Suppression of both ELIP1 and ELIP2 in Arabidopsis does not affect tolerance to photoinhibition and photooxidative stress

ELIPs (early light-induced proteins) are thylakoid proteins transiently induced during greening of etiolated seedlings and during exposure to high light stress conditions. This expression pattern suggests that these proteins may be involved in the protection of the photosynthetic apparatus against photooxidative damage. To test this hypothesis, we have generated Arabidopsis (Arabidopsis thaliana) mutant plants null for both elip genes (Elip1 and Elip2) and have analyzed their sensitivity to light during greening of seedlings and to high light and cold in mature plants. In particular, we have evaluated the extent of damage to photosystem II, the level of lipid peroxidation, the presence of uncoupled chlorophyll molecules, and the nonphotochemical quenching of excitation energy. The absence of ELIPs during greening at moderate light intensities slightly reduced the rate of chlorophyll accumulation but did not modify the extent of photoinhibition. In mature plants, the absence of ELIP1 and ELIP2 did not modify the sensitivity to photoinhibition and photooxidation or the ability to recover from light stress. This raises questions about the photoprotective function of these proteins. Moreover, no compensatory accumulation of other ELIP-like proteins (SEPs, OHPs) was found in the elip1/elip2 double mutant during high light stress. elip1/elip2 mutant plants show only a slight reduction in the chlorophyll content in mature leaves and greening seedlings and a lower zeaxanthin accumulation in high light conditions, suggesting that ELIPs could somehow affect the stability or synthesis of these pigments. On the basis of these results, we make a number of suggestions concerning the biological function of ELIPs.     

UV radiation-responsive proteins in rice leaves: a proteomic analysis

Depletion of stratospheric ozone has led to increased UV radiation reaching the surface of the Earth. This may damage plants. Using physiological, proteomic and quantitative real-time PCR (qPCR) methods, we systematically studied the response of 16-day-old rice seedlings to UV [0.67 W m(-2) biologically effective UVB (UVB(BE)) and 0.28 W m(-2) UVA] exposure for 6, 12 and 24 h. UV exposure resulted in the appearance of light brown patches on leaves, a decrease in the net photosynthetic rate (Pn), lipid peroxidation, accumulation of UV-absorbing compounds (including flavonoids and other phenolic pigments) and differential expression of 22 proteins. Both physiological and molecular responses became stronger with increasing UV exposure time, indicating the effects of UV accumulation on plants. UV-induced responses included (i) phytohormone-regulative responses (up-regulation of proteins related to phytohormone synthesis such as IAA and ethylene); (ii) injurious responses (photosynthesis suppression, lipid peroxidation and visible injury); and (iii) protective responses (accumulation of UV-absorbing compounds and differential expression of proteins involved in detoxification/antioxidation, defense, protein processing, RNA processing, carbohydrate metabolism and secondary metabolism). The identification of UV-responsive proteins provided a better understanding of the molecular mechanism of plant responses to UV stress. Proteomic and qPCR analysis identified one up-regulated and two induced proteins with important functions: tryptophan synthase α chain (production of radical oxygen species), glyoxalase I (detoxification/antioxidation) and a Bet v I family protein (defense). These results will contribute to future research into their roles in UV stress responses in plants.     

Photo-oxidative stress in a xanthophyll-deficient mutant of Chlamydomonas

When there is an imbalance between the light energy absorbed by a photosynthetic organism and that which can be utilized in photosynthesis, photo-oxidative stress can damage pigments, proteins, lipids, and nucleic acids. In this work we compared the wild type and a xanthophyll-deficient mutant of Chlamydomonas reinhardtii in their response to high amounts of light. Wild-type Chlamydomonas cells were able to acclimate to high amounts of light following transfer from low light conditions. In contrast, the npq1 lor1 double mutant, which lacks protective xanthophylls (zeaxanthin and lutein) in the chloroplast, progressively lost viability and photosynthetic capacity along with destruction of thylakoid membrane protein-pigment complexes and accumulation of reactive oxygen species and membrane lipid peroxides. Loss of viability was partially rescued by lowered oxygen tension, suggesting that the high sensitivity of the mutant to light stress is caused by the production of reactive oxygen species in the chloroplast. Cell death was not prevented by the addition of an organic carbon source to the growth medium, demonstrating that the photo-oxidative damage can target other essential chloroplast processes besides photosynthesis. From the differential sensitivity of the mutant to exogenously added pro-oxidants, we infer that the reactive oxygen species produced during light stress in npq1 lor1 may be singlet oxygen and/or superoxide but not hydrogen peroxide. The bleaching phenotype of npq1 lor1 was not due to enhanced photodamage to photosystem II but rather to a less localized phenomenon of accumulation of photo-oxidation products in chloroplast membranes.     

In vitro synthesis, assembly and function of a photosynthetic membrane protein

Cell-free translation of Chlamydomonas reinhardtii RNA in the presence of photosynthetic membranes resulted in association of the herbicide binding (Qb) protein with membranes. Incubation of recovered membranes with high salt did not extract the polypeptide from membranes. Tryptic digestion of in vivo labeled membranes or membranes recovered from in vitro translation mixtures showed that Qb had similar orientation. In vitro translation in the presence of chloroplast membranes from cells exposed to high light intensity restored the membrane associated kinase activity lost by photoinhibition. Thus, in vitro synthesis resulted in functional integration of the Qb protein within the photosynthetic membrane.     

Biosynthetic cause of in vivo acquired thermotolerance of photosynthetic light reactions and metabolic responses of chloroplasts to heat stress

Thermotolerance of photosynthetic light reactions in vivo is correlated with a decrease in the ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol and an increased incorporation into thylakoid membranes of saturated digalactosyl diacylglycerol species. Although electron transport remains virtually intact in thermotolerant chloroplasts, thylakoid protein phosphorylation is strongly inhibited. The opposite is shown for thermosensitive chloroplasts in vivo. Heat stress causes reversible and irreversible inactivation of chloroplast protein synthesis in heat-adapted and nonadapted plants, respectively, but doe not greatly affect formation of rapidly turned-over 32 kilodalton proteins of photosystem II. The formation on cytoplasmic ribosomes and import by chloroplasts of thylakoid and stroma proteins remain preserved, although decreased in rate, at supraoptimal temperatures. Thermotolerant chloroplasts accumulate heat shock proteins in the stroma among which 22 kilodalton polypeptides predominate. We suggest that interactions of heat shock proteins with the outer chloroplast envelope membrane might enhance formation of digalactosyl diacylglycerol species. Furthermore, a heat-induced recompartmentalization of the chloroplast matrix that ensures effective transport of ATP from thylakoid membranes towards those sites inside the chloroplast and the cytoplasm where photosynthetically indispensable components and heat shock proteins are being formed is proposed as a metabolic strategy of plant cells to survive and recover from heat stress.     

光质对植物光合作用的调控及其机理

光合作用是植物生长发育的基础.光质对植物光合作用的调控主要包括可见光对植物气孔器运动、叶片生长、叶绿体结构、光合色素、D1蛋白及其编码基因和光合碳同化等的调节,以及紫外光对植物光系统Ⅱ的影响.蓝光和红光能促进气孔的开张,而绿光能够逆转这种作用.蓝光有利于叶绿体的发育,红、蓝、绿复合光有利于叶面积的扩展,而红光更有利于光合产物的积累;不同光质对不同植物、不同组织器官叶绿素积累的影响不同.蓝光和远红光可以促进psbA基因转录物质的积累.大多数高等植物和绿藻在橙、红光下光合速率最高,蓝紫光其次,绿光最低.紫外光可以导致光系统Ⅱ的电子传递活性下降.此外,针对光质与光合作用研究领域中存在的问题,对今后的研究方向进行了讨论.     

The light stress-induced protein ELIP2 is a regulator of chlorophyll synthesis in Arabidopsis thaliana

The early light-induced proteins (ELIPs) belong to the multigenic family of pigment-binding light-harvesting complexes. ELIPs accumulate transiently and are believed to play a protective role in plants exposed to high levels of light. Constitutive expression of the ELIP2 gene in Arabidopsis resulted in a marked reduction of the pigment content of the chloroplasts, both in mature leaves and during greening of etiolated seedlings. The chlorophyll loss was associated with a decrease in the number of photosystems in the thylakoid membranes, but the photosystems present were fully assembled and functional. A detailed analysis of the chlorophyll-synthesizing pathway indicated that ELIP2 accumulation downregulated the level and activity of two important regulatory steps: 5-aminolevulinate synthesis and Mg-protoporphyrin IX (Mg-Proto IX) chelatase activity. The contents of glutamyl tRNA reductase and Mg chelatase subunits CHLH and CHLI were lowered in response to ELIP2 accumulation. In contrast, ferrochelatase activity was not affected and the inhibition of Heme synthesis was null or very moderate. As a result of reduced metabolic flow from 5-aminolevulinic acid, the steady state levels of various chlorophyll precursors (from protoporphyrin IX to protochlorophyllide) were strongly reduced in the ELIP2 overexpressors. Taken together, our results indicate that the physiological function of ELIPs could be related to the regulation of chlorophyll concentration in thylakoids. This seems to occur through an inhibition of the entire chlorophyll biosynthesis pathway from the initial precursor of tetrapyrroles, 5-aminolevulinic acid. We suggest that ELIPs work as chlorophyll sensors that modulate chlorophyll synthesis to prevent accumulation of free chlorophyll, and hence prevent photooxidative stress.     

Molecular mechanisms of stress resistance of the photosynthetic apparatus

The mechanisms of action of environmental stress-inducing factors on the photosynthetic apparatus (PA) of plants are considered. The basic targets for stress produced by heat, cold, salinity, osmotic imbalance, and high irradiance are analyzed. It is suggested that stress factors have an influence on the composition of thylakoid membranes and inhibit photosynthetic processes. However, recent studies demonstrated that strong light induces the photodamage to photosystem II (PS II) due to direct action of light on the oxygen-evolving complex. Stress-induced accumulation of reactive oxygen species (ROS) leads to inhibition of the recovery of the PSII by suppressing thede novo synthesis of photosynthetic proteins. In addition, stress stimulates the synthesis of protective low-molecular weight compounds (e.g., glycine betaine) and stress proteins. The major mechanisms of acclimation and protection of the PA against damaging effects of environmental stress-inducing factors are analyzed with special reference to cyanobacterial cells and mutants with high or low stress resistance.     

Chloroplast‐targeted bacterial RecA proteins confer tolerance to chloroplast DNA damage by methyl viologen or UV‐C radiation in tobacco (Nicotiana tabacum) plants

The nature and importance of the DNA repair system in the chloroplasts of higher plants under oxidative stress or UV radiation‐induced genotoxicity was investigated via gain‐of‐functional approaches exploiting bacterial RecAs. For this purpose, transgenic tobacco (Nicotiana tabacum) plants and cell suspensions overexpressing Escherichia coli or Pseudomonas aeruginosa RecA fused to a chloroplast‐targeting transit peptide were first produced. The transgenic tobacco plants maintained higher amounts of chloroplast DNA compared with wild‐type (WT) upon treatments with methyl viologen (MV), a herbicide that generates reactive oxygen species (ROS) in chloroplasts. Consistent with these results, the transgenic tobacco leaves showed less bleaching than WT following MV exposure. Similarly, the MV‐treated transgenic Arabidopsis plants overexpressing the chloroplast RecA homologue RECA1 showed weak bleaching, while the recA1 mutant showed opposite results upon MV treatment. In addition, when exposed to UV‐C radiation, the dark‐grown E. coli RecA‐overexpressing transgenic tobacco cell suspensions, but not their WT counterparts, resumed growth and greening after the recovery period under light conditions. Measurements of UV radiation‐induced chloroplast DNA damage using DraI assays (Harlow et al. 1994) with the chloroplast rbcL DNA probe and quantitative PCR analyses showed that the transgenic cell suspensions better repaired their UV‐C radiation‐induced chloroplast DNA lesions compared with WT. Taken all together, it was concluded that RecA‐overexpressing transgenic plants are endowed with an increased chloroplast DNA maintenance capacity and enhanced repair activities, and consequently have a higher survival tolerance to genotoxic stresses. These observations are made possible by the functional compatibility of the bacterial RecAs in chloroplasts.     

Absence of nitrate reductase activity in San 9789 bleached leaves of barley seedlings (Hordeum vulgare cv. Midas)

Abstract San 9789 (norflurazone) blocks carotenoid synthesis which allows chlorophyll bleaching in the light, and has been used recently as a tool to study phytochrome responses without interference from photosynthetic pigments. By using this herbicide, we have found that nitrate reductase activity and light dependent nitrite reduction were lost simultaneously from achlorophyllous areas of barley leaves, with the green areas of the leaf tip still showing high activities. By contrast nitrate reductase is still present in the roots of herbicide treated plants. We suggest that intact chloroplasts are required for the presence of nitrate reductase in barley leaves.     

Differential responses to different light spectral ranges of violaxanthin de-epoxidation and accumulation of Cbr,an algal homologue of plant early light inducible proteins,in two strains of Dunaliella

Unicellular green algae of the genus Dunaliella, similar to higher plants, respond to light stress by enhanced de-epoxidation of violaxanthin and accumulation of Cbr, a protein homologous to early light inducible proteins (Elips) in plants. These proteins belong to the superfamily of chlorophyll a/b binding proteins. Two Dunaliella strains, D. bardawil and D. salina, were compared for these two responses under light in the UVA, blue, green and red spectral ranges. In D. bardawil, the two stress responses were similarly induced under UVA, blue or red light and to a lesser extent under green light. In D. salina, a similar spectral range dependence was exhibited for violaxanthin de-epoxidation. However, Cbr accumulated only under UVA or blue light but not under green or red light. A strong synergistic effect of a low dose of blue light superimposed on red light resulted in Cbr accumulation. These results reveal strain-specific differences in spectral range requirements of the two light-stress responses. In the two strains, violaxanthin de-epoxidation is triggered under photosynthetically-active spectral ranges but at least in D. salina, Cbr accumulation appears to require a specific light signal additionally to a signal(s) generated by light stress.     

Transgenic tobacco plants expressing antisense ferredoxin-NADP(H) reductase transcripts display increased susceptibility to photo-oxidative damage

Ferredoxin-NADP(H) reductase (FNR) catalyses the final step of the photosynthetic electron transport in chloroplasts. Using an antisense RNA strategy to reduce expression of this flavoenzyme in transgenic tobacco plants, it has been demonstrated that FNR mediates a rate-limiting step of photosynthesis under both limiting and saturating light conditions. Here, we show that these FNR-deficient plants are abnormally prone to photo-oxidative injury. When grown under autotrophic conditions for 3 weeks, specimens with 20-40% extant reductase undergo leaf bleaching, lipid peroxidation and membrane damage. The magnitude of the effect was proportional to the light intensity and to the extent of FNR depletion, and was accompanied by morphological changes involving accumulation of aberrant plastids with defective thylakoid stacking. Damage was initially confined to chloroplast membranes, whereas Rubisco and other stromal proteins began to decline only after several weeks of autotrophic growth, paralleled by partial recovery of NADPH levels. Exposure of the transgenic plants to moderately high irradiation resulted in rapid loss of photosynthetic capacity and accumulation of singlet oxygen in leaves. The collected results suggest that the extensive photo-oxidative damage sustained by plants impaired in FNR expression was caused by singlet oxygen building up to toxic levels in these tissues, as a direct consequence of the over-reduction of the electron transport chain in FNR-deficient chloroplasts.