拟南芥固醇C-4-甲基氧化酶2（SMO2）在雌雄配子体发育中的功能研究

The Arabidopsis sterol C-4-methyl oxidase 2 (SMO2) specifically acts on 4-methyl sterols to oxidize the methyl group at C-4 position to produce the C-4-carboxy-sterol derivative. In Arabidopsis, there are two genes encoding SMO2 enzyme, termed SMO2-1 and SMO2-2. In our previous study, we demonstrated that SMO2s affect embryo development via auxin-associated mechanisms. We also found that both female and male gametophytic developments were defective in the smo2-1 smo2-2/+ mutants, but normal in the smo2-1/+ smo2-2 mutants. This finding suggests that a threshold of SMO2 enzyme level was required for gametogenesis and SMO2 loss of function caused sporophytic female and male defect. Cell biological analysis showed that development of smo2-1 smo2-2/+ female gametophyte was arrested at FG1 stage; and its pollen development was aborted prior to bicellular stage. These results indicate that development of both female and male gametophytes of the smo2-1 smo2-2/+ mutant were still normal when meiosis was finished, but were arrested during the following mitosis. Therefore SMO2 enzyme is required for the mitosis process of gametophyte development. In this study we will perform cell biology, genetic and transcriptomic experiments to investigate the mechanisms of gametogenesis defects of smo2-1 smo2-2/+ mutants. We will determine that: (1) Whether SMO2s affect gametophyte development via auxin-related mechanisms. (2) Whether accumulation of 4α-methyl-sterols, or reduction of the major sterols such as sitosterol has an effect on gametogenesis. (3) Which genes expression levels were affected in smo2-1 smo2-2/+ mutants, and identify the major SMO2 down-stream genes by RNA-sequencing, bioinformatics and genetic studies. Our research will reveal the mechanism of gametophytic abortion of the smo2-1 smo2-2/+ mutant, and help to identify previously unrecognized roles of sterols in cellular processes and plant growth and development, and further reveal its functional mechanism.

固醇C-4-甲基氧化酶2（SMO2）以4-甲基固醇为底物，将C-4位的甲基氧化成羧基。拟南芥有两个SMO2基因：SMO2-1和SMO2-2。我们已发表的工作证明SMO2通过生长素相关的机制影响胚的发育。我们发现smo2-1 smo2-2/+杂合双突变体的雌、雄配子体都存在缺陷，而smo2-1/+ smo2-2的配子体正常。初步的研究表明smo2-1 smo2-2/+的雌、雄配子都在完成减数分裂后，进行有丝分裂的时期败育。这暗示SMO2对配子体的有丝分裂是必须的。本项目将通过细胞生物学、遗传学和转录组学等手段研究：1）smo2-1 smo2-2/+配子体的缺陷是否与生长素有关；2）SMO2的底物4-甲基固醇及终产物谷甾醇等是否影响配子体的发育；3）SMO2缺陷影响哪些配子体相关基因的表达，找出关键基因并进行遗传学验证。综合上述研究结果阐明smo2-1 smo2-2/+配子体败育的机制。

拟南芥固醇C-4-甲基氧化酶2（SMO2）以4-甲基固醇为底物，将C-4位的甲基氧化成羧基。拟南芥有两个SMO2编码基因：SMO2-1和SMO2-2。我们前期研究发现smo2-1 smo2-2双突变体胚胎致死，阐明了SMO2通过生长素相关的机制影响胚发育。此外，smo2-1 smo2-2/+杂合双突变体雌、雄配子体发育都存在缺陷，而smo2-1/+ smo2-2的配子体正常。这表明SMO2主要通过孢子体效应影响配子发育。本项目对smo2-1 smo2-2/+雌、雄配子体败育的机制进行了研究。首先通过回补实验确定smo2-1 smo2-2/+突变体花粉和胚珠败育的表型是由于SMO2功能缺失造成的。接着分析了SMO2-1和SMO2-2基因在雌雄配子体发育过程中的表达情况。发现SMO2-1在小孢子母细胞、四分体、小孢子、二核花粉粒及成熟花粉粒均有表达，在绒毡层细胞也有表达，而SMO2-2只在成熟花粉粒里有表达。此外，二者在胚珠发育过程中均有表达，且二者的表达模式存在互补。SMO2-1在胚囊有较强的表达，而SMO2-2在珠被有较强的表达。随后分析了smo2-1 smo2-2/+雌、雄配子体败育的细胞学机制。发现smo2-1 smo2-2/+花粉发育在从单核小孢子向二核花粉粒转变的时期出现败育。在花粉发育的早期，虽然突变体的花粉粒没有明显的细胞器降解和胞质塌陷，但其液泡的大小和分布与野生型相比存在明显不同，暗示突变体花粉粒的败育可能是由于液泡发育缺陷造成的。进一步发现smo2-1 smo2-2/+花药中生长素响应标记基因的表达下调，用SMO2-1启动子驱动生长素合成相关基因YUC9的表达，以及外源施加生长素均能部分回补其花粉败育的表型。同样，smo2-1 smo2-2/+的胚囊能正常完成减数分裂，但是有丝分裂过程不能正常进行。smo2-1 smo2-2/+胚珠中，生长素响应标记基因的表达也降低，但内源表达生长合成相关基因不能回补其败育的表型。为了深入分析SMO2在雌雄配子体发育中的作用机制，进行了酵母双杂交筛库及验证实验，发现SMO2-1与脂类合成相关的IPCS、CYP450家族成员、小G蛋白ROP2及其激活因子ROPGEF1均有互作。目前正在进行遗传学及生化实验，揭示SMO2通过与ROP2和ROPGEF1互作，影响雌雄配子体发育的分子机制。