This study aimed to evaluate the effects of phosphorus (P) deficiency on the lateral root growth of Fraxinus mandshurica seedlings and to reveal the potential molecular mechanisms involved. F. mandshurica seedlings were treated with various concentrations of P (0, 0.5, 1, and 2 mM KH2PO4) for 15 d. The growth of lateral roots was quantified by the number of lateral roots, total root length, total root surface area, and root : shoot ratio. Additionally, the levels of endogenous hormones and acid phosphatase were determined in the lateral roots of F. mandshurica seedlings using enzyme‐linked immunosorbent assay (ELISA). The transcription of 17 FmWRKYs and FmPHR1 was detected in the lateral roots of F. mandshurica seedlings using quantitative real‐time PCR (qRT‐PCR). F. mandshurica seedlings under P deficiency (0 mM) exhibited significantly higher number of lateral roots, total root length, total root surface area, and root : shoot ratio than those under P supply. P deficiency significantly decreased the levels of indole‐3‐acetic acid, brassinolide, and ethylene, and increased the levels of gibberellins 3, cytokinin, abscisic acid, and acid phosphatase in the lateral roots of F. mandshurica seedlings when compared to seedlings with P supply. In addition, the transcription of four FmWRKYs, including FmWRKY6, FmWRKY23, FmWRKY44, and FmWRKY71, as well as FmPHR1 were significantly higher in the lateral roots of F. mandshurica seedlings under P deficiency compared to seedlings treated with 1 mM P. Phosphorus deficiency promoted the lateral root growth of F. mandshurica seedlings and this process may be associated with the up‐regulation of FmWRKY6, FmWRKY23, FmWRKY44, FmWRKY71, and FmPHR1.

The TEOSINTE BRANCHED1, CYCLOIDEA, and PROLIFERATION CELL FACTOR (TCP) transcription factor is a plant-specific gene family and acts on multiple functional genes in controlling growth, development, stress response, and the circadian clock. In this study, a class I member of the TCP family from Fraxinus mandshurica Rupr. was isolated and named FmTCP15, which encoded a protein of 362 amino acids. Protein structures were analyzed and five ligand binding sites were predicted. The phylogenetic relationship showed that FmTCP15 was most closely related to Solanaceae and Plantaginaceae. FmTCP15 was localized in the nuclei of F. mandshurica protoplast cells and highly expressed in cotyledons. The expression pattern revealed the FmTCP15 response to multiple abiotic stresses and hormone signals. Downstream genes for transient overexpression of FmTCP15 in seedlings were also investigated. A yeast two-hybrid assay confirmed that FmTCP15 could interact with DELLA proteins. FmTCP15 participated in the GA-signaling pathway, responded to abiotic stresses and hormone signals, and regulated multiple genes in these biological processes. Our study revealed the potential value of FmTCP15 for understanding the molecular mechanisms of stress and hormone signal responses.

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The basic helix–loop–helix (bHLH) transcription factors (TFs) are involved in plant morphogenesis and various abiotic and biotic stress responses. However, it requires further exploration of drought-responsive bHLH family members and their detailed regulatory mechanisms in Populus. Two bHLH TF genes, PxbHLH01/02, were identified in Populus simonii × P. nigra and cloned. The aim of this study was to examine the role of bHLH TFs in drought tolerance in P. simonii × P. nigra. The results showed that the amino acid sequences of the two genes were homologous to Arabidopsis thaliana UPBEAT1 (AtUPB1) and overexpression of PxbHLH01/02 restored normal root length in the AtUPB1 insertional mutant (upb1–1). The PxbHLH01/02 gene promoter activity analysis suggested that they were involved in stress responses and hormone signaling. Furthermore, Arabidopsis transgenic lines overexpressing PxbHLH01/02 exhibited higher stress tolerance compared with the wild-type. P. simonii × P. nigra overexpressing PxbHLH02 increased drought tolerance and exhibited higher superoxide dismutase and peroxidase activities, lower H2O2 and malondialdehyde (MDA) content, and lower relative conductivity. The results of transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) suggested that the response of PxbHLH02 to drought stress was related to ABA signal transduction. Overall, the findings of this study suggest that PxbHLH02 from P. simonii × P. nigra functions as a positive regulator of drought stress responses by regulating stomatal aperture and promoting ABA signal transduction.

Fraxinus mandshurica Rupr. (hereafter “F. mandshurica”) is known as one of northeast China′s important, valuable hardwood timber species. However, tissue culture and micropropagation of the species are difficult and have low efficiency, limiting asexual propagation. In this manuscript, stem explants were utilized to establish an effective regeneration system through adventitious bud organogenesis. The factors influencing callus regeneration in vitro were determined, and callus regeneration technology was established. The mechanism of adventitious bud formation was analyzed. Thidiazuron (TDZ) played a crucial role in the formation of adventitious buds. Elevated concentrations of TDZ were beneficial to callus induction and low concentrations of 6-benzyladenine (BA) led to loose state callus formation. The order of callus induction rates for different explants was stem cotyledon (100%) > segment (98.54%) > hypocotyl (92.56%) > root (50.71%). The effects of exogenous addition of 6-BA and TDZ on the endogenous hormone content of plants during the regeneration of adventitious buds were also assessed, as well as the expression characteristics of genes related to the regeneration pathway. The comprehensive analysis results showed that the suitable medium for callus induction and adventitious bud differentiation was c12 medium (MSB5 + 30 g/L sucrose + 7 g/L Agar + 5 mg/L 6-BA + 8 mg/L TDZ + 2 mg/L glycine + 0.1 mg/L IBA + 5% coconut water). The induction rates of callus and adventitious buds were 99.15% and 33.33%. The addition of 2.4 mg/L of the DNA demethylation reagent 5-azacytidine (5-aza) and 0.15 mg/L of the histone deacetylase inhibitor trichostatin A (TSA) increased the rates of adventitious bud induction by 17.78% over the control. This further laid the foundation for large-scale cultivation of excellent varieties and genetic transformation techniques.

RELATED TO AP2.12 (RAP2.12) is one of the Ethylene Response Factors (ERF) transcription factor and plays a key role in controlling plant root bending and responding to multiple abiotic stresses including hypoxia stress. In this study, FmRAP2.12 gene was isolated and characterized from Fraxinus mandshurica Rupr. The open reading frame (ORF) of FmRAP2.12 was 1170 bp and encoded a protein of 389 amino acids. The conserved domains, three-dimensional phylogenetic relationship of FmRAP2.12 was also investigated. Quantitative real-time (qRT-PCR) analyzed the expression of FmRAP2.12 in different tissues. The expression level of FmRAP2.12 was highest in roots followed by leaves, and lowest in male flowers. Abiotic stress and hormone signal-induced expression was established using qRT-PCR. Salt stress induced FmRAP2.12 to a double peak pattern: the first peak value was at 6 h and the second at 72 h. Drought stress also induced FmRAP2.12 to a double peak pattern: the first at 6 h and the second at 48 h. FmRAP2.12 was up-regulated after initiation of gibberellic acid (GA3) treatment, with a one peak pattern at 24 h. FmRAP2.12 may not respond to cold stress and Abscisic acid (ABA) treatment. The transient overexpression of FmRAP2.12 caused the up-expression of downstream key genes of abiotic stress response and gibberellin pathway. Our research reveals the molecular characteristic and expression patterns under abiotic stress and hormone condition of FmRAP2.12, providing support for the genetic improvement of F. mandshurica at a molecular level.

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