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In previous studies, GA20 oxidase (GA20ox) has been identified to be an important enzyme in the biosynthesis of GA, and SHOOTMERISTEMLESS (STM) can repress the expression of GA20ox. In this study, the GATA transcription factor PdeGATA3 was identified in the poplar line NL895, and its overexpression (OE) transgenic lines showed a dwarf phenotype. RNA sequencing (RNA-Seq) analysis suggested that OE PdeGATA3 could promote the expression of PdeSTM and repress the expression of PdeGA20ox. Therefore, we hypothesized that PdeGATA3 would directly promote the expression of PdeSTM and that PdeSTM would repress the expression of PdeGA20ox. Four experiments, a dual-luciferase reporter assay, GUS transient coexpression assay, yeast one-hybrid assay and electrophoretic mobility shift assay, were conducted and verified that PdeGATA3 could promote the expression of PdeSTM by binding GATA-Boxes in its promoter. OE PdeSTM in poplar resulted in a dwarf phenotype and repressed the expression of PdeGA20ox. GA measurement of the OE PdeSTM and PdeGATA3 lines showed that GA3 and GA4 contents were significantly lower than those in the wild type (WT). Accordingly, we put forward a regulation model involving plant height regulation by PdeGATA3 , PdeSTM and PdeGA20ox. [ABSTRACT FROM AUTHOR]

Peroxidase (Prx)-related genes are reported to be involved in the metabolism of hydrogen peroxide (H2O2) in plants. Here, we found that the expression of the PdePrx12 gene was upregulated in wild-type (WT) poplar line NL895 infected with the pathogens Botryosphaeria dothidea strain 3C and Alternaria alternata strain 3E. The PdePrx12 gene was cloned in the poplar line NL895 and its overexpression (OE) and reduced-expression (RE) vectors were constructed. OE and RE transgenic lines were then generated. The H2O2 content in the leaves was measured by DAB staining and spectrophotometric analysis, and the data revealed that the OE line had a reduced H2O2 content, whereas the RE line had an increased H2O2 content. These transgenic and WT plants were also inoculated with the 3C/3E pathogens. The leaf area infected by pathogen 3C/3E was determined and the OE line was found to have a larger area of infection, whereas the RE line was found to have a smaller area of infection. This result suggested PdePRX12 is involved in disease resistance in poplar. Given these results, this study demonstrated that when poplar is infected by pathogens, the expression of PdePrx12 is inhibited, leading to an increase in H2O2 content, thereby enhancing disease resistance. [ABSTRACT FROM AUTHOR]

Summary: Callus formation is important for numerous biological processes in plants. Previously, we revealed that the PdeWRKY75–PdeRBOHB module positively regulates hydrogen peroxide (H2O2) accumulation, thereby affecting callus formation in poplar.In this study, we identified and confirmed a transcription factor, PdeERF114, that interacts with PdeWRKY75 both in vitro and in vivo. Gene expression analysis identified both PdeRBOHB and PdeEXPB2 as downstream genes of PdeERF114 and PdeWRKY75. Overexpression (OE) and reduced‐expression (RE) transgenic poplar lines for these four genes were generated, and the observation of callus formation was also performed in all plant materials.We demonstrated that PdeERF114 and PdeWRKY75 formed a protein complex and that this complex could bind W‐Box motifs in the promoters of PdeRBOHB and PdeEXPB2, thereby positively regulating the expression of PdeRBOHB and PdeEXPB2. The OE/RE transgenic lines for these four genes also showed enhanced/reduced callus formation.Overall, we revealed a novel gene regulatory network for the regulation of callus formation in plants that involves four genes and regulates callus formation through two pathways: the accumulation of H2O2 in explants and the relaxation of cell walls. In the future, the four genes could be used to enhance transformation effectiveness in genetic engineering. [ABSTRACT FROM AUTHOR]

SUMMARY: Phosphorus (P) is an important nutrient for plants. Here, we identify a WRKY transcription factor (TF) in poplar (Populus deltoides × Populus euramericana) (PdeWRKY65) that modulates tissue phosphate (Pi) concentrations in poplar. PdeWRKY65 overexpression (OE) transgenic lines showed reduced shoot Pi concentrations under both low and normal Pi availabilities, while PdeWRKY65 reduced expression (RE) lines showed the opposite phenotype. A gene encoding a Pi transporter (PHT), PdePHT1;9, was identified as the direct downstream target of PdeWRKY65 by RNA sequencing (RNA‐Seq). The negative regulation of PdePHT1;9 expression by PdeWRKY65 was confirmed by DNA–protein interaction assays, including yeast one‐hybrid (Y1H), electrophoretic mobility shift assay (EMSA), co‐expression of the promoters of PdePHT1;9 and PdeWRKY65 in tobacco (Nicotiana benthamiana) leaves, and chromatin immunoprecipitation–quantitative PCR. A second WRKY TF, PdeWRKY6, was subsequently identified and confirmed to positively regulate the expression of PdePHT1;9 by DNA–protein interaction assays. PdePHT1;9 and PdeWRKY6 OE and RE poplar transgenic lines were used to confirm their positive regulation of shoot Pi concentrations, under both normal and low Pi availabilities. No interaction between PdeWRKY6 and PdeWRKY65 was observed at the DNA or protein levels. Collectively, these data suggest that the low Pi‐responsive TFs PdeWRKY6 and PdeWRKY65 independently regulate the expression of PHT1;9 to modulate tissue Pi concentrations in poplar. Significance Statement: The genes PdeWRKY65 and PdeWRKY6, encoding WRKY transcription factors, and PdePHT1;9, encoding a phosphate (Pi) transporter, form a regulatory network to modulate tissue Pi concentrations in response to low Pi availability in poplar (Populus deltoides × Populus euramericana). In the regulatory network, PdeWRKY65 and PdeWRKY6 negatively and positively regulate the expression of PdePHT1;9 by binding to W‐Boxes within the PdePHT1;9 promoter, respectively. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide (H2O2) plays important roles in plant development. Adventitious roots (AR), lateral buds (LB) and callus formation are important traits for plants. Here, a gene encoding RESPIRATORY BURST OXIDASE HOMOLOG B (PdeRBOHB) from poplar line 'NL895' (Populus. deltoides × P. euramericana) was predicted to be involved in H2O2 accumulation, and lines with reduced expression were generated. H2O2 content was decreased, and the development of adventitious roots, lateral buds, and callus was inhibited in reduced expression PdeRBOHB lines. A gene encoding PdeWRKY75 was identified as the upstream transcription factor positively regulating PdeRBOHB. This regulation was confirmed by dual luciferase reporter assay, GUS transient expression analysis and electrophoretic mobility shift assay. In the reduced expression PdeWRKY75 lines, H2O2 content was decreased and the development of adventitious roots, lateral buds, and callus development was inhibited, while in the overexpression lines, H2O2 content was increased and the development of adventitious roots and lateral buds was inhibited, but callus formation was enhanced. Additionally, reduced expression PdeRBOHB lines showed lowered expression of PdeWRKY75 , while exogenous application of H2O2 showed the opposite effect. Together, these results suggest that PdeWRKY75 and PdeRBOHB are part of a regulatory module in H2O2 accumulation, which is involved in the regulation of multiple biological processes. [ABSTRACT FROM AUTHOR]

Lignin is made up of three main p-hydroxycinamyl alcohol precursors or monolignols. Lignin biosynthesis is not only important for plant growth, but also the key factor for human use of plants as biofuels. In past decades, there were numerous studies have focused on lignification in plant stems; however, little has been uncovered about lignification in petioles. In this study, a gene encoding a glycosyltransferase was identified in poplar and named UGT75L28. A relatively higher expression of UGT75L28 could be observed in petioles. Transgenic lines overexpressing UGT75L28 showed reduced lignification in petioles, while reduced expression (RE) of this gene displayed opposite phenotype. The protein of UGT75L28 fused with six His tags was expressed in E. coli and purified. In vitro enzyme reactions were conducted for a number of substrates for this fused protein. The results revealed that UGT75L28 could only glycosylate coniferyl aldehyde, which is a substrate in lignin biosynthesis in plants. The identification of the upstream regulator of UGT75L28 found that the transcription factor PdeWRKY65 could bind to the promoter of UGT75L28 and activate its expression both in vivo and in vitro by the yeast one-hybrid (Y1H) assay, the coexpression of the promoters of UGT75L28 and PdeWRKY65 in tobacco, and chromatin immunoprecipitation PCR (ChIP-PCR). Overexpression (OE) and RE PdeWRKY65 transgenic lines in poplar were also generated. The phenotype assay also showed that OE and RE PdeWRKY65 could reduce and enhance lignification in poplar petioles, respectively. The expression examination of PdeWRKY65 also showed that it was mainly expressed in petioles. Collectively, these results suggest that the gene module PdeWRKY65 - UGT75L28 negatively regulates petiole lignification by modulating the glycosylation of coniferyl aldehyde in poplar. • UGT75L28 can glycosylate a substrate in lignin biosynthesis in poplar. • PdeWRKY65 TF directly activates the expression of UGT75L28. • PdeWRKY65 - UGT75L28 negatively regulates lignin biosynthesis in poplar petioles. [ABSTRACT FROM AUTHOR]