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

Translation elongation factor Tu (EF-Tu) is a conserved GTP-binding protein essential for protein translation in prokaryotes and eukaryotic organelles of mitochondria and plastids. EF-Tu also has a GTP/GDP-independent chaperone activity implicated in heat adaptation. Here, we report that Arabidopsis plastid EF-Tu, Rabe1b, rapidly became insoluble in vitro at a temperature as low as 35{degree sign}, but remained largely soluble in vivo at the elevated temperature. At a temperature above 41{degree sign}C, insolubilization of Rabe1b also occurred in vivo, with more than 90% becoming aggregated in 9 hours at 45{degree sign}C. Heat-induced aggregation would inactivate Rabe1b based on its established mode in protein translation. To determine the impact of heat-induced aggregation, we isolated an Arabidopsis rabe1b knockdown mutant and discovered that it was highly compromised in heat tolerance. Overexpression of constitutive GTP- or GDP-bound mutant Rabe1b in Arabidopsis or virus-induced silencing of Rabe1b in tomato also reduced heat tolerance. Compromised heat tolerance in Arabidopsis rabe1b mutant and in overexpression lines for constitutive GTP- or GDP-bound mutant Rabe1b proteins was associated with reduced plastid translation under heat stress. Arabidopsis rabe1b mutant was also compromised in 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 heat-sensitive plastid EF-Tu is a critical limiting factor of plant heat stress responses primarily as a plastid protein translation but also through a positive role in protein folding and retrograde signaling of nuclear heat-responsive gene expression.