Glycolate Oxidase Isozymes Are Coordinately Controlled by GLO1 and GLO4 in Rice

Glycolate oxidase (GLO) is a key enzyme in photorespiratory metabolism. Four putative GLO genes were identified in the rice genome, but how each gene member contributes to GLO activities, particularly to its isozyme profile, is not well understood. In this study, we analyzed how each gene plays a role in isozyme formation and enzymatic activities in both yeast cells and rice tissues. Five GLO isozymes were detected in rice leaves. GLO1 and GLO4 are predominately expressed in rice leaves, while GLO3 and GLO5 are mainly expressed in the root. Enzymatic assays showed that all yeast-expressed GLO members except GLO5 have enzymatic activities. Further analyses suggested that GLO1, GLO3 and GLO4 interacted with each other, but no interactions were observed for GLO5. GLO1/GLO4 co-expressed in yeast exhibited the same isozyme pattern as that from rice leaves. When either GLO1 or GLO4 was silenced, expressions of both genes were simultaneously suppressed and most of the GLO activities were lost, and consistent with this observation, little GLO isozyme protein was detected in the silenced plants. In contrast, no observable effect was detected when GLO3 was suppressed. Comparative analyses between the GLO isoforms expressed in yeast and the isozymes from rice leaves indicated that two of the five isozymes are homo-oligomers composed of either GLO1 or GLO4, and the other three are hetero-oligomers composed of both GLO1 and GLO4. Our current data suggest that GLO isozymes are coordinately controlled by GLO1 and GLO4 in rice, and the existence of GLO isozymes and GLO molecular and compositional complexities implicate potential novel roles for GLO in plants

Young’s modulus of multi-layer microcantilevers

A theoretical model for calculating the Young’s modulus of multi-layer microcantilevers with a coating is proposed, and validated by a three-dimensional (3D) finite element (FE) model using ANSYS parametric design language (APDL) and atomic force microscopy (AFM) characterization. Compared with typical theoretical models (Rayleigh-Ritz model, Euler-Bernoulli (E-B) beam model and spring mass model), the proposed theoretical model can obtain Young’s modulus of multi-layer microcantilevers more precisely. Also, the influences of coating’s geometric dimensions on Young’s modulus and resonant frequency of microcantilevers are discussed. The thickness of coating has a great influence on Young’s modulus and resonant frequency of multi-layer microcantilevers, and the coating should be considered to calculate Young’s modulus more precisely, especially when fairly thicker coating is employed

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

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

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

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