H+-pyrophosphatases enhance low nitrogen stress tolerance in transgenic Arabidopsis and wheat by interacting with a receptor-like protein kinase

Abstract

Introduction: Nitrogen is a major abiotic stress that affects plant productivity. Previous studies have shown that plant H+-pyrophosphatases (H+-PPases) enhance plant resistance to low nitrogen stress. However, the molecular mechanism underlying H+-PPase-mediated regulation of plant responses to low nitrogen stress is still unknown. In this study, we aimed to investigate the regulatory mechanism of AtAVP1 in response to low nitrogen stress.

Methods and results: AtAVP1 in Arabidopsis thaliana and EdVP1 in Elymus dahuricus belong to the H+-PPase gene family. In this study, we found that AtAVP1 overexpression was more tolerant to low nitrogen stress than was wild type (WT), whereas the avp1-1 mutant was less tolerant to low nitrogen stress than WT. Plant height, root length, aboveground fresh and dry weights, and underground fresh and dry weights of EdVP1 overexpression wheat were considerably higher than those of SHI366 under low nitrogen treatment during the seedling stage. Two consecutive years of low nitrogen tolerance experiments in the field showed that grain yield and number of grains per spike of EdVP1 overexpression wheat were increased compared to those in SHI366, which indicated that EdVP1 conferred low nitrogen stress tolerance in the field. Furthermore, we screened interaction proteins in Arabidopsis; subcellular localization analysis demonstrated that AtAVP1 and Arabidopsis thaliana receptor-like protein kinase (AtRLK) were located on the plasma membrane. Yeast two-hybrid and luciferase complementary imaging assays showed that the AtRLK interacted with AtAVP1. Under low nitrogen stress, the Arabidopsis mutants rlk and avp1-1 had the same phenotypes.

Discussion: These results indicate that AtAVP1 regulates low nitrogen stress responses by interacting with AtRLK, which provides a novel insight into the regulatory pathway related to H+-pyrophosphatase function in plants.

Keywords: H+-pyrophosphatase; arabidopsis; low nitrogen stress; receptor-like protein kinase; transgenic wheat.

Figure 1

Functions of AtAVP1 in Arabidopsis under normal conditions and different levels of nitrogen stress Phenotypic analysis of the transgenic AtAVP1 Arabidopsis line OE-AVP1 grown under (A) 6 mM ${\text{NO}}_3^{-}$ (control), (B) 1 mM ${\text{NO}}_3^{-}$ , and (C) 0.2 mM ${\text{NO}}_3^{-}$ (low nitrogen) conditions. Images were taken after 5 d of growth on different media. DIAN Win RHIZO Pro 2012 root scanning software and EPSON Flatbed and Expression 10000XL scanners were used to scan and measure the roots. (D) Root length, (E) total root surface, and (F) lateral root number of OE-AVP1 lines grown under different nitrogen treatment conditions. Phenotypic analysis of the mutant avp1-1 under (G) 6 mM ${\text{NO}}_3^{-}$ (control), (H) 1 mM ${\text{NO}}_3^{-}$ , and (I) 0.2 mM ${\text{NO}}_3^{-}$ medium conditions. Images were taken after 7 d of growth; the (J) root length, (K) total root surface, and (L) lateral root number of avp1-1 lines grown under different nitrogen treatments were measured. All data represent mean ± standard deviation (n ≥ 6). (* p< 0.05, ** p< 0.01, *** p< 0.001, Duncan’s multiple range test). Scale bars = 0.5 cm.

Figure 2

EdVP1 overexpression in wheat seedlings enhances low nitrogen stress tolerance (A) Phenotypes of the WT (SHI366) and transgenic EdVP1-overexpressing wheat lines OE-8, OE-28, and OE-32 grown under 0.2 mM low nitrogen (0.2 mM ${\text{NO}}_3^{-}$ ) conditions. Scale bars = 5 cm. (B) EdVP1 expression in seedling roots of WT and T₃ transgenic EdVP1-overexpressing wheat lines grown under 0.2 mM ${\text{NO}}_3^{-}$ and normal conditions. (C) Plant height, (D) root length, (E) aboveground fresh weight, (F) aboveground dry weight, (G) underground fresh weight, and (H) underground dry weight of WT (SHI366) and the transgenic EdVP1-overexpressing wheat lines OE-8, OE-28, OE-32 grown under 0.2 mM low nitrogen condition. Data represent mean ± standard deviation (n = 5), (*p< 0.05, Duncan’s multiple range test).

Figure 3

Low nitrogen stress tolerance analysis of EdVP1 transgenic wheat grown in the field Phenotypes of OE-28 and SHI366 wheat plants grown under (A) normal and (B) low nitrogen conditions in 2022. (C) Phenotypes of OE-32 and SHI366 wheat plants grown under low nitrogen conditions in 2021. Scale bars = 10 cm (D) The 1000-grain weight, (E) spike number per m², (F) grain number per spike, and (G) grain yield of the OE-28 and OE-32-overexpressing lines and wild-type SHI366 plants grown under normal and low nitrogen conditions in 2021 and 2022. Data represent the mean of values from three biological replicates ± standard deviation. Asterisks indicate significant differences between the OE-28, OE-32, and SHI366 wheat plants, as assessed using Duncan’s multiple range test (*p< 0.05, Duncan’s multiple range test).

Figure 4

Subcellular AtAVP1 and AtRLK localization in Arabidopsis protoplasts The 35S:AtAVP1-GFP, 35S:AtRLK-GFP, and 35S:GFP control vectors were transiently expressed in Arabidopsis protoplasts. Compared to that of the 35S:GFP construct, the expression of 35S:AtAVP1-GFP and 35S:AtRLK-GFP was localized to the plasma membrane of the protoplasts. Fluorescence was observed using a Zeiss LSM980 confocal laser scanning microscope 16 h after transformation. Membrane marker protein (mCherry) was co-expressed with 35S:AtAVP1-GFP. Scale bars = 5 μm.

Figure 5

Identification of AtAVP1 interactions with AtRLK (A) Identification of an interaction between AtRLK and AtAVP1 using a luciferase complementary imaging (LCI) assay. The nLUC : AtAVP1/cLUC : AtRLK and negative control constructs were transfected into Agrobacterium tumefaciens and then injected into four different regions of a tobacco leaf. The leaves were analyzed for luminescence signal by Night SHADE LB 985. (B, C) Identification of an interaction between AtRLK and AtAVP1 using a yeast two-hybrid system. (B) The pBT3-STE : AtAVP1/pPR3N:AtRLK and negative control constructs were co-transformed into yeast cells and grown on SD/-Leu-Trp double deficiency medium. (C) The co-transformed pBT3-STE : AtAVP1/pPR3N:AtRLK and negative control into yeast cells were grown on SD/-Leu-Trp-His-Ade selective medium at 1×, 10×, 100×, and 1000× dilution gradients to test the interaction.

Figure 6

AtRLK plays a role in low nitrogen stress tolerance in Arabidopsis (A–C) Phenotypic analysis of mutant rlk and WT plants under control and nitrogen stress conditions. The rlk-01 and rlk-02 represent the same line plated out twice. (D) Root length, (E) total root surface, and (F) lateral root number in rlk mutant lines grown on 6 mM ${\text{NO}}_3^{-}$ (control), 1 mM ${\text{NO}}_3^{-}$ , and 0.2 mM ${\text{NO}}_3^{-}$ media. All data represent mean ± standard deviation (n ≥ 6). Asterisks indicate significant differences compared to WT (* p< 0.05, ** p< 0.01, Duncan’s multiple range test). Scale bars = 0.5 cm.