Functional Analysis Of PtoVNS11 And PtoMYB156 Transcription Factors Involved In Secondary Wall Formation And Flavonoid Biosynthetic Pathway In Poplar

Phenylpropanoid biosynthesis pathway generates numbers of phenylalanine- derived components involved in secondary metabolism, which are quite important during the whole life cycle of plants. The metabolic flux of carbon skeletons forms different branches of the phenylpropanoid pathway, under the action of catalysis of phenylalanine ammonia-lyase(PAL), 4-Hydroxycinnamic acid(C4H), and synthetize compounds of phenylalanine-derived components including lignin, anthocyanidin, and tannin. These secondary metabolites play important roles in plant growth and development, as well as resistance to biotic or abiotic stresses. For example, lignin is an important component of plant cell wall, which plays a critical role in the normal growth and development of vascular tissues. Flavonoids, Anthocyanidin and tannin, could help resist biotic stresses(such as pests and diseases) and abiotic stresses(such as ultraviolet radiation). Therefore, revealing the mechanism of how phenylpropanoid pathway-related transcription factors involve in lignin and flavonoids biosynthesis is very significant.In this dissertation, PtoMYB156 and PtoVNS11,two genes that each encode a transcription factor(TF) from the MYB and NAC family, respectively, were cloned from Populus tomentosa Carr., and detailed functional studies were carried out on their involvement of lignin or flavonoids biosynthesis regulation in the phenylpropanoid pathway. The main results are as follows.1. We isolated a NAC domain transcription factor named PtoVNS11 from Populus tomentosa Carr. which shares high identity with SND1 of Arabidopsis thaliana. Amino acid sequence alignment showed that Pto VNS11 contained an open reading frame(ORF) encoding a protein of 416 amino acids which shares high identity with SND1 of Arabidopsis thaliana(59.6%) and PtrWND1B(97.9%) of P. trichocarpa. The characteristic amino acid sequences are almost consistent with others in the conserved region of NAC domain(A~E domain). Therefore, it is quite likely that Pto VNS11 belongs to the same subtype of NAC transcription factors as SND1 and PtrWND1 B.Gene expression analyses of PtoVNS11 by real time fluorescence quantitative-PCR showed that PtoVNS11 was mainly expressed in xylem of poplar, suggesting that it may be involved in the regulation of plant secondary wall formation. Subcellular localization assay of PtoVNS11:GFP fusion protein demonstrated that PtoVNS11 has the characteristics of nuclear localization, which was consistent with previous prediction. Transcriptional activation assay showed that Pto VNS11 was a transcription activator. Further, GUS expression driven by the PtoVNS11 promoter of 2069 bp length was observed in various vascular tissues of vegetative and reproductive organs in transgenic Arabidopsis, all of which further imp ly that PtoVNS11 may affect the whole plant growth cycle of vascular tissue development processes. Promoter deletion analysis revealed that the fragment with the secondary wall NAC binding element(SN BE) was crucially required for GUS expression.In order to understand the biological function of PtoVNS11, a plant expression vector Pro35S:PtoVNS11 was introduced into the Arabidopsis snd1nst1 double mutant strain and it could effectively complement the secondary wall defects in interfascicular fibers and xylary fibers, and recovered the stiffness of the inflorescence stems. When the Pro35S:ProVNS11 vector was expressed in wildtype Arabidopsis thaliana, ectopic deposition of lignin in epidermal cells, increased thickness of secondary walls in xylem along with delayed growth were observed. The results were similar to the results reported previously about At SND1, At NST1 and PtrWNDs genes, which proved that PtoVNS11 was a master switch activating the biosynthetic program for secondary wall formation and lignin biosynthetic pathway of poplar.Moreover, transgenic lines in poplar showed higher content of lignin in histochemical staining of stem sections. Transcript analysis showed that the expression levels of a series of wood-associated genes were up-regulated in transgenic plants overexpressing PtoVNS11. The above results showed that Pto VNS11 was involved in the development of xylem through the regulation of lignin metabolism pathway.2. Overexpression of PtoVNS11 in poplar notably repressed the expression of PtoMYB156, a gene with unknown function, which is a close homologue of AtMYB4 that functioned as a negative regulator of lignin biosynthetic metabolism. Therefore, it is reasonable to suppose that PtoMYB156 was a regulatory factor downstream of PtoVNS11 involved in lignin biosynthesis during the development of secondary wall in poplar.3. In order to elucidate the role of PtoMYB156, we isolated the c DNA of PtoMYB156 from Populus tomentosa. Subcellular localization using the fluorescent protein-tagging approach demonstrated that GFP-tagged PtoMYB156 was exclusively targeted to the nucleus, consistent with its predicted function as a transcription factor. Quantitative RT-PCR analysis showed that PtoMYB156 was widely expressed in all tissues examined, including stem, root, xylem and bark, and especially highly in leaves. It was found that Pto MYB156 was not able to activate the expression of reporter genes even when it was fused with a VP16 activating sequence in yeast one-hybrid system, indicating that it was likely to be a transcriptional repressor. For further demonstrating the function of PtoMYB156 transcription factor, we constructed a binary pYLCRIPSR/Cas9 multiplex genome targeting vector carrying CAS9-PtoMYB156 and an overexpression vector harboring Pro35S:PtoMYB156 and obtained knock-out and overexpression transgenic poplar strains with the two vectors, respectively. Morphological analysis of the overexpression plants revealed flattened stem and curled leaves. Lignin autofluorescence of a transgenic leaf showed reduced lignified secondary wall thickening in veins compared with the control. O n the other hand, a significant increase in lignin content o f the vessel and the fiber secondary cell walls was found in knock-out transgenic poplar, with the expression levels of a number of wood-associated genes significantly increased compared to the control. The results above proved that PtoMYB156 gene is indeed involved in the phenylpropanoid biosynthesis pathway, and it influences the processes of lignin biosynthesis, leading to abnormal formation of secondary wall in the vascular tissues of poplar.3. The expression of key enzymes C4 H, CHS, DFR in the metabolic pathway of phenylpropanoid pathway were down regulated in Pro35S:PtoMYB156 transgenic poplar, suggesting that PtoMYB156 may be involved in the synthesis of flavonoids. In order to further study the effect of PtoMYB156 on flavonoid synthesis branch of the phenylpropanoid pathway, the PtoMYB156 transcription factor was investigated by overexpressing in Arabidopsis thaliana. The transgenic lines were obviously shorter than the controls and displayed obviously upward curled leaves and weakened resistance to UV-B radiation. We therefore analyzed extracts of 2-week-old rosette leaves by high-performance liquid chromatography-diode array detection(HPLC-DAD) and the results confirmed that transgenic plants show a strong reduction of quercetin and kaempferol which were UV-B-absorbing compounds derived from flavonoid biosynthesis. Meanwhile, overexpression of PtoMYB156 caused a pale yellow color seed coat rather than dark brown seen in wild-type Arabidopsis. Quantitative analysis of HPLC as well as DMAC A staining proved that the content of soluble tannin was decreased, and the expression level of main genes involved in flavonoid biosynthesis(such as tannin, quercetin and kaempferol) were decreased. Finally, the reporter and effector constructs were co-transfected into tobacco leaves, and subsequent assay of the GUS activity in the transfected tobacco leaves demonstrated that PtoMYB156 effectively repressed the promoters of secondary cell wall-associated genes, for instence, C4H(involved in lignin synthesis), CesA17(involved in cellulose synthesis), GT43B(involved in xylan biosynthesis), as well as genes of flavonoid biosynthesis such as LAR and FLS. Together, these results showed that Pto MYB156 was a general repressor of the phenylpropanoid pathway affecting flavonoid biosynthesis and caused decreased resistance to UV-B radition.All together, these results illustrated the function of PtoMYB156 in seco ndary cell wall formation, as well as its regulatory mechanism involved in the regulation of phenylalanine-derived components in secondary metabolism at the level of cell biology, histochemistry, and molecular biology, all of which shed light on genetic improvement of poplar.