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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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POR activity, ALA content and steady state Chl biosynthesis intermediates of WT and PORCx plants.(A) Photoperiodically (14 h L/10 h D) grown 4-week-old WT and PORCx (T-12, T-13) plants were incubated in dark for 14 h and their protochlorophyllide (Pchlide) contents were determined in dark. After dark incubation, plants were exposed to light (100 µmoles photons m−2 s−1) for 10 min and their Pchlide contents were monitored and phtotransformation of Pchlide to chlorophyllide was determined. (B) Net accumulation of ALA from endogenous substrates of leaves harvested from WT and PORCx (T-12, T-13) plants. (C) Steady state tetrapyrrole contents of WT and PORCx plants. Leaf samples were harvested from photoperiodically (14 h L/10 h D) grown plants during the light phase (7 h after beginning of light cycle), homogenized immediately in light and the chlorophyll biosynthetic tetrapyrroles (Pchlide, Proto IX and MP(E)) contents were estimated. The experiments were repeated for 3 times and each data point is the average of 6 replicates. The error bar represents ± SD.
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pone-0026532-g003: POR activity, ALA content and steady state Chl biosynthesis intermediates of WT and PORCx plants.(A) Photoperiodically (14 h L/10 h D) grown 4-week-old WT and PORCx (T-12, T-13) plants were incubated in dark for 14 h and their protochlorophyllide (Pchlide) contents were determined in dark. After dark incubation, plants were exposed to light (100 µmoles photons m−2 s−1) for 10 min and their Pchlide contents were monitored and phtotransformation of Pchlide to chlorophyllide was determined. (B) Net accumulation of ALA from endogenous substrates of leaves harvested from WT and PORCx (T-12, T-13) plants. (C) Steady state tetrapyrrole contents of WT and PORCx plants. Leaf samples were harvested from photoperiodically (14 h L/10 h D) grown plants during the light phase (7 h after beginning of light cycle), homogenized immediately in light and the chlorophyll biosynthetic tetrapyrroles (Pchlide, Proto IX and MP(E)) contents were estimated. The experiments were repeated for 3 times and each data point is the average of 6 replicates. The error bar represents ± SD.

Mentions: To measure the PORC activity, four-week-old light-grown WT and PORCx plants were kept in dark for 14 h followed by 10 min light exposure (100 µmoles photons m−2 s−1) and then the photo-transformation of Pchlide was measured. Although a single saturating flash illumination is quite efficient for the transformation of photo-transformable Pchlide pool to Chlide in etiolated tissues, it is not sufficient in light-grown green tissues, for the conversion of non-photo- transformable Pchlide. Actually, exposure of plants to light for several minutes/hours was used before to monitor the disappearance of Pchlide [2], [5], [7], [28]. After 10 min light exposure the Pchlide content was reduced to a greater extent in PORCx plants compared to WT plants; the percentage of phototransformation in WT was 61%, whereas in PORCx T-12 and T-13 plants it was 82% and 89% respectively (Figure 3A).


Overexpression of protochlorophyllide oxidoreductase C regulates oxidative stress in Arabidopsis.

Pattanayak GK, Tripathy BC - PLoS ONE (2011)

POR activity, ALA content and steady state Chl biosynthesis intermediates of WT and PORCx plants.(A) Photoperiodically (14 h L/10 h D) grown 4-week-old WT and PORCx (T-12, T-13) plants were incubated in dark for 14 h and their protochlorophyllide (Pchlide) contents were determined in dark. After dark incubation, plants were exposed to light (100 µmoles photons m−2 s−1) for 10 min and their Pchlide contents were monitored and phtotransformation of Pchlide to chlorophyllide was determined. (B) Net accumulation of ALA from endogenous substrates of leaves harvested from WT and PORCx (T-12, T-13) plants. (C) Steady state tetrapyrrole contents of WT and PORCx plants. Leaf samples were harvested from photoperiodically (14 h L/10 h D) grown plants during the light phase (7 h after beginning of light cycle), homogenized immediately in light and the chlorophyll biosynthetic tetrapyrroles (Pchlide, Proto IX and MP(E)) contents were estimated. The experiments were repeated for 3 times and each data point is the average of 6 replicates. The error bar represents ± SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198771&req=5

pone-0026532-g003: POR activity, ALA content and steady state Chl biosynthesis intermediates of WT and PORCx plants.(A) Photoperiodically (14 h L/10 h D) grown 4-week-old WT and PORCx (T-12, T-13) plants were incubated in dark for 14 h and their protochlorophyllide (Pchlide) contents were determined in dark. After dark incubation, plants were exposed to light (100 µmoles photons m−2 s−1) for 10 min and their Pchlide contents were monitored and phtotransformation of Pchlide to chlorophyllide was determined. (B) Net accumulation of ALA from endogenous substrates of leaves harvested from WT and PORCx (T-12, T-13) plants. (C) Steady state tetrapyrrole contents of WT and PORCx plants. Leaf samples were harvested from photoperiodically (14 h L/10 h D) grown plants during the light phase (7 h after beginning of light cycle), homogenized immediately in light and the chlorophyll biosynthetic tetrapyrroles (Pchlide, Proto IX and MP(E)) contents were estimated. The experiments were repeated for 3 times and each data point is the average of 6 replicates. The error bar represents ± SD.
Mentions: To measure the PORC activity, four-week-old light-grown WT and PORCx plants were kept in dark for 14 h followed by 10 min light exposure (100 µmoles photons m−2 s−1) and then the photo-transformation of Pchlide was measured. Although a single saturating flash illumination is quite efficient for the transformation of photo-transformable Pchlide pool to Chlide in etiolated tissues, it is not sufficient in light-grown green tissues, for the conversion of non-photo- transformable Pchlide. Actually, exposure of plants to light for several minutes/hours was used before to monitor the disappearance of Pchlide [2], [5], [7], [28]. After 10 min light exposure the Pchlide content was reduced to a greater extent in PORCx plants compared to WT plants; the percentage of phototransformation in WT was 61%, whereas in PORCx T-12 and T-13 plants it was 82% and 89% respectively (Figure 3A).

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