Clinal variation in PHY (PAS domain) and CRY (CCT domain)-Signs of local adaptation to light quality in Norway spruce.

Detection of the genomic basis of local adaptation to environmental conditions is challenging in forest trees. Phytochromes (PHY) and cryptochromes (CRY) perceive the red (R)/far-red (FR) and blue light respectively, thus playing a fundamental role in regulating plant growth and development. PHYO and PHYP from conifers are the equivalents of PHYA/PHYC and PHYB in angiosperms, respectively. Norway spruce shows an adaptive latitudinal cline for shade (low R:FR or FR-enriched light) tolerance and requirement of FR light for its growth. We analyzed the exome capture data that included a uniquely large data set of 1654 Norway spruce trees sampled across many latitudes in Sweden to capture the natural clines for photoperiod and FR light exposure during the growth season. Statistically significant clinal variation was detected in allele and genotype frequencies of missense mutations in coding regions belonging to well-defined functional domains of PHYO (PAS-B), PHYP2 (PAS fold-2), CRY1 (CCT1) and CRY2 (CCT2) that strongly correlates with the latitudinal gradient in response to variable light quality in Norway spruce. The missense SNP in PHYO resulting in Asn835Ser, displayed the steepest cline among all other polymorphisms. We propose that these variations in the photoreceptors represent signs of local adaptation to light quality.

Analysis of the ASR and LP3 homologous gene families reveal positive selection acting on LP3-3 gene.

Drought has long been established as a major environmental stress for plants which have in turn developed several coping strategies, ranging from physiological to molecular mechanisms. LP3 that was first discovered in loblolly pine (Pinus taeda L.) is a homolog of the Abscisic Acid, Stress and Ripening (ASR) gene belonging to the ABA/WDS gene family that was first detected in tomato. LP3 has been shown to be present in four different paralogs in loblolly pine called LP3-0, LP3-1, LP3-2 and LP3-3. LP3 in loblolly pine has not been as extensively studied as the ASR in tomato. Similar to ASR, the different LP3 paralogs have been shown to be upregulated in response to water deficit stress and to act as transcription factors for genes likely involved in hexose transport. In the current study, we have investigated the evolutionary history of LP3 gene family, with the aim of relating it to that of ASR from a phylogenetic perspective and comparing the differences in selective pressure and codon usage. Phylogenetic trees revealed that LP3 is less divergent across species than ASR even when the trees were solely based on the different sub-sections of the gene. Phylogenetic, GC content, codon usage and selective pressure analyses suggest that LP3-3 is undergoing positive selection.

Adaptive strategies of Scots pine under shade: Increase in lignin synthesis and ecotypic variation in defense-related gene expression.

Shade is a stressful condition for plants characterized by low Red:Far-Red (R:FR) ratio. The northern latitudes in Sweden daily receive more hours of FR-enriched light (twilight) or shade-like conditions compared to southern forests during the growing season. Scots pine (Pinus sylvestris L.) is a shade-intolerant species. Yet, it is well adapted to this latitudinal variation in light, which is evident by a northward increase in FR requirement to maintain growth. Shade adversely affects plant growth; it makes the plant weak and, therefore, susceptible to pathogen attack. Lignin is involved in plant protection against pathogen invasion mainly by forming a physical barrier. We studied lignin synthesis and expression of defense-related genes (growth-defense trade-offs) under a low R:FR (shade) ratio in Scots pine. A higher number of immunity/defense-related genes were up-regulated in response to shade in northern populations compared to southern ones, which can be viewed as a local adaptation to light quality for optimal growth and survival. Light quality regulates lignin metabolism; light stimulates lignin synthesis, while shade causes a decrease in lignin synthesis in most angiosperms. In contrast, Scots pine shows an increase in lignin synthesis supported by the higher expression of a few key genes in the lignin biosynthetic pathway, a novel finding reported by our study. These findings can be applied to future breeding strategies in forestry to produce disease-resilient trees.

Enhanced lignin synthesis and ecotypic variation in defense-related gene expression in response to shade in Norway spruce.

During the growth season, northern forests in Sweden daily receive more hours of far-red (FR)-enriched light or twilight (shade) as compared to southern forests. Norway spruce (shade-tolerant) are adapted to latitudinal variation in twilight characterized by a northward increase in FR requirement to maintain growth. Shade is a stressful condition that affects plant growth and increases plant's susceptibility to pathogen attack. Lignin plays a central role in plant defense and its metabolism is regulated by light wavelength composition (light quality). In the current work, we studied regulation of lignin synthesis and defense-related genes (growth-defense trade-offs) in response to shade in Norway spruce. In most angiosperms, light promotes lignin synthesis, whereas shade decreases lignin production leading to weaker stem, which may make plants more disease susceptible. In contrast, enhanced lignin synthesis was detected in response to shade in Norway spruce. We detected a higher number of immunity/defense-related genes up-regulated in northern populations as compared to south ones in response to shade. Enhanced lignin synthesis coupled with higher defense-related gene expression can be interpreted as an adaptive strategy for better survival in northern populations. Findings will contribute to ensuring deployment of well-adapted genetic material and identifying tree families with enhanced disease resistance.

Protocol development for somatic embryogenesis, SSR markers and genetic modification of Stipagrostis pennata (Trin.) De Winter.

BACKGROUND: Stipagrostis pennata (Trin.) De Winter is an important species for fixing sand in shifting and semi-fixed sandy lands, for grazing, and potentially as a source of lignocellulose fibres for pulp and paper industry. The seeds have low viability, which limits uses for revegetation. Somatic embryogenesis offers an alternative method for obtaining large numbers of plants from limited seed sources. RESULTS: A protocol for plant regeneration from somatic embryos of S. pennata was developed. Somatic embryogenesis was induced on Murashige & Skoog (MS) medium supplemented with 3 mg·L-1 2,4-D subsequently shoots were induced on MS medium and supplemented with 5 mg·L-1 zeatin riboside. The highest shoots induction was obtained when embryogenic callus derived from mature embryos (96%) in combination with MS filter-sterilized medium was used from Khuzestan location. The genetic stability of regenerated plants was analysed using ten simple sequence repeats (SSR) markers from S. pennata which showed no somaclonal variation in regenerated plants from somatic embryos of S. pennata. The regenerated plants of S. pennata showed genetic stability without any somaclonal variation for the four pairs of primers that gave the expected amplicon sizes. This data seems very reliable as three of the PCR products belonged to the coding region of the genome. Furthermore, stable expression of GUS was obtained after Agrobacterium-mediated transformation using a super binary vector carried by a bacterial strain LBA4404. CONCLUSION: To our knowledge, the current work is the first attempt to develop an in vitro protocol for somatic embryogenesis including the SSR marker analyses of regenerated plants, and Agrobacterium-mediated transformation of S. pennata that can be used for its large-scale production for commercial purposes.

In silico characterization of putative gene homologues involved in somatic embryogenesis suggests that some conifer species may lack LEC2, one of the key regulators of initiation of the process.

BACKGROUND: Somatic embryogenesis (SE) is the process in which somatic embryos develop from somatic tissue in vitro on medium in most cases supplemented with growth regulators. Knowledge of genes involved in regulation of initiation and of development of somatic embryos is crucial for application of SE as an efficient tool to enable genetic improvement across genotypes by clonal propagation. RESULTS: Current work presents in silico identification of putative homologues of central regulators of SE initiation and development in conifers focusing mainly on key transcription factors (TFs) e.g. BBM, LEC1, LEC1-LIKE, LEC2 and FUSCA3, based on sequence similarity using BLASTP. Protein sequences of well-characterised candidates genes from Arabidopsis thaliana were used to query the databases (Gymno PLAZA, Congenie, GenBank) including whole-genome sequence data from two representative species from the genus Picea (Picea abies) and Pinus (Pinus taeda), for finding putative conifer homologues, using BLASTP. Identification of corresponding conifer proteins was further confirmed by domain search (Conserved Domain Database), alignment (MUSCLE) with respective sequences of Arabidopsis thaliana proteins and phylogenetic analysis (Phylogeny.fr). CONCLUSIONS: This in silico analysis suggests absence of LEC2 in Picea abies and Pinus taeda, the conifer species whose genomes have been sequenced. Based on available sequence data to date, LEC2 was also not detected in the other conifer species included in the study. LEC2 is one of the key TFs associated with initiation and regulation of the process of SE in angiosperms. Potential alternative mechanisms that might be functional in conifers to compensate the lack of LEC2 are discussed.

Molecular signatures of local adaptation to light in Norway spruce.

MAIN CONCLUSION: Transcriptomic and exome capture analysis reveal an adaptive cline for shade tolerance in Norway spruce. Genes involved in the lignin pathway and immunity seem to play a potential role in contributing towards local adaptation to light. The study of natural variation is an efficient method to elucidate how plants adapt to local climatic conditions, a key process for the evolution of a species. Norway spruce is a shade-tolerant conifer in which the requirement of far-red light for growth increases latitudinally northwards. The objective of the study is to characterize the genetic control of local adaptation to light enriched in far-red in Norway spruce, motivated by a latitudinal gradient for the Red:Far-red (R:FR) ratio to which Norway spruce has been proven to be genetically adapted. We have established the genomic signatures of local adaptation by conducting transcriptomic (total RNA-sequencing) and genomic analyses (exome capture), for the identification of genes differentially regulated along the cline. RNA-sequencing revealed 274 differentially expressed genes in response to SHADE (low R:FR light), between the southern and northern natural populations in Sweden. Exome capture included analysis of a uniquely large data set (1654 trees) that revealed missense variations in coding regions of nine differentially expressed candidate genes, which followed a latitudinal cline in allele and genotype frequencies. These genes included five transcription factors involved in vital processes like bud-set/bud-flush, lignin pathway, and cold acclimation and other genes that take part in cell-wall remodeling, secondary cell-wall thickening, response to starvation, and immunity. Based on these results, we suggest that the northern populations might not only be able to adjust their growing season in response to low R:FR light, but they may also be better adapted towards disease resistance by up-regulation of the lignin pathway that is linked to immunity. This forms a concrete basis for local adaptation to light quality in Norway spruce, one of the most economically important conifer tree species in Sweden.

Global gene expression analysis in etiolated and de-etiolated seedlings in conifers.

Plant life cycle begins with germination of seed below the ground. This is followed by seedling's development in the dark: skotomorphogenesis; and then a light-mediated growth: photomorphogenesis. After germination, hypocotyl grows rapidly to reach the sun, which involves elongation of shoot at the expense of root and cotyledons. Upon reaching ground level, seedling gets exposed to sunlight following a switch from the etiolated (skotomorphogenesis) to the de-etiolated (photomorphogenesis) stage, involving a series of molecular and physiological changes. Gymnosperms have evolved very differently and adopted diverse strategies as compared to angiosperms; with regards to response to light quality, conifers display a very mild high-irradiance response as compared to angiosperms. Absence of apical hook and synthesis of chlorophyll during skotomorphogenesis are two typical features in gymnosperms which differentiate them from angiosperms (dicots). Information regarding etiolation and de-etiolation processes are well understood in angiosperms, but these mechanisms are less explored in conifer species. It is, therefore, interesting to know how similar these processes are in conifers as compared to angiosperms. We performed a global expression analysis (RNA sequencing) on etiolated and de-etiolated seedlings of two economically important conifer species in Sweden to review the differentially expressed genes associated with the two processes. Based on the results, we propose that high levels of HY5 in conifers under DARK condition coupled with expression of few other genes associated with de-etiolation in angiosperms e.g. SPA, DET1 (lower expression under DARK) and CRY1 (higher expression under DARK), leads to partial expression of photomorphogenic genes in the DARK phenotype in conifers as displayed by absence of apical hook, opening of cotyledons and synthesis of chlorophyll.

Transcriptome analysis of shade avoidance and shade tolerance in conifers.

MAIN CONCLUSION: Gymnosperms respond differently to light intensity and R:FR; although some aspects of shade response appear conserved, yet underlying mechanisms seem to be diverse in gymnosperms as compared to angiosperms. Shade avoidance syndrome (SAS) is well-characterized in the shade intolerant model species Arabidopsis thaliana whereas much less is known about shade tolerance response (STR), yet regulation of SAS and STR with reference to conifers remains poorly understood. We conducted a comparative study of two conifer species with contrasting responses to shade, Scots pine (shade-intolerant) and Norway spruce (shade-tolerant), with the aim to understand mechanisms behind SAS and STR in conifers. Pine and spruce seedlings were grown under controlled light and shade conditions, and hypocotyl and seedling elongation following different light treatments were determined in both species as indicators of shade responses. Red to far-red light ratio (R:FR) was shown to trigger the shade response in Norway spruce. In Scots pine, we observed an interaction between R:FR and light intensity. RNA sequencing (RNA-Seq) data revealed that SAS and STR responses included changes in expression of genes involved primarily in hormone signalling and pigment biosynthesis. From the RNA-Seq analysis, we propose that although some aspects of shade response appear to be conserved in angiosperms and gymnosperms, yet the underlying mechanisms may be different in gymnosperms that warrants further research.

Differential response of Scots pine seedlings to variable intensity and ratio of red and far-red light.

We investigated the response to increasing intensity of red (R) and far-R (FR) light and to a decrease in R:FR ratio in Pinus sylvestris L. (Scots pine) seedling. The results showed that FR high-irradiance response for hypocotyl elongation may be present in Scots pine and that this response is enhanced by increasing light intensity. However, both hypocotyl inhibition and pigment accumulation were more strongly affected by the R light compared with FR light. This is in contrast to previous reports in Arabidopsis thaliana (L.) Heynh. In the angiosperm, A. thaliana R light shows an overall milder effect on inhibition of hypocotyl elongation and on pigment biosynthesis compared with FR suggesting conifers and angiosperms respond very differently to the different light regimes. Scots pine shade avoidance syndrome with longer hypocotyls, shorter cotyledons and lower chlorophyll content in response to shade conditions resembles the response observed in A. thaliana. However, anthocyanin accumulation increased with shade in Scots pine, which again differs from what is known in angiosperms. Overall, the response of seedling development and physiology to R and FR light in Scots pine indicates that the regulatory mechanism for light response may differ between gymnosperms and angiosperms.

Microbial phenomics information extractor (MicroPIE): a natural language processing tool for the automated acquisition of prokaryotic phenotypic characters from text sources.

BACKGROUND: The large-scale analysis of phenomic data (i.e., full phenotypic traits of an organism, such as shape, metabolic substrates, and growth conditions) in microbial bioinformatics has been hampered by the lack of tools to rapidly and accurately extract phenotypic data from existing legacy text in the field of microbiology. To quickly obtain knowledge on the distribution and evolution of microbial traits, an information extraction system needed to be developed to extract phenotypic characters from large numbers of taxonomic descriptions so they can be used as input to existing phylogenetic analysis software packages. RESULTS: We report the development and evaluation of Microbial Phenomics Information Extractor (MicroPIE, version 0.1.0). MicroPIE is a natural language processing application that uses a robust supervised classification algorithm (Support Vector Machine) to identify characters from sentences in prokaryotic taxonomic descriptions, followed by a combination of algorithms applying linguistic rules with groups of known terms to extract characters as well as character states. The input to MicroPIE is a set of taxonomic descriptions (clean text). The output is a taxon-by-character matrix-with taxa in the rows and a set of 42 pre-defined characters (e.g., optimum growth temperature) in the columns. The performance of MicroPIE was evaluated against a gold standard matrix and another student-made matrix. Results show that, compared to the gold standard, MicroPIE extracted 21 characters (50%) with a Relaxed F1 score > 0.80 and 16 characters (38%) with Relaxed F1 scores ranging between 0.50 and 0.80. Inclusion of a character prediction component (SVM) improved the overall performance of MicroPIE, notably the precision. Evaluated against the same gold standard, MicroPIE performed significantly better than the undergraduate students. CONCLUSION: MicroPIE is a promising new tool for the rapid and efficient extraction of phenotypic character information from prokaryotic taxonomic descriptions. However, further development, including incorporation of ontologies, will be necessary to improve the performance of the extraction for some character types.

Non-functional plastid ndh gene fragments are present in the nuclear genome of Norway spruce (Picea abies L. Karsch): insights from in silico analysis of nuclear and organellar genomes.

Many genes have been lost from the prokaryote plastidial genome during the early events of endosymbiosis in eukaryotes. Some of them were definitively lost, but others were relocated and functionally integrated to the host nuclear genomes through serial events of gene transfer during plant evolution. In gymnosperms, plastid genome sequencing has revealed the loss of ndh genes from several species of Gnetales and Pinaceae, including Norway spruce (Picea abies). This study aims to trace the ndh genes in the nuclear and organellar Norway spruce genomes. The plastid genomes of higher plants contain 11 ndh genes which are homologues of mitochondrial genes encoding subunits of the proton-pumping NADH-dehydrogenase (nicotinamide adenine dinucleotide dehydrogenase) or complex I (electron transport chain). Ndh genes encode 11 NDH polypeptides forming the Ndh complex (analogous to complex I) which seems to be primarily involved in chloro-respiration processes. We considered ndh genes from the plastidial genome of four gymnosperms (Cryptomeria japonica, Cycas revoluta, Ginkgo biloba, Podocarpus totara) and a single angiosperm species (Arabidopsis thaliana) to trace putative homologs in the nuclear and organellar Norway spruce genomes using tBLASTn to assess the evolutionary fate of ndh genes in Norway spruce and to address their genomic location(s), structure, integrity and functionality. The results obtained from tBLASTn were subsequently analyzed by performing homology search for finding ndh specific conserved domains using conserved domain search. We report the presence of non-functional plastid ndh gene fragments, excepting ndhE and ndhG genes, in the nuclear genome of Norway spruce. Regulatory transcriptional elements like promoters, TATA boxes and enhancers were detected in the upstream regions of some ndh fragments. We also found transposable elements in the flanking regions of few ndh fragments suggesting nuclear rearrangements in those regions. These evidences support the hypothesis that, at least in Picea, ndh translocations from the plastid to the nuclear genome have occurred, and that there might have been a functional machinery at some time during evolution to accommodate them within a nuclear-encoded environment, or attempts to form it.

Comparative in silico analysis of SSRs in coding regions of high confidence predicted genes in Norway spruce (Picea abies) and Loblolly pine (Pinus taeda).

BACKGROUND: Microsatellites or simple sequence repeats (SSRs) are DNA sequences consisting of 1-6 bp tandem repeat motifs present in the genome. SSRs are considered to be one of the most powerful tools in genetic studies. We carried out a comparative study of perfect SSR loci belonging to class I (≥20) and class II (≥12 and <20 bp) types located in coding regions of high confidence genes in Picea abies and Pinus taeda. SSRLocator was used to retrieve SSRs from the full length CDS of predicted genes in both species. RESULTS: Trimers were the most abundant motifs in class I followed by hexamers in Picea abies, while trimers and hexamers were equally abundant in Pinus taeda class I SSRs. Hexamers were most frequent within class II SSRs followed by trimers, in both species. Although the frequency of genes containing SSRs was slightly higher in Pinus taeda, SSR counts per Mbp for class I was similar in both species (P-value = 0.22); while for class II SSRs, it was significantly higher in Picea abies (P-value = 0.00009). AT-rich motifs were higher in abundance than the GC-rich motifs, within class II SSRs in both the species (P-values = 10(-9) and 0). With reference to class I SSRs, AT-rich and GC-rich motifs were detected with equal frequency in Pinus taeda (P-value = 0.24); while in Picea abies, GC-rich motifs were detected with higher frequency than the AT-rich motifs (P-value = 0.0005). CONCLUSIONS: Our study gives a comparative overview of the genome SSRs composition based on high confidence genes in the two recently sequenced and economically important conifers and, also provides information on functional molecular markers that can be applied in genetic studies in Pinus and Picea species.

Fungal Infection Increases the Rate of Somatic Mutation in Scots Pine (Pinus sylvestris L.).

Somatic mutations are transmitted during mitosis in developing somatic tissue. Somatic cells bearing the mutations can develop into reproductive (germ) cells and the somatic mutations are then passed on to the next generation of plants. Somatic mutations are a source of variation essential to evolve new defense strategies and adapt to the environment. Stem rust disease in Scots pine has a negative effect on wood quality, and thus adversely affects the economy. It is caused by the 2 most destructive fungal species in Scandinavia: Peridermium pini and Cronartium flaccidum. We studied nuclear genome stability in Scots pine under biotic stress (fungus-infected, 22 trees) compared to a control population (plantation, 20 trees). Stability was assessed as accumulation of new somatic mutations in 10 microsatellite loci selected for genotyping. Microsatellites are widely used as molecular markers in population genetics studies of plants, and are particularly used for detection of somatic mutations as their rate of mutation is of a much higher magnitude when compared with other DNA markers. We report double the rate of somatic mutation per locus in the fungus-infected trees (4.8×10(-3) mutations per locus), as compared to the controls (2.0×10(-3) mutations per locus) when individual samples were analyzed at 10 different microsatellite markers. Pearson's chi-squared test indicated a significant effect of the fungal infection which increased the number of mutations in the fungus-infected trees (χ(2) = 12.9883, df = 1, P = 0.0003134).

Comparative in silico analysis of EST-SSRs in angiosperm and gymnosperm tree genera.

BACKGROUND: Simple Sequence Repeats (SSRs) derived from Expressed Sequence Tags (ESTs) belong to the expressed fraction of the genome and are important for gene regulation, recombination, DNA replication, cell cycle and mismatch repair. Here, we present a comparative analysis of the SSR motif distribution in the 5'UTR, ORF and 3'UTR fractions of ESTs across selected genera of woody trees representing gymnosperms (17 species from seven genera) and angiosperms (40 species from eight genera). RESULTS: Our analysis supports a modest contribution of EST-SSR length to genome size in gymnosperms, while EST-SSR density was not associated with genome size in neither angiosperms nor gymnosperms. Multiple factors seem to have contributed to the lower abundance of EST-SSRs in gymnosperms that has resulted in a non-linear relationship with genome size diversity. The AG/CT motif was found to be the most abundant in SSRs of both angiosperms and gymnosperms, with a relative increase in AT/AT in the latter. Our data also reveals a higher abundance of hexamers across the gymnosperm genera. CONCLUSIONS: Our analysis provides the foundation for future comparative studies at the species level to unravel the evolutionary processes that control the SSR genesis and divergence between angiosperm and gymnosperm tree species.

Next-generation phenomics for the Tree of Life.

The phenotype represents a critical interface between the genome and the environment in which organisms live and evolve. Phenotypic characters also are a rich source of biodiversity data for tree building, and they enable scientists to reconstruct the evolutionary history of organisms, including most fossil taxa, for which genetic data are unavailable. Therefore, phenotypic data are necessary for building a comprehensive Tree of Life. In contrast to recent advances in molecular sequencing, which has become faster and cheaper through recent technological advances, phenotypic data collection remains often prohibitively slow and expensive. The next-generation phenomics project is a collaborative, multidisciplinary effort to leverage advances in image analysis, crowdsourcing, and natural language processing to develop and implement novel approaches for discovering and scoring the phenome, the collection of phentotypic characters for a species. This research represents a new approach to data collection that has the potential to transform phylogenetics research and to enable rapid advances in constructing the Tree of Life. Our goal is to assemble large phenomic datasets built using new methods and to provide the public and scientific community with tools for phenomic data assembly that will enable rapid and automated study of phenotypes across the Tree of Life.

Ecotypic variation in response to light spectra in Scots pine (Pinus sylvestris L.).

We investigated Scots pine adaptive responses to the light spectra by measuring hypocotyl length in seeds sampled from three natural Scots pine ecotypes across a latitudinal cline ranging from 63° to 68° N in Sweden where the adaptive cline is known to be steeper. Seeds were germinated under dark (D) and three monochromatic continuous light wavelengths: blue (B), red (R) and far-red (FR). Analysis of variance revealed a northward decrease in the inhibitory effect of FR with respect to D, the so-called far red high irradiance response. Ecotypic variation for hypocotyl development was observed under the FR and D treatments, while the trends for the B and R treatments were not statistically significant. Under FR the ecotypic variation showed an increase in hypocotyl length northwards, in contrast to the treatment under D which showed a decrease in the hypocotyl length northwards. These results could be interpreted in view of the previously reported northward increase in FR requirement to maintain growth in Norway spruce and Scots pine. Prior to the performance of the main light experiment, the maternal effect on progeny performance was investigated, which showed the absence of maternal environment effect on the performance of the seedlings.