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Journal of Integrative Agriculture  2025, Vol. 24 Issue (5): 1831-1843    DOI: 10.1016/j.jia.2025.03.003
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Genome-wide analysis of RAD23 gene family and a functional characterization of AcRAD23D1 in drought resistance in Actinidia

Xiaoli Zhang, Daolin Ye, Xueling Wen, Xinling Liu, Lijin Lin, Xiulan Lü, Jin Wang, Qunxian Deng, Hui Xia, Dong Liang#

College of Horticulture, Sichuan Agricultural University, Chengdu 611130, China

 Highlights 

Six AcRAD23 genes were identified in kiwifruit.
All AcRAD23 genes showed differential expression patterns under various stress conditions.
AcRAD23D1 may play a positive role in regulating kiwifruit’s response to drought stress.  

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摘要  

UBL-UBA蛋白作为26 S泛素蛋白降解途径中的转运蛋白,在植物生长和发育以及应对各种生物和非生物胁迫中发挥关键作用。尽管RAD23(一种UBL-UBA蛋白)在多种植物中得到了广泛研究,但目前在猕猴桃中还未见报道。本研究中,我们在猕猴桃中鉴定了六个AcRAD23基因,分析了它们的系统发育关系、基因结构、保守基序组成和启动子中的顺式作用元件。亚细胞定位实验表明,所有AcRAD23都定位在细胞核和细胞膜中。实时定量PCR(qRT-PCR)分析证明了AcRAD23基因在不同组织和各种逆境(干旱、涝渍、盐等)下的差异表达模式,AcRAD23D1对非生物胁迫表现出强烈的响应。此外,我们在干旱胁迫条件下使用VIGS介导的基因沉默方法研究了AcRAD23D1的生物学功能。与对照系相比,AcRAD23D1表达的抑制导致D1-VIGS株系的相对含水量(RWC)降低,但导致丙二醛(MDA)含量和相对电解质渗漏(REL)水平增加。此外,D1-VIGS株系表现出更高的活性氧(RoS)积累,同时超氧化物歧化酶(SOD)和酶(POD)活性降低。这些发现表明AcRAD23 D1可能在调节猕猴桃对干旱胁迫的反应方面发挥积极作用。我们的结果为AcRAD23在非生物胁迫条件下的潜在参与提供了新的见解,同时为理解猕猴桃适应胁迫的分子机制提供了理论基础。



Abstract  

UBL-UBA protein functions as a shuttle factor in the 26S ubiquitin degradation pathway, playing a critical role in plant growth and development, and responding to various biotic and abiotic stresses.  Although RAD23, a type of UBL-UBA protein, has been extensively studied in several plants, there is currently no comprehensive analysis available for kiwifruit (Actinidia chinensis).  In this study, we identified six AcRAD23 genes in kiwifruit and further analyzed their phylogenetic relationships, gene structure, conserved motif composition and cis-acting element in the promoter.  Subcellular localization experiments revealed that all AcRAD23 were localized in the nucleus and the cell membranes.  Quantitative real-time PCR (qRT-PCR) analysis demonstrated differential expression patterns of these AcRAD23 genes across different tissues and under various stress conditions (drought, waterlogging, salt stress, etc.), with AcRAD23D1 showing the highest responsiveness to abiotic stress.  Additionally, we investigated the biological function of AcRAD23D1 using VIGS-mediated gene silencing methods under drought stress conditions.  Suppression of AcRAD23D1 expression resulted in reduced relative water content (RWC) but increased malondialdehyde (MDA) content and relative electrolyte leakage (REL) levels in D1-VIGS lines compared to control lines.  Furthermore, D1-VIGS lines exhibited a higher accumulation of reactive oxygen species (ROS) along with decreased superoxide dismutase (SOD) and peroxidase (POD) enzyme activities.  These findings suggest that AcRAD23D1 may play a positive role in regulating kiwifruit’s response to drought stress.  Our results provide new insights into the potential involvement of AcRAD23 under abiotic stress conditions while offering a theoretical foundation for understanding the molecular mechanisms underlying kiwifruit’s adaptation to stresses. 

Keywords:  kiwifruit       RAD23        expression profile        drought stress        ubiquitin degradation  
Received: 12 December 2023   Online: 13 March 2025   Accepted: 17 October 2024
Fund: 

This study was financially supported by the National Natural Science Foundation of China (32472679), the Sichuan Science and Technology Department Projects (2023ZHCG0098, 2024JDRC0011), the Chengdu Science and Technology Department Project, China (2024-YF05-00408-SN), the Postdoctoral Fellowship Program of China Postdoctoral Science Foundation (GZC20231871) and the Special Project for the Double Support Plan of Discipline Construction at Sichuan Agricultural University, China (2024ZYTS021).

About author:  #Correspondence Dong Liang, Tel: +86-28-86291136, E-mail: liangeast@sicau.edu.cn

Cite this article: 

Xiaoli Zhang, Daolin Ye, Xueling Wen, Xinling Liu, Lijin Lin, Xiulan Lü, Jin Wang, Qunxian Deng, Hui Xia, Dong Liang. 2025. Genome-wide analysis of RAD23 gene family and a functional characterization of AcRAD23D1 in drought resistance in Actinidia. Journal of Integrative Agriculture, 24(5): 1831-1843.

Bulduklu Y, Ersoy F, Akkaya, M. 2018. RAD23 as a negative regulator of barley powdery mildew disease resistance. Fresenius Environmental Bulletin, 27, 6239-6244.

Choudhury F K, Rivero R M, Blumwald E, Mittler R. 2007. Reactive oxygen species, abiotic stress and stress combination. Plant Journal, 90, 856-867. 

Farmer L M, Book A J, Lee K H, Lin Y L, Fu H Y, Vierstra R D. 2010. The RAD23 family provides an essential connection between the 26S proteasome and ubiquitylated proteins in Arabidopsis. Plant Cell, 22, 124–142. 

Ferguson A R, Huang H. 2007. Genetic Resources of Kiwifruit: Domestication and Breeding. Horticultural Reviews33, 1-121.

Gill S S, Tuteja N. 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry, 48, 909-930. 

Guzder S N, Sung P, Prakash L, Prakash S. 1998. Affinity of yeast nucleotide excision repair factor 2, consisting of the Rad4 and Rad23 proteins, for ultraviolet damaged DNA. Journal of Biological Chemistry, 273, 31541-31546.

Huang H, Liu Y. 2014. Natural hybridization, introgression breeding, and cultivar improvement in the genus Actinidia. Tree Genetics and Genomes, 10, 1113-1122.

Ishii T, Funakoshi M, Kobayashi H, Sekiguchi T. 2014. Yeast Irc22 is a novel Dsk2-interacting protein that is involved in salt tolerance. Cell, 3, 180–198.

Jia X, Mao K, Wang P, Wang Y, Jia X M, Huo L Q, Sun X, Che R M, Gong X Q, Ma F W. 2021. Overexpression of MdATG8i improves water use efficiency in transgenic apple by modulating photosynthesis, osmotic balance, and autophagic activity under moderate water deficit. Horticulture Research, 8, 81.

Jing Y Y, Liu C H, Liu B B, Pei T T, Zhan M H, Li C R, Wang D N, Li P M, Ma F W. 2022. Overexpression of the FERONIA receptor kinase MdMRLK2 confers apple drought tolerance by regulating energy metabolism and free amino acids production. Tree Physiology, 43, 154-168.

Jogawat A, Yadav B, Chhaya, Lakra N, Singh A K, Narayan O P. 2021. Crosstalk between phytohormones and secondary metabolites in the drought stress tolerance of crop plants: A review. Physiologia plantarum, 172, 1106-1132.

Laxa M, Liebthal M, Telman W, Chibani K, Dietz K J. 2009. The role of the plant antioxidant system in drought tolerance. Antioxidants8, 94-125.

Lambertson D, Chen L, Madura K. 1999. Pleiotropic defects caused by loss of the proteasome-interacting factors Rad23 and Rpn10 of saccharomyces cerevisiae. Genetics, 153, 69-79. 

Li X S, Zhang J H, Zhao X Y, Liu X M. 2013. Protective effects of Rad23 protein on ultraviolet damage to HeLa cells. Journal of Central South University, 20, 2974-2980.

Li L, Li B, Xie C, Zhang T, Borassi C, Estevez J M, Li X S, Liu X M. 2020. Arabidopsis RAD23B regulates pollen development by mediating degradation of KRP1. Journal of Experimental Botany, 71, 4010-4019.

Liu R D, Shen Y H, Wang M X, Liu R H, Cui Z Q, Li P Z, Wu Q D, Shen Q, Chen J, Zhang S P, Liu S D, Ma H J, Pang C Y, Ge C W. 2023. GhMYB102 promotes drought resistance by regulating drought-responsive genes and ABA biosynthesis in cotton (Gossypium hirsutum L.). Plant Science, 329, 111608.

Li D, Liu Y, Li X, Rao J, Yao X, Zhong C. 2014. Genetic diversity in kiwifruit polyploid complexes: insights into cultivar evaluation, conservation, and utilization. Tree Genetics and Genomes, 10, 1451-1463.

Li T, Huang Y, Khadr A, Wang Y H, Xu Z S, Xiong A S. 2020. DcDREB1A, a DREB-binding transcription factor from Daucus carota, enhances drought tolerance in transgenic Arabidopsis thaliana and modulates lignin levels by regulating lignin-biosynthesis-related genes. Environmental and Experimental Botany, 169, 103896.

Liang B W, Gao T T, Zhao Q, Ma C Q, Chen Q, Wei Z W, Li C Y, Li C, Ma F W. 2018. Effects of exogenous dopamine on the uptake, transport, and resorption of apple ionome under moderate drought. Frontiers in Plant Science, 9, 755.

Ning D L, Lu T C, Liu G F, Yang C P, Wang B C. 2013. Proteomic analysis points to a role for RAD23 in apical dominance in Pinus sylvestris var. mongolica. Plant Molecular Biology Reporter, 31, 1283-1292.

Schultz T F, Quatrano R S. 1997. Characterization and expression of a rice RAD23 gene. Plant Molecular Biology, 34, 557-562.

Sturm A, Lienhard S. 1998. Two isoforms of plant RAD23 complement a UV-sensitive rad23 mutant in yeast. Plant Journal, 13, 815-821.

Tsuchiya H, Ohtake F, Arai N, Kaiho A, Yasuda S, Tanaka K, Saeki Y. 2017. In vivo ubiquitin Linkage-type analysis reveals that the Cdc48-Rad23/Dsk2 axis contributes to K48-Linked chain specificity of the proteasome. Molecular Cell, 66, 488-+.

Tian R, Yang Y, Chen M. 2022. Genome-wide survey of the amino acid transporter gene family in wheat (Triticum aestivum L.): identification, expression analysis and response to abiotic stress. International Journal of Biological Macromolecules, 162, 1372–1387.

Wang Y P, Chen Q, Zheng J Z, Zhang Z J, Gao T T, Li C, Ma F W. 2021. Overexpression of the tyrosine decarboxylase gene MdTyDC in apple enhances long-term moderate drought tolerance and WUE. Plant Science, 313, 111064.

Wang N, Gong X Q, Ma F W. 2017. Genome-wide identification of the radiation sensitivity protein-23 (RAD23) family members in apple (Malus×domestica Borkh.) and expression analysis of their stress responsiveness. Journal Of Integrative Agriculture, 16, 820-827.

Watkins J F, Sung P, Prakash L, Prakash S. 1993. The Saccharomyces cerevisiae DNA repair gene RAD23 encodes a nuclear protein containing a ubiquitin-like domain required for biological function. Molecular and Cellular Biology, 13, 7757-7765.

Wang X, Zhang X Y , Song C P , Gong Z Z , Yang S H , Ding Y L. 2023. PUB25 and PUB26 dynamically modulate ICE1 stability via differential ubiquitination during cold stress in Arabidopsis. Plant Cell, 35, 3585-3603.

Wang J, Qin H, Zhou S R,Wei P C, Zhang H W, Zhou Y, Miao Y C, Huang R F. 2020. The ubiquitin-binding protein OsDSK2a mediates seedling growth and salt responses by regulating gibberellin metabolism in rice. Plant Cell, 32, 414-428.

Wang H, Song C, Fang S, Wang Z, Song S, Jiao J, Wang M, Zheng X, Bai T. 2022. Genome-wide identification and expression analysis of the ASMT gene family reveals their role in abiotic stress tolerance in apple. Scientia Horticulturae, 293, 110683.

Xia L J, Sun S M, Han B, Yang X Y. 2023. NAC domain transcription factor gene GhNAC3 confers drought tolerance in plants. Plant Physiology and Biochemistry, 195, 114-123.

Xu Z, Zhou G. 2008. Responses of leaf stomatal density to water status and its relationship with photosynthesis in a grass. Journal of Experimental Botany, 59, 3317-25. 

Yamaguchi-Shinozaki K, Shinozaki K. 2005. Organization of cis-acting regulatory elements in osmotic- and cold-stress-responsive promoters. Trends in Plant Science, 10, 88-94.

Ye D L, Liu J N, Tian X B, Wen X L, Zhang Y Y, Zhang X L, Sun G C, Xia H, Liang D. 2024. Genome-wide identification of bHLH gene family and screening of candidate gene in response to salt stress in kiwifruit. Environmental and Experimental Botany, 222, 105774.

Zhao S, Gao H B, Jia X M, Wang H B, Mao K, Ma F W. 2020. The HD-Zip I transcription factor MdHB-7 regulates drought tolerance in transgenic apple (Malus domestica). Plant and Soil, 180, 104246.

Zhang D N, Chen T, Ziv I, Rosenzweig R, Matiuhin Y, Bronner V, Glickman M H, Fushman D. 2009. Together, Rpn10 and Dsk2 can serve as a polyubiquitin chain-length sensor. Molecular Cell, 36, 1018-1033.

Zhang C W, Zhou Q, Liu W S, Wu XT, Li Z B, Xu Y Y, Li Y, Imaizumi T, Hou X L, Liu T K. 2022. BrABF3 promotes flowering through the direct activation of CONSTANS transcription in pak choi. The Plant Journal, 111, 134-148.

Zhang X L, Gong X Q, Yu H X, Su X J, Cheng S Y, Huang J W, Lei Z L, Li M J, Ma F W. 2023c. The proline-rich protein MdPRP6 confers tolerance to salt stress in transgenic apple (Malus domestica). Scientia Horticulturae, 308 , 111581.

Zhang X L, Gong X Q, Su X J, Yu H X, Cheng S Y, Huang J W, Li D Y, Lei Z L, Li M J, Ma F W. 2023a. The ubiquitin-binding protein MdRAD23D1 mediates drought response by regulating degradation of the proline-rich protein MdPRP6 in apple (Malus domestica). Plant Biotechnology Journal, 21, 1560-1576.

Zhang X L, Gong X Q, Zhao B Z, Huang J W, Zhou H Y, Li M J, Ma F W. 2023b. The ubiquitin-binding protein MdRAD23D1 affects WUE under long-term moderate drought stress in transgenic apple (Malus domestica). Scientia Horticulturae, 319, 112164.

Zhou Y, Zeng L, Chen R, Wang Y, Song J. 2018. Genome-wide identification and characterization of stress-associated protein (SAP) gene family encoding A20/AN1 zinc-finger proteins in Medicago truncatula. Proceedings of the Royal Society B: Biological Sciences, 70, 87–98.

Zhou Y B, Chen M, Guo J K, Wang Y X, Min D H, Jiang Q Y, Ji H T, Huang C Y, Wei W, Xu H J, Chen X, Li L C, Xu Z S, Cheng X G, Wang C X, Wang C S, Ma Y Z. 2020. Overexpression of soybean DREB1 enhances drought stress tolerance of transgenic wheat in the field. Journal of Experimental Botany, 71, 1842-1857.

Zhu L C, Li B Y, Wu L M, Li H X, Wang Z Y, Wei X Y, Ma B Q, Zhang Y F, Ma F W, Ruan Y L, Li M J. 2022. MdERDL6-mediated glucose efflux to the cytosol promotes sugar accumulation in the vacuole through up-regulating TSTs in apple and tomato. Proceedings of the National Academy of Sciences of the United States of America, 118, e2022788118.

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