Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (1): 156-169.doi: 10.3864/j.issn.0578-1752.2025.01.012

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Functional Analysis of the Autophagy Gene ATG8f in the Grapevine

GE Yi1,2(), ZHENG QiuLing3,4, CHEN MengXia1,2, XIA JiaXin1,2, FANG Xiang2,5,*(), TANG MeiLing3,4, FANG JingGui1,2,6, SHANGGUAN LingFei1,2,6,*()   

  1. 1 Department of Horticulture, Nanjing Agricultural University, Nanjing 210095
    2 Fruit Crop Genetic Improvement and Seedling Propagation Engineering Research Center of Jiangsu Province, Nanjing 210095
    3 Yantai Academy of Agricultural Sciences in Shandong Province, Yantai 264003, Shandong
    4 Yantai Key Laboratory of Quality Safety and Nutrition of Characteristic Fruits, Yantai 264003, Shandong
    5 School of Agronomy and Horticulture, Jiangsu Vocational College of Agriculture and Forestry, Jurong 212400, Jiangsu
    6 Collaborative Innovation Center for Wine Industry Technology of Ningxia Helan Mountain Eastern Foothills, Yinchuan 750299
  • Received:2024-06-04 Accepted:2024-07-30 Online:2025-01-01 Published:2025-01-07
  • Contact: FANG Xiang, SHANGGUAN LingFei

Abstract:

【Objective】This study aims to investigate the role of the grape autophagy gene ATG8f in regulating copper stress tolerance in plants, thereby laying the foundation for elucidating copper tolerance mechanisms and constructing regulatory networks. 【Method】Based on multi-omics sequencing analysis of Shine Muscat grapevine under copper stress, the autophagy gene VlATG8f was identified and its sequence characteristics were analyzed. The CDS sequence of VlATG8f was cloned and an overexpression vector was constructed. The subcellular localization of the VlATG8f protein was studied through transient expression in tobacco. Heterologous expression of VlATG8f in Arabidopsis was achieved using the floral dip method. Seed germination rate and root length were measured to compare the growth and development of wild-type and transgenic lines under copper stress. Real-time quantitative PCR was used to analyze the expression levels of related antioxidant genes, and yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays were used to preliminarily screen for VlATG8f- interacting proteins. 【Result】The CDS of VlATG8f was 393 bp, encoding 130 amino acids. Bioinformatics analysis revealed that the ATG8f protein sequences in Arabidopsis, tomato, apple, pear, peach, and grapevine were relatively conserved, all containing a Ubl_ATG8 conserved domain, indicating potential functional were conserved. Subcellular localization showed that the VlATG8f protein was located on both the nucleus and cell membrane. Under normal conditions, both wild-type and VlATG8f-overexpressing lines exhibited healthy growth with green leaves. Under copper stress, the transgenic lines were less stunted and exhibited milder symptoms of chlorosis, leaf curling, reduced root hair size, and increased lateral roots compared to wild-type plants. Overexpression of VlATG8f in Arabidopsis resulted in less growth inhibition under copper stress, higher seed germination rate, longer root, and lower accumulation of hydrogen peroxide (H2O2), indicating enhanced copper tolerance. Additionally, the expression of antioxidant genes AtCSD1 and AtCCS1 increased in VlATG8f-overexpressing plants under copper stress, while the expression of the key ROS synthesis gene RbohB decreased, promoting ROS scavenging. Yeast two-hybrid screening identified 23 potential interacting proteins with VlATG8f. BiFC further confirmed strong interactions between VlATG8f and proteins such as PYL8, MnSOD, AUX22D, bZIP17, and COT44. 【Conclusion】Overexpression of VlATG8f may enhance copper tolerance in transgenic lines by strengthening the antioxidant system, reducing ROS synthesis, and promoting the synthesis of plant hormones, thereby mitigating copper toxicity in plants.

Key words: grapevine, autophagy, copper stress, VlATG8f, antioxidant-related genes

Table 1

The primers used for this experiment"

引物名称
Primers name
正向引物
Forward primers (5′-3′)
反向引物
Reverse primers (5′-3′)
用途
Application
VlATG8f-F/R TTTGCTGATGAAGGAGGAATGGC TCGACAATCACCGGAATCCTGT 拟南芥定量引物
Arabidopsis thaliana quantitative primers
AtRbohB-F/R CCGGGGATGATTACCTCAGC CGATGTCAATGCCGCTCTTG
AtGPX6-F/R CAAAGCTGCCCCAGTCTACA GGTGAGGTAGTTGGTGCGAA
AtCSD1-F/R CCCTGAGGATGCTAATCGACAT TGGCAATCAGTGATTGTGAAGG
AtTRX1-F/R ACATGGAACGAGCAGCTTCA GCCTGTATCGCCCAATCACT
AtCCS1-F/R GCAAACTGGTCGAAAAGCTC GTCAGGGCCTTTGAATTCTG
AtP5CS1-F/R GCAGCTTTGCGGATCTTCAG GATATGGGGCTCTTCGGGTG
AtMDAR4-F/R GGAATGCGGTTGTTATCGGC ACCAAGTCGGCTGGTAAGTG
AtPEX4-F/R CTCCTTACGAAGGCGGTGTT TTCTTAGGCATAGCGGCGAG 拟南芥定量引物(Actin)
Arabidopsis thaliana quantitative primers
Hyg-F/R GGTCGCGGAGGCTATGGATGC GCTTCTGCGGGCGATTTGTGT 转基因拟南芥鉴定
Identification of transgenic Arabidopsis thaliana
1302-VlATG8f GAAGATCTATGATTTTTGCTGATGAAGGAGGA GGACTAGTATGTGGTATCTCATATCCAAACGT 构建植物过表达载体
Construction of plant overexpression vectors
1305-VlATG8f AGGACAGCCCAGATCACTAGTATGATTTTTGCTGATGAAGGAGGA GCTCACCATGGATCCCCCGGGATGTGGTATCTCATATCCAAACGT 构建亚细胞定位载体
Construction of subcellular localized vectors
BD-VlATG8f ATGGCCATGGAGGCCGAATTCATGATTTTTGCTGATGAAGGAGGA CCGCTGCAGGTCGACGGATCCATGTGGTATCTCATATCCAAACGT 构建BD载体
Construction of the BD vector

Fig. 1

Observation of autophagy in grapevine leaves under high-ECS and the expression levels of related gene and protein A: Observation of autophagosomes by transmission electron microscopy. Arrows indicate autophagosomes, Bar=2 μm; B: Counts of the number of autophagosomes; C: Expression levels of autophagy genes under high copper stress; D: ATG8f protein content under high-ECS. Different lowercase letters indicate significant differences at the P<0.05 level. The same as below"

Fig. 2

The bioinformatic analysis of VlATG8f and its subcellular localization A: Phylogenetic tree analysis; B: Homology analysis. VlATG8f was marked with an asterisk; C: Subcellular localization. At the far left of images show cells with green GFP signal. mCherry was red mCherry signal. Light was bright-field images of the same cells, merged images of a cinfocal fluorescence image and a bright field image. The red triangles point to the nucleus. Bar=50 μm"

Fig. 3

Screening and identification of Arabidopsis overexpression lines A and B: Selection of overexpressing transgenic plants; C: RT-qPCR analysis of overexpressed transgenic plants"

Fig. 4

Phenotypes of WT and transgenic Arabidopsis overexpression lines under Cu stress A: Phenotypes of different lines; B: The statistical analysis of germination rate; C: The statistical analysis of root length"

Fig. 5

Effect of overexpression of VlATG8f on the antioxidant system A: DAB staining results; B: Expression levels of antioxidant-related genes in overexpressed VlATG8f lines"

Fig. 6

Screening and validation of the candidate VlATG8f interaction proteins A: Toxicity and transcriptional activation detection assays of VlATG8f protein; B: Y2H validation of proteins interacting with VlATG8f; C: BiFC assay of VlATG8f interaction candidate proteins in B. The red triangles were represented the nuclei of cells. An irregular polygon surrounded by a yellow curve was the cell membrane. Bar=20 μm"

Table 2

The screened candidate interaction proteins of VlATG8f"

编号
Code
基因
Gene
蛋白ID
Protein ID
描述
Description
1 VvDRIP2 XP_010651942.1 E3泛素蛋白连接酶E3 ubiquitin protein ligase DRIP2
2 VvsGR1 XP_002282183.1 滞绿蛋白1,叶绿体Protein STAY-GREEN 1, chloroplastic
3 VvPsaD XP_002281825.1 光系统Ⅰ反应中心亚基Ⅱ,叶绿体Photosystem Ⅰ reaction center subunit Ⅱ, chloroplastic
4 VvGST XP_002262842.1 谷胱甘肽S-转移酶Glutathione S-transferase
5 VvRHF2a XP_010653244.1 E3泛素蛋白连接酶RHF2A亚型X2 E3 ubiquitin-protein ligase RHF2A isoform X2
6 VvAUX22D XP_002281696.1 生长素诱导蛋白22D Auxin-induced protein 22D
7 VvPYL8 XP_010659134.1 脱落酸受体PYL8 Abscisic acid receptor PYL8
8 VvHIPP39 XP_002274512.1 重金属相关异戊二烯化植物蛋白39 Heavy metal-associated isoprenylated plant protein 39
9 VvbZIP17 XP_010653644.1 bZIP转录因子17 bZIP transcription factor 17
10 VvGOLS1 XP_002265947.1 半乳糖醇合酶1 Galactinol synthase 1
11 VvCOT44 XP_002283263.1 半胱氨酸蛋白酶COT44 Cysteine proteinase COT44
12 VvsDH6 XP_002268190.1 琥珀酸脱氢酶亚基6,线粒体Succinate dehydrogenase subunit 6, mitochondrial
13 VvALS1 XP_002274073.1 乙酰乳酸合酶1,叶绿体Acetolactate synthase 1, chloroplastic
14 VvPGDH3 XP_002273552.1 D-3-磷酸甘油酸脱氢酶3,叶绿体D-3-phosphoglycerate dehydrogenase 3, chloroplastic
15 VvEXO XP_002285762.1 磷酸诱导酶Phosphoinductase 1
16 VvPP6-ARS-C XP_002268442.1 丝氨酸/苏氨酸蛋白磷酸酶6调节锚蛋白重复亚基C
Serine/threonine-protein phosphatase 6 regulatory ankyrin repeat subunit C
17 VvDRM homolog 4 XP_003632757.1 休眠相关蛋白同源物4亚型X2 Dormancy-associated protein homolog 4 isoform X2
18 VvOEE1 XP_002274796.1 放氧增强蛋白1,叶绿体Oxygen-evolving enhancer protein 1, chloroplastic
19 VvFKBP13 XP_002272628.1 肽基脯氨酰顺反异构酶FKBP13,叶绿体Peptidyl-prolyl cis-trans isomerase FKBP13, chloroplastic
20 VvMnSOD NP_001268135.1 锰超氧化物歧化酶Manganese superoxide dismutase
21 VvVAMP711 XP_002285207.1 囊泡相关膜蛋白711 Vesicle-associated membrane protein 711
22 VvPAT2 XP_019072271.1 蛋白驱动蛋白轻链相关2 Protein kinesin light chain-related 2
23 VvHCF136 XP_002278958.1 光系统Ⅱ稳定性/组装因子HCF136,叶绿体
Photosystem Ⅱ stability/assembly factor HCF136, chloroplastic
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