Plant materials and treatment

The garlic cultivar “PiZi” used in this study were obtained from the Sanbao garlic planting base in Xuzhou (Jiangsu, China). These garlic cultivars were grown in pots containing soil in a greenhouse under short-day conditions (8 h light/16 h dark, 25 °C/18 °C, day/night) with a relative humidity of 65%. Leaves, stalks, roots, and bulbs were collected from two-mount old garlic seedling for gene expression analysis. Tissue specimens were collected from the third true leaves of garlic, grown under identical conditions. The leaves were gently pinched with tweezers to cause slight damage, and the treated area was collected after 3, 6, and 12 h. All samples were immediately frozen in liquid nitrogen and stored at – 80 °C until RNA extraction. The tobacco used in this study were grown in a greenhouse under normal-day conditions (12 h light/12 h dark, 22 °C, and 65% relative humidity). Callus were cultured at 22 °C in the dark and subcultured at 3-week intervals on the above solid medium.

Callus tissue culture preparation

To initiate garlic callus tissue culture, “PiZi” garlic bulb scales were first washed with water for 30 min, followed by immersion in 75% ethanol for 60 s, and then rinsed 5 times with water, each rinse lasting 1 min. Subsequently, the scales were soaked in 0.1% mercuric chloride solution for 15 min and rinsed 7 times with sterile water, each rinse also lasting 1 min. The sterilized garlic scales were cut into 1 mm pieces and inoculated onto MS medium with varying hormonal concentrations. The concentrations of 2,4-D were varied across 6 gradients (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/L) and NAA across 4 gradients (0.1, 0.5, 0.9, 1.3 mg/L), as detailed in the Additional file 1: Table. S1, with 15 scale tissues used per gradient. The cultures were incubated at 25 °C with 65% humidity under a light cycle of 16 h light/8 h dark for 30 days to identify the optimal medium for inducing callus in “PiZi” garlic scales.

Activation and transformation of target bacterial strain

The plasmids pHB-AsWRKY9-GFP and pHB-GFP (control) were transformed into Agrobacterium tumefaciens strain GV3101. Five microliters of bacterial suspension was added to 5 mL of LB medium supplemented with 5 μL of 50 mg/mL kanamycin and 5 μL of 50 mg/mL rifampicin and incubated overnight at 28 °C with shaking at 200 r/min. The next day, 2 mL of the overnight culture was transferred to 50 mL of LB medium containing 50 μL of 50 mg/mL kanamycin and 50 μL of 50 mg/mL rifampicin and incubated again overnight at 28 °C with shaking at 200 r/min. On the following day, an equal volume of LB medium with the same antibiotics concentrations was added, and the culture was shaken at 28 °C at 200 r/min for 4 h. The bacterial suspension was then centrifuged at 4 °C at 4000 r/min for 5 min, the supernatant was discarded, and the bacterial pellet was obtained. The pellet was resuspended in MS liquid medium, and when the OD550 = 0.6, acetosyringone was added to a final concentration of 0.1 μM. Subsequently, 100 pieces of fresh garlic callus tissues were prepared 2 h in advance by grinding into fine particles and washed with sterile water until clear. The callus particles were suspended in 20 mL of MS liquid medium and gently shaken at 100 r/min at 28 °C to serve as the genetic transformation receptors. The calluses were immersed in the bacterial suspension for 30 min, after which they were rinsed three times with distilled water, dried on sterile filter paper, and transferred to MS medium containing 1.5 mg/L 2,4-D, 0.5 mg/L NAA and 200 mg/L cefotaxime for 7 days. Subsequently, the callus tissues were transferred to MS medium supplemented with 1.5 mg/L 2,4-D, 0.5 mg/L NAA, 10 mg/L hygromycin, and 200 mg/L cefotaxime for selection, and the transformation efficiency was assessed after 2–3 weeks.

Isolation and sequence analysis of AsWRKY9

RNA isolation was performed by the EASYspin plant RNA extraction kit (Aidlab Bio., China) according to the instructions. The RNA quality and quantity were determined using the NanoDrop2000c spectrophotometer (Thermo Fisher Scientific, USA), and RNA integrity was identified by electrophoresis on 1.0% agarose gels. cDNA was synthesized from 1 μg total RNA using the HiScript II QRT SuperMix for qPCR kit with gDNase (Vazyme, China) according to the manufacture protocols. Total RNA was extracted from the leaves of “PiZi” garlic using the Plant RNA Extraction Kit (Aidlab, Beijing, China). Based on our transcriptome data [30], the AsWRKY9 F/R primer (Additional file 3: Table. S2) pairs were designed and used for cloning AsWRKY9 coding sequence from the cDNA of garlic. Then, the open reading frame (ORF) of AsWRKY9 was cloned into a pHB-GFP vector to create the 35S:GFP-AsWRKY9. The molecular weight and amino acid composition of AsWRKY9 were computed using the online website ExPASy ProtParam (https://web.expasy.org/protparam/). A BLAST search (https://www.ncbi.nlm.nih.gov/) was used for a homology search from the SWISS-PROT protein database. A phylogenetic tree was constructed using the MEGA 11 with the Neighbor-Joining algorithm, and the reliability of the branching pattern was tested with 1000 bootstrap repetitions. The online tool NCBI CD-Search was used to predict conserved domains in the encoded proteins (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi).

Quantitative real-time PCR

To determine the expression levels of AsWRKY9 in different tissues or treatments, qRT-PCR analysis was performed using the ABI StepOne Plus Real-time PCR systems (Thermol fisher, USA). One microliter of synthesized cDNA (diluted 1:5) was used as template for qRT-PCR. The AsGAPDH was selected as a reference gene (Additional file 3: Table. S2). Amplification cycles consisted of 30 s at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 30 s. Each measurement was performed using three biological replicates. The data was analyzed using the 2−ΔΔCT method.

Construction of overexpression vectors

The coding region of AsWRKY9 was derived from our previous transcriptome datasets of garlic. Specific primers were designed to amplify AsWRKY9 DNA segment from the cDNA of garlic using the following PCR parameters: initial denaturation at 94 °C for 90 s, 30 cycles of denaturation at 94 °C for 20 s, annealing at 55 °C for 20 s, extension at 72 °C for 90 s, and a final extension at 72 °C for 5 min. The PCR products were subcloned into pHB-GFP vector with Hind III restriction sites to form 35S:GFP-AsWRKY9.

Subcellular localization analysis

Transient expression in tobacco leaves was carried out as described in the literature [38]. To determine the subcellular localization of AsWRKY9, its full-length open reading frame (ORF) was constructed into the pCAMBIA1300-35S-GFP. The constructed fusion vector, containing green fluorescent protein (GFP), along with a nuclear marker, NLS-mCherry (red fluorescent protein), was transiently expressed in 4-week-old N. benthamiana leaves using the Agrobacterium tumefaciens GV3101 strain. The fluorescence signal was observed under a confocal laser scanning microscope (Leica SP8, Leica, Germany).

Cis-elements analysis of promoter sequences

Genome DNA was extracted using the Plant Genomic DNA extraction Kit (Aidlab Biotech, China) and used as a template. The specific primers for AsFMO1 promoters were designed based on previous literature report [15], and the CDS region of AsFMO1 was amplified using the genome walking technique. The primers used are listed in Additional file 3: Table. S2. Then, the online database PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to identify the cis-acting elements of promoters.

Chromatin Immunoprecipitation PCR

ChIP was performed with the transgenic garlic callus harboring 35S:GFP-AsWRKY9 using the method reported in the literature [39]. The ChIP DNA products were analyzed by qRT-PCR using primers designed to amplify DNA fragments in the promoter regions of AsFMO1 genes. The primer sequences for W-box were used for AsFMO1 promoters respectively, and the primer sequences AsFMO1 CDS served as an internal control (Additional file 3: Table. S2). These experiments were repeated more than three times.

Yeast one-hybrid assays

To confirm the interaction between AsWRKY9 and the promoter of AsFMO1, Y1H assays were performed using the blue-white selection. The full-length cDNA of AsWRKY9 was cloned into the EcoR I sites of the pB42AD activation vector to form pB42AD-AsWRKY9. The promoter fragment containing the putative W-box of AsFMO1 promoter was amplified from the genome DNA of garlic with primers in Additional file 3: Table. S2. This fragment was then divided into three parts, and these fragments were cloned into the Xho I sites of the p178 vector to form p178-AsFMO1-F1/F2/F3, p178-mAsFMO1-F3 using ClonExpress II One step Clonging Kit (Vazyme, China). Then, the vectors were co-transformed into the yeast strain EGY48Gold using LiAc conversion protocols. Transformed yeast cells were dropped onto a selective medium containing synthetic dextrose (SD) without Ura and Trp (SD/-Trp/-Ura) and then screening for blue and white spots.

Electrophoretic mobility shift assays

AsWRKY9 CDS segment was subcloned into pGEX-4 T-1 vector to form GST-AsWRKY9 in which GST-tag was fused into the N-terminal of the AsWRKY9. The resulting plasmid was transformed into Escherichia coli Rosetta (DE3). The 3′-end biotin W-box biotin probe corresponding to the W-box site was prepared (Sangon Bio, China) (Additional file 3: Table. S2). The W-box biotin was without biotin label and seted as competitor probe. The EMSA were performed using LightShift Chemiluminescent EMSA Kit (Thermo Scientific, USA) according to the manufacturer’s instructions.

Dual-luciferase reporter assays

To further investigate the regulation of AsFMO1 expression by AsWRKY9 protein, LUC reporter assays were performed. For transcriptional activity analysis, the coding region of AsWRKY9 was cloned into the pHB-GFP vector with Hind III under the control of the 35S promoter as effector (35Spro::AsWRKY9). The promoter sequence of AsFMO1 was inserted into a pGreenII 0800-Luc vector and then co-transformed with 35Spro::AsWRKY9 or free pHB vector (35Spro, set as a negative control) into the tobacco leaves using an Agrobacterium-mediated method as described previously. After being cultivated in darkness for 6 h and under long-day conditions (16 h/8 h, day/night) for 36 h, the transformed leaves were sprayed with D-luciferin sodium salt (Solarbio, Beijing, China) and then were examined by using a Tanon 5200 multi-imaging apparatus (Tanon, Shanghai, China). Each assay was performed with three biological replicates. The sequences of primers were listed in Additional file 3: Table. S2.

Enzyme activity determination

To evaluate the enzyme activity of FMO using the consumption of NADPH, prepare a standard curve by measuring the OD340 values at NADPH concentrations of 0.02, 0.04, 0.06, 0.14, 0.18, and 0.20 mM. Fresh garlic callus tissue was collected, immediately frozen in liquid nitrogen, and ground to a fine powder. The ground tissue was then suspended in chilled extraction buffer and gently mixed. The suspension was then centrifuged at high speed at 4 °C to remove insoluble cell debris and impurities, and the supernatant was collected as the crude extract. The enzyme activity reaction components include the following: 50 mM Tris–HCl (pH = 7.8), 500 μM NADPH, 1 mM DTT, and 0.1 mM EDTA. The OD340 value changes over 5 min were recorded using a multifunctional microplate reader, and the enzyme activity was calculated based on these absorbance changes. Under specific conditions, the amount of enzyme that consumes 1 μM of NADPH per minute is defined as one enzyme activity unit. The calculation formula is as follows:

Δc:change in substrate concentration; Δt: reaction time; V: total volume of the reaction system.

Determination and analysis of alliin content

Garlic callus was collected, rapidly frozen in liquid nitrogen, and finely ground to prevent alliin degradation during processing. The powdered garlic was then mixed thoroughly with 4% sulfosalicylic acid (contains protease inhibitor cocktail), which helps protect alliin from oxidation. Then stored at 25 °C for 30 min. Centrifugation at 12,000 r/min for 20 min was then performed, and the content of alliin in the supernatant was determined by S-4330D amino acid analyzer. The alliin content is calculated using an automated process by the amino acid analyzer, which considers the standard concentrations, the peak areas from chromatographic analysis, the volumes of the samples introduced, and the dilution factors applied. This approach ensures precise quantification of the alliin mass fraction in the sample. To ensure the accuracy of the data, at least three replicates were obtained per tissue sample. Values represent the mean ± standard error.