Crafts-Brandner S J, Klein R R, Klein P, Hölzer R, Feller U
USDA-ARS Western Cotton Research Laboratory, Phoenix, AZ 85040-8830, USA.
Planta. 1996;200(3):312-8. doi: 10.1007/BF00200298.
Our objective was to determine the coordination of transcript and/or protein abundances of stromal enzymes during leaf senescence. First trifolioliate leaves of Phaseolus vulgaris L. plants were sampled beginning at the time of full leaf expansion; at this same time, half of the plants were switched to a nutrient solution lacking N. Total RNA and soluble protein abundances decreased after full leaf expansion whereas chlorophyll abundance remained constant; N stress enhanced the decline in these traits. Abundances of ribulose-1,5-bisposphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39), Rubisco activase and phosphoribulokinase (Ru5P kinase; EC 2.7.1.19) decreased after full leaf expansion in a coordinated manner for both treatments. In contrast, adenosine diphosphate glucose (ADPGlc) pyrophosphorylase (EC 2.7.7.27) abundance was relatively constant during natural senescence but did decline similar to the other enzymes under N stress. Northern analyses indicated that transcript abundances for all enzymes declined markedly on a fresh-weight basis just after full leaf expansion. This rapid decline was particularly strong for the Rubisco small subunit (rbcS) transcript. The decline was enhanced by N stress for rbcS and Rubisco activase (rca), but not for Ru5P kinase (prk) and ADPGlc pyrophosphorylase (agp). Transcripts of the Clp protease subunits clpC and clpP declined in abundance just after full leaf expansion, similar to the other mRNA species. When Northern blots were analyzed using equal RNA loads, rbcS transcripts still declined markedly just after full leaf expansion whereas rca and clpC transcripts increased over time. The results indicated that senescence was initiated near the time of full leaf expansion, was accelerated by N stress, and was characterized by large decline in transcripts of stromal enzymes. The decreased mRNA abundances were in general associated with steadily declining stromal protein abundances, with ADPGlc pyrophosphorylase being the notable exception. Transcript analyses for the Clp subunits supported a recent report (Shanklin et al., 1995, Plant Cell 7: 1713-1722) indicating that the Clp protease subunits were constitutive throughout development and suggested that ClpC and ClpP do not function as a senescence-specific proteolytic system in Phaseolus.
我们的目标是确定叶片衰老过程中基质酶转录本和/或蛋白质丰度的协调性。从菜豆植株完全展开叶片时开始采集其三出复叶；此时，将一半植株转移至缺氮的营养液中。完全展开叶片后，总RNA和可溶性蛋白质丰度下降，而叶绿素丰度保持不变；氮胁迫加剧了这些性状的下降。两种处理下，完全展开叶片后，1,5-二磷酸核酮糖羧化酶/加氧酶（Rubisco；EC 4.1.1.39）、Rubisco活化酶和磷酸核酮糖激酶（Ru5P激酶；EC 2.7.1.19）的丰度以协调的方式下降。相反，在自然衰老过程中，二磷酸腺苷葡萄糖（ADPGlc）焦磷酸化酶（EC 2.7.7.27）的丰度相对恒定，但在氮胁迫下其下降情况与其他酶类似。Northern分析表明，完全展开叶片后，所有酶的转录本丰度以鲜重计均显著下降。这种快速下降在Rubisco小亚基（rbcS）转录本中尤为明显。氮胁迫增强了rbcS和Rubisco活化酶（rca）的下降，但对Ru5P激酶（prk）和ADPGlc焦磷酸化酶（agp）没有影响。Clp蛋白酶亚基clpC和clpP的转录本在完全展开叶片后丰度下降，与其他mRNA种类类似。当使用等量RNA上样量分析Northern印迹时，完全展开叶片后rbcS转录本仍显著下降，而rca和clpC转录本随时间增加。结果表明，衰老在完全展开叶片时开始，氮胁迫加速衰老，其特征是基质酶转录本大幅下降。mRNA丰度降低通常与基质蛋白丰度稳步下降相关，ADPGlc焦磷酸化酶是明显的例外。对Clp亚基的转录本分析支持了最近的一份报告（Shanklin等人，1995年，《植物细胞》7: 1713 - 1722），该报告表明Clp蛋白酶亚基在整个发育过程中都是组成型的，并表明ClpC和ClpP在菜豆中不是作为衰老特异性蛋白水解系统发挥作用。
