- Open Access
Identification of ABC transporter G subfamily in white lupin and functional characterization of L.albABGC29 in phosphorus use
BMC Genomics volume 22, Article number: 723 (2021)
White lupin (Lupinus albus) is a leguminous crop with elite adaptive ability in phosphorus-deficient soil and used as a model plant for studying phosphorus (P) use. However, the genetic basis of its adaptation to low P (LP) remains unclear. ATPase binding cassette (ABC) transports G subfamily play a crucial role in the transportation of biological molecules across the membrane. To date, identification of this subfamily has been analyzed in some plants, but no systematic analysis of these transporters in phosphorus acquisition is available for white lupin.
This study identified 66 ABCG gene family members in the white lupin genome using comprehensive approaches. Phylogenetic analysis of white lupin ABCG transporters revealed six subclades based on their counterparts in Arabidopsis, displaying distinct gene structure and motif distribution in each cluster. Influences of the whole genome duplication on the evolution of L.albABCGs were investigated in detail. Segmental duplications appear to be the major driving force for the expansion of ABCGs in white lupin. Analysis of the Ka/Ks ratios indicated that the paralogs of the L.albABCG subfamily members principally underwent purifying selection. However, it was found that L.albABCG29 was a result of both tandem and segmental duplications. Overexpression of L.albABCG29 in white lupin hairy root enhanced P accumulation in cluster root under LP and improved plant growth. Histochemical GUS staining indicated that L.albABCG29 expression increased under LP in white lupin roots. Further, overexpression of L.albABCG29 in rice significantly improved P use under combined soil drying and LP by improving root growth associated with increased rhizosheath formation.
Through systematic and comprehensive genome-wide bioinformatics analysis, including conserved domain, gene structures, chromosomal distribution, phylogenetic relationships, and gene duplication analysis, the L.albABCG subfamily was identified in white lupin, and L.albABCG29 characterized in detail. In summary, our results provide deep insight into the characterization of the L.albABCG subfamily and the role of L.albABCG29 in improving P use.
The ATPase binding cassette (ABC) proteins constitute nucleotide binding domain (NBD) and transmembrane domain (TMD) conserved domains  containing superfamily ubiquitous across all living organisms, including plants. ABC transporters gene family encode for a membrane-bounded transporter protein that mediates molecular transportation of soluble proteins across the plasma membrane or among different organelles . With the advent of advanced genomic and bioinformatics techniques, a plethora of ABC transporters has been identified in numerous plant species such as Arabidopsis, soybean, tomato, rice, peppers, pineapple, and Lotus [3,4,5,6,7,8,9,10]. Plant ABC transporter gene family typically distributed to ABCA-ABCI subfamilies, except ABCH subfamily . However, the ABCG subfamily is unique in fungi and plants. Like other ABC subfamilies, the ABCG subfamily is also categorized into full-size ABCG transporters, containing two NBD and two TMD (NBD-TMD2) and half-size transporters containing only one NBD and TMD. The former probably originated by a single duplication of later ABCG transporters group .
ABCG is known to be one of the most extensive subfamilies in the plant kingdom and reported to be involved in diverse biological processes such as pathogenicity, cuticle formation , transportation of various biological molecules, and hormone transport . ATP-binding cassette (ABC) transporter genes from Arabidopsis, AtABCG25 and AtABCD40 exhibited ATP-dependent ABA transport . Mutations in these ABCGs lead to reduced ABA-dependent stomatal closure and severe phenotypes to drought stress . We also reported two white lupin ABCG genes, L.albABCG36 and L.albABCG37, potentially involved in auxin-mediated cluster root (CR) formation under P deficiency . In rice, RCN1/OsABCG5 involved in ABA-mediated stomatal closure in guard cells . In addition, a half-size AtABCG22 is involved in the regulation of stomatal responsiveness to change in air humidity in both opening and closing directions . Arabidopsis DSO/ABCG11 transporter affects cutin and suberin Metabolism in reproductive organs and roots tissues . Similarly, ABCG37 and ABCG33 functioned as caesium influx carriers in Arabidopsis roots . These suggested the crucial roles of ABCG subfamily members to improve plant growth, development, and nutrients acquisition. However, the identification and characterization of the white lupin L.albABCG transporters subfamily remain to be determined.
White lupin (Lupinus albus) is an economically important legume crop belonging to the family Fabaceae due to its high nutritious value, high protein and low oil contents . It gained a lot of research attention due to its significant role in improving nutrient mobilization, soil exploration and fertility, and nitrogen-fixing ability . Rhizosheath formation (soil strictly attached with root) is an important root adaptive trait that alleviates nutrient uptake under soil drying (SD) . Phosphorus is an essential macronutrient for plant growth and productivity [25, 26]. It has been reported that rhizosheath with long root hairs having a competitive advantage for P acquisition to relieve P deficiency in barley and wheat [27, 28]. Therefore, a robust rhizosheath can improve P uptake efficiency via root hair growth in water-limited soils. White lupin L.albABCG37 reported in stimulating CR formation by modulating IBA transport under P deficiency , suggesting its role in P acquisition. Rice is an important staple food crop, and 1/3rd of the world’s population relies on it . However, it consumed a massive amount of water and nutrients from the soil, becoming an alarming situation for agriculture sustainability . Several studies reported that rice biomass and yield depend on P availability under acidic soil [31,32,33]. However, the role of genes involved in improving P uptake efficiency from the soil is scarce. Therefore, an urgency to identify novel candidate genes or plant traits directly involved in improving phosphorus use efficiency would support sustainable agriculture.
This study provided comprehensive information on the white lupin ABCG transporter subfamily and investigated their expression under LP conditions. We analyzed the evolutionary importance of L. albus ABCG subfamily with A. thaliana, G. max, L. angustifolius, and P. vulgaris. Furthermore, we overexpressed L.albABCG29 in white lupin and rice to investigate its putative role in improving phosphorus use efficiency under LP hydroponic solution and SD conditions, respectively. Our findings provide cues for future investigation on crop P use improvement.
Identification of white lupin ABCG subfamily members
Whole-genome sequence of white lupin was used to identify the ABCG subfamily transporters genes. Arabidopsis ABCG protein sequences were used to search against the white lupin genome to screen out candidate ABCG subfamily members. In total, 66 ABCG transporter genes (Additional file 1) in the whole white lupin genome. The protein length of L. albus ABCG genes ranged from 130 (L.albABCG22) to 1498 amino acids (L.albABCG35). L. albus ABCG transporter genes were unevenly distributed across 24 chromosomes except for 18 chromosomes. Of 66 ABCGs, 8 members were located on chromosome 2, accounted for 12.2% of the total LalbABCG transporter gene, followed by 7 on chromosome 20 (10.6%), 6 on chromosome 25 (9.0%), and only 1 gene was located on chromosome 4, 8, 10, 13, 15, 16, 17, and 23 accounted for 1.5%. Moreover, 4 sets of genes were located on chromosome 12, 19, and 24 (6.0%) each, and 3 were found on chromosome 1, 3, 9, 14, and 22 (4.5%), and 2 were found on chromosome 5, 6, 7, 11, and 21 (3.0%) (Additional file 1). Detailed characteristics of the L. albus ABCG transporter subfamily, including transcript IDs, gene name, domain type, and in silico subcellular location, is provided in Additional file 2.
ABCG subfamily is the most diverse in white lupin
Evolutionary analysis of ABCG transporter proteins reveals that the ABCG subfamily is among the most abundant groups among all species (Fig. 1a). ABCG subfamily accounted for 66 members in L. albus, 56 L. angustifolius, 77 P. vulgaris, 117 G. max, and 43 A. thaliana in the plant lineages. As ABCG subfamily is tremendously diverse in the plants, we are curious to know how this diversity evolved. Phylogenetic analysis of the ABCG subfamily in a broad range of legumes and A. thaliana was conducted. The white lupin ABCG transporters proteins clustered into seven groups (I to IV, VI-VII), with other plant homologs. However, group V is absent in white lupin. A. thaliana; Group-III shared seven L.albABCG with AtABCG1, AtABCG2, AtABCG6, and AtABCG16; Group-IV contained two A. thaliana AtABCG (AtABCG9 and AtABCG26) along with 14 ABCG from white lupin. Similarly, Group-VII includes those L.albABCG, which share homology with diverse functional ABCG of A. thaliana (Fig. 1a). However, phylogenetic analysis of the white lupin ABCG subfamily (Fig. 1b) follows the above-mentioned phylogenetic clustering (Fig. 1a). Our data suggested that ABCG genes evolved in white lupin to acquire new functions in a very diverse way.
Gene structure analysis and motif composition of ABCG subfamily
The conserved domain analysis of ABCG transporter proteins revealed four conserved domains exist in two complexes, the first complex as NBD or TMD or contain both (Fig. 2a). These genes have 1–24 exons and scattered over many different chromosomes (Fig. 2b). Intron-exon distribution revealed that closely related ABCG genes were generally displayed a similar gene structure. Eight of 66 ABC transporter genes contained single or 4 exon, including L.albABCG06, L.albABCG17, L.albABCG21, L.albABCG22, L.albABCG32, L.albABCG44, L.albABCG52, and L.albABCG62 or L.albABCG02, L.albABCG16, L.albABCG23, L.albABCG31, L.albABCG38, L.albABCG41, L.albABCG45, and L.albABCG63, respectively. Moreover, 9 exons are present in L.albABCG01, L.albABCG13, L.albABCG26, L.albABCG43, L.albABCG47, L.albABCG46, and L.albABCG50, or L.albABCG20, LalbABCG29, LalbABCG33, and LalbABCG39 contained highest number of exons (24) among ABCGs each (Additional file 1 and Fig. 2b). Several closely related genes display similar exons distribution pattern, indicating that these genes belong to the same subclade.
Conserved motifs were analyzed to understand better the global conservation and diversification of white lupin ABCG transporter proteins. A total of 10 conserved motifs were identified and displayed a very diverse distribution pattern validating their phylogenetic classification (Fig. 2c). For instance, ABCG genes in all phylogenetic clades except for G-VI and G-VII contained five and six motifs with some exceptions, respectively. For example, G-I had six motifs with the exceptions of L.albABCG13, L.albABCG31, and L.albABCG63 containing 5, 1, and 4 motifs, respectively. While L.alABCG62 of G-II contained 3 motifs, and L.albABCG05 of G-IV contained 5 motifs. All ABCG members in G-VI had 5 motifs, except for L.albABCG42 of G-VI contained 5 motifs, but motif 4 is exchanged with motif 5. Lastly, G-VII contained all the motifs except for L.albABCG27 and L.albABCG28 having 7 and 5 motifs each, respectively. In a few ABCGs some motifs occur twice, including motif 2, 6, and 7 (Fig. 2c). MEME analysis also indicated that 8 of the total 10 conserved motifs overlapping to ABC_trans (NBD) and ABC2_membrane (TMD) domains. Motif-1, motif-3, motif-5, and motif-9 belong to the ABC_trans domain, while motif-2, motif-6, motif-7, and motif-10 belong to the ABC2_membrane domain (Additional file 3).
Cis-regulatory element analysis of L. albus ABC transporters
Plant evolves complex signalling mechanisms, including promoter cis-regulatory elements related to stress, mediate adaptation to rapidly changing environmental variabilities . The cis-regulating elements associated with transcription factors regulate the expression of ABCG transporter genes upon stress conditions. To determine the potential role of cis-regulatory elements in L. albus ABCGs, 2 kb region upstream promoter of each gene was subjected to cis-regulatory elements prediction from the online database PlantCare. The resulting cis-regulatory elements were then compared with available literature. A few key components were selected to draw their distribution.
ABCG transporters subfamily had at least one important cis-regulatory element related to stresses. Some important elements involved in stress response or hormone regulation include Abscisic acid-responsive element (ABRE), Auxin responsive element (ARE), Salicylic acid-responsive elements (TCA-element), Wound responsive elements (WRE3), WUN-motif, Ethylene responsive element (ERE), Myb3 binding promoter motif (MBS, Myb), and Auxin responsive element (ARE) were identified in L.albABCG promoter sequences (Fig. 2d). ABRE elements are predicted in 69.70% ABCG genes promoters sequences, while ARE accounted for 86.36%, ERE 63.64%, MBS 56.06%, 69 TCA elements 39.39%, WUN motifs 43.94%, and WRE3 39.39% in all L.albABCGs.
Synteny and colinearity analysis of L. albus ABC transporters genes
Gene duplication plays a pivotal role in gene family expansion . In the current study, a total of 31 paralogues gene pairs in the L.albABCG subfamily were identified (Fig. 3a). Out of 31 L.albABCG transporter gene pairs, 29 L.albABCGs were segmental duplication pairs (involving 38 L.albABCG genes), and 2 L.albABCG were tandemly duplicated pairs (involving 4 L.albABCG genes) in the whole L. albus genome (Additional file 4). These segmental duplication pairs were randomly distributed on all chromosomes, and the maximum number of genes were located on chromosomes 20 and 24. Contrastingly, chromosomes 3 and 20 contained single pairs of tandemly duplicated L.albABCG genes (Additional file 1). These results suggest that segmental duplication was the major driving force for ABCG gene expansion in the L. albus genome.
Moreover, we constructed comparative synteny maps of L. albus with A. thaliana, L. angustifolius, P. vulgaris, and G. max. A total of 25 orthologous pairs of segmentally duplicated L.albABCG were identified by comparing with A. thaliana genome (Additional file 5a & 6), distributed on 13 white lupin chromosomes. The maximum number of L.albABCG genes of orthologous pairs were located on chromosomes 2 and 25. Moreover, 31 and 44 orthologous pairs of segmental duplications were detected in G. max (Additional file 5b & 7) and L. angustifolius genome (Additional file 5c & 8), respectively, whereas no tandem duplication was found. Additionally, L.albABCG29 was paired with two L. angustifolius genes (XP_019455889.1, XP_019463336.1) (Additional file 9), while no duplication pair was found with A. thaliana and G. max may imply that L.albABCG29 may exist before ancestral divergence. Furthermore, the Ka and Ks substitution rate was calculated to determine the selection pressure of gene pairs. We found that all segmental and tandem duplication pairs of different species had a Ka/Ks ratio < 1, implying that L. albus ABCG transporters subfamily genes may experience purifying selection during evolution (Additional files 4 & 6, 7 and 8).
To further infer the effect of evolutionary pressure on L. albus ABCGs, we constructed comparative colinearity maps of L. albus among five different representative plant species, including L. angustifolius, P. vulgaris, G. max, and A. thaliana (Additional file 9). A total of 130 orthologous pairs exhibited a colinear relationship between L. albus and G. max, followed by 96 with L. angustifolius, 71 with P. vulgaris, and 34 with A. thaliana. Moreover, to further infer the evolutionary significance of L.albABCG29, a collinearity map between L. albus L.albABCG29 with G. max, L. angustifolius, and P. vulgaris was generated. L.albABCG29 form two collinear pairs with G. max (Glyma07g36160, Glyma17g04350), and single pair with L. angustifolius (XM_019600344.1), and P. vulgaris (XM_007154390.1). Whereas no collinear block was detected with A. thaliana (Fig. 3b). The functional characterization of L.albABCG29 can provide a more valuable gene functional reference for legumes growing under phosphorus deficient conditions.
L.albABCG genes are differentially expressed under low phosphorus
To elucidate the biological significance of white lupin ABCG in phosphorus transport, we examined the gene expression pattern of ABCG subfamily members among different L. albus tissues grown under CK and LP conditions using our previously generated RNA-seq data. The expression pattern of L.albABCG in different tissues revealed that the majority of genes were sensitive to LP (Additional file 10). For instance, L.albABCG46, L.albABCG47, and L.albABCG48 showed higher expression in leaf (L) under CK compared to LP conditions. Most of the genes had a relatively lower expression in the shoot (S), while few genes were expressed under both CK and LP conditions. In CR, L.albABCG29 was highly expressed under LP conditions, while L.albABCG17, L.albABCG52, L.albABCG58, and L.albABCG66 had moderate expression levels. Four genes L.albABCG3, L.albABCG20, L.albABCG26, and L.albABCG35 were highly expressed in L and L.albABCG38, and L.albABCG64 has only a highly expressed root tip (RT) under LP condition. Contrastingly, L.albABCG2, L.albABCG5, L.albABCG7, L.albABCG9, L.albABCG14, L.albABCG15, L.albABCG16, L.albABCG18, L.albABCG25, L.albABCG37, L.albABCG43, L.albABCG44, L.albABCG60, and L.albABCG65 in S under CK and LP stresses, respectively (Additional file 11). To further validate the expression of L.albABCG, L.albABCGfour duplication pairs and others from phylogenetic clade G-VII were selected to investigate their expression in different white lupin tissues under low P. The results showed that overall expression trends of these genes by RT-qPCR were consistent with that of RNA-seq analysis. All the L.albABCG genes showed the highest expression levels in the lateral root (LR) and CR except L.albABCG20, which had the highest expression in the leaf (Fig. 4). RNA-seq data and RT-qPCR analysis showed that most genes showed higher expression in CR and lower in LR under LP, indicating ABCG transporters may be involved in mitigating P related stresses.
Overexpression of L.albABCG29 improves phosphorus uptake in white lupin
Previously, we reported that L.albABCG29 might act as a critical gene in P acquisition under low P. Here, we also found continuous increasing expression of L.albABCG29 in different tissues and peaked expression in CR. We generated transgenic hairy roots overexpressing L.albABCG29 in white lupin. We found significantly increased expression under LP conditions compared to the non-transgenic roots (Fig. 5a). To precisely analyze the expression of L.albABCG29 in the roots under P variability, transgenic lines resulting in the expression of L.albABCG29 promoter::GUS was generated. Staining of transgenic plant root revealed elevated expression of L.albABCG29 under low P (Fig. 5b). After 6-weeks of transformation, transgenic CR and LR of L. albus were collected to measure phosphorus content. We found that CR had a higher phosphorus concentration followed by LR under LP conditions (Fig. 5c). LP condition showed enlarged root growth in both transgenic lines (Fig. 5d) and improved overall plant biomass (Fig. 5e), indicating that transgenic roots have a role in nutrient translocation from root (transgenic) to shoot (non-transgenic), ultimately improves plant growth.
L.albABCG29 encourage phosphorus uptake in rice
Due to the lack of L. albus stable transformation protocol, we then overexpressed L.albABCG29 in rice. L.albABCG29 overexpression transgenic rice plants were used to grow under four different (WW + P, SD + P, WW–P, and SD–P) soil treatments. Plants were harvested after 45 days of growth, and rhizosheath formation was observed only under SD treatments (SD + P and SD-P) (Fig. 6a), no rhizosheath was developed under WW conditions. We found that SD–P showed higher expression of L.albABCG29 in transgenic rice lines than WT among all four soil treatments (Fig. 6b). SD–P treatment showed increased rhizosheath formation in transgenic lines compared to SD + P (Fig. 6e), suggesting that OE-L.albABCG29 involved enhanced rhizosheath formation via extended root structure and hair growth (Fig. 6c-d). Additionally, we observed that OE-L.albABCG29 showed a higher P concentration compared to WT among all tissues. Root showed higher P uptake under SD–P (Fig. 6g). Shoot P uptake was also higher under SD–P in the overexpression line (Fig. 6f), indicating the phosphorus uptake improved under combined SD and LP conditions. Significant differences were noted among rhizosheath P concentration of OE-L.albABCG29 and WT plants under four different soil treatments (Fig. 6h). The data suggested that OE-L.albABCG29 is associated with increased P uptake in O. sativa by improved rhizosheath formation and root growth, especially under SD–P soil treatment.
ABC transporters are ubiquitous membrane-bound proteins [2, 36] present in all prokaryotes, from plants to animals [3, 37]. Comprehensive genome-wide investigation of the ABC transporter gene family among several plant species provides deep insight in identifying their regulatory mechanism and functional characterization to stress responses . Phylogenetically ABC gene family is divided into distinct subfamilies, including the ABCA-ABCI subfamilies, except the ABCH subfamily [8, 9, 11]. Among these subfamilies, the ABCG subfamily is tremendously diverse and encode either 577–1107 amino acids (aa) for half-size or 1382–1469 aa for full-size ABCG transporters proteins . ABCG transporter proteins are functionally diverse and ubiquitous in all organisms, including animals and plants. Several studies have been reported that ABCG was the largest subfamily of the ABC gene family in plants but needed to be functionally characterized . These genes have 1–24 exons and distributed on different chromosomes. In this study, we identified ABCG subfamily members in white lupin (Additional file 2). Phylogeny of the ABCG subfamily in plant lineage reveals several exciting insights . Our phylogenetic study validated those findings; for instance, white lupin ABCG transporter proteins were clustered with A. thaliana ABCG proteins. Unlike A. thaliana, L.albABCG proteins were diversified into six clusters (Fig. 1a). We speculated that proteins sharing a similar ancestral origin might have similar functions. This suggested that L.albABCG clustered with A. thaliana homologs may share some functional similarities which need to validate.
Duplication of the gene on the individual chromosome, between different chromosomes, or even entire genomes may be a major driving force in building up gene diversity during genome evolution . Our findings showed that segmental duplication events of the ABCG subfamily were more frequent within the L. albus genome (Additional file 4 & Fig. 3a) comparing with G. max, L. angustifolius, and A. thaliana (Additional file 5). For instance, we found a total of 66 ABCG members in L. albus (Additional file 4), while 43 in A. thaliana (Additional file 6) were previously reported [9, 41], indicating expansion of ABCG subfamily in legumes. Additionally, we compared gene duplication events of ABCG subfamily genes within the L. albus genome and with A. thaliana, G. max, and L. angustifolius genomes (Additional file 5). A total of 29 segmental duplication pairs (involved 38 L.albABCG genes) and 2 tandem duplication pairs (involved 4 L.albABCG genes) were identified within the L. albus genome (Additional file 4). A total of 31 segmental duplication pairs were found between L. albus and G. max genome (Additional file 7), while 44 segmental pairs by comparing with L. albus with L. angustifolius genome (Additional file 8). Contrastingly, 25 segmental duplication pairs were shared by L. albus and A. thaliana genome (Additional file 6). More specifically, the L.albABCG29 pair was found in both tandem and segmental duplication, indicating that duplication events could be a major cause of ABCG subfamily expansion. Most of the members of ABCG transporter subfamily originated by segmental gene duplications, which suggests that segmental duplication may contribute as a major driving force for ABCG transporters evolution in the L. albus genome.
ABCG subfamily members are involved in regulating many biological processes. For instance, ABCG transporters are known to be involved in the metal detoxification process. Overexpression of ABCB25 showed enhanced resistance to polluted cadmium conditions in A. thaliana . AtABCG37 is a close homolog of the L.albABCG29, showed IBA and 2,4-D emission into the soil due to its locality on the plasma membrane . Pighin et al.  reported that ABCG subfamily involves the transportation of lipids. Gene function prediction and regulation are mainly determined by identifying cis-regulatory elements in the promoter region . Several transcription factors and RNA polymerase II enzyme binds with the TATA box that forms a transcription initiation complex to regulate the transcription process . TCA element involved in salicylic acid responsiveness and WUN motif related to wound responsiveness. Several other stress-related elements were identified, involving the DRE element involved in dehydration responses. Additionally, cis-regulatory elements related to hormonal responses, such as ABRE and ARE responsive elements, were identified (Fig. 2d). Our finding collectively showed the diverse functionality of the ABCG subfamily among different cellular, hormone signalling mechanisms to improve plant growth, development, and stress response.
The release of the high-quality L. albus genome makes it readily available for researchers to discover important candidate genes related to specific agronomic traits [17, 47]. Transcript expression of most of the root regulatory network related genes was significantly increased under LP conditions, particularly in CR tissues , suggesting that LP activates several genes involved in alleviating nutrient and water stress. Similarly, we performed RNA-seq analysis  of different L. albus tissues grown under LP revealed a total of 2128 DEGs (differentially expressed genes). A total of 904 genes were found in P-deficient CR compared to P-sufficient R to understand plant acclimation responses. For example, members of the bHLH transcription factor family (14 genes) showed increased expression in P-deficient CR . We also found that L.albABCG37 (rename as L.albABCG29 in the present study) was also upregulated under LP in white lupin root . Our RNA-seq data showed that L.albABCG subfamily genes have higher expressions in root tissues than shoot under LP conditions (Additional file 12), which implies that these ABCGs play a significant role in plant adaptation to limited nutrient/P stress. These findings indicate that plants require ABCG proteins for maintaining root growth and nutrient uptake related processes. Notably, L.albABCG29 had higher CR expression under LP conditions than other plant tissues, which was further validated by overexpression in rice. Overexpression of L.albABCG29 in white lupin hairy root improved plant biomass under LP conditions (Fig. 5e), while in O. sativa its overexpression displayed increase rhizosheath formation via extending root length and root hair growth (Fig. 6c-d). Enhanced rhizosheath formation is directly associated with improved PUE under SD-P soil compared to WW-P (Fig. 6f-h). Our study indicates that L.albABCG29 promote root growth, increase rhizosheath formation, which is associated with improved phosphorus acquisition from the soil, and ultimately improved overall plant biomass. L. albus ABCG subfamily analysis provides comprehensive information on gene function, which may serve as a base for their role in generating P efficient crop that may emerge as a promising approach to meet plant nutritional demand under LP conditions.
ABC transporter gene families are more diverse and ubiquitous gene families among all living organisms. We identified L. albus ABCG transporter genes classified into six clusters. Variation in genes structural features supports evolutionary relationships based on their domain homology and phylogeny. Furthermore, gene duplication played a vital role in the expansion of L.albABCG subfamily in L. albus. RT-qPCR validation revealed that L.albABCG29 showed higher expression in CR under low P conditions, indicating its role in plant nutrients acclimation responses to starvation. Overexpression of L.albABCG29 improves P uptake in transgenic rice by improving root growth associated with increased rhizosheath formation. However, our data suggest that ABCGs show function redundancy when exposed to specific environmental stress. Collectively, this study provides a comprehensive role of L.albABCG29 in improving P uptake in plants. Functional characterization of essential ABCG subfamily transporters genes in response to nutrient and water stress will give helpful information for the generation of nutrient efficient and stress-resistant crop production.
Materials and methods
Identification of L.albABCG subfamily
To identify the ABCG gene subfamily in L. albus, the reported ABCG peptide sequences of A. thaliana  retrieved from the Phytozome (V12) database  were used as query sequences to perform BLASTP searches against white lupin genome (https://www.whitelupin.fr/) [52,53,54]. The deduced protein sequences were validated in HMMER  for the presence of ABC transporter domain (PF00005) , manually verified in Simple Modular Architecture Research Tool (SMART)  and NCBI Conserved Domain Tool (Batch CD-Search Tool) . Finally, all the non-redundant sequences and those lacking conserved domain or motif were removed, and the remaining sequences were used for further analysis. For each L. albus ABCG protein, in silico subcellular localization was predicted using WoLF PSORT .
ABC transporter protein sequences of G. max, [8, 50], L. angustifolius, and P. vulgaris were retrieved from Phytozome. L. albus protein sequences and A. thaliana, G. max, L. angustifolius, and P. vulgaris were aligned using a muscle program . The resultant multiple sequence alignment was used to construct an unrooted maximum-likelihood using IQ-TREE  and bootstrap adjusted to 1000 replicates. Finally, the tree was visualized and annotated in the MEGA program .
Gene structure and cis-regulatory element analysis
For ABCG genes intron-exon distribution, the genomic and corresponding nucleotide coding sequences were downloaded from the L. albus genome database  and submitted to the Gene Structure Display Server (GSDS) . Conserved motifs were predicted using MEME server  with the following parameters; a maximum number of motifs was 10, the optimum width was adjusted between 6 to 100 bp (base pairs). To predict cis-regulatory elements in L.albABCG genes promoter sequences, 2 kb upstream nucleotide sequences from the transcription activation site (ATG) were downloaded and submitted to PlantCARE .
Chromosomal distribution and gene duplication analysis
The white lupin genome is mapped across 25 chromosomes . The chromosomal distribution of L.albABCG subfamily genes was visualized using MapGene2chromosome (http://mg2c.iask.in/mg2cv2.1/). To examine gene duplication events, the ABCG transporters gene in white lupin and other plants, including A. thaliana, G. max, and L. angustifolius, MCScanX  was used. The gene duplication pairs were visualized in Circos . The whole-genome sequences of different plant species, including L. albus, L. angustifolius, P. vulgaris, G. max, and A. thaliana, were used to analyze the collinear relationship. The detected syntenic blocks were visualized using Dual Synteny Plotter . Furthermore, Ka and Ks substitution rates were calculated each syntenic pair using Ka/Ks calculator .
Expression analysis of ABCG subfamily in white lupin
White lupin seeds were grown under phosphorus-sufficient (CK, 0.25 mM KH2PO4) and low phosphorus (LP, 0 mM KH2PO4) hydroponic solution (1.75 mM K2SO4, 1.25 mM MgSO4, 0.5 mM Ca(NO3)2, 20 μM Fe(III)-EDTA, 25 μM H3BO3, 1.5 μM MnSO4, 1.5 μM ZnSO4, 0.5 μM CuSO4, 0.025 μM (NH4)6Mo7O24, and 0.25 mM KH2PO4). To determine the expression profile of ABCGs in L. albus tissues, different plant parts such as leaf (L), stem (S), root (R), root tip (RT), and cluster root (CR) were collected from 45 days hydroponically grown L. albus plants. The harvested tissues/parts were stored at − 80 °C for further analysis. The expressions of the L.albABCG subfamily in L. albus were analyzed from previously obtained RNA-seq data deposited to NCBI GEO (Gene expression omnibus) number GSE31132 . Genes expression was visualized in heatmap generated with R (version 3.8) using the RColorBrewer package.
Hairy root transformation of white lupin
The full-length coding sequence of L. albus L.albABCG29 (Lalb_Chr12g0200641, 4,362 bp) was amplified with forwarding primer 5`-BamHI-CGGGATCCATGGCTCAGCTGGTTGGT-3` and reverse primer 5`-SmaI-CCCCCGGGTTATCTTTTCTGGAAGTTGAGGTTT-3`. The amplicon was digested with BamHI and SmaI restriction enzymes and cloned into corresponding multiple cloning sites in modified pFGC5941 vector harboring 35S promoter and kanamycin and bar (glufosinate) gene for selection. Additionally, a 2 kb native promoter sequence of L.albABCG29 was amplified using forward 5`-PstI-AACTGCAGGAATGAGATGAAGAGCCTTCC-3` and reverse 5`-BamHI-CGGGATCCCTC AACACAATACTAAGGACC-3` primers. The amplified promoter region was then subsequently cloned into the pCAMBIA1301 vector by replacing its 35S promoter harboring the GUS gene and hygromycin as a stable marker. Both constructs were then transformed into Agrobacterium rhizogenic strain (K599) (Shanghai Weidi Biotechnology, China) using the freeze and thaw method as described by Liu et al. .
The surface-sterilized seeds of L. albus (cv. Amiga) were grown on ½ MS medium (pH 5.6) supplemented with 1% sucrose. After 3-days of germination, the elongated root was carefully excised with sterilized scalpels and sharp blades. The injured point was immediately dipped into Agrobacterium suspension solution for 30 mins. The infected seedling was then shifted to co-culture media (½ MS media augmented with 1% sucrose (w/v), 150 μmol AS, 0.8% agar, at pH 5.5) and kept in the dark chamber for 3-days. Explants were washed with sterilized water mixed with Carbenicillin 400 mg ml− 1 and dried on sterilized filter paper inside the laminar flow chamber. The transformed seedlings were shifted to ½ MS solid media and 25 mg ml− 1 kanamycin in capped glass jars and kept in a sterilized growth chamber. After 14 days, hairy roots were beginning to develop, and transgenic roots were confirmed by performing qPCR using bar gene forward 5`-ATATCCGAGCGCCTCGTG-3` and reverse 5`-CACGCAACGCCTACGACT-3` primers. After 20 days of transformation, hairy roots were transferred to CK (0.25 mM KH2PO4) or LP (0 mM KH2PO4) hydroponic solution. Finally, CRs and LRs were sampled after 4-weeks of treatment to proceed with further analysis.
GUS staining and transient expression
Transgenic white lupin hairy roots grown under CK and LP nutrient solutions were collected and dipped into GUS staining buffer augmented with 5-Bromo-4-chloro-3-indolyl β-D-glucuronide (X-Gluc) substrate (Jefferson, 1989) and kept at 37 °C for overnight. GUS staining was observed under Differential interference contrast (DIC) microscope, and the images were captured with a DXM1200 digital camera (Nikon).
Rice transformation and water/phosphorus treatments
The full-length cDNA sequence (4362 bp) of L.albABCG29 was amplified (using the same primers mentioned in section 2.5) and then cloned into pBWA(V)HS overexpression vector harboring 35S promoter and hygromycin gene as a selection marker. Finally, pBWA(V)HS::L.albABCG29 transformed into A. tumefaciens (EHA105, BioRun, Wuhan, China). Transgenic plants were screened by performing RT-qPCR analysis. For phenotypic characterization of L.albABCG29 transgenic homozygous lines (T3 lines) were generated in rice plants. The soil used to conduct this experiment was acquired from paddy rice field of Huayang, Jiangxi Province, China (115°09′32′′E, 28°32′29′′N). The physicochemical properties of collected soil were as follows: total K, 27.7 g kg− 1; total N, 1.75 g kg− 1; total P, 0.65 g kg− 1; organic C, 20.5 g kg− 1; exchangeable K, 92.0 mg kg− 1; and Olsen P, 42.6 mg kg− 1. The plant pots (designated as +P, with phosphorus) were supplemented with nutrient solution containing 1.75 mM K2SO4, 1.25 mM MgSO4, 0.5 mM Ca(NO3)2, 20 μM Fe(III)-EDTA, 25 μM H3BO3, 1.5 μM MnSO4, 1.5 μM ZnSO4, 0.5 μM CuSO4, 0.025 μM (NH4)6Mo7O24, and 0.25 mM KH2PO4. The -P (without P) soil was supplied with the same nutrient solution as stated above, but only with 0 mM KH2PO4 throughout the growth period. WW treatment represents well-watered pot (top 5 cm layer on pot), and SD represents the soil moisture (20%) throughout the study. Soil treatments were designed as WW + P (well-watered and with P), WW–P (well-watered and without P), SD + P (soil drying with P), and SD-P (soil drying without P). The soil was completely dried and stained through a 4 mm mesh to ensure the soil moisture percentage and homogeneity. A total of 1.8 kg of soil was added to each pot, WW treatments were performed every day to maintain a water level up to 5 cm of the pot, and in SD treatments, 100 ml water was added every third day. Water moisture and phosphorus status were maintained under all treatments throughout the experiment.
cDNA synthesis and quantitative real-time polymerase chain reaction
To investigate the expression variation of L.albABCG29 under phosphorus variability supplemented in liquid culture and soil, L. albus and O. sativa root samples were collected, respectively. The total RNA of selected plant tissues was extracted using Trizol reagent (Invitrogen) as described by Yockteng et al. . RNA integrity and quantity were confirmed by running on 2% gel electrophoresis and Nanodrop (ND-1000) spectrophotometer, respectively. The first strand of cDNA was synthesized from purified RNA samples using PrimeScriptTM RT Reagent Kit (TaKaRa, DALIAN) following the manufacturer’s instructions. Gene-specific primers for L.albABCG29 and other selected ABCG genes were designed in Prime Quest online database https://www.idtdna.com/PrimerQuest/Home/Index (Additional file 12). The RT-qPCR reaction was performed in a 20 μL reaction volume containing 1 μL of cDNA, 1 μL of each primer, 10 μL of SYBR Green mix (TAKARA), and ddH2O up to 20 μL under the following program: 95 °C for 3 min; 32 cycles at 95 °C for 15 s, 60 °C for 15 s and 72 °C for 30 s; 72 °C for 10 min (Bio-Rad, CFX Connect Real-Time PCR Detection System).
Plant biomass measurement
To determine the effect of each treatment on plant biomass in overexpression transgenic (OE-L.albABCG29) and wild type rice plants (Zhonghua 11, Zh-11) shoot and root samples (after 45 days of growth) were harvested and kept at 60 °C incubator for 3 days to dry completely. The dried plant tissues were weighed to calculate the total plant dry weight.
Rhizosheath collection and phosphorous measurements
Rice plants were carefully disassembled from pots as described in Aslam et al. . The plant roots were systematically shaken so that only soil adhering to roots was collected and designated as rhizosheath soil. Roots attached with rhizosheath soil were washed in a tray, and the soil was dried in an oven at 60 °C for 3–4 days. After drying, soil weight was calculated and designated as total dry rhizosheath weight. To carefully determine the phosphorus concentration in different plant tissues such as roots, stem, and leaf. All the collected samples were washed with ddH2O and dried at 60 °C incubator for 3 days. The dried samples were then ground into a fine powder and digested in H2O2 and H2SO4. Phosphorous concentration in digested tissues was measured spectrophotometrically at 700 nm using an ammonium molybdate method . PUE (phosphorus uptake efficiency) was determined using this formula presented by Irfan et al. .
Availability of data and materials
All data generated or analyzed during this study are included in this published article [and its supplementary information files]. However, RNAseq data used in this study is available at NCBI GEO (Gene expression omnibus) repository database under GSE31132 series number at this link (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE31132).
Feng Y, Sun Q, Zhang G, Wu T, Zhang X, Xu X, et al. Genome-wide identification and characterization of ABC transporters in nine Rosaceae species identifying MdABCG28 as a possible Cytokinin transporter linked to dwarfing. Int J Mol Sci. 2019;20(22):5783.
Verrier PJ, Bird D, Burla B, Dassa E, Forestier C, Geisler M, et al. Plant ABC proteins–a unified nomenclature and updated inventory. Trends Plant Sci. 2008;13(4):151–9.
Chen P, Li Y, Zhao L, Hou Z, Yan M, Hu B, et al. Genome-wide identification and expression profiling of ATP-binding cassette (ABC) transporter gene family in pineapple (Ananas comosus (L.) Merr.) reveal the role of AcABCG38 in pollen development. Front. Plant Sci. 2017;8:2150.
Garcia O, Bouige P, Forestier C, Dassa E. Inventory and comparative analysis of rice and Arabidopsis ATP-binding cassette (ABC) systems. J Mol Biol. 2004;343(1):249–65.
Lopez-Ortiz C, Dutta SK, Natarajan P, Pena-Garcia Y, Abburi V, Saminathan T, et al. Genome-wide identification and gene expression pattern of ABC transporter gene family in Capsicum spp. PLoS One. 2019;14(4):e0215901.
Mishra AK, Choi J, Rabbee MF, Baek K-H. In silico genome-wide analysis of the ATP-binding cassette transporter gene family in soybean (Glycine max L.) and their expression profiling. BioMed Res Int. 2019;2019:8150523.
Moon S, Jung KH. Genome-wide expression analysis of rice ABC transporter family across spatio-temporal samples and in response to abiotic stresses. J Plant Physiol. 2014;171(14):1276–88.
Ofori PA, Mizuno A, Suzuki M, Martinoia E, Reuscher S, Aoki K, et al. Genome-wide analysis of ATP binding cassette (ABC) transporters in tomato. PLoS One. 2018;13(7):e0200854.
Sanchez-Fernandez RO, Davies TE, Coleman JO, Rea PA. The Arabidopsis thaliana ABC protein superfamily, a complete inventory. J Biol Chem. 2001;276(32):30231–44.
Sugiyama A, Shitan N, Sato S, Nakamura Y, Tabata S, Yazaki K. Genome-wide analysis of ATP-binding cassette (ABC) proteins in a model legume plant, Lotus japonicus: comparison with Arabidopsis ABC protein family. DNA Res. 2006;13(5):205–28.
Yan L, Zhang J, Chen H, Luo H. Genome-wide analysis of ATP-binding cassette transporter provides insight to genes related to bioactive metabolite transportation in Salvia miltiorrhiza. BMC Genomics. 2021;22:315.
Jasinski M, Banasiak J, Radom M, Kalitkiewicz A, Figlerowicz M. Full-size ABC transporters from the ABCG subfamily in Medicago truncatula. Mol Plant-Microbe Interact. 2009;22(8):921–31.
Elejalde-Palmett C, Martinez San Segundo I, Garroum I, Charrier L, De Bellis D, Mucciolo A, Guerault A, Liu J, Zeisler-Diehl V, Aharoni A, et al. ABCG transporters export cutin precursors for the formation of the plant cuticle. Curr Biol 2021;31(10):2111–2123.
Zhu B, Li H, Xia X, Meng Y, Wang N, Li L, et al. ATP-binding cassette G transporters SGE1 and MtABCG13 control stigma Exsertion. Plant Physiol. 2020;184(1):223–35.
Kuromori T, Miyaji T, Yabuuchi H, Shimizu H, Sugimoto E, Kamiya A, et al. ABC transporter AtABCG25 is involved in Abscisic acid transport and responses. PNAS. 2010;107:2361–6.
Osakabe Y, Yamaguchi-Shinozaki K, Shinozaki K, Tran LP. ABA control of plant macroelement membrane transport systems in response to water deficit and high salinity. New Phytol. 2014;202(1):35–49.
Xu W, Zhang Q, Yuan W, Xu F, Aslam MM, Miao R, et al. The genome evolution and low-phosphorus adaptation in white lupin. Nat Commun. 2020;11(1):1–13.
Matsuda S, Takano S, Sato M, Furukawa K, Nagasawa H, Yoshikawa S, et al. Rice Stomatal closure requires guard cell plasma membrane ATP-binding cassette transporter RCN1/OsABCG5. Mol Plant. 2016;9(3):417–27.
Merilo E, Jalakas P, Kollist H, Brosche M. The role of ABA recycling and transporter proteins in rapid Stomatal responses to reduced air humidity, elevated CO2, and exogenous ABA. Mol Plant. 2015;8(4):657–9.
Panikashvili D, Shi JX, Bocobza S, Franke RB, Schreiber L, Aharoni A. The Arabidopsis DSO/ABCG11 transporter affects cutin metabolism in reproductive organs and suberin in roots. Mol Plant. 2010;3(3):563–75.
Ashraf MA, Akihiro T, Ito K, Kumagai S, Sugita R, Tanoi K, et al. ATP binding cassette proteins ABCG37 and ABCG33 function as potassium-independent cesium uptake carriers in Arabidopsis roots. Mol Plant. 2021;14(4):664–78.
Aslam MM, Karanja JK, Zhang Q, Lin H, Xia T, Akhtar K, et al. In vitro regeneration potential of White Lupin (Lupinus albus) from Cotyledonary nodes. Plants. 2020;9(3):318.
Uhde-Stone C. White lupin: a model system for understanding plant adaptation to low phosphorus availability. In: Legume Nitrogen Fixation in Soils with Low Phosphorus Availability: Springer Nature Switzerland AG; 2017. p. 243–80.
Haling RE, Brown LK, Bengough AG, Valentine TA, White PJ, Young IM, et al. Root hair length and rhizosheath mass depend on soil porosity, strength and water content in barley genotypes. Planta. 2014;239(3):643–51.
Aslam MM, Karanja J, Bello SK. Piriformospora indica colonization reprograms plants to improved P-uptake, enhanced crop performance, and biotic/abiotic stress tolerance. PMPP. 2019;106:232–7.
Aslam MM, Akhtar K, Karanja JK, Haider FU. Understanding the Adaptive Mechanisms of Plant in Low Phosphorous Soil. In: Plant Stress Physiology: IntechOpen London, United kingdom; 2020.
Haling RE, Brown LK, Bengough AG, Young IM, Hallett PD, White PJ, et al. Root hairs improve root penetration, root–soil contact, and phosphorus acquisition in soils of different strength. J Exp Bot. 2013;64(12):3711–21.
Brown L, George T, Thompson J, Wright G, Lyon J, Dupuy L, et al. What are the implications of variation in root hair length on tolerance to phosphorus deficiency in combination with water stress in barley (Hordeum vulgare). Ann Bot. 2012;110(2):319–28.
Zhang Y, Wang X, Xu F, Song T, Du H, Gui Y, et al. Combining irrigation scheme and phosphorous application levels for grain yield and their impacts on rhizosphere microbial communities of two rice varieties in a field trial. J Agric Food Chem. 2019;67(38):10577–86.
Zhang Y, Du H, Gui Y, Xu F, Liu J, Zhang J, et al. Moderate water stress in rice induces rhizosheath formation associated with abscisic acid and auxin responses. J Exp Bot. 2020;71(9):2740–51.
Dogbe W, Sogbedji J, Buah S. Site-specific nutrient management for lowland rice in the northern Savannah zones of Ghana. Curr Agric Res J. 2015;3(2):109–17.
Kone B, Fofana M, Sorho F, Diatta S, Ogunbayo A, Sie M, et al. Nutrient constraint of rainfed rice production in foot slope soil of Guinea Forest in CotedIvoire. Arch Agron. 2014;60(6):735–46.
Koné B, Amadji GL, Aliou S, Diatta S, Akakpo C, Science S. Nutrient constraint and yield potential of rice on upland soil in the south of the Dahoumey gap of West Africa. Arch Agron. 2011;57(7):763–74.
Arefin S, Bhuiyan MFH, Akther J, Prodhan SH, Hoque HJ. The dynamics of cis-regulatory elements in promoter regions of tomato sucrose transporter genes. Int J Veg Sci. 2020;27:1–20.
Cannon SB, Mitra A, Baumgarten A, Young ND, May G. The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol. 2004;4(1):10.
Song W-Y, Park J, Eisenach C, Maeshima M, Lee Y, Martinoia E. ABC transporters and heavy metals. In: Plant ABC transporters: Springer Nature Switzerland AG; 2014. p. 1–17.
Vasiliou V, Vasiliou K, Nebert DW. Human ATP-binding cassette (ABC) transporter family. Hum Genet. 2009;3(3):1–10.
Kang J, Park J, Choi H, Burla B, Kretzschmar T, Lee Y, et al. Plant ABC transporters. Am Soc Plant Biol. 2011;9:e0153.
Cho CH, Jang S, Choi BY, Hong D, Choi DS, Choi S, et al. Phylogenetic analysis of ABCG subfamily proteins in plants: functional clustering and coevolution with ABCGs of pathogens. Physiol Plant. 2019.
Yu J, Wang J, Lin W, Li S, Li H, Zhou J, et al. The genomes of Oryza sativa: a history of duplications. PLoS Biol. 2005;3(2):e38.
Gupta BB, Selter LL, Baranwal VK, Arora D, Mishra SK, Sirohi P, et al. Updated inventory, evolutionary and expression analyses of G (PDR) type ABC transporter genes of rice. Plant Physiol. 2019;142:429–39.
Kim DY, Bovet L, Kushnir S, Noh EW, Martinoia E, Lee Y. AtATM3 is involved in heavy metal resistance in Arabidopsis. Plant Physiol. 2006;140(3):922–32.
Ruzicka K, Strader LC, Bailly A, Yang H, Blakeslee J, Langowski L, et al. Arabidopsis PIS1 encodes the ABCG37 transporter of auxinic compounds including the auxin precursor indole-3-butyric acid. PNAS. 2010;107(23):10749–53.
Pighin JA, Zheng H, Balakshin LJ, Goodman IP, Western TL, Jetter R, et al. Plant cuticular lipid export requires an ABC transporter. Science. 2004;306(5696):702–4.
Ricci WA, Lu Z, Ji L, Marand AP, Ethridge CL, Murphy NG, et al. Widespread long-range cis-regulatory elements in the maize genome. Nat Plants. 2019;5(12):1237–49.
Kwak H, Lis JT. Control of transcriptional elongation. Annu Rev Genet. 2013;47:483–508.
Hufnagel B, Marques A, Soriano A, Marquès L, Divol F, Doumas P, Sallet E, Mancinotti D, Carrere S, Marande W. High-quality genome sequence of white lupin provides insight into soil exploration and seed quality. Nat Commun 2020;11(1):1–12.
Secco D, Shou H, Whelan J, Berkowitz O. RNA-seq analysis identifies an intricate regulatory network controlling cluster root development in white lupin. BMC Genomics. 2014;15(1):230.
Rourke JA, Yang SS, Miller SS, Bucciarelli B, Liu J, Rydeen A, et al. An RNA-Seq transcriptome analysis of orthophosphate-deficient white lupin reveals novel insights into phosphorus acclimation in plants. Plant Physiol. 2013;161(2):705–24.
Jasinski M, Ducos E, Martinoia E, Boutry M. The ATP-binding cassette transporters: structure, function, and gene family comparison between rice and Arabidopsis. Plant Physiol. 2003;131(3):1169–77.
Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, et al. Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res. 2012;4:D1178–86.
Hufnagel B, Marques A, Soriano A, Marques L, Divol F, Doumas P, et al. High-quality genome sequence of white lupin provides insight into soil exploration and seed quality. Nat Commun. 2020;11(1):492.
Vlasova A, Capella-Gutierrez S, Rendón-Anaya M, Hernandez OM, Minoche AE, Erb I, et al. Genome and transcriptome analysis of the Mesoamerican common bean and the role of gene duplications in establishing tissue and temporal specialization of genes. Genome Biol. 2016;17:32.
Hane JK, Ming Y, Kamphuis LG, Nelson MN, Garg G, Atkins CA, et al. A comprehensive draft genome sequence for lupin (Lupinus angustifolius), an emerging health food: insights into plant-microbe interactions and legume evolution. Plant Biotechnol J. 2017;15(3):318–30.
Eddy SR. Profile hidden Markov models. Bioinformatics. 1998;14(9):755–63.
Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, et al. Pfam: the protein families database. Nucleic Acids Res. 2014;42:D222–30.
Schultz J, Copley RR, Doerks T, Ponting CP, Bork P. SMART: a web-based tool for the study of genetically mobile domains. Nucleic Acids Res. 2000;28(1):231–4.
Lu S, Wang J, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, et al. CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Res. 2020;48(D1):D265–d268.
Horton P, Park KJ, Obayashi T, Fujita N, Harada H, Adams-Collier CJ, et al. WoLF PSORT: protein localization predictor. Nucleic Acids Res. 2007;35:W585–7.
Edgar RC. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics. 2004;5(1):113.
Trifinopoulos J, Nguyen LT, von Haeseler A, Minh BQ. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res. 2016;44(W1):W232–5.
Hall BG. Building phylogenetic trees from molecular data with MEGA. Mol Biol Evol. 2013;30(5):1229–35.
Guo A-Y, Zhu Q-H, Chen X, Luo JC. GSDS: a gene structure display server. Yi chuan=. Hereditas. 2007;29(8):1023–6.
Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009;37:W202–8.
Lescot M, Dehais P, Thijs G, Marchal K, Moreau Y, Peer Y, et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002;30(1):325–7.
Wang Y, Tang H, Debarry JD, Tan X, Li J, Wang X, et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012;40(7):e49.
Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020;13(8):1194–202.
Zhang Z, Li J, Zhao XQ, Wang J, Wong GK, Yu J. KaKs_Calculator: calculating Ka and Ks through model selection and model averaging. Genomics Proteomics Bioinformatics. 2006;4(4):259–63.
Xu W, Zhang Q, Yuan W, Xu F, Muhammad Aslam M, Miao R, et al. The genome evolution and low-phosphorus adaptation in white lupin. Nate Comm. 2020;11(1):1069.
Liu S, Su L, Liu S, Zeng X, Zheng D, Hong L, et al. Agrobacterium rhizogenes-mediated transformation of Arachis hypogaea: an efficient tool for functional study of genes. Biotechnol Biotechnol Equip. 2016;30(5):869–78.
Yockteng R, Almeida AM, Yee S, Andre T, Hill C, Specht CD. A method for extracting high-quality RNA from diverse plants for next-generation sequencing and gene expression analyses. APPS. 2013;1(12):1300070.
Aslam MM, Karanja JK, Yuan W, Zhang Q, Zhang J, Xu W. Phosphorus uptake is associated with the rhizosheath formation of mature cluster roots in white lupin under soil drying and phosphorus deficiency. Plant Physiol Biochem. 2021;166:531–9.
Xu W, Shi W, Jia L, Liang J, Zhang J. TFT6 and TFT7, two different members of tomato 14-3-3 gene family, play distinct roles in plant adaption to low phosphorus stress. Plant Cell Eviron. 2012;35(8):1393–406.
Irfan M, Aziz T, Maqsood MA, Bilal HM, Siddique KH, Xu M. Phosphorus (P) use efficiency in rice is linked to tissue-specific biomass and P allocation patterns. Sci Rep. 2020;10(1):1–14.
We are grateful for the financial support from the National Key R&D Program of China and the National Natural Science Foundation of China. We are thankful to Professor Feng Yan at the Justus Liebig University, Germany, for providing us with white lupin seeds.
This work was supported by the National Key R&D Program of China (2017YFE0118100) and the National Natural Science Foundation of China (31761130073, 31422047, 31872169, 31770123 and 31901428).
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Chromosomal distribution of White lupin ABCG transporter subfamily. Names of chromosomes are mentioned on left side, genes linked with purple line indicates tandem duplication, and the chromosome size scale is in Mb. Different colours inside the chromosome bar represent genes density of each chromosome.
Detailed information of L. albus ABCG subfamily genes
Conserved protein sequences of ten motifs identifed in ABCG subfamily of white lupin. Motif scan and sequence logos were generated in linux based MEME program.
Whole genome duplication of ABCG subfamily and Ka/Ks ratios in the L. albus genome
Duplication gene pairs identified with Synteny analysis in L. albus ATPase-binding cassette transporters (ABC) gene family. a duplicated gene pairs in L. albus vs A. thaliana b duplicated gene pairs in L. albus vs G. max. c duplicated gene pairs in L. albus vs L. angustifolius. Different color lines exhibited paralogous pairs of ABC transporter genes and their subsequent location on chromosomes. The shaded gray background shows the synteny pairs of the whole-genome and respective chromosome numbers are labeled outside the circle. Pink blocks represent L. albus chromosomes, green blocks represent A. thaliana chromosomes, brown represent G.max, and blue blocks represent L. angustifolius chromosomes.
Duplication of ABCG subfamily and Ka/Ks ratios of L. albus and A. thaliana
ABCG subfamily whole genome duplication and Ka/Ks ratios of L. albus and G.max
ABCG subfamily whole genome duplication and Ka/Ks ratios of L. albus and L. angustifolius
Collinearity relationship of the ABCG subfamily members of L. albus compared with A. thaliana, G. max, L. angustifolius, and P. vulgaris. Background shaded gray lines indicate the collinearity blocks of L. albus genome with targeted plant genome, while red lines indicate the syntenic pairs of only ABCG transporters gene subfamily between two genomes.
Raw data for tissue specific expression of LaABCG subfamily under phosphorus sufficient (CK) and low phosphorus (LP) condition
Expression pattern of ABCG subfamily members among different plant tissues under control (CK: phosphorus sufficient) and low phosphorus (LP) conditions. Fragment per kilobase of exon model per million mapped read (FPKM) values were used to visualize the gene expression on the heat map using R statistical online tool using RColorBrewer package. All the values are log2 transformed. LP-L/CK-L; leaf, CK-S/LP-S; stem, LP-RT; root tip, LP-CR; cluster root, and CK-R; root.
Primers used for qPCR
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Aslam, M.M., Waseem, M., Zhang, Q. et al. Identification of ABC transporter G subfamily in white lupin and functional characterization of L.albABGC29 in phosphorus use. BMC Genomics 22, 723 (2021). https://doi.org/10.1186/s12864-021-08015-0
- ABCG subfamily
- White lupin