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

INTRODUCTION: Cell wall degradation and remodeling is the key factor causing fruit softening during ripening. OBJECTIVES: To explore the mechanism underlying postharvest cell wall metabolism, a transcriptome analysis method for more precious prediction on functional genes was needed. METHODS: 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. RESULTS: As a result, six cell wall metabolisms related structural genes AdGAL1, AdMAN1, AdPL1, AdPL5, Adß-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 Adß-Gal5 were recognized and trans-activated by transcription factor AdZAT5. The relatively higher enzyme activities of PL and ß-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 Adß-Gal5 expression, which confirmed the in vivo regulations between transcription factor and pectin related genes. CONCLUSION: 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 Adß-Gal5.

Transcriptome Analysis Revealed the Roles of Carbohydrate Metabolism on Differential Acetaldehyde Production Capacity in Persimmon Fruit in Response to High-CO2 Treatment.

Persimmon (Diospyros kaki Thunb.) fruit is unique due to the continuous accumulation of soluble tannins during fruit development in most cultivars, which causes undesired astringency. High-CO2 treatment was the most effective widely used method for astringency removal. However, differential effects of high-CO2 treatment between cultivars were observed and the molecular basis remained inclusive. Previously, one cultivar ("Luoyangfangtianshengshi," LYFTSS) showed rapid deastringency, while two cultivars ("Shijiazhuanglianhuashi," SJZLHS; "Laopige," LPG) showed slow deastringency in response to high-CO2 (95% CO2) treatment. In this study, the metabolites (acetaldehyde and ethanol) related to deastringency were further analyzed and both acetaldehyde and ethanol were higher in SJZLHS and LYFTSS than that in LPG, where acetaldehyde was undetectable. Based on the RNA-seq data, the weighted gene coexpression network analysis (WGCNA) revealed that one module, comprised of 1773 unigenes, significantly correlated with the contents of acetaldehyde and ethanol (P < 0.001). Further analysis based on the acetaldehyde metabolism pathway indicated that the differentially expressed structural genes, including previously characterized DkADH and DkPDC and also their upstream members (e.g., PFK, phosphofructokinase), showed positive correlations with acetaldehyde production. Quantitative analysis of the precursor substances indicated that sucrose, glucose, and fructose exhibited limited differences between cultivar except for malic acid. However, the content of malic acid is much less than the total soluble sugar content. To verify the correlations between these genes and acetaldehyde production, the fruit from 14 more cultivars were collected and treated with high CO2. After the treatment, acetaldehyde contents in different cultivars ranked in 30.4-255.5 µg/g FW. Real-time polymerase chain reaction (PCR) and correlation analysis indicated that the EVM0002315 (PFK) gene, belonging to carbohydrate metabolism, was significantly correlated with acetaldehyde content in fruit. Thus, it could be proposed that the differentially expressed carbohydrate metabolism related genes (especially PFK) are the basis for the variance of acetaldehyde production among different persimmon cultivars.