The functional analysis of ABCG transporters in the adaptation of pigeon pea (Cajanus cajan) to abiotic stresses

Identification of ABCG transporters in the pigeon pea genome

One hundred-twenty nine Arabidopsis ABC protein sequences were downloaded from the Phytozome v12.1 database (https://phytozome.jgi.doe.gov/pz/portal.html). All of the Arabidopsis ABCs were used to identify the ABC transporters in the Cajanus cajan (L.) Millsp (taxid: 3821) database using BLASTP search in the National Center for Biotechnology Information (NCBI) with an initial cut-off e-value of 1.0 e⁻¹⁰ and max target sequence of 500. The Hidden Markov Model (HMM) profiles of ABC transporters (such as PF00005, PF00664, and PF10614) were downloaded from the Pfam database (http://pfam.xfam.org/search#tabview=tab1) and the HMMER search server was used against the pigeon pea proteome with an E-value setting of 1.0 e⁻⁵ (https://www.ebi.ac.uk/Tools/hmmer/) (Potter et al., 2018). The resulting protein sequences were further identified by a conserved domain of ABC based on a conserved domain search (CD-search) on the NCBI website with a threshold e-value of 1.0 e⁻⁵. We identified members of the ABC transporters using the above approach.

Multiple-sequence alignments were performed on the ABC transporters identified above and several ABC proteins of Arabidopsis thaliana, rice, and soybean, using clustalW with default settings. The ABC transporters’ phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1000 replications of bootstrap and p-distance of a model in MEGA6.0 (https://www.megasoftware.net/) (Tamura et al., 2013). The phylogenetic tree was visualized using the iTOL website (http://itol.embl.de/help.cgi) (Sugiyama et al., 2011). Finally, ABCG transporters were identified through the phylogenetic analysis of the ABC family of the pigeon pea.

Phylogenetic tree construction and chromosome localization

The ABCG genes’ chromosomal information was obtained through the NCBI website (https://www.ncbi.nlm.nih.gov/) and were systematically named based on the number and relative position of the ABCG genes in the pigeon pea chromosome. The phylogenetic tree of the pigeon pea ABCG transporters was constructed in MEGA6.0, as described above.

The chromosomal location of the CcABCGs was mapped using the MapChart software (https://www.wur.nl/en/show/Mapchart.htm) based on the chromosomal location information of the ABCG transporters (Voorrips, 2002).

Protein property prediction, subcellular localization

The exon information of CcABCGs was collected on the NCBI website. The number of amino acid sequences, relative molecular weight, and the isoelectric point (pI) of CcABCGs was predicted in the ExPASy ProtParam server database (http://expasy.org/). We predicted the subcellular localization of ABCG proteins using the Cell-PLoc 2.0 database (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) (Chou & Shen, 2008).

To verify our predictions of subcellular localization, two-week-old pigeon pea seedlings were used as a cDNA template to amplify the full-length coding region of the CcABCG7 gene. The primers were: F: 5′-ATGGTGATGATATGGGAAAATGTTAC- 3′ and R: 5′-TTATATTGGAAGGTTTGGGGACA- 3′. The recovered PCR production was ligated to the T vector pMD19-T (TaKaRa, Japan). Subsequently, it was cloned into the eGFP-pROK II vector by double digestion using Kpn I/Xb I to construct the CcABCG7-eGFP-pROK II expression vector, and was transformed into the agrobacterium strain GV3101 (Shanghai Weidi Biotechnology, China). One-month-old tobacco seedlings were injected into the agrobacterium liquid on the back of tobacco leaves with a disposable syringe and placed in the dark for 3 days for observation. Approximately 0.5 cm² of the material was taken around the injection site, observed, and photographed under a Leica SP8 laser confocal microscope (Leica, Germany).

Analysis of motifs and conserved domain

To examine the characteristics and properties of the pigeon pea ABCG subfamily protein domain, the conserved motif of CcABCGs was analyzed on the MEME software http://meme-suite.org/tools/meme with following parameters: the number of motifs was set 10 and the optimum motif width was set between 20 and 200, the other parameters were set to their default (Bailey & Elkan, 1994). Then InterPro program http://www.ebi.ac.uk/interpro/scan.html was used to annotate all 10 motifs.

The conserved domain of CcABCGs was performed using the HMMER servers (https://www.ebi.ac.uk/Tools/hmmer/) and visualized with TBtools software (Chen, 2018). We selected representative WBC and PDR transporter amino acid sequences as target sequences and chose the template with higher homology and better coverage using the Swiss-model server set to automatic (https://swissmodel.expasy.org/interactive). The model results were evaluated using the SAVES server (https://servicesn.mbi.ucla.edu/SAVES/), and the homology model of the CcABCG proteins was visualized by the PyMol software.

Gene structure and cis-elements analysis of CcABCGs

The intron/exon structures of the ABCG genes were analyzed using the gene annotation file (GFF), C.cajan_V1.0, and visualized using TBtools software (Chen, 2018), based on the evolution analysis of CcABCGs. The 2,000 bp upstream region of all identified CcABCGs was extracted from the pigeon pea genome to identify the stress-related or other functional cis-acting regulatory elements of the promoter sequences using TBtools software. All promoter sequences were analyzed using PlantCARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Rombauts et al., 1999).

GO annotation and function prediction

GO (Gene Ontology) integrates and unifies the description and standards of gene products in a database and provides the most comprehensive description of gene functions and gene products. The ABCG protein sequences in the pigeon pea were used for blast alignment in the Swiss-prot database (https://www.uniprot.org/blast/) and the alignment results were annotated and classified by the pigeon pea ABCG gene (Ashburner et al., 2000; Consortium, 2019; Ehlert et al., 2006; Yang et al., 2016).

Plant materials and treatments

Pigeon pea seeds (ICPL87119) were sown in a soil mixture of nutrient soil, vermiculite, and perlite (1:1:1) in the greenhouse at Beijing Forestry University in China with natural light for 10-14 h/day, a temperature of 18−28 °C and a relative humidity of 45–70%. After three months, the roots, stems, leaves, and flowers from pigeon pea trees were selected and stored at −80 °C to extract RNA and further analyze their tissue-specific expression.

The pigeon pea seeds were surface-sterilized with 75% ethanol for 30 s, then soaked in sodium hypochlorite solution for 6 min, and finally washed 5 times with sterile water for 30 s durations. These sterilized seeds were grown in Murashige and Skoog (MS) medium at pH=5.8 in growth chambers at 24  ± 2 °C with a photoperiod of 16 h light/8 h dark, 400 µM m⁻² s⁻¹ light intensity. To analyze the expression of the CcABCG genes in various abiotic stresses, the 14-day old pigeon pea seedlings were assigned to different treatments. The treatments were as follows: heat and cold stress, in which the seedlings were cultured at 4 °C and 42 °C in incubators, respectively; salt and metal stress, in which the seedlings were grown in solid MS medium with 200 nmol/L NaCl and 100 µmo/L AlCl₃, respectively; drought stress, in which the pigeon pea seedlings were treated with 250 nmol/L mannitol in solid MS medium. The roots and leaves of these seedlings were sampled at 0 h, 6 h, and 12 h after various treatments. Three biological replicates of each sample were immediately frozen in liquid nitrogen and stored at −80 °C until expression analysis of variously abiotic stresses was conducted.

RNA isolation and quantitative real-time PCR analysis

We selected 10 genes containing elements that responded to adversity for tissue specificity analysis and abiotic stress analysis based on the above analysis of cis-elements in the promoter region. Total RNA was isolated using the CTAB method and first-strand cDNA was synthesized from 1 µg of total RNA using a PrimeScript RT kit (Takara) according to the manufacturer’s instructions. The quality and concentration of cDNA were assessed using a Nano Photometer N50 (Implen GmbH, Munich, Germany). We performed expression analysis of the ABCG transporter under different tissues and abiotic stresses using qRT-PCR. Real-time RT-PCR analysis was performed using CFX connect (Bio-Rad, California, USA) with the SYBR Green PCR Master Mix (TaKaRa, Tokyo, Japan) with CcActin as a reference gene. The gene primers selected were synthesized on the Sangon Biotech website (http://www.sangon.com/newPrimerDesign) and are shown in Table S1. All analyses were performed with three biological replicates and qRT-PCR data was analyzed using IBM SPSS 22 software (IBM Corporation, USA).

Identification of ABCG transporter genes in the pigeon pea genome

One hundred-twenty nine Arabidopsis ABC proteins were downloaded and used as queries in the pigeon pea genome database using BLASTP server to identify all members of the pigeon pea ABCG transporters. A total of 222 ABC proteins were identified in the pigeon pea after removing redundancies. The HMMER search was performed using the HMM of ABC transporters (such as PF00005, PF00664, and PF10614) to screen for ABC transporters identified by BLASTP. One hundred fifty-five ABC proteins were eventually confirmed to have NBD/TMD domains (Table S2). To explore the phylogenetic evolutionary relationship between the pigeon pea and other species including Arabidopsis, rice, and soybean, a Neighbor-Joining (NJ) tree was constructed (Fig. S1) based on ABC proteins. A phylogenetic relationship indicated that the pigeon pea ABC gene family was classified into 8 different groups (ABCA-ABCI), and the ABCG subfamily, which contained 51 members, was the largest group of ABC transporters in the pigeon pea.

Phylogenetic tree construction and chromosome localization

All of the ABCG transporters identified above were named CcABCG1-CcABCG51 based on their chromosomal location. A Neighbor-Joining (NJ) tree was constructed to analyze the phylogenetic relationship of ABCG transporters (Fig. 1). Phylogenetic analysis indicated that the ABCG transporters were further divided into WBC and PDR, in which WBC contained 33 ABCG transporters and PDR contained 18 transporters.

The chromosome localization of ABCG transporters was determined in the pigeon pea genome and all identified CcABCGs were found to be diversely distributed on pigeon pea chromosomes, except for chromosome 7. Chromosomes 1 and 8 both contained one ABCG transporter and all chromosomes of the pigeon pea had the least ABCG transporters. Chromosomes 2, 9, and 11 included 5, 5, and 8 transporters, respectively. Seventeen genes were distributed on unplaced scaffolds and were mapped into a single chromosome, ChrUn (Fig. 2). Two pairs of genes (CcABCG8/9, CcABCG32/33) were closely linked on the chromosome and belonged to the paralogous genes. The ABCG gene locations were annotated using the NCBI website and the information in shown in Table 1.

Protein property prediction, subcellular localization of CcABCGs

Fifty-one CcABCGs were identified and their protein properties were predicted (Table 1), including their amino acid lengths, relative molecular weights, and isoelectric points (pIs). Results indicated that the lengths of all ABCG proteins ranged from 609 aa (CcABCG34) to 1500 aa (CcABCG29). Similarly, their relative molecular mass ranged from 68159.95 Kd (CcABCG34) to 170370.83 Kd (CcABCG29) and the theoretical isoelectric points ranged from 6.75 (CcABCG29) to 9.47(CcABCG4). The ABCG genes’ exon numbers changed from 1 to 26.

We performed the subcellular localization of CcABCGs to determine the active sites of the CcABCGs. The prediction of the subcellular localization indicated that all CcABCGs were localized in the cell membrane. Three ABCG transporters (CcABCG26, 28, 29) were found to be localized in the chloroplast and not just on the cell membranes (Table 1). To confirm our prediction, we verified the subcellular localization of the ABCG transporter by transiently expressing CcABCG7-eGFP in tobacco leaves. The results showed that the green fluorescent signal of CcABCG7-eGFP-pROK II was mainly detected in the cell membrane (Fig. S2).

Analysis of motifs and conserved domain of CcABCGs

To identify the structure and function of the ABCGs in the pigeon pea, motif analysis was performed based on the phylogenetic analysis (Fig. 3A). A total of 10 conserved motifs were predicted and the width of those motifs ranged from 32 to 123 amino acids. The number of motifs in each amino acid varied from 2 to 10. Results showed that the number of motifs in the WBC was significantly less than the number of motifs detected in the PDR. All 10 motifs were annotated using the InterPro program to identify the motif structure. Annotation analysis demonstrated that Motifs 3, 7, 9, and 10 did not annotate at all; Motif 6 and Motif 8 annotated in the ABC_2_trans structure (IPR013525); Motif 1,2,4,5 annotated in the P_loop_NTPase structure (IPR027417); Motifs 1, 4, and 5 were annotated in the ABC_transporter_like structure (IPR003439). Our results indicated that the motifs of the same annotation were relatively conservative in structure, such as Motifs 1, 4, and 5 (Table S3).

We examined the conserved domain of CcABCGs to explore the ABCG transporter’s domain function using HMMER servers (Fig. 3B). The HMME model, “ABC2_membrane” indicated the TMD domain, and “ABC_tran” indicated the NBD domain. The results showed that the ABCG transporters were different from other members of the ABC transporter family with an inverted “TMD-NBD” domain arrangement pattern. All ABCG transporters contained the NBD domain, but several ABCG transporters did not contain the TMD domain. The WBC subgroup contained one NBD domain with a length that increased from 97 to 153 and one or no TMD domains with a length of approximately 200, while the PDR subgroup contained two NBD domain with an amino acid length of 138 to 200 and a domain approximately 200 TMD long.

CcABCG35 was selected for homology modeling to explore the molecular functions of CcABCGs. The best template of CcABCG35 was 6hij.1A (Seq Identity:33.05%, GMQE:0.58, Coverage:0.85) using the Swiss-model program. According to the PROCHECK evaluation results, Ramachandran Plot results in the model evaluation revealed that in each ABCG model >90% of the amino acid residues were distributed in the allowed region, indicating that the quality of the CcABCG protein model obtained by homology modeling was reliable (Fig. S3A). A model of CcABCG35 revealed that Walker A and Walker B were located in the NBD domain, while the TMD domain contained six alpha helices (Fig. S3B).

Gene structure and cis-elements analysis of CcABCGs

Gene structure was analyzed based on the phylogenetic relationship of the ABCG gene family to better understand their structural evolution (Fig. 4A). The highly conserved exon sequence is an essential sequence for the ABCG transporter to perform gene functions, and the differences in introns may be based on dissimilar regulatory mechanisms for the existence of genes. The results indicated that all ABCG transporters in the pigeon pea contained different numbers of exons and introns. The number of exons in CcPDRs was significantly higher than that in CcWBCs. The number of exons of CcPDRs ranged from 5 to 25, while the number of exons of CcWBCs varied from 1 to 14 and there were large differences in the number of introns. There were approximately 20 introns in CcPDRs and about 5 in CcWBCs.

A 2,000 bp region upstream of the promoter was selected for analysis of the cis-acting element of the pigeon pea ABCG gene family in order to explore the expression elements of the promoter region (Fig. 4B). Three cis-acting elements were screened, and focused on hormone-responsive elements, light-responsive elements, and stress-responsive elements. The number of light-responsive elements in the promoter region of the pigeon pea ABCG transporter was found to be relatively large. Hormonal response elements had a large number of copies in the promoter region of the ABCG transporter, including auxin response elements (AuxRR-core, TGA-element, etc.) and gibberellin response elements. There were fewer stress response elements than hormone response elements and some genes did not contain stress response elements (CcABCG14, CcABCG10, etc.). Drought-related cis-acting elements were identified simultaneously in CcABCG7 and CcABCG24. Low-temperature stress-related cis-acting elements (LTR) were identified in CcABCG28 and their elements were highly conserved with short sequences composed of CCGAAA.

Go annotation and function prediction

The ABCG gene in pigeon pea was annotated and divided into three categories: molecular function, biological processes, and cell components. The annotation results showed that all 50 genes were annotated into three major categories, but CcABCG48 did not show results from the blast alignment method and UniProtKB ID and all other usable data are provided in Data S5. The transmembrane transport (GO:0055085) had the highest prominence and 45 genes were annotated in the transmembrane transport. CcABCG28 and CcABCG29 were blasted in AB36G_ARATH (UniProtKB ID), and annotated in 42 GO annotations, which included “response to salt stress” (GO:0009651) and “root development” (GO:0048364). Fifty ABCG genes were annotated to the broad category of molecular functions related to ATPase activity and ATP binding. The major category of cellular components found most of the ABCG genes to be annotated on the membrane (Fig. 5).

Expression analysis of the pigeon pea ABCG gene family in different organs

To explore the expression pattern of the ABCG gene in different organs during the growth and development of pigeon peas, the organs (roots, stems, leaves, and flowers) of one-year-old pigeon peas were collected to extract RNA for qRT-PCR analysis for the expression of the ABCG genes. Tissue-specific expression analysis found that the expression of ABCG transporters in different tissues was not significantly different. However, there was an obvious difference in the expression level of the ABCG transporter gene in flowers, which was significantly lower than that in the roots, stems, and leaves. The expression of CcABCG24 was significantly higher than that of any other gene (Fig. 6).

Expression analysis of the pigeon pea ABCG transporters under different abiotic stresses

To explore the expression level of the pigeon pea ABCG gene family under different abiotic stresses, two-week-old pigeon pea seedlings were selected and transplanted into the culture environments at 4 °C, 42 °C, 200 nmol/L NaCl, 100 µmol/L AlCl₃ and 250 nmol/L mannitol in solid MS medium, respectively. CcABCG5,7,14,19,21 belonged to the WBC subgroup, and CcABCG10,24,28,29,32 belonged to the PDR subfamily. We found that the expression difference of 10 genes in the roots was significantly higher than in the leaves (Fig. 7).

The two major subfamilies of ABCG transporters had significant differences in expression in the roots (Figs. 7A, 7C, 7E, 7G and 7I) under 4 °C stress and NaCl stress. The PDR family genes were significantly up-regulated under both stressors. CcABCG24 was up-regulated 40-fold after 12 h at 4 °C and CcABCG28 increased 65 times. Under the initial NaCl treatment, CcABCG24 increased 10 times. However, it is worth noting that with an increase of the treatment time, the expression of CcABCG24 decreased to below the initial level at 12 h. We found that the relative expression changes of the WBC subgroups under drought and aluminum stress treatments were opposite, as shown in Figs. 7E and 7G. The expression level of CcABCG5 increased under drought stress at 6 h but did not change significantly under aluminum stress. CcABCG19 also exhibited the same pattern of opposite expression in the treatments of mannitol stress and AlCl₃ stress. Under high-temperature stress, we observed that the expression of CcABCG7 was down-regulated but the expression of CcABCG7 in leaves was up-regulated.

There was a relatively mild difference in the expression levels in leaves (Figs. 7B, 7D, 7F, 7H and 7J). CcABCG28 experienced up-regulation under 4 °C treatment. However, CcABCG7 was significantly up-regulated after 6 h of drought stress. In the same situation, CcABCG7 was up-regulated 11.3 times after 12 h of aluminum stress treatment. However, under sodium stress, CcABCG7, 19, 21 were down-regulated. The ABCG transporter was found to have different expressions in response to different environmental stresses.