Arabidopsis mutants Atisa1 and Atisa2 have identical phenotypes and lack the same multimeric isoamylase, which influences the branch point distribution of amylopectin during starch synthesis
Arabidopsis mutants Atisa1 and Atisa2 have identical phenotypes and lack the same multimeric isoamylase, which influences the branch point distribution of amylopectin during starch synthesis
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DOI：
10.1111/j.1365-313x.2005.02348.x
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发表时间：
2005-03-01
期刊：
影响因子：
7.2
通讯作者：
Zeeman, SC
中科院分区：
文献类型：
--
作者：
Delatte, T;Trevisan, M;Zeeman, SC
本研究旨在探讨异淀粉酶在拟南芥叶片淀粉粒生物合成中的作用。一个反向遗传的方法被用来敲除AtISA 1，在拟南芥编码异淀粉酶型脱支酶的三个基因之一。突变体（Atisa 1 -1）缺乏功能AtISA 1转录和主要的异淀粉酶活性（天然凝胶检测）在叶的粗提取物。DBE 1基因座的突变也会消除相同的活性，DBE 1基因座编码第二种异淀粉酶型蛋白AtISA 2。这与ISA 1和ISA 2蛋白是体内相同酶的亚基的想法一致。Atisa 1 -1、Atisa 2 -1（dbe 1）和Atisa 1 -1/Atisa 2 -1双突变体都具有相同的表型。与野生型相比，淀粉含量减少，但产生大量的可溶性葡聚糖植物糖原。剩余淀粉的支链淀粉和突变体中的植物糖原在结构上彼此相关，并且不同于野生型支链淀粉。电子显微照片显示，植物糖原积累表型是高度组织特异性的。植物糖原主要积累在栅栏和海绵叶肉细胞的质体中。值得注意的是，其他类型的细胞似乎只积累淀粉，这是正常的外观，但在结构上发生了变化。随着植物糖原在白天积累，其积累速率降低，其结构发生变化，葡聚糖分解的中间产物积累，这表明降解与合成同时发生。我们得出的结论是，AtISA 1/AtISA 2异淀粉酶影响葡聚糖分支模式，但这可能不是结晶淀粉和可溶性植物糖原之间分配的主要决定因素。
The aim of this work was to evaluate the function of isoamylase in starch granule biosynthesis in Arabidopsis leaves. A reverse-genetic approach was used to knockout AtISA1, one of three genes in Arabidopsis encoding isoamylase-type debranching enzymes. The mutant (Atisa1-1) lacks functional AtISA1 transcript and the major isoamylase activity (detected by native gels) in crude extracts of leaves. The same activity is abolished by mutation at the DBE1 locus, which encodes a second isoamylase-type protein, AtISA2. This is consistent with the idea that ISA1 and ISA2 proteins are subunits of the same enzyme in vivo. Atisa1-1, Atisa2-1 (dbe1), and the Atisa1-1/Atisa2-1 double mutant all have identical phenotypes. Starch content is reduced compared with the wild type but substantial quantities of the soluble glucan phytoglycogen are produced. The amylopectin of the remaining starch and the phytoglycogen in the mutants are structurally related to each other and differ from wild-type amylopectin. Electron micrographs reveal that the phytoglycogen-accumulating phenotype is highly tissue-specific. Phytoglycogen accumulates primarily in the plastids of the palisade and spongy mesophyll cells. Remarkably, other cell types appear to accumulate only starch, which is normal in appearance but is altered in structure. As phytoglycogen accumulates during the day, its rate of accumulation decreases, its structure changes and intermediates of glucan breakdown accumulate, suggesting that degradation occurs simultaneously with synthesis. We conclude that the AtISA1/AtISA2 isoamylase influences glucan branching pattern, but that this may not be the primary determinant of partitioning between crystalline starch and soluble phytoglycogen.