The improvement of the in vitro plant regeneration in barley with the epigenetic modifier of histone acetylation, trichostatin A.

Genotype-limited plant regeneration is one of the main obstacles to the broader use of genetic transformation in barley breeding. Thus, developing new approaches that might improve responses of in vitro recalcitrant genotypes remains at the center of barley biotechnology. Here, we analyzed different barley genotypes, including "Golden Promise," a genotype commonly used in the genetic transformation, and four malting barley cultivars of poor regenerative potential. The expression of hormone-related transcription factor (TF) genes with documented roles in plant regeneration was analyzed in genotypes with various plant-regenerating capacities. The results indicated differential expression of auxin-related TF genes between the barley genotypes in both the explants and the derived cultures. In support of the role of auxin in barley regeneration, distinct differences in the accumulation of free and oxidized auxin were observed in explants and explant-derived callus cultures of barley genotypes. Following the assumption that modifying gene expression might improve plant regeneration in barley, we treated the barley explants with trichostatin A (TSA), which affects histone acetylation. The effects of TSA were genotype-dependent as TSA treatment improved plant regeneration in two barley cultivars. TSA-induced changes in plant regeneration were associated with the increased expression of auxin biosynthesis-involved TFs. The study demonstrated that explant treatment with chromatin modifiers such as TSA might provide a new and effective epigenetic approach to improving plant regeneration in recalcitrant barley genotypes.

Contribution of cultural heritage values to steppe conservation on ancient burial mounds of Eurasia.

Civilizations, including ancient ones, have shaped global ecosystems in many ways through coevolution of landscapes and humans. However, the cultural legacies of ancient and lost civilizations are rarely considered in the conservation of the Eurasian steppe biome. We used a data set containing more than 1000 records on localities, land cover, protection status, and cultural values related to ancient steppic burial mounds (kurgans); we evaluated how these iconic and widespread landmarks can contribute to grassland conservation in the Eurasian steppes, which is one of the most endangered biomes on Earth. Using Bayesian logistic generalized regressions and proportional odds logistic regressions, we examined the potential of mounds to preserve grasslands in landscapes with different levels of land-use transformation. We also compared the conservation potential of mounds inside and outside protected areas and assessed whether local cultural values support the maintenance of grasslands on them. Kurgans were of great importance in preserving grasslands in transformed landscapes outside protected areas, where they sometimes acted as habitat islands that contributed to habitat conservation and improved habitat connectivity. In addition to steep slopes hindering ploughing, when mounds had cultural value for local communities, the probability of grassland occurrence on kurgans almost doubled. Because the estimated number of steppic mounds is about 600,000 and similar historical features exist on all continents, our results may be applicable at a global level. Our results also suggested that an integrative socioecological approach in conservation might support the positive synergistic effects of conservation, landscape, and cultural values.

Contribución de los valores culturales para la conservación esteparia en los antiguos montículos funerarios de Eurasia Resumen Las civilizaciones modernas y antiguas han moldeado de muchas maneras los ecosistemas globales mediante la coevolución del paisaje y la humanidad. Sin embargo, pocas veces se considera el legado cultural de las civilizaciones perdidas o antiguas para la conservación del bioma de la estepa euroasiática. Usamos un conjunto de datos que contiene más de 1,000 registros de las localidades, cobertura del suelo, estado de protección y valores culturales relacionados con los antiguos montículos funerarios de esta estepa (kurgans). Después analizamos cómo estos símbolos icónicos y distribuidos extensamente pueden contribuir a la conservación de los pastizales en la estepa euroasiática, uno de los biomas en mayor peligro de extinción. Analizamos el potencial de conservación de los montículos en paisajes con diferentes niveles de transformación en el uso de suelo mediante regresiones logísticas generalizadas bayesianas y regresiones logísticas de probabilidades proporcionales. También comparamos el potencial de conservación de los montículos dentro y fuera de las áreas protegidas y evaluamos si los valores culturales locales conservan los pastizales dentro de estas mismas áreas. Los kurgans fueron de gran importancia para la conservación de los pastizales en los paisajes transformados ubicados fuera de las áreas protegidas, en donde llegaron a fungir como hábitats aislados que contribuyeron a la conservación y conectividad del hábitat. Además de que las pendientes pronunciadas impiden el arado, cuando los montículos contaban con valor cultural para las comunidades locales, la probabilidad de que el pastizal se ubicara sobre un kurgan casi se duplicó. Ya que se estima que el número de montículos esteparios ronda los 6,000 y que rasgos históricos similares existen en todos los continentes, nuestros resultados pueden aplicarse a nivel global. Nuestros resultados también sugieren que una estrategia socio-ecológica integradora para la conservación podría respaldar los efectos sinérgicos positivos de la conservación, el paisaje y los valores culturales.

Game of thrones among AUXIN RESPONSE FACTORs-over 30 years of MONOPTEROS research.

For many years, research has been carried out with the aim of understanding the mechanism of auxin action, its biosynthesis, catabolism, perception, and transport. One central interest is the auxin-dependent gene expression regulation mechanism involving AUXIN RESPONSE FACTOR (ARF) transcription factors and their repressors, the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins. Numerous studies have been focused on MONOPTEROS (MP)/ARF5, an activator of auxin-dependent gene expression with a crucial impact on plant development. This review summarizes over 30 years of research on MP/ARF5. We indicate the available analytical tools to study MP/ARF5 and point out the known mechanism of MP/ARF5-dependent regulation of gene expression during various developmental processes, namely embryogenesis, leaf formation, vascularization, and shoot and root meristem formation. However, many questions remain about the auxin dose-dependent regulation of gene transcription by MP/ARF5 and its isoforms in plant cells, the composition of the MP/ARF5 protein complex, and, finally, all the genes under its direct control. In addition, information on post-translational modifications of MP/ARF5 protein is marginal, and knowledge about their consequences on MP/ARF5 function is limited. Moreover, the epigenetic factors and other regulators that act upstream of MP/ARF5 are poorly understood. Their identification will be a challenge in the coming years.

Ancient settlements as natural heritage sites: the first occurrence dataset on vascular plant species from ancient settlements in the Lower Dnipro region (Ukraine).

Background: This work is a long-term outcome of an international Ukrainian-Polish teamwork, aiming to assess the role of ancient settlements for steppe conservation and protection. The dataset contains georeferenced occurrences of vascular plant species on 18 ancient settlements (Lower Dnipro, southern Ukraine), collected during the 2015-2020 period. Additionally, to the total species list, the publication presents the taxonomic coverage (according to GBIF Backbone Taxonomy), the frequency classes of occurrences of the total taxa and the floristic differences amongst studied sites. The report also shows the high sozological value of the studied ancient settlements, the high levels of vascular plant species richness and the various means of the plant species protection (according to the Bern Convention, the Red Data Book of Ukraine and regional Red Lists). New information: This work provides the first occurrence dataset from ancient settlements in Ukraine. The dataset includes 3,210 occurrences of vascular plants recorded during the study period of 2015-2020 conducted in the Lower Dnipro region. As ancient settlements were generally considered as steppe refuges, great attention was paid to the native steppe species, as well as to the rare components of the flora. The dataset includes 1,525 occurrences of steppe species and 87 occurrences of rare species, respectively. The dataset could be useful for further research of ancient settlements` floristic richness, but also analyses and comparison with other objects of cultural origin (e.g. kurgans, hillforts, old cemeteries, forgotten parks, sacred groves etc.).

The first dataset of vascular plant species occurrences on kurgans in Southern Ukraine.

Background: The dataset contains the records of vascular plant species occurrences and distribution on Ukrainian kurgans (burial mounds, barrows), located in various zones of steppe vegetation: desert steppe, grass steppe, herb-rich grass steppe and forest steppe. Much of the studied kurgans belongs to the territory historically known as the "Wild Fields". Besides the occurrence data, the publication presents a comparison of the floristic richness amongst five microhabitats distinguished on kurgans (top, northern slope, northern bottom, southern slope, and southern bottom) and amongst kurgans located in different steppe zones. The Original publication includes 721 species of vascular plants) within four vegetation zone (desert steppe, grass steppe, herb-rich grass steppe and forest steppe). The report shows also sozological value of kurgans in southern Ukraine, as they play a role of steppe habitat islands in a landscape almost completely transformed to arable land. The obtained flora inventory was analyzed in various aspects. This occurrence dataset is the first public record of species from kurgans in Ukraine. New information: This is the first occurrence dataset from kurgans in Ukraine. The dataset includes 28,456 occurrences of vascular plants recorded in the years 2004-2009 on Ukrainian kurgans. The dataset includes information about 1446 occurrences of rare species on kurgans (69 species). It contains information on the kurgan flora within four vegetation zone (desert steppe, grass steppe, herb-rich grass steppe and forest steppe) on the area ca. 32000 km2. Of the approximately 450 mounds visited, the ones with the best preserved vegetation cover were selected. For each of 106 investigated mounds, floristic lists from five microhabitats were compiled - 530 lists in total.

Vascular plants of old cemeteries in the Lower Dnipro region (Southern Ukraine).

Background: The dataset contains the records of vascular plant species occurrences and distribution in old cemeteries (OC) of the Lower Dnipro region (Southern Ukraine). The analysed cemeteries were located in different types of landscapes (agricultural, rural and urban) and represent various ways of using their area (currently used, closed, abandoned). The floristic list includes 440 species of vascular plants (437 in situ, 3 ex situ). The dataset demonstrates a sozological (Red-lists species) value of old cemeteries in Southern Ukraine. The cemeteries constitute refuges of native, rare and steppe flora and play a role of steppe habitat islands in a landscape almost completely transformed to arable land or urbanised. New information: This is the first dataset which contains information about flora of old cemeteries in Lower Dnipro region (Southern Ukraine). The dataset comprises 2118 occurrences of vascular plants (440 species) recorded in the years 2008-2021 in 13 old cemeteries of the Lower Dnipro region. The dataset includes information about 85 occurrences of rare species (23 species in situ, 3 ex situ) and 652 occurrences of 117 steppe species.

Insights into the Histone Acetylation-Mediated Regulation of the Transcription Factor Genes That Control the Embryogenic Transition in the Somatic Cells of Arabidopsis.

Somatic embryogenesis (SE), which is a process that involves the in vitro-induced embryogenic reprogramming of plant somatic cells, requires dynamic changes in the cell transcriptome. These changes are fine-tuned by many genetic and epigenetic factors, including posttranslational histone modifications such as histone acetylation. Antagonistically acting enzymes, histone acetyltransferases (HATs) and deacetylases (HDACs), which control histone acetylation in many developmental processes, are believed to control SE. However, the function of specific HAT/HDACs and the genes that are subjected to histone acetylation-mediated regulation during SE have yet to be revealed. Here, we present the global and gene-specific changes in histone acetylation in Arabidopsis explants that are undergoing SE. In the TSA (trichostatin A)-induced SE, we demonstrate that H3 and H4 acetylation might control the expression of the critical transcription factor (TF) genes of a vital role in SE, including LEC1, LEC2 (LEAFY COTYLEDON 1; 2), FUS3 (FUSCA 3) and MYB118 (MYB DOMAIN PROTEIN 118). Within the HATs and HDACs, which mainly positively regulate SE, we identified HDA19 as negatively affecting SE by regulating LEC1, LEC2 and BBM. Finally, we provide some evidence on the role of HDA19 in the histone acetylation-mediated regulation of LEC2 during SE. Our results reveal an essential function of histone acetylation in the epigenetic mechanisms that control the TF genes that play critical roles in the embryogenic reprogramming of plant somatic cells. The results implicate the complexity of Hac-related gene regulation in embryogenic induction and point to differences in the regulatory mechanisms that are involved in auxin- and TSA-induced SE.

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.

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 different 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.

Trichostatin A Triggers an Embryogenic Transition in Arabidopsis Explants via an Auxin-Related Pathway.

Auxin is an important regulator of plant ontogenies including embryo development and the exogenous application of this phytohormone has been found to be necessary for the induction of the embryogenic response in plant explants that have been cultured in vitro. However, in the present study, we show that treatment of Arabidopsis explants with trichostatin A (TSA), which is a chemical inhibitor of histone deacetylases, induces somatic embryogenesis (SE) without the exogenous application of auxin. We found that the TSA-treated explants generated somatic embryos that developed efficiently on the adaxial side of the cotyledons, which are the parts of an explant that are involved in auxin-induced SE. A substantial reduction in the activity of histone deacetylase (HDAC) was observed in the TSA-treated explants, thus confirming a histone acetylation-related mechanism of the TSA-promoted embryogenic response. Unexpectedly, the embryogenic effect of TSA was lower on the auxin-supplemented media and this finding further suggests an auxin-related mechanism of TSA-induced SE. Congruently, we found a significantly increased content of indolic compounds, which is indicative of IAA and an enhanced DR5::GUS signal in the TSA-treated explants. In line with these results, two of the YUCCA genes (YUC1 and YUC10), which are involved in auxin biosynthesis, were found to be distinctly up-regulated during TSA-induced SE and their expression was colocalised with the explant sites that are involved in SE. Beside auxin, ROS were extensively accumulated in response to TSA, thereby indicating that a stress-response is involved in TSA-triggered SE. Relevantly, we showed that the genes encoding the transcription factors (TFs) that have a regulatory function in auxin biosynthesis including LEC1, LEC2, BBM, and stress responses (MYB118) were highly up-regulated in the TSA-treated explants. Collectively, the results provide several pieces of evidence about the similarities between the molecular pathways of SE induction that are triggered by TSA and 2,4-D that involve the activation of the auxin-responsive TF genes that have a regulatory function in auxin biosynthesis and stress responses. The study suggests the involvement of histone acetylation in the auxin-mediated release of the embryogenic program of development in the somatic cells of Arabidopsis.

Maternal auxin supply contributes to early embryo patterning in Arabidopsis.

The angiosperm seed is composed of three genetically distinct tissues: the diploid embryo that originates from the fertilized egg cell, the triploid endosperm that is produced from the fertilized central cell, and the maternal sporophytic integuments that develop into the seed coat1. At the onset of embryo development in Arabidopsis thaliana, the zygote divides asymmetrically, producing a small apical embryonic cell and a larger basal cell that connects the embryo to the maternal tissue2. The coordinated and synchronous development of the embryo and the surrounding integuments, and the alignment of their growth axes, suggest communication between maternal tissues and the embryo. In contrast to animals, however, where a network of maternal factors that direct embryo patterning have been identified3,4, only a few maternal mutations have been described to affect embryo development in plants5-7. Early embryo patterning in Arabidopsis requires accumulation of the phytohormone auxin in the apical cell by directed transport from the suspensor8-10. However, the origin of this auxin has remained obscure. Here we investigate the source of auxin for early embryogenesis and provide evidence that the mother plant coordinates seed development by supplying auxin to the early embryo from the integuments of the ovule. We show that auxin response increases in ovules after fertilization, due to upregulated auxin biosynthesis in the integuments, and this maternally produced auxin is required for correct embryo development.

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. 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.

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.

LEAFY COTYLEDON2 (LEC2) promotes embryogenic induction in somatic tissues of Arabidopsis, via YUCCA-mediated auxin biosynthesis.

The LEAFY COTYLEDON2 (LEC2) transcription factor with a plant-specific B3 domain plays a central role in zygotic and somatic embryogenesis (SE). LEC2 overexpression induced in planta leads to spontaneous somatic embryo formation, but impairs the embryogenic response of explants cultured in vitro under auxin treatment. The auxin-related functions of LEC2 appear during SE induction, and the aim of the present study was to gain further insights into this phenomenon. To this end, the effect of LEC2 overexpression on the morphogenic responses of Arabidopsis explants cultured in vitro under different auxin treatments was evaluated. The expression profiles of the auxin biosynthesis genes were analysed in embryogenic cultures with respect to LEC2 activity. The results showed that LEC2 overexpression severely modifies the requirement of cultured explants for an exogenous auxin concentration at a level that is effective in SE induction and suggested an increase in the auxin content in 35S::LEC2-GR transgenic explants. The assumption of an LEC2 promoted increase in endogenous auxin in cultured explants was further supported by the expression profiling of the genes involved in auxin biosynthesis. The analysis indicated that YUCCAs and TAA1, working in the IPA-YUC auxin biosynthesis pathway, are associated with SE induction, and that the expression of three YUCCA genes (YUC1, YUC4 and YUC10) is associated with LEC2 activity. The results also suggest that the IAOx-mediated auxin biosynthesis pathway involving ATR1/MYB34 and CYP79B2 does not seem to be involved in SE induction. We conclude that de novo auxin production via the tryptophan-dependent IPA-YUC auxin biosynthesis pathway is implicated in SE induction, and that LEC2 plays a key role in this mechanism.