Yevtushenko, Dmytro

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https://hdl.handle.net/10133/6823

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Browsing Yevtushenko, Dmytro by Author "Misra, Santosh"

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    Douglas-fir LEAFY COTYLEDON1 (PmLEC1) is an active transcription factor during zygotic and somatic embryogenesis
    (Wiley, 2021) Vetrici, Mariana A.; Yevtushenko, Dmytro P.; Misra, Santosh
    Douglas-fir (Pseudotsuga menziesii) is one of the world's premier lumber species and somatic embryogenesis (SE) is the most promising method for rapid propagation of superior tree genotypes. The development and optimization of SE protocols in conifers is hindered by a lack of knowledge of the molecular basis of embryogenesis and limited sequence data. In Arabidopsis, the LEAFY COTYLEDON1 (AtLEC1) gene is a master regulator of embryogenesis that induces SE when expressed ectopically. We isolated the LEC1 homologue from Douglas-fir, designated as PmLEC1. PmLEC1 expression in somatic embryos and developing seeds demonstrated a unique, alternating pattern of expression with the highest levels during early stages of embryogenesis. PmLEC1 protein accumulation during seed development correlated with its transcriptional levels during early embryogenesis; however, substantial protein levels persisted until 2 weeks on germination medium. Treatment of mature, stratified seeds with 2,4-epibrassinolide, sorbitol, mannitol, or NaCl upregulated PmLEC1 expression, which may provide strategies to induce SE from mature tissues. Sequence analysis of the PmLEC1 gene revealed a 5′ UTR intron containing binding sites for transcription factors (TFs), such as ABI3, LEC2, FUS3, and AGL15, which are critical regulators of embryogenesis in angiosperms. Regulatory elements for these and other seed-specific TFs and biotic and abiotic signals were identified within the PmLEC1 locus. Most importantly, functional analysis of PmLEC1 showed that it rescued the Arabidopsis lec1-1 null mutant and, in the T2 generation, led to the development of embryo-like structures, indicating a key role of PmLEC1 in the regulation of embryogenesis.
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    Overexpression of Douglas-Fir LEAFY COTYLEDON1 (PmLEC1) in Arabidopsis induces embryonic programs and embryo-like structures in the lec1-1 mutant but not in wild type plants
    (MDPI, 2021) Vetrici, Mariana A.; Yevtushenko, Dmytro P.; Misra, Santosh
    Somatic embryogenesis (SE) is the most promising method for the quick propagation of desirable plant genotypes. However, application of SE to conifers remains challenging due to our limited knowledge about the genes involved in embryogenesis and the processes that lead to somatic embryo formation. Douglas-fir, an economically important lumber species, possesses a homolog of the angiosperm embryo-regulatory LEC1 gene. In the present study, we analyzed the potential of Douglas-fir PmLEC1 to induce embryonic programs in the vegetative cells of a heterologous host, Arabidopsis thaliana. PmLEC1 complemented the Arabidopsis lec1-1 null mutant and led to a variety of phenotypes ranging from normal morphology to developmental arrest at various stages in the T1 generation. PmLEC1 did not affect the morphology of wild type Arabidopsis T1 plants. More profound results occurred in T2 generations. PmLEC1 expression induced formation of recurrent somatic embryo-like structures in vegetative tissues of the rescued lec1-1 mutant but loss of apical dominance (bushy phenotype) in wild type plants. The activation of embryonic programs in the lec1-1PmLEC1 T2 plants was confirmed by the presence of the embryo-specific transcripts, OLEOSIN and CRUCIFERIN. In contrast, no embryo-like structures, and no OLEOSIN or CRUCIFERIN were observed in PmLEC1-expressing bushy wild type T2 plants.