Plant material and growth conditions
All Arabidopsis thaliana plants used in this study belonged to the Columbia-0 (Col-0) ecotype, including wild-type, T-DNA insertion mutant, and transgenic plants. The AtVHA-d2 T-DNA insertion mutant SAIL_141_G06 (CS806840) was obtained from the Arabidopsis Biological Resource Center (ABRC: http://www.arabidopsis.org/). Homozygous plants were selected by PCR using the specific primers At3g28715-LP, At3g28715-RP, and left-border LB-3. The expression levels of AtVHA-d2 and AtVHA-d1 in mutant SAIL_141_G06 were analyzed by real-time quantitative PCR using their respective primers (AtVHA-d1-qF, AtVHA-d1-qR, AtVHA-d2-qF and AtVHA-d2-qR) (Additional file 3: Table S3). The sterilized Arabidopsis seeds were sown on 1/2 Murashige and Skoog (MS) medium. After sowing, the seeds were incubated at 4 °C for 3 days and then germinated in a growth chamber at 22 °C under a 12 h/12 h light/dark photoperiod.
Vector Construction And Arabidopsis Transformation
To construct proAtVHA-d1:GUS and proAtVHA-d2:GUS, 1644 and 1657 bp of the AtVHA-d1 and AtVHA-d2 promoter regions were amplified using Col-0 genomic DNA and cloned into the HindIII/BamHI or KpnI/XhoI sites of pBI121-GUS or pBI121-GUS (modified, more enzyme digestion sites were added) vectors. To construct the AtVHA-d2-GFP fusion genes, the open reading frame (ORF) of AtVHA-d2, without the stop codon, was amplified using PCR and cloned into the XbaI/KpnI sites of the pBI121-GFP vector. The specific primers used in this study are listed in Table S1. The constructs were stably transformed into Arabidopsis via the Agrobacterium tumefaciens-mediated floral dip method . The T3 transgenic plants were identified by reverse transcription PCR (RT-PCR).
Confocal Laser Scanning Microscopy
Roots of transgenic Arabidopsis seedlings stably expressing AtVHA-d2-GFP were washed twice with liquid 1/2 MS medium immediately before visualizing via confocal laser scanning microscopy (Nikon, A1, Japan). GFP signals were detected using a 500–530 nm emission filter.
Histochemical β-glucuronidase Staining
Seedlings and different organs of transgenic Arabidopsis (proAtVHA-d1:GUS and proAtVHA-d2:GUS) were immersed in the staining buffer (100 mM sodium phosphate, pH 7.0, 10 mM EDTA, 0.5 mM K3[Fe(CN)6], 0.5 mM K4[Fe(CN)6], 0.1% Triton X-100) supplemented with 0.5 mM 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (X-Gluc) for 12 h at 37 °C. Chlorophyll in green parts was removed by repeated washing in 95% ethanol.
Quantitative Real-time Pcr Analyses
For abiotic stress treatments, 7-day-old Arabidopsis seedlings were exposed to 150 mM NaCl, 300 mM mannitol, 5 mM H2O2, 100 µM abscisic acid (ABA), cold (4 °C), or heat (37 °C). The seedlings were collected at different time points (0, 3, 6, 12, and 24 h) after treatment, and then frozen immediately in liquid nitrogen for RNA extraction.
Total RNA was extracted using the RNAprep pure plant kit (Tiangen, Beijing, China), and cDNA was synthesized from 1 µg total RNA using the M-MLV RTase cDNA synthesis kit (TaKaRa, Shiga, Japan), according to the manufacturer’s instructions. qPCR was performed on a Mx3000P QPCR system (Agilent Technologies, Palo Alto, CA, USA). The Arabidopsis AtActin2 gene was used as an internal control. Three biological repeats and three technical repeats were performed for qPCR analysis. The primers used in this study are shown in Additional file 4: Table S3.
Stress Tolerant Phenotype
For stress tolerant assay, the 30 seeds of Col-0 and the atvha-d2 mutant were treated at 4 °C for 3 days and then grown vertically on 1/2 MS (control) or 1/2 MS medium supplemented with different concentrations of NaCl (75 and 100 mM), H2O2 (1 and 2 mM), and mannitol (175 and 200 mM). After 14 days, seedling phenotypes were photographed, and the root length, relative root length and fresh weight of the seedlings were measured.
Net H+ Flux Measurement
Net H+ flux was measured using the Non-invasive Micro-test Technology (NMT100 Series, YoungerUSA LLC, Amherst, MA, USA) as described previously [12, 24]. Seven-day-old seedlings of Col-0 and the atvha-d2 mutant were grown on 1/2 MS medium, and were exposed to mannitol (200 mM), NaCl (100 mM), and H2O2 (2 mM) for 24 h. Root segments were immobilized in the measuring solution (0.1 mM KCl, 0.1 mM CaCl2, 0.1 mM MgCl2, 0.5 mM NaCl, and 0.3 mM MES, pH 5.8) to measure the H+ flux. Six biological repeats were performed for each analysis.
Rna-seq And Degs Analysis
Seedlings of Col-0 and the atvha-d2 mutant were treated in 1/2 MS medium supplemented with H2O2 (2 mM) for 0, 12, and 24 h. Total RNA from the seedlings was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Subsequently, the RNA samples were sent to the Beijing Genomic Institute (BGI, Shenzhen, China) for RNA-seq.
DEGs were screened with a false discovery rate (FDR) threshold of 0.01 and an absolute log2 ratio of 1. All DEGs were mapped to each term of KEGG module from KEGG databases, and significant pathways were defined based on a corrected P value ≤ 0.05.