PeMYB26, an R2R3-MYB transcription factor, positively regulates Lignin deposition in Moso bamboo

PeMYB26, an R2R3-MYB transcription factor, positively regulates Lignin deposition in Moso bamboo | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article PeMYB26, an R2R3-MYB transcription factor, positively regulates Lignin deposition in Moso bamboo Shanglian Hu, Boya Wang, Sen Chen, Ze Zhu, Yuan Li, Chaopeng Li, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3495971/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Nov, 2024 Read the published version in Plant Growth Regulation → Version 1 posted 6 You are reading this latest preprint version Abstract Moso bamboo ( Phyllostachys edulis ) is a highly valuable woody bamboo species. A better understanding of its regulation of lignin deposition would significantly benefit its cultivation and breeding. Here, we identified PeMYB26, an transcription factor gene that is primarily expressed in the vascular system. PeMYB26 encodes an R2R3-MYB transcriptional activator that localizes to the nucleus. Heterologous expression of PeMYB26 under control of the cauliflower mosaic virus 35S promoter caused widening of xylem, thickening of vessel elements, and deposition of lignin in transgenic tobacco ( Nicotiana tabacum ) plants. Moreover, transcript abundances of the lignin biosynthesis genes PAL （ PHENYLALANINE AMMONIA-LYASE ）, CAD （CINNAMYL ALCOHOL DEHYDROGENASE）, COMT （CAFFEATE O-METHYLTRANSFERASE） and CCR （CINNAMOYL CoA REDUCTASE ）were markedly higher in N. tabacum lines overexpressing PeMYB26 than in control lines. In particular, the expression of PeCCR was highly promoted by PeMYB26. These results indicate that PeMYB26 plays a positive role in regulating lignin accumulation and xylem formation. Bamboo R2R3-MYBs xylem widen lignin monomer synthesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Moso bamboo ( Phyllostachys edulis )is the most economically woody bamboo species with more than 3.87 million ha area in China providing 70% of the bamboo-production (Song et al. 2011; Song et al. 2016). The rapid growth rate of bamboo shoots and the huge biomass of bamboo plants make bamboo an important bioenergy source. Moso bamboo shoots can grow 1 m per day during the most rapid growth period (Peng et al. 2013 ; Dixon and Gibson 2014 ). The fast-growth and huge output, made moso bamboo as the raw materials of paper-making, building and industry. The cellulose in cell wall of bamboo culm is the valuable constituents in bamboo woody production. However, the tight combination of cellulose and lignin in bamboo culm always bothering the bamboo industrial producing. Lignin accumulation in the SCW of vascular cells along with cellulose increases rigidity and enables plants to stand upright and conduct water more efficiently. Altered expression of genes encoding lignin biosynthetic enzymes usually results in changes in the width of xylem cells and altered drought tolerance. For example, overexpression of DfCCoAOMT14 (CAFFEOYL COENZYME A ESTER O-METHYLTRANSFERASE) from the bamboo species Dendrocalamus farinosus in Nicotiana tabacum increases xylem thickness and improves drought tolerance, and enhances accumulation of lignin (Wei et al. 2023 ). Because xylem is the main tissue involved in long-distance water transport in plants, its anatomical structure is highly related to plant drought tolerance (Weithmann et al. 2022 ; Lens et al. 2022 ). In Atemoyas ( Annona cherimola × Annona squamosa ), the presence of wider xylem cells and larger vessel elements results in better performance when soil water is scarce (Losada et al. 2023 ). Genes involved in drought responses are likely to affect the development of the vascular system. VASCULAR RELATED NAC-DOMAIN PROTEIN 7 (VND7), a master switch in the transcriptional regulatory network in Arabidopsis ( Arabidopsis thaliana ), responds to abscisic acid (ABA) signaling induced upon drought stress by promoting xylem differentiation (Ramachandran et al. 2021 ; Yamaguchi et al. 2011 ; Yamaguchi et al. 2010 ). PdOLP1 , encoding an osmotin-like protein in Eastern cottonwood ( Populus deltoides ), is co-expressed with genes involved in secondary cell wall (SCW) biosynthesis and regulates plant defenses against water deficit (Li et al. 2020 ). Likewise, the altered accumulation of lignin in the plant SCW leads to different responses to drought. For instance, the microRNA CamiR397 in chickpea ( Cicer arietinum ) can target the transcripts of lignin biosynthesis genes, reducing plant sensitivity to drought and pathogen infection by decreasing deposition of lignin (Sharma et al. 2023 ). Overexpression of PtoMYB170 in transgenic poplar leads to stronger lignification and thicker SCW in xylem (Xu et al. 2017 ). In many plant species, R2R3-MYB proteins are the directly targets of VNDs that affects SCW formation in SCW transcriptional regulatory networks. In Arabidopsis, MYB46 and MYB83 are the second main switch in SCW regulatory network that promote the synthesis of cellulose, lignin and hemicellulos, respectively (Xiao et al. 2021 ; Ko et al. 2014 ). Arabidopsis MYB proteins target lignin biosynthesis genes to regulate lignin content and xylem development. For example, AtMYB15 activates the transcription of lignin biosynthesis genes such as PHENYLALANINE AMMONIA-LYASE (PAL), CAFFEATE O-METHYLTRANSFERASE (COMT), and CINNAMYL ALCOHOL DEHYDROGENASE (CAD) to increase the production of lignin monomers (Kim et al. 2020 ). MYB8 promotes PAL expression in vascular cells in maritime pine ( Pinus pinaster Ait.) (Craven-Bartle et al. 2013 ), and MYB103 is involved in lignin biosynthesis in Arabidopsis and Chinese silver grass ( Miscanthus sinensis ) (Ohman et al. 2013; Golfier et al. 2021 ). MYBS3 in agricultural crops like maize ( Zea mays ), rice ( Oryza sativa ) and sorghum ( Sorghum bicolor ) positively regulates plant tolerance to drought (Liu et al. 2023 ). In rice, ROLLING-LEAF MUTANT 1 (RLM1) interacts with MITOGEN-ACTIVATED PROTEIN KINASE 10 (OsMAPK10) and increases lignin accumulation in vessel elements by targeting the CAD promoter (Chen et al. 2022c ). PeMYB83L in moso bamboo was also reported positively regulating SCW fast deposition through ABA signaling pathway (Chen et al. 2022d). In this study, we identified an R2R3-type MYB protein of Moso bamboo, named PeMYB26, positively regulated lignin deposition and xylem formation. PeMYB26 was expressed specifically in branches and was induced by water deficit. Tabacoo ( Nicotiana tabacum ) plants overexpressing PeMYB26 showed more developed xylem and increased expression levels of lignin biosynthesis genes. In addition, expression of CINNAMOYL CoA REDUCTASE ( PeCCR ) was promoted by the accumulation of PeMYB26. These results indicated that PeMYB26 is a functional R2R3-MYB protein that played positive role in lignin deposition, xylem development by promoting the expression of lignin biosynthesis genes. Materials and methods Plant growth conditions and drought stress treatment All plant materials used in this study were from the Institute of Bamboo Research, Southwest University of Science and Technology, Mianyang, Sichuan province, China. For drought stress treatment, 2-month-old Moso bamboo ( Phyllostachys edulis ) seedlings were treated with 0.8 M mannitol and frozen in liquid nitrogen before treatment and after 24 h of treatment. Nicotiana benthamiana plants were grown in a greenhouse with a photoperiod of 16-h light/8-h dark at a light intensity of ~ 110 mol m − 2 s − 1 . Nicotiana tabacum seeds were surface sterilized for 15 min in a 20% (v/v) bleach solution in water, and were subsequently washed six times with sterile water. The disinfected seeds were then kept at 4°C for 72 h and then transferred onto Murashige and Skoog (MS) medium (PhytoTechnology Laboratories™) containing 1% (w/v) sucrose and 0.8% (w/v) phytagel (pH 5.7, Sigma-Aldrich). Plants were then grown in a greenhouse as described above. RNA isolation and RT-qPCR Total RNA was extracted from P. edulis leaves using TRIzol reagent (Takara, Dalian, China), and RNA quality was monitored using 1% (w/v) agarose gels and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Beijing, China). First-strand cDNAs were synthesized using 2 µg of total RNA and a FastKing cDNA First Strand Synthesis Kit (TIANGEN, Beijing, China). Quantitative PCR (qPCR) was performed using a CFX connect Real-time PCR Detection System and a SYBR qPCR Master MIX kit (Vazyme, Nanjing, China); amplification profiles were analyzed using CFX Manager software version 3.1 (Bio-Rad, Hercules, CA, USA). The relative expression levels of genes were calculated using the 2 –ΔΔCt method (Livak and Schmittgen 2001 ). All RT-qPCR experiments were performed with two technical replicates and three independent biological replicates. Transactivation activity assay The coding sequence of PeMYB26 was subcloned into pGBKT7 keeped in our institute to obtain the pGBKT7 -PeMYB26 vector. This construct, along with the empty pGADT7 vector, were co-transformed into the yeast ( Saccharomyces cerevisiae ) AH109 strain. After transformation, positive transformants were selected on synthetic defined (SD) medium –Trp–Leu (SD/–Trp/–Leu). Positive transformants were then plated onto SD/–Trp/–Leu/–His/–Ade medium and SD/–Trp/–Leu–His/–Ade/+X-α-GAL medium, followed by incubation at 30℃ for approximately four to six days. Heterologous overexpression of PeMYB26 in N. tabacum Overexpressing N. tabacum lines were obtained by introducing pCAMBIA1303 containing PeMYB26 driven by the CaMV 35S promoter ( 35S :: PeMYB26-GFP ) into Agrobacterium ( Agrobacterium tumefaciens ) strain EHA105 using the leaf disc method (Wang et al. 2022 ). The empty pCAMBIA1303 vector was introduced into Agrobacterium strain EHA105 as a negative control. Infected leaf discs were incubated on MS medium containing 0.1 mg/L 1-Naphthaleneacetic acid (NAA, Sigma-Aldrich), 0.5 mg/L 6-Benzylaminopurine (6-BA, Sigma-Aldrich), 500 mg/L acetosyringone (Sigma-Aldrich) and 50 mg/L kanamycin (TIANGEN, Beijing) in the dark at 27 ± 1°C for 2 days. Regenerated buds were transferred to MS plates containing 0.1 mg/L NAA and 50 mg/L kanamycin for selection and to promote the formation of complete plants. Genomic DNA was extracted from the leaves of transgenic plants, and then amplified using specific primers for pCAMBIA1303 and PeMYB26 . Primers used were listed in Supplement Table 1. PCR products were checked by 1% (w/v) agarose gel electrophoresis. The expression of PeMYB26 in transgenic plants was assessed by RT-qPCR and Actin was used as the internal reference for normalization (Wei et al. 2023 ). Histochemical staining Intact thin sections of PeMYB26 overexpression Tabacco plants’ stem were stained in 2% (w/v) phloroglucinol solution for 5 min, and were then stained with 30% (v/v) hydrochloric acid for 1 min, washed with water, observed and photographed under a Leica microscope. Determination of lignin content in transgenic N. tabacum plants Determination of lignin content was based on the method of Li et al (Li et al. 2015 ). and was improved as follows: After 0.5 g of fresh sample was ground to a fine powder in liquid nitrogen, 5 mL of 0.1 M phosphate buffer (pH 7.2) was added, and the mixture was incubated at 37°C for 30 min, followed by centrifugation at 4000 rpm for 5 min. Then, 5 mL of 80% (v/v) ethanol was added, and the sample was incubated at 80°C for 1 h. After this, the sample was centrifuged at 4000 rpm for 5 min, 10 mL of acetone was added to extract lignin, and the sample was centrifuged at 5000 rpm for 5 min and then dried at 60°C. For lignin analysis, approximately 20 mg of dried plant cell wall material was incubated at 80°C in a mixture of 750 µL of water and 250 µL of concentrated HCl and 100 µL of thioglycolic acid for 3 h. The mixture was centrifuged at 4000 rpm for 10 min and the pellet was washed with 1 mL of water and resuspended in 1 mL of 1 M NaOH and placed on a rocking plate at room temperature overnight. The mixture was centrifuged again, and the supernatant was collected and mixed with 200 µL of concentrated HCl. After being vortexed and incubated at 4°C for 4 h, the mixture was centrifuged and the pellet was dissolved in 1 mL of 1 M NaOH. The absorbance of a 50-fold dilution of the supernatant in 1 M NaOH was measured at 280 nm to determine lignin content. Dual-luciferase reporter assay A promoter fragment of 1.8 kb upstream of the PeCCR start codon was amplified from P. edulis genomic DNA inserted into the multiple cloning site of pGreenII 0800-LUC, yielding the PeCCRpro:LUC reporter construct. The promoter construct was transformed into Agrobacterium strain GV3101 containing the p19 helper plasmid. Bacteria were resuspended in 10 mL of infiltration buffer consisting of 10 mM MgCl 2 , 200 µM acetosyringone and 10 mM MES (pH 5.7), and the cell suspensions were incubated in the dark for 2 to 3 h before infiltration. The Agrobacterium cell suspension containing the PeCCRpro:LUC construct was mixed at a ratio of 1 : 1 (v/v) with Agrobacterium cell suspension harboring the effector construct 35S::PeMYB26 generated above. The cell mixture was infiltrated into young leaves of 35-day-old N. benthamiana plants. Two days after infiltration, the ratio of firefly luciferase (LUC) to Renilla luciferase (Ren) activity was measured using a Dual-Glo® Luciferase Assay System (Promega) and an Infinite M200 luminometer (Tecan, Mannerdorf, Switzerland). Phylogenetic analysis The MYBs of Moso Bamboo ( P. edulis ), rice ( Oryza sativa ) and maize ( Zea may ) were downloaded from NCBI ( https://www.ncbi.nlm.nih.gov/ ). The evolutionary history was inferred using the Neighbor-Joining method. The analysis involved 29 amino acid sequences. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA7. The protoplast transient assay For subcellular localization, approximately 1-month-old N.tabacum plants were used for protoplast transfection. The PeMYB26-GFP and the 35S:GFP construct (positive control) were transformed into Agrobacterium strain GV3101 and individually infiltrated into N. benthamiana leaves. 48 h after injection, leaves express PeMYB26-GFP and 35S:GFP were digested protoplasts respectively, and GFP signal was investigated after 16 h. Photos were capture by a confocal fluorescence microscope (TCS SP5 system, Leica, Germany). Statistical analysis The data in this study were analyzed using SPSS 25.0 and Origin 2018 softwares. Data are presented as means ± standard deviation (SD) of three independent biological replicates and two technical replicates. Asterisks indicate significant differences from the control ( * , P < 0.05; * , P < 0.01) according to Student’s t test. Results PeMYB26 encodes an R2R3-MYB transcription factor and responds to drought treatment In previous research, we noticed a gene whose expression was induced by drought in Moso bamboo. To further explore this observation, we treated 2-month-old Moso bamboo seedlings with 0.8 M mannitol to simulate drought stress. We determined that the expression level of this gene increases 5-fold compared to untreated control seedlings after a 48-h treatment (Fig. 1 a). Analysis using the SMART online database ( https://smart.embl.de/ ) revealed the presence of two conserved SANT domains in the N-terminal region of the encoded protein (Fig. S1 a). Because these domains are structurally homologous to those in the helix-turn-helix MYB family of DNA-binding proteins, we named this gene PeMYB26 . We detected PeMYB26 transcripts by RT-qPCR in different tissues of P. edulis , including unrolled leaves, branches and internodes, with the expression of PeMYB26 being higher in branches and internodes (Fig. 1 b). Compared to leaves, the expression level of PeMYB26 was 2 to 2.4 times higher in branches and internodes. Therefore, we speculated that PeMYB26 might be specifically expressed in vascular tissue and induced by conditions of water deficit. In addition, PeMYB26 is orthologous to several plant R2R3-type MYB transcription factors that regulate SCW deposition (Fig. 2 a). The N-terminal region of PeMYB26 has conserved R2 and R3 repeats (Fig. S1 b), which are also found in R2R3-MYB proteins functioning in SCW accumulation (Fig. S2). Taken together, these results suggest that PeMYB26 participates the formation of SCW. To analyze the subcellular location of PeMYB26, we cloned the PeMYB26 sequence in-frame and upstream of the green fluorescent protein ( GFP ) sequence. We then infiltrated the resulting construct 35S:PeMYB26-GFP and the empty GFP vector ( 35S:GFP ) into N. benthamiana leaves. We detected fluorescence signals predominantly in the nucleus (Fig. 2 b, PeMYB26-GPF), whereas the 35S::GFP vector produced green fluorescence throughout the cell (Fig. 2 b, positive control). This result demonstrates that PeMYB26 is located in the nucleus. Because PeMYB26 appeared to encode a transcription factor, we used the yeast two-hybrid (Y2H) system to determine whether it acted as a transcriptional activator or a transcriptional repressor. To this end, we cloned the PeMYB26 coding sequence in-frame and upstream of the sequence encoding the DNA-binding domain of yeast GAL4 in the pGBKT7 vector. Only yeast clones harboring the pGBKT7- PeMYB26 plasmid showed transcriptional activity and could grow on synthetic defined medium lacking Leu, Trp, His, and Ade (SD/–Trp/–Leu/–His/–Ade). We examined the authenticity and strength of PeMYB26 transcriptional activity by titration and X-gal staining. The Y2H assay revealed that PeMYB26 can activate the expression of GAL4 indicating that PeMYB26 is a transcriptional activator (Fig. 2 c). Heterologous expression of PeMYB26 in N. tabacum results in more highly developed vascular tissue To determine the role of PeMYB26 in SCW biosynthesis and response to drought, we generated transgenic N. tabacum lines overexpressing PeMYB26 under the control of the cauliflower mosaic virus 35S promoter. Using kanamycin as a selectable marker, we obtained three independent 35S::PeMYB26 transgenic lines ( ox-1 , ox-2 and ox-4 ). RT-qPCR analysis demonstrated that the transcript abundance of PeMYB26 is significantly higher in these overexpressing lines relative to control lines (Fig. 3 ). Anatomical analysis of culm cross-sections from the control and transgenic lines showed that overexpression of PeMYB26 results in increased the entire xylem width compared to the control line (Fig. 4 a). In addition, the xylem width of the overexpression lines was almost double that of control lines (Fig. 4 b). At the same time, overexpression of PeMYB26 also resulted in production of more vessel elements with larger diameters (Fig. 4 c and 4 d), suggesting that the overexpression of PeMYB26 may help plants to grow a wider xylem and thicker SCW. Altered expression of PeMYB26 changes the content of lignin in transgenic plants Along with wider xylem, the lignin content of culms from overexpression lines also increased (Fig. 5 a). In the ox-1 and ox-4 transgenic lines, lignin content was 20% higher than in control lines. Lignin content in the ox-2 transgenic line was not significantly different from that in control lines. Because of the increased lignin content in some PeMYB26 overexpression lines, we suspected that PeMYB26 affected lignin biosynthesis. Considering that the lignin monomer biosynthesis pathway is relatively well conserved in seed plants, we measured the expression of several key lignin biosynthesis genes in control and PeMYB26 overexpression plants. Along with the higher expression of PeMYB26 , the expression level of lignin biosynthesis genes increased. Compared to the control line, CCR expression was 75-fold higher in the ox-1 line and more than 10-fold higher in ox-2 and ox-4 lines (Fig. 5 b). To test whether PeMYB26 can activate PeCCR expression. we used a dual-luciferase reporter (DLR) assay system in N. benthamiana protoplasts transfected with a construct consisting of the firefly luciferase ( LUC ) gene driven by a 1.8-kb fragment of the PeCCR promoter (Fig. 6 a). The DLR assay revealed a 5-fold increase in transcriptional activation in protoplasts co-transfected with the 35S:PeMYB26 effector and the PeCCRpro:LUC reporter construct (Fig. 6 b). The DLR assay result, along with the highly induced expression of CCR in PeMYB26 transgenic lines, suggests that PeMYB26 is a transcriptional activator that promotes PeCCR expression. Discussion It is widely known that SCW formation is regulated by a NAC-MYB transcriptional network. In Arabidopsis, MYB46 and MYB83 are targeted by VND7 and regulate other MYB’s expression to switch the accumulation of cellulose and lignin in plant SCW (Ko et al. 2014 ; Ohtani and Demura 2019 ). There are also evidences that MYB transcriptional factors affect plant stress tolerance by regulating wax and flavonoid biosynthesis (Chen et al. 2023 ; Gao et al. 2023 ; Liu et al. 2022a ; Yang et al. 2022 ). Similarly, we showed that PeMYB26 was also highly expressed in internodes of Moso bamboo, indicating that PeMYB26 might be involved in both modulating the rate of plant growth and responding to drought (Fig. 1 ). Therefore, we selected PeMYB26 for functional analysis. Transcriptional assays, protein sequence analysis and subcellular localization results demonstrated that PeMYB26 encodes a nucleus-localized R2R3-type MYB activator (Figs. 2 and S1). The results of phylogenetic analysis showed that PeMYB26 is homologous to Arabidopsis AtMYB4, rice OsMYB108 and maize ZmMYB42 and ZmMYB31. All four of these R2R3-MYB proteins participate in cell wall formation, in addition to affecting the expression of stress-related genes. Both ZmMYB31 and OsMYB108 can directly repress lignin biosynthesis genes that negatively regulate SCW biosynthesis (Fornale et al. 2010; Miyamoto et al. 2019 ). Importantly, overexpression of PeMYB26 in tobacco not only improved lignin content in transgenic plants but also promoted expression of lignin biosynthesis genes (Fig. 5 and Fig. 6 ). It has been reported that MYB transcriptional factor participate in regulation of bamboo fast-growing, lignification and cell differentiation. BmMYB83 is specifically exprssed in protoxylem vessels and fiber cells and can promotes lignification in overexpression plants (Guo et al. 2022 ). PeMYB4.1 and PeMYB20 directly bind to the promoter of PeLAC20 to regulate lignin biosynthesis (Yang et al. 2021 ). The magnitude of changes of CCR in transgenic tobacco lines was more highly affected by PeMYB26 expression than that of other enzyme genes detected. The DLR assay confirmed that PeMYB26 positively promotes the expression of PeCCR . Unfortunately, using probes designed based on cis -element analysis, an electrophoretic mobility shift assay (EMSA) showed that PeMYB26 did not directly bind to the PeCCR promoter (Data is not shown). For further study, DNA affinity purification followed by deep sequencing (DAP-seq) analysis could help find the direct target gene(s) of PeMYB26. Lignin is a major component of the SCW. Changing the lignin content results in an altered structure of the xylem, and it also affects overall water transport in the plant. SiMYB16 positively regulates plant salt tolerance by affecting the biosynthesis of lignin (Yu et al. 2023 ), and CcMYB107 in the legume pigeon pea ( Cajanus cajan ) functioned as a positive regulator of stress-related genes and lignin biosynthesis genes under drought stress (Li et al. 2023). In purple false brome ( Brachypodium distachyon ), SECONDARY WALL ASSOCIATED MYB1 (SWAM1) activated the expression of cellulose and lignin biosynthesis genes, leading to a thicker SCW and more biomass accumulation (Handakumbura et al. 2018 ). In the present study, overexpression of PeMYB26 in N. tabacum also led to wider xylem and an increased number of vessel elements (Fig. 4 ). These results are in accordance with the relationship between xylem structure and drought tolerance (Isasa et al. 2023 ), indicating that PeMYB26 regulates the plant response to drought by affecting xylem formation. Although transgenic approaches in Moso bamboo and other bamboo species have been reported in recent years, stable genetic transformation systems for bamboo have not yet been established (Huang et al. 2022 ; Zheng et al. 2022 ). In conclusion, we find that PeMYB26, an R2R3-MYB transcriptional factor, positively regulates lignin biosynthesis by promoting the expression of PeCCR . Declarations Acknowledgments This study was funded by 2022NSFC1766 (B.W.), 2021YFD2200505-2 (G. X.). Author contributions Constructs generation, molecular and physiological analysis, Hitochemical staining: B.W., S.C., Z.Z., Dual-luciferase reporter assay, qRT-PCR and subcellular location: B.W. Y.L., C.L., Y.Z., Y.H., G.X., X.Z. Project design and writing: Y.C., S.H. and B.W. All authors read and approve the manuscript. Conflict or Interest statement The authors declare that we have no conflict of interest. References Chen Q, Peng L, Wang A, Yu L, Liu Y, Zhang X, Wang R, Li X, Yang Y, Li X, Wang J (2023) An R2R3-MYB FtMYB11 from Tartary buckwheat has contrasting effects on abiotic tolerance in Arabidopsis . Journal of plant physiology 280:153842. doi:10.1016/j.jplph.2022.153842. Chen Y, Feng P, Zhang X, Xie Q, Chen G, Zhou S, Hu Z (2022a) Silencing of SlMYB50 affects tolerance to drought and salt stress in tomato. 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Supplementary Files Supportinformation.docx Cite Share Download PDF Status: Published Journal Publication published 08 Nov, 2024 Read the published version in Plant Growth Regulation → Version 1 posted Editorial decision: Major revisions 14 Jun, 2024 Reviewers agreed at journal 23 Feb, 2024 Reviewers invited by journal 23 Jan, 2024 Editor invited by journal 03 Nov, 2023 Editor assigned by journal 31 Oct, 2023 First submitted to journal 30 Oct, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3495971","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":268939094,"identity":"519f2acc-2d01-4585-8483-12c2124e1a1a","order_by":0,"name":"Shanglian 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Engineering","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Cao","suffix":""}],"badges":[],"createdAt":"2023-10-26 18:46:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3495971/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3495971/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10725-024-01236-9","type":"published","date":"2024-11-08T15:57:40+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50174053,"identity":"33b91563-6297-493d-936a-562a66910d3e","added_by":"auto","created_at":"2024-01-25 16:05:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":32848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression patterns of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePeMYB26 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ein \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. edulis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e RT-qPCR analysis of \u003cem\u003ePeMYB26 \u003c/em\u003eexpression under drought treatment. Two-month-old Moso bamboo seedlings were treated with 0.8 M mannitol for 24 h to simulate drought. The expression level of \u003cem\u003ePeMYB26 \u003c/em\u003ewas normalized to \u003cem\u003ePeTIP40 \u003c/em\u003eas an internal reference. \u003cstrong\u003eb\u003c/strong\u003e RT-qPCR analysis of \u003cem\u003ePeMYB26 \u003c/em\u003eexpression in different tissues. The value in mature leaves was set to 1. Data represent means ± SD (\u003cem\u003en \u003c/em\u003e= 3) from three independent biological replicates. Asterisks indicate significant differences (**, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01) according to Student’s t test compared with controls or leaves.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3495971/v1/360bc0a8bb077d0fefdc430e.png"},{"id":50174054,"identity":"2106c8bd-619b-4374-a364-b929f21538f1","added_by":"auto","created_at":"2024-01-25 16:05:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":137603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacteristics of PeMYB26.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Phylogenetic tree representing species of \u003cem\u003eArabidopsis thaliana \u003c/em\u003e(AtMYB4, encoding by At4g38620, AtMYB7 [At2g16720], AtMYB32 [At4g34990]\u003cem\u003e, \u003c/em\u003eAtMYB26 [At3g13890], AtMYB46 [At5g12870], AtMYB83 [At3g08500], AtMYB55 [At4g01680], AtMYB20 [At1g66230], AtMYB58 [At1g16490], AtMYB63 [At1g79180]), \u003cem\u003eOryza sativa\u003c/em\u003e (OsMYB103 [LOC_Os04g39470], OsMYB61 [LOC_Os01g18240], OsMYB20 [LOC_Os03g20090], OsMYB60 [LOC_Os12g03150], OsMYB58 [LOC_Os04g46384], OsMYB55 [LOC_Os05g48010], OsMYB108 [LOC_Os09g36730]), \u003cem\u003eZea mays\u003c/em\u003e (ZmMYB42-1 [GRMZM2G419239], ZmMYB31 [GRMZM2G050305]) and \u003cem\u003ePhyllostachys edulis \u003c/em\u003ePeMYB26. The neighbor-joining method was used to calculate evolutionary history. Phylogenetic analysis was conducted in MEGA 7. \u003cstrong\u003eb\u003c/strong\u003eSubcellular localization of the PeMYB26-GFP fusion protein. The \u003cem\u003e35S:PeMYB26-GFP\u003c/em\u003econstruct and the \u003cem\u003e35S:GFP\u003c/em\u003e construct (positive control) were transformed into Agrobacterium\u003cem\u003e \u003c/em\u003estrain GV3101 and individually infiltrated into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Protoplasts prepared from the infiltrated leaves were monitored by confocal fluorescence microscopy and photographed 2 days after injection. Bars = 10 μm. \u003cstrong\u003ec\u003c/strong\u003e Analysis of transactivational activity of PeMYB26. AH109 yeast cells harboring the pGBKT7-PeMYB26 construct were grown on selective (SD-Leu/-Trp/-His/-Ade) media, followed by β-galactosidase assays (X-gal staining). BD (binding domain) represents the empty pGBKT7 vector and AD (activation domain) represents the empty pGADT7 vector.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3495971/v1/0cf8489bca52eef70bb4900e.png"},{"id":50172723,"identity":"3dae1771-18d0-43c4-ad78-338191f68c72","added_by":"auto","created_at":"2024-01-25 15:57:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":24001,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePeMYB26 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression level in overexpressing \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eN. tabacum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e plants and in control lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReal-time fluorescence quantification analysis of the transcription level of \u003cem\u003ePeMYB26\u003c/em\u003e overexpression transgenic lines and control. Actin was used as an internal control. Data are means ± SD (\u003cem\u003en \u003c/em\u003e= 3) from three independent replicates. Asterisks indicate significant differences (\u003cem\u003e**\u003c/em\u003e, \u003cem\u003eP \u0026lt; \u003c/em\u003e0.01) according to Student’s t test compared with untreated sample.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3495971/v1/a6240597c88a8b39f7eff20b.png"},{"id":50172724,"identity":"f5fdeeaa-05ca-4acf-81f7-6f92771a7485","added_by":"auto","created_at":"2024-01-25 15:57:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":387123,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePeMYB26 positively regulates xylem formation in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eN. tabacum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Phloroglucinol-Cl staining of stem transverse sections from transgenic \u003cem\u003eN. tabacum\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003elines overexpressing \u003cem\u003ePeMYB26\u003c/em\u003e. \u003cstrong\u003eb\u003c/strong\u003e Xylem width in sections shown in \u003cstrong\u003ea\u003c/strong\u003e. \u003cstrong\u003ec\u003c/strong\u003e Morphology of vessel cells in xylem of control lines and PeMYB26 ox lines. \u003cstrong\u003ed\u003c/strong\u003eStatistical analysis of vessel cell area cross-sections in transgenic plants. Data in \u003cstrong\u003eb\u003c/strong\u003e and \u003cstrong\u003ed\u003c/strong\u003e represent means ± SD (\u003cem\u003en \u003c/em\u003e= 3) from three independent biological replicates. Asterisks indicate significant differences from untreated control samples (\u003cem\u003e**\u003c/em\u003e, \u003cem\u003eP \u0026lt; \u003c/em\u003e0.01) according to Student’s t test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3495971/v1/7a03e9bfb6cf97074c0742c3.png"},{"id":50172726,"identity":"add7575e-5267-4034-b503-103081dd25e0","added_by":"auto","created_at":"2024-01-25 15:57:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":32463,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLignin content and expression of lignin biosynthesis genes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eN. tabacum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e plants overexpressing\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e PeMYB26\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Lignin content of transgenic lines and control lines. \u003cstrong\u003eb\u003c/strong\u003e Changes in the expression levels of downstream genes in the lignin biosynthesis pathway in \u003cem\u003ePeMYB26\u003c/em\u003e-overexpressing plants. Data are means ± SD (\u003cem\u003en \u003c/em\u003e= 3) from three independent biological replicates. Asterisks indicate significant differences from controls (\u003cem\u003e*\u003c/em\u003e, \u003cem\u003eP \u0026lt; \u003c/em\u003e0.05) according to Student’s t test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3495971/v1/7ef52d5eb803dd9b1d5fda0f.png"},{"id":50174055,"identity":"e588958e-30db-47cd-a5a0-c634cd83f17f","added_by":"auto","created_at":"2024-01-25 16:05:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":40818,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePeMYB26 promotes \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePeCCR \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression to regulate the response to drought.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Diagram of effector and reporter plasmids. \u003cstrong\u003eb\u003c/strong\u003eDual-luciferase assay using \u003cem\u003e35S:PeMYB26\u003c/em\u003e as effector and \u003cem\u003ePeCCRpro:LUC\u003c/em\u003eas reporter.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3495971/v1/a425693c1822d9657cf412d5.png"},{"id":68749941,"identity":"2f2a9034-c30c-4815-8241-bde844f3e9ca","added_by":"auto","created_at":"2024-11-11 16:07:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1303337,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3495971/v1/db62e360-057f-4240-bd2e-58cecfda83fc.pdf"},{"id":50172728,"identity":"297efb32-0ab9-4b34-bc3b-6a27b792d33f","added_by":"auto","created_at":"2024-01-25 15:57:56","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1752159,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-3495971/v1/f4a4be93b723d5aa807fbbf7.docx"}],"financialInterests":"","formattedTitle":"PeMYB26, an R2R3-MYB transcription factor, positively regulates Lignin deposition in Moso bamboo","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMoso bamboo (\u003cem\u003ePhyllostachys edulis\u003c/em\u003e)is the most economically woody bamboo species with more than 3.87\u0026nbsp;million ha area in China providing 70% of the bamboo-production (Song et al. 2011; Song et al. 2016). The rapid growth rate of bamboo shoots and the huge biomass of bamboo plants make bamboo an important bioenergy source. Moso bamboo shoots can grow 1 m per day during the most rapid growth period (Peng et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Dixon and Gibson \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The fast-growth and huge output, made moso bamboo as the raw materials of paper-making, building and industry. The cellulose in cell wall of bamboo culm is the valuable constituents in bamboo woody production. However, the tight combination of cellulose and lignin in bamboo culm always bothering the bamboo industrial producing.\u003c/p\u003e \u003cp\u003eLignin accumulation in the SCW of vascular cells along with cellulose increases rigidity and enables plants to stand upright and conduct water more efficiently. Altered expression of genes encoding lignin biosynthetic enzymes usually results in changes in the width of xylem cells and altered drought tolerance. For example, overexpression of \u003cem\u003eDfCCoAOMT14\u003c/em\u003e (CAFFEOYL COENZYME A ESTER O-METHYLTRANSFERASE) from the bamboo species \u003cem\u003eDendrocalamus farinosus\u003c/em\u003e in Nicotiana tabacum increases xylem thickness and improves drought tolerance, and enhances accumulation of lignin (Wei et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Because xylem is the main tissue involved in long-distance water transport in plants, its anatomical structure is highly related to plant drought tolerance (Weithmann et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lens et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In Atemoyas (\u003cem\u003eAnnona cherimola\u003c/em\u003e \u0026times; \u003cem\u003eAnnona squamosa\u003c/em\u003e), the presence of wider xylem cells and larger vessel elements results in better performance when soil water is scarce (Losada et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Genes involved in drought responses are likely to affect the development of the vascular system. VASCULAR RELATED NAC-DOMAIN PROTEIN 7 (VND7), a master switch in the transcriptional regulatory network in Arabidopsis (\u003cem\u003eArabidopsis thaliana\u003c/em\u003e), responds to abscisic acid (ABA) signaling induced upon drought stress by promoting xylem differentiation (Ramachandran et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yamaguchi et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Yamaguchi et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). \u003cem\u003ePdOLP1\u003c/em\u003e, encoding an osmotin-like protein in Eastern cottonwood (\u003cem\u003ePopulus deltoides\u003c/em\u003e), is co-expressed with genes involved in secondary cell wall (SCW) biosynthesis and regulates plant defenses against water deficit (Li et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Likewise, the altered accumulation of lignin in the plant SCW leads to different responses to drought. For instance, the microRNA CamiR397 in chickpea (\u003cem\u003eCicer arietinum\u003c/em\u003e) can target the transcripts of lignin biosynthesis genes, reducing plant sensitivity to drought and pathogen infection by decreasing deposition of lignin (Sharma et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Overexpression of \u003cem\u003ePtoMYB170\u003c/em\u003e in transgenic poplar leads to stronger lignification and thicker SCW in xylem (Xu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn many plant species, R2R3-MYB proteins are the directly targets of VNDs that affects SCW formation in SCW transcriptional regulatory networks. In Arabidopsis, MYB46 and MYB83 are the second main switch in SCW regulatory network that promote the synthesis of cellulose, lignin and hemicellulos, respectively (Xiao et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ko et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Arabidopsis MYB proteins target lignin biosynthesis genes to regulate lignin content and xylem development. For example, AtMYB15 activates the transcription of lignin biosynthesis genes such as PHENYLALANINE AMMONIA-LYASE (PAL), CAFFEATE O-METHYLTRANSFERASE (COMT), and CINNAMYL ALCOHOL DEHYDROGENASE (CAD) to increase the production of lignin monomers (Kim et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). MYB8 promotes PAL expression in vascular cells in maritime pine (\u003cem\u003ePinus pinaster\u003c/em\u003e Ait.) (Craven-Bartle et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and MYB103 is involved in lignin biosynthesis in Arabidopsis and Chinese silver grass (\u003cem\u003eMiscanthus sinensis\u003c/em\u003e) (Ohman et al. 2013; Golfier et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). MYBS3 in agricultural crops like maize (\u003cem\u003eZea mays\u003c/em\u003e), rice (\u003cem\u003eOryza sativa\u003c/em\u003e) and sorghum (\u003cem\u003eSorghum bicolor\u003c/em\u003e) positively regulates plant tolerance to drought (Liu et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In rice, ROLLING-LEAF MUTANT 1 (RLM1) interacts with MITOGEN-ACTIVATED PROTEIN KINASE 10 (OsMAPK10) and increases lignin accumulation in vessel elements by targeting the \u003cem\u003eCAD\u003c/em\u003e promoter (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022c\u003c/span\u003e). PeMYB83L in moso bamboo was also reported positively regulating SCW fast deposition through ABA signaling pathway (Chen et al. 2022d).\u003c/p\u003e \u003cp\u003eIn this study, we identified an R2R3-type MYB protein of Moso bamboo, named PeMYB26, positively regulated lignin deposition and xylem formation. \u003cem\u003ePeMYB26\u003c/em\u003e was expressed specifically in branches and was induced by water deficit. Tabacoo (\u003cem\u003eNicotiana tabacum\u003c/em\u003e) plants overexpressing \u003cem\u003ePeMYB26\u003c/em\u003e showed more developed xylem and increased expression levels of lignin biosynthesis genes. In addition, expression of \u003cem\u003eCINNAMOYL CoA REDUCTASE\u003c/em\u003e (\u003cem\u003ePeCCR\u003c/em\u003e) was promoted by the accumulation of PeMYB26. These results indicated that PeMYB26 is a functional R2R3-MYB protein that played positive role in lignin deposition, xylem development by promoting the expression of lignin biosynthesis genes.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant growth conditions and drought stress treatment\u003c/h2\u003e \u003cp\u003eAll plant materials used in this study were from the Institute of Bamboo Research, Southwest University of Science and Technology, Mianyang, Sichuan province, China. For drought stress treatment, 2-month-old Moso bamboo (\u003cem\u003ePhyllostachys edulis\u003c/em\u003e) seedlings were treated with 0.8 M mannitol and frozen in liquid nitrogen before treatment and after 24 h of treatment. \u003cem\u003eNicotiana benthamiana\u003c/em\u003e plants were grown in a greenhouse with a photoperiod of 16-h light/8-h dark at a light intensity of ~\u0026thinsp;110 mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. \u003cem\u003eNicotiana tabacum\u003c/em\u003e seeds were surface sterilized for 15 min in a 20% (v/v) bleach solution in water, and were subsequently washed six times with sterile water. The disinfected seeds were then kept at 4\u0026deg;C for 72 h and then transferred onto Murashige and Skoog (MS) medium (PhytoTechnology Laboratories\u0026trade;) containing 1% (w/v) sucrose and 0.8% (w/v) phytagel (pH 5.7, Sigma-Aldrich). Plants were then grown in a greenhouse as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation and RT-qPCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from \u003cem\u003eP. edulis\u003c/em\u003e leaves using TRIzol reagent (Takara, Dalian, China), and RNA quality was monitored using 1% (w/v) agarose gels and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Beijing, China). First-strand cDNAs were synthesized using 2 \u0026micro;g of total RNA and a FastKing cDNA First Strand Synthesis Kit (TIANGEN, Beijing, China). Quantitative PCR (qPCR) was performed using a CFX connect Real-time PCR Detection System and a SYBR qPCR Master MIX kit (Vazyme, Nanjing, China); amplification profiles were analyzed using CFX Manager software version 3.1 (Bio-Rad, Hercules, CA, USA). The relative expression levels of genes were calculated using the 2\u003csup\u003e\u0026ndash;ΔΔCt\u003c/sup\u003e method (Livak and Schmittgen \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). All RT-qPCR experiments were performed with two technical replicates and three independent biological replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTransactivation activity assay\u003c/h2\u003e \u003cp\u003eThe coding sequence of \u003cem\u003ePeMYB26\u003c/em\u003e was subcloned into pGBKT7 keeped in our institute to obtain the pGBKT7\u003cem\u003e-PeMYB26\u003c/em\u003e vector. This construct, along with the empty pGADT7 vector, were co-transformed into the yeast (\u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e) AH109 strain. After transformation, positive transformants were selected on synthetic defined (SD) medium \u0026ndash;Trp\u0026ndash;Leu (SD/\u0026ndash;Trp/\u0026ndash;Leu). Positive transformants were then plated onto SD/\u0026ndash;Trp/\u0026ndash;Leu/\u0026ndash;His/\u0026ndash;Ade medium and SD/\u0026ndash;Trp/\u0026ndash;Leu\u0026ndash;His/\u0026ndash;Ade/+X-α-GAL medium, followed by incubation at 30℃ for approximately four to six days.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHeterologous overexpression of\u003c/b\u003e \u003cb\u003ePeMYB26\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eN. tabacum\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOverexpressing \u003cem\u003eN. tabacum\u003c/em\u003e lines were obtained by introducing pCAMBIA1303 containing \u003cem\u003ePeMYB26\u003c/em\u003e driven by the CaMV 35S promoter (\u003cem\u003e35S :: PeMYB26-GFP\u003c/em\u003e) into Agrobacterium (\u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e) strain EHA105 using the leaf disc method (Wang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The empty pCAMBIA1303 vector was introduced into Agrobacterium strain EHA105 as a negative control. Infected leaf discs were incubated on MS medium containing 0.1 mg/L 1-Naphthaleneacetic acid (NAA, Sigma-Aldrich), 0.5 mg/L 6-Benzylaminopurine (6-BA, Sigma-Aldrich), 500 mg/L acetosyringone (Sigma-Aldrich) and 50 mg/L kanamycin (TIANGEN, Beijing) in the dark at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C for 2 days. Regenerated buds were transferred to MS plates containing 0.1 mg/L NAA and 50 mg/L kanamycin for selection and to promote the formation of complete plants. Genomic DNA was extracted from the leaves of transgenic plants, and then amplified using specific primers for pCAMBIA1303 and \u003cem\u003ePeMYB26\u003c/em\u003e. Primers used were listed in Supplement Table\u0026nbsp;1. PCR products were checked by 1% (w/v) agarose gel electrophoresis. The expression of \u003cem\u003ePeMYB26\u003c/em\u003e in transgenic plants was assessed by RT-qPCR and \u003cem\u003eActin\u003c/em\u003e was used as the internal reference for normalization (Wei et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHistochemical staining\u003c/h2\u003e \u003cp\u003eIntact thin sections of PeMYB26 overexpression Tabacco plants\u0026rsquo; stem were stained in 2% (w/v) phloroglucinol solution for 5 min, and were then stained with 30% (v/v) hydrochloric acid for 1 min, washed with water, observed and photographed under a Leica microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of lignin content in transgenic\u003c/b\u003e \u003cb\u003eN. tabacum\u003c/b\u003e \u003cb\u003eplants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDetermination of lignin content was based on the method of Li et al (Li et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). and was improved as follows: After 0.5 g of fresh sample was ground to a fine powder in liquid nitrogen, 5 mL of 0.1 M phosphate buffer (pH 7.2) was added, and the mixture was incubated at 37\u0026deg;C for 30 min, followed by centrifugation at 4000 rpm for 5 min. Then, 5 mL of 80% (v/v) ethanol was added, and the sample was incubated at 80\u0026deg;C for 1 h. After this, the sample was centrifuged at 4000 rpm for 5 min, 10 mL of acetone was added to extract lignin, and the sample was centrifuged at 5000 rpm for 5 min and then dried at 60\u0026deg;C. For lignin analysis, approximately 20 mg of dried plant cell wall material was incubated at 80\u0026deg;C in a mixture of 750 \u0026micro;L of water and 250 \u0026micro;L of concentrated HCl and 100 \u0026micro;L of thioglycolic acid for 3 h. The mixture was centrifuged at 4000 rpm for 10 min and the pellet was washed with 1 mL of water and resuspended in 1 mL of 1 M NaOH and placed on a rocking plate at room temperature overnight. The mixture was centrifuged again, and the supernatant was collected and mixed with 200 \u0026micro;L of concentrated HCl. After being vortexed and incubated at 4\u0026deg;C for 4 h, the mixture was centrifuged and the pellet was dissolved in 1 mL of 1 M NaOH. The absorbance of a 50-fold dilution of the supernatant in 1 M NaOH was measured at 280 nm to determine lignin content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDual-luciferase reporter assay\u003c/h2\u003e \u003cp\u003eA promoter fragment of 1.8 kb upstream of the \u003cem\u003ePeCCR\u003c/em\u003e start codon was amplified from \u003cem\u003eP. edulis\u003c/em\u003e genomic DNA inserted into the multiple cloning site of pGreenII 0800-LUC, yielding the \u003cem\u003ePeCCRpro:LUC\u003c/em\u003e reporter construct. The promoter construct was transformed into Agrobacterium strain GV3101 containing the p19 helper plasmid. Bacteria were resuspended in 10 mL of infiltration buffer consisting of 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 200 \u0026micro;M acetosyringone and 10 mM MES (pH 5.7), and the cell suspensions were incubated in the dark for 2 to 3 h before infiltration. The Agrobacterium cell suspension containing the \u003cem\u003ePeCCRpro:LUC\u003c/em\u003e construct was mixed at a ratio of 1 : 1 (v/v) with Agrobacterium cell suspension harboring the effector construct \u003cem\u003e35S::PeMYB26\u003c/em\u003e generated above. The cell mixture was infiltrated into young leaves of 35-day-old \u003cem\u003eN. benthamiana\u003c/em\u003e plants. Two days after infiltration, the ratio of firefly luciferase (LUC) to \u003cem\u003eRenilla\u003c/em\u003e luciferase (Ren) activity was measured using a Dual-Glo\u0026reg; Luciferase Assay System (Promega) and an Infinite M200 luminometer (Tecan, Mannerdorf, Switzerland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe MYBs of Moso Bamboo (\u003cem\u003eP. edulis\u003c/em\u003e), rice (\u003cem\u003eOryza sativa\u003c/em\u003e) and maize (\u003cem\u003eZea may\u003c/em\u003e) were downloaded from NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The evolutionary history was inferred using the Neighbor-Joining method. The analysis involved 29 amino acid sequences. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA7.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eThe protoplast transient assay\u003c/h2\u003e \u003cp\u003eFor subcellular localization, approximately 1-month-old \u003cem\u003eN.tabacum\u003c/em\u003e plants were used for protoplast transfection. The \u003cem\u003ePeMYB26-GFP\u003c/em\u003e and the \u003cem\u003e35S:GFP\u003c/em\u003e construct (positive control) were transformed into Agrobacterium strain GV3101 and individually infiltrated into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. 48 h after injection, leaves express \u003cem\u003ePeMYB26-GFP\u003c/em\u003e and \u003cem\u003e35S:GFP\u003c/em\u003e were digested protoplasts respectively, and GFP signal was investigated after 16 h. Photos were capture by a confocal fluorescence microscope (TCS SP5 system, Leica, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data in this study were analyzed using SPSS 25.0 and Origin 2018 softwares. Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of three independent biological replicates and two technical replicates. Asterisks indicate significant differences from the control (\u003cem\u003e*\u003c/em\u003e, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003cem\u003e*\u003c/em\u003e, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) according to Student\u0026rsquo;s t test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePeMYB26\u003c/b\u003e \u003cb\u003eencodes an R2R3-MYB transcription factor and responds to drought treatment\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn previous research, we noticed a gene whose expression was induced by drought in Moso bamboo. To further explore this observation, we treated 2-month-old Moso bamboo seedlings with 0.8 M mannitol to simulate drought stress. We determined that the expression level of this gene increases 5-fold compared to untreated control seedlings after a 48-h treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Analysis using the SMART online database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://smart.embl.de/\u003c/span\u003e\u003cspan address=\"https://smart.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) revealed the presence of two conserved SANT domains in the N-terminal region of the encoded protein (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea). Because these domains are structurally homologous to those in the helix-turn-helix MYB family of DNA-binding proteins, we named this gene \u003cem\u003ePeMYB26\u003c/em\u003e. We detected \u003cem\u003ePeMYB26\u003c/em\u003e transcripts by RT-qPCR in different tissues of \u003cem\u003eP. edulis\u003c/em\u003e, including unrolled leaves, branches and internodes, with the expression of \u003cem\u003ePeMYB26\u003c/em\u003e being higher in branches and internodes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Compared to leaves, the expression level of \u003cem\u003ePeMYB26\u003c/em\u003e was 2 to 2.4 times higher in branches and internodes. Therefore, we speculated that \u003cem\u003ePeMYB26\u003c/em\u003e might be specifically expressed in vascular tissue and induced by conditions of water deficit. In addition, PeMYB26 is orthologous to several plant R2R3-type MYB transcription factors that regulate SCW deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The N-terminal region of PeMYB26 has conserved R2 and R3 repeats (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb), which are also found in R2R3-MYB proteins functioning in SCW accumulation (Fig. S2). Taken together, these results suggest that PeMYB26 participates the formation of SCW.\u003c/p\u003e \u003cp\u003eTo analyze the subcellular location of PeMYB26, we cloned the \u003cem\u003ePeMYB26\u003c/em\u003e sequence in-frame and upstream of the green fluorescent protein (\u003cem\u003eGFP\u003c/em\u003e) sequence. We then infiltrated the resulting construct \u003cem\u003e35S:PeMYB26-GFP\u003c/em\u003e and the empty GFP vector (\u003cem\u003e35S:GFP\u003c/em\u003e) into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. We detected fluorescence signals predominantly in the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, PeMYB26-GPF), whereas the \u003cem\u003e35S::GFP\u003c/em\u003e vector produced green fluorescence throughout the cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, positive control). This result demonstrates that PeMYB26 is located in the nucleus. Because \u003cem\u003ePeMYB26\u003c/em\u003e appeared to encode a transcription factor, we used the yeast two-hybrid (Y2H) system to determine whether it acted as a transcriptional activator or a transcriptional repressor. To this end, we cloned the \u003cem\u003ePeMYB26\u003c/em\u003e coding sequence in-frame and upstream of the sequence encoding the DNA-binding domain of yeast GAL4 in the pGBKT7 vector. Only yeast clones harboring the pGBKT7-\u003cem\u003ePeMYB26\u003c/em\u003e plasmid showed transcriptional activity and could grow on synthetic defined medium lacking Leu, Trp, His, and Ade (SD/\u0026ndash;Trp/\u0026ndash;Leu/\u0026ndash;His/\u0026ndash;Ade). We examined the authenticity and strength of PeMYB26 transcriptional activity by titration and X-gal staining. The Y2H assay revealed that PeMYB26 can activate the expression of \u003cem\u003eGAL4\u003c/em\u003e indicating that PeMYB26 is a transcriptional activator (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003cb\u003eHeterologous expression of\u003c/b\u003e \u003cb\u003ePeMYB26\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eN. tabacum\u003c/b\u003e \u003cb\u003eresults in more highly developed vascular tissue\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine the role of PeMYB26 in SCW biosynthesis and response to drought, we generated transgenic \u003cem\u003eN. tabacum\u003c/em\u003e lines overexpressing \u003cem\u003ePeMYB26\u003c/em\u003e under the control of the cauliflower mosaic virus 35S promoter. Using kanamycin as a selectable marker, we obtained three independent \u003cem\u003e35S::PeMYB26\u003c/em\u003e transgenic lines (\u003cem\u003eox-1\u003c/em\u003e, \u003cem\u003eox-2\u003c/em\u003e and \u003cem\u003eox-4\u003c/em\u003e). RT-qPCR analysis demonstrated that the transcript abundance of \u003cem\u003ePeMYB26\u003c/em\u003e is significantly higher in these overexpressing lines relative to control lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Anatomical analysis of culm cross-sections from the control and transgenic lines showed that overexpression of \u003cem\u003ePeMYB26\u003c/em\u003e results in increased the entire xylem width compared to the control line (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In addition, the xylem width of the overexpression lines was almost double that of control lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). At the same time, overexpression of \u003cem\u003ePeMYB26\u003c/em\u003e also resulted in production of more vessel elements with larger diameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), suggesting that the overexpression of \u003cem\u003ePeMYB26\u003c/em\u003e may help plants to grow a wider xylem and thicker SCW.\u003c/p\u003e\u003cp\u003e \u003cb\u003eAltered expression of\u003c/b\u003e \u003cb\u003ePeMYB26\u003c/b\u003e \u003cb\u003echanges the content of lignin in transgenic plants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAlong with wider xylem, the lignin content of culms from overexpression lines also increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In the \u003cem\u003eox-1\u003c/em\u003e and \u003cem\u003eox-4\u003c/em\u003e transgenic lines, lignin content was 20% higher than in control lines. Lignin content in the \u003cem\u003eox-2\u003c/em\u003e transgenic line was not significantly different from that in control lines. Because of the increased lignin content in some \u003cem\u003ePeMYB26\u003c/em\u003e overexpression lines, we suspected that PeMYB26 affected lignin biosynthesis. Considering that the lignin monomer biosynthesis pathway is relatively well conserved in seed plants, we measured the expression of several key lignin biosynthesis genes in control and \u003cem\u003ePeMYB26\u003c/em\u003e overexpression plants. Along with the higher expression of \u003cem\u003ePeMYB26\u003c/em\u003e, the expression level of lignin biosynthesis genes increased. Compared to the control line, \u003cem\u003eCCR\u003c/em\u003e expression was 75-fold higher in the \u003cem\u003eox-1\u003c/em\u003e line and more than 10-fold higher in \u003cem\u003eox-2\u003c/em\u003e and \u003cem\u003eox-4\u003c/em\u003e lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eTo test whether PeMYB26 can activate \u003cem\u003ePeCCR\u003c/em\u003e expression. we used a dual-luciferase reporter (DLR) assay system in \u003cem\u003eN. benthamiana\u003c/em\u003e protoplasts transfected with a construct consisting of the firefly luciferase (\u003cem\u003eLUC\u003c/em\u003e) gene driven by a 1.8-kb fragment of the \u003cem\u003ePeCCR\u003c/em\u003e promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The DLR assay revealed a 5-fold increase in transcriptional activation in protoplasts co-transfected with the \u003cem\u003e35S:PeMYB26\u003c/em\u003e effector and the \u003cem\u003ePeCCRpro:LUC\u003c/em\u003e reporter construct (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The DLR assay result, along with the highly induced expression of \u003cem\u003eCCR\u003c/em\u003e in \u003cem\u003ePeMYB26\u003c/em\u003e transgenic lines, suggests that PeMYB26 is a transcriptional activator that promotes \u003cem\u003ePeCCR\u003c/em\u003e expression.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt is widely known that SCW formation is regulated by a NAC-MYB transcriptional network. In Arabidopsis, MYB46 and MYB83 are targeted by VND7 and regulate other MYB\u0026rsquo;s expression to switch the accumulation of cellulose and lignin in plant SCW (Ko et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ohtani and Demura \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). There are also evidences that MYB transcriptional factors affect plant stress tolerance by regulating wax and flavonoid biosynthesis (Chen et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Gao et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similarly, we showed that \u003cem\u003ePeMYB26\u003c/em\u003e was also highly expressed in internodes of Moso bamboo, indicating that \u003cem\u003ePeMYB26\u003c/em\u003e might be involved in both modulating the rate of plant growth and responding to drought (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, we selected \u003cem\u003ePeMYB26\u003c/em\u003e for functional analysis.\u003c/p\u003e \u003cp\u003eTranscriptional assays, protein sequence analysis and subcellular localization results demonstrated that \u003cem\u003ePeMYB26\u003c/em\u003e encodes a nucleus-localized R2R3-type MYB activator (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and S1). The results of phylogenetic analysis showed that PeMYB26 is homologous to Arabidopsis AtMYB4, rice OsMYB108 and maize ZmMYB42 and ZmMYB31. All four of these R2R3-MYB proteins participate in cell wall formation, in addition to affecting the expression of stress-related genes. Both ZmMYB31 and OsMYB108 can directly repress lignin biosynthesis genes that negatively regulate SCW biosynthesis (Fornale et al. 2010; Miyamoto et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Importantly, overexpression of \u003cem\u003ePeMYB26\u003c/em\u003e in tobacco not only improved lignin content in transgenic plants but also promoted expression of lignin biosynthesis genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). It has been reported that MYB transcriptional factor participate in regulation of bamboo fast-growing, lignification and cell differentiation. BmMYB83 is specifically exprssed in protoxylem vessels and fiber cells and can promotes lignification in overexpression plants (Guo et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). PeMYB4.1 and PeMYB20 directly bind to the promoter of PeLAC20 to regulate lignin biosynthesis (Yang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe magnitude of changes of \u003cem\u003eCCR\u003c/em\u003e in transgenic tobacco lines was more highly affected by \u003cem\u003ePeMYB26\u003c/em\u003e expression than that of other enzyme genes detected. The DLR assay confirmed that PeMYB26 positively promotes the expression of \u003cem\u003ePeCCR\u003c/em\u003e. Unfortunately, using probes designed based on \u003cem\u003ecis\u003c/em\u003e-element analysis, an electrophoretic mobility shift assay (EMSA) showed that PeMYB26 did not directly bind to the \u003cem\u003ePeCCR\u003c/em\u003e promoter (Data is not shown). For further study, DNA affinity purification followed by deep sequencing (DAP-seq) analysis could help find the direct target gene(s) of PeMYB26.\u003c/p\u003e \u003cp\u003eLignin is a major component of the SCW. Changing the lignin content results in an altered structure of the xylem, and it also affects overall water transport in the plant. \u003cem\u003eSiMYB16\u003c/em\u003e positively regulates plant salt tolerance by affecting the biosynthesis of lignin (Yu et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and CcMYB107 in the legume pigeon pea (\u003cem\u003eCajanus cajan\u003c/em\u003e) functioned as a positive regulator of stress-related genes and lignin biosynthesis genes under drought stress (Li et al. 2023). In purple false brome (\u003cem\u003eBrachypodium distachyon\u003c/em\u003e), SECONDARY WALL ASSOCIATED MYB1 (SWAM1) activated the expression of cellulose and lignin biosynthesis genes, leading to a thicker SCW and more biomass accumulation (Handakumbura et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the present study, overexpression of \u003cem\u003ePeMYB26\u003c/em\u003e in \u003cem\u003eN. tabacum\u003c/em\u003e also led to wider xylem and an increased number of vessel elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results are in accordance with the relationship between xylem structure and drought tolerance (Isasa et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), indicating that PeMYB26 regulates the plant response to drought by affecting xylem formation. Although transgenic approaches in Moso bamboo and other bamboo species have been reported in recent years, stable genetic transformation systems for bamboo have not yet been established (Huang et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zheng et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In conclusion, we find that PeMYB26, an R2R3-MYB transcriptional factor, positively regulates lignin biosynthesis by promoting the expression of \u003cem\u003ePeCCR\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by 2022NSFC1766 (B.W.), 2021YFD2200505-2 (G. X.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConstructs generation, molecular and physiological analysis, Hitochemical staining: B.W., S.C., Z.Z., Dual-luciferase reporter assay, qRT-PCR and subcellular location: B.W. Y.L., C.L., Y.Z., Y.H., G.X., X.Z. Project design and writing: Y.C., S.H. and B.W. All authors read and approve the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict or Interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that we have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChen Q, Peng L, Wang A, Yu L, Liu Y, Zhang X, Wang R, Li X, Yang Y, Li X, Wang J (2023) An R2R3-MYB FtMYB11 from \u003cem\u003eTartary buckwheat\u003c/em\u003e has contrasting effects on abiotic tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e. Journal of plant physiology 280:153842. doi:10.1016/j.jplph.2022.153842.\u003c/li\u003e\n\u003cli\u003eChen Y, Feng P, Zhang X, Xie Q, Chen G, Zhou S, Hu Z (2022a) Silencing of \u003cem\u003eSlMYB50 \u003c/em\u003eaffects tolerance to drought and salt stress in tomato. 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Journal of integrative plant biology. doi:10.1111/jipb.13217.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-growth-regulation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"grow","sideBox":"Learn more about [Plant Growth Regulation](https://www.springer.com/journal/10725)","snPcode":"10725","submissionUrl":"https://submission.nature.com/new-submission/10725/3","title":"Plant Growth Regulation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bamboo, R2R3-MYBs, xylem widen, lignin monomer synthesis","lastPublishedDoi":"10.21203/rs.3.rs-3495971/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3495971/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMoso bamboo (\u003cem\u003ePhyllostachys edulis\u003c/em\u003e) is a highly valuable woody bamboo species. A better understanding of its regulation of lignin deposition would significantly benefit its cultivation and breeding. Here, we identified PeMYB26, an transcription factor gene that is primarily expressed in the vascular system. \u003cem\u003ePeMYB26 \u003c/em\u003eencodes an R2R3-MYB transcriptional activator that localizes to the nucleus. Heterologous expression of PeMYB26 under control of the cauliflower mosaic virus 35S promoter caused widening of xylem, thickening of vessel elements, and deposition of lignin in transgenic tobacco (\u003cem\u003eNicotiana tabacum\u003c/em\u003e) plants. Moreover, transcript abundances of the lignin biosynthesis genes \u003cem\u003ePAL\u003c/em\u003e（\u003cem\u003ePHENYLALANINE AMMONIA-LYASE\u003c/em\u003e）, \u003cem\u003eCAD\u003c/em\u003e（CINNAMYL ALCOHOL DEHYDROGENASE）, \u003cem\u003eCOMT\u003c/em\u003e（CAFFEATE O-METHYLTRANSFERASE）\u003cem\u003e \u003c/em\u003eand \u003cem\u003eCCR \u003c/em\u003e（CINNAMOYL CoA REDUCTASE ）were markedly higher in \u003cem\u003eN. tabacum\u003c/em\u003e lines overexpressing \u003cem\u003ePeMYB26 \u003c/em\u003ethan in control lines. In particular, the expression of \u003cem\u003ePeCCR \u003c/em\u003ewas highly promoted by PeMYB26. These results indicate that PeMYB26 plays a positive role in regulating lignin accumulation and xylem formation.\u003c/p\u003e","manuscriptTitle":"PeMYB26, an R2R3-MYB transcription factor, positively regulates Lignin deposition in Moso bamboo","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-25 15:57:51","doi":"10.21203/rs.3.rs-3495971/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2024-06-14T20:45:13+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-02-23T08:40:37+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-23T10:13:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant Growth Regulation","date":"2023-11-03T11:26:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-10-31T08:07:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Growth Regulation","date":"2023-10-30T09:38:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-growth-regulation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"grow","sideBox":"Learn more about [Plant Growth Regulation](https://www.springer.com/journal/10725)","snPcode":"10725","submissionUrl":"https://submission.nature.com/new-submission/10725/3","title":"Plant Growth Regulation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7f2bd0a3-6d89-4391-9f5d-ef4915571d9c","owner":[],"postedDate":"January 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-11T16:01:37+00:00","versionOfRecord":{"articleIdentity":"rs-3495971","link":"https://doi.org/10.1007/s10725-024-01236-9","journal":{"identity":"plant-growth-regulation","isVorOnly":false,"title":"Plant Growth Regulation"},"publishedOn":"2024-11-08 15:57:40","publishedOnDateReadable":"November 8th, 2024"},"versionCreatedAt":"2024-01-25 15:57:51","video":"","vorDoi":"10.1007/s10725-024-01236-9","vorDoiUrl":"https://doi.org/10.1007/s10725-024-01236-9","workflowStages":[]},"version":"v1","identity":"rs-3495971","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3495971","identity":"rs-3495971","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}