Effects of BrMYC2/3/4 on Plant Development, Glucosinolate Metabolism, and Sclerotinia sclerotiorum Resistance in Transgenic Arabidopsis thaliana

MYC2/3/4, known as a basic helix–loop–helix (bHLH) transcription factor, directly activate the genes involved in diverse plant development and secondary metabolites biosynthesis. In this study, we identified and cloned five MYC paralogs (BrMYC2/3-1/3-2/4-1/4-2) from Chinese cabbage (Brassica rapa ssp. pekinensis). In-silico analyses for the physicochemical properties suggested that BrMYC2/3-1/3-2/4-2/4-3 are unstable hydrophobic and acidic proteins, while BrMYC4-1 is an unstable hydrophobic and basic protein. BrMYC2/3/4 belong to the bHLH superfamily and are closely related to AthMYC2/3/4 orthologs that mediate the regulation of various secondary metabolites. It was demonstrated that BrMYC2/3/4-GFP fusion protein localized in the nucleus and expression levels of five BrMYC2/3/4 homologous genes all elevated relative to control (Ctrl). When expressed in Arabidopsis under the control of 35S promoter, each of the BrMYC2/3-1/3-2/4-1/4-2 transgenes differentially influenced root and shoot elongation, vegetative phase change, flowering time, plant height and tiller number after flowering, and seed production. Despite the variation of phenotypes between the transgenic lines, all the lines except for BrMYC4-2 exhibited shorter seed length, less seed weight, higher accumulation of glucosinolates (GSs), and resistance to Sclerotinia sclerotiorum than Ctrl. Notably, BrMYC2 overexpression (OE) line significantly reduced the lengths of root and hypocotyl, seed length, and weight, along with faster bolting time and strikingly higher accumulation of total GSs. Accumulation of GSs at the highest levels in the BrMYC2OE line conferred the highest resistance to S. sclerotiorum. Unlike BrMYC3OE and BrMYC4OE, BrMYC2OE stimulated the growth of plant height after fluorescence. The results of this study point to the BrMYC2 overexpression that may provide a beneficial effect on plant growth and development via plant resistance to the fungal pathogen.

Glucosinolates are a group of secondary metabolites found in Brassicaceae family and are composed of β-D-thioglucose and sulfonated oxime moieties (Tiwari, 2018). They have received considerable interest because of their commercial properties of anti-cancer agents (Arumugam and Razis, 2018), bio-pesticides (Malka and Cheng, 2017), and flavor condiment (Burow et al., 2015). They are typically classified as aliphatic GS (from alanine, valine, leucine, isoleucine, or methionine), aromatic GS (from phenylalanine or tyrosine), or indole GS (from tryptophan) on the basis of their modified side chains (R) derived from amino acids through a long chain lengthening process and hydroxylation or oxidation (Rahimi and Rahmanpour, 2020).
Evolutionary data suggest that Chinese cabbage is closely related to A. thaliana and that it underwent a whole-genome triplication since it has diverged from Arabidopsis thaliana (Wang et al., 2011). There are one, two, and three copies of BrMYC2/3/4 family genes in Chinese cabbage, respectively. Functions of MYC2/3/4 orthologs have been mainly investigated in A. thaliana, and they showed a functional divergence (Schweizer et al., 2013). However, the functions and regulatory roles of multiple paralogous copies of BrMYC2/3/4 family genes have remained unresolved.
In the current study, we identified, cloned five MYC2/3/4 paralogs (BrMYC2, BrMYC3-1, BrMYC3-2, BrMYC4-1, and BrMYC4-2) in Chinese cabbage, and conducted in-silico comparative studies on physicochemical properties, domains, and evolutionary relationships among the deduced BrMYC2/3/4 proteins. In order to study the phenotypes conferred by expression of BrMYC2/3/4, each of the cloned coding sequence of five MYC2/3/4 paralogs was introduced into a binary vector pCAMBIA2302, and each chimeric transgene expression construct was transformed into Arabidopsis. Subsequently, T2 lines showing 3:1 segregation ratio of selectable marker gene were selected, and T3 lines were used to analyze various phenotypes of plants at juvenile and adult stage, GSs contents, and resistance to white mold caused by necrotrophic Sclerotinia sclerotiorum.

In-silico Analyses
The amino sequences deduced from coding sequences were used to predict the functional properties of BrMYC2/3/4 proteins. Physiochemical properties were inferred using ProtParam software (http://web.expacy.org/protparam/). The percentages of sequence identities among and between MYC2/3/4 in terms of coding sequence and deduced amino acid sequence were analyzed using DNAMAN software version 6.0 (Lynnon Biosoft, Montreal, Canada). The domains were predicted using the conserved domain database (CDD, https://www.ncbi.nlm.nih. gov/Structure/cdd/wrpsb.cgi), which has a domain prediction tool provided by the National Center for Biotechnology Information (NCBI, US National Library of Medicine). Structural and evolutionary analyses of MYC2/3/4 coding sequences in the U's triangle species were conducted with homologous sequences on Brassica database (http://brassicadb.cn/#/BLAST/) according to the neighbor-joining method (Tarahomi et al., 2020) using molecular evolutionary genetics analysis software (MEGA7 software) (Kumar et al., 2016). The main parameter settings were distance model, p-distance method (Sáez-López et al., 2019), gene tree robustness detection, bootstrap test (1,000 replicates) (Elateek et al., 2020), processing of gap missing data, and deletion between pairs.

Expression Constructs
All cloning experiments were carried out through homologous recombination using the One Step Cloning Kit (Vazyme, Nanjing, China). The coding sequences of BrMYC2, BrMYC3-1, BrMYC3-2, BrMYC4-1, and BrMYC4-2 were put under control of the constitutive CaMV 35S promoter in the binary vector pCAMBIA2302 with green fluorescent protein (GFP). The integrity of the cloned coding sequence was verified by colony PCR, followed by DNA sequencing.

Plant Materials and Growth Conditions
Arabidopsis ecotype Col-0 was used as the wild-type for all experiments. Plant seeds were sown in a mixture of grass charcoal soil and vermiculite as 1:1 ratio and cultivated in a greenhouse for 2 months under well-controlled conditions of 65% relative humidity and 600 µmol·m −2 ·s −1 maximum light intensity.
Arabidopsis seeds from T0 lines were first surface sterilized using 75% (v/v) ethanol for 90 s and then washed three times with double-distilled water (ddH 2 O). The seeds were further sterilized in 10% bleach for 10 min and washed three times with ddH 2 O. They were subsequently sown onto 1× MS medium (Phyto Technology Laboratories, Shawnee Mission, KS, USA) supplemented with 3% sucrose (pH 5.8 and 0.8% (w/v) plant tissue culture agar) and 50 µg·ml −1 kanamycin (Phyto Technology Laboratories, Shawnee Mission, KS, USA). Seedlings were chilled at 4 • C for 2 days and transferred to a growth room with controlled conditions (25 • C, 16:8 h, light:dark regime) grown vertically for 4 days. Healthy seedlings were then transferred to the soil and cultivated in a greenhouse with controlled conditions as described above. Homozygous plants were selected at the T3 generation based on the 3:1 segregation ratio of T2 line in the presence of kanamycin.
In order to observe GFP fluorescence, 5-day-old kanamycin resistant seedlings were used under a confocal laser scanning microscope, Olympus FluoView FV10i (Olympus, Tokyo, Japan). Images were recorded and contrast-enhanced using ImageJ software (ImageJ, 1.47v, NIH, Bethesda, USA). Some seedlings were subjected to plasmolysis in 1 M mannitol for 5 min (Xiao et al., 2014). GFP fluorescence was detected using a 488-nm excitation laser and 525/50-nm emission filter.
Leaf samples obtained from different T3 transgenic lines were harvested, frozen in liquid nitrogen, and stored at −80 • C until later use.

Semi-quantitative RT-PCR for Detection of Gene Expression Levels
Semi-quantitative RT-PCR to detect RNAs was carried out using total RNA isolated from 8-day-old seedlings. Total RNAs were isolated using the TRIzol reagent kit (Invitrogen) and treated with RNase-free DNase to remove any genomic DNA contaminants. All RNA samples were quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and were adjusted to the same concentration with diethylpyrocarbonate-treated water. Complementary DNA (cDNA) was synthesized using the PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara Bio, Inc, Otsu, Japan) following the protocol of the manufacturer using an oligo dT primer and 1 µg of total RNA. PCR was then performed using gene-specific primers and KOD Plus Neo (Toyobo, Osaka, Japan). The reaction was initiated by predenaturating at 94 • C for 2 min, followed by 35 cycles of denaturation (98 • C for 10 s), annealing (50 • C for 30 s), and extension (68 • C for 30 s), and was terminated with a final extension of 10 min at 68 • C. The amplification products were analyzed by 1.2% agarose gel electrophoresis.
AtActin2, a housekeeping gene of A. thaliana, was used as an internal control to adjust the amount of cDNA template for PCR. Stably expressed AtActin2 (AT3G18780) was used for normalization. MYC2/3/4 and Atactin2 primers are shown in Supplementary Table 1. Gel images were quantified using Gelpro 3.2 software (Media Cybernetics, Inc., Rockville, MD, USA). Three biological replicates were carried out for each sample.

Seed Production Evaluations
In order to determine the seed yield, 30 T3 lines were taken from each genotype. After the siliques matured, the first five incompletely developed siliques were removed from the main inflorescence axis as described before (Jiang et al., 2020). The first intact siliques were then used to assess the number of seeds per silique. The mature siliques were tiled on A4 paper, and the carpel wall was removed with a dissecting needle, photographed with a Leica stereomicroscope (MZ16FA, Leica, Germany) to count the seed number per silique. The remaining siliques were allowed to mature, and seeds from the siliques located on the basis of a major inflorescence were selected for observation. Approximately 2,000 mature seeds from Ctrl and BrMYC2/3-1/3-2/4-1/4-2 OE of each line were randomly selected, observed, and photographed using a Leica stereomicroscope. The length and width of seeds were estimated using ImageJ software. Seed weight was measured using an electronic scale. Data points represent the average of 300 biological replicates.

Plant Phase Change Evaluations
To evaluate the phase change among different genotypes, ∼30 seedlings were randomly selected from each T3 line, transferred to an autoclaved mixture of grass charcoal soil and vermiculite a 1:1 ratio, and cultivated in the greenhouse for about 2-3 weeks. In order to assess whether the juvenile seedlings have reached the mature stage, abaxial trichomes as a hallmark of vegetative phase change were scored with a Leica stereomicroscope. For leaf shape analysis, fully expanded leaves were removed, attached to a cardboard with double-sided tape, flattened with transparent tape, and then scanned using the Epson V700 Professional scanner (Epson, Suwa, Japan). The bolting time and rosette leaf number at bolting time were recorded to determine the flowering time in A. thaliana (Xing et al., 2018). Plant height and tiller number were determined just after flowering. Pictures were taken from the plant adjacent to a ruler that was used to calibrate the ImageJ software for precise measurement of distance. Maximum height (from the growing point to the highest leaf tip as depicted by the vertical line) was estimated from the silhouette of A. thaliana differing in size. Measurements were obtained from 12 independent biological replicates.

GSs Contents Determination
The leaves of 8-week-old homozygous T3 generation transgenic A. thaliana lines were used for determining GSs contents. Six seedlings were randomly selected from one transgenic line. GSs were essentially quantified as previously described (Zang et al., 2015). High-performance liquid chromatography (HPLC) analysis was performed using an Agilent1200 system (Agilent Technologies, Inc., Santa Clara, USA) with a C18 reversephase column (250 mm × 4 mm, 5 µm, Bischoff, Germany). Chromatography was performed over 60 min at a flow rate of 1 ml·min −1 in the following order: 100% H 2 O (2 min), a linear gradient of 0-20% ACN (32 min), 20% ACN (6 min), followed by 20-100% ACN (5 min), and 0% ACN before injecting the next sample. Eluents were monitored with a UV detector at 229 nm. Three biological and three technical replicates were performed.

Antifungal Activity Bioassay
Lyophilized leaf powder of each transgenic A. thaliana line was used to study the effect of different GSs on the visible growth of S. sclerotiorum. Anti-fungal activity was essentially assayed as previously described with minor modifications (Kelemu et al., 2004). S. sclerotiorum was preserved at 4 • C and then reactivated in a Petri dish containing potato dextrose agar (PDA) medium (Becton Dickinson, Columbia, MD). The mycelium was inoculated into the center using a 5 mm puncher; the Petri dishes were then incubated at 22 • C for 72 h to provide actively growing mycelium for subsequent experiments. New marginal hyphae were excised with a puncher from the Petri dishes. Filter paper disks of 10 mm in diameter were then placed onto the surface of PDA. Each filter-paper disk received 25 mg lyophilized powder and 100 µl ddH 2 O, along with 100 µl ddH 2 O only as a control. None of the three filter paper disks was a PDA Petri dish, and each of the three Petri dishes was a biological repeat. The filterpaper disks carrying lyophilized powder and S. sclerotiorum were incubated at 22 • C. Fungal growth was assessed by observing visible mycelium growth and sclerotia number per disk after 72 h of incubation. Three technical replicates were performed.

Statistical Analysis
The data were evaluated using analysis of variance to determine statistical significance and were represented as means ± SD, as calculated by SPSS20.0 analysis software (IBM, Chicago, IL, USA). A difference was considered significant at the 95% confidence level (p < 0.05). All photographs were taken with a Nikon D5300 camera (Nikon Corporation, Tokyo, Japan) and edited in ImageJ. Graphs were plotted using Excel 2019 (Microsoft, Redmond, WA, USA) and figures were assembled using Microsoft PowerPoint.

Structural Analyses of BrMYC2/3/4
Analysis of physicochemical properties showed that BrMYC2 consist of 605 amino acid residues, with a relative molecular mass of 65,848.54 and theoretical isoelectric point (pI) of 5.21, which suggests an acidic protein. Total average hydrophilicity and instability index are −0.548 and 45.84, respectively. Based on these values, BrMYC2 is an unstable hydrophobic and protein. BrMYC3-1 and BrMYC3-2 consist of 563 and 580 amino acid residues, with relative molecular masses of 61,940.76 and 63512.36, theoretical pI of 5.05 and 5.15, respectively.

Domain Prediction of BrMYC2/3/4
The protein domains encoded by BrMYC2/3/4 coding sequences were predicted using CDD, an NCBI online domain analysis software. The results showed that BrMYC2, BrMYC3-1, BrMYC3-2, and BrMYC4-2 have a conserved domain named bHLH-MYC_N superfamily; BrMYC2, BrMYC3-1, BrMYC3-2, and BrMYC4-1 have a conserved domain named bHLH_AtABAinducible bHLH-TYPE_like in the C-terminal region; and BrMYC4-3 contains a single domain named bHLH-MYC_N (Figure 1). A bHLH_AtAIB_like is the bHLH-type domain found in AIB and MYC proteins (MYC2, MYC3, and MYC4) of A. thaliana. AIB is an abscisic acid (ABA)-inducible transcriptional repressor that negatively regulates JA signaling (Nakata et al., 2013). These domain features implicate that BrMYC2/3/4 belongs to the bHLH superfamily of TFs mediating the positive and negative regulation of plant development and various secondary metabolites synthesis.

Evolutionary Analyses of BrMYC2/3/4
A cladogram of MYC2/3/4 in the U's triangle species genomes was constructed according to the neighbor-joining method. As shown in Figure 2, Brassicaceae MYC2/3/4 family members exhibited a divergent evolution. BraMYC2 (B. rapa) forms a clade with BnaMYC2 (B. napus), and they form a clade with AthMYC2 (A. thaliana) by a high nodal support value. BraMYC3-1 forms a clade with BnaMYC3-1, BjuMYC3-2 (B. juncea), and BjuMYC3-1. BraMYC3-2 forms a clade with BnaMYC3-2. BraMYC3-1 and BraMYC3-2 make up a clade with AthMYC3 by a high nodal support value. BraMYC4-1, 4-2, and 4-3 share an evolutionary lineage with the middle part, the first half, and a small part of the second half of AthMYC4, respectively. All these phylogenetic relationships are well in accordance with the classification based on morphological and biochemical characteristics of Brassicaceae plants.

Transgenic Plants Obtain and Differential Expression of BrMYC2/3/4 Genes
To determine the obtain of transgenic plants, the root tip of resistant Arabidopsis was observed using spinning disk confocal microscopy. BrMYC2/3/4-GFP fusion protein fluorescence signal was observed under the control of CaMV 35S promoter (Figure 3).
Semi-quantitative RT-PCR was used to estimate the expression levels of five BrMYC2/3/4 homologous genes in BrMYC OE transgenic lines relative to Ctrl. Primers were designed from the gene-specific region (Supplementary Table 1). In the randomly selected three BrMYC2/3/4 OE transgenic lines, BrMYC2/3/4 expression levels were all elevated (Supplementary Table 2). In BrMYC2 OE , BrMYC2 expression levels elevated from 8.61 to 11.07 folds relative to Ctrl. In BrMYC3 OE , BrMYC3-1 expression levels elevated from 5.72 to 7.50 folds, and BrMYC3-2 expression levels elevated from 2.97 to 4.43 folds. In BrMYC4 OE , BrMYC4-1 expression levels elevated from 9.32 to 10.49 folds, and BrMYC4-2 expression levels elevated from 11.32 to 11.43 folds.

Effects of BrMYC2/3/4 Overexpression on Seed Production
Since BrMYC4-3 was only 63 bp and its predicted domain function was completely different from others, only BrMYC2/3-1/3-2/4-1/4-2 were cloned and transformed for further expression analysis. Transgenic Arabidopsis plants of Ctrl and BrMYC2/3-1/3-2/4-1/4-2 OE were analyzed for seed yield in terms of seed size (length and width), 1,000 seed weight, and seed number per silique. In order to minimize the effects of environmental factors on seed development, all plants were kept under identical growing conditions, such as temperature, light, water, and nutrition. As shown in Figure 4, there was a significant difference in seed production among different transgenic plants. BrMYC2 OE plant had the smallest seed length; there was no significant difference in seed length among BrMYC3-2 OE , BrMYC4-1 OE , and BrMYC4-2 OE , all of which were significantly smaller than that of Ctrl ( Figure 4C). BrMYC3-2 OE and BrMYC4-1 OE plants possessed the smallest seed width; there was no significant difference in seed width between BrMYC2 OE and BrMYC4-2 OE , as well as between BrMYC3-1 OE and Ctrl (Figure 4D). Thousand seed weight significantly decreased in all the transgenic lines as compared with Ctrl ( Figure 4E). BrMYC2 OE line contained the smallest seed weight, followed by BrMYC3-2 OE , BrMYC4-1 OE , BrMYC4-2 OE , and BrMYC3-1 OE . Thousand seed weight of BrMYC3-1 OE increased more than those of BrMYC3-2 OE and BrMYC4-1 OE , while there was no significant difference Tested transgenic lines are Ctrl, BrMYC2 OE , BrMYC3-1 OE , BrMYC3-2 OE , BrMYC4-1 OE , and BrMYC4-2 OE . Seeds and siliques were randomly selected and pictures were taken under the same conditions. Data are shown as mean ± SD from three independent biological replicates. ANOVA was used for statistical analysis, followed by Tukey's multiple comparison test (p < 0.05). Bar = 500 µm. among those of BrMYC3-2 OE , BrMYC4-1 OE , and BrMYC4-2 OE , as well as between BrMYC4-2 OE and BrMYC3-1 OE . BrMYC2 OE line contained the lowest seed number per silique, while all other transgenic lines had significantly higher seed numbers than Ctrl (Figures 4B,F). BrMYC4-1 OE line produced the highest seed number per silique followed by BrMYC3-2 OE , BrMYC3-1 OE , and BrMYC4-2 OE . Taken the results together, constitutive expression of BrMYC2/3/4 TFs differentially affected the seed production with respect to the size, weight, and number of seed. Overexpression of BrMYC3-1/3-2/4-1/4-2 had a negative effect on both the seed length and seed weight and a positive effect on seed number per silique. In contrast, overexpression of BrMYC2 led to a negative effect on the seed length, seed weight, and seed number per silique.

Effects of BrMYC2/3/4 Overexpression on Plant Development
To determine if BrMYC2/3/4 expression influences vegetative and reproductive development, we examined the phenotypes of transgenic BrMYC2/3/4 plants at the seedling and bolting stage.
About 3-4 weeks after seeds were sown, Arabidopsis seedlings transitioned from juvenile to adult vegetative growth phase (Figure 6). The leaf shape of transgenic plants was normal and indistinguishable from that of Ctrl. BrMYC2/3-1/3-2/4-1 OE lines produced smaller number of the first leaves having trichomes than Ctrl, whereas BrMYC4-2 OE produced a larger number of them on leaf than Ctrl (Figure 6B). For the leaf initiation rate, BrMYC2/3-1/4-1/4-2 OE was significantly faster than Ctrl, but BrMYC3-2 OE was similar to Ctrl ( Figure 6C).
As for the reproductive stage, we studied bolting time, rosette leaf number at the bolting time, plant height, and tiller number (Figure 7). BrMYC2 OE line had significantly faster bolting time, while BrMYC3-2 OE and BrMYC4-2 OE lines had significantly slower bolting time than Ctrl. Both BrMYC3-1 OE and BrMYC4-1 OE were similar to Ctrl in bolting time (Figures 7A,B). The number of rosette leaves at bolting time significantly decreased in BrMYC2/3-1/4-1 OE lines but increased in BrMYC3-2/4-2 OE lines as compared with Ctrl (Figures 7A,C). Previously, flowering time was recorded as the number of rosette leaves at bolting, with the observation of late flowering  Leaf initiation rate of transgenic Arabidopsis in short days. Leaf numbers were scored at 12, 16, 20, 24, and 28 days after planting. Transgenic lines are Ctrl, BrMYC2 OE , BrMYC3-1 OE , BrMYC3-2 OE , BrMYC4-1 OE , and BrMYC4-2 OE . Data are shown as mean ± SD from three independent biological replicates. ANOVA was used for statistical analysis, followed by Tukey's multiple comparison test (p < 0.05). Bar = 1 cm.
plants having more rosette leaves (Lopez-Vernaza et al., 2012). Thus, it appears that BrMYC2 OE , BrMYC3-1 OE , and BrMYC4-1 OE promoted flowering time, while BrMYC3-2 OE and BrMYC4-2 OE inhibited it in Arabidopsis. For the plant height, BrMYC2 OE significantly increased, while BrMYC3-2 OE , BrMYC4-1 OE , and BrMYC4-2 OE significantly decreased as compared with Ctrl. And there was no significant difference between BrMYC3-2 OE and BrMYC4-2 OE , as well as between BrMYC3-1 OE and Ctrl (Figures 7A,E). Tiller number of BrMYC2 OE and BrMYC3-1 OE was significantly higher than Ctrl and no significant difference was noted among BrMYC3-2 OE , BrMYC4-1 OE , BrMYC4-2 OE , and Ctrl (Figures 7A,F). Thus, expression of BrMYC2 only promoted the growth of both shoot elongation and plant height after fluorescence with faster bolting time.

Anti-fungal Activity
To study the extent of resistance of transgenic lines to S. sclerotiorum, 25 mg of the lyophilized powder of each transgenic line's leaf tissue was uniformly sprinkled around the hyphae. The visible growth of cottony mycelium of plaque was observed after 72 h of S. sclerotiorum incubation (Figure 8). The thinnest plaque was noted in BrMYC2 OE line, followed by BrMYC3-1 OE , BrMYC4-1 OE , BrMYC3-2 OE , and BrMYC4-2 OE . Ctrl displayed much thinner plaque than water control, suggesting that endogenous basal levels of GSs present in the vector control line inhibited the growth of S. sclerotiorum to a certain degree. However, Ctrl showed thicker plaques than BrMYC2/3/4 OE lines. This may result from the suppression of the mycelium growth by higher contents of GSs accumulated in BrMYC2/3/4 OE lines.

DISCUSSION
Our in-silico analysis indicated that BrMYC2/3-1/3-2/4-2/4-3 are unstable hydrophobic and acidic proteins, whereas BrMYC4-1 is an unstable hydrophobic and acidic basic protein. BrMYC2/3/4 TFs were predicted to contain the bHLH AtAIB-like domain whose protein is involved in the positive regulation of ABA signaling in Arabidopsis (Figure 1). Notably, the molecular weight of BrMYC4-3 was far smaller and its domain function was very different from others. Phylogenetic relationship analyses indicated that BrMYC2/3/4 were closely related to AthMYC2/3/4 orthologs from the Brassicaceae family.
When expressed in Arabidopsis under the control of 35S promoter, each member of the BrMYC2/3-1/3-2/4-1/4-2 transgenes differentially influenced root and shoot elongation, vegetative phase change, flowering time, plant height and tiller number after fluorescence, and seed production. Seed size is a key agronomic trait that determines the grain yield and breeding of plants (Shirley et al., 2019). The number and weight of the seed ultimately determine seed yield (Jiang et al., 2020). In this study, we found that as a result of overexpression of each of BrMYC2/3-1/3-2/4-1/4-2, both the seed length and seed weight decreased (Figures 4C,E), while seed number per silique increased except for BrMYC2 OE as compared to Ctrl (Figure 4F). The results appear to be consistent with a finding that MYC2, MYC3, and MYC4 act additively during seed development and   that triple mutants produced the largest seeds with more seed storage proteins, while the seeds of single and double mutants were much larger than those of wild type (Gao et al., 2016). Hence, overexpression of the individual BrMYC2/3-1/3-2/4-1/4-2 family genes appears to have a negative effect on seed size and seed weight.
To examine the effect of BrMYC expression on plant development, the lengths of root and hypocotyl of seedlings were measured. We found that BrMYC2 expression significantly inhibited both root and hypocotyl elongation, whereas BrMYC3-2/4-1/4-2 expression significantly promoted both root and hypocotyl elongation when compared to Ctrl (Figure 5). In contrast, BrMYC3-1 expression significantly inhibited root elongation but promoted hypocotyl elongation. Thus, expression of the duplicated copies of BrMYC3, BrMYC3-1, and BrMYC3-2, did not lead to the same effect on the root length but on the hypocotyl length. This is an interesting observation, suggesting that they have divergent functions and regulatory roles in plant development. In A. thaliana, the transition from juvenile to adult vegetative phase referred to as "vegetative phase change" is signified by the formation of trichomes on the abaxial side of leaf blade and an increase in the leaf length/width ratio during shoot development (Xu et al., 2019). Our studies on abaxial trichome production and leaf initiation rate as shown in Figure 6 suggested that BrMYC2/3-1/4-1 OE positively regulate the time of vegetative phase change in Arabidopsis, since the less number of the firstformed leaves having abaxial trichomes relative to control was correlated with promoting floral transition (Chien and Sussex, 1996). Previous studies showed that the plant height determines its ability to compete for light and therefore often correlates with the leaf mass and seed production, and that stem growth is initiated once the plant becomes reproductive and continues until termination of the inflorescence meristems (Serrano-Mislata et al., 2017;Moles et al., 2019). Based on the bolting time and rosette leaf number at bolting time, it appears that BrMYC2/3-1/4-1 OE promoted flowering time, while BrMYC3-2/4-2 OE inhibited it (Figure 7). Hence, expression of each set of the duplicated copies of BrMYC3 (3-1/3-2) and BrMYC4 (4-1/4-2) led to the opposite action on flowering time, suggesting that they have divergent function and regulatory roles in fluorescence. After flowering, BrMYC2 OE promoted the growth of the plant height, whereas BrMYC3-2/4-1/4-2 OE repressed it, suggesting that the three MYC TFs play different roles in signaling pathways. Notably, BrMYC2/3-1 OE displayed a remarkable increase in tiller number after fluorescence. The earlier flowering phenotype of overexpressed BrMYC2 seems to be consistent with a previous finding that expression of all three MYC2/3/4 was required to inhibit flowering in Arabidopsis .
Along this line, it was reported that MYC2 regulates diverse functions within the JA signaling pathway and that MYC2 forms homo-and/or heterodimers with MYC3 and MYC4 and binds to the conserved G-box present in the promoters of JA-responsive genes (Kazan and Manners, 2013). Moreover, Zhai et al. (2013) reported that high and low accumulation of the MYC2 protein correlated with positive regulation of early wound-responsive genes and negative regulation of late pathogen-responsive genes, respectively, and that MYC activity was further regulated by phosphorylation and the ubiquitinproteasome system-mediated proteolysis. Accordingly, a wide range of differential effects of each BrMYC2/3/4 expression on the root and shoot elongation, vegetative phase change, flowering time, and seed production implicates MYC-mediated complex signaling networks for positive and negative regulation of plant growth and development to connect environmental stresses with developmental signals at both transcriptional and posttranscription levels.
Modulation of GS content affected plant resistance to pathogenic infection in Arabidopsis (Madloo et al., 2019). Brassicaceae plants typically produce GSs which are hydrolyzed by myrosinases upon tissue damage to generate ITCs, toxic bioactive compounds acting against broad pathogens. 4MeGBC was activated by the atypical PEN2 myrosinase (a type of βthioglucoside glucohydrolase) for antifungal defense (Bednarek et al., 2009). Fungal infection triggered the accumulation of both IGS and AGS in B. rapa (Abdel-Farid et al., 2010). In B. rapa, S. sclerotiorum inoculation in vivo induced GS accumulation and increased GS biosynthesis-related proteins (Teng et al., 2021). In our study, the lyophilized leaf powder of each Arabidopsis transgenic line was uniformly sprinkled around the hyphae in order to assess anti-fungal activity against S. sclerotiorum (Figure 8). PDA plate sprinkled with lyophilized powder of BrMYC2 OE line showed the thinnest fungal hyphae plaque, followed by those of BrMYC3-1 OE and BrMYC4-1 OE lines. Such a varying degree of fungal resistance exhibited by the overexpression lines seemed to be in line with the accumulation levels of GS. IGSs such as GBC, 4MeGBC, and NeoGBC, as well as GHT accumulated at a higher level in the order of BrMYC2 OE , BrMYC3-1 OE , and BrMYC3-1 OE lines (Table 2). GBC, 4MeGBC, and NeoGBC were not metabolized by S. sclerotiorum (Pedras and Hossain, 2011). Besides, a previous study showed that aliphatic ITCs inhibited the growth of S. sclerotiorum in vitro, and that anti-microbial activity of AGS-derived isothiocyanates was dependent on side chain elongation and modification, with glucohirsutin (GHT) being most toxic to S. sclerotiorum (Stotz et al., 2011).

CONCLUSIONS
In summary, Arabidopsis plants expressing each of the five MYC paralogous genes of Chinese cabbage exhibited a wide range of differing phenotypes with respect to the root and shoot elongation, vegetative phase change, flowering time, plant height and tiller number right after flowering, and seed production. Despite the wide variation of phenotypes between the transgenic lines, all of the lines except for BrMYC4-2 OE exhibited shorter seed length, less seed weight, higher accumulation of GSs, and resistance to S. sclerotiorum than Ctrl. Notably, the highest GSs level accumulated in BrMYC2 OE line was correlated with the highest extent of resistance to the necrotic fungal pathogen S. sclerotiorum. Unlike BrMYC3-1/3-2/4-/4-2, BrMYC2 expression stimulated the growth of plant height after fluorescence with a faster bolting time. The results presented here indicate that despite the complexity of GSs biosynthesis and metabolism regulated in the positive and negative manner by many other TFs, BrMYC2 expression alone was far more effective to positively regulate the biosynthesis of both AGS and IGC than BrMYC3 and BrMYC4, and thus may provide the beneficial effects on the plant growth and development via resistance to fungal pathogens.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

AUTHOR CONTRIBUTIONS
YZ conceived and designed the experiments and performed the analysis with discussions. ZT performed the experiment and data analysis. ZT, YY, and ZZ contributed to manuscript preparation. ZT and YZ wrote the original draft. S-BH, WZ, and ZZ edited the manuscript. All authors contributed to the article and approved the submitted version.