Overexpression of Protochlorophyllide Oxidoreductase C Regulates Oxidative Stress in Arabidopsis

Light absorbed by colored intermediates of chlorophyll biosynthesis is not utilized in photosynthesis; instead, it is transferred to molecular oxygen, generating singlet oxygen (1O2). As there is no enzymatic detoxification mechanism available in plants to destroy 1O2, 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 1O2 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 1O2 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 1O2-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

Controlling the Cyanobacterial Clock by Synthetically Rewiring Metabolism

SummaryCircadian clocks are oscillatory systems that allow organisms to anticipate rhythmic changes in the environment. Several studies have shown that circadian clocks are connected to metabolism, but it is not generally clear whether metabolic signaling is one voice among many that influence the clock or whether metabolic cycling is the major clock synchronizer. To address this question in cyanobacteria, we used a synthetic biology approach to make normally autotrophic cells capable of growth on exogenous sugar. This allowed us to manipulate metabolism independently from light and dark. We found that feeding sugar to cultures blocked the clock-resetting effect of a dark pulse. Furthermore, in the absence of light, the clock efficiently synchronizes to metabolic cycles driven by rhythmic feeding. We conclude that metabolic activity, independent of its source, is the primary clock driver in cyanobacteria

Chlorophyll biosynthetic pathway intermediate contents and ¹O₂ production in ALA-treated WT and <i>PORCx</i> plants.

<p>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. (<b>A</b>) Pchlide, Proto IX and MP(E) contents of ALA-treated (3 mM) and 14 h-dark-incubated WT and T-13 plants. (<b>B</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. (<b>C</b>) 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.</p

<i>PORCx</i> plants are tolerant to ALA-inducd oxidative damage.

<p>WT and <i>PORCx</i> (T-13) plants were treated with ALA and exposed to light for different time periods. (<b>A</b>) Photographs of T-13 plants under ALA-induced oxidative damage. Notice the death of the WT plants after 24 h of light exposure, whereas T-13 plants are slightly damaged. (<b>B</b>) Survival of light-exposed T-13 plants treated with different concentration of ALA. Both WT and T-13 plants grown under the same condition as described above were treated with different concentration of ALA (from 1 mM to 5 mM) and their dose dependent tolerance was observed. Notice the WT plants were killed by 3 mM or 5 mM ALA-treatment.</p

POR activity, ALA content and steady state Chl biosynthesis intermediates of WT and <i>PORCx</i> plants.

<p>(<b>A</b>) Photoperiodically (14 h L/10 h D) grown 4-week-old WT and <i>PORCx</i> (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>B</b>) Net accumulation of ALA from endogenous substrates of leaves harvested from WT and <i>PORCx</i> (T-12, T-13) plants. (<b>C</b>) Steady state tetrapyrrole contents of WT and <i>PORCx</i> 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.</p

Transformation of Arabidopsis by using <i>AtPORC</i> cDNA.

<p>(<b>A</b>) TDNA region of modified pCAMBIA1304 - 35SΩ <i>AtporC</i>. (<b>B</b>) Western blot of PORC in WT and <i>PORCx</i> plants (T-9, T-12, T-13). Thylakoids were isolated from WT and <i>PORCx</i> 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. (<b>C</b>) Quantification of band intensities of PORC and Ribulose-1, 5-bisphosphate carboxylase oxygenase large subunit (RbcL) presented in ‘B’. (<b>D</b>) WT and <i>PORCx</i> plants were grown as mentioned above and their chlorophyll and carotenoid contents were measured. (<b>E</b>) 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. (<b>F</b>) 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. (<b>G</b>) 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. (<b>H</b>) Leaves were excised from 18 days old and 28 days old WT and T-13 plants and photographed.</p

Accelerated cell death 2 suppresses mitochondrial oxidative bursts and modulates cell death in Arabidopsis

The Arabidopsis ACCELERATED CELL DEATH 2 (ACD2) protein protects cells from programmed cell death (PCD) caused by endogenous porphyrin-related molecules like red chlorophyll catabolite or exogenous protoporphyrin IX. We previously found that during bacterial infection, ACD2, a chlorophyll breakdown enzyme, localizes to both chloroplasts and mitochondria in leaves. Additionally, acd2 cells show mitochondrial dysfunction. In plants with acd2 and ACD2 (+) sectors, ACD2 functions cell autonomously, implicating a pro-death ACD2 substrate as being cell non-autonomous in promoting the spread of PCD. ACD2 targeted solely to mitochondria can reduce the accumulation of an ACD2 substrate that originates in chloroplasts, indicating that ACD2 substrate molecules are likely to be mobile within cells. Two different light-dependent reactive oxygen bursts in mitochondria play prominent and causal roles in the acd2 PCD phenotype. Finally, ACD2 can complement acd2 when targeted to mitochondria or chloroplasts, respectively, as long as it is catalytically active: the ability to bind substrate is not sufficient for ACD2 to function in vitro or in vivo. Together, the data suggest that ACD2 localizes dynamically during infection to protect cells from pro-death mobile substrate molecules, some of which may originate in chloroplasts, but have major effects on mitochondria