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During crop cultivation, water-deficit conditions retard growth, thus reducing crop productivity. Therefore, uncovering the mechanisms behind drought tolerance is a critical task for crop improvement. Here, we show that the rice (Oryza sativa) WRKY transcription factor OsWRKY5 negatively regulates drought tolerance. We determined that OsWRKY5 was mainly expressed in developing leaves at the seedling and heading stages, and that its expression was reduced by drought stress and by treatment with NaCl, mannitol, and abscisic acid (ABA). Notably, the genome-edited loss-of-function alleles oswrky5-2 and oswrky5-3 conferred enhanced drought tolerance, measured as plant growth under water-deficit conditions. Conversely, the overexpression of OsWRKY5 in the activation-tagged line oswrky5-D resulted in higher susceptibility under the same conditions. The loss of OsWRKY5 activity increased sensitivity to ABA, thus promoting ABA-dependent stomatal closure. Transcriptome deep sequencing and reverse transcription quantitative polymerase chain reaction analyses demonstrated that the expression of abiotic stress-related genes including rice MYB2 (OsMYB2) was upregulated in oswrky5 knockout mutants and downregulated in oswrky5-D mutants. Moreover, dual-luciferase, yeast one-hybrid, and chromatin immunoprecipitation assays showed that OsWRKY5 directly binds to the W-box sequences in the promoter region of OsMYB2 and represses OsMYB2 expression, thus downregulating genes downstream of OsMYB2 in the ABA signaling pathways. Our results demonstrate that OsWRKY5 functions as a negative regulator of ABA-induced drought stress tolerance, strongly suggesting that inactivation of OsWRKY5 or manipulation of key OsWRKY5 targets could be useful to improve drought tolerance in rice cultivars.

In most plants, abscisic acid (ABA) induces premature leaf senescence; however, the mechanisms of ABA signaling during leaf senescence remain largely unknown. Here, we show that the rice (Oryza sativa) NAM/ATAF1/2/CUC2 (NAC) transcription factor ONAC054 plays an important role in ABA-induced leaf senescence. The onac054 knockout mutants maintained green leaves, while ONAC054-overexpressing lines showed early leaf yellowing under dark- and ABA-induced senescence conditions. Genome-wide microarray analysis showed that ABA signaling-associated genes, including ABA INSENSITIVE5 (OsABI5) and senescence-associated genes, including STAY-GREEN and NON-YELLOW COLORING1 (NYC1), were significantly down-regulated in onac054 mutants. Chromatin immunoprecipitation and protoplast transient assays showed that ONAC054 directly activates OsABI5 and NYC1 by binding to the mitochondrial dysfunction motif in their promoters. ONAC054 activity is regulated by proteolytic processing of the C-terminal transmembrane domain (TMD). We found that nuclear import of ONAC054 requires cleavage of the putative C-terminal TMD. Furthermore, the ONAC054 transcript (termed ONAC054α) has an alternatively spliced form (ONAC054β), with seven nucleotides inserted between intron 5 and exon 6, truncating ONAC054α protein at a premature stop codon. ONAC054β lacks the TMD and thus localizes to the nucleus. These findings demonstrate that the activity of ONAC054, which is important for ABA-induced leaf senescence in rice, is precisely controlled by multilayered regulatory processes.

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].

Leaf senescence is the final stage of leaf development and in cereal crops, the timing of senescence relative to grain filling has major effects on agronomic traits such as yield. Although many genetic factors are involved in the regulation of leaf senescence in cereals, the key regulators remain to be determined. Plant transcription factors with a conserved DOF (DNA-binding one zinc finger) domain play roles in multiple physiological processes. Here, we show a novel function for OsDOF24 as a repressor of leaf senescence in rice (Oryza sativa). In wild-type leaves, OsDOF24 expression rapidly decreased during natural senescence (NS) and dark-induced senescence (DIS). The gain-of-function mutant osdof24-D, which contains an enhancer-trap T-DNA in the OsDOF24 promoter, exhibited delayed leaf yellowing during NS and DIS. Transgenic plants overexpressing OsDOF24 showed the same phenotype during DIS. Reverse-transcription quantitative real-time PCR analysis revealed that senescence-associated genes (Osl85, Osl57 and OsNAP) and chlorophyll degradation genes (NYC1, NYC3 and SGR) were downregulated in the osdof24-D mutant during dark incubation. Among the phytohormones, only methyl jasmonate induced OsDOF24 expression. Furthermore, the reduced expression of jasmonate biosynthesis-related genes (OsLOX2, OsLOX8, OsHI-LOX, OsAOS1 and OsAOS2) in osdof24-D decreased endogenous jasmonate levels, resulting in delayed leaf senescence under DIS conditions. Yeast one-hybrid assays showed that OsDOF24 binds to the promoter region of OsAOS1. Taken together, our results demonstrate that OsDOF24 suppresses the induction of leaf senescence during vegetative growth by deactivating jasmonate biosynthetic pathways.

MYB-type transcription factors (TFs) play important roles in plant growth and development, and in the responses to several abiotic stresses. In rice (Oryza sativa), the roles of MYB-related TFs in leaf senescence are not well documented. Here, we examined rice MYB TF gene OsMYB102 and found that an OsMYB102 T-DNA activation-tagged line (termed osmyb102-D), which constitutively expresses OsMYB102 under the control of four tandem repeats of the 35S promoter, and OsMYB102-overexpressing transgenic lines (35S:OsMYB102 and 35S:GFP-OsMYB102) maintain green leaves much longer than the wild-type under natural, dark-induced, and abscisic acid (ABA)-induced senescence conditions. Moreover, an osmyb102 knockout mutant showed an accelerated senescence phenotype under dark-induced and ABA-induced leaf senescence conditions. Microarray analysis showed that a variety of senescence-associated genes (SAGs) were down-regulated in the osmyb102-D line. Further studies demonstrated that overexpression of OsMYB102 controls the expression of SAGs, including genes associated with ABA degradation and ABA signaling (OsABF4, OsNAP, and OsCYP707A6), under dark-induced senescence conditions. OsMYB102 inhibits ABA accumulation by directly activating the transcription of OsCYP707A6, which encodes the ABA catabolic enzyme ABSCISIC ACID 8′-HYDROXYLASE. OsMYB102 also indirectly represses ABA-responsive genes, such as OsABF4 and OsNAP. Collectively, these results demonstrate that OsMYB102 plays a critical role in leaf senescence by down-regulating ABA accumulation and ABA signaling responses., Rice transcription factor OsMYB102 delays leaf senescence by decreasing ABA accumulation through up-regulation of OsCYP707A6, which encodes an ABA catalytic enzyme.

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.

Leaf senescence is the final stage of leaf development and an important step that relocates nutrients for grain filling in cereal crops. Senescence occurs in an age-dependent manner and under unfavorable environmental conditions such as deep shade, water deficit, and high salinity stresses. Although many transcription factors that modulate leaf senescence have been identified, the mechanisms that regulate leaf senescence in response to environmental conditions remain elusive. Here, we show that rice (Oryza sativa) ETHYLENE RESPONSE FACTOR 101 (OsERF101) promotes the onset and progression of leaf senescence. OsERF101 encodes a predicted transcription factor and OsERF101 transcript levels rapidly increased in rice leaves during dark-induced senescence (DIS), indicating that OsERF101 is a senescence-associated transcription factor. Compared with wild type, the oserf101 T-DNA knockout mutant showed delayed leaf yellowing and higher chlorophyll contents during DIS and natural senescence. Consistent with its delayed-yellowing phenotype, the oserf101 mutant exhibited downregulation of genes involved in chlorophyll degradation, including rice NAM, ATAF1/2, and CUC2 (OsNAP), STAY-GREEN (SGR), NON-YELLOW COLORING 1 (NYC1), and NYC3 during DIS. After methyl jasmonate treatment to induce rapid leaf de-greening, the oserf101 leaves retained more chlorophyll compared with wild type, indicating that OsERF101 is involved in promoting jasmonic acid (JA)-induced leaf senescence. Consistent with the involvement of JA, the expression of the JA signaling genes OsMYC2/JA INSENSITIVE 1 (OsJAI1) and CORONATINE INSENSITIVE 1a (OsCOI1a), was downregulated in the oserf101 leaves during DIS. Transient transactivation and chromatin immunoprecipitation assays revealed that OsERF101 directly binds to the promoter regions of OsNAP and OsMYC2, which activate genes involved in chlorophyll degradation and JA signaling-mediated leaf senescence. These results demonstrate that OsERF101 promotes the onset and progression of leaf senescence through a JA-mediated signaling pathway.

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.

In higher plants, abscisic acid (ABA) biosynthesis and ABA signaling are associated with seed germination, leaf senescence, and abiotic stress tolerance. Here, we demonstrate that the MYB-related transcription factor Oryza sativa RADIALIS-LIKE3 (OsRL3) promotes dark-induced leaf senescence and reduces susceptibility to salt stress in rice. OsRL3 expression was upregulated in detached leaves under dark-induced senescence conditions and in seedlings under salt-stress conditions. In the dark, osrl3 null mutants exhibited a stay-green phenotype, with increased chlorophyll retention and photosynthetic capacity. RT-qPCR analysis demonstrated that, compared with wild type (WT), the expression levels of chlorophyll-degradation and senescence-associated genes were lower in osrl3 mutants; this was likely responsible for their stay-green phenotype. Under salt stress, proline levels were lower in osrl3 mutants than WT due to the reduced expression of proline biosynthesis genes, thereby leading to hypersensitivity to salt stress. Among phytohormones, OsRL3 expression was induced by only ABA, and osrl3 mutants were less sensitive to exogenous ABA treatment than WT. RT-qPCR analysis indicated that ABA signaling genes were downregulated in osrl3 mutants under both dark and salt-stress treatment. Taken together, these results indicate that OsRL3 promotes leaf senescence and delays the salt-stress response via ABA signaling pathways.

Soil degradation affects agriculture worldwide. Soils with high salt can result from local geological conditions or accumulation of salt from irrigation. Salt limits water uptake and reduces crop yields; therefore, salt tolerance is an important trait for crops grown in high-salt soils. Here, we show that the rice (Oryza sativa) phytochrome B (osphyB) mutant has greater tolerance to salt stress than its parent japonica rice (cv. Dongjin). We found that the osphyB mutant showed a higher survival rate, fresh weight, and levels of total chlorophylls and carotenoids, as well as enhanced membrane integrity under salt stress compared to the wild type. OsPHYB transcripts increased in tissues of the wild type after salt treatment; OsPHYB expression was much higher in the leaf blade than in the stem and root. The osphyB mutant accumulated less Na+ in the shoot and considerably more K+ in both the shoot and root, maintaining a significantly lower Na+ to K+ ratio, possibly due to a lower rate of Na+ uptake and a higher rate of K+ uptake. To elucidate the possible mechanism of salt tolerance in the osphyB mutant, we performed quantitative reverse transcription PCR analysis, which indicated that salt stress-associated genes, including transcription factors and high-affinity K+ transporters, are upregulated in the osphyB mutant under high-salinity conditions. Taken together, our findings show that the null mutation of OsPHYB contributes to a decrease in the Na+/K+ ratio and enhances cell membrane integrity through upregulation of salt stress-associated genes, resulting in improved tolerance to salt stress.

In the tightly regulated chlorophyll biosynthesis pathway, the transcription factor Rice Phytochrome-Interacting Factor-Like1 promotes chlorophyll biosynthesis by up-regulating chlorophyll biosynthetic genes through feed-forward regulatory loops involving GOLDEN2-LIKE1 (OsGLK1) and OsGLK2., In phototrophic plants, the highly conserved and tightly regulated process of chlorophyll (Chl) biosynthesis comprises multi-step reactions involving more than 15 enzymes. Since the efficiency of Chl biosynthesis strongly affects plant productivity, understanding the underlying regulatory mechanisms in crop plants can be useful for strategies to increase grain and biomass yields. Here, we show that rice (Oryza sativa) Phytochrome-Interacting Factor-Like1 (OsPIL1), a basic helix-loop-helix transcription factor, promotes Chl biosynthesis. The T-DNA insertion knockdown ospil1 mutant showed a pale-green phenotype when grown in a natural paddy field. Transcriptome analysis revealed that several genes responsible for Chl biosynthesis and photosynthesis were significantly down-regulated in ospil1 leaves. Using promoter binding and transactivation assays, we found that OsPIL1 binds to the promoters of two Chl biosynthetic genes, OsPORB and OsCAO1, and promotes their transcription. In addition, OsPIL1 directly up-regulates the expression of two transcription factor genes, GOLDEN2-LIKE1 (OsGLK1) and OsGLK2. OsGLK1 and OsGLK2 both bind to the promoters of OsPORB and OsCAO1, as well as some of genes encoding the light-harvesting complex of photosystems, probably promoting their transcription. Thus, OsPIL1 is involved in the promotion of Chl biosynthesis by up-regulating the transcription of OsPORB and OsCAO1 via trifurcate feed-forward regulatory loops involving two OsGLKs.

Expansins are key regulators of cell-wall extension and are also involved in the abiotic stress response. In this study, we evaluated the function of OsEXPA7 involved in salt stress tolerance. Phenotypic analysis showed that OsEXPA7 overexpression remarkably enhanced tolerance to salt stress. OsEXPA7 was highly expressed in the shoot apical meristem, root, and the leaf sheath. Promoter activity of OsEXPA7:GUS was mainly observed in vascular tissues of roots and leaves. Morphological analysis revealed structural alterations in the root and leaf vasculature of OsEXPA7 overexpressing (OX) lines. OsEXPA7 overexpression resulted in decreased sodium ion (Na+) and accumulated potassium ion (K+) in the leaves and roots. Under salt stress, higher antioxidant activity was also observed in the OsEXPA7-OX lines, as indicated by lower reactive oxygen species (ROS) accumulation and increased antioxidant activity, when compared with the wild-type (WT) plants. In addition, transcriptional analysis using RNA-seq and RT-PCR revealed that genes involved in cation exchange, auxin signaling, cell-wall modification, and transcription were differentially expressed between the OX and WT lines. Notably, salt overly sensitive 1, which is a sodium transporter, was highly upregulated in the OX lines. These results suggest that OsEXPA7 plays an important role in increasing salt stress tolerance by coordinating sodium transport, ROS scavenging, and cell-wall loosening.

Exploring genetic methods to improve yield in grain crops such as rice (Oryza sativa) is essential to help meet the needs of the increasing population. Here, we report that rice ONAC096 affects grain yield by regulating leaf senescence and panicle number. ONAC096 expression increased rapidly in rice leaves upon the initiation of aging- and dark-induced senescence. Two independent T-DNA insertion mutants (onac096-1 and onac096-2) with downregulated ONAC096 expression retained their green leaf color during natural senescence in the field, thus extending their photosynthetic capacity. Reverse-transcription quantitative PCR analysis showed that ONAC096 upregulated genes controlling chlorophyll degradation and leaf senescence. Repressed OsCKX2 (encoding cytokinin oxidase/dehydrogenase) expression in the onac096 mutants led to a 15% increase in panicle number without affecting grain weight or fertility. ONAC096 mediates abscisic acid (ABA)-induced leaf senescence by upregulating the ABA signaling genes ABA INSENSITIVE5 and ENHANCED EM LEVEL. The onac096 mutants showed a 16% increase in grain yield, highlighting the potential for using this gene to increase grain production.

he onset of leaf senescence is triggered by external cues and internal factors such as phytohormones and signaling pathways involving transcription factors (TFs). Abscisic acid (ABA) strongly induces senescence and endogenous ABA levels are finely tuned by many senescence-associated TFs. Here, we report on the regulatory function of the senescence-induced TF OsWRKY5 TF in rice (Oryza sativa). OsWRKY5 expression was rapidly upregulated in senescing leaves, especially in yellowing sectors initiated by aging or dark treatment. A T-DNA insertion activation-tagged OsWRKY5-overexpressing mutant (termed oswrky5-D) promoted leaf senescence under natural and dark-induced senescence (DIS) conditions. By contrast, a T-DNA insertion oswrky5-knockdown mutant (termed oswrky5) retained leaf greenness during DIS. Reverse-transcription quantitative PCR (RT-qPCR) showed that OsWRKY5 upregulates the expression of genes controlling chlorophyll degradation and leaf senescence. Furthermore, RT-qPCR and yeast one-hybrid analysis demonstrated that OsWRKY5 indirectly upregulates the expression of senescence-associated NAM/ATAF1/2/CUC2 (NAC) genes including OsNAP and OsNAC2. Precocious leaf yellowing in the oswrky5-D mutant might be caused by elevated endogenous ABA concentrations resulting from upregulated expression of ABA biosynthesis genes OsNCED3, OsNCED4, and OsNCED5, indicating that OsWRKY is a positive regulator of ABA biosynthesis during leaf senescence. Furthermore, OsWRKY5 expression was suppressed by ABA treatment. Taken together, OsWRKY5 is a positive regulator of leaf senescence that upregulates senescence-induced NAC, ABA biosynthesis, and chlorophyll degradation genes.

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.

Changes in plant architecture, such as leaf size, leaf shape, leaf angle, plant height, and floral organs, have been major factors in improving the yield of cereal crops. Moreover, changes in grain size and weight can also increase yield. Therefore, screens for additional factors affecting plant architecture and grain morphology may enable additional improvements in yield. Among the basic Helix-Loop-Helix (bHLH) transcription factors in rice (Oryza sativa), we found an enhancer-trap T-DNA insertion mutant of OsbHLH079 (termed osbhlh079-D). The osbhlh079-D mutant showed a wide leaf angle phenotype and produced long grains, similar to the phenotypes of mutants with increased brassinosteroid (BR) levels or enhanced BR signaling. Reverse transcription-quantitative PCR analysis showed that BR signaling-associated genes are largely upregulated in osbhlh079-D, but BR biosynthesis-associated genes are not upregulated, compared with its parental japonica cultivar &lsquo, Dongjin&rsquo, Consistent with this, osbhlh079-D was hypersensitive to BR treatment. Scanning electron microscopy revealed that the expansion of cell size in the adaxial side of the lamina joint was responsible for the increase in leaf angle in osbhlh079-D. The expression of cell-elongation-associated genes encoding expansins and xyloglucan endotransglycosylases/hydrolases increased in the lamina joints of leaves in osbhlh079-D. The regulatory function of OsbHLH079 was further confirmed by analyzing 35S::OsbHLH079 overexpression and 35S::RNAi-OsbHLH079 gene silencing lines. The 35S::OsbHLH079 plants showed similar phenotypes to osbhlh079-D, and the 35S::RNAi-OsbHLH079 plants displayed opposite phenotypes to osbhlh079-D. Taking these observations together, we propose that OsbHLH079 functions as a positive regulator of BR signaling in rice.

The plant-specific WUSCHEL-related homeobox (WOX) nuclear proteins have important roles in the transcriptional regulation of many developmental processes. Among the rice (Oryza sativa) WOX proteins, a loss of OsWOX3A function in narrow leaf2 (nal2) nal3 double mutants (termed nal2/3) causes pleiotropic effects, such as narrow and curly leaves, opened spikelets, narrow grains, more tillers, and fewer lateral roots, but almost normal plant height. To examine OsWOX3A function in more detail, transgenic rice overexpressing OsWOX3A (OsWOX3A-OX) were generated; unexpectedly, all of them consistently exhibited severe dwarfism with very short and wide leaves, a phenotype that resembles that of gibberellic acid (GA)-deficient or GA-insensitive mutants. Exogenous GA3 treatment fully rescued the developmental defects of OsWOX3A-OX plants, suggesting that constitutive overexpression of OsWOX3A downregulates GA biosynthesis. Quantitative analysis of GA intermediates revealed significantly reduced levels of GA20 and bioactive GA1 in OsWOX3A-OX, possibly due to downregulation of the expression of KAO, which encodes ent-kaurenoic acid oxidase, a GA biosynthetic enzyme. Yeast one-hybrid and electrophoretic mobility shift assays revealed that OsWOX3A directly interacts with the KAO promoter. OsWOX3A expression is drastically and temporarily upregulated by GA3 and downregulated by paclobutrazol, a blocker of GA biosynthesis. These data indicate that OsWOX3A is a GA-responsive gene and functions in the negative feedback regulation of the GA biosynthetic pathway for GA homeostasis to maintain the threshold levels of endogenous GA intermediates throughout development.

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

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.

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.

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.

To improve our understanding of the molecular–genetic mechanisms governing leaf and root development in rice (Oryza sativa), we investigated narrow leaf1 (nal1), a pleiotropic mutant with short and narrow leaves, semi-dwarf stature, and fewer adventitious (or crown) roots. The narrow leaf5 (nal5) and nal1 mutants display similar defects in leaf and root development. Sequence analysis and complementation tests showed that nal5 is allelic to nal1. NAL1 encodes a putative trypsin-like serine/cysteine protease; the coding region of nal5 contains a missense mutation and nal1 harbors a 30-bp deletion. Quantitative real-time PCR revealed that nal1 mutants have altered expression levels of many OSHB, YABBY, and PIN-FORMED genes associated with leaf development and auxin transport. In addition, expression levels of CROWN ROOTLESS genes are markedly down-regulated in nal1. These results indicate that NAL1 functions in the regulation of both leaf and adventitious root development at the transcriptional level. Notably, exogenous auxin treatment rescued the reduced number of adventitious roots in nal1. Based on our results, we propose that NAL1 plays important roles in adventitious root development in rice.

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.

During natural or dark-induced senescence, chlorophyll degradation causes leaf yellowing. Recent evidence indicates that chlorophyll catabolic enzymes (CCEs) interact with the photosynthetic apparatus; for example, five CCEs (NYC1, NOL, PPH, PAO and RCCR) interact with LHCII. STAY-GREEN (SGR) and CCEs interact with one another in senescing chloroplasts; this interaction may allow metabolic channeling of potentially phototoxic chlorophyll breakdown intermediates. 7-Hydroxymethyl chlorophyll a reductase (HCAR) also acts as a CCE, but HCAR functions during leaf senescence remain unclear. Here we show that in Arabidopsis, HCAR-overexpressing plants exhibited accelerated leaf yellowing and, conversely, hcar mutants stayed green during dark-induced senescence. Moreover, HCAR interacted with LHCII in in vivo pull-down assays, and with SGR, NYC1, NOL and RCCR in yeast two-hybrid assays, indicating that HCAR is a component of the proposed SGR-CCE-LHCII complex, which acts in chlorophyll breakdown. Notably, HCAR and NOL are expressed throughout leaf development and are drastically down-regulated during dark-induced senescence, in contrast with SGR, NYC1, PPH and PAO, which are up-regulated during dark-induced senescence. Moreover, HCAR and NOL are highly up-regulated during greening of etiolated seedlings, strongly suggesting a major role for NOL and HCAR in the chlorophyll cycle during vegetative stages, possibly in chlorophyll turnover.

In field conditions, the zebra2 (z2) mutant in rice (Oryza sativa) produces leaves with transverse pale-green/yellow stripes. It was recently reported that ZEBRA2 encodes carotenoid isomerase (CRTISO) and that low levels of lutein, an essential carotenoid for non-photochemical quenching, cause leaf variegation in z2 mutants. However, we found that the z2 mutant phenotype was completely suppressed by growth under continuous light (CL; permissive) conditions, with concentrations of chlorophyll, carotenoids and chloroplast proteins at normal levels in z2 mutants under CL. In addition, three types of reactive oxygen species (ROS; superoxide [O₂⁻], hydrogen peroxide [H₂O₂], and singlet oxygen [¹O₂]) accumulated to high levels in z2 mutants grown under short-day conditions (SD; alternate 10-h light/14-h dark; restrictive), but do not accumulate under CL conditions. However, the levels of lutein and zeaxanthin in z2 leaves were much lower than normal in both permissive CL and restrictive SD growth conditions, indicating that deficiency of these two carotenoids is not responsible for the leaf variegation phenotype. We found that the CRTISO substrate tetra-Cis-lycopene accumulated during the dark periods under SD, but not under CL conditions. Its accumulation was also positively correlated with ¹O₂ levels generated during the light period, which consequently altered the expression of ¹O₂-responsive and cell death-related genes in the variegated z2 leaves. Taking these results together, we propose that the z2 leaf variegation can be largely attributed to photoperiodic accumulation of tetra-cis-lycopene and generation of excessive ¹O₂ under natural day-night conditions.

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.

Highlight Among the lesion mimic mutants in rice, the spotted leaf3 (spl3) locus was identified by map-based cloning that encodes OsMAPKKK1. SPL3 was found to be involved in ABA-responsive signalling and to promote leaf yellowing during senescence., Lesion mimic mutants commonly display spontaneous cell death in pre-senescent green leaves under normal conditions, without pathogen attack. Despite molecular and phenotypic characterization of several lesion mimic mutants, the mechanisms of the spontaneous formation of cell death lesions remain largely unknown. Here, the rice lesion mimic mutant spotted leaf3 (spl3) was examined. When grown under a light/dark cycle, the spl3 mutant appeared similar to wild-type at early developmental stages, but lesions gradually appeared in the mature leaves close to heading stage. By contrast, in spl3 mutants grown under continuous light, severe cell death lesions formed in developing leaves, even at the seedling stage. Histochemical analysis showed that hydrogen peroxide accumulated in the mutant, likely causing the cell death phenotype. By map-based cloning and complementation, it was shown that a 1-bp deletion in the first exon of Oryza sativa Mitogen-Activated Protein Kinase Kinase Kinase1 (OsMAPKKK1)/OsEDR1/OsACDR1 causes the spl3 mutant phenotype. The spl3 mutant was found to be insensitive to abscisic acid (ABA), showing normal root growth in ABA-containing media and delayed leaf yellowing during dark-induced and natural senescence. Expression of ABA signalling-associated genes was also less responsive to ABA treatment in the mutant. Furthermore, the spl3 mutant had lower transcript levels and activities of catalases, which scavenge hydrogen peroxide, probably due to impairment of ABA-responsive signalling. Finally, a possible molecular mechanism of lesion formation in the mature leaves of spl3 mutant is discussed.

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.

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.

Photosynthetic organisms exhibit a green color due to the accumulation of chlorophyll pigments in chloroplasts. Mg-protoporphyrin IX chelatase (Mg-chelatase) comprises three subunits (ChlH, ChlD and ChlI) and catalyzes the insertion of Mg(2+) into protoporphyrin IX, the last common intermediate precursor in both chlorophyll and heme biosyntheses, to produce Mg-protoporphyrin IX (MgProto). Chlorophyll deficiency in higher plants results in chlorina (yellowish-green) phenotype. To date, 10 chlorina (chl) mutants have been isolated in rice, but the corresponding genes have not yet been identified. Rice Chl1 and Chl9 genes were mapped to chromosome 3 and isolated by map-based cloning. A missense mutation occurred in a highly conserved amino acid of ChlD in the chl1 mutant and ChlI in the chl9 mutant. Ultrastructural analyses have revealed that the grana are poorly stacked, resulting in the underdevelopment of chloroplasts. In the seedlings fed with aminolevulinate-dipyridyl in darkness, MgProto levels in the chl1 and chl9 mutants decreased up to 25% and 31% of that in wild-type, respectively, indicating that the Mg-chelatase activity is significantly reduced, causing the eventual decrease in chlorophyll synthesis. Furthermore, Northern blot analysis indicated that the nuclear genes encoding the three subunits of Mg-chelatase and LhcpII in chl1 mutant are expressed about 2-fold higher than those in WT, but are not altered in the chl9 mutant. This result indicates that the ChlD subunit participates in negative feedback regulation of plastid-to-nucleus in the expression of nuclear genes encoding chloroplast proteins, but not the ChlI subunit.

Leaf color turns yellow during senescence due to the degradation of chlorophylls and photosynthetic proteins. A stay green mutant was isolated from the glutinous japonica rice Hwacheong- wx through N-methyl-N-nitrosourea mutagenesis. Leaves of the mutant remained green, while turning yellow in those of the wild-type rice during senescence. The stay green phenotype was controlled by a single recessive nuclear gene, tentatively symbolized as sgr(t). All the phenotypic characteristics of the mutant were the same as those of the wild-type lines except for the stay green trait. The leaf chlorophyll concentration of the mutant was similar to that of the wild-type before heading, but decreased steeply in the wild-type during grain filling, while very slowly in the mutant. However, no difference in photosynthetic activity was observed between the stay green mutant and the yellowing wild-type leaves, indicating that senescence is proceeding normally in the mutant leaves and that the mutation affects the rate of chlorophyll degradation during the leaf senescence. Using phenotypic and molecular markers, we mapped the sgr(t) locus to the long arm of chromosome 9 between RFLP markers RG662 and C985 at 1.8- and 2.1-cM intervals, respectively.

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.

Protein quality of soybean [Glycine max (L.) Merr.] seed could be enhanced by increasing the concentration of S-containing amino acids. The N:S ratio of soybean seed tissue is an indicator of protein quality. The objective of this study was to compare N and S accumulation and distribution in the soybean plant under varying levels of S availability. Soil S levels were varied in two greenhouse trials and tissue S and N contents were monitored. A field experiment with 0 and 60 kg ha -1 applied S was also conducted. In the two greenhouse trials, seed yield, rate of dry matter increase, and rate of N accrual by shoots all increased at least five-fold as S availability increased from less than 15 (zero added S) to 60 mg available S per plant, but showed little response to higher levels of S. Rate of S accrual was strongly related to S availability. In the field trial, there were no responses to added S. Leaf, pod, and seed tissue appeared to accumulate S in proteins, whereas, root and stem tissue appeared to accumulate S as sulfate. Harvest index values for S were consistently less than those for N, indicating S is not remobilized to seed as efficiently as is N. Seed S:N ratio was linearly related to the rate of S versus N accrual on a whole-plant basis. We estimate that a 50% increase in the amount of S-containing amino acids in soybean seed may require a 65 to 80% increase in S accrual by shoots to satisfy the increased demand for S.

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.

WUSCHEL-related homeobox (WOX) transcription factors play important roles in cell fate determination during plant development and function in the signaling pathways of phytohormones such as gibberellic acid (GA). In a recent study, we demonstrated that OsWOX3A directly binds to the promoter of the KAO gene, which encodes ent-kaurenoic acid oxidase, an enzyme involved in GA biosynthesis, and represses KAO expression. Interestingly, we observed that OsWOX3A overexpression causes not only severe dwarfism, but also an increase in the number of lateral roots. Moreover, the expression of PIN-FORMED 1 (PIN1), involved in polar auxin transport, is significantly upregulated in OsWOX3A-OX plants. These findings indicate that OsWOX3A may be involved in modulation of GA-auxin crosstalk in rice root development.

· In order to understand the molecular genetic mechanisms of rice (Oryza sativa) organ development, we studied the narrow leaf2 narrow leaf3 (nal2 nal3; hereafter nal2/3) double mutant, which produces narrow-curly leaves, more tillers, fewer lateral roots, opened spikelets and narrow-thin grains. · We found that narrow-curly leaves resulted mainly from reduced lateral-axis outgrowth with fewer longitudinal veins and more, larger bulliform cells. Opened spikelets, possibly caused by marginal deformity in the lemma, gave rise to narrow-thin grains. · Map-based cloning revealed that NAL2 and NAL3 are paralogs that encode an identical OsWOX3A (OsNS) transcriptional activator, homologous to NARROW SHEATH1 (NS1) and NS2 in maize and PRESSED FLOWER in Arabidopsis. · OsWOX3A is expressed in the vascular tissues of various organs, where nal2/3 mutant phenotypes were displayed. Expression levels of several leaf development-associated genes were altered in nal2/3, and auxin transport-related genes were significantly changed, leading to pin mutant-like phenotypes such as more tillers and fewer lateral roots. OsWOX3A is involved in organ development in rice, lateral-axis outgrowth and vascular patterning in leaves, lemma and palea morphogenesis in spikelets, and development of tillers and lateral roots.

Summary • To expand our understanding of cell death in plant defense responses, we isolated a novel rice (Oryza sativa) spotted leaf mutant (spl28) that displays a lesion mimic phenotype in the absence of pathogen attack through treatment of Hwacheongbyeo (an elite Korean japonica cultivar) with N-methyl-N-nitrosourea (MNU). • Early stage development of the spl28 mutant was normal. However, after flowering, spl28 mutants exhibited a significant decrease in chlorophyll content, soluble protein content, and photosystem II efficiency, and high concentrations of reactive oxygen species (ROS), phytoalexin, callose, and autofluorescent phenolic compounds that localized in or around the lesions. The spl28 mutant also exhibited significantly enhanced resistance to rice blast and bacterial blight. • Using a map-based cloning approach, we determined that SPL28 encodes a clathrin-associated adaptor protein complex 1, medium subunit μ1 (AP1M1), which is involved in the post-Golgi trafficking pathway. A green fluorescent protein (GFP) fusion protein of SPL28 (SPL28::GFP) localized to the Golgi apparatus, and expression of SPL28 complemented the membrane trafficking defect of apm1-1Δ yeast mutants. SPL28 was ubiquitously expressed and contained a highly conserved adaptor complex medium subunit (ACMS) family domain. • SPL28 appears to be involved in the regulation of vesicular trafficking, and SPL28 dysfunction causes the formation of hypersensitive response (HR)-like lesions, leading to the initiation of leaf senescence.

During leaf senescence, the most characteristic visible change is leaf yellowing due to the preferential breakdown of chlorophyll (Chl) with concomitant chloroplast degradation. In this study, we examined the characteristics for the staygreenness during dark-induced senescence (DIS) in two stay-green varieties, SNU-SG1 and sgr;. During DIS, Chl loss was delayed in SNU-SG1 and sgr; compared with that in wild type (WT), but the photochemical efficiency of PSII (Fv/Fm) was not. The content of the functional PSII during DIS, estimated as (1/Fo 1/Fm) was high in WT, but low in SNU-SG1 and sgr;. In both varieties, Fo parameter increased during DIS, indicating the detachment of LHCII in PSII. In western blot analysis, the D1 and LHCII of WT were detected even when leaves turned yellowed. However, D1 protein in sgr; completely disappeared after 2 days without significant decrease in LHCII. These results suggest that both of the SNU-SG1 variety and sgr; mutant are non-functional stay-green species and largely attributed to the high stability of LHCII with an early degradation of D1 protein as a key protein of PSII.

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.

Coordinated regulation of the many genes controlling leaf, flower, and root development determines the phenotypes of plants; this regulation requires exquisite control of many transcription factors, including the WUSCHEL-related homeobox (WOX) family. We recently reported that rice (Oryza sativa) WUSCHEL-related homeobox 3A (OsWOX3A) plays important roles in organ development, including lateral-axis outgrowth and vasculature patterning in leaves, lemma and palea morphogenesis in spikelets, and the numbers of tillers and lateral roots. OsWOX3A is encoded by NARROW LEAF2 (NAL2) and NAL3, a pair of duplicated genes. In this study, further analysis of nal2 nal3 (hereafter nal2/3) double mutants revealed that, in addition to its role in lateral root development, OsWOX3A also acts in the control of root hair formation. Based on this new finding, we describe a possible mechanism by which OsWOX3A regulation of auxin transport genes acts in root development.

Background NADPH: protochlorophyllide oxidoreductase (POR) is an essential enzyme that catalyzes the photoreduction of protochlorophyllide to chlorophyllide, which is ultimately converted to chlorophyll in developing leaves. Rice has two POR isoforms, OsPORA and OsPORB. OsPORA is expressed in the dark during early leaf development; OsPORB is expressed throughout leaf development regardless of light conditions. The faded green leaf (fgl) is a loss-of-function osporB mutant that displays necrotic lesions and variegation in the leaves due to destabilized grana thylakoids, and has increased numbers of plastoglobules in the chloroplasts. To investigate whether the function of OsPORA can complement that of OsPORB, we constitutively overexpressed OsPORA in fgl mutant. Results In the 35S:OsPORA/fgl (termed OPAO) transgenic plants, the necrotic lesions of the mutant disappeared and the levels of photosynthetic pigments and proteins, as well as plastid structure, were recovered in developing leaves under natural long days in the paddy field and under short days in an artificially controlled growth room. Under constant light conditions, however, total chlorophyll and carotenoid levels in the developing leaves of OPAO plants were lower than those of wild type. Moreover, the OPAO plants exhibited mild defects in mature leaves beginning at the early reproductive stage in the paddy field. Conclusions The physiological function of OsPORB in response to constant light or during reproductive growth cannot be completely replaced by constitutive activity of OsPORA, although the biochemical functions of OsPORA and OsPORB are redundant. Therefore, we suggest that the two OsPORs have differentiated over the course of evolution, playing distinct roles in the adaptation of rice to the environment. Electronic supplementary material The online version of this article (doi:10.1186/s12284-016-0141-2) contains supplementary material, which is available to authorized users.