Arabidopsis BBX14 negatively regulates nitrogen starvation- and dark-induced leaf senescence.

Senescence is a highly regulated process driven by developmental age and environmental factors. Although leaf senescence is accelerated by nitrogen (N) deficiency, the underlying physiological and molecular mechanisms are largely unknown. Here, we reveal that BBX14, a previously uncharacterized BBX-type transcription factor in Arabidopsis, is crucial for N starvation-induced leaf senescence. We find that inhibiting BBX14 by artificial miRNA (amiRNA) accelerates senescence during N starvation and in darkness, while BBX14 overexpression (BBX14-OX) delays it, identifying BBX14 as a negative regulator of N starvation- and dark-induced senescence. During N starvation, nitrate and amino acids like glutamic acid, glutamine, aspartic acid, and asparagine were highly retained in BBX14-OX leaves compared to the wild type. Transcriptome analysis showed a large number of senescence-associated genes (SAGs) to be differentially expressed between BBX14-OX and wild-type plants, including ETHYLENE INSENSITIVE3 (EIN3) which regulates N signaling and leaf senescence. Chromatin immunoprecipitation (ChIP) showed that BBX14 directly regulates EIN3 transcription. Furthermore, we revealed the upstream transcriptional cascade of BBX14. By yeast one-hybrid screen and ChIP, we found that MYB44, a stress-responsive MYB transcription factor, directly binds to the promoter of BBX14 and activates its expression. In addition, Phytochrome Interacting Factor 4 (PIF4) binds to the promoter of BBX14 to repress BBX14 transcription. Thus, BBX14 functions as a negative regulator of N starvation-induced senescence through EIN3 and is directly regulated by PIF4 and MYB44.

Dof1.7 and NIGT1 transcription factors mediate multilayered transcriptional regulation for different expression patterns of NITRATE TRANSPORTER2 genes under nitrogen deficiency stress.

Elucidating the mechanisms regulating nitrogen (N) deficiency responses in plants is of great agricultural importance. Previous studies revealed that decreased expression of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) transcriptional repressor genes upon N deficiency is involved in N deficiency-inducible gene expression in Arabidopsis thaliana. However, our knowledge of the mechanisms controlling N deficiency-induced changes in gene expression is still limited. Through the identification of Dof1.7 as a direct target of NIGT1 repressors and a novel N deficiency response-related transcriptional activator gene, we here show that NIGT1 and Dof1.7 transcription factors (TFs) differentially regulate N deficiency-inducible expression of three high-affinity nitrate transporter genes, NRT2.1, NRT2.4, and NRT2.5, which are responsible for most of the soil nitrate uptake activity of Arabidopsis plants under N-deficient conditions. Unlike NIGT1 repressors, which directly suppress NRT2.1, NRT2.4, and NRT2.5 under N-sufficient conditions, Dof1.7 directly activated only NRT2.5 but indirectly and moderately activated NRT2.1 and NRT2.4 under N-deficient conditions, probably by indirectly decreasing NIGT1 expression. Thus, Dof1.7 converted passive transcriptional activation into active and potent transcriptional activation, further differentially enhancing the expression of NRT2 genes. These findings clarify the mechanism underlying different expression patterns of NRT2 genes upon N deficiency, suggesting that time-dependent multilayered transcriptional regulation generates complicated expression patterns of N deficiency-inducible genes.

A Jasmonate-Activated MYC2-Dof2.1-MYC2 Transcriptional Loop Promotes Leaf Senescence in Arabidopsis.

DNA binding-with-one-finger (Dof) proteins are plant-specific transcription factors closely associated with a variety of physiological processes. Here, we show that the Dof protein family in Arabidopsis (Arabidopsis thaliana) functions in leaf senescence. Disruption of Dof2 1, a jasmonate (JA)-inducible gene, led to a marked reduction in promotion of leaf senescence and inhibition of root development as well as dark-induced and age-dependent leaf senescence, while overexpression of Dof2 1 promoted these processes. Additionally, the dof2 1 knockout mutant showed almost no change in the transcriptome in the absence of JA; in the presence of JA, expression of many senescence-associated genes, including MYC2, which encodes a central regulator of JA responses, was induced to a lesser extent in the dof2 1 mutant than in the wild type. Furthermore, direct activation of the MYC2 promoter by Dof2.1, along with the results of epistasis analysis, indicated that Dof2.1 enhances leaf senescence mainly by promoting MYC2 expression. Interestingly, MYC2 was also identified as a transcriptional activator responsible for JA-inducible expression of Dof2 1 Based on these results, we propose that Dof2.1 acts as an enhancer of JA-induced leaf senescence through the MYC2-Dof2.1-MYC2 feedforward transcriptional loop.

Multilayered Regulation of Membrane-Bound ONAC054 Is Essential for Abscisic Acid-Induced Leaf Senescence in Rice.

In most plants, abscisic acid (ABA) induces premature leaf senescence; however, the mechanisms of ABA signaling during leaf senescence remain largely unknown. Here, we show that the rice (Oryza sativa) NAM/ATAF1/2/CUC2 (NAC) transcription factor ONAC054 plays an important role in ABA-induced leaf senescence. The onac054 knockout mutants maintained green leaves, while ONAC054-overexpressing lines showed early leaf yellowing under dark- and ABA-induced senescence conditions. Genome-wide microarray analysis showed that ABA signaling-associated genes, including ABA INSENSITIVE5 (OsABI5) and senescence-associated genes, including STAY-GREEN and NON-YELLOW COLORING1 (NYC1), were significantly down-regulated in onac054 mutants. Chromatin immunoprecipitation and protoplast transient assays showed that ONAC054 directly activates OsABI5 and NYC1 by binding to the mitochondrial dysfunction motif in their promoters. ONAC054 activity is regulated by proteolytic processing of the C-terminal transmembrane domain (TMD). We found that nuclear import of ONAC054 requires cleavage of the putative C-terminal TMD. Furthermore, the ONAC054 transcript (termed ONAC054α) has an alternatively spliced form (ONAC054ß), with seven nucleotides inserted between intron 5 and exon 6, truncating ONAC054α protein at a premature stop codon. ONAC054ß lacks the TMD and thus localizes to the nucleus. These findings demonstrate that the activity of ONAC054, which is important for ABA-induced leaf senescence in rice, is precisely controlled by multilayered regulatory processes.

NIGT1 family proteins exhibit dual mode DNA recognition to regulate nutrient response-associated genes in Arabidopsis.

Fine-tuning of nutrient uptake and response is indispensable for maintenance of nutrient homeostasis in plants, but the details of underlying mechanisms remain to be elucidated. NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1) family proteins are plant-specific transcriptional repressors that function as an important hub in the nutrient signaling network associated with the acquisition and use of nitrogen and phosphorus. Here, by yeast two-hybrid assays, bimolecular fluorescence complementation assays, and biochemical analysis with recombinant proteins, we show that Arabidopsis NIGT1 family proteins form a dimer via the interaction mediated by a coiled-coil domain (CCD) in their N-terminal regions. Electrophoretic mobility shift assays defined that the NIGT1 dimer binds to two different motifs, 5'-GAATATTC-3' and 5'-GATTC-N38-GAATC-3', in target gene promoters. Unlike the dimer of wild-type NIGT1 family proteins, a mutant variant that could not dimerize due to amino acid substitutions within the CCD had lower specificity and affinity to DNA, thereby losing the ability to precisely regulate the expression of target genes. Thus, expressing the wild-type and mutant NIGT1 proteins in the nigt1 quadruple mutant differently modified NIGT1-regulated gene expression and responses towards nitrate and phosphate. These results suggest that the CCD-mediated dimerization confers dual mode DNA recognition to NIGT1 family proteins, which is necessary to make proper controls of their target genes and nutrient responses. Intriguingly, two 5'-GATTC-3' sequences are present in face-to-face orientation within the 5'-GATTC-N38-GAATC-3' sequence or its complementary one, while two 5'-ATTC-3' sequences are present in back-to-back orientation within the 5'-GAATATTC-3' or its complementary one. This finding suggests a unique mode of DNA binding by NIGT1 family proteins and may provide a hint as to why target sequences for some transcription factors cannot be clearly determined.

RWP-RK domain-containing transcription factors in the Viridiplantae: biology and phylogenetic relationships.

The RWP-RK protein family is a group of transcription factors containing the RWP-RK DNA-binding domain. This domain is an ancient motif that emerged before the establishment of the Viridiplantae-the green plants, consisting of green algae and land plants. The domain is mostly absent in other kingdoms but widely distributed in Viridiplantae. In green algae, a liverwort, and several angiosperms, RWP-RK proteins play essential roles in nitrogen responses and sexual reproduction-associated processes, which are seemingly unrelated phenomena but possibly interdependent in autotrophs. Consistent with related but diversified roles of the RWP-RK proteins in these organisms, the RWP-RK protein family appears to have expanded intensively, but independently, in the algal and land plant lineages. Thus, bryophyte RWP-RK proteins occupy a unique position in the evolutionary process of establishing the RWP-RK protein family. In this review, we summarize current knowledge of the RWP-RK protein family in the Viridiplantae, and discuss the significance of bryophyte RWP-RK proteins in clarifying the relationship between diversification in the RWP-RK protein family and procurement of sophisticated mechanisms for adaptation to the terrestrial environment.

Environmental Control of Phosphorus Acquisition: A Piece of the Molecular Framework Underlying Nutritional Homeostasis.

Homeostasis of phosphorus (P), an essential macronutrient, is vital for plant growth under diverse environmental conditions. Although plants acquire P from the soil as inorganic phosphate (Pi), its availability is generally limited. Therefore, plants employ mechanisms involving various Pi transporters that facilitate efficient Pi uptake against a steep concentration gradient across the plant-soil interface. Among the different types of Pi transporters in plants, some members of the PHOSPHATE TRANSPORTER 1 (PHT1) family, present in the plasma membrane of root epidermal cells and root hairs, are chiefly responsible for Pi uptake from the rhizosphere. Therefore, accurate regulation of PHT1 expression is crucial for the maintenance of P homeostasis. Previous investigations positioned the Pi-dependent posttranslational regulation of PHOSPHATE STARVATION RESPONSE 1 (PHR1) transcription factor activity at the center of the regulatory mechanism controlling PHT1 expression and P homeostasis; however, recent studies indicate that several other factors also regulate the expression of PHT1 to modulate P acquisition and sustain P homeostasis against environmental fluctuations. Together with PHR1, several transcription factors that mediate the availability of other nutrients (such as nitrogen and zinc), light, and stress signals form an intricate transcriptional network to maintain P homeostasis under highly diverse environments. In this review, we summarize this intricate transcriptional network for the maintenance of P homeostasis under different environmental conditions, with a main focus on the mechanisms identified in Arabidopsis.

Light-Mediated Regulation of Leaf Senescence.

Light is the primary regulator of various biological processes during the plant life cycle. Although plants utilize photosynthetically active radiation to generate chemical energy, they possess several photoreceptors that perceive light of specific wavelengths and then induce wavelength-specific responses. Light is also one of the key determinants of the initiation of leaf senescence, the last stage of leaf development. As the leaf photosynthetic activity decreases during the senescence phase, chloroplasts generate a variety of light-mediated retrograde signals to alter the expression of nuclear genes. On the other hand, phytochrome B (phyB)-mediated red-light signaling inhibits the initiation of leaf senescence by repressing the phytochrome interacting factor (PIF)-mediated transcriptional regulatory network involved in leaf senescence. In recent years, significant progress has been made in the field of leaf senescence to elucidate the role of light in the regulation of nuclear gene expression at the molecular level during the senescence phase. This review presents a summary of the current knowledge of the molecular mechanisms underlying light-mediated regulation of leaf senescence.

Light signalling-induced regulation of nutrient acquisition and utilisation in plants.

Light is the foremost regulator of plant growth and development, and the critical role of light signalling in the promotion of nutrient uptake and utilisation was clarified in recent decades. Recent studies with Arabidopsis demonstrated the molecular mechanisms underlying such promotive effects and uncovered the pivotal role of the transcription factor ELONGATED HYPOCOTYL5 (HY5) whose activity is under the control of multiple photoreceptors. Together with a recent finding that phytochrome B, one of photoreceptors, is activated in subterranean plant parts, the discovery that HY5 directly promotes the transcription of genes involved in nutrient uptake and utilisation, including several nitrogen and sulphur assimilation-related genes, expands our understanding of the ways in which light signalling effectively and co-ordinately modulates uptake and utilisation of multiple nutrients in plants. This review presents a summary of the current knowledge regarding light signalling-induced regulation of nutrient uptake and utilisation.

Rice transcription factor OsMYB102 delays leaf senescence by down-regulating abscisic acid accumulation and signaling.

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

Arabidopsis EARLY FLOWERING3 increases salt tolerance by suppressing salt stress response pathways.

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

The Arabidopsis Transcription Factor NAC016 Promotes Drought Stress Responses by Repressing AREB1 Transcription through a Trifurcate Feed-Forward Regulatory Loop Involving NAP.

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.

Rice Phytochrome-Interacting Factor-Like1 (OsPIL1) is involved in the promotion of chlorophyll biosynthesis through feed-forward regulatory loops.

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

Mutation of Rice Early Flowering3.1 (OsELF3.1) delays leaf senescence in rice.

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.

Arabidopsis NAC016 promotes chlorophyll breakdown by directly upregulating STAYGREEN1 transcription.

KEY MESSAGE: 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.

Rice ONAC106 Inhibits Leaf Senescence and Increases Salt Tolerance and Tiller Angle.

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.

Mutation of SPOTTED LEAF3 (SPL3) impairs abscisic acid-responsive signalling and delays leaf senescence in rice.

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.

STAY-GREEN and chlorophyll catabolic enzymes interact at light-harvesting complex II for chlorophyll detoxification during leaf senescence in Arabidopsis.

During leaf senescence, plants degrade chlorophyll to colorless linear tetrapyrroles that are stored in the vacuole of senescing cells. The early steps of chlorophyll breakdown occur in plastids. To date, five chlorophyll catabolic enzymes (CCEs), NONYELLOW COLORING1 (NYC1), NYC1-LIKE, pheophytinase, pheophorbide a oxygenase (PAO), and red chlorophyll catabolite reductase, have been identified; these enzymes catalyze the stepwise degradation of chlorophyll to a fluorescent intermediate, pFCC, which is then exported from the plastid. In addition, STAY-GREEN (SGR), Mendel's green cotyledon gene encoding a chloroplast protein, is required for the initiation of chlorophyll breakdown in plastids. Senescence-induced SGR binds to light-harvesting complex II (LHCII), but its exact role remains elusive. Here, we show that all five CCEs also specifically interact with LHCII. In addition, SGR and CCEs interact directly or indirectly with each other at LHCII, and SGR is essential for recruiting CCEs in senescing chloroplasts. PAO, which had been attributed to the inner envelope, is found to localize in the thylakoid membrane. These data indicate a predominant role for the SGR-CCE-LHCII protein interaction in the breakdown of LHCII-located chlorophyll, likely to allow metabolic channeling of phototoxic chlorophyll breakdown intermediates upstream of nontoxic pFCC.

CONSTITUTIVE PHOTOMORPHOGENIC 10 (COP10) Contributes to Floral Repression under Non-Inductive Short Days in Arabidopsis.

In Arabidopsis, CONSTITUTIVE PHOTOMORPHOGENIC/DE-ETIOLATED/FUSCA (COP/DET/FUS) genes act in repression of photomorphogenesis in darkness, and recent reports revealed that some of these genes, such as COP1 and DET1, also have important roles in controlling flowering time and circadian rhythm. The COP/DET/FUS protein COP10 interacts with DET1 and DNA DAMAGE-BINDING PROTEIN 1 (DDB1) to form a CDD complex and represses photomorphogenesis in darkness. The cop10-4 mutants flower normally in inductive long days (LD) but early in non-inductive short days (SD) compared with wild type (WT); however, the role of COP10 remains unknown. Here, we investigate the role of COP10 in SD-dependent floral repression. Reverse transcription-quantitative PCR revealed that in SD, expression of the LD-dependent floral inducers GI, FKF1, and FT significantly increased in cop10-4 mutants, compared with WT. This suggests that COP10 mainly regulates FT expression in a CO-independent manner. We also show that COP10 interacts with GI in vitro and in vivo, suggesting that COP10 could also affect GI function at the posttranslational level. Moreover, FLC expression was repressed drastically in cop10-4 mutants and COP10 interacts with MULTICOPY SUPPRESSOR OF IRA1 4 (MSI4)/FVE (MSI4/FVE), which epigenetically inhibits FLC expression. These data suggest that COP10 contributes to delaying flowering in the photoperiod and autonomous pathways by downregulating FT expression under SD.