Gene structure, motif composition, and promoter element analysis
All the 117 BnaCPA members were divided into three subfamilies, including 77 BnaCHXs, 20 BnaKEAs, and 20 BnaNHXs (Fig. 1A). Most BnaCPA members in the same subfamily have similar motif compositions, suggesting that these proteins might have similar functions (Fig. 1B). The motif 10, motif 14, motif 15, and motif 20 were found in all three subfamilies, which might be used as conserved domains for the identification of CPA-type proteins in B. napus.
The putative promoter sequences (2000 bp upstream region of transcription initiation site) of BnaCPAs were submitted to PlantCARE to search for the prediction of cis-acting regulatory elements. A total of 45 cis-acting elements were identified: except common elements, such as the CAAT box and TATA box, 20 other important cis-acting elements related to plant growth, development and various stresses were explored (Fig. 1C). The results showed that the cis-acting elements of BnaCPAs were diverse, suggesting that the expression of BnaCPAs might be regulated in a variety of ways. In terms of phytohormone-involving regulation, there were more responsive elements of abscisic acid, auxin, gibberellin acid and methyl jasmonate in the promoter regions. In terms of defense responses, there were anaerobic induction, defense and stress, hypothermia and trauma response elements. In addition, zein metabolic regulatory elements existed in the BnaCPA promoter elements.
As it shows in the three subfamilies of BnaCPAs, the structure of the same subfamily was similar, while the lengths of the exon and intron of the gene was significantly different (Fig. 1D). The genic structure of BnaNHXs and BanKEAs were relatively complex, while it was relatively simple the BnaCHX subfamily. Specifically, the number of exons in the CHX subfamily ranged from 2 to 9, while the number of exons in the KEA subfamily ranged from 9 to 21, and the number of exons in the NHX subfamily ranged from 10 to 23.
Phylogenetic analysis of CPAs
In order to further study the subfamily classification of BnaCPAs and their association with other species, we used query sequences (PF00999) to identify the CPA homologs in B. rapa and B. oleracea (Tables S2, S3). The phylogenetic tree showed that all the CPA members of the four Brassica species, including A. thaliana, B. rapa, B. oleracea, and B. napus were obviously divided into three subfamilies: NHXs, KEAs, and CHXs (Fig. 2). We found that the number of CPAs in B. napus, which was most in the identified several species was relatively smaller than the sum of CPAs in B. rapa and B. oleracea (Table 1), which might imply some CPA homologs were lost during the evolution and formation of B. napus.
Chromosomal localization analysis
To have a knowledge of the genomic distribution of the BnaCPA family, we mapped all the identified 117 members onto the rapeseed chromosomes. Members of the BnaCPAs were unevenly mapped to 19 chromosomes (A subgenome: ChrA01-A10; C subgenome: ChrC01-C09) (Fig. S2). Chromosome C09 and chromosome C04, having the most BnaCAP, contained 10 and 11 BnaCPA members, respectively. However, chromosome A05 and chromosome C01 contained the fewest BnaCPAs (only two). The patterns of gene expansion consist of whole-genome duplication, segmental duplication, and tandem duplication (Zhang et al., 2021), which contribute to the diversity of biological genes. To investigate the expansion patterns of BnaCPAs, we used the multicollinearity scanning toolkit (MCScanX) to study the types of BnaCPA expansion. According to the physical location information, each BnaCPA was mapped onto the chromosomes of B. napus, and its evolutionary relationship was deduced accordingly (Fig. 3). Gene duplication, an indispensable mechanism, can expand new genes that share similar or different function, therefore, we analyzed the duplication events that occurred in the BnaCPA family. A total of 149 CPA pairs were detected (Fig. 3, Table S4), with 3 being tandem duplications, and 146 as segmental duplication events. Ka/Ks, the nonsynonymous to synonymous substitution ratio, determines the selection pressure of duplicated genes. Thus, the protein and CDS sequences of each duplicated gene pair were compared, and Ka/Ks ratios and divergence times were calculated. According to the results, the Ka/Ks ratios of all BnaCPA pairs are < 1.0 (Table S4), indicating that the evolution of BnaCPAs was accompanied by intense purifying selection.
Gene duplication and synteny analysis of BanCPA s
To better understand the origin of the BnaCPA family, we detected the colinearity of B. napus with other species at a genome-wide level. To further infer the origin and evolutionary history of CPAs, the synthetic regions between B. napus and Arabidopsis were analyzed and compared. According to the synthetic map, there were 128 pairs of collinear genes in B. napus and Arabidopsis, containing 26 NHXs, 14 KEAs, and 88 CHXs (Fig. S3A, Table S3). Among the two synthetic orthologous gene pairs, one B. napus gene corresponded to multiple Arabidopsis genes, such as BnaA01.CHX17-AtCHX16/AtCHX17, BnaA03.CHX9-AtCHX4/AtCHX9. Accordingly, there also existed syntenic orthologous gene pairs with one Arabidopsis gene corresponding to multiple B. napus genes, for instance, AtNHX8-BnaA06.NHX8/BnaA09.NHX7/BnaC05.NHX8/BnaC09.NHX7, AtKEA1- BnaA10.KEA1a/BnaC05.KEA1a/BnaC08.KEA1. There are also some overlaps between the two events above, the gene pairs of two or more Arabidopsis genes corresponding to the same two gene pairs were also found, such as AtNHX8/AtNHX7-BnaA03.CHX13/BnaA04.CHX13/BnaA08.CHX14/BnaC03.CHX13/BnaC04.CHX13/BnaC08.CHX14, AtCHX23/AtCHX21- BnaA10.CHX23/BnaC05.CHX23. A series of synteny events indicated that many CPAs appeared before the divergence of the Arabidopsis and Brassica napus lineages. In addition, we also detected 160, and 152 gene pairs between B. napus and B. rapa/B. oleracea, respectively (Fig. S3B-C, Table S4-5).
Expression analysis under multiple nutrient stress conditions
According to previous studies, we know that CPAs are greatly affected by salt stress in wheat, radish, and some other plant species (Wang et al., 2020). In this study, we studied the responsive patterns of CPAs in B. napus under salt stress. In the roots, the expression of BnaA08.CHX7, BnaC03.CHX17, BnaC07.CHX17, BnaA05.NHX4, and BnaC05.NHX4 were significantly reduced under salt stress compared with the NaCl-free condition; however, the expression levels of BnaA04.CHX20, BnaC03.CHX10, BnaA05.NHX2, BnaC09.NHX3b, BnaC05.NHX2, and BnaC06.NHX6 were significantly increased. In the shoots, the expression abundances of BnaA04.CHX20 and BnaC04.CHX20b were significantly decreased under salt stress; however, the expression of BnaA01.CHX19, BnaA04.CHX18, BnaC06.CHX20, BnaC03.CHX17, BnaA05.NHX4, BnaA05.NHX2, BnaA09.NHX1, BnaA09.NHX7, BnaC09.NHX7, BnaC09.NHX1, BnaC02.NHX1, BnaC05.NHX2, BnaC06.NHX6, and BnaC06.NHX6 were significantly upregulated under salt stress compared with the control condition (Fig. 5C).
To full understand the transcriptional responses of BnaCPAs to multiple nutrient stresses and their potential biological function, we also identified the expression profiling of BnaCPAs under several other nutrient stresses, including K deficiency, cadmium toxicity, phosphate starvation, and different forms of nitrogen nutrients. Potassium plays an important role in plant growth and development, and it can promote plant stem robust, improve fruit quality, enhance plant cold resistance, and improve fruit sugar and vitamin C content (Templalexis et al., 2022). In this study, we also studied the effect of potassium deficiency on expression of CPAs in B. napus. Under low potassium condition, in the roots, all of BnaC06.CHX20, BnaC02.NHX1a, BnaC05.NHX4, and BnaA09.KEA3 were significantly down-regulated, while both BnaA01.CHX19 and BnaC07.CHX17 were significantly up-regulated. In the shoots, BnaC07.CHX17 were significantly down-regulated; however, all the expression levels of BnaA01.CHX19, BnaA04.CHX18, BnaA07.NHX6, BnaA09.NHX7b, BnaC05.NHX4, and BnaC09.NHX7 were significantly enhanced (Fig. 5B).
Cadmium (Cd) as a heavy metal with high biotoxicity can cause physiological and metabolic disorders of plants, normal growth disorders, development disorders and agricultural product quality decline. In this study, we found that under cadmium stress, all of BnaC09.CHX17, BnaA09.CHX17, BnaC02.CHX20, BnaC04.CHX17, BnaA08.NHX1, BnaA09.NHX1, BnaC05.NHX3, BnaC02.KEA1, and BnaA05.KEA6 were down-regulated in the roots, all the expression levels of BnaA09.CHX18, BnaC07.NHX2, BnaA02.NHX1, and BnaA03.NHX7 were significantly increased. In the shoots, all of BnaA09.CHX17, BnaC04.CHX17, and BnaC05.NHX3 were down-regulated, while all of BnaA01.CHX19, BnaA09.CHX18, BnaA09.CHX17, BnaC02.CHX20, and BnaA03.NHX7 were significantly up-regulated (Fig. 5A).
Phosphate can promote flower bud differentiation, early flowering and fruiting, promote seedling root growth, and improve fruit quality. When phosphorus is lacking, the buds and roots grow slowly, and the plant is stunted, with dark green, dull leaves and purple underside (Yi et al., 2021). In this study, we found that in the roots, BnaA03.CHX9 were down-regulated; all the expression of BnaC01.CHX17, BnaC04.CHX20a, BnaA08.CHX17, BnaC06.CHX20, BnaA05.NHX2, and BnaC05.NHX2 was significantly increased under phosphate starvation condition. However, in the shoots, all the expression of BnaA03.CHX9, BnaA06.NHX8, BnaC09.KEA3, and BnaA09.KEA3 were reduced, while all the expression levels of BnaA01.CHX17, BnaC01.CHX17, BnaC04.CHX20a, BnaA08.CHX17, BnaC06.CHX20, BnaA05.NHX2, BnaA09.NHX1, BnaC02.NHX1, BnaC05.NHX2, and BnaC09.NHX1 were significantly increased (Fig. 5E).
Nitrogen is a main component of proteins, which plays an important role in the growth of stem and leaf, and the development of fruit, then the nutrient element most closely related to yield. In nature, the sources of N absorbed by plants are mainly nitrate nitrogen or ammonia nitrogen (Abassi and Ki, 2022). This study studied the effects of two different forms of nitrogen nutrients (NH4+ and NO3−) on the expression of CPAs in B. napus. In the shoots, compared with the treatment of NH4+ as the sole nitrogen source, nitrate significantly repressed the expression of BnaC03.CHX28, BnaC01.CHX13, BnaA04.CHX16, BnaC06.CHX19, and BnaC09.NHX3; however, all the expression of BnaC05.CHX27, BnaA10.CHX18, BnaC09.KEA1 was significantly increased. In the roots, all of BnaA03.CHX27, BnaC05.CHX27, and BnaA10.CHX18 were significantly down-regulated, while all the expression levels of BnaA04.CHX16, BnaC09.NHX3a, BnaC03.NHX6, BnaA05.KEA1, and BnaC09.KEA1 were significantly increased (Fig. 5D).
Preliminary verification of the function of BnaA05.NHX2 gene
From the analysis of the transcriptomic data above, we proposed that BnaA5.NHX2 might play a pivotal role in the resistance against salt stress and other nutrient stresses. Previous studies have shown that the NHX family plays an important regulatory role in salt stress in numerous plant species (Huang et al., 2022). In this study, two selected from several positive lines of rapeseed plants overexpressing BnaA05.NHX2 were constructed to study the roles of BnaA05.NHX2 in regulating salt tolerance (Fig. 6A). The phenotypic and physiological differences between the identified positive plants and wild-type rapeseed Westar were identified after 10 days of salt treatment. We found that under salt treatment, compared with the overexpressed BnaA05.NHX2 plants, the shoots and roots of wild-type plants were smaller, and the leaves were wilted and yellow. Obviously, the growth of the overexpressed lines was significantly better than that of the wild type under salt treatment (Fig. 6B).
Subsequently, we determined the phenotypic characteristics of the wild-type and overexpressed lines under salt treatment. We found that the biomasses in the shoots and roots of the overexpressed lines were significantly higher than those of the wild-type under salt treatment, whereas there was no significant difference in water content between the wild type and transgenic plants (Fig. 6C-E). We also found that the leaf specific weight and leaf areas of the overexpressed lines were significantly larger than those of the wild type (Fig. 6F, Table S9). The length of primary roots in the BnaA05.NHX2-overexpressing rapeseed plants under salt treatment was not significantly different from that of wild type. The total root length, root surface area, root volume, and root tip number of the overexpressed lines were significantly higher than those of the wild type. However, the root diameter of the overexpressed lines was significantly smaller than that of the wild type (Fig. 6G-I, Table S9). Overall, the overexpressed lines had stronger salt tolerance under salt treatment.
Previous studies have shown that the photosynthetic system of plants will be damaged to a certain extent under salt stress (Ahmad et al., 2022). Through our observation, we found that the leaves of wild-type showed etiolation under salt treatment (Fig. 6B). Therefore, we measured the differences in the photosynthetic pigments between wild-type and transgenic plants under salt treatment. We found that the contents of chlorophyll a, chlorophyll b, and carotene in transgenic lines were higher than those of wild type under salt treatment (Fig. 7A-C, Table S9). Subsequently, we also measured other photosynthetic parameters, including net photosynthetic rate, intercellular CO2 concentration, transpiration rate, stomatal conductance. We found that the photosynthetic rate and transpiration rate of the plants overexpressing BnaA05.NHX2 were significantly higher than those of the wild type, while the intercellular CO2 concentration was significantly lower than that of the wild type, and the stomatal conductance was similar to that of the wild type (Fig. 7D-G, Table S9).
Various physiological substances also play important roles in salt stress in plants. Previous studies have shown that soluble sugars can improve the tolerance of crops and play an important role in the mechanism of plant salt stress resistance (Saddhe et al., 2021). Malondialdehyde is one of the most important products of membrane lipid peroxidation, which can indirectly reflect the damage degree of membrane system and stress resistance of plants (Zhan et al., 2019). Proline is an important molecule in regulating plant resilience, which can regulate osmotic pressure, maintain protein and membrane stability and remove reactive oxygen species (Gupta et al., 2022). Therefore, we measured soluble sugars, malondialdehyde and proline in the wild-type and overexpressed plants under salt stress. We found that the content of proline and soluble sugar in the overexpressed lines was significantly higher than that of the wild type under salt treatment, while the content of malondialdehyde was significantly lower than that of the wild type (Fig. 8A-C, Table S9).
Previous studies have shown that AtNHX2 is localized in the tonoplast, responsible for sequestrating Na+ from the cytoplasm into the vacuole, reducing the concentrations of cytoplasmic Na+, alleviating the toxicity of Na+ to cell metabolism (Barragan et al., 2012; Bassil et al., 2011). Therefore, we measured the ion content in the shoots and roots of the rapeseed plants overexpressing BnaA05.NHX2 and wild-type, and we found that the Na+ content of wild-type rapeseed and overexpressed rape was consistent at the tissue level (Fig. 8D, Table S9).
To further explore the biological function of BnaA05.NHX2, we investigated its subcellular localization and its physiological effect on the vacuolar Na+ compartmentation. Subcellular localization analysis showed that BnaA05.NHX2 was localized on the tonoplast (Fig. 9A), which indicated its potential roles in sequestrating Na+ into vacuoles. Observation of leaf cell ultrastructure revealed the wild type presented severe degradation of chloroplasts compared with the two overexpression lines (Fig. 9B). Further, combing TEM and X-ray energy spectrum analysis showed that the transgenic lines had significantly higher vacuolar Na+ abundances (Fig. 9C-E).
In order to further verify the expression efficiency of overexpressed rapeseed, we performed RT-qPCR assays on BnaA05.NHX2 in the overexpressed rapeseed lines. The expression of BnaA05.NHX2 in the OE-1 and OE-2 lines was 8 and 4 folds higher than that in the wild type, respectively. Through testing the effect of BnaA05.NHX2 overexpression on the expression of other vacuolar Na+ transporters in the transgenic plants, we found that the expression of other NHX1 and NHX2 family genes was increased to different degrees in the transgenic plants overexpressing BnaA05.NHX2 (Fig. 10A-G, Table S10).