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.

Summary 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.

C. (2023). PuHox52 promotes coordinated uptake of nitrate, phosphate, and iron under nitrogen deficiency in Populus ussuriensis.

Populus ussuriensis is an important tree species with high economic and ecologic values. However, traditional sexual propagation is time-consuming and inefficient, challenging afforestation and wood production using P. ussuriensis, and requires a rapid and efficient regeneration system. The present study established a rapid, efficient, and stable shoot regeneration method from root explants in P. ussuriensis using several plant growth regulators. Most shoot buds (15.2 per explant) were induced at high efficiency under WPM medium supplemented with 221.98 μM 6-BA, 147.61 μM IBA, and 4.54 μM TDZ within two weeks. The shoot buds were further multiplicated and elongated under WPM medium supplemented with 221.98 μM 6-BA, 147.61 μM IBA, and 57.74 μM GA3 for four weeks. The average number and efficiency of elongation of multiplication and elongation for induced shoot buds were 75.2 and 78%, respectively. All the shoots were rooted within a week and none of them showed abnormality in rooting. The time spent for the entire regeneration of this direct shoot organogenesis was seven weeks, much shorter than conventional indirect organogenesis with the callus induction phase, and no abnormal growth was observed. This novel regeneration system will not only promote the massive propagation, but also accelerate the genetic engineering studies for trait improvement of P. ussuriensis species.

The GRAS family transcription factors play important roles in regulating plant growth and responses to abiotic stress, which can be utilized to breed novel plants with improved abiotic stress resistance. However, the GRAS gene family has been largely unexplored for tree species, particularly for Larix kaempferi, which has high economic and ecological values, challenging practices for breeding abiotic stress-resistant L. kaempferi. In order to improve the stress resistance by regulating the transcription factors in L. kaempferi, we identified 11 GRAS genes in L. kaempferi and preliminarily characterized them through comprehensive analyses of phylogenetic relationships, conserved motifs, promoter cis-elements, and expression patterns, as well as protein interaction network prediction. The phylogenetic analysis showed that the LkGRAS family proteins were classified into four subfamilies, including DELLA, HAM, SCL, and PAT1, among which the SCL subfamily was the largest one. Conserved motif analysis revealed many putative motifs such as LHRI-VHIID-LHRII-PFYRE-SAW at C-terminals of the LkGRAS proteins; we discovered a unique motif of the LkGRAS genes. Promoter cis-acting element analysis exhibited several putative elements associated with abiotic stresses and phytohormones; the abscisic acid-responsive elements (ABRE) and G-box are the most enriched elements in the promoters. Through expression profiles of LkGRAS genes in different tissues and under drought-stress and phytohormones (GA3 and ABA) treatments, it was demonstrated that LkGRAS genes are most active in the needles, and they rapidly respond to environmental cues such as drought-stress and phytohormone treatments within 24 h. Protein interaction network prediction analysis revealed that LkGRAS proteins interact with various proteins, among which examples are the typical GA, ABA, and drought-stress signaling factors. Taken together, our work identifies the novel LkGRAS gene family in L. kaempferi and provides preliminary information for further in-depth functional characterization studies and practices of breeding stress-resistant L. kaempferi.