Yongmei Bao

Overexpression of a TFIIIA-type zinc finger protein gene ZFP252 enhances drought and salt tolerance in rice (Oryza sativa L.).

We previously identified a salt and drought stress‐responsive TFIIIA‐type zinc finger protein gene ZFP252 from rice. Here we report the functional analysis of ZFP252 using gain‐ and loss‐of‐function strategies. We found that overexpression of ZFP252 in rice increased the amount of free proline and soluble sugars, elevated the expression of stress defense genes and enhanced rice tolerance to salt and drought stresses, as compared with ZFP252 antisense and non‐transgenic plants. Our findings suggest that ZFP252 plays an important role in rice response to salt and drought stresses and is useful in engineering crop plants with enhanced tolerance to salt and drought stresses.

Functional analysis of a novel Cys2/His2-type zinc finger protein involved in salt tolerance in rice

The Cys2/His2-type zinc finger proteins have been implicated in different cellular processes involved in plant development and stress responses. Through microarray analysis, a salt-responsive zinc finger protein gene ZFP179 was identified and subsequently cloned from rice seedlings. ZFP179 encodes a 17.95 kDa protein with two C2H2-type zinc finger motifs having transcriptional activation activity. The real-time RT-PCR analysis showed that ZFP179 was highly expressed in immature spikes, and markedly induced in the seedlings by NaCl, PEG 6000, and ABA treatments. Overexpression of ZFP179 in rice increased salt tolerance and the transgenic seedlings showed hypersensitivity to exogenous ABA. The increased levels of free proline and soluble sugars were observed in transgenic plants compared to wild-type plants under salt stress. The ZFP179 transgenic rice exhibited significantly increased tolerance to oxidative stress, the reactive oxygen species (ROS)-scavenging ability, and expression levels of a number of stress-related genes, including OsDREB2A, OsP5CS OsProT, and OsLea3 under salt stress. Our studies suggest that ZFP179 plays a crucial role in the plant response to salt stress, and is useful in developing transgenic crops with enhanced tolerance to salt stress.

Increased tolerance of rice to cold, drought and oxidative stresses mediated by the overexpression of a gene that encodes the zinc finger protein ZFP245.

ZFP245 is a cold- and drought-responsive gene that encodes a zinc finger protein in rice. The ZFP245 protein localizes in the nucleus and exhibits trans-activation activity. Transgenic rice plants overexpressing ZFP245 were generated and found to display high tolerance to cold and drought stresses. The transgenic plants did not exhibit growth retardation, but showed growth sensitivity against exogenous abscisic acid, increased free proline levels and elevated expression of rice pyrroline-5-carboxylatesynthetase and proline transporter genes under stress conditions. Overproduction of ZFP245 enhanced the activities of reactive oxygen species-scavenging enzymes under stress conditions and increased the tolerance of rice seedlings to oxidative stress. Our data suggest that ZFP245 may contribute to the tolerance of rice plants to cold and drought stresses by regulating proline levels and reactive oxygen species-scavenging activities, and therefore may be useful for developing transgenic crops with enhanced tolerance to abiotic stress.

Expression analysis of rice A20/AN1-type zinc finger genes and characterization of ZFP177 that contributes to temperature stress tolerance

The A20/AN1-type zinc finger protein family is conserved in animals and plants. Using human AWP1 protein as a query, we identified twelve A20/AN1-type zinc finger proteins in japonica rice. Most of these genes were constitutively expressed in leaves, roots, culms and spikes. Through microarray analysis, it was found that four genes (ZFP177, ZFP181, ZFP176, ZFP173), two genes (ZFP181 and ZFP176) and one gene (ZFP157) were significantly induced by cold, drought and H(2)O(2) treatments, respectively. Further expression analysis showed that ZFP177 was responsive to both cold and heat stresses, but down-regulated by salt. The subcellular localization assay indicated that ZFP177 was localized in cytoplasm in tobacco leaf and root cells. Yeast-one hybrid assay showed that ZFP177 lacked trans-activation potential in yeast cells. Overexpression of ZFP177 in tobacco conferred tolerance of transgenic plants to both low and high temperature stresses, but increased sensitivity to salt and drought stresses. Further we found expression levels of some stress-related genes were inhibited in ZFP177 transgenic plants. These results suggested that ZFP177 might play crucial but differential roles in plant responses to various abiotic stresses.

QTL Analysis of Na+ and K+ Concentrations in Roots and Shoots under Different Levels of NaCl Stress in Rice (Oryza sativa L.)

The key to plant survival under NaCl salt stress is maintaining a low Na+ level or Na+/K+ ratio in the cells. A population of recombinant inbred lines (RILs, F2∶9) derived from a cross between the salt-tolerant japonica rice variety Jiucaiqing and the salt-sensitive indica variety IR26, was used to determine Na+ and K+ concentrations in the roots and shoots under three different NaCl stress conditions (0, 100 and 120 mM NaCl). A total of nine additive QTLs were identified by QTL Cartographer program using single-environment phenotypic values, whereas eight additive QTLs were identified by QTL IciMapping program. Among these additive QTLs, five were identified by both programs. Epistatic QTLs and QTL-by-environment interactions were detected by QTLNetwork program in the joint analyses of multi-environment phenotypic values, and one additive QTL and nine epistatic QTLs were identified. There were three epistatic QTLs identified for Na+ in roots (RNC), three additive QTLs and two epistatic QTLs identified for Na+ in shoots (SNC), four additive QTLs identified for K+ in roots (RKC), four additive QTLs and three epistatic QTLs identified for K+ in shoots (SKC) and one additive QTL and one epistatic QTL for salt tolerance rating (STR). The phenotypic variation explained by each additive, epistatic QTL and QTL×environment interaction ranged from 8.5 to 18.9%, 0.5 to 5.3% and 0.7 to 7.5%, respectively. By comparing the chromosomal positions of these additive QTLs with those previously identified, five additive QTLs, qSNC9, qSKC1, qSKC9, qRKC4 and qSTR7, might represent novel salt tolerance loci. The identification of salt tolerance in selected RILs showed that a major QTL qSNC11 played a significant role in rice salt tolerance, and could be used to improve salt tolerance of commercial rice varieties with marker-assisted selection (MAS) approach.

A TFIIIA-type zinc finger protein confers multiple abiotic stress tolerances in transgenic rice (Oryza sativa L.).

The TFIIIA-type zinc finger transcription factors are involved in plant development and abiotic stress responses. Most TFIIIA-type zinc finger proteins are transcription repressors due to existence of an EAR-motif in their amino acid sequences. In this work, we found that ZFP182, a TFIIIA-type zinc finger protein, forms a homodimer in the nucleus and exhibits trans-activation activity in yeast cells. The deletion analysis indicated that a Leu-rich region at C-terminus is required for the trans-activation. Overexpression of ZFP182 significantly enhanced multiple abiotic stress tolerances, including salt, cold and drought tolerances in transgenic rice. Overexpression of ZFP182 promotes accumulation of compatible osmolytes, such as free proline and soluble sugars, in transgenic rice. ZFP182 activates the expression of OsP5CS encoding pyrroline-5-carboxylate synthetase and OsLEA3 under stress conditions, while OsDREB1A and OsDREB1B were regulated by ZFP182 under both normal and stress conditions. Interestingly, site-directed mutagenesis assay showed that DRE-like elements in ZFP182 promoter are involved in dehydration-induced expression of ZFP182. The yeast two-hybrid assay revealed that ZFP182 interacted with several ribosomal proteins including ZIURP1, an ubiquitin fused to ribosomal protein L40. The in vivo and in vitro interactions of ZFP182 and ZIURP1 were further confirmed by bimolecular fluorescence complementation and His pull-down assays. Our studies provide new clues in the understanding of the mechanisms for TFIIIA-type zinc finger transcription factor mediated stress tolerance and a candidate gene for improving stress tolerance in crops.

Quantitative trait loci analysis for rice seed vigor during the germination stage

Seed vigor is an important characteristic of seed quality, and rice cultivars with strong seed vigor are desirable in direct-sowing rice production for optimum stand establishment. In the present study, the quantitative trait loci (QTLs) of three traits for rice seed vigor during the germination stage, including germination rate, final germination percentage, and germination index, were investigated using one recombinant inbred line (RIL) population derived from a cross between japonica Daguandao and indica IR28, and using the multiple interval mapping (MIM) approach. The results show that indica rice presented stronger seed vigor during the germination stage than japonica rice. A total of ten QTLs, and at least five novel alleles, were detected to control rice seed vigor, and the amount of variation (R2) explained by an individual QTL ranged from 7.5% to 68.5%, with three major QTLs with R2>20%. Most of the QTLs detected here are likely to coincide with QTLs for seed weight, seed size, or seed dormancy, suggesting that the rice seed vigor might be correlated with seed weight, seed size, and seed dormancy. At least five QTLs are novel alleles with no previous reports of seed vigor genes in rice, and those major or minor QTLs could be used to significantly improve the seed vigor by marker-assisted selection (MAS) in rice.

SRWD: a novel WD40 protein subfamily regulated by salt stress in rice (OryzasativaL.).

By analysis with microarray data, we found that a gene encoding a novel protein containing five WD40 repeats, was regulated by salt stress in rice and named as SRWD1 (Salt responsive WD40 protein 1). By database searching, additional four SRWD1-like genes (SRWD2-SRWD5) were found in rice genome, and these five SRWD genes formed a novel WD40 subfamily. Phylogenetic analysis showed that plant SRWD proteins divided into four groups. The significant functional divergences during SRWD evolution were found. The tissue-specific and salt responsive expression profiling for SRWD genes was investigated based on microarray data. It was found that all five SRWD genes in rice were regulated by salt stress. Further, we found that SRWD1 was regulated with different patterns by salt stress in two rice cultivars responding differently to salt stress. Our study correlates WD40 proteins with salt stress in plants and provides fundamental information for the further investigation of plant SRWD proteins.

Genetic Control of Germination Ability under Cold Stress in Rice

An F9 recombinant inbred lines (RIL) population, derived from a cross between IR28 (Oryza sativa L. spp. indica) and Daguandao (O. sativa L. spp. japonica), was used to construct a molecular linkage map and to identify germination ability including the traits of imbibition rate, germination rate, germination index, root length, shoot length and seed vigor at 14°C for 23 d. A composite interval mapping approach was applied to conduct genetic analysis for germination ability. The frequency distributions of the germination ability traits under the cold stress in the RIL population showed continuous segregation, suggesting they were quantitative traits controlled by several genes. A total of seven QTLs were identified on chromosomes 4, 6 and 9, including two for imbibition rate (qIR-6, qIR-9), one for germination rate (qGR-4), two for germination index (qGI-4-1, qGI-4-2) and two for root length (qRL-4-1, qRL-4-2). There were no detected QTLs controlling shoot length and seed vigor. The phenotypic variance explained by a single QTL ranged from 9.1% to 37.0%, and two major QTLs, qIR-6 and qGI-4-2, accounted for over 30% of the phenotypic variance. The expressions of QTLs were developmentally regulated and growth stage-specific. Most of the QTLs observed here were located in the regions similar to the QTLs for rice cold tolerance reported previously, indicating that these QTLs were reliable. However, qRL-4-2 is not reported before.

Cloning and characterization of three genes encoding Qb-SNARE proteins in rice

Qb-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and function as important components of the vesicle trafficking machinery in eukaryotic cells. Here, we report three novel plant SNARE (NPSN) genes isolated from rice and named OsNPSN11, OsNPSN12 and OsNPSN13. They have about 70% nucleotide identity over their entire coding regions and similar genomic organization with ten exons and nine introns in each gene. Multiple alignment of deduced amino acid sequences indicate that the OsNPSNs proteins are homologous to AtNPSNs from Arabidopsis, containing a Qb-SNARE domain and a membrane-spanning domain in the C-terminal region. Semi-quantitative RT-PCR assays showed that the OsNPSNs were ubiquitously and differentially expressed in roots, culms, leaves, immature spikes and flowering spikes. The expression of OsNPSNs was significantly activated in rice seedlings treated with H2O2, but down-regulated under NaCl and PEG6000 stresses. Transient expression method in onion epidermal cells revealed that OsNPSNs were located in the plasma membrane. Transformed yeast cells with OsNPSNs had better growth rates than empty-vector transformants when cultured on either solid or liquid selective media containing various concentrations of H2O2, but more sensitive to NaCl and mannitol stresses. The 35S:OsNPSN11 transgenic tobacco also showed more tolerance to H2O2 and sensitivity to NaCl and mannitol than non-transgenic tobacco. These results indicate that OsNPSNs may be involved in different aspects of the signal transduction in plant and yeast responses to abiotic stresses.