Mutation in CSA creates a new photoperiod-sensitive genic male sterile line applicable for hybrid rice seed production.

Rice is a major staple food worldwide. Making hybrid rice has proved to be an effective strategy to significantly increase grain yield. Current hybrid rice technologies rely on male sterile lines and have been used predominantly in indica cultivars. However, intrinsic problems exist in the implementation of these technologies, such as limited germplasms and unpredictable conversions from sterility to fertility in the field. Here, we describe a photoperiod-controlled male sterile line, carbon starved anther (csa), which contains a mutation in an R2R3 MYB transcription regulator of pollen development. This mutation was introduced into indica and japonica rice, and it rendered male sterility under short-day conditions and male fertility under long-day conditions in both lines. Furthermore, F(1) plants of csa and a restorer line JP69 exhibited heterosis (hybrid vigor), suggesting the feasibility of using this mutation to create hybrid rice. The csa-based photoperiod-sensitive male sterile line allows the establishment of a stable two-line hybrid system, which promises to have a significant impact on agriculture.

The putative auxin efflux carrier OsPIN3t is involved in the drought stress response and drought tolerance.

The phytohormone auxin plays a critical role in plant growth and development, and its spatial distribution largely depends on the polar localization of the PIN-FORMED (PIN) auxin efflux carrier family members. In this study, we identify a putative auxin efflux carrier gene in rice, OsPIN3t, which acts in auxin polar transport but is also involved in the drought stress response in rice. We show that OsPIN3t-GFP fusion proteins are localized in plasma membranes, and this subcellular localization changes under 1-N-naphthylphthalamic acid (NPA) treatment. The tissue-specific expression patterns of OsPIN3t were also investigated using a ß-glucuronidase (GUS) reporter, which showed that OsPIN3t was mainly expressed in vascular tissue. The GUS activity in OsPIN3tpro::GUS plants increased by NAA treatment and decreased by NPA treatment. Moreover, knockdown of OsPIN3t caused crown root abnormalities in the seedling stage that could be phenocopied by treatment of wild-type plants with NPA, which indicated that OsPIN3t is involved in the control of polar auxin transport. Overexpression of OsPIN3t led to improved drought tolerance, and GUS activity significantly increased when OsPIN3tpro::GUS plants were subjected to 20% polyethylene glycol stress. Taken together, these results suggest that OsPIN3t is involved in auxin transport and the drought stress response, which suggests that a polar auxin transport pathway is involved in the regulation of the response to water stress in plants.

Characterization and fine mapping of a novel rice narrow leaf mutant nal9.

A narrow leaf mutant was isolated from transgenic rice (Oryza sativa L.) lines carrying a T-DNA insertion. The mutant is characterized by narrow leaves during its whole growth period, and was named nal9 (narrow leaf 9). The mutant also has other phenotypes, such as light green leaves at the seedling stage, reduced plant height, a small panicle and increased tillering. Genetic analysis revealed that the mutation is controlled by a single recessive gene. A hygromycin resistance assay showed that the mutation was not caused by T-DNA insertion, so a map-based cloning strategy was employed to isolate the nal9 gene. The mutant individuals from the F2 generations of a cross between the nal9 mutant and Longtepu were used for mapping. With 24 F2 mutants, the nal9 gene was preliminarily mapped near the marker RM156 on the chromosome 3. New INDEL markers were then designed based on the sequence differences between japonica and indica at the region near RM156. The nal9 gene was finally located in a 69.3 kb region between the markers V239B and V239G within BAC OJ1212_C05 by chromosome walking. Sequence and expression analysis showed that an ATP-dependent Clp protease proteolytic subunit gene (ClpP) was most likely to be the nal9 gene. Furthermore, the nal9 mutation was rescued by transformation of the ClpP cDNA driven by the 35S promoter. Accordingly, the ClpP gene was identified as the NAL9 gene. Our results provide a basis for functional studies of NAL9 in future work.

Small RNAs from MITE-derived stem-loop precursors regulate abscisic acid signaling and abiotic stress responses in rice.

Small silencing RNAs (sRNAs) are non-coding RNA regulators that negatively regulate gene expression by guiding mRNA degradation, translation repression or chromatin modification. Plant sRNAs play crucial roles in various developmental processes, hormone signaling, immune responses and adaptation to a variety of abiotic stresses. miR441 and miR446 were previously annotated as microRNAs (miRNAs) because their precursors can form typical stem-loop structures, but are not considered as real miRNAs because of inconsistency with some ancillary criteria of the recent guidelines for annotation of plant miRNAs. We tentatively rename them small interfering (si)R441 and siR446, respectively, in this study. It has recently been shown that the precursors of siR441 and siR446 might originate from the miniature inverted-repeat transposable element (MITE) Stowaway1. In this report, we show that, in contrast with Dicer-like (DCL)3- and RNA-dependent RNA polymerase (RDR)2-dependent MITE-derived ra-siRNAs, siR441 and siR446 are processed by OsDCL3a but independent of OsRDR2, indicating that siR441 and siR446 are generated from single-stranded stem-loop precursors. We also show that abscisic acid (ABA) and abiotic stresses downregulate the expression of siR441 and siR446 but, surprisingly, increase the accumulation of their precursors in rice plants, implying that processing of siRNA precursors is inhibited. We provide evidence to show that this defective processing is due to increased precursor accumulation per se, possibly by intermolecular self-pairing of the processing intermediate sequences, thus hindering their normal processing. Functional examinations indicate that siR441 and siR446 are positive regulators of rice ABA signaling and tolerance to abiotic stress, possibly by regulating MAIF1 expression.

Expression of an apoplast-localized BURP-domain protein from soybean (GmRD22) enhances tolerance towards abiotic stress.

The BURP-domain protein family comprises a diverse group of plant-specific proteins that share a conserved BURP domain at the C terminus. However, there have been only limited studies on the functions and subcellular localization of these proteins. Members of the RD22-like subfamily are postulated to associate with stress responses due to the stress-inducible nature of some RD22-like genes. In this report, we used different transgenic systems (cells and in planta) to show that the expression of a stress-inducible RD22-like protein from soybean (GmRD22) can alleviate salinity and osmotic stress. We also performed detailed microscopic studies using both fusion proteins and immuno-electron microscopic techniques to demonstrate the apoplast localization of GmRD22, for which the BURP domain is a critical determinant of the subcellular localization. The apoplastic GmRD22 interacts with a cell wall peroxidase and the ectopic expression of GmRD22 in both transgenic Arabidopsis thaliana and transgenic rice resulted in increased lignin production when subjected to salinity stress. It is possible that GmRD22 regulates cell wall peroxidases and hence strengthens cell wall integrity under such stress conditions.

OsCD1 encodes a putative member of the cellulose synthase-like D sub-family and is essential for rice plant architecture and growth.

The cell wall plays important roles in plant architecture and morphogenesis. The cellulose synthase-like super-families were reported to contain glycosyltransferases motif and are required for the biosynthesis of cell wall polysaccharides. Here, we describe a curled leaf and dwarf mutant, cd1, in rice, which exhibits multiple phenotypic traits such as the reduction of plant height and leaf width, curled leaf morphology and a decrease in the number of grains and in the panicle length. Map-based cloning indicates that a member of the cellulose synthase-like D (CSLD) group is a candidate for OsCD1. RNAi transgenic plants with the candidate CSLD gene display a similar phenotype to the cd1 mutant, suggesting that OsCD1 is a member of the CSLD sub-family. Furthermore, sequence analysis indicates that OsCD1 contains the common D,D,D,QXXRW motif, which is a feature of the cellulose synthase-like super-family. Analysis of OsCD1 promoter with GUS fusion expression shows that OsCD1 exhibits higher expression in young meristem tissues such as fresh roots, young panicle and stem apical meristem. Cell wall composition analysis reveals that cellulose content and the level of xylose are significantly reduced in mature culm owing to loss of OsCD1 function. Take together, the work presented here is useful for expanding the understanding of cell wall biosynthesis.

Rice hypersensitive induced reaction protein 1 (OsHIR1) associates with plasma membrane and triggers hypersensitive cell death.

BACKGROUND: In plants, HIR (Hypersensitive Induced Reaction) proteins, members of the PID (Proliferation, Ion and Death) superfamily, have been shown to play a part in the development of spontaneous hypersensitive response lesions in leaves, in reaction to pathogen attacks. The levels of HIR proteins were shown to correlate with localized host cell deaths and defense responses in maize and barley. However, not much was known about the HIR proteins in rice. Since rice is an important cereal crop consumed by more than 50% of the populations in Asia and Africa, it is crucial to understand the mechanisms of disease responses in this plant. We previously identified the rice HIR1 (OsHIR1) as an interacting partner of the OsLRR1 (rice Leucine-Rich Repeat protein 1). Here we show that OsHIR1 triggers hypersensitive cell death and its localization to the plasma membrane is enhanced by OsLRR1. RESULT: Through electron microscopy studies using wild type rice plants, OsHIR1 was found to mainly localize to the plasma membrane, with a minor portion localized to the tonoplast. Moreover, the plasma membrane localization of OsHIR1 was enhanced in transgenic rice plants overexpressing its interacting protein partner, OsLRR1. Co-localization of OsHIR1 and OsLRR1 to the plasma membrane was confirmed by double-labeling electron microscopy. Pathogen inoculation studies using transgenic Arabidopsis thaliana expressing either OsHIR1 or OsLRR1 showed that both transgenic lines exhibited increased resistance toward the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. However, OsHIR1 transgenic plants produced more extensive spontaneous hypersensitive response lesions and contained lower titers of the invading pathogen, when compared to OsLRR1 transgenic plants. CONCLUSION: The OsHIR1 protein is mainly localized to the plasma membrane, and its subcellular localization in that compartment is enhanced by OsLRR1. The expression of OsHIR1 may sensitize the plant so that it is more prone to HR and hence can react more promptly to limit the invading pathogens' spread from the infection sites.

Characterization of the genome expression trends in the heading-stage panicle of six rice lineages.

To study how changes in gene regulation shape phenotypic variations in rice, we performed a comparative analysis of genome expression in the heading-stage panicle from six lineages of cultivated and wild rice, including Oryza sativa subsp. indica, japonica and javanica, O. nivara , O. rufipogon and O. glaberrima. While nearly three-quarters of the genes are expressed at a constant level in all six lineages, a large portion of the genome, ranging from 1767 to 4489 genes, exhibited differential expression between Asian domesticated and wild rice with repression or down-regulation of genome expression in Asian cultivated rice as the dominant trend. Importantly, we found this repression was achieved to a large extent by the differential expression of a single member of paralogous gene families. Functional analysis of the differentially expressed genes revealed that genes related to catabolism are repressed while genes related to anabolism up-regulated. Finally, we observed that distinct evolutionary forces may have acted on gene expression and the coding sequences in the examined rice lineages.

Identification and characterization of HTD2: a novel gene negatively regulating tiller bud outgrowth in rice.

Tiller number is highly regulated by controlling the formation of tiller bud and its subsequent outgrowth in response to endogenous and environmental signals. Here, we identified a rice mutant htd2 from one of the 15,000 transgenic rice lines, which is characterized by a high tillering and dwarf phenotype. Phenotypic analysis of the mutant showed that the mutation did not affect formation of tiller bud, but promoted the subsequent outgrowth of tiller bud. To isolate the htd2 gene, a map-based cloning strategy was employed and 17 new insertions-deletions (InDels) markers were developed. A high-resolution physical map of the chromosomal region around the htd2 gene was made using the F(2) and F(3) population. Finally, the gene was mapped in 12.8 kb region between marker HT41 and marker HT52 within the BAC clone OSJNBa0009J13. Cloning and sequencing of the target region from the mutant showed that the T-DNA insertion caused a 463 bp deletion between the promoter and first exon of an esterase/lipase/thioesterase family gene in the 12.8 kb region. Furthermore, transgenic rice with reduced expression level of the gene exhibited an enhanced tillering and dwarf phenotype. Accordingly, the esterase/lipase/thioesterase family gene (TIGR locus Os03g10620) was identified as the HTD2 gene. HTD2 transcripts were expressed mainly in leaf. Loss of function of HTD2 resulted in a significantly increased expression of HTD1, D10 and D3, which were involved in the strigolactone biosynthetic pathway. The results suggest that the HTD2 gene could negatively regulate tiller bud outgrowth by the strigolactone pathway.

Relationship between proline and Hg2+-induced oxidative stress in a tolerant rice mutant.

There has been little agreement regarding the mechanism by which proline reduces heavy metal stress. The present work examines the relationship between Hg(2+)-induced oxidative stress and proline accumulation in rice and explores the possible mechanisms through which proline protects against Hg(2+) stress. The effect of proline on alleviation of Hg(2+) toxicity was studied by spectrophotography and enzymatic methods. Hg(2+) induced oxidative stress in rice by increasing lipid peroxidation. Pretreatment of the rice with 2 mM proline for 12 h profoundly alleviated Hg(2+)-induced lipid peroxidation and minimized H(2)O(2) accumulation. Proline pretreatment significantly reduced (p < 0.01) the Hg(2+) content in rice leaves. A comparison of the effects of proline pretreatment on H(2)O(2) accumulation by Hg(2+) and aminotrazole suggested that proline protected cells from Hg(2+)-induced oxidative stress by scavenging reactive oxygen species. The present work demonstrates a protective effect of proline on Hg(2+) toxicity through detoxifying reactive oxygen species, rather than chelating metal ions or maintaining the water balance under Hg(2+) stress.

Genome-wide analysis of the auxin response factors (ARF) gene family in rice (Oryza sativa).

Auxin response factors (ARFs) are transcription factors that bind with specificity to TGTCTC-containing auxin response elements (AuxREs) found in promoters of primary/early auxin response genes and mediate responses to the plant hormone auxin. The ARF genes are represented by a large multigene family in plants. A comprehensive genome-wide analysis was carried out in this study to find all ARFs in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa subsp. japonica), 23 and 25 ARF genes, named as AtARFs and OsARFs, were identified, respectively. Chromosomal locations of all OsARFs were presented and it was found that the duplication of OsARFs was associated with only the chromosomal block duplications but not local tandem duplications. A phylogenetic tree was generated from alignments of the full-length protein sequences of 25 OsARFs and 23 AtARFs to examine the phylogenetic relationships of rice and Arabidopsis ARF proteins. All 48 members of ARF gene families fell into three major classes, a total of 13 sister pairs, including 9 OsARF-OsARF, 2 AtARF-AtARF and 2 AtARF-OsARF sister pairs were formed, showing different orthologous relationships between AtARFs and OsARFs. EST analysis and RT-PCR assays demonstrated that 24 of all 25 OsARF genes were active and the transcript abundance of some OsARF genes was affected by auxin treatment or light- and dark-grown conditions. The outcome of the present study provides basic genomic information for the rice ARF gene family and will pave the way for elucidating the precise role of OsARFs in plant growth and development in the future.

Application of TILLING in plant improvement.

TILLING (Targeting induced local lesions in genomes) is a general reverse-genetic strategy that is used to locate an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and cost-effective detection of induced point mutations in populations of chemically mutagenized individuals. The technique can be applied not only to model organisms but also to economically important organisms in plants. Owing to its full of advantages such as simple procedure, high sensitivity, and high efficiency, TILLING provides a powerful approach for gene discovery, DNA polymorphism assessment, and plant improvement. Coupled with other genomic resources, TILLING and EcoTILLING can be used immediately as a haplotyping tool in plant breeding for identifying allelic variation in genes exhibiting expression correlating with phenotypes and establishing an allelic series at genetic loci for the traits of interest in germplasm or induced mutants.

Isolation and physiological characteristics of a premature senescence mutant in rice (Oryza sativa L.).

A rice pse(t) (premature senescence, tentatively) mutant line, was isolated from 4,500 independent T-DNA inserted transgenic lines. The symptoms of premature senescence appeared more severely than those of the control plants (Zhonghua 11, japonica) at the last development stage. To characterize the mutant and provide basic information on the candidate genes by mapping to a physical region of 220-kb, experiments were carried out in two phytotrons under controlled temperature of 24 degrees C and 28 degrees C, respectively. The content of chlorophyll, soluble protein and MDA (malondialdehyde), net photosynthesis, the antioxidant enzyme activities of SOD (superoxide dismuase) (EC 1.15.1.1) and POD (peroxidase) (EC 1.11.1.7) and the peptidase activities of leaves were measured from top to bottom according to the leaf positions at the flowering stage. Compared with the control plant, the mutant showed the following characteristics: (1) Higher net photosynthesis rate (P(n)) appeared in the 1st and 2nd leaves, contents of chlorophyll and soluble protein were also higher in the 1st leaf; (2) The activities of SOD, POD and peptidase were higher according to the leaf position from top to bottom; (3) The symptom of premature senescence was accelerated in the mutant at 28 degrees C treatment. The MDA content and the SOD and POD activities between the 24 degrees C and 28 degrees C treatment mutants were not significantly different. Content of chlorophyll and soluble protein of leaves mutant decreased rapidly at 28 degrees C treatment. The results show that pse(t) is sensitive to high temperature. The probable function of PSE(T) is discussed.

[Ac/Ds tagging system and functional genomics in rice].

With the completion of the rice genome sequence in 2002, the study on functional genomics in rice has become a major task. Establishment of rice mutant library is an essential approach for rice functional genomics study. At present, utilizing maize transposable element Ac/Ds (Activator/Dissociation) is a promising method to construct insertional mutagenesis library of rice. Ac/Ds tagging system has received extensive application in rice during the past several years, but it is still confronted with practical problems. In this paper, constructing rice insertional mutagenesis library using Ac/Ds-tagging system and transpositional behaviors of an Ac/Ds-tagging system, difficulties and advantages are reviewed. The research advances and challenge in rice functional genomics study using Ac/Ds tagging system are also discussed and summarized in the paper.

Preliminary study on a gravity-insensitive rice mutant.

A gravity-insensitive mutant was isolated from rice (Oryza sativa L. cv. Zhonghua 11) transformed by Agrobacterium tumefaciens. The mutant's shoot growth (prostrate growth) was insensitive to gravity; whereas root growth displayed a normal positive gravitropism. Histological observation of root caps and leaf sheaths indicated that there was no significant difference in the number and size of amyloplasts in cells of the mutant and cells of the wild type.

Enhancing rice resistance to fungal pathogens by transformation with cell wall degrading enzyme genes from Trichoderma atroviride.

Three genes encoding for fungal cell wall degrading enzymes (CWDEs), ech42, nag70 and gluc78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possible combinations and transformed to rice plants. More than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. The ech42 gene encoding for an endochitinase increased resistance to sheath blight caused by Rhizoctonia solani, while the exochitinase-encoding gene, nag70, had lesser effect. The expression level of endochitinase but exochitinase was correlated with disease resistance. Nevertheless, exochitinase enhanced the effect of endochitinase on disease resistance when the two genes co-expressed in transgenics. Resistance to Magnaporthe grisea was found in all kinds of regenerated plants including that with single gluc78. A few lines expressing either ech42 or nag70 gene were immune to the disease. Transgenic plants are being tested to further evaluate disease resistance at field level. This is the first report of multiple of expression of genes encoding CWDEs from Trichoderma atroviride that result in resistance to blast and sheath blight in rice.

[Comparison between two methods of establishing DH population for mapping lox-3 gene in rice (Oryza sativa L.)].

Crosses were produced by using a storable rice variety (Daw Dam) and 2070, Ming Hui 63, as well as Dulur/Xie Qing Zao B as parents, among these materials there are abundant polymorphisms of DNA molecular markers. Their F1's were applied to generate DH lines by an improved method of direct induction of pollen plant. The method of the direct induction of pollen plant was used as check treatment to evaluate the improved method. The results showed that the improved method increased the re-differentiation percentage of pollen callus, the percentage of regeneration plantlets, and the percentage of green regeneration plantlets and produced more vigorous seedlings. The improved method was a competent method of establishing DH population for mapping lox-3 gene. It was also showed that the efficiency of the new method was different among genotypes and out of three experimental crosses Daw Dam/2070 and Daw Dam/Minghui 63 showed more efficient. More than 60 DH lines have been obtained in this experiment.

Dlf1, a WRKY transcription factor, is involved in the control of flowering time and plant height in rice.

Flowering time and plant height are important agronomic traits for crop production. In this study, we characterized a semi-dwarf and late flowering (dlf1) mutation of rice that has pleiotropic effects on these traits. The dlf1 mutation was caused by a T-DNA insertion and the cloned Dlf1 gene was found to encode a WRKY transcription factor (OsWRKY11). The dlf1 mutant contains a T-DNA insertion at the promoter region, leading to enhanced accumulation of Dlf1 transcripts, resulting in a semidominant mutation. The dlf1 mutation suppressed the transcription of Ehd2/RID1/OsId1 and its downstream flowering-time genes including Hd1, Ehd1 and Hd3a under both long-day (LD) and short-day (SD) conditions. Knock-down of Dlf1 expression exhibited early flowering at LD condition related to the wild-type plants. Accumulation of Dlf1 mRNA was observed in most tissues, and two splicing forms of Dlf1 cDNAs were obtained (OsWRKY11.1 and OsWRKY11.2). These two proteins showed transactivation activity in yeast cells. Dlf1 protein was found to be localized in the nucleus. Enhanced expression of OsWRKY11.2 or its 5' truncated gene showed similar phenotypes to the dlf1 mutant, suggesting that it might function as a negative regulator. We conclude that Dlf1 acts as a transactivator to downregulate Ehd2/RID1/OsId1 in the signal transduction pathway of flowering and plays an important role in the regulation of plant height in rice.

A novel rice gene, NRR responds to macronutrient deficiency and regulates root growth.

To better understand the response of rice to nutrient stress, we have taken a systematic approach to identify rice genes that respond to deficiency of macronutrients and affect rice growth. We report here the expression and biological functions of a previously uncharacterized rice gene that we have named NRR (nutrition response and root growth). NRR is alternatively spliced, producing two 5'-coterminal transcripts, NRRa and NRRb, encoding two proteins of 308 and 223 aa, respectively. Compared to NRRb, NRRa possesses an additional CCT domain at the C-terminus. Expression of NRR in rice seedling roots was significantly influenced by deficiency of macronutrients. Knock-down of expression of NRRa or NRRb by RNA interference resulted in enhanced rice root growth. By contrast, overexpression of NRRa in rice exhibited significantly retarded root growth. These results revealed that both NRRa and NRRb played negative regulatory roles in rice root growth. Our findings suggest that NRRa and NRRb, acting as the key components, modulate the rice root architecture with the availability of macronutrients.

Overexpression of an F-box protein gene reduces abiotic stress tolerance and promotes root growth in rice.

As one of the largest gene families, F-box domain proteins have important roles in regulating various developmental processes and stress responses. In this study, we have investigated a rice F-box domain gene, MAIF1. The MAIF1 protein is mainly localized in the plasma membrane and nucleus. MAIF1 expression is induced rapidly and strongly by abscisic acid (ABA) and abiotic stresses. MAIF1 expression is also induced in root tips by sucrose, independent of its hydrolytic hexose products, glucose and fructose, and the plant hormones auxin and cytokinin. Overexpression of MAIF1 reduces rice ABA sensitivity and abiotic stress tolerance and promotes rice root growth. These results suggest that MAIF1 is involved in multiple signaling pathways in regulating root growth. Growth restraint in plants is an acclimatization strategy against abiotic stress. Our results also suggest that MAIF1 plays the negative role in response to abiotic stress possibly by regulating root growth.