The BpMYB4 Transcription Factor From Betula platyphylla Contributes Toward Abiotic Stress Resistance and Secondary Cell Wall Biosynthesis

The MYB (v-myb avian myeloblastosis viral oncogene homolog) family is one of the largest transcription factor families in plants, and is widely involved in the regulation of plant metabolism. In this study, we show that a MYB4 transcription factor, BpMYB4, identified from birch (Betula platyphylla Suk.) and homologous to EgMYB1 from Eucalyptus robusta Smith and ZmMYB31 from Zea mays L. is involved in secondary cell wall synthesis. The expression level of BpMYB4 was higher in flowers relative to other tissues, and was induced by artificial bending and gravitational stimuli in developing xylem tissues. The expression of this gene was not enriched in the developing xylem during the active season, and showed higher transcript levels in xylem tissues around sprouting and near the dormant period. BpMYB4 also was induced express by abiotic stress. Functional analysis indicated that expression of BpMYB4 in transgenic Arabidopsis (Arabidopsis thaliana) plants could promote the growth of stems, and result in increased number of inflorescence stems and shoots. Anatomical observation of stem sections showed lower lignin deposition, and a chemical contents test also demonstrated increased cellulose and decreased lignin content in the transgenic plants. In addition, treatment with 100 mM NaCl and 200 mM mannitol resulted in the germination rate of the over-expressed lines being higher than that of the wild-type seeds. The proline content in transgenic plants was higher than that in WT, but MDA content was lower than that in WT. Further investigation in birch using transient transformation techniques indicated that overexpression of BpMYB4 could scavenge hydrogen peroxide and O2.– and reduce cell damage, compared with the wild-type plants. Therefore, we believe that BpMYB4 promotes stem development and cellulose biosynthesis as an inhibitor of lignin biosynthesis, and has a function in abiotic stress resistance.

The MYB (v-myb avian myeloblastosis viral oncogene homolog) family is one of the largest transcription factor families in plants, and is widely involved in the regulation of plant metabolism. In this study, we show that a MYB4 transcription factor, BpMYB4, identified from birch (Betula platyphylla Suk.) and homologous to EgMYB1 from Eucalyptus robusta Smith and ZmMYB31 from Zea mays L. is involved in secondary cell wall synthesis. The expression level of BpMYB4 was higher in flowers relative to other tissues, and was induced by artificial bending and gravitational stimuli in developing xylem tissues. The expression of this gene was not enriched in the developing xylem during the active season, and showed higher transcript levels in xylem tissues around sprouting and near the dormant period. BpMYB4 also was induced express by abiotic stress. Functional analysis indicated that expression of BpMYB4 in transgenic Arabidopsis (Arabidopsis thaliana) plants could promote the growth of stems, and result in increased number of inflorescence stems and shoots. Anatomical observation of stem sections showed lower lignin deposition, and a chemical contents test also demonstrated increased cellulose and decreased lignin content in the transgenic plants. In addition, treatment with 100 mM NaCl and 200 mM mannitol resulted in the germination rate of the over-expressed lines being higher than that of the wild-type seeds. The proline content in transgenic plants was higher than that in WT, but MDA content was lower than that in WT. Further investigation in birch using transient transformation techniques indicated that overexpression of BpMYB4 could scavenge hydrogen peroxide and O 2 .− and reduce cell damage, compared with the wild-type plants. Therefore, we believe that BpMYB4 promotes stem development and cellulose biosynthesis as an inhibitor of lignin biosynthesis, and has a function in abiotic stress resistance.

INTRODUCTION
As a ubiquitous transcription factor in plants and animals, MYB proteins are widely involved in the regulation of developmental and metabolic changes in plants. The first MYB gene in plants, ZmMYBC1, was isolated from maize (Paz-ares et al., 1987). Subsequently, a large number of MYB functional genes were isolated and identified from various plants (Dubos et al., 2010). There are at least 196 MYB genes in Arabidopsis (Arabidopsis thaliana), at least 197 in Populus (Populus trichocarpa), and 124 in grapevine (Vitis vinifera L.) (Wilkins et al., 2009). NAC-MYB-based transcriptional regulation of secondary cell wall biosynthesis in land plants is widely known. Plant secondary growth processes are affected by both MYB transcriptional activators and MYB transcriptional repressors, most of which belong to the fourth sub-group of the MYB family (Chen et al., 2002;Vannini et al., 2004). As transcriptional activators, AtMYB46 has been well-studied. Two highly homologous Arabidopsis genes, AtMYB46 and AtMYB83, can be activated by SND1 (SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 1) and its homologous proteins, and its overexpression can activate the biosynthesis of lignin, cellulose and hemicellulose (Zhong et al., 2007;Mccarthy et al., 2009). AtMYB26 regulates the synthesis of secondary cell walls through NST1 (NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1) and NST2, and its overexpression can lead to ectopic deposition of secondary cell walls (Mitsuda et al., 2005;Yang et al., 2007). PtMYB4 from pine (Pinus taeda) and EgMYB2 from eucalyptus (Eucalyptus robusta Smith) belong to the same phylogenetic group as AtMYB46. PtMYB1 and PtMYB4 can bind with AC elements and are expressed in developing wood, causing secondary wall thickening and lignin biosynthesis (Patzlaff et al., 2003b). Overexpression of PtMYB4 in tobacco (Nicotiana tabacum L.) plants can induce the expression of some lignin biosynthesis genes and cause abnormal deposition of lignin and secondary wall thickening (Patzlaff et al., 2003a). PtrMYB003 and PtrMYB020 in Populus are homologous with Arabidopsis AtMYB46 and are specifically expressed in secondary xylem; when overexpressed in Arabidopsis they activate the biosynthesis of cellulose, hemicellulose, and lignin (Mccarthy et al., 2010). Overexpression of AtMYB58 and AtMYB63 promoted the expression of lignin biosynthesis genes, resulting in ectopic deposition of lignin; in contrast, inhibiting their expression reduced the lignin content, resulting in secondary cell wall developmental defects (Zhong et al., 2008;Zhou et al., 2009). In addition, the MYB transcription factor regulates the synthesis of secondary walls by participating in other phenylpropanoid pathways. For example, AtMYB75 mainly regulates the biosynthesis of anthocyanins, but overexpression of this protein also leads to a slight increase in lignin accumulation, indicating that the protein is also involved in the regulation of lignin biosynthesis (Borevitz et al., 2000).
MYBs can also act as transcriptional inhibitors of lignin biosynthesis. For example, transfer of the Antirrhinum (Antirrhinum majus L.) AmMYB308 gene into tobacco resulted in a significant decrease in the expression of C4H (cinnamate-4-hydroxylase), 4CL (4-coumarate:coenzyme A ligase) and CAD (cinnamyl alcohol dehydrogenase) genes, there by effectively inhibiting the biosynthesis of lignin. Transfer of AmMYB300 into tobacco resulted in the inhibition of expression of 4CL (Tamagnone et al., 1998). In Arabidopsis, overexpression of the maize (Zea mays L.) transcriptional repressors ZmMYB31 and ZmMYB42 resulted in the inhibition of expression of COMT (caffeic acid 3-O-methyltransferase), thereby reducing lignin content (Fornalé et al., 2006). EgMYB1 from eucalyptus is an inhibitor of lignin biosynthesis (Legay et al., 2007). Overexpression of PvMYB4 in switchgrass (Panicum virgatum) inhibits lignin biosynthesis (Shen et al., 2011). Overexpression of AtMYB32 causes anther distortion and affects the formation of anther cell walls (Preston et al., 2004). MYB189 negatively regulates secondary cell wall biosynthesis in Populus (Jiao et al., 2019). Given that there is a little information on the function of negative regulators in woody plants, the screening and identification of MYB transcription factors with transcriptional activation or transcriptional inhibition has broad research significance.
Following a stress stimulus, the expression of some transcription factors increases, resulting in signal amplification. Thus, the expression of downstream related genes are also regulated, so that overall stress resistance is improved to some extent. According to existing reports, MYB transcription factors are involved in a variety of plant stress responses and are key in regulating the cell's response to stress. Examples include a high temperature resistance gene MYB68 (Feng et al., 2004); genes that respond to drought and salt stress, GmMYB177 (Liao et al., 2008); genes that respond to drought and cold stress, OsMYB4; genes that respond to drought, salt and radiation stress, OsMYB4 (Pasquali et al., 2008); genes that respond to cold, salt and drought stress, AtMYB2 (Hoeren et al., 1998); and OsMYB2 (Yang et al., 2012), to name a few.
Secondary growth and stress resistance are the important physiological processes for tree radial growth and wood formation. The properties of wood are determined by the composition and characteristics of xylem secondary cell wall. For pulping industry the lignin content in secondary cell wall is an important factor affecting pulping yield. The birch (Betula platyphylla Suk.) is one of the main pulpwood species. Therefore, it is of great significance to study the lignin biosynthesis and stress resistance for the genetic improvement of forest trees. In this study, a MYB transcription factor homologous to the genes inhibiting lignin synthesis was identified. The expression pattern of this gene in tissues, under artificial bending or abiotic stress treatment were analyzed. The functions related to secondary cell wall biosynthesis were identified by overexpressing BpMYB4 in Arabidopsis thaliana. Besides, the function of this gene in abiotic stress tolerance was investigated through transient transformation in birch. The expression patterns of genes involved in abiotic stress response and cell wall biosynthesis in transgenic plants were analyzed. The results can help further understand the molecular mechanism of secondary growth processes and abiotic stress responses regulated by MYB transcription factors in woody plants.

Plant Materials and Treatment
Under aseptic conditions, the epidermal cells of onion were torn and plated in 1/2 MS solid medium for sub-cellular localization studies (Wang et al., 2019).
The vernalized birch (B. platyphylla) seeds were washed with running water for 3 days and planted on the soil surface; the soil contained a mixture of perlite: vermiculite: soil (1: 1: 4). The plants were grown in a greenhouse under controlled conditions (16 h/8 h light/dark, 25 • C, 70-75% relative humidity). Twomonth-old seedlings were used to test the express levels between leaves, stems and roots. Developing xylem of 2-years-old birch seedlings were subjected to artificial bending experiments and temporal expression analysis (Wang et al., 2014). The birch seedlings were irrigated with 200 mM NaCl or 300 mM Mannitol solutions for 12 h, 24 h, and 48 h, and the birch irrigated with water was used as a control group for expression analysis under abiotic stress. Three biological replicates were performed.
Wild-type (WT) Arabidopsis thaliana (ecotype Col-0) seeds were surface-sterilized with 50% sodium hypochlorite for 6 min, washed six times with sterile distilled water, the seeds plated on solid 1/2 MS medium and incubated at 22 • C under light. After growing the cotyledons, the seedlings were transferred to a mixture of perlite: vermiculite: soil (1: 1: 4) and grown in a greenhouse under controlled conditions (16 h/8 h light/dark, 25 • C, 70-75% relative humidity). The plants were thoroughly watered every 5 days (Wen and Chang, 2002).

Gene Identification and Bioinformatics Analysis
A cDNA sequence (GenBank accession number KA257119.1) annotated as a MYB transcription factor was screened from birch tension wood transcriptome (Wang et al., 2014). The open reading frame (ORF) was determined using the NCBI blastX procedures 1 . The molecular weight and isoelectric point of the protein were predicted using Expasy 2 . The similarities in amino acid sequences were checked using BioEdit for multiple sequence alignments, and finally the MEGA version 4 software (Kubo et al., 1998) was used for phylogenetic tree analysis.

Sub-Cellular Localization
Total RNA of birch was extracted using CTAB method (Chang et al., 1993) and treated with DNase I to remove DNA contamination. Total RNA was reverse transcribed into cDNA using a PrimeScriptTMRT reagent Kit (Takara Bio Inc., Kusatsu, Shiga, Japan). BpMYB4 CDS without a stop codon was fused to the N-terminus of green fluorescent protein (GFP) into the pROKII vector and driven by a CaMV 35S promoter. Vector construct primers are shown in Supplementary Table 1. The microcarriers (tungsten powder) embedded with the 35S: GFP plasmid and the 35S: gene-GFP plasmid were transformed into onion epidermal cells with high pressure helium gas using a PDS-1000 benchtop gene gun (Bio-Rad laboratories, Inc., Hercules, CA, United States). After 2 days of culture under dark conditions, the expression pattern was observed and captured using a LSM700 laser confocal microscope (Zeiss, Jena, Germany).

Real-Time PCR
Total RNA of plant was extracted by CTAB method for semiquantitative RT-PCR and real-time RT-PCR analysis. Realtime RT-PCR was performed with a TransStart Top Green qPCR SuperMix kit (TransGen Biotech, Beijing, China) using the primer sequences listed in Supplementary Table 2. The amplification procedure was conducted using the following parameters: 94 • C for 30 s; 45 cycles at 94 • C for 12 s, 58 • C for 30 s and 72 • C for 45 s; and 79 • C for 1 s for plate reading. The semi-quantitative PCR enzyme by rTaq (TaKaLa), which amplification procedure was conducted using the following parameters: 94 • C for 2 min; 28 cycles at 94 • C for 30 s, 58 • C for 30 s and 72 • C for 45 s. Three independent experiments were performed. The tubulin (GenBank accession number: FG067376) and ubiquitin (GenBank accession number: FG065618) genes were used as the internal controls. The relative expression level of each gene was calculated using the delta-delta CT method (Livak and Schmittgen, 2001).

Construction of Overexpression and Knock-Down Expression Vectors
The CDS of genes was inserted into the pROKII vector to construct the overexpression vector (OE) driven by a CaMV 35S promoter. A short sequence inverted repeat of 200 bp was inserted in the RNAi vector pFGC5941 to generate the RNAisilencing knock down vector (SE). Vector construct primers are shown in Supplementary Table 1. The recombinant plasmids of the overexpression vector or the RNAi-silencing vector were transformed into Agrobacterium tumefaciens strain EHA105 using the freeze-thaw method .

Transformation of Arabidopsis
For transformation, WT Arabidopsis was used at flowering time with the ripe pods removed. Transformation of 35S: MYB4-GFP into A. thaliana was performed using the Arabidopsis flower dipping method (Clough and Bent, 1998). The obtained seeds were screened for resistance with 1/2 MS solid medium containing 50 mg/L kanamycin, until T3 generation seeds were obtained. The semi-quantitative PCR was performed for expression identification of BpMYB4 in transgenetic Arabidopsis. Primers are shown in Supplementary Table 2.

Histochemical Analysis
Transgenic A. thaliana growing for about 50 days was selected as the experimental group, and WT A. thaliana in the same growth cycle under the same environment was used as the control group. The mature stems of A. thaliana in the control group and the experimental group were sectioned by hand, and histochemically stained with a 1% phloroglucinol solution. The sections were observed with an optical microscope (Olympus BX53, Japan).

Chemical Analysis of Secondary Cell Wall
Lignin and cellulose content were determined by the Soxhlet extraction method (Thurbide and Hughes, 2000). The transgenic and WT Arabidopsis plants were harvested at about 50 days growth stage, dried in an oven at 105 • C, pulverized, and bake to a constant weight at 55 • C. A sample weight of exactly 0.5 g (weight to 0.0001 g) was used to determine the content of lignin in transgenic Arabidopsis, and a sample weight of exactly 1.0 g (weight to 0.0001 g) was used to determine the content of cellulose. Three technical replicas were made. All samples were wrapped with filter paper and tied with cotton thread.

Seed Germination Rate Under Abiotic Stress
Seeds of WT A. thaliana and from the overexpression lines of transgenic A. thaliana T3 generation (OE-7, OE-9, OE-25) were washed with sodium hypochlorite solution and sequentially sown on 1/2 MS, 1/2 MS + 100 mM NaCl, and 1/2 MS + 200 mM mannitol (Ji et al., 2013). The seeds were germinated in an artificial climate culture chamber for 10-15 days, and the germination rate was counted.

MDA and PRO Content Determination
Using PRO kit (Nanjing Jiancheng, China) to determine the content of L-proline (Pro) in plant materials under abiotic stress. Malondialdehyde (MDA) determination of plants under treatment was conducted by thiobarbituric acid method (Wang et al., 2010b). Three biological replicates were performed.

Transient Transformation in Birch and Treatment of Plants
Agrobacterium tumefaciens with pROKII, pFGC5941 empty vector, and the recombinant vectors were cultured to OD 600 value of 0.5, and the cells were collected and resuspended in 5% sucrose, 1.5 mg/L KT (6-Furfurylaminopurine), 0.5 mg/L NAA (1-naphthylacetic acid), 100 µM AS (acetosyringone) and 0.02% Tween 20 in 50 mL of 1/2 MS liquid medium (pH 5.6). Two month-old birch seedlings were soaked in the medium containing resuspended bacterial cells under 25 • C, with constant shaking at 100 rpm for 2 h; the cultured birch seedlings were then washed 1-2 times with sterile deionized water, and the water on the birch seedlings was dried with sterile absorbent paper. The infected birch seedlings were transplanted into artificial nutrient soil (The ratio of soil, perlite and vermiculite was 5:3:2) and moisturized by mulching. The gene expression was detected by RT-PCR after culture at 25 • C and light (approximately 150 µmol m −2 s −1 ) for 3 days .
The transiently transformed birch seedlings were replanted into the artificial nutrient soil in the greenhouse. After 3 days of culture, the transiently transformed birch and the untreated WT plants were treated with 100 mM NaCl and 200 mM mannitol for 24 h, respectively (Wang et al., 2019). Total RNA was extracted from birch seedlings under stress and transcribed into cDNA.

DAB, NBT, and Evan's Blue Staining
DAB (3-3'-diaminobenzidine), NBT (nitro blue tetrazolium) and Evan's Blue staining were performed on birch leaves after stress treatment . The leaves were extracted and stained with DAB, NBT and Evan's Blue stains. At 37 • C, tissues were stained with DAB overnight, stained with NBT for 4 h, and stained with Evan's Blue for 8 h. After staining, the stain was removed and decolorized. The solution (95% ethanol + 5% glycerol) was dehydrated in a boiling water bath.

BpMYB4 Is Homologous to Lignin Biosynthetic Inhibitory Factor
An mRNA sequence, KA257119.1, 4258 bp in length, from reaction wood of B. platyphylla was found in the Transcriptome Shotgun Assembly database (Wang et al., 2014). According to BLASTX and ORF analysis, a MYB transcription factor was identified with an open reading region 654 bp in length, encoding a total of 217 amino acids. The molecular weight of this MYB protein is predicted to be approximately 53.661 kDa.

Sub-Cellular Localization of BpMYB4
Recombinant plasmid 35S:MYB4-GFP was transfected into onion epidermal cells using the gene gun transformation technique, and the 35S: GFP plasmid was used as a positive control. Observations using a laser confocal microscope showed a green fluorescent signal in the nucleus of the onion epidermis, indicating that BpMYB4 is a nuclear protein (Figure 2).

Expression Analysis of BpMYB4 Gene
To further investigate the biological role of BpMYB4 in secondary growth of birch, its gene expression pattern in different tissues, development stages in a growth season and during tension wood development, were analyzed using real-time quantitative PCR. The results (Figure 3A) showed that the expression level of BpMYB4 was the highest in the inflorescence than other  Each experiment was replicated independently for at least three times. Asterisk indicates significant difference (*p < 0.05).
tissues, followed by buds, petioles, stems and leaves of birch. Its expression level was nearly 100 times higher in inflorescence than that in leaves, and 40-60 times higher in petioles and stems, compared to leaves. In a growth season the expression level of BpMYB4 was different in different stages of xylem development in birch ( Figure 3C); its expression was abundant in late April and middle September, but decreased during active stages of cambium development. When the stems of birch were subjected to artificial bending and gravity stimulation, the transcript of BpMYB4 was increased during tension wood (xylem formed above the area of the bending) development, compared to those in opposite wood (xylem formed below the area of the bending) and normal wood (xylem of the straight tree) (Figure 3B). Temporal expression patterns of the BpMYB4 gene were carried out to test BpMYB4 response to salt stress and drought stress using qRT-PCR. The results showed the expression of BpMYB4 were induced at 24 h after stress treatment (Figures 3D,E).

Phenotypic Analysis by Ectopic Expression of BpMYB4 in Transgenic Arabidopsis
In order to identify the function of BpMYB4 in secondary growth, transgenic Arabidopsis plants overexpressing BpMYB4

Analysis of Lignin and Cellulose Content in Transgenic A. thaliana
To verify the inhibitory role of BpMYB4 in lignin biosynthesis, chemical analysis was performed to detect the changes in lignin and cellulose content of transgenic Arabidopsis. Chemical composition analysis suggested that the content of lignin in transgenic lines was reduced relative to WT, with decreasement of 3.85-8.23%. However, compared with WT, the content of cellulose in transgenic plants increased significantly (P < 0.05), with a averange increasment of 5.29-13.15% (Table 1). These results indicated that BpMYB4 overexpression has a negative effect on lignin biosynthesis and deposition but a positive effect on cellulose content.

Analysis of Germination Rate in Response to Salt and Osmotic Stress
When the seeds of transgenic lines (OE-7, OE9, OE-25) and WT were exposed to salt or mannitol, it was found that BpMYB4 overexpression conferred salt and osmotic stress tolerance to the seeds. There was no substantial difference in germination rates between transgenic Arabidopsis and WT lines under control conditions (Figure 5). Following 100 mM NaCl or 200 mM mannitol treatment, compared with WT plants both OE-7, OE-9 and OE-25 lines displayed higher germination rates. These results suggest that BpMYB4 overexpression improves abiotic stress tolerance in Arabidopsis.

MDA and PRO Content Determination
Malondialdehyde is the product of membrane lipid peroxidation in plant tissues subjected to oxidative stress under adversity, reflecting the degree of cell membrane lipid peroxidation and the strength of plant response to adversity (Ma et al., 2015). Pro plays an important role in osmotic regulation in plants.
Under adversity conditions, the Pro content reflects the stress resistance of plants to a certain extent (Vendruscolo et al., 2007). We tested the content of MDA and Pro to study the resistance of BpMYB4 transgenic plants to salt and drought stress. The WT and transgenic Arabidopsis were treated with 200 mM NaCl and 300 mM Mannitol solutions for 24 h, and the control plants were treated with water. Compared with the control group, the content of MDA in transgenic Arabidopsis was reduced, and the content of Pro was increased.
The above results indicate that the BpMYB4 gene has certain stress resistance ability.

Generation of Transiently Transgenic Birch Plants With Knocked Down or Overexpressing BpMYB4
BpMYB4 was over-expressed or knocked down in birch using an Agrobacterium-mediated transient expression system, and the expression levels of BpMYB4 in transgenic plants were detected by qRT-PCR (Figure 6). The results showed that expression of BpMYB4 was highly up-regulated in the transiently transformed plants with 35S: MYB4 vectors, which was approximately 20fold higher than that of the control, while expression of BpMYB4 in the transiently transformed knock-down plants (pFGC5941: MYB4) was down-regulated, with only 1/4th of the transcripts present in the control. The results showed that BpMYB4 was overexpressed or knocked down in the transiently transformed birch seedlings. These transformed lines were used in further functional analysis.

BpMYB4 Functions to Scavenge ROS and Maintain Cell Membrane Integrity in Transgenic Birch Subjected to Abiotic Stress
Abiotic stress can induce the generation of reactive oxygen species (ROS), such as hydrogen peroxide (H 2 O 2 ) and superoxide anion O 2 .− , and accumulation of ROS could damage cell membranes by oxidation of proteins, lipids and DNA (Mittler et al., 2004). To analyze the physiological mechanisms by which BpMYB4 increases the ability to tolerate stress, birch plants were transformed transiently with 35S: BpMYB4, pROKII plasmid (control), pFGC5941: BpMYB4 or pFGC5941 plasmid (control), and WT as well as transgenic plants were irrigated with water (contro), 100 mM NaCl or 200 mM mannitol for 24 h. DAB staining in plants following abiotic stress have previously shown reduced hydrogen peroxide accumulation, leading to increased resistance (Sela et al., 2013). In the present results, DAB staining showed that under non-stress condition the over-expression (OE) plants, silencing expression (SE) plants, control plants and WT plants were not stained, with no obvious difference between them ( Figure 7A). These results indicate that the content of hydrogen peroxide in these lines was similar in the controls. Under abiotic stress conditions, hydrogen peroxide accumulation in OE plants were at the lowest levels, and the hydrogen peroxide accumulation levels in the SE plants were the highest. This indicates that BpMYB4 can reduce hydrogen peroxide accumulation in OE birch seedlings subjected to abiotic stress, and plays a role in stress tolerance of birch plants.
We performed NBT staining on the transiently transformed birch plants after abiotic stress treatment. Studies have shown that NBT staining can detect the accumulation of O 2 .− in cells under stress, thereby demonstrating the resistance ability of plants (Yu et al., 2018). The results are shown in Figure 7B.  (Singh and Upadhyay, 2014). Evan's Blue staining was performed on the transiently transformed birch plants after abiotic stress treatment. The results are shown in Figure 7C. Under the water treatment, there was no obvious color difference between OE plants, SE plants, the control group, and the WT, indicating that there was no remarkable difference in the degree of cell damage. However, under abiotic stress conditions, the damage of OE plant cell membranes was the lowest, and the damage of SE plant cell membranes was the highest. Our results indicate that BpMYB4 can reduce the damage to cells in OE birch plants under abiotic stress. The Evan's Blue staining results again showed that BpMYB4 has a certain function of stress resistance in birch.

BpMYB4 Regulates the Expression of Genes Related to Resistance and Cell Wall Biosynthesis
To analyze the genes regulated by BpMYB4 in stress resistance and cell well formation, we analyzed the expression levels of putative downstream resistance-related and cell wall biosynthesis-related genes in overexpressing and knockout transgenic lines. The results in Figure 8A show that most resistance-related genes were highly up-regulated in overexpressing lines (pROKII-BpMYB4) compared to the empty vector (pROK II) control line, including SOD1, SOD2, SOD3, SOD4, SOD5, POD1, POD2, POD3 POD5, POD12, P5CDh1, P5CDh2, and P5CS2 genes. Among them, the maximum differential expression amount can reach as much as 27.4 fold, and the smallest is only about 1 flod. Therefore, most of the resistance-related genes were up-regulated in overexpressing lines, suggesting that overexpression of BpMYB4 (pROKII-BpMYB4) can induce the expression of resistance-related genes. The results in Figure 8B show that most resistance genes are negatively regulated by the suppressed expression of BpMYB4 (RNAi-BpMYB4), compared to the empty vector (RNAi) control line. Among them, POD1, POD3, POD6, POD7, POD8, POD9, POD10, POD11, POD12, P5CDh1, P5CDh2, P5CS1, and P5CS2 expression were remarkably highly down-regulated. Among them, POD12 had the largest down-regulated expression level, and the down-regulated expression level could reach as much as 84.9 fold. These data indicate that knock-down expression of BpMYB4 (RNAi-BpMYB4) can negatively affect the expression of resistance-related genes. The results in Figure 9A show that compared with the empty vector (pROKII) control line, in the OE lines (pROKII-BpMYB4), CCO, CCR and C4H genes which are the key regulator in lignin biosynthesis were down-regulated. CESA gene relate to cellulose biosynthesis was up-regulated, expression of CAD and 4CL were also slight increased. Among them, the transcripts increase of CESA reached 3.61 fold, and the decreased expression level of C4H reached 8 fold. The results in Figure 9B show that compared with the RNAi empty vector (pFGC5941) control line, in the suppressed expression lines (pFGC5941-BpMYB4), CAD and C4H genes were up-regulated, and CCO, CESA and CCR genes were down-regulated. Among them, the increased expression level of CAD reached 5.86 fold, and the transcripts decrease of CESA reached 30.27 flod. These data suggest that BpMYB4 transcript factor can negatively affect the expression of some lignin biosynthesis gene and positive regulate the cellulose biosynthesis gene in OE plants compare with the WT. Therefore, BpMYB4 has a certain function in regulating the expression of cell wall biosynthesis-related and resistance-related genes.

DISCUSSION
Multiple sequence alignment and phylogenetic tree analysis demonstrated that BpMYB4 is similar to EgMYB1, AmMYB308, ZmMYB42 and ZmMYB31 at the amino acid level, all of which have been identified as lignin biosynthetic inhibitory factors (Tamagnone et al., 1998;Fornalé et al., 2006Fornalé et al., , 2010Legay et al., 2007). Therefore, we speculated that BpMYB4 might also play a negative role in lignin biosynthesis in birch.
According to relevant reports, genes encoding transcription factors that inhibit vascular development are usually not highly or specifically expressed in vascular tissues (Zhao and Dixon, 2011). In this study BpMYB4 was expressed at high levels in flowers, at levels higher than that seen in stems. In addition, BpMYB4 was abundantly expressed during the dormancy seasons of April and September. This expression pattern was unlike that seen in some genes whose products are involved in secondary cell wall formation, which were highly expressed in June or July (Wang et al., 2010a), and might imply the role of inhibitors in secondary growth. BpMYB4 transcript levels increased during tension wood development compared with those seen in opposite wood and normal wood. In tension wood of birch, the cellulose content was higher and lignin content was lower than those in opposite wood and normal wood (Wang et al., 2014). Together with the expression level changes and phylogenetic tree analysis, our results suggest that BpMYB4 might be an inhibitor of lignin biosynthesis in birch.
Studies in populations of forest tree hybrids have shown that when the lignin content is greatly reduced and the cellulose content is increased in transgenic plants, the growth rate of roots and stems is significantly increased (Hu et al., 1999). There is a negative correlation between biomass growth and lignin content (Kirst et al., 2004;Novaes et al., 2009Novaes et al., , 2010. In the present study, the inflorescence stems of the transgenic plants was higher than the WT. So we detected the secondary xylem development and lignin deposition using anatomical observations (Figure 4) and chemical analysis. The stem sections stained with phloroglucinol-HCl showed decreased lignin deposition in transgenic plants, relative to WT A. thaliana. The content of lignin in transgenic lines was reduced, but the content of cellulose in transgenic plants was increased relative to WT (Table 1). Therefore, we hypothesized that the reason of growth increase in stems of transgenic plants is that BpMYB4 inhibited lignin biosynthesis. These results confirmed that BpMYB4 has a negative effect on lignin biosynthesis and deposition but a positive effect on cellulose content. The express analysis of cell wall biosynthesis relate genes in transgenetic birch with BpMYB4 transiently over-expressing suggested that BpMYB4 transcript factor can decreased the expression of some lignin biosynthesis gene and positive regulate the cellulose biosynthesis gene in OE plants compare with the WT. These data might explain the mechanism of cell wall components modification in transgenetic plants.
The transgenetic birch with BpMYB4 transiently expression were used to investigate the resistance ability of BpMYB4. On the one hand, functional analysis in birch was used to verify the results of heterologous expression of BpMYB4 in Arabidopsis experiment, on the other hand, it is convenient to analyze the genes that can be regulated in birch by BpMYB4. ROS are produced when plants face adverse conditions (Scandalios, 1993). Levels of H 2 O 2 in plant cells may accumulate and cause oxidative damage when plants undergo stress (Larrigaudière et al., 2001). The amount of H 2 O 2 released from the cells can be detected by the intensity of DAB staining. Previous studies have shown that reduced H 2 O 2 accumulation (detected via DAB) following abiotic stress can increase stress resistance (Sela et al., 2013). In this study, after treatment with 100 mM NaCl or 200 mM mannitol, DAB staining showed that H 2 O 2 accumulation in OE plants was lower than WT, but higher in SE plants compared to WT. This indicates that BpMYB4 can reduce H 2 O 2 accumulation in OE birch seedlings subjected to abiotic stress. Another way ROS is produced in cells is via stress signaling, the cellular level of O 2 .− (Mittler et al., 2004). Staining with NBT indicates the activity of superoxide dismutase (Buc-Calderon and Roberfroid, 1988), which reflects the content of O 2 .− in cells (Yu et al., 2018 (Mittler et al., 2004). Studies have shown that Evan's Blue staining can detect cell death and demonstrate the degree of stress resistance of plants (Singh and Upadhyay, 2014). Therefore, we performed Evan's Blue staining on the transiently transformed birch plants after abiotic stress treatment. The results indicate that BpMYB4 can reduce the damage done by abiotic stress in OE lines of birch. BpMYB4 also induces the expression of most resistance-related genes. All the above results showed that BpMYB4 activates the metabolic pathway of ROS clearance by regulating the expression of resistance-related genes in transgenic birch trees subjected to abiotic stress, thereby reducing the level of ROS to maintain cell membrane integrity and playing a role in the resistance of birch trees.

CONCLUSION
In this study, we identified a BpMYB4 gene which is homologous to other transcription factors that negatively regulate lignin biosynthesis. The expression analysis of BpMYB4 in different tissues, under artificial bending treatment and at different stages in a growing season also imply that it might be an inhibitory transcription factor in secondary growth. Functional analysis in transgenic Arabidopsis further demonstrated that BpMYB4 can promote height growth of inflorescence stems, increase cellulose and decrease lignin content in the transgenic plants. Analysis of the stable transformation of Arabidopsis and transiently transformed birch with BpMYB4 also indicated a certain stress resistance function. These results can be useful for further understanding the molecular mechanism of secondary growth procesess and abiotic stress responses regulated by MYB transcription factors.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.

AUTHOR CONTRIBUTIONS
CW: conceptualization and methodology. HL and NZ: software. YY, HL, and NZ: formal analysis and investigation. YY and CW: writing-review and editing. YY: visualization. CW: funding acquisition. All authors contributed to the article and approved the submitted version.