Plastid Translation Elongation Factor Tu Is Prone to Heat-Induced Aggregation Despite Its Critical Role in Plant Heat Tolerance

Abstract

Translation elongation factor Tu (EF-Tu) is a conserved GTP-binding protein essential for protein translation in prokaryotes and in eukaryotic mitochondria and plastids. EF-Tu also has a GTP/GDP-independent chaperone activity that may function in acclimation to heat. Here, we report that the Arabidopsis (<i>Arabidopsis thaliana</i>) plastid EF-Tu, Rabe1b, rapidly becomes insoluble at temperatures as low as 35°C in vitro and 41°C in vivo, with more than 90% aggregation after 9 h at 45°C in vivo. Based on its established function in protein translation, heat-induced aggregation likely inactivates Rabe1b. To determine the effect of heat-induced aggregation, we isolated an Arabidopsis <i>rabe1b</i> knockdown mutant and discovered it to be highly compromised in heat tolerance. Overexpression of constitutive GTP- or GDP-bound mutant forms of Rabe1b in Arabidopsis and virus-induced silencing of <i>Rabe1b</i> in tomato (<i>Solanum lycopersicum</i>) also reduced heat tolerance. Compromised heat tolerance in the Arabidopsis <i>rabe1b</i> mutant and in the lines overexpressing constitutive GTP- or GDP-bound mutant Rabe1b proteins was associated with reduced plastid translation under heat stress. The Arabidopsis <i>rabe1b</i> mutant also showed compromised heat-induced expression of <i>HsfA2</i> and its target genes. Constitutive overexpression of HsfA2 activated its target genes but only partially restored the heat tolerance of the <i>rabe1b</i> mutant. These results strongly suggest that the plastid protein EF-Tu is heat sensitive and acts as a critical limiting factor in plant heat stress responses, primarily functioning in plastid protein translation but also in protein folding and retrograde signaling of nuclear heat-responsive gene expression.