Two ethylene-responsive transcription factors, AdVAL2 and AdKAN2, regulate early steps in kiwifruit starch degradation

Abstract

Starch degradation is considered as an early step in the initiation of kiwifruit postharvest ripening and softening after ethylene treatment, which is driven by various transcription factors (TFs) and genes in the carbohydrate pathway. Studies regarding starch degradation have been conducted after ethylene treatment then removal, but early ethylene responsive genes and their function in triggering starch degradation remains unclear. In this study, a continuous sampling approach was conducted with 2 h intervals throughout 24 h treatment. As a result, starch degradation and accumulation of total soluble solid were accelerated after 10 h ethylene treatment. Based on RNA-seq and Real-time quantitative PCR (RT-qPCR) data, three β-Amylase genes, AdBAM3, AdBAM3.1 and AdBAM3L, were significantly up-regulated in ethylene treated samples. Weighted Gene Co-expression Network Analysis (WGCNA) identified TFs that are highly correlated with these three BAM genes, based on gene expression patterns. AdVAL2 (a B3 domain-containing transcription factor) and AdKAN2 (a MYB-like domain-containing transcription factor) were found to transcriptionally repress the promoter activity of AdBAM3 and AdBAM3L, respectively. Additionally, the expression of both AdVAL2 and AdKAN2 was observed to be repressed by ethylene treatment. Transient overexpression analysis confirmed the inhibitory effect of AdVAL2 and AdKAN2 on the expression of BAM genes, resulting in starch accumulation in kiwifruit. Altogether, continuous sampling during ethylene exposure has revealed that these early ethylene responsive TFs could be the potential triggers for starch degradation.