Transcript profiling for early stages during embryo development in Scots pine

Background: Characterization of the expression and function of genes regulating embryo development in conifers is interesting from an evolutionary point of view. However, our knowledge about the regulation of embryo development in conifers is limited. During early embryo development in Pinus species the proembyo goes through a cleavage process, named cleavage polyembryony, giving rise to four embryos. One of these embryos develops to a dominant embryo, which will develop further into a mature, cotyledonary embryo, while the other embryos, the subordinate embryos, are degraded. The main goal of this study has been to identify processes that might be important for regulating the cleavage process and for the development of a dominant embryo. Results: RNA samples from embryos and megagametophytes at four early developmental stages during seed development in Pinus sylvestris were subjected to high-throughput sequencing. A total of 6.6 million raw reads was generated, resulting in 121,938 transcripts, out of which 36.106 contained ORFs. 18,638 transcripts were differentially expressed (DETs) in embryos and megagametophytes. GO enrichment analysis of transcripts up-regulated in embryos showed enrichment for different cellular processes, while those up-regulated in megagametophytes were enriched for accumulation of storage material and responses to stress. The highest number of DETs was detected during the initiation of the cleavage process. Transcripts related to embryogenic competence, cell wall modifications, cell division pattern, axis specification and response to hormones and stress were highly abundant and differentially expressed during early embryo development. The abundance of representative DETs was confirmed by qRT-PCR analyses. Conclusion: Based on the processes identified in the GO enrichment analyses and the expression of the selected transcripts we suggest that (i) processes related to embryogenic competence and cell wall loosening are involved in activating the cleavage process; (ii) apical-basal polarization is strictly regulated in dominant embryos but not in the subordinate embryos; (iii) the transition from the morphogenic phase to the maturation phase is not completed in subordinate embryos. This is the first genome-wide transcript expression profiling of the earliest stages during embryo development in a Pinus species. Our results can serve as a framework for future studies to reveal the functions of identified genes

Programmed cell death and genetic stability in conifer embryogenesis

Somatic embryogenesis, the generation of embryos from somatic cells, is a valuable tool for studying embryology. In addition, somatic embryos can be used for large-scale vegetative propagation, an application of great interest for forestry. A critical event during early embryo differentiation in conifers is the apical basal polarization, which proceeds through the establishment of two embryonic parts: the proliferating embryonal mass and the terminally differentiated suspensor. The development of both parts is strictly coordinated and imbalance causes embryonic defects. The suspensor cells are eliminated by programmed cell death (PCD). In animals, caspase family proteases are the main executioners of PCD. In this work we have used synthetic peptide substrates containing caspase recognition sites and corresponding specific inhibitors to analyse the role of caspase-like activity during early embryo differentiation in Norway spruce (Picea abies L. Karst.). We found that VEIDase is the principal caspase-like activity. This activity is localized specifically in suspensor cells, and its inhibition prevents normal embryo development by blocking the suspensor differentiation. The in vitro VEIDase activity was shown to be highly sensitive to pH, ionic strength, temperature and zinc concentration. In vivo studies with Zinquin, a zinc-specific fluorescent probe, revealed a high accumulation of intracellular free zinc in the embryonal masses and an abrupt decrease in the suspensor. Increased zinc concentration in the culture medium suppresses terminal differentiation and PCD of the suspensor. In accordance, exposure of early embryos to TPEN, a zinc-specific chelator, induces ectopic cell death affecting embryonal masses. This establishes zinc as an important factor affecting cell fate specification during plant embryogenesis. Before somatic embryos can be accepted for clonal propagation it is important to show that the regenerated plants have similar growth to that of seedlings and are genetically uniform. The genetic integrity during zygotic and somatic embryogenesis in Norway spruce and Scots pine (Pinus sylvestris L.) was investigated by comparing the stability of variable nuclear microsatellite loci. The stability varied significantly among families in both species during somatic embryogenesis. Scots pine families showing low genetic stability during establishment of embryogenic cultures had a higher embryogenic potential than those that were genetically more stable. In contrast, embryo development was suppressed in genetically unstable families. The stability of microsatellites was in general higher in zygotic embryos than in somatic embryos. No deviation in growth was observed in somatic embryo plants of Norway spruce carrying mutated microsatellites

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

Evidence of WUSCHEL (WOX2) gene expression during induction of somatic embryogenesis from apical shoot buds of mature trees of Pinus roxburghii

This study highlights for the first time the expression of transcription factor, WOX2(WUSCHEL homeobox 2) in the embryogenic tissue derived from the apical shoot buds of mature trees of 3 genotypes of P. roxburghii. Therefore, PrWOX2 could be used as a potential molecular signature for the identification of embryogenic cultures during the early development of somatic embryos in conifers. On the other hand, the non-embryogenic tissue of shoot bud cultures which were initiated without cold treatment (control) failed to showthe expression of WOX2. This clearly demonstrates the role of WOX2 in the somatic embryogenesis pathway and might be directly related to the stress conditions

insights from the coding and non-coding transcriptomes

"In a context of increasing wood demand from the forest industry worldwide, maritime pine (Pinus pinaster) breeding programs have been putting their focus on wood traits, resilience to abiotic stresses and to biotic agents. Ultimately, one of the main goals is to produce large numbers of clonal trees from the ones selected with the improved traits. Currently, the clonal propagation of the genotypes selected in breeding programs is already being applied to several forest tree species for commercial purposes, namely by using a type of vegetative propagation technology called somatic embryogenesis. P. pinaster is not yet among the forest tree species which have been successfully clonally propagated on a large commercial scale by somatic embryogenesis.(...)

Population history and selection in closely related mountain pine species (genus Pinus) in Europe

Wydział BiologiiSosna górska (Pinus mugo Turra) i sosna hakowata (P. uncinata Ramond ex DC.) stanowią parę taksonów siostrzanych o słabo rozpoznanej historii ewolucyjnej i relacjach pokrewieństwa. Oba gatunki zasiedlają stanowiska rozrzucone w subalpejskim paśmie europejskich masywów górskich, niemniej różnią się pod względem pewnych cech fenotypowych i ekologicznych. Razem z blisko spokrewnionym taksonem referencyjnym – sosną zwyczajną (Pinus sylvestris L.), stanowiły układ porównawczy w badaniach genetycznych podstaw przystosowań drzew leśnych do warunków wysokogórskich. Pierwszym etapem badań były analizy filogeograficzne dotyczące relacji istniejących pomiędzy i w obrębie naturalnych populacji tych taksonów. Rekonstrukcje oparte na polimorfizmie nowo opracowanych markerów mtDNA rzuciły światło na obecną strukturę genetyczną populacji oraz możliwe historyczne drogi ich migracji. Pozwoliły także, po raz pierwszy, na rozróżnienie P. mugo i P. uncinata za pomocą markerów olekularnych. Zmienność genomu jądrowego, eksplorowana w kolejnych etapach badań, polegała na analizie danych genotypowych otrzymanych dzięki zastosowaniu macierzy SNP oraz informacji uzyskanych z sekwencjonowania transkryptomów reprezentatywnej grupy osobników tych trzech taksonów. Badania te wyłoniły szereg genów odbiegających wzorcem zróżnicowania pomiędzy taksonami od zmienności ich neutralnego tła genetycznego. Funkcje wielu z tych genów kandydackich można wiązać z przystosowaniami do wymagań środowisk wysokogórskich, co czyni je wartościowym punkt odniesienia dla dalszych analiz w tym zakresie.Dwarf mountain pine (Pinus mugo Turra) and Pyrenean pine (P. uncinata Ramond ex DC.) constitute a pair of sister taxa with poorly understood evolutionary history and relationships. Both species inhabit sites scattered in the subalpine belt of European mountain ranges, but they differ in phenotypically and ecologically. Together with a closely related reference taxon - Scots pine (Pinus sylvestris L.), they formed comparative system for study of the genetic basis of the adaptation of forest trees to high mountain conditions. The phylogeographic analysis of the relationships between and within natural populations of these taxa was first stage of the research. Reconstructions based on the polymorphism of a set of newly developed mtDNA markers shed light on the current genetic structure of their populations and possible historical routes of their migration. They also allowed, for the first time, to distinguish P. mugo and P. uncinata using molecular markers. The nuclear genome variation was explored in subsequent stages of the research. It involved the analysis of data obtained through sequence genotyping with the use of the SNP matrix and of information gained from transcriptome sequencing in a representative group of individuals from the three taxa. These studies revealed a number of genes that departed in pattern of differentiation between taxa from the variation observed in neutral genetic background. Functions of many of these candidate genes can be associated with adaptations to the requirements of high mountain environments, which makes them a valuable reference point for further analyzes in this area.Wykonanie niniejszej pracy odbyło się w ramach realizacji projektu badawczego nr 2015/19/B/NZ9/00024, pt. „Genomika środowiskowa wybranych gatunków sosen (rodzaj Pinus) w Europie”, przyznanego prof. dr. hab. Witoldowi Wachowiakowi w ramach konkursu OPUS 10, finansowanego ze środków Narodowego Centrum Nauki, w którym autorka była zatrudniona w roli wykonawcy na stanowisku doktoranta-stypendysty. Autorka uzyskała również środki finansowe na ukończenie rozprawy doktorskiej w ramach projektu „Paszport do przyszłości – Interdyscyplinarne studia doktoranckie na Wydziale Biologii UAM” (POWR.03.02.00-00-I006/17); stypendium to było współfinansowane ze środków Uniwersytetu im. Adama Mickiewicza w Poznaniu oraz Europejskiego Funduszu Społecznego. W badaniach przedstawionych w niniejszej pracy wykorzystano również materiał badawczy i/ lub dane uzyskane przy wsparciu następujących projektów: - 2017/27/B/NZ9/00159 pt. Rewizja wzorów migracji-selekcji sosny zwyczajnej (Pinus sylvestris L.) z wykorzystaniem nowatorskich podejść genomicznych; projekt finansowany ze środków Narodowego Centrum Nauki - NE/K012177/1 pt. Population genomics and evolution of adaptive traits in Pines; projekt finansowany ze środków Natural Environment Research Council, UK - BB/L012243/1 pt. Promoting resilience of UK tree species to novel pests and pathogens: ecological and evolutionary solutions; projekt finansowany ze środków Biotechnology and Biological Sciences Research Council, U

The effects of abiotic and biotic factors on somatic embryogenesis and seedlings of Pinus sylvestris (L.)

Somatic embryogenesis (SE) is an asexual form of plant propagation that occurs in nature and mimics many of the events of sexual reproduction. Pinus sylvestris (L.) is an important source of timber in Northern Eurasia but it is recalcitrant to somatic embryogenesis. Several factors important for the success of the P. sylvestris embryogenic cultures have not been thoroughly investigated. In this study, we examined the effects of parental genotypes on the SE in P. sylvestris, the involvement of the gaseous plant growth regulator, ethylene in SE, and also biotic effects on somatic embryos as well as on seedlings. We tested parental effects on immature embryo initiation for different media, storage periods, and on the maturation process. Maternal effects were found to be crucial for SE in the absence of paternal effects. No maternal-paternal interaction was observed at any stage of somatic embryo production. Additionally the role of ethylene at different developmental stages of SE was investigated. Two ACC synthase genes, PsACS1 and PsACS2, were isolated and characterized. PsACS1 was expressed during the proliferation stage in all tested genotypes, whereas PsACS2 was only expressed in somatic embryos of each genotype. Ethylene production in embryos at stage 3 was significantly higher than the other stages. In a parallel study, the response of somatic embryos to fungal elicitors was investigated. Three fungi, a mutualistic ectomycorrhizal (ECM) fungus (Suillus bovinus), a weak Scots pine pathogen (Heterobasidion parviporum) and a strong pathogen (H. annosum) were used. The gene expression patterns for embryos exposed to the H. parviporum elicitor were found to be similar to that documented for S. bovinus among the tested genes. By contrast somatic embryos exposed to the H. annosum elicitor had a different pattern of regulation which was marked by a delayed response, and in some cases death of the embryos. Furthermore, interaction without direct contact between P. sylvestris seedlings and microbes (mutualistic and pathogenic fungus, cyanobacterium) were investigated. Several novel genes expressed in seedlings treated with ECM fungus were isolated which suggested that physical contact is not necessary for elicitation of host responses. The results suggest that somatic embryos and seedlings of P. sylvestris are genetically well equipped to respond to fungal elicitor/exudates and could serve as a suitable model for reproducible molecular studies in conifer tree patho- and symbiotic systems.Abioottisten ja bioottisten tekijöiden vaikutukset männyn (Pinus sylvestris L.) somaattiseen embryogeneesiin ja siementaimiin Somaattinen embryogeneesi (SE) on kasvin suvuton lisääntymistapa, jota esiintyy luonnossa ja joka jäljittelee monia suvulliseen lisääntymiseen liittyviä tapahtumia. Pinus sylvestris (L.) on merkittävä tuotantopuu pohjoisessa Euraasiassa, mutta sen somaattinen embryogeneesi on hankalasti toteutettavissa. Monia SE:n onnistumiselle tärkeitä tekijöitä ei ole tutkittu tarpeeksi. Tässä tutkimuksessa me tarkastelemme vanhempien genotyypin vaikutusta männyn SE:iin, kaasumaisen kasvunsäätäjän etyleenin osallistumista SE:iin ja etyleenin bioottisia vaikutuksia sekä somaattisiin alkioihin että siementaimiin. Testasimme vanhempien genotyypin vaikutusta epäkypsien alkioiden kasvuunlähtöön eri kasvatusalustoilla eri pituisten varastoiniaikojen jälkeen ja somaattisten alkioiden kypsymisvaiheessa. Isä-äiti yhteisvaikutusta ei havaittu missään somaattisten alkioiden kehitysvaiheessa. Tutkimme myös etyleenin roolia SE:n eri vaiheissa. Kaksi ACC-syntaasi geeniä, PsACS1 ja PsACS2, eristettiin ja karakterisoitiin. PsACS1 ekspressoitui proliferaatiovaiheessa kaikilla testatuilla genotyypeillä, kun taas PsACS2 ekspressoitui kaikkien genotyyppien somaattisissa alkioissa. Etyleenin tuotanto 3. vaiheen alkioissa oli huomattavasti korkeampi kuin muissa kehitysvaiheissa. Rinnakkaisessa kokeessa tutkimme myös somaattisten alkioiden reaktiota sienielisitoreihin. Kokeessa käytettiin kolmea sientä, symbionttista ektomykorritsasientä (ECM) Suillus bovinus, lievää männyn patogeenia Heterobasidion parviporum ja haitallista tuhosientä H. annosum. H. parviporum elisitorille altistettujen alkioiden geeniekspressiot todettiin samanlaisiksi kuin S. bovinus sienen aiheuttamat samoilla geeneillä. Sen sijaan somaattisilla alkioilla, jotka oli käsitelty S. bovinus sienen elisitoreilla oli erilainen toimintamalli, jota kuvaa hitaampi vastine ja joissain tapauksissa alkioiden kuolema. Lisäksi tutkittiin vuorovaikutusta ilman suoraa kontaktia männyn siementaimien ja mikrobien (symbionttiset ja patogeeniset sienet, syanobakteeri) välillä. Pystyimme eristämään useita uusia geenejä, jotka ekspressoituivat siementaimissa kun ne altistettiin ECM sienelle ilman suoraa kontaktia, mikä viittaa siihen että fyysinen kontakti ei ole tarpeen jotta saadaan aikaan vastine isäntäkasvissa. Saadut tulokset antavat viitteitä siitä, että männyn somaattiset alkiot ja siementaimet ovat geneettisesti kykeneviä vastaamaan sienten elisitoreihin/eritteisiin ja ne voivat tarjota sopivan mallin toistettaviin molekyylitason tutkimuksiin havupuiden ja patogeenien sekä symbionttisten sienten vuorovaikutuksista

Hormonal signals involved in the regulation of cambial activity, xylogenesis and vessel patterning in trees

The radial growth of plant stem is based on the development of cribro-vascular cambium tissues. It affects the transport efficiency of water, mineral nutrients and photoassimilates and, ultimately, also plant height. The rate of cambial cell divisions for the assembly of new xylem and phloem tissue primordia and the rate of differentiation of the primordia into mature tissues determine the amount of biomass produced and, in the case of woody species, the wood quality. These complex physiological processes proceed at a rate which depends on several factors, acting at various levels: growth regulators, resource availability and environmental factors. Several hormonal signals and, more recently, further regulatory molecules, have been shown to be involved in the induction and maintenance of cambium and the formation of secondary vascular tissues. The control of xylem cell patterning is of particular interest, because it determines the diameter of xylem vessels, which is central to the efficiency of water and nutrient transport from roots to leaves through the stem and may strongly influence the growth in height of the tree. Increasing scientific evidence have proved the role of other hormones in cambial cell activities and the study of the hormonal signals and their crosstalking in cambial cells may foster our understanding of the dynamics of xylogenesis and of the mechanism of vessel size control along the stem. In this article, the role of the hormonal signals involved in the control of cambium and xylem development in trees and their crosstalking are reviewed