Korth K L, Jaggard D A, Dixon R A
Department of Plant Pathology, 217 Plant Science Building, University of Arkansas, Fayetteville, AR 72701, USA.
Plant J. 2000 Aug;23(4):507-16. doi: 10.1046/j.1365-313x.2000.00821.x.
In plants, the first committed step in the cytosolic pathway for biosynthesis of isoprenoids is catalysed by 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). We have added an eight amino-acid-residue epitope tag to a potato (Solanum tuberosum L.) HMGR isoform and expressed this novel protein (HMGR-FLAG) in transgenic plants. Despite high levels of transcript accumulation in all leaf stages of transgenic plants, high levels of HMGR-FLAG protein were found only in apical meristematic tissue, suggesting post-translational regulation of potato HMGR affected by plant development. Protein immunoblots, and determination of enzymatic activity and transcript accumulation in the HMGR-FLAG transgenic and the non-transgenic parental plant lines, show that HMGR levels decrease dramatically in the dark. Again, the mechanism of this control occurs at a post-translational level. After 2.5 h in darkness, levels of HMGR-FLAG are approximately one-half of those in plants in the light; protein levels recover rapidly when dark-treated plants are returned to the light. In non-transgenic plants, hmg transcript levels are reduced in the dark, whereas dark treatments do not affect transgene hmg transcripts expressed under the control of a constitutive promoter. Furthermore, transcripts for HMGR-FLAG remain associated with polyribosomes in dark-treated tissues. Addition of inhibitors of cysteine proteases during microsomal protein extraction is required for recovery of immunoreactive HMGR-FLAG. The epitope-tagged isozyme has been used to show for the first time that a regulated decrease in plant HMGR activity correlates closely with a loss of the HMGR protein. We have used whole plants to demonstrate that developmental and light-regulated control of HMGR occurs post-translationally in vivo.
在植物中，异戊二烯类生物合成的胞质途径中的第一个关键步骤由3-羟基-3-甲基戊二酰辅酶A还原酶（HMGR）催化。我们在一种马铃薯（Solanum tuberosum L.）HMGR同工型上添加了一个八氨基酸残基的表位标签，并在转基因植物中表达了这种新型蛋白质（HMGR-FLAG）。尽管转基因植物在所有叶片发育阶段都有高水平的转录物积累，但仅在顶端分生组织中发现了高水平的HMGR-FLAG蛋白，这表明马铃薯HMGR的翻译后调控受植物发育的影响。蛋白质免疫印迹以及对HMGR-FLAG转基因和非转基因亲本株系中酶活性和转录物积累的测定表明，HMGR水平在黑暗中急剧下降。同样，这种调控机制发生在翻译后水平。在黑暗中处理2.5小时后，HMGR-FLAG的水平约为光照下植物中水平的一半；当黑暗处理的植物重新置于光照下时，蛋白质水平迅速恢复。在非转基因植物中，hmg转录物水平在黑暗中降低，而黑暗处理不影响在组成型启动子控制下表达的转基因hmg转录物。此外，HMGR-FLAG的转录物在黑暗处理的组织中仍与多核糖体相关联。在微粒体蛋白质提取过程中添加半胱氨酸蛋白酶抑制剂是恢复免疫反应性HMGR-FLAG所必需的。带有表位标签的同工酶首次被用于表明植物HMGR活性的调节性降低与HMGR蛋白的丧失密切相关。我们利用整株植物证明了HMGR的发育和光调控在体内发生在翻译后水平。
