<em>Zea mays</em> Taxilin Protein Negatively Regulates Opaque-2 Transcriptional Activity by Causing a Change in Its Sub-Cellular Distribution

<div><p><em>Zea mays</em> (maize) Opaque-2 (ZmO2) protein is an important bZIP transcription factor that regulates the expression of major storage proteins (22-kD zeins) and other important genes during maize seed development. ZmO2 is subject to functional regulation through protein-protein interactions. To unveil the potential regulatory network associated with ZmO2, a protein-protein interaction study was carried out using the truncated version of ZmO2 (O2-2) as bait in a yeast two-hybrid screen with a maize seed cDNA library. A protein with homology to Taxilin was found to have stable interaction with ZmO2 in yeast and was designated as ZmTaxilin. Sequence analysis indicated that ZmTaxilin has a long coiled-coil domain containing three conserved zipper motifs. Each of the three zipper motifs is individually able to interact with ZmO2 in yeast. A GST pull-down assay demonstrated the interaction between GST-fused ZmTaxilin and ZmO2 extracted from developing maize seeds. Using onion epidermal cells as <em>in vivo</em> assay system, we found that ZmTaxilin could change the sub-cellular distribution of ZmO2. We also demonstrated that this change significantly repressed the transcriptional activity of ZmO2 on the 22-kD zein promoter. Our study suggests that a Taxilin-mediated change in sub-cellular distribution of ZmO2 may have important functional consequences for ZmO2 activity.</p> </div

Table_1_Ae1/Sbe1 maize-derived high amylose improves gut barrier function and ameliorates type II diabetes in high-fat diet-fed mice by increasing Akkermansia.docx

Type II diabetes mellitus (T2DM) has its origins in chronic inflammation due to immune dysregulation. Improving chronic inflammation can significantly reduce the probability of T2DM and the rate of disease progression. Resistance to starch 2 (RSII) high-amylose maize starch (HAMS) has been widely implicated in the improvement and regulation of T2DM. However, its exact molecular mechanisms have not been fully discovered. Here, we used CRISPR/Cas9 technology to knock out two starch-branching enzyme genes, Ae1 and Sbe1, in maize to obtain mutants containing higher levels of HAMS. In experiments in which HAMS was fed to mice on a high-fat diet (HFD), we confirmed the function of HAMS in ameliorating hyperglycemia. Mechanistically, we found that HAMS improves the gut barrier function by increasing the Akkermansia abundance in the gut. This increase led to the alleviation of chronic inflammation in mice on a HFD, resulting in improved insulin sensitivity and a decrease in blood glucose.</p

Table_2_Ae1/Sbe1 maize-derived high amylose improves gut barrier function and ameliorates type II diabetes in high-fat diet-fed mice by increasing Akkermansia.docx

Type II diabetes mellitus (T2DM) has its origins in chronic inflammation due to immune dysregulation. Improving chronic inflammation can significantly reduce the probability of T2DM and the rate of disease progression. Resistance to starch 2 (RSII) high-amylose maize starch (HAMS) has been widely implicated in the improvement and regulation of T2DM. However, its exact molecular mechanisms have not been fully discovered. Here, we used CRISPR/Cas9 technology to knock out two starch-branching enzyme genes, Ae1 and Sbe1, in maize to obtain mutants containing higher levels of HAMS. In experiments in which HAMS was fed to mice on a high-fat diet (HFD), we confirmed the function of HAMS in ameliorating hyperglycemia. Mechanistically, we found that HAMS improves the gut barrier function by increasing the Akkermansia abundance in the gut. This increase led to the alleviation of chronic inflammation in mice on a HFD, resulting in improved insulin sensitivity and a decrease in blood glucose.</p

Improved ethanol production in the presence of cadmium ions by a <i>Saccharomyces cerevisiae</i> transformed with a novel cadmium-resistance gene <i>DvCRP1</i>

<p>The <i>DvCRP1</i> gene obtained from <i>Dunaliella viridis</i> is a cadmium-resistance gene that induces cadmium accumulation in microbial and plant cells. In the present study, <i>Saccharomyces cerevisiae</i> was used as a model system to investigate the effect of <i>DvCRP1</i> on both cadmium detoxification and ethanol production. Inhibitory effects of cadmium (50–300 µmol/L) on growth (29–92%), glucose consumption (23–89%), and ethanol production (17–92%) were observed at 24 h by <i>S. cerevisiae</i>. <i>DvCRP1</i> alleviated the inhibitory effect of cadmium, with increase in the ethanol production. The established mathematical model showed that the initial inoculation concentration, cadmium concentration, and transformation of <i>DvCRP1</i> were the most important factors for cell growth, glucose consumption, and ethanol production. Cadmium detoxification of yeast was also enhanced by increasing the initial concentration of yeast cells. Transforming with <i>DvCRP1</i> further enhanced detoxification, especially at high cadmium concentrations. Transforming with DvCRP1 further enhanced detoxification, especially at high cadmium concentrations (200 µmol/L). The present results evidenced the potential of the insertion of the <i>DvCRP1</i> gene into yeast for use in bio-refineries during fermentation of heavy metals-contaminated substrates. In addition, this is a promising method for phytoremediation of agricultural soils highly contaminated by heavy metals.</p

ZmTaxilin and ZmO2 interact in a GST pull-down assay.

<p>(<b>A</b>) Western blot detection of the GST pull-down sample with a GST antibody. The kernel sample was a pool of equal amounts of RNA from different developmental stages between 3 and 36 days after pollination (DAP). (<b>B</b>) Western blot detection of the GST pull-down sample with the ZmO2 antibody. Lane 1 in (<b>A</b>) and (<b>B</b>): <i>E. coli</i> lysate containing the GST-Taxilin protein and the maize seed protein containing ZmO2. Lane 2 in (<b>A</b>) and (<b>B</b>): <i>E. coli</i> lysate containing GST and maize seed protein containing ZmO2. The expected molecular weight of the GST-Taxilin fusion protein, the GST tag and ZmO2 are 75.397, 27.895 and 47.075 kDa, respectively. The apparent molecular weight of the ZmO2 protein was approximately 68–72 kDa.</p

<i>ZmTaxilin</i> clones identified by yeast two-hybrid.

<p><i>ZmTaxilin</i> clones identified by yeast two-hybrid.</p

Additional file 1: Table S1. of High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize

The primers used in the experiments. (PDF 7 kb

ZmTaxilin can repress the transcriptional activity of ZmO2.

<p>(<b>A</b>) Structure of effecter and reporters. (<b>B</b>) GUS/luciferase values of different reporter and effecter combinations. The values are the averages with SD of three independent experiments, after normalisation to the internal control. Statistical significance between YFP-O2 and YFP-O2+CFP-Taxilin was calculated using a two-tailed T-test. *p≤0.05.</p

Model depicting the function of O10.

<p>In the wild-type kernel, O10 was first synthesized in the cytoplasm; HIP and HSP70 may have offered assistance when O10 was transported into the ER lumen. The TMD of O10 assisted with its retention in the ER. Finally, O10 was deposited in the interior of PB mainly depending on the aid of 16-kD γ-zein. In PB, O10 might determine the ring-shaped distribution of 16-kD γ-zein and 22-kD α-zein at the interface between the α-zein-rich core and the γ-zein-rich periphery of PB. The ring-shaped structure of 16-kD γ-zein and 22-kD α-zein is essential for maintaining the morphology of PB. In the o10 kernel, o10 was also transported inside the ER lumen, but it could not anchor onto the ER membrane as a result of the transmembrane deletion; thus, it might be degraded but not deposited in PB. This could affect the normal zein assembly, especially the 16-kD γ-zein and 22-kD α-zein, generating the misshapen PBs.</p

The distribution and accumulation of O10 in the wild-type and <i>o10</i> developing kernels.

<p>(A) The subcellular localization of YFP-O10 fusion protein in <i>N</i>. <i>benthamiana</i> leaves. Bars = 20 μm. (B) O10 could co-localize with ER around the nucleus. Confocal microscopic images were taken from <i>N</i>. <i>benthamiana</i> leaves in which YFP-O10 and the ER maker (mCherry-HDEL) were co-expressed. Bars = 20 μm. (C) The subcellular localization of YFP-o10 fusion protein in <i>N</i>. <i>benthamiana leaves</i>. Bars = 20 μm. (D) o10 could co-localize with ER throughout the cells. Confocal microscopic images were taken from <i>N</i>. <i>benthamiana</i> leaves in which YFP-o10 and the ER maker (mCherry-HDEL) were co-expressed. Bars = 20 μm. (E) Immunoblot comparing accumulation of O10 in the wild-type and <i>o10</i> developing kernels. Anti-tubulin was used as a sample loading control. (F) Immunoblot analysis of O10 in the wild-type and <i>o10</i> cytoplasm fractions with anti-O10 antibody (50 μg of protein per lane). (G) Immunoblot analysis of O10 in the wild-type and <i>o10</i> total membrane fractions with anti-O10 antibody. Anti-BIP was used as a sample loading control. (H) Immunoblot analysis of O10 in the ER and PB fractions from the wild-type and <i>o10</i> developing kernel cells (21 DAP) with anti-O10 antibody. Anti-BIP was used as an ER marker, and Anti-22-kd α-zein was used as a PB marker.</p