Zheng Bo, MacDonald Tara M, Sutinen Sirkka, Hurry Vaughan, Clarke Adrian K
Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, 901 87 Umea, Sweden.
Planta. 2006 Oct;224(5):1103-15. doi: 10.1007/s00425-006-0292-2. Epub 2006 May 17.
ClpP4 is a nuclear-encoded plastid protein that functions as a proteolytic subunit of the ATP-dependent Clp protease of higher plants. Given the lack of viable clpP4 knockout mutants, antisense clpP4 repression lines were prepared to study the functional importance of ClpP4 in Arabidopsis thaliana. Screening of transformants revealed viable lines with up to 90% loss of wild type levels of ClpP4 protein, while those with > 90% were severely bleached and strongly retarded in vegetative growth, failing to reach reproductive maturity. Of the viable antisense plants, repression of clpP4 expression produced a pleiotropic phenotype, of which slow growth and leaf variegation were most prominent. Chlorosis was most severe in younger leaves, with the affected regions localized around the mid-vein and exhibiting impaired chloroplast development and mesophyll cell differentiation. Chlorosis lessened during leaf expansion until all had regained the wild type appearance upon maturity. This change in phenotype correlated with the developmental expression of ClpP4 in the wild type, in which ClpP4 was less abundant in mature leaves due to post-transcriptional/translational regulation. Repression of ClpP4 caused a concomitant down-regulation of other nuclear-encoded ClpP paralogs in the antisense lines, but no change in other chloroplast-localized Clp proteins. Greening of the young chlorotic antisense plants upon maturation was accelerated by increased light, either by longer photoperiod or by higher growth irradiance; conditions that both raised levels of ClpP4 in wild type leaves. In contrast, shift to low growth irradiance decreased the relative amount of ClpP4 in wild type leaves, and caused newly developed leaves of fully greened antisense lines to regain the chlorotic phenotype.
ClpP4是一种核编码的质体蛋白，作为高等植物ATP依赖型Clp蛋白酶的蛋白水解亚基发挥作用。鉴于缺乏可行的clpP4敲除突变体，制备了反义clpP4抑制系以研究ClpP4在拟南芥中的功能重要性。对转化体的筛选揭示了ClpP4蛋白水平损失高达90%的可行株系，而那些损失>90%的株系严重白化且营养生长严重受阻，无法达到生殖成熟。在可行的反义植物中，clpP4表达的抑制产生了多效性表型，其中生长缓慢和叶片杂色最为突出。黄化在较嫩的叶片中最为严重，受影响的区域位于中脉周围，表现出叶绿体发育受损和叶肉细胞分化受损。黄化在叶片扩展过程中减轻，直到成熟时所有叶片都恢复了野生型外观。这种表型变化与野生型中ClpP4的发育表达相关，其中由于转录后/翻译调控，ClpP4在成熟叶片中的丰度较低。ClpP4的抑制导致反义株系中其他核编码的ClpP旁系同源物伴随下调，但其他叶绿体定位蛋白没有变化。通过延长光周期或提高生长辐照度增加光照，加速了幼嫩黄化反义植物成熟时的变绿；这两种条件都会提高野生型叶片中ClpP4的水平。相反，转向低生长辐照度会降低野生型叶片中ClpP4的相对含量，并导致完全变绿的反义株系新长出的叶片恢复黄化表型。
