Wang Yan, Mortimer Jennifer C, Davis Jonathan, Dupree Paul, Keegstra Kenneth
Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, MI, 48824, USA.
Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI, 48824, USA.
Plant J. 2013 Jan;73(1):105-17. doi: 10.1111/tpj.12019. Epub 2012 Oct 19.
Galactomannans comprise a β-1,4-mannan backbone substituted with α-1,6-galactosyl residues. Genes encoding the enzymes that are primarily responsible for backbone synthesis and side-chain addition of galactomannans were previously identified and characterized. To identify additional genes involved in galactomannan biosynthesis, we previously performed deep EST profiling of fenugreek (Trigonella foenum-graecum L.) seed endosperm, which accumulates large quantities of galactomannans as a reserve carbohydrate during seed development. One of the candidate genes encodes a protein that is likely to be a glycosyltransferase. Because this protein is involved in mannan biosynthesis, we named it 'mannan synthesis-related' (MSR). Here, we report the characterization of a fenugreek MSR gene (TfMSR) and its two Arabidopsis homologs, AtMSR1 and AtMSR2. TfMSR was highly and specifically expressed in the endosperm. TfMSR, AtMSR1 and AtMSR2 proteins were all determined to be localized to the Golgi by fluorescence confocal microscopy. The level of mannosyl residues in stem glucomannans decreased by approximately 40% for Arabidopsis msr1 single T-DNA insertion mutants and by more than 50% for msr1 msr2 double mutants, but remained unchanged for msr2 single mutants. In addition, in vitro mannan synthase activity from the stems of msr1 single and msr1 msr2 double mutants also decreased. Expression of AtMSR1 or AtMSR2 in the msr1 msr2 double mutant completely or partially restored mannosyl levels. From these results, we conclude that the MSR protein is important for mannan biosynthesis, and offer some ideas about its role.
半乳甘露聚糖由一个被α-1,6-半乳糖基残基取代的β-1,4-甘露聚糖主链组成。先前已鉴定并表征了编码主要负责半乳甘露聚糖主链合成和侧链添加的酶的基因。为了鉴定参与半乳甘露聚糖生物合成的其他基因，我们之前对胡芦巴（Trigonella foenum - graecum L.）种子胚乳进行了深度EST分析，胡芦巴种子胚乳在种子发育过程中积累大量半乳甘露聚糖作为储备碳水化合物。其中一个候选基因编码一种可能是糖基转移酶的蛋白质。由于该蛋白质参与甘露聚糖生物合成，我们将其命名为“甘露聚糖合成相关”（MSR）。在此，我们报告了胡芦巴MSR基因（TfMSR）及其两个拟南芥同源基因AtMSR1和AtMSR2的表征。TfMSR在胚乳中高度且特异性表达。通过荧光共聚焦显微镜确定TfMSR、AtMSR1和AtMSR2蛋白均定位于高尔基体。拟南芥msr1单T-DNA插入突变体茎中的甘露糖基残基水平降低了约40%，msr1 msr2双突变体降低了超过50%，但msr2单突变体保持不变。此外，msr1单突变体和msr1 msr2双突变体茎中的体外甘露聚糖合酶活性也降低。在msr1 msr2双突变体中表达AtMSR1或AtMSR2可完全或部分恢复甘露糖基水平。根据这些结果，我们得出结论，MSR蛋白对甘露聚糖生物合成很重要，并对其作用提出了一些见解。
