CmMYB8 encodes an R2R3 MYB transcription factor which represses lignin and flavonoid synthesis in chrysanthemum

Plant Physiology and Biochemistry 149 (2020) 217–224

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Research article

CmMYB8 encodes an R2R3 MYB transcription factor which represses lignin and flavonoid synthesis in chrysanthemum

T

Lu Zhu, Yunxiao Guan, Zhaohe Zhang, Aiping Song, Sumei Chen, Jiafu Jiang, Fadi Chen∗ State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, College of Horticulture, Nanjing Agricultural University, Nanjing, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Chrysanthemum R2R3-MYB factors Lignin Flavonoids

R2R3-MYB transcription factors are important regulators of the growth and development of plants. Here, CmMYB8 a chrysanthemum gene encoding an R2R3-MYB transcription factor, was isolated and functionally characterized. The gene was transcribed throughout the plant, but most strongly in the stem. When CmMYB8 was over-expressed, a number of genes encoding components of lignin synthesis were down-regulated, and the plants’ lignin content was reduced. The composition of the lignin in the transgenic plants was also altered, and its S/G ratio was reduced. A further consequence of the over-expression of CmMYB8 was to lessen the transcript abundance of key genes involved in flavonoid synthesis, resulting in a reduced accumulation of flavonoids. The indication is that the CmMYB8 protein participates in the negative regulation of both lignin and flavonoid synthesis.

1. Introduction Lignin is a major component of the vascular plant cell wall (Boerjan et al., 2003). The lignin polymer is generated from p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol (Jorge et al., 2011; Ruben et al., 2012), compounds which are synthesized via the phenylpropanoid pathway (Boerjan et al., 2003; Douglas, 1996). The key enzymes involved in lignin synthesis are L-phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-hydroxycinnamate-CoA ligase (4CL), p-coumarate 3-hydroxylase (C3H) and shikimate hydroxycinnamoyl transferase (HCT) (Huang et al., 2010; Sykes et al., 2015; Voelker et al., 2011; Shadle et al., 2007), while the synthesis of the lignin monomers relies on the enzymes caffeoyl-CoA O-methyltransferase (CCoAOMT), cinnamoyl-CoA reductase (CCR), caffeic acid 3-O-methyltransferase (COMT), ferulate 5-hydroxylase (F5H) and cinnamoyl alcohol dehydrogenase (CAD) (Shafrin et al., 2015; Wagner et al., 2011; Ragauskas et al., 2013; Meester et al., 2018; Richard et al., 2005). The large family of MYB transcription factors comprises three subclasses, namely MYB1R, R2R3-MYB and MYB3R; of these, the largest group in plants is formed by the R2R3s (Dubos et al., 2010; Stracke et al., 2001). The genome of the model plant Arabidopsis thaliana, for example, harbors 126 genes encoding an R2R3-MYB (Stracke et al., 2001), while that of maize harbors 173 such genes (Yilmaz et al., 2009),

that of apple 222 (Cao et al., 2013), that of tomato 121 (Zhao et al., 2014) and that of black cottonwood 192 (Wilkins et al., 2009). Some of these transcription factors are known to regulate both lignin and flavonoid synthesis (Liu et al., 2015; Zhong and Ye, 2009; Zhao and Dixon, 2011; Rogers and Campbell, 2004; Xu et al., 2014; Nemesio-Gorriz et al., 2017; Zhao et al., 2013). A number of them have proven to be negative regulators: for example, the products of both the Antirrhinum majus genes AmMYB308 and AmMYB330 negatively regulate activity in the phenylpropanoid pathway (Tamagnone et al., 1998). The over-expression of the A. thaliana gene AtMYB4, as well as its constitutive expression in tobacco, suppresses plant growth by restricting the synthesis of both lignin and flavonoids (Jin et al., 2014). Over-expressing the banana MYB31 gene down-regulates many of the genes implicated in the synthesis of both lignin and flavonoids, thereby limiting the accumulation of lignin in the vascular elements' cell wall (Tak et al., 2017). Constitutively expressing either of the maize genes ZmMYB31 and ZmMYB42 in A. thaliana also represses lignin and flavonoid synthesis, leading to an altered composition of lignin, a reduced syringyl to guaiacyl (S/G) ratio and a lowered flavonol content (Silvia et al., 2010; Sonbol et al., 2009). In grape vine, phenylpropanoid metabolism can be down-regulated by over-expressing VvMYB5b, producing plants of shortened stature featuring a reduced content of flavonoids (Mahjoub et al., 2009). According to Zhu et al. (2013), the product of the chrysanthemum gene CmMYB1 acts as a negative

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Corresponding author. College of Horticulture, Nanjing Agricultural University, Nanjing, 210095, China. E-mail addresses: [email protected] (L. Zhu), [email protected] (Y. Guan), [email protected] (Z. Zhang), [email protected] (A. Song), [email protected] (S. Chen), [email protected] (J. Jiang), [email protected] (F. Chen). https://doi.org/10.1016/j.plaphy.2020.02.010 Received 20 November 2019; Received in revised form 16 January 2020; Accepted 11 February 2020 0981-9428/ © 2020 Elsevier Masson SAS. All rights reserved.

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Fig. 1. The deduced peptide sequence of CmMYB8 and related MYBs. (a) Polypeptide alignment: the R2 and R3 MYB DNA binding domains are indicated by underlining. (b) Phylogeny of CmMYB8 and related MYBs. LsMYB8: XP_023745626.1, HaMYB8: XP_022022105.1, CcMYB8: XP_024994176.1, CtMYB1: AJE68930.1, GhMYB9A: CAD87009.1, HaMYB308: XP_022002072.1, DzMYB308: XP_022773545.1, MtMYB6: XP_013450665.1, VaMYB6: XP_017405792.1, GaMYB308: XP_017624260.1, AtMYB6: AT4G09460, AtMYB8: AT1G35515.1, CcMYB308: XP_020212715.1, GaMYB3: KHG00323.1, VrMYB8: XP_014491519.1, GsMYB8: XP_028226877.1, ApMYB8: XP_027334676.1, MpMYB4: RDX69244.1, AtMYB3: AT1G22640.1, AtMYB4: AT4G38620.1.

MdMYBA and MdMYB10 from apple (Ban et al., 2007; Jiang et al., 2014), and GMYB10 from Gerbera hybrida (Laitinen et al., 2008). Chrysanthemum (Chrysanthemum morifolium) is a commercially important ornamental species. A number of commercially important traits, including lodging, aphid resistance and pedicel rigidity, are dependent on the quantity and quality of the lignin deposited in the plants’ cell walls (Peng et al., 2014; An et al., 2019; Lv et al., 2011). The purpose of the present research was to isolate and characterize CmMYB8, a gene encoding an R2R3-MYB transcription factor. The research was intended to add to the current level of understanding of how R2R3 transcription factors regulate phenylpropanoid synthesis in chrysanthemum. 2. Materials and methods 2.1. Growing conditions Cuttings of the chrysanthemum cultivar ‘Jinba’, maintained by Nanjing Agricultural University's Chrysanthemum Germplasm Resource Preserving Centre, were raised in a greenhouse using a 1:1:1 mixture of perlite, vermiculite and soilrite. Plants were cultured in plant growth incubator (RDN-1000D, Dongnan, Ningbo, China) set to 23/18 °C (day/ night) under long days (16 h photoperiod/8 h dark) and 200 μmol/m2/s and relative humidity 70%.

Fig. 2. Expression of CmMYB8 in different tissues of chrysanthemum plants. (a) Complete chrysanthemum plant containing root, stem, leaf and flower. Bar: 1 cm. (b) Tubular florets and ray florets. Bar: 1 cm. (c) Ray florets. Bar: 1 cm. (d) Transcriptional profiling of CmMYB8 in the chrysanthemum plant. Values derived from qRT-PCR data and shown in the form mean ± SE (n=3).

regulator of both lignin and flavonoid synthesis. However, some R2R3MYB transcription factors act to positively regulate the synthesis of lignin and/or flavonoids. Thus, for example, the constitutive expression of VvMYB5a in tobacco promotes the accumulation of flavonol (Deluc et al., 2006), while the over-expression of either AtMYB58 or AtMYB63 results in an ectopic pattern of lignin deposition (Zhou et al., 2009). The products of the poplar genes PtrMYB152 and PtoMYB216 act as transcriptional activators in the lignin synthesis pathway (Li et al., 2014; Tian et al., 2013). In chrysanthemum, the over-expression of two MYB genes (CmMYB15 and CmMYB19) promotes lignin synthesis, thereby boosting the plants’ ability to resist aphid infestation (An et al., 2019; Wang et al., 2017). Other MYB products identified as activators of flavonol synthesis include AtMYB11, AtMYB12 and AtMYB111 from A. thaliana (Luo et al., 2008; Misra et al., 2010; Pandey et al., 2014, 2015),

2.2. Isolation of cDNA and transcriptional profiling RNA was extracted from the root, stem, leaf, tubular floret and ray floret sampled from three cultivar ‘Jinba’ plants, using the RNAiso reagent (TaKaRa, Tokyo, Japan). To obtain single-stranded cDNA, the RNA samples were treated with M-MLV reverse transcriptase (TaKaRa). Primers created to amplify portions of CmMYB8 were designed from its full length cDNA sequence (accession SRP070731 in the cultivar ‘Jinba’ chrysanthemum transcriptome database, see http://trace.ncbi.nlm.nih. gov/Traces/sra_sub/sub.cgi?) and the resulting amplicons were cloned into the pMD19-T easy plasmid (TaKaRa) for sequencing. Transcriptional profiling was based on a quantitative real time PCR (qRT-PCR) platform, for which the chrysanthemum EF1α gene 218

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Fig. 3. Sub-cellular localization and transcriptional activation of CmMYB8. (a) GFP activity in onion epidermal cells transiently expressing a p35S::CmMYB8-GFP construct. As a control, cells were transformed with the construct p35S::GFP. Bar: 50 μm. GFP: green fluorescence channel, Marker: red fluorescence channel, DIC: bright light channel, Merge: the GFP and RFP overlap. Arrows are the sites of GFP and RFP overlap. (b) Transcriptional activation by CmMYB8 in yeast. The pCL1 and pGBKT7 plasmids were employed as, respectively, a positive and a negative control. SD/-Ade-His: SD medium lacking both adenine and histidine. X-α-gal activity: medium including X-α-gal. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4. CmMYB8 over-expression results in a suppression of lignin synthesis. (a) A PCR assay directed at hygromycin resistant gene, using as template genomic DNA extracted from the leaves confirms the incorporation of the transgene, while an RT-PCR assay of CmMYB8 from the leaves confirms that the CmMYB8 transgene was transcribed at a higher level than in wild type plants. The reference sequence was CmEF1α. WT: wild type, CmMYB8-OX-1 and -2: two independent transgenic lines. (b) Phloroglucinol staining of stem cross-sections used to quantify lignin content in wild type (WT) plants and the two selected CmMYB8 over-expressors. Xy: xylem, Ph: phloem. Bar: 1 mm. (c) Growth performance of wild type and two CmMYB8 transgenic lines. Bar: 1 cm. (d) Transcriptional profiling of genes encoding components of lignin synthesis in wild type and the two selected CmMYB8 over-expressors. Values shown in the form mean ± SE (n=3). **: mean performance of the transgenic differed significantly (p < 0.01) from that of the wild type.

transcript abundances were calculated using the 2−ΔΔCt method. Each qRT-PCR result was based on the mean performance of three biological replicates. The identical primers described by An et al. (2019) and Wang et al. (2017) were used to assay the transcriptional activity of

(KF305681) was used as the reference sequence (Ding et al., 2019). Each qRT-PCR consisted of 10 μL of SYBR Premix Ex Taq™ II (TaKaRa), 0.4 μL of each primer (10 μM) and 5 μL of template cDNA (1 ng/μL). Each PCR was performed as described by Zhu et al. (2013). Relative 219

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thence into cultivar ‘Jinba’ plants using Agrobacterium-mediated transformation. The method adopted for transgenesis largely followed that of Aida et al. (2004). Sterile cultivar ‘Jinba’ leaves were cut into 1 cm2 squares, which were cultured for three days on a solidified MS medium containing 1 mg/L 6-benzyl aminopurine (6BA) and 0.5 mg/L 1naphthylacetic acid (NAA). Ag. tumefaciens cells harboring pMDC43CmMYB8 were grown in vitro until an OD of 0.5–0.6 had been reached, were harvested by centrifugation, then re-suspended in equal volume liquid MS medium. The leaf squares were immersed in the cell suspension for 10 min, then cultured in the dark for three days on solidified MS medium containing 1 mg/L 6BA and 0.5 mg/L NAA, after which they were transferred onto a solidified MS medium containing 1 mg/L 6BA, 0.5 mg/L NAA and 0.5 g/L carbenicillin for a further seven days. The explants were subsequently re-plated onto a solidified MS medium containing 1 mg/L 6BA, 0.5 mg/L NAA, 0.35 g/L carbenicillin and 10 mg/L hygromycin for 14 days. Hygromycin concentration gradually reduced until the seedlings grown.

Table 1 Lignin content in the stems of wild type and the two selected CmMYB8 overexpressors.

Klason lignin content Vanillic acid Vanillin Syringic acid Syringaldehyde p-Hydroxybenzaldehyde p-Coumaric acid Ferulic acid H G S S/G

Wild type

35S::CmMYB8-1

35S::CmMYB8-2

100.07 ± 6.2 7.45 ± 0.31 5.64 ± 0.20 7.21 ± 0.36 2.63 ± 0.09 3.93 ± 0.12 0.98 ± 0.03 1.61 ± 0.07 3.93 (100%) 13.03 (100%) 9.84 (100%) 0.76

55.49 ± 3.3 7.23 ± 0.38 3.48 ± 0.34 3.75 ± 0.20 0.90 ± 0.08 1.32 ± 0.02 0.71 ± 0.03 0.95 ± 0.05 1.32 (34%) 10.71 (82%) 7.23 (73%) 0.68

42.33 ± 2.6 6.09 ± 0.37 2.33 ± 0.27 3.47 ± 0.24 0.25 ± 0.02 1.18 ± 0.03 0.59 ± 0.05 1.42 ± 0.13 1.18 (30%) 8.42 (65%) 3.72 (38%) 0.44

H: p-hydroxybenzaldehyde, G: vanillin and vanillic acid, S: syringaldehyde and syringic acid. Each data point represents the mean (mg/g dry tissue) ± SE (n=3). The percentages represent the content of H, G and S subunits in the transgenic lines relative to that in the wild type.

2.6. Histological sectioning Cross-sections prepared from top second part stem of height 10 cm seedling were fixed in 6 M HCl for 5 min, then incubated in 5% (w/v) phloroglucinol in 95% (v/v) ethanol for 1 min. The stained sections were analyzed using light microscopy.

genes involved in lignin synthesis. The sequences of all the primers mentioned above are given in Table S1. 2.3. Sub-cellular localization of CmMYB8 expression

2.7. Lignin quantification and compositional analysis

The CmMYB8 open reading frame (ORF) was amplified using the primer pair CmMYB8-pENTR1A-F/R (sequences given in Table S1), and the resulting amplicon was double-digested with Sal I and Not I, as was the pENTR1A vector (Invitrogen, Carlsbad, CA, USA). The two sequences were then ligated to one another using T4 DNA ligase (TaKaRa) and the ligated product was inserted into the pMDC43 vector (Curtis and Grossniklaus, 2003) using the LR ClonaseTM II enzyme system (Invitrogen). The resulting pMDC43-CmMYB8 construct comprised the CaMV 35S promoter, the sequence encoding green fluorescent protein (GFP) and CmMYB8. The construct (or an empty pMDC43 plasmid to serve as a negative control) and the nuclear marker p35S::D53-RFP (Zhou et al., 2013) were transiently co-transformed into onion epidermal cells using a PDS-1000 particle bombardment device (Bio-Rad, Hercules, CA, USA). Transformed onion epidermal cells were cultured for 16 h on Murashige and Skoog (1962) medium in the dark at 23 °C, after which GFP and RFP activity was detected by confocal laser scanning microscopy.

The Klason method (Kirk and Obst, 1988) was used to quantify the lignin present in mature stem samples. In brief, whole stems harvested from seedlings of height 10 cm were ground to a powder and extracted four times in methanol. After drying under vacuum, a 0.1 g aliquot of the extracted powder was hydrolyzed by immersion in 2 mL 72% (v/v) H2SO4. The mixture was then held in a 30 °C water bath for 1 h, after which 56 mL of water was added and the mixture autoclaved (121 °C, 1 h). The cooled solution was vacuum-filtered through a fritted glass crucible, and the residue was rinsed in water to remove any residual acid and sugar, then left to dry at 105 °C. The acid-insoluble lignin content was measured. The composition of lignin monomers in the mature stem samples was derived using the CuO oxidation method given by Sonbol et al. (2009) and the subsequent steps applied for the HPLC separation of the methanolic extract followed the protocols given by the same authors. 2.8. Flavonoid compositional analysis

2.4. Transcriptional activation activity assay of CmMYB8 The analysis of flavonoids was adapted from the method given by Burbulis et al. (1996). A 0.2 mg aliquot of freshly harvested stem of seedlings of height 10 cm was snap-frozen in liquid nitrogen and ground to a powder, which was extracted in 1 mL methanol, then centrifuged (10,000 rpm, 3 min). After removal of the supernatant, the pellet was re-extracted in 0.4 mL 70% (v/v) methanol; following a second centrifugation, the two supernatants were combined, mixed with an equal volume 2 M HCl and held at 70 °C for 40 min. The hydrolysis reaction was terminated by the addition of 0.7 mL methanol and the samples were dried under nitrogen. The residue was re-dissolved in 0.5 mL methanol, and separated by HPLC (Agilent 1260), employing the following conditions: the injection volume was 20 μL and the mixture was separated by passing through a 4.6 × 250 mm, 5 μm pore size ZORBAX Eclipse Plus C18 column (Agilent, Santa Clara, CA, USA) held at 25 °C. The mobile phase comprised a combination of 0.05% (v/v) acetic acid (A) and methanol (B) at a flow rate of 1 ml/ min. From 0 to 5 min, the mobile phase was 90% A, 10% B, rising to 50% A, 50% B by 10 min, to 10% A, 90% B by 20 min and to 100% B by 25 min, at which point it was held for a further 5 min. Flavonoids (rutin, kaempferol, quercetin and isorhamnetin) were detected by scanning the eluate at a wavelength of 254 nm.

The CmMYB8 ORF was amplified by the primer pair CmMYB8-BDF/R (Table S1), and the resulting amplicon inserted into the yeast (Saccharomyces cereviseae) expression vector pGBKT7 (Clontech, Mountain View, CA, USA). For the subsequent transcriptional activation activity assay, the pCL1 plasmid (which contains a full length copy of GAL4) and an empty pGBKT7 plasmid were used as, respectively, positive and negative controls. All constructs were transformed into yeast strain Y2HGold (Clontech) following the Yeastmaker Yeast Transformation System 2 manufacturer's protocol. Transformants harboring pCL1 were incubated on synthetic drop-out (SD) medium lacking leucine, while those harboring pGBKT7 or pGBKT7-CmMYB8 were incubated on SD medium lacking tryptophan. After holding at 30 °C for three days, the cultures were transferred onto SD medium lacking both histidine and adenine. X-α-gal was added to the medium to assay for the transcriptional activation activity of CmMYB8. 2.5. Chrysanthemum transformation The pMDC43-CmMYB8 construct was transferred into Agrobacterium tumefaciens EHA105 using the freezing transformation method and from 220

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Fig. 5. CmMYB8 over-expression results in a suppression of flavonoid synthesis. The flavonoid content in the stems of wild type (WT) and the two selected CmMYB8 over-expressors: (a) rutin, (b) quercetin, (c) kaempferol, (d) isorhamnetin. Values shown in the form mean ± SE (n=3). *and**: mean performance of the transgenic differed significantly (respectively p < 0.05 and < 0.01) from that of the wild type. (e) Transcriptional profiling of genes encoding components of flavonoid synthesis in wild type and the two selected CmMYB8 over-expressors. Values shown in the form mean ± SE (n=3). **: mean performance of the transgenic differed significantly (p < 0.01) from that of the wild type.

in the root, stem, leaf, tubular florets and ray florets of cultivar ‘Jinba’; the gene was most abundantly transcribed in the stem, and least abundantly in the ray florets (Fig. 2).

3. Results 3.1. Isolation and characterization of CmMYB8 The full length CmMYB8 cDNA sequence harbored by cultivar ‘Jinba’ comprised an 831 nt ORF predicted to encode a 276 residue polypeptide of molecular mass 31.01 kDa and pI 6.63. The sequence included two conserved MYB domains: one was an R2 domain lying between residues 16 and 61, and the other an R3 domain lying between residues 67 and 112 (Fig. 1a). A phylogenetic analysis implied that the protein most closely related to CmMYB8 was the sunflower protein HaMYB8 (Fig. 1b).

3.3. Localization of the expression of CmMYB8 and its transcriptional activation When the p35S::GFP-CmMYB8 construct was transiently expressed in onion epidermal cells along with the nuclear marker p35S::D53-RFP, GFP activity co-localized with RFP activity, showing that CmMYB8 expression was restricted to the nucleus; in contrast, GFP activity generated by the control p35S::GFP transgene was distributed throughout the cells (Fig. 3a). In the yeast-based transactivation assay, cells harboring the positive control pCL1 plasmid were able to grow on SD medium lacking both histidine and adenine, while the negative control cells harboring pGBKT7 were only able to grow on SD medium

3.2. Transcription profiling of CmMYB8 According to a qRT-PCR analysis, CmMYB8 transcript was detected 221

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synthesis has been characterized (Tak et al., 2017; Ge et al., 2017; Yan et al., 2013; Negishi et al., 2011). Several members of the MYB transcription factor family have been shown to act as negative regulators of lignin synthesis (Tamagnone et al., 1998; Jin et al., 2014; Tak et al., 2017; Omer et al., 2013; Ohman et al., 2013; Silvia et al., 2010; Sonbol et al., 2009). Here, a chrysanthemum R2R3-MYB transcription factor has been isolated and functionally characterized. The CmMYB8 protein harbors two repeat MYB domains and is closely related to the sunflower homolog HaMYB8. Its encoding gene has been shown to be transcribed throughout the plant, but particularly strongly so in the stem. At the sub-cellular level, it has been experimentally demonstrated that its expression was restricted to the nucleus and that its product exhibited transcriptional activation activity in yeast. Although the function of MYB8 is uncertain in many species, it is known that AtMYB6 and AtMYB8 both belong to R2R3-MYB subgroup 4, which also includes the genes AtMYB3, AtMYB4, AtMYB7 and AtMYB32; the products of the latter four genes act to suppress phenylpropane synthesis (Stracke et al., 2001). Transcription repressors belonging to R2R3-MYB subgroup 4 have also been identified in a number of other species (Tamagnone et al., 1998; Jin et al., 2014; Tak et al., 2017; Zhu et al., 2013). It is plausible that CmMYB8 too acts to regulate phenylpropane synthesis, because the over-expression of CmMYB8 reduced the tissue content of both lignin and flavonoids, while simultaneously down-regulating a number of genes encoding components of lignin and flavonoid synthesis. The implication is that CmMYB8 acts by activating negative regulators of both lignin and flavonoid synthesis. MYB proteins comprise one of the largest family of plant transcription factors, within which the R2R3s represent the most common sub-group (Dubos et al., 2010; Stracke et al., 2001). A number of these proteins have been implicated in lignin synthesis: examples include the products of the loquat (Eriobotrya japonica) gene EjMYB2 (Xu et al., 2014), the poplar (Populus deltoides) gene PdMYB221 (Tang et al., 2015), the banana gene MusaMYB31 (Tak et al., 2017) and the switchgrass (Panicum virgatum) gene PvMYB4 (Rao et al., 2019). Plants over-expressing CmMYB8 exhibited no visible growth abnormalities, but histochemical staining was able to show that their stems contained a reduced amount of lignin. As is also the effect of over-expressing the genes ZmMYB31 (Silvia et al., 2010), ZmMYB42 (Sonbol et al., 2009) and PvMYB4 (Shen et al., 2011), an additional consequence of CmMYB8 over-expression was an alteration in the composition of lignin, specifically inducing a reduction in the content of H, S and G subunits, as well as a fall in the S/G ratio. A number of R2R3-MYB transcription factors active in the lignin synthesis pathway are known to bind either directly or indirectly to AC elements (Tang et al., 2015; Zhou et al., 2009; Deluc et al., 2006), but the present experiments were not designed to test whether this is the case for CmMYB8 as well. Flavonoids are a class of ubiquitous and versatile plant secondary metabolites (Allan et al., 2008; Treutter, 2006; Lewis et al., 2011; Peer and Murphy, 2007). Flavonoids are synthesized via a branch of the phenylpropanoid pathway, and the production of some of these compounds is regulated by R2R3-MYB transcription factors. Examples include the grapevine protein VvMYB4 (Pérez-Díaz et al., 2015), the A. thaliana protein AtMYB7 (Fornalé et al., 2014) and the strawberry (Fragaria × ananassa) protein FaMYB1 (Aharoni et al., 2010). Since the over-expression of CmMYB8 was accompanied by a reduced abundance of transcripts generated from the genes PAL, C4H and 4CL, the products of which act early in the phenylpropanoid pathway, the suggestion is that CmMYB8 is a negative regulator of flavonoid synthesis, consistent with the lowered tissue content of each of four flavonoid compounds (rutin, quercetin, kaempferol and isorhamnetin) in the CmMYB8 overexpressing plants. In summary, CmMYB8, a chrysanthemum R2R3-MYB transcription factor, acts as a negative regulator of both lignin and flavonoid synthesis. A future priority will be to test for the effect of CmMYB8 overexpression on resistance to lodging and aphid infestation, as well as the mechanical strength of the pedicel, which are all traits of importance to

lacking tryptophan. In contrast, cells harboring CmMYB8 displayed transcriptional activation of each of the His3, Ade2 and Mel1 reporter genes, were able to grow on the SD medium lacking both histidine and adenine, and accumulated blue pigment when the medium was supplemented with X-α-gal (Fig. 3b). 3.4. The over-expression of CmMYB8 reduced lignin content and altered lignin composition Two independent transgenic lines harboring the p35S::CmMYB8 construct were selected on the basis of their exhibiting a high level of CmMYB8 transcription (Fig. 4a). Both transgenics grew normally. When phloroglucinol staining was used to characterize lignification, the xylem in the stem accepted less of the stain in the two transgenics than was the case for wild type plants (Fig. 4b). The effect of over-expressing CmMYB8 on the transcription of a set of genes implicated in lignin synthesis (CmPAL, CmC4H, Cm4CL1, CmHCT, CmC3H1, CmCCoAOMT1, CmCCR1, CmF5H1 and CmCOMT) was then investigated. CmPAL, CmC4H and Cm4CL1 (the products of each of which contribute to the early steps of phenylpropanoid metabolism) were all down-regulated in both of the CmMYB8 transgenics (Fig. 4c), consistent with the observed reduction in their capacity to synthesize lignin: the lignin content of one of transgenics was 55% of the wild type's level, while that of the other was 42% (Table 1). The level of transcription of CmHCT, CmC3H1, CmCCoAOMT1, CmCCR1, CmF5H1 and CmCOMT (each of which encode enzymes involved in the synthesis of lignin monomers) was also lower in the transgenics than in the wild type (Fig. 4c). The composition of the lignin in both the wild type and transgenic plants was then investigated using an HPLC platform (Table 1). Compared with wild type plants, the two transgenics exhibited, respectively, a 66% and a 70% reduction in the presence of H subunits. There was also a major reduction in the production of vanillin and vanillic acid (G subunits): the estimated content of G subunits in the lignin formed by the two transgenics was, respectively, 82% and 65% of that present in wild type lignin. The over-expression of CmMYB8 additionally was compromised with respect to the synthesis of S subunits: the levels in the transgenic lignin were, respectively, 73% and 38% of that in wild type lignin (Table 1). There was also a fall in the S/G ratio (Table 1). 3.5. CmMYB8 represses flavonoid synthesis The investigation of the effect of CmMYB8 over-expression on flavonoid synthesis focused on the accumulation of rutin, quercetin, kaempferol and isorhamnetin. Of these, the content of rutin was the one most altered by the presence of the transgene, falling to, respectively, 32% and 19% of the wild type level (Fig. 5a). The least affected flavonoid was isorhamnetin, the content of which in the two transgenics was, respectively, 94% and 64% of the control (Fig. 5d). The accumulation of both quercetin and kaempferol was reduced significantly in the transgenic plants (Fig. 5b and c). When a set of genes associated with the flavonoid pathway were transcriptionally profiled, the indication was that CHS, CHI, F3H, F3′H and DRF were all down-regulated by the presence of the CmMYB8 transgene (Fig. 5e), consistent with the reduced capacity of the transgenics to synthesize flavonoids. 4. Discussion Lignin is a core component of plant cell walls. Its mechanical strength not only supports the structure of the plant, but also provides protection from attack by a range of both microbial pathogens and insect pests (Ruben et al., 2012; Weng and Chapple, 2010). Furthermore, its hydrophobicity facilitates the transport of water via the xylem. The key enzymes required for synthesis of lignin have been largely identified (Shafrin et al., 2015; Wagner et al., 2011; Ragauskas et al., 2013; Meester et al., 2018; Richard et al., 2005), and the influence of numerous transcription factors over the regulation of lignin 222

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chrysanthemum improvement.

Jin, H., Cominelli, E., Bailey, P., Parr, A., Mehrtens, F., Jones, J., Tonelli, C., Weisshaar, B., Martin, C., 2014. Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis. EMBO J. 19, 6150–6161. Jorge, R., Ana, G., Lidia, N., Jiménez-Barbero, J., Faulds, C.B., Hoon, K., John, R., Martínez, A.T., Río, J.C. Del, 2011. Lignin composition and structure in young versus adult Eucalyptus globulus plants. Plant Physiol. 155, 667–682. Kirk, T.K., Obst, J.R., 1988. Lignin determination. Methods Enzymol. 161, 87–101. Laitinen, R.A.E., Ainasoja, M., Broholm, S.K., Teeri, T.H., Elomaa, P., 2008. Identification of target genes for a MYB-type anthocyanin regulator in Gerbera hybrida. J. Exp. Bot. 59, 3691–3703. Lewis, D.R., Ramirez, M.V., Miller, N.D., Prashanthi, V.W., Keith, R., Helm, R.F., Winkel, B.S.J., Muday, G.K., 2011. Auxin and ethylene induce flavonol accumulation through distinct transcriptional networks. Plant Physiol. 156, 144–164. Li, C.F., Wang, X.Q., Lu, W.X., Liu, R., Tian, Q.Y., Sun, Y.M., Luo, K.M., 2014. A poplar R2R3-MYB transcription factor, PtrMYB152, is involved in regulation of lignin biosynthesis during secondary cell wall formation. Plant Cell Tiss. Org. 119, 553–563. Liu, J., Osbourn, A., Ma, P., 2015. MYB transcription factors as regulators of phenylpropanoid metabolism in plants. Mol. Plant 8, 689–708. Luo, J., Butelli, E., Hill, L., Parr, A., Niggeweg, R., Bailey, P., Weisshaar, B., Martin, C., 2008. AtMYB12 regulates caffeoyl quinic acid and flavonol synthesis in tomato: expression in fruit results in very high levels of both types of polyphenols. Plant J. 56, 316–326. Lv, G.S., Tang, D.J., Chen, F.D., Sun, Y., Fang, W.M., Guan, Z.Y., Liu, Z.L., Chen, S.M., 2011. The anatomy and physiology of spray cut chrysanthemum pedicels, and expression of a caffeic acid 3-O-methyltransferase homologue. Postharvest Biol. Technol. 60, 244–250. Mahjoub, A., Hernould, M., Joubès, J., Decendit, A., Mars, M., Barrieu, F., Hamdi, S., Delrot, S., 2009. Overexpression of a grapevine R2R3-MYB factor in tomato affects vegetative development, flower morphology and flavonoid and terpenoid metabolism. Plant Physiol. Biochem. (Paris) 47, 551–561. Meester, B.D., Vries, L.D., Özparpucu, M., Gierlinger, N., Corneillie, S., Pallidis, A., Goeminne, G., Morreel, K., Bruyne, M.D., Rycke, R.D., 2018. Vessel-specific reintroduction of CINNAMOYL-COA REDUCTASE1 (CCR1) in dwarfed ccr1 mutants restores vessel and xylary fiber integrity and increases biomass. Plant Physiol. 176, 611. Misra, P., Pandey, A., Tiwari, M., Chandrashekar, K., Sidhu, O.P., Asif, M.H., Chakrabarty, D., Singh, P.K., Trivedi, P.K., Nath, P., Tuli, R., 2010. Modulation of transcriptome and metabolome of tobacco by Arabidopsis transcription factor, AtMYB12, leads to insect resistance. Plant Physiol. 152, 2258–2268. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plantarum 15, 473–497. Negishi, N., Nanto, K., Hayashi, K., Onogi, S., Kawaoka, A., 2011. Transcript abundances of LIM transcription factor, 4CL, CAld5H and CesAs affect wood properties in Eucalyptus globulus. Silvae Genet. 60, 288–296. Nemesio-Gorriz, M., Blair, P.B., Dalman, K., Hammerbacher, A., Arnerup, J., Stenlid, J., Mukhtar, S.M., Elfstrand, M., 2017. Identification of Norway spruce MYB-bHLH-WDR transcription factor complex members linked to regulation of the flavonoid pathway. Front. Plant Sci. 8, 305. Ohman, D., Demedts, B., Kumar, M., Gerber, L., Gorzsás, A., Goeminne, G., Hedenström, M., Ellis, B., Boerjan, W., Sundberg, B., 2013. MYB103 is required for FERULATE-5HYDROXYLASE expression and syringyl lignin biosynthesis in Arabidopsis stems. Plant J. 73, 63–76. Omer, S., Kumar, S., Khan, B.M., 2013. Over-expression of a subgroup 4 R2R3 type MYB transcription factor gene from Leucaena leucocephala reduces lignin content in transgenic tobacco. Plant Cell Rep. 32, 161–171. Pandey, A., Misra, P., Bhambhani, S., Bhatia, C., Trivedi, P.K., 2014. Expression of Arabidopsis MYB transcription factor, AtMYB111, in tobacco requires light to modulate flavonol content. Sci. Rep. 4, 5018. Pandey, A., Misra, P., Trivedi, P.K., 2015. Constitutive expression of Arabidopsis MYB transcription factor, AtMYB11, in tobacco modulates flavonoid biosynthesis in favor of flavonol accumulation. Plant Cell Rep. 34, 1515–1528. Peer, W.A., Murphy, A.S., 2007. Flavonoids and auxin transport: modulators or regulators? Trends Plant Sci. 12, 556–563. Peng, D.L., Chen, X.G., Yin, Y.P., Lu, K.L., Yang, W.B., Tang, Y.H., Wang, Z.L., 2014. Lodging resistance of winter wheat (Triticum aestivum L.): lignin accumulation and its related enzymes activities due to the application of paclobutrazol or gibberellin acid. Field Crop. Res. 157, 1–7. Pérez-Díaz, J.R., Pérez-Díaz, J., Madrid-Espinoza, J., González-Villanueva, E., Moreno, Y., Ruiz-Lara, S., 2015. New member of the R2R3-MYB transcription factors family in grapevine suppresses the anthocyanin accumulation in the flowers of transgenic tobacco. Plant Mol. Biol. 90, 63–76. Ragauskas, A., Pu, Y., Samuel, R., Jiang, N., Fu, C., Wang, Z.Y., 2013. Structural characterization of lignin in wild-type versus COMT down-regulated Switchgrass. Front. Energy Res. 1, 1–9. Rao, X.L., Chen, X., Shen, H., Ma, Q., Li, G.F., Tang, Y.H., Pena, M., York, W., Frazier, T.P., Lenaghan, S., Xiao, X.R., Chen, F., Dixon, R.A., 2019. Gene regulatory networks for lignin biosynthesis in switchgrass (Panicum virgatum). Plant Biotechnol. J. 17, 580–593. Richard, S., Aymerick, E., Gregory, M., Brigitte, P., Catherine, L., Lise, J., Armand, S., 2005. CINNAMYL ALCOHOL DEHYDROGENASE-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis. Plant Cell 17, 2059–2076. Rogers, L.A., Campbell, M.M., 2004. The genetic control of lignin deposition during plant growth and development. New Phytol. 164, 17–30. Ruben, V., Kris, M., Chiarina, D., Paula, O., Grabber, J.H., John, R., Wout, B., 2012. Metabolic engineering of novel lignin in biomass crops. New Phytol. 196, 978–1000.

Contributions LZ and FC designed the experiments. LZ, YG, ZZ and AS performed all experiments and analyzed the data. LZ and FC wrote the manuscript. SC and JJ reviewed the draft of the manuscript. All the authors have read and approved the manuscript. Declaration of competing interest The authors declare that they have no competing interests. Acknowledgments This research was financially supported by grants from the National Natural Science Foundation of China (31672192) and a Priority Academic Program Development project for Jiangsu Higher Education Institutions. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.plaphy.2020.02.010. References Aharoni, A., Vos, De C.H., Wein, M., Sun, Z.K., Greco, R., Kroon, A., Mol, J.N. O'Connell A.P., 2010. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. Plant J. 28, 319–332. Aida, R., Ohira, K., Tanaka, Y., Yoshida, K., Kishimoto, S., Shibata, M., Ohmiya, A., 2004. Efficient transgene expression in chrysanthemum, Dendranthema grandiflorum (Ramat.) Kitamura, by using the promoter of a gene for chrysanthemum chlorophylla/b binding protein. Breed Sci. 54, 51–58. Allan, A.C., Hellens, R.P., Laing, W.A., 2008. MYB transcription factors that colour our fruit. Trends Plant Sci. 13, 99–102. An, C., Sheng, L.P., Du, X.P., Wang, Y.J., Zhang, Y., Song, A.P., Jiang, J.F., Guan, Z.Y., Fang, W.M., Chen, F.D., Chen, S.M., 2019. Overexpression of CmMYB15 provides chrysanthemum resistance to aphids by regulating the biosynthesis of lignin. Hortic. Res. 6, 1–10. Ban, Y., Honda, C., Hatsuyama, Y., Igarashi, M., Bessho, H., Moriguchi, T., 2007. Isolation and functional analysis of a MYB transcription factor gene that is a keyregulator for the development of red coloration in apple skin. Plant Cell Physiol. 48, 958–970. Boerjan, W., Ralph, J., Baucher, M., 2003. Lignin biosynthesis. Annu. Rev. Plant Biol. 54, 519–546. Burbulis, I.E., Iacobucci, M., Shirley, B.W., 1996. A null mutation in the first enzyme of flavonoid biosynthesis does not affect male fertility in Arabidopsis. Plant Cell 8, 1013–1025. Cao, Z.H., Zhang, S.Z., Wang, R.K., Zhang, R.F., Hao, Y.J., 2013. Genome wide analysis of the apple MYB transcription factor family allows the identification of MdoMYB121 gene confering abiotic stress tolerance in plants. PloS One 8, e69955. Curtis, M.D., Grossniklaus, U.A., 2003. Gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 133, 462–469. Deluc, L., Barrieu, F., Marchive, C., Lauvergeat, V., Decendit, A., Richard, T., Carde, J.P., Mérillon, J.M., Hamdi, S., 2006. Characterization of a grapevine R2R3-MYB transcription factor that regulates the phenylpropanoid pathway. Plant Physiol. 140, 499–511. Ding, L., Zhao, K.K., Zhang, X., Song, A.P., Su, J.S., Hu, Y.H., Zhao, W.Q., Jiang, J.F., Chen, F.D., 2019. Comprehensive characterization of a floral mutant reveals the mechanism of hooked petal morphogenesis in Chrysanthemum morifolium. Plant Biotechnol. J. 17, 2325–2340. Dubos, C., Stracke, R., Grotewold, E., Weisshaar, B., Martin, C., Lepiniec, L., 2010. MYB transcription factors in Arabidopsis. Trends Plant Sci. 15, 573–581. Douglas, C.J., 1996. Phenylpropanoid metabolism and lignin biosynthesis: from weeds to trees. Trends Plant Sci. 1, 171–178. Fornalé, S., Lopez, E., Salazarhenao, J.E., Fernándeznohales, P., Rigau, J., Caparrosruiz, D., 2014. AtMYB7, a new player in the regulation of UV-sunscreens in Arabidopsis thaliana. Plant Cell Physiol. 55, 507–516. Ge, H., Zhang, J., Zhang, Y.J., Li, X., Yin, X.R., Grierson, D., Chen, K.S., 2017. EjNAC3 transcriptionally regulates chilling-induced lignification of loquat fruit via physical interaction with an atypical CAD-like gene. J. Exp. Bot. 68, 5129–5136. Huang, J., Gu, M., Lai, Z., Fan, B., Shi, K., Zhou, Y.H., Yu, J.Q., Chen, Z., 2010. Functional analysis of the Arabidopsis PAL gene family in plant growth, development, and response to environmental stress. Plant Physiol. 153, 1526–1538. Jiang, R., Tian, J., Song, T.T., Zhang, J., Yao, Y.C., 2014. The Malus crabapple transcription factor McMYB10 regulates anthocyanin biosynthesis during petal coloration. Sci. Hortic. Amsterdam 166, 42–49.

223

Plant Physiology and Biochemistry 149 (2020) 217–224

L. Zhu, et al.

Wagner, A., Tobimatsu, Y., Phillips, L., Flint, H., Torr, K., Donaldson, L., Pears, L., Ralph, J., 2011. CCoAOMT suppression modifies lignin composition in Pinus radiata. Plant J. 67, 119–129. Wang, Y.J., Sheng, L.P., Zhang, H.R., Du, X.P., An, C., Xia, X.L., Chen, F.D., Jiang, J.F., Chen, S.M., 2017. CmMYB19 over-expression improves aphid tolerance in chrysanthemum by promoting lignin synthesis. Int. J. Mol. Sci. 18, 619. Weng, J.K., Chapple, C., 2010. The origin and evolution of lignin biosynthesis. New Phytol. 187, 273–285. Wilkins, O., Nahal, H., Foong, J., Provart, N.J., Campbell, M.M., 2009. Expansion and diversification of the Populus R2R3-MYB family of transcription factors. Plant Physiol. 149, 981–993. Xu, Q., Yin, X.R., Zeng, J.K., Ge, H., Song, M., Xu, C.J., Li, X., Ferguson, I.B., Chen, K.S., 2014. Activator- and repressor-type MYB transcription factors are involved in chilling injury induced flesh lignification in loquat via their interactions with the phenylpropanoid pathway. J. Exp. Bot. 65, 4349–4359. Yan, L., Xu, C.H., Kang, Y.L., Gu, T.W., Wang, D.X., Zhao, S.Y., Xia, G.M., 2013. The heterologous expression in Arabidopsis thaliana of sorghum transcription factor SbbHLH1 downregulates lignin synthesis. J. Exp. Bot. 64, 3021–3032. Yilmaz, A., Nishiyama Jr., M.Y., Fuentes, B.G., Souza, G.M., Janies, D., Gray, J., Grotewold, E., 2009. GRASSIUS: a platform for comparative regulatory genomics across the grasses. Plant Physiol. 149, 171–180. Zhao, L., Gao, L.P., Wang, H.X., Chen, X.T., Wang, Y.S., Yang, H., Wei, C.L., Wan, X.C., Xia, T., 2013. The R2R3-MYB, bHLH, WD40, and related transcription factors in flavonoid biosynthesis. Funct. Integr. Genom. 13, 75–98. Zhao, P.P., Li, Q., Li, J., Wang, L.N., Ren, Z.H., 2014. Genome-wide identification and characterization of R2R3MYB family in Solanum lycopersicum. Mol. Genet. Genom. 289, 1183–1207. Zhao, Q., Dixon, R.A., 2011. Transcriptional networks for lignin biosynthesis: more complex than we thought? Trends Plant Sci. 16, 227–233. Zhong, R.Q., Ye, Z.H., 2009. Transcriptional regulation of lignin biosynthesis. Plant Signal. Behav. 4, 1028–1034. Zhou, J., Lee, C., Zhong, R., Ye, Z.H., 2009. MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis. Plant Cell 21, 248–266. Zhou, F., Lin, Q.B., Zhu, L.H., Ren, Y.L., Zhou, K.N., Shabek, N., Wu, F.Q., Mao, H.B., Dong, W., Gan, L., Ma, W.W., Gao, H., Chen, J., Yang, C., Wang, D., Tan, J.J., Zhang, X., Guo, X.P., Wang, J.L., Jiang, L., Liu, X., Chen, W.Q., Chu, J.F., Yan, C.Y., Ueno, K., Ito, S., Asami, T., Cheng, Z.J., Wang, J., Lei, C.L., Zhai, H.Q., Wu, C.Y., Wang, H.Y., Zheng, N., Wan, J.M., 2013. D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling. Nature 504, 406. Zhu, L., Shan, H., Chen, S.M., Jiang, J.F., Gu, C.S., Zhou, G.Q., Chen, Y., Song, A.P., Chen, F.D., 2013. The heterologous expression of the chrysanthemum R2R3-MYB transcription factor CmMYB1 alters lignin composition and represses flavonoid synthesis in Arabidopsis thaliana. PloS One 8, e65680.

Shadle, G., Chen, F., Reddy, M.S.S., Jackson, L., Jin, N., Dixon, R.A., 2007. Down-regulation of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase in transgenic alfalfa affects lignification, development and forage quality. Phytochemistry 68, 1521–1529. Shafrin, F., Das, S.S., Sanan-Mishra, N., Khan, H., 2015. Artificial miRNA-mediated downregulation of two monolignoid biosynthetic genes (C3H and F5H) cause reduction in lignin content in jute. Plant Mol. Biol. 89, 511–527. Shen, H., He, X.Z., Poovaiah, C.R., Wuddineh, W.A., Ma, J., Mann, D.G., Wang, H., Jackson, L., Tang, Y., Stewart Jr., C.N., Chen, F., Dixon, R.A., 2011. Functional characterization of the switchgrass (Panicum virgatum) R2R3-MYB transcription factor PvMYB4 for improvement of lignocellulosic feedstocks. New Phytol. 193, 121–136. Silvia, F., Xinhui, S., Chenglin, C., Antonio, E., Sami, I., Montserrat, C., Elisabet, F., JosepLluís, T., Pere, R., Pere, P., 2010. ZmMYB31 directly represses maize lignin genes and redirects the phenylpropanoid metabolic flux. Plant J. 64, 633–644. Sonbol, F.M., Fornalé, S., Capellades, M., Encina, A., Touriño, S., Torres, J.L., Rovira, P., Ruel, K., Puigdomènech, P., Rigau, J., 2009. The maize ZmMYB42 represses the phenylpropanoid pathway and affects the cell wall structure, composition and degradability in Arabidopsis thaliana. Plant Mol. Biol. 70, 283–296. Stracke, R., Werber, M., Weisshaar, B., 2001. The R2R3-MYB gene family in Arabidopsis thaliana. Curr. Opin. Plant Biol. 4, 447–456. Sykes, R.W., Gjersing, E.L., Foutz, K., Rottmann, W.H., Kuhn, S.A., Foster, C.E., Ziebell, A., Turner, G.B., Decker, S.R., Hinchee, M.A.W., 2015. Down-regulation of p-coumaroyl quinate/shikimate 3′-hydroxylase (C3′H) and cinnamate 4-hydroxylase (C4H) genes in the lignin biosynthetic pathway of Eucalyptus urophylla×E. grandis leads to improved sugar release. Biotechnol. Biofuels 8, 128. Tak, H., Negi, S., Ganapathi, T.R., 2017. Overexpression of MusaMYB31, a R2R3 type MYB transcription factor gene indicate its role as a negative regulator of lignin biosynthesis in banana. PloS One 12, e0172695. Tamagnone, L.A., Parr, A., Mackay, S., Culianez, Macia F.A., Roberts, K., Martin, C., 1998. The AmMYB308 and AmMYB330 transcription factors from Antirrhinum regulated phenylpropanoid and lignin biosynthesis in transgenic tobacco. Plant Cell 10, 135–154. Tang, X.F., Zhuang, Y.M., Qi, G., Wang, D., Liu, H.H., Wang, K.R., Chai, G.H., Zhou, G.K., 2015. Poplar PdMYB221 is involved in the direct and indirect regulation of secondary wall biosynthesis during wood formation. Sci. Rep. 5, 12240. Tian, Q.Y., Wang, X.Q., Li, C.F., Lu, W.X., Yang, Li, Jiang, Y.Z., Luo, K.M., 2013. Functional characterization of the poplar R2R3-MYB transcription factor PtoMYB216 involved in the regulation of lignin biosynthesis during wood formation. PloS One 8 (10), e76369. Treutter, D., 2006. Significance of flavonoids in plant resistance: a review. Environ. Chem. Lett. 4, 147. Voelker, S.L., Barbara, L., Meinzer, F.C., Strauss, S.H., 2011. Reduced wood stiffness and strength, and altered stem form, in young antisense 4CL transgenic poplars with reduced lignin contents. New Phytol. 189, 1096–1109.

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