Jasmonates and Histone deacetylase 6 activate Arabidopsis genome-wide histone acetylation and methylation during the early acute stress response.

BACKGROUND: Jasmonates (JAs) mediate trade-off between responses to both biotic and abiotic stress and growth in plants. The Arabidopsis thaliana HISTONE DEACETYLASE 6 is part of the CORONATINE INSENSITIVE1 receptor complex, co-repressing the HDA6/COI1-dependent acetic acid-JA pathway that confers plant drought tolerance. The decrease in HDA6 binding to target DNA mirrors histone H4 acetylation (H4Ac) changes during JA-mediated drought response, and mutations in HDA6 also cause depletion in the constitutive repressive marker H3 lysine 27 trimethylation (H3K27me3). However, the genome-wide effect of HDA6 on H4Ac and much of the impact of JAs on histone modifications and chromatin remodelling remain elusive. RESULTS: We performed high-throughput ChIP-Seq on the HDA6 mutant, axe1-5, and wild-type plants with or without methyl jasmonate (MeJA) treatment to assess changes in active H4ac and repressive H3K27me3 histone markers. Transcriptional regulation was investigated in parallel by microarray analysis in the same conditions. MeJA- and HDA6-dependent histone modifications on genes for specialized metabolism; linolenic acid and phenylpropanoid pathways; and abiotic and biotic stress responses were identified. H4ac and H3K27me3 enrichment also differentially affects JAs and HDA6-mediated genome integrity and gene regulatory networks, substantiating the role of HDA6 interacting with specific families of transposable elements in planta and highlighting further specificity of action as well as novel targets of HDA6 in the context of JA signalling for abiotic and biotic stress responses. CONCLUSIONS: The findings demonstrate functional overlap for MeJA and HDA6 in tuning plant developmental plasticity and response to stress at the histone modification level. MeJA and HDA6, nonetheless, maintain distinct activities on histone modifications to modulate genetic variability and to allow adaptation to environmental challenges.

Jasmonates induce Arabidopsis bioactivities selectively inhibiting the growth of breast cancer cells through CDC6 and mTOR.

Phytochemicals are used often in vitro and in vivo in cancer research. The plant hormones jasmonates (JAs) control the synthesis of specialized metabolites through complex regulatory networks. JAs possess selective cytotoxicity in mixed populations of cancer and normal cells. Here, direct incubation of leaf explants from the non-medicinal plant Arabidopsis thaliana with human breast cancer cells, selectively suppresses cancer cell growth. High-throughput LC-MS identified Arabidopsis metabolites. Protein and transcript levels of cell cycle regulators were examined in breast cancer cells. A synergistic effect by methyljasmonate (MeJA) and by compounds upregulated in the metabolome of MeJA-treated Arabidopsis leaves, on the breast cancer cell cycle, is associated with Cell Division Cycle 6 (CDC6), Cyclin-dependent kinase 2 (CDK2), Cyclins D1 and D3, indicating that key cell cycle components mediate cell viability reduction. Bioactives such as indoles, quinolines and cis-(+)-12-oxophytodienoic acid, in synergy, could act as anticancer compounds. Our work suggests a universal role for MeJA-treatment of Arabidopsis in altering the DNA replication regulator CDC6, supporting conservation, across kingdoms, of cell cycle regulation, through the crosstalk between the mechanistic target of rapamycin, mTOR and JAs. This study has important implications for the identification of metabolites with anti-cancer bioactivities in plants with no known medicinal pedigree and it will have applications in developing disease treatments.

The mitogen-activated protein kinase 4-phosphorylated heat shock factor A4A regulates responses to combined salt and heat stresses.

Heat shock factors regulate responses to high temperature, salinity, water deprivation, or heavy metals. Their function in combinations of stresses is, however, not known. Arabidopsis HEAT SHOCK FACTOR A4A (HSFA4A) was previously reported to regulate responses to salt and oxidative stresses. Here we show, that the HSFA4A gene is induced by salt, elevated temperature, and a combination of these conditions. Fast translocation of HSFA4A tagged with yellow fluorescent protein from cytosol to nuclei takes place in salt-treated cells. HSFA4A can be phosphorylated not only by mitogen-activated protein (MAP) kinases MPK3 and MPK6 but also by MPK4, and Ser309 is the dominant MAP kinase phosphorylation site. In vivo data suggest that HSFA4A can be the substrate of other kinases as well. Changing Ser309 to Asp or Ala alters intramolecular multimerization. Chromatin immunoprecipitation assays confirmed binding of HSFA4A to promoters of target genes encoding the small heat shock protein HSP17.6A and transcription factors WRKY30 and ZAT12. HSFA4A overexpression enhanced tolerance to individually and simultaneously applied heat and salt stresses through reduction of oxidative damage. Our results suggest that this heat shock factor is a component of a complex stress regulatory pathway, connecting upstream signals mediated by MAP kinases MPK3/6 and MPK4 with transcription regulation of a set of stress-induced target genes.

COI1-dependent jasmonate signalling affects growth, metabolite production and cell wall protein composition in arabidopsis.

Background and Aims: Cultured cell suspensions have been the preferred model to study the apoplast as well as to monitor metabolic and cell cycle-related changes. Previous work showed that methyl jasmonate (MeJA) inhibits leaf growth in a CORONATINE INSENSITIVE 1 (COI1)-dependent manner, with COI1 being the jasmonate (JA) receptor. Here, the effect of COI1 overexpression on the growth of stably transformed arabidopsis cell cultures is described. Methods: Time-course experiments were carried out to analyse gene expression, and protein and metabolite levels. Key Results: Both MeJA treatment and the overexpression of COI1 modify growth, by altering cell proliferation and expansion. DNA content as well as transcript patterns of cell cycle and cell wall remodelling markers were altered. COI1 overexpression also increases the protein levels of OLIGOGALACTURONIDE OXIDASE 1, BETA-GLUCOSIDASE/ENDOGLUCANASES and POLYGALACTURONASE INHIBITING PROTEIN2, reinforcing the role of COI1 in mediating defence responses and highlighting a link between cell wall loosening and growth regulation. Moreover, changes in the levels of the primary metabolites alanine, serine and succinic acid of MeJA-treated Arabidopsis cell cultures were observed. In addition, COI1 overexpression positively affects the availability of metabolites such as ß-alanine, threonic acid, putrescine, glucose and myo-inositol, thereby providing a connection between JA-inhibited growth and stress responses. Conclusions: This study contributes to the understanding of the regulation of growth and the production of metabolic resources by JAs and COI1. This will have important implications in dissecting the complex relationships between hormonal and cell wall signalling in plants. The work also provides tools to uncover novel mechanisms co-ordinating cell division and post-mitotic cell expansion in the absence of organ developmental control.

The low oxygen, oxidative and osmotic stress responses synergistically act through the ethylene response factor VII genes RAP2.12, RAP2.2 and RAP2.3.

The ethylene response factor VII (ERF-VII) transcription factor RELATED TO APETALA2.12 (RAP2.12) was previously identified as an activator of the ALCOHOL DEHYDROGENASE1 promoter::luciferase (ADH1-LUC) reporter gene. Here we show that overexpression of RAP2.12 and its homologues RAP2.2 and RAP2.3 sustains ABA-mediated activation of ADH1 and activates hypoxia marker genes under both anoxic and normoxic conditions. Inducible expression of all three RAP2s conferred tolerance to anoxia, oxidative and osmotic stresses, and enhanced the sensitivity to abscisic acid (ABA). Consistently, the rap2.12-2 rap2.3-1 double mutant showed hypersensitivity to both submergence and osmotic stress. These findings suggest that the three ERF-VII-type transcription factors play roles in tolerance to multiple stresses that sequentially occur during and after submergence in Arabidopsis. Oxygen-dependent degradation of RAP2.12 was previously shown to be mediated by the N-end rule pathway. During submergence the RAP2.12, RAP2.2 and RAP2.3 are stabilized and accumulates in the nucleus affecting the transcription of stress response genes. We conclude that the stabilized RAP2 transcription factors can prolong the ABA-mediated activation of a subset of osmotic responsive genes (e.g. ADH1). We also show that RAP2.12 protein level is affected by the REALLY INTERESTING GENE (RING) domain containing SEVEN IN ABSENTIA of Arabidopsis thaliana 2 (SINAT2). Silencing of SINAT1/2 genes leads to enhanced RAP2.12 abundance independently of the presence or absence of its N-terminal degron. Taken together, our results suggest that RAP2.12 and its homologues RAP2.2 and RAP2.3 act redundantly in multiple stress responses. Alternative protein degradation pathways may provide inputs to the RAP2 transcription factors for the distinct stresses.

The heat shock factor A4A confers salt tolerance and is regulated by oxidative stress and the mitogen-activated protein kinases MPK3 and MPK6.

Heat shock factors (HSFs) are principal regulators of plant responses to several abiotic stresses. Here, we show that estradiol-dependent induction of HSFA4A confers enhanced tolerance to salt and oxidative agents, whereas inactivation of HSFA4A results in hypersensitivity to salt stress in Arabidopsis (Arabidopsis thaliana). Estradiol induction of HSFA4A in transgenic plants decreases, while the knockout hsfa4a mutation elevates hydrogen peroxide accumulation and lipid peroxidation. Overexpression of HSFA4A alters the transcription of a large set of genes regulated by oxidative stress. In yeast (Saccharomyces cerevisiae) two-hybrid and bimolecular fluorescence complementation assays, HSFA4A shows homomeric interaction, which is reduced by alanine replacement of three conserved cysteine residues. HSFA4A interacts with mitogen-activated protein kinases MPK3 and MPK6 in yeast and plant cells. MPK3 and MPK6 phosphorylate HSFA4A in vitro on three distinct sites, serine-309 being the major phosphorylation site. Activation of the MPK3 and MPK6 mitogen-activated protein kinase pathway led to the transcriptional activation of the HEAT SHOCK PROTEIN17.6A gene. In agreement that mutation of serine-309 to alanine strongly diminished phosphorylation of HSFA4A, it also strongly reduced the transcriptional activation of HEAT SHOCK PROTEIN17.6A. These data suggest that HSFA4A is a substrate of the MPK3/MPK6 signaling and that it regulates stress responses in Arabidopsis.

The Arabidopsis F-box protein FBW2 targets AGO1 for degradation to prevent spurious loading of illegitimate small RNA.

RNA silencing is a conserved mechanism in eukaryotes involved in development and defense against viruses. In plants, ARGONAUTE1 (AGO1) protein plays a central role in both microRNA- and small interfering RNA-directed silencing, and its expression is regulated at multiple levels. Here, we report that the F-box protein FBW2 assembles an SCF complex that selectively targets for proteolysis AGO1 when it is unloaded and mutated. Although FBW2 loss of function does not lead to strong growth or developmental defects, it significantly increases RNA-silencing activity. Interestingly, under conditions in which small-RNA accumulation is affected, the failure to degrade AGO1 in fbw2 mutants becomes more deleterious for the plant. Accordingly, the non-degradable AGO1 protein assembles high-molecular-weight complexes and binds illegitimate small RNA, leading to off-target cleavage. Therefore, control of AGO1 homeostasis by FBW2 plays an important role in quality control of RNA silencing.

Stable Transformation of Arabidopsis thaliana Cell Suspension Cultures: A Case Study for The Overexpression of the COI1 Receptor.

Cell suspension cultures have been studied for decades to produce natural molecules. However, the difficulty in generating stably transformed cell lines has limited their use to produce high value chemicals reproducibly and in elevated quantities. In this protocol, a method to stably transform and maintain Arabidopsis cell suspension cultures is devised and presented in detail. Arabidopsis cell cultures were directly transformed with A. tumefaciens for the overexpression of the CORONATINE INSENSITIVE 1 (COI1) jasmonate receptor. Cell cultures were established after transformation and continuously maintained and tested for the overexpression of COI1. The protocol was also previously used to silence Arabidopsis peroxidases and allows for long term maintenance of transformed cells. Details on culture maintenance, both in liquid and solid media are provided, alongside with evidence of protein expression to confirm transformation. The system described provides a powerful tool for synthetic biology to study signaling independent of developmental control and to obtain metabolites of interest for the biotechnological and medical sectors.

Screening Stress Tolerance Traits in Arabidopsis Cell Cultures.

Screening for tolerance traits in plant cell cultures can combine the efficiency of microbial selection and plant genetics. Agrobacterium-mediated transformation can efficiently introduce cDNA library to cell suspension cultures generating population of randomly transformed microcolonies. Transformed cultures can subsequently be screened for tolerance to different stress conditions such as salinity, high osmotic, or oxidative stress conditions. cDNA inserts in tolerant cell lines can be easily identified by PCR amplification and homology search of the determined nucleotide sequences. The described methods have been tested and used to identify regulatory genes controlling salt tolerance in Arabidopsis. As cDNA libraries can be prepared from any plants, natural diversity can be explored by using extremophile plants as gene source.