| As a poplar species specific and widely distributed in China, Populous tomentosa is not only an ideal tree for ecological environment reservation but also an important resource for wood material. With the completion of the genome sequencing of P. trichocarpa in 2006, poplar became a model organism for tree research and is widely employed in studies on secondary wall biosynthesis, especially lignin biosynthesis.According to analysis on transcription factors involved in lignin biosynthesis in Arabidopsis, Eucalyptus and Pinus taeda, we have noticed that At MYB61 is not only involved in lignin biosynthesis, but also specifically expressed in stomata. It is not clear whether this kind of transcription factors contribute to both secondary wall biosynthesis and drought resistance. To answer these questions, we cloned Pto MYB170, the homologue gene of At MYB61 in P. tomentosa and carried out the following study:(1) Bioinformatics analysis has revealed that Pto MYB170 contains conserved R2R3 motif and nuclear localization signal and is closely relate to known MYB transcription factors involved in second wall biosynthesis. Therefore, Pto MYB170 may be also involved in the regulation of lignin biosynthesis.(2) Yeast transcription activation assay and subcellular localization experiment have shown that Pto MYB170 is a transcription activator localized in the nucleus.(3) Quantitative RT-PCR has shown that Pto MYB170 expressed in the stem of P. tomentosa with the highest level, suggesting its involvement in secondary cell wall biosynthesis.(4) We obtained Pto MYB170-overexpression and Pto MYB170-knock-out transgenic poplar plant and conducted morphological analysis. In comparison with WT plants, overexpression poplar carried curved dark green leaves and showed short strong stem, while knock-out plants showed dwarf and curved phenotype, indicating that Pto MYB170 may play a role in secondary wall synthesis(5) Microscopic and histochemical analysis on leaf and stem slices have revealed that the overexpression plants showed significantly higher lignification degree than WT, while knock-out plants showed lower lignification degree with some collapse in the secondary wall. Further observation with scanning electronic microscope and laser confocal microscope has also revealed dramatically different secondary cell wall structure in transgenic plants compared with WT plants, suggesting Pto MYB170 is a transcription factor that influences secondary wall structure.(6) Quantitative real time RT-PCR analysis of structural genes involved in secondary cell wall biosynthesis showed that the expression level of genes involved in lignin, xylan and cellulose biosynthesis were all elevated in overexpression lines while downregulated in knock-out transgenic poplars, indicating that Pto MYB170 may influence the expression of key enzymes genes involved in secondary wall biosynthesis.(7) Transient co-infection experiment in tobacco confirmed that Pto MYB170 was able to activate the promoters of key enzymes genes involved in secondary wall biosynthesis.(8) Detection of secondary wall components showed that the secondary wall composition was upregulated in Pto MYB170-overexpression lines and downregulated in Pto MYB170-knock-out plants, in concert with the active role of Pto MYB170 in secondary wall biosynthesis.(9) GUS staining of the promoter has shown that Pto MYB170 specifically expressed in the stomata of rosette leaves in Arabidopsis, similar with At MYB61. Drought treatment and stomata opening/closure assay of transgenic Arabidopsis has revealed that Pto MYB170 does confer drought resistance to the plant.The above results have shown that: Pto MYB170 regulates plant secondary wall biosynthesis through regulation of key enzymes involved in secondary wall biosynthesis, thereby influencing the contents of lignin, xylan and cellulose in secondary wall. On the other hand, Pto MYB170 could improve drought resistance via regulating stomata opening/closure in the leaf. |
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