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Journal of Integrative Agriculture  2024, Vol. 23 Issue (03): 863-875    DOI: 10.1016/j.jia.2023.11.023
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Alternative splicing of the PECTINESTERASE gene encoding a cell wall-degrading enzyme affects postharvest softening in grape

Hainan Liu1, 2*, Maosong Pei1, 2*, Charles Ampomah-Dwamena3, Yaxin Shang1, 2, Yihe Yu1, 2, Tonglu Wei1, 2, Qiaofang Shi1, 2, Dalong Guo1, 2#

1 College of Horticulture and Plant Protection, Henan University of Science and Technology, Luoyang 471000, China

2 Henan Engineering Technology Research Center of Quality Regulation and Control of Horticultural Plants, Luoyang 471000, China

3 The New Zealand Institute for Plant & Food Research Limited (PFR), Auckland 1010, New Zealand

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摘要  

果实硬度是鲜食葡萄最重要的品质性状之一,但果实采后软化分子机制尚不完全清楚。为进一步解析采后软化调控机制,我们对采后巨峰葡萄进行叶酸(Folic AcidFA)处理(以水处理为对照,CK),通过比较各处理组间果实的转录组数据,筛选出差异表达基因(Differentially Expressed GenesDEGs),并鉴定采后软化相关可变剪接(Alternative splicingAS)事件及相关差异表达基因。共鉴定出2559个差异表达基因,根据表达模式将其分为四个亚簇,其中亚簇4中的差异表达基因在CK组中高表达。FACK处理组果实中特异性AS相关基因数分别为1045个和1042个。GO注释结果表明,CK处理组果实中的AS相关基因主要富集在细胞壁代谢过程中,尤其是细胞壁降解过程。通过比较AS相关基因与亚簇4的差异表达基因,筛选出了8个发生AS事件的差异表达基因,其中包括一个编码细胞壁降解酶的基因(果胶酯酶2VvPE2Vitvi15g00704),分析结果显示该基因可能发生了A3SS事件。RT-PCR进一步证实在FA处理组的葡萄果实中存在较高比例的VvPE2的截短转录本变体。本研究运用转录组测序技术分析葡萄采后果实的AS事件,并结合实验验证进一步明确细胞壁降解酶基因的AS事件在葡萄采后果实软化调控中发挥了重要作用。



Abstract  

The firmness of table grape berries is a crucial quality parameter.  Despite extensive research on postharvest fruit softening, its precise molecular mechanisms remain elusive.  To enhance our comprehension of the underlying molecular factors, we initially identified differentially expressed genes (DEGs) by comparing the transcriptomes of folic acid (FA)-treated and water-treated (CK) berries at different time points.  We then analyzed the sequences to detect alternatively spliced (AS) genes associated with postharvest softening.  A total of 2,559 DEGs were identified and categorized into four subclusters based on their expression patterns, with subcluster-4 genes exhibiting higher expression in the CK group compared with the FA treatment group.  There were 1,045 AS-associated genes specific to FA-treated berries and 1,042 in the CK-treated berries, respectively.  Gene Ontology (GO) annotation indicated that the AS-associated genes in CK-treated berries were predominantly enriched in cell wall metabolic processes, particularly cell wall degradation processes.  Through a comparison between treatment-associated AS genes and subcluster-4 DEGs, we identified eight genes, including Pectinesterase 2 (VvPE2, Vitvi15g00704), which encodes a cell wall-degrading enzyme and was predicted to undergo an A3SS event.  The reverse transcription polymerase chain reaction further confirmed the presence of a truncated transcript variant of VvPE2 in the FA-treated berries.  Our study provides a comprehensive analysis of AS events in postharvest grape berries using transcriptome sequencing and underscores the pivotal role of VvPE2 during the postharvest storage of grape berries.

Keywords:  Grape       Postharvest softening        Folic Acid        Alternative splicing        Pectinesterase 2        Alternative 3′ splice site (A3SS)   
Received: 17 April 2023   Accepted: 07 October 2023
Fund: This work was financially supported by the National Natural Science Foundation of China (32202560 and 32302470), the Program for Innovative Research Team (in Science and Technology) in University of Henan Province, China (21IRTSTHN021), the Natural Science Foundation of Henan, China (232300421112), the Program for Science & Technology Innovation Talents in Universities of Henan Province, China (21HASTIT035), and the PhD Research Startup Foundation of Henan University of Science and Technology, China (13480068 and 13480067).
About author:  Hainan Liu, E-mail: liuhainan0995@163.com; Maosong Pei, E-mail: peimaosong@163.com; #Correspondence Dalong Guo, E-mail: guodalong@haust.edu.cn * These authors contributed equally to this study.

Cite this article: 

Hainan Liu, Maosong Pei, Charles Ampomah-Dwamena, Yaxin Shang, Yihe Yu, Tonglu Wei, Qiaofang Shi, Dalong Guo. 2024.

Alternative splicing of the PECTINESTERASE gene encoding a cell wall-degrading enzyme affects postharvest softening in grape . Journal of Integrative Agriculture, 23(03): 863-875.

Aghdam M S, Mukherjee S, Flores F B, Arnao M B, Luo Z, Corpas F J. 2023. Functions of melatonin during postharvest of horticultural crops. Plant and Cell Physiology, 63, 1764–1786.

Atkinson R G, Sutherland P W, Johnston S L, Gunaseelan K, Hallett I C, Mitra D, Brummell D A, Schröder R, Johnston J W, Schaffer R J. 2012. Down-regulation of POLYGALACTURONASE1 alters firmness, tensile strength and water loss in apple (Malus × domestica) fruit. BMC Plant Biology, 12, 129–142.

Brummell D A, Harpster M H. 2001. Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Molecular Biology, 47, 311–340.

Castillejo C, de la Fuente J I, Iannetta P, Botella M A, Valpuesta V. 2004. Pectin esterase gene family in strawberry fruit: Study of FaPE1, a ripening-specific isoform. Journal of Experimental Botany, 55, 909–918.

Castellarin S D, Gambetta G A, Wada H, Krasnow M N, Cramer G R, Peterlunger E, Shackel K A, Matthews M A. 2016. Characterization of major ripening events during softening in grape: Turgor, sugar accumulation, abscisic acid metabolism, colour development, and their relationship with growth. Journal of Experimental Botany, 67, 709–722.

Chamala S, Feng G, Chavarro C, Barbazuk W B. 2015. Genome-wide identification of evolutionarily conserved alternative splicing events in flowering plants. Frontiers in Bioengineering and Biotechnology, 3, 33.

Chen C, Liu C H, Shi L J, Jiang A L, Hu W Z. 2021. An analysis of alternative splicing events during browning inhibition of fresh-cut apples by hydrogen sulfide treatment. Acta Horticulturae Sinica, 48, 2121–2132. (in Chinese)

Chen Y, Chen F, Lai S, Yang H, Liu H, Liu K, Bu G, Deng Y. 2013. In vitro study of the interaction between pectinase and chelate-soluble pectin in postharvest apricot fruits. European Food Research and Technology, 237, 987–993.

Cingolani P, Platts A, Wang le L, Coon M, Nguyen T, Wang L, Land S J, Lu X, Ruden D M. 2012. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin), 6, 80–92.

Clark S, Yu F, Gu L, Min X J. 2019. Expanding alternative splicing identification by integrating multiple sources of transcription data in tomato. Frontiers in Plant Science, 10, 689.

Correa J, Mamani M, Muñoz-Espinoza C, González-Agüero M, Defilippi B G, Campos-Vargas R, Pinto M, Hinrichsen P. 2016. New stable QTLs for berry firmness in table grapes. American Journal of Enology and Viticulture, 67, 212–217.

Cosgrove D J. 2005. Growth of the plant cell wall. Nature Reviews Molecular Cell Biology, 6, 850–861.

Dantas L L B, Calixto, C P G, Dourado M M, Carneiro M S, Brown J W S, Hotta C T. 2019. Alternative splicing of circadian clock genes correlates with temperature in field-grown sugarcane. Frontiers in Plant Science, 10, 1614.

Filichkin S, Priest H D, Megraw M, Mockler T C. 2015. Alternative splicing in plants: Directing traffic at the crossroads of adaptation and environmental stress. Current Opinion in Plant Biology, 24, 125–135.

Ge T, Boris L. 2016. TFBSTools: An R/bioconductor package for transcription factor binding site analysis. Bioinformatics, 32, 1555–1556.

Kalyna M, Simpson C G, Syed N H, Lewandowska D, Marquez Y, Kusenda B, Marshall J, Fuller J, Cardle L, McNicol J, Dinh H Q, Barta A, Brown J W. 2012. Alternative splicing and nonsense-mediated decay modulate expression of important regulatory genes in Arabidopsis. Nucleic Acids Research, 40, 2454–2469.

Laloum T, Martín G, Duque P. 2018. Alternative splicing control of abiotic stress responses. Trends in Plant Science, 23, 140–150.

Liu H N, Pei M S, Wei T L, Yu Y H, Guo D L. 2021. Molecular cloning and expression analysis of hydrogen peroxide sensors under H2O2 and ROS inhibitor treatment in ‘Kyoho’ grape berry. Postharvest Biology and Technology, 180, 1–9.

Liu H N, Pei M S, Wei T L, Yu Y H, Guo D L. 2022. ROS scavenger hypotaurine delays postharvest softening of ‘Kyoho’ grape by regulating pectin and cell metabolism pathway. Postharvest Biology and Technology, 186, 1–12.

Love M I, Huber W, Anders S. 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 15, 550.

Luo Z S. 2006. Extending shelf-life of persimmon (Diospyros kaki L.) fruit by hot air treatment. European Food Research and Technology, 222, 149–154.

Luo Z S, Xie J, Xu T Q, Zhang L. 2009. Delay ripening of ‘Qingnai’ plum (Prunus salicina Lindl.) with 1-methylcyclopropene. Plant Science, 177, 705–706.

Paniagua C, Posé S, Morris V J, Kirby A R, Quesada M A, Mercado JA. 2014. Fruit softening and pectin disassembly: An overview of nanostructural pectin modifications assessed by atomic force microscopy. Annals of Botany, 114, 1375–1383.

Payasi A, Mishra N N, Chaves A L S, Singh R. 2009. Biochemistry of fruit softening: An overview. Physiology and Molecular Biology of Plants, 15, 103–113.

Pei M S, Liu H N, Wei T L, Yu Y H, Guo D L. 2023. Folic acid delays postharvest quality deterioration of table grape by regulating cell wall metabolism-associated hub WRKY31 transcription factor. Postharvest Biology and Technology, 197, 1–11.

Peng Z, Liu G, Li H, Wang Y, Gao H, Jemrić T, Fu D. 2022. Molecular and genetic events determining the softening of fleshy fruits: A comprehensive review. International Journal of Molecular Sciences, 23, 1–19.

Pertea M, Pertea G M, Antonescu C M, Chang T C, Mendell J T, Salzberg S L. 2015. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nature Biotechnology, 33, 290–295.

Phan T D, Bo W, West G, Lycett G W, Tucker G A. 2007. Silencing of the major salt-dependent isoform of pectinesterase in tomato alters fruit softening. Plant Physiology, 144, 1960–1967.

Reddy A S, Marquez Y, Kalyna M, Barta A. 2013. Complexity of the alternative splicing landscape in plants. Plant Cell, 25, 3657–3683.

Romero I, Vazquez-Hernandez M, Maestro-Gaitan I, Escribano M I, Merodio C, Sanchez-Ballesta M T. 2020. Table grapes during postharvest storage: A review of the mechanisms implicated in the beneficial effects of treatments applied for quality retention. International Journal of Molecular Sciences, 21, 1–19.

Santiago-Doménech N, Jiménez-Bemúdez S, Matas A J, Rose J K, Muñoz-Blanco J, Mercado J A, Quesada M A. 2008. Antisense inhibition of a pectate lyase gene supports a role for pectin depolymerization in strawberry fruit softening. Journal of Experimental Botany, 59, 2769–2779.

Staiger D, Brown J W. 2013. Alternative splicing at the intersection of biological timing, development, and stress responses. Plant Cell, 25, 3640–3656.

Syed N H, Kalyna M, Marquez Y, Barta A, Brown J W. 2012. Alternative splicing in plants-coming of age. Trends in Plant Science, 17, 616–623.

Maillot P, Velt A, Rustenholz C, Butterlin G, Merdinoglu D, Duchêne E. 2021. Alternative splicing regulation appears to play a crucial role in grape berry development and is also potentially involved in adaptation responses to the environment. BMC Plant Biology, 21, 487.

Nasser M A, El-Mogy M M, Samaan M S F, Hassan K M, El-Sayed S M, Alsubeie M S, Darwish D B E, Mahmoud S F, Al-Harbi N A, Al-Qahtani S M, Alzuaibr F M, El-Gawad H A. 2022. Postharvest exogenous melatonin treatment of table grape berry enhances quality and maintains bioactive compounds during refrigerated storage. Horticulturae, 8, 860.

Ni P Y, Ji X R, Guo D L. 2020. Genome-wide identification, characterization, and expression analysis of GDSL-type esterases/lipases gene family in relation to grape berry ripening. Scientia Horticulturae, 264, 109162.

Niklas K J, Bondos S E, Dunker A K, Newman S A. 2015. Rethinking gene regulatory networks in light of alternative splicing, intrinsically disordered protein domains, and post-translational modifications. Frontiers in Cell and Developmental Biology, 3, 8.

Wang D, Yeats T H, Uluisik S, Rose J K C, Seymour G B. 2018. Fruit softening: Revisiting the role of pectin. Trends in Plant Science, 23, 302–310.

Wang H, Yan A, Sun L, Zhang G, Wang X, Ren J, Xu H. 2020. Novel stable QTLs identification for berry quality traits based on high-density genetic linkage map construction in table grape. BMC Plant Biology, 20, 411.

Wang J, Mujumdar A S, Deng L Z, Gao Z J, Xiao H W, Raghavan G S V. 2018. High-humidity hot air impingement blanching alters texture, cell-wall polysaccharides, water status and distribution of seedless grape. Carbohydrate Polymers, 194, 9–17.

Wang X, Wilson L, Cosgrove D J. 2020. Pectin methylesterase selectively softens the onion epidermal wall yet reduces acid-induced creep. Journal of Experimental Botany, 71, 2629–2640.

Wen B, Ström A, Tasker A, West G, Tucker G A. 2013. Effect of silencing the two major tomato fruit pectin methylesterase isoforms on cell wall pectin metabolism. Plant Biology, 15, 1025–1032.

Wong D C, Lopez Gutierrez R, Dimopoulos N, Gambetta G A, Castellarin S D. 2016. Combined physiological, transcriptome, and cis-regulatory element analyses indicate that key aspects of ripening, metabolism, and transcriptional program in grapes (Vitis vinifera L.) are differentially modulated accordingly to fruit size. BMC Genomics, 17, 1–22.

Xu D, Zuo J, Fang Y, Yan Z, Shi J, Gao L, Wang Q, Jiang A. 2021. Effect of folic acid on the postharvest physiology of broccoli during storage. Food Chemistry, 339, 1–11.

Xue C, Guan S, Chen J, Wen C, Cai J, Chen X. 2020. Genome wide identification and functional characterization of strawberry pectin methylesterases related to fruit softening. BMC Plant Biology, 20, 13.

Xue C, Yao J L, Qin M F, Zhang M Y, Allan A C, Wang D F, Wu J. 2019. PbrmiR397a regulates lignification during stone cell development in pear fruit. Plant Biotechnology Journal, 17, 103–117.

Yang M, Luo Z, Li D, Ma C, Li L. 2023. Role of epicuticular wax involved in quality maintenance of table grapes: Evidence from transcriptomic data. Postharvest Biology and Technology, 196, 1–16.

Young M D, Wakefield M J, Smyth G K, Oshlack A. 2010. Gene ontology analysis for RNA-seq: Accounting for selection bias. Genome Biology, 11, 1–12.

Zhou Q, Zhang F, Ji S, Dai H, Zhou X, Wei B, Cheng S, Wang A. 2021. Abscisic acid accelerates postharvest blueberry fruit softening by promoting cell wall metabolism. Scientia Horticulturae, 288, 1–19.

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