Interaction Network Construction and Functional Analysis of the Plasma Membrane H+-ATPase in Bangia fuscopurpurea (Rhodophyta)

Abstract

Salinity is a serious threat to most land plants. Although seaweeds adapt to salty environments, intertidal species experience wide fluctuations in external salinities, including hyper- and hypo-saline stress. Bangia fuscopurpurea is an economic intertidal seaweed with a strong tolerance to hypo-salinity. Until now, the salt stress tolerance mechanism has remained elusive. Our previous study showed that the expression of B. fuscopurpurea plasma membrane H⁺-ATPase (BfPMHA) genes were the most upregulated under hypo-salinity. In this study, we obtained the complete sequence of BfPMHA, traced the relative expression of this BfPMHA gene in B. fuscopurpurea under hypo-salinity, and analyzed the protein structure and properties based on the gene's sequence. The result showed that the expression of BfPMHA in B. fuscopurpurea increased significantly with varying hypo-salinity treatments, and the higher the degree of low salinity stress, the higher the expression level. This BfPMHA had typical PMHA structures with a Cation-N domain, an E1-E2 ATPase domain, a Hydrolase domain, and seven transmembrane domains. In addition, through the membrane system yeast two-hybrid library, three candidate proteins interacting with BfPMHA during hypo-saline stress were screened, fructose-bisphosphate aldolase (BfFBA), glyceraldehyde 3-phosphate dehydrogenase (NADP⁺) (phosphorylating) (BfGAPDH), and manganese superoxide dismutase (BfMnSOD). The three candidates and BfPMHA genes were successfully transferred and overexpressed in a BY4741 yeast strain. All of them significantly enhanced the yeast tolerance to NaCl stress, verifying the function of BfPMHA in salt stress response. This is the first study to report the structure and topological features of PMHA in B. fuscopurpurea and its candidate interaction proteins in response to salt stress.

Keywords: hypo-salinity; interaction proteins; plasma membrane H+-ATPase; salt stress; seaweed; yeast two-hybrid.

Figure 1

Schematic depiction of the structures of BfPMHA protein in B. fuscopurpurea (a) and PMHA in different organisms (b). SP: signal peptide.

Figure 1

Schematic depiction of the structures of BfPMHA protein in B. fuscopurpurea (a) and PMHA in different organisms (b). SP: signal peptide.

Figure 2

Phylogenetics of subsets of PMHA and H⁺-ATPase amino acids. (a) Phylogenetic analysis of PMHA protein in different organisms. (b) Phylogenetic analysis of H⁺-ATPase in algae.

Figure 3

The relative expression of the BfPMHA gene under hypo-saline stress with ef1γ gene as reference. The values are represented as mean ± SD of three replicates. Error bars indicate standard error of the mean. Different letters indicate significant differences (p-value < 0.05).

Figure 4

Screening of yeast two-hybrid system. (a) PCR amplification of inserted fragments in secondary library. (b) The result of BfPMHA auto-activation and function verification. (c) The transformation efficiency of the library screened by BfPMHA. (d) Yeast two-hybrid assay of interesting proteins in B. fuscopurpurea hypo-saline stress response, manganese superoxide dismutase (BfMnSOD), fructose-bisphosphate aldolase (BfFBA), glyceraldehyde 3-phosphate dehydrogenase (BfGAPDH, NADP⁺, phosphorylating). 1: positive control, 2: negative control, 3: no activation, 4: functional. SD-TL: SD/-Leu/-Trp, SD-TLH: SD/-Leu/-Trp/-His, SD-TLHA: SD/-Leu/-Trp/-His/-Ade (+X-α-gal), +: positive control, -: negative control.

Figure 5

Schematic depiction of the structures of three proteins, BfMnSOD, BfFBA, and BfGAPDH. Scale bar is 50 amino acids. BS, bind site. SP, signal peptide. AS, active site. DH, dehydrogenase.

Figure 6

BfPMHA separately interacts with BfMnSOD, BfFBA, and BfGAPDH in rice protoplasts by BIFC assays. mVenus, yellow fluorescence. Chloroplast, red fluorescence. Scale bar, 10 μm. The negative control of BiFC is shown in Supplementary Figure S1.

Figure 7

The salt tolerance ability of transgenic yeast. The salt tolerance ability of pYES2 strain with overexpressed genes of BfPMHA (a), BfMnSOD (b), BfFBA (c) and BfGAPDH (d) under the different NaCl concentrations, with the empty pYES2 strain as control. The concentrations of NaCl include 0 M, 0.1 M, 0.25 M, 0.5 M, and 1 M, each NaCl concentration with 5 times gradient dilution. M, mol/L.

Figure 8

Expression pattern analysis of BfPMHA (a), BfMnSOD (b), BfFBA (c), and BfGAPDH (d) in yeast under various NaCl concentrations. M: mol L⁻¹. The values are represented as mean ± SD of three replicates. Error bars indicate standard error of the mean, *, **, and *** indicate significant differences (p-value < 0.05, p-value < 0.01 and p-value < 0.001).