Heyl Alexander, Ramireddy Eswar, Brenner Wolfram G, Riefler Michael, Allemeersch Joke, Schmülling Thomas
Institute of Biology/Applied Genetics, Free University of Berlin, D-14195 Berlin, Germany.
Plant Physiol. 2008 Jul;147(3):1380-95. doi: 10.1104/pp.107.115436. Epub 2008 May 23.
The signal transduction of the phytohormone cytokinin is mediated by a multistep histidine-to-aspartate phosphorelay system. One component of this system are B-type response regulators, transcription factors mediating at least part of the response to cytokinin. In planta functional analysis of this family is hampered by the high level of functional redundancy of its 11 members. We generated a dominant repressor version of the Arabidopsis (Arabidopsis thaliana) response regulator ARR1 (ARR1-SRDX) using chimeric repressor silencing technology in order to study the extent of the contribution of B-type response regulators to cytokinin activities. In a protoplast test system, ARR1-SRDX suppressed ARR6:beta-glucuronidase reporter gene activation by different B-type ARRs. 35S:ARR1-SRDX transgenic Arabidopsis plants showed phenotypic changes reminiscent of plants with a reduced cytokinin status, such as a strongly reduced leaf size, an enhanced root system, and larger seeds. Several bioassays showed that 35S:ARR1-SRDX plants have an increased resistance toward cytokinin. The rapid induction of a large part of the cytokinin response genes was dampened. The transcript levels of more than 500 genes were more than 2.5-fold reduced in 35S:ARR1-SRDX transgenic seedlings, suggesting a broad function of B-type ARRs. Collectively, the suppression of pleiotropic cytokinin activities by a dominant repressor version of a B-type ARR indicates that this protein family is involved in mediating most, if not all, of the cytokinin activities in Arabidopsis. In addition, a role for B-type ARRs in mediating cross talk with other pathways is supported by the resistance of 35S:ARR1-SRDX seeds to phytochrome B-mediated inhibition of germination by far-red light. This study demonstrates the usefulness of chimeric repressor silencing technology to overcome redundancy in transcription factor families for functional studies.
植物激素细胞分裂素的信号转导由一个多步骤的组氨酸 - 天冬氨酸磷酸化传递系统介导。该系统的一个组成部分是B型响应调节因子，它们是介导对细胞分裂素至少部分响应的转录因子。由于该家族11个成员的功能冗余程度高，对其进行植物体内功能分析受到阻碍。我们利用嵌合阻遏物沉默技术生成了拟南芥响应调节因子ARR1的显性阻遏物版本（ARR1-SRDX），以研究B型响应调节因子对细胞分裂素活性贡献的程度。在原生质体测试系统中，ARR1-SRDX抑制了不同B型ARR对ARR6：β-葡萄糖醛酸酶报告基因的激活。35S：ARR1-SRDX转基因拟南芥植物表现出类似于细胞分裂素水平降低的植物的表型变化，如叶片大小大幅减小、根系增强和种子变大。几种生物测定表明，35S：ARR1-SRDX植物对细胞分裂素的抗性增加。大部分细胞分裂素响应基因的快速诱导受到抑制。在35S：ARR1-SRDX转基因幼苗中，500多个基因的转录水平降低了2.5倍以上，这表明B型ARR具有广泛的功能。总体而言，B型ARR的显性阻遏物版本对多效性细胞分裂素活性的抑制表明，该蛋白家族参与介导拟南芥中大部分（如果不是全部）细胞分裂素活性。此外，35S：ARR1-SRDX种子对远红光介导的光敏色素B抑制发芽的抗性支持了B型ARR在介导与其他途径的相互作用中的作用。这项研究证明了嵌合阻遏物沉默技术在克服转录因子家族冗余以进行功能研究方面的有用性。
