Jianqing Zhu

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.

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 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.

Fine mapping and candidate gene analysis of the dwarf gene d162(t) in rice (Oryza sativa L.)

In our previous study, d162(t), a single recessive gene, which caused rice dwarf mutant, had been mapped on the short arm of chromosome 3. In this study, the d162(t) gene was fine mapped to a confined region about 0.82 cM by RM14641 and RM3134, and co-segregated with InDel361-2, InDel361-3, InDel361-5, RM14645, RM1022 and RM14643, where no known gene involved in plant height has been identified. Based on the annotation results of TIGR, dozens of open reading frames (ORFs) were predicted in this region, among them, five ORFs were the most possible genes related to the phenotype. In these ORFs, Os03g13010, related to U-box domain containing protein, had a 62bp segment deletion in the coding region in 162d (mutant type, MT). The results of RT-PCR showed that the transcriptional level of Os03g13010 was significantly different between Shuhui162 (wild type, WT) and 162d (MT). Therefore, the gene (Os03g13010) encoding a U-box domain containing protein was considered as the candidate gene of d162(t).

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.

Genome-wide identification and analyses of the rice OsDUF936 family

ABSTRACT The domain of unknown function DUF936 family consists of proteins with an average length of around 328 residues that have no characterized function in rice. In this study, we report nine OsDUF936 (OsDUF936.1–OsDUF936.9) family members in rice Nipponbare with three distinct motifs. All the OsDUF936 proteins were classified into four major groups (I, II, III and IV) by phylogenetic analysis. The expression level of OsDUF936.3, OsDUF936.5 and OsDUF936.6 displayed significant increase under salt stress conditions. Overexpression of OsDUF936.6 in Escherichia coli significantly improved the tolerance to salt, while overexpression of OsDUF936.3 and OsDUF936.5 in E. coli did not have such an effect. The results presented here would be helpful for further study on the function of the OsDUF936 family in rice.

Overexpression of a thylakoid membrane protein gene OsTMP14 improves indica rice cold tolerance

ABSTRACT Cold has a major impact on rice, so rice can only grow in specific climate zones. TMP14 (thylakoid membrane protein of 14 kDa) is a nuclear genome-encoded chloroplast thylakoid membrane protein, which is involved in a wide range of abiotic stress responses. However, the knowledge of TMP14 roles in response to adverse stimuli is still very limited in rice. Based on the results of expression profiles, OsTMP14 (GenBank accession: BAF27084.1) was highly induced in both leaf and panicle at all the developmental stages of rice analyses under cold and drought stresses. Sequence analyses of OsTMP14 putative promoter regions identified nine cis-elements related to stress responses. Furthermore, transgenic rice plants with overexpression of OsTMP14 showed more tolerance to cold stress. Taken together, these results indicate that OsTMP14 is involved in stress tolerance in rice, which is useful in developing transgenic rice with enhancing tolerance to cold stress.