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. 2022 Sep:40:59-68.
doi: 10.1016/j.jare.2021.11.019. Epub 2021 Dec 4.

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

Qiu-Yun Zhang  1 , Jun Ge  2 , Xin-Cheng Liu  1 , Wen-Qiu Wang  1 , Xiao-Fen Liu  1 , Xue-Ren Yin  3
Affiliations

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

Qiu-Yun Zhang et al. J Adv Res. 2022 Sep.

Abstract

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, 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.

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 Adβ-Gal5.

Keywords: AdPL5; AdZAT5; Adβ-Gal5; CCNA; Cell wall remodeling.

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Conflict of interest statement

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

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Graphical abstract
Fig. 1
Fig. 1
Transcriptome analysis and cluster-trait associations. (A) Dendrogram and CCNA clusters of transcriptome data. (B) Gene expression profiles of 85 clusters in 12 transcriptome libraries. (C) Interactions between 85 clusters and traits, including firmness, cell wall materials (CWM), cellulose, hemicellulose, water soluble pectin (WSP), covalent binding pectin (CBP) and ionic soluble pectin (ISP). (D) Cluster correlations of firmness trait. The others were listed in Fig. S2. (E and F) Different expressed genes in one day after ethylene treatment /one day after air control (ETH1d/CK1d) and four days after ethylene treatment /four days after air control (ETH4d/CK4d).
Fig. 2
Fig. 2
Gene significance of individual gene in candidate clusters with physiological traits. Physiological traits include firmness, cell wall materials (CWM), cellulose, water soluble pectin (WSP), covalent binding pectin (CBP) and ionic soluble pectin (ISP), with one for each subplot. The x axis in each subplot indicates the number of consensus cluster, while y axis indicates gene significance (GS). In GS.FIRMNESS, GS.CWM, GS.WSP and GS.CBP subplots, the red triangles refer to candidate genes, green dots refer to genes with GS higher than 0.9 or 0.8, blue dots refer to genes with GS lower than −0.9 or −0.8, purple dots refer to genes with GS between −0.8 to 0.8 or between −0.9 to 0.9. In GS.CELLULOSE and GS.ISP subplots, the red triangles refer to candidate genes, blue dots represent genes with GS higher than 0.8, green dots represent genes with GS lower than 0.8. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3
Fig. 3
Criteria of candidate genes screening. Including correlation coefficients among 40 candidate genes, their belongings to clusters, fold-change of one day after ethylene treatment /one day after air control (FC(ETH1d/CK1d)) and four days after ethylene treatment /four days after air control (FC(ETH4d/CK4d)), FPKM in ETH 4d. Gene names with red color and highlighted by red dots indicate 10 candidate genes. The grey dots refer to correlation coefficients between six candidate transcription factors and four candidate structural genes. The values are indicated by color bars. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4
Relative expression profiles and FPKM of candidate genes in ethylene treatment (ETH, 100 µL L−1 for 24 h at 20 °C), in air control (CK, 20 °C) and in 1-Methylcyclopropene treatment (1-MCP, 1 µL L−1, 20 °C). The expression profiles of AdDof3 and AdPL1, AdMAN1 were verified by Zhang et al., 2019. Error bars represent SE based on three replicates. Least Significant Difference (LSD) values represent LSD at p < 0.05.
Fig. 5
Fig. 5
Investigation and confirmation of regulations between candidate transcription factors and structural genes. (A) Regulatory verified by dual-luciferase assay. (B) Subcellular localization of AdZAT5. (C and D) Electrophoretic mobility shift assays (EMSA) of recombinant AdZAT5 protein binding cis-elements on AdPL5 and Adβ-Gal5 promoters. (E) Enzyme activities of pectate lyase and β-galactosidase. (F) Relative expression of AdZAT5, AdPL5, Adβ-Gal5 in transient overexpression ‘Xuxiang’ kiwifruit core tissue. The two ends of core tissue were injected with empty vector as control or AdZAT5 recombinant vector. Error bars in A indicate SE from three replicates (*, p < 0.05; **, p < 0.01; and ***, p < 0.001). Error bars represent SE based on three replicates. Least Significant Difference (LSD) values represent LSD at p < 0.05.
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