Transcriptomic analysis of <i>Arabidopsis thaliana</i> plants treated with the Ky-9 and Ky-72 histone deacetylase inhibitors

<p>Histone acetylation plays a pivotal role in plant growth and development, and is regulated by the antagonistic relationship between histone acetyltransferase (HAT) and histone deacetylase (HDAC). We previously revealed that some HDAC inhibitors confer high-salinity stress tolerance in plants. In this study, we identified two HDAC inhibitors, namely Ky-9 and Ky-72, which enhanced the high-salinity stress tolerance of <i>Arabidopsis thaliana</i>. Ky-9 and Ky-72 are structurally similar chlamydocin analogs. However, the <i>in vitro</i> inhibitory activity of Ky-9 against mammalian HDAC is greater than that of Ky-72. A western blot indicated that Ky-9 and Ky-72 increased the acetylation levels of histone H3, suggesting they exhibit HDAC inhibitory activities in plants. We conducted a transcriptomic analysis to investigate how Ky-9 and Ky-72 enhance high-salinity stress tolerance. Although Ky-9 upregulated the expression of more genes than Ky-72, similar gene expression patterns were induced by both HDAC inhibitors. Additionally, the expression of high-salinity stress tolerance-related genes, such as anthocyanin-related genes and a small peptide-encoding gene, increased by Ky-9 and Ky-72. These data suggest that slight structural differences in chemical side chain between HDAC inhibitors can alter inhibitory effect on HDAC protein leading to influence gene expression, thereby enhancing high-salinity stress tolerance in different extent.</p

Development of Gateway Binary Vector Series with Four Different Selection Markers for the Liverwort <i>Marchantia polymorpha</i>

<div><p>We previously reported <i>Agrobacterium</i>-mediated transformation methods for the liverwort <i>Marchantia polymorpha</i> using the hygromycin phosphotransferase gene as a marker for selection with hygromycin. In this study, we developed three additional markers for <i>M</i>. <i>polymorpha</i> transformation: the gentamicin 3'-acetyltransferase gene for selection with gentamicin; a mutated acetolactate synthase gene for selection with chlorsulfuron; and the neomycin phosphotransferase II gene for selection with G418. Based on these four marker genes, we have constructed a series of Gateway binary vectors designed for transgenic experiments on <i>M</i>. <i>polymorpha</i>. The 35S promoter from cauliflower mosaic virus and endogenous promoters for constitutive and heat-inducible expression were used to create these vectors. The reporters and tags used were Citrine, 3×Citrine, Citrine-NLS, TagRFP, tdTomato, tdTomato-NLS, GR, SRDX, SRDX-GR, GUS, ELuc(PEST), and 3×FLAG. These vectors, designated as the pMpGWB series, will facilitate molecular genetic analyses of the emerging model plant <i>M</i>. <i>polymorpha</i>.</p></div

Schematic illustrations of pMpGWBs.

<p>A. Outline of pMpGWBs (pMpGWB101-136, pMpGWB201-236, pMpGWB301-336, and pMpGWB401-436 containing selection markers for Hyg, Gen, CS, and G418, respectively). Markers were placed in tail-to-tail orientation in relation to genes cloned by LR reaction. B. Structures of “<i>Gateway</i>” regions shown in (A). Only general structures are shown for vectors harboring fusion construct; <i>i</i>.<i>e</i>., no promoter and C-fusion (No pro/C), CaMV 35S promoter and C-fusion (_pro35S/C), CaMV 35S promoter and N-fusion (_pro35S/N), <i>EF</i> promoter and C-fusion (_proEF/C), <i>EF</i> promoter and N-fusion (_proEF/N), <i>MpHSP17</i>.<i>8A1</i> promoter and C-fusion (_proHSP/C). C. Reporters and tags used in pMpGWBs illustrated in (B). Vector number corresponds to combination of reporter/tag and fusion type. Citrine, synthetic yellow fluorescent protein; 3×Citrine, three repeats of Citrine fluorescent protein; TagRFP, tag red fluorescent protein; tdTomato; tandem dimer Tomato fluorescent protein; GR, ligand-binding domain of rat glucocorticoid receptor; SRDX, modified EAR motif plant-specific repression domain with strong repression activity; SRDX-GR, SRDX fused with GR; 3×FLAG, three repeats of FLAG tag; GUS, β-glucuronidase; ELuc(PEST), Emerald Luc (luciferase from click beetle) fused with PEST sequence; Citrine-NLS, Citrine fused with nuclear localization signal sequence; tdTomato-NLS, tdTomato fused with nuclear localization signal sequence. <i>RB</i>, right border; <i>LB</i>, left border; <i>sta</i>, region conferring stability in <i>Agrobacterium</i>; <i>rep</i>, broad host-range replication origin; <i>bom</i>, <i>cis</i>-acting element for conjugational transfer; <i>ori</i>, ColE1 replication origin. <i>Cm</i>^<i>r</i>, chloramphenicol-resistance marker (chloramphenicol acetyl transferase) used to select bacteria; <i>aadA</i>, spectinomycin-resistance marker (<i>Spc</i>^<i>r</i>) used to select bacteria; <i>ccdB</i>, negative selection marker.</p

Transformation of <i>M</i>. <i>polymorpha</i> using pMpGWB vectors developed in this study.

<p>Sporelings co-cultivated with <i>A</i>. <i>tumefaciens</i> strain GV2260 containing the binary vector pMpGWB100 (A), pMpGWB200 (C), pMpGWB300 (E), pMpGWB400 (G), or no binary vector (B, D, F, and H) at 7 d after transfer onto selection medium containing 10 mg/l hygromycin (Hyg; A, B), 100 mg/l gentamicin (Gen; C, D), 0.5 μM chlorsulfuron (CS; E, F), or 5 mg/l G418 (G, H). Insets show magnified view of antibiotic/herbicide-resistant transformants. Bars = 1 mm.</p

Comparison of transformation efficiencies of <i>M</i>. <i>polymorpha</i> among four vectors with different selection markers and selective antibiotics/herbicide.

<p>Hyg, hygromycin; Gen, gentamicin; CS, chlorsulfuron. Values are means ± SD from three independent experiments.</p

Noncanonical Function of a Small-Molecular Virulence Factor Coronatine against Plant Immunity: An <i>In Vivo</i> Raman Imaging Approach

Coronatine (<b>1</b>), a small-molecular virulence factor produced by plant-pathogenic bacteria, promotes bacterial infection by inducing the opening of stomatal pores, the major route of bacterial entry into the plant, via the jasmonate-mediated COI1-JAZ signaling pathway. However, this pathway is also important for multiple plant functions, including defense against wounding by herbivorous insects. Thus, suppression of the COI1-JAZ signaling pathway to block bacterial infection would concomitantly impair plant defense against herbivorous wounding. Here, we report additional, COI1-JAZ-independent, action of <b>1</b> in <i>Arabidopsis thaliana</i> guard cells. First, we found that a stereoisomer of <b>1</b> regulates the movement of <i>Arabidopsis</i> guard cells without affecting COI1-JAZ signaling. Second, we found using alkyne-tagged Raman imaging (ATRI) that <b>1</b> is localized to the endoplasmic reticulum (ER) of living guard cells of <i>Arabidopsis</i>. The use of <i>arc6</i> mutant lacking chloroplast formation was pivotal to circumvent the issue of autofluorescence during ATRI. These findings indicate that <b>1</b> has an ER-related action on <i>Arabidopsis</i> stomata that bypasses the COI1-JAZ signaling module. It may be possible to suppress the action of <b>1</b> on stomata without impairing plant defense responses against herbivores

Cytokine and chemokine expression in the kidney.

<p>The protein levels of MIP-2 (A), IL-1β (B), and MCP-1 (C) were determined by quantitative sandwich ELISA and corrected for the total amount of protein. Values are presented as mean ±SD; SHED group, day1 (n = 9), day2 (n = 8), day7 (n = 8); control group, day1 (n = 9), day2 (n = 7), day 7 (n = 7).* Significant difference (<i>P</i><0.05 vs. control).</p

Flow cytometry analysis of SHED.

<p>Values are presented as mean ±SD; (n = 4).</p><p>Flow cytometry analysis of SHED.</p

MCP-1 expression subsequent to stimulation with 0.4 mM H₂O₂.

<p>The expression of MCP-1 protein was determined by quantitative sandwich ELISA at 24 h. (A) The level of MCP-1 secreted from TEC. (B) The level of MCP-1 secreted from vascular endothelial cells. Each assay was performed in triplicate, and experiments were repeated five times. Values are presented as mean ±SD; SHED group (n = 5); control group (n = 5).* Significant difference (<i>P</i><0.05 vs. control).</p

Scratch wound healing assays of TEC.

<p>Repair of TEC is accelerated by SHED-CM and anti-HGF antibody blunted the effects in scratch wound-healing assays. Injured TEC monolayers were incubated with SHED-CM (SHED), SHED-CM with anti HGF antibody (anti HGF Ab+ SHED), SHED-CM with normal goat IgG (Goat IgG+ SHED) or control medium (Control). (A) Representative images of the progression of wound closure at 0 h, 9 h, and 24 h (original magnification, x5 Scale bar: 300 μmm), black dotted lines indicate initial injury. Cells growing over the lines represent injury closure. Each assay was performed in triplicate and experiments were repeated five times. (B) Evaluation of the scratch wound healing assay. The percentage of cell-free area at indicated time points compared with the area at the start of experiment (0 h) was determined. Values are presented as mean ±SD; SHED group (n = 5); anti HGF antibody group (n = 5); normal goat IgG group (n = 5); control group (n = 5).* <i>P</i><0.01 SHED vs. control, normal goat IgG vs. control, SHED vs. anti HGF antibody, and normal goat IgG vs. anti HGF antibody. **<i>P</i><0.001 SHED vs. control, normal goat IgG vs. control, SHED vs. anti HGF antibody, and normal goat IgG vs. anti HGF antibody. (C) Expression of HGF in conditioned media from BMMSC, FB, and SHED at 48 h. Each assay was performed in triplicate and experiments were repeated three times. Values are presented as mean ±SD.</p