Differences in Transcriptional Regulatory Mechanisms Functioning for Free Lysine Content and Seed Storage Protein Accumulation in Rice Grain
Differences in Transcriptional Regulatory Mechanisms Functioning for Free Lysine Content and Seed Storage Protein Accumulation in Rice Grain
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DOI：
10.1093/pcp/pcq164
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发表时间：
2010-12-01
影响因子：
4.9
通讯作者：
Takaiwa, Fumio
中科院分区：
文献类型：
--
作者：
Kawakatsu, Taiji;Takaiwa, Fumio
赖氨酸是谷物中最缺乏的必需氨基酸。赖氨酸酮戊二酸还原酶/糖胺脱氢酶(LKR/SDH)是一种双功能赖氨酸降解酶，是决定植物体内游离赖氨酸含量的关键调节因子之一。在水稻(Oryza sativa.)L)，一个双功能的OsLKR/SDH主要存在于种子中。在这里，我们证明了OsLKR/SDH直接受种子储存蛋白(SSP)基因的主要转录调控因子：碱性亮氨酸拉链(BZIP)转录因子(Tf)RISBZ1和DNA结合单指(DOF)转录因子RPBF的调控。OsLKR/SDH在胚乳的糊粉层和糊粉层中高表达。植物突变分析、体内反式激活报告分析和体外凝胶迁移率改变分析表明，识别RPBF的醇溶蛋白盒(AAAG)和识别RISBZ1的Gcn4基序(TGAG/CTCA)是OsLKR/SDH样SSP基因正确表达的重要顺式元件。然而，ProOsLKR/SDH中Gcn4基序的突变并没有改变空间表达模式，而ProGluB-1中Gcn4基序的突变确实改变了空间表达模式。降低RISBZ1或RPBF都会降低OsLKR/SDH水平，导致水稻籽粒中游离赖氨酸含量增加。这一结果与只有当这些转录因子同时被还原时才能观察到SSP显著减少的事实相反，这表明RISBZ1和RPBF分别以高和有限的冗余调控SSP基因和OsLKR/SDH。在OsLKR/SDH和SSP基因的调控中，Tf和顺式元件的组合是相同的，但它们的调控机制有明显的不同。
Lysine is the most deficient essential amino acid in cereal grains. A bifunctional lysine-degrading enzyme, lysine ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH), is one of the key regulators determining free lysine content in plants. In rice (Oryza sativa. L), a bifunctional OsLKR/SDH is predominantly present in seeds. Here, we show that OsLKR/SDH is directly regulated by major transcriptional regulators of seed storage protein (SSP) genes: the basic leucine zipper (bZIP) transcription factor (TF), RISBZ1, and the DNA-binding with one finger (DOF) transcription factor, RPBF. OsLKR/SDH was highly expressed in the aleurone and subaleurone layers of the endosperm. Mutation analyses in planta, trans-activation reporter assays in vivo and electrophorestic mobility shift assays in vitro showed that the RPBF-recognizing prolamin box (AAAG) and the RISBZ1-recognizing GCN4 motif (TGAG/CTCA) act as important cis-elements for proper expression of OsLKR/SDH like SSP genes. However, mutation of the GCN4 motif within ProOsLKR/SDH did not alter the spatial expression pattern, whereas mutation of the GCN4 motif within ProGluB-1 did alter spatial expression. Reducing either RISBZ1 or RPBF decreased OsLKR/SDH levels, resulting in an increase in free lysine content in rice grain. This result was in contrast to the fact that a significant reduction of SSP was observed only when these transcription factors were simultaneously reduced, suggesting that RISBZ1 and RPBF regulate SSP genes and OsLKR/SDH with high and limited redundancy, respectively. The same combinations of TF and cis-elements are involved in the regulation of OsLKR/SDH and SSP genes, but there is a distinct difference in their regulation mechanisms.