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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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Chlorophyll biosynthetic pathway intermediate contents and 1O2 production in ALA-treated WT and PORCx plants.WT and T-13 plants grown for 28–32 days at 22°C±2°C under 14 h L/10 h D photoperiod (100 µmoles photons m−2 s−1) were sprayed with ALA (3 mM), dark incubaed for 14 h and exposed to light (100 µmoles photons m−2 s−1) for 10 min. Leaves were harvested both from dark incubated and light exposed plants, homogenized and their tetrapyrrole contents were monitored by spectrofluorometrically. (A) Pchlide, Proto IX and MP(E) contents of ALA-treated (3 mM) and 14 h-dark-incubated WT and T-13 plants. (B) After dark incubation both WT and T-13 plants were exposed to light (10 min) and their Pchlide, Proto IX and MP(E) were determined. (C) 1O2 contents in ALA-treated (+ALA) and untreated (-ALA) WT and T-13 plants. The experiments were repeated 5 times and each data point is the average of 5 replicates. The error bar represents ± SD.
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pone-0026532-g005: Chlorophyll biosynthetic pathway intermediate contents and 1O2 production in ALA-treated WT and PORCx plants.WT and T-13 plants grown for 28–32 days at 22°C±2°C under 14 h L/10 h D photoperiod (100 µmoles photons m−2 s−1) were sprayed with ALA (3 mM), dark incubaed for 14 h and exposed to light (100 µmoles photons m−2 s−1) for 10 min. Leaves were harvested both from dark incubated and light exposed plants, homogenized and their tetrapyrrole contents were monitored by spectrofluorometrically. (A) Pchlide, Proto IX and MP(E) contents of ALA-treated (3 mM) and 14 h-dark-incubated WT and T-13 plants. (B) After dark incubation both WT and T-13 plants were exposed to light (10 min) and their Pchlide, Proto IX and MP(E) were determined. (C) 1O2 contents in ALA-treated (+ALA) and untreated (-ALA) WT and T-13 plants. The experiments were repeated 5 times and each data point is the average of 5 replicates. The error bar represents ± SD.

Mentions: ALA is the precursor of Pchlide, which acts like a herbicide in presence of light [2], [4]. Exogenous application of 3 mM ALA to both WT and PORCx plants (28-32 days old plants grown under 14 h light/10 h dark photoperiod) at sunset resulted in overnight (14 h) over-accumulation of Chl biosynthetic intermediates i.e., Pchlide, Proto IX and MP(E) (Figure 5A). Pchlide regulates its own accumulation via feedback inhibition of ALA biosynthesis [29], [30]. Treatment of plants with ALA bypasses this regulatory site. Therefore, the feed-back regulation of ALA synthesis by Pchlide is ineffective in the presence of exogenous ALA. In contrast to data reported in Figure 3A, where overnight Pchlide accumulation in non-treated WT and PORCx plants is almost equal (because of tight feedback regulation of ALA synthesis by Pchilde), the ALA-treated transgenic plants accumulated higher amounts of Pchlide than that of WT as ALA treatment bypasses the feedback regulatory site and PORCx plants have higher Chl biosynthesis potential, as revealed by higher gene and protein expression of their Chl biosynthetic pathway enzymes. Angiosperms are incapable of converting Pchlide to Chlide in dark. Therefore, only after 10 min of light (100 µmoles photons m−2 s−1) exposure, the Pchlide content of both WT and T-13 plants declined due to light-dependent transformation of Pchlide to Chlide (Figure 5B). However, Pchlide content of ALA-treated and light exposed T-13 plants were significantly lower than that of WT (Figure 5B). This was due to higher efficiency of phototransformation of Pchlide in PORCx plants (76%) as compared to that for WT (30%).


Overexpression of protochlorophyllide oxidoreductase C regulates oxidative stress in Arabidopsis.

Pattanayak GK, Tripathy BC - PLoS ONE (2011)

Chlorophyll biosynthetic pathway intermediate contents and 1O2 production in ALA-treated WT and PORCx plants.WT and T-13 plants grown for 28–32 days at 22°C±2°C under 14 h L/10 h D photoperiod (100 µmoles photons m−2 s−1) were sprayed with ALA (3 mM), dark incubaed for 14 h and exposed to light (100 µmoles photons m−2 s−1) for 10 min. Leaves were harvested both from dark incubated and light exposed plants, homogenized and their tetrapyrrole contents were monitored by spectrofluorometrically. (A) Pchlide, Proto IX and MP(E) contents of ALA-treated (3 mM) and 14 h-dark-incubated WT and T-13 plants. (B) After dark incubation both WT and T-13 plants were exposed to light (10 min) and their Pchlide, Proto IX and MP(E) were determined. (C) 1O2 contents in ALA-treated (+ALA) and untreated (-ALA) WT and T-13 plants. The experiments were repeated 5 times and each data point is the average of 5 replicates. The error bar represents ± SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198771&req=5

pone-0026532-g005: Chlorophyll biosynthetic pathway intermediate contents and 1O2 production in ALA-treated WT and PORCx plants.WT and T-13 plants grown for 28–32 days at 22°C±2°C under 14 h L/10 h D photoperiod (100 µmoles photons m−2 s−1) were sprayed with ALA (3 mM), dark incubaed for 14 h and exposed to light (100 µmoles photons m−2 s−1) for 10 min. Leaves were harvested both from dark incubated and light exposed plants, homogenized and their tetrapyrrole contents were monitored by spectrofluorometrically. (A) Pchlide, Proto IX and MP(E) contents of ALA-treated (3 mM) and 14 h-dark-incubated WT and T-13 plants. (B) After dark incubation both WT and T-13 plants were exposed to light (10 min) and their Pchlide, Proto IX and MP(E) were determined. (C) 1O2 contents in ALA-treated (+ALA) and untreated (-ALA) WT and T-13 plants. The experiments were repeated 5 times and each data point is the average of 5 replicates. The error bar represents ± SD.
Mentions: ALA is the precursor of Pchlide, which acts like a herbicide in presence of light [2], [4]. Exogenous application of 3 mM ALA to both WT and PORCx plants (28-32 days old plants grown under 14 h light/10 h dark photoperiod) at sunset resulted in overnight (14 h) over-accumulation of Chl biosynthetic intermediates i.e., Pchlide, Proto IX and MP(E) (Figure 5A). Pchlide regulates its own accumulation via feedback inhibition of ALA biosynthesis [29], [30]. Treatment of plants with ALA bypasses this regulatory site. Therefore, the feed-back regulation of ALA synthesis by Pchlide is ineffective in the presence of exogenous ALA. In contrast to data reported in Figure 3A, where overnight Pchlide accumulation in non-treated WT and PORCx plants is almost equal (because of tight feedback regulation of ALA synthesis by Pchilde), the ALA-treated transgenic plants accumulated higher amounts of Pchlide than that of WT as ALA treatment bypasses the feedback regulatory site and PORCx plants have higher Chl biosynthesis potential, as revealed by higher gene and protein expression of their Chl biosynthetic pathway enzymes. Angiosperms are incapable of converting Pchlide to Chlide in dark. Therefore, only after 10 min of light (100 µmoles photons m−2 s−1) exposure, the Pchlide content of both WT and T-13 plants declined due to light-dependent transformation of Pchlide to Chlide (Figure 5B). However, Pchlide content of ALA-treated and light exposed T-13 plants were significantly lower than that of WT (Figure 5B). This was due to higher efficiency of phototransformation of Pchlide in PORCx plants (76%) as compared to that for WT (30%).

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