Phosphate (Pi) stress-responsive transcription factors PdeWRKY6 and PdeWRKY65 regulate the expression of PdePHT1;9 to modulate tissue Pi concentration in poplar.

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.

The transcription factor WRKY75 regulates the development of adventitious roots, lateral buds and callus by modulating hydrogen peroxide content in poplar.

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.

Comparative transcriptomic analysis uncovers conserved pathways involved in adventitious root formation in poplar.

Cutting propagation is widely used in establishing poplar plantations, and this approach requires efficient adventitious root (AR) forming capacities. Although poplar species are considered to form roots easily, interspecific variations in AR formation are still observed. To better understand the gene regulatory network underlying the conserved modified pathways that are essential for AR formation in poplar species, comparative transcriptomic approaches were applied to identify the conserved common genes that were differentially expressed during the AR formation processes in two poplar species (Populus × euramericana and P. simonii) in woody plant medium (WPM). A total of 2146 genes were identified as conserved genes that shared similar gene expression profiles in at least one comparison. These conserved genes were enriched in diverse hormone signaling pathways, as well as the mitogen-associated protein kinase (MAPK) signaling pathway, suggesting an important role for signaling transduction in coordinating external stimuli and endogenous physiological status during AR regulation in poplar. Furthermore, the co-expression network analysis of conserved genes allowed identification of several co-expressed modules (CM) that are co-expressed with distinct biological functions, for instance, CM1 was enriched in defense response and hormone signaling, CM2 and CM3 were overrepresented in defense response-related pathways and for cell cycle, respectively. These results suggest that the AR formation processes in poplar were finely tuned at the transcriptomic level by integrating multiple biological processes essential for AR formation. Our results suggest conserved machinery for AR formation in poplar and generated informative gene co-expression networks that describe the basis of AR formation in these species. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01054-7.

The bZIP53-IAA4 module inhibits adventitious root development in Populus.

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.

Combining QTL Mapping with Genome Resequencing Identifies an Indel in an R Gene that is Associated with Variation in Leaf Rust Disease Resistance in Poplar.

Poplar trees (Populus spp.) are important and are widely grown worldwide. However, the extensive occurrence of leaf rust disease caused by Melampsora spp. seriously inhibits their growth and reduces their biomass. In our previous study, a high-quality genetic map was constructed for the poplar F1 population I-69 × XYY by using next-generation sequencing-based genotyping-by-sequencing. Here, we collected phenotypic data on leaf rust disease resistance on three different dates for all 300 progenies of the F1 population. Combining a high-quality genetic map and phenotypic data, we were able to detect 11 major quantitative trait loci (QTLs) for leaf rust disease resistance. Among these 11 QTLs, two pairs were detected on at least two dates. In the corresponding genomic sequence, we found that resistance (R) gene clusters were located in these two QTL regions. By using genome resequencing, PCR confirmation and statistical analysis, a 611-bp deletion within an R gene in one QTL region was found to be associated with variation in leaf rust disease resistance. A PCR-based examination of this 611-bp deletion was performed. This 611-bp deletion was also found to affect mRNA splicing and form a new protein with the loss of some key protein domains. Based on this study, we were able to determine the genetic architecture of variation in poplar leaf rust disease resistance, and the 611-bp deletion in the R gene could be used as a diagnostic marker for future poplar molecular breeding.

Coexpression analysis of a large-scale transcriptome identified a calmodulin-like protein regulating the development of adventitious roots 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 regulate 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 indole acetic acid (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 was involved in AR development in poplar. We also predicted that PdeCML23-1 possibly regulates AR development by modulating IAA content in poplar.