Dataset Information

Cytological, Biochemical and Molecular Events of the Embryogenic State in Douglas-fir (Pseudotsuga menziesii [Mirb.]).

ABSTRACT: Somatic embryogenesis techniques have been developed for most coniferous species, but only using very juvenile material. To extend the techniques' scope, better integrated understanding of the key biological, physiological and molecular characteristics of embryogenic state is required. Therefore, embryonal masses (EMs) and non-embryogenic calli (NECs) have been compared during proliferation at multiple levels. EMs and NECs originating from a single somatic embryo (isogenic lines) of each of three unrelated genotypes were used in the analyses, which included comparison of the lines' anatomy by transmission light microscopy, transcriptomes by RNAseq Illumina sequencing, proteomes by free-gel analysis, contents of endogenous phytohormones (indole-3-acetic acid, cytokinins and ABA) by LC-MS analysis, and soluble sugar contents by HPLC. EMs were characterized by upregulation (relative to levels in NECs) of transcripts, proteins, transcription factors and active cytokinins associated with cell differentiation accompanied by histological, carbohydrate content and genetic markers of cell division. In contrast, NECs were characterized by upregulation (relative to levels in EMs) of transcripts, proteins and products associated with responses to stimuli (ABA, degradation forms of cytokinins, phenols), oxidative stress (reactive oxygen species) and carbohydrate storage (starch). Sub-Network Enrichment Analyses that highlighted functions and interactions of transcripts and proteins that significantly differed between EMs and NECs corroborated these findings. The study shows the utility of a novel approach involving integrated multi-scale transcriptomic, proteomic, biochemical, histological and anatomical analyses to obtain insights into molecular events associated with embryogenesis and more specifically to the embryogenic state of cell in Douglas-fir.

SUBMITTER: Gautier F

PROVIDER: S-EPMC6403139 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Cytological, Biochemical and Molecular Events of the Embryogenic State in Douglas-fir (<i>Pseudotsuga menziesii</i> [Mirb.]).

Gautier Florian F Label Philippe P Eliášová Kateřina K Leplé Jean-Charles JC Motyka Václav V Boizot Nathalie N Vondráková Zuzana Z Malbeck Jiří J Trávníčková Alena A Le Metté Claire C Lesage-Descauses Marie-Claude MC Lomenech Anne-Marie AM Trontin Jean-François JF Costa Guy G Lelu-Walter Marie-Anne MA Teyssier Caroline C

Frontiers in plant science 20190228

Somatic embryogenesis techniques have been developed for most coniferous species, but only using very juvenile material. To extend the techniques' scope, better integrated understanding of the key biological, physiological and molecular characteristics of embryogenic state is required. Therefore, embryonal masses (EMs) and non-embryogenic calli (NECs) have been compared during proliferation at multiple levels. EMs and NECs originating from a single somatic embryo (isogenic lines) of each of thre ...[more]

PMID: 30873184

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Project description:BACKGROUND:To explore poorly understood differences between primary and subsequent somatic embryogenic lines of plants, we induced secondary (2ry) and tertiary (3ry) lines from cotyledonary somatic embryos (SEs) of two Douglas-fir genotypes: SD4 and TD17. The 2ry lines exhibited significantly higher embryogenic potential (SE yields) than the 1ry lines initiated from zygotic embryos (SD4, 2155 vs 477; TD17, 240 vs 29 g-?1 f.w.). Moreover, we observed similar differences in yield between 2ry and 3ry lines of SD4 (2400 vs 3921 g-?1 f.w.). To elucidate reasons for differences in embryogenic potential induced by repetitive somatic embryogenesis we then compared 2ry vs 1ry and 2ry vs 3ry lines at histo-cytological (using LC-MS/MS) and proteomic levels. RESULTS:Repetitive somatic embryogenesis dramatically improved the proliferating lines' cellular organization (genotype SD4's most strongly). Frequencies of singulated, bipolar SEs and compact polyembryogenic centers with elongated suspensors and apparently cleavable embryonal heads increased in 2ry and (even more) 3ry lines. Among 2300-2500 identified proteins, 162 and 228 were classified significantly differentially expressed between 2ry vs 1ry and 3ry vs 2ry lines, respectively, with special emphasis on "Proteolysis" and "Catabolic process" Gene Ontology categories. Strikingly, most of the significant proteins (>?70%) were down-regulated in 2ry relative to 1ry lines, but up-regulated in 3ry relative to 2ry lines, revealing a down-up pattern of expression. GO category enrichment analyses highlighted the opposite adjustments of global protein patterns, particularly for processes involved in chitin catabolism, lignin and L-phenylalanine metabolism, phenylpropanoid biosynthesis, oxidation-reduction, and response to karrikin. Sub-Network Enrichment Analyses highlighted interactions between significant proteins and both plant growth regulators and secondary metabolites after first (especially jasmonic acid, flavonoids) and second (especially salicylic acid, abscisic acid, lignin) embryogenesis cycles. Protein networks established after each induction affected the same "Plant development" and "Defense response" biological processes, but most strongly after the third cycle, which could explain the top embryogenic performance of 3ry lines. CONCLUSIONS:This first report of cellular and molecular changes after repetitive somatic embryogenesis in conifers shows that each cycle enhanced the structure and singularization of EMs through modulation of growth regulator pathways, thereby improving the lines' embryogenic status.

Project description:Key message Douglas-fir growth correlates with the climate, the soil moisture regime, and the soil nutrient status, reflecting a broad physiological amplitude. Even though planting this non-native tree species is suggested as a viable strategy to improve adaptiveness of European forests to a more extreme climate and to assure future productivity, the expected temperature increase may induce a decline in forest stand productivity for Douglas-fir in already warm and dry regions. ContextTree species selection is one of the most important forest management decisions to enhance forest productivity and stand stability on a given site. Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco var. menziesii), a non-native species from north-western America, is seen as an important additional species option for adapting Central European forests to a changing climate.AimsThis study assesses Douglas-fir forest productivity derived from site conditions. We investigate climatic and physico-chemical soil characteristics and productivity of 28 mature Douglas-fir stands growing on siliceous, as well as carbonate bedrock material in southern Germany and north-eastern Austria.MethodsThe importance of climatic and physico-chemical soil characteristics was analyzed with the machine learning method Random Forests.ResultsThe results show that Douglas-fir growth correlates with climate, soil moisture, and soil nutrient availability derived from ten climatic and physico-chemical soil parameters.ConclusionThe broad pH optimum between 4.5 and 7.2 reflects the broad physiological amplitude of Douglas-fir, and no significant differences were detectable between carbonate and siliceous bedrock. We also conclude that climate change may induce a forest stand productivity decline, because lower productivity with the highest mean summer temperature across our study range was observed at the warmest sites in Eastern Austria.Electronic supplementary materialThe online version of this article (10.1007/s13595-019-0805-3) contains supplementary material, which is available to authorized users.

Project description:BACKGROUND: Douglas-fir (Pseudotsuga menziesii) extends over a wide range of contrasting environmental conditions, reflecting substantial local adaptation. For this reason, it is an interesting model species to study plant adaptation and the effects of global climate change such as increased temperatures and significant periods of drought on individual trees and the forest landscape in general. However, genomic data and tools for studying genetic variation in natural populations to understand the genetic and physiological mechanisms of adaptation are currently missing for Douglas-fir. This study represents a first step towards characterizing the Douglas-fir transcriptome based on 454 sequencing of twelve cDNA libraries. The libraries were constructed from needle and wood tissue of coastal and interior provenances subjected to drought stress experiments. RESULTS: The 454 sequencing of twelve normalized cDNA libraries resulted in 3.6 million reads from which a set of 170,859 putative unique transcripts (PUTs) was assembled. Functional annotation by BLAST searches and Gene Ontology mapping showed that the composition of functional classes is very similar to other plant transcriptomes and demonstrated that a large fraction of the Douglas-fir transcriptome is tagged by the PUTs. Based on evolutionary conservation, we identified about 1,000 candidate genes related to drought stress. A total number of 187,653 single nucleotide polymorphisms (SNPs) were detected by three SNP detection tools. However, only 27,688 SNPs were identified by all three methods, indicating that SNP detection depends on the particular method used. The two alleles of about 60% of the 27,688 SNPs are segregating simultaneously in both coastal and interior provenances, which indicates a high proportion of ancestral shared polymorphisms or a high level of gene flow between these two ecologically and phenotypically different varieties. CONCLUSIONS: We established a catalogue of PUTs and large SNP database for Douglas-fir. Both will serve as a useful resource for the further characterization of the genome and transcriptome of Douglas-fir and for the analysis of genetic variation using genotyping or resequencing methods.

Dataset Information

Cytological, Biochemical and Molecular Events of the Embryogenic State in Douglas-fir (Pseudotsuga menziesii [Mirb.]).

Publications

Cytological, Biochemical and Molecular Events of the Embryogenic State in Douglas-fir (<i>Pseudotsuga menziesii</i> [Mirb.]).

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