An E3 ubiquitin ligase,ERECT LEAF1, functions in brassinosteroid signaling of rice

A spontaneous rice mutant, erect leaf1 (elf1–1), produced a dwarf phenotype with erect leaves and short grains. Physiological analyses suggested that elf1–1 is brassinosteroid-insensitive, so we hypothesized that ELF1 encodes a positive regulator of brassinosteroid signaling. ELF1, identified by means of positional cloning, encodes a protein with both a U-box domain and ARMADILLO (ARM) repeats. U-box proteins have been shown to function as E3 ubiquitin ligases; in fact, ELF1 possessed E3 ubiquitin ligase activity in vitro. However, ELF1 itself does not appear to be polyubiquitinated. Mutant phenotypes of 2 more elf1 alleles indicate that the entire ARM repeats is indispensable for ELF1 activity. These results suggest that ELF1 ubiquitinates target proteins through an interaction mediated by ARM repeats. Similarities in the phenotypes of elf1 and d61 mutants (mutants of brassinosteroid receptor gene OsBRI1), and in the regulation of ELF1 and OsBRI1 expression, imply that ELF1 functions as a positive regulator of brassinosteroid signaling in rice.     

The U-Box E3 Ubiquitin Ligase TUD1 Functions with a Heterotrimeric G α Subunit to Regulate Brassinosteroid-Mediated Growth in Rice

Heterotrimeric G proteins are an important group of signaling molecules found in eukaryotes. They function with G-protein-coupled-receptors (GPCRs) to transduce various signals such as steroid hormones in animals. Nevertheless, their functions in plants are not well-defined. Previous studies suggested that the heterotrimeric G protein α subunit known as D1/RGA1 in rice is involved in a phytohormone gibberellin-mediated signaling pathway. Evidence also implicates D1 in the action of a second phytohormone Brassinosteroid (BR) and its pathway. However, it is unclear how D1 functions in this pathway, because so far no partner has been identified to act with D1. In this study, we report a D1 genetic interactor Taihu Dwarf1 (TUD1) that encodes a functional U-box E3 ubiquitin ligase. Genetic, phenotypic, and physiological analyses have shown that tud1 is epistatic to d1 and is less sensitive to BR treatment. Histological observations showed that the dwarf phenotype of tud1 is mainly due to decreased cell proliferation and disorganized cell files in aerial organs. Furthermore, we found that D1 directly interacts with TUD1. Taken together, these results demonstrate that D1 and TUD1 act together to mediate a BR-signaling pathway. This supports the idea that a D1-mediated BR signaling pathway occurs in rice to affect plant growth and development.     

Fine Mapping and Candidate Gene Analysis of qSTL3, a Stigma Length-Conditioning Locus in Rice (Oryza sativa L.)

The efficiency of hybrid seed production can be improved by increasing the percentage of exserted stigma, which is closely related to the stigma length in rice. In the chromosome segment substitute line (CSSL) population derived from Nipponbare (recipient) and Kasalath (donor), a single CSSL (SSSL14) was found to show a longer stigma length than that of Nipponbare. The difference in stigma length between Nipponbare and SSSL14 was controlled by one locus (qSTL3). Using 7,917 individuals from the SSSL14/Nipponbare F₂ population, the qSTL3 locus was delimited to a 19.8-kb region in the middle of the short arm of chromosome 3. Within the 19.8-kb chromosome region, three annotated genes (LOC_Os03g14850, LOC_Os03g14860 and LOC_Os03g14880) were found in the rice genome annotation database. According to gene sequence alignments in LOC_Os03g14850, a transition of G (Nipponbare) to A (Kasalath) was detected at the 474-bp site in CDS. The transition created a stop codon, leading to a deletion of 28 amino acids in the deduced peptide sequence in Kasalath. A T-DNA insertion mutant (05Z11CN28) of LOC_Os03g14850 showed a longer stigma length than that of wild type (Zhonghua 11), validating that LOC_Os03g14850 is the gene controlling stigma length. However, the Kasalath allele of LOC_Os03g14850 is unique because all of the alleles were the same as that of Nipponbare at the 474-bp site in the CDS of LOC_Os03g14850 among the investigated accessions with different stigma lengths. A gene-specific InDel marker LQ30 was developed for improving stigma length during rice hybrid breeding by marker-assisted selection.     

The RhoGAP SPIN6 Associates with SPL11 and OsRac1 and Negatively Regulates Programmed Cell Death and Innate Immunity in Rice

The ubiquitin proteasome system in plants plays important roles in plant-microbe interactions and in immune responses to pathogens. We previously demonstrated that the rice U-box E3 ligase SPL11 and its Arabidopsis ortholog PUB13 negatively regulate programmed cell death (PCD) and defense response. However, the components involved in the SPL11/PUB13-mediated PCD and immune signaling pathway remain unknown. In this study, we report that SPL11-interacting Protein 6 (SPIN6) is a Rho GTPase-activating protein (RhoGAP) that interacts with SPL11 in vitro and in vivo. SPL11 ubiquitinates SPIN6 in vitro and degrades SPIN6 in vivo via the 26S proteasome-dependent pathway. Both RNAi silencing in transgenic rice and knockout of Spin6 in a T-DNA insertion mutant lead to PCD and increased resistance to the rice blast pathogen Magnaporthe oryzae and the bacterial blight pathogen Xanthomonas oryzae pv. oryzae. The levels of reactive oxygen species and defense-related gene expression are significantly elevated in both the Spin6 RNAi and mutant plants. Strikingly, SPIN6 interacts with the small GTPase OsRac1, catalyze the GTP-bound OsRac1 into the GDP-bound state in vitro and has GAP activity towards OsRac1 in rice cells. Together, our results demonstrate that the RhoGAP SPIN6 acts as a linkage between a U-box E3 ligase-mediated ubiquitination pathway and a small GTPase-associated defensome system for plant immunity.     

Functional molecular markers and high-resolution melting curve analysis of low phytic acid mutations for marker-assisted selection in rice

Phytic acid (PA, myo-inositol-1,2,3,4,5,6-hexakis-phosphate) and its salt form (phytate) are the principal storage forms of phosphorus in cereal grains. Since PA and phytates cannot be efficiently digested by monogastric animals, the abundance of PA in cereal and legume grains causes nutritional and environmental problems. The present study aimed at developing breeder-friendly functional molecular markers of five low phytic acid (LPA) mutant alleles of three rice (Oryza sativa L.) genes: viz., XQZ-lpa (a 1,475-bp deletion) and KBNT-lpa (a C→T single nucleotide polymorphism [SNP]) of LOC_Os02g57400, Z9B-lpa (a 6-bp deletion) and MH-lpa (a 1-bp deletion) of LOC_Os04g55800, and XS-lpa (a C→T SNP) of LOC_Os03g04920. First, markers for gel-based length polymorphism analysis were developed: viz., two insertion–deletion markers for XQZ-lpa and Z9B-lpa, two cleaved amplified polymorphic sequence (CAPS) markers for KBNT-lpa and XS-lpa, and one derived CAPS marker for MH-lpa. Second, the high-resolution melting (HRM) curve analysis method was explored for distinguishing plants with wild-type (WT) and LPA alleles (except XQZ-lpa). Plants of genotypes with homozygous mutant allele and WT, and with heterozygous alleles, could be directly differentiated by HRM for KBNT-lpa, XS-lpa and MH-lpa; only heterozygous individuals could be directly distinguished from homozygous WT and mutant plants for Z9B-lpa. However, by adding 15 % WT DNA templates to test samples before PCR, amplicons of three genotypes of the Z9B-lpa allele could also be differentiated by HRM analysis. Third, it was demonstrated that these markers could be effectively used for marker-assisted selection of LPA rice, and breeding lines with two non-allelic LPA mutations were developed with PA contents significantly lower than their respective parental LPA lines. Taken together, the present study developed functional molecular markers for efficient selection of LPA plants and demonstrated that double mutant LPA lines with significantly lower PA levels than primary LPA mutants (with single mutations) could be developed by pyramiding two non-allelic LPA mutations.     

Molecular cloning and characterization of OsUPS,a U-box containing E3 ligase gene that respond to phosphate starvation in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>)

The ubiquitin-26S proteasome system is important in the quality control of intracellular proteins. The ubiquitin-26S proteasome system includes the E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligase) enzymes. U-box proteins are a derived version of RING-finger domains, which have E3 enzyme activity. Here, we present the isolation of a novel U-box protein, U-box containing E3 ligase induced by phosphate starvation (OsUPS), from rice (Oryza sativa). The cDNA encoding the O. sativa U-box protein (OsUPS) comprises 1338 bp, with an open reading frame of 445 amino acids. The amino acid sequence of OsUPS cDNA shows 41–79% identity with other plant U-box homologous genes. The open reading frame of the OsUPS protein is comprised of notable domains: a single ~70-amino acid domain and a GKL domain that contains conserved glycine, lysine/arginine residues and leucine-rich feature. We found that full-length expression of OsUPS was up-regulated in both rice plants and cell culture in the absence of inorganic phosphate (P_i). A self-ubiquitination assay indicated that the bacterially expressed OsUPS protein had E3 ligase activity, and subcellular localization results showed that OsUPS was located in the chloroplast. These results support the notion that OsUPS plays an important role in the P_i signaling pathway through the ubiquitin-26S proteasome system.     

Fine mapping of a dominant minute-grain gene, Mi3, in rice

Grain weight is a major determinant of rice grain yield and is widely believed to be controlled by quantitative trait loci (QTL). We have previously reported a new major gene, Mi3, regulating grain length in rice, and that the Mi3 allele from Y34 functioned in a dominant manner. In this paper we report the fine mapping and candidate analysis of Mi3. By employing a chromosome walking strategy in the F₂ population of 9311/Y34, the Mi3 gene was finally narrowed to an interval of ~?41.6?kb between the markers RM6881 and LM9 in the pericentromeric region of rice chromosome 3. According to the rice genome annotations, five putative gene loci, LOC_Os03g_29614, LOC_Os03g_29630, LOC_Os03g_29650, LOC_Os03g_29660 and LOC_Os03g_29680, were located in this candidate region. Mi3 was also determined to be a new gene for grain size in rice by allelic analysis with the previously reported genes. Our results will facilitate the cloning and functional characterization of the Mi3 gene and targeted marker-assisted breeding.     

PHOR1: A U-Box GA Signaling Component With a Role in Proteasome Degradation?

Recent identification of U-box proteins as E3 ubiquitin ligases suggests that the U-box arm-repeat protein PHOR1, for which we have demonstrated a role in GA signal transduction, may play a role in GA signaling by ubiquitinating one or more components of the GA response pathway to target them for proteasome degradation. Here we show that PHOR1 function in GA signaling is not exclusive of potato plants, but it is also conserved in Arabidopsis. Three PHOR1-homologs have been identified in this plant species, which would correspond to PHOR1-orthologs. Experimental evidence has recently been obtained for the involvement of proteasome-dependent protein degradation in GA-mediated destabilization of the SLN1 DELLA protein, thus pointing to this repressor as a likely substrate for ubiquitination by the PHOR1 ubiquitin ligase activity.     

Analysis of the rice mutant dwarf and gladius leaf 1. Aberrant katanin-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling

Molecular genetic studies of plant dwarf mutants have indicated that gibberellin (GA) and brassinosteroid (BR) are two major factors that determine plant height; dwarf mutants that are caused by other defects are relatively rare, especially in monocot species. Here, we report a rice (Oryza sativa) dwarf mutant, dwarf and gladius leaf 1 (dgl1), which exhibits only minimal response to GA and BR. In addition to the dwarf phenotype, dgl1 produces leaves with abnormally rounded tip regions. Positional cloning of DGL1 revealed that it encodes a 60-kD microtubule-severing katanin-like protein. The protein was found to be important in cell elongation and division, based on the observed cell phenotypes. GA biosynthetic genes are up-regulated in dgl1, but the expression of BR biosynthetic genes is not enhanced. The enhanced expression of GA biosynthetic genes in dgl1 is not caused by inappropriate GA signaling because the expression of these genes was repressed by GA3 treatment, and degradation of the rice DELLA protein SLR1 was triggered by GA3 in this mutant. Instead, aberrant microtubule organization caused by the loss of the microtubule-severing function of DGL1 may result in enhanced expression of GA biosynthetic genes in that enhanced expression was also observed in a BR-deficient mutant with aberrant microtubule organization. These results suggest that the function of DGL1 is important for cell and organ elongation in rice, and aberrant DGL1-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling.     

PUB13, a U-box/ARM E3 ligase,regulates plant defense,cell death,and flowering time

The ubiquitination pathway is involved in a variety of cellular processes in plant growth, development, and immune responses. However, the function of this pathway in connecting plant development and innate immunity is still largely unknown. Recently, we characterized the U-box/ARM E3 ubiquitin ligase PUB13, which regulates both immune responses and flowering time in Arabidopsis. Here, we show that the rice Spl11 gene can complement the cell death and flowering functions of PUB13 in the pub13 mutant. In addition, HFR1, which functions mainly in photomorphogenesis, was identified as one of the PUB13-interacting proteins through yeast two-hybrid screening and pull-down assays. Because the flowering phenotype of pub13 depends on photoperiod, we propose that PUB13 may regulate HFR1 to fine-tune photomorphogenesis and flowering time in Arabidopsis.     

Phenotypic and Candidate Gene Analysis of a New Floury Endosperm Mutant (osagpl2-3) in Rice

A floury endosperm mutant, osagpl2-3, was isolated from the M₂ generation of japonica rice cultivar Nipponbare following ethyl methane sulfonate mutagenesis. The osagpl2-3 mutant produced a white-core endosperm compared to the transparent endosperm of the wild type (WT). The results from scanning electron microscope showed that the osagpl2-3 mutant grains comprised of round and loosely packed starch granules, some of which were compounded. The analysis for cooking and nutrition quality traits indicated that the values of gel consistency, gelatinization temperature, and rapid viscosity analysis profile of osagpl2-3 grains were lower than those of the WT. Besides, the protein content, the contents of nine different amino acids, and the thermodynamic parameters of T _p and ??T _1/2 in osagpl2-3 were also different from those of the WT. Genetic analysis revealed that osagpl2-3 mutation was controlled by a single recessive gene. The osagpl2-3 gene was mapped between InDel markers R1M30 and ID1-12 on rice chromosome 1. In the candidate region of the Nipponbare genome, an annotated gene, LOC_Os01g44220 which encodes a large subunit of putative ADP-glucose pyrophosphrylase named OsAPL2 was considered the optimal candidate. Cloning and sequencing of LOC_Os01g44220 in different plants of the osagpl2-3 mutants revealed a single nucleotide mutation (G??A) in the open reading frame region, which led to a substitution of an acidic amino acid Glu (E) by a basic amino acid Lys (K) accordingly. Furthermore, the mutant site is close to the functional domain which interacts with the ADP-Glc. In brief, these results suggested that the osagpl2-3 is a new mutant of OsAPL2.     

A key ABA hydrolase gene,OsABA8ox3 is involved in rice resistance to Nilaparvata lugens by affecting callose deposition

Abscisic acid (ABA) is an important plant hormone in regulating abiotic and biotic stresses. OsABA8ox3 is the key gene in ABA hydrolase genes, and plays an important role in controlling ABA level, but little is known in rice resistance to insects. We used rice osaba8ox3 T-DNA insertion mutant (knocking down the OsABA8ox3 gene) to elucidate rice resistance to the insect. There were obvious phenotype differences between the osaba8ox3 T-DNA insertion mutant and wild-type (WT), and the relative expression of synthetase genes in the osaba8ox3 mutant was higher, while the relative expression of hydrolase genes was lower than that of WT, respectively. The electrical penetration graph (EPG) recording indicated that the osaba8ox3 mutant had the less sucking phloem sap duration compared with WT, which indicated a significant increase in rice resistance to brown planthopper (Nilaparvata lugens; BPH). The callose deposition in the osaba8ox3 mutant increased by 60.39%, 52.2%, 26.6% and 31.7% than that of WT after BPH feeding for 0, 24, 48, and 72 h, respectively. These results showed OsABA8ox3 gene played an important role in rice resistance to BPH, and also provided new insights into the mechanism of callose deposition regulation in response to the piercing-sucking pest.     

Identification and characterization of dwarf 62, a loss-of-function mutation in DLT/OsGRAS-32 affecting gibberellin metabolism in rice

A dwarf mutant, dwarf 62 (d62), was isolated from rice cultivar 93-11 by mutagenesis with γ-rays. Under normal growth conditions, the mutant had multiple abnormal phenotypes, such as dwarfism, wide and dark-green leaf blades, reduced tiller numbers, late and asynchronous heading, short roots, partial male sterility, etc. Genetic analysis indicated that the abnormal phenotypes were controlled by the recessive mutation of a single nuclear gene. Using molecular markers, the D62 gene was fine mapped in 131-kb region at the short arm of chromosome 6. Positional cloning of D62 gene revealed that it was the same locus as DLT/OsGRAS-32, which encodes a member of the GRAS family. In previous studies, the DLT/OsGRAS-32 is confirmed to play positive roles in brassinosteroid (BR) signaling. Sequence analysis showed that the d62 carried a 2-bp deletion in ORF region of D62 gene which led to a loss-of-function mutation. The function of D62 gene was confirmed by complementation experiment. RT-PCR analysis and promoter activity analysis showed that the D62 gene expressed in all tested tissues including roots, stems, leaves and panicles of rice plant. The d62 mutant exhibited decreased activity of α-amylase in endosperm and reduced content of endogenous GA₁. The expression levels of gibberellin (GA) biosynthetic genes including OsCPS1, OsKS1, OsKO1, OsKAO, OsGA20ox2/SD1 and OsGA2ox3 were significantly increased in d62 mutant. Briefly, these results demonstrated that the D62 (DLT/OsGRAS-32) not only participated in the regulation of BR signaling, but also influenced GA metabolism in rice.     

ErbB2 degradation mediated by the co-chaperone protein CHIP

ErbB2 overexpression contributes to the evolution of a substantial group of human cancers and signifies a poor clinical prognosis. Thus, down-regulation of ErbB2 signaling has emerged as a new anti-cancer strategy. Ubiquitinylation, mediated by the Cbl family of ubiquitin ligases, has emerged as a physiological mechanism of ErbB receptor down-regulation, and this mechanism appears to contribute to ErbB2 down-regulation induced by therapeutic anti-ErbB2 antibodies. Hsp90 inhibitory ansamycin antibiotics such as geldanamycin (GA) induce rapid ubiquitinylation and down-regulation of ErbB2. However, the ubiquitin ligase(s) involved has not been identified. Here, we show that ErbB2 serves as an in vitro substrate for the Hsp70/Hsp90-associated U-box ubiquitin ligase CHIP. Overexpression of wild type CHIP, but not its U-box mutant H260Q, induced ubiquitinylation and reduction in both cell surface and total levels of ectopically expressed or endogenous ErbB2 in vivo, and this effect was additive with that of 17-allylamino-geldanamycin (17-AAG). The CHIP U-box mutant H260Q reduced 17-AAG-induced ErbB2 ubiquitinylation. Wild type ErbB2 and a mutant incapable of association with Cbl (ErbB2 Y1112F) were equally sensitive to CHIP and 17-AAG, implying that Cbl does not play a major role in geldanamycin-induced ErbB2 down-regulation. Both endogenous and ectopically expressed CHIP and ErbB2 coimmunoprecipitated with each other, and this association was enhanced by 17-AAG. Notably, CHIP H260Q induced a dramatic elevation of ErbB2 association with Hsp70 and prevented the 17-AAG-induced dissociation of Hsp90. Our results demonstrate that ErbB2 is a target of CHIP ubiquitin ligase activity and suggest a role for CHIP E3 activity in controlling both the association of Hsp70/Hsp90 chaperones with ErbB2 and the down-regulation of ErbB2 induced by inhibitors of Hsp90.     

Rice DWARF AND LOW-TILLERING and the homeodomain protein OSH15 interact to regulate internode elongation via orchestrating brassinosteroid signaling and metabolism

Brassinosteroid (BR) phytohormones play crucial roles in regulating internode elongation in rice (Oryza sativa). However, the underlying mechanism remains largely unclear. The dwarf and low-tillering (dlt) mutant is a mild BR-signaling-defective mutant. Here, we identify two dlt enhancers that show more severe shortening of the lower internodes compared to the uppermost internode (IN1). Both mutants carry alleles of ORYZA SATIVA HOMEOBOX 15 (OSH15), the founding gene for dwarf6-type mutants, which have shortened lower internodes but not IN1. Consistent with the mutant phenotype, OSH15 expression is much stronger in lower internodes, particularly in IN2, than IN1. The osh15 single mutants have impaired BR sensitivity accompanied by enhanced BR synthesis in seedlings. DLT physically interacts with OSH15 to co-regulate many genes in seedlings and internodes. OSH15 targets and promotes the expression of the BR receptor gene BR INSENSITIVE1 (OsBRI1), and DLT facilitates this regulation in a dosage-dependent manner. In osh15, dlt, and osh15 dlt, BR levels are higher in seedlings and panicles, but unexpectedly lower in internodes compared with the wild-type. Taken together, our results suggest that DLT interacts with OSH15, which functions in the lower internodes, to modulate rice internode elongation via orchestrating BR signaling and metabolism.

DWARF AND LOW-TILLERING interacts with the homeodomain protein OSH15, which directly targets the brassinosteroid receptor gene OsBRI1 and is expressed in lower internodes, to regulate the internode elongation via modulating brassinosteroid signaling and metabolism.

IN A NUTSHELL Background: Rice culms consist of five to seven internodes and the length of these internodes determines plant height and resistance to wind, which is crucial for field performance. Brassinosteroid (BR) plant hormones are involved in regulating plant height because defects in BR synthesis or signaling (such as mutants in the BR receptor gene BRASSINOSTEROID INSENSITIVE 1 (OsBRI1)) usually result in dwarfism with specific shortening of the lower internodes or the second internode (IN2) compared to that of the uppermost/first internode (IN1). This pattern is known as d6 or dm-type dwarfism. Question: We wanted to know how BRs are involved in organizing the different internodes and therefore, we carried out a large-scale screen for mutants with altered internode organization pattern using the mild BR signaling-defective mutant dwarf and low-tillering (dlt). Findings: We identified two mutants showing specific shortening of the lower internodes, that is d6-type dwarfism. Both mutants have the same causal gene, namely, OSH15, which encodes a homeodomain-containing protein. OSH15 can directly interact with DLT, forming a protein complex to regulate BR contents and BR signaling. For example, DLT–OSH15 directly binds the promoter of OsBRI1 to promote gene expression. OSH15 expression is strong in the lower internodes, particularly in IN2, and DLT shows an opposite expression pattern. Therefore, the protein complex has different levels in different internodes, exerting different effects on BR levels and signaling to modulate internode organization. Next steps: Scientists aim to use BR-related genes to engineer plant height and grain size and thus produce new crops having improved grain yield and lodging resistance. The discovery of the DLT–OSH15–OsBRI1 module could help achieve this goal. Next, we will try to uncover how BRs coordinate internode elongation with panicle development.     

Genetic mapping and salt tolerance of a novel <Emphasis Type="Italic">D1</Emphasis>-allelic mutant of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Plant height and grain shape are important traits that may affect yield in rice, and they therefore have enormous importance in breeding. A dwarf small-grain mutant (S525) was identified among progeny of the Indica rice restorer line ‘Xida 1B’ (wild type) raised from seeds treated with ethyl methanesulfonate. The dwarf and small-grain phenotypes were stably inherited after multi-generation selfing. Field-grown mutant plants showed the phenotypes of dwarfism, broad leaves, and small round grains. Genetic mapping and sequencing confirmed that S525 was a novel d1-allelic mutant. A single-base transition (G to A) in the functional dwarfism gene D1 at the conjunction site of the 11th intron caused excision or duplication of the 11th exon in the mRNA and resulted in translation of a defective G_α protein. The S525 showed enhanced salt tolerance compared with the wild type (WT), and the expression of genes associated with salt tolerance quantitatively increased in response to treatment with 200 mM NaCl. The S525 may be useful for future investigation of G_α functions and in the breeding of new dwarf rice cultivars.     

Green-revertible Chlorina 1 (grc1) is required for the biosynthesis of chlorophyll and the early development of chloroplasts in rice

The nuclear genes involved in chloroplast development and chlorophyll biosynthesis must be investigated to understand their functions in plant growth and development. In this study, we isolated and identified a unique leaf-color mutant of rice with a green-yellow phenotype before the four-leaf stage and named the mutation green-revertible chlorina 1 (grc1). The mutants had significantly lower plant height, number of tillers, and panicle length and headed significantly earlier than the wild type. The levels of chlorophylls, carotenoids, and chlorophyll precursors were also lower. The mutation in grc1 affected chloroplast ultrastructure, particularly thylakoid development. Genetic analysis indicated that the green-yellow phenotype was controlled by a single recessive gene. We mapped the grc1 gene to a 32.4-kb region on the long arm of chromosome 6. Through map-based cloning, we identified a 45-bp insertion in the genomic region of LOC_Os06g40080, which encoded a heme oxygenase. Expression of LOC_Os06g40080 was significantly down-regulated in the grc1 mutant. Subcellular localization showed that this heme oxygenase was localized in the chloroplast. In summary, we isolated and identified the gene for grc1, which plays an important role in chlorophyll biosynthesis and chloroplast development in rice.