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

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.

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

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

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

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

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

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

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.

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

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

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.

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.

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.

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.

Flowering time (or heading date) is controlled by intrinsic genetic programs in response to environmental cues, such as photoperiod and temperature. Rice, a facultative short-day (SD) plant, flowers early in SD and late in long-day (LD) conditions. Casein kinases (CKs) generally act as positive regulators in many signaling pathways in plants. In rice, Heading date 6 (Hd6) and Hd16 encode CK2α and CKI, respectively, and mainly function to delay flowering time. Additionally, the major LD-dependent floral repressors Hd2/Oryza sativa Pseudo-Response Regulator 37 (OsPRR37; hereafter PRR37) and Ghd7 also confer strong photoperiod sensitivity. In floral induction, Hd16 acts upstream of Ghd7 and CKI interacts with and phosphorylates Ghd7. In addition, Hd6 and Hd16 also act upstream of Hd2. However, whether CKI and CK2α directly regulate the function of PRR37 remains unclear. Here, we use in vitro pull-down and in vivo bimolecular fluorescence complementation assays to show that CKI and CK2α interact with PRR37. We further use in vitro kinase assays to show that CKI and CK2α phosphorylate different regions of PRR37. Our results indicate that direct posttranslational modification of PRR37 mediates the genetic interactions between these two protein kinases and PRR37. The significance of CK-mediated phosphorylation for PRR37 and Ghd7 function is discussed.

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

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

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.

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.

Development of specialized epidermal cells and structures plays a key role in plant tolerance to biotic and abiotic stresses. In the paddy field, the bright green leaf (bgl) mutants of rice (Oryza sativa) exhibit a luminous green color that is clearly distinguishable from the normal green of wild-type plants. Transmission and scanning electron microscopy revealed that small cuticular papillae (or small papillae; SP), nipple-like structures, are absent on the adaxial and abaxial leaf surfaces of bgl mutants, leading to more direct reflection and less diffusion of green light. Map-based cloning revealed that the bgl locus encodes OsRopGEF10, one of eleven OsRopGEFs in rice. RopGEFs (guanine nucleotide exchange factors for Rop) activate Rop/Rac GTPases, acting as molecular switches in eukaryotic signal transduction by replacing the bound GDP (inactive form) with GTP (active form) in response to external or internal cues. In agreement with the timing of SP initiation on the leaf epidermis, OsRopGEF10 is most strongly expressed in newly developing leaves before emergence from the leaf sheath. In yeast two-hybrid assays, OsRopGEF10 interacts with OsRac1, one of seven OsRac proteins; consistent with this, both proteins are localized in the plasma membrane. These results suggest that OsRopGEF10 activates OsRac1 to turn on the molecular signaling pathway for SP development. Together, our findings provide new insights into the molecular genetic mechanism of SP formation during early leaf morphogenesis.

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

The zebra-necrosis (zn) mutant of rice (Oryza sativa) produces transversely green/yellow-striped leaves. The mutant phenotype is formed by unequal impairment of chloroplast biogenesis before emergence from the leaf sheath under alternate light/dark or high/low temperatures (restrictive), but not under constant light and temperature (permissive) conditions. Map-based cloning revealed that ZN encodes a thylakoid-bound protein of unknown function. Virus-induced gene silencing of a ZN homolog in Nicotiana benthamiana causes leaf variegation with sporadic green/yellow sectors, indicating that ZN is essential for chloroplast biogenesis during early leaf development. Necrotic lesions often occur in the yellow sectors as a result of an excessive accumulation of reactive oxygen species (ROS). The phenotypic severity (leaf variegation and necrosis) and ROS levels are positively correlated with an increase in light intensity under restrictive conditions. In the mutant leaves, chlorophyll (Chl) metabolism, ROS scavenging activities, maximum quantum yield of photosystem II (PSII), and structures and functions of the photosynthetic complexes are normal in the Chl-containing cells, suggesting that ROS are mainly generated from the defective plastids of the Chl-free cells. The PSII activity of normal chloroplasts is hypersensitive to photoinhibition because the recovery rates of PSII are much slower. In the PSII repair, the degradation of damaged D1 is not impaired, suggesting a reduced activity of new D1 synthesis, possibly because of higher levels of ROS generated from the Chl-free cells by excess light. Together, we propose that ZN is required for protecting developing chloroplasts, especially during the assembly of thylakoid protein complexes, from incidental light after darkness.

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.

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

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.

Symbiotic nitrogen fixation with nitrogen-fixing bacteria in the root nodules is a distinctly beneficial metabolic process in legume plants. Legumes control the nodule number and nodulation zone through a systemic negative regulatory system between shoot and root. Mutation in the soybean NTS gene encoding GmNARK, a CLAVATA1-like serine/threonine receptor-like kinase, causes excessive nodule development called hypernodulation. To examine the effect of nts mutation on the gene expression profile in the leaves, suppression subtractive hybridization was performed with the trifoliate leaves of nts mutant 'SS2-2' and the wild-type (WT) parent Sinpaldalkong2, and 75 EST clones that were highly expressed in the leaves of the SS2-2 mutant were identified. Interestingly, the expression of jasmonate (JA)-responsive genes such as vspA, vspB, and Lox2 were upregulated, whereas that of a salicylate-responsive gene PR1a was suppressed in the SS2-2 mutant. In addition, the level of JA was about two-fold higher in the leaves of the SS2-2 mutant than in those of the WT under natural growth conditions. Moreover, the JA-responsive gene expression persists in the leaves of SS2-2 mutant without rhizobia infection in the roots. Taken together, our results suggest that the nts mutation increases JA synthesis in mature leaves and consequently leads to constitutive expression of JA-responsive genes which is irrelevant to hypernodulation in the root.

During monocarpic senescence in higher plants, functional stay-green delays leaf yellowing, maintaining photosynthetic competence, whereas nonfunctional stay-green retains leaf greenness without sustaining photosynthetic activity. Thus, functional stay-green is considered a beneficial trait that can increase grain yield in cereal crops. A stay-green japonica rice 'SNU-SG1' had a good seed-setting rate and grain yield, indicating the presence of a functional stay-green genotype. SNU-SG1 was crossed with two regular cultivars to determine the inheritance mode and identify major QTLs conferring stay-green in SNU-SG1. For QTL analysis, linkage maps with 100 and 116 DNA marker loci were constructed using selective genotyping with F2 and RIL (recombinant inbred line) populations, respectively. Molecular marker-based QTL analyses with both populations revealed that the functional stay-green phenotype of SNU-SG1 is regulated by several major QTLs accounting for a large portion of the genetic variation. Three main-effect QTLs located on chromosomes 7 and 9 were detected in both populations and a number of epistatic-effect QTLs were also found. The amount of variation explained by several digenic interactions was larger than that explained by main-effect QTLs. Two main-effect QTLs on chromosome 9 can be considered the target loci that most influence the functional stay-green in SNU-SG1. The functional stay-green QTLs may help develop low-input high-yielding rice cultivars by QTL-marker-assisted breeding with SNU-SG1.

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.

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

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

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.

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

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.