Rice requires a chromatin remodeler for Polymerase IV-small interfering RNA production and genomic immunity.

Transgenes are often spontaneously silenced, which hinders the application of genetic modifications to crop breeding. While gene silencing has been extensively studied in Arabidopsis (Arabidopsis thaliana), the molecular mechanism of transgene silencing remains elusive in crop plants. We used rice (Oryza sativa) plants silenced for a 35S::OsGA2ox1 (Gibberellin 2-oxidase 1) transgene to isolate five elements mountain (fem) mutants showing restoration of transgene expression. In this study, we isolated multiple fem2 mutants defective in a homolog of Required to Maintain Repression 1 (RMR1) of maize (Zea mays) and CLASSY (CLSY) of Arabidopsis. In addition to failing to maintain transgene silencing, as occurs in fem3, in which mutation occurs in NUCLEAR RNA POLYMERASE E1 (OsNRPE1), the fem2 mutant failed to establish transgene silencing of 35S::OsGA2ox1. Mutation in FEM2 eliminated all RNA POLYMERASE IV (Pol-IV)-FEM1/OsRDR2 (RNA-DEPENDENT RNA POLYMERASE 2)-dependent small interfering RNAs (siRNAs), reduced DNA methylation on genome-wide scale in rice seedlings, caused pleiotropic developmental defects, and increased disease resistance. Simultaneous mutation in 2 FEM2 homologous genes, FEM2-Like 1 (FEL1) and FEL2, however, did not affect DNA methylation and rice development and disease resistance. The predominant expression of FEM2 over FEL1 and FEL2 in various tissues was likely caused by epigenetic states. Overexpression of FEL1 but not FEL2 partially rescued hypomethylation of fem2, indicating that FEL1 maintains the cryptic function. In summary, FEM2 is essential for establishing and maintaining gene silencing; moreover, FEM2 is solely required for Pol IV-FEM1 siRNA biosynthesis and de novo DNA methylation.

Gibberellin signaling regulates lignin biosynthesis to modulate rice seed shattering.

The elimination of seed shattering was a key step in rice (Oryza sativa) domestication. In this paper, we show that increasing the gibberellic acid (GA) content or response in the abscission region enhanced seed shattering in rice. We demonstrate that SLENDER RICE1 (SLR1), the key repressor of GA signaling, could physically interact with the rice seed shattering-related transcription factors quantitative trait locus of seed shattering on chromosome 1 (qSH1), O. sativa HOMEOBOX 15 (OSH15), and SUPERNUMERARY BRACT (SNB). Importantly, these physical interactions interfered with the direct binding of these three regulators to the lignin biosynthesis gene 4-COUMARATE: COENZYME A LIGASE 3 (4CL3), thereby derepressing its expression. Derepression of 4CL3 led to increased lignin deposition in the abscission region, causing reduced rice seed shattering. Importantly, we also show that modulating GA content could alter the degree of seed shattering to increase harvest efficiency. Our results reveal that the "Green Revolution" phytohormone GA is important for regulating rice seed shattering, and we provide an applicable breeding strategy for high-efficiency rice harvesting.

OsWR2 recruits HDA704 to regulate the deacetylation of H4K8ac in the promoter of OsABI5 in response to drought stress.

Drought stress is a major environmental factor that limits the growth, development, and yield of rice (Oryza sativa L.). Histone deacetylases (HDACs) are involved in the regulation of drought stress responses. HDA704 is an RPD3/HDA1 class HDAC that mediates the deacetylation of H4K8 (lysine 8 of histone H4) for drought tolerance in rice. In this study, we show that plants overexpressing HDA704 (HDA704-OE) are resistant to drought stress and sensitive to abscisic acid (ABA), whereas HDA704 knockout mutant (hda704) plants displayed decreased drought tolerance and ABA sensitivity. Transcriptome analysis revealed that HDA704 regulates the expression of ABA-related genes in response to drought stress. Moreover, HDA704 was recruited by a drought-resistant transcription factor, WAX SYNTHESIS REGULATORY 2 (OsWR2), and co-regulated the expression of the ABA biosynthesis genes NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3), NCED4, and NCED5 under drought stress. HDA704 also repressed the expression of ABA-INSENSITIVE 5 (OsABI5) and DWARF AND SMALL SEED 1 (OsDSS1) by regulating H4K8ac levels in the promoter regions in response to polyethylene glycol 6000 treatment. In agreement, the loss of OsABI5 function increased resistance to dehydration stress in rice. Our results demonstrate that HDA704 is a positive regulator of the drought stress response and offers avenues for improving drought resistance in rice.

The Rise and Fall of Billionaire siRNAs during Reproductive Development in Rice.

RNA polymerase IV-dependent siRNAs, usually 24 nt in length, function in the RNA-directed DNA methylation that is responsible for de novo methylation in plants. We analyzed 24 nt siRNAs in inflorescences and found that among the 20,200 24 nt siRNA clusters, the top 0.81% highly expressed clusters accounted for more than 68% of the 24 nt siRNA reads in inflorescences. We named the highly expressed siRNAs as billionaire siRNAs (bill-siRNAs) and the less-expressed siRNAs as pauper siRNAs (pau-siRNAs). The bill-siRNAs in inflorescences are mainly derived from the ovary. Female gametes produced more bill-siRNAs than male gametes. In embryos and seedlings developed from fertilized egg cells, the bill-siRNAs from gametes disappeared. The endosperm, which develops from the fertilized central cell, also contained no bill-siRNAs from gametes but did contain newly and highly expressed siRNAs produced in different regions. In contrast, bill-siRNAs from the ovaries were maintained in the seed coat. The biosynthesis of bill-siRNAs in various tissues and cells is dependent on OsRDR2 (RNA-dependent RNA polymerase 2) and Pol IV (DNA-dependent RNA polymerase IV). Similar to the pau-siRNAs, the first base of bill-siRNAs is enriched at adenine, and bill-siRNAs can direct DNA methylation in various tissues.

Reinforcement of CHH methylation through RNA-directed DNA methylation ensures sexual reproduction in rice.

DNA methylation is an important epigenetic mark that regulates the expression of genes and transposons. RNA-directed DNA methylation (RdDM) is the main molecular pathway responsible for de novo DNA methylation in plants. Although the mechanism of RdDM has been well studied in Arabidopsis (Arabidopsis thaliana), most mutations in RdDM genes cause no remarkable developmental defects in Arabidopsis. Here, we isolated and cloned Five Elements Mountain 1 (FEM1), which encodes RNA-dependent RNA polymerase 2 (OsRDR2) in rice (Oryza sativa). Mutation in OsRDR2 abolished the accumulation of 24-nt small interfering RNAs, and consequently substantially decreased genome-wide CHH (H = A, C, or T) methylation. Moreover, male and female reproductive development was disturbed, which led to sterility in osrdr2 mutants. We discovered that OsRDR2-dependent DNA methylation may regulate the expression of multiple key genes involved in stamen development, meiosis, and pollen viability. In wild-type (WT) plants but not in osrdr2 mutants, genome-wide CHH methylation levels were greater in panicles, stamens, and pistils than in seedlings. The global increase of CHH methylation in reproductive organs of the WT was mainly explained by the enhancement of RdDM activity, which includes OsRDR2 activity. Our results, which revealed a global increase in CHH methylation through enhancement of RdDM activity in reproductive organs, suggest a crucial role for OsRDR2 in the sexual reproduction of rice.

The effect of RNA polymerase V on 24-nt siRNA accumulation depends on DNA methylation contexts and histone modifications in rice.

RNA-directed DNA methylation (RdDM) functions in de novo methylation in CG, CHG, and CHH contexts. Here, we performed map-based cloning of OsNRPE1, which encodes the largest subunit of RNA polymerase V (Pol V), a key regulator of gene silencing and reproductive development in rice. We found that rice Pol V is required for CHH methylation on RdDM loci by transcribing long noncoding RNAs. Pol V influences the accumulation of 24-nucleotide small interfering RNAs (24-nt siRNAs) in a locus-specific manner. Biosynthesis of 24-nt siRNAs on loci with high CHH methylation levels and low CG and CHG methylation levels tends to depend on Pol V. In contrast, low methylation levels in the CHH context and high methylation levels in CG and CHG contexts predisposes 24-nt siRNA accumulation to be independent of Pol V. H3K9me1 and H3K9me2 tend to be enriched on Pol V-independent 24-nt siRNA loci, whereas various active histone modifications are enriched on Pol V-dependent 24-nt siRNA loci. DNA methylation is required for 24-nt siRNAs biosynthesis on Pol V-dependent loci but not on Pol V-independent loci. Our results reveal the function of rice Pol V for long noncoding RNA production, DNA methylation, 24-nt siRNA accumulation, and reproductive development.

Karrikin Signaling Acts Parallel to and Additively with Strigolactone Signaling to Regulate Rice Mesocotyl Elongation in Darkness.

Seedling emergence in monocots depends mainly on mesocotyl elongation, requiring coordination between developmental signals and environmental stimuli. Strigolactones (SLs) and karrikins are butenolide compounds that regulate various developmental processes; both are able to negatively regulate rice (Oryza sativa) mesocotyl elongation in the dark. Here, we report that a karrikin signaling complex, DWARF14-LIKE (D14L)-DWARF3 (D3)-O. sativa SUPPRESSOR OF MAX2 1 (OsSMAX1) mediates the regulation of rice mesocotyl elongation in the dark. We demonstrate that D14L recognizes the karrikin signal and recruits the SCFD3 ubiquitin ligase for the ubiquitination and degradation of OsSMAX1, mirroring the SL-induced and D14- and D3-dependent ubiquitination and degradation of D53. Overexpression of OsSMAX1 promoted mesocotyl elongation in the dark, whereas knockout of OsSMAX1 suppressed the elongated-mesocotyl phenotypes of d14l and d3 OsSMAX1 localizes to the nucleus and interacts with TOPLESS-RELATED PROTEINs, regulating downstream gene expression. Moreover, we showed that the GR24 enantiomers GR245DS and GR24 ent-5DS specifically inhibit mesocotyl elongation and regulate downstream gene expression in a D14- and D14L-dependent manner, respectively. Our work revealed that karrikin and SL signaling play parallel and additive roles in modulating downstream gene expression and negatively regulating mesocotyl elongation in the dark.

A genome-wide survey of copy number variations reveals an asymmetric evolution of duplicated genes in rice.

BACKGROUND: Copy number variations (CNVs) are an important type of structural variations in the genome that usually affect gene expression levels by gene dosage effect. Understanding CNVs as part of genome evolution may provide insights into the genetic basis of important agricultural traits and contribute to the crop breeding in the future. While available methods to detect CNVs utilizing next-generation sequencing technology have helped shed light on prevalence and effects of CNVs, the complexity of crop genomes poses a major challenge and requires development of additional tools. RESULTS: Here, we generated genomic and transcriptomic data of 93 rice (Oryza sativa L.) accessions and developed a comprehensive pipeline to call CNVs in this large-scale dataset. We analyzed the correlation between CNVs and gene expression levels and found that approximately 13% of the identified genes showed a significant correlation between their expression levels and copy numbers. Further analysis showed that about 36% of duplicate pairs were involved in pseudogenetic events while only 5% of them showed functional differentiation. Moreover, the offspring copy mainly contributed to the expression levels and seemed more likely to become a pseudogene, whereas the parent copy tended to maintain the function of ancestral gene. CONCLUSION: We provide a high-accuracy CNV dataset that will contribute to functional genomics studies and molecular breeding in rice. We also showed that gene dosage effect of CNVs in rice is not exponential or linear. Our work demonstrates that the evolution of duplicated genes is asymmetric in both expression levels and gene fates, shedding a new insight into the evolution of duplicated genes.

A Strigolactone Biosynthesis Gene Contributed to the Green Revolution in Rice.

Plant architecture is a complex agronomic trait and a major factor of crop yield, which is affected by several important hormones. Strigolactones (SLs) are identified as a new class hormoneinhibiting branching in many plant species and have been shown to be involved in various developmental processes. Genetical and chemical modulation of the SL pathway is recognized as a promising approach to modify plant architecture. However, whether and how the genes involved in the SL pathway could be utilized in breeding still remain elusive. Here, we demonstrate that a partial loss-of-function allele of the SL biosynthesis gene, HIGH TILLERING AND DWARF 1/DWARF17 (HTD1/D17), which encodes CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), increases tiller number and improves grain yield in rice. We found that the HTD1 gene had been widely utilized and co-selected with Semidwarf 1 (SD1), both contributing to the improvement of plant architecture in modern rice varieties since the Green Revolution in the 1960s. Understanding how phytohormone pathway genes regulate plant architecture and how they have been utilized and selected in breeding will lay the foundation for developing the rational approaches toward improving crop yield.

Copy number variation at the GL7 locus contributes to grain size diversity in rice.

Copy number variants (CNVs) are associated with changes in gene expression levels and contribute to various adaptive traits. Here we show that a CNV at the Grain Length on Chromosome 7 (GL7) locus contributes to grain size diversity in rice (Oryza sativa L.). GL7 encodes a protein homologous to Arabidopsis thaliana LONGIFOLIA proteins, which regulate longitudinal cell elongation. Tandem duplication of a 17.1-kb segment at the GL7 locus leads to upregulation of GL7 and downregulation of its nearby negative regulator, resulting in an increase in grain length and improvement of grain appearance quality. Sequence analysis indicates that allelic variants of GL7 and its negative regulator are associated with grain size diversity and that the CNV at the GL7 locus was selected for and used in breeding. Our work suggests that pyramiding beneficial alleles of GL7 and other yield- and quality-related genes may improve the breeding of elite rice varieties.

A Rare Allele of GS2 Enhances Grain Size and Grain Yield in Rice.

Grain size determines grain weight and affects grain quality. Several major quantitative trait loci (QTLs) regulating grain size have been cloned; however, our understanding of the underlying mechanism that regulates the size of rice grains remains fragmentary. Here, we report the cloning and characterization of a dominant QTL, grain size on chromosome 2 (GS2), which encodes Growth-Regulating Factor 4 (OsGRF4), a transcriptional regulator. GS2 localizes to the nucleus and may act as a transcription activator. A rare mutation of GS2 affecting the binding site of a microRNA, OsmiR396c, causes elevated expression of GS2/OsGRF4. The increase in GS2 expression leads to larger cells and increased numbers of cells, which thus enhances grain weight and yield. The introduction of this rare allele of GS2/OsGRF4 into rice cultivars could significantly enhance grain weight and increase grain yield, with possible applications in breeding high-yield rice varieties.

Quantitative trait locus analysis and fine mapping of the qPL6 locus for panicle length in rice.

KEY MESSAGE: Two QTLs were identified to control panicle length in rice backcross lines, and one QTL qPL6 was finely mapped with potential in high yield breeding. Panicle length (PL) is the key determinant of panicle architecture in rice, and strongly affects yield components, such as grain number per panicle. However, this trait has not been well studied genetically nor its contribution to yield improvement. In this study, we performed quantitative trait locus (QTL) analysis for PL in four backcross populations derived from the cross of Nipponbare (japonica) and WS3 (indica), a new plant type (NPT) variety. Two QTLs were identified on chromosome 6 and 8, designated as qPL6 and qPL8, respectively. Near-isogenic lines (NILs) were developed to evaluate their contribution to important agronomic traits. We found that qPL6 and qPL8 had additive effects on PL trait. For the qPL6 locus, the WS3 allele also increased panicle primary and secondary branches and grain number per panicle. Moreover, this allele conferred wide and strong culms, a character of lodging resistance. By analyzing key recombinants in two steps, the qPL6 locus was finely mapped to a 25-kb interval, and 3 candidate genes were identified. According to the single nucleotide polymorphisms (SNPs) within candidate genes, 5 dCaps markers were designed and used to get haplotypes of 96 modern Chinese varieties, which proved that qPL6 locus is differentiated between indica and temperate japonica varieties. Taken together, the superior qPL6 allele can be applied in rice breeding programs for large sink size, particularly for japonica varieties that originally lack the allele.

Curved chimeric palea 1 encoding an EMF1-like protein maintains epigenetic repression of OsMADS58 in rice palea development.

Floral organ specification is controlled by various MADS-box genes in both dicots and monocots, whose expression is often subjected to both genetic and epigenetic regulation in Arabidopsis thaliana. However, little information is known about the role of epigenetic modification of MADS-box genes during rice flower development. Here, we report the characterization of a rice gene, curved chimeric palea 1 (CCP1) that functions in palea development. Mutation in CCP1 resulted in abnormal palea with ectopic stigmatic tissues and other pleiotropic phenotypes. We found that OsMADS58, a C-class gene responsible for carpel morphogenesis, was ectopically expressed in the ccp1 palea, indicating that the ccp1 palea was misspecified and partially acquired carpel-like identity. Constitutive expression of OsMADS58 in the wild-type rice plants caused morphological abnormality of palea similar to that of ccp1, whereas OsMADS58 knockdown by RNAi in ccp1 could rescue the abnormal phenotype of mutant palea, suggesting that the repression of OsMADS58 expression by CCP1 is critical for palea development. Map-based cloning revealed that CCP1 encodes a putative plant-specific emBRYONIC flower1 (EMF1)-like protein. Chromatin immunoprecipitation assay showed that the level of the H3K27me3 at the OsMADS58 locus was greatly reduced in ccp1 compared with that in the wild-type. Taken together, our results show that CCP1 plays an important role in palea development through maintaining H3K27me3-mediated epigenetic silence of the carpel identity-specifying gene OsMADS58, shedding light on the epigenetic mechanism in floral organ development.

Sugar homeostasis mediated by cell wall invertase GRAIN INCOMPLETE FILLING 1 (GIF1) plays a role in pre-existing and induced defence in rice.

Sugar metabolism and sugar signalling are not only critical for plant growth and development, but are also important for stress responses. However, how sugar homeostasis is involved in plant defence against pathogen attack in the model crop rice remains largely unknown. In this study, we observed that the grains of gif1, a loss-of-function mutant of the cell wall invertase gene GRAIN INCOMPLETE FILLING 1 (GIF1), were hypersusceptible to postharvest fungal pathogens, with decreased levels of sugars and a thinner glume cell wall in comparison with the wild-type. Interestingly, constitutive expression of GIF1 enhanced resistance to both the rice bacterial pathogen Xanthomonas oryzae pv. oryzae and the fungal pathogen Magnaporthe oryzae. The GIF1-overexpressing (GIF1-OE) plants accumulated higher levels of glucose, fructose and sucrose compared with the wild-type plants. More importantly, higher levels of callose were deposited in GIF1-OE plants during pathogen infection. Moreover, the cell wall was much thicker in the infection sites of the GIF1-OE plants when compared with the wild-type plants. We also found that defence-related genes were constitutively activated in the GIF1-OE plants. Taken together, our study reveals that sugar homeostasis mediated by GIF1 plays an important role in constitutive and induced physical and chemical defence.

Control of rice embryo development, shoot apical meristem maintenance, and grain yield by a novel cytochrome p450.

Angiosperm seeds usually consist of two major parts: the embryo and the endosperm. However, the molecular mechanism(s) underlying embryo and endosperm development remains largely unknown, particularly in rice, the model cereal. Here, we report the identification and functional characterization of the rice GIANT EMBRYO (GE) gene. Mutation of GE resulted in a large embryo in the seed, which was caused by excessive expansion of scutellum cells. Post-embryonic growth of ge seedling was severely inhibited due to defective shoot apical meristem (SAM) maintenance. Map-based cloning revealed that GE encodes a CYP78A subfamily P450 monooxygenase that is localized to the endoplasmic reticulum. GE is expressed predominantly in the scutellar epithelium, the interface region between embryo and endosperm. Overexpression of GE promoted cell proliferation and enhanced rice plant growth and grain yield, but reduced embryo size, suggesting that GE is critical for coordinating rice embryo and endosperm development. Moreover, transgenic Arabidopsis plants overexpressing AtCYP78A10, a GE homolog, also produced bigger seeds, implying a conserved role for the CYP78A subfamily of P450s in regulating seed development. Taken together, our results indicate that GE plays critical roles in regulating embryo development and SAM maintenance.

Beak-shaped grain 1/TRIANGULAR HULL 1, a DUF640 gene, is associated with grain shape, size and weight in rice.

Grain shape and size both determine grain weight and therefore crop yield. However, the molecular mechanisms controlling grain shape and size are still largely unknown. Here, we isolated a rice mutant, beak-shaped grain1 (bsg1), which produced beak-shaped grains of decreased width, thickness and weight with a loosely interlocked lemma and palea that were unable to close tightly. Starch granules were also irregularly packaged in the bsg1 grains. Consistent with the lemma and palea shapes, the outer parenchyma cell layers of these bsg1 tissues developed fewer cells with decreased size. Map-based cloning revealed that BSG1 encoded a DUF640 domain protein, TRIANGULAR HULL 1, of unknown function. Quantitative PCR and GUS fusion reporter assays showed that BSG1 was expressed mainly in the young panicle and elongating stem. The BSG1 mutation affected the expression of genes potentially involved in the cell cycle and GW2, an important regulator of grain size in rice. Our results suggest that BSG1 determines grain shape and size probably by modifying cell division and expansion in the grain hull.

Plant hormone jasmonate prioritizes defense over growth by interfering with gibberellin signaling cascade.

Plants must effectively defend against biotic and abiotic stresses to survive in nature. However, this defense is costly and is often accompanied by significant growth inhibition. How plants coordinate the fluctuating growth-defense dynamics is not well understood and remains a fundamental question. Jasmonate (JA) and gibberellic acid (GA) are important plant hormones that mediate defense and growth, respectively. Binding of bioactive JA or GA ligands to cognate receptors leads to proteasome-dependent degradation of specific transcriptional repressors (the JAZ or DELLA family of proteins), which, at the resting state, represses cognate transcription factors involved in defense (e.g., MYCs) or growth [e.g. phytochrome interacting factors (PIFs)]. In this study, we found that the coi1 JA receptor mutants of rice (a domesticated monocot crop) and Arabidopsis (a model dicot plant) both exhibit hallmark phenotypes of GA-hypersensitive mutants. JA delays GA-mediated DELLA protein degradation, and the della mutant is less sensitive to JA for growth inhibition. Overexpression of a selected group of JAZ repressors in Arabidopsis plants partially phenocopies GA-associated phenotypes of the coi1 mutant, and JAZ9 inhibits RGA (a DELLA protein) interaction with transcription factor PIF3. Importantly, the pif quadruple (pifq) mutant no longer responds to JA-induced growth inhibition, and overexpression of PIF3 could partially overcome JA-induced growth inhibition. Thus, a molecular cascade involving the COI1-JAZ-DELLA-PIF signaling module, by which angiosperm plants prioritize JA-mediated defense over growth, has been elucidated.

The rice hydroperoxide lyase OsHPL3 functions in defense responses by modulating the oxylipin pathway.

As important signal molecules, jasmonates (JAs) and green leaf volatiles (GLVs) play diverse roles in plant defense responses against insect pests and pathogens. However, how plants employ their specific defense responses by modulating the levels of JA and GLVs remains unclear. Here, we describe identification of a role for the rice HPL3 gene, which encodes a hydroperoxide lyase (HPL), OsHPL3/CYP74B2, in mediating plant-specific defense responses. The loss-of-function mutant hpl3-1 produced disease-resembling lesions spreading through the whole leaves. A biochemical assay revealed that OsHPL3 possesses intrinsic HPL activity, hydrolyzing hydroperoxylinolenic acid to produce GLVs. The hpl3-1 plants exhibited enhanced induction of JA, trypsin proteinase inhibitors and other volatiles, but decreased levels of GLVs including (Z)-3-hexen-1-ol. OsHPL3 positively modulates resistance to the rice brown planthopper [BPH, Nilaparvata lugens (Stål)] but negatively modulates resistance to the rice striped stem borer [SSB, Chilo suppressalis (Walker)]. Moreover, hpl3-1 plants were more attractive to a BPH egg parasitoid, Anagrus nilaparvatae, than the wild-type, most likely as a result of increased release of BPH-induced volatiles. Interestingly, hpl3-1 plants also showed increased resistance to bacterial blight (Xanthomonas oryzae pv. oryzae). Collectively, these results indicate that OsHPL3, by affecting the levels of JA, GLVs and other volatiles, modulates rice-specific defense responses against different invaders.

A novel protein RLS1 with NB-ARM domains is involved in chloroplast degradation during leaf senescence in rice.

Leaf senescence, a type of programmed cell death (PCD) characterized by chlorophyll degradation, is important to plant growth and crop productivity. It emerges that autophagy is involved in chloroplast degradation during leaf senescence. However, the molecular mechanism(s) involved in the process is not well understood. In this study, the genetic and physiological characteristics of the rice rls1 (rapid leaf senescence 1) mutant were identified. The rls1 mutant developed small, yellow-brown lesions resembling disease scattered over the whole surfaces of leaves that displayed earlier senescence than those of wild-type plants. The rapid loss of chlorophyll content during senescence was the main cause of accelerated leaf senescence in rls1. Microscopic observation indicated that PCD was misregulated, probably resulting in the accelerated degradation of chloroplasts in rls1 leaves. Map-based cloning of the RLS1 gene revealed that it encodes a previously uncharacterized NB (nucleotide-binding site)-containing protein with an ARM (armadillo) domain at the carboxyl terminus. Consistent with its involvement in leaf senescence, RLS1 was up-regulated during dark-induced leaf senescence and down-regulated by cytokinin. Intriguingly, constitutive expression of RLS1 also slightly accelerated leaf senescence with decreased chlorophyll content in transgenic rice plants. Our study identified a previously uncharacterized NB-ARM protein involved in PCD during plant growth and development, providing a unique tool for dissecting possible autophagy-mediated PCD during senescence in plants.