Identification of VPE Gene Family and Their Functional Analysis under Abiotic Stress in Tomato

doi:10.13560/j.cnki.biotech.bull.1985.2026-0160

Abstract

Abstract:

Objective To systematically identify the members of vacuolar processing enzyme (VPE) gene family in tomato, and elucidate its evolutionary relationships, expression patterns, and potential functions in response to abiotic stress. Method Members of the SlVPE family were identified using bioinformatics methods. Systematic analyses were performed on their physicochemical properties, phylogenetic relationships, gene structures, conserved domains, chromosomal distribution, cis-acting elements, and interspecific collinearity. Public transcriptome data and RT-qPCR were integrated to analyze the tissue-specific expression patterns of the SlVPE family and their expression responses to drought, high/low temperature, salt, alkali, and combined salt-alkali stress. Result A total of 13 SlVPE genes containing both Peptidase_C13 and legumain_C domains were identified in tomato and classified into three subfamilies. With the exception of SlVPE4 and SlVPE5, all members were clustered in a gene cluster on chromosome 8. Expression analysis revealed significant tissue and spatiotemporal specificity within the SlVPE family: SlVPE3 and SlVPE5 were constitutively highly expressed, while SlVPE2 and SlVPE7 were fruit-specific highly expressed genes. Under abiotic stress, the family’s response was predominantly characterized by transcriptional suppression. Notably, SlVPE3 emerged as a core responsive gene to drought, salt, and combined salt-alkali stress, SlVPE7 was specifically induced by alkali stress, and SlVPE8 primarily responded to high and low-temperature stress. Based on sequence variations between wild tomato (Solanum pimpinellifolium) and cultivated tomato (S. lycopersicum), four polymorphic molecular markers were developed in the promoter regions or downstream regions of SlVPE gene. Conclusion The tomato SlVPE gene family consists of 13 members, 11 of which are clustered on chromosome 8. SlVPE3 and SlVPE5 are constitutively highly expressed genes, while SlVPE2 and SlVPE7 exhibit specific high expression during early fruit development. In contrast, abiotic stresses primarily induce the expressions of SlVPE3, SlVPE7, and SlVPE8.

Key words: tomato, vacuolar processing enzyme, cis-acting element, abiotic stress, gene cluster, promoter, molecular marker, Solanum pimpinellifolium

LI Ying-hui, WANG Yang-bo-han, ZHOU Hao-bo, LU Xin-ru, ZHANG Ke-xin, YU Yang, LI Chuan-you, SUN Chuan-long. Identification of VPE Gene Family and Their Functional Analysis under Abiotic Stress in Tomato[J]. Biotechnology Bulletin, 2026, 42(3): 263-274.

Figures/Tables 8

Table 1 Basic information about SlVPEs

基因名称 Gene name	基因ID Gene ID	氨基酸数量 Number of amino acids	分子量 Molecular weight （kD）	疏水性 GRAVY	等电点 pI
SlVPE1	Solyc08g065790.2.1	460	51.77	-0.49	6.92
SlVPE2	Solyc08g065780.1.1	451	50.09	-0.12	5.76
SlVPE3	Solyc08g065610.2.1	470	52.11	-0.36	7.27
SlVPE4	Solyc08g079160.2.1	469	52.00	-0.33	7.59
SlVPE5	Solyc12g095910.1.1	463	51.04	-0.20	5.05
SlVPE6	Solyc08g065530.1.1	483	53.88	-0.31	5.83
SlVPE7	Solyc08g065570.1.1	428	47.54	-0.28	7.49
SlVPE8	Solyc08g065590.1.1	446	49.35	-0.30	7.26
SlVPE9	Solyc08g065690.1.1	450	49.50	-0.20	7.27
SlVPE10	Solyc08g065710.1.1	451	50.68	-0.44	7.78
SlVPE11	Solyc08g065720.1.1	482	53.22	-0.34	5.67
SlVPE12	Solyc08g065740.1.1	497	55.21	-0.37	5.49
SlVPE13	Solyc08g065750.1.1	480	52.97	-0.27	6.10

Fig. 1 Phylogenetic analysis of VPEs protein in tomato (Solanum lycopersicum) and other species

Fig. 2 Motifs, conserved domains and gene structure analysis of SlVPE proteinA: Phylogenetic tree of SlVPE proteins. B: Analysis of conserved motifs in SlVPE proteins. C: Analysis of conserved domains of SlVPE proteins, with green and yellow boxes representing Peptidase_C13 and legumain_C domains, respectively. D: Analysis of SlVPE gene structures. CDS, UTRs, and introns are indicated by green boxes, yellow boxes, and gray lines, respectively

Fig. 3 Analysis of chromosomal localization, interspecies collinearity, and cis-acting elements of SlVPE genesA: Chromosomal locations of the SlVPE genes. B: Collinear relationships of SlVPE genes among tomato, Arabidopsis, potato, and peanut genomes. Gray and blue lines indicate the collinearity of all genes and VPE genes, respectively. C: Heatmap of cis-acting elements in SlVPEs promoters. D: Visualization of the types, positions, and numbers of cis-acting elements in SlVPEs promoters

Fig. 4 Expression profile analysis of SlVPE gene in different tissues of tomatoData are presented as mean ± SE from four biological replicates (n=4). Different lowercase letters indicate significant differences (P<0.05)

Fig. 5 Predicted interaction network of SlVPE proteins

Fig. 6 Expression analysis of SlVPE genes under various abiotic stressesDrought stress： 20% PEG6000； salt stress： 150 mmol/L NaCl； alkali stress： 100 mmol/L NaHCO₃； salt-alkali combined stress： 60 mmol/L NaCl+60 mmol/L NaHCO3； low-temperature stress： 12 ℃； high-temperature stress： 38 ℃. Samples were collected at 7 d after stress treatment. Data are presented as mean±SE of three biological replicates （n=3）， each with three technical replicates. *P<0.05， **P<0.01

Fig. 7 Polymorphism detection of SlVPE gene molecular markersAgarose gel electrophoresis was used to examine polymorphisms between Solanum pimpinellifolium and cultivated tomato in the promoter region of SlVPE6 (A), the downstream region of SlVPE11 (B), the promoter region of SlVPE13 (C), and the promoter region of SlVPE10 (D). Lane 1-6: S. pimpinellifolium accessions, LA0533, LA1375, LA1420, LA2873, LA4763, and LA1589; lane 7-12: cultivated tomato materials, AC, M82, TS-418, Hawaii7996, MoneyMaker, and Heinz1706

References 39

[1]	Hara-Nishimura I, Hatsugai N. The role of vacuole in plant cell death [J]. Cell Death Differ, 2011, 18(8): 1298-1304.
[2]	Teper-Bamnolker P, Danieli R, Peled-Zehavi H, et al. Vacuolar processing enzyme translocates to the vacuole through the autophagy pathway to induce programmed cell death [J]. Autophagy, 2021, 17(10): 3109-3123.
[3]	Sarwar R, Yu J, Zekraoui M, et al. Unraveling the molecular functions of multifaced plant-vacuolar processing enzymes [J]. Physiol Plant, 2024, 176: e14131.
[4]	Gao XX, Tang YL, Shi QY, et al. Vacuolar processing enzyme positively modulates plant resistance and cell death in response to Phytophthora parasitica infection [J]. J Integr Agric, 2023, 22(5): 1424-1433.
[5]	Duan XB, Zhang K, Yu Y, et al. Genome-wide analysis of soybean vacuolar processing enzyme gene family reveals their roles in plant development and response to stress conditions [J]. J Plant Growth Regul, 2024, 43(8): 2817-2829.
[6]	Shimada T, Yamada K, Kataoka M, et al. Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana [J]. J Biol Chem, 2003, 278(34): 32292-32299.
[7]	Tang CN, Wan Abdullah WMAN, Wee CY, et al. Promoter cis-element analyses reveal the function of αVPE in drought stress response of Arabidopsis [J]. Biology, 2023, 12(3): 430.
[8]	Nakaune S, Yamada K, Kondo M, et al. A vacuolar processing enzyme, δVPE, is involved in seed coat formation at the early stage of seed development [J]. Plant Cell, 2005, 17(3): 876-887.
[9]	Kuroyanagi M, Yamada K, Hatsugai N, et al. Vacuolar processing enzyme is essential for mycotoxin-induced cell death in Arabidopsis thaliana [J]. J Biol Chem, 2005, 280(38): 32914-32920.
[10]	Lu WY, Deng MJ, Guo F, et al. Suppression of OsVPE3 enhances salt tolerance by attenuating vacuole rupture during programmed cell death and affects stomata development in rice [J]. Rice, 2016, 9: 65.
[11]	Deng HB, Cao S, Zhang GL, et al. OsVPE2, a member of vacuolar processing enzyme family, decreases chilling tolerance of rice [J]. Rice, 2024, 17: 5.
[12]	Zhang JH, Yue Y, Hu MJ, et al. Dynamic transcriptome landscape of maize pericarp development [J]. Plant J, 2024, 117(5): 1574-1591.
[13]	Wang TH, Guan MH, Zheng YH, et al. Genome-wide molecular characterization and expression profiling of the cysteine protease gene family in maize [J]. BMC Genomics, 2025, 26(1): 789.
[14]	Quadros IPS, Madeira NN, Loriato VAP, et al. Cadmium-mediated toxicity in plant cells is associated with the DCD/NRP-mediated cell death response [J]. Plant Cell Environ, 2022, 45(2): 556-571.
[15]	Radchuk V, Tran V, Radchuk R, et al. Vacuolar processing enzyme 4 contributes to maternal control of grain size in barley by executing programmed cell death in the pericarp [J]. New Phytol, 2018, 218(3): 1127-1142.
[16]	Wang BK, Li N, Huang SY, et al. Enhanced soluble sugar content in tomato fruit using CRISPR/Cas9-mediated SlINVINH1 and SlVPE5 gene editing [J]. PeerJ, 2021, 9: e12478.
[17]	Wang WH, Cai JH, Wang PW, et al. Post-transcriptional regulation of fruit ripening and disease resistance in tomato by the vacuolar protease SlVPE3 [J]. Genome Biol, 2017, 18: 47.
[18]	Zhu LF, Wang XP, Tian J, et al. Genome-wide analysis of VPE family in four Gossypium species and transcriptional expression of VPEs in the upland cotton seedlings under abiotic stresses [J]. Funct Integr Genomics, 2022, 22(2): 179-192.
[19]	Song JF, Yang F, Xun M, et al. Genome-wide identification and characterization of vacuolar processing enzyme gene family and diverse expression under stress in apple (Malus × domestic) [J]. Front Plant Sci, 2020, 11: 626.
[20]	Bombarely A, Menda N, Tecle IY, et al. The Sol Genomics Network (solgenomics.net): growing tomatoes using Perl [J]. Nucleic Acids Res, 2011, 39(): D1149-D1155.
[21]	Consortium The Tomato Genome. The tomato genome sequence provides insights into fleshy fruit evolution [J]. Nature, 2012, 485(7400): 635-641.
[22]	Finn RD, Bateman A, Clements J, et al. Pfam: the protein families database [J]. Nucleic Acids Res, 2014, 42(D1): D222-D230.
[23]	Berardini TZ, Reiser L, Li DH, et al. The Arabidopsis information resource: Making and mining the “gold standard” annotated reference plant genome [J]. Genesis, 2015, 53(8): 474-485.
[24]	Bertioli DJ, Cannon SB, Froenicke L, et al. The genome sequences of Arachis duranensis and Arachis ipaensis, the diploid ancestors of cultivated peanut [J]. Nat Genet, 2016, 48(4): 438-446.
[25]	Kumar S, Stecher G, Li M, et al. MEGA X: molecular evolutionary genetics analysis across computing platforms [J]. Mol Biol Evol, 2018, 35(6): 1547-1549.
[26]	Chen CJ, Chen H, Zhang Y, et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data [J]. Mol Plant, 2020, 13(8): 1194-1202.
[27]	Bailey TL, Boden M, Buske FA, et al. MEME Suite: tools for motif discovery and searching [J]. Nucleic Acids Res, 2009, 37(): W202-W208.
[28]	Lescot M, Déhais P, Thijs G, et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences [J]. Nucleic Acids Res, 2002, 30(1): 325-327.
[29]	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method [J]. Methods, 2001, 25(4): 402-408.
[30]	Wleklik K, Borek S. Vacuolar processing enzymes in plant programmed cell death and autophagy [J]. Int J Mol Sci, 2023, 24(2): 1198.
[31]	Pat Heslop-Harrison JS, Schwarzacher T, Liu Q. Polyploidy: its consequences and enabling role in plant diversification and evolution [J]. Ann Bot, 2023, 131(1): 1-10.
[32]	Fang YP, Jiang JM, Hou XL, et al. Plant protein-coding gene families: Their origin and evolution [J]. Front Plant Sci, 2022, 13: 995746.
[33]	Zheng P, Zheng CY, Otegui MS, et al. Endomembrane mediated-trafficking of seed storage proteins: from Arabidopsis to cereal crops [J]. J Exp Bot, 2022, 73(5): 1312-1326.
[34]	Baral R, Vainer A, Melzer S, et al. ‘Bud to fruit’—hormonal interactions governing early fruit development [J]. J Exp Bot, 2025, 76(22): 6657-6673.
[35]	Wang RF, de Maagd RA. Transcriptional control of tomato fruit development and ripening [J]. J Exp Bot, 2025, 76(21): 6311-6326.
[36]	Wei SB, Li X, Lu ZF, et al. A transcriptional regulator that boosts grain yields and shortens the growth duration of rice [J]. Science, 2022, 377(6604): eabi8455.
[37]	Tan WR, Chen JH, Yue XL, et al. The heat response regulators HSFA1s promote Arabidopsis thermomorphogenesis via stabilizing PIF4 during the day [J]. Sci Adv, 2023, 9(44): eadh1738.
[38]	Xu L, Yang LJ, Li AP, et al. An AP2/ERF transcription factor confers chilling tolerance in rice [J]. Sci Adv, 2024, 10(35): eado4788.
[39]	Zhu JK. Abiotic stress signaling and responses in plants [J]. Cell, 2016, 167(2): 313-324.