Revealing cis- and trans-regulatory elements underlying nuclear distribution and function of the Arabidopsis histone H2B.8 variant.

The H2B.8 variant has been diverged from other variants by its extended N-terminal region that possesses a conserved domain. We generated transgenic Arabidopsis plants expressing H2B.9 (class I), H2B.5 (class II) and H2B.8 (class III) fused to GFP under the 35 S promoter and studied their nuclear distribution and function. H2B.8-GFP showed peculiar nuclear localization at chromocenters in all cell types examined, while H2B.5-GFP and H2B.9-GFP displayed various patterns often dependent on cell types. H2B variants faithfully assembled onto nucleosomes showing no effect on nuclear organization; H2B.8-GFP appeared as three distinct isoforms in which one isoform appeared to be SUMOylated. Interestingly, transient expression in protoplasts revealed H2B.8 nuclear localization distinct from transgenic plants as it was restricted to the nuclear periphery generating a distinctive ring-like appearance accompanied by nuclear size reduction. This unique appearance was abolished by deletion of the N-terminal conserved domain or when H2B.8-GFP is transiently expressed in ddm1 protoplasts. GFP-TRAP-coupled proteome analysis uncovered H2B.8-partner proteins including H2A.W.12, which characterizes heterochromatin. Thus, our data highlight H2B.8 as a unique variant evolved in angiosperms to control chromatin compaction/aggregation and uncover cis- and trans-regulatory elements underlying its nuclear distribution and function.

Mass spectrometry-based metabolite profiling reveals functional seasonal shifts in the metabolome of Zygophyllum dumosum Boiss and its relation to environmental conditions.

MAIN CONCLUSION: A multi-year study of perennial Z. dumosum shows a consistent seasonal pattern in the changes of petiole metabolism, involving mainly organic acids, polyols, phenylpropanoids, sulfate conjugates, and piperazines. GC-MS and UPLC-QTOF-MS-based metabolite profiling was performed on the petioles of the perennial desert shrub Zygophyllum dumosum Boiss (Zygophyllaceae). The petioles, which are physiologically functional throughout the year and, thus, exposed to seasonal rhythms, were collected every month for 3 years from their natural ecosystem on a southeast-facing slope. Results showed a clear multi-year pattern following seasonal successions, despite different climate conditions, i.e., rainy and drought years, throughout the research period. The metabolic pattern of change encompassed an increase in the central metabolites, including most polyols, e.g., stress-related D-pinitol, organic and sugar acids, and in the dominant specialized metabolites, which were tentatively identified as sulfate, flavonoid, and piperazine conjugates during the summer-autumn period, while significantly high levels of free amino acids were detected during the winter-spring period. In parallel, the levels of most sugars (including glucose and fructose) increased in the petioles at the flowering stage at the beginning of the spring, while most of the di- and tri-saccharides accumulated at the beginning of seed development (May-June). Analysis of the conserved seasonal metabolite pattern of change shows that metabolic events are mostly related to the stage of plant development and its interaction with the environment and less to environmental conditions per se.

What Worth the Garlic Peel.

Plants have two types of reproduction: sexual, resulting in embryo production, and asexual, resulting in vegetative bodies commonly derived from stems and roots (e.g., bulb, tuber). Dead organs enclosing embryos (DOEEs, such as seed coat and pericarp) are emerging as central components of the dispersal unit acting to nurture the embryo and ensure its survival in the habitat. Here we wanted to investigate the properties of dead organs enclosing plant asexual reproductive bodies, focusing on the garlic (Allium sativum) bulb. We investigated the biochemical and biological properties of the outer peel enclosing the bulb and the inner peel enclosing the clove using various methodologies, including bioassays, proteomics, and metabolomics. The garlic peels differentially affected germination and post-germination growth, with the outer peel demonstrating a strong negative effect on seed germination of Sinapis alba and on post-germination growth of Brassica juncea. Proteome analysis showed that dead garlic peels possess 67 proteins, including chitinases and proteases, which retained their enzymatic activity. Among primary metabolites identified in garlic peels, the outer peel accumulated multiple sugars, including rhamnose, mannitol, sorbitol, and trehalose, as well as the modified amino acid 5-hydroxylysine, known as a major component of collagen, at a higher level compared to the clove and the inner peel. Growth of Escherichia coli and Staphylococcus aureus was promoted by garlic peel extracts but inhibited by clove extract. All extracts strongly inhibited spore germination of Fusarium oxysporum f.sp. melonis. Thus, the garlic peels not only provide physical protection to vegetative offspring but also appear to function as a refined arsenal of proteins and metabolites for enhancing growth and development, combating potential pathogens, and conferring tolerance to abiotic stresses.

Differential Response to Single and Combined Salt and Heat Stresses: Impact on Accumulation of Proteins and Metabolites in Dead Pericarps of Brassica juncea.

Dead organs enclosing embryos, such as seed coats and pericarps, are emerging as important maternally-derived components of the dispersal unit that affect seed performance and fate. In the face of climate change and increased incidents of heatwaves, we sought to investigate the effect of salinity (S), short episodes of high temperature (HS), and combination of S + HS (SHS), at the reproductive phase, on the properties of dead pericarps of Brassica juncea. Proteome and metabolome analyses revealed multiple proteins and metabolites stored in dead pericarps whose levels and composition were altered under single and combined stress conditions. The protein profile of SHS showed a higher correlation with salt than with HS indicating the dominant effect of salt over heat stress. On the other hand, the analysis of metabolites showed that the profile of SHS has better correlation with HS than with salt. The integration of metabolic and proteomic data showed that changes in TCA cycle intermediates and certain amino acids (e.g., proline) under salt treatments (S and SHS) are highly correlated with changes in proteins involved in their biosynthetic pathways. Thus, accumulation of proteins and metabolites in dead pericarps is differently affected by single and combination of salt and heat stresses. Salinity appears to dominate plant response to combined stresses at the protein level, while heat appears to be the major factor affecting metabolite accumulation in dead pericarps.

Extreme drought alters progeny dispersal unit properties of winter wild oat (Avena sterilis L.).

MAIN CONCLUSION: The dead husk is a vital component of the dispersal unit whose biochemical properties can be modified following exposure to drought. This might affect seed performance and fate, soil properties and consequently plant biodiversity. We investigated the effects of extreme drought on the dispersal unit (DU) properties of winter wild oat (Avena sterilis L.) in the Mediterranean ecosystems focusing on a commonly ignored component of the DU, namely the dead floral bracts (husk). DUs were collected from a climate change experimental research station in the Judean Hills, Israel, simulating extreme drought and from two additional sites differing in the rainfall amounts. Our results showed that drought conditions significantly affected A. sterilis reproductive traits displaying reduced DUs and caryopses weights. The husk contributes profoundly to seed performance showing that germination from the intact DUs or the intact florets 1 was higher, faster and more homogenous compared to naked caryopses; no effect of drought on germination properties was observed. The husk stored hundreds of proteins that retain enzymatic activity and multiple metabolites including phytohormones. Changes in rainfall amounts affected the composition and levels of proteins and other metabolites accumulated in the husk, with a notable effect on abscisic acid (ABA). The husk of both control and drought plants released upon hydration substances that selectively inhibited other species seed germination as well as substances that promoted microbial growth. Our data showed that the dead husk represents a functional component of the DU that have been evolved to nurture the embryo and to ensure its success in its unique habitat. Furthermore, drought conditions can modify husk biochemical properties, which in turn might affect seed performance and fate, soil microbiota and soil fertility and consequently plant species diversity.

Dead but Not Dead End: Multifunctional Role of Dead Organs Enclosing Embryos in Seed Biology.

Dry fruits consist of two types, dehiscent and indehiscent, whereby the fruit is splitting open or remains closed at maturity, respectively. The seed, the dispersal unit (DU) of dehiscent fruits, is composed of three major parts, the embryo and the food reserve, encapsulated by the maternally-derived organ, the seed coat. Indehiscent fruit constitutes the DU in which the embryo is covered by two protective layers (PLs), the seed coat and the fruit coat. In grasses, the caryopsis, a one-seeded fruit, can be further enclosed by the floral bracts to generate two types of DUs, florets and spikelets. All protective layers enclosing the embryo undergo programmed cell death (PCD) at maturation and are thought to provide mainly a physical shield for embryo protection and a means for dispersal. In this review article, I wish to highlight the elaborate function of these dead organs enclosing the embryo as unique storage structures for beneficial substances and discuss their potential role in seed biology and ecology.

Epigenetic aspects of floral homeotic genes in relation to sexual dimorphism in the dioecious plant Mercurialis annua.

In plants, dioecy characterizes species that carry male and female flowers on separate plants and it occurs in about 6% of angiosperms; however, the molecular mechanisms that underlie dioecy are essentially unknown. The ability for sex-reversal by hormone application raises the hypothesis that the genes required for the expression of both sexes are potentially functional but are regulated by epigenetic means. In this study, proteomic analysis of nuclear proteins isolated from flower buds of females, males, and feminized males of the dioecious plant Mercurialis annua revealed differential expression of nuclear proteins that are implicated in chromatin structure and function, including floral homeotic proteins. Focusing on floral genes, we found that class B genes were mainly expressed in male flowers, while class D genes, as well as SUPERMAN-like genes, were mainly expressed in female flowers. Cytokinin-induced feminization of male plants was associated with down-regulation of male-specific genes concomitantly with up-regulation of female-specific genes. No correlation was found between the expression of class B and D genes and the changes in DNA methylation or chromatin conformation of these genes. Thus, we could not confirm DNA methylation or chromatin conformation of floral genes to be the major determinant regulating sexual dimorphisms. Instead, determination of sex in M. annua might be controlled upstream of floral genes by one or more sex-specific factors that affect hormonal homeostasis. A comprehensive model is proposed for sex-determination in M. annua.

The Dead Can Nurture: Novel Insights into the Function of Dead Organs Enclosing Embryos.

Plants have evolved a variety of dispersal units whereby the embryo is enclosed by various dead protective layers derived from maternal organs of the reproductive system including seed coats (integuments), pericarps (ovary wall, e.g., indehiscent dry fruits) as well as floral bracts (e.g., glumes) in grasses. Commonly, dead organs enclosing embryos (DOEEs) are assumed to provide a physical shield for embryo protection and means for dispersal in the ecosystem. In this review article, we highlight recent studies showing that DOEEs of various species across families also have the capability for long-term storage of various substances including active proteins (hydrolases and ROS detoxifying enzymes), nutrients and metabolites that have the potential to support the embryo during storage in the soil and assist in germination and seedling establishment. We discuss a possible role for DOEEs as natural coatings capable of "engineering" the seed microenvironment for the benefit of the embryo, the seedling and the growing plant.

Activation of Tag1 transposable elements in Arabidopsis dedifferentiating cells and their regulation by CHROMOMETHYLASE 3-mediated CHG methylation.

Dedifferentiation, that is, the acquisition of stem cell-like state, commonly induced by stress (e.g., protoplasting), is characterized by open chromatin conformation, a chromatin state that could lead to activation of transposable elements (TEs). Here, we studied the activation of the Arabidopsis class II TE Tag1, in which two copies, situated close to each other (near genes) on chromosome 1 are found in Landsberg erecta (Ler) but not in Columbia (Col). We first transformed protoplasts with a construct in which a truncated Tag1 (ΔTag1 non-autonomous) blocks the expression of a reporter gene AtMBD5-GFP and found a relatively high ectopic excision of ΔTag1 accompanied by expression of AtMBD5-GFP in protoplasts derived from Ler compared to Col; further increase was observed in ddm1 (decrease in DNA methylation1) protoplasts (Ler background). Ectopic excision was associated with transcription of the endogenous Tag1 and changes in histone H3 methylation at the promoter region. Focusing on the endogenous Tag1 elements we found low level of excision in Ler protoplasts, which was slightly and strongly enhanced in ddm1 and cmt3 (chromomethylase3) protoplasts, respectively, concomitantly with reduction in Tag1 gene body (GB) CHG methylation and increased Tag1 transcription; strong activation of Tag1 was also observed in cmt3 leaves. Notably, in cmt3, but not in ddm1, Tag1 elements were excised out from their original sites and transposed elsewhere in the genome. Our results suggest that dedifferentiation is associated with Tag1 activation and that CMT3 rather than DDM1 plays a central role in restraining Tag1 activation via inducing GB CHG methylation.

Stress induces cell dedifferentiation in plants.

Accumulating evidence lends support to the proposal that a major theme in plant responses to stresses is dedifferentiation, whereby mature cells acquire stem cell features (e.g. open chromatin conformation) prior to acquisition of a new cell fate. In this review, we discuss data addressing plant cell plasticity and provide evidence linking stress, dedifferentiation and a switch in cell fate. We emphasize the epigenetic modifications associated with stress-induced global changes in chromatin structure and conclude with the implications for genetic variation and for induced pluripotent stem cells in animals. It appears that stress is perceived as a signal that directs plant cells to undergo reprogramming (dedifferentiation) as a means for adaptation and in preparation for a stimulus-based acquisition of a new cell fate. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.