The Over-Expression of Two R2R3-MYB Genes, PdMYB2R089 and PdMYB2R151, Increases the Drought-Resistant Capacity of Transgenic Arabidopsis.

The R2R3-MYB genes in plants play an essential role in the drought-responsive signaling pathway. Plenty of R2R3-MYB S21 and S22 subgroup genes in Arabidopsis have been implicated in dehydration conditions, yet few have been covered in terms of the role of the S21 and S22 subgroup genes in poplar under drought. PdMYB2R089 and PdMYB2R151 genes, respectively belonging to the S21 and S22 subgroups of NL895 (Populus deltoides × P. euramericana cv. 'Nanlin895'), were selected based on the previous expression analysis of poplar R2R3-MYB genes that are responsive to dehydration. The regulatory functions of two target genes in plant responses to drought stress were studied and speculated through the genetic transformation of Arabidopsis thaliana. PdMYB2R089 and PdMYB2R151 could promote the closure of stomata in leaves, lessen the production of malondialdehyde (MDA), enhance the activity of the peroxidase (POD) enzyme, and shorten the life cycle of transgenic plants, in part owing to their similar conserved domains. Moreover, PdMYB2R089 could strengthen root length and lateral root growth. These results suggest that PdMYB2R089 and PdMYB2R151 genes might have the potential to improve drought adaptability in plants. In addition, PdMYB2R151 could significantly improve the seed germination rate of transgenic Arabidopsis, but PdMYB2R089 could not. This finding provides a clue for the subsequent functional dissection of S21 and S22 subgroup genes in poplar that is responsive to drought.

Comprehensive Genome-Wide Analyses of Poplar R2R3-MYB Transcription Factors and Tissue-Specific Expression Patterns under Drought Stress.

R2R3-type MYB transcription factors are implicated in drought stress, which is a primary factor limiting the growth and development of woody plants. The identification of R2R3-MYB genes in the Populus trichocarpa genome has been previously reported. Nevertheless, the diversity and complexity of the conserved domain of the MYB gene caused inconsistencies in these identification results. There is still a lack of drought-responsive expression patterns and functional studies of R2R3-MYB transcription factors in Populus species. In this study, we identified a total of 210 R2R3-MYB genes in the P. trichocarpa genome, of which 207 genes were unevenly distributed across all 19 chromosomes. These poplar R2R3-MYB genes were phylogenetically divided into 23 subgroups. Collinear analysis demonstrated that the poplar R2R3-MYB genes underwent rapid expansion and that whole-genome duplication events were a dominant factor in the process of rapid gene expansion. Subcellular localization assays indicated that poplar R2R3-MYB TFs mainly played a transcriptional regulatory role in the nucleus. Ten R2R3-MYB genes were cloned from P. deltoides × P. euramericana cv. Nanlin895, and their expression patterns were tissue-specific. A majority of the genes showed similar drought-responsive expression patterns in two out of three tissues. This study provides a valid cue for further functional characterization of drought-responsive R2R3-MYB genes in poplar and provides support for the development of new poplar genotypes with elevated drought tolerance.

Genome-Wide Characterization and Evolutionary Expansion of Poplar NAC Transcription Factors and Their Tissue-Specific Expression Profiles under Drought.

The NAC (NAM, ATAF1/2 and CUC2) is a large gene family of plant-specific transcription factors that play a pivotal role in various physiological processes and abiotic stresses. Due to the lack of genome-wide characterization, intraspecific and interspecific synteny, and drought-responsive expression pattern of NAC genes in poplar, the functional characterization of drought-related NAC genes have been scarcely reported in Populus species. Here, we identified a total of 170 NAC domain-containing genes in the P. trichocarpa genome, 169 of which were unevenly distributed on its nineteen chromosomes. These NAC genes were phylogenetically divided into twenty subgroups, some of which exhibited a similar pattern of exon-intron architecture. The synteny and Ka/Ks analysis indicated that the expansion of NAC genes in poplar was mainly due to gene duplication events occurring before and after the divergence of Populus and Salix. Ten PdNAC (P. deltoids × P. euramericana cv.'Nanlin895') genes were randomly selected and cloned. Their drought-responsive expression profiles showed a tissue-specific pattern. The transcription factor PdNAC013 was verified to be localized in the nucleus. Our research results provide genomic information for the expansion of NAC genes in the poplar genome, and for further characterizing putative poplar NAC genes associated with water-deficit.

Study of spontaneous mutations in the transmission of poplar chloroplast genomes from mother to offspring.

BACKGROUND: Chloroplasts have their own genomes, independent from nuclear genomes, that play vital roles in growth, which is a major targeted trait for genetic improvement in Populus. Angiosperm chloroplast genomes are maternally inherited, but the chloroplast' variation pattern of poplar at the single-base level during the transmission from mother to offspring remains unknown. RESULTS: Here, we constructed high-quality and almost complete chloroplast genomes for three poplar clones, 'NL895' and its parents, 'I69' and 'I45', from the short-read datasets using multi-pass sequencing (15-16 times per clone) and ultra-high coverage (at least 8500× per clone), with the four-step strategy of Simulation-Assembly-Merging-Correction. Each of the three resulting chloroplast assemblies contained contigs covering > 99% of Populus trichocarpa chloroplast DNA as a reference. A total of 401 variant loci were identified by a hybrid strategy of genome comparison-based and mapping-based single nucleotide polymorphism calling. The genotypes of 94 variant loci were different among the three poplar clones. However, only 1 of the 94 loci was a missense mutation, which was located in the exon region of rpoC1 encoding the ß' subunit of plastid-encoded RNA polymerase. The genotype of the loci in NL895 and its female parent (I69) was different from that of its male parent (I45). CONCLUSIONS: This research provides resources for further chloroplast genomic studies of a F1 full-sibling family derived from a cross between I69 and I45, and will improve the application of chloroplast genomic information in modern Populus breeding programs.

Two-stage identification of SNP effects on dynamic poplar growth.

This project proposes an approach to identify significant single nucleotide polymorphism (SNP) effects, both additive and dominant, on the dynamic growth of poplar in diameter and height. The annual changes in yearly phenotypes based on regular observation periods are considered to represent multiple responses. In total 156,362 candidate SNPs are studied, and the phenotypes of 64 poplar trees are recorded. To address this ultrahigh dimensionality issue, this paper adopts a two-stage approach. First, the conventional genome-wide association studies (GWAS) and the distance correlation sure independence screening (DC-SIS) methods (Li et al., 2012) were combined to reduce the model dimensions at the sample size; second, a grouped penalized regression was applied to further refine the model and choose the final sparse SNPs. The multiple response issue was also carefully addressed. The SNP effects on the dynamic diameter and height growth patterns of poplar were systematically analyzed. In addition, a series of intensive simulation studies was performed to validate the proposed approach.

How trees allocate carbon for optimal growth: insight from a game-theoretic model.

How trees allocate photosynthetic products to primary height growth and secondary radial growth reflects their capacity to best use environmental resources. Despite substantial efforts to explore tree height-diameter relationship empirically and through theoretical modeling, our understanding of the biological mechanisms that govern this phenomenon is still limited. By thinking of stem woody biomass production as an ecological system of apical and lateral growth components, we implement game theory to model and discern how these two components cooperate symbiotically with each other or compete for resources to determine the size of a tree stem. This resulting allometry game theory is further embedded within a genetic mapping and association paradigm, allowing the genetic loci mediating the carbon allocation of stemwood growth to be characterized and mapped throughout the genome. Allometry game theory was validated by analyzing a mapping data of stem height and diameter growth over perennial seasons in a poplar tree. Several key quantitative trait loci were found to interpret the process and pattern of stemwood growth through regulating the ecological interactions of stem apical and lateral growth. The application of allometry game theory enables the prediction of the situations in which the cooperation, competition or altruism is an optimal decision of a tree to fully use the environmental resources it owns.

Molecular cloning, expression analysis and subcellular localization of four DELLA genes from hybrid poplar.

Gibberellic acid (GA) signaling regulates diverse aspects of plant growth and developmental processes. The DELLA repressors of GA signaling are named for an N-terminal conserved DELLA domain. In this study, four genes encoding DELLA proteins, PeRGA1, PeRGA2, PeGAI1 and PeGAI2, were isolated and characterized in poplar. A gene structural analysis revealed that the DELLA genes were all intron-free. Multiple protein sequence alignments revealed that these proteins contained seven highly conserved domains: the DELLA domain, the TVHYNP domain, leucine heptad repeat I (LHR I), the VHIID domain, leucine heptad repeat II (LHR II), the PFYRE domain, and the SAM domain. Temporal expression patterns of these genes were profiled during the adventitious root development of poplar. The four DELLA genes were expressed in root, stem and leaf in a dynamic manner. The subcellular localization demonstrated that these DELLA genes were mainly localized to the nucleus. These results suggest that the four DELLA genes may play diverse regulatory roles in the adventitious root, stem and leaf development of poplar, and contribute to improving our understanding of conserved and divergent aspects of DELLA proteins that restrain GA signaling in various species.

Computational identification of genes modulating stem height-diameter allometry.

The developmental variation in stem height with respect to stem diameter is related to a broad range of ecological and evolutionary phenomena in trees, but the underlying genetic basis of this variation remains elusive. We implement a dynamic statistical model, functional mapping, to formulate a general procedure for the computational identification of quantitative trait loci (QTLs) that control stem height-diameter allometry during development. Functional mapping integrates the biological principles underlying trait formation and development into the association analysis of DNA genotype and endpoint phenotype, thus providing an incentive for understanding the mechanistic interplay between genes and development. Built on the basic tenet of functional mapping, we explore two core ecological scenarios of how stem height and stem diameter covary in response to environmental stimuli: (i) trees pioneer sunlit space by allocating more growth to stem height than diameter and (ii) trees maintain their competitive advantage through an inverse pattern. The model is equipped to characterize 'pioneering' QTLs (piQTLs) and 'maintaining' QTLs (miQTLs) which modulate these two ecological scenarios, respectively. In a practical application to a mapping population of full-sib hybrids derived from two Populus species, the model has well proven its versatility by identifying several piQTLs that promote height growth at a cost of diameter growth and several miQTLs that benefit radial growth at a cost of height growth. Judicious application of functional mapping may lead to improved strategies for studying the genetic control of the formation mechanisms underlying trade-offs among quantities of assimilates allocated to different growth parts.

A computational framework for mapping the timing of vegetative phase change.

Phase change plays a prominent role in determining the form of growth and development. Although considerable attention has been focused on identifying the regulatory control mechanisms of phase change, a detailed understanding of the genetic architecture of this phenomenon is still lacking. We address this issue by deriving a computational model. The model is founded on the framework of functional mapping aimed at characterizing the interplay between quantitative trait loci (QTLs) and development through biologically meaningful mathematical equations. A multiphasic growth equation was implemented into functional mapping, which, via a series of hypothesis tests, allows the quantification of how QTLs regulate the timing and pattern of vegetative phase transition between independently regulated, temporally coordinated processes. The model was applied to analyze stem radial growth data of an interspecific hybrid family derived from two Populus species during the first 24 yr of ontogeny. Several key QTLs related to phase change have been characterized, most of which were observed to be in the adjacent regions of candidate genes. The identification of phase transition QTLs, whose expression is regulated by endogenous and environmental signals, may enhance our understanding of the evolution of development in changing environments.

A Comprehensive Analysis of the Transcriptomes of Marssonina brunnea and Infected Poplar Leaves to Capture Vital Events in Host-Pathogen Interactions.

BACKGROUND: Understanding host-pathogen interaction mechanisms helps to elucidate the entire infection process and focus on important events, and it is a promising approach for improvement of disease control and selection of treatment strategy. Time-course host-pathogen transcriptome analyses and network inference have been applied to unravel the direct or indirect relationships of gene expression alterations. However, time series analyses can suffer from absent time points due to technical problems such as RNA degradation, which limits the application of algorithms that require strict sequential sampling. Here, we introduce an efficient method using independence test to infer an independent network that is exclusively concerned with the frequency of gene expression changes. RESULTS: Highly resistant NL895 poplar leaves and weakly resistant NL214 leaves were infected with highly active and weakly active Marssonina brunnea, respectively, and were harvested at different time points. The independent network inference illustrated the top 1,000 vital fungus-poplar relationships, which contained 768 fungal genes and 54 poplar genes. These genes could be classified into three categories: a fungal gene surrounded by many poplar genes; a poplar gene connected to many fungal genes; and other genes (possessing low degrees of connectivity). Notably, the fungal gene M6_08342 (a metalloprotease) was connected to 10 poplar genes, particularly including two disease-resistance genes. These core genes, which are surrounded by other genes, may be of particular importance in complicated infection processes and worthy of further investigation. CONCLUSIONS: We provide a clear framework of the interaction network and identify a number of candidate key effectors in this process, which might assist in functional tests, resistant clone selection, and disease control in the future.

Two WUSCHEL-related HOMEOBOX genes, PeWOX11a and PeWOX11b, are involved in adventitious root formation of poplar.

The plant-specific WUSCHEL-related HOMEOBOX (WOX) transcription factors play important roles in key developmental processes, but knowledge regarding functional characterization of WOX genes in poplar remains limited. To reveal genes and signaling pathways associated with adventitious rooting in poplar, here we isolated and characterized two WOX genes through the rapid amplification of cDNA ends (RACE), sequence aligning, expression profiling, protoplast transfection and poplar transformation. Detailed information about the sequence similarity, structural features, evolutionary relationships, expression patterns and subcellular localization of the two genes were revealed. Overexpression of either PeWOX11a or PeWOX11b not only increased the number of adventitious roots on the cuttings but also induced ectopic roots in the aerial parts of transgenic poplars. Meanwhile, their overexpression in transgenic poplars affected axillary bud and leaf development. These results suggest that PeWOX11a and PeWOX11b were involved in multiple developmental processes of poplar, especially in adventitious root formation. Our results provide new insights into the molecular mechanisms underlying adventitious root formation of poplar.

Discovery of a novel small secreted protein family with conserved N-terminal IGY motif in Dikarya fungi.

BACKGROUND: Small secreted proteins (SSPs) are employed by plant pathogenic fungi as essential strategic tools for their successful colonization. SSPs are often species-specific and so far only a few widely phylogenetically distributed SSPs have been identified. RESULTS: A novel fungal SSP family consisting of 107 members was identified in the poplar tree fungal pathogen Marssonina brunnea, which accounts for over 17% of its secretome. We named these proteins IGY proteins (IGYPs) based on the conserved three amino acids at the N-terminus. In spite of overall low sequence similarity among IGYPs; they showed conserved N- and C-terminal motifs and a unified gene structure. By RT-PCR-seq, we analyzed the IGYP gene models and validated their expressions as active genes during infection. IGYP homologues were also found in 25 other Dikarya fungal species, all of which shared conserved motifs and the same gene structure. Furthermore, 18 IGYPs from 11 fungi also shared similar genomic contexts. Real-time RT-PCR showed that 8 MbIGYPs were highly expressed in the biotrophic stage. Interestingly, transient assay of 12 MbIGYPs showed that the MbIGYP13 protein induced cell death in resistant poplar clones. CONCLUSIONS: In total, 154 IGYPs in 26 fungi of the Dikarya subkingdom were discovered. Gene structure and genomic context analyses indicated that IGYPs originated from a common ancestor. In M. brunnea, the expansion of highly divergent MbIGYPs possibly is associated with plant-pathogen arms race.

Identification and expression analysis of twenty ARF genes in Populus.

The auxin response factor (ARF) family of transcription factors is a crucial component of auxin signaling and plays important roles regulating numerous growth and developmental processes in plants. We isolated and characterized 20 ARF genes involved in adventitious root development of Populus. Multiple protein sequence alignments revealed that the PeARF proteins contained a highly conserved region in their N-terminal portion corresponding to the DNA-binding domain of the Arabidopsis ARF family. Except for PeARF3.1, PeARF3.2, PeARF17.1 and PeARF17.2, the PeARF proteins contained a carboxyl-terminal domain related to the Arabidopsis domains III and IV, which are involved in homo- and heterodimerization. The exon-intron structures of the PeARF genes were determined by aligning cDNA and genomic sequences. As expected, most PeARF genes had a similar distribution of exon-intron structures. Temporal expression patterns of these genes were profiled during adventitious root development. All 20 PeARF genes were expressed in root, stem and leaf in a dynamic manner. Transient expression assays with Populus protoplasts demonstrated that these PeARFs were localized to the nucleus. These results suggest that PeARFs may play diverse regulatory roles in adventitious root development of Populus and contribute to improving our understanding of conserved and divergent aspects of auxin signaling in various species.

Untangling the transcriptome from fungus-infected plant tissues.

The development of sequencing technology allows low-cost generation of sequence data. The huge amount of raw sequence data now available has introduced many challenges associated with analysis of these large-scale data banks. For example, it is very important to distinguish materials of plant and fungal origin in fungus-infected plant tissue. The origin of transcripts that were sequenced from Library 895-M6 (poplar tissue infected by Marssonina brunnea) on Illumina/Solexa GA IIx was determined by combining three methods: (1) based on the taxonomic information of homologous sequences; (2) based on the reference genome sequence; (3) based on the transcriptome sequence of the host and its pathogen obtained from Library 895 (poplar) and Library M6 (M. brunnea) as well as Library 895-M6 (mixture of poplar and M. brunnea). We idenified accurately the origin of 80,978 (99.5%) contigs in the mixed poplar and M. brunnea sample (Library 895-M6) by integrating the results from the three methods. The results of this study demonstrate that a combination of these three approaches described here is an effective strategy for determining the origin of sequences in a mixed pool, and provides a basis for further transcriptome analysis of the mixed sample.

A quantitative model of transcriptional differentiation driving host-pathogen interactions.

Despite our expanding knowledge about the biochemistry of gene regulation involved in host-pathogen interactions, a quantitative understanding of this process at a transcriptional level is still limited. We devise and assess a computational framework that can address this question. This framework is founded on a mixture model-based likelihood, equipped with functionality to cluster genes per dynamic and functional changes of gene expression within an interconnected system composed of the host and pathogen. If genes from the host and pathogen are clustered in the same group due to a similar pattern of dynamic profiles, they are likely to be reciprocally co-evolving. If genes from the two organisms are clustered in different groups, this means that they experience strong host-pathogen interactions. The framework can test the rates of change for individual gene clusters during pathogenic infection and quantify their impacts on host-pathogen interactions. The framework was validated by a pathological study of poplar leaves infected by fungal Marssonina brunnea in which co-evolving and interactive genes that determine poplar-fungus interactions are identified. The new framework should find its wide application to studying host-pathogen interactions for any other interconnected systems.

Sequencing the genome of Marssonina brunnea reveals fungus-poplar co-evolution.

BACKGROUND: The fungus Marssonina brunnea is a causal pathogen of Marssonina leaf spot that devastates poplar plantations by defoliating susceptible trees before normal fall leaf drop. RESULTS: We sequence the genome of M. brunnea with a size of 52 Mb assembled into 89 scaffolds, representing the first sequenced Dermateaceae genome. By inoculating this fungus onto a poplar hybrid clone, we investigate how M. brunnea interacts and co-evolves with its host to colonize poplar leaves. While a handful of virulence genes in M. brunnea, mostly from the LysM family, are detected to up-regulate during infection, the poplar down-regulates its resistance genes, such as nucleotide binding site domains and leucine rich repeats, in response to infection. From 10,027 predicted proteins of M. brunnea in a comparison with those from poplar, we identify four poplar transferases that stimulate the host to resist M. brunnea. These transferas-encoding genes may have driven the co-evolution of M. brunnea and Populus during the process of infection and anti-infection. CONCLUSIONS: Our results from the draft sequence of the M. brunnea genome provide evidence for genome-genome interactions that play an important role in poplar-pathogen co-evolution. This knowledge could help to design effective strategies for controlling Marssonina leaf spot in poplar.

Overexpression of PeRHD3 alters the root architecture in Populus.

Adventitious rooting is essential for the vegetative propagation of economically important woody species. A better understanding of the genetic and physiological mechanisms that promote or hinder rooting will enhance the potential for successful commercial deployment of trees. ROOT HAIR DEFECTIVE 3 (RHD3), a large GTP-binding protein, is ubiquitously expressed in plants. Our previous microarray study identified differential expression patterns of genes belonging to the RHD3 family during adventitious root development from hardwood cuttings, and indicated that the RHD3 genes were involved in adventitious rooting in Populus. In this study, we cloned and characterized cDNAs of the two Populus RHD3 genes, designated as PeRHD3a and PeRHD3b. Transcripts encoded by the two genes were detected in roots, stems, leaves and petioles. To characterize the cellular functions of the genes, Agrobacterium tumifaciens was used to transform poplar with a vector that places expression of the target gene under the control of the strong constitutive promoter, Cauliflower Mosaic Virus 35S (Pro35S) promoter. Both PeRHD3a transgenic lines and PeRHD3b transgenic lines showed very similar phenotypic characteristics. Overexpression of PeRHD3a or PeRHD3b in poplar plants resulted in the formation of only a single prominent adventitious root with well-developed lateral roots, characteristic abnormalities in the root tip, and longer and more plentiful root hairs. These results imply that RHD3 may control adventitious and lateral root formation, as well as root hair development by regulating anisotropic cell expansion.

Development of polymorphic microsatellite markers in Camellia chekiangoleosa (Theaceae) using 454-ESTs.

PREMISE OF THE STUDY: A set of microsatellite markers for Camellia chekiangoleosa was developed and characterized using 454 sequencing technology to study the population genetic structure and the diversity of germplasm collections. METHODS AND RESULTS: Eighteen polymorphic microsatellite markers were identified and tested in 150 individuals from three natural populations of C. chekiangoleosa. Alleles numbered from two to seven, and the observed and expected heterozygosities ranged from 0.100 to 0.760 and 0.133 to 0.809, respectively. CONCLUSIONS: These markers will potentially be conducive to further genetic studies on C. chekiangoleosa.

Multiallelic epistatic model for an out-bred cross and mapping algorithm of interactive quantitative trait loci.

BACKGROUND: Genetic mapping has proven to be powerful for studying the genetic architecture of complex traits by characterizing a network of the underlying interacting quantitative trait loci (QTLs). Current statistical models for genetic mapping were mostly founded on the biallelic epistasis of QTLs, incapable of analyzing multiallelic QTLs and their interactions that are widespread in an outcrossing population. RESULTS: Here we have formulated a general framework to model and define the epistasis between multiallelic QTLs. Based on this framework, we have derived a statistical algorithm for the estimation and test of multiallelic epistasis between different QTLs in a full-sib family of outcrossing species. We used this algorithm to genomewide scan for the distribution of multiallelic epistasis for a rooting ability trait in an outbred cross derived from two heterozygous poplar trees. The results from simulation studies indicate that the positions and effects of multiallelic QTLs can well be estimated with a modest sample and heritability. CONCLUSIONS: The model and algorithm developed provide a useful tool for better characterizing the genetic control of complex traits in a heterozygous family derived from outcrossing species, such as forest trees, and thus fill a gap that occurs in genetic mapping of this group of important but underrepresented species.

Natural variation in petal color in Lycoris longituba revealed by anthocyanin components.

Lycoris longituba is one of the species belonging to the Amaryllidaceae family. Despite its limited distribution, endemic to central eastern China, this species displays an exceptionally wide diversity of flower colors from purple, red, orange, to yellow, in nature. We study the natural variation of floral color in L. longituba by testing the components of water-soluble vacuolar pigments--anthocyanins--in its petals using high-performance liquid chromatography coupled with photodiode array detection and electrospray ionization mass spectrometry. Four anthocyanins were identified, cyanidin-3-sophoroside (Cy3So), cyanidin-3-xylosylglucoside (Cy3XyGlc), cyanidin-3-sambubioside (Cy3Sa), and pelargonidin-3-xylosylglucoside (Pg3XyGlc), which occur at various amounts in L. longituba petals of different colors. A multivariate analysis was used to explore the relationship between pigments and flower color. Anthocyanins have been thought to play a major role in acting as a UV screen that protects the plant's DNA from sunlight damage and attracting insects for the purpose of pollination. Thus, knowledge about the content and type of anthocyanins determining the petal coloration of Lycoris longituba will help to study the adaptive evolution of flowers and provide useful information for the ornamental breeding of this species.