition, we show that AtMYB21 directly regulates two genes that are also expressed more abundantly in the cop1 mutant. The results indicate that COP1 is required to repress the AtMYB21 gene in seedlings, and the pleiotropic phenotypes shown in the cop1 mutant are due to the combination of misregulation of genuine light-signalling components and other tissue-specific factors.

Summary Under mild abiotic-stress conditions, Arabidopsis atg mutants showed a functional stay-green phenotype which is probably caused by the lack of chloroplastic autophagy and the retrograde regulation of senescence-associated gene expression., 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.