Jianbo Xie

Population genomic analysis of gibberellin-responsive long non-coding RNAs in Populus

Long non-coding RNAs (lncRNAs) participate in a wide range of biological processes, but lncRNAs in plants remain largely unknown; in particular, we lack a systematic identification of plant lncRNAs involved in hormone responses. Moreover, allelic variation in lncRNAs remains poorly characterized at a large scale. Here, we conducted high-throughput RNA-sequencing of leaves from control and gibberellin (GA)-treated Populus tomentosa and identified 7655 reliably expressed lncRNAs. Among the 7655 lncRNAs, the levels of 410 lncRNAs changed in response to GA. Seven GA-responsive lncRNAs were predicted to be putative targets of 18 miRNAs, and one GA-responsive lncRNA (TCONS_00264314) was predicted to be a target mimic of ptc-miR6459b. Computational analysis predicted 939 potential cis-regulated target genes and 965 potential trans-regulated target genes for GA-responsive lncRNAs. Functional annotation of these potential target genes showed that they participate in many different biological processes, including auxin signal transduction and synthesis of cellulose and pectin, indicating that GA-responsive lncRNAs may influence growth and wood properties. Finally, single nucleotide polymorphism (SNP)-based association analysis showed that 112 SNPs from 52 GA-responsive lncRNAs and 1014 SNPs from 296 potential target genes were significantly associated with growth and wood properties. Epistasis analysis also provided evidence for interactions between lncRNAs and their potential target genes. Our study provides a comprehensive view of P. tomentosa lncRNAs and offers insights into the potential functions and regulatory interactions of GA-responsive lncRNAs, thus forming the foundation for future functional analysis of GA-responsive lncRNAs in P. tomentosa.

Adaptive evolution and functional innovation of Populus‐specific recently evolved microRNAs

Lineage-specific microRNAs (miRNAs) undergo rapid turnover during evolution; however, their origin and functional importance have remained controversial. Here, we examine the origin, evolution, and potential roles in local adaptation of Populus-specific miRNAs, which originated after the recent salicoid-specific, whole-genome duplication. RNA sequencing was used to generate extensive, comparable miRNA and gene expression data for six tissues. A natural population of Populusxa0trichocarpa and closely related species were used to study the divergence rates, evolution, and adaptive variation of miRNAs. MiRNAs that originated in 5 untranslated regions had higher expression levels and their expression showed high correlation with their host genes. Compared with conserved miRNAs, a significantly higher proportion of Populus-specific miRNAs appear to target genes that were duplicated in salicoids. Examination of single nucleotide polymorphisms in Populus-specific miRNA precursors showed high amounts of population differentiation. We also characterized the newly emerged MIR6445 family, which could trigger the production of phased small interfering RNAs from NAC mRNAs, which encode a transcription factor with primary roles in a variety of plant developmental processes. Together, these observations provide evolutionary insights into the birth and potential roles of Populus-specific miRNAs in genome maintenance, local adaptation, and functional innovation.

Association genetics and transcriptome analysis reveal a gibberellin-responsive pathway involved in regulating photosynthesis

Gibberellins (GAs) regulate a wide range of important processes in plant growth and development, including photosynthesis. However, the mechanism by which GAs regulate photosynthesis remains to be understood. Here, we used multi-gene association to investigate the effect of genes in the GA-responsive pathway, as constructed by RNA sequencing, on photosynthesis, growth, and wood property traits, in a population of 435 Populus tomentosa By analyzing changes in the transcriptome following GA treatment, we identified many key photosynthetic genes, in agreement with the observed increase in measurements of photosynthesis. Regulatory motif enrichment analysis revealed that 37 differentially expressed genes related to photosynthesis shared two essential GA-related cis-regulatory elements, the GA response element and the pyrimidine box. Thus, we constructed a GA-responsive pathway consisting of 47 genes involved in regulating photosynthesis, including GID1, RGA, GID2, MYBGa, and 37 photosynthetic differentially expressed genes. Single nucleotide polymorphism (SNP)-based association analysis showed that 142 SNPs, representing 40 candidate genes in this pathway, were significantly associated with photosynthesis, growth, and wood property traits. Epistasis analysis uncovered interactions between 310 SNP-SNP pairs from 37 genes in this pathway, revealing possible genetic interactions. Moreover, a structural gene-gene matrix based on a time-course of transcript abundances provided a better understanding of the multi-gene pathway affecting photosynthesis. The results imply a functional role for these genes in mediating photosynthesis, growth, and wood properties, demonstrating the potential of combining transcriptome-based regulatory pathway construction and genetic association approaches to detect the complex genetic networks underlying quantitative traits.

Poplar CBF1 functions specifically in an integrated cold regulatory network

The C-repeat binding factors (CBFs), also termed dehydration-responsive element-binding protein 1 (DREB1) family members, play crucial roles in the acquisition of stress tolerance, but in trees, the underlying mechanisms of stress tolerance remain elusive. To gain insight into these mechanisms, we isolated five CBF1 orthologs from four poplar sections (Populus spp.) and assessed their expression under drought, cold, heat and salt stress conditions. Globally induced expression in response to cold suggested a correlation between poplar CBF1 expression and the acquisition of cold tolerance. Responses that varied between sections may reflect section-specific stress tolerance mechanisms, suggesting an effect of ecological context on the development of CBF1-mediated stress tolerance in poplar. We then used a genome-wide search strategy in Populus trichocarpa to predict 2263 putative CBF target genes; the identified genes participate in multiple biological processes and pathways. Almost all of the putative target genes contained multiple cis-acting elements that mediate responses to various environmental and endogenous signals, consistent with an important role of CBF1s in an integrated cold regulatory network. Finally, analysis of an association population of 528 individuals of Populus simonii identified six single-nucleotide polymorphisms (false discovery rate Q < 0.10) significantly (P < 0.005) associated with malondialdehyde production and electrolyte leakage, suggesting the potential importance of PsCBF1 in the regulation of some membrane-related functions. Our findings provide new insights into the function of PsCBF1 and shed light on the CBF-mediated regulatory network in poplar.

PDM3, a pentatricopeptide repeat-containing protein, affects chloroplast development

The chloroplast, as the photosynthetic organelle of plants, plays a crucial role in plant development. Extensive studies have been conducted on chloroplast development; however, the related regulatory mechanism still remains elusive. Here, we characterized a mutant with defective chloroplasts in Arabidopsis, termed pigment-defective mutant3 (pdm3), which exhibits a distinct albino phenotype in leaves, eventually leading to pdm3 seedling lethality under autotrophic growth conditions. Electron microscopy demonstrated that the number of thylakoids was reduced and the structure of those thylakoids was disrupted in the pdm3 mutant, which eventually led to the breakdown of chloroplasts. Sequence analysis showed that PDM3 encodes a chloroplast protein consisting of 12 pentratricopeptide repeat domains that belongs to the P subgroup. Both confocal microscopic analysis and immunoblotting in the chloroplast protein fraction showed that PDM3 was located in the stroma. Furthermore, analysis of the transcript profiles of chloroplast genes revealed that plastid-encoded polymerase-dependent transcript levels were markedly reduced, while nuclear-encoded polymerase-dependent transcript levels were increased in pdm3 mutants. In addition, we found that the splicing of introns in trnA, ndhB, and clpP-1 is also affected in pdm3. Taken together, we propose that PDM3 plays an essential role in chloroplast development in Arabidopsis.

Genetic variations and miRNA-target interactions contribute to natural phenotypic variations in Populus.

Variation in regulatory factors, including microRNAs (miRNAs), contributes to variation in quantitative and complex traits. However, in plants, variants in miRNAs and their target genes that contribute to natural phenotypic variation, and the underlying regulatory networks, remain poorly characterized. We investigated the associations and interactions of single-nucleotide polymorphisms (SNPs) in miRNAs and their target genes with phenotypes in 435 individuals from a natural population of Populus. We used RNA-seq to identify 217 miRNAs differentially expressed in a tension wood system, and identified 1196 candidate target genes; degradome sequencing confirmed 60 of the target sites. In addition, 72 miRNA-target pairs showed significant co-expression. Gene ontology (GO) term analysis showed that most of the genes in the co-regulated pairs participate in biological regulation. Genome resequencing found 5383 common SNPs (frequencyxa0≥xa00.05) in 139 miRNAs and 31xa0037 SNPs in 819 target genes. Single-SNP association analyses identified 232 significant associations between wood traits (Pxa0≤xa00.05) and SNPs in 102 miRNAs and 1387 associations with 478 target genes. Among these, 102 miRNA-target pairs associated with the same traits. Multi-SNP associations found 102 epistatic pairs associated with traits. Furthermore, a reconstructed regulatory network contained 12 significantly co-expressed pairs, including eight miRNAs and nine targets associated with traits. Lastly, both expression and genetic association showed that miR156i, miR156j, miR396a and miR6445b were involved in the formation of tension wood. This study shows that variants in miRNAs and target genes contribute to natural phenotypic variation and annotated roles and interactions of miRNAs and their target genes by genetic association analysis.

The genetic regulatory network centered on Pto-Wuschela and its targets involved in wood formation revealed by association studies.

Transcription factors (TFs) regulate gene expression and can strongly affect phenotypes. However, few studies have examined TF variants and TF interactions with their targets in plants. Here, we used genetic association in 435 unrelated individuals of Populus tomentosa to explore the variants in Pto-Wuschela and its targets to decipher the genetic regulatory network of Pto-Wuschela. Our bioinformatics and co-expression analysis identified 53 genes with the motif TCACGTGA as putative targets of Pto-Wuschela. Single-marker association analysis showed that Pto-Wuschela was associated with wood properties, which is in agreement with the observation that it has higher expression in stem vascular tissues in Populus. Also, SNPs in the 53 targets were associated with growth or wood properties under additive or dominance effects, suggesting these genes and Pto-Wuschela may act in the same genetic pathways that affect variation in these quantitative traits. Epistasis analysis indicated that 75.5% of these genes directly or indirectly interacted Pto-Wuschela, revealing the coordinated genetic regulatory network formed by Pto-Wuschela and its targets. Thus, our study provides an alternative method for dissection of the interactions between a TF and its targets, which will strength our understanding of the regulatory roles of TFs in complex traits in plants.

Genetic variation in transcription factors and photosynthesis light-reaction genes regulates photosynthetic traits

Transcription factors (TFs) play crucial roles in regulating the production of the components required for photosynthesis; elucidating the mechanisms by which underlying genetic variation in TFs affects complex photosynthesis-related traits may improve our understanding of photosynthesis and identify ways to improve photosynthetic efficiency. Promoter analysis of 96 nuclear-encoded Populus tomentosa Carr. genes within this pathway revealed 47 motifs responsive to light, stress, hormones and organ-specific regulation, as well as 86 TFs that might bind these motifs. Using phenotype-genotype associations, we identified 244 single-nucleotide polymorphisms (SNPs) within 105 genes associated with 12 photosynthesis-related traits. Most (30.33%) of these SNPs were located in intronic regions and these SNPs explained 18.66% of the mean phenotypic variation in the photosynthesis-related traits. Additionally, expression quantitative trait loci (eQTL) mapping identified 216 eQTLs associated with 110 eGenes (genes regulated by eQTLs), explaining 14.12% of the variability of gene expression. The lead SNPs of 12.04% of the eQTLs also contributed to phenotypic variation. Among these, a SNP in zf-Dof 5.6 (G120_9287) affected photosynthesis by modulating the expression of a sub-regulatory network of eight other TFs, which in turn regulate 55 photosynthesis-related genes. Furthermore, epistasis analysis identified a large interacting network representing 732 SNP-SNP pairs, of which 354 were photosynthesis gene-TF pairs, emphasizing the important roles of TFs in affecting photosynthesis-related traits. We combined eQTL and epistasis analysis and found 32 TFs harboring eQTLs being epistatic to their targets (identified by eQTL analysis), of which 15 TFs were also associated with photosynthesis traits. We therefore constructed a schematic model of TFs involved in regulating the photosynthetic light reaction pathway. Taken together, our results provide insight into the genetic regulation of photosynthesis, and may drive progress in the marker-assisted selection of desirable P. tomentosa genotypes with more efficient photosynthesis.

Genetic architecture underlying the lignin biosynthesis pathway involves noncoding RNAs and transcription factors for growth and wood properties in Populus

Summary Lignin provides structural support in perennial woody plants and is a complex phenolic polymer derived from phenylpropanoid pathway. Lignin biosynthesis is regulated by coordinated networks involving transcription factors (TFs), microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). However, the genetic networks underlying the lignin biosynthesis pathway for tree growth and wood properties remain unknown. Here, we used association genetics (additive, dominant and epistasis) and expression quantitative trait nucleotide (eQTN) mapping to decipher the genetic networks for tree growth and wood properties in 435 unrelated individuals of Populus tomentosa. We detected 124 significant associations (P ≤ 6.89E‐05) for 10 growth and wood property traits using 30 265 single nucleotide polymorphisms from 203 lignin biosynthetic genes, 81 TF genes, 36 miRNA genes and 71 lncRNA loci, implying their common roles in wood formation. Epistasis analysis uncovered 745 significant pairwise interactions, which helped to construct proposed genetic networks of lignin biosynthesis pathway and found that these regulators might affect phenotypes by linking two lignin biosynthetic genes. eQTNs were used to interpret how causal genes contributed to phenotypes. Lastly, we investigated the possible functions of the genes encoding 4‐coumarate: CoA ligase and cinnamate‐4‐hydroxylase in wood traits using epistasis, eQTN mapping and enzymatic activity assays. Our study provides new insights into the lignin biosynthesis pathway in poplar and enables the novel genetic factors as biomarkers for facilitating genetic improvement of trees.

Time-specific and pleiotropic quantitative trait loci coordinately modulate stem growth in Populus

Summary In perennial woody plants, the coordinated increase of stem height and diameter during juvenile growth improves competitiveness (i.e. access to light); however, the factors underlying variation in stem growth remain unknown in trees. Here, we used linkage‐linkage disequilibrium (linkage‐LD) mapping to decipher the genetic architecture underlying three growth traits during juvenile stem growth. We used two Populus populations: a linkage mapping population comprising a full‐sib family of 1,200 progeny and an association mapping panel comprising 435 unrelated individuals from nearly the entire natural range of Populus tomentosa. We mapped 311 quantitative trait loci (QTL) for three growth traits at 12 timepoints to 42 regions in 17 linkage groups. Of these, 28 regions encompassing 233 QTL were annotated as 27 segmental homology regions (SHRs). Using SNPs identified by whole‐genome re‐sequencing of the 435‐member association mapping panel, we identified significant SNPs (P ≤ 9.4 × 10−7) within 27 SHRs that affect stem growth at nine timepoints with diverse additive and dominance patterns, and these SNPs exhibited complex allelic epistasis over the juvenile growth period. Nineteen genes linked to potential causative alleles that have time‐specific or pleiotropic effects, and mostly overlapped with significant signatures of selection within SHRs between climatic regions represented by the association mapping panel. Five genes with potential time‐specific effects showed species‐specific temporal expression profiles during the juvenile stages of stem growth in five representative Populus species. Our observations revealed the importance of considering temporal genetic basis of complex traits, which will facilitate the molecular design of tree ideotypes.