Hydrogen peroxide (H2O2) plays important roles in plant development. Adventitious root (AR), lateral bud (LB) and callus formation are important traits for plants. Here, a gene encoding respiratory burst oxidase homologs B (PdeRBOHB) from poplar line “NL895” (Populus. deltoides × P. euramericana) was predicted to be involved in H2O2 accumulation and its reduced expression (RE) transgenic lines were generated. H2O2 content was decreased, and the development of AR, LB and callus was inhibited, in RE PdeRBOHB lines. A gene encoding PdeWRKY75 was identified as the positive upstream transcription factor (TF) for PdeRBOHB. This regulation was confirmed by dual luciferase reporter assay, GUS transient expression analysis and electrophoretic mobility shift assay (EMSA). In the RE PdeWRKY75 lines, H2O2 content was decreased and the development of AR, LB and callus development was inhibited, while in the over expression (OE) lines, H2O2 content was increased and the development of AR and LB was inhibited, but callus formation was enhanced. Additionally, RE PdeRBOHB inhibited the expression of PdeWRKY75, while exogenous application of H2O2 showed the opposite effect. All of these results suggested that PdeWRKY75 and PdeRBOHB are part of a regulatory module in H2O2 accumulation, which is involved in the regulation of multiple biological processes.
Adventitious roots (ARs) are important for some plants that depend on clonal propagation. In this study, we demonstrate that a salt-responsive gene module is involved in the negative regulation of AR development in poplar. In this module, the expression of bZIP53 is induced by salt stress and it encodes a transcription factor with transactivation activity. Overexpression or induced expression of bZIP53 in poplar lines resulted in inhibition of AR growth, while heterologous overexpression of bZIP53 in Arabidopsis resulted in a similar phenotype. Results from RNA-seq and RT-qPCR assays predicted IAA4-1 and IAA4-2 to be downstream genes that were regulated by bZIP53. Further investigation of protein–DNA interactions using yeast one-hybrid, electrophoretic mobility shift, dual luciferase reporter, and GUS co-expression assays also showed that IAA4-1/2 were the genes that were directly regulated by bZIP53. Induced-expression IAA4-1/2 transgenic poplar lines also showed inhibited AR growth. In addition, both poplar bZIP53 and IAA4-1/2 showed a response to salt stress. On the basis of these results, we conclude that the bZIP53–IAA4 module is involved in the negative regulation of AR development in poplar.
Poplars are important woody plants, and the ability to form adventitious roots (ARs) is the key factor for their cultivation because most poplars are propagated by cloning. In previous studies, Ca2+ was confirmed to regulate AR formation in poplar. In this study, wild type poplar cuttings grown in 1.0 mM Ca2+ solution showed the best visible performance of AR development. Coexpression analysis of a large-scale RNA-Seq transcriptome was conducted to identify Ca2+-related genes that regulates AR development in poplar. A total of 15 coexpression modules (CMs) were identified and two CMs showed high association with AR development. Functional analysis identified a number of biological pathways, including “oxidation-reduction process”, “response to biotic stimulus” and “metabolic process”, in tissues of AR development. The Ca2+-related pathway was specifically selected, and its regulation in poplar AR development was predicted. A Ca2+ sensor, PdeCML23–1, which is a member of the calmodulin-like protein (CML) family, was found to promote AR development by phenotypic assay of overexpressed PdeCML23–1 transgenic lines at various growing conditions. By measuring cytosolic Ca2+ in AR tips, PdeCML23–1 seemed to play a role in decreasing cytosolic Ca2+ concentration. Additionally, the expression profiles of some genes and phytohormone IAA were also changed in the overexpressed PdeCML23–1 transgenic lines. According to this study, we were able to provide a global view of gene regulation for poplar AR development. Moreover, we also observed the regulation of cytosolic Ca2+ concentration by PdeCML23–1, and this regulation were involved in AR development in poplar. We also predicted that PdeCML23–1 possibly regulates AR development by modulating IAA content in poplar.
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