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.

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.

Comprehensive analysis of dynamic gene expression and investigation of the roles of hydrogen peroxide during adventitious rooting in poplar.

BACKGROUND: Adventitious roots (ARs) are roots that are generated from nonrooting tissues. ARs are usually produced both during normal development and in response to stress conditions, such as flooding, nutrient deprivation, heavy metal stress and wounding. The ability of plants to form ARs is a key trait that enables plant propagation, especially for most tree species. RESULTS: Here, the kinetics of AR formation in a tissue culture of a hybrid variety of poplar were investigated. AR formation mainly occurred during the first 8 days and both pre- and newly- formed primordia contributed to AR formation in poplar by histological study. RNA-Seq-based transcriptome analysis was performed for stem bases collected at 0, 2, 4, 6 and 8 days after excision (DAE). Based on the data, the expression patterns of 8 phytohormone-related genes were investigated, and their influences on AR formation were considered. Subsequent gene expression cluster analysis showed a number of biological processes involved in AR formation. Among these biological pathways, genes involved in H2O2 homeostasis showed enrichment in one cluster that was highly upregulated from DAE0 to DAE8. Pharmacological assay confirmed that an appropriate content of H2O2 in stem bases could accelerate the formation of ARs in poplar. CONCLUSIONS: Based on the results of this study, we were able to predict a regulatory network for 7 phytohormones that are involved in poplar AR formation. The influence of H2O2 on AR formation was also confirmed. These results enhance our understanding of the regulation of AR formation in tree species.

Integrated Transcriptome Analysis Reveals Plant Hormones Jasmonic Acid and Salicylic Acid Coordinate Growth and Defense Responses upon Fungal Infection in Poplar.

Plants have evolved a sophisticated system to respond to various stresses. Fungal attack or infection is one of the most important biotic stresses for most plants. During the defense response to fungal infection, the plant hormones jasmonic acid (JA) and salicylic acid (SA) play critical roles. Here, gene expression data on JA/SA treatments and Melampsora larici-populina (MLP) infection were generated. Integrated transcriptome analyses of these data were performed, and 943 genes in total were identified as common responsive genes (CRG). Gene ontology (GO) term analysis revealed that the genes from CRG are generally involved in the processes of stress responses, metabolism, and growth and development. The further cluster analysis of the CRG identified a set of core genes that are involved in the JA/SA-mediated response to fungal defense with distinct gene expression profiles upon JA/SA treatment, which highlighted the different effects of these two hormones on plant fungal defenses. The modifications of several pathways relative to metabolism, biotic stress, and plant hormone signal pathways suggest the possible roles of JA/SA on the regulation of growth and defense responses. Co-expression modules (CMs) were also constructed using the poplar expression data on JA, SA, M. larici-populina, Septoria musiva, and Marssonina brunnea treatment or infection. A total of 23 CMs were constructed, and different CMs clearly exhibited distinct biological functions, which conformably regulated the concerted processes in response to fungal defense. Furthermore, the GO term analysis of different CMs confirmed the roles of JA and SA in regulating growth and defense responses, and their expression profiles suggested that the growth ability was reduced when poplar deployed defense responses. Several transcription factors (TFs) among the CRG in the co-expression network were proposed as hub genes in regulating these processes. According to this study, our data finely uncovered the possible roles of JA/SA in regulating the balance between growth and defense responses by integrating multiple hormone signaling pathways. We were also able to provide more knowledge on how the plant hormones JA/SA are involved in the regulation of the balance between growth and plant defense.

Construction of a high-density genetic map and its application for leaf shape QTL mapping in poplar.

MAIN CONCLUSION: High-quality and dense genetic maps were constructed, and leaf shape variation was dissected by QTL mapping in poplar. Species in the genus Populus, also known as poplars, are important woody species and considered model plants for perennial trees. High-density genetic maps are valuable genomic resources for population genetics. Here, we generated a high-quality and dense genetic map for an F1 poplar population using high-throughput NGS-based genotyping. A total of 92,097 high-quality SNP markers were developed by stringent filtering and identification. In total, 889 and 1650 SNPs formed the female and male genetic maps, respectively. To test the application of the genetic maps, QTL mapping of leaf shape was conducted for this F1 population. A total of nine parameters were scored for leaf shape variation in three different environments. Combining genetic maps and measurements of the nine leaf shape parameters, we mapped a total of 42 significant QTLs. The highest LOD score of all QTLs was 9.2, and that QTL explained the most (15.13%) trait variation. A total of nine QTLs could be detected in at least two environments, and they were located in two genomic regions. Within these two QTL regions, some candidate genes for regulating leaf shape were predicted through functional annotation. The successful mapping of leaf shape QTLs demonstrated the utility of our genetic maps. According to the performance of this study, we were able to provide high-quality and dense genetic maps and dissect the leaf shape variation in poplar.