CaBBX9, an interaction partner of autophagy-related protein CaATG8c, negatively regulates the heat tolerance of pepper

● Pepper zinc-finger protein CaBBX9 interacts with autophagy-related protein CaATG8c and exhibits a transcriptional activity. ● Silencing and overexpression of CaBBX9 indicate that it negatively regulates heat tolerance of pepper plants. To explore the molecular mechanisms by which autophagy contributes to pepper’s heat tolerance, we previously identified the zinc-finger protein B-BOX 9/CONSTANS-LIKE 13 (CaBBX9/CaCOL13) as an interaction partner of the autophagy related protein (ATG) CaATG8c, a core component in autophagy. However, the involvement of CaBBX9 in both autophagy and heat tolerance remains unclear. In this study, we further confirmed the interaction between CaBBX9 and CaATG8c and defined the interaction regions of CaBBX9 as CONSTANS, CONSTANS-Like, and TOC1 (CCT) domain and the fragment region. The expression of CaBBX9 can be induced by heat treatment. CaBBX9 is co-localized with CaATG8c in the nucleus and exhibits a transcriptional activity. When the expression of CaBBX9 is silenced, the heat tolerance of pepper is enhanced, shown by the decrement of MDA content, H 2 O 2 and dead cells accumulation, and relative electrolyte leakage, along with the increment of chlorophyll content and expression level of heat-tolerance-related genes. Overexpression of CaBBX9 in tomatoes displays the opposite effects. Taken together, we demonstrate that CaBBX9 negatively regulates the heat tolerance of peppers by exacerbating oxidative damage and inhibiting the expression of heat-related genes. Our findings provide a new clue for guiding crop breeding for pepper tolerance to heat stress.