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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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Transformation of Arabidopsis by using AtPORC cDNA.(A) TDNA region of modified pCAMBIA1304 - 35SΩ AtporC. (B) Western blot of PORC in WT and PORCx plants (T-9, T-12, T-13). Thylakoids were isolated from WT and PORCx plants grown for 4-weeks under 14 h L/10 h D photoperiod (100 µmoles photons m−2 s−1) at 22°C±2°C and thirty µg of thylakoid proteins were loaded in each lane of SDS-PAGE. Western blot was done using PORC (1:1000) monoclonal antiserum (top panel). The bottom panel shows coomassie-stained gel for equal loading. Before coomassie staining, the membrane was probed with RbcL antibody (1:20000) and the respective protein was identified by Western blot. (C) Quantification of band intensities of PORC and Ribulose-1, 5-bisphosphate carboxylase oxygenase large subunit (RbcL) presented in ‘B’. (D) WT and PORCx plants were grown as mentioned above and their chlorophyll and carotenoid contents were measured. (E) Western blot of LHC II (1:5000) in WT and T-12, T-13 plants (upper panel). RbcL protein was identified by Western blot analysis as described above and was shown in the bottom panel to check for the equal loading of the thylakoid proteins. (F) Quantification of band intensities of LHCII with the RbcL control presented in ‘E’. Signal intensity for each protein was expressed relative to WT. All the above experiments were repeated three times and each data point is the average of three replicates and the error bar represents SD. (G) Phenotypic differences of Arabidopsis WT and T-13 plants after 4-weeks of growth at 22°C±2°C under 14 h L/10 h D photoperiod (100 µmoles photons m−2 s−1). As the T-13 plant has higher PORC amount and higher Chl content, we have only shown its picture. Notice the T-13 plants were greener and slightly smaller than the WT plants. (H) Leaves were excised from 18 days old and 28 days old WT and T-13 plants and photographed.
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pone-0026532-g001: Transformation of Arabidopsis by using AtPORC cDNA.(A) TDNA region of modified pCAMBIA1304 - 35SΩ AtporC. (B) Western blot of PORC in WT and PORCx plants (T-9, T-12, T-13). Thylakoids were isolated from WT and PORCx plants grown for 4-weeks under 14 h L/10 h D photoperiod (100 µmoles photons m−2 s−1) at 22°C±2°C and thirty µg of thylakoid proteins were loaded in each lane of SDS-PAGE. Western blot was done using PORC (1:1000) monoclonal antiserum (top panel). The bottom panel shows coomassie-stained gel for equal loading. Before coomassie staining, the membrane was probed with RbcL antibody (1:20000) and the respective protein was identified by Western blot. (C) Quantification of band intensities of PORC and Ribulose-1, 5-bisphosphate carboxylase oxygenase large subunit (RbcL) presented in ‘B’. (D) WT and PORCx plants were grown as mentioned above and their chlorophyll and carotenoid contents were measured. (E) Western blot of LHC II (1:5000) in WT and T-12, T-13 plants (upper panel). RbcL protein was identified by Western blot analysis as described above and was shown in the bottom panel to check for the equal loading of the thylakoid proteins. (F) Quantification of band intensities of LHCII with the RbcL control presented in ‘E’. Signal intensity for each protein was expressed relative to WT. All the above experiments were repeated three times and each data point is the average of three replicates and the error bar represents SD. (G) Phenotypic differences of Arabidopsis WT and T-13 plants after 4-weeks of growth at 22°C±2°C under 14 h L/10 h D photoperiod (100 µmoles photons m−2 s−1). As the T-13 plant has higher PORC amount and higher Chl content, we have only shown its picture. Notice the T-13 plants were greener and slightly smaller than the WT plants. (H) Leaves were excised from 18 days old and 28 days old WT and T-13 plants and photographed.

Mentions: We generated transgenic Arabidopsis plants overexpressing PORC cDNA (PORCx) under the control of the cauliflower mosaic virus (CaMV) 35S promoter having omega (Ω) translational enhancer (Figure 1A). Protein levels of PORC in four-week-old WT and PORCx (T9, T12, T13) plants were analyzed using the PORC monoclonal antibody (Figure 1B). The PORCx lines had higher protein expression (1.5–3 folds) of the trans-gene (Figure 1C). They had increased Chl content i.e., up to 28% over that of WT plants and had also a little higher carotenoid content (Figure 1D). The PORCx plants/leaves look greener than WT plants (Figure 1G, H). LHC II protein was increased in PORCx plants by 33% and 58% respectively (Figure 1E, F).


Overexpression of protochlorophyllide oxidoreductase C regulates oxidative stress in Arabidopsis.

Pattanayak GK, Tripathy BC - PLoS ONE (2011)

Transformation of Arabidopsis by using AtPORC cDNA.(A) TDNA region of modified pCAMBIA1304 - 35SΩ AtporC. (B) Western blot of PORC in WT and PORCx plants (T-9, T-12, T-13). Thylakoids were isolated from WT and PORCx plants grown for 4-weeks under 14 h L/10 h D photoperiod (100 µmoles photons m−2 s−1) at 22°C±2°C and thirty µg of thylakoid proteins were loaded in each lane of SDS-PAGE. Western blot was done using PORC (1:1000) monoclonal antiserum (top panel). The bottom panel shows coomassie-stained gel for equal loading. Before coomassie staining, the membrane was probed with RbcL antibody (1:20000) and the respective protein was identified by Western blot. (C) Quantification of band intensities of PORC and Ribulose-1, 5-bisphosphate carboxylase oxygenase large subunit (RbcL) presented in ‘B’. (D) WT and PORCx plants were grown as mentioned above and their chlorophyll and carotenoid contents were measured. (E) Western blot of LHC II (1:5000) in WT and T-12, T-13 plants (upper panel). RbcL protein was identified by Western blot analysis as described above and was shown in the bottom panel to check for the equal loading of the thylakoid proteins. (F) Quantification of band intensities of LHCII with the RbcL control presented in ‘E’. Signal intensity for each protein was expressed relative to WT. All the above experiments were repeated three times and each data point is the average of three replicates and the error bar represents SD. (G) Phenotypic differences of Arabidopsis WT and T-13 plants after 4-weeks of growth at 22°C±2°C under 14 h L/10 h D photoperiod (100 µmoles photons m−2 s−1). As the T-13 plant has higher PORC amount and higher Chl content, we have only shown its picture. Notice the T-13 plants were greener and slightly smaller than the WT plants. (H) Leaves were excised from 18 days old and 28 days old WT and T-13 plants and photographed.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026532-g001: Transformation of Arabidopsis by using AtPORC cDNA.(A) TDNA region of modified pCAMBIA1304 - 35SΩ AtporC. (B) Western blot of PORC in WT and PORCx plants (T-9, T-12, T-13). Thylakoids were isolated from WT and PORCx plants grown for 4-weeks under 14 h L/10 h D photoperiod (100 µmoles photons m−2 s−1) at 22°C±2°C and thirty µg of thylakoid proteins were loaded in each lane of SDS-PAGE. Western blot was done using PORC (1:1000) monoclonal antiserum (top panel). The bottom panel shows coomassie-stained gel for equal loading. Before coomassie staining, the membrane was probed with RbcL antibody (1:20000) and the respective protein was identified by Western blot. (C) Quantification of band intensities of PORC and Ribulose-1, 5-bisphosphate carboxylase oxygenase large subunit (RbcL) presented in ‘B’. (D) WT and PORCx plants were grown as mentioned above and their chlorophyll and carotenoid contents were measured. (E) Western blot of LHC II (1:5000) in WT and T-12, T-13 plants (upper panel). RbcL protein was identified by Western blot analysis as described above and was shown in the bottom panel to check for the equal loading of the thylakoid proteins. (F) Quantification of band intensities of LHCII with the RbcL control presented in ‘E’. Signal intensity for each protein was expressed relative to WT. All the above experiments were repeated three times and each data point is the average of three replicates and the error bar represents SD. (G) Phenotypic differences of Arabidopsis WT and T-13 plants after 4-weeks of growth at 22°C±2°C under 14 h L/10 h D photoperiod (100 µmoles photons m−2 s−1). As the T-13 plant has higher PORC amount and higher Chl content, we have only shown its picture. Notice the T-13 plants were greener and slightly smaller than the WT plants. (H) Leaves were excised from 18 days old and 28 days old WT and T-13 plants and photographed.
Mentions: We generated transgenic Arabidopsis plants overexpressing PORC cDNA (PORCx) under the control of the cauliflower mosaic virus (CaMV) 35S promoter having omega (Ω) translational enhancer (Figure 1A). Protein levels of PORC in four-week-old WT and PORCx (T9, T12, T13) plants were analyzed using the PORC monoclonal antibody (Figure 1B). The PORCx lines had higher protein expression (1.5–3 folds) of the trans-gene (Figure 1C). They had increased Chl content i.e., up to 28% over that of WT plants and had also a little higher carotenoid content (Figure 1D). The PORCx plants/leaves look greener than WT plants (Figure 1G, H). LHC II protein was increased in PORCx plants by 33% and 58% respectively (Figure 1E, F).

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