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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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Morphological and physiological responses of WT and PORCx plants to light stress.Both WT and PORCx (T-13) plants were grown in light (100 µmoles photons m−2 s−1) for 22–24 days and subsequently transferred to low-light (LL) (50 µmoles photons m−2 s−1, 16 h light/8 h dark) or high-light (HL) (330 µmoles photons m−2 s−1, 16 h light/8 h dark) regimes for 6-7 d as described in experimental procedures. (A) Photographs of WT and T-13 plants after 6–7 d of transfer to LL and HL. (B) Photosynthetic efficiency (Fv/Fm) of leaves of LL- and HL- exposed plants was monitored by PAM 2100 fluorometer. Values are mean ± SD (n = 20). (C) Anthocyanin contents of WT and T-13 plants grown under HL. (D) The gene expression study of CHS in HL-grown WT and T-13 plants was done by RT-PCR as described in experimental procedures. AtACT1 was used as an internal control. (E) Pchlide contents of HL-treated WT and T-13 plants measured 10 min after the end of dark period. (F) Singlet oxygen (1O2) contents in WT and T-13 plants. Thylakoid membranes were isolated in complete darkness from HL- exposed plants and the 1O2 production was determined in terms of RNO bleaching using histidine as a trap. (G) Malondealdehyde (MDA) production in HL- treated WT and T-13 plants. Each data point represented in all the above experiments is the average of 6 replicates. The error bar represents SD.
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pone-0026532-g004: Morphological and physiological responses of WT and PORCx plants to light stress.Both WT and PORCx (T-13) plants were grown in light (100 µmoles photons m−2 s−1) for 22–24 days and subsequently transferred to low-light (LL) (50 µmoles photons m−2 s−1, 16 h light/8 h dark) or high-light (HL) (330 µmoles photons m−2 s−1, 16 h light/8 h dark) regimes for 6-7 d as described in experimental procedures. (A) Photographs of WT and T-13 plants after 6–7 d of transfer to LL and HL. (B) Photosynthetic efficiency (Fv/Fm) of leaves of LL- and HL- exposed plants was monitored by PAM 2100 fluorometer. Values are mean ± SD (n = 20). (C) Anthocyanin contents of WT and T-13 plants grown under HL. (D) The gene expression study of CHS in HL-grown WT and T-13 plants was done by RT-PCR as described in experimental procedures. AtACT1 was used as an internal control. (E) Pchlide contents of HL-treated WT and T-13 plants measured 10 min after the end of dark period. (F) Singlet oxygen (1O2) contents in WT and T-13 plants. Thylakoid membranes were isolated in complete darkness from HL- exposed plants and the 1O2 production was determined in terms of RNO bleaching using histidine as a trap. (G) Malondealdehyde (MDA) production in HL- treated WT and T-13 plants. Each data point represented in all the above experiments is the average of 6 replicates. The error bar represents SD.

Mentions: After 6–7 days of low light (LL; 50 μmoles photons m−2 s−1) exposure there was no significant difference in the phenotype of WT and T-13 plants (Figure 4A). However, in high light (HL; 350 μmoles photons m−2 s−1) several leaves of the WT plants looked purple (Figure 4A).


Overexpression of protochlorophyllide oxidoreductase C regulates oxidative stress in Arabidopsis.

Pattanayak GK, Tripathy BC - PLoS ONE (2011)

Morphological and physiological responses of WT and PORCx plants to light stress.Both WT and PORCx (T-13) plants were grown in light (100 µmoles photons m−2 s−1) for 22–24 days and subsequently transferred to low-light (LL) (50 µmoles photons m−2 s−1, 16 h light/8 h dark) or high-light (HL) (330 µmoles photons m−2 s−1, 16 h light/8 h dark) regimes for 6-7 d as described in experimental procedures. (A) Photographs of WT and T-13 plants after 6–7 d of transfer to LL and HL. (B) Photosynthetic efficiency (Fv/Fm) of leaves of LL- and HL- exposed plants was monitored by PAM 2100 fluorometer. Values are mean ± SD (n = 20). (C) Anthocyanin contents of WT and T-13 plants grown under HL. (D) The gene expression study of CHS in HL-grown WT and T-13 plants was done by RT-PCR as described in experimental procedures. AtACT1 was used as an internal control. (E) Pchlide contents of HL-treated WT and T-13 plants measured 10 min after the end of dark period. (F) Singlet oxygen (1O2) contents in WT and T-13 plants. Thylakoid membranes were isolated in complete darkness from HL- exposed plants and the 1O2 production was determined in terms of RNO bleaching using histidine as a trap. (G) Malondealdehyde (MDA) production in HL- treated WT and T-13 plants. Each data point represented in all the above experiments is the average of 6 replicates. The error bar represents SD.
© Copyright Policy
Related In: Results  -  Collection

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pone-0026532-g004: Morphological and physiological responses of WT and PORCx plants to light stress.Both WT and PORCx (T-13) plants were grown in light (100 µmoles photons m−2 s−1) for 22–24 days and subsequently transferred to low-light (LL) (50 µmoles photons m−2 s−1, 16 h light/8 h dark) or high-light (HL) (330 µmoles photons m−2 s−1, 16 h light/8 h dark) regimes for 6-7 d as described in experimental procedures. (A) Photographs of WT and T-13 plants after 6–7 d of transfer to LL and HL. (B) Photosynthetic efficiency (Fv/Fm) of leaves of LL- and HL- exposed plants was monitored by PAM 2100 fluorometer. Values are mean ± SD (n = 20). (C) Anthocyanin contents of WT and T-13 plants grown under HL. (D) The gene expression study of CHS in HL-grown WT and T-13 plants was done by RT-PCR as described in experimental procedures. AtACT1 was used as an internal control. (E) Pchlide contents of HL-treated WT and T-13 plants measured 10 min after the end of dark period. (F) Singlet oxygen (1O2) contents in WT and T-13 plants. Thylakoid membranes were isolated in complete darkness from HL- exposed plants and the 1O2 production was determined in terms of RNO bleaching using histidine as a trap. (G) Malondealdehyde (MDA) production in HL- treated WT and T-13 plants. Each data point represented in all the above experiments is the average of 6 replicates. The error bar represents SD.
Mentions: After 6–7 days of low light (LL; 50 μmoles photons m−2 s−1) exposure there was no significant difference in the phenotype of WT and T-13 plants (Figure 4A). However, in high light (HL; 350 μmoles photons m−2 s−1) several leaves of the WT plants looked purple (Figure 4A).

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