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Glycolate oxidase isozymes are coordinately controlled by GLO1 and GLO4 in rice.

Zhang Z, Lu Y, Zhai L, Deng R, Jiang J, Li Y, He Z, Peng X - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China.

ABSTRACT
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.

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GLO1 and GLO4 are the major contributors to GLO activities in rice.(A) 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) GLO enzyme activities in transgenic plants. RiGLO1, RiGLO3, RiGLO4 represent the specific GLO1, GLO3 and GLO4 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.
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pone-0039658-g005: GLO1 and GLO4 are the major contributors to GLO activities in rice.(A) 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) GLO enzyme activities in transgenic plants. RiGLO1, RiGLO3, RiGLO4 represent the specific GLO1, GLO3 and GLO4 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.

Mentions: We then compared the isozyme profiles between the isoforms expressed in yeast and the GLO from rice leaves. As shown in Figure 4, when either GLO1 or GLO4 was expressed in yeast, a single band was seen, whose position was comparable to the first and fifth bands for the rice GLO. When the genes were pair by pair co-expressed in yeast, only GLO1/GLO4 exhibited the same isozyme pattern as the GLO pattern seen in rice. When GLO4 was over-expressed in rice, the fifth band became much stronger, whose position was comparable to GLO4 expressed in yeast. GLO3 could barely move through the gel in this system, and the isozyme patterns for GLO3/GLO4 and GLO3/GLO1 were obviously different from the rice GLO pattern (Figure 4). Up to this point, our results suggest that GLO1 and GLO4 control GLO activities and GLO isozyme formation. To further verify this observation in vivo, an RNAi approach was used to specifically silence each GLO gene. The interfering sequences were carefully designed to guarantee the specificity of silencing (Table S2). When either GLO1 or GLO4 was down-regulated, expression of both genes was simultaneously suppressed, and most of the GLO enzymatic activities were lost. (Figure 5). Consistent with this observation, little GLO isozyme proteins were detected in these plants (data not shown).


Glycolate oxidase isozymes are coordinately controlled by GLO1 and GLO4 in rice.

Zhang Z, Lu Y, Zhai L, Deng R, Jiang J, Li Y, He Z, Peng X - PLoS ONE (2012)

GLO1 and GLO4 are the major contributors to GLO activities in rice.(A) 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) GLO enzyme activities in transgenic plants. RiGLO1, RiGLO3, RiGLO4 represent the specific GLO1, GLO3 and GLO4 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3383670&req=5

pone-0039658-g005: GLO1 and GLO4 are the major contributors to GLO activities in rice.(A) 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) GLO enzyme activities in transgenic plants. RiGLO1, RiGLO3, RiGLO4 represent the specific GLO1, GLO3 and GLO4 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.
Mentions: We then compared the isozyme profiles between the isoforms expressed in yeast and the GLO from rice leaves. As shown in Figure 4, when either GLO1 or GLO4 was expressed in yeast, a single band was seen, whose position was comparable to the first and fifth bands for the rice GLO. When the genes were pair by pair co-expressed in yeast, only GLO1/GLO4 exhibited the same isozyme pattern as the GLO pattern seen in rice. When GLO4 was over-expressed in rice, the fifth band became much stronger, whose position was comparable to GLO4 expressed in yeast. GLO3 could barely move through the gel in this system, and the isozyme patterns for GLO3/GLO4 and GLO3/GLO1 were obviously different from the rice GLO pattern (Figure 4). Up to this point, our results suggest that GLO1 and GLO4 control GLO activities and GLO isozyme formation. To further verify this observation in vivo, an RNAi approach was used to specifically silence each GLO gene. The interfering sequences were carefully designed to guarantee the specificity of silencing (Table S2). When either GLO1 or GLO4 was down-regulated, expression of both genes was simultaneously suppressed, and most of the GLO enzymatic activities were lost. (Figure 5). Consistent with this observation, little GLO isozyme proteins were detected in these plants (data not shown).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China.

ABSTRACT
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.

Show MeSH