Plant materials and growth conditions
The seed sources and seedlings cultivation of A. canescens were carried out according to the methods of our previous study (Pan et al. 2016; Guo et al. 2019). A. canescens seedlings with similar size were selected and cultured in plastic pots (5 cm × 5 cm × 5 cm; two seedlings/pot) containing sterilized vermiculite, and irrigated with 1/2 strength Hoagland nutrient solution (Ma et al. 2012).
A. thaliana ecotype Columbia (Col) was used for genetic transformation material in this study. The seeds of A. thaliana were sterilized with 75% (V/V) ethanol in a 1.5 mL centrifuge tube for 5 min, then treated with 1% (W/V) NaClO for 8 min, and finally rinsed with distilled water for six times. The sterilized seeds were incubated at 4°C for 4 d before germination. The temperature of the greenhouse for seedlings growth was 22 ± 3°C, the light duration was 16 h light /8 h dark, the light intensity was about 150 µmol·m− 2·s− 1, and the relative humidity of the air was 63 ± 2%.
Fluorescence observation and content determination of flavonoids in roots and leaves of A. canescens
To observe the distribution of flavonoids in different tissues of A. canescens, four-day-old seedlings were treated with different concentrations of NaCl (0, 100, 300 mM) for 2 d, and then 2 cm of root tips were cut for diphenylboric acid-2-aminoethyl ester (DPBA) staining. Similarly, two-week-old seedlings were treated with 1/2 strength Hoagland nutrient solutions supplemented with 0 (control), 100 and 300 mM NaCl for 2 d, then the first and second true leaves were excised for DPBA staining. The DPBA staining was performed according to the method of Murphy et al. (2000) with slight modification. The epidermis of leaves and root tips were immersed in DPBA dyeing liquid (DPBA, 2.5 mg mL− 1; Triton X-100, 0.005%; pH, 7) for 45 min and 15 min for staining, respectively, then were washed in deionized water and mounted on microscope slides. The fluorescence signal was visualized on a confocal microscope (Leica, DM6B, Germany) using a FITC filter set (excitation 450–490 nm, suppression long pass 520, 4 or 10X Plan-Apo objective lens).
To measure the content of flavonoids in A. canescens, four-week-old seedlings were treated with 1/2 strength Hoagland nutrient solutions supplemented with 0 (control), 100 and 300 mM NaCl for 2 d, the mature leaves and roots of A. canescens were harvested for 6 repetitions, respectively. After the samples were dried and ground, the total flavonoids content was extracted using Cominbio Plant Flavonoid Extraction kit (Suzhou Comin biotechnology Co., Ltd., Suzhou, China). Briefly, approximately 0.018 g (dry weight) of the crushed plant sample was added to 1.8 mL of 60% ethanol and combined with aluminum ions in an alkaline nitrite solution, and then incubated with oscillation at 60°C for 2 h. After that, the mixtures were centrifuged at 10,000 × g for 10 min at 25°C. The absorbance (at 510 nm) of the supernatant was measured using a spectrophotometer (UV-6100PCS; Mapada Instruments Co. Ltd, Shanghai, P.R. China), and the content of flavonoids was estimated as the following formula: flavonoids content [mg/g dry weight (DW)] = 0.398 × (ΔA510 − 0.0007) / DW (Li et al. 2020).
Expression pattern analysis of genes encoding three key enzymes of flavonoids biosynthesis in A. canescens
According to our previous transcriptome data (Feng et al. 2023), three genes encoding chalcone synthase (CHS) (Gene ID CL5595.Contig2_All), chalcone isomerase (CHI) (Gene ID CL990.Contig3_All) and flavanone 3-hydroxylase (F3H) (Gene ID Unigene9407_All) were identified and found all of them were upregulated by NaCl. To determine the expression abundance of AcCHS, AcCHI and AcF3H in various tissues of A. canescens under saline conditions, four-week-old seedlings were treated with 1/2 strength Hoagland nutrient solutions supplemented with 0 (control), 100 and 300 mM NaCl for 0, 3, 6, 12, 24 and 48 h. After that, the roots, stems, young leaves, mature leaves and old leaves were harvested, respectively (Guo et al. 2022), and the expression levels of AcCHS, AcCHI and AcF3H in above tissues were analyzed by real-time quantitative PCR with reference gene AcACTIN. The primers used in this experiment were shown in Supplementary Table S1.
Cloning of A. canescens AcCHI and sequence analysis
Based on sequence of AcCHI gene (Gene ID CL990.Contig3_All) in our previous transcriptome sequencing data, the open reading frame (ORF) of this gene was predicted through the NCBI ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). The nested PCR method was used to amplify the full-length cDNA of AcCHI. All the primers for the nested amplification of cDNA ends by PCR were shown in Supplementary Table S2. Total RNA of the A. canescens samples was extracted following the instructions of TaKaRa Mini BEST Plant RNA Extraction Kit (TaKaRa, Beijing, China), and then was reverse-transcribed into cDNA by using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Beijing, China). Then the full-length cloning was synthesized using Phusion® High-Fidelity DNA Polymerase kit (Thermo Fisher Scientific, Waltham, MA, USA). The PCRs were conducted in a total volume of 20 µL, containing 0.8 µL of cDNA, 0.8 µL of forward and reverse primers, 4 µL of 5 × Phusion GC buffer, 1.6 µL of dNTP Mixture, 0.2 µL of Phusion DNA Polymerase, and 11.8 µL of ddH2O. PCR reactions were performed according to following procedure: initial denaturation at 98°C for 30 s min followed by 35 cycles of 10 s at 98°C, 10 s at 55°C and 40 s at 72°C, and final extension of 10 min at 72°C. Then the PCR products were collected and sequenced.
After the homologous amino acid sequences sharing high similarity with AcCHI being retrieved by NCBI BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi), multiple-sequence alignment and phylogenetic analysis were performed according to the previously described method (Guo et al. 2022).
Generation of transgenic A. thaliana overexpressing AcCHI
The open reading frame (ORF) of AcCHI was amplified by using the primers of Supplementary Table S3 containing NcoI restriction sites and then was fused into the vector of pCAMBIA1302-Basta-35S by In-Fusion® HD Cloning Kits (Takara, Beijing, China). Subsequently, the recombinant plasmid pCAMBIA1302-Basta-35S-AcCHI was mobilized into Agrobacterium tumefaciens strain GV3101 and was transformed into Col-0 Arabidopsis seedlings according to floral dip method (Clough and Bent 1998). T1 transgenic plants were selected by spraying the 0.1‰ (v/v) Basta on the seedlings three times at 3 or 4 d intervals. After screening for the next two generations, homozygous plants of T3 generation were obtained. Then, we randomly selected two lines (named as I1 and I2) for further experiments in this study.
The expression of AcCHI in above two transgenic lines was evaluated by RT-PCR using the primers listed in Supplementary Table S4. AtActin2 gene was used as an internal control. The PCR reaction was carried out at 94°C for 2 min for initial denaturation, followed by 35 cycles of 94°C for 45 s, 60°C for 30 s, 72°C for 90 s of extension, and 10 min of final extension at 72°C. The reaction mixture containing 1 µL of cDNA from A. thaliana as template, 10 µL of Premix Taq (Takara, Beijing, China), 0.5 µL each of forward and reverse primers, and 8 µL of ddH2O to the final volume of 20 µL. Finally, the PCR products were separated by agarose gel electrophoresis and visualized by AlphaImager (ProteinSimple Inc., Santa Clara, CA, USA).
Fluorescence observation and content determination of flavonoids in transgenic A. thaliana overexpressing AcCHI
To observe the distribution of flavonoids in A. thaliana, the first and second true leaves of hydroponic 6-day-old wild type (WT) and transgenic seedlings were excised and stained with diphenylboric acid-2-aminoethyl ester (DPBA). To measure the content of flavonoids in A. thaliana, the shoots of hydroponic 4-week-old WT and transgenic seedlings were harvested for 6 repetitions, respectively. The visualization and quantification of flavonoids were performed according to the procedures described previously.
Analysis on expression of the genes encoding key enzyme related to flavonoids biosynthesis in leaf of A. thaliana
To investigate whether the overexpression of AcCHI affected the expression of genes encoding other key enzymes related to flavonoids biosynthesis in transgenic A. thaliana, the leaves from hydroponic 3-week-old seedlings of WT and transgenic line I2 were collected separately, and the relative expression levels of 8 genes that encode phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL1), chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), flavonol synthase (FLS1), dihydroflavonol-4-reductase (DFR) and anthocyanidin synthase (ANS) were analyzed by qRT-PCR, respectively. AtActin2 was used as reference gene. All the primers used in qRT-PCR for these genes were shown in Supplementary Table S5.
Real-time quantitative PCR analysis
Total RNA of all mentioned samples was obtained by the TaKaRa Mini BEST Plant RNA Extraction Kit (TaKaRa, Beijing, China) and cDNA was synthesized using PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Beijing, China). The expression pattern of above-mentioned genes was determined using SYBR® Premix Ex Taq™ II kit (TaKaRa, Dalian, China) and finished on a StepOnePlus real-time system (ABI PRISM 7500, USA). Finally, the relative expression levels of each gene were analyzed using the 2−ΔΔCT method (Chmittgen and Livak 2008). Three replicates were conducted in each tested sample.
Assessment of salt and osmotic stress tolerance of transgenic A. thaliana overexpressing AcCHI
To evaluate the salt and osmotic stress tolerance of transgenic A. thaliana, a comparative analysis of growth phenotype under salt or osmotic stress conditions was conducted between WT and transgenic plants using an agar plate culture system. The seeds of WT and two transgenic lines were sown on 1/2 MS medium (1.5% sucrose and 0.8% agar, pH = 5.8), respectively. The plates were placed at 4°C for 2 d of vernalization and then germinated at 22°C for 4 d. Three uniform seedlings of each line were transferred into 1/2 MS plates (control) and 1/2 MS plates containing 100 mM NaCl or 200 mM Sorbitol. After 10 d of vertical culture, the seedlings were photographed and their root lengths were measured using image J software (v.1.31; https://imagej.nih.gov/ij/). All plate experiments were repeated for six times independently.
The hydroponic experiment was conducted to verify the salt and drought tolerance of transgenic plants. For salt stress treatment, the uniform 3-week-old seedlings of WT and transgenic lines were treated with 1/2 strength Hoagland nutrient solution without (control) or with additional 50 mM NaCl for 7 d, respectively. For simulating drought stress treatment, the uniform 3-week-old seedlings of each line were treated with 1/2 strength Hoagland nutrient solution without (control) or with additional 150 mM sorbitol where the osmotic potential was − 0.15 MPa for 3 d, respectively. Finally, the seedlings were photographed and harvested to measure the growth and physiological indicators.
To determine the biomass of WT and transgenic plants, the roots and shoots from hydroponic treatment were excised, and the fresh weights were weighted immediately. After that, the samples were dried in an oven (DGG-9426A, Shanghai, China) at 80°C for 48 h, and their dry weights were obtained.
Measurement of H2O2 content and relative plasma permeability in the leaf of transgenic A. thaliana overexpressing AcCHI
The hydroponic 3-week-old WT and transgenic seedlings were treated with 50 mM NaCl for 7 d or 150 mM sorbitol for 3 d, then the diaminobenzidine (DAB) staining was used to visualize the H2O2 in leaves according to the method of Daudi and O’Brien (2012). Finally, the contents of H2O2 in leaves were quantified by using the hydrogen peroxide content determination kit (Suzhou Comin biotechnology Co., Ltd., Suzhou, China). The experiments were repeated six times.
The fresh leaves of WT and transgenic plants from hydroponic experiment were excised, and the relative plasma permeability (RPP) of leaf cells was measured by using a conductivity meter (REX, DDSJ-319L, Shanghai, China) according to the method of Bao et al. 2014.
Data analysis
One-way analysis of variance (ANOVA) of SPSS v 26.0 (IBM, Armonk, NY, USA) was used for statistical analysis, and Duncan’s multiple range tests were performed to evaluate the differences between means at a significance level of P < 0.05. Values were presented as means ± SE (n = 3 or 6).