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Secondary growth is a key characteristic of trees, which requires the coordination of multiple regulatory mechanisms including transcriptional regulators and microRNAs (miRNAs). However, the roles of microRNAs in the regulation of secondary growth need to be explored in depth. Here, the role of miR319a and its target, PtoTCP20, in the secondary growth of Populus tomentosa stem was investigated using genetic and molecular analyses. The expression level of miR319a gradually decreased from primary to secondary growth in P. tomentosa, while that of PtoTCP20 gradually increased. MiR319a overexpression in seedlings resulted in delayed secondary growth and decreased xylem production, while miR319a knockdown and PtoTCP20 overexpression promoted secondary growth and increased xylem production. Further analysis showed that PtoTCP20 interacted with PtoWOX4a and activated PtoWND6 transcription in vitro and in vivo. Our data show that PtoTCP20 controls vascular cambium proliferation by binding to PtoWOX4a, and promotes secondary xylem differentiation by activating PtoWND6 transcription, thereby regulating secondary growth in P. tomentosa. Our findings provide insight into the molecular mechanisms underlying secondary growth in trees.

Trichomes are specialized epidermal cells that contribute to plant resistance against herbivores. Their formation is controlled precisely by multiple genetic and environmental signals. Previous studies have shown that microRNA319 (miR319) and gibberellin (GA) signaling are involved in trichome development in Arabidopsis, but little is known about their interaction between these factors. Here we reported that the miR319a/TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) module participates in trichome initiation synergistically with GA signaling in Populus tomentosa. We demonstrated that overexpression of miR319a decreased transcription levels of its targeted TCPs and significantly elevated leaf trichome density in transgenic poplar, resulting in decreasing insect herbivory. Conversely, repressing miR319a by short tandem target mimics (STTM) elevated TCP expression levels and decreased trichome density in transgenic plants. The trichome phenotype of 35S:miR319a plants could be abolished by introducing a miR319a-resistant form of TCP19. Furthermore, the miR319a-targeted TCP19 interacted directly with REPRESSOR OF ga1-3 (RGA), a downstream repressor of GA signaling. TCP19 and RGA synergistically inhibited the GLABROUS1 (GL1)-induced expression of trichome marker gene GLABRA2 (GL2), thereby repressing leaf trichome initiation. Our results provide an insight into the molecular mechanism by which miR319/TCP19 module and GA signaling coordinated regulating trichome initiation in P. tomentosa.

The secondary cell wall is an important carbon sink in higher plants and its biosynthesis requires coordination of metabolic fluxes in the phenylpropanoid pathway. In Arabidopsis (Arabidopsis thaliana), MYB75 and the KNOX transcription factor KNAT7 form functional complexes to regulate secondary cell wall formation in the inflorescence stem. However, the molecular mechanism by which these transcription factors control different branches of the phenylpropanoid pathway remains poorly understood in woody species. We isolated an R2R3-MYB transcription factor MYB6 from Populus tomentosa and determined that it was expressed predominately in young leaves. Overexpression of MYB6 in transgenic poplar upregulated flavonoid biosynthetic gene expression, resulting in significantly increased accumulation of anthocyanin and proanthocyanidins. MYB6-overexpression plants showed reduced secondary cell wall deposition, accompanied by repressed expression of secondary cell wall biosynthetic genes. We further showed that MYB6 interacted physically with KNAT7 and formed functional complexes that acted to repress secondary cell wall development in poplar and Arabidopsis. The results provide an insight into the transcriptional mechanisms involved in the regulation of the metabolic fluxes between the flavonoid and lignin biosynthetic pathways in poplar.

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.

An approach for restoring recombination activity of complementation split-Cre was developed to excise the transgene in hybrid progeny of GM crops. Growing concerns about the biosafety of genetically modified (GM) crops has currently become a limited factor affecting the public acceptance. Several approaches have been developed to generate selectable-marker-gene-free GM crops. However, no strategy was reported to be broadly applicable to hybrid crops. Previous studies have demonstrated that complementation split-Cre recombinase restored recombination activity in transgenic plants. In this study, we found that split-Cre mediated by split-intein Synechocystis sp. DnaE had high recombination efficiency when Cre recombinase was split at Asp232/Asp233 (866 bp). Furthermore, we constructed two plant expression vectors, pCA-NCre-In and pCA-Ic-CCre, containing NCre866-In and Ic-CCre866 fragments, respectively. After transformation, parent lines of transgenic Arabidopsis with one single copy were generated and used for hybridization. The results of GUS staining demonstrated that the recombination activity of split-Cre could be reassembled in these hybrid progeny of transgenic plants through hybridization and the foreign genes flanked by two loxP sites were efficiently excised. Our strategy may provide an effective approach for generating the next generation of GM hybrid crops without biosafety concerns.

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.

Some R2R3 MYB transcription factors have been shown to be major regulators of phenylpropanoid biosynthetic pathway and impact secondary wall formation in plants. In this study, we describe the functional characterization of PtoMYB156, encoding a R2R3-MYB transcription factor, from Populus tomentosa. Expression pattern analysis showed that PtoMYB156 is widely expressed in all tissues examined, but predominantly in leaves and developing wood cells. PtoMYB156 localized to the nucleus and acted as a transcriptional repressor. Overexpression of PtoMYB156 in poplar repressed phenylpropanoid biosynthetic genes, leading to a reduction in the amounts of total phenolic and flavonoid compounds. Transgenic plants overexpressing PtoMYB156 also displayed a dramatic decrease in secondary wall thicknesses of xylem fibers and the content of cellulose, lignin and xylose compared with wild-type plants. Transcript accumulation of secondary wall biosynthetic genes was down-regulated by PtoMYB156 overexpression. Transcriptional activation assays revealed that PtoMYB156 was able to repress the promoter activities of poplar CESA17, C4H2 and GT43B. By contrast, knockout of PtoMYB156 by CRISPR/Cas9 in poplar resulted in ectopic deposition of lignin, xylan and cellulose during secondary cell wall formation. Taken together, these results show that PtoMYB156 may repress phenylpropanoid biosynthesis and negatively regulate secondary cell wall formation in poplar.