Consensus co-expression network analysis identifies AdZAT5 regulating pectin degradation in ripening kiwifruit

Cell wall degradation and remodeling is the key factor causing fruit softening during ripening.To explore the mechanism underlying postharvest cell wall metabolism, a transcriptome analysis method for more precious prediction on functional genes was needed.Kiwifruits treated by ethylene (a conventional and effective phytohormone to accelerate climacteric fruit ripening and softening as kiwifruits) or air were taken as materials. Here, Consensus Coexpression Network Analysis (CCNA), a procedure evolved from Weighted Gene Co-expression Network Analysis (WGCNA) package in R, was applied and generated 85 consensus clusters from twelve transcriptome libraries. Advanced and comprehensive modifications were achieved by combination of CCNA and WGCNA with introduction of physiological traits, including firmness, cell wall materials, cellulose, hemicellulose, water soluble pectin, covalent binding pectin and ionic soluble pectin.As a result, six cell wall metabolisms related structural genes AdGAL1, AdMAN1, AdPL1, AdPL5, Adβ-Gal5, AdPME1 and four transcription factors AdZAT5, AdDOF3, AdNAC083, AdMYBR4 were identified as hub candidate genes for pectin degradation. Dual-luciferase system and electrophoretic mobility shift assays validated that promoters of AdPL5 and Adβ-Gal5 were recognized and trans-activated by transcription factor AdZAT5. The relatively higher enzyme activities of PL and β-Gal were observed in ethylene treated kiwifruit, further emphasized the critical roles of these two pectin related genes for fruit softening. Moreover, stable transient overexpression AdZAT5 in kiwifruit significantly enhanced AdPL5 and Adβ-Gal5 expression, which confirmed the in vivo regulations between transcription factor and pectin related genes.Thus, modification and application of CCNA would be powerful for the precious phishing the unknown regulators. It revealed that AdZAT5 is a key factor for pectin degradation by binding and regulating effector genes AdPL5 and Adβ-Gal5.