Zhengjun Xu

Divinyl Chlorophyll(ide) a Can Be Converted to Monovinyl Chlorophyll(ide) a by a Divinyl Reductase in Rice

3,8-Divinyl (proto)chlorophyll(ide) a 8-vinyl reductase (DVR) catalyzes the reduction of 8-vinyl group on the tetrapyrrole to an ethyl group, which is indispensable for monovinyl chlorophyll (Chl) synthesis. So far, three 8-vinyl reductase genes (DVR, bciA, and slr1923) have been characterized from Arabidopsis (Arabidopsis thaliana), Chlorobium tepidum, and Synechocystis sp. PCC6803. However, no 8-vinyl reductase gene has yet been identified in monocotyledonous plants. In this study, we isolated a spontaneous mutant, 824ys, in rice (Oryza sativa). The mutant exhibited a yellow-green leaf phenotype, reduced Chl level, arrested chloroplast development, and retarded growth rate. The phenotype of the 824ys mutant was caused by a recessive mutation in a nuclear gene on the short arm of rice chromosome 3. Map-based cloning of this mutant resulted in the identification of a gene (Os03g22780) showing sequence similarity with the Arabidopsis DVR gene (AT5G18660). In the 824ys mutant, nine nucleotides were deleted at residues 952 to 960 in the open reading frame, resulting in a deletion of three amino acid residues in the encoded product. High-performance liquid chromatography analysis of Chls indicated the mutant accumulates only divinyl Chl a and b. A recombinant protein encoded by Os03g22780 was expressed in Escherichia coli and found to catalyze the conversion of divinyl chlorophyll(ide) a to monovinyl chlorophyll(ide) a. Therefore, it has been confirmed that Os03g22780, renamed as OsDVR, encodes a functional DVR in rice. Based upon these results, we succeeded to identify an 8-vinyl reductase gene in monocotyledonous plants and, more importantly, confirmed the DVR activity to convert divinyl Chl a to monovinyl Chl a.

Identification of a Geranylgeranyl reductase gene for chlorophyll synthesis in rice

Geranylgeranyl reductase (CHL P) catalyzes the reduction of geranylgeranyl diphosphate to phytyl diphosphate, and provides phytol for both Chlorophyll (Chl) and tocopherol synthesis. In this study, we isolated a yellow-green leaf mutant, 502ys, in rice (Oryza sativa). The mutant exhibited reduced level of Chls, arrested development of chloroplasts, and retarded growth rate. The phenotype of the 502ys mutant was controlled by by a recessive mutation in a nuclear gene on the long arm of rice chromosome 2. Map-based cloning of the mutant resulted in the identification of an OsChl P gene (LOC_Os02g51080). In the 502ys mutant, a single base pair mutation was detected at residue 1279 in DNA sequence of the gene, resulting in an amino acid change (Gly-206 to Ser) in the encoded protein. HPLC analysis of Chls indicated that the majority of Chl molecules are conjugated with an unsaturated geranylgeraniol side chain, in addition to small amount of normal Chls in the mutant. Furthermore, the mutant phenotype was complemented by transformation with the wild-type gene. Therefore, this study has confirmed the 502ys mutant resulted from a single base pair mutation in OsChl P gene.

OsPOP5, a prolyl oligopeptidase family gene from rice confers abiotic stress tolerance in Escherichia coli.

The prolyl oligopeptidase family, which is a group of serine peptidases, can hydrolyze peptides smaller than 30 residues. The prolyl oligopeptidase family in plants includes four members, which are prolyl oligopeptidase (POP, EC3.4.21.26), dipeptidyl peptidase IV (DPPIV, EC3.4.14.5), oligopeptidase B (OPB, EC3.4.21.83), and acylaminoacyl peptidase (ACPH, EC3.4.19.1). POP is found in human and rat, and plays important roles in multiple biological processes, such as protein secretion, maturation and degradation of peptide hormones, and neuropathies, signal transduction and memory and learning. However, the function of POP is unclear in plants. In order to study POP function in plants, we cloned the cDNA of the OsPOP5 gene from rice by nested-PCR. Sequence analysis showed that the cDNA encodes a protein of 596 amino acid residues with Mw ≈ 67.29 kD. In order to analyze the protein function under different abiotic stresses, OsPOP5 was expressed in Escherichia coli. OsPOP5 protein enhanced the tolerance of E. coli to high salinity, high temperature and simulated drought. The results indicate that OsPOP5 is a stress-related gene in rice and it may play an important role in plant tolerance to abiotic stress.

Mutation of FdC2 gene encoding a ferredoxin-like protein with C-terminal extension causes yellow-green leaf phenotype in rice.

Ferredoxins (Fds) are small iron-sulfur proteins that mediate electron transfer in a wide range of metabolic reactions. Besides Fds, there is a type of Fd-like proteins designated as FdC, which have conserved elements of Fds, but contain a significant C-terminal extension. So far, only two FdC genes of Arabidopsis (Arabidopsis thaliana) have been identified in higher plants and thus the functions of FdC proteins remain largely unknown. In this study, we isolated a yellow-green leaf mutant, 501ys, in rice (Oryza sativa). The mutant exhibited yellow-green leaf phenotype and reduced chlorophyll level. The phenotype of 501ys was caused by mutation of a gene on rice chromosome 3. Map-based cloning of this mutant resulted in identification of OsFdC2 gene (LOC_Os03g48040) showing high identity with Arabidopsis FdC2 gene (AT1G32550). OsFdC2 was expressed most abundantly in leaves and its encoded protein was targeted to the chloroplast. In 501ys mutant, a missense mutation was detected in DNA sequence of the gene, resulting in an amino acid change in the encoded protein. The mutant phenotype was rescued by introduction of the wild-type gene. Therefore, we successfully identified FdC2 gene via map-based cloning approach, and demonstrated that mutation of this gene caused yellow-green leaf phenotype in rice.

GRY79 encoding a putative metallo-β-lactamase-trihelix chimera is involved in chloroplast development at early seedling stage of rice.

Key messageThegreen-revertible yellow79mutant resulting from a single-base mutation suggested that theGRY79gene encoding a putative metallo-β-lactamase-trihelix chimera is involved in chloroplast development at early seedling stage of rice.AbstractFunctional studies of metallo-β-lactamases and trihelix transcription factors in higher plants remain very sparse. In this study, we isolated the green-revertible yellow79 (gry79) mutant in rice. The mutant developed yellow-green leaves before the three-leaf stage but recovered to normal green at the sixth-leaf stage. Meanwhile, the mutant exhibited reduced level of chlorophylls and arrested development of chloroplasts in the yellow leaves. Genetic analysis suggested that the mutant phenotype was controlled by a single recessive nuclear gene on rice chromosome 2. Map-based cloning revealed that the candidate gene was Os02g33610 encoding a putative metallo-β-lactamase-trihelix chimera. In the gry79 mutant, a single-base mutation occurred in coding region of the gene, resulting in an amino acid change in the encoded protein. Furthermore, the mutant phenotype was rescued by transformation with the wild-type gene. Therefore, we have confirmed that the gry79 mutant phenotype resulted from a single-base mutation in GRY79 (Os02g33610) gene, suggesting that the gene encoding a putative metallo-β-lactamase-trihelix chimera is involved in chloroplast development at early seedling stage of rice. In addition, we considered that the gry79 mutant gene could be applicable as a leaf-color marker gene for efficient identification and elimination of false hybrids in commercial hybrid rice production.

Molecular characterization, expression pattern and function analysis of the OsHSP90 family in rice

ABSTRACT The HSP90 is an abundant chaperone protein that is conserved in all eukaryotes. The main function of HSP90 is to assist other proteins to fold properly. In this study, we uncovered and analysed nine OsHSP90 (OsHSP90-1--OsHSP90-9) family members in rice Nipponbare, in which three distinct motifs were identified. All the HSP90 proteins were classified into three major groups (I, II, III) by phylogenetic analysis. The expression of OsHSP90 family in 10 tissues was examined by real-time polymerase chain reaction (PCR). OsHSP90-4, OsHSP90-6 and OsHSP90-7 had high expression, while OsHSP90-5 and OsHSP90-8 had very low expression across almost all 10 samples. The gene that encodes OsHSP90-1 was preferentially expressed in embryo at 14 days after flowering. It has been reported that some heat shock proteins were up-regulated in response to heat or other stresses. However, in our study the expression pattern of OsHSP90 genes is heterogeneous under a range of stress conditions. The expression of OsHSP90-2 and OsHSP90-4 was up-regulated under drought, salt, cold and heat conditions, while the expression of OsHSP90-3 and OsHSP90-5 was down-regulated under salt and drought conditions. OsHSP90-7 and OsHSP90-9 were down-regulated only under drought conditions. OsHSP90-6 did not change its expression across all conditions compared to control. Overexpression of OsHSP90-2 in E. coli could enhance cell viability and significantly improved resistance to heat, high salinity and drought stress conditions. The results presented here may provide new insights into the function of OsHSP90 family in rice.

Molecular characterization, expression pattern and function analysis of the rice OsDUF866 family

ABSTRACT Domain-of-unknown function (DUF) proteins represent a number of gene families with no functional annotation in the Pfam database. So far, the DUF866 family has not been characterized, and no member has been functionally studied. In this study, we uncovered and analysed four OsDUF866 (OsDUF866.1–OsDUF866.4) family members in rice Nipponbare, in which three distinct motifs were identified. The expression of OsDUF866 family in nine tissues was examined by real-time polymerase chain reaction (PCR), and the highest expression of four genes members was found in embryos at 14 days after flowering. We performed real-time PCR to examine the expression of OsDUF866 family under abiotic stress and abscisic acid (ABA) treatment conditions. The expression level of OsDUF866.1 displayed significant decrease under drought and salt conditions, while significant increase under heat conditions. The expression level of OsDUF866.2 displayed significant decrease under drought conditions. The expression level of OsDUF866.3 was significantly elevated under drought and cold, while lowered under heat conditions. The expression level of OsDUF866.4 was increased under cold and heat conditions, while decreased under drought conditions. Interestingly, the expression level of OsDUF866 members was approximately constant under ABA treatment conditions. Overexpression of OsDUF866.1 in Escherichia coli could enhance cell viability and significantly improve the resistance to heat stress conditions. The results may provide new insights into the function of OsDUF866 family in rice.

A single nucleotide mutation of IspF gene involved in the MEP pathway for isoprenoid biosynthesis causes yellow-green leaf phenotype in rice

Key messageWe identified IspF gene through yellow-green leaf mutant 505ys in rice. OsIspF was expressed in all tissues detected, and its encoded protein was targeted to the chloroplast. On expression levels of genes in this mutant, OsIspF itself and the genes encoding other enzymes of the MEP pathway and chlorophyll synthase were all up-regulated, however, among eight genes associated with photosynthesis, only psaA, psaN and psbA genes for three reaction center subunits of photosystem obviously changed.AbstractIsoprenoids are the most abundant natural compounds in all organisms, which originate from the basic five-carbon units isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In plants, IPP and DMAPP are synthesized through two independent pathways, the mevalonic acid pathway in cytoplasm and the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in plastids. The MEP pathway comprises seven enzymatic steps, in which IspF is the fifth enzyme. So far, no IspF gene has been identified in monocotyledonous plants. In this study, we isolated a leaf-color mutant, 505ys, in rice (Oryza sativa). The mutant displayed yellow-green leaf phenotype, reduced level of photosynthetic pigments, and arrested development of chloroplasts. By map-based cloning of this mutant, we identified OsIspF gene (LOC_Os02g45660) showing significant similarity to IspF gene of Arabidopsis, in which a missense mutation occurred in the mutant, resulting in an amino acid change in the encoded protein. OsIspF gene was expressed in all tissues detected, and its encoded protein was targeted to the chloroplast. Further, the mutant phenotype of 505ys was complemented by transformation with the wild-type OsIspF gene. Therefore, we successfully identified an IspF gene in monocotyledonous plants. In addition, real-time quantitative RT-PCR implied that a positive regulation could exist between the OsIspF gene and the genes encoding other enzymes of the MEP pathway and chlorophyll synthase. At the same time, it also implied that the individual genes involved in the MEP pathway might differentially regulated expression levels of the genes associated with photosynthesis.

Molecular characterization and function analysis of the rice OsDUF829 family

ABSTRACT The domain of unknown function DUF946 family consists of plant proteins with an average length of around 239 residues that have no characterized function. The gene family needs deeper characterization and functional analysis of its members. In this study, we report five OsDUF946 (OsDUF946.1–OsDUF946.5) family members in rice Nipponbare with three distinct motifs. All the OsDUF946 proteins were classified into three major groups (I, II, III) by phylogenetic analysis. Real-time polymerase chain reaction showed that the expression patterns of the five OsDUF946 family members were different in 15 different rice tissues as well as under various stress conditions and abscisic acid treatment. The expression of OsDUF946.1 was significantly downregulated under salt, cold and heat stress, while the expression of OsDUF946.4 and OsDUF946.5 was significantly upregulated under drought and salt conditions. Overexpression of OsDUF946.4 in Escherichia coli significantly improved the resistance to salt and drought, while overexpression of OsDUF946.5 in E. coli did not have such an effect. The obtained results provide a starting point for further research into the function of the OsDUF946 family in rice.

The Rice OsDUF810 Family: OsDUF810.7 May be Involved in the Tolerance to Salt and Drought

With the advance of sequencing technology, the number of sequenced plant genomes has been rapidly increasing. However, understanding of the gene function in these sequenced genomes lags far behind; as a result, many coding plant sequences in public databases are annotated as proteins with domains of unknown function (DUF). Function of a protein family DUF810 in rice is not known. In this study, we analysed seven members of OsDU810 (OsDUF810.1–OsDUF810.7) family with three distinct motifs in rice Nipponbare. By phylogenetic analysis, OsDUF810 proteins fall into three major groups (I, II, III). Expression patterns of the seven corresponding OsDUF810 protein-encoding genes in 15 different rice tissues vary. Under drought, salt, cold and heat stress conditions and ABA treatment, the expression of OsDUF810.7 significantly increases. Overexpression of this protein in E. coli lead to a significant enhancement of catalase (CAT) and peroxidase (POD) activities, and improved bacterial resistance to salt and drought.