Baishnab C. Tripathy

Reactive oxygen species generation and signaling in plants

The introduction of molecular oxygen into the atmosphere was accompanied by the generation of reactive oxygen species (ROS) as side products of many biochemical reactions. ROS are permanently generated in plastids, peroxisomes, mitochiondria, the cytosol and the apoplast. Imbalance between ROS generation and safe detoxification generates oxidative stress and the accumulating ROS are harmful for the plants. On the other hand, specific ROS function as signaling molecules and activate signal transduction processes in response to various stresses. Here, we summarize the generation of ROS in the different cellular compartments and the signaling processes which are induced by ROS.

Root-Shoot Interaction in the Greening of Wheat Seedlings Grown under Red Light

Wheat seedlings grown with roots exposed to constant red light (300–500 [mu]mol m-2 s-1) did not accumulate chlorophyll in the leaves. In contrast, seedlings grown with their roots shielded from light accumulated chlorophylls. Chlorophyll biosynthesis could be induced in red-light-grown chlorophyll-deficient yellow plants by either reducing the red-light intensity at the root surface to 100 [mu]mol m-2 s-1 or supplementing with 6% blue light. The inhibition of chlorophyll biosynthesis was due to impairment of the Mg-chelatase enzyme working at the origin of the Mg-tetrapyrrole pathway. The root-perceived photomorphogenic inhibition of shoot greening demonstrates root-shoot interaction in the greening process.

Inactivation of chloroplast photosynthetic electron-transport activity by Ni2+

Abstract Ni2+ inhibits electron-transport activity of isolated barley chloroplasts and this inhibition of electron transport by Ni2+ is distinctly different from other heavy metal ion (e.g., Pb2+, Cd2+, Zn2+)-induced inhibition of chloroplast function. Ni2+ inactivates Photosystem II (PS II) activity at a lower concentration than that required for the same extent of inhibition of Photosystem I (PS I)-mediated electron flow. Ni2+ induces changes in chlorophyll a (Chl a) emission characteristics and brings about a lowering of the Chl a fluorescence yield, and this lowering of Chl a fluorescence intensity is not relieved by the exogenously supplied electron donor NH2OH which donates electrons very close to the PS II reaction centres. Immobilization of the chloroplast membrane structure with glutaraldehyde fails to arrest the Ni2+-induced loss of PS II activity. Also, Ni2+-treated chloroplasts do not regain the ability to photoreduce 2,6-dichlorophenolindophenol even after washing of chloroplasts with buffer. These results indicate that unlike Zn2+ or Pb2+, Ni2+ induces alterations in the chloroplast photosynthetic apparatus resulting in an irreversible loss of electron-transport activity.

Role of Temperature Stress on Chloroplast Biogenesis and Protein Import in Pea

Modulation of photosynthesis and chloroplast biogenesis, by low and high temperatures, was studied in 12-d-old pea (Pisum sativum) plants grown at 25°C and subsequently exposed to 7°C or 40°C up to 48 h. The decline in variable chlorophyll a fluorescence/maximum chlorophyll a fluorescence and estimated electron transport rate in temperature-stressed plants was substantially restored when they were transferred to room temperature. The ATP-driven import of precursor of small subunit of Rubisco (pRSS) into plastids was down-regulated by 67% and 49% in heat-stressed and chill-stressed plants, respectively. Reduction in binding of the pRSS to the chloroplast envelope membranes in heat-stressed plants could be due to the down-regulation of Toc159 gene/protein expression. In addition to impaired binding, reduced protein import into chloroplast in heat-stressed plants was likely due to decreased gene/protein expression of certain components of the TOC complex (Toc75), the TIC complex (Tic20, Tic32, Tic55, and Tic62), stromal Hsp93, and stromal processing peptidase. In chill-stressed plants, the gene/protein expression of most of the components of protein import apparatus other than Tic110 and Tic40 were not affected, suggesting the central role of Tic110 and Tic40 in inhibition of protein import at low temperature. Heating of intact chloroplasts at 35°C for 10 min inhibited protein import, implying a low thermal stability of the protein import apparatus. Results demonstrate that in addition to decreased gene and protein expression, down-regulation of photosynthesis in temperature-stressed plants is caused by reduced posttranslational import of plastidic proteins required for the replacement of impaired proteins coded by nuclear genome.

Light and dark modulation of chlorophyll biosynthetic genes in response to temperature

Temperature and light significantly influence chloroplast development and chlorophyll biosynthesis. To understand the mechanism of the modulation of chlorophyll biosynthesis, the levels of transcripts and proteins of many enzymatic steps of tetrapyrrole biosynthesis in wheat and cucumber were simultaneously examined. The effect of low (chill-stress) as well as high (heat-stress) temperatures on dark- and light-grown seedlings was monitored. The protochlorophyllide oxidoreductase (POR) content was greatly reduced in response to light in control and heat-stressed seedlings. However, the POR level was not reduced in light-exposed chill-stressed seedlings. The genes for glutamate semialdehyde aminotransferase (gsa; cucumber), glutamyl-tRNA reductase (GluTR; cucumber), 5-aminolevulinic acid dehydratase (Ala D; cucumber and wheat) and for a subunit of Mg-chelatase (Chl I; wheat) showed a reduced expression in cold stress compared to controls and heat-stress conditions. Although expression of the ferrochelatase gene (Fch) and geranylgeranyl reductase gene (Chl P) was upregulated in light, they were downregulated by both chill- and heat-stress. Interestingly, gsa and uroporphyrinogen decarboxylase gene (UroD) and gene product abundance was stimulated by light and heat-stress implying the presence of both light and heat-inducible elements in their promoters. This observation corroborates with the previous report of increased enzymatic activity of UroD in heat-stressed cucumber seedlings. The gsa and Uro D may play an important role in tolerance of the greening process of plants to heat-stress.

Light Intensity-Dependent Modulation of Chlorophyll b Biosynthesis and Photosynthesis by Overexpression of Chlorophyllide a Oxygenase in Tobacco

Chlorophyll b is synthesized by the oxidation of a methyl group on the B ring of a tetrapyrrole molecule to a formyl group by chlorophyllide a oxygenase (CAO). The full-length CAO from Arabidopsis (Arabidopsis thaliana) was overexpressed in tobacco (Nicotiana tabacum) that grows well at light intensities much higher than those tolerated by Arabidopsis. This resulted in an increased synthesis of glutamate semialdehyde, 5-aminolevulinic acid, magnesium-porphyrins, and chlorophylls. Overexpression of CAO resulted in increased chlorophyll b synthesis and a decreased chlorophyll a/b ratio in low light-grown as well as high light-grown tobacco plants; this effect, however, was more pronounced in high light. The increased potential of the protochlorophyllide oxidoreductase activity and chlorophyll biosynthesis compensated for the usual loss of chlorophylls in high light. Increased chlorophyll b synthesis in CAO-overexpressed plants was accompanied not only by an increased abundance of light-harvesting chlorophyll proteins but also of other proteins of the electron transport chain, which led to an increase in the capture of light as well as enhanced (40%–80%) electron transport rates of photosystems I and II at both limiting and saturating light intensities. Although the quantum yield of carbon dioxide fixation remained unchanged, the light-saturated photosynthetic carbon assimilation, starch content, and dry matter accumulation increased in CAO-overexpressed plants grown in both low- and high-light regimes. These results demonstrate that controlled up-regulation of chlorophyll b biosynthesis comodulates the expression of several thylakoid membrane proteins that increase both the antenna size and the electron transport rates and enhance carbon dioxide assimilation, starch content, and dry matter accumulation.

Modulation of chlorophyll biosynthesis by water stress in rice seedlings during chloroplast biogenesis

To understand the impact of water stress on the greening process, water stress was applied to 6-day-old etiolated seedlings of a drought-sensitive cultivar of rice (Oryza sativa), Pusa Basmati-1 by immersing their roots in 40 mm polyethylene glycol (PEG) 6000 (-0.69 MPa) or 50 mm PEG 6000 (-1.03 MPa) dissolved in half-strength Murashige and Skoog (MS)-nutrient-solution, 16 h prior to transfer to cool-white-fluorescent + incandescent light. Chlorophyll (Chl) accumulation substantially declined in developing water-stressed seedlings. Reduced Chl synthesis was due to decreased accumulation of chlorophyll biosynthetic intermediates, that is, glutamate-1-semialdehyde (GSA), 5-aminolevulinic acid, Mg-protoporphyrin IX monomethylester and protochlorophyllide. Although 5-aminolevulinic acid synthesis decreased, the gene expression and protein abundance of the enzyme responsible for its synthesis, GSA aminotransferase, increased, suggesting its crucial role in the greening process in stressful environment. The biochemical activities of Chl biosynthetic enzymes, that is, 5-aminolevulinic acid dehydratase, porphobilinogen deaminase, coproporphyrinogen III oxidase, porphyrinogen IX oxidase, Mg-chelatase and protochlorophyllide oxidoreductase, were down-regulated due to their reduced protein abundance/gene expression in water-stressed seedlings. Down-regulation of protochlorophyllide oxidoreductase resulted in impaired Shibata shift. Our results demonstrate that reduced synthesis of early intermediates, that is, GSA and 5-aminolevulinic acid, could modulate the gene expression of later enzymes of Chl biosynthesis pathway.

Chlorophyll biosynthetic reactions during senescence of excised Barley (Hordeum vulgare L. cv IB 65) leaves

The chlorophyll (Chl) biosynthetic reactions were monitored during senescence of dark-incubated excised barley (Hordeum vulgare L. cv IB 65) leaves floated in double-distilled water or kinetin solution. Kinetin abolished the degradation of Chl but failed to check the net degradation of protochlorophyllide (Pchlide), suggesting that different sets of enzymes, i.e. kinetin sensitive and insensitive, are responsible for the degradation of Chl and Pchlide, respectively. Upon exposure of the leaves to light, the dark-accumulated Pchlide was efficiently phototransformed to chorophyllide (Chlide), even on the 7th d of dark incubation, demonstrating that the activity of Pchlide reductase, one of the late enzymes of the Chl biosynthetic pathway, is not substantially affected during senescence. The senescing leaves continued to synthesize Pchlide and Chlide until the 7th d, although at a reduced rate (20% of the 1st d). The decline of the rate of synthesis of Pchlide and Chlide is due to the loss of activity of two early enzymes of the Chl biosynthetic pathway, i.e. 5-aminolevulinic acid dehydratase and porphobilinogen deaminase. Kinetin substantially checked the loss of activity of these two enzymes.

Acclimation of chlorophyll biosynthetic reactions to temperature stress in cucumber (Cucumis sativus L.)

Abstract. The adaptive responses of the greening process of plants to temperature stress were studied in cucumber (Cucumis sativus L. cv. Poinsette) seedlings grown at ambient (25 °C), low (7 °C) and high (42 °C) temperatures. Plastids isolated from these seedlings were incubated at different temperatures and the net syntheses of various tetrapyrroles were monitored. In plastids isolated from control seedlings grown at 25 °C, the optimum temperature for synthesis of Mg-protoporphyrin IX monoester or protochlorophyllide was 35 °C. Temperature maxima for Mg-protoporphyrin IX monoester and protochlorophyllide syntheses were shifted to 30 °C in chill-stressed seedlings. The net synthesis of total tetrapyrroles was severely reduced in heat-stressed seedlings and the optimum temperature for Mg-protoporphyrin IX monoester or protochlorophyllide synthesis shifted slightly towards higher temperatures, i.e. a broader peak was observed. To further study the temperature acclimation of seedlings with respect to the greening process, tetrapyrrole biosynthesis was monitored at 25 °C after pre-heating the plastids (28–70 °C) isolated from control, chill- and heat-stressed seedlings. In comparison to 28 °C-pre-heated plastids the percent inhibition of protochlorophyllide synthesis in 40 °C-pre-heated plastids was higher than for the control (25 °C-grown) in chill-stressed seedlings and lower than for the control in heat-stressed seedlings. Maximum synthesis of total tetrapyrroles and protoporphyrin IX was observed when chloroplasts were heated at 50 °C, which was probably due to heat-induced activation of the enzymes involved in protoporphyrin IX synthesis. Prominent shoulders towards lower or higher temperatures were seen in chill-stressed or heat-stressed seedlings, respectively. The shift in optimum temperature for tetrapyrrole biosynthesis in chill- and heat-stressed seedlings was probably due to acclimation of membranes possibly undergoing desaturation or saturation of membrane lipids. Proteins synthesized in response to temperature-stress may also play an important role in conferring stress-tolerance in plants.

Cobalt ions inhibit electron-transport activity of Photosystem II without affecting Photosystem I

Abstract Investigations on photosynthesis have greatly benefited by the use of specific inhibitors that affect a specific site of inhibition on the electron-transport chain. We show here for the first time that cobalt (Co 2+ ) ions can be used specifically to inactivate electron donation to the reaction centre of Photosystem (PS) II without affecting PS I reactions. This conclusion is based on the following observations: (1) addition of exogenous electron donors such as NH 2 OH does not relieve Co 2+ -induced inactivation of photoelectron transport or the lowering of steady-state chlorophyll a fluorescence yield; this suggests that the inhibition is beyond the NH 2 OH donation site and before the fluorescence quencher Q, i.e., on the reaction centre complex itself. (2) Washing of Co 2+ -pretreated chloroplasts with isolation buffer to remove Co 2+ does not relieve Co 2+ -induced inhibition of Hill activity, suggesting that the Co 2+ effect is irreversible. (3) Co 2+ did not alter the PS I reactions. Thus, Co 2+ -treated chloroplasts can be used to study PS I functions free from PS II reactions in isolated chloroplasts.