A dramatic increase of chlorophyll (Chl) degradation occurs during senescence of vegetative plant organs and fruit ripening. Although the biochemical pathway of Chl degradation has long been proposed, little is known about its regulatory mechanism. Identification of Chl degradation-disturbed mutants and subsequently isolation of responsible genes would greatly facilitate the elucidation of the regulation of Chl degradation. Here, we describe a nonyellowing mutant of Arabidopsis (Arabidopsis thaliana), nye1-1, in which 50% Chl was retained, compared to less than 10% in the wild type (Columbia-0), at the end of a 6-d dark incubation. Nevertheless, neither photosynthesis-nor senescence-associated process was significantly affected in nye1-1. Characteristically, a significant reduction in pheophorbide a oxygenase activity was detected in nye1-1. However, no detectable accumulation of either chlorophyllide a or pheophorbide a was observed. Reciprocal crossings revealed that the mutant phenotype was caused by a monogenic semidominant nuclear mutation. We have identified AtNYE1 by positional cloning. Dozens of its putative orthologs, predominantly appearing in higher plant species, were identified, some of which have been associated with Chl degradation in a few crop species. Quantitative polymerase chain reaction analysis showed that AtNYE1 was drastically induced by senescence signals. Constitutive overexpression of AtNYE1 could result in either pale-yellow true leaves or even albino seedlings. These results collectively indicate that NYE1 plays an important regulatory role in Chl degradation during senescence by modulating pheophorbide a oxygenase activity.

Degreening, caused by chlorophyll degradation, is the most obvious symptom of senescing leaves. Chlorophyll degradation can be triggered by endogenous and environmental cues, and ethylene is one of the major inducers. ETHYLENE INSENSITIVE3 (EIN3) is a key transcription factor in the ethylene signaling pathway. It was previously reported that EIN3, miR164, and a NAC (NAM, ATAF, and CUC) transcription factor ORE1/NAC2 constitute a regulatory network mediating leaf senescence. However, how this network regulates chlorophyll degradation at molecular level is not yet elucidated. Here we report a feed-forward regulation of chlorophyll degradation that involves EIN3, ORE1, and chlorophyll catabolic genes (CCGs). Gene expression analysis showed that the induction of three major CCGs, NYE1, NYC1 and PAO, by ethylene was largely repressed in ein3 eil1 double mutant. Dual-luciferase assay revealed that EIN3 significantly enhanced the promoter activity of NYE1, NYC1 and PAO in Arabidopsis protoplasts. Furthermore, Electrophoretic mobility shift assay (EMSA) indicated that EIN3 could directly bind to NYE1, NYC1 and PAO promoters. These results reveal that EIN3 functions as a positive regulator of CCG expression during ethylene-mediated chlorophyll degradation. Interestingly, ORE1, a senescence regulator which is a downstream target of EIN3, could also activate the expression of NYE1, NYC1 and PAO by directly binding to their promoters in EMSA and chromatin immunoprecipitation (ChIP) assays. In addition, EIN3 and ORE1 promoted NYE1 and NYC1 transcriptions in an additive manner. These results suggest that ORE1 is also involved in the direct regulation of CCG transcription. Moreover, ORE1 activated the expression of ACS2, a major ethylene biosynthesis gene, and subsequently promoted ethylene production. Collectively, our work reveals that EIN3, ORE1 and CCGs constitute a coherent feed-forward loop involving in the robust regulation of ethylene-mediated chlorophyll degradation during leaf senescence in Arabidopsis.

MicroRNAs (miRNAs) are regulators of gene expression in plants and animals. The biogenesis of miRNAs is precisely controlled to secure normal development of organisms. Here we report that TOUGH (TGH) is a component of the DCL1-HYL1-SERRATE complex that processes primary transcripts of miRNAs [i.e., primary miRNAs (pri-miRNAs)] into miRNAs in Arabidopsis. Lack of TGH impairs multiple DCL activities in vitro and reduces the accumulation of miRNAs and siRNAs in vivo. TGH is an RNA-binding protein, binds pri-miRNAs and precursor miRNAs in vivo, and contributes to pri-miRNA-HYL1 interaction. These results indicate that TGH might regulate abundance of miRNAs through promoting DCL1 cleavage efficiency and/or recruitment of pri-miRNAs.S mall RNAs, including microRNAs (miRNAs) and siRNAs, are sequence-specific regulators of gene expression in plants and animals (1). miRNAs are derived from imperfect stem-loop transcripts, called primary miRNAs (pri-miRNAs), which are predominately produced by DNA-dependent RNA polymerase II, whereas siRNAs are processed from perfect or near-perfect long dsRNAs (2). After generation, miRNA and siRNA are loaded into an RNA-induced silencing complex containing the Argonaute protein to guide posttranscriptional or transcriptional gene silencing (1).In animals, pri-miRNAs are first processed to precursor miRNAs (pre-miRNAs) in the nucleus by the microprocessor containing Drosha and a dsRNA-binding protein DGCR8 (1). The resulting pre-miRNAs are then processed by Dicer in the cytoplasm to produce mature miRNAs (1). It has emerged that the activities of Drosha and Dicer are controlled to regulate miRNA expression in response to developmental and environmental signals (3). In Arabidopsis, DCL1, a dsRNA-binding protein, HYL1, and a zinc finger protein, SERRATE (SE), form a complex to process pri-miRNAs in the nucleus to pre-miRNAs and then to mature miRNAs (4-6). The accumulation of miRNAs in Arabidopsis also requires DDL, which was proposed to stabilize pri-miRNAs and to facilitate their processing (7). Recently, two cap-binding proteins, CBP80/ABH1 and CBP20, were found to be required for pre-mRNA splicing and primiRNA processing (8, 9). Plants also encode several classes of endogenous siRNAs, including the natural antisense transcriptderived siRNA, siRNA derived from repetitive DNA sequences (rasiRNA), and transacting siRNA (ta-siRNA) (10). In Arabidopsis, the generation of these siRNAs from long dsRNAs involves DCL1 homologues DCL2, DCL3, and DCL4, which produce 22-nt, 24-nt, and 21-nt siRNAs, respectively (11-13).In this report, we show that TOUGH (TGH) is an important factor for miRNA and siRNA biogenesis. Loss-of-function TGH in tgh-1 reduces the activity of multiple DCLs in vitro and the accumulation of miRNA and siRNAs in vivo. In the miRNA pathway, TGH associates with the DCL1 complex and binds primiRNAs and pre-miRNAs. TGH is required for the efficient in vivo interaction between pri-miRNA and HYL1. These data suggest that TGH assists DCLs to efficiently process and/or recruit the prec...

Chlorophyll (Chl) degradation is an integral process of leaf senescence, and NYE1/SGR1 has been demonstrated as a key regulator of Chl catabolism in diverse plant species. In this study, using yeast one-hybrid screening, we identified three abscisic acid (ABA)-responsive element (ABRE)-binding transcription factors, ABF2 (AREB1), ABF3, and ABF4 (AREB2), as the putative binding proteins of the NYE1 promoter. Through the transactivation analysis, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we demonstrated that ABF2, ABF3, and ABF4 directly bound to and activated the NYE1 promoter in vitro and in vivo. ABA is a positive regulator of leaf senescence, and exogenously applied ABA can accelerate Chl degradation. The triple mutant of the ABFs, abf2abf3abf4, as well as two ABA-insensitive mutants, abi1-1 and snrk2.2/2.3/2.6, exhibited stay-green phenotypes after ABA treatment, along with decreased induction of NYE1 and NYE2 expression. In contrast, overexpression of ABF4 accelerated Chl degradation upon ABA treatment. Interestingly, ABF2/3/4 could also activate the expression of two Chl catabolic enzyme genes, PAO and NYC1, by directly binding to their promoters. In addition, abf2abf3abf4 exhibited a functional stay-green phenotype, and senescence-associated genes (SAGs), such as SAG29 (SWEET15), might be directly regulated by the ABFs. Taken together, our results suggest that ABF2, ABF3, and ABF4 likely act as key regulators in mediating ABA-triggered Chl degradation and leaf senescence in general in Arabidopsis.

Summary HEN1-mediated 2′-O-methylation has been shown to be a key mechanism to protect plant microRNAs (miRNAs) and small interfering RNAs (siRNAs) as well as animal piwi-interacting RNAs (piRNAs) from degradation and 3′ terminal uridylation [1–8]. However, enzymes uridylating unmethylated miRNAs, siRNAs, or piRNAs in hen1 are unknown. In this study, a genetic screen identified a second-site mutation hen1 suppressor1-2 (heso1-2) that partially suppresses the morphological phenotypes of the hypomorphic hen1-2 allele and the null hen1-1 allele in Arabidopsis. HESO1 encodes a terminal nucleotidyl transferase that prefers to add untemplated uridine to the 3′ end of RNA, which is completely abolished by 2′-O-methylation. heso1-2 affects the profile of u-tailed miRNAs and siRNAs and increases the abundance of truncated and/or normal sized ones in hen1, which often results in increased total amount of miRNAs and siRNAs in hen1. In contrast, overexpressing HESO1 in hen1-2 causes more severe morphological defects and less accumulation of miRNAs. These results demonstrate that HESO1 is an enzyme uridylating unmethylated miRNAs and siRNAs in hen1. These observations also suggest that uridylation may destabilize unmethylated miRNAs through an unknown mechanism and compete with 3′-to-5′ exoribonuclease activities in hen1. This study shall have implications on piRNA uridylation in hen1 in animals.

Summary Degreening caused by rapid chlorophyll (Chl) degradation is a characteristic event during green organ senescence or maturation. Pheophorbide a oxygenase gene (PAO) encodes a key enzyme of Chl degradation, yet its transcriptional regulation remains largely unknown. Using yeast one‐hybrid screening, coupled with in vitro and in vivo assays, we revealed that Arabidopsis MYC2/3/4 basic helix‐loop‐helix proteins directly bind to PAO promoter. Overexpression of the MYCs significantly enhanced the transcriptional activity of PAO promoter in Arabidopsis protoplasts, and methyl jasmonate (MeJA) treatment greatly induced PAO expression in wild‐type Arabidopsis plants, but the induction was abolished in myc2 myc3 myc4. In addition, MYC2/3/4 proteins could promote the expression of another Chl catabolic enzyme gene, NYC1, as well as a key regulatory gene of Chl degradation, NYE1/SGR1, by directly binding to their promoters. More importantly, the myc2 myc3 myc4 triple mutant showed a severe stay‐green phenotype, whereas the lines overexpressing the MYCs showed accelerated leaf yellowing upon MeJA treatment. These results suggest that MYC2/3/4 proteins may mediate jasmonic acid (JA)‐induced Chl degradation by directly activating these Chl catabolic genes (CCGs). Three NAC family proteins, ANAC019/055/072, downstream from MYC2/3/4 proteins, could also directly promote the expression of a similar set of CCGs (NYE1/SGR1, NYE2/SGR2 and NYC1) during Chl degradation. In particular, anac019 anac055 anac072 triple mutant displayed a severe stay‐green phenotype after MeJA treatment. Finally, we revealed that MYC2 and ANAC019 may interact with each other and synergistically enhance NYE1 expression. Together, our study reveals a hierarchical and coordinated regulatory network of JA‐induced Chl degradation.

CDC5 is a MYB-related protein that exists in plants, animals, and fungi. In Arabidopsis, CDC5 regulates both growth and immunity through unknown mechanisms. Here, we show that CDC5 from Arabidopsis positively regulates the accumulation of microRNAs (miRNAs), which control many biological processes including development and adaptations to environments in plants. CDC5 interacts with both the promoters of genes encoding miRNAs (MIR) and the DNA-dependent RNA polymerase II. As a consequence, lack of CDC5 reduces the occupancy of polymerase II at MIR promoters, as well as MIR promoter activities. In addition, CDC5 is associated with the DICER-LIKE1 complex, which generates miRNAs from their primary transcripts and is required for efficient miRNA production. These results suggest that CDC5 may have dual roles in miRNA biogenesis: functioning as a positive transcription factor of MIR and/or acting as a component of the DICER-LIKE1 complex to enhance primary miRNA processing.M icroRNAs (miRNAs) and small interfering RNAs (siRNAs) are ∼22-nucleotide (nt) noncoding RNAs that regulate various biological processes including development, metabolism, and immunity in plants and animals (1-3). miRNAs are generated from primary miRNA transcripts (pri-miRNAs) containing stem-loop structure, whereas siRNAs are derived from long, perfect, doublestranded RNAs (dsRNAs) (1-3). They are associated with members of the Argonaute protein family to repress gene expression at posttranscriptional and/or transcriptional levels (1-3). In addition to miRNAs, plants encode two major classes of siRNAs: siRNAs derived from repeated DNAs (ra-siRNAs) and transacting siRNAs (ta-siRNAs) (4-6).Studies in Arabidopsis have established the framework of miRNA biogenesis in plants (1-3). In Arabidopsis, pri-miRNAs are primarily transcribed by DNA-dependent RNA polymerase II (Pol II), with assistance from the mediator complex and the transcription factor Negative on TATA less2 (NOT2) (7,8). After transcription, pri-miRNAs are processed by an RNase III enzyme called DICER-LIKE1 (DCL1) to miRNA precursors and then to mature miRNAs (9, 10). The efficient processing of pri-miRNA requires SERRATE (SE; a zinc finger protein), TOUGH (an RNA-binding protein), and a dephosphorylated HYPONASTIC LEAVES1 (HYL1; a double-stranded RNA binding protein) that form a complex with DCL1 (11-18). SE and HYL1 also promote the processing accuracy of pri-miRNAs (19). Four other proteins, DAWDLE (DDL; an RNA binding protein), Cap-binding protein 20, Cap-binding protein 80, and NOT2, which are associated with the DCL1 complex (8,(20)(21)(22), also function in miRNA biogenesis. Recent studies also reveal that the correct localization of DCL1 requires NOT2 and MODIFIER OF SNC1, 2 (an RNA binding protein) (8,23). In addition, the accumulation of a subset of miRNAs requires a proline-rich protein named SICKLE (24).The cell division cycle 5 (CDC5) protein is a conserved protein in animals, plants, and fungi (25). It was first isolated from Schizosaccharomyces pombe as a cell cycle regulat...

MicroRNAs (miRNAs), a class of endogenous, tiny, non-coding RNAs, are master regulators of gene expression among most eukaryotes. Intracellular miRNA abundance is regulated under multiple levels of control including transcription, processing, RNA modification, RNA-induced silencing complex (RISC) assembly, miRNA-target interaction, and turnover. In this review, we summarize our current understanding of the molecular components and mechanisms that influence miRNA biogenesis, homeostasis, and degradation in plants. We also make comparisons with findings from other organisms where necessary.