DNA methylation analysis of floral parts revealed dynamic changes during the development of homostylous Fagopyrum tataricum and heterostylous F. esculentum flowers.

BACKGROUND: Proper flower development is essential for plant reproduction, a crucial aspect of the plant life cycle. This process involves precisely coordinating transcription factors, enzymes, and epigenetic modifications. DNA methylation, a ubiquitous and heritable epigenetic mechanism, is pivotal in regulating gene expression and shaping chromatin structure. Fagopyrum esculentum demonstrates anti-hypertensive, anti-diabetic, anti-inflammatory, cardio-protective, hepato-protective, and neuroprotective properties. However, the heteromorphic heterostyly observed in F. esculentum poses a significant challenge in breeding efforts. F. tataricum has better resistance to high altitudes and harsh weather conditions such as drought, frost, UV-B radiation damage, and pests. Moreover, F. tataricum contains significantly higher levels of rutin and other phenolics, more flavonoids, and a balanced amino acid profile compared to common buckwheat, being recognised as functional food, rendering it an excellent candidate for functional food applications. RESULTS: This study aimed to compare the DNA methylation profiles between the Pin and Thrum flower components of F. esculentum, with those of self-fertile species of F. tataricum, to understand the potential role of this epigenetic mechanism in Fagopyrum floral development. Notably, F. tataricum flowers are smaller than those of F. esculentum (Pin and Thrum morphs). The decline in DNA methylation levels in the developed open flower components, such as petals, stigmas and ovules, was consistent across both species, except for the ovule in the Thrum morph. Conversely, Pin and Tartary ovules exhibited a minor decrease in DNA methylation levels. The highest DNA methylation level was observed in Pin stigma from closed flowers, and the most significant decrease was in Pin stigma from open flowers. In opposition, the nectaries of open flowers exhibited higher levels of DNA methylation than those of closed flowers. The decrease in DNA methylation might correspond with the downregulation of genes encoding methyltransferases. CONCLUSIONS: Reduced overall DNA methylation and the expression of genes associated with these epigenetic markers in fully opened flowers of both species may indicate that demethylation is necessary to activate the expression of genes involved in floral development.

Efficient Agrobacterium-mediated transformation and genome editing of Fagopyrum tataricum.

Fagopyrum tataricum (L.) Gaertn. is an exceptional crop known for its remarkable health benefits, high levels of beneficial polyphenols and gluten-free properties, making it highly sought-after as a functional food. Its self-fertilisation capability and adaptability to challenging environments further contribute to its potential as a sustainable agricultural option. To harness its unique traits, genetic transformation in F. tataricum is crucial. In this study, we optimised the Agrobacterium-mediated transformation protocol for F. tataricum callus, resulting in a transformation rate of regenerated plants of approximately 20%. The protocol's effectiveness was confirmed through successful GUS staining, GFP expression, and the generation of albino plants via FtPDS gene inactivation. These results validate the feasibility of genetic manipulation and highlight the potential for trait enhancement in F. tataricum.

Promotive effect of phytosulfokine - peptide growth factor - on protoplast cultures development in Fagopyrum tataricum (L.) Gaertn.

BACKGROUND: Fagopyrum tataricum (Tartary buckwheat) is a valuable crop of great nutritional importance due to its high level of bioactive compounds. Excellent opportunities to obtain plants with the high level or the desired profile of valuable metabolites may be provided by in vitro cultures. Among known in vitro techniques, protoplast technology is an exciting tool for genetic manipulation to improve crop traits. In that context, protoplast fusion may be applied to generate hybrid cells between different species of Fagopyrum. To apply protoplast cultures to the aforementioned approaches in this research, we established the protoplast-to-plant system in Tartary buckwheat. RESULTS: In this work, cellulase and pectinase activity enabled protoplast isolation from non-morphogenic and morphogenic callus (MC), reaching, on average, 2.3 × 106 protoplasts per g of fresh weight. However, to release protoplasts from hypocotyls, the key step was the application of driselase in the enzyme mixture. We showed that colony formation could be induced after protoplast embedding in agarose compared to the alginate matrix. Protoplasts cultured in a medium based on Kao and Michayluk supplemented with phytosulfokine (PSK) rebuilt cell walls, underwent repeated mitotic division, formed aggregates, which consequently led to callus formation. Plating efficiency, expressing the number of cell aggregate formed, in 10-day-old protoplast cultures varied from 14% for morphogenic callus to 30% for hypocotyls used as a protoplast source. However plant regeneration via somatic embryogenesis and organogenesis occurred only during the cultivation of MC-derived protoplasts. CONCLUSIONS: This study demonstrated that the applied protoplast isolation approach facilitated the recovery of viable protoplasts. Moreover, the embedding of protoplasts in an agarose matrix and supplementation of a culture medium with PSK effectively stimulated cell division and further development of Tartary buckwheat protoplast cultures along with the plant regeneration. Together, these results provide the first evidence of developing a protoplast-to-plant system from the MC of Fagopyrum tataricum used as source material. These findings suggest that Tartary buckwheat's protoplast cultures have potential implications for the species' somatic hybridization and genetic improvement.

The Chromosome Number and rDNA Loci Evolution in Onobrychis (Fabaceae).

The evolution of chromosome number and ribosomal DNA (rDNA) loci number and localisation were studied in Onobrychis Mill. Diploid and tetraploid species, as well as two basic chromosome numbers, x = 7 and x = 8, were observed among analysed taxa. The chromosomal distribution of rDNA loci was presented here for the first time using fluorescence in situ hybridisation (FISH) with 5S and 35S rDNA probes. Onobrychis species showed a high polymorphism in the number and localisation of rDNA loci among diploids, whereas the rDNA loci pattern was very similar in polyploids. Phylogenetic relationships among the species, inferred from nrITS sequences, were used as a framework to reconstruct the patterns of basic chromosome number and rDNA loci evolution. Analysis of the evolution of the basic chromosome numbers allowed the inference of x = 8 as the ancestral number and the descending dysploidy and polyploidisation as the major mechanisms of the chromosome number evolution. Analyses of chromosomal patterns of rRNA gene loci in a phylogenetic context resulted in the reconstruction of one locus of 5S rDNA and one locus of 35S rDNA in the interstitial chromosomal position as the ancestral state in this genus.

Plant Cell and Organism Development 2.0.

In the editorial summarising the first edition of the Special Issue on "Plant Cell and Organism Development", we listed the key features that make plants a unique and fascinating group of living organisms [...].

Buckwheat in Tissue Culture Research: Current Status and Future Perspectives.

Buckwheat is a member of a genus of 23 species, where the two most common species are Fagopyrum esculentum (common buckwheat) and Fagopyrum tataricum (Tartary buckwheat). This pseudocereal is a source of micro and macro nutrients, such as gluten-free proteins and amino acids, fatty acids, bioactive compounds, dietary fibre, fagopyrins, vitamins and minerals. It is gaining increasing attention due to its health-promoting properties. Buckwheat is widely susceptible to in vitro conditions which are used to study plantlet regeneration, callus induction, organogenesis, somatic embryogenesis, and the synthesis of phenolic compounds. This review summarises the development of buckwheat in in vitro culture and describes protocols for the regeneration of plantlets from various explants and differing concentrations of plant growth regulators. It also describes callus induction protocols as well as the role of calli in plantlet regeneration. Protocols for establishing hairy root cultures with the use of Agrobacterium rhizogens are useful in the synthesis of secondary metabolites, as well as protocols used for transgenic plants. The review also focuses on the future prospects of buckwheat in tissue culture and the challenges researchers are addressing.

Changes in the Cell Wall Proteome of Leaves in Response to High Temperature Stress in Brachypodium distachyon.

High temperature stress leads to complex changes to plant functionality, which affects, i.a., the cell wall structure and the cell wall protein composition. In this study, the qualitative and quantitative changes in the cell wall proteome of Brachypodium distachyon leaves in response to high (40 °C) temperature stress were characterised. Using a proteomic analysis, 1533 non-redundant proteins were identified from which 338 cell wall proteins were distinguished. At a high temperature, we identified 46 differentially abundant proteins, and of these, 4 were over-accumulated and 42 were under-accumulated. The most significant changes were observed in the proteins acting on the cell wall polysaccharides, specifically, 2 over- and 12 under-accumulated proteins. Based on the qualitative analysis, one cell wall protein was identified that was uniquely present at 40 °C but was absent in the control and 24 proteins that were present in the control but were absent at 40 °C. Overall, the changes in the cell wall proteome at 40 °C suggest a lower protease activity, lignification and an expansion of the cell wall. These results offer a new insight into the changes in the cell wall proteome in response to high temperature.

3,4-Dehydro-L-proline Induces Programmed Cell Death in the Roots of Brachypodium distachyon.

As cell wall proteins, the hydroxyproline-rich glycoproteins (HRGPs) take part in plant growth and various developmental processes. To fulfil their functions, HRGPs, extensins (EXTs) in particular, undergo the hydroxylation of proline by the prolyl-4-hydroxylases. The activity of these enzymes can be inhibited with 3,4-dehydro-L-proline (3,4-DHP), which enables its application to reveal the functions of the HRGPs. Thus, to study the involvement of HRGPs in the development of root hairs and roots, we treated seedlings of Brachypodium distachyon with 250 µM, 500 µM, and 750 µM of 3,4-DHP. The histological observations showed that the root epidermis cells and the cortex cells beneath them ruptured. The immunostaining experiments using the JIM20 antibody, which recognizes the EXT epitopes, demonstrated the higher abundance of this epitope in the control compared to the treated samples. The transmission electron microscopy analyses revealed morphological and ultrastructural features that are typical for the vacuolar-type of cell death. Using the TUNEL test (terminal deoxynucleotidyl transferase dUTP nick end labelling), we showed an increase in the number of nuclei with damaged DNA in the roots that had been treated with 3,4-DHP compared to the control. Finally, an analysis of two metacaspases' gene activity revealed an increase in their expression in the treated roots. Altogether, our results show that inhibiting the prolyl-4-hydroxylases with 3,4-DHP results in a vacuolar-type of cell death in roots, thereby highlighting the important role of HRGPs in root hair development and root growth.

Plant Cell and Organism Development.

Plants represent a unique and fascinating group of living organisms [...].

A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum.

The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits.

From Single Cell to Plants: Mesophyll Protoplasts as a Versatile System for Investigating Plant Cell Reprogramming.

Plants are sessile organisms that have a remarkable developmental plasticity, which ensures their optimal adaptation to environmental stresses. Plant cell totipotency is an extreme example of such plasticity, whereby somatic cells have the potential to form plants via direct shoot organogenesis or somatic embryogenesis in response to various exogenous and/or endogenous signals. Protoplasts provide one of the most suitable systems for investigating molecular mechanisms of totipotency, because they are effectively single cell populations. In this review, we consider the current state of knowledge of the mechanisms that induce cell proliferation from individual, differentiated somatic plant cells. We highlight initial explant metabolic status, ploidy level and isolation procedure as determinants of successful cell reprogramming. We also discuss the importance of auxin signalling and its interaction with stress-regulated pathways in governing cell cycle induction and further stages of plant cell totipotency.

In Vitro Tissue Culture in Brachypodium: Applications and Challenges.

Brachypodium distachyon has become an excellent model for plant breeding and bioenergy grasses that permits many fundamental questions in grass biology to be addressed. One of the constraints to performing research in many grasses has been the difficulty with which they can be genetically transformed and the generally low frequency of such transformations. In this review, we discuss the contribution that transformation techniques have made in Brachypodium biology as well as how Brachypodium could be used to determine the factors that might contribute to transformation efficiency. In particular, we highlight the latest research on the mechanisms that govern the gradual loss of embryogenic potential in a tissue culture and propose using B. distachyon as a model for other recalcitrant monocots.

CDKG1 Is Required for Meiotic and Somatic Recombination Intermediate Processing in Arabidopsis.

The Arabidopsis (Arabidopsis thaliana) cyclin-dependent kinase G1 (CDKG1) is necessary for recombination and synapsis during male meiosis at high ambient temperature. In the cdkg1-1 mutant, synapsis is impaired and there is a dramatic reduction in the number of class I crossovers, resulting in univalents at metaphase I and pollen sterility. Here, we demonstrate that CDKG1 is necessary for the processing of recombination intermediates in the canonical ZMM recombination pathway and that loss of CDKG1 results in increased class II crossovers. While synapsis and events associated with class I crossovers are severely compromised in a cdkg1-1 mutant, they can be restored by increasing the number of recombination intermediates in the double cdkg1-1 fancm-1 mutant. Despite this, recombination intermediates are not correctly resolved, leading to the formation of chromosome aggregates at metaphase I. Our results show that CDKG1 acts early in the recombination process and is necessary to stabilize recombination intermediates. Finally, we show that the effect on recombination is not restricted to meiosis and that CDKG1 is also required for normal levels of DNA damage-induced homologous recombination in somatic tissues.

Comparatively Barcoded Chromosomes of Brachypodium Perennials Tell the Story of Their Karyotype Structure and Evolution.

The Brachypodium genus is an informative model system for studying grass karyotype organization. Previous studies of a limited number of species and reference chromosomes have not provided a comprehensive picture of the enigmatic phylogenetic relationships in the genus. Comparative chromosome barcoding, which enables the reconstruction of the evolutionary history of individual chromosomes and their segments, allowed us to infer the relationships between putative ancestral karyotypes of extinct species and extant karyotypes of current species. We used over 80 chromosome-specific BAC (bacterial artificial chromosome) clones derived from five reference chromosomes of B. distachyon as probes against the karyotypes of twelve accessions representing five diploid and polyploid Brachypodium perennials. The results showed that descending dysploidy is common in Brachypodium and occurs primarily via nested chromosome fusions. Brachypodium distachyon was rejected as a putative ancestor for allotetraploid perennials and B. stacei for B. mexicanum. We propose two alternative models of perennial polyploid evolution involving either the incorporation of a putative x = 5 ancestral karyotype with different descending dysploidy patterns compared to B. distachyon chromosomes or hybridization of two x = 9 ancestors followed by genome doubling and descending dysploidy. Details of the karyotype structure and evolution in several Brachypodium perennials are revealed for the first time.

Hydroxyproline-Rich Glycoproteins as Markers of Temperature Stress in the Leaves of Brachypodium distachyon.

Plants frequently encounter diverse abiotic stresses, one of which is environmental thermal stress. To cope with these stresses, plants have developed a range of mechanisms, including altering the cell wall architecture, which is facilitated by the arabinogalactan proteins (AGP) and extensins (EXT). In order to characterise the localisation of the epitopes of the AGP and EXT, which are induced by the stress connected with a low (4 °C) or a high (40 °C) temperature, in the leaves of Brachypodium distachyon, we performed immunohistochemical analyses using the antibodies that bind to selected AGP (JIM8, JIM13, JIM16, LM2 and MAC207), pectin/AGP (LM6) as well as EXT (JIM11, JIM12 and JIM20). The analyses of the epitopes of the AGP indicated their presence in the phloem and in the inner bundle sheath (JIM8, JIM13, JIM16 and LM2). The JIM16 epitope was less abundant in the leaves from the low or high temperature compared to the control leaves. The LM2 epitope was more abundant in the leaves that had been subjected to the high temperatures. In the case of JIM13 and MAC207, no changes were observed at the different temperatures. The epitopes of the EXT were primarily observed in the mesophyll and xylem cells of the major vascular bundle (JIM11, JIM12 and JIM20) and no correlation was observed between the presence of the epitopes and the temperature stress. We also analysed changes in the level of transcript accumulation of some of the genes encoding EXT, EXT-like receptor kinases and AGP in the response to the temperature stress. In both cases, although we observed the upregulation of the genes encoding AGP in stressed plants, the changes were more pronounced at the high temperature. Similar changes were observed in the expression profiles of the EXT and EXT-like receptor kinase genes. Our findings may be relevant for genetic engineering of plants with increased resistance to the temperature stress.

Defining the Genetic Basis of Plant⁻Endophytic Bacteria Interactions.

Endophytic bacteria, which interact closely with their host, are an essential part of the plant microbiome. These interactions enhance plant tolerance to environmental changes as well as promote plant growth, thus they have become attractive targets for increasing crop production. Numerous studies have aimed to characterise how endophytic bacteria infect and colonise their hosts as well as conferring important traits to the plant. In this review, we summarise the current knowledge regarding endophytic colonisation and focus on the insights that have been obtained from the mutants of bacteria and plants as well as 'omic analyses. These show how endophytic bacteria produce various molecules and have a range of activities related to chemotaxis, motility, adhesion, bacterial cell wall properties, secretion, regulating transcription and utilising a substrate in order to establish a successful interaction. Colonisation is mediated by plant receptors and is regulated by the signalling that is connected with phytohormones such as auxin and jasmonic (JA) and salicylic acids (SA). We also highlight changes in the expression of small RNAs and modifications of the cell wall properties. Moreover, in order to exploit the beneficial plant-endophytic bacteria interactions in agriculture successfully, we show that the key aspects that govern successful interactions remain to be defined.

Ribosomal DNA loci derived from Brachypodium stacei are switched off for major parts of the life cycle of Brachypodium hybridum.

Nucleolar dominance is an epigenetic phenomenon that occurs in some plant and animal allopolyploids and hybrids, whereby only one ancestral set of 35S rRNA genes retains the ability to form the nucleolus while the rDNA loci derived from the other progenitor are transcriptionally silenced. There is substantial evidence that nucleolar dominance is regulated developmentally. This study focuses upon the establishment and/or maintenance of nucleolar dominance during different stages of development in the model grass allotetraploid Brachypodium hybridum. Fluorescence in situ hybridization with a 25S rDNA probe to cells in three-dimensional cytogenetic preparations showed that nucleolar dominance is present not only in root meristematic and differentiated cells of this species, but also in male meiocytes at prophase I, tetrads of microspores, and different embryonic tissues. The inactive state of Brachypodium stacei-originated rDNA loci was confirmed by silver staining. Only B. distachyon-derived 35S rDNA loci formed nucleoli in the aforementioned tissues, whereas B. stacei-like loci remained highly condensed and thus transcriptionally suppressed. The establishment of nucleolar dominance during earlier stages of B. hybridum embryo development cannot be ruled out. However, we propose that gradual pseudogenization of B. stacei-like loci in the evolution of the allotetraploid seems to be more likely.

Cell Wall Epitopes and Endoploidy as Reporters of Embryogenic Potential in Brachypodium Distachyon Callus Culture.

Effective regeneration of callus tissue into embryos and then into whole plants is essential for plant biotechnology. The embryonic potential is often low and can further decrease with time in culture, which limits the utilisation of calli for transformation procedures and in vitro propagation. In this study, we show that the loss of embryogenic potential in callus cultures of Brachypodium distachyon is progressive over time. Flow cytometry analyses indicated endoploidy levels increased in 60- and 90-day-old calli with effective loss of the 2C DNA content peak in the latter. Analysis of indolic compounds content revealed a decrease in 60- and 90-day-old calli compared to either freshly isolated explants or 30-day-old calli. Immunohistochemical analysis revealed a decrease in arabinogalactan proteins (AGP) signal with the time of culture, but extensin (EXT) epitopes either increased (JIM12 epitopes) or decreased (JIM11 epitopes). The transcript accumulation levels of AGPs and EXTs confirmed these results, with most of AGP and EXT transcripts gradually decreasing. Some chimeric EXT transcripts significantly increased on the 30th day of culture, perhaps because of an increased embryogenic potential. Selected somatic embryogenesis-related genes and cyclins demonstrated a gradual decrease of transcript accumulation for YUCCA (YUC), AINTEGUMENTA-LIKE (AIL), BABY BOOM (BBM), and CLAVATA (CLV3) genes, as well as for most of the cyclins, starting from the 30th day of culture. Notably, WUSCHEL (WUS) transcript was detectable only on the 30th and 60th day and was not detectable in the zygotic embryos and in 90-day-old calli.

Germination and the Early Stages of Seedling Development in Brachypodium distachyon.

Successful germination and seedling development are crucial steps in the growth of a new plant. In this study, we investigated the course of the cell cycle during germination in relation to grain hydration in the model grass Brachypodium distachyon (Brachypodium) for the first time. Flow cytometry was performed to monitor the cell cycle progression during germination and to estimate DNA content in embryo tissues. The analyses of whole zygotic embryos revealed that the relative DNA content was 2C, 4C, 8C, and 16C. Endoreplicated nuclei were detected in the scutellum and coleorhiza cells, whereas the rest of the embryo tissues only had nuclei with a 2C and 4C DNA content. This study was accompanied by a spatiotemporal profile analysis of the DNA synthetic activity in the organs of Brachypodium embryos during germination using EdU labelling. Upon imbibition, nuclear DNA replication was initiated in the radicle within 11 h and subsequently spread towards the plumule. The first EdU-labelled prophases were observed after 14 h of imbibition. Analysis of selected genes that are involved in the regulation of the cell cycle, such as those encoding cyclin-dependent kinases and cyclins, demonstrated an increase in their expression profiles.