Overexpression of PtrMYB119, a R2R3-MYB transcription factor from Populus trichocarpa, promotes anthocyanin production in hybrid poplar.

Anthocyanins are a group of colorful and bioactive natural pigments with important physiological and ecological functions in plants. We found an MYB transcription factor (PtrMYB119) from Populus trichocarpa that positively regulates anthocyanin production when expressed under the control of the CaMV 35S promoter in transgenic Arabidopsis Amino acid sequence analysis revealed that PtrMYB119 is highly homologous to Arabidopsis PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT1), a well-known transcriptional activator of anthocyanin biosynthesis. Independently produced transgenic poplars overexpressing PtrMYB119 or PtrMYB120 (a paralogous gene to PtrMYB119) (i.e., 35S::PtrMYB119 and 35S::PtrMYB120, respectively) showed elevated accumulation of anthocyanins in the whole plants, including leaf, stem and even root tissues. Using a reverse-phase high-performance liquid chromatography, we confirmed that the majority of the accumulated anthocyanin in our transgenic poplar is cyanidin-3-O-glucoside. Gene expression analyses revealed that most of the genes involved in the anthocyanin biosynthetic pathway were highly upregulated in 35S::PtrMYB119 poplars compared with the nontransformed control poplar. Among these genes, expression of PtrCHS1 (Chalcone Synthase1) and PtrANS2 (Anthocyanin Synthase2), which catalyze the initial and last steps of anthocyanin biosynthesis, respectively, was upregulated by up to 350-fold. Subsequent transient activation assays confirmed that PtrMYB119 activated the transcription of both PtrCHS1 and PtrANS2 Interestingly, expression of MYB182, a repressor of both anthocyanin and proanthocyanidin (PA) biosynthesis, was largely suppressed in 35S::PtrMYB119 poplars, while expression of MYB134, an activator of PA biosynthesis, was not changed significantly. More interestingly, high-level accumulation of anthocyanins in 35S::PtrMYB119 poplars did not have an adverse effect on plant growth. Taken together, our results demonstrate that PtrMYB119 and PtrMYB120 function as transcriptional activators of anthocyanin accumulation in both Arabidopsis and poplar.

Evaluation of a novel promoter from Populus trichocarpa for mature xylem tissue specific gene delivery.

Wood (i.e., secondary xylem) is an important raw material for many industrial applications. Mature xylem (MX) tissue-specific genetic modification offers an effective means to improve the chemical and physical properties of the wood. Here, we describe a promoter that drives strong gene expression in a MX tissue-specific manner. Using whole-transcriptome genechip analyses of different tissue types of poplar, we identified five candidate genes that had strong expression in the MX tissue. The putative promoter sequences of the five MX-specific genes were evaluated for their promoter activity in both transgenic Arabidopsis and poplar. Among them, we found the promoter of Potri.013G007900.1 (called the PtrMX3 promoter) had the strongest activity in MX and thus was further characterized. In the stem and root tissues of transgenic Arabidopsis plants, the PtrMX3 promoter activity was found exclusively in MX tissue. MX-specific activity of the promoter was reproduced in the stem tissue of transgenic poplar plants. The PtrMX3 promoter activity was not influenced by abiotic stresses or exogenously applied growth regulators, indicating the PtrMX3 promoter is bona fide MX tissue-specific. Our study provides a strong MX-specific promoter for MX-specific modifications of woody biomass.

Overexpression of gibberellin 20-oxidase1 from Pinus densiflora results in enhanced wood formation with gelatinous fiber development in a transgenic hybrid poplar.

Gibberellins (GAs) are important regulators of plant shoot biomass growth, and GA 20-oxidase (GA20ox) is one of the major regulatory enzymes in the GA biosynthetic pathway. Previously, we showed that the expression levels of a putative GA20ox1 (i.e., PdGA20ox1) in stem tissue of 3-month-old seedlings of 12 families of Pinus densiflora were positively correlated with stem diameter growth across those same families growing in an even-aged 32-year-old pine forest (Park EJ, Lee WY, Kurepin LV, Zhang R, Janzen L, Pharis RP (2015) Plant hormone-assisted early family selection in Pinus densiflora via a retrospective approach. Tree Physiol 35:86-94). To further investigate the molecular function of this gene in the stem wood growth of forest trees, we produced transgenic poplar lines expressing PdGA20ox1 under the control of the 35S promoter (designated as 35S::PdGA20ox1). By age 3 months, most of the 35S::PdGA20ox1 poplar trees were showing an exceptional enhancement of stem wood growth, i.e., up to fourfold increases in stem dry weight, compared with the nontransformed control poplar plants. Significant increases in endogenous GA1, its immediate precursor (GA20) and its catabolite (GA8) in elongating internode tissue accompanied the increased stem growth in the transgenic lines. Additionally, the development of gelatinous fibers occurred in vertically grown stems of the 35S::PdGA20ox1 poplars. An analysis of the cell wall monosaccharide composition of the 35S::PdGA20ox1 poplars showed significant increases in xylose and glucose contents, indicating a qualitative increase in secondary wall depositions. Microarray analyses led us to find a total of 276 probe sets that were upregulated (using threefold as a threshold) in the stem tissues of 35S::PdGA20ox1 poplars relative to the controls. 'Cell organization or biogenesis'- and 'cell wall'-related genes were overrepresented, including many of genes that are involved in cell wall modification. Several transcriptional regulators, which positively regulate cell elongation through GA signaling, were also upregulated. In contrast, genes involved in defense signaling were appreciably downregulated in the 35S::PdGA20ox1 stem tissues, suggesting a growth versus defense trade-off. Taken together, our results suggest that PdGA20ox1 functions to promote stem growth and wood formation in poplar, probably by activating GA signaling while coincidentally depressing defense signaling.