Phosphatases Generate Signal Specificity Downstream of Ssp1 Kinase in Fission Yeast

AMPK-related protein kinases (ARKs) coordinate cell growth, proliferation, and migration with environmental status. It is unclear how specific ARKs are activated at specific times. In the fission yeast Schizosaccharomyces pombe, the CaMKK-like protein kinase Ssp1 promotes cell cycle progression by activating the ARK Cdr2 according to cell growth signals. Here, we demonstrate that Ssp1 activates a second ARK, Ssp2/AMPKα, for cell proliferation in low environmental glucose. Ssp1 activates these two related targets by the same biochemical mechanism: direct phosphorylation of a conserved residue in the activation loop (Cdr2-T166 and Ssp2-T189). Despite a shared upstream kinase and similar phosphorylation sites, Cdr2 and Ssp2 have distinct regulatory input cues and distinct functional outputs. We investigated this specificity and found that distinct protein phosphatases counteract Ssp1 activity toward its different substrates. We identified the PP6 family phosphatase Ppe1 as the primary phosphatase for Ssp2-T189 dephosphorylation. The phosphatase inhibitor Sds23 acts upstream of PP6 to regulate Ssp2-T189 phosphorylation in a manner that depends on energy but not on the intact AMPK heterotrimer. In contrast, Cdr2-T166 phosphorylation is regulated by protein phosphatase 2A but not by the Sds23-PP6 pathway. Thus, our study provides a phosphatase-driven mechanism to induce specific physiological responses downstream of a master protein kinase

Characterization of fab2 T-DNA insertion mutants in terms of fatty acid composition and plant phenotype

Fatty acid biosynthesis 2 (FAB2) is an essential enzyme responsible for the synthesis of unsaturated fatty acids in chloroplast membrane lipids found in leaves and triacylglycerols (TAG) in seeds. FAB2 functions at the junction of saturated to unsaturated fatty acid conversion in chloroplasts by converting 18:0-ACP to 18:1-ACP. In the present study, plant growth and seed phenotypes were examined in three Arabidopsis T-DNA mutants (fab2–1, fab2–2, and fab2–3). The three fab2 T-DNA mutants exhibited increased 18:0 fatty acid content in both the leaves and seeds. The degree of growth inhibition of the fab2 mutant was proportional to the increase in 18:0 and decrease in 18:3 fatty acids present in the leaves. The FAB2 mutation affected seed yield but not the seed phenotype. This result indicates that FAB2 affects the fatty acid composition of the leaf chloroplast membrane more than seed TAG. In summary, the characteristics of these three fab2 mutants provide information for studying leaf membrane lipid and seed oil biosynthesis

Oxidative balance score as a useful predictive marker for new-onset type 2 diabetes mellitus in Korean adults aged 60 years or older: The Korean Genome and Epidemiologic Study–Health Examination (KoGES-HEXA) cohort

Background: The oxidative balance score (OBS) is a comprehensive pro- and anti-oxidative marker for assessing the risk of various metabolic diseases and cancers. However, it is not well established whether OBS is related to type 2 diabetes mellitus (T2DM), particularly in elderly populations. Therefore, our objective was to investigate the longitudinal effect of OBS on T2DM in a large cohort of Korean adults aged 60 years and older. Methods: We assessed the data for 3516 participants aged 60 years and older without diabetes mellitus from the Health Examinees cohort of the Korean Genome and Epidemiology Study. We classified the participants into three groups according to OBS tertiles. We prospectively assessed hazard ratios (HRs) with 95 % confidence intervals (CIs) for new-onset T2DM using multivariable Cox proportional-hazard regression models during the mean 3.5 years following the baseline survey. Results: A total of 109 participants (3.1 %) developed T2DM during a mean follow-up of 3.5 years. The incidence rates per 1000 person-years were 11.73 for the lowest OBS tertile (T1), 8.19 for the second tertile (T2), and 6.23 for the highest tertile (T3). Adjusting for all confounding factors, compared with the referent T1, the HR (95 % CI) of new-onset T2DM was not significant in T2 (0.71 [0.47–1.07]) but was significant in T3 at (0.47 [0.30–0.75]) (p for trend = 0.002). Conclusions: The study suggests that a OBS could serve as a valuable predictive marker for new-onset T2DM in older adults. Our study suggests that maintaining an appropriate body weight through healthy lifestyle modification has the potential to lower T2DM incidence in elderly. This implies that the OBS may be a useful tool for assessing the incidence of T2DM even in older individuals

The Arabidopsis MYB96 Transcription Factor Mediates ABA-Dependent Triacylglycerol Accumulation in Vegetative Tissues under Drought Stress Conditions

Triacylglycerols (TAGs), a major lipid form of energy storage, are involved in a variety of plant developmental processes. While carbon reserves mainly accumulate in seeds, significant amounts of TAG have also been observed in vegetative tissues. Notably, the accumulation of leaf TAGs is influenced by environmental stresses such as drought stress, although underlying molecular networks remain to be fully elucidated. In this study, we demonstrate that the R2R3-type MYB96 transcription factor promotes TAG biosynthesis in Arabidopsis thaliana seedlings. Core TAG biosynthetic genes were up-regulated in myb96-ox seedlings, but down-regulated in myb96-deficient seedlings. In particular, ABA stimulates TAG accumulation in the vegetative tissues, and MYB96 plays a fundamental role in this process. Considering that TAG accumulation contributes to plant tolerance to drought stress, MYB96-dependent TAG biosynthesis not only triggers plant adaptive responses but also optimizes energy metabolism to ensure plant fitness under unfavorable environmental conditions

Transcriptome Analysis and Identification of Lipid Genes in <i>Physaria lindheimeri</i>, a Genetic Resource for Hydroxy Fatty Acids in Seed Oil

Hydroxy fatty acids (HFAs) have numerous industrial applications but are absent in most vegetable oils. Physaria lindheimeri accumulating 85% HFA in its seed oil makes it a valuable resource for engineering oilseed crops for HFA production. To discover lipid genes involved in HFA synthesis in P. lindheimeri, transcripts from developing seeds at various stages, as well as leaf and flower buds, were sequenced. Ninety-seven percent clean reads from 552,614,582 raw reads were assembled to 129,633 contigs (or transcripts) which represented 85,948 unique genes. Gene Ontology analysis indicated that 60% of the contigs matched proteins involved in biological process, cellular component or molecular function, while the remaining matched unknown proteins. We identified 42 P. lindheimeri genes involved in fatty acid and seed oil biosynthesis, and 39 of them shared 78–100% nucleotide identity with Arabidopsis orthologs. We manually annotated 16 key genes and 14 of them contained full-length protein sequences, indicating high coverage of clean reads to the assembled contigs. A detailed profiling of the 16 genes revealed various spatial and temporal expression patterns. The further comparison of their protein sequences uncovered amino acids conserved among HFA-producing species, but these varied among non-HFA-producing species. Our findings provide essential information for basic and applied research on HFA biosynthesis

Polarization of Diploid Daughter Cells Directed by Spatial Cues and GTP Hydrolysis of Cdc42 in Budding Yeast

<div><p>Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast exhibit a characteristic pattern of budding, which depends on cell-type-specific cortical markers, reflecting a genetic programming for the site of cell polarization. The Cdc42 GTPase plays a key role in cell polarization in various cell types. Although previous studies in budding yeast suggested positive feedback loops whereby Cdc42 becomes polarized, these mechanisms do not include spatial cues, neglecting the normal patterns of budding. Here we combine live-cell imaging and mathematical modeling to understand how diploid daughter cells establish polarity preferentially at the pole distal to the previous division site. Live-cell imaging shows that daughter cells of diploids exhibit dynamic polarization of Cdc42-GTP, which localizes to the bud tip until the M phase, to the division site at cytokinesis, and then to the distal pole in the next G1 phase. The strong bias toward distal budding of daughter cells requires the distal-pole tag Bud8 and Rga1, a GTPase activating protein for Cdc42, which inhibits budding at the cytokinesis site. Unexpectedly, we also find that over 50% of daughter cells lacking Rga1 exhibit persistent Cdc42-GTP polarization at the bud tip and the distal pole, revealing an additional role of Rga1 in spatiotemporal regulation of Cdc42 and thus in the pattern of polarized growth. Mathematical modeling indeed reveals robust Cdc42-GTP clustering at the distal pole in diploid daughter cells despite random perturbation of the landmark cues. Moreover, modeling predicts different dynamics of Cdc42-GTP polarization when the landmark level and the initial level of Cdc42-GTP at the division site are perturbed by noise added in the model.</p> </div

Positions of the first bud in a/α daughter cells of wild type and mutants deleted for Cdc42 GAPs.

<p><b>A.</b> Time-lapse DIC images of diploid cells of wild type (YEF473) and <i>rga1Δ</i> (YEF1233). Arrows indicate budding events from daughter cells. Numbers indicate times (in min) from the first image. Size bars, 5 µm. (Histogram) The position of the first bud of daughter cells was scored in wild type (HPY1680), <i>rga1Δ</i> (HPY2205), <i>rga2Δ</i> (HPY2246), <i>bem2Δ</i> (HPY2384), and <i>bem3Δ</i> (HPY2426). The mean percentage ± SD of each budding pattern is shown from three or four independent countings of wild type (n = 106), <i>rga1Δ</i> (n = 144), <i>rga2Δ</i> (n = 56), <i>bem2Δ</i> (n = 108), and <i>bem3Δ</i> (n = 53). Statistical significance was determined by Student's t-test between proximal-pole buddings in wild type and <i>rga1Δ</i> or <i>bem2Δ</i> (marked with asterisks): *p<10⁻⁵ (<i>rga1Δ</i>) and **p = 0.02 (<i>bem2Δ</i>). <b>B.</b> The position of the first bud relative to the birth scar in diploid daughter cells. Cells were double stained with Calcoflour white and WGA-FITC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056665#pone.0056665-Frydlov1" target="_blank">[44]</a> from wild type (YEF473), <i>rga1Δ</i> (YEF1233), <i>bud8Δ</i> (YHH415), and <i>rga1Δ bud8Δ</i> (HPY2385). Arrows indicate birth scars. Size bar, 3 µm.</p

Time-lapse microscopy of Cdc42-GTP polarization in wild-type a/α diploids.

<p><b>A.</b> A schematics diagram of the bipolar budding pattern. M and D stand for mother and daughter cells, respectively. Red arrows depict the axis of cell polarity. <b>B.</b> Localization of Gic2-PBD-RFP and Cdc3-GFP in diploid wild-type cells (HPY2353). An arrowhead marks Gic2-PBD-RFP localized to the proximal pole in the daughter cell. Numbers indicate time (in min) from the first image. Size bars, 3 µm.</p

Localization of Bud8 in large-budded cells of the wild type (HPY1680) and <i>rga1Δ</i> (HPY2205) carrying YEpGFP-BUD8F.

<p>Representative images are shown for each pattern (A-D) and the percentage (mean ± SD) of each pattern is shown from three independent experiments (n = 160–230). Student's t-test was performed to compare the distal-pole localization in wild type and <i>rga1Δ</i> (P = 0.006).</p

Localization of Gic2-PBD-RFP and Cdc3-GFP in <i>rga1Δ</i> cells.

<p><b>A.</b> In <i>rga1Δ</i> cells (HPY2204), Gic2-PBD-RFP localized continuously to (a) the proximal pole or (b) the distal pole from cytokinesis to the next G1 phase. Arrows in (a) & (b) denote the Cdc3 ring splitting and an arrowhead in (a) denotes Gic2-PBD-RFP enriched at the division site (as well as the bud tip). Numbers indicate times (in min) from the first image. Size bars, 3 µm. <b>B.</b> Localization pattern of Gic2-PBD-RFP (red) prior to, during, and after cytokinesis (Cdc3-GFP in green) is summarized from time-lapse imagings of wild type (n = 15), <i>rga1Δ</i> (n = 19), <i>bud8Δ</i> (n = 7) and <i>rga1Δ bud8Δ</i> (n = 8). The proximal-pole localization pattern (marked with 2*) of <i>rga1Δ</i> or <i>rga1Δ bud8Δ</i> daughter cells is different from those seen in wild type and <i>bud8Δ</i> cells (see text for details).</p