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 (Arabidopsis thaliana) 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 rabe1b 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 Rabe1b in tomato (Solanum lycopersicum) also reduced heat tolerance. Compromised heat tolerance in the Arabidopsis rabe1b 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 rabe1b mutant also showed compromised heat-induced expression of HsfA2 and its target genes. Constitutive overexpression of HsfA2 activated its target genes but only partially restored the heat tolerance of the rabe1b 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.