Identification and Functional Characterization of PtoMYB055 Involved in the Regulation of the Lignin Biosynthesis Pathway in Populus tomentosa.

Wood, which is mainly composed of lignified secondary cell wall, is the most abundant biomass in woody plants. Previous studies have revealed that R2R3-type MYB transcription factors are important regulators of the formation of the secondary cell wall in vascular plants. In this study, we isolated the R2R3-type MYB transcription factor gene PtoMYB055, which is mainly expressed in xylem and phloem tissue, from Populus tomentosa and demonstrate that PtoMYB055 is a key regulator of lignin biosynthesis. PtoMYB055 as a transcriptional activator is localized to the nucleus. Overexpression of PtoMYB055 upregulates expression of lignin biosynthetic genes in transgenic poplar plants, resulting in ectopic deposition of lignin in phloem tissue and an increase in thickness of the secondary cell wall. In sum, PtoMYB055 is a transcriptional activator that is involved in regulating lignin biosynthesis during the formation of the secondary cell wall in poplar.

The transcription factor MYB115 contributes to the regulation of proanthocyanidin biosynthesis and enhances fungal resistance in poplar.

Proanthocyanidins (PAs) are major defense phenolic compounds in the leaves of poplar (Populus spp.) in response to abiotic and biotic stresses. Transcriptional regulation of PA biosynthetic genes by the MYB-basic helix-loop-helix (bHLH)-WD40 complexes in poplar is not still fully understood. Here, an Arabidopsis TT2-like gene MYB115 was isolated from Populus tomentosa and characterized by various molecular, genetic and biochemical approaches. MYB115 restored PA productions in the seed coat of the Arabidopsis tt2 mutant. Overexpression of MYB115 in poplar activated expression of PA biosynthetic genes, resulting in a significant increase in PA concentrations. By contrast, the CRISPR/Cas9-generated myb115 mutant exhibited reduced PA content and decreased expression of PA biosynthetic genes. MYB115 directly activated the promoters of PA-specific structural genes. MYB115 interacted with poplar TT8. Coexpression of MYB115, TT8 and poplar TTG1 significantly enhanced the expression of ANR1 and LAR3. Additionally, transgenic plants overexpressing MYB115 had increased resistance to the fungal pathogen Dothiorella gregaria, whereas myb115 mutant exhibited greater sensitivity compared with wild-type plants. Our data provide insight into the regulatory mechanisms controlling PA biosynthesis by MYB115 in poplar, which could be effectively employed for metabolic engineering of PAs to improve resistance to fungal pathogens.

PtoMYB92 is a Transcriptional Activator of the Lignin Biosynthetic Pathway During Secondary Cell Wall Formation in Populus tomentosa.

Wood is the most abundant biomass in perennial woody plants and is mainly made up of secondary cell wall. R2R3-MYB transcription factors are important regulators of secondary wall biosynthesis in plants. In this study, we describe the identification and characterization of a poplar MYB transcription factor PtoMYB92, a homolog of Arabidopsis MYB42 and MYB85, which is involved in the regulation of secondary cell wall biosynthesis. PtoMYB92 is specifically expressed in xylem tissue in poplar. Subcellular localization and transcriptional activation analysis suggest that PtoMYB92 is a nuclear-localized transcriptional activator. Overexpression of PtoMYB92 in poplar resulted in an increase in secondary cell wall thickness in stems and ectopic deposition of lignin in leaves. Quantitative real-time PCR showed that PtoMYB92 specifically activated the expression of lignin biosynthetic genes. Furthermore, transient expression assays using a ß-glucuronidase (GUS) reporter gene revealed that PtoMYB92 is an activator in the lignin biosynthetic pathway during secondary cell wall formation. Taken together, our results suggest that PtoMYB92 is involved in the regulation of secondary cell wall formation in poplar by controlling the biosynthesis of monolignols.

Isolation and promoter analysis of a chalcone synthase gene PtrCHS4 from Populus trichocarpa.

As perennial plants, Populus species are constantly exposed to environmental stresses, such as wounding and pathogen attack, which lead to production of compounds including lignin, flavonoids and phytoalexins. Chalcone synthase (CHS) is a key enzyme in the flavonoid biosynthesis pathway. In this study, a cDNA clone encoding CHS was isolated from Populus trichocarpa by reverse transcription-polymerase chain reaction (RT-PCR). The full-length cDNA, named PtrCHS4, was 1,314 bp with a 1,173 bp open reading frame that corresponded to a deduced protein of 391 amino acid residues. Multiple sequence alignments showed that PtrCHS4 shared high homology with CHS proteins from other plants. Phylogenetic analysis revealed that PtrCHS4 was most closely related to PhCHS from Petunia hybrida and NaCHS from Nicotiana attenuata. Semi-quantitative RT-PCR analysis identified that the PtrCHS4 gene was abundantly expressed in the leaves and stems, while its expression was drastically reduced in the roots. Transcript abundance of PtrCHS4 was stimulated by 2.5-fold within 24 h of wounding treatment. Promoter analysis confirmed that the PtrCHS4 promoter was capable of directing expression of the GUS reporter in both wounded and unwounded leaves of transgenic Chinese white poplar (P. tomentosa Carr.), indicating that the PtrCHS4 promoter is systemically responsive to wounding stimuli. Furthermore, promoter deletion analysis showed that the proximal 1,592 bp from the transcription start site were required for promoter activation by jasmonic acid and the -1,096 to -148 region was proved to be necessary for establishing wound-induced pattern of expression.

The chitinase gene (Bbchit1) from Beauveria bassiana enhances resistance to Cytospora chrysosperma in Populus tomentosa Carr.

The Chinese white poplar (Populus tomentosa Carr.) is susceptible to infection by plant diseases which severely affect its growth and substantially decrease its economic value. A chitinase gene (Bbchit1) from Beauveria bassiana was introduced into Chinese white poplar (Populus tomentosa Carr.) by Agrobacterium-mediated transformation. The T-DNA of plant transformation vector contained the beta-glucuronidase reporter gene (GUS) under the control of CaMV 35S promoter and the neomycin phosphotransferase selection marker gene (NPTII) driven by the nos promoter. GUS activity was detected in most of the kanamycin-resistant plants tested. Stable integration of transgenes in the plant genome was confirmed using PCR. RT-PCR analysis showed that the Bbchit1 gene was transcribed in the transformed plants. When evaluated for resistance to poplar fungal pathogens with an in vitro assay, crude extracts from leaves and shoots of transgenic lines were inhibitory against the pathogenic fungus Cytospora chrysosperma (Pers.) Fr. Similarly, Bbchit1 overexpression enhanced disease resistance to C. chrysosperma in the transformed poplar plants, indicating that is gene is potentially useful to protect the trees against fungal diseases.

PtoMYB170 positively regulates lignin deposition during wood formation in poplar and confers drought tolerance in transgenic Arabidopsis.

Wood formation is a complex developmental process under multi-level transcriptional control executed by a large set of transcription factors. However, only limited members have been characterized to be key regulators of lignin biosynthesis in poplar. Here we report the conserved and unique functions of PtoMYB170, a transcription factor identified from Populus tomentosa (Chinese white poplar), in lignin deposition and drought tolerance in comparison with its duplicate paralog PtoMYB216. PtoMYB170 is preferentially expressed in young leaves and xylem tissues. Overexpression of PtoMYB170 in transgenic poplar plants resulted in stronger lignification and more thickened secondary wall in xylem compared with wild-type plants, whereas the CRISPR/Cas9-generated mutation of PtoMYB170 weakened lignin deposition, thereby leading to a more flexible and collapsed xylem phenotype. Transient expression experiments demonstrated that PtoMYB170 specifically activated the expression of lignin biosynthetic genes, consistent with the function of PtoMYB216. However, GUS staining assays revealed that PtoMYB170 was specifically expressed in guard cells of transgenic Arabidopsis while PtoMYB216 was not. Heterologous expression of PtoMYB170 in Arabidopsis enhanced stomatal closure in the dark and resulted in drought tolerance of the transgenic plants through reduced water loss, indicating a diversified role from PtoMYB216. These results revealed the PtoMYB170-dependent positive transcriptional regulation on lignin deposition in poplar and its coordinated function in enhancing drought tolerance by promoting dark-induced stomatal closure.

Functional characterization of the poplar R2R3-MYB transcription factor PtoMYB216 involved in the regulation of lignin biosynthesis during wood formation.

Because of the importance of wood in many industrial applications, tremendous studies have been performed on wood formation, especially in lignin biosynthesis. MYB transcription factors (TFs), which consist of a large family of plant TFs, have been reported to directly regulate lignin biosynthetic genes in a number of plants. In this study, we describe the cloning and functional characterization of PtoMYB216, a cDNA isolated from Chinese white poplar (Populus tomentosa Carr.). PtoMYB216 encodes a protein belonging to the R2R3-MYB family and displays significant similarity with other MYB factors shown to regulate lignin synthesis in Arabidopsis. Gene expression profiling studies showed that PtoMYB216 mRNA is specifically expressed during secondary wall formation in wood. The 1.8-kb promoter sequence of PtoMYB216 was fused to the GUS coding sequence and introduced into wild-type A. thaliana. GUS expression was shown to be restricted to tissues undergoing secondary cell wall formation. Overexpression of PtoMYB216 specifically activated the expression of the upstream genes in the lignin biosynthetic pathway and resulted in ectopic deposition of lignin in cells that are normally unligninified. These results suggest that PtoMYB216 is specific transcriptional activators of lignin biosynthesis and involved in the regulation of wood formation in poplar.