Lizhong Xiong

Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice

Drought and salinity are major abiotic stresses to crop production. Here, we show that overexpression of stress responsive gene SNAC1 (STRESS-RESPONSIVE NAC 1) significantly enhances drought resistance in transgenic rice (22–34% higher seed setting than control) in the field under severe drought stress conditions at the reproductive stage while showing no phenotypic changes or yield penalty. The transgenic rice also shows significantly improved drought resistance and salt tolerance at the vegetative stage. Compared with WT, the transgenic rice are more sensitive to abscisic acid and lose water more slowly by closing more stomatal pores, yet display no significant difference in the rate of photosynthesis. SNAC1 is induced predominantly in guard cells by drought and encodes a NAM, ATAF, and CUC (NAC) transcription factor with transactivation activity. DNA chip analysis revealed that a large number of stress-related genes were up-regulated in the SNAC1-overexpressing rice plants. Our data suggest that SNAC1 holds promising utility in improving drought and salinity tolerance in rice.

Patterns of cytosine methylation in an elite rice hybrid and its parental lines, detected by a methylation-sensitive amplification polymorphism technique.

Abstract DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we assessed the extent and pattern of cytosine methylation in the rice genome, using the technique of methylation-sensitive amplified polymorphism (MSAP), which is a modification of the amplified fragment length polymorphism (AFLP) method that makes use of the differential sensitivity of a pair of isoschizomers to cytosine methylation. The tissues assayed included seedlings and flag leaves of an elite rice hybrid, Shanyou 63, and the parental lines Zhenshan 97 and Minghui 63. In all, 1076 fragments, each representing a recognition site cleaved by either or both of the isoschizomers, were amplified using 16 pairs of selective primers. A total of 195 sites were found to be methylated at cytosines in one or both parents, and the two parents showed approximately the same overall degree of methylation (16.3%), as revealed by the incidence of differential digestion by the isoschizomers. Four classes of patterns were identified in a comparative assay of cytosine methylation in the parents and hybrid; increased methylation was detected in the hybrid compared to the parents at some of the recognition sites, while decreased methylation in the hybrid was detected at other sites. A small proportion of the sites was found to be differentially methylated in seedlings and flag leaves; DNA from young seedlings was methylated to a greater extent than that from flag leaves. Almost all of the methylation patterns detected by MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrate that the MSAP technique is highly efficient for large-scale detection of cytosine methylation in the rice genome. We believe that the technique can be adapted for use in other plant species.

Characterization of OsbZIP23 as a Key Player of the Basic Leucine Zipper Transcription Factor Family for Conferring Abscisic Acid Sensitivity and Salinity and Drought Tolerance in Rice

OsbZIP23 is a member of the basic leucine zipper (bZIP) transcription factor family in rice (Oryza sativa). Expression of OsbZIP23 is strongly induced by a wide spectrum of stresses, including drought, salt, abscisic acid (ABA), and polyethylene glycol treatments, while other stress-responsive genes of this family are slightly induced only by one or two of the stresses. Transactivation assay in yeast demonstrated that OsbZIP23 functions as a transcriptional activator, and the sequences at the N terminus (amino acids 1–59) and a region close to the C terminus (amino acids 210–240) are required for the transactivation activity. Transient expression of OsbZIP23-green fluorescent protein in onion (Allium cepa) cells revealed a nuclear localization of the protein. Transgenic rice overexpressing OsbZIP23 showed significantly improved tolerance to drought and high-salinity stresses and sensitivity to ABA. On the other hand, a null mutant of this gene showed significantly decreased sensitivity to a high concentration of ABA and decreased tolerance to high-salinity and drought stress, and this phenotype can be complemented by transforming the OsbZIP23 back into the mutant. GeneChip and real-time polymerase chain reaction analyses revealed that hundreds of genes were up- or down-regulated in the rice plants overexpressing OsbZIP23. More than half of these genes have been annotated or evidenced for their diverse functions in stress response or tolerance. In addition, more than 30 genes that are possible OsbZIP23-specific target genes were identified based on the comparison of the expression profiles in the overexpressor and the mutant of OsbZIP23. Collectively, these results indicate that OsbZIP23 functions as a transcriptional regulator that can regulate the expression of a wide spectrum of stress-related genes in response to abiotic stresses through an ABA-dependent regulation pathway. We propose that OsbZIP23 is a major player of the bZIP family in rice for conferring ABA-dependent drought and salinity tolerance and has high potential usefulness in genetic improvement of stress tolerance.

Genomic Organization, Differential Expression, and Interaction of SQUAMOSA Promoter-Binding-Like Transcription Factors and microRNA156 in Rice

Transcription factors play essential roles in the developmental processes of plants. Many such factors are regulated by microRNAs (miRNAs). SQUAMOSA (SQUA) promoter-binding-like (SPL) genes encode plant-specific transcription factors, some of which contain complementary sequences of miRNA156. In this study, 19 rice (Oryza sativa) SPL (OsSPL) genes and 12 rice miRNA156 (OsmiR156) precursors were identified in the rice genome. Sequence and experimental analysis suggested that 11 OsSPL genes were putative targets of OsmiR156. Plant SPL proteins were classified into six subgroups based on the phylogenetic analysis of SQUA promoter-binding protein domain. Diverse exon-intron structures and distinct organizations of putative motifs beyond the SQUA promoter-binding protein domains were identified in the OsSPL gene family. Transcript level analysis of OsSPL genes in various rice tissues and organs revealed different tempospatial expression patterns. More than half of the OsSPL genes including most OsmiR156-targeted genes are predominantly expressed in the young panicles, whereas OsmiR156 genes are predominantly expressed in the young shoots and leaves of rice. Overexpression of two OsmiR156 genes (OsmiR156b and OsmiR156h) in rice resulted in severe dwarfism, strongly reduced panicle size, and delayed flowering, suggesting that OsmiR156 and OsSPL target genes are involved in various developmental processes, especially the flower development of rice. Different patterns of transcript changes (decreased or unchanged) of different target genes in same tissue and of same target gene in different tissues detected in the OsmiR156-overexpressing plants suggested diverse interactions between OsmiR156 and OsSPL target genes in a tissue-specific manner.

Over-expression of a LEA gene in rice improves drought resistance under the field conditions

Late embryogenesis abundant (LEA) proteins have been implicated in many stress responses of plants. In this report, a LEA protein gene OsLEA3-1 was identified and over-expressed in rice to test the drought resistance of transgenic lines under the field conditions. OsLEA3-1 is induced by drought, salt and abscisic acid (ABA), but not by cold stress. The promoter of OsLEA3-1 isolated from the upland rice IRAT109 exhibits strong activity under drought- and salt-stress conditions. Three expression constructs consisting of the full-length cDNA driven by the drought-inducible promoter of OsLEA3-1 (OsLEA3-H), the CaMV 35S promoter (OsLEA3-S), and the rice Actin1 promoter (OsLEA3-A) were transformed into the drought-sensitive japonica rice Zhonghua 11. Drought resistance pre-screening of T1 families at anthesis stage revealed that the over-expressing families with OsLEA3-S and OsLEA3-H constructs had significantly higher relative yield (yield under drought stress treatment/yield under normal growth conditions) than the wild type under drought stress conditions, although a yield penalty existed in T1 families under normal growth conditions. Nine homozygous families, exhibiting over-expression of a single-copy of the transgene and relatively low yield penalty in the T1 generation, were tested in the field for drought resistance in the T2 and T3 generations and in the PVC pipes for drought tolerance in the T2 generation. Except for two families (transformed with OsLEA3-A), all the other families (transformed with OsLEA3-S and OsLEA3-H constructs) had higher grain yield than the wild type under drought stress in both the field and the PVC pipes conditions. No significant yield penalty was detected for these T2 and T3 families. These results indicate that transgenic rice with significantly enhanced drought resistance and without yield penalty can be generated by over-expressing OsLEA3-1 gene with appropriate promoters and following a bipartite (stress and non-stress) in-field screening protocol.

Systematic sequence analysis and identification of tissue-specific or stress-responsive genes of NAC transcription factor family in rice

NAM, ATAF, and CUC (NAC) transcription factors comprise a large plant-specific gene family and a few members of this family have been characterized for their roles in plant growth, development, and stress tolerance. In this study, systematic sequence analysis revealed 140 putative NAC or NAC-like genes (ONAC) in rice. Phylogenetic analysis suggested that NAC family can be divided into five groups (I–V). Among them, all the published development-related genes fell into group I, and all the published stress-related NAC genes fell into the group III (namely stress-responsive NAC genes, SNAC). Distinct compositions of the putative motifs were revealed on the basis of NAC protein sequences in rice. Most members contained a complete NAC DNA-binding domain and a variable transcriptional regulation domain. Sequence analysis, together with the organization of putative motifs, indicated distinct structures and potential diverse functions of NAC family in rice. Yeast one-hybrid analysis confirmed that 12 NAC proteins representing different motif compositions can bind the NAC core DNA-binding site. Real-time polymerase chain reaction (PCR) analysis revealed 12 genes with different tissue-specific (such as callus, root, stamen, or immature endosperm) expression patterns, suggesting that these genes may play crucial regulatory roles during growth and development of rice. The expression levels of this family were also checked under various abiotic stresses including drought, salinity, and low temperature. A preliminary check based on our microarray data suggested that more than 40 genes of this family were responsive to drought and/or salt stresses. Among them, 20 genes were further investigated for their stress responsiveness in detail by real-time PCR analysis. Most of these stress-responsive genes belonged to the group III (SNAC). Considering the fact that a very limited number of genes of the NAC family have been characterized, our data provide a very useful reference for functional analysis of this family in rice.

Genetic Basis of Drought Resistance at Reproductive Stage in Rice: Separation of Drought Tolerance From Drought Avoidance

Drought tolerance (DT) and drought avoidance (DA) are two major mechanisms in drought resistance of higher plants. In this study, the genetic bases of DT and DA at reproductive stage in rice were analyzed using a recombinant inbred line population from a cross between an indica lowland and a tropical japonica upland cultivar. The plants were grown individually in PVC pipes and two cycles of drought stress were applied to individual plants with unstressed plants as the control. A total of 21 traits measuring fitness, yield, and the root system were investigated. Little correlation of relative yield traits with potential yield, plant size, and root traits was detected, suggesting that DT and DA were well separated in the experiment. A genetic linkage map consisting of 245 SSR markers was constructed for mapping QTL for these traits. A total of 27 QTL were resolved for 7 traits of relative performance of fitness and yield, 36 QTL for 5 root traits under control, and 38 for 7 root traits under drought stress conditions, suggesting the complexity of the genetic bases of both DT and DA. Only a small portion of QTL for fitness- and yield-related traits overlapped with QTL for root traits, indicating that DT and DA had distinct genetic bases.

Characterization of Stress-Responsive CIPK Genes in Rice for Stress Tolerance Improvement

Plants respond to adverse environments by initiating a series of signaling processes that often involves diverse protein kinases, including calcineurin B-like protein-interacting protein kinases (CIPKs). In this study, putative CIPK genes (OsCIPK01–OsCIPK30) in the rice (Oryza sativa) genome were surveyed for their transcriptional responses to various abiotic stresses. The results showed that 20 OsCIPK genes were differentially induced by at least one of the stresses, including drought, salinity, cold, polyethylene glycol, and abscisic acid treatment. Most of the genes induced by drought or salt stress were also induced by abscisic acid treatment but not by cold. A few CIPK genes containing none of the reported stress-responsive cis-elements in their promoter regions were also induced by multiple stresses. To prove that some of these stress-responsive OsCIPK genes are potentially useful for stress-tolerance improvement, three CIPK genes (OsCIPK03, OsCIPK12, and OsCIPK15) were overexpressed in japonica rice ‘Zhonghua 11’. Transgenic plants overexpressing the transgenes OsCIPK03, OsCIPK12, and OsCIPK15 showed significantly improved tolerance to cold, drought, and salt stress, respectively. Under cold and drought stresses, OsCIPK03- and OsCIPK12-overexpressing transgenic plants accumulated significantly higher contents of proline and soluble sugars than the wild type. Putative proline synthetase and transporter genes had significantly higher expression level in the transgenic plants than in the wild type. The differentially induced expression of OsCIPK genes by different stresses and the examples of improved stress tolerance of the OsCIPK transgenic rice suggest that rice CIPK genes have diverse roles in different stress responses and some of them may possess potential usefulness in stress-tolerance improvement of rice.

Identification of genetic factors controlling domestication-related traits of rice using an F2 population of a cross between Oryza sativa and O. rufipogon

Abstract Domesticated rice differs from the wild progenitor in large arrays of morphological and physiological traits. The present study was conducted to identify the genetic factors controlling the differences between cultivated rice and its wild progenitor, with the intention to assess the genetic basis of the changes associated with the processes of rice domestication. A total of 19 traits, including seven qualitative and 12 quantitative traits, that are related to domestication were scored in an F2 population from a cross between a variety of the Asian cultivated rice (Oryza sativa) and an accession of the common wild rice (O. rufipogon). Loci controlling the inheritance of these traits were determined by making use of a molecular linkage map consisting of 348 molecular-marker loci (313 RFLPs, 12 SSRs and 23 AFLPs) based on this F2 population. All seven qualitative traits were each controlled by a single Mendelian locus. Analysis of the 12 quantitative traits resolved a total of 44 putative QTLs with an average of 3.7 QTLs per trait. The amount of variation explained by individual QTLs ranged from a low of 6.9% to a high of 59.8%, and many of the QTLs accounted for more than 20% of the variation. Thus, genes of both major and minor effect were involved in the differences between wild and cultivated rice. The results also showed that most of the genetic factors (qualitative or QTLs) controlling the domestication-related traits were concentrated in a few chromosomal blocks. Such a clustered distribution of the genes may provide explanations for the genetic basis of the “domestication syndrome” observed in evolutionary studies and also for the “linkage drag” that occurs in many breeding programs. The information on the genetic basis of some desirable traits possessed by the wild parent may also be useful for facilitating the utilization of these traits in rice-breeding programs.

A Raf-Like MAPKKK Gene DSM1 Mediates Drought Resistance through Reactive Oxygen Species Scavenging in Rice

Mitogen-activated protein kinase (MAPK) cascades have been identified in various signaling pathways involved in plant development and stress responses. We identified a drought-hypersensitive mutant (drought-hypersensitive mutant1 [dsm1]) of a putative MAPK kinase kinase (MAPKKK) gene in rice (Oryza sativa). Two allelic dsm1 mutants were more sensitive than wild-type plants to drought stress at both seedling and panicle development stages. The dsm1 mutants lost water more rapidly than wild-type plants under drought stress, which was in agreement with the increased drought-sensitivity phenotype of the mutant plants. DSM1-RNA interference lines were also hypersensitive to drought stress. The predicted DSM1 protein belongs to a B3 subgroup of plant Raf-like MAPKKKs and was localized in the nucleus. By real-time PCR analysis, the DSM1 gene was induced by salt, drought, and abscisic acid, but not by cold. Microarray analysis revealed that two peroxidase (POX) genes, POX22.3 and POX8.1, were sharply down-regulated compared to wild type, suggesting that DSM1 may be involved in reactive oxygen species (ROS) signaling. Peroxidase activity, electrolyte leakage, chlorophyll content, and 3,3′-diaminobenzidine staining revealed that the dsm1 mutant was more sensitive to oxidative stress due to an increase in ROS damage caused by the reduced POX activity. Overexpression of DSM1 in rice increased the tolerance to dehydration stress at the seedling stage. Together, these results suggest that DSM1 might be a novel MAPKKK functioning as an early signaling component in regulating responses to drought stress by regulating scavenging of ROS in rice.