Chuxiong Zhuang

Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA

Photoperiod- and thermo-sensitive genic male sterility (PGMS and TGMS) are the core components for hybrid breeding in crops. Hybrid rice based on the two-line system using PGMS and TGMS lines has been successfully developed and applied widely in agriculture. However, the molecular mechanism underlying the control of PGMS and TGMS remains obscure. In this study, we mapped and cloned a major locus, p/tms12-1 (photo- or thermo-sensitive genic male sterility locus on chromosome 12), which confers PGMS in the japonica rice line Nongken 58S (NK58S) and TGMS in the indica rice line Peiai 64S (PA64S, derived from NK58S). A 2.4-kb DNA fragment containing the wild-type allele P/TMS12-1 was able to restore the pollen fertility of NK58S and PA64S plants in genetic complementation. P/TMS12-1 encodes a unique noncoding RNA, which produces a 21-nucleotide small RNA that we named osa-smR5864w. A substitution of C-to-G in p/tms12-1, the only polymorphism relative to P/TMS12-1, is present in the mutant small RNA, namely osa-smR5864m. Furthermore, overexpression of a 375-bp sequence of P/TMS12-1 in transgenic NK58S and PA64S plants also produced osa-smR5864w and restored pollen fertility. The small RNA was expressed preferentially in young panicles, but its expression was not markedly affected by different day lengths or temperatures. Our results reveal that the point mutation in p/tms12-1, which probably leads to a loss-of-function for osa-smR5864m, constitutes a common cause for PGMS and TGMS in the japonica and indica lines, respectively. Our findings thus suggest that this noncoding small RNA gene is an important regulator of male development controlled by cross-talk between the genetic networks and environmental conditions.

Rice OsGL1-6 Is Involved in Leaf Cuticular Wax Accumulation and Drought Resistance

Cuticular wax is a class of organic compounds that comprises the outermost layer of plant surfaces. Plant cuticular wax, the last barrier of self-defense, plays an important role in plant growth and development. The OsGL1-6 gene, a member of the fatty aldehyde decarbonylase gene family, is highly homologous to Arabidopsis CER1, which is involved in cuticular wax biosynthesis. However, whether OsGL1-6 participates in cuticular wax biosynthesis remains unknown. In this study, an OsGL1-6 antisense-RNA vector driven by its own promoter was constructed and introduced into the rice variety Zhonghua11 by Agrobacterium-mediated transformation to obtain several independent transgenic plants with decreased OsGL1-6 expression. These OsGL1-6 antisense-RNA transgenic plants showed droopy leaves at the booting stage, significantly decreased leaf cuticular wax deposition, thinner cuticle membrane, increased chlorophyll leaching and water loss rates, and enhanced drought sensitivity. The OsGL1-6 gene was constitutively expressed in all examined organs and was very highly expressed in leaf epidermal cells and vascular bundles. The transient expression of OsGL1-6-GFP fusion indicated that OsGL1-6 is localized in the endoplasmic reticulum. Qualitative and quantitative analysis of the wax composition using gas chromatography-mass spectrometry revealed a significantly reduced total cuticular wax load on the leaf blades of the OsGL1-6 antisense-RNA transgenic plants as well as markedly decreased alkane and aldehyde contents. Their primary alcohol contents increased significantly compared with those in the wild type plants, suggesting that OsGL1-6 is associated with the decarbonylation pathways in wax biosynthesis. We propose that OsGL1-6 is involved in the accumulation of leaf cuticular wax and directly impacts drought resistance in rice.

Development of Commercial Thermo-sensitive Genic Male Sterile Rice Accelerates Hybrid Rice Breeding Using the CRISPR/Cas9-mediated TMS5 Editing System.

Hybrid rice breeding offers an important strategy to improve rice production, in which the cultivation of a male sterile line is the key to the success of cross-breeding. CRISPR/Cas9 systems have been widely used in target-site genome editing, whereas their application for crop genetic improvement has been rarely reported. Here, using the CRISPR/Cas9 system, we induced specific mutations in TMS5, which is the most widely applied thermo-sensitive genic male sterility (TGMS) gene in China, and developed new “transgene clean” TGMS lines. We designed 10 target sites in the coding region of TMS5 for targeted mutagenesis using the CRISPR/Cas9 system and assessed the potential rates of on- and off-target effects. Finally, we established the most efficient construct, the TMS5ab construct, for breeding potentially applicable “transgene clean” TGMS lines. We also discussed factors that affect the editing efficiency according to the characteristics of different target sequences. Notably, using the TMS5ab construct, we developed 11 new “transgene clean” TGMS lines with potential applications in hybrid breeding within only one year in both rice subspecies. The application of our system not only significantly accelerates the breeding of sterile lines but also facilitates the exploitation of heterosis.

Expression of RNA-Interference/Antisense Transgenes by the Cognate Promoters of Target Genes Is a Better Gene-Silencing Strategy to Study Gene Functions in Rice

Antisense and RNA interference (RNAi)-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.

Efficient culture protocol for plant regeneration from petiole explants of physiologically mature trees of Jatropha curcas L.

An efficient and reproducible protocol for induction of adventitious shoot buds and plant regeneration from petiole explant cultures of Jatropha curcas, an important biofuel crop, is described. Physiologically mature trees of three J. curcas genotypes were selected and explants were prepared from young petioles. Treating the explants with high concentrations (5 to 120 mg/L) of thidiazuron (TDZ) solution for short time periods (5 to 80 min) helped increase the regeneration frequency and improved the quality of the regenerated buds significantly. The age of the petioles and inoculation methods were found to influence the culture results. The best shoot buds induction (65.78%) and number of buds (6.77) per explant was seen in the second petiole explants of genotype M-1 treated with 20 mg/L TDZ solution for 20 min, followed by 35-day culture on hormone-free Murashige and Skoog medium. The regenerated buds could elongate to become shoots in a medium containing gibberellic acid. The elongated shoots initiated roots to become intact plantlets in rooting medium containing indole-3-butyric acid and L-glutamine (Gln), and supplementing 16 mg/L Gln into the rooting medium effectively stimulated the initiation and growth of roots, with the best rooting rate (51.72%). After acclimatization, these plantlets were transplanted to soil wherein normal growth was observed. Therefore, an intact plantlet could usually be obtained at 60 days of culture by using the culture protocol described in this study. This protocol can be used for mass production of true-to-type plants and the production of transgenic plants through Agrobacterium/biolistic-mediated transformation.

Overexpression of miR164b-resistant OsNAC2 improves plant architecture and grain yield in rice

Plant architecture affects grain yield. Overexpressing a version of OsNAC2 resistant to miRNA-mediated degradation increased panicle number and grain yield under field conditions, identifying a new strategy to improve yield.

OsCER1 Plays a Pivotal Role in Very-Long-Chain Alkane Biosynthesis and Affects Plastid Development and Programmed Cell Death of Tapetum in Rice (Oryza sativa L.)

Cuticle waxes, which are primarily comprised of very-long-chain (VLC) alkanes, play an important role in plant reproductive development. ECERIFERUM1 (CER1) is recognized as the core element for VLC alkane biosynthesis in Arabidopsis (Arabidopsis thaliana). However, genes involved in the VLC alkane biosynthesis in rice remain unclear, and the alkane-form pathway in rice has still to be further explored. Here, we show that OsCER1, a homology of CER1, functions in VLC alkanes biosynthesis, which also could regulate anther development and plastids differentiation in rice. OsCER1 was highly expressed in the tapetum (stage 10) and bicellular pollen cells (stage 11). The decreased content of VLC alkanes (C25 and C27) in the OsCER1 knocked down plants as well as the increased content of C27 alkanes in the OsCER1 overexpression plants indicates that OsCER1 participates in VLC alkane biosynthesis. Downregulation of OsCER1 in rice led to sterility, and fewer amyloplasts within the mature pollen grains. In addition, the downregulation of OsCER1 in rice caused delayed tapetal programmed cell death and abnormal development of plastids in the tapetal cells. Furthermore, significantly altered levels of expression of genes involved in the pollen development were exhibited in the OsCER1 knocked down plants. These results indicate that OsCER1 is critical for VLC alkanes biosynthesis, plastids differentiation, and pollen development. This work provides insights into the VLC alkanes biosynthesis in anther development in rice.

Effect of Stresses on Leaf Cuticular Wax Accumulation and Its Relationship to Expression of OsGL1 -Homologous Genes in Rice: Effect of Stresses on Leaf Cuticular Wax Accumulation and Its Relationship to Expression of OsGL1 -Homologous Genes in Rice

植物角质层蜡质在抵抗各种生物和非生物胁迫中起着非常重要的作用。本试验以水稻( Oryza sativa L.)幼苗为材料, 分别以200 mmol L -1 NaCl、12% PEG、1.0% H 2 O 2 、40℃高温和8℃低温为逆境, 研究叶角质层蜡质的积累情况以及其与水稻蜡质合成相关基因 OsGL1 表达的关系。扫描电镜观察以及叶角质层蜡质总量测定结果表明, 12% PEG、1.0% H 2 O 2 和8℃低温处理下水稻幼苗叶角质层蜡质的积累明显增加, 而200 mmol L -1 NaCl和40℃高温处理下叶角质层蜡质覆盖量略有下降。RT-PCR分析显示, 逆境处理下水稻蜡质合成相关基因 OsGL1 的表达量变化与水稻幼苗叶角质层蜡质的积累存在相关性。