Phenotypic analysis of the survival rate of 400 common bean accessions
The seedling SR can reflect plant drought resistance mechanisms and cellular responses. It is less affected by environmental fluctuation, which helps to identify the underlying genetic determinants (Mao et a., 2015). Therefore, the drought resistance of 400 common bean accessions was assayed by calculating the SR after severe drought stress at the seedling stage. A large range of variation was detected, with the coefficient of variation (CV) varying from 40.90% for SR3 to 56.22% for SR2 (Table S3). The SR at different time points ranged from 0.00 to 100.00%, and the means were 50.81, 52.04, and 58.22, respectively (Table S3). All SR data showed a uniform distribution (Fig. S2). These results indicated that the drought resistance of the 400 common bean accessions was different at the seedling stage. Correlation analysis showed that pairwise positive correlations (p < 0.01) were detected for SR1, SR2 and SR3 (Table S4), with correlation coefficients of 0.72, 0.56 and 0.54, respectively.
Association analysis of drought resistance at the seedling stage
To identify drought resistance-related association loci, a reported genotypic dataset consisting of 3832340 SNPs was used for conducting GWAS for the SR. According to the definition described in the methods, a total of 12 associated loci containing 89 SNPs were identified (Fig. 1, Table S5), and 12 leak SNPs were detected, which were distributed on Chr. 2, 3, 5, 6, 7, 10, and 11 (Table S6). For SR1, 4 associated loci were detected, which were distributed on Chr. 6, 10, and 11 (Table S6). For SR2, 5 associated loci were detected, which were distributed on Chr. 5, 7, and 11 (Table S6). For SR3, 5 associated loci were detected, which were distributed on Chr. 2, 3, and 11 (Table S6). Among these associated loci, four loci collocated with QTLs previously reported to be related to drought resistance (Villordo-Pineda et al., 2015; Wu et al., 2021, Table S6). It is worth noting that Locus_10, located on Chr. 11, was simultaneously associated with SR1, SR2 and SR3 and contained a significant SNP with the lowest P value of 4.36E-08. These results indicated that these associated loci were related to drought resistance.
Prediction of candidate genes associated with drought resistance
Among all physical positions of the 12 associated loci, 199 genes were annotated (Table S7), including protein kinase genes, RING/U-box superfamily protein genes, MYB domain genes, and WRKY family transcription factors, which may be related to drought response (Fang et al., 2015; Mao et al., 2015; Li et al., 2019; Li et al., 2021). Combining the expression level changes after drought stress treatment (Wu et al., 2014; Pereira et al., 2020), 57 genes among these 199 genes were responsive to drought stress, of which 22 were upregulated and 35 were downregulated (Table S7). Phvul.003G124000, located at Locus_3, is associated with SR3 and encodes a WRKY family transcription factor, and the expression of the gene was downregulated in common bean after drought treatment with a Log2(FC) = -2.76 (Table S7, Wu et al., 2014). We subsequently searched the NCBI database to determine the functions of these genes in A. thaliana. Two genes, Phvul.002G278200 and Phvul.002G279300, located at Locus_1 and associated with SR3 (Fig. 1, Table S7) encode a protein kinase superfamily protein and calcium-dependent protein kinase 2, whose homologous genes in Arabidopsis are AtSNRK2.4 and AtCDPK2, respectively. AtSNRK2.4 is involved in root development and the response to osmotic stress (McLoughlin et al., 2012; Fujii et al., 2011), and AtCDPK2 is involved in the ABA-activated signalling pathway (Zhu et al., 2007). A total of six genes were annotated in Locus_2; among these genes, Phvul.003G067700 encodes a homeobox-leucine zipper protein, whose homologous gene in Arabidopsis (AtHB13) is involved in drought resistance (Cabello et al., 2012), and Phvul.003G067800 encodes MYB domain protein 88, whose homologous gene in Arabidopsis (AtMYB88) is also involved in drought resistance (Xie et al., 2010) (Fig. 1, Table S7). Another gene (Phvul.007G220900) located at Loucs_8 (Fig. 1, Table S7) encodes a RING/U-box superfamily protein whose homologous gene in Arabidopsis (LOG2) is involved in drought resistance (Kim et al., 2013). There were 39 candidate genes screened from all annotated genes (Table S8) according to the following rules: (1) it was a stress-responsive transcription factor, such as an NAC, MYB, or bHLH transcription factor; (2) its homologous genes in A. thaliana were identified in a previous study as being involved in the drought and ABA response and ABA signal transduction; and (3) its expression was upregulated or downregulated after drought treatment with |Log2(FC)| ≥ 2. These results further showed that these significant SNPs were related to drought resistance, and potential genes associated with drought resistance were identified.
A major locus on Chromosome No.11
A stable locus (Locus_10) was detected at Chr. 11 and was simultaneously associated with SR1, SR2 and SR3 (Fig. 1). There are 41 genes contained at Locus_10 (Fig. 2a-c). Combining the expression level changes after drought stress treatment (Wu et al., 2014; Pereira et al., 2020), among these 41 genes, 4 genes were upregulated and 9 genes were downregulated (Table S7, Fig. 2d). Phvul.011G024800 encodes expansin-like A2, whose homologous gene in Arabidopsis (AtEXLA2) responds to ABA (Abuqamar et al., 2013), and the expression of the gene was downregulated in common bean after drought treatment (Table S7, Fig. 2b, d). Phvul.011G025500 encodes rho guanyl-nucleotide exchange factor 1, whose homologous gene in Arabidopsis (AtROPGEF1) was involved in the ABA-activated signalling pathway and lateral root development (Li et al., 2016; Li et al., 2018), and the expression of the gene was downregulated in common bean after drought treatment (Table S7, Fig. 2b, d). Phvul.011G025200 encodes a late embryogenesis abundant protein (LEA), Phvul.011G025700 and Phvul.011G025800 encode plasma membrane intrinsic proteins (XIP1;1, XIP1;2), and Phvul.011G026700 encodes a proline-rich protein (PRP2) (Table S7, Fig. 2b). All these genes mentioned above were generally considered to be associated with drought resistance (Fang et al., 2015). Combining the expression level changes after drought stress treatment (Wu et al., 2014; Pereira et al., 2020), Phvul.011G025200 and Phvul.011G026700 were downregulated with Log2(FC) of -3.07 and -5.99, respectively, in common bean after drought treatment (Table S7). Phvul.011G025800 was upregulated with a high Log2(FC) of 8.2 and was assumed to be the most promising candidate gene. Phvul.011G025800 was located 74 kb upstream of the lead SNP (Chr11__2209628, P = 4.36E-08), which was annotated as aquaporin (named PvXIP1;2). A phylogenetic tree based on XIPs from different species was constructed (Fig. S3a), indicated that the XIPs from legumes clustered together. The results of sequence alignment (Fig. S3b) showed that two highly conserved ‘NPA signature motifs’ were found in PvXIP1;2, but the first NPA motif was replaced with SPV, similar to that in soybean. The four residues forming the ar/R selectivity filter of PvXIP1;2 are valine from TMH2, isoleucine from TMH5, and alanine and arginine from R3 and R4, which are identical to those in soybean (Bienert et al., 2011, Fig. S3b). The results demonstrated that PvXIP1;2 is an aquaporin and belongs to the X-intrinsic protein (XIP) subfamily, which is absent in Arabidopsis (Reuscher et al., 2013; Ariani et al., 2015). Aquaporins are involved in almost every physiological process in plants and in the response to environmental stresses (Maurel et al., 2008; Li et al., 2014). Most aquaporins have been identified to be involved in resistance to drought, such as MaPIP1;1 (Xu et al., 2014), ScPIP1;1 (Wang et al., 2019), RWC3 (Lian et al., 2004) and TaAQP7 (Zhou et al., 2012). However, the function of PvXIP1;2 remains to be elucidated. Therefore, Phvul.011G025800 was assumed to be a promising candidate gene for further functional investigation.
Expression patterns of PvXIP1;2
To investigate the expression of PvXIP1;2 in different organs of common bean, total RNA was extracted from roots, leaves, trifoliates and stems for quantitative real-time RT-qPCR analysis. The results showed that PvXIP1;2 was expressed in various tissues of common bean (Fig. 3a).
The expression file data indicated that PvXIP1;2 transcript levels were strongly induced by drought stress (Wu et al., 2014; Pereira et al., 2020). To verify the expression data, we performed quantitative real-time RT-qPCR using RNA isolated from drought-treated common bean, and the results confirmed the expression data (Fig. 4a). Furthermore, various stress treatments were applied to common bean with trifoliates, including salt, mannitol and ABA treatments, to determine the transcriptional response of PvXIP1;2 to abiotic stress. The results demonstrated that the expression of PvXIP1;2 was induced in leaves and roots after salinity stress, simulated drought and ABA treatments (Fig. 4b-c). These results suggested that the PvXIP1;2 transcript level was affected by various stress treatments.
To determine the subcellular localization of the PvXIP1;2 protein, its ORF was introduced into the pCAMBIA1300-GFP vector upstream of the GFP gene to create a PvXIP1;2-GFP fusion construct. Then, we transformed the PvXIP1;2-GFP fusion construct into Nicotiana benthamiana by injection. A strong fluorescent signal derived from GFP alone was observed in the cytoplasm and nuclei (Fig. 3b), whereas transformed cells carrying PvXIP1;2-GFP showed a strong green fluorescence signal in the plasma membrane, indicating the plasma membrane localization of PvXIP1;2.
Overexpression of PvXIP1;2 increases the drought resistance of Arabidopsis
To further understand the function of PvXIP1;2, we constructed a 35S::PvXIP1;2 vector. After floral-dip transformation of Arabidopsis, three homozygous lines (L6, L8 and L10) were selected from the T4 generation for further functional investigation (Fig. 5a). To investigate the drought resistance of transgenic Arabidopsis overexpressing PvXIP1;2, WT and transgenic Arabidopsis were grown for 3 weeks in soils before water was withheld for 13 d and then rewatered. Most transgenic plants remained turgid, and their leaves remained green, whereas the WT plants wilted, and their leaves became yellow (Fig. 5b). When data from three different experiments were analysed, approximately 80% of the transgenic plants survived stress after rewatering, which was approximately four times higher than that of WT plants (Fig. 5c). Osmotic adjustment and antioxidant defence systems are the main pathways of drought tolerance-associated mechanisms (Fang et al., 2015). Therefore, the MDA and proline contents and IL were measured in WT and transgenic plants under drought stress and well-watered environments to identify the function of PvXIP1;2 in these physiological processes. MDA, which causes oxidative injury to the cytomembrane, accumulates in leaves when plants experience stress. No evident difference was observed in the MDA content of WT and transgenic plants under well-watered conditions, whereas a significantly lower MDA content was observed in transgenic plants than in the WT under drought treatment (Fig. 5d). Consistent with these results, a lower IL was observed in transgenic plants than in the WT under drought treatment, whereas there was no significant difference in WT and transgenic plants under well-watered conditions (Fig. 5e), suggesting that the more severe membrane damage after water was withheld in the WT could at least partly be attributed to the inability of these plants to efficiently eliminate MDA due to the disruption of antioxidant defence systems under severe drought stress. In addition, proline, which is an essential osmotic substance, accumulated more in transgenic plants than in WT plants under drought stress conditions, while no significant difference was observed in WT and transgenic plants (Fig. 5f). These results indicated that heterologously overexpressing PvXIP1;2 in Arabidopsis improved resistance to drought stress due to reduced lipid peroxidation and membrane injury and increased accumulation of osmotic substances.
35S::PvXIP1;2 transgenic Arabidopsis is resistant to osmotic, salt and ABA stresses
PvXIP1;2 is a mannitol-, salt- and ABA-induced gene (Fig. 4b-c), indicating that PvXIP1;2 might also be necessary for plant responses to osmotic, salt and ABA stresses. To further identify the results, four-day-old WT and transgenic seedlings were transformed on MS medium containing different concentrations of mannitol, NaCl and ABA for 10 days. There was no significant difference in WT and transgenic plants (Fig. 6a) on MS medium without treatments. The addition of mannitol, NaCl and ABA significantly inhibited root development, and the degree of inhibition increased with increasing concentrations of mannitol, NaCl and ABA (Fig. 6b-f). The root development of WT plants was more inhibited than that of transgenic plants in all treatments except 150 mM NaCl (Fig. 6b-f). These results demonstrated that the overexpression of PvXIP1;2 improved the resistance of Arabidopsis to osmotic, salt and ABA stresses.
PvXIP1;2 improves stress resistance in transgenic common bean hairy roots
An Agrobacterium rhizogenes-mediated hairy root transformation system was used to investigate the function of PvXIP1;2 in common bean under osmotic stress conditions. The K599 strain harbouring the PvXIP1;2-overexpression (PvXIP1;2-OE) and PvXIP1;2-RNA interference (PvXIP1;2-RNAi) constructs was injected into the hypocotyls of common bean seedlings for the generation of transgenic hairy roots. The transgenic hairy roots grew out of the injection sites approximately 10 days later. PvXIP1;2 expression in PvXIP1;2-OE roots was 4 times higher than that in control hairy roots, whereas that in PvXIP1;2-RNAi roots was only half of that in the control (Fig. 7a). The original roots were removed before the plants with hairy roots were subjected to 200 mM mannitol to simulate osmotic stress. After keeping in mannitol for 24 h, the leaves of the plants with PvXIP1;2-OE roots performed better than the control leaves; however, the leaves from the plants with PvXIP1;2-RNAi roots were more wilted than the control leaves (Fig. 7b-c). The results of the RWC showed that seedlings with PvXIP1;2-OE roots had a higher water content than the control seedlings, whereas seedlings with PvXIP1;2-RNAi roots had a lower water content (Fig. 7d). After keeping in mannitol for 72 h, the relative hairy root growth rates were measured and compared. The results demonstrated that the relative growth rate of PvXIP1;2-OE hairy roots was significantly higher, whereas this parameter in PvXIP1;2-RNAi roots was significantly lower than that of control roots (Fig. 7e-f). These results indicated that PvXIP1;2 confers resistance to drought in common bean and further demonstrated that these loci detected in this study were related to drought resistance.