Genome-wide identification and characterization of glutathione S-transferase gene family in quinoa (Chenopodium quinoa Willd.)

Abstract

The present investigation was envisaged for large scale in-silico genome wide identification and characterization of glutathione S-transferases (GSTs) in Chenopodium quinoa. In this study, a total of 120 GST genes (CqGSTs) were identified and divided into 11 classes of which tau and phi were highest in numbers. The average protein length of protein was found to be 279.06 with their corresponding average molecular weight of 31,819.4 kDa. The subcellular localization analysis results showed that proteins were centrally localized in the cytoplasm followed by chloroplast, mitochondria and plastids. Structural analysis revealed the presence of 2 -14 exons in CqGST genes. Most of the proteins possessed two exon one intron organization. MEME analysis identified 15 significantly conserved motifs with a width of 6–50 amino acids. Motifs 1, 3, 2, 5, 6, 8, 9 and 13 were found specifically in tau class family; motifs 3, 4, 5, 6, 7 and 9 were found in phi class gene family, while motifs 3, 4, 13 and 14 were found in metaxin class. Multiple sequence alignment revealed highly conserved N-terminus with active site serine (Ser; S) or cysteine (Cys; C) residue for the activation of GSH binding and GST catalytic activity. The gene loci were found to be unevenly distributed across 18 different chromosomes with a maximum of 17 genes located on chromosome number 7. Dominance of alpha helix was followed by coil, extended strand and beta turns. Gene duplication analysis revealed that segmental duplication and purifying type selection were highest in number and found to be main source of expansion of GST gene family. Cis acting regulatory elements analysis showed the presence of 21 different elements involved in stress, hormone and light response and cellular development. The evolutionary relationship of CqGST proteins carried out using maximum likelihood method revealed that all the tau and phi class GSTs were closely associated with those of G. max, O. sativa and A. thaliana. Molecular docking of GST molecules with the fungicide metalaxyl showed that the CqGSTF1 had the lowest binding energy. The comprehensive study of CqGST gene family in quinoa provides groundwork for further functional analysis of CqGST genes in the species at molecular level and has potential applications in plant breeding.

Introduction

Quinoa (Chenopodium quinoa Willd.) is an allotetraploid (2n = 4x = 36) pseudocereal of Amaranthaceae family that originated in the Andean region of South America (Hong et al. 2017). Quinoa produces gluten free nutritious grains that have exceptional balance between carbohydrates, vitamins, minerals, essential amino acids, dietary fibers and oils (Yasui et al. 2016; Dakhili et al. 2019). It is usually cultivated in clay loam or sandy loam soils, with approximate pH of 5.5–8.5 with good drainage and moderate slopes (Fuentes and Bhargava 2011). The optimum temperature for quinoa is around 8–15 °C, although it can withstand up to − 4 °C (Alvar-Beltrán et al. 2020). Quinoa is recognized as a crop of great value due to its nutritious grain and high tolerance towards abiotic stresses (Yasui et al. 2016). Quinoa is adapted to a wide range of marginal soils, including drought prone ones and those with high salinity, which help it to thrive under adverse agroclimatic conditions (Morales et al. 2017; Vita et al. 2021). Due to its potential health benefits and adaptability to adverse climatic conditions, the year 2013 was declared by the FAO as the International Year of Quinoa (Bhargava and Ohri 2015; Jarvis et al. 2017). Considering its economic and nutritional significance, it could be a fascinating challenge for plant breeders to use transgenic approaches to develop varieties resistant against diverse biotic and abiotic stresses. The availability of sequenced genome of quinoa provides an opportunity to search and characterize diverse gene families which are functionally important. Recently, several gene families like HSP 70 (Liu et al. 2018), WRKY (Yue et al. 2019), trihelix transcription factor (Li et al. 2022), PYL (Pizzio 2022) and CIPK CBL (Xiaolin et al. 2022) have been identified and well characterized in quinoa.

Environmental stresses such as high salt, drought, extreme temperatures are often very harmful to plants, and they produce reactive oxygen species (ROS) to cause oxidative stress and cell damage (Czarnocka and Karpiński 2018). In order to maintain the redox balance within cells, plants have a series of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) and non-enzymatic mechanisms like glutathione, alpha-tocopherol, carotenoids and flavonoids to maintain the balance between production and elimination of ROS (Mittler et al. 2004).

GSTs (EC 2.5.1.8) constitute ancient protein superfamily of multifunctional proteins and are an integral part of the antioxidant enzymatic defense system in plants that works downstream of Cyt P450 by catalyzing the nucleophilic conjugation of reduced tripeptide glutathione (GSH) (g-Glu–Cys–Gly) into wide variety of hydrophobic and electrophilic substrates to make a chemical compound more hydrophilic to be expelled from the cell (Marimo et al. 2016; Vaish et al. 2020; Song et al. 2021). GSTs are characterized by the presence of canonical thioredoxin fold in the highly conserved N-terminal domain which dominantly consists of α-helices and β-strands with a β1α1β2α2β3β4α3 topology. It possesses a G-site for glutathione binding. The variable C-terminal domains consisting of all the α-helices and possesses an H-site for secondary hydrophobic substrate binding (Vaish et al. 2020).

In plants, GSTs play vital roles in the detoxification of xenobiotics and toxic lipid peroxides (Chronopoulou et al. 2014; Fafián-Labora et al. 2020), glucosinolate biosynthesis (Czerniawski and Bednarek 2018) and in metabolism (Liu et al. 2014). The GST proteins of plants have been classified into 14 distinct classes, namely tau, phi, theta, zeta, lambda, γ-subunit of the eukaryotic translation elongation factor 1B (EF1B), dehydroascorbate reductase (DHAR), metaxin, tetrachlorohydroquinone dehalogenase (TCHQD), Ure2p, microsomal prostaglandin E synthase type 2 (mPGES2), hemerythrin, iota, and glutathionyl-hydroquinone reductases (GHR) (Nianiou-Obeidat et al. 2017). Given the important detoxification effects of GSTs during plant stress, a number of studies have been carried out on GSTs in many plants. With the advent of whole genome sequencing, a large number of GSTs have been reported and characterized through genome-wide analyses in a number of plants like Physcomitrella patens (Liu et al. 2013), Solanum Lycopersicum (Islam et al. 2017), Ipomoea batatas (Ding et al. 2017), Cucurbita maxima (Kayum et al. 2018), Vigna radiata (Vaish et al. 2018), Malus domestica (Fang et al. 2020), Raphanus sativus (Gao et al. 2020), Cicer arietinum (Ghangal et al. 2020), Medicago truncatula (Han et al. 2018; Hasan et al. 2021), Triticum aestivum (Hao et al. 2021) and Cucumis melo (Song et al. 2021).

Despite the availability of whole genome sequence of quinoa, large scale in-silico genome wide identification and characterization of GSTs have not been carried out in this potential crop. Till date, there is no report of GST gene family identification and characterization in quinoa which provided us with an opportunity to perform in-silico genome-wide analysis of the GST gene family in this underutilized crop.

Material and methods

The in-silico analysis was carried out using 1 Gbps LAN speed on 8 GB RAM personal computer having AMD Ryzen 5 5500 U processor with Radeon Graphics 2.10 Ghz and 64-Bit operating system.

Protein sequence retrieval and search in quinoa database available at Phytozome

C. quinoa genome database available at Phytozome (https://phytozome-next.jgi.doe.gov/) was used to conduct in-silico genome identification and characterization of GST genes. GST protein sequences of Arabidopsis thaliana were obtained from The Arabidopsis Information Resource (TAIR) (http://www.arabidopsis.org/). Glycine max, Physcomitrella patens and Medicago truncatula GST sequences were retrieved from NCBI by the locus ID or the accession number published by Liu et al. (2013), Liu et al. (2015) and Hasan et al. (2021), respectively, while sequences of Oryza sativa were obtained from Rice Genome Annotation Project (RGAP) by the accession number published by Jain et al. (2010). pBLAST searches of each GST class was performed separately using retrieved GST protein sequences of five different species as query with an e-value of 0.0001. The identified amino acid, genomic and coding sequences were downloaded and subjected to NCBI Batch CD (conserved domain) search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (Marchler-Bauer et al. 2017), SMART (Simple Modular Architecture Research Tool) database (http://smart.embl-heidelberg.de/) (Letunic et al. 2021) and Pfam search (http://pfam.xfam.org/search) for confirmation of conserved C-terminal domain and conserved N-terminal domain with the thioredoxin fold.

Subcellular localization and in-silico physicochemical characterization of identified GSTs

Subcellular localization of the identified GSTs were predicted by using three different tools namely CELLO online tool v.2.5 (http://cello.life.nctu.edu.tw/) (Yu et al. 2006), WoLF PSORT (www.genscript.com/wolf-psort.html) (Horton et al. 2007) and DeepLoc (Armenteros et al. 2017) (http://www.cbs.dtu.dk/services/DeepLoc/). The physicochemical parameters such as isoelectric point (pI), molecular weight, aliphatic index and Grand Average of Hydropathy (GRAVY) were analysed using default parameters of ProtParam tool at Expasy server (http://web.expasy.org/protparam/) (Gasteiger et al. 2005).

Gene structure visualization

The exon/intron organization of quinoa GSTs were analysed using Gene Structure Display Server 2.0 (GSDS, https://gsds.cbi.pku.edu.cn/) (Hu et al. 2015) by aligning CDS and genomic sequences that were retrieved from Phytozome (https://phytozome-next.jgi.doe.gov/).

Motif identification

Conserved motifs of quinoa GSTs were identified using the Mutiple Em for Motif Elicitation (MEME) analysis (http://meme-suite.org/) (Bailey et al. 2009). The parameters for analysis were 20 as motif number while the motif width was 6- 50. The results were visualized using TBtool software.

Protein sequence alignment and catalytic residue position prediction

The CqGSTs protein sequences were aligned with protein sequences of Arabidopsis thaliana, O. sativa, G. max, P. patens and M. truncatula using Clustal Omega (Sievers et al. 2011). The protein sequence alignments were visualized in ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) (Robert and Gouet 2014).

Chromosomal localization and gene duplication analysis of CqGST genes

The genomic locations of the CqGST genes were retrieved from the genomic data (Jarvis et al. 2017). The locations of these genes were diagrammatically depicted on their respective chromosomes using online TBtools software v0.667 (https://github.com/CJ-Chen/TBtools). Gene duplication events were analysed by pBLAST search of CqGSTs against each other on NCBI pBLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins). The CqGSTs having sequence similarity > 80% were assumed to be duplicated genes (Kong et al. 2013). The homologous pair genes present on same chromosome were considered as tandem duplicated (TD), while those located at different chromosomal locations were depicted as segmental duplicated (SD) genes (Holub et al. 2001). PAL2NAL online tool (https://bio.tools/pal2nal) (Suyama et al. 2006) was used for the estimation of synonymous rate (dS), non-synonymous rate (dN), and evolutionary constraint (dN/dS) between the duplicated CqGST gene pairs. This was carried out using sequences that were aligned using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) along with their respective mRNA sequences. dN/dS ratio is the basis of mode of selection between duplicated genes. Positive, neutral and purifying selection was considered based on the values > 1, = 1 and < 1, respectively. The divergence time T (million years ago i.e., Mya) of each duplicated gene pair was calculated using the formula: (T = dS/2λ), where T is divergence time, dS is the number of synonymous substitutions per site, and λ is the fixed rate of 1.5 × 10^–8 synonymous substitutions per site per year for dicotyledonous plants (Koch et al. 2000).

Protein secondary structure prediction

The components of protein secondary structure like random coil, beta turn, extended strand and alpha helix of CqGSTs were predicted using Self-Optimized Prediction Method with Alignment (SOPMA), a secondary structure prediction tool (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html) (Combet et al. 2000).

Evolutionary or phylogenetic analysis

The evolutionary relationship of CqGST proteins was carried out with P. patens (a bryophyte), A. thaliana (an angiosperm), O. sativa, G. max, M. truncatula GST proteins in MEGA 7 (Molecular Evolutionary Genetics Analysis) (Kumar et al. 2016) using maximum likelihood method. Bootstrap analysis was performed with 1000 replicates for the accuracy of the constructed tree.

Promoter analysis in CqGST genes

In order to analyze cis acting elements in promoter region, 2000 base pair upstream genomic sequences of initiation codon (ATG) of the identified CqGSTs were retrieved from Chenopodium database (https://www.cbrc.kaust.edu.sa/chenopodiumdb/). To identify various elements, the extracted genomic sequences were subjected to online software PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Lescot et al. 2002).

Homology modelling and molecular docking analysis

One member from each GST family was selected for docking against the acylalanine fungicide, metalaxyl (methyl N-(methoxyacetyl)-N-(2,6-xylyl)-dl-alaninate) which is used to control downy mildew infestation in a number of crops, including quinoa (Danielsen et al. 2003). Docking was not performed for metaxin and EF1B since the catalytic residue was not predicted for these two families. Homology modelling of protein was done by using I-TASSER (Zheng et al. 2021) (https://zhanggroup.org/I-TASSER/). It selects template having best identity from protein data bank hit and gives predicted model. The 3D structure of the ligand was downloaded from PubChem database (http://www.pubchem.ncbi.nlm.nih.gov) in SDF format. These structures were further converted into PDB file format using the PyMol tool and used for docking studies with identified protein members of quinoa. Molecular docking study was carried out using AutoDock v.4 tool (Morris et al. 2009).

Results

Quinoa GST genes

Comprehensive searches using Phytozome database led to the identification of total 120 GST genes in quinoa. To classify GSTs, all protein sequences were retrieved from Phytozome and analyzed through SMART, Pfam and NCBI Batch CD search database. A total of 120 full length GST genes containing both the domain were named as CqGSTs and classified into 11 different classes: tau (65 members), phi (19 members), lambda (2 members), theta (3 members), zeta (4 members), DHAR (2 members), hemerythrin (4 members), metaxin (2 members), mPGES (6 members), EF1B (7 members), GHR (6 members). Plant GSTs tau and phi were highest in numbers. All the classes of CqGSTs were named CqGSTU, CqGSTF, CqGSTL, CqGSTT, CqGSTZ, CqDHAR, CqGSTH, CqGSTM, CqGSTmi, CqGSTEF1B and CqGSTGHR. The numbering for each member of the class was done based on their chromosomal localization in the ascending order (Table 1).

Table 1.

List of identified GST members in Chenopodium quinoa along with their detailed genomic information and physicochemical features

Sr no.	Locus ID	Gene name	Chr. no.	Start	End	Base pair	Strand	Protein (aa)	pI	Mol.Wt. (kDa)	GRAVY	Aliphatic\Index
1	AUR62037578	CqGSTU1	Chr05	22,258,067	22,265,792	7725	Forward	229	5.64	24,913.84	− 0.4	65.76
2	AUR62037579	CqGSTU2	Chr05	22,266,529	22,267,465	936	Reverse	222	5.92	25,784.89	− 0.216	98.78
3	AUR62037930	CqGSTU3	Chr05	31,850,247	31,851,615	1368	Reverse	172	5.21	20,184.12	− 0.459	79.24
4	AUR62037936	CqGSTU4	Chr05	32,278,507	32,279,315	808	Forward	155	6.21	18,104.97	− 0.166	90.65
5	AUR62037937	CqGSTU5	Chr05	32,279,645	32,281,429	1784	Reverse	225	5.18	26,005.1	− 0.257	90.18
6	AUR62037938	CqGSTU6	Chr05	32,288,793	32,295,251	6458	Forward	387	4.97	42,858.16	− 0.055	97.26
7	AUR62037941	CqGSTU7	Chr05	32,449,451	32,450,850	1399	Forward	222	4.89	26,250.07	− 0.345	90.45
8	AUR62037944	CqGSTU8	Chr05	32,654,240	32,654,593	353	Forward	117	4.58	13,507.64	0.144	121.62
9	AUR62017444	CqGSTU9	Chr06	43,484,072	43,484,836	764	Reverse	220	6	24,952	− 0.125	96.5
10	AUR62044663	CqGSTU10	Chr00	45,020,735	45,021,025	290	Reverse	96	4.84	11,060.67	− 0.051	94.38
11	AUR62042823	CqGSTU11	Chr06	57,771,916	57,775,409	3493	Reverse	220	6.84	24,997.03	− 0.138	97.45
12	AUR62026482	CqGSTU12	Chr06	66,508,196	66,510,448	2252	Reverse	227	4.98	26,087.02	− 0.171	85.07
13	AUR62026484	CqGSTU13	Chr06	66,489,506	66,492,004	2498	Reverse	229	5.99	25,748.75	− 0.18	89.08
14	AUR62017445	CqGSTU14	Chr06	43,533,054	43,534,970	1916	Reverse	210	6.83	24,091.14	− 0.039	100.24
15	AUR62026483	CqGSTU15	Chr06	66,493,346	66,495,958	2612	Reverse	229	4.87	26,220.13	− 0.187	92.05
16	AUR62042824	CqGSTU16	Chr06	57,779,096	57,780,971	1875	Forward	222	5.54	25,364.37	− 0.112	90.45
17	AUR62035903	CqGSTU17	Chr07	24,669,985	24,671,288	1303	Forward	224	5.43	26,261.42	− 0.176	96.56
18	AUR62035904	CqGSTU18	Chr07	24,672,990	24,673,832	842	Forward	149	6.3	17,470.16	− 0.317	79.87
19	AUR62001302	CqGSTU19	Chr07	98,865,582	98,867,040	1458	Reverse	235	6.07	27,207.71	− 0.319	92.64
20	AUR62001701	CqGSTU20	Chr07	103,459,631	103,460,839	1208	Reverse	202	6.54	23,733.44	− 0.39	89.26
21	AUR62025397	CqGSTU21	Chr07	106,343,591	106,346,764	3173	Forward	161	5.02	18,404.18	− 0.111	96.21
22	AUR62025473	CqGSTU22	Chr07	107,763,570	107,765,588	2018	Forward	223	5.58	25,745.54	− 0.473	87.44
23	AUR62021697	CqGSTU23	Chr08	8,943,013	8,945,332	2319	Forward	347	7.63	38,973.37	− 0.131	95.19
24	AUR62021696	CqGSTU24	Chr08	8,946,584	8,947,569	985	Forward	226	6.42	26,211.54	− 0.25	105.18
25	AUR62021695	CqGSTU25	Chr08	8,966,413	8,970,065	3652	Reverse	228	8.28	26,524	− 0.232	91.45
26	AUR62021692	CqGSTU26	Chr08	9,050,946	9,051,821	875	Forward	200	5.9	23,015.75	− 0.149	106.25
27	AUR62021691	CqGSTU27	Chr08	9,068,011	9,069,374	1363	Forward	230	6.54	26,555.9	− 0.24	91.52
28	AUR62021690	CqGSTU28	Chr08	9,072,050	9,075,464	3414	Reverse	348	6.11	40,313.64	− 0.261	93.56
29	AUR62021689	CqGSTU29	Chr08	9,077,561	9,078,839	1278	Forward	232	5.74	26,702.89	− 0.316	98.32
30	AUR62021688	CqGSTU30	Chr08	9,080,067	9,081,578	1511	Forward	230	5.39	26,466.62	− 0.287	92.87
31	AUR62021687	CqGSTU31	Chr08	9,085,491	9,087,253	1762	Forward	231	5.61	26,657.75	− 0.383	90.78
32	AUR62018920	CqGSTU32	Chr10	11,529,705	11,531,271	1566	Reverse	224	5.28	25,766.81	− 0.114	96.16
33	AUR62018919	CqGSTU33	Chr10	11,531,703	11,533,254	1551	Reverse	214	4.96	24,449.53	− 0.057	100.61
34	AUR62033918	CqGSTU34	Chr10	47,430,348	47,431,888	1540	Reverse	224	5.5	25,805.84	− 0.287	89.6
35	AUR62035336	CqGSTU35	Chr11	11,262,390	11,263,905	1515	Reverse	224	5.42	26,026.11	− 0.163	98.3
36	AUR62035335	CqGSTU36	Chr11	11,336,767	11,337,934	1167	Reverse	149	6.52	17,588.21	− 0.462	73.36
37	AUR62041794	CqGSTU37	Chr12	34,308,452	34,310,039	1587	Forward	224	6.23	26,150.29	− 0.261	98.35
38	AUR62026287	CqGSTU38	Chr14	25,331,081	25,334,093	3012	Reverse	197	4.75	22,448.84	− 0.072	88.17
39	AUR62026289	CqGSTU39	Chr14	25,349,451	25,353,218	3767	Reverse	228	5.26	25,944.95	− 0.131	96.36
40	AUR62026290	CqGSTU40	Chr14	25,355,749	25,356,129	380	Reverse	126	5.01	13,945	− 0.13	81.51
41	AUR62026291	CqGSTU41	Chr14	25,369,752	25,370,216	464	Reverse	107	5.88	12,293.33	− 0.172	101.12
42	AUR62037811	CqGSTU42	Chr14	44,238,894	44,240,398	1504	Forward	219	5.3	25,413.43	− 0.295	94.75
43	AUR62037813	CqGSTU43	Chr14	44,344,324	44,348,405	4081	Forward	203	5.56	23,561.36	− 0.114	99.9
44	AUR62037814	CqGSTU44	Chr14	44,358,959	44,360,695	1736	Forward	227	8.19	25,675.99	− 0.076	96.61
45	AUR62025080	CqGSTU45	Chr15	50,947,192	50,947,825	633	Reverse	114	6.15	13,639.66	− 0.455	75.26
46	AUR62025085	CqGSTU46	Chr15	50,990,916	50,992,018	1102	Forward	202	7.7	23,848.52	− 0.473	84.95
47	AUR62025088	CqGSTU47	Chr15	51,029,526	51,035,545	6019	Forward	371	5.72	42,037.14	− 0.231	88.54
48	AUR62025095	CqGSTU48	Chr15	51,102,015	51,111,215	9200	Reverse	187	4.95	21,387.47	− 0.212	84.97
49	AUR62008398	CqGSTU49	Chr16	4,065,234	4,070,967	5733	Reverse	429	5.29	49,549.39	− 0.236	101.72
50	AUR62008404	CqGSTU50	Chr16	4,153,451	4,154,576	1125	Forward	221	5.34	25,390.38	− 0.237	89.5
51	AUR62008405	CqGSTU51	Chr16	4,162,275	4,163,834	1559	Forward	228	5.17	26,813.09	− 0.289	96.62
52	AUR62008406	CqGSTU52	Chr16	4,165,237	4,166,614	1377	Forward	232	5.22	26,717.93	− 0.18	105.86
53	AUR62008407	CqGSTU53	Chr16	4,169,081	4,169,886	805	Reverse	226	5.53	25,852.93	− 0.208	97.57
54	AUR62008408	CqGSTU54	Chr16	4,171,850	4,173,231	1381	Reverse	234	5.91	27,070.29	− 0.344	92.52
55	AUR62008409	CqGSTU55	Chr16	4,179,428	4,181,751	2323	Reverse	225	5.61	26,142.13	− 0.322	92.27
56	AUR62008410	CqGSTU56	Chr16	4,186,653	4,188,495	1842	Reverse	230	5.65	26,513.68	− 0.289	89.04
57	AUR62041790	CqGSTU57	Chr17	40,148,842	40,149,664	822	Forward	221	5.75	25,605.64	− 0.209	95.7
58	AUR62041791	CqGSTU58	Chr17	40,150,004	40,151,521	1517	Reverse	225	5.34	26,205.29	− 0.274	88.44
59	AUR62041792	CqGSTU59	Chr17	40,156,851	40,179,701	22,850	Forward	290	6.38	33,610.25	− 0.592	75.31
60	AUR62008765	CqGSTU60	Chr17	66,283,593	66,287,858	4265	Forward	236	6.27	26,596.27	− 0.813	80.59
61	AUR62008847	CqGSTU61	Chr17	67,490,100	67,491,129	1029	Reverse	222	5.39	25,660.54	− 0.285	92.79
62	AUR62008848	CqGSTU62	Chr17	67,491,834	67,492,999	1165	Reverse	223	5.36	26,018.82	− 0.35	86.91
63	AUR62025684	CqGSTU63	Chr18	23,464,056	23,477,054	12,998	Reverse	240	6.66	27,708.19	− 0.363	92.75
64	AUR62020080	CqGSTU64	Chr18	30,540,853	30,542,115	1262	Forward	220	6.85	25,895.92	− 0.392	82.86
65	AUR62020081	CqGSTU65	Chr18	30,542,691	30,544,606	1915	Forward	221	6.91	25,739.75	− 0.309	89.1
1	AUR62026020	CqGSTF1	Chr07	80,504,079	80,505,139	1060	Forward	216	7.74	24,561.57	− 0.161	86.71
2	AUR62033160	CqGSTF2	Chr07	94,403,967	94,405,978	2011	Forward	217	5.5	24,562.2	− 0.19	93.5
3	AUR62033161	CqGSTF3	Chr07	94,433,227	94,443,156	9929	Forward	240	6.67	27,399.39	− 0.403	88.21
4	AUR62033162	CqGSTF4	Chr07	94,485,894	94,489,106	3212	Forward	214	6.24	24,012.56	− 0.265	87.52
5	AUR62033175	CqGSTF5	Chr07	95,026,283	95,027,659	1376	Reverse	242	5.9	27,546.77	− 0.126	91.57
6	AUR62033176	CqGSTF6	Chr07	95,036,182	95,036,815	633	Reverse	184	5.56	21,203.59	− 0.15	94.51
7	AUR62033177	CqGSTF7	Chr07	95,037,534	95,039,279	1745	Reverse	214	5.75	24,289.96	− 0.233	93.5
8	AUR62033178	CqGSTF8	Chr07	95,040,874	95,041,248	374	Reverse	124	5.34	14,487.85	− 0.091	103.95
9	AUR62033179	CqGSTF9	Chr07	95,057,377	95,058,945	1568	Reverse	214	5.31	24,037.62	− 0.08	83.46
10	AUR62033180	CqGSTF10	Chr07	95,061,685	96,064,148	1E + 06	Reverse	218	5.04	25,000.56	− 0.29	91.7
11	AUR62013858	CqGSTF11	Chr10	15,176,795	15,179,961	3166	Reverse	223	6.12	25,280.79	− 0.409	83.5
12	AUR62008598	CqGSTF12	Chr17	62,528,187	62,530,381	2194	Forward	197	5.75	22,882.2	− 0.434	89.14
13	AUR62008599	CqGSTF13	Chr17	62,534,839	62,536,278	1439	Forward	214	5.3	24,008.56	− 0.071	82.99
14	AUR62008602	CqGSTF14	Chr17	62,632,018	62,638,824	6806	Forward	430	6.32	48,830.46	− 0.209	92.14
15	AUR62008604	CqGSTF15	Chr17	62,658,237	62,669,988	11,751	Forward	218	5.91	24,720.42	− 0.248	83.21
16	AUR62008607	CqGSTF16	Chr17	62,679,846	62,681,734	1888	Forward	214	5.91	24,108.69	− 0.19	91.17
17	AUR62008609	CqGSTF17	Chr17	62,850,817	62,853,467	2650	Forward	214	6.25	24,060.62	− 0.286	85.28
18	AUR62008610	CqGSTF18	Chr17	62,859,591	62,861,331	1740	Forward	218	5.8	24,992.13	− 0.136	100.14
19	AUR62008630	CqGSTF19	Chr17	63,533,471	63,533,854	383	Forward	127	8.52	15,012.66	− 0.134	99.13
1	AUR62040536	CqGSTL1	Chr00	13,469,432	13,473,006	3574	Reverse	260	5.21	29,085.43	− 0.139	92.73
2	AUR62041623	CqGSTL2	Chr01	100,372,908	100,385,913	13,005	Reverse	1028	6.82	113,884.86	− 0.115	84.37
1	AUR62010591	CqGSTT1	Chr13	10,768,543	10,777,898	9355	Forward	230	9.22	26,192.53	− 0.188	100.52
2	AUR62010590	CqGSTT2	Chr13	10,781,037	10,783,997	2960	Forward	233	6.5	26,741.66	− 0.279	90.86
3	AUR62017234	CqGSTT3	Chr16	68,183,290	68,195,926	12,636	Forward	292	9.14	33,000.1	− 0.276	94.93
1	AUR62024246	CqGSTZ1	Chr06	1,069,058	1,070,356	1298	Reverse	229	5.14	26,235.94	− 0.335	93.28
2	AUR62013634	CqGSTZ2	Chr10	12,347,463	12,349,907	2444	Forward	223	5.24	25,317.09	− 0.246	85.25
3	AUR62032505	CqGSTZ3	Chr14	42,313,607	42,321,587	7980	Forward	242	8.44	27,732.89	− 0.404	92.81
4	AUR62004214	CqGSTZ4	Chr01	116,540,900	116,543,258	2358	Reverse	224	5.36	25,201.91	− 0.205	83.57
1	AUR62035961	CqGSTDHAR1	Chr04	22,089,257	22,094,551	5294	Reverse	174	5.95	19,123.12	− 0.03	97.47
2	AUR62022530	CqGSTDHAR2	Chr01	93,473,248	93,477,932	4684	Reverse	212	6.09	23,533.29	− 0.099	96.57
1	AUR62008342	CqGSTH1	Chr16	3,358,572	3,371,304	12,732	Forward	1143	6.09	131,200.91	− 0.364	77.02
2	AUR62021756	CqGSTH2	Chr08	8,201,575	8,214,048	12,473	Reverse	1120	5.8	128,670.9	− 0.376	76.5
3	AUR62037305	CqGSTH3	Chr14	10,282,014	10,295,553	13,539	Forward	1246	5.7	139,397.54	− 0.275	79.26
4	AUR62002840	CqGSTH4	Chr06	19,255,868	19,269,917	14,049	Forward	1247	5.81	139,438.58	− 0.288	78.97
1	AUR62021331	CqGSTM1	Chr01	16,537,256	16,542,557	5301	Reverse	289	5.77	32,460.06	− 0.192	87.4
2	AUR62033246	CqGSTM2	Chr01	52,855,100	52,861,286	6186	Reverse	329	5.22	36,922.8	− 0.257	86.53
1	AUR62006187	CqGSTMi1	Chr15	3,433,426	3,442,275	8849	Forward	363	8.2	39,964	− 0.146	86.47
2	AUR62040567	CqGSTMi2	Chr00	14,528,356	14,539,835	11,479	Forward	328	8.89	36,427.4	− 0.278	76.46
3	AUR62013931	CqGSTMi3	Chr10	16,899,006	16,906,074	7068	Reverse	377	9.1	41,690.79	− 0.183	80
4	AUR62037507	CqGSTMi4	Chr12	26,058,521	26,062,960	4439	Forward	151	9.28	17,148.39	0.35	85.83
5	AUR62028521	CqGSTMi5	Chr17	44,448,009	44,456,799	8790	Forward	315	8.55	34,578.84	− 0.131	85.11
6	AUR62004745	CqGSTMi6	Chr05	61,536,667	61,541,109	4442	Reverse	151	9.06	17,112.38	0.407	85.23
1	AUR62035146	CqGSTEF1B1	Chr03	1,343,991	1,349,912	5921	Reverse	225	5.78	26,332.32	− 0.794	64.49
2	AUR62022367	CqGSTEF1B2	Chr10	27,104,863	27,109,452	4589	Reverse	415	5.74	47,454.59	− 0.343	78.51
3	AUR62022369	CqGSTEF1B3	Chr10	27,140,328	27,159,888	19,560	Reverse	491	6.6	55,025.98	− 0.465	74.73
4	AUR62034332	CqGSTEF1B4	Chr03	51,421,029	51,424,593	3564	Forward	416	5.79	47,231.4	− 0.354	79.5
5	AUR62034333	CqGSTEF1B5	Chr03	51,481,446	51,485,168	3722	Forward	416	5.45	47,374.58	− 0.324	81.83
6	AUR62042585	CqGSTEF1B6	Chr10	58,953,083	58,953,451	368	Reverse	122	4.59	14,489.55	− 0.481	67.05
7	AUR62042586	CqGSTEF1B7	Chr10	58,955,993	58,959,931	3938	Reverse	311	8.83	34,887.53	− 0.221	92.15
1	AUR62033029	CqGSTGHR1	Chr01	8,677,338	8,680,969	3631	Forward	329	6.56	37,869.89	− 0.488	72.61
2	AUR62033030	CqGSTGHR2	Chr01	8,731,951	8,736,630	4679	Forward	348	6.27	39,855.23	− 0.437	78.48
3	AUR62040763	CqGSTGHR3	Chr02	14,209,838	14,214,347	4509	Reverse	348	7.07	40,147.73	− 0.435	82.1
4	AUR62040764	CqGSTGHR4	Chr02	14,222,320	14,225,704	3384	Reverse	296	6.32	33,928.12	− 0.611	69.19
5	AUR62008787	CqGSTGHR5	Chr17	66,538,547	66,543,242	4695	Forward	408	8	46,176.97	− 0.257	88.6
6	AUR62025453	CqGSTGHR6	Chr07	107,323,805	107,335,636	11,831	Forward	408	7.09	46,048.76	− 0.236	88.6

Subcellular localization analysis

Subcellular localization analysis revealed that most of the proteins were found majorly in cytoplasm. The average protein length of protein was found to be 279.06 with their corresponding average molecular weight of 31,819.4 kDa. The estimated mean isoelectric point (pI) was found to be 6.14. The highest pI value was 9.2 in theta and mPGES class. The grand average of hydropathy values of most of the proteins of all the classes were negative indicating that all the CqGST proteins were hydrophilic having good interaction with water. Mean aliphatic index of proteins was 89.33. The subcellular localization analysis results showed that proteins were centrally localized in the cytoplasm followed by chloroplast, mitochondria, plastid, plasma membrane, nucleus, extracellular, ER and cytoskeleton (Table 2).

Table 2.

Subcellular location of various CqGSTs

Sr no.	Locus ID	Gene name	Subcellular localization by WoLFPSORT	Subcellular localization by DeepLoc	Subcellular localization by CELLO
1	AUR62037578	CqGSTU1	Cytoskeleton	Soluble cytoplasmic	Cytoplasmic
2	AUR62037579	CqGSTU2	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
3	AUR62037930	CqGSTU3	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
4	AUR62037936	CqGSTU4	Nucleus	Soluble cytoplasmic	Cytoplasmic
5	AUR62037937	CqGSTU5	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
6	AUR62037938	CqGSTU6	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
7	AUR62037941	CqGSTU7	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
8	AUR62037944	CqGSTU8	Cytoplasm	Soluble cytoplasmic	Extracellular
9	AUR62017444	CqGSTU9	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
10	AUR62044663	CqGSTU10	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
11	AUR62042823	CqGSTU11	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
12	AUR62026482	CqGSTU12	Chloroplast	Soluble cytoplasmic	Cytoplasmic
13	AUR62026484	CqGSTU13	Chloroplast	Soluble cytoplasmic	Cytoplasmic
14	AUR62017445	CqGSTU14	Nucleus	Soluble cytoplasmic	Cytoplasmic
15	AUR62026483	CqGSTU15	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
16	AUR62042824	CqGSTU16	Nucleus	Soluble cytoplasmic	Cytoplasmic
17	AUR62035903	CqGSTU17	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
18	AUR62035904	CqGSTU18	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
19	AUR62001302	CqGSTU19	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
20	AUR62001701	CqGSTU20	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
21	AUR62025397	CqGSTU21	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
22	AUR62025473	CqGSTU22	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
23	AUR62021697	CqGSTU23	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
24	AUR62021696	CqGSTU24	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
25	AUR62021695	CqGSTU25	Cytoplasm	Soluble cytoplasmic	Extracellular
26	AUR62021692	CqGSTU26	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
27	AUR62021691	CqGSTU27	Mitochondria	Soluble cytoplasmic	Cytoplasmic
28	AUR62021690	CqGSTU28	Chloroplast	Soluble cytoplasmic	Cytoplasmic
29	AUR62021689	CqGSTU29	Chloroplast	Soluble cytoplasmic	Cytoplasmic
30	AUR62021688	CqGSTU30	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
31	AUR62021687	CqGSTU31	Mitochondria	Soluble cytoplasmic	Cytoplasmic
32	AUR62018920	CqGSTU32	Chloroplast	Soluble cytoplasmic	Cytoplasmic
33	AUR62018919	CqGSTU33	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
34	AUR62033918	CqGSTU34	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
35	AUR62035336	CqGSTU35	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
36	AUR62035335	CqGSTU36	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
37	AUR62041794	CqGSTU37	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
38	AUR62026287	CqGSTU38	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
39	AUR62026289	CqGSTU39	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
40	AUR62026290	CqGSTU40	Chloroplast	Soluble mitochondrial	Cytoplasmic
41	AUR62026291	CqGSTU41	Chloroplast	Soluble cytoplasmic	Mitochondrial
42	AUR62037811	CqGSTU42	Nucleus	Soluble cytoplasmic	Cytoplasmic
43	AUR62037813	CqGSTU43	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
44	AUR62037814	CqGSTU44	Nucleus	Soluble cytoplasmic	Chloroplast
45	AUR62025080	CqGSTU45	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
46	AUR62025085	CqGSTU46	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
47	AUR62025088	CqGSTU47	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
48	AUR62025095	CqGSTU48	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
49	AUR62008398	CqGSTU49	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
50	AUR62008404	CqGSTU50	Chloroplast	Soluble cytoplasmic	Cytoplasmic
51	AUR62008405	CqGSTU51	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
52	AUR62008406	CqGSTU52	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
53	AUR62008407	CqGSTU53	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
54	AUR62008408	CqGSTU54	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
55	AUR62008409	CqGSTU55	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
56	AUR62008410	CqGSTU56	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
57	AUR62041790	CqGSTU57	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
58	AUR62041791	CqGSTU58	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
59	AUR62041792	CqGSTU59	Cytoplasm	Soluble plastid	Cytoplasmic
60	AUR62008765	CqGSTU60	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
61	AUR62008847	CqGSTU61	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
62	AUR62008848	CqGSTU62	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
63	AUR62025684	CqGSTU63	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
64	AUR62020080	CqGSTU64	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
65	AUR62020081	CqGSTU65	Cytoskeleton	Soluble cytoplasmic	Cytoplasmic
1	AUR62026020	CqGSTF1		Soluble cytoplasmic	Cytoplasmic
2	AUR62033160	CqGSTF2	Chloroplast	Soluble cytoplasmic	Cytoplasmic
3	AUR62033161	CqGSTF3	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
4	AUR62033162	CqGSTF4	Chloroplast	Soluble cytoplasmic	Cytoplasmic
5	AUR62033175	CqGSTF5	Cytoplasm	Soluble mitochondrial	Cytoplasmic
6	AUR62033176	CqGSTF6	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
7	AUR62033177	CqGSTF7	Chloroplast	Soluble cytoplasmic	Cytoplasmic
8	AUR62033178	CqGSTF8	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
9	AUR62033179	CqGSTF9	Chloroplast	Soluble cytoplasmic	Cytoplasmic
10	AUR62033180	CqGSTF10	Extracellular	Soluble cytoplasmic	Cytoplasmic
11	AUR62013858	CqGSTF11	Chloroplast	Soluble cytoplasmic	Cytoplasmic
12	AUR62008598	CqGSTF12	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
13	AUR62008599	CqGSTF13	Chloroplast	Soluble cytoplasmic	Cytoplasmic
14	AUR62008602	CqGSTF14	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
15	AUR62008604	CqGSTF15	Chloroplast	Soluble cytoplasmic	Cytoplasmic
16	AUR62008607	CqGSTF16	Chloroplast	Soluble cytoplasmic	Cytoplasmic
17	AUR62008609	CqGSTF17	Chloroplast	Soluble cytoplasmic	Cytoplasmic
18	AUR62008610	CqGSTF18	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
19	AUR62008630	CqGSTF19	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
1	AUR62040536	CqGSTL1	Chloroplast	Soluble plastid	Cytoplasmic
2	AUR62041623	CqGSTL2	Plastid	Membrane plastid	Plasma membrane
1	AUR62010591	CqGSTT1	Mitochondria	Soluble nucleus	Cytoplasmic
2	AUR62010590	CqGSTT2	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
3	AUR62017234	CqGSTT3	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
1	AUR62024246	CqGSTZ1	Mitochondria	Soluble cytoplasmic	Cytoplasmic
2	AUR62013634	CqGSTZ2	Chloroplast	Soluble cytoplasmic	Chloroplast
3	AUR62032505	CqGSTZ3	Mitochondria	Soluble mitochondrial	Mitochondrial
4	AUR62004214	CqGSTZ4	Chloroplast	Soluble cytoplasmic	Chloroplast
1	AUR62035961	CqGSTDHAR1	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
2	AUR62022530	CqGSTDHAR2	Cytoplasm	Soluble cytoplasmic	Cytoplasmic
1	AUR62008342	CqGSTH1	Nucleus	Soluble nucleus	Nuclear
2	AUR62021756	CqGSTH2	Nucleus	Soluble nucleus	Nuclear
3	AUR62037305	CqGSTH3	cytoplasm	Soluble nucleus	Nuclear
4	AUR62002840	CqGSTH4	Nucleus	Soluble nucleus	Nuclear
1	AUR62021331	CqGSTM1	Nucleus	Membrane mitochondrial	Plasma membrane
2	AUR62033246	CqGSTM2	Cytoplasm	Membrane mitochondrial	Plasma membrane
1	AUR62006187	CqGSTMi1	Chloroplast	Membrane plastid	Chloroplast
2	AUR62040567	CqGSTMi2	Chloroplast	Membrane mitochondrial	Chloroplast
3	AUR62013931	CqGSTMi3	Chloroplast	Membrane mitochondrial	Chloroplast
4	AUR62037507	CqGSTMi4	Cytoplasm	Membrane ER	Plasma membrane
5	AUR62028521	CqGSTMi5	Chloroplast	Membrane plastid	Chloroplast
6	AUR62004745	CqGSTMi6	Cytoplasm	Membrane ER	Plasma membrane
1	AUR62035146	CqGSTEF1B1	Mitochondria	Soluble cytoplasmic	Cytoplasmic
2	AUR62022367	CqGSTEF1B2	Chloroplast	Soluble cytoplasmic	Cytoplasmic
3	AUR62022369	CqGSTEF1B3	Cytoplasm	Soluble nucleus	Chloroplast
4	AUR62034332	CqGSTEF1B4	Chloroplast	Soluble cytoplasmic	Cytoplasmic
5	AUR62034333	CqGSTEF1B5	Chloroplast	Soluble cytoplasmic	Cytoplasmic
6	AUR62042585	CqGSTEF1B6	Chloroplast	Soluble cytoplasmic	Cytoplasmic
7	AUR62042586	CqGSTEF1B7	Cytoplasm	Membrane peroxisome	Cytoplasmic
1	AUR62033029	CqGSTGHR1	Cytoplasm	Soluble cytoplasmic	Mitochondrial
2	AUR62033030	CqGSTGHR2	Mitochondria	Soluble mitochondrial	Mitochondrial
3	AUR62040763	CqGSTGHR3	Chloroplast	Soluble mitochondrial	Mitochondrial
4	AUR62040764	CqGSTGHR4	Cytoplasm	Soluble cytoplasmic	Mitochondrial
5	AUR62008787	CqGSTGHR5	Chloroplast	Soluble plastid	Mitochondrial
6	AUR62025453	CqGSTGHR6	Chloroplast	Soluble plastid	Extracellular

Gene structure organization

Gene structure organization having number of exons and introns were analyzed using coding and genomic sequences in Gene Structure Display server 2.0 to investigate possible structural evolution of GST gene family. Structural analysis revealed the presence of 2–14 exons in CqGST genes (Fig. 1). Most of the proteins possessed two exon one intron organization. The classes showed greater intron numbers with mixed conservation of splice site sequence.

Fig. 1.

Gene structure analyses of quinoa CqGST genes drawn using GSDS tool. Color description; CDS—blue color, intron—black thread, upstream and downstream—green

Motif analysis

Further, we analyzed quinoa GST proteins for motif discovery via MEME suite (v4.11.2) using parameters, motif discovery mode: Normal; site distribution: 0 or 1 occurrence per sequence; motif length 10–50; number of motifs: 15. Top fifteen motifs based on lowest e-value were identified and selected. Motifs 1, 3, 2, 5, 6, 8, 9 and 13 were found specifically in tau class family, motifs 3, 4, 5, 6, 7 and 9 were found in phi class gene family, while motif 3, 4, 13 and 14 were found in metaxin class. Motif 15 and motif 11 were highly conserved for GHR and EF1B class respectively, while motif 4 was present in almost all classes (Fig. 2).

Fig. 2.

Conserved motif analyses of CqGSTs were identified with MEME suite by inputting complete CqGST protein sequence with 15 motifs. Individual motifs were represented by different colored boxes

Multiple sequence alignment

The multiple sequence alignment was performed with GST protein sequences of A. thaliana, O. sativa, G. max, P. patens and M. truncatula to identify the conserved and catalytic residue among different GST classes (Fig. 3). It revealed highly conserved N-terminus with active site serine (Ser; S) or cysteine (Cys; C) residue for the activation of GSH binding and GST catalytic activity. Tau, phi, theta and zeta possessed serine active site residues, whereas lambda, GHR, DHAR mPGES had cysteine active site residues.

Fig. 3.

Catalytic residue depiction in CqGST proteins using ESpript. Multiple sequence alignment was performed with A. thaliana, G. max, P. patens, M. truncatula and O. sativa GST sequences. The red colour indicate the active site cysteine in GHR, DHAR and Lambda while serine in theta, zeta and tau CqGSTs

Chromosomal localization and gene duplication analysis of CqGST genes

All 120 CqGST gene loci were found to be unevenly distributed across 18 different chromosomes. Seventeen genes were located on chromosome 7, 16 on chromosome 17, 10 on chromosome 08, chromosome 10 and chromosome 16, 9 genes on chromosome 14, chromosome 5 and chromosome 6. Only two genes were found to be present on chromosomes 2, 11, 12 and 13; and a single gene on chromosome 4. No genes were found on chromosome 9 (Fig. 4). Tandem and segmental duplication play an important role in gene family expansion. Further analysis revealed that segmental duplication and purifying type selection were highest in number and found to be main source of expansion of GST gene family as compared to tandem duplication and positive type selection. We found 92 duplicated gene pairs (Fig. 5) with percent identity of more than 80% against each other using blast search and further calculated the ratio of non-synonymous substitutions and synonymous substitutions (Ka/Ks) for all duplicated CqGST genes to examine that genes were naturally favored or not. Twenty-one duplicated gene pairs were found to be tandem duplicated while 71 were duplicated segmentally. Ka/Ks ratio of gene pairs was less than 1 indicating that non-synonymous substitutions were not favored among duplicated genes and purifying selection was more prevalent. We also calculated the divergence time for these genes. The mean average divergence time for these genes was approximately 9.27 million years ago (MYA) (Table 3). CqGSTU genes were majorly involved in gene duplication event which played a major role in quinoa GST gene family expansion.

Fig. 4.

Chromosomal distribution of 120 CqGST genes on 18 different chromosomes using TB Tool software. CqGST Genes present on different chromosome were represented by different colors. Color description: genes present on chr1—rust orange, chr2—light green, chr3 and chr4—grey, chr5 and chr11—aqua, chr6—brown, chr7 and chr 16—green, chr8—dark brown, chr10—red, chr 12 and chr14—mustard yellow, chr 13 and chr 15—yellow, chr 17—pink and chr 18—sky blue

Fig. 5.

A circular plot depicting duplication of 92 gene pairs present in quinoa with different colors using TB tool software

Table 3.

Estimated Ka/Ks ratios and divergence times of the duplicated CqGST genes

S no.	Gene name 1	Chr no.	Gene name 2	Chr no.	Percent identity	Ka	Ks	Ka/Ks	Duplication time (Mya)	Duplication type	Selection type
1	CqGSTU1	Chr05	CqGSTU5	Chr05	89.43%	0.4158	0.7940	0.5236	26.46	Tandem	Purifying
2	CqGSTU1	Chr05	CqGSTU58	Chr17	88.62%	0.4201	0.7123	0.5898	23.74	Segmental	Purifying
3	CqGSTU2	Chr05	CqGSTU4	Chr05	93.59%	0.0316	0.1078	0.2932	3.593	Tandem	Purifying
4	CqGSTU2	Chr05	CqGSTU57	Chr16	94.62%	0.0321	0.0972	0.3306	3.24	Segmental	Purifying
5	CqGSTU5	Chr05	CqGSTU58	Chr17	92.04%	0.0381	0.1432	0.2663	4.77	Segmental	Purifying
6	CqGSTU6	Chr05	CqGSTU59	Chr17	93%	0.5209	0.9775	0.5328	32.58	Segmental	Purifying
7	CqGSTU8	Chr05	CqGSTU37	Chr11	90.48%	0.1207	0.1955	0.6178	6.51	Segmental	Purifying
8	CqGSTU9	Chr06	CqGSTU44	Chr14	92.76%	0.0324	0.1090	0.2976	3.63	Segmental	Purifying
9	CqGSTU10	Chr00	CqGSTU17	Chr07	100%	0.0000	0.0000	0.2035	0	Segmental	Purifying
10	CqGSTU10	Chr00	CqGSTU35	Chr10	92.78%	0.0303	0.1070	0.2829	3.56	Segmental	Purifying
11	CqGSTU12	Chr06	CqGSTU38	Chr12	93.79%	0.0785	0.1124	0.6981	3.74	Segmental	Purifying
12	CqGSTU12	Chr06	CqGSTU41	Chr14	84.31%	0.1181	0.5356	0.2205	17.85	Segmental	Purifying
13	CqGSTU13	Chr06	CqGSTU40	Chr14	92.80%	0.0418	0.1305	0.3204	4.35	Segmental	Purifying
14	CqGSTU13	Chr06	CqGSTU41	Chr14	97.14%	0.0269	0.0848	0.3170	2.82	Segmental	Purifying
15	CqGSTU14	Chr06	CqGSTU43	Chr14	91.67%	0.0386	0.1567	0.2462	5.22	Segmental	Purifying
16	CqGSTU15	Chr06	CqGSTU39	Chr14	92.95%	0.0363	0.1519	0.2387	5.06	Segmental	Purifying
17	CqGSTU17	Chr07	CqGSTU35	Chr10	95.96%	0.0190	0.0717	0.2653	2.39	Segmental	Purifying
18	CqGSTU18	Chr07	CqGSTU36	Chr11	90.67%	0.0470	0.0334	1.4071	1.11	Segmental	Positive
19	CqGSTU19	Chr07	CqGSTU63	Chr17	95.74%	0.0222	0.1866	0.1189	6.22	Segmental	Purifying
20	CqGSTU20	Chr07	CqGSTU45	Chr14	92.98%	0.0350	0.0281	1.2486	0.93	Segmental	Positive
21	CqGSTU20	Chr07	CqGSTU46	Chr15	94.58%	0.0255	0.0511	0.4987	1.7	Segmental	Purifying
22	CqGSTU20	Chr07	CqGSTU48	Chr15	89.74%	0.3383	0.3252	1.0404	10.84	Segmental	Positive
23	CqGSTU20	Chr07	CqGSTU64	Chr18	91.13%	0.0398	0.1847	0.2155	6.15	Segmental	Purifying
24	CqGSTU20	Chr07	CqGSTU65	Chr18	87.19%	0.0704	0.0754	0.9331	2.51	Segmental	Purifying
25	CqGSTU21	Chr07	CqGSTU62	Chr17	93.12%	0.0374	0.0907	0.4128	3.02	Segmental	Purifying
26	CqGSTU22	Chr07	CqGSTU60	Chr17	92.91%	0.3185	0.8690	0.3665	28.97	Segmental	Purifying
27	CqGSTU23	Chr07	CqGSTU24	Chr08	88.16%	0.0639	0.1998	0.3197	6.66	Segmental	Purifying
28	CqGSTU23	Chr07	CqGSTU49	Chr15	87.50%	0.0788	0.2402	0.3281	8.01	Segmental	Purifying
29	CqGSTU24	Chr08	CqGSTU49	Chr15	80.34%	0.0925	0.2220	0.4166	7.40	Segmental	Purifying
30	CqGSTU27	Chr08	CqGSTU50	Chr16	88.74%	0.0492	0.1251	0.3934	4.17	Segmental	Purifying
31	CqGSTU29	Chr08	CqGSTU52	Chr16	88.41%	0.0642	0.1255	0.5120	4.18	Segmental	Purifying
32	CqGSTU30	Chr08	CqGSTU31	Chr08	84.48%	0.0841	0.2455	0.3426	8.18	Tandem	Purifying
33	CqGSTU30	Chr08	CqGSTU53	Chr16	86.58%	0.0604	0.1497	0.4035	4.99	Segmental	Purifying
34	CqGSTU30	Chr08	CqGSTU54	Chr16	82.13%	0.0943	0.3304	0.2853	11.01	Segmental	Purifying
35	CqGSTU31	Chr08	CqGSTU53	Chr16	80.17%	0.0994	0.2779	0.3576	9.26	Segmental	Purifying
36	CqGSTU31	Chr08	CqGSTU54	Chr16	92.34%	0.0333	0.1984	0.1677	6.61	Segmental	Purifying
37	CqGSTU33	Chr10	CqGSTU34	Chr10	87.11%	0.0654	0.0611	1.0700	2.04	Tandem	Positive
38	CqGSTU38	Chr12	CqGSTU41	Chr14	92.45%	0.2519	1.2935	0.1948	43.12	Segmental	Purifying
39	CqGSTU45	Chr15	CqGSTU46	Chr15	96.49%	0.0192	0.0574	0.3342	1.91	Tandem	Purifying
40	CqGSTU45	Chr15	CqGSTU47	Chr15	92.31%	0.5645	0.9946	0.5675	33.15	Tandem	Purifying
41	CqGSTU45	Chr15	CqGSTU48	Chr15	93.86%	0.0365	0.0245	1.4908	0.82	Tandem	Positive
42	CqGSTU45	Chr15	CqGSTU64	Chr18	91.23%	0.0427	0.1953	0.2184	6.51	Segmental	Purifying
43	CqGSTU45	Chr15	CqGSTU65	Chr18	84.21%	0.0989	0.1047	0.9445	3.49	Segmental	Purifying
44	CqGSTU46	Chr15	CqGSTU64	Chr18	90.64%	0.0409	0.2245	0.1822	7.48	Segmental	Purifying
45	CqGSTU46	Chr15	CqGSTU65	Chr18	86.21%	0.0719	0.1264	0.5691	4.21	Segmental	Purifying
46	CqGSTU46	Chr15	CqGSTU48	Chr15	91.45%	0.3091	0.3581	0.8631	11.94	Tandem	Purifying
47	CqGSTU47	Chr15	CqGSTU48	Chr15	97.87%	0.5602	0.7139	0.7847	23.80	Tandem	Purifying
48	CqGSTU48	Chr15	CqGSTU64	Chr18	88.03%	0.3132	0.6329	0.4949	21.10	Segmental	Purifying
49	CqGSTU48	Chr15	CqGSTU65	Chr18	81.20%	0.3798	0.4596	0.8264	15.32	Segmental	Purifying
50	CqGSTU55	Chr16	CqGSTU56	Chr16	89.04%	0.0852	0.1898	0.4486	6.33	Tandem	Purifying
51	CqGSTF2	Chr07	CqGSTF16	Chr17	96.26%	0.0175	0.0888	0.1973	2.96	Segmental	Purifying
52	CqGSTF3	Chr07	CqGSTF4	Chr07	91.39%	0.2246	0.3272	0.6866	10.91	Tandem	Purifying
53	CqGSTF3	Chr07	CqGSTF17	Chr17	90.07%	0.2242	0.3650	0.6142	12.17	Segmental	Purifying
54	CqGSTF4	Chr07	CqGSTF17	Chr17	97.67%	0.0115	0.1161	0.0991	3.87	Segmental	Purifying
55	CqGSTF5	Chr07	CqGSTF7	Chr07	88.43%	0.0780	0.1430	0.5455	4.77	Tandem	Purifying
56	CqGSTF5	Chr07	CqGSTF14	Chr17	93.64%	0.1004	0.2293	0.4378	7.64	Segmental	Purifying
57	CqGSTF6	Chr07	CqGSTF8	Chr07	95.92%	0.0314	0.0452	0.6948	1.51	Tandem	Purifying
58	CqGSTF6	Chr07	CqGSTF19	Chr17	85.71%	0.0955	0.1053	0.9065	3.51	Segmental	Purifying
59	CqGSTF7	Chr07	CqGSTF14	Chr17	87.04%	0.0618	0.1391	0.4444	4.64	Segmental	Purifying
60	CqGSTF8	Chr07	CqGSTF19	Chr17	83.87%	0.0887	0.1014	0.8748	3.38	Segmental	Purifying
61	CqGSTF9	Chr07	CqGSTF13	Chr17	96.28%	0.0170	0.1201	0.1411	4.00	Segmental	Purifying
62	CqGSTF10	Chr07	CqGSTF12	Chr17	91.37%	0.0515	0.1630	0.3159	5.43	Segmental	Purifying
63	CqGSTT2	Chr13	CqGSTT3	Chr16	80.69%	0.1201	0.3129	0.3837	10.43	Segmental	Purifying
64	CqGSTZ2	Chr10	CqGSTZ4	Chr01	95.09%	0.0176	0.1252	0.1410	4.17	Segmental	Purifying
65	CqGSTDHAR1	Chr04	CqGSTDHAR2	Chr01	81.13%	0.0075	0.1168	0.0643	3.89	Segmental	Purifying
66	CqGSTH1	Chr16	CqGSTH2	Chr08	92.42%	0.0135	0.0597	0.2262	1.99	Segmental	Purifying
67	CqGSTH3	Chr14	CqGSTH4	Chr06	98.56%	0.0071	0.0587	0.1216	1.96	Segmental	Purifying
68	CqGSTM1	Chr01	CqGSTM2	Chr01	85.20%	0.0111	0.0750	0.1474	2.50	Tandem	Purifying
69	CqGSTMi1	Chr15	CqGSTMi5	Chr17	83.52%	0.0301	0.1066	0.2829	3.55	Segmental	Purifying
70	CqGSTMi2	Chr00	CqGSTMi3	Chr10	84.13%	0.0171	0.0695	0.2463	2.32	Segmental	Purifying
71	CqGSTMi4	Chr12	CqGSTMi6	Chr05	92.76%	0.0324	0.0646	0.5008	2.15	Segmental	Purifying
72	CqGSTEF1B1	Chr03	CqGSTEF1B2	Chr10	82.74%	0.0917	0.7587	0.1209	25.29	Segmental	Purifying
73	CqGSTEF1B1	Chr03	CqGSTEF1B3	Chr10	83.63%	0.0851	0.8136	0.1046	27.12	Segmental	Purifying
74	CqGSTEF1B1	Chr03	CqGSTEF1B4	Chr03	84.07%	0.0828	0.8725	0.0949	29.08	Tandem	Purifying
75	CqGSTEF1B1	Chr03	CqGSTEF1B5	Chr03	83.63%	0.0847	0.8571	0.0988	28.57	Tandem	Purifying
76	CqGSTEF1B1	Chr03	CqGSTEF1B6	Chr10	99.19%	0.0036	0.1574	0.0232	5.25	Segmental	Purifying
77	CqGSTEF1B1	Chr03	CqGSTEF1B7	Chr10	97.00%	0.1271	0.2749	0.4622	9.16	Segmental	Purifying
78	CqGSTEF1B2	Chr10	CqGSTEF1B3	Chr10	90.41%	0.0490	0.2030	0.2414	6.77	Tandem	Purifying
79	CqGSTEF1B2	Chr10	CqGSTEF1B4	Chr03	91.61%	0.0437	0.2507	0.1743	8.36	Segmental	Purifying
80	CqGSTEF1B2	Chr10	CqGSTEF1B5	Chr03	90.41%	0.0473	0.2175	0.2175	7.25	Segmental	Purifying
81	CqGSTEF1B2	Chr10	CqGSTEF1B6	Chr10	91.06%	0.0407	0.6499	0.0626	21.66	Tandem	Purifying
82	CqGSTEF1B3	Chr10	CqGSTEF1B4	Chr03	98.56%	0.0066	0.1303	0.0503	4.34	Segmental	Purifying
83	CqGSTEF1B3	Chr10	CqGSTEF1B5	Chr03	96.40%	0.0188	0.1438	0.1306	4.79	Segmental	Purifying
84	CqGSTEF1B3	Chr10	CqGSTEF1B6	Chr10	92.68%	0.0328	0.7165	0.0457	23.88	Tandem	Purifying
85	CqGSTEF1B4	Chr03	CqGSTEF1B5	Chr03	96.16%	0.0186	0.1663	0.1120	5.54	Tandem	Purifying
86	CqGSTEF1B4	Chr03	CqGSTEF1B6	Chr10	92.62%	0.0322	0.7986	0.0404	26.62	Segmental	Purifying
87	CqGSTEF1B5	Chr03	CqGSTEF1B6	Chr10	92.68%	0.0328	0.6440	0.0510	21.47	Segmental	Purifying
88	CqGSTGHR1	Chr01	CqGSTGHR2	Chr01	84.66%	0.0884	0.4827	0.1832	16.09	Tandem	Purifying
89	CqGSTGHR1	Chr01	CqGSTGHR3	Chr02	83.44%	0.0998	0.4451	0.2241	14.84	Segmental	Purifying
90	CqGSTGHR1	Chr01	CqGSTGHR4	Chr02	84.05%	0.0413	0.0960	0.4302	3.20	Segmental	Purifying
91	CqGSTGHR2	Chr01	CqGSTGHR3	Chr02	95.42%	0.0232	0.0416	0.5579	1.39	Segmental	Purifying
92	CqGSTGHR5	Chr17	CqGSTGHR6	Chr07	97.07%	0.0130	0.0556	0.2346	1.85	Segmental	Purifying

Protein secondary structure prediction

Dominance of alpha helix was followed by coil, extended strand and beta turns. Percentage of alpha helix was found to be higher in all CqGSTs except CqGSTU60, CqGSTZ3, CqGSTMi1, CqGSTMi5, CqGSTEF1B1, CqGSTEF1B2, CqGSTEF1B3, CqGSTEF1B6 and in all GHR genes in which high coil percentage was found. Highest alpha helix and coil percentage was present in CqGSTU40 and in CqGSTMi5 which is 69.84 and 54.6 respectively (Fig. 6) (Table 4).

Fig. 6.

Secondary structure prediction of all CqGSTs using SOPMA showed the dominance of alpha helices followed by coils

Table 4.

Quinoa GSTs secondary structure prediction using SOPMA

Gene name	Coil (C) (%)	Beta-Turn (T) (%)	Extended strand (E) (%)	Alpha helix (H) (%)
CqGSTU1	37.12	4.8	14.41	43.67
CqGSTU2	23.87	5.41	13.51	57.21
CqGSTU3	26.16	4.65	11.05	58.14
CqGSTU4	21.29	1.29	9.03	68.39
CqGSTU5	29.33	7.56	13.33	49.78
CqGSTU6	30.75	7.24	16.54	45.48
CqGSTU7	26.13	3.6	14.86	55.41
CqGSTU8	31.62	5.98	11.97	50.43
CqGSTU9	29.09	4.55	14.09	52.27
CqGSTU10	20.83	7.29	10.42	61.46
CqGSTU11	29.55	3.64	13.64	53.18
CqGSTU12	27.31	3.52	11.01	58.15
CqGSTU13	25.33	4.37	10.48	59.83
CqGSTU14	27.14	3.33	13.81	55.71
CqGSTU15	24.45	3.06	11.35	61.14
CqGSTU16	27.03	5.41	13.51	54.05
CqGSTU17	25.89	6.7	12.95	54.46
CqGSTU18	24.16	7.38	9.4	59.06
CqGSTU19	28.51	4.68	11..06	55.74
CqGSTU20	26.73	2.97	12.38	57.92
CqGSTU21	27.33	7.45	16.15	49.07
CqGSTU22	29.6	5.38	12.11	52.91
CqGSTU23	27.38	8.65	15.85	48.13
CqGSTU24	25.66	4.87	9.29	60.18
CqGSTU25	29.39	5.26	9.21	56.14
CqGSTU26	28.5	7.5	13	51
CqGSTU27	29.57	4.35	12.17	53.91
CqGSTU28	28.16	6.9	11.78	53.16
CqGSTU29	30.17	5.6	9.05	55.17
CqGSTU30	26.09	6.96	12.61	54.35
CqGSTU31	23.38	8.23	12.99	55.41
CqGSTU32	25.45	5.36	12.05	57.14
CqGSTU33	28.04	6.07	13.55	52.34
CqGSTU34	25.89	6.25	11.16	56.7
CqGSTU35	28.57	4.46	11.61	55.36
CqGSTU36	25.5	4.03	6.71	63.76
CqGSTU37	26.34	4.46	16.52	52.68
CqGSTU38	25.38	4.57	11.68	58.38
CqGSTU39	25.44	4.82	9.65	60.09
CqGSTU40	15.87	3.97	10.32	69.84
CqGSTU41	36.45	7.48	18.69	37.38
CqGSTU42	28.31	5.02	12.33	54.34
CqGSTU43	28.08	4.43	9.85	57.64
CqGSTU44	30.84	4.41	14.1	50.66
CqGSTU45	21.05	6.14	17.54	55.26
CqGSTU46	25.74	5.45	9.9	58.91
CqGSTU47	34.77	6.74	17.52	40.97
CqGSTU48	22.46	6.42	14.97	56.15
CqGSTU49	26.34	7.93	11.42	54.31
CqGSTU50	31.22	7.69	10.41	50.68
CqGSTU51	28.51	4.39	10.53	56.58
CqGSTU52	26.29	5.17	11.21	57.33
CqGSTU53	26.99	4.87	12.39	55.75
CqGSTU54	29.49	6.41	11.97	52.14
CqGSTU55	28.89	4.89	12.44	53.78
CqGSTU56	29.13	3.91	9.57	57.39
CqGSTU57	27.15	4.07	14.48	54.3
CqGSTU58	32	4.44	11.56	52
CqGSTU59	39.66	3.45	13.1	43.79
CqGSTU60	41.53	6.36	15.25	36.86
CqGSTU61	27.48	4.5	13.51	54.5
CqGSTU62	26.91	5.83	11.66	55.61
CqGSTU63	27.92	6.25	11.25	54.58
CqGSTU64	27.73	6.82	14.09	51.36
CqGSTU65	29.41	4.52	14.03	52.04
CqGSTF1	32.41	6.02	15.28	46.3
CqGSTF2	31.8	6.45	14.75	47
CqGSTF3	32.8	4.58	15	48.33
CqGSTF4	31.31	7.94	13.55	47.2
CqGSTF5	29.75	7.02	11.57	51.65
CqGSTF6	23.91	6.52	8.7	60.87
CqGSTF7	29.91	7.01	13.55	49.53
CqGSTF8	27.42	6.45	9.68	56.45
CqGSTF9	28.04	6.54	14.95	50.47
CqGSTF10	32.32	5.05	14.22	45.41
CqGSTF11	37.22	5.83	14.35	42.6
CqGSTF12	32.99	7.61	13.2	46.19
CqGSTF13	32.24	7.48	15.42	44.86
CqGSTF14	30.93	7.67	15.81	45.58
CqGSTF15	31.19	5.96	14.68	48.17
CqGSTF16	32.24	6.54	13.55	47.66
CqGSTF17	33.18	5.61	14.95	46.26
CqGSTF18	27.06	7.8	14.68	50.46
CqGSTF19	23.62	7.09	4.72	64.57
CqGSTL1	38.46	3.85	14.62	43.08
CqGSTL2	31.81	8.95	14.4	44.84
CqGSTT1	32.61	6.09	8.26	53.04
CqGSTT2	35.19	4.72	10.73	49.36
CqGSTT3	28.77	7.53	10.96	52.74
CqGSTZ1	37.12	3.93	11.79	47.16
CqGSTZ2	38.57	4.48	10.76	46.19
CqGSTZ3	42.15	3.72	12.4	41.74
CqGSTZ4	35.71	4.91	12.5	46.88
CqGSTDHAR1	41.38	2.3	5.17	51.15
CqGSTDHAR2	38.68	3.77	14.15	43.4
CqGSTH1	37.45	2.01	6.12	54.42
CqGSTH2	39.55	2.14	6.7	51.61
CqGSTH3	40.05	3.69	7.62	48.64
CqGSTH4	40.34	3.45	6.98	49.24
CqGSTM1	39.79	2.08	13.15	44.98
CqGSTM2	37.69	5.17	12.16	44.98
CqGSTMi1	47.38	5.79	17.36	29.48
CqGSTMi2	31.4	3.05	8.23	57.32
CqGSTMi3	33.95	3.98	11.41	50.66
CqGSTMi4	25.17	3.97	9.93	60.93
CqGSTMi5	54.6	4.13	13.33	27.94
CqGSTMi6	23.84	3.31	13.91	58.94
CqGSTEF1B1	48	4	21.33	26.67
CqGSTEF1B2	42.65	2.89	14.94	39.52
CqGSTEF1B3	43.79	4.07	12.83	39.31
CqGSTEF1B4	40.38	2.4	14.9	42.31
CqGSTEF1B5	39.42	3.61	14.66	42.31
CqGSTEF1B6	31.97	5.74	29.51	31.79
CqGSTEF1B7	40.51	2.89	10.93	45.66
CqGSTGHR1	44.38	2.43	11.85	41.34
CqGSTGHR2	44.83	3.16	11.21	40.8
CqGSTGHR3	44.54	3.16	11.49	40.8
CqGSTGHR4	47.64	3.38	11.49	37.5
CqGSTGHR5	46.32	2.45	13.73	37.5
CqGSTGHR6	45.59	3.68	11.27	39.46

Evolutionary or phylogenetic analysis

To understand evolutionary relationship between GST gene family members, an unrooted phylogenetic tree was generated between quinoa and other species including A. thaliana, O. sativa, G. max, P. patens and M. truncatula. The phylogenetic analysis showed that CqGSTs were grouped into 11 GST classes including tau, phi, theta, zeta, lambda, DHAR, mPGES, GHR, EF1B, metaxin and hemerythrin. In quinoa, tau and phi are the largest class of GSTs with 65 and 19 members respectively followed by EF1B (7 members), GHR and mPGES (6 member each), hemerythrin (4 members), theta (3 members), metaxin, DHAR and lambda (2 member each). The occurrence of tau and phi as major classes of GST gene family is in accordance with GSTs reported in G. max, O. sativa and A. thaliana. All the tau and phi class CqGSTs were found to be closely associated with G. max, O. sativa and A. thaliana. The analysis clearly represents that CqGSTs of specific class clustered with their respective class GSTs with the exception of lambda class which clustered with DHAR and metaxin that clustered with tau and mPGES class GSTs (Fig. 7).

Fig. 7.

Phylogenetic relationship among GST proteins of A. thaliana, G. max, O. sativa, M. truncatula, P. patens and C. quinoa using MEGA 7. A total of 11 different clades were depicted in different colors (Color description: Blue—Tau; Brown—Hemerythrin; Light green—Lambda; Purple—DHAR; Red—GHR; Light blue—Zeta; Yellow—Theta; Orange—EF1B; Dark green—Phi; Grey—Microsomal; Light orange—Metaxin)

Promoter analysis

Various cis- acting regulatory elements (CAREs) are found to be present in promoter region of the genes. This study identified 21 cis-elements which are responsible for various responses including light, hormone, stress, cellular development and other elements. After the core promoters (AT ~ TATA box, TATA box and CAAT box), MYB (395) was highest in number followed by STRE (280), ARE (279), ABRE (231), ERE (160), AAGAA (112), TCT (112), TGA (57), TC rich repeat (52), P box (44), Circadian (36), AE and CCAAT (35), TCCC (29), DRE (28), GARE (27), CTAG (15) and ACA (1). CqGSTL2 (275) and CqGSTU7 (243) possessed highest number of cis-acting elements while CqGSTU4 (49), CqGSTL1 and CqGSTT3 (50) have least (Fig. 8). Presence of highest number of stress responsive element (MYB) can be linked with their role in stress responses.

Fig. 8.

Cis-acting regulatory element in upstream promoter region of CqGSTs using PlantCARE. The scale represents the number of particular CARE elements in corresponding genes. Grey color is indicative of absence of cis acting regulatory elements

Molecular docking

One candidate member from each identified family was selected for molecular docking study with metalaxyl. Docking study of molecules (CqGSTU1, CqGSTF1, CqGSTL1, CqGSTT1, CqGSTZ1, CqGSTDHAR1, CqGSTH1, CqGSTMi1, CqGSTGHR1) showed that metalaxyl binds with CqGSTF1 with lowest binding energy among all (Fig. 9) (Table 5).

Fig. 9.

Molecular docking study of one member of each quinoa GST gene family proteins with metalaxyl

Table 5.

Binding energies of CqGST molecules with metalaxyl

Protein molecules	Run	Binding energy	Cluster RMSD	Reference RMSD
CqGSTU1	2	− 3.63	0.00	99.10
CqGSTF1	25	− 5.21	0.00	97.63
CqGSTL1	24	− 4.77	0.00	90.22
CqGSTT1	18	− 3.25	0.00	98.47
CqGSTZ1	22	− 2.66	0.00	93.71
CqGSTDHAR1	13	− 2.52	0.00	106.64
CqGSTH1	10	− 3.26	0.00	179.61
CqGSTMi1	7	+ 6.7	0.00	115.18
CqGSTGHR1	1	− 3.7	0.00	113.82

Discussion

GST genes have been found to be involved in various physiological activities in plants that include plant growth and development, signal transduction pathways, tetrapyrrole signaling, hormone responses and importantly in plant’s biotic and abiotic stress metabolism (Csiszár et al. 2019). Therefore, GSTs have become a potential target for plant breeders. The comprehensive genome-wide identification studies of various gene families using the genomic information available at different databases is precise and highly significant (Vaish et al. 2022). In the present investigation, genome wide analysis of GST genes in C. quinoa have been reported for the first time. This study identified 120 GST genes in quinoa that are higher in number as compared to those reported in several other species like 31 in V. radiata (Vaish et al. 2018), 60 in A. thaliana (Lallement et al. 2014a, b), 65 in Brassica oleracea (Vijayakumar et al. 2016), 81 in S. lycopersicum (Csiszár et al. 2014), 82 in O. sativa (Jain et al. 2010), 84 in H. vulgare (Rezaei et al. 2013), 85 in C. annuum L. (Islam et al. 2019) and 101 in G. max (Liu et al. 2015), but fewer than reported in Triticum aestivum and B. napus having 346 and 179 GST genes, respectively (Wei et al. 2019; Hao et al. 2021).

Among the 11 identified classes in quinoa, tau and phi class GSTs were most represented with 65 and 19 members, respectively followed by EF1B (7), GHR and microsomal (6), hemerythrin and zeta (4) and theta (3). Metaxin, DHAR and lambda were represented by 2 members each. The occurrence of tau and phi class was most abundant that was in accordance with those reported for other crops like O. sativa and C. arietinum (Jain et al. 2010; Ghangal et al. 2020). The high number of tau and phi genes reflect their functional importance in plant growth and development, and therefore can be termed as ‘plant specific GST’s’ due to their dominance over other classes (Kumar et al. 2013). The CqGSTs varied in size, sequence and physicochemical parameters like isoelectric point, molecular weight, GRAVY, aliphatic index which were comparable with GSTs of other plant species.

Subcellular location prediction of proteins is vital for having an in-depth understanding of its function and physicochemical characteristics (Liao et al. 2021; Cong et al. 2022). In the present investigation, subcellular localization prediction showed dominance of proteins in cytoplasm, followed by its presence in other cellular compartments including chloroplast, mitochondria, plastid, plasma membrane, nucleus, extracellular, ER and cytoskeleton. The identified GST genes were found to be distributed over 18 chromosomes at different locations. To study the expanded mechanism of GST genes in quinoa, gene duplication events were analyzed which showed that the origin of new members in a gene family was majorly due to gene duplication. The ratio of non-synonymous to synonymous substitutions (Ka/Ks value) < 1 indicates that a gene pair has purifying selection, the Ka/Ks value > 1 indicates positive selection, while Ka/Ks = 1 indicates neutral selection. In the current study, Ka/Ks was less than 1 for most of the genes which was indicative of dominance of purifying selection over positive selection. In quinoa, tandem and segmental duplication were two major driving force for gene family expansion of GST. Chromosome 7 possessed highest seventeen genes while chromosome 4 possessed the lowest i.e., only one CqGST gene. Segmental duplication has major contribution for rapid expansion of GSTs in quinoa as well as reported in other crops like C. annum (Islam et al. 2019), B. napus (Wei et al. 2019), C. arietinum (Ghangal et al. 2020), S. lycopersicum (Islam et al. 2017) and P. bretschneideri (Wang et al. 2018) while tandem duplication was reported as major event for expansion of GSTs in M. accuminata (Vaish et al. 2022), T. aestivum (Hao et al. 2021), B. oleracea and B. rapa (Wei et al. 2019). Secondary structure prediction of GSTs showed highest percentage of alpha helix, followed by coiled and extended strands.

Phylogenetic relationship revealed that all the tau and phi class GSTs were closely associated with those of G. max, O. sativa and A. thaliana. Well-conserved signature motifs were identified in CqGSTs i.e. W(A/V)S(P/M) in tau, SQPS/C in theta, SSCS/A in zeta, CPF/YA in lambda, CPFC/S in DHAR, and CPWA in GHR (Vaish et al. 2020). Similar results have also been reported in S. lycopersicum (Islam et al. 2017), C. annum (Islam et al. 2019) and T. aestivum (Wang et al. 2019a, b). The presence of conserved motifs validates them as GST proteins and confers their functions in plants. There are 2 exons in most of tau class members, 3- 6 in phi, 5–6 in DHAR, 6–8 in EF1B, 3–6 in GHR, 11–14 in hemerythrin, 6 in metaxin, 4–12 in microsomal prostaglandin E synthetase and 2–9 in zeta class. Similar gene organization (2 exons and 1 intron) have also been reported in cotton and mung bean (Dong et al. 2016; Vaish et al. 2018). Structural heterogeneity was observed in almost all classes along with variable number of exons/introns within the members of the same class. A number of previous studies have also reported the presence of multiple introns in zeta and lambda classes, while fewer in tau (Ding et al. 2017; Islam et al. 2017; Kayum et al. 2018; Han et al. 2018; Vaish et al. 2018).

CARE, a specific motif that exists in the promoter region of a gene, has the unique ability to combine with transcription factors and aid in regulation of the downstream genes (Zhu et al. 2021). Thus, the identification and analysis of CAREs in gene promoter regions is of immense utility in understanding the molecular regulation of these genes (Xiaolin et al. 2022). Promoter analysis revealed the presence of different CAREs in response to hormone, light, cellular and stress (Kaur et al. 2017) as well as core promoters including AT ~ TATA box, TATA box and CAAT box (Rahman et al. 2021). Various regulatory elements like ERE motif, GARE motif, ABRE, AAGAA, TCCC, TCT, TGA, MYB, P box were found in the promoter region of CqGSTs that are responsive to light and various hormones like auxin, gibberellins, abscisic acid, thus playing an important role in plant growth and metabolism. The presence of different stress responsive element like STRE, DRE, TC rich repeats confirmed the role of CqGSTs in biotic and abiotic stress. Circadian elements responsible for circadian control were also found in CqGSTs. Sporadic reports are available regarding the role of this element in plant metabolism. Alderete et al. (2018) extensively studied putative circadian regulation while analyzing the NtGST gene (phi) in tobacco seedlings.

Downy mildew caused by Peronospora farinosa (Fr.) Fr. f. sp. chenopodii is a serious disease in quinoa that leads to considerable drop in crop yields across continents (Colque-Little et al. 2021). Metalaxyl, a member of the phenylamides group of systemic fungicides, has been traditionally used for the control of downy mildew in several economically important crops. But there have been reports of health hazards associated with this fungicide (Gupta 2018). Molecular docking study showed that CqGSTF1 possessed highest affinity with metalaxyl, and therefore can be effectively applied to enhance in vivo GST activity in quinoa. Binding of the fungicide metalaxyl with phi family members could be a potential target for enhancing GST activity for detoxification of fungicides. Although safeners induced GST activity has been reported in variety of crops including arabidopsis, maize, and wheat but there is no such report of fungicide induced GST activity is available in quinoa till date. Results of the present study identified metalaxyl as potential GST inducer that could be beneficial for crop development and stress modulation. Availability of this information might encourage researchers for further functional validation.

Conclusion

The in-silico genome wide identification and characterization of GST genes in quinoa assume significance since a number of these assist the plant in their physiological functions, and enables it to grow through biotic and abiotic stresses. Modern biotechnological tools coupled with conventional breeding approaches can help raise plants adapted to various abiotic stresses. These findings can be used for developing stress tolerant plants with enhance productivity through molecular cloning and characterization, as well as their expression studies under stress conditions. The role of GSTs in crop improvement with special emphasis on plant growth and productivity will open new avenues for genetic improvement of plants.

Author contributions

Conceptualization, ST, MB and AB. Methodology, ST, SV, NS and MB. Analysis, ST, SV, MB and AB. Writing- original draft preparation, ST, MB and AB. Writing- review and editing, ST, SV and MB. Supervision, AB. All authors have read and agreed to the published version of the manuscript.

Funding

No funding, financial or non-financial interests were involved.

Data availability

The datasets were derived from sources in the public domain.

Declarations

Conflict of interest

The authors report there are no competing interests to declare.

Ethical approval

Not applicable.

Research involving human participants and/or animals

Not applicable. No human or animal trials were conducted.

Informed consent

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