Summary: Plant MYB transcription factors control diverse biological processes, such as differentiation, development and abiotic stress responses. In this study, we characterized BplMYB46, an MYB gene from Betula platyphylla (birch) that is involved in both abiotic stress tolerance and secondary wall biosynthesis. BplMYB46 can act as a transcriptional activator in yeast and tobacco. We generated transgenic birch plants with overexpressing or silencing of BplMYB46 and subjected them to gain‐ or loss‐of‐function analysis. The results suggest that BplMYB46 improves salt and osmotic tolerance by affecting the expression of genes including SOD, POD and P5CS to increase both reactive oxygen species scavenging and proline levels. In addition, BplMYB46 appears to be involved in controlling stomatal aperture to reduce water loss. Overexpression of BplMYB46 increases lignin deposition, secondary cell wall thickness and the expression of genes in secondary cell wall formation. Further analysis indicated that BplMYB46 binds to MYBCORE and AC‐box motifs and may directly activate the expression of genes involved in abiotic stress responses and secondary cell wall biosynthesis whose promoters contain these motifs. The transgenic BplMYB46‐overexpressing birch plants, which have improved salt and osmotic stress tolerance, higher lignin and cellulose content and lower hemicellulose content than the control, have potential applications in the forestry industry.

The MYB (v-myb avian myeloblastosis viral oncogene homolog) family is one of the largest transcription factor families in plants, and is widely involved in the regulation of plant metabolism. In this study, we show that a MYB4 transcription factor, BpMYB4, identified from birch (Betula platyphyllaSuk.) and homologous to EgMYB1 fromEucalyptus robustaSmith and ZmMYB31 fromZea maysL. is involved in secondary cell wall synthesis. The expression level ofBpMYB4was higher in flowers relative to other tissues, and was induced by artificial bending and gravitational stimuli in developing xylem tissues. The expression of this gene was not enriched in the developing xylem during the active season, and showed higher transcript levels in xylem tissues around sprouting and near the dormant period.BpMYB4also was induced express by abiotic stress. Functional analysis indicated that expression ofBpMYB4in transgenic Arabidopsis (Arabidopsis thaliana) plants could promote the growth of stems, and result in increased number of inflorescence stems and shoots. Anatomical observation of stem sections showed lower lignin deposition, and a chemical contents test also demonstrated increased cellulose and decreased lignin content in the transgenic plants. In addition, treatment with 100 mM NaCl and 200 mM mannitol resulted in the germination rate of the over-expressed lines being higher than that of the wild-type seeds. The proline content in transgenic plants was higher than that in WT, but MDA content was lower than that in WT. Further investigation in birch using transient transformation techniques indicated that overexpression ofBpMYB4could scavenge hydrogen peroxide and O₂^.–and reduce cell damage, compared with the wild-type plants. Therefore, we believe that BpMYB4 promotes stem development and cellulose biosynthesis as an inhibitor of lignin biosynthesis, and has a function in abiotic stress resistance.

Transcription factors (TFs) play vital roles in various biological processes by binding to cis‐acting elements to control expressions of their target genes. The MYB TF BplMYB46, from Betula platyphylla, is involved in abiotic stress responses and secondary wall deposition. In the present study, we used a TF‐centered yeast one‐hybrid technology (TF‐centered Y1H) to identify the cis‐acting elements bound by BplMYB46. We screened a short‐insert random library and identified three cis‐elements bound by BplMYB46: an E‐box (CA(A/T/C)(A/G/C)TG) and two novel motifs, a TC‐box (T(G/A)TCG(C/G)) and a GT‐box (A(G/T)T(A/C)GT(T/G)C). Chromatin immunoprecipitation (ChIP) and effector‐reporter coexpression assays in Nicotiana tabacum confirmed binding of BplMYB46 to the TC‐box, GT‐box, and E‐box motifs in the promoters of the phenylalanine ammonia lyase (PAL), peroxidase (POD), and superoxide dismutase (SOD) genes, which function in abiotic stress tolerance and secondary wall biosynthesis. This finding improves our understanding of potential regulatory mechanisms in the response to abiotic stress and secondary wall deposition of BplMYB46 in B. platyphylla.

Background: Populus davidiana × P. bollenais a species of poplar from northeastern China that is characterized by cold resistance and fast growth but now suffers from pathogen infections. Leaf blight caused byAlternaria alternatahas become a common poplar disease that causes serious economic impacts, but the molecular mechanisms of resistance toA. alternatainP. davidiana × P. bollenaare still unclear.Results: In this study, the transcriptomic response ofP. davidiana × P. bollenatoA. alternatainfection was determined via RNA-Seq. Twelve cDNA libraries were generated from RNA isolated from three biological replicates at four time points (0, 2, 3, and 4 d post inoculation), and a total of 5,930 differentially expressed genes (DEGs) were detected (| log₂fold change |≥ 1 and FDR values < 0.05). Functional analysis revealed that the DEGs were mainly enriched for the “plant hormone signal transduction” pathway, followed by the “phenylpropanoid biosynthesis” pathway. In addition, DEGs that encode defense-related proteins and are related to ROS metabolism were also identified. Numerous transcription factors, such as the bHLH, WRKY and MYB families, were also induced byA. alternatainfection. Among these DEGs, those related to JA biosynthesis and JA signal transduction were consistently activated. Therefore, the lipoxygenase genePdbLOX2, which is involved in JA biosynthesis, was selected for functional characterization. Overexpression ofPdbLOX2enhanced the resistance ofP. davidiana × P. bollenatoA. alternata,whereas silencing this gene enhanced susceptibility toA. alternatainfection.Conclusions: These results provide new insight into the molecular mechanisms of poplar resistance toA. alternatainfection and provide candidate genes for breeding resistant cultivars using genetic engineering.

MYB proteins play important roles in the regulation of plant growth, development, and stress responses. Overexpression of BplMYB46 from Betula platyphylla improved plant salt and osmotic tolerances. In the present study, the interaction of eight avian myeloblastosis viral oncogene homolog (MYB) transcription factors with BplMYB46 was investigated using the yeast two-hybrid system, which showed that BplMYB46 could form homodimers and heterodimers with BplMYB6, BplMYB8, BplMYB11, BplMYB12, and BplMYB13. Relative beta-glucuronidase activity and chromatin immunoprecipitation assays showed that the interaction between BplMYB46 and the five MYBs increased the binding of BplMYB46 to the MYBCORE motif. A subcellular localization study showed that these MYBs were all located in the nucleus. Real-time fluorescence quantitative PCR results indicated that the expressions of BplMYB46 and the five MYB genes could be induced by salt and osmotic stress, and the BplMYB46 and BplMYB13 exhibited the most similar expression patterns. BplMYB46 and BplMYB13 co-overexpression in tobacco using transient transformation technology improved tobacco’s tolerance to salt and osmotic stresses compared with overexpressing BplMYB13 or BplMYB46 alone. Taken together, these results demonstrated that BplMYB46 could interact with five other MYBs to form heterodimers that activate the transcription of target genes via an enhanced binding ability to the MYBCORE motif to mediate reactive oxygen species scavenging in response to salt and osmotic stresses.

Heat shock transcription factors (HSFs) play critical roles in several types of environmental stresses. However, the detailed regulatory mechanisms in response to salt stress are still largely unknown. In this study, we examined the salt-induced transcriptional responses of ThHSFA1-ThWRKY4 in Tamarix hispida and their functions and regulatory mechanisms in salt tolerance. ThHSFA1 protein acts as an upstream regulator that can directly activate ThWRKY4 expression by binding to the heat shock element (HSE) of the ThWRKY4 promoter using yeast one-hybrid (Y1H), chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays. ThHSFA1 and ThWRKY4 expression was significantly induced by salt stress and abscisic acid (ABA) treatment in the roots and leaves of T. hispida. ThHSFA1 is a nuclear-localized protein with transactivation activity at the C-terminus. Compared to nontransgenic plants, transgenic plants overexpressing ThHSFA1 displayed enhanced salt tolerance and exhibited reduced reactive oxygen species (ROS) levels and increased antioxidant enzyme activity levels under salt stress. Therefore, we further concluded that ThHSFA1 mediated the regulation of ThWRKY4 in response to salt stress in T. hispida.