Brassinosteroid Signaling Converges With Auxin-Mediated C3H17 to Regulate Xylem Formation in Populus.

Brassinosteroid (BR) signaling has long been reported to have an effect on xylem development, but the detailed mechanism remains unclear, especially in tree species. In this study, we find PdC3H17, which was demonstrated to mediate xylem formation driven by auxin in our previous report, is also involved in BR-promoted xylem development. Y1H analysis, EMSA, and transcription activation assay confirmed that PdC3H17 was directly targeted by PdBES1, which is a key transcriptional regulator in BR signaling. Tissue specificity expression analysis and in situ assay revealed that PdC3H17 had an overlapping expression profile with PdBES1. Hormone treatment examinations verified that xylem phenotypes in PdC3H17 transgenic plants, which were readily apparent in normal condition, were attenuated by treatment with either brassinolide or the BR biosynthesis inhibitor propiconazole. The subsequent quantitative real-time polymerase chain reaction (qRT-PCR) analyses further revealed that BR converged with PdC3H17 to influence transcription of downstream xylem-related genes. Additionally, the enhancement of xylem differentiation by auxin in PdC3H17 overexpression plants was significantly attenuated compared with wild-type and dominant negative plants due to BR deficiency, which suggested that the BR- and auxin-responsive gene PdC3H17 acted as an mediation of these two hormones to facilitate xylem development. Taken together, our results demonstrate that BR signaling converges with auxin-mediated PdC3H17 to regulate xylem formation in Populus and thus provide insight into the regulation mechanism of BRs and the crosstalk with auxin signaling on xylem formation.

Overexpression of PdC3H17 Confers Tolerance to Drought Stress Depending on Its CCCH Domain in Populus.

Plant CCCH zinc finger proteins control growth, development, and stress responses mainly at the post-transcriptional level. Currently, limited reports are available about the roles of plant CCCH proteins in drought tolerance. In this study, we provided evidence showing that PdC3H17 from Populus deltoides × P. euramericana involves drought tolerance and response. Overexpression of PdC3H17 in poplar caused dwarf, resulted in higher stem water potential, and showed increased photosynthetic and ROS-scavenging abilities, thereby enhancing tolerance to drought stress, compared to controls. Accordingly, after drought treatment the stem elongation and thickening rates of these overexpression lines were higher than those of the controls. However, overexpression of the coding region excluding the CCCH domain of PdC3H17 roughly exhibited WT-like physiological and drought-resistant phenotypes, indicating the requirement of the CCCH domain for PdC3H17 controlling these processes. In addition, N-terminal sequence of PdC3H17 was found to possess transcriptional activity ability in yeast cells. Together, our results suggest that PdC3H17 may depend on its CCCH domain to control drought tolerance in Populus.

Poplar PdC3H17 and PdC3H18 are direct targets of PdMYB3 and PdMYB21, and positively regulate secondary wall formation in Arabidopsis and poplar.

Wood biomass is mainly made of secondary cell walls, whose formation is controlled by a multilevel network. The tandem CCCH zinc finger (TZF) proteins involved in plant secondary wall formation are poorly understood. Two TZF genes, PdC3H17 and PdC3H18, were isolated from Populus deltoides and functionally characterized in Escherichia coli, tobacco, Arabidopsis and poplar. PdC3H17 and PdC3H18 are predominantly expressed in cells of developing wood, and the proteins they encode are targeted to cytoplasmic foci. Transcriptional activation assays showed that PdMYB2/3/20/21 individually activated the PdC3H17 and PdC3H18 promoters, but PdMYB3/21 were most significant. Electrophoretic mobility shift assays revealed that PdMYB3/21 bound directly to the PdC3H17/18 promoters. Overexpression of PdC3H17/18 in poplar increased secondary xylem width and secondary wall thickening in stems, whereas dominant repressors of them had the opposite effects on these traits. Similar alteration in secondary wall thickening was observed in their transgenic Arabidopsis plants. qRT-PCR results showed that PdC3H17/18 regulated the expression of cellulose, xylan and lignin biosynthetic genes, and several wood-associated MYB genes. These results demonstrate that PdC3H17 and PdC3H18 are the targets of PdMYB3 and PdMYB21 and are an additional two components in the regulatory network of secondary xylem formation in poplar.