Reverse genetic characterization of two paralogous acetoacetyl CoA thiolase genes in Arabidopsis reveals their importance in plant growth and development
Reverse genetic characterization of two paralogous acetoacetyl CoA thiolase genes in Arabidopsis reveals their importance in plant growth and development
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DOI：
10.1111/j.1365-313x.2012.04942.x
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发表时间：
2012-06-01
期刊：
影响因子：
7.2
通讯作者：
Nikolau, Basil J.
中科院分区：
文献类型：
--
作者：
Jin, Huanan;Song, Zhihong;Nikolau, Basil J.
乙酰辅酶A硫解酶(AACT，EC 2.3.1.9)催化两个乙酰辅酶A分子缩合生成乙酰辅酶A。通过直接酶学分析和在酵母中的功能表达，在拟南芥基因组中鉴定了两个编码AACT的基因At5g47720(AACT1)和At5g48230(AACT2)。启动子：：GUS融合实验表明，AACT1主要在维管系统中表达，AACT2在根尖、幼叶、顶茎和花药中高表达。对每个AACT基因座的T-DNA插入突变等位基因的鉴定表明，AACT2功能是胚胎发育和正常雄配子传递所必需的。相反，缺乏AACT1功能的植株是完全存活的，没有表现出明显的生长表型，这表明AACT1功能相对于AACT2功能是多余的。表达AACT2水平降低的RNAi品系表现出多效性，包括顶端优势降低、寿命延长和开花持续时间延长、不育、矮化、种子产量下降和根长缩短。显微分析表明，身高下降是由于细胞尺寸减小和细胞减少造成的，而雄性不育是由于花粉被丢失和绒毛细胞过早退化造成的。生化分析表明，AACT2 RNAi植物的根在植物甾醇谱中表现出定量和定性的变化。当AACT2 RNAi植物在甲羟戊酸存在下生长时，这些表型和生化变化被逆转，这与AACT2在产生大量乙酰乙酰辅酶A前体方面的作用是一致的，这是胞浆定位的甲羟戊酸衍生的异戊二烯生物合成途径所必需的。
Acetoacetyl CoA thiolase (AACT, EC 2.3.1.9) catalyzes the condensation of two acetyl CoA molecules to form acetoacetyl CoA. Two AACT-encoding genes, At5g47720 (AACT1) and At5g48230 (AACT2), were functionally identified in the Arabidopsis genome by direct enzymological assays and functional expression in yeast. Promoter::GUS fusion experiments indicated that AACT1 is primarily expressed in the vascular system and AACT2 is highly expressed in root tips, young leaves, top stems and anthers. Characterization of T-DNA insertion mutant alleles at each AACT locus established that AACT2 function is required for embryogenesis and for normal male gamete transmission. In contrast, plants lacking AACT1 function are completely viable and show no apparent growth phenotypes, indicating that AACT1 is functionally redundant with respect to AACT2 function. RNAi lines that express reduced levels of AACT2 show pleiotropic phenotypes, including reduced apical dominance, elongated life span and flowering duration, sterility, dwarfing, reduced seed yield and shorter root length. Microscopic analysis reveals that the reduced stature is caused by a reduction in cell size and fewer cells, and male sterility is caused by loss of the pollen coat and premature degeneration of the tapetal cells. Biochemical analyses established that the roots of AACT2 RNAi plants show quantitative and qualitative alterations in phytosterol profiles. These phenotypes and biochemical alterations are reversed when AACT2 RNAi plants are grown in the presence of mevalonate, which is consistent with the role of AACT2 in generating the bulk of the acetoacetyl CoA precursor required for the cytosol-localized, mevalonate-derived isoprenoid biosynthetic pathway.