Proposition pour un tiroir de sélection de référence pour les alimentations programmables utilisables à hautes et à basses énergies: cas particulier des alimentations PFW

(a) Effect of varying pH on activities of GLO isozymes. Each buffer (50 mM) was made of respective PBS (pH 6.0-8.0) and Tris-HCl (pH 8.0-9.0). (The highest activity of each GLO isozyme at pH 7.8 was set as 1). (b) Effect of temperature on activities of GLO isozymes. Enzymes in 50 mM PBS buffer (pH 7.8) were pre-incubated at various temperatures (22-60 °C) for 5 min, and then activities were measured at the same temperature (For GLO1 and GLO1 + 4, the highest activity at 45 °C was set as 1; For GLO3, the highest activity at 47 °C was set as 1; For GLO4, the highest activity at 42 °C was set as 1). Values are means ± SD (n = 3). (TIFF 7340 kb

GLO isozyme patterns and the expression of various <i>GLO</i> members in rice.

<p>(<b>A</b>) Enzymatic activity staining showing patterns of GLO isozymes. GLO enzyme was extracted from rice leaves and separated by 6% CN-PAGE at a running pH of 10.2. The number above each lane indicates activity units (µmol H₂O₂ min⁻¹ mg⁻¹ protein) loaded. Arrows point to each isozyme band. This result is representative of five independent experiments. (<b>B</b>) mRNA transcript abundance of the four <i>GLO</i> genes (<i>GLO1, GLO3, GLO4,</i> and <i>GLO5</i>) in rice leaves and roots was determined by real-time quantitative RT-PCR. The second leaf from the top and 5 cm of roots were detached from plants for RNA isolation. Relative mRNA levels were graphed based on the mRNA level of Leaf-<i>GLO1</i> as 1. The data represent means ±SD of 3 independent experiments.</p

Catalytic activities of the GLO isoforms expressed in yeast.

<p>Y-GLO1S2, Y-GLO3S2, Y-GLO4S2, Y-GLO5S2 represent the crude enzyme extracted from yeast cells expressing pYES2-<i>GLO1</i>, pYES2-<i>GLO3</i>, pYES2-<i>GLO4</i>, pYES2-<i>GLO5</i>, respectively. Y-GLO1S3, Y-GLO3S3, Y-GLO4S3, represent the crude enzyme extracted from yeast cells expressing pYES3-<i>GLO1</i>, pYES3-<i>GLO3</i>, pYES3-<i>GLO4</i>, respectively. Y-GLO1S3+3S2, Y-GLO1S3+4S2, Y-GLO1S3+5S2, Y-GLO4S3+3S2, Y-GLO4S3+5S2, Y-GLO3S3+5S2, represent the crude enzyme extracted from yeast cells co-expressing pYES3-<i>GLO1</i> and pYES2-<i>GLO3</i>, pYES3-<i>GLO1</i> and pYES2-<i>GLO4</i>, pYES3-<i>GLO1</i> and pYES2-<i>GLO5</i>, pYES3-<i>GLO4</i> and pYES2-<i>GLO3</i>, pYES3-<i>GLO4</i> and pYES2-<i>GLO5</i>, pYES3-<i>GLO3</i> and pYES2-<i>GLO5</i>, respectively. R-GLO represents the crude enzyme extracted from leaves of rice. The data represent means ±SD of 3 independent experiments.</p

Interactions of the GLOs determined by His-tag pull down assay.

<p>GLO-his means the 6 amino acids on the C-terminus of GLO was mutated to a 6×his-tag. The interactions between every two GLOs are evaluated by calculating the activity recovery rate. The data are means of 3 independent experiments.</p

BiFC detection of interactions among the proteins encoded by the four <i>GLO</i> genes.

<p>Every two <i>GLO</i> genes were co-expressed in rice protoplasts and BiFC visualization assays were carried out to test the interaction as indicated: (A) NYFP-tagged GLO1 and CYFP-tagged GLO3; (B) NYFP-tagged GLO1 and CYFP-tagged GLO4; (C) NYFP-tagged GLO1 and CYFP-tagged GLO5; (D) NYFP-tagged GLO3 and CYFP-tagged GLO4; (E) NYFP-tagged GLO3 and CYFP-tagged GLO5; (F) NYFP-tagged GLO4 and CYFP-tagged GLO5; (G) NYFP and CYFP. This result is representative of three independent experiments.</p

Subcellular localization of GLO isoforms.

<p>The YFP-tagged GLOs and CFP-tagged PTS1 fusion constructs were used in protoplast transient expression system to determine the subcellular localization of GLOs, the CFP-tagged PTS1 was used as the peroxisome marker. A1–A4: transfection of rice protoplasts; B1–B4: transfection of <i>Arabidopsis</i> protoplasts. This result is representative of three independent experiments.</p

Comparison between the GLO isoforms expressed in yeast and those extracted from rice leaves.

<p>GLO isozymes were separated in 6% uniform CN-PAGE at a running pH of 10.2. Y-GLO1, Y-GLO3, Y-GLO4, represent the enzyme extracted from yeast cells expressing pYES3-<i>GLO1</i>, pYES3-<i>GLO3</i>, pYES3-<i>GLO4</i>, respectively. Y-GLO3+4, Y-GLO3+1, Y-GLO1+4, represent the enzyme extracted from yeast cells co-expressing pYES3-<i>GLO3</i> and pYES2-<i>GLO4</i>, pYES3-<i>GLO3</i> and pYES2-<i>GLO1</i>, pYES3-<i>GLO1</i> and pYES2-<i>GLO4</i>, respectively. R-GLO represents the enzyme extracted from leaves of rice, and R-OxGLO4 represents the enzyme extracted from leaves of <i>GLO4</i>-overexpressed transgenic rice. This result is representative of five independent experiments.</p

Molecular weights of the GLO isofroms extracted from yeast and rice leaves.

<p>(<b>A</b>) The molecular weights of the subunits determined by uniform SDS-PAGE (12.5%). Y-GLO represents the enzyme purified from yeast cells by immobilized metal affinity chromatography, and R-GLO represents the enzyme purified from leaves of rice. (<b>B</b>) The molecular weights of the holoenzymes determinated by gradient CN-PAGE (3–12%, running pH 8.3). Y-GLO represents the crude enzyme extracted from yeast cells, R-GLO represents the partially purified enzyme extracted from leaves of rice. This result is representative of five independent experiments.</p

<i>GLO1</i> and <i>GLO4</i> are the major contributors to GLO activities in rice.

<p>(<b>A</b>) Semi-quantitative RT-PCR analysis of each GLO gene in the transgenic plants carrying the silencing construct. This result is representative of three independent experiments. (<b>B</b>) GLO enzyme activities in transgenic plants. Ri<i>GLO1</i>, Ri<i>GLO3</i>, Ri<i>GLO4</i> represent the specific <i>GLO1</i>, <i>GLO3</i> and <i>GLO4</i> RNA-silencing transgenic plants, respectively. The second leaf from the top was detached from plants at vegetative stage. Relative GLO activity was graphed based on the GLO activity of wild type (WT) as 1. The data represent means ±SD of 3 independent experiments.</p

Final DNA sequence used for phylogenetic inference

Final DNA sequence used for phylogenetic inferenc