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

<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_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

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

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

ZmMADS47 Regulates Zein Gene Transcription through Interaction with Opaque2

<div><p>Zeins, the predominent storage proteins in maize endosperm, are encoded by multiple genes and gene families. However, only a few transcriptional factors for zein gene regulation have been functionally characterized. In this study, a MADS-box protein, namely ZmMADS47, was identified as an Opaque2 (O2) interacting protein via yeast two-hybrid screening. The N-terminal portion of ZmMADS47 contains a nuclear localization signal (NLS), and its C-terminal portion contains a transcriptional activation domain (AD). Interestingly, the transcriptional activation activity is blocked in its full length form, suggesting conformational regulation of the AD. Molecular and RNA-seq analyses of Zm<i>MADS47 RNAi</i> lines revealed down regulation of α-zein and 50-kD γ-zein genes. ZmMADS47 binds the CATGT motif in promoters of these zein genes, but ZmMADS47 alone is not able to transactivate the promoters. However, when both O2 and ZmMADS47 are present, the transactivation of these promoters was greatly enhanced. This enhancement was dependent on the AD function of ZmMADS47 and the interaction between ZmMADS47 and O2, but it was independent from the AD function of O2. Therefore, it appears interaction with O2 activates ZmMADS47 on zein gene promoters.</p></div

Illustrtion of how the O2/ZmMADS47 complex may regulate zein gene expression.

<p>The upper illustration shows ZmMADS47 alone (square) binds the CATGT motif but does not induce transactivation. The middle illustration shows O2 binding is able to transactivate its targets. The bottom illustration shows ZmMADS47 (oval) and O2 bind their respective motifs and the O2/ZmMADS47 complex enhances transactivation of gene targets.</p

List of nutrient reservoir activity-related DEGs.

<p>List of nutrient reservoir activity-related DEGs.</p

Characterization of the ZmMADS47 binding motif.

<p><b>A.</b> Schematic representation of z1A promoter fragmentation. Black arrow points to the ZmMADS47-indused shift bands. <b>B.</b> Deletion analysis to narrow down the conserved domain recognized by ZmMADS47. The upper black arrow identifies the shifted bands due to the ZmMADS47/DNA complex. The bases highlighted by red letters represent the sharing sequences in both two fragments. <b>C.</b> Mutagenesis assay of CATGT motifs. The upper black arrow identified the shifted bands due to the ZmMADS47/DNA complex. The bases highlighted by red letters represent the mutational bases compared with wild-type bases. <b>D.</b> Effect of competitive probe on the band shift reaction. The upper black arrow identifies the shifted bands due to ZmMADS47/DNA complex. <b>E.</b> The binding ability of His-ZmMADS47 and His-O2 to CATGT motifs in the z1A promoter and 50-kD zein promoter. The experiment was performed by adding ZmMADS47 or O2 protein. The upper black arrow identifies shifted bands due to ZmMADS47/DNA complex or O2/DNA complex. <b>F.</b> Schematic representation of ZmMADS47 binding site (M) and O2 DNA binding site (O) in down-regulated zein promoters.</p