FLOWERING LOCUS T duplication coordinates reproductive and vegetative growth in perennial poplar.

Annual plants grow vegetatively at early developmental stages and then transition to the reproductive stage, followed by senescence in the same year. In contrast, after successive years of vegetative growth at early ages, woody perennial shoot meristems begin repeated transitions between vegetative and reproductive growth at sexual maturity. However, it is unknown how these repeated transitions occur without a developmental conflict between vegetative and reproductive growth. We report that functionally diverged paralogs FLOWERING LOCUS T1 (FT1) and FLOWERING LOCUS T2 (FT2), products of whole-genome duplication and homologs of Arabidopsis thaliana gene FLOWERING LOCUS T (FT), coordinate the repeated cycles of vegetative and reproductive growth in woody perennial poplar (Populus spp.). Our manipulative physiological and genetic experiments coupled with field studies, expression profiling, and network analysis reveal that reproductive onset is determined by FT1 in response to winter temperatures, whereas vegetative growth and inhibition of bud set are promoted by FT2 in response to warm temperatures and long days in the growing season. The basis for functional differentiation between FT1 and FT2 appears to be expression pattern shifts, changes in proteins, and divergence in gene regulatory networks. Thus, temporal separation of reproductive onset and vegetative growth into different seasons via FT1 and FT2 provides seasonality and demonstrates the evolution of a complex perennial adaptive trait after genome duplication.

Characterization of the basal angiosperm Aristolochia fimbriata: a potential experimental system for genetic studies.

BACKGROUND: Previous studies in basal angiosperms have provided insight into the diversity within the angiosperm lineage and helped to polarize analyses of flowering plant evolution. However, there is still not an experimental system for genetic studies among basal angiosperms to facilitate comparative studies and functional investigation. It would be desirable to identify a basal angiosperm experimental system that possesses many of the features found in existing plant model systems (e.g., Arabidopsis and Oryza). RESULTS: We have considered all basal angiosperm families for general characteristics important for experimental systems, including availability to the scientific community, growth habit, and membership in a large basal angiosperm group that displays a wide spectrum of phenotypic diversity. Most basal angiosperms are woody or aquatic, thus are not well-suited for large scale cultivation, and were excluded. We further investigated members of Aristolochiaceae for ease of culture, life cycle, genome size, and chromosome number. We demonstrated self-compatibility for Aristolochia elegans and A. fimbriata, and transformation with a GFP reporter construct for Saruma henryi and A. fimbriata. Furthermore, A. fimbriata was easily cultivated with a life cycle of just three months, could be regenerated in a tissue culture system, and had one of the smallest genomes among basal angiosperms. An extensive multi-tissue EST dataset was produced for A. fimbriata that includes over 3.8 million 454 sequence reads. CONCLUSIONS: Aristolochia fimbriata has numerous features that facilitate genetic studies and is suggested as a potential model system for use with a wide variety of technologies. Emerging genetic and genomic tools for A. fimbriata and closely related species can aid the investigation of floral biology, developmental genetics, biochemical pathways important in plant-insect interactions as well as human health, and various other features present in early angiosperms.

Genome wide identification of chilling responsive microRNAs in Prunus persica.

BACKGROUND: MicroRNAs (miRNAs) are small RNAs (sRNAs) approximately 21 nucleotides in length that negatively control gene expression by cleaving or inhibiting the translation of target gene transcripts. Within this context, miRNAs and siRNAs are coming to the forefront as molecular mediators of gene regulation in plant responses to annual temperature cycling and cold stress. For this reason, we chose to identify and characterize the conserved and non-conserved miRNA component of peach (Prunus persica (L.) Batsch) focusing our efforts on both the recently released whole genome sequence of peach and sRNA transcriptome sequences from two tissues representing non-dormant leaves and dormant leaf buds. Conserved and non-conserved miRNAs, and their targets were identified. These sRNA resources were used to identify cold-responsive miRNAs whose gene targets co-localize with previously described QTLs for chilling requirement (CR). RESULTS: Analysis of 21 million peach sRNA reads allowed us to identify 157 and 230 conserved and non-conserved miRNA sequences. Among the non-conserved miRNAs, we identified 205 that seem to be specific to peach. Comparative genome analysis between peach and Arabidopsis showed that conserved miRNA families, with the exception of miR5021, are similar in size. Sixteen of these conserved miRNA families are deeply rooted in land plant phylogeny as they are present in mosses and/or lycophytes. Within the other conserved miRNA families, five families (miR1446, miR473, miR479, miR3629, and miR3627) were reported only in tree species (Populustrichocarpa, Citrus trifolia, and Prunus persica). Expression analysis identified several up-regulated or down-regulated miRNAs in winter buds versus young leaves. A search of the peach proteome allowed the prediction of target genes for most of the conserved miRNAs and a large fraction of non-conserved miRNAs. A fraction of predicted targets in peach have not been previously reported in other species. Several conserved and non-conserved miRNAs and miRNA-regulated genes co-localize with Quantitative Trait Loci (QTLs) for chilling requirement (CR-QTL) and bloom date (BD-QTL). CONCLUSIONS: In this work, we identified a large set of conserved and non-conserved miRNAs and describe their evolutionary footprint in angiosperm lineages. Several of these miRNAs were induced in winter buds and co-localized with QTLs for chilling requirement and bloom date thus making their gene targets potential candidates for mediating plant responses to cold stress. Several peach homologs of genes participating in the regulation of vernalization in Arabidopsis were identified as differentially expressed miRNAs targets, potentially linking these gene activities to cold responses in peach dormant buds. The non-conserved miRNAs may regulate cellular, physiological or developmental processes specific to peach and/or other tree species.

Chestnut resistance to the blight disease: insights from transcriptome analysis.

BACKGROUND: A century ago, Chestnut Blight Disease (CBD) devastated the American chestnut. Backcross breeding has been underway to introgress resistance from Chinese chestnut into surviving American chestnut genotypes. Development of genomic resources for the family Fagaceae, has focused in this project on Castanea mollissima Blume (Chinese chestnut) and Castanea dentata (Marsh.) Borkh (American chestnut) to aid in the backcross breeding effort and in the eventual identification of blight resistance genes through genomic sequencing and map based cloning. A previous study reported partial characterization of the transcriptomes from these two species. Here, further analyses of a larger dataset and assemblies including both 454 and capillary sequences were performed and defense related genes with differential transcript abundance (GDTA) in canker versus healthy stem tissues were identified. RESULTS: Over one and a half million cDNA reads were assembled into 34,800 transcript contigs from American chestnut and 48,335 transcript contigs from Chinese chestnut. Chestnut cDNA showed higher coding sequence similarity to genes in other woody plants than in herbaceous species. The number of genes tagged, the length of coding sequences, and the numbers of tagged members within gene families showed that the cDNA dataset provides a good resource for studying the American and Chinese chestnut transcriptomes. In silico analysis of transcript abundance identified hundreds of GDTA in canker versus healthy stem tissues. A significant number of additional DTA genes involved in the defense-response not reported in a previous study were identified here. These DTA genes belong to various pathways involving cell wall biosynthesis, reactive oxygen species (ROS), salicylic acid (SA), ethylene, jasmonic acid (JA), abscissic acid (ABA), and hormone signalling. DTA genes were also identified in the hypersensitive response and programmed cell death (PCD) pathways. These DTA genes are candidates for host resistance to the chestnut blight fungus, Cryphonectria parasitica. CONCLUSIONS: Our data allowed the identification of many genes and gene network candidates for host resistance to the chestnut blight fungus, Cryphonectria parasitica. The similar set of GDTAs in American chestnut and Chinese chestnut suggests that the variation in sensitivity to this pathogen between these species may be the result of different timing and amplitude of the response of the two to the pathogen infection. Resources developed in this study are useful for functional genomics, comparative genomics, resistance breeding and phylogenetics in the Fagaceae.

Phylogeny and expression profiling of CAD and CAD-like genes in hybrid Populus (P. deltoides x P. nigra): evidence from herbivore damage for subfunctionalization and functional divergence.

BACKGROUND: Cinnamyl Alcohol Dehydrogenase (CAD) proteins function in lignin biosynthesis and play a critical role in wood development and plant defense against stresses. Previous phylogenetic studies did not include genes from seedless plants and did not reflect the deep evolutionary history of this gene family. We reanalyzed the phylogeny of CAD and CAD-like genes using a representative dataset including lycophyte and bryophyte sequences. Many CAD/CAD-like genes do not seem to be associated with wood development under normal growth conditions. To gain insight into the functional evolution of CAD/CAD-like genes, we analyzed their expression in Populus plant tissues in response to feeding damage by gypsy moth larvae (Lymantria dispar L.). Expression of CAD/CAD-like genes in Populus tissues (xylem, leaves, and barks) was analyzed in herbivore-treated and non-treated plants by real time quantitative RT-PCR. RESULTS: CAD family genes were distributed in three classes based on sequence conservation. All the three classes are represented by seedless as well as seed plants, including the class of bona fide lignin pathway genes. The expression of some CAD/CAD-like genes that are not associated with xylem development were induced following herbivore damage in leaves, while other genes were induced in only bark or xylem tissues. Five of the CAD/CAD-like genes, however, showed a shift in expression from one tissue to another between non-treated and herbivore-treated plants. Systemic expression of the CAD/CAD-like genes was generally suppressed. CONCLUSIONS: Our results indicated a correlation between the evolution of the CAD gene family and lignin and that the three classes of genes may have evolved in the ancestor of land plants. Our results also suggest that the CAD/CAD-like genes have evolved a diversity of expression profiles and potentially different functions, but that they are nonetheless co-regulated under stress conditions.

Comparison of gene order of GIGANTEA loci in yellow-poplar, monocots, and eudicots.

GIGANTEA plays an important role in the control of circadian rhythms and photoperiodic flowering. The GIGANTEA gene has been studied in various species, but not in basal angiosperms. Moreover, to the best of our knowledge, no study of the genome organization of a basal angiosperm has yet been published. In this study, we sequenced a bacterial artificial chromosome (BAC) harboring GIGANTEA from yellow-poplar (Liriodendron tulipifera L.) and compared the genomic organization of this gene in yellow-poplar with that in other species from various angiosperm clades. This is the first report on the gene structure and organization of a large contig in any basal angiosperm species. The BAC clone, covering a region of approximately 122 kb from the yellow-poplar genome, was sequenced and assembled by coupling the 454 pyrosequencing technology with ABI capillary sequencing. In addition to GIGANTEA, the gene RPS18.A (encoding ribosomal protein S18.A) was found in this segment of the genome. We found that gene content and order in this region of the yellow-poplar genome were similar to those in the corresponding region in eudicots but not in Oryza sativa and Sorghum bicolor, implying that clustering of the GIGANTEA and RPS18.A genes is ancestral and separation of the genes occurred after the phylogenetic split of monocots from dicots. Phylogenetic analysis of GIGANTEA amino acid sequences placed yellow-poplar closer to eudicots than to monocots. In addition, evidence for transposition and large insertions and duplications was found, suggesting multiple and complex mechanisms of basal angiosperm genome evolution.

Comparison of next generation sequencing technologies for transcriptome characterization.

BACKGROUND: We have developed a simulation approach to help determine the optimal mixture of sequencing methods for most complete and cost effective transcriptome sequencing. We compared simulation results for traditional capillary sequencing with "Next Generation" (NG) ultra high-throughput technologies. The simulation model was parameterized using mappings of 130,000 cDNA sequence reads to the Arabidopsis genome (NCBI Accession SRA008180.19). We also generated 454-GS20 sequences and de novo assemblies for the basal eudicot California poppy (Eschscholzia californica) and the magnoliid avocado (Persea americana) using a variety of methods for cDNA synthesis. RESULTS: The Arabidopsis reads tagged more than 15,000 genes, including new splice variants and extended UTR regions. Of the total 134,791 reads (13.8 MB), 119,518 (88.7%) mapped exactly to known exons, while 1,117 (0.8%) mapped to introns, 11,524 (8.6%) spanned annotated intron/exon boundaries, and 3,066 (2.3%) extended beyond the end of annotated UTRs. Sequence-based inference of relative gene expression levels correlated significantly with microarray data. As expected, NG sequencing of normalized libraries tagged more genes than non-normalized libraries, although non-normalized libraries yielded more full-length cDNA sequences. The Arabidopsis data were used to simulate additional rounds of NG and traditional EST sequencing, and various combinations of each. Our simulations suggest a combination of FLX and Solexa sequencing for optimal transcriptome coverage at modest cost. We have also developed ESTcalc http://fgp.huck.psu.edu/NG_Sims/ngsim.pl, an online webtool, which allows users to explore the results of this study by specifying individualized costs and sequencing characteristics. CONCLUSION: NG sequencing technologies are a highly flexible set of platforms that can be scaled to suit different project goals. In terms of sequence coverage alone, the NG sequencing is a dramatic advance over capillary-based sequencing, but NG sequencing also presents significant challenges in assembly and sequence accuracy due to short read lengths, method-specific sequencing errors, and the absence of physical clones. These problems may be overcome by hybrid sequencing strategies using a mixture of sequencing methodologies, by new assemblers, and by sequencing more deeply. Sequencing and microarray outcomes from multiple experiments suggest that our simulator will be useful for guiding NG transcriptome sequencing projects in a wide range of organisms.

The cinnamyl alcohol dehydrogenase gene family in Populus: phylogeny, organization, and expression.

BACKGROUND: Lignin is a phenolic heteropolymer in secondary cell walls that plays a major role in the development of plants and their defense against pathogens. The biosynthesis of monolignols, which represent the main component of lignin involves many enzymes. The cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis as it catalyzes the final step in the synthesis of monolignols. The CAD gene family has been studied in Arabidopsis thaliana, Oryza sativa and partially in Populus. This is the first comprehensive study on the CAD gene family in woody plants including genome organization, gene structure, phylogeny across land plant lineages, and expression profiling in Populus. RESULTS: The phylogenetic analyses showed that CAD genes fall into three main classes (clades), one of which is represented by CAD sequences from gymnosperms and angiosperms. The other two clades are represented by sequences only from angiosperms. All Populus CAD genes, except PoptrCAD 4 are distributed in Class II and Class III. CAD genes associated with xylem development (PoptrCAD 4 and PoptrCAD 10) belong to Class I and Class II. Most of the CAD genes are physically distributed on duplicated blocks and are still in conserved locations on the homeologous duplicated blocks. Promoter analysis of CAD genes revealed several motifs involved in gene expression modulation under various biological and physiological processes. The CAD genes showed different expression patterns in poplar with only two genes preferentially expressed in xylem tissues during lignin biosynthesis. CONCLUSION: The phylogeny of CAD genes suggests that the radiation of this gene family may have occurred in the early ancestry of angiosperms. Gene distribution on the chromosomes of Populus showed that both large scale and tandem duplications contributed significantly to the CAD gene family expansion. The duplication of several CAD genes seems to be associated with a genome duplication event that happened in the ancestor of Salicaceae. Phylogenetic analyses associated with expression profiling and results from previous studies suggest that CAD genes involved in wood development belong to Class I and Class II. The other CAD genes from Class II and Class III may function in plant tissues under biotic stresses. The conservation of most duplicated CAD genes, the differential distribution of motifs in their promoter regions, and the divergence of their expression profiles in various tissues of Populus plants indicate that genes in the CAD family have evolved tissue-specialized expression profiles and may have divergent functions.

Comparison of the transcriptomes of American chestnut (Castanea dentata) and Chinese chestnut (Castanea mollissima) in response to the chestnut blight infection.

UNLABELLED: BACKGROUND1471-2229-9-51: American chestnut (Castanea dentata) was devastated by an exotic pathogen in the beginning of the twentieth century. This chestnut blight is caused by Cryphonectria parasitica, a fungus that infects stem tissues and kills the trees by girdling them. Because of the great economic and ecological value of this species, significant efforts have been made over the century to combat this disease, but it wasn't until recently that a focused genomics approach was initiated. Prior to the Genomic Tool Development for the Fagaceae project, genomic resources available in public databases for this species were limited to a few hundred ESTs. To identify genes involved in resistance to C. parasitica, we have sequenced the transcriptome from fungal infected and healthy stem tissues collected from blight-sensitive American chestnut and blight-resistant Chinese chestnut (Castanea mollissima) trees using ultra high throughput pyrosequencing. RESULTS: We produced over a million 454 reads, totaling over 250 million bp, from which we generated 40,039 and 28,890 unigenes in total from C. mollissima and C. dentata respectively. The functions of the unigenes, from GO annotation, cover a diverse set of molecular functions and biological processes, among which we identified a large number of genes associated with resistance to stresses and response to biotic stimuli. In silico expression analyses showed that many of the stress response unigenes were expressed more in canker tissues versus healthy stem tissues in both American and Chinese chestnut. Comparative analysis also identified genes belonging to different pathways of plant defense against biotic stresses that are differentially expressed in either American or Chinese chestnut canker tissues. CONCLUSION: Our study resulted in the identification of a large set of cDNA unigenes from American chestnut and Chinese chestnut. The ESTs and unigenes from this study constitute an important resource to the scientific community interested in the discovery of genes involved in various biological processes in Chestnut and other species. The identification of many defense-related genes differentially expressed in canker vs. healthy stem in chestnuts provides many new candidate genes for developing resistance to the chestnut blight and for studying pathways involved in responses of trees to necrotrophic pathogens. We also identified several candidate genes that may underline the difference in resistance to Cryphonectria parasitica between American chestnut and Chinese chestnut.

Genome reorganization of the GmSHMT gene family in soybean showed a lack of functional redundancy in resistance to soybean cyst nematode.

In soybeans, eighteen members constitute the serine hydroxymethyltransferase (GmSHMT) gene family, of which the cytosolic-targeted GmSHMT08c member has been reported to mediate resistance to soybean cyst nematode (SCN). This work presents a comprehensive study of the SHMT gene family members, including synteny, phylogeny, subcellular localizations, haplotypes, protein homology modeling, mutational, and expression analyses. Phylogenetic analysis showed that SHMT genes are divided into four classes reflecting their subcellular distribution (cytosol, nucleus, mitochondrion, and chloroplast). Subcellular localization of selected GmSHMT members supports their in-silico predictions and phylogenetic distribution. Expression and functional analyses showed that GmSHMT genes display many overlapping, but some divergent responses during SCN infection. Furthermore, mutational analysis reveals that all isolated EMS mutants that lose their resistance to SCN carry missense and nonsense mutations at the GmSHMT08c, but none of the Gmshmt08c mutants carried mutations in the other GmSHMT genes. Haplotype clustering analysis using the whole genome resequencing data from a collection of 106 diverse soybean germplams (15X) was performed to identify allelic variants and haplotypes within the GmSHMT gene family. Interestingly, only the cytosolic-localized GmSHMT08c presented SNP clusters that were associated with SCN resistance, supporting our mutational analysis. Although eight GmSHMT members respond to the nematode infestation, functional and mutational analysis has shown the absence of functional redundancy in resistance to SCN. Structural analysis and protein homology modeling showed the presence of spontaneous mutations at important residues within the GmSHMT proteins, suggesting the presence of altered enzyme activities based on substrate affinities. Due to the accumulation of mutations during the evolution of the soybean genome, the other GmSHMT members have undergone neofunctionalization and subfunctionalization events.

Conservation and divergence of microRNAs in Populus.

BACKGROUND: MicroRNAs (miRNAs) are small RNAs (sRNA) approximately 21 nucleotides in length that negatively control gene expression by cleaving or inhibiting the translation of target gene transcripts. miRNAs have been extensively analyzed in Arabidopsis and rice and partially investigated in other non-model plant species. To date, 109 and 62 miRNA families have been identified in Arabidopsis and rice respectively. However, only 33 miRNAs have been identified from the genome of the model tree species (Populus trichocarpa), of which 11 are Populus specific. The low number of miRNA families previously identified in Populus, compared with the number of families identified in Arabidopsis and rice, suggests that many miRNAs still remain to be discovered in Populus. In this study, we analyzed expressed small RNAs from leaves and vegetative buds of Populus using high throughput pyrosequencing. RESULTS: Analysis of almost eighty thousand small RNA reads allowed us to identify 123 new sequences belonging to previously identified miRNA families as well as 48 new miRNA families that could be Populus-specific. Comparison of the organization of miRNA families in Populus, Arabidopsis and rice showed that miRNA family sizes were generally expanded in Populus. The putative targets of non-conserved miRNA include both previously identified targets as well as several new putative target genes involved in development, resistance to stress, and other cellular processes. Moreover, almost half of the genes predicted to be targeted by non-conserved miRNAs appear to be Populus-specific. Comparative analyses showed that genes targeted by conserved and non-conserved miRNAs are biased mainly towards development, electron transport and signal transduction processes. Similar results were found for non-conserved miRNAs from Arabidopsis. CONCLUSION: Our results suggest that while there is a conserved set of miRNAs among plant species, a large fraction of miRNAs vary among species. The non-conserved miRNAs may regulate cellular, physiological or developmental processes specific to the taxa that produce them, as appears likely to be the case for those miRNAs that have only been observed in Populus. Non-conserved and conserved miRNAs seem to target genes with similar biological functions indicating that similar selection pressures are acting on both types of miRNAs. The expansion in the number of most conserved miRNAs in Populus relative to Arabidopsis, may be linked to the recent genome duplication in Populus, the slow evolution of the Populus genome, or to differences in the selection pressure on duplicated miRNAs in these species.

Molecular evolutionary analyses of the Arabidopsis L7 ribosomal protein gene family.

Cytoplasmic ribosomal protein (r-protein) genes in Arabidopsis thaliana are encoded by 80 multigene families that contain between two and seven members. Gene family members are typically similar at the protein sequence level, with the most divergent members of any gene family retaining 94% identity, on average. However, three Arabidopsis r-protein families - S15a, L7 and P2 - contain highly divergent family members. Here, we investigated the organization, structure, expression and molecular evolution of the L7 r-protein family. Phylogenetic analyses showed that L7 r-protein gene family members constitute two distinct phylogenetic groups. The first group including RPL7B, RPL7C and RPL7D has homologs in plants, animals and fungi. The second group represented by RPL7A is found in plants but has no orthologs from other fully-sequenced eukaryotic genomes. These two groups may have derived from a duplication event prior to the divergence of animals and plants. All four L7 r-protein genes are expressed and all exhibit a differential expression in inflorescence and flowers. RPL7A and RPL7B are less expressed than the other genes in all tissues analyzed. Molecular characterization of nucleic and protein sequences of L7 r-protein genes and analysis of their codon usage did not indicate any functional divergence. The probable evolution of an extra-ribosomal function of group 2 genes is discussed.

Characterization of the Soluble NSF Attachment Protein gene family identifies two members involved in additive resistance to a plant pathogen.

Proteins with Tetratricopeptide-repeat (TPR) domains are encoded by large gene families and distributed in all plant lineages. In this study, the Soluble NSF-Attachment Protein (SNAP) subfamily of TPR containing proteins is characterized. In soybean, five members constitute the SNAP gene family: GmSNAP18, GmSNAP11, GmSNAP14, GmSNAP02, and GmSNAP09. Recently, GmSNAP18 has been reported to mediate resistance to soybean cyst nematode (SCN). Using a population of recombinant inbred lines from resistant and susceptible parents, the divergence of the SNAP gene family is analysed over time. Phylogenetic analysis of SNAP genes from 22 diverse plant species showed that SNAPs were distributed in six monophyletic clades corresponding to the major plant lineages. Conservation of the four TPR motifs in all species, including ancestral lineages, supports the hypothesis that SNAPs were duplicated and derived from a common ancestor and unique gene still present in chlorophytic algae. Syntenic analysis of regions harbouring GmSNAP genes in soybean reveals that this family expanded from segmental and tandem duplications following a tetraploidization event. qRT-PCR analysis of GmSNAPs indicates a co-regulation following SCN infection. Finally, genetic analysis demonstrates that GmSNAP11 contributes to an additive resistance to SCN. Thus, GmSNAP11 is identified as a novel minor gene conferring resistance to SCN.

Effect of Drought on Herbivore-Induced Plant Gene Expression: Population Comparison for Range Limit Inferences.

Low elevation "trailing edge" range margin populations typically face increases in both abiotic and biotic stressors that may contribute to range limit development. We hypothesize that selection may act on ABA and JA signaling pathways for more stable expression needed for range expansion, but that antagonistic crosstalk prevents their simultaneous co-option. To test this hypothesis, we compared high and low elevation populations of Boechera stricta that have diverged with respect to constitutive levels of glucosinolate defenses and root:shoot ratios; neither population has high levels of both traits. If constraints imposed by antagonistic signaling underlie this divergence, one would predict that high constitutive levels of traits would coincide with lower plasticity. To test this prediction, we compared the genetically diverged populations in a double challenge drought-herbivory growth chamber experiment. Although a glucosinolate defense response to the generalist insect herbivore Spodoptera exigua was attenuated under drought conditions, the plastic defense response did not differ significantly between populations. Similarly, although several potential drought tolerance traits were measured, only stomatal aperture behavior, as measured by carbon isotope ratios, was less plastic as predicted in the high elevation population. However, RNAseq results on a small subset of plants indicated differential expression of relevant genes between populations as predicted. We suggest that the ambiguity in our results stems from a weaker link between the pathways and the functional traits compared to transcripts.

Characterization of drought- and heat-responsive microRNAs in switchgrass.

Recent investigations revealed that microRNAs (miRNAs) play crucial roles in plant acclimation to stress conditions. Switchgrass, one of the important biofuel crop species can withstand hot and dry climates but the molecular basis of stress tolerance is relatively unknown. To identify miRNAs that are important for tolerating drought or heat, small RNAs were profiled in leaves of adult plants exposed to drought or heat. Sequence analysis enabled the identification of 29 conserved and 62 novel miRNA families. Notably, the abundances of several conserved and novel miRNAs were dramatically altered following drought or heat. Using at least one fold (log2) change as cut off, we observed that 13 conserved miRNA families were differentially regulated by both stresses, and, five and four families were specifically regulated by drought and heat, respectively. Similarly, using a more stringent cut off of two fold (log2) regulation, we found 5 and 16 novel miRNA families were upregulated but 6 and 7 families were downregulated under drought and heat, respectively. The stress-altered expression of a subset of miRNAs and their targets was confirmed using quantitative PCR. Overall, the switchgrass plants exposed to drought or heat revealed similarities as well as differences with respect to miRNA regulation, which could be important for enduring different stress conditions.

Plant genome archaeology: evidence for conserved ancestral chromosome segments in dicotyledonous plant species.

We have developed genetic maps, based on expressed sequence tags (ESTs) that are homologous to Arabidopsis genes, in four dicotyledonous crop plant species from different families. A comparison of these maps with the physical map of Arabidopsis reveals common genome segments that appear to have been conserved throughout the evolution of the dicots. In the four crop species analysed these segments comprise between 16 and 33% of the Arabidopsis genome. Our findings extend the synteny patterns previously observed only within plant families, and indicate that structural and functional information from the model species will be, at least in part, applicable in crop plants with large genomes.

Lignin and lignans in plant defence: insight from expression profiling of cinnamyl alcohol dehydrogenase genes during development and following fungal infection in Populus.

Cinnamyl alcohol dehydrogenase (CAD) catalyses the final step in the biosynthesis of monolignol, the main component of lignin. Lignins, deposited in the secondary cell wall, play a role in plant defence against pathogens. We re-analysed the phylogeny of CAD/CAD-like genes using sequences from recently sequenced genomes, and analysed the temporal and spatial expression profiles of CAD/CAD-like genes in Populus trichocarpa healthy and infected plants. Three fungal pathogens (Rhizoctonia solani, Fusarium oxysporum, and Cytospora sp.), varying in lifestyle and pathogenicity, were used for plant infection. Phylogenetic analyses showed that CAD/CAD-like genes were distributed in classes represented by all members from angiosperm lineages including basal angiosperms and Selaginella. The analysed genes showed different expression profiles during development and demonstrated that three genes were involved in primary xylem maturation while five may function in secondary xylem formation. Expression analysis following inoculation with fungal pathogens, showed that five genes were induced in either stem or leaves. These results add further evidence that CAD/CAD-like genes have evolved specialised functions in plant development and defence against various pest and pathogens. Two genes (PoptrCAD11 and PoptrCAD15), which were induced under various stresses, could be treated as universal markers of plant defence using lignification or lignan biosynthesis.

The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution.

Rosaceae is the most important fruit-producing clade, and its key commercially relevant genera (Fragaria, Rosa, Rubus and Prunus) show broadly diverse growth habits, fruit types and compact diploid genomes. Peach, a diploid Prunus species, is one of the best genetically characterized deciduous trees. Here we describe the high-quality genome sequence of peach obtained from a completely homozygous genotype. We obtained a complete chromosome-scale assembly using Sanger whole-genome shotgun methods. We predicted 27,852 protein-coding genes, as well as noncoding RNAs. We investigated the path of peach domestication through whole-genome resequencing of 14 Prunus accessions. The analyses suggest major genetic bottlenecks that have substantially shaped peach genome diversity. Furthermore, comparative analyses showed that peach has not undergone recent whole-genome duplication, and even though the ancestral triplicated blocks in peach are fragmentary compared to those in grape, all seven paleosets of paralogs from the putative paleoancestor are detectable.

Overexpression of CONSTANS homologs CO1 and CO2 fails to alter normal reproductive onset and fall bud set in woody perennial poplar.

CONSTANS (CO) is an important flowering-time gene in the photoperiodic flowering pathway of annual Arabidopsis thaliana in which overexpression of CO induces early flowering, whereas mutations in CO cause delayed flowering. The closest homologs of CO in woody perennial poplar (Populus spp.) are CO1 and CO2. A previous report showed that the CO2/FLOWERING LOCUS T1 (FT1) regulon controls the onset of reproduction in poplar, similar to what is seen with the CO/FLOWERING LOCUS T (FT) regulon in Arabidopsis. The CO2/FT1 regulon was also reported to control fall bud set. Our long-term field observations show that overexpression of CO1 and CO2 individually or together did not alter normal reproductive onset, spring bud break, or fall dormancy in poplar, but did result in smaller trees when compared with controls. Transcripts of CO1 and CO2 were normally most abundant in the growing season and rhythmic within a day, peaking at dawn. Our manipulative experiments did not provide evidence for transcriptional regulation being affected by photoperiod, light intensity, temperature, or water stress when transcripts of CO1 and CO2 were consistently measured in the morning. A genetic network analysis using overexpressing trees, microarrays, and computation demonstrated that a majority of functionally known genes downstream of CO1 and CO2 are associated with metabolic processes, which could explain their effect on tree size. In conclusion, the function of CO1 and CO2 in poplar does not appear to overlap with that of CO from Arabidopsis, nor do our data support the involvement of CO1 and CO2 in spring bud break or fall bud set.

Comparative genomics and evolutionary analyses of the O-methyltransferase gene family in Populus.

S-adenosyl-l-methionine (SAM) dependent O-methyltransferases (OMTs) proteins are involved in the methylation of various secondary metabolites. The OMT genes have been studied in various plants, but these studies focused either on a single or a small set of genes. Moreover, no comprehensive study was published yet on the OMT gene family in a tree species. To investigate the evolutionary history of this gene family and the functional diversification of its members, phylogenetic and several comparative genomics analyses were performed. Phylogeny across land plant lineages showed that OMT genes were distributed in two main classes deeply rooted in the phylogeny of land plants, suggesting that they have evolved by a gene duplication that had happen in the ancestor of land plants. COMT and COMT-like genes were clustering with few flavonoid and multifunctional OMT genes in class II. Class I included flavonoid, simple phenol, and multifunctional OMT genes. All 26 Populus OMT genes were located in segmental duplication blocks and two third of them were tandem duplicated, indicating the role of duplication processes in the expansion of this gene family. Expression profiling of OMT genes in Populus showed that only PoptrOMT25 was differentially expressed in xylem. The other genes were differentially expressed in leaves, bark, or both. Some OMT genes showed differential expression patterns under various biotic and abiotic stresses. The divergence of protein sequences, the phylogenetic distribution, and the expression of COMT and COMT-like genes suggest that they have evolved different functions or tissue specificities following duplications.