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. 2018 Oct;178(2):850-863.
doi: 10.1104/pp.18.00427. Epub 2018 Aug 22.

Transcriptome Analysis Identifies a Zinc Finger Protein Regulating Starch Degradation in Kiwifruit

Ai-di Zhang  1   2 , Wen-Qiu Wang  1   2 , Yang Tong  1 , Ming-Jun Li  3 , Donald Grierson  1   4 , Ian Ferguson  1   5 , Kun-Song Chen  1   2 , Xue-Ren Yin  6   2
Affiliations

Transcriptome Analysis Identifies a Zinc Finger Protein Regulating Starch Degradation in Kiwifruit

Ai-di Zhang et al. Plant Physiol. 2018 Oct.

Abstract

Ripening, including softening, is a critical factor in determining the postharvest shelf-life of fruit and is controlled by enzymes involved in cell wall metabolism, starch degradation, and hormone metabolism. Here, we used a transcriptomics-based approach to identify transcriptional regulatory components associated with texture, ethylene, and starch degradation in ripening kiwifruit (Actinidia deliciosa). Twelve differentially expressed structural genes, including seven involved in cell wall metabolism, four in ethylene biosynthesis, and one in starch degradation, and 14 transcription factors (TFs) induced by exogenous ethylene treatment and inhibited by the ethylene signaling inhibitor 1-methylcyclopropene were identified as changing in transcript levels during ripening. Moreover, analysis of the regulatory effects of differentially expressed genes identified a zinc finger TF, DNA BINDING WITH ONE FINGER (AdDof3), which showed significant transactivation on the AdBAM3L (β-amylase) promoter. AdDof3 interacted physically with the AdBAM3L promoter, and stable overexpression of AdBAM3L resulted in lower starch content in transgenic kiwifruit leaves, suggesting that AdBAM3L is a key gene for starch degradation. Moreover, transient overexpression analysis showed that AdDof3 up-regulated AdBAM3L expression in kiwifruit. Thus, transcriptomics analysis not only allowed the prediction of some ripening-regulating genes but also facilitated the characterization of a TF, AdDof3, and a key structural gene, AdBAM3L, in starch degradation.

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Figures

Figure 1.
Figure 1.
Effects of ethylene and 1-MCP treatment on kiwifruit ripening and softening. Fruit were treated with 100 μL L−1 ethylene (ETH), 1 μL L−1 1-MCP, or air (control [CK]) for 24 h at 20°C. A, Ethylene production of cv Hayward kiwifruit during storage. Error bars represent se from three replicates. B, TSS and starch content in cv Hayward fruit. For TSS and starch content, error bars represent se from 10 and three replicates, respectively. C, Firmness, cell wall material (CWM) content, cellulose content, hemicellulose content, and pectin content (covalent binding pectin [CBP], water soluble pectin [WSP], and ionic soluble pectin [ISP]) of fruit in storage. Error bars for firmness represent se based on 12 replicates; all others were from three replicates. FW, Fresh weight. lsd values represent lsd at P = 0.05.
Figure 2.
Figure 2.
Comparison of DEGs between control, ethylene-treated, and 1-MCP-treated kiwifruit. Fruit were treated with 100 μL L−1 ethylene (ETH), 1 μL L−1 1-MCP, or air (control [CK]) for 24 h at 20°C, and comparisons were made at 1 and 4 d. A, DEGs of 13 structural genes with putative function in kiwifruit ethylene biosynthesis, cell wall modification, and starch degradation. B, DEGs of 14 transcriptional factors. There were three replicates at each point. Transcript abundance is indicated by color. The names in black represent new genes, which were not included in the cv Hong Yang genome database; those in blue and red are published structural and TF genes, respectively.
Figure 3.
Figure 3.
Expression of structural genes in response to ethylene or 1-MCP treatment during kiwifruit ripening. Fruit were treated with 100 μL L−1 ethylene (ETH), 1 μL L−1 1-MCP, or air (control [CK]) for 24 h at 20°C. Gene expression was analyzed by RT-qPCR. Error bars represent se based on three replications. lsd values represent lsd at P = 0.05.
Figure 4.
Figure 4.
Expression of TFs in response to ethylene or 1-MCP treatment during kiwifruit ripening. Fruit were treated with 100 μL L−1 ethylene (ETH), 1 μL L−1 1-MCP, or air (control [CK]) for 24 h at 20°C. Gene expression was analyzed by RT-qPCR. A, Expression of putative activators. B, Expression of putative repressors. BEE, Brassinosteroid enhanced expression; GT, Trihelix TF. Error bars represent se based on three replications. lsd values represent lsd at P = 0.05.
Figure 5.
Figure 5.
Regulatory effects of TFs on promoters of ethylene biosynthesis, cell wall-modifying, and starch degradation genes as determined by dual-luciferase assays. The ratio of firefly luciferase and Renilla luciferase (LUC/REN) of the empty vector plus promoter was set as 1. SK represents the empty pGreen II 0029 62-SK vector. Error bars indicate se from three replicates (**, P < 0.01 and ***, P < 0.001).
Figure 6.
Figure 6.
Subcellular localization of AdDof3 and EMSA. A, Subcellular localization of AdDof3-GFP in transgenic Nicotiana benthamiana leaves (expressed with nucleus-located mCherry). AdDof3 was inserted into the pCAMBIA1300-sGFP vector. The GFP fluorescence of AdDof3-GFP is indicated. Bars = 25 μm. B, Oligonucleotides used for the EMSA with the Dof core sequences are in red. The mutated bases are indicated in green. C, Core sequences (AAAG/CTTT) of Dof protein-binding sites in the AdBAM3L promoter. D, EMSA of 3′ biotin-labeled dsDNA probes with the AdDof3 DNA-binding domain proteins. Recombinant AdDof3 was purified from E. coli cells and used for DNA-binding assays with P-abc, P-a, P-bc, P-c, P-ab, and mutated P-ΔaΔbc, P-Δabc, and P-aΔbc together with cold unlabeled competitor as the probes. Water was added in place of AdDof3 protein as a control.
Figure 7.
Figure 7.
Overexpression of AdBAM3L in kiwifruit plants. A, Five-month-old plants on MS medium. B, Schematic map of the AdBAM3L-pCAMBIA1301 construct and PCR analysis of the wild type (WT) and two independently regenerated transgenic lines. The positive control used a plasmid containing the AdBAM3L-pCAMBIA1301 construct as a template. C, Expression of AdBAM3L in the wild type and transgenic lines. D, GUS staining of wild-type and AdBAM3L transgenic plants. E, Starch content reflected by quinoneimine dye. The color intensity represents starch concentration. Positive control, d-Glc standard (Megazyme International Ireland); negative control, water. F, Starch content in wild-type and transgenic plant leaves. FW, Fresh weight. Error bars in C and F indicate se from three replicates (*, P < 0.05; **, P < 0.01; and ***, P < 0.001).
Figure 8.
Figure 8.
Transient overexpression of AdDof3 and its up-regulation of AdBAM3L in the core tissue of cv Hayward fruit. A, Schematic diagram for injection with differential color inks. The arrows show injection sites. B, GUS staining of kiwifruit core tissue segments injected with AdDof3-pCAMBIA1301-EHA105 or EHA105 at 1 d after injection. The segments were photographed separately. Bars = 100 μm. C, Gene expression of endogenous AdDof3 and AdBAM3L in immature kiwifruit at 80 DAFB. Injection of A. tumefaciens strain (GV3101) with the empty SK vector was the control, and the AdDof3 recombined SK vector was the treatment. D, Gene expression of endogenous AdDof3 and AdBAM3L in mature kiwifruit harvested at 170 DAFB. Error bars in C and D indicate se from three replicates (*, P < 0.05 and **, P < 0.01).
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