Abstract
As sessile organisms, plants are constantly exposed to a wide spectrum of stress conditions such as high temperature, which causes protein misfolding. Misfolded proteins are highly toxic and must be efficiently removed to reduce cellular proteotoxic stress if restoration of native conformations is unsuccessful. Although selective autophagy is known to function in protein quality control by targeting degradation of misfolded and potentially toxic proteins, its role and regulation in heat stress responses have not been analyzed in crop plants. In the present study, we found that heat stress induced expression of autophagy-related (ATG) genes and accumulation of autophagosomes in tomato plants. Virus-induced gene silencing (VIGS) of tomato ATG5 and ATG7 genes resulted in increased sensitivity of tomato plants to heat stress based on both increased development of heat stress symptoms and compromised photosynthetic parameters of heat-stressed leaf tissues. Silencing of tomato homologs for the selective autophagy receptor NBR1, which targets ubiquitinated protein aggregates, also compromised tomato heat tolerance. To better understand the regulation of heat-induced autophagy, we found that silencing of tomato ATG5, ATG7, or NBR1 compromised heat-induced expression of not only the targeted genes but also other autophagy-related genes. Furthermore, we identified two tomato genes encoding proteins highly homologous to Arabidopsis WRKY33 transcription factor, which has been previously shown to interact physically with an autophagy protein. Silencing of tomato WRKY33 genes compromised tomato heat tolerance and reduced heat-induced ATG gene expression and autophagosome accumulation. Based on these results, we propose that heat-induced autophagy in tomato is subject to cooperative regulation by both WRKY33 and ATG proteins and plays a critical role in tomato heat tolerance, mostly likely through selective removal of heat-induced protein aggregates.
Highlights
Autophagy is a highly conserved intracellular degradation system in eukaryotes for removal and recycling of cytoplasmic components including damaged proteins and organelles (Klionsky, 2005)
IDENTIFICATION OF TOMATO ATG5, ATG7, AND NBR1 GENES To analyze the role of autophagy in tomato heat tolerance, we chose first to focus on tomato ATG5 and ATG7 as potential targets for gene silencing as their products are required for the core process of autophagy and mutants of their Arabidopsis homologs, which are single-copy genes, have been widely used for functional analysis of autophagy (Yoshimoto, 2010; Lai et al, 2011b; Zhou et al, 2013)
The heat tolerance of autophagy-suppressed tomato plants due to silencing of ATG5 and ATG7 genes was compromised based on their increased morphological symptoms associated with enhanced defects in the efficiency and capacity of photosynthesis after heat stress (Figures 5–7)
Summary
Autophagy is a highly conserved intracellular degradation system in eukaryotes for removal and recycling of cytoplasmic components including damaged proteins and organelles (Klionsky, 2005). Autophagy is active at very low levels but is highly inducible in responses to stress and extracellular cues (He and Klionsky, 2009). In yeast and animal systems, the serine/threonine protein kinase TOR (target of rapamycin) functions as a central inhibitor of autophagosome formation. Inhibition of TOR leads to activation of ATG1, which can bind ATG13 and ATG17 with increased affinities to promote assembly of the ATG1-ATG13-ATG17 scaffold and initiation of autophagosome formation through recruitment of multiple ATG proteins (He and Klionsky, 2009). A delayed and protracted autophagic response, relies on activation of specific transcription programs involving stress-responsive transcription factors
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