Ballas N, Wong L M, Theologis A
Plant Gene Expression Center, Albany, CA 94710.
J Mol Biol. 1993 Oct 20;233(4):580-96. doi: 10.1006/jmbi.1993.1537.
The plant hormone auxin transcriptionally activates early genes in pea. We have developed a transient assay system using protoplasts of auxin-responsive pea seedling cells to define the auxin-responsive element, AuxRE, of the early auxin-induced PS-IAA4/5 gene. The auxin responsive protoplasts show an authentic hormonal response identical to that observed in intact pea tissue, with respect to rapidity, specificity and cycloheximide (CHX) inducibility of the PS-IAA4/5 transcript. The hormone also mediates rapid and specific induction of chloramphenicol acetyltransferase (CAT) activity in protoplasts transfected with a chimeric IAA4/5-CAT gene. The IAA-induced CAT activity is developmentally regulated and is observed only in protoplasts derived from auxin-responsive regions of the pea seedling. Extensive deletion analysis of the PS-IAA4/5 promoter defined a promoter region between -318 and -154 that confers auxin inducibility. This AuxRE mediates auxin-inducible CAT activity in pea cells driven by the non auxin-responsive CaMV 35S minimal promoter. The functionality of this promoter region as an AuxRE was further verified in tobacco plants using IAA4/5-GUS gene fusions. The AuxRE contains two domains: Domain A acts as an auxin switch; domain B has an enhancer-like activity. The A and B domains contain the highly conserved sequences found in various auxin-regulated genes (T/GGT-CCCAT (domain A) and C/AACATGGNC/AA/GTGTT/CT/CC/A (domain B)). DNase I footprinting reveals binding of nuclear proteins to the highly conserved sequence found in A and B domains. The sequence of the A domain does not correspond to any known regulatory elements found in other eukaryotic genes, and the data suggest that this conserved motif functions as an AuxRE. A model for the early transcriptional activation of the PS-IAA4/5 gene by IAA is discussed.
植物激素生长素可通过转录激活豌豆中的早期基因。我们利用生长素响应型豌豆幼苗细胞的原生质体开发了一种瞬时检测系统，以确定早期生长素诱导的PS-IAA4/5基因的生长素响应元件（AuxRE）。生长素响应型原生质体在PS-IAA4/5转录本的快速性、特异性和环己酰亚胺（CHX）诱导性方面，表现出与完整豌豆组织中观察到的真实激素反应相同。该激素还介导了用嵌合IAA4/5-CAT基因转染的原生质体中氯霉素乙酰转移酶（CAT）活性的快速和特异性诱导。IAA诱导的CAT活性受发育调控，仅在源自豌豆幼苗生长素响应区域的原生质体中观察到。对PS-IAA4/5启动子的广泛缺失分析确定了一个位于-318至-154之间的启动子区域，该区域赋予生长素诱导性。这个AuxRE在由非生长素响应型CaMV 35S最小启动子驱动的豌豆细胞中介导生长素诱导的CAT活性。使用IAA4/5-GUS基因融合在烟草植物中进一步验证了该启动子区域作为AuxRE的功能。AuxRE包含两个结构域：结构域A作为生长素开关；结构域B具有增强子样活性。A和B结构域包含在各种生长素调节基因中发现的高度保守序列（T/GGT-CCCAT（结构域A）和C/AACATGGNC/AA/GTGTT/CT/CC/A（结构域B））。DNase I足迹分析揭示了核蛋白与A和B结构域中发现的高度保守序列的结合。A结构域的序列与其他真核基因中发现的任何已知调控元件都不对应，数据表明这个保守基序作为AuxRE发挥作用。文中讨论了IAA对PS-IAA4/5基因早期转录激活的模型。
