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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.

The 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 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 significantly suppressed by ABA treatment, indicating negative feedback regulation of OsWRKY5 expression by ABA. OsWRKY5 is a positive regulator of leaf senescence that upregulates senescence-induced NAC genes leading to expression of 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.

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.

The previously published version of this Article contained errors in Figure 5. In panel c, the second and fourth blot images were incorrectly labeled ‘α-Myc’ and should have been labelled ‘α-HA’. These errors have been corrected in both the PDF and HTML versions of the Article.

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.

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.

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.

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.

Transmission electron microscopy analysis of chloroplasts in the z3 mutant leaves. a-c Chloroplasts in the green leaves of the WT (a) and in the dark-green (b) and green (c) sectors of the z3 mutant leaves. Flag leaves of the 150-day-old WT and z3 mutant grown under natural long day conditions were sampled for analysis. G, grana thylakoid. Scale bars = 0.5 μm. (PDF 955 kb)

Primers used in this study. (PDF 1453 kb)

Phenotypes of the homozygous T-DNA null mutants of Z3. a Schematic diagrams of the T-DNA insertion. The z3–2 and z3–3 mutants have a T-DNA insertion in the first exon, but in different locations. These T-DNA knockout mutants of Z3 were derived from the japonica rice cv ‘Hwayoung’. Filled boxes and thick lines indicate exons and introns, respectively. Primers LP1 (left primer 1), RP1 (right primer 1), LP2 (left primer 2), RP2 (right primer 2), and RB (right border primer) used for genotyping are represented as arrows and listed in Additional file 8: Table S1. b Phenotypes of leaf blades from four-month-old WT (Hwayoung), and z3–2 and z3–3 mutants grown under natural long day (NLD) conditions in the paddy field. c–d Panicle and grain phenotypes of the WT (Hwayoung) and two z3-knockout mutants at 150 DAS grown in a paddy field. e Days to heading data of the WT, and the z3–2, and z3–3 mutants in natural long-day conditions. Means and standard deviations were obtained from 5 plants of each genotype. Error bars indicate SD. Differences between means were compared using Student’s t-test (*** P

Statistical analysis of agronomic traits in the z3 mutant. a–f Agronomic traits were investigated between the WT and the z3 mutant plants grown under natural conditions. Investigated traits are as follows: a main panicle length, b number of panicles per plant, c number of spikelets per main panicle, d seed setting rate, e 500-grain weight, and f yield per plant. Ten plants were used to measure each trait. Values are shown as means. Error bars indicate SD. Student’s t-test was performed for statistical analysis (* P

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.

Late flowering phenotypes of the z3 mutant. a Flowering phenotypes of the 117-day-old WT and z3 mutant grown under natural long day conditions (14 h light/day, 37o N latitude) in the paddy field. b Days to heading of the WT and the z3 mutant in natural long day conditions. Means and SD were obtained from 15 plants of each genotype. Error bars indicate SD. Differences between means were compared using Student’s t-test (*** P

Analysis of reactive oxygen species (ROS) in the z3 mutant leaves. a-b Hydrogen peroxide (H2O2) and superoxide anion radicals (O2-) in flag leaves of the WT and z3 mutants at 160 DAS grown under natural long day conditions were visualized by staining with DAB (a) and NBT (b), respectively. Leaves before (left) and after (right) staining are shown. (PDF 497 kb)

Confirmation of the overexpression of Z3 by the CaMV 35S promoter in six independent complementation lines. a Genomic PCR analysis for confirmation of rice transformation. The genomic region between the vector and the Z3 transgene was amplified in the transformed lines. All six independent lines were confirmed to be transformed. Primer information is listed in Additional file 8: Table S1. EV, empty vector; Control, vector used for transformation without the Z3 gene. b The overexpression of Z3 by the CaMV 35S promoter in six independent complementation lines. Primers were designed to amplify the transgene mRNA, and are listed in Additional file 8: Table S1. The GAPDH mRNA level was measured as a loading control. (PDF 647 kb)

Amino acid alignment of Z3 homologs in higher plants. The amino acid sequences of Z3 homologs in higher plants were acquired from NCBI ( http://www.ncbi.nlm.nih.gov /), and the amino acid alignment was obtained using the Clustal Omega EMBL-EBI ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ) and BoxShade 3.21 Server ( https://www.ch.embnet.org/software/BOX_form.html ). O. brachyantha, Z. mays_1, Z. mays_2, H. vulgare, S. bicolor, S. tuberosum, G. max, and A. thaliana have 99, 96, 95, 87, 72, 65, 64, and 62% sequence similarity to Z3, respectively. The mutated residue (S542P in z3) is indicated by a red arrowhead. O. sativa_ZEBRA3 (Oryza sativa ZEBRA3, LOC_Os03g05390, NP_001048962.1); O. brachyantha (Oryza brachyantha, XP_006649400.1); Z. mays_1 (Zea mays, NP_001151517.1); Z. mays_2 (Zea mays, ACG43196.1); H. vulgare (Hordeum vulgare, BAK05230.1); S. bicolor (Sorghum bicolor, XP_002467148.1); S. tuberosum (Solanum tuberosum, XP_006363328.1); G. max (Glycine max, XP_003533988.1); A. thaliana (Arabidopsis thaliana, NP_171728.2). (PDF 2499 kb)

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.

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.

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.

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.

Natural variation in heading-date genes enables rice, a short-day (SD) plant, to flower early under long-day (LD) conditions at high latitudes. Through analysis of heading-date quantitative trait loci (QTL) with F7 recombinant inbred lines from the cross of early heading 'H143' and late heading 'Milyang23 (M23)', we found a minor-effect Early Heading3 (EH3) QTL in the Hd16 region on chromosome 3. We found that Early flowering1 (EL1), encoding casein kinase I (CKI), is likely to be responsible for the EH3/Hd16 QTL, because a missense mutation occurred in the highly conserved serine/threonine kinase domain of EL1 in H143. A different missense mutation was found in the EL1 kinase domain in Koshihikari. In vitro kinase assays revealed that EL1/CKI in H143 and Koshihikari are non-functional. In F7:9 heterogeneous inbred family-near isogenic lines (HNILs), HNIL(H143) flowered 13 days earlier than HNIL(M23) in LD, but not in SD, in which EL1 mainly acts as a LD-dependent flowering repressor, down-regulating Ehd1 expression. In the world rice collection, two types of non-functional EL1 variants were found in japonica rice generally cultivated at high latitudes. These results indicate that natural variation in EL1 contributes to early heading for rice adaptation to LD in 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.

Total Pchlide and photoactive Pchlide levels determined by UV/VIS spectrophotometry. (PDF 175 kb)

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.

Phenotypes of WT, fgl mutant, and OPAO homozygous T2 line #11 in the paddy field. (PDF 573 kb)

Phenotypic characterization of OPAO line #11 under SD conditions. (PDF 339 kb)

Primers used in this study. (PDF 125 kb)

PCR-based confirmation of OsPORA insertion in genomic DNA of three independent OPAO T0 lines. (PDF 136 kb)

Expression levels of OsPORA in the 2nd leaves of OPAO T1 lines. (PDF 125 kb)

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 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.

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.

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 virescent3 (v3) and stripe1 (st1) mutants in rice (Oryza sativa) produce chlorotic leaves in a growth stage-dependent manner under field conditions. They are temperature-conditional mutants that produce bleached leaves at a constant 20°C or 30°C but almost green leaves under diurnal 30°C/20°C conditions. Here, we show V3 and St1, which encode the large and small subunits of ribonucleotide reductase (RNR), RNRL1, and RNRS1, respectively. RNR regulates the rate of deoxyribonucleotide production for DNA synthesis and repair. RNRL1 and RNRS1 are highly expressed in the shoot base and in young leaves, and the expression of the genes that function in plastid transcription/translation and in photosynthesis is altered in v3 and st1 mutants, indicating that a threshold activity of RNR is required for chloroplast biogenesis in developing leaves. There are additional RNR homologs in rice, RNRL2 and RNRS2, and eukaryotic RNRs comprise α 2 β 2 heterodimers. In yeast, RNRL1 interacts with RNRS1 (RNRL1:RNRS1) and RNRL2:RNRS2, but no interaction occurs between other combinations of the large and small subunits. The interacting activities are RNRL1:RNRS1 > RNRL1:rnrs1(st1) > rnrl1(v3):RNRS1 > rnrl1(v3):rnrs1(st1), which correlate with the degree of chlorosis for each genotype. This suggests that missense mutations in rnrl1(v3) and rnrs1(st1) attenuate the first αβ dimerization. Moreover, wild-type plants exposed to a low concentration of an RNR inhibitor, hydroxyurea, produce chlorotic leaves without growth retardation, reminiscent of v3 and st1 mutants. We thus propose that upon insufficient activity of RNR, plastid DNA synthesis is preferentially arrested to allow nuclear genome replication in developing leaves, leading to continuous plant growth.

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.