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

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

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

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

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

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. Abstract: 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.Bio4Energ