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

Data_for_Descriptions

Chloroplast number in leaf stomatal GCs of Col, Ler, and Ws. Measurements of chloroplast number in arbitrarily selected pairs of GCs ('cell A' and 'cell B') are shown

Intracellular localization of bHLH106.

<p>The construct of <i>bHLH106</i> fused with <i>GFP</i> was bombarded into onion epidermal cells. Transient expression of <i>GFP</i> was observed. (A) Bombarded with <i>GFP</i> alone construct, pTH2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126872#pone.0126872.ref049" target="_blank">49</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126872#pone.0126872.ref064" target="_blank">64</a>]. (B, and C) Bombarded with <i>bHLH106</i> fused with <i>GFP</i>.</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

Expression of <i>bHLH106</i> with regard to salt-response and organ-specificity.

<p>(A) <i>bHLH106</i> transcripts in response to salt. Plants were transferred to salt-containing medium 4 days after germination, and kept vertical for 2 weeks. Total RNA was extracted from whole plants and subjected to real-time RT-PCR with normalization using <i>ACTIN2</i> (<i>ACT2</i>). Error bars represent ±SEM from three experimental replicates. All the <i>P</i> values are less than 0.05 between 0 mM NaCl and 100 mM or 125 mM NaCl. (B) <i>bHLH106</i> transcripts in different organs. The wild-type plants were grown under standard conditions. RNA was extracted from mature plants. Expression was determined by real-time RT-PCR and normalized using <i>ACTIN2</i> (<i>ACT2</i>). Error bars represent ±SEM from three experimental replicates.</p

Phenotypes of OX lines of <i>bHLH106</i>.

<p>(A) Expression of <i>bHLH106</i> in OX lines. <i>bHLH106</i> was driven by the CaVM 35S promoter and F₂ homozygous lines used for analysis. Total RNA was extracted from whole plants grown on standard MS. Expression was determined by real-time RT-PCR and normalized using <i>ACTIN2</i> (<i>ACT2</i>). Error bars represent ±SEM from three experimental replicates. (B) Phenotypes of <i>bHLH106</i>-OX grown on culture medium containing different concentrations of NaCl. The culture and treatment of plants with NaCl was as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126872#pone.0126872.g003" target="_blank">Fig 3</a>. (C) Statistic data of shoot weight of plants growth with (red column) or without (blue column) 100 mM NaCl in the same experiments shown in panel B. 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 is * for <i>P</i> < 0.05 in ANOVA.</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