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. 2024 Sep 5;30(9):1493–1515. doi: 10.1007/s12298-024-01506-w

Comparative omics-based characterization, phylogeny and melatonin-mediated expression analyses of GDSL genes in pitaya (Selenicereus undatus L.) against multifactorial abiotic stresses

Obaid Ullah Shah 1, Jiantao Peng 1, Lingling Zhou 1, Wasi Ullah Khan 1, Zhang Shanshan 1, Pan Zhuyu 1, Pingwu Liu 1,✉, Latif Ullah Khan 1,✉
PMCID: PMC11413313  PMID: 39310703

Abstract

The GDSL gene family plays diverse roles in plant growth and development. Despite its significance, the functions of the GDSL in the pitaya plant are still unknown. Pitaya (Selenicereus undatus L.) also called Hylocereus undatus (Hu), belongs to the family Cactaceae and is an important tropical plant that contains high dietary fibers and antioxidants. In the present investigation, we screened 91 HuGDSL genes in the pitaya genome by conducting a comprehensive computational analysis. The phylogenetic tree categorized HuGDSL genes into 9 distinct clades in combination with four other species. Further, 29 duplicate events were identified of which 12 were tandem, and 17 were segmental. The synteny analysis revealed that segmental duplication was more prominent than tandem duplication among these genes. The majority of duplicated gene pairs (95%) indicate their Ka/Ks ratios ranging from 0.1 to 0.3, which shows that maximum HuGDSL genes were under purifying selection pressure. The cis-acting element in the promotor region contains phytohormones such as auxin, gibberellin, jasmonic acid, and abscisic acid abundantly. Finally, the HuGDSL gene expression pattern under single and multiple stresses was analyzed via; RNA-seq. We select ten stress-responsive HuGDSL genes for RT-qPCR validation. After careful investigation, we identified five HuGDSL candidate genes (HuGDSL-1/3/55/59, and HuGDSL-78) based on RNA-seq, and RT-qPCR data that showed enhanced expression in stress and melatonin-applied seedlings. This study represents valuable insights into maintaining pitaya growth and development by preparing stress-resilient pitaya genotypes through modern biotechnological techniques.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-024-01506-w.

Keywords: Pitaya/dragon fruit; Selenicereus undatus L, In-silico analysis; melatonin, HuGDSL gene family; growth and development, Abiotic stresses

Introduction

Pitaya, commonly referred to as dragon fruit, belongs to the Cactaceae family, which comprises 127 genera and 1750 species (Mercado-Silva 2018). Among these, Selenicereus undatus (S. undatus), formerly known as Hylocereus undatus (H. undatus) (2n = 2x = 22), is a diploid perennial climbing plant that originated in rainforests in the tropical regions of Mexico, Australia, Colombia (Chen et al. 2021). Pitaya possesses high nutritional properties, efficient Agri-commodity, and industrial condiments, which is why it is broadly cultivated in the tropical and subtropical regions of the USA, China, and Vietnam (Trivellini et al. 2020; Chen et al. 2021), and also the agronomists from the Mediterranean areas (Italy), and now South Asia (India, Pakistan) also take interest in growing pitaya. The presence of high phenolic, beta-cyanine contents and vitamin C enhanced the antioxidant scavenging potential of pitaya (Mercado-Silva 2018; Cai et al. 2022). Global production of pitaya has risen in recent years, and the demand for this fruit has increased in many countries due to its nutritional benefits and unique flavor (Barthlott and Hunt 1993). Previously several gene families have been reported in pitaya, such as bHLH (Chen et al. 2023), MYB (Xie et al. 2021), APX (Zaman et al. 2023a), GRAS (Zaman et al. 2022), HMA (Zaman et al. 2023b) and Dof gene family (Alam et al. 2024) while in the present study, we explored the GDSL genes biological and regulatory network roles in pitaya.

GDSL-type esterase/lipase proteins (GELPs) belong to the SGNH hydrolase superfamily and contain a conserved GDSL motif at their N-terminus (Upton and Buckley 1995). GELPs are hydrolytic enzymes that display broad substrate specificity and regio-specificity (Ding et al. 2023). It is well indicated that the GDSL genes play a significant role against abiotic stressors (Zhang 2020). Previously research on the GDSL family genes was accomplished on a variety of crops, including Brassica napus, Zea mays, and Oryza sativa. The GDSL family genes play important roles in abiotic stresses, pathogen defense, seed development, and lipid metabolism (Ling et al. 2006; Ren et al. 2021). For instance, the OsGELP34, OsGELP110, and OsGELP115, were proven to regulate the pollen development in O. sativa (Park et al. 2010; Zhang et al. 2020). Overexpression of AtGDSL1 in B. napus enhanced resistance to Sclerotinia sclerotiorum (Ding et al. 2020). Until now different omics technologies have been used to up-regulate the OsGDSL genes in O. sativa to improve the ability to withstand the drought and salinity stresses (Ding et al. 2019).

The comprehensive genome-wide analysis has predicted different numbers of GDSL proteins in different plant species such as, it has been reported that the GDSL family of A. thaliana is composed of 108 members (Ling 2008), Vitus viniferous (96) members, Sorghum bicolor (130) members, Populous trichocarpa (126) members, and 57 members in Physcomitrella patens (Volokita et al. 2011), 114 GDSL members in Oryza sativa, 53 members in Zea mays, 90 members in Selaginella moellendorffii, 88 members in Medicago truncatula, 102 members in Chlamydomonas reinhardtii, and 75 members in Phaeodactylum tricornutum (Ouyang et al. 2007; Youens-Clark et al. 2011), while in the present study, we investigated 91 GDSL genes in pitaya. These findings demonstrate the broad spectrum presence and varied quantity of GDSL genes across diverse plant species.

In addition to genetic regulators, growth and development stimulators are essential for reducing the effects of several biotic and abiotic stressors, such as melatonin (MT) (Altaf et al. 2021; Ahmad et al. 2023). MT is a natural, non-hazardous, universal biomolecule having diverse functions against plant stresses. The MT is a low molecular weight biomolecule with pleiotropic properties in the plant (Tiwari et al. 2021; Altaf et al. 2022c). It promotes seed and vegetative growth and upregulates the osmo-protectants and leaf photosynthesis. MT's biochemical and physiological properties have been investigated comprehensively in different studies (Zhang et al. 2015; Altaf et al. 2021, 2022b). The MT foliar application can promote tolerance against radiation, drought, heavy metals, salinity, and high-temperature plant stresses by alleviating the antioxidant defense machinery (Altaf et al. 2023; Raza et al. 2023b, a).

The present study is a comprehensive Genome-wide study of the GDSL gene family in pitaya. The investigation revealed 91 GDSL genes in the pitaya genome, where these genes were mapped on 11 chromosomes of the pitaya. The phylogenetic relationships of HuGDSL genes were established with the GDSL proteins of O. sativa, Brassica oleracea, Gossypium barbadense, and A. thaliana. In addition, we also comparatively analyzed the expression profile of HuGDSL genes among different single and multiple abiotic stresses, i.e., high temperature (H), drought (D), vanadium (V), vanadium + drought (VD), vanadium + heat (VH), vanadium + heat + drought (VHD) and MT applied plants. The MT application mitigates the effects of these stresses by alleviating the production of antioxidant genes. Based on transcriptome analyses of HuGDSL during various stresses and different plant tissues, we selected ten genes for RT-qPCR to validate the RNA-seq data. Our findings provide the foundation for the development of stress resistance pitaya genotypes under different climate conditions.

Materials and methods

Identification of HuGDSL gene family members and domain analysis

The Protein sequences of HuGDSL genes were obtained from the pitaya genome database (http://www.Pitayagenomic.com/), accessed on March 9, 2023 (Chen et al. 2022). The NCBI website (https://www.ncbi.nlm.nih.gov/protein), accessed on March 16, 2023, and the Phytozome (https://phytozome-next.jgi.doe.gov/), accessed on March 23, 2023 (Goodstein et al. 2012), was used for the collection of previously published information about the GDSL protein. To find the domain of the GDSL protein, the InterPro tool (https://www.ebi.ac.uk/interpro/), accessed on March 17, 2023, was employed. The characterized GDSL protein sequences of B. oleracea (Dong et al. 2016), O. sativa (Chepyshko et al. 2012), A. thaliana (Ling 2008), and G. barbadense (Liu et al. 2023), were obtained from previous information. The expasy ProtParam tool (https://web.expasy.org/protparam/) accessed on March 21, 2023, was used to download the physical and chemical properties such as molecular weight (kDa), isoelectric point (pI), and grand average of hydropathicity (GRAVY) based on amino acid sequence. The Protein length and CDS (bp) were computed from the pitaya genome database. The NCBI conserved domain tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) against Pfamv34.0-19178pSSMs, accessed on March 24, 2023, was employed to identify and annotate the conserved domains of the HuGDSLs. The visualization of the domain were done by TBTool (Chen et al. 2020). Moreover, the identification of motifs in HuGDSLs were indicated through motif finder tool (https://www.genome.jp/tools/motif/), accessed on March 26, 2023.

Phylogenetic analysis and Ka/Ks analysis of duplications of the HuGDSL family genes

To observe the evolutionary relationship of HuGDSL proteins with the GDSL proteins of other plant species we constructed the phylogenetic tree. The GDSL protein sequences of A. thaliana, G. barbadens, B. oleracea, and O. sativa, were downloaded from Phytozome version 13 (https://phytozome-next.jgi.doe.gov/), accessed on March 28, 2023 (Goodstein et al. 2012) and the pitaya HuGDSLs from the "pitaya-genome-website (http://www.Pitayagenomic.com/)" (Chen et al. 2022), respectively. By using the molecular evolutionary genetic analysis (MEGA-11) software (https://www.megasoftware.net/) accessed on 28 March 2023, all the GDSL proteins (194 protein sequences) were aligned (Tamura et al. 2021). The aligned protein sequences were subsequently used for phylogenetic analysis, and the maximum likelihood tree method was used with 1000 bootstrap replicates. The resulting phylogenetic tree was visualized using Interactive Tree Of Life (iTOL) software (https://itol.embl.de/), accessed on 1 April 2023 (Letunic and Bork 2019), and further modified through Adobe Illustrator. The Ka and Ks substitutions between the gene pairs were also calculated as described by Aslam et al. (Aslam et al. 2023). The divergence time (T, MYA; millions of years ago) was calculated as follows: T = Ks/2y (y = 6.56 × 10 − 9) and is synonymous with the number of substitutions per site per year (He et al. 2016).

The HuGDSL genes distribution on pitaya chromosomes

The pitaya genome database was used to obtain the data and genomic DNA for the HuGDSLs. Additionally, the pitaya chromosome length was obtained by using TBTool. An online web tool called as Phonogram plot tool (http://visualisation.ritchielab.org/phenograms/plot), accessed on April 9, 2023, was used to visualize the gene structure.

Distribution of conserved motifs and their patterns

For the identification of conserved motifs within the HuGDSLs, we employ MEME Suit (https://meme-suite.org/meme/tools/meme), accessed April 15, 2023, (Bailey et al. 2009). Default parameters were used, with a maximum of 10 conserved motifs.

Network analysis of the HuGDSL protein

To investigate the co-expression of all the 91 HuGDSL genes with other genes, a comprehensive analysis was conducted using the pitaya genome database (http://www.Pitayagenomic.com/coexpression), accessed April 17, 2023 (Chen et al. 2022). To visualize the interaction of all the identified and interacting HuGDSL genes, the bioinformatics Cytoscape tool (https://cytoscape.org/), accessed on 17 April 2023, was employed. (Shannon et al. 2003).

Cis-regulatory elements analysis

The PlantCare database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/), accessed on April 18, 2023, was used to examine the potential cis-regulatory elements located within 2 k bp upstream from the transcription start site of HuGDSL genes. To retrieve the promoter sequences from the pitaya genome file, the TBTools software is used with 2 Kbp upstream of the start codon (Chen et al. 2020). The PlantCARE database discovered the cis-acting regulatory elements (Lescot 2002).

Expression analysis of HuGDSL genes in different tissues

The expression data of the HuGDSL genes were retrieved from the pitaya genome database (http://www.pitayagenomic.com/) accessed on 18 April 2023 (Chen et al. 2022). We evaluated the temporal and spatial expression data of the 91 HuGDSL genes using RNA-seq data for various pitaya tissues, including, four flower bud stages, five flower phases, three pericarp stages, and three fruit pulp stages as well as at different developmental stages.

Plant material and stress treatments

The Shuangse Dahong" pitaya variety was selected as a plant material for this experiment. Flower buds were collected from the germplasm resource of Hainan-Shengda Modern Agriculture Development Company, located in Qionghai, Hainan, China. The seedlings were kept in a hydroponic condition with a half-strength Hoagland solution (pH 5.4). For comparative gene expression analyses of the HuGDSL genes in pitaya among multiple abiotic stresses, the following stresses H (Heat) (42 °C), D (Drought) (30%, PEG 6000), V (Vanadium), VD (Vanadium + Drought, VH (Vanadium + Heat), VHD (Vanadium + Heat + Drought), were applied to 40 days seedling in growth chamber having perfect growth conditions (16/8 h Light/Dark, for 40 days at 24 °C) along with control healthy seedlings. Pre-melatonin (M) was applied for one week, and the final treatment was then MVHD. The MT and V concentrations were applied following the procedure described by (Altaf et al. 2022c, a; Zaman et al. 2023a). The entire seedling and control were harvested after one week of stress application and kept at − 80 oC until total RNA extraction.

Transcriptome analyses of HuGDSL genes in multiple abiotic stress treatments and melatonin-applied seedlings

The plant samples of each treatment, as mentioned above, were subjected to RNA-seq analyses. The FPKM (Fragment per kilobase of exon per million) calculation was used for HuGDSL gene expression analyses. The heat expression map was constructed using TBTools (Chen et al. 2020). The number of DEGs under single and multiple stress conditions was also calculated via Venn diagram, (Oliveros, J.C. (2007–2015) Venny. An interactive tool for comparing lists with Venn's diagrams. https://bioinfogp.cnb.csic.es/tools/venny/index.html).

Total RNA extraction, cDNA synthesis, and real-time quantitative PCR (RT-qPCR) analyses

The Solarbio Total RNA Extraction Kit, Cat No: R1200 (Beijing, China) was used to extract the total RNA from each sample, following the manufacturer's guidelines. A Nano-drop spectrophotometer and gel electrophoresis were employed to check the concentration and integrity of total RNA. The cDNA was constructed using HiScript 1st Strand cDNA Synthesis Kit, Cat No. R111 (Vazyme, China) following company guidelines. After cDNA construction, the RT-qPCR gene analyses were accomplished using ChamQ Universal SYBER qPCR Master Mix Cat No.: Q711 (Vazyme, China) following the given protocol. The expression data was computed using the 2−ΔΔ CT method (Livak and Schmittgen 2001).

Results

Genome-wide identification of the HuGDSLs

The comprehensive genome-wide analysis identified 91 potential genes from the pitaya genome, designated as HuGDSL-1 to HuGDSL-91. The basic physical and chemical properties of HuGDSL genes, including the chromosome number of each gene, position on the chromosome (start and end), the length of the coding sequence of the gene (CDS length), the length of the protein encoded by the gene (protein length), the protein molecular weight, isoelectric point (pI), and the grand average of hydropathicity (GRAVY), were summarized in Table 1. There was significant variation in the protein length and molecular weight of the HuGDSL proteins. All the HuGDSL genes in pitaya demonstrate protein lengths varying between 106 amino acids (HuGDSL-79) and 333 amino acids (HuGDSL-20), and the molecular weight of the HuGDSL genes ranged from 11,845.13 to 36,384.81 kilo Dalton (kDa). The GRAVY value was 0, which indicated that pitaya HuGDSL proteins were hydrophilic, moreover, the values ranged from − 0.496 (HuGDSL-70) to 0.143 (HuGDSL-77). All HuGDSL proteins have a diversity in pI values, ranging from 4.68 (HuGDSL-91) to 9.31 (HuGDSL-31), whereas the average of the pI values was 6.4.

Table 1.

Physio-chemical properties of HuGDSL genes in pitaya

Transcript ID Renamed ID ChrNo Start
Position		 End
Position		 CDS
(bp)		 Protein
Length
(A.A)		 MW (kDa) PI Gravy
HU02G00677.1 HuGDSL-1 2 8,650,528 8,652,722 1146 319 34,433.73 5.78 − 0.103
HU02G00684.1 HuGDSL-2 2 8,781,541 8,783,559 1137 319 34,390.7 5.62 − 0.086
HU05G00641.1 HuGDSL-3 5 18,278,915 18,307,417 1221 322 35,749.58 6.48 − 0.178
HU08G02225.1 HuGDSL-4 8 105,993,830 105,995,906 1098 322 35,379.2 7.25 − 0.048
HU08G00489.1 HuGDSL-5 8 26,491,529 26,498,118 1098 323 35,700.8 6.62 − 0.094
HU02G02586.1 HuGDSL-6 2 116,449,505 116,454,304 1140 323 35,935.63 5.81 − 0.232
HU07G01768.1 HuGDSL-7 7 103,708,568 103,728,901 1125 322 35,179 5.79 − 0.121
HU07G01775.1 HuGDSL-8 7 104,089,156 104,101,486 1128 319 35,069.86 5.93 − 0.072
HU07G01771.1 HuGDSL-9 7 103,834,255 103,837,595 1062 316 35,165.76 5.61 − 0.185
HU07G01770.1 HuGDSL-10 7 103,797,439 103,801,233 1119 319 35,675.5 5.81 − 0.157
HU07G01776.1 HuGDSL-11 7 104,117,886 104,123,529 1104 324 36,130.95 5.14 − 0.109
HU08G00488.1 HuGDSL-12 8 26,055,200 26,055,200 1128 323 35,816.61 5.98 − 0.089
HU06G02039.1 HuGDSL-13 6 115,332,784 115,333,938 1155 318 35,981.99 6.32 − 0.214
HU06G02218.1 HuGDSL-14 6 118,766,358 118,767,518 1161 320 35,611.69 6.7 − 0.16
HU09G01255.1 HuGDSL-15 9 94,809,099 94,810,232 1134 318 35,520.49 5.93 − 0.168
HU03G01227.1 HuGDSL-16 3 13,091,226 13,094,319 1110 324 35,682.54 5.16 − 0.004
HU02G00135.1 HuGDSL-17 2 1,627,851 1,631,133 1143 327 36,046.75 7.74 − 0.167
HU01G02437.1 HuGDSL-18 1 138,461,578 138,465,742 1113 311 34,173.7 5.59 − 0.186
HU09G01106.1 HuGDSL-19 9 90,774,967 90,778,770 900 275 30,335.82 8.04 0.021
HU11G01714.1 HuGDSL-20 11 94,345,537 94,347,358 1158 331 35,830.52 5.66 0.078
HU03G01454.1 HuGDSL-21 3 16,858,509 16,861,838 1110 310 33,680.19 6.2 − 0.046
HU08G01404.1 HuGDSL-22 8 95,600,334 95,602,441 1140 315 34,331.81 5.11 − 0.073
HU04G02164.1 HuGDSL-23 4 128,520,896 128,524,006 1152 327 36,384.81 6.36 − 0.386
HU07G01909.1 HuGDSL-24 7 107,524,541 107,527,351 1194 310 35,050.26 9.24 − 0.177
HU03G01153.1 HuGDSL-25 3 12,359,271 12,361,462 1065 326 35,760.44 5 − 0.092
HU03G01266.1 HuGDSL-26 3 13,552,119 13,554,391 1146 326 35,789.44 5.1 − 0.117
HU09G00909.1 HuGDSL-27 9 76,223,676 76,226,497 1143 320 35,809.06 5.4 − 0.225
HU02G00136.1 HuGDSL-28 2 1,640,556 1,643,495 1170 327 35,985.94 8.35 − 0.096
HU02G00134.1 HuGDSL-29 2 1,623,340 1,627,115 1236 321 36,181.02 6.12 − 0.278
HU06G00437.1 HuGDSL-30 6 4,713,935 4,715,941 1098 316 34,914.45 5.73 − 0.078
HU02G00584.1 HuGDSL-31 2 7,524,445 7,527,547 1155 312 35,139.29 9.31 − 0.182
HU03G01746.1 HuGDSL-32 3 26,086,160 26,088,928 996 310 33,384.72 8.56 − 0.073
HU05G02137.1 HuGDSL-33 5 125,861,822 125,874,832 1089 306 33,569.14 5.5 − 0.06
HU07G02334.1 HuGDSL-34 7 112,672,920 112,674,773 1095 311 34,845.54 5.33 − 0.239
HU04G00326.1 HuGDSL-35 4 3,834,474 3,836,599 1209 310 34,365.91 5.49 − 0.054
HU09G00411.1 HuGDSL-36 9 9,604,124 9,606,998 1104 307 34,872.09 8.72 − 0.176
HU03G01338.1 HuGDSL-37 3 14,794,064 14,798,211 1104 309 34,292.96 8.41 − 0.126
HU06G00158.1 HuGDSL-38 6 1,507,391 1,509,198 1134 310 34,143.95 5.33 − 0.039
HU01G01427.1 HuGDSL-39 1 101,029,663 101,032,168 1095 308 33,956.6 5.1 − 0.092
HU05G00035.1 HuGDSL-40 5 1,066,653 1,069,833 1245 309 34,635.41 5.62 − 0.04
HU11G01342.1 HuGDSL-41 11 90,596,055 90,601,276 1194 306 33,099.43 5.79 − 0.006
HU07G01911.1 HuGDSL-42 7 107,554,267 107,557,666 1086 301 33,702.69 8.96 − 0.298
HU01G01425.1 HuGDSL-43 1 100,782,654 100,790,386 1014 306 33,554.06 5.57 − 0.147
HU11G00983.1 HuGDSL-44 11 84,179,187 84,184,162 1113 305 33,300.62 5.14 − 0.042
HU02G01525.1 HuGDSL-45 2 21,522,114 21,526,965 1023 309 34,042.86 4.77 0.096
HU02G03256.1 HuGDSL-46 2 142,470,937 142,474,535 1107 311 33,861.58 7.05 0.035
HU02G01278.1 HuGDSL-47 2 17,533,674 17,564,615 1968 309 34,042.86 4.77 0.096
HU02G00582.1 HuGDSL-48 2 7,482,850 7,488,125 1080 300 33,518.36 8.88 − 0.233
HU10G01611.1 HuGDSL-49 10 73,376,919 73,385,714 1068 314 35,196.02 6 − 0.108
HU03G02210.1 HuGDSL-50 3 88,968,605 88,985,281 1068 309 34,860.5 4.91 − 0.261
HU02G03360.1 HuGDSL-51 2 143,719,999 143,724,103 1107 302 34,119.01 6.5 − 0.185
HU06G00728.1 HuGDSL-52 6 8,439,938 8,442,704 1113 309 34,510 7.71 − 0.174
HU06G00814.1 HuGDSL-53 6 9,962,861 9,964,627 1149 310 34,050.77 7.72 − 0.166
HU08G02217.1 HuGDSL-54 8 105,922,575 105,924,779 1134 311 34,454.57 6.65 − 0.022
HU08G01890.1 HuGDSL-55 8 101,295,523 101,298,781 1101 309 34,239.25 6.19 − 0.317
HU02G01276.1 HuGDSL-56 2 17,510,416 17,516,041 1098 308 33,977.55 5.61 − 0.166
HU02G01277.1 HuGDSL-57 2 17,525,454 17,531,631 990 268 30,272.7 8.19 − 0.096
HU07G01773.1 HuGDSL-58 7 103,928,999 103,941,034 795 180 20,132.77 5.01 − 0.285
HU08G00214.1 HuGDSL-59 8 6,197,306 6,202,553 1107 309 33,740.3 8.56 − 0.174
HU08G00213.1 HuGDSL-60 8 6,093,645 6,098,834 1107 309 33,721.25 8.56 − 0.194
HU01G01679.1 HuGDSL-61 1 117,988,156 117,992,346 1056 310 34,782.27 4.83 − 0.255
HU03G01813.1 HuGDSL-62 3 30,618,699 30,622,238 1062 310 34,184.43 4.74 − 0.061
HU08G01891.1 HuGDSL-63 8 101,303,555 101,303,555 1059 297 32,704.09 8.54 − 0.196
HU11G00982.1 HuGDSL-64 11 84,170,145 84,172,393 1020 306 34,221.32 5.96 − 0.288
HU05G01862.1 HuGDSL-65 5 122,572,984 122,574,697 1107 316 34,291.88 6.57 − 0.119
HU10G01479.1 HuGDSL-66 10 46,201,010 46,204,374 918 244 27,354.44 9.22 − 0.152
HU05G01789.1 HuGDSL-67 5 120,855,192 120,868,680 1119 309 34,320.34 8.29 − 0.159
HU03G00853.1 HuGDSL-68 3 8,365,262 8,370,630 1020 289 32,187.63 8.81 − 0.175
HU01G01882.1 HuGDSL-69 1 127,320,193 127,322,731 1053 289 32,535.79 9.3 − 0.136
HU07G01772.1 HuGDSL-70 7 103,876,305 103,879,899 693 113 12,819.22 4.79 − 0.496
HU04G02214.1 HuGDSL-71 4 129,087,015 129,088,662 1104 309 34,251.91 5.86 − 0.262
HU01G02407.1 HuGDSL-72 1 138,148,828 138,150,244 936 309 34,779.56 6.25 − 0.269
HU03G01312.1 HuGDSL-73 3 14,306,857 14,309,733 1101 309 33,907.44 7.1 − 0.078
HU05G01128.1 HuGDSL-74 5 90,858,153 90,865,774 999 270 29,727.72 8.21 − 0.083
HU10G01610.1 HuGDSL-75 10 73,079,060 73,081,244 398 131 14,335.59 5.21 0.07
HU07G00280.1 HuGDSL-76 7 2,937,713 2,941,163 1056 299 32,051.78 9.03 0.036
HU11G01515.1 HuGDSL-77 11 92,300,742 92,307,020 1134 303 32,913.42 4.89 0.143
HU08G01555.1 HuGDSL-78 8 97,382,288 97,385,585 1110 309 33,935.51 5.03 − 0.019
HU07G01774.1 HuGDSL-79 7 104,011,579 104,012,165 336 106 11,845.13 4.87 − 0.397
HU05G00305.1 HuGDSL-80 5 4,340,932 4,343,657 924 260 28,151.72 7.16 − 0.055
HU05G01790.1 HuGDSL-81 5 120,869,139 120,873,236 1140 327 36,369.37 7.73 − 0.215
HU09G00178.1 HuGDSL-82 9 1,893,900 1,896,560 1155 272 30,481.76 5.62 − 0.137
HU08G01350.1 HuGDSL-83 8 94,816,852 94,818,876 964 253 28,105.98 4.68 − 0.024
HU07G01522.1 HuGDSL-84 7 91,859,647 91,861,913 1053 308 33,977.61 6.37 − 0.144
HU03G01760.1 HuGDSL-85 3 26,987,085 26,995,524 744 227 25,051.24 5.79 − 0.232
HU08G01978.1 HuGDSL-86 8 102,281,339 102,283,536 912 267 29,305.39 5.95 − 0.055
HU07G01645.1 HuGDSL-87 7 99,689,781 99,691,839 732 174 19,339.84 5.18 − 0.168
HU03G01764.1 HuGDSL-88 3 27,190,104 27,191,135 435 133 14,885.1 8.17 − 0.302
HU07G02136.1 HuGDSL-89 7 111,284,529 111,285,580 525 148 16,710.99 5.28 − 0.225
HU03G00937.1 HuGDSL-90 3 9,258,300 9,259,550 816 244 26,648.55 8.45 0.047
HU05G01431.1 HuGDSL-91 5 111,378,905 111,380,467 678 150 16,317.49 4.68 − 0.019
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Furthermore, all 91 HuGDSL proteins underwent domain-based analysis using an NCBI-conserved domain search, and the resulting data from the CDD website were employed through TBTools software to construct the HuGDSL structure of the domain. The result indicated the presence of the GDSL domain in all of the HuGDSL genes except HuGDSL-70, and 79 (Fig. 1).

Fig. 1.

Fig. 1

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GDSL family protein domains, all 91 HuGDSL sequences (HuGDSL‐1 to HuGDSL-91) contained GDSL domains except HuGDSL-70, and 79

Phylogenetic and Ka/Ks analysis of the HuGDSLs

A total of 194 characterized protein sequences of GDSL, including the 91 HuGDSL protein sequences from the pitaya genome and 103 protein sequences from the other four species, i.e., A. thaliana, G. barbadense, B. oleracea, and O. sativa, (Supplementary File S1), were collected from previously published data to construct the phylogenetic tree. The resulting phylogenetic tree divided the GDSL genes into nine Clades (Clade1-Clade9). Clade-3 contained 20 HuGDSL proteins and was the largest clade among all the nine clades of GDSL protein, while Clade-4 contained only one HuGDSL protein (HuGDSL-36) and was one of the smallest clades among all, while surprisingly HuGDSL59-60 make no clade with outgroups (Clade-8).

The Ka, Ks, and Ka/Ks values are key indicators of selection pressures. In general, a Ka/Ks ratio of > 1 refers to positive selection; a Ka/Ks ratio of < 1 refers to purifying selection, and a Ka/Ks ratio equal to 1 refers to neutral selection. The Ka/Ks ratio varied between 0.13 and 0.95, indicating that all the HuGDSL gene pairs exhibited negative or purifying selection (Ka/Ks < 1). These findings demonstrated that the purifying or negative selection was involved in maintaining the conservation of the HuGDSL gene structure during the domestication or evolution process. Further, we estimated the divergence time between duplication events, and the results showed that the estimated divergence time ranged from 0.29 to 225.04 million years ago (MYA), with an average of ~ 71.89 MYA (Table 2). The results shed light on the selection force and the molecular mechanisms during the evaluation of the HuGDSL genes (Fig. 2).

Table 2.

Parameters related to gene duplication and evolution of HuGDSL genes in pitaya

Gene name Gene name Ka Ks Ka_Ks T(MYA) Selection Events
HuGDSL-55 HuGDSL-64 0.401135 2.952603 0.135858 225.0459779 Purifying Segmental duplication
HuGDSL-73 HuGDSL-78 0.145361 1.019953 0.142517 77.74032039 Purifying Segmental duplication
HuGDSL-14 HuGDSL-15 0.125632 0.838327 0.149861 63.89691098 Purifying Segmental duplication
HuGDSL-17 HuGDSL-28 0.30673 1.857186 0.165158 141.5538445 Purifying Segmental duplication
HuGDSL-67 HuGDSL-81 0.257555 1.517037 0.169775 115.627795 Purifying Tandem duplication
HuGDSL-6 HuGDSL-19 0.169891 0.953405 0.178194 72.66809314 Purifying Segmental duplication
HuGDSL-32 HuGDSL-62 0.333501 1.823972 0.182843 139.022273 Purifying Tandem duplication
HuGDSL-71 HuGDSL-72 0.192129 1.04628 0.183631 79.74691925 Purifying Segmental duplication
HuGDSL-49 HuGDSL-54 0.239196 1.279746 0.186909 97.54163557 Purifying Segmental duplication
HuGDSL-33 HuGDSL-38 0.210229 1.096631 0.191704 83.58465423 Purifying Segmental duplication
HuGDSL-53 HuGDSL-65 0.189212 0.959377 0.197224 73.12328245 Purifying Segmental duplication
HuGDSL-35 HuGDSL-40 0.17879 0.883671 0.202326 67.35298697 Purifying Segmental duplication
HuGDSL-21 HuGDSL-22 0.187809 0.88839 0.211404 67.71265906 Purifying Segmental duplication
HuGDSL-59 HuGDSL-60 0.005914 0.027947 0.211601 2.130133015 Purifying Tandem duplication
HuGDSL-5 HuGDSL-12 0.281872 1.23973 0.227366 94.49161863 Purifying Tandem duplication
HuGDSL-82 HuGDSL-89 0.191909 0.801164 0.239538 61.0643604 Purifying Segmental duplication
HuGDSL-7 HuGDSL-11 0.15629 0.636247 0.245644 48.49445042 Purifying Tandem duplication
HuGDSL-24 HuGDSL-31 0.2705 1.084578 0.249406 82.66600156 Purifying Segmental duplication
HuGDSL-30 HuGDSL-91 0.242576 0.94625 0.256355 72.12272616 Purifying Segmental duplication
HuGDSL-76 HuGDSL-77 0.47104 1.56324 0.301323 119.1494148 Purifying Tandem duplication
HuGDSL-63 HuGDSL-86 0.440986 1.4338 0.307565 109.2835583 Purifying Tandem duplication
HuGDSL-1 HuGDSL-2 0.020804 0.064996 0.320078 4.953947925 Purifying Tandem duplication
HuGDSL-68 HuGDSL-69 0.324939 0.964153 0.33702 73.48728965 Purifying Segmental duplication
HuGDSL-48 HuGDSL-51 0.267045 0.757653 0.352463 57.74794006 Purifying Tandem duplication
HuGDSL-41 HuGDSL-80 0.149142 0.42308 0.352516 32.24695832 Purifying Segmental duplication
HuGDSL-85 HuGDSL-88 0.116294 0.258446 0.449974 19.69861241 Purifying Tandem duplication
HuGDSL-45 HuGDSL-47 0.016392 0.019475 0.841728 1.484343639 Purifying Tandem duplication
HuGDSL-25 HuGDSL-26 0.003715 0.003924 0.946749 0.299096217 Purifying Tandem duplication
HuGDSL-29 HuGDSL-87 0.011011 0.011578 0.951061 0.882443135 Purifying Segmental duplication
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Fig. 2.

Fig. 2

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The phylogeny of pitaya GDSL genes with other four plant species (A. thaliana, B. oleracea, O. sativa, and G. barbadense). The phylogenetic tree was divided into nine clades (clade 1– clade 9), shown with different colors: blue, red, green, yellow, steel, pink, sky blue, grey, and gold—the square red dots in the figure indicated the HuGDSL genes

HuGDSL protein sequence alignments and conserved motifs

The Molecular Evolutionary Genetics Analysis Version 11 (MEGA-11) was used to construct a rectangle phylogenetic tree of the 91 HuGDSL proteins. The maximum likelihood method was used to create the phylogenetic tree, which had a bootstrap value of 1000 replicates and was classified into 9 distinct clades (Fig. 3A). The bioinformatics software MEME tool was used to perform a conserved motif analysis on all GDSL proteins of pitaya, which resulted in the discovery of 10 conserved motifs, and the length of each motif ranges from 15 to 40 amino acids. The C terminal (the carboxyl-terminus, carboxy-terminus, C-terminal tail, C-terminal end, or COOH-terminus) Of the HuGDSL proteins exhibit highly conserved domains. Motifs 1, 2, 6, and 8 were found in nearly all of the HuGDSL proteins, indicating the functional relevance of these motifs. Motif 5 was not found in HuGDSL-33, HuGDSL-55, HuGDSL-64, and HuGDSL-44 (Fig. 3B), which suggests potential variation among the genes of the HuGDSL gene family in pitaya. However, the remaining motifs were present in very small amounts, which indicates the limited occurrence of these motifs in the HuGDSL gene family (Fig. 3B). These findings suggested the close relationship among all the HuGDSL genes in pitaya. All the known motifs of the HuGDSL are shown in Fig. 3 with different colors, the sequences and lengths are shown in Table 3, and the logos of these identified motifs are shown in Figure S1.

Fig. 3.

Fig. 3

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The motif distribution in each HuGDSL protein sequence. A The phylogeny of the HuGDSL genes B The identified conserved motifs in HuGDSL genes. Motifs 1 to 10 are highlighted in differently colored boxes

Table 3.

Detail information about the 10 conserved motifs of pitaya GDSL proteins

Motif Width (aa) Sequences
Motif-1 21 VCPBPSKYVFWDGIHPTEAAN
Motif-2 23 PTGRFSBGRLIIDFJAEALGLPL
Motif-3 29 FLKELYNLGARKFVVPGLGPJGCLPSQLA
Motif-4 21 PAIFVFGDSJVDTGNNNYJFT
Motif-5 16 BFLHGVNFASAGAGIL
Motif-6 40 DDPSMYDELGCLKSYNEFAQFHNDQLQAAIQDLQKZHPDA
Motif-7 15 AKANYPPYGIDFFHG
Motif-8 15 LYLIEIGSNDYANNY
Motif-9 23 LDLIQNPSKYGFEEVKTACCGTG
Motif-10 15 ISLSVQLDYFKEYLN
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Chromosomal locations and synteny analysis of HuGDSL genes

The HuGDSL genes were unevenly distributed in all the chromosomes in the pitaya genome. For example, chr02, chr03, chr07 had 14, 13 and 15 HuGDSL genes. The chr01 and chr06 comprised of same 6 HuGDSL genes, whereas the chr04 and chr10 contained the least number of HuGDSL genes i.e. 3 genes (Fig. 4). The majority of HuGDSL genes were found in chr02, chr03, and chr07 i.e. 14, 13, and 15, respectively. The expansion of a gene family occurs owing to the duplication events arising at the whole genome, and we identified duplicate gene pairs among the HuGDSL based on sequence similarity. As a result, a total of 29 gene duplications were identified with a > 80% sequence similarity. Gene pairs were selected as tandemly duplicated if the sequence was found within a 100 kb window of the duplicated genomic region. In our study, we found 12 duplicated gene pairs including, HuGDSL-32–62, HuGDSL-76–77, HuGDSL-67–81, HuGDSL-63–86, HuGDSL-5–12, HuGDSL-48–51, HuGDSL-7–11, HuGDSL-85–88, HuGDSL-1–2, HuGDSL-59- 60, HuGDSL-45–47, and HuGDSL-25–26. The remaining 17 HuGDSL gene pairs including HuGDSL-55–64, HuGDSL-17–28, HuGDSL-49–54, HuGDSL-33–38, HuGDSL-24–31, HuGDSL-71–72, HuGDSL-73–78, HuGDSL-68–69, HuGDSL-53–65, HuGDSL-6–19, HuGDSL-30–91, HuGDSL-21–22, HuGDSL-35–40, HuGDSL-14–15, HuGDSL-82–89, HuGDSL-29–87, and HuGDSL-41–80, were identified as a segmental duplication gene pairs. Additionally, chr02, chr03, and chr07 contained the greatest number of segmental and tandem duplicated genes, suggesting the importance of these genes in the evaluation of the HuGDSL gene family. The supplementary file S2 contained detailed information about each gene's chromosome length and position in the pitaya genome.

Fig. 4.

Fig. 4

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All 91 HuGDSL genes were distributed on the 11 chromosomes of pitaya. chr02, chr07, chr08, and chr03 contain a high number of HuGDSL genes, and chr04 and chr10 contain very few HuGDSL genes. The Gene names from HuGDSL-1 to HuGDSL-91 are mentioned in different colors

A collinearity investigation revealed strong orthologs of GDSL genes between pitaya and A. thaliana (Fig. 5), which indicates a high degree of similarity between their genomes and may perform similar biological functions. (Fig. 6). In summary, the genes presented on chr01, chr02, chr03, chr04, chr05, chr06, chr07, chr08, and chr011 demonstrated syntenic relations with A. thaliana, respectively (Fig. 6). In contrast, chr09 and chr10 showed no syntenic relations with A. thaliana (Fig. 6). The results suggested the importance of collinear blocks during the evaluation of the GDSL gene family in pitaya.

Fig. 5.

Fig. 5

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The inter-chromosomal association and the chromosomal location of the HuGDSL genes in pitaya. The red, pink, brown, green, and blue lines display syntenic GDSL gene pairs

Fig. 6.

Fig. 6

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Collinearity analysis between pitaya and the A. thaliana chromosomes of the GDSL genes Collinear blocks within the genomes of pitaya and A. thaliana are shown by grey, and the red lines showed the syntenic GDSL gene pairs between pitaya and A. thaliana

Analysis of exon–intron structure of HuGDSL genes

To get insight into the structural organization of HuGDSLs, we used all the identified HuGDSL genes for the detection of the exon–intron organization as shown in Fig. 7. The organizational patterns of the HuGDSL genes were found to have varied exon–intron distributions, with exons ranging from 1 (HuGDSL-13) to 10 (HuGDSL-47). The number of introns in the HuGDSL genes also varies, ranging from 0 (HuGDSL-14) to 9 (HuGDSL-47). Three HuGDSL genes (HuGDSL-13, HuGDSL-14, and HuGDSL-15) had only one exon, and no introns were found. Generally, GDSL genes in the same subgroup showed similar exon–intron features, providing further evidence of their phylogenetic relationship (Fig. 7).

Fig. 7.

Fig. 7

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The gene structure of 91 HuGDSL genes showed conserved exon/intron organization

Identification of Cis-acting elements and subcellular localization of HuGDSL genes in pitaya

To identify the biological functions of the HuGDSL, such as stress response, growth, and development in pitaya. All 91 HuGDSL gene sequences (2 Kbp upstream of the start codon) were chosen for cis-element analysis using the PlantCARE online tool. The cis-regulatory elements are involved in gene expression regulation. In this study, all HuGDSL genes harbored cis-elements in their upstream regions (Fig. 8). All the identified promoters were classified into hormone-responsive elements. Among the hormone-responsive elements, we identified ABRE (abscisic-acid-responsive element), the CGTCA/TGACG motif (methyl jasmonate-responsive element), the TGA element (auxin-responsive element), and the TCA element (salicylic-acid-responsive element) (Fig. 8). These findings indicate the diverse roles of GDSL genes based on their cis-acting elements.

Fig. 8.

Fig. 8

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The cis-acting elements of the promoter regions (2000 bp upstream of the start codon) of 91 HuGDSL genes. All the hormone-responsive elements, such as auxin-responsive elements, gibberellin-responsive elements, MeJA-responsiveness, salicylic acid responsiveness, and abscisic acid responsiveness, are shown as green, pink, red, steal, and yellow, respectively

The subcellular localization of all 91 GDSL proteins in pitaya showed that GDSL genes were mainly distributed in the nucleus, endoplasmic reticulum, plasmid, Golgi apparatus, peroxisomes, chloroplasts, mitochondria, cytoskeletons, vacuoles, and cytoplasm (Fig. 9, Table S4). However, the distribution of each member in different cellular parts varies. The majority of the genes were found in the nucleus and chloroplast.

Fig. 9.

Fig. 9

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The heat map shows the presence of all 91 HuGDSL genes in the subcellular parts of pitaya, which contain the nucleus, endoplasmic reticulum, plasmid, Golgi apparatus, peroxisomes, chloroplasts, mitochondria, cytoskeletons, vacuoles, and cytoplasm. The red color showed high expression, the yellow color indicated medium expression, and the blue color indicated minimum expression of the genes in subcellular parts of the pitaya

HuGDSL protein network analysis

The analysis of HuGDSL proteins and their interaction network revealed a significant difference in the number of proteins regulated by each predicted gene. Out of 91 HuGDSL genes in pitaya, 61 genes were involved in 375 possible interactions. We classified the interacting genes into four categories based on network analysis: Blue (2–5 interactions), Light Green (6–10 interactions), red (11–15 interactions), and pink (16–20 interactions). The HuGDSL-35, HuGDSL-23, HuGDSL-91, HuGDSL-76, and HuGDSL-49 were indicated to be involved in maximum interaction, as shown in pink and red (Fig. 10). Our results also indicated that HuGDSL-18, HuGDSL-41, and HuGDSL-57 interact significantly with other genes in the light green category.

Fig. 10.

Fig. 10

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Protein interaction network. The key HuGDSL genes based on maximum interaction are shown in Pink and red as they interact with more than ten genes. The different interactions of the HuGDSL genes are shown in other colors as follows: pink (16 to 20), red (11 to 15), light green (6 to 10), and blue (2 to 5). Most of the genes showed 2–5 interactions

Expression analysis of HuGDSL genes in different tissues of pitaya

The in-silico expression analysis of HuGDSL genes revealed a significant expression in various tissues of pitaya, including the flower bud at four different stages (FB1 to FB4), the lowers at five different stages (F1 to F5), the pericarp at three different stages (45 days, 65 days, and 85 days), and the pulp of the pitaya fruit at three different stages (29 days, 35 days, and 49 days). Across these tissues and different stages, most of the HuGDSL genes were not expressed (Fig. 11). HuGDSL-57, HuGDSL-58, and HuGDSL-70 were expressed significantly in periC-65d, whereas HuGDSL-1, HuGDSL-2, and HuGDSL-3 were expressed in the flower bud at every stage. Additionally, the HuGDSL-23 was partially expressed in the Pulp stages but in the flower bud stages, it was expressed notably. The HuGDSL-89, HuGDSL-56, and HuGDSL-59 were expressed higher in the five flower phases. From the expression data of all the 91 HuGDSL genes, we selected 10 genes that exhibited significant differential expression across all tissues, such as HuGDSL-1, HuGDSL-2, HuGDSL-3, HuGDSL-16, HuGDSL-23, HuGDSL-42, HuGDSL-48, HuGDSL-55, HuGDSL-59, and HuGDSL-78 for RT-qPCR validation.

Fig. 11.

Fig. 11

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The heat map of HuGDSL genes provides a visual representation of the gene expression across 15 pitaya tissues. The four flower bud’s stages have been designated as FB1, FB2, FB3, and FB4, while the five flower phases are marked as F1, F2, F3, F4, and F5. The three pericarp stages are recognized as periC-45d, periC-65d, and periC-85d. However, the three fruit pulp stages are indicated as pul-29d, pul-35d, and pul-49d. The color gradient showed expression from high to low

The RNA-seq expression analyses of HuGDSL genes under multiple abiotic stresses

The RNA-seq data of the HuGDSL genes in single H (heat), D (drought), V (vanadium), combined stress conditions VD, VH, VHD, and melatonin applied-MVHD samples exhibited vast HuGDSL gene expression patterns, as compared to control. Among 91 HuGDSL genes, eleven genes (HuGDSL-6/24/33/47/53/54/59/60/61/62/91) showed higher expression in separate, combined, and melatonin-applied seedlings as compared to CK. Six genes (HuGDSL-3/8/21/23/42/57) showed moderate expression pattern, as compared to CK, while thirteen genes (HuGDSL-5/7/12/13/14/15/24/35/37/67/69/80–90) exhibit low or no expression in all applications, as compared to CK. Besides these all, some key genes i.e. twelve genes (HuGDSL-1/2/3/31/18/29/32/41/43/44/55/91), showed enhanced expression in melatonin-applied seedlings (MVHD) as compared to single (D, H, V) combined (VD, VH, VHD) and CK which suggests that melatonin may play a key role in stress tolerance pathways in pitaya seedlings by upregulating the expression of HuGDSL genes (Fig. 12) (Table S5). Finally, the comparison of common and differentially expressed gene numbers under single, and multiple stress conditions was calculated using the Venn diagram as shown in Fig. 13, where, Fig. 13A, showed the comparison of the number of common and exclusive gene numbers between single stress conditions i.e. high temperature (H), drought (D), vanadium (V), while the Fig. 13B, showed the comparison of the number of common and exclusive gene numbers among multiple stress conditions i.e. vanadium + drought (VD), vanadium + heat (VH), vanadium + heat + drought (VHD) and MT applied plants. The majority of commonly expressed genes were 52 and 55, in single and multiple stresses, respectively, and the majority of DEGs 10, were expressed between V and D stresses (Fig. 13B).

Fig. 12.

Fig. 12

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The expression pattern of the RNA-seq data of HuGDSL genes during multiple abiotic stresses. The heatmap showed the gene expression in H (heat), D (drought), V (vanadium), VD, VH, VHD, and melatonin-MVHD as compared to CK. The expression bar is red to blue, demonstrating high to low expression levels. Up-and down-regulated genes showed (log2FoldChange) of FPKM values and three biological replications were used for RNA-seq analyses (n = 3), P < 0.05

Fig. 13.

Fig. 13

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Comparison of differentially expressed gene number under (A) single i.e. high temperature (H), drought (D), vanadium (V), and (B) multiple stress conditions i.e. vanadium + drought (VD), vanadium + heat (VH), vanadium + heat + drought (VHD) and MT applied plants, using Venn diagram. Overlapping areas represent common gene numbers. Up- and down-regulated genes (|log2FoldChange|≥ 1) were examined for common genes using the Venn diagram

HuGDSL genes expression analyses and validation of RNA-seq; data through RT-qPCR

The comparative expression analyses of 10 HuGDSL genes in separate, double, triple stresses, and melatonin-applied seedlings against the triple stresses were accomplished viz RT-qPCR to analyze the melatonin-mediated upregulation of the stress tolerance in pitaya and also to validate the RNA-seq data during the aforesaid mentioned stress conditions. In the present study, 10 genes were selected for expression validation based on the in-silico expression data in different tissues and plant parts, as shown in Fig. 8, and RNA-seq data of HuGDSL genes (Fig. 12). The same expression patterns of the selected genes were observed via; RT-qPCR as obtained from RNA-seq analyses. Under single (H, D, V), double (VH, VD), and multiple stress (VDH) conditions, all genes exhibit relatively high expression levels as compared to CK, on the other hand, Melatonin elevates the expression of each gene against these stresses that support the melatonin-based tolerance against different stresses in pitaya (Fig. 13).

Discussion

Pitaya (Selenicereus undatus L.) is a tropical fruit plant known for its distinctive appearance, vibrant colors, and sweet-tasting fruit (Arredondo et al. 2022). It has a unique adaptation in the presence of cladodes, which have modified stems that have replaced leaves as photosynthetic organs. The flowers and fruits of S. undatus are safe for consumption, and both are edible. The pitaya fruit's components are known to be very important because of the presence of polyphenols, tannins, betalains, and non-betalain antioxidants (Ortiz and Takahashi 2015). These compounds increase the nutritional value by mitigating oxidative damage (Ling 2008).

GDSL esterase/lipase proteins (GELP) are a subfamily of lipolytic enzymes that have been discovered recently (Chepyshko et al. 2012). GELPs have broad substrate specificity and have a high number of family members within a genome. The GDSL family contains many functional genes that perform important biological functions in growth and development, morphogenesis, seed oil synthesis, and defense responses in plants (Yao-guang et al. 2022). The GDSL-type esterase/lipase gene named Enod8 is found in Medicago truncatula (Model legume), and Medicago sativa (Alfalfa) has butyl and acetyl esterase function but lacks aliphatic esterase activity (Dickstein 1993; Coque et al. 2008). The GDSL26 is extensively distributed in lipids in cotton, which are responsive to variations in osmotic pressure during drought stress. The GDSL26 triggers the RD29 expression in MAPK pathway, which results in enhanced accumulation of wax layer that in turn decreases the water loss via stomata, and in that way, GDSL26 enhances the plant drought resistance capabilities (Liu et al. 2023).

Previously GDSL family genes were studied in different crops, for instance, A. thaliana (Lai et al. 2017), G. max (Su et al. 2020), G. barbadense (Wang et al. 2023), Solanum lycopersicum (Yao-guang et al. 2022), and Carya illinoinensis (Jiao et al. 2022). Nevertheless, our study is the first comprehensive study of GDSL family genes against different abiotic stresses and their response to melatonin against these stresses in pitaya. The GDSL family genes have been determined to play an important role in biotic and abiotic stress resistance in plants (Ling et al. 2006);(Yao-guang et al. 2022). According to Hong et al. the GDSL-type pepper lipase gene “GaGLIP1” plays a key role in drought tolerance in pepper plants (Hong et al. 2008). In B. napus a GDSL lipase gene “BnGLIP” triggers the plant immunity against pathogen resistance (Tan et al. 2014). Arabidopsis lipase-1 (Arab-1) shares similarities with the GDSL protein found in B. napus. It is classified as an extracellular protein and it is speculated that it is involved significantly in various plant developmental stages. The extracellular lipase 4 (EXL4) improved hydration in the early pollination stage of Arabidopsis (Updegraff et al. 2009).

In the present study, we successfully identified 91 GDSL gene family members in the pitaya genome, named HuGDSL-1 to HuGDSL-91 (Table S1). Interestingly, all 91 HuGDSL genes were found to be distributed on all of the 11 chromosomes of the pitaya (Fig. 4). In contrast, in O. sativa, the genes were located on 12 chromosomes and distributed on 20 chromosomes in soybeans (Su et al. 2020). Most HuGDSL genes are located towards the ends of the chromosomes, similar to the discoveries in other plant species such as O. sativa (Jiang et al. 2012), and A. thaliana (Lai et al. 2017). The conserved motif structures (Fig. 3) and HuGDSL gene sequences revealed a similar pattern of conserved motifs and exon–intron sequences, respectively. Our results showed that the GDSL genes in pitaya may have the same function as described previously by (Wang et al. 2020). The GLIP2 (GDSL-motif lipase 2) is involved in the immunity of plants against pathogens, such as enhancing the resistance to Erwinia carotovora by playing the role of a negative regulator of auxin signaling (Lee et al. 2009). The study discovered that the excessive production of five GDSL-type Seed Fatty Acid Reducer (SFAR) in Arabidopsis resulted in a decrease in the overall amount of fatty acids in the seeds and caused alterations in the composition of fatty acids in the seeds (Huang et al. 2015).

The phylogeny of the GDSL genes in pitaya with other four plant species revealed that all the identified genes were divided into nine clades designated as clade 1 to clade 09 (Fig. 2); similarly, there were varying numbers of GDSL genes in each clade. The previous finding of GDSL genes in different species revealed that the A. thaliana GDSL genes were divided into four clades (Lai et al. 2017), and O. sativa GDSL family genes were divided into three groups (Jiang et al. 2012). Gene duplication, such as segmental duplication, tandem duplication, and whole-genome duplication, is responsible for the evolution and expansion of the gene family (Wu et al. 2019) Next, 29 paralogous GDSL genes were identified in pitaya, indicating that segmental duplication contributed to the HuGDSL expansion. Accumulated evidence has demonstrated that duplication events have been important for gene expansion in the GDSL family. The results of our study were consistent with GDSL expansion in O. sativa (Jiang et al. 2012). The expression patterns of the HuGDSLs in pitaya further provided insight into the selection of genes that play an important role in response to abiotic stress conditions. (Fig. 11).

In the network analysis the candidate genes, are involved in many interaction modules (Jiang et al. 2012). We also investigated the cis-regulatory elements in the promotor region of the HuGDSLs. The analyses revealed that the growth regulatory gibberellin, auxin, abscisic acid (ABA), and salicylic acid cis-elements were found to be in large quantity (Fig. 8). These cis-acting elements were previously found to be involved in diverse signaling pathways and plant regulatory processes, which have been demonstrated to have significant functions in plant growth, development, and stress responses (Lai et al. 2017). The presence of these cis-acting elements in the promoter region of HuGDSL genes sheds light on their putative regulatory mechanisms and indicates that they participate in pitaya hormone-mediated activities.

To validate further the functional contribution of these cis-regulatory elements in growth and development, we analyzed the expression pattern of HuGDSL genes via RNA-seq under various abiotic stresses, i.e. (H, V, D, VD, VH, VHD), and MT applied seedlings (MVHD). The transcriptome data of the HuGDSL gene family in the stress mentioned above exhibited a vast HuGDSL gene expression pattern compared to the control. Among 91 HuGDSL genes, some genes (HuGDSL-6/24/33/47/53/54/59/60/61/62/91) showed higher expression in separate, combined, and melatonin-applied seedlings than healthy control. Few genes (HuGDSL-3/8/21/23/42/57) showed moderate expression pattern, while some (HuGDSL-5/7/12/13/14/15/35/37/67/69/80–90) exhibit low or no expression in all applications. Besides these, some essential genes showed enhanced expression in melatonin-applied seedlings (MVHD) as compared to single (D, H, V) combined (VD, VH, VHD) and control (HuGDSL-1/2/3/31/18/29/32/41/43/44/55/59/78/91), which showed that melatonin may play a key role in stress tolerance pathways in pitaya seedlings (Fig. 12), (Table S5).

We selected 10 highly expressed genes among pitaya different tissues (HuGDSL-1/2/3/16/23/42/48/55/59 and HuGDSL-78) to investigate and validate the transcriptome expression pattern viz RT-qPCR under different abiotic stresses (Fig. 13). The current research outcomes showed that HuGDSL genes simultaneously play a critical role against various abiotic stresses. At the same time, the melatonin application exhibits enhanced accumulation of these genes to resist the stress effects. We identified five candidate genes (HuGDSL-1, HuGDSL-3, HuGDSL-55, HuGDSL-59, HuGDSL-78) based on heat expression (Fig. 8), cis-elements (Fig. 10), transcriptome analyses (Fig. 12), and RT-qPCR (Fig. 14). These genes can be used for the functional characterization of specific traits by producing transgenes using the CRISPR-CAS system (Zaman et al. 2019, 2021; Varshney et al. 2021; Tariq et al. 2023). The Mobile-CRISPR system of genome editing technique, which is newly introduced, can be used to enhance the stress tolerance in pitaya and to improve the agronomic traits in pitaya (Yang et al. 2023; Zaman et al. 2023a).

Fig. 14.

Fig. 14

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The expression analyses of HuGDSL genes under multiple abiotic stresses via RT-qPCR. In stress-applied seedlings, all genes showed high expression as compared to CK, except HuGDSL-59, which is downregulated in all stresses compared to CK. Melatonin-applied seedlings showed enhanced expression of all genes as compared to single, combined stresses and control (CK). The bars denoted the standard error of the means (n = 6). Different lower-case letters represent significant differences among treatments at P ≤ 0.05, according to the LSD test

Conclusions

This is the first-ever comprehensive investigation of the GDSL gene family in pitaya (S. undatus L.) at the genome-wide level. The comprehensive analyses and by using many bioinformatics approaches such as sequence analysis, physio-chemical properties, phylogeny analysis, gene structure analyses, and expression profiling of GDSL genes in different tissues helped us to discover and annotate the GDSL family genes in pitaya. The comparative study across five crops of GDSL gene families, including pitaya, followed by constructing a phylogenetic tree, analysis of expression patterns, and gene network analysis, establishes a solid foundation for the functional characterization of these genes in pitaya. This comparative study also facilitates the identification of potential orthologues, paralogs, and evolutionary trends of the GDSL gene family in growth and development by improving the defense mechanisms against different abiotic stresses.

Based on heatmap expression (Fig. 11), cis-elements in the promotor region (Fig. 8), transcriptome analyses (Fig. 12), and RT-qPCR (Fig. 14), we identified 5 candidate genes (HuGDSL-1, HuGDSL-3, HuGDSL-55, HuGDSL-59, and HuGDSL-78), that possibly can play a pivotal role against these stresses, and enhanced accumulation of these genes were recorded in melatonin-applied seedlings in combination with these stresses. The RT-qPCR validation under separate and combined abiotic stresses set a foundation for improving pitaya growth and development programs. Our findings could be applied to future conventional and molecular breeding projects to develop climate-smart pitaya genotypes. The Investigation and characterization of the GDSL gene family in pitaya are crucial in determining the molecular genetic basis of essential traits, and this information can be leveraged in breeding programs to select and develop improved pitaya genotypes with desired characteristics such as improved fruit quality, enhanced yield, and tolerance to both abiotic and biotic stresses.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (RAR 3974 KB) (3.9MB, rar)

Acknowledgements

The authors thank CSC (Chinese Scholarship Council) for giving us the opportunity to study in China. We are also thankful to Dr. Qamar U Zaman (School of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication), School of Tropical Agriculture and Forestry Hainan University, Sanya 572025, Hainan, China), for providing us the transcriptomic data under different stress conditions.

Author contributions

L.U.K. conceived and designed the experiments.; O.U.S. and L.U.K in-silico study, perform experiments, and interpret the data.; M.W, W.U.K, M.I, and M.E analyzed and review the data, O.U.S. and L.U.K write the manuscript.; L.Z, M.E, J.P, Z.S, P.Z, and S.B managed the multiple stresses and seedlings protection; L.P revised and approved the final paper.; L.P. supervised and provided funding for the whole research work.

Funding

This work was supported by grants from the China NSFC Research Fund for International Young Scientists (grant number: 32250410291), Key Research Program of Hainan Province (grant number: ZDYF2022XDNY185), and Special Project for the Academician Team Innovation Center of Hainan Province (grant number: YSPTZX202206).

Data availability

All the data is available in the manuscript and supplementary files.

Declarations

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Informed consent

Not applicable.

Footnotes

Publisher's Note

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Contributor Information

Pingwu Liu, Email: hnulpw@hainanu.edu.cn.

Latif Ullah Khan, Email: latif.hainu3103@gmail.com.

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