Auxin-related functions of LEAFY COTYLEDON2 gene in the induction of somatic embryogenesis in Arabidopsis

Sesja posterowa - XIV Overall Polish in vitro Culture and Plant Biotechnology Conference Structural, physiological and molecular bases of plant differentiation September 14-17, 2015, Pozna

Expression profiling of AUXIN RESPONSE FACTOR genes during somatic embryogenesis induction in Arabidopsis

Key message: Extensive modulation of numerous ARF transcripts in the embryogenic culture of Arabidopsis indicates a substantial role of auxin signaling in the mechanism of somatic embryogenesis induction. Abstract: Somatic embryogenesis (SE) is induced by auxin in plants and auxin signaling is considered to play a key role in the molecular mechanism that controls the embryogenic transition of plant somatic cells. Accordingly, the expression of AUXIN RESPONSE FACTOR (ARF) genes in embryogenic culture of Arabidopsis was analyzed. The study revealed that 14 of the 22 ARFs were transcribed during SE in Arabidopsis. RT-qPCR analysis indicated that the expression of six ARFs (ARF5, ARF6, ARF8, ARF10, ARF16, and ARF17) was significantly up-regulated, whereas five other genes (ARF1, ARF2, ARF3, ARF11, and ARF18) were substantially down-regulated in the SE-induced explants. The activity of ARFs during SE was also monitored with GFP reporter lines and the ARFs that were expressed in areas of the explants engaged in SE induction were detected. A functional test of ARFs transcribed during SE was performed and the embryogenic potential of the arf mutants and overexpressor lines was evaluated. ARFs with a significantly modulated expression during SE coupled with an impaired embryogenic response of the relevant mutant and/or overexpressor line, including ARF1, ARF2, ARF3, ARF5, ARF6, ARF8, and ARF11 were indicated as possibly being involved in SE induction. The study provides evidence that embryogenic induction strongly depends on ARFs, which are key regulators of the auxin signaling. Some clues on the possible functions of the candidate ARFs, especially ARF5, in the mechanism of embryogenic transition are discussed. The results provide guidelines for further research on the auxin-related functional genomics of SE and the developmental plasticity of somatic cells.NC

LEAFY COTYLEDON2-mediated control of the endogenous hormone content : implications for the induction of somatic embryogenesis in Arabidopsis

The overexpression of LEAFY COTYLEDON2 (LEC2) in Arabidopsis, results in the induction of somatic embryogenesis (SE) in an auxin-free environment and the stimulation of auxin biosynthesis was postulated as being involved in this response. To gain further insight into the hormone-related functions of LEC2 in SE, the effect of LEC2 overexpression on the hormone content in Arabidopsis plants and in vitro cultured explants was analysed. In addition to indole-3-acetic acid (IAA) and cytokinins (CKs), which are hormones that play a key role in plant development, the stress-related hormones, abscisic acid (ABA) and salicylic acid (SA), which are involved in the stress response that is related to SE-induction, were analysed. Together with the observations that LEC2 activity can compensate for the auxin treatment required for SE induction (Ledwoń and Gaj in Plant Tissue Cell Org Cult 28:1677–1688, 2009) and LEC2 may control auxin biosynthesis pathway during SE induction (Wójcikowska et al. in Planta 238:425–440, 2013), a significant increase in the IAA content in response to LEC2 overexpression found in the present study supply further evidence that LEC2-controlled auxin biosynthesis may be involved in the mechanism that triggers embryogenic development in somatic cells. Moreover, LEC2-controlled SE induction was shown to be associated with a decrease in the total content of CKs and an accumulation of some specific CK types, including isopentenyl-adenin and cis-zeatin. Additionally, an increase in SA and a decrease in ABA content were also found to be related to LEC2 activity in embryogenically induced tissue. The obtained results provide further proof of the close link between LEC2 and the establishment of the hormonal environment that is required for the promotion of SE

ERF022 impacts the induction of somatic embryogenesis in Arabidopsis through the ethylene-related pathway

Main conclusion The ERF022 gene was found to affect embryogenic transition in somatic cells in Arabidopsis via the ethylene-related pathway. The study provides evidence that ERF022-LEC2 interaction is involved in the auxin–ethylene crosstalk that operates in somatic embryogenesis induction. The ERF022 gene of the ERF family was previously identified among the transcription factor genes that were differentially expressed in an embryogenic culture of Arabidopsis. A strong inhibition of the gene was found to be associated with the induction of somatic embryogenesis (SE) and an erf022 mutant was indicated to display a substantially impaired capacity for SE. Therefore, the molecular function of ERF022 in the induction of SE was studied in the present work. A phenotype of an erf022 mutant was indicated as being related to an increased content of ethylene. The results further suggest that the ERF022 controls the genes that are involved in both the biosynthesis (ACS7) and signalling (ERF1, ETR1) of ethylene and indicate that the ERF022 is a new regulatory element in ethylene-related responses that negatively control the ethylene content and perception. It is proposed that the negative impact of ethylene on the induction of SE may result from a modulation of the auxin-related genes that control the embryogenic transition in somatic cells. Among them, the LEC2, which is a key regulator of the induction of SE through the stimulation of auxin synthesis, was possibly related to ERF022. The results of the study provide new hormone-related clues to define the genetic network that governs SE. A putative model of the regulatory pathway is proposed that is involved in the induction of SE in which the auxin–ethylene interactions are controlled by ERF022 and LEC2 and their targets

Current Perspectives on the Auxin-Mediated Genetic Network that Controls the Induction of Somatic Embryogenesis in Plants

Auxin contributes to almost every aspect of plant development and metabolism as well as the transport and signalling of auxin-shaped plant growth and morphogenesis in response to endo- and exogenous signals including stress conditions. Consistently with the common belief that auxin is a central trigger of developmental changes in plants, the auxin treatment of explants was reported to be an indispensable inducer of somatic embryogenesis (SE) in a large number of plant species. Treating in vitro-cultured tissue with auxins (primarily 2,4-dichlorophenoxyacetic acid, which is a synthetic auxin-like plant growth regulator) results in the extensive reprogramming of the somatic cell transcriptome, which involves the modulation of numerous SE-associated transcription factor genes (TFs). A number of SE-modulated TFs that control auxin metabolism and signalling have been identified, and conversely, the regulators of the auxin-signalling pathway seem to control the SE-involved TFs. In turn, the di erent expression of the genes encoding the core components of the auxin-signalling pathway, the AUXIN/INDOLE-3-ACETIC ACIDs (Aux/IAAs) and AUXIN RESPONSE FACTORs (ARFs), was demonstrated to accompany SE induction. Thus, the extensive crosstalk between the hormones, in particular, auxin and the TFs, was revealed to play a central role in the SE-regulatory network. Accordingly, LEAFY COTYLEDON (LEC1 and LEC2), BABY BOOM (BBM), AGAMOUS-LIKE15 (AGL15) and WUSCHEL (WUS) were found to constitute the central part of the complex regulatory network that directs the somatic plant cell towards embryogenic development in response to auxin. The revealing picture shows a high degree of complexity of the regulatory relationships between the TFs of the SE-regulatory network, which involve direct and indirect interactions and regulatory feedback loops. This review examines the recent advances in studies on the auxin-controlled genetic network, which is involved in the mechanism of SE induction and focuses on the complex regulatory relationships between the down- and up-stream targets of the SE-regulatory TFs. In particular, the outcomes from investigations on Arabidopsis, which became a model plant in research on genetic control of SE, are presented

Directions of changes in the flora structure in the abandoned railway areas

In 2007-2008, floristic studies were carried out in active railway areas in NE Poland, as well as on railway tracks, which had been abandoned at different times. Only homogeneous habitats were surveyed (the area between the tracks and a narrow transect running parallel to the tracks). Trends in the transformation of the flora in abandoned railway areas were discussed: the retreat of alien species with a short life cycle, the encroachment of native perennial plants and an increase in the number of trees

Epigenetic Regulation of Auxin-Induced Somatic Embryogenesis in Plants

Somatic embryogenesis (SE) that is induced in plant explants in response to auxin treatment is closely associated with an extensive genetic reprogramming of the cell transcriptome. The significant modulation of the gene transcription profiles during SE induction results from the epigenetic factors that fine-tune the gene expression towards embryogenic development. Among these factors, microRNA molecules (miRNAs) contribute to the post-transcriptional regulation of gene expression. In the past few years, several miRNAs that regulate the SE-involved transcription factors (TFs) have been identified, and most of them were involved in the auxin-related processes, including auxin metabolism and signaling. In addition to miRNAs, chemical modifications of DNA and chromatin, in particular the methylation of DNA and histones and histone acetylation, have been shown to shape the SE transcriptomes. In response to auxin, these epigenetic modifications regulate the chromatin structure, and hence essentially contribute to the control of gene expression during SE induction. In this paper, we describe the current state of knowledge with regard to the SE epigenome. The complex interactions within and between the epigenetic factors, the key SE TFs that have been revealed, and the relationships between the SE epigenome and auxin-related processes such as auxin perception, metabolism, and signaling are highlighted

Azacitidine (5-AzaC)-treatment and mutations in DNA methylase genes affect embryogenic response and expression of the genes that are involved in somatic embryogenesis in Arabidopsis

Epigenetic processes including DNA methylation play a pivotal role in regulating the genes that control plant development. In contrast to in planta development, the contribution of DNA methylation to the morphogenic processes that are induced in vitro are much less recognised. Hence, in the present study, we analysed the impact of DNA methylation on somatic embryogenesis (SE) that was induced in Arabidopsis. The results demonstrated a decrease in the global DNA methylation level during SE that contrasted with the up-regulation of MET1 and CMT3 DNA methylases and the down-regulation of DNA demethylases (ROS1, DME and DML2). Hence, the global DNA methylation level appears not to correlate with the transcriptional activity of the genes encoding DNA methylases/demethylases, thereby implying the complexity of the regulatory mechanism that controls the DNA methylation status of the SE-epigenome. Moreover, distinct changes in the expression level of the SE-regulatory genes were indicated in the 5-AzaC-treated and DNA methylase mutant cultures. Accordingly, a significant repression of the LEC2, LEC1 and BBM genes was found in the 5-AzaC-treated culture that was incapable of SE induction. In contrast, the distinct up-regulation of these genes was observed in the drm1drm2 and drm1drm2cmt3 mutant cultures with an improved embryogenic response. The modulated expression of DNA methylase genes and the significantly modified embryogenic response of the met1 and drm mutants imply that both the maintenance and the de novo pathway of DNA methylation are engaged in the regulation of SE in Arabidopsis

Immature zygotic embryo cultures of Arabidopsis. Amodel system for molecular studies on morphogenic pathways induced in vitro

To understand the molecular mechanism controlling in vitro plant morphogenesis, a culture system enabling induction of alternative morphogenic pathways (somatic embryogenesis, SE; shoot organogenesis, ORG) in a well defined population of somatic cells is needed. Arabidopsis is the most useful model plant for genomic studies, but a system in which SE or ORG can be induced alternatively in the same type of explant has not been proposed. Immature zygotic embryos (IZEs) of Arabidopsis provide the only explants with embryogenic potential, and have been recommended for studying mechanisms of SE induced in vitro. This study was aimed at defining culture conditions promoting induction of alternative morphogenic pathways: shoot ORG in IZE explants. The established protocol involves pretreatment of IZE explants with liquid auxin-rich callus induction (CIM) medium, followed by subculture on solid cytokinin-rich shoot induction medium (SIM). The method enables efficient shoot induction in Columbia (Col-0) and Wassilewskija (Ws), genotypes commonly used in molecular studies. During 3 weeks of culture up to 90% of Col-0 and 70% of Ws explants regenerated shoots via an indirect morphogenic pathway. We analyzed the qRT-PCR expression patterns of the LEC (LEC1, LEC2 and FUS3) genes, the key regulators of Arabidopsis embryogenesis, in the IZE explants induced to promote shoot ORG. The sharp decline of LEC expression on SIM medium confirmed that culture of Arabidopsis IZE explants enables experimental manipulation of the morphogenic response of somatic cells. A scheme illustrating various in vitro morphogenic responses of IZEs in relation to hormonal treatment is presented