Isolation of putativeKT/HAK/KUP family genes from mango
By BLAST searching of the Mango Genome Data Resources (Wang et al., 2020), 18 putative strawberry MiHAK genes were identified, which were originally entitled as MiHAK1 to MiHAK18. Protein domain verification demonstrated that all of them possess the K+ transporter domain (PF02705, K03549). Detailed information about these MiHAK genes, including gene ID, gene location, coding sequence (CDS) length, and peptide length, were listed in Table 1. Physical and chemical properties of MiHAK proteins were listed in supplemental Table 1. All MiHAK genes were distributed on 10 distinct chromosomes, in which 3 genes on Chr3, Chr8 and Chr10 chromosomes, respectively, and 2 genes on Chr9 and Chr19 chromosome, and 1 gene on each of the other 5 chromosomes. The amino acid sequences of MiHAK proteins shared an overall identity of 55% (Supplemental Figure 1). Interestingly, 9 (MiHAK1, 6, 7, 8, 11, 2, 3, 5 and 16) and 4 (MiHAK4, 5, 14 and 18) MiHAK transporters in mango exhibited identical tertiary structures, respectively, while MiHAK9 and MiHAK17 exhibited quite distinct tertiary structures (Fig. 1).
Phylogenetic tree construction showed that the KT/HAK/KUP family proteins of known higher plants were classified into 4 groups (I- IV), including 3, 6, 6 and 3 members, respectively (Fig. 2). Notably, KT/HAK/KUP homologs from Rosaceae (peach, pear and strawberry) or Salicaceae (poplar and purple osier) may have the closest genetic relationship, and mango MiHAK transporters (like MiHAK3, 6, 7, 8, 9, 12, 14, 16, and 17) are prone to be closely clustered with poplar and purple osier homologs (Fig. 2).
Expression profiles and response of MiHAK genes to abiotic stresses
qRT-PCR investigation showed that MiHAK genes were unevenly expressed in different ‘Guire 82’ tissues or organs under control conditions (Fig. 3). The maximum expression of 9 genes (MiHAK1, 5, 9, 10, 11, 13, 14, 17, 18) was observed in roots and 4 genes (MiHAK3, 6, 8 and 15) in leaves, more than that of the other tissues, while the maximum expression of MiHAK4 and MiHAK12 was observed in phloem tissues and full-bloom flowers, respectively. In particular, MiHAK14 was the most abundant KT/HAK/KUP family gene throughout the whole plant of mango, especially in roots. Both MiHAK7 and MiHAK16 were not detected in all tested mango tissues. Although expression level was relatively low, MiKUP2 was evenly expressed among different tissues (Fig. 3).
To investigate the role of MiHAK genes in maintaining K+homeostasis in mango, we analyzed the expression profiles of MiHAK genes in roots under different abiotic stresses. Results showed that expression of MiHAKs were differentially responded to K+deficiency, PEG and NaCl treatment in mango roots (Fig. 4). In details, 10 (MiHAK1, 3, 4, 6, 8, 9, 12, 13, 17 and 18) and 8 (MiHAK1, 2, 3, 5, 8, 9, 12, 17 and 18) genes were responsive to K+deficiency and PEG treatment, respectively, which were significantly increased. Expression of 8 genes were sensitive to NaCl treatment, in which 6 genes (MiHAK1, 3, 5, 9, 12, 17 and 18) were decreased while MiHAK11 and MiHAK14 were significantly enhanced (Fig. 4). Notably, MiHAK1, 3, 12, 17 and 18 were the most sensitive genes, whose expression was easily affected under all tested treatments, whereas expression of MiHAK7, 10, 15 and 16 were quite steady that had no response to any treatment.
Functional analysis of MiHAK14 in bacterial TK2420 mutant
As the most abundant KT/HAK/KUP family gene in mango, the maximum expression of MiHAK14 was observed in roots that had no response to external K+ variation. We further verified its function in the E. coli TK2420 mutant. Heterologous expre\ssion of MiHAK14 restored the normal growth of TK2420 mutants on KML medium that supplied with 0.2 mmol·L-1 K+ and 0.5, mmol·L-1 IPTG (Fig. 5), while TK2420 mutants harboring the empty vector could not grow (Fig. 5). Without IPTG induction, both the wild type and TK2420 mutants hardly grew on KML medium that containing 0.2 mmol·L-1 KCl, but grew well under 10 mmol·L-1 KCl conditions. These findings implies that MiHAK14 is a functional K+ transporter that can uptake or utilize external K+.
Heterologous expression of MiHAK14 in Arabidopsis
The CDS of MiKUP14 was subcloned into the binary expression vector pBH (Fig. 6A). Putative T1 generation transgenic Arabidopsis seedlings were verified using semi-quantitative RT-PCR for the presence of a 2.2 kb fragment of MiHAK14, which was expressed throughout the whole plant of #1, #2, and #3 transgenic seedlings (Fig. 6B). And the T2 generation of #1 transgenic Arabidopsis lines were randomly selected for subsequent studies.
Under control conditions, there was no difference in growth status between WT and #1 transgenic lines, and the total fresh weight, dry weight, root length, and lateral root number was non-significantly changed (Table 2). Under both K+depletion and NaCl treatment, #1transgenic lines exhibited better growth status, either shoots or roots, compared to WT seedlings (Fig. 6C). Under K+depletion treatment, the total fresh weight, dry weight, root length, and lateral root number of #1transgenic lines was increased by 78.82%, 79.41%, 127.65%, and 138.78%, respectively (Table 2). Under NaCl treatment, the total fresh weight, dry weight, root length, and lateral root number of #1transgenic lines was increased by 772.84%, 45.19%, 65.42%, and 55.93%, respectively (Table 2). There was no difference in tissue K+ concentration under control conditions, and both K+depletion and NaCl treatment significantly reduced the K+ concentration in all tested seedlings. In particular, there was an increase of K+ concentration in #1transgenic lines under both K+depletion and NaCl treatment, at the whole plant level (Table 2). Although there was no difference between WT and #1transgenic lines under control conditions, tissue Na+ concentration was significantly increased under NaCl treatment but changed little under K+depletion, and Na+concentration was significantly lower in #1transgenic lines than WT seedlings under NaCl treatment (Table 2).
Further antioxidant enzyme activity analysis showed that the activities of SOD, POD, CAT and APX were significantly increased in both WT and transgenic Arabidopsis seedlings, respectively, under both K+depletion and NaCl treatments for 7 days. In particular, higher activities of endogenous SOD and POD were observed in #1 transgenic lines under K+ depletion, but no difference was detected in CAT and APX activities between WT and #1 transgenic seedlings. While the activities of SOD, POD, CAT and APX were all significantly higher in #1 transgenic lines under NaCl treatment (Table 2). Simultaneously, the endogenous H2O2 content was significantly enhanced under either K+depletion or NaCl treatment, and the H2O2 content in #1 transgenic lines was significantly lower than that of WT seedlings (Table 2).