The FLOWERING LOCUS T (FT) gene is the essential integrator of flowering regulatory pathways in angiosperms. The paralogs of the FT gene may perform antagonistic functions, as exemplified by BvFT1, that suppresses flowering in Beta vulgaris, unlike the paralogous activator BvFT2. The roles of FT genes in other amaranths were less investigated. Here, we transformed Arabidopsis thaliana with the FLOWERING LOCUS T like (FTL) genes of Chenopodium ficifolium and found that both CfFTL1 and CfFTL2-1 accelerated flowering, despite having been the homologs of the Beta vulgaris floral promoter and suppressor, respectively. The floral promotive effect of CfFTL2-1 was so strong that it caused lethality when overexpressed under the 35S promoter. CfFTL2-1 placed in an inducible cassette accelerated flowering after induction with methoxyphenozide. The spontaneous induction of CfFTL2-1 led to precocious flowering in some primary transformants even without chemical induction. The CqFT2-1 homolog from Chenopodium quinoa had the same impact on viability and flowering as CfFTL2-1 when transferred to A. thaliana. After the FTL gene duplication in Amaranthaceae, the FTL1 copy maintained the role of floral activator. The second copy FTL2 underwent subsequent duplication and functional diversification, which enabled it to control the onset of flowering in amaranths to adapt to variable environments.
- MeSH
- Arabidopsis * genetika metabolismus MeSH
- Chenopodium * genetika metabolismus MeSH
- květy genetika metabolismus MeSH
- proteiny huseníčku * genetika metabolismus MeSH
- regulace genové exprese u rostlin genetika MeSH
- semenáček metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
MAIN CONCLUSION: Chenopodium ficifoliumflowered under long days despite much lower expression ofFLOWERING LOCUS Thomolog than under short days. Frequent duplications of the FLOWERING LOCUS T (FT) gene across various taxonomic lineages resulted in FT paralogs with floral repressor function, whereas others duplicates maintained their floral-promoting role. The FT gene has been confirmed as the inducer of photoperiodic flowering in most angiosperms analyzed to date. We identified all FT homologs in the transcriptome of Chenopodium ficifolium and in the genome of Chenopodium suecicum, which are closely related to diploid progenitors of the tetraploid crop Chenopodium quinoa, and estimated their expression during photoperiodic floral induction. We found that expression of FLOWERING LOCUS T like 1 (FTL1), the ortholog of the sugar beet floral activator BvFT2, correlated with floral induction in C. suecicum and short-day C. ficifolium, but not with floral induction in C. ficifolium with accelerated flowering under long days. This C. ficifolium accession was induced to flowering without the concomitant upregulation of any FT homolog.
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.
- MeSH
- genové regulační sítě MeSH
- hmotnostní spektrometrie MeSH
- proteomika MeSH
- Pseudotsuga embryologie růst a vývoj metabolismus MeSH
- rostlinné proteiny metabolismus fyziologie MeSH
- semena rostlinná růst a vývoj metabolismus MeSH
- somatická embryogeneze rostlin metody MeSH
- Publikační typ
- časopisecké články MeSH
Our study focused on the possible association between the cryotolerance of Norway spruce (Picea abies (L.) Karst.) embryogenic cultures and the anatomical structures of their embryogenic suspensor mass (ESM), their growth rate and their content of endogenous polyamines (PAs). The anatomical characteristics and PA content during cryopreservation and regrowth were studied in the ESMs of AFO 541 and C110 cultures, which have comparable ESM anatomy but diverse growth rates, PA content and regeneration abilities after cryopreservation. Different levels of tolerance to exogenous treatment were already apparent after transfer of the ESMs to liquid media. The endogenous free PAs were maintained at high levels, with spermidine being the predominant PA in the ESM of AFO 541, while in the ESM of C110 the content of putrescine and spermidine was almost identical and rather low, the content of spermidine being approximately one-third that in the ESM of AFO 541. Osmotic pretreatment, using a double application of sorbitol followed by an application of dimethyl sulfoxide (DMSO) resulted in the continual disintegration of polyembryogenic centers and suspensors in both cell lines. A continual decrease in the level of PAs was observed during the cell osmotic pretreatment. The cells that retained their viability and regrowth ability after cryopreservation were the meristematic cells inside the embryonal heads and the cells in the intermediate area between suspensor and meristems. Restoration of AFO 541 growth after cryopreservation was almost immediate; however, the C110 ESM culture regrew with difficulty, often exhibiting callogenesis. High levels of PA-soluble conjugates and an increase in the amount of PAs bound to high-molecular-mass substances was observed in cells of AFO 541 on Day 6 after thawing and also to some extent on Day 11. On Day 21 after thawing, the amount of free putrescine and spermidine in the AFO 541 cells reached the level observed in the suspension culture before the cryotreatment. The extremely low level of PAs determined in the ESM of C110 3 weeks after thawing agreed with the cell viability and rate of regrowth observed in this culture. The possible role of PAs in the process of cryopreservation of Norway spruce cultures is discussed.
- MeSH
- buněčné linie MeSH
- dimethylsulfoxid farmakologie MeSH
- kryoprezervace metody MeSH
- polyaminy metabolismus MeSH
- semena rostlinná anatomie a histologie cytologie účinky léků fyziologie MeSH
- smrk anatomie a histologie embryologie genetika růst a vývoj MeSH
- sorbitol farmakologie MeSH
- studené klima MeSH
- zmrazování MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Somatic embryogenesis in spruce is a process of high importance for biotechnology, yet it comprises of orchestrated series of events whose cellular and molecular details are not well understood. In this study, we examined the role of actin cytoskeleton during somatic embryogenesis in Norway spruce line AFO 541 by means of anti-actin drugs. RESULTS: Application of low doses (50-100 nM) of latrunculin B (Lat B) during the maturation of somatic embryos predominantly killed suspensor cells while leaving the cells in meristematic centres alive, indicating differential sensitivity of actin in the two cell types. The treatment resulted in faster development of more advanced embryos into mature somatic embryos and elimination of insufficiently developed ones. In searching for the cause of the differential actin sensitivity of the two cell types, we analysed the composition of actin isoforms in the culture and isolated four spruce actin genes. Analysis of their expression during embryo maturation revealed that one actin isoform was expressed constitutively in both cell types, whereas three actin isoforms were expressed predominantly in suspensor cells and their expression declined during the maturation. The expression decline was greatly enhanced by Lat B treatment. Sequence analysis revealed amino-acid substitutions in the Lat B-binding site in one of the suspensor-specific actin isoforms, which may result in a different binding affinity for Lat B. CONCLUSIONS: We show that manipulating actin in specific cell types in somatic embryos using Lat B treatment accelerated and even synchronized the development of somatic embryos and may be of practical use in biotechnology.
- MeSH
- aktiny antagonisté a inhibitory metabolismus MeSH
- bicyklické sloučeniny heterocyklické farmakologie MeSH
- cytoskelet účinky léků MeSH
- embryonální vývoj MeSH
- fylogeneze MeSH
- protein - isoformy metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- RNA rostlin genetika MeSH
- rostlinné geny MeSH
- sekvenční seřazení MeSH
- smrk embryologie růst a vývoj MeSH
- substituce aminokyselin MeSH
- techniky tkáňových kultur MeSH
- thiazolidiny farmakologie MeSH
- vazebná místa MeSH
- vývojová regulace genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
