PuHox52 promotes coordinated uptake of nitrate, phosphate, and iron under nitrogen deficiency in Populus ussuriensis.

It is of great importance to better understand how trees regulate nitrogen (N) uptake under N deficiency conditions which severely challenge afforestation practices, yet the underlying molecular mechanisms have not been well elucidated. Here, we functionally characterized PuHox52, a Populus ussuriensis HD-ZIP transcription factor, whose overexpression greatly enhanced nutrient uptake and plant growth under N deficiency. We first conducted an RNA sequencing experiment to obtain root transcriptome using PuHox52-overexpression lines of P. ussuriensis under low N treatment. We then performed multiple genetic and phenotypic analyses to identify key target genes of PuHox52 and validated how they acted against N deficiency under PuHox52 regulation. PuHox52 was specifically induced in roots by N deficiency, and overexpression of PuHox52 promoted N uptake, plant growth, and root development. We demonstrated that several nitrate-responsive genes (PuNRT1.1, PuNRT2.4, PuCLC-b, PuNIA2, PuNIR1, and PuNLP1), phosphate-responsive genes (PuPHL1A and PuPHL1B), and an iron transporter gene (PuIRT1) were substantiated to be direct targets of PuHox52. Among them, PuNRT1.1, PuPHL1A/B, and PuIRT1 were upregulated to relatively higher levels during PuHox52-mediated responses against N deficiency in PuHox52-overexpression lines compared to WT. Our study revealed a novel regulatory mechanism underlying root adaption to N deficiency where PuHox52 modulated a coordinated uptake of nitrate, phosphate, and iron through 'PuHox52-PuNRT1.1', 'PuHox52-PuPHL1A/PuPHL1B', and 'PuHox52-PuIRT1' regulatory relationships in poplar roots.

Two high hierarchical regulators, PuMYB40 and PuWRKY75, control the low phosphorus driven adventitious root formation in Populus ussuriensis.

Adventitious rooting is an essential biological process in the vegetative propagation of economically important horticultural and forest tree species. It enables utilization of the elite genotypes in breeding programmes and production. Promotion of adventitious root (AR) formation has been associated with starvation of inorganic phosphate and some factors involved in low phosphorus (LP) signalling. However, the regulatory mechanism underlying LP-mediated AR formation remains largely elusive. We established an efficient experimental system that guaranteed AR formation through short-term LP treatment in Populus ussuriensis. We then generated a time-course RNA-seq data set to recognize key regulatory genes and regulatory cascades positively regulating AR formation through data analysis and gene network construction, which were followed by experimental validation and characterization. We constructed a multilayered hierarchical gene regulatory network, from which PuMYB40, a typical R2R3-type MYB transcription factor (TF), and its interactive partner, PuWRKY75, as well as their direct targets, PuLRP1 and PuERF003, were identified to function upstream of the known adventitious rooting genes. These regulatory genes were functionally characterized and proved their roles in promoting AR formation in P. ussuriensis. In conclusion, our study unveiled a new hierarchical regulatory network that promoted AR formation in P. ussuriensis, which was activated by short-term LP stimulus and primarily governed by PuMYB40 and PuWRKY75.

PuC3H35 confers drought tolerance by enhancing lignin and proanthocyanidin biosynthesis in the roots of Populus ussuriensis.

Since the roots are the very organ where plants first sense and respond drought stress, it is of great importance to better understand root responses to drought. Yet the underlying molecular mechanisms governing root responses to drought stress have been poorly understood. Here, we identified and functionally characterized a CCCH type transcription factor, PuC3H35, and its targets, anthocyanin reductase (PuANR) and early Arabidopsis aluminum induced1 (PuEARLI1), which are involved in mediating proanthocyanidin (PA) and lignin biosynthesis in response to drought stress in Populus ussuriensis root. PuC3H35 was root-specifically induced upon drought stress. Overexpressing PuC3H35 promoted PA and lignin biosynthesis and vascular tissue development, resulting in enhanced tolerance to drought stress by the means of anti-oxidation and mechanical supporting. We further demonstrated that PuC3H35 directly bound to the promoters of PuANR and PuEARLI1 and overexpressing PuANR or PuEARLI1 increased root PA or lignin levels, respectively, under drought stress. Taken together, these results revealed a novel regulatory pathway for drought tolerance, in which PuC3H35 mediated PA and lignin biosynthesis by collaboratively regulating 'PuC3H35-PuANR-PA' and 'PuC3H35-PuEARLI1-PuCCRs-lignin' modules in poplar roots.