Plant materials and growth conditions
Limonium bicolor seeds were obtained from plants grown in saline soil (N37°20'; E118°36') in the Yellow River Delta (Shandong Province, China) with the permission of the Dongying government. The author Baoshan Wang had formally identified Limonium bicolor, and the seeds harvesting process is in full compliance with relevant government guidelines. Unfortunately, we were unable to find a voucher specimen of Limonium bicolor stored in any publicly available herbarium. The dried seeds were stored at 4°C for at least six months . Before use, the seeds were surface-disinfected with 70% ethanol (5 min), followed by 6% (v/v) NaClO (Sigma, USA) for 17 min with shaking. The seeds were washed five times with sterile distilled water and sown on MS basal medium. The plants were cultured at 28 ± 3°C/23 ± 3°C (day/night) at a light intensity of 600 µmol/m2/s (15 h photoperiod) and 65% relative humidity.
The Arabidopsis thaliana ecotype Col-0 (Columbia-0) seeds used are originally preserved in our laboratory. The author Baoshan Wang had formally identified Col-0 before. The collection of seeds and the performance of experimental research on such plant were complied with the national guidelines of China. Arabidopsis thaliana ecotype Col-0 (Columbia-0) seeds were sterilized three times with 70% alcohol for four minutes each time and three times with 95% alcohol for four minutes each time. The sterilized seeds were washed with sterile distilled water and sown on 1/2MS medium. After two days of stratification at 4°C, the plants were cultivated at 22°C/18°C (day/night) under a 16 h/8 h light/dark cycle with a light level of 150 µmol/m2/s and 70% relative humidity . To facilitate infection and transformation by Agrobacterium tumefaciens, seedlings were cultivated for one week on 1/2MS medium and transplanted into pots (9 cm height × 9 cm diameter) filled with nutrient-rich soil (soil:vermiculite:perlite, 3:1:1).
Cloning and bioinformatic analysis of LbHLH
The first true leaves of L. bicolor were collected at different stages of leaf development, including the undifferentiated stage (stage A; 4–5 days after sowing, 5000 leaves), salt gland differentiation stage (stage B; 6–7 days after sowing, 4000 leaves), stomata differentiation stage (stage C; 8–10 days after sowing, 3000 leaves), pavement cell differentiation stage (stage D; 11–16 days after sowing, 1000 leaves), and mature stage (stage E; more than 17 days after sowing, 1000 leaves) according to Yuan .
The total RNA was extracted from pooled leaves of each stage using a FastPure Plant Total RNA Isolation kit (RC401-01; Vazyme Biotech Co., Ltd.). cDNA was reverse transcribed from the RNA with a ReverTra Ace quantitative PCR (qPCR) RT kit (TOYOBO Co., Ltd, Japan) according to the manufacturer’s instructions. The reference sequence of LbHLH was obtained from the assembled sequence from a previously reported transcriptome . Full-length LbHLH was cloned using the primers LbHLH-S and LbHLH-A (Table S1), which were designed with Primer Premier 5.0. The conserved domain of LbHLH was predicted using the online tool SMART (http://smart.embl-heidelberg.de/).
Subcellular localization of LbHLH
The subcellular localization of LbHLH was determined using transformed onion epidermal cells harboring a GFP expression vector . The pCAMBIA1300 vector was digested with SalI to form a linear vector. To obtain LbHLH cDNA, primers LbHLH OE-S and LbHLH OE-A were designed with SalI digestion sites (Table S1). The full-length coding sequence (CDS) of LbHLH carrying a SalI digestion site was introduced into the pCAMBIA1300 vector under the control of the CaMV 35S promoter by homologous recombination using a ClonExpress II One Step Cloning Kit (Vazyme Biotech Co., Ltd., China). Agrobacterium tumefaciens GV3101 was used to transform the pCAMBIA1300-LbHLH recombinant vector into onion epidermal cells . After two days of cultivation in the light, fluorescent signals of GFP-labeled LbHLH were detected under a TCS S8 MP two-photon laser-scanning confocal microscope (Leica, Germany). DAPI was used to locate the nucleus and was observed under excitation at 358 nm . FM4 − 64 (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino)Phenyl) Hexatrienyl) Pyridinium Dibromide, Invitrogen) was used to locate the plasma membrane and observed under excitation at 559 nm .
Expression analysis and in situ hybridization of L. bicolor
A-E stage leaves, stems, roots, and aged leaves of L. bicolor grown on MS medium were collected for RNA extraction. Seedlings grown under different treatments (100 mM NaCl, 0.04 mg/L 6-BA and 0.1 mg/L ABA added in MS medium) were also collected for RNA extraction. Quantitative RT-PCR primers LbHLH-RT-S and LbHLH-RT-A were designed using Beacon Designer software (version 7.8) (Table S1). RT-PCR was performed in a 20 µl reaction system including 10 µl SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.), 0.2 µM primers, and 300 ng cDNA in a fluorometric thermal cycler (Bio-Rad CFX96 ™ Realtime PCR System) under the following conditions: 95°C for 30 s, 40 cycles (95°C for 5 s, 60°C for 30 s). Lbtubulin (primers Lbtubulin-RT-S and Lbtubulin-RT-A, Table S1) was used as an internal control . The expression level of LbHLH in different tissues was calculated relative to the expression level in roots (which was set to 1). Three biological replicates (separate experiments) were performed. Relative expression levels were calculated using the formula 2−ΔΔC(T).
To further explore the expression patterns of LbHLH in L. bicolor, developing leaves (the first true leaf at 5–8 days of germination) were isolated from L. bicolor for in situ hybridization. Briefly, the leaves were fixed in 4% paraformaldehyde, embedded in paraffin, and dehydrated through an alcohol series. Thin sections (8 µm) of tissue were treated with proteinase K and hybridized in 6 ng/µL hybridization solution at 37°C overnight. Digoxin-labeled LbHLH probe (5’-DIG-CUCCCUAACAUUAACCUUCAGAUCCAGCCC-3’, purified by HPLC) appeared blue-violet.
Cloning of the LbHLH promoter and histochemical analysis
Genomic DNA was extracted from L. bicolor using a FastPure Plant DNA Isolation Mini Kit (Vazyme Biotech Co., Ltd.) to obtain the full-length LbHLH promoter. The reference sequence of the LbHLH promoter was obtained from the L. bicolor genome (unpublished). The promoter sequence was cloned using primers LbHLH-P-S and LbHLH-P-A (Table S1). Elements in the promoter were predicted using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/), and maps were drawn using CSDS 2.0 ( http://gsds.gao-lab.org/).
To replace the 35S promoter with the LbHLH promoter in pCAMBIA3301, HindIII and NcoI were used to excise the CaMV 35S promoter from pCAMBIA3301 and to obtain a linear vector. The promoter was cloned into the vector using primers 3301-LbHLH-P-S and 3301-LbHLH-P-A (Table S1) to add HindIII and NcoI digestion sites in advance. The linear vector pCAMBIA3301 and the inserted LbHLH promoter were ligated together using an In-Fusion HD Cloning Kit (Takara) to construct the recombinant vector.
Agrobacterium tumefaciens GV3101 cells were transformed with the recombinant plasmid and used to infect Arabidopsis thaliana Col-0 to generate Col::pLbHLH-GUS. The transgenic seedlings were continuously screened with herbicides (0.1%, v/v), and homozygous plants of the T3 generation were subjected to histochemical staining. Ten-day-old seedlings were immersed in GUS staining solution and incubated at 37°C overnight with shaking. The stained plant materials were decolorized by incubating in 70% ethanol 2–3 times and observed under a dissecting microscope (Nikon, Japan).
Generation of Col-35 S::LbHLH plants
The full-length CDS of LbHLH was cloned using primers LbHLH OEAt-S and LbHLH OEAt-A (Table S1) containing NcoI digestion sites for cloning into different vectors. A ClonExpress II One Step Cloning Kit (Vazyme Biotech Co., Ltd.) was used to generate p35S::LbHLH via homologous recombination. p35S::LbHLH was introduced into Agrobacterium tumefaciens GV3101 cells, which were used to transform Arabidopsis. After screening for three generations with herbicides (0.1%, v/v), the Col-35S::LbHLH overexpression lines were subjected to physiological measurements.
Three Col-35S::LbHLH overexpression lines were selected for physiological characterization based on LbHLH expression (low, medium, and high). Specifically, positive transgenic plants were first identified using primers LbHLH OEAt-S and LbHLH OEAt-A based on the genomic sequences of the transgenic lines. mRNA was then extracted from different Col-35S::LbHLH lines using a FastPure Plant Total RNA Isolation kit (Vazyme Biotech Co., Ltd.) according to the manufacturer’s instructions. LbHLH expression levels in different Col-35S::LbHLH lines were analyzed by qRT-PCR using primers LbHLH RT-S and LbHLH RT-A (Table S1). Given that no homologs of LbHLH were detected in Arabidopsis, the line with the lowest LbHLH expression level (OE35) was used as a control (relative expression level set to 1) to calculate the expression levels of LbHLH in the Col-35S::LbHLH lines. Three biological replicates were performed for each group. Lines with high (OE40), medium (OE26), and low expression (OE4) levels were retained for analysis.
Phenotypic observation and expression analysis of trichome/root hair-related genes in Col-35S::LbHLH
Trichome and root hair development were measured in three overexpression lines (OE4, OE26, and OE40) and the wild type. The trichomes on the first true leaves of one-week-old seedlings were counted under a dissecting microscope (Nikon, Japan). The root hairs 0.5 cm–1.5 cm from the tip of the roots of 5-day-old seedlings were counted. Trichome and root hair numbers were calculated with ImageJ software. Twenty seedlings were examined per line.
RNA was extracted from seedlings grown on 1/2MS medium for one week. The expression levels of ten genes involved in trichome differentiation and root hair fate determination were identified by qRT-PCR, including AtTTG1, AtTRY, AtCPC, AtEGL3, AtGL1, AtGL3, AtSAD2, GLABRA 2 (AtGL2), MYB DOMAIN PROTEIN 23 (AtMYB23), and ZINC FINGER PROTEIN 5 (AtZFP5). The expression levels of genes related to root hair development, including the root hair initiation genes ROOT HAIR DEFECTIVE 6 (AtRHD6) and RING FINGER OF SEED LONGEVITY 1 (AtRSL1) and the root hair elongation gene LJRHL1-LIKE 1 (AtLRL1) were also measured by qRT-PCR. All primers used in qRT-PCR are listed in Table S1 as gene name-RT-Sense (gene name-RT-S) and gene name-RT-Antisense (gene name-RT-A) (e.g., AtEGL3-RT-S and AtEGL3-RT-A). AtActin (amplified with primers Atactin-RT-S and Atactin-RT-A) was used as an internal control ; three replicate biological experiments were performed.
Yeast two-hybrid assay to examine self-activation of LbHLH and identify candidate LbHLH-interacting proteins
The full-length CDS of LbHLH was cloned into pGBKT7 (BD) to generate BD-LbHLH using an NdeI digestion site by homologous recombination using a ClonExpress® II One Step Cloning Kit (Vazyme Biotech Co., Ltd.) and the primers BD-LbHLH-S and BD-LbHLH-A (Table S1). The same method was used to construct AD-AtGL1 and AD-AtGL3 using pGADT7 (AD) and primers AD-AtGL1-S, AD-AtGL1-A, AD-AtGL3-S, and AD-AtGL3-A with NdeI digestion sites (Table S1).
Seven groups were designed to test interactions between LbHLH and AtGL1 or AtGL3: BD-53&AD-T (positive control), BD-Lam&AD-T (negative control), BD&AD-AtGL1 (to verify AtGL1 self-activation), BD&AD-AtGL3 (to verify AtGL3 self-activation), BD-LbHLH&AD (to verify LbHLH self-activation), BD-LbHLH&AD-AtGL1 (to verify the interaction between LbHLH and AtGL1), and BD-LbHLH&AD-AtGL3 (to verify the interaction between LbHLH and AtGL3). The plasmids were transformed into Y2H Gold yeast (Saccharomyces cerevisiae) cells separately using Yeastmaker Yeast Transformation System 2 (Clontech Code No. 630439) following the manufacturer’s instructions. All groups were first cultured on SD/-Leu/-Trp medium to determine successful transformation based on the presence of colonies. Further interaction experiments were performed on both SD/-Leu/-Trp/X-a-gal/Aba (200 ng/ml) and SD/-Ade/-His/-Leu/-Trp/X-a-gal/Aba (200 ng/ml) media. The self-activation of LbHLH and verification of possible interactions were determined based on the growth status and blue color of colonies after 2 days of culture.
Measuring LbHLH expression in L. bicolor during a time course of NaCl treatment, salt tolerance indices, and physiological indicators
L. bicolor seedlings were cultured in soil for 20 days and treated with 100 mM NaCl for 0, 12, 24, 48, and 72 hours to generate samples for time course analysis. Total RNA was extracted from each sample and used for qRT-PCR with primers LbHLH-RT-S and LbHLH-RT-A (Table S1) to analyze the expression pattern of LbHLH over a time course of 100 mM NaCl treatment.
To investigate salt resistance among different transgenic lines, OE4, OE26, OE40, and wild-type (WT) seeds were germinated on 1/2MS medium (containing 1% agar) with different concentrations of NaCl (0, 50, 100, or 150 mM). The germinated seeds were counted each day for five days: a seed containing a radicle > 1 mm long that had emerged from the seed coat was considered to be germinated. The germination percentage (%) was calculated as the number of germinated seeds / total number of seeds × 100%. Thirty seeds were sown per line for each treatment, and three biological replicates were performed.
The emergence of green cotyledons was used as an indicator of cotyledon growth. The cotyledon growth rate of each line was measured after three days of germination. Cotyledon growth rate (%) = (number of seeds with cotyledons / number of all tested seeds) × 100%. After continuous cultivation for five days on different media, the root lengths of different lines were measured using ImageJ software. Thirty replicates were performed per treatment.
Five-day-old seedlings on 1/2MS medium were transplanted into a matrix irrigated with different concentrations of NaCl (0 or 100 mM NaCl dissolved in Hoagland solution, pH 6.2). The leaves (0.5 g) of two-week-old seedlings subjected to control or 100 mM NaCl conditions were harvested separately. The Na+, K+, proline, and MDA contents were measured as described previously [30, 31]. Ion concentrations were measured with a flame photometer (Cole-Parmer, USA). Five replicates per measurement were performed for each line.
To verify the effect of LbHLH on alleviating salt stress, all lines were cultured in 180 mM mannitol (causing the same osmotic pressure as 100 mM NaCl) and on 10 mM LiCl medium (causing the same ionic effect as 100 mM NaCl). After five days of culture, the germination rate and root length were measured to compare the effects of ionic stress and osmotic stress on LbHLH expression.
qRT-PCR of marker genes related to salt stress in transgenic Arabidopsis
To investigate the expression of genes under salt stress, all lines were cultured for ~ 10 days in 1/2MS medium containing 0 or 100 mM NaCl and subjected to RNA extraction using a FastPure Plant Total RNA Isolation kit (RC401-01; Vazyme Biotech Co., Ltd.). The RNA was reverse-transcribed into cDNA and used for qRT-PCR.
Four marker genes involved in stress resistance were selected for qRT-PCR analysis: SALT OVERLY SENSITIVE 1 (AtSOS1), AtSOS3, DELTA1-PYRROLINE-5-CARBOXYLATE SYNTHASE 1 (AtP5CS1), and AtP5CS2 (Table S1). AtACTIN was used as the internal control. Three biological replicates were performed.
Statistical significance at P = 0.05 (Duncan’s multiple range tests) was determined using SPSS. ANOVA with orthogonal contrasts and mean comparison procedures was used to detect significant differences between treatments.