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Overexpression of protochlorophyllide oxidoreductase C regulates oxidative stress in Arabidopsis.

Pattanayak GK, Tripathy BC - PLoS ONE (2011)

Bottom Line: Further, PORCx plants treated with 5-aminolevulinicacid when exposed to light, photo-converted over-accumulated protochlorophyllide to chlorophyllide, reduced the generation of (1)O(2) and malonedialdehyde production and reduced plasma membrane damage.Reduced protochlorophyllide content in PORCx plants released the protochlorophyllide-mediated feed-back inhibition of 5-aminolevulinicacid biosynthesis that resulted in higher 5-aminolevulinicacid production.Increase of 5-aminolevulinicacid synthesis upregulated the gene and protein expression of several downstream chlorophyll biosynthetic enzymes elucidating a regulatory net work of expression of genes involved in 5-aminolevulinicacid and tetrapyrrole biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delphi, India.

ABSTRACT
Light absorbed by colored intermediates of chlorophyll biosynthesis is not utilized in photosynthesis; instead, it is transferred to molecular oxygen, generating singlet oxygen ((1)O(2)). As there is no enzymatic detoxification mechanism available in plants to destroy (1)O(2), its generation should be minimized. We manipulated the concentration of a major chlorophyll biosynthetic intermediate i.e., protochlorophyllide in Arabidopsis by overexpressing the light-inducible protochlorophyllide oxidoreductase C (PORC) that effectively phototransforms endogenous protochlorophyllide to chlorophyllide leading to minimal accumulation of the photosensitizer protochlorophyllide in light-grown plants. In PORC overexpressing (PORCx) plants exposed to high-light, the (1)O(2) generation and consequent malonedialdehyde production was minimal and the maximum quantum efficiency of photosystem II remained unaffected demonstrating that their photosynthetic apparatus and cellular organization were intact. Further, PORCx plants treated with 5-aminolevulinicacid when exposed to light, photo-converted over-accumulated protochlorophyllide to chlorophyllide, reduced the generation of (1)O(2) and malonedialdehyde production and reduced plasma membrane damage. So PORCx plants survived and bolted whereas, the 5-aminolevulinicacid-treated wild-type plants perished. Thus, overexpression of PORC could be biotechnologically exploited in crop plants for tolerance to (1)O(2)-induced oxidative stress, paving the use of 5-aminolevulinicacid as a selective commercial light-activated biodegradable herbicide. Reduced protochlorophyllide content in PORCx plants released the protochlorophyllide-mediated feed-back inhibition of 5-aminolevulinicacid biosynthesis that resulted in higher 5-aminolevulinicacid production. Increase of 5-aminolevulinicacid synthesis upregulated the gene and protein expression of several downstream chlorophyll biosynthetic enzymes elucidating a regulatory net work of expression of genes involved in 5-aminolevulinicacid and tetrapyrrole biosynthesis.

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RT-PCR and Western blot analysis of Chl biosynthetic enzymes in PORCx plants.(A) The gene expression study of the Chl biosynthetic pathway enzymes in T-12 and T-13 plants was done by semi-quantitative RT-PCR analysis using the gene specific primer pairs for each enzyme as described in materials and methods. ACT1 was used as an internal control. Fifteen µl of the PCR products were loaded and separated on 1% agarose Tris-acetate EDTA gel. Ethidium bromide-stained PCR products were quantified using the Alpha Imager 3400. (B) Bar diagram of gene expression (%). Rate of expression is represented as percentage of control (WT). The data presented are representative of three independent experiments. (C) Immunoblot analysis of Chl biosynthetic pathway proteins was carried out using plastid proteins isolated from WT and T-12, T-13 plants. (D) Bar diagram of protein expression (%) of different enzymes and the rate of expression is represented as percentage of control (WT). Each data point is the average of three replicates and the error bar represents SD.
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pone-0026532-g002: RT-PCR and Western blot analysis of Chl biosynthetic enzymes in PORCx plants.(A) The gene expression study of the Chl biosynthetic pathway enzymes in T-12 and T-13 plants was done by semi-quantitative RT-PCR analysis using the gene specific primer pairs for each enzyme as described in materials and methods. ACT1 was used as an internal control. Fifteen µl of the PCR products were loaded and separated on 1% agarose Tris-acetate EDTA gel. Ethidium bromide-stained PCR products were quantified using the Alpha Imager 3400. (B) Bar diagram of gene expression (%). Rate of expression is represented as percentage of control (WT). The data presented are representative of three independent experiments. (C) Immunoblot analysis of Chl biosynthetic pathway proteins was carried out using plastid proteins isolated from WT and T-12, T-13 plants. (D) Bar diagram of protein expression (%) of different enzymes and the rate of expression is represented as percentage of control (WT). Each data point is the average of three replicates and the error bar represents SD.

Mentions: The increased amount of Chl in PORCx led us to study if the over-expression of PORC modulated the gene expression and protein abundance of other Chl biosynthetic pathway enzymes. We performed semi-quantitative reverse transcription (RT)-PCR and found the transcript abundance of GluTR, GSAT, UROD and CHLP increased respectively by 38%, 80%, 67% and 36% in T-12 and 90%, 110%, 130% and 60% in T-13 PORCx lines (Figure 2A, B).


Overexpression of protochlorophyllide oxidoreductase C regulates oxidative stress in Arabidopsis.

Pattanayak GK, Tripathy BC - PLoS ONE (2011)

RT-PCR and Western blot analysis of Chl biosynthetic enzymes in PORCx plants.(A) The gene expression study of the Chl biosynthetic pathway enzymes in T-12 and T-13 plants was done by semi-quantitative RT-PCR analysis using the gene specific primer pairs for each enzyme as described in materials and methods. ACT1 was used as an internal control. Fifteen µl of the PCR products were loaded and separated on 1% agarose Tris-acetate EDTA gel. Ethidium bromide-stained PCR products were quantified using the Alpha Imager 3400. (B) Bar diagram of gene expression (%). Rate of expression is represented as percentage of control (WT). The data presented are representative of three independent experiments. (C) Immunoblot analysis of Chl biosynthetic pathway proteins was carried out using plastid proteins isolated from WT and T-12, T-13 plants. (D) Bar diagram of protein expression (%) of different enzymes and the rate of expression is represented as percentage of control (WT). Each data point is the average of three replicates and the error bar represents SD.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198771&req=5

pone-0026532-g002: RT-PCR and Western blot analysis of Chl biosynthetic enzymes in PORCx plants.(A) The gene expression study of the Chl biosynthetic pathway enzymes in T-12 and T-13 plants was done by semi-quantitative RT-PCR analysis using the gene specific primer pairs for each enzyme as described in materials and methods. ACT1 was used as an internal control. Fifteen µl of the PCR products were loaded and separated on 1% agarose Tris-acetate EDTA gel. Ethidium bromide-stained PCR products were quantified using the Alpha Imager 3400. (B) Bar diagram of gene expression (%). Rate of expression is represented as percentage of control (WT). The data presented are representative of three independent experiments. (C) Immunoblot analysis of Chl biosynthetic pathway proteins was carried out using plastid proteins isolated from WT and T-12, T-13 plants. (D) Bar diagram of protein expression (%) of different enzymes and the rate of expression is represented as percentage of control (WT). Each data point is the average of three replicates and the error bar represents SD.
Mentions: The increased amount of Chl in PORCx led us to study if the over-expression of PORC modulated the gene expression and protein abundance of other Chl biosynthetic pathway enzymes. We performed semi-quantitative reverse transcription (RT)-PCR and found the transcript abundance of GluTR, GSAT, UROD and CHLP increased respectively by 38%, 80%, 67% and 36% in T-12 and 90%, 110%, 130% and 60% in T-13 PORCx lines (Figure 2A, B).

Bottom Line: Further, PORCx plants treated with 5-aminolevulinicacid when exposed to light, photo-converted over-accumulated protochlorophyllide to chlorophyllide, reduced the generation of (1)O(2) and malonedialdehyde production and reduced plasma membrane damage.Reduced protochlorophyllide content in PORCx plants released the protochlorophyllide-mediated feed-back inhibition of 5-aminolevulinicacid biosynthesis that resulted in higher 5-aminolevulinicacid production.Increase of 5-aminolevulinicacid synthesis upregulated the gene and protein expression of several downstream chlorophyll biosynthetic enzymes elucidating a regulatory net work of expression of genes involved in 5-aminolevulinicacid and tetrapyrrole biosynthesis.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delphi, India.

ABSTRACT
Light absorbed by colored intermediates of chlorophyll biosynthesis is not utilized in photosynthesis; instead, it is transferred to molecular oxygen, generating singlet oxygen ((1)O(2)). As there is no enzymatic detoxification mechanism available in plants to destroy (1)O(2), its generation should be minimized. We manipulated the concentration of a major chlorophyll biosynthetic intermediate i.e., protochlorophyllide in Arabidopsis by overexpressing the light-inducible protochlorophyllide oxidoreductase C (PORC) that effectively phototransforms endogenous protochlorophyllide to chlorophyllide leading to minimal accumulation of the photosensitizer protochlorophyllide in light-grown plants. In PORC overexpressing (PORCx) plants exposed to high-light, the (1)O(2) generation and consequent malonedialdehyde production was minimal and the maximum quantum efficiency of photosystem II remained unaffected demonstrating that their photosynthetic apparatus and cellular organization were intact. Further, PORCx plants treated with 5-aminolevulinicacid when exposed to light, photo-converted over-accumulated protochlorophyllide to chlorophyllide, reduced the generation of (1)O(2) and malonedialdehyde production and reduced plasma membrane damage. So PORCx plants survived and bolted whereas, the 5-aminolevulinicacid-treated wild-type plants perished. Thus, overexpression of PORC could be biotechnologically exploited in crop plants for tolerance to (1)O(2)-induced oxidative stress, paving the use of 5-aminolevulinicacid as a selective commercial light-activated biodegradable herbicide. Reduced protochlorophyllide content in PORCx plants released the protochlorophyllide-mediated feed-back inhibition of 5-aminolevulinicacid biosynthesis that resulted in higher 5-aminolevulinicacid production. Increase of 5-aminolevulinicacid synthesis upregulated the gene and protein expression of several downstream chlorophyll biosynthetic enzymes elucidating a regulatory net work of expression of genes involved in 5-aminolevulinicacid and tetrapyrrole biosynthesis.

Show MeSH
Related in: MedlinePlus