The heat shock transcription factors (Hsfs) play critical roles in plant responses to abiotic stresses. However, the mechanism of Hsfs in the regulation of pollen thermotolerance and their specific biological functions and signaling remain unclear. Herein, we demonstrated that HsfA1a played a key role in tomato pollen thermotolerance. hsfA1a mutants reduced, while its overexpression increased pollen themotolerance based on the pollen viability and germination. Analyzing the whole transcriptome by RNA-Seq data, we found that HsfA1a mainly regulated the genes involved in oxidative stress protection, protein homeostasis regulation and protein modification, as well as the response to biological stress in anthers under heat stress. The accumulation of reactive oxygen species in anthers were enhanced in hsfA1a mutants, while were decreased in HsfA1a overexpressing lines. Furthermore, the bounding of HsfA1a to the promoter region of genes involved in redox regulation (Cu/Zn-SOD, GST8 and MDAR1), protein repair (HSP17.6A, HSP70–2, HSP90–2 and HSP101) and degradation (UBP5, UBP18, RPN10a and ATG10) and regulated the expression of these genes in tomato anthers under heat stress. Our findings suggest that HsfA1a maintains pollen thermotolerance and cellular homeostasis by enhancing the antioxidant capacity and protein repair and degradation, ultimately improving pollen viability and fertility.

The Bcl-2-associated athanogene (BAG) family, a group of co-chaperones that share conservative domains in flora and fauna, is involved in plant growth, development, and stress tolerance. However, the function of tomato BAG genes on thermotolerance remains largely unknown. Herein, we found that the expression of BAG9 was induced during heat stress in tomato plants. Knockout of the BAG9 gene by CRISPR/Cas9 reduced, while its overexpression increased thermotolerance in tomato plants as reflected by the phenotype, photosynthesis rate, and membrane peroxidation. Heat-induced reactive oxygen species and oxidative/oxidized proteins were further increased in bag9 mutants and were normalized in BAG9 overexpressing plants. Furthermore, the activities of antioxidant enzymes, ascorbic acid (AsA)/dehydroascorbic acid (DHA), and reduced glutathione (GSH)/oxidized glutathione (GSSG) were reduced in bag9 mutants and were increased in BAG9 overexpressing plants under heat stress. Additionally, BAG9 interacted with Hsp20 proteins in vitro and in vivo. Accumulation of Hsp proteins induced by heat showed a reduction in bag9 mutants; meanwhile, it was increased in BAG9 overexpressing plants. Thus, BAG9 played a crucial role in response to heat stress by regulating cellular redox homeostasis and the stability of heat shock proteins.