Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H⁺-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8

<p>Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H⁺-ATPase inhibitors concanamycin A and bafilomycin A₁, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H⁺-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.</p

Data_for_Fig6

Chloroplast number in leaf stomatal GCs of WT, arc5, arc6, and atminE1. Measurements of chloroplast number in arbitrarily selected pairs of GCs ('cell A' and 'cell B') are shown

Phenotypes of <i>bHLH106</i>-KO line growing on culture medium containing NaCl, KCl, or LiCl.

<p>(A) <i>bHLH106</i>-KO1 and control plants were germinated and grown on standard MS medium for 4 days, and then transferred to media containing different concentrations of NaCl, 110 mM KCl, or 10 mM LiCl. Plants were then kept vertical in rectangular culture plates for 2 weeks. <i>SUF4</i>-KO is a KO line of <i>SUF4</i> (At1g30970) for suppressor of FRI4 as an unrelated gene, and was used as a control for KO lines generated by T-DNA at the other loci. (B) Statistic data of plant growth with (red column) or without (blue column) NaCl in the same experiments shown in panel A. Fresh weights of whole plants were measured with or without 125 mM NaCl. Fresh weights of shoots and roots were measured with or without 100 mM NaCl. Error bars represent ±SEM from six experimental replicates. The numbers above columns are ratios of fresh weight of each line with salt to that without salt. Here are “n.s.” for no significant difference and * for <i>P</i> < 0.05 in ANOVA of the ratios. (C) Statistic data of root length in the same experiments shown in panel A. Here are “n.s.” for no significant difference and * for <i>P</i> < 0.05 in ANOVA.</p

<i>bHLH106</i>-knockout (KO) lines.

<p>(A) Integrated locations of T-DNA for gene disruption at the <i>bHLH106</i> locus in SALK_109295 and GABI_560F05, designated as <i>bHLH106</i>-KO1 and <i>bHLH106</i>-KO2, respectively. The pair of opposing arrowheads shows the primers employed for RT-PCR. (B) <i>bHLH106</i> gene expression in KO plants. Expression was determined by real-time RT-PCR and normalized using <i>ACTIN2</i> (<i>ACT2</i>). Error bars represent ±SEM from four experimental replicates.</p

Interaction of bHLH106 with a variety of G-box sequences.

<p>Interactions were examined by electrophoresis mobility shift assay (EMSA). (A) Nucleotide sequences of regions containing a possible G-box were employed for EMSA as probes. (B) Thirteen kinds of 20-mer G-box sequences consisting of 5′-^C/_G^A/_GNN^T/G/_A^G/_C-3′ (M1 to M13) and arbitrary degenerate probes (C1 and C2). The “+” and “-”denote the presence or absence of bHLH106 protein in EMSA. (C) Competition experiments using the 5′-CACGTG-3′ sequences against the bHLH106 protein.</p

Plastid phenotypes in leaf mesophyll cells of <i>Arabidopsis</i> WT, <i>arc5</i>, <i>arc6</i>, and <i>atminE1</i>.

(A–D) Images of plastid-targeted YFP in the third-fourth leaf petioles of 4-week-old WT (A), arc5 (B), arc6 (C), and atminE1 (D) seedlings. CLSM images of maximal intensity projection are shown. Arrowheads and arrows indicate epidermal PC plastids and stromules from enlarged plastids, respectively. Bar = 10 μm.</p

Development of transgenic <i>A. thaliana</i>.

<p>A. Schematic representation of the dimeric hybrid-IgG/IgA expression vector. P<i>_CAB</i>, chlorophyll <i>a/b</i>-binding protein promoter; T<i>_CAB1</i> and T<i>_CAB2</i>, chlorophyll <i>a/b</i>-binding protein terminators; <i>Hc</i>, hybrid-IgG/IgA heavy chain; <i>Lc</i>, hybrid-IgG/IgA light chain; <i>Jc</i>, immunoglobulin J chain; P<i>_35S</i>, cauliflower mosaic virus <i>35S</i> promoter; T<i>_NOS</i>, nopaline synthase terminator. B. Incorporation of the dimeric hybrid-IgG/IgA transgene into the genome of <i>A. thaliana</i>, as revealed by PCR analysis. <i>ACTIN2</i> is a house-keeping gene of <i>A. thaliana</i>. C. RT-PCR analysis of the mRNA for the dimeric hybrid-IgG/IgA. D. Appearance of a transgenic plant grown on soil for 7 wk. dimer Tg, <i>A. thaliana</i> transgenic for H, L and J chains; WT, wild-type <i>A. thaliana</i>.</p

Activation-tagged locus and activated genes in the <i>stc8</i> mutant.

<p>(A) Integrated location point of T-DNA for activation, in chromosome 2. (B) Expression of At2g41130, encoding <i>bHLH106</i> and (C) At2g41140 encoding <i>CRK1</i>. Total RNA was extracted from calli grown on CIM supplemented with 150 mM NaCl. Expression was determined by real-time RT-PCR and normalized using <i>ACTIN2</i> (<i>ACT2</i>) and shown as ratios of transcript levels to those of the wild-type without salt. Error bars represent ±SEM from four experimental replicates. Here are “n.s.” for no significant difference and * for <i>P</i> < 0.05 in ANOVA.</p

Confirmation of T-DNA integration in mutants.

<p>(A) The 200-bp genomic DNA product corresponding to P<i>35S</i>-<i>ALS-SU</i>^r was detected in chlorsulfuron-resistant calli. Lane 1, M5 Marker (NipponGene); lane 2, positive control (vector pRi35ADEn4); lane 3, negative control (wild-type, <i>Arabidopsis</i> Col-0); lanes 4–9 <i>stc1</i>, <i>stc2</i>, <i>stc3</i>, <i>stc4</i>, <i>stc12</i> and <i>stc17</i>, respectively. (B) Transcript levels of <i>MIPS1</i> in calli. Total cellular RNA was extracted from 3-week-old calli grown on CIM without NaCl or with 150 mM NaCl. Transcript levels were determined by real-time PCR using the LightCycler (Roche) and normalized using the internal standard (<i>ACT2</i>). Error bars represent ± standard error (SEM) from three experimental replicates. Here is * for significant difference with <i>P</i> < 0.05. (C) A map around the insertion of <i>stc1</i> on chromosome 4.</p

Neutralization of Stx1 holotoxin by the plantibody.

<p>Stx1 holotoxin was pre-treated with an extract of dimer Tg plants, an extract of a wild-type plants (WT), or control IgA (TEPC 15) for 1 h, and then the mixture was added to Stx1-sensitive cells. A. Cell viability assay. Vero cells were cultured for 48 h in the presence of 20 pg/ml of Stx1 that had been pre-incubated with a plant extract or control IgA at various concentrations (abscissa). The IgA concentration is effective for Tg and TEPC 15, and TSP for Tg and WT. Cell viability was exhibited as a percentage of the control level (without toxin exposure). Data are expressed as means ± SD of triplicate determinations. Error bars underneath the symbols are not visible. B. DNA fragmentation. Vero cells were cultured for 48 h in the presence of 10 pg/ml of Stx1 that had been pre-incubated with a plant extract or control IgA. DNA ladder formation was observed on agarose gel electrophoresis. Lane 1, untreated; lane 2, Stx1 only; lane 3, Stx1 + TEPC 15 (300 ng/ml IgA); lane 4, Stx1 + Tg (30 ng/ml IgA); lane 5, Stx1 + Tg (300 ng/ml IgA; 270 µg/ml TSP); lane 6, Stx1 + WT (270 µg/ml TSP). C. Caspase 3 activation. Ramos cells were cultured for 5 h in the presence of 10 pg/ml of Stx1 that had been pre-treated as indicated. Activated caspase 3 was probed with FITC-DEVD-FMK (abscissa), and analyzed with a flow cytometer. The number in each graph indicates the percentage of cells with activated caspase 3. D. Annexin V binding. Ramos cells were cultured as described for panel C. The binding of FITC-annexin V (abscissa) revealed cell surface exposure of phosphatidylserine. The number in each graph indicates the percentage of cells labeled with annexin V. The results are representative of three experiments.</p