Accumulation Of Protochlorophyllide Research Articles

Plant competition cues activate a singlet oxygen signaling pathway in Arabidopsis thaliana.

Oxidative stress responses of Arabidopsis to reflected low red to far-red signals (R:FR ≈ 0.3) generated by neighboring weeds or an artificial source of FR light were compared with a weed-free control (R:FR ≈1.6). In the low R:FR treatments, induction of the shade avoidance responses (SAR) coincided with increased leaf production of singlet oxygen (1O2). This 1O2 increase was not due to protochlorophyllide accumulation and did not cause cell death. Chemical treatments, however, with 5-aminolevulinic acid (the precursor of tetrapyrrole biosynthesis) and glutathione (a quinone A reductant) enhanced cell death and growth inhibition. RNA sequencing revealed that transcriptome responses to the reflected low R:FR light treatments minimally resembled previously known Arabidopsis 1O2 generating systems that rapidly generate 1O2 following a dark to light transfer. The upregulation of only a few early 1O2 responsive genes (6 out of 1931) in the reflected low R:FR treatments suggested specificity of the 1O2 signaling. Moreover, increased expression of two enzyme genes, the SULFOTRANSFERASE ST2A (ST2a) and the early 1O2-responsive IAA-LEUCINE RESISTANCE (ILR)-LIKE6 (ILL6), which negatively regulate jasmonate level, suggested that repression of bioactive JAs may promote the shade avoidance (versus defense) and 1O2 acclimation (versus cell death) responses to neighboring weeds.

PIF3/HY5 module regulates BBX11 to suppress protochlorophyllide levels in dark and promote photomorphogenesis in light.

Greening of cotyledons during de-etiolation is critical for harvesting light energy and sustaining plant growth. PIF3 and HY5 antagonistically regulate protochlorophyllide synthesis in the dark. However, the mechanism by which the PIF3/HY5 module regulates genes involved in protochlorophyllide synthesis is not clear. Using genetic, molecular and biochemical techniques we identified that the B-BOX protein BBX11 acts directly downstream of PIF3 and HY5 to transcriptionally modulate genes involved in protochlorophyllide synthesis. Dark-grown bbx11 and 35S:BBX11 seedlings exhibit an enhanced and reduced ability to green, respectively, when exposed to light. Transcript levels of HEMA1 and CHLH are upregulated in 35S:BBX11 seedlings that accumulate high levels of protochlorophyllide in the dark and undergo photobleaching upon illumination. PIF3 inhibitsBBX11in the dark by directly binding to its promoter. bbx11 suppresses the cotyledon greening defect ofpif3after prolonged dark, indicating that the PIF3-mediated regulation of greening is dependent on BBX11. The enhanced greening of hy5 is also suppressed in hy5 lines overexpressing BBX11. In light, HY5 directly binds to the promoter of BBX11 and activates its expression to regulate BBX11-mediated hypocotyl inhibition. We show that a PIF3/HY5 module regulates BBX11 expression in opposite ways to optimise protochlorophyllide accumulation in the dark and promote photomorphogenesis in light.

Soybean (Glycine max) PHYTOCHROME-INTERACTION FACTOR 1 induced skotomorphogenesis and de-etiolation in Arabidopsis

Skotomorphogenesis occurs after germination and before excavation in plants. It inhibits excessive absorbed energy in cells and can prevent the lethal photooxidative damage caused by transitioning from skotomorphogenesis to photomorphogenesis for light energy utilization. To investigate the mechanisms underlying photoreactions in soybean [Glycine max (L.) Merr.], we identified and isolated soybean phytochrome-interaction factor 1 (GmPIF1). A yeast two-hybrid (Y2H) assay showed that GmPIF1 interacted with photoactive PHYTOCHROME A (PHYA) and B (PHYB) in both soybean and Arabidopsis (GmPHYA, GmPHYB, AtPHYA, and AtPHYB). To analyze its function, we ectopically over-expressed GmPIF1 in wild type and pif1 mutant Arabidopsis. In etiolated seedlings, GmPIF1 caused hypocotyl elongation, cotyledon closed, apical hooks folded, and less accumulation of protochlorophyllide. In Y2H, GmPIF1 interacted with AtHDA15 that inhibited chlorophyll synthesis under dark conditions. After transition from darkness to white light, GmPIF1 promotes the reduction of photobleaching and induced de-etiolation. Moreover, GmPIF1 inhibited PHYA- and PHYB-mediated seed germination. Our findings increase our understanding of the regulatory network of light response in soybean and provide useful gene resources for soybean breeding in programs and genetics engineering.

The miR396-GRFs Module Mediates the Prevention of Photo-oxidative Damage by Brassinosteroids during Seedling De-Etiolation in Arabidopsis.

The switch from dark- to light-mediated development is critical for the survival and growth of seedlings, but the underlying regulatory mechanisms are incomplete. Here, we show that the steroids phytohormone brassinosteroids play crucial roles during this developmental transition by regulating chlorophyll biosynthesis to promote greening of etiolated seedlings upon light exposure. Etiolated seedlings of the brassinosteroids-deficient det2-1 (de-etiolated2) mutant accumulated excess protochlorophyllide, resulting in photo-oxidative damage upon exposure to light. Conversely, the gain-of-function mutant bzr1-1D (brassinazole-resistant 1-1D) suppressed the protochlorophyllide accumulation of det2-1, thereby promoting greening of etiolated seedlings. Genetic analysis indicated that phytochrome-interacting factors (PIFs) were required for BZR1-mediated seedling greening. Furthermore, we reveal that GROWTH REGULATING FACTOR 7 (GRF7) and GRF8 are induced by BZR1 and PIF4 to repress chlorophyll biosynthesis and promote seedling greening. Suppression of GRFs function by overexpressing microRNA396a caused an accumulation of protochlorophyllide in the dark and severe photobleaching upon light exposure. Additionally, BZR1, PIF4, and GRF7 interact with each other and precisely regulate the expression of chlorophyll biosynthetic genes. Our findings reveal an essential role for BRs in promoting seedling development and survival during the initial emergence of seedlings from subterranean darkness into sunlight.

Nuclear-encoded sigma factor 6 (SIG6) is involved in the block of greening response in Arabidopsis thaliana.

Light is critical in the ability of plants to accumulate chlorophyll. When exposed to far-red (FR) light and then grown in white light in the absence of sucrose, wild-type seedlings fail to green in a response known as the FR block of greening (BOG). This response is controlled by phytochrome A through repression of protochlorophyllide reductase-encoding (POR) genes by FR light coupled with irreversible plastid damage. Sigma (SIG) factors are nuclear-encoded proteins that contribute to plant greening and plastid development through regulating gene transcription in chloroplasts and impacting retrograde signaling from the plastid to nucleus. SIGs are regulated by phytochromes, and the expression of some SIG factors is reduced in phytochrome mutant lines, includingphyA. Given the association of phyA with the FR BOG and its regulation of SIG factors, we investigated the potential regulatory role of SIG factors in the FR BOG response. We examined FR BOG responses in sig mutants, phytochrome-deficient lines, and mutant lines for several phy-associated factors. We quantified chlorophyll levels and examined expression of key BOG-associated genes. Among six sig mutants, onlythe sig6mutant significantly accumulated chlorophyll after FR BOG treatment, similar tothe phyA mutant. SIG6 appears to control protochlorophyllide accumulation by contributing to the regulation of tetrapyrrole biosynthesis associated with glutamyl-tRNA reductase (HEMA1) function, select phytochrome-interacting factor genes (PIF4 and PIF6), and PENTA1, which regulates PORA mRNA translation after FR exposure. Regulation of SIG6 plays a significant role in plant responses to FR exposure during the BOG response.

Execution of programmed cell death by singlet oxygen generated inside the chloroplasts of Arabidopsis thaliana.

Absorption of excess excitation energy induces overproduction of singlet oxygen (1O2) in plants. The major sources of singlet oxygen production are chlorophyll and its intermediates located in the chloroplast. Over-accumulation of the chlorophyll biosynthetic intermediate protochlorophyllide by the exogenous application of 5-aminolevulinic acid (ALA), the precursor of tetrapyrrole, induced singlet oxygen production in the plastidic membranes. Over-expression of protochlorophyllide oxidoreductase C (PORC) in Arabidopsis thaliana resulted in efficient light-induced photo-transformation of protochlorophyllide to chlorophyllide that limited the accumulation of protochlorophyllide. Consequently, the 1O2 generation decreased in the PORC overexpressors (PORCx) and their cell death was minimal. Conversely, porC-2 over-accumulated protochlorophyllide in response to ALA treatment and generated higher amounts of 1O2 in light and had highest cell death as monitored by Evans blue staining. The protoplasts isolated from PORCx plants, when treated with ALA, generated minimal amounts of 1O2 as revealed by singlet oxygen sensor green (SOSG) fluorescence emission from chloroplasts. Conversely, the protoplasts of porC-2 mutants under identical conditions generated the maximum SOSG fluorescence in their chloroplasts and cytosol surrounding the chloroplasts most likely due to the leakage from the organelle. The membrane blebbing, a hallmark of programmed cell death, was clearly visible in WT and porC-2 protoplasts. Similarly, the nick end labelling (TUNEL) assay revealed nicks in the DNA. The TUNEL-positive nuclei after 30min of light exposure were highest in porC-2 and lowest in PORCx protoplasts. The results demonstrate that higher amounts of singlet oxygen produced in the chloroplasts play an important role in programmed cell death.

GluTR2 Complements a hema1 Mutant Lacking Glutamyl-tRNA Reductase 1, but is Differently Regulated at the Post-Translational Level

Arabidopsis HEMA1 and HEMA2 encode glutamyl-tRNA reductase (GluTR) 1 and 2, the two isoforms of the initial enzyme of tetrapyrrole biosynthesis. HEMA1 is dominantly expressed in photosynthetic tissue, while HEMA2 shows low constitutive expression and is induced upon stress treatments. We introduce a new HEMA1 knockout mutant which grows only heterotrophically on MS (Murashige and Skoog) medium at low light, indicating that the remaining GluTR2 does not sufficiently compensate for the extensive needs of metabolic precursors for Chl. While hema1 accumulates low amounts of Chl, it contains more than half of the wild-type heme content. The functional diversity of the two GluTR isoforms was analyzed by means of complementation studies of the hema1 mutant by expression of pHEMA1::HEMA2 and p35S::HEMA1, respectively. Expression of both transgenes complements hema1, indicating that GluTR2 can likewise be involved in the synthesis of Chl and is not exclusively assigned to heme synthesis. In comparison with p35S::HEMA1-complemented hema1 and the wild type, GluTR2 expression under control of the HEMA1 promoter (pHEMA1) in pHEMA1::HEMA2-complemented hema1 mutants causes elevated protochlorophyllide levels under extended dark periods as well as in short-day-grown adult plants, resulting in the formation of necrotic leaf tissue. Although both GluTR isoforms have similar activity and contribute to 5-aminolevulinic acid synthesis for adequate accumulation of Chl and heme, it is proposed that the two proteins experience a different post-translational control in darkness and light. While GluTR2 continues 5-aminolevulinic acid synthesis in darkness, GluTR1 is efficiently inactivated by the interaction with the FLU (FLUORESCENT) protein, thereby preventing an accumulation of protochlorophyllide.

RNAi based simultaneous silencing of all forms of light-dependent NADPH:protochlorophyllide oxidoreductase genes result in the accumulation of protochlorophyllide in tobacco (Nicotiana tabacum)

Conversion of protochlorophyllide (Pchlide) into chlorophyllide (Chlide), a key step in chlorophyll biosynthesis, is mediated by a light-dependent NADPH:protochlorophyllide oxidoreductase (POR). POR exists in multiple isoforms that share high degree of homology. RNAi-mediated gene silencing approach was used to suppress the expression of POR genes in order to study its role in the Chls biosynthesis in tobacco (Nicotiana tabacum L.). The transgenic plants were devoid of chlorophyll pigments and resembled albino plants. Northern blot analysis confirmed the degradation of POR transcripts into 21-23 bp fragments. Pigment analysis showed the accumulation of various intermediate compounds of Chl biosynthesis pathway including Pchlide. However, no trace of chlorophyll was observed. As compared to wild type, POR-silenced plants accumulated larger (60%) amounts of Pchlide from its endogenous substrate. When leaf discs of WT and POR-silenced plants were treated with exogenous ALA both WT and POR-silenced plants accumulated large amounts of tetrapyrrolic intermediates demonstrating that Pchlide biosynthesis potential was not suppressed in POR-silenced plants. Upon illumination, WT plants photo-transformed large amounts of Pchlide to Chlide. However, POR-silenced plants almost completely failed to do so. Results demonstrate that the antisense approaches to drop expression of individual POR isoforms have provided valuable insights into the role of distinct PORs during greening. Moreover, data illustrate that the POR is the only enzyme that can convert the Pchlide to Chlide and there is no alternate enzyme that can substitute the POR in higher plants. Thus, this investigation describes ideal mechanism for the silencing of POR isozymes in tobacco.

Variations in xanthophyll composition in etiolated seedlings of Arabidopsis thaliana correlate with protochlorophyllide accumulation.

Protochlorophyllide (Pchlide) accumulation and xantophyll composition were studied in 5-day old etiolated seedlings of three ecotypes of Arabidopsis thaliana: Columbia (Col-0), Landsberg erecta (Ler) and Wassiliewska (Ws). The total Pchlide level as measured by fluorescence spectroscopy varied significantly between ecotypes. A rapid HPLC method revealed quantitative differences in carotenoid composition. It was found that in the Ler ecotype any enhanced accumulation of Pchlide correlates with an increased level of lutein, suggesting the role of enzymes involved in lutein synthesis in cross-regulation between chlorophyll and carotenoid biosynthetic pathways. The function of the dark-accumulated carotenoid pool in seedling de-etiolation is discussed.

High biological variability of plastids, photosynthetic pigments and pigment forms of leaf primordia in buds

To study the formation of the photosynthetic apparatus in nature, the carotenoid and chlorophyllous pigment compositions of differently developed leaf primordia in closed and opening buds of common ash (Fraxinus excelsior L.) and horse chestnut (Aesculus hippocastanum L.) as well as in closed buds of tree of heaven (Ailanthus altissima P. Mill.) were analyzed with HPLC. The native organization of the chlorophyllous pigments was studied using 77 K fluorescence spectroscopy, and plastid ultrastructure was investigated with electron microscopy. Complete etiolation, i.e., accumulation of protochlorophyllide, and absence of chlorophylls occurred in the innermost leaf primordia of common ash buds. The other leaf primordia were partially etiolated in the buds and contained protochlorophyllide (0.5-1 μg g(-1) fresh mass), chlorophyllides (0.2-27 μg g(-1) fresh mass) and chlorophylls (0.9-643 μg g(-1) fresh mass). Etio-chloroplasts with prolamellar bodies and either regular or only low grana were found in leaves having high or low amounts of chlorophyll a and b, respectively. After bud break, etioplast-chloroplast conversion proceeded and the pigment contents increased in the leaves, similarly to the greening processes observed in illuminated etiolated seedlings under laboratory conditions. The pigment contents and the ratio of the different spectral forms had a high biological variability that could be attributed to (i) various light conditions due to light filtering in the buds resulting in differently etiolated leaf primordia, (ii) to differences in the light-exposed and inner regions of the same primordia in opening buds due to various leaf folding, and (iii) to tissue-specific slight variations of plastid ultrastructure.

Deficiency in riboflavin biosynthesis affects tetrapyrrole biosynthesis in etiolated Arabidopsis tissue

Tetrapyrrole biosynthesis is controlled by multiple environmental and endogenous cues. Etiolated T-DNA insertion mutants were screened for red fluorescence as result of elevated levels of protochlorophyllide and four red fluorescent in the dark (rfd) mutants were isolated and identified. rfd3 and rfd4 belong to the group of photomorphogenic cop/det/fus mutants. rfd1 and rfd2 had genetic lesions in RIBA1 and FLU encoding the dual-functional protein GTP cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase and a negative regulator of tetrapyrrole biosynthesis, respectively. RIBA1 catalyses the initial reaction of the metabolic pathway of riboflavin biosynthesis and rfd1 contains reduced contents of riboflavin and the flavo-coenzymes FMN and FAD. Transcriptome analysis of rfd1 revealed up-regulated genes encoding nucleus-localized factors involved in cytokinin signalling and numerous down-regulated LEA genes as well as an auxin-inducible GH3 gene. Alteration of cytokinin metabolism of rfd1was confirmed by elevated contents of active forms of cytokinin and stimulated expression of an ARR6::GUS reporter construct. An etiolated quadruple ckx (cytokinin oxidase) mutant with impaired cytokinin degradation as well as different knockout mutants for the negative AUX/IAA regulators shy2-101 (iaa3), axr2-1 (iaa7) and slr-1 (iaa14) showed also excessive protochlorophyllide accumulation. The transcript levels of CHLH and HEMA1 encoding Mg chelatase and glutamyl-tRNA reductase were increased in rfd1 and the AUX/IAA loss-of-function mutants. It is proposed that reduced riboflavin synthesis impairs the activity of the flavin-containing cytokinin oxidase, increases cytokinin contents and de-represses synthesis of 5-aminolevulinic acid of tetrapyrrole metabolism in darkness. As result of the mutant analyses, the antagonistic cytokinin and auxin signalling is required for a balanced tetrapyrrole biosynthesis in the dark.

Overexpression of HEMA1 encoding glutamyl-tRNA reductase

5-Aminolevulinic acid (ALA) synthesis has been shown to be the rate limiting step of tetrapyrrole biosynthesis. Glutamyl-tRNA reductase (GluTR) is the first committed enzyme of plant ALA synthesis and is controlled by interacting regulators, such as heme and the FLU protein. Induced inactivation of the HEMA1 gene encoding GluTR by RNAi expression in tobacco resulted in a reduced activity of Mg chelatase and Fe chelatase indicating a feed-forward regulatory mechanism that links ALA synthesis posttranslationally with late enzymes of tetrapyrrole biosynthesis (Hedtke et al., 2007). Here, the regulatory impact of GluTR was investigated by overexpression of AtHEMA1 in Arabidopsis and tobacco plants. Light-dependent ALA synthesis cannot benefit from an up to 7-fold induced expression of GluTR in Arabidopsis. While constitutive AtHEMA1 overexpression in tobacco stimulates ALA synthesis by 50–90% during light-exposed growth of seedlings, no increase in heme and chlorophyll contents is observed. HEMA1 overexpression in etiolated and dark-grown Arabidopsis and tobacco seedlings leads to additional accumulation of protochlorophyllide. As excessive accumulation of GluTR does not correlate with increased ALA formation, it is hypothesized that ALA synthesis is additionally limited by other effectors that balance the allocation of ALA with the activity of enzymes of chlorophyll and heme biosynthesis.

EIN3/EIL1 cooperate with PIF1 to prevent photo-oxidation and to promote greening of Arabidopsis seedlings

The ability to switch from skotomorphogenesis to photomorphogenesis is essential for seedling development and plant survival. Recent studies revealed that COP1 and phytochrome-interacting factors (PIFs) are key regulators of this transition by repressing the photomorphogenic responses and/or maintaining the skotomorphogenic state of etiolated seedlings. Here we report that the plant hormone ethylene plays a crucial role in the transition from skotomorphogenesis to photomorphogenesis by facilitating greening of etiolated seedlings upon light irradiation. Activation of EIN3/EIL1 is both necessary and sufficient for ethylene-induced enhancement of seedling greening, as well as repression of the accumulation of protochlorophyllide, a phototoxic intermediate of chlorophyll synthesis. EIN3/EIL1 were found to induce gene expression of two key enzymes in the chlorophyll synthesis pathway, protochlorophyllide oxidoreductase A and B (PORA/B). ChIP and EMSA assays demonstrated that EIN3 directly binds to the specific elements present in the PORA and PORB promoters. Genetic studies revealed that EIN3/EIL1 function in cooperation with PIF1 in preventing photo-oxidative damage and promoting cotyledon greening. Moreover, activation of EIN3 reverses the blockage of greening triggered by cop1 mutation or far-red light irradiation. Consistently, EIN3 acts downstream of COP1 and its protein accumulation is enhanced by COP1 but decreased by light. Taken together, EIN3/EIL1 represent a new class of transcriptional regulators along with PIF1 to optimize de-etiolation of Arabidopsis seedlings. Our study highlights the essential role of ethylene in enhancing seedling development and survival through protecting etiolated seedlings against photo-oxidative damage.

Silencing of a plant gene by transcriptional interference

Integration of foreign DNA into eukaryotic genomes results frequently in a total or partial loss of gene function, caused by the interruption of indispensable structures of the gene itself. Using T-DNA insertions in Arabidopsis we screened for mutants with deregulated chlorophyll precursor accumulation in etiolated seedlings. A mutant designated rfd1 (red fluorescent in darkness) with increased protochlorophyllide accumulation showed a fluorescent phenotype that was associated with a lack of transcript initiation from the AtRibA1 promoter situated downstream of the integrated T-DNA. Complementation experiments confirmed rfd1 to be a knockout phenotype. Comparison with two SALK insertion lines bearing T-DNA integrations in the 5′UTR of AtRibA1 demonstrated that the insertion event in rfd1 itself does not explain the complete lack of transcript initiation. A 35S tetrameric enhancer sequence present on the rfd1 T-DNA causes the overaccumulation of a large polycistronic transcript originating inside the T-DNA. This 5.5-kb RNA runs over the downstream situated AtRibA1 promoter, which was shown by 5′RACE analyses to be consequently silenced. Hence, a transcription process that starts upstream and overlaps AtRibA1 blocks the initiation at the AtRibA1 promoter in rfd1. This regulatory mechanism has recently been introduced in yeast as transcriptional interference and is described here for the first time in a plant system.

No single way to understand singlet oxygen signalling in plants

When plant cells are under environmental stress, several chemically distinct reactive oxygen species (ROS) are generated simultaneously in various intracellular compartments and these can cause oxidative damage or act as signals. The conditional flu mutant of Arabidopsis, which generates singlet oxygen in plastids during a dark-to-light transition, has allowed the biological activity of singlet oxygen to be determined, and the criteria to distinguish between cytotoxicity and signalling of this particular ROS to be defined. The genetic basis of singlet-oxygen-mediated signalling has been revealed by the mutation of two nuclear genes encoding the plastid proteins EXECUTER (EX)1 and EX2, which are sufficient to abrogate singlet-oxygen-dependent stress responses. Conversely, responses due to higher cytotoxic levels of singlet oxygen are not suppressed in the ex1/ex2 background. Whether singlet oxygen levels lower than those that trigger genetically controlled cell death activate acclimation is now under investigation.

Misregulation of tetrapyrrole biosynthesis in transgenic tobacco seedlings expressing mammalian biliverdin reductase.

Previous studies have established that the expression of mammalian biliverdin IXalpha reductase (BVR) in transgenic tobacco (Nicotiana tabacum cv. Maryland Mammoth) resulted in the loss of photoregulatory activity of all phytochromes together with a pronounced chlorophyll deficiency. This study was undertaken to assess the contribution of BVR-mediated alteration of tetrapyrrole metabolism to the observed phenotypes of BVR transgenic plants. BVR expression in dark-grown plants led to the reduced accumulation of protochlorophyllide and transcripts for the two committed enzymes for 5-aminolevulinic acid (ALA) synthesis despite the marked increased capacity for synthesis of ALA. Together with the observation that Mg-porphyrin accumulation in dark-grown seedlings treated with an iron chelator was unaffected by BVR expression, these results indicate that BVR diverts tetrapyrrole metabolism toward heme synthesis while also reducing heme levels to de-repress ALA synthesis. By contrast with dark-grown seedlings, light-grown BVR plants showed a marked inhibition of ALA synthesis compared with wild-type plants - a result that was correlated with the disappearance of the CHL I subunit of Mg-chelatase and an increase in heme oxygenase protein levels. As transcript levels of all tetrapyrrole biosynthetic genes tested were not strongly affected by BVR expression, these results implicate misregulated tetrapyrrole metabolism to be a major mechanism for BVR-dependent inhibition of chlorophyll biosynthesis in light-grown plants.

Rice plants with a high protochlorophyllide accumulation show oxidative stress in low light that mimics water stress

Summary This report describes the possible involvement of singlet oxygen ( 1 O 2 ) as the causative prooxidant in water stress i n plants. Rice seedlings were loaded with 5-aminolevulinic acid (ALA), incubated in the dark, exposed to light, and then subjected to measurements of oxidative and antioxidative responses of cells to such treatment. The responses were very similar to those observed earlier in seedlings under water stress conditions. It was particularly noteworthy that both ALA treatment and water deficit treatment gave rise to virtually the same pattern of antioxidative enzymatic responses in low light. That is, the activities of enzymes which directly remove superoxide and H 2 O 2 were not enhanced to any significant extent, whereas, the last three enzymes of the ascorbate-glutathione cycle, whose collective function is to regenerate ascorbic acid (AA) from its oxidized forms, showed markedly increased activities. Because ALA feeding resulted in a high accumulation of protochlorophyllide P-627, a potent photodynamic generator of 1 O 2 , the results suggest not only the intermediacy of 1 O 2 in stress-developing processes in photosynthetic cells encountering water-limited environments, but also the crucial roles of AA, in combination with a-tocopherol action, and the AA-regenerating enzyme system in defense against the detrimental effects of 1 O 2 overproduction in chloroplast membranes.

Analysis of the chlorophyll biosynthetic system in a chlorophyll b-less barley mutant

The chlorophyll b-less barley (Hordeum vulgare L.) mutant chlorina 2807 allelic to the well-known barley mutant chlorina f2 was studied. 5-Aminolevulinic acid at saturating concentration (40 mM) was introduced into postetiolated leaves of the mutant and its wild type, and the protochlorophyllide accumulation in the dark was measured. It was found that the activity of the enzyme system transforming 5-aminolevulinic acid into protochlorophyllide was the same in both types of plants. The activity of esterifying enzymes that catalyze attachment of phytol to chlorophyllide was analyzed by infiltration of exogenous chlorophyllides a and b into etiolated leaves. The reaction was shown to have close rates in the mutant and wild-type plants. In very early stages of greening of etiolated leaves, when the apoproteins of the light-harvesting complexes are not yet formed, appearance of chlorophyll b was clearly recorded in the wild-type plants, while in the mutant chlorina 2807 no indications of chlorophyll b were detected in any stage of greening. On the other hand, in the mutant as well as in the wild type an active reverse conversion of chlorophyll b into chlorophyll a was possible. It is concluded that (a) in the mutant chlorina 2807 the ability of the biosynthetic system to transform 5-aminolevulinic acid to chlorophyll a is fully preserved, (b) in the mutant the enzymes converting chlorophyll a into chlorophyll b are most likely absent or damaged, (c) the conversion of chlorophyll a into chlorophyll b and the reverse conversion of chlorophyll b into chlorophyll a are performed by different enzymes.

Response of magnesium chelatase activity in green pea ( Pisum sativum L.) leaves to light, 5-aminolevulinic acid and dipyridyl supply

The role of extra-porphyrin production in the magnesium branch of the porphyrin pathway was investigated in green pea ( Pisum sativum L.) leaves treated with 5-aminolevulinic acid (ALA) and 2,2′-dipyridyl (DP). The magnesium chelatase activity was estimated during protoporphyrin IX, Mg-protoporphyrin IX (monomethyl ester) (MgProto(E)) and protochlorophyllide accumulation in the presence of exogenous ALA and/or DP. The results showed a close correlation only between the enzyme activity and MgProto(E) levels. The inverse dependence of the magnesium chelatase activity on the amount of MgProto(E) accumulated supports the hypothesis that a mechanism is involved which controls the magnesium branch of chlorophyll biosynthesis in vivo through inhibition of the enzymes by their products. Illumination of intact pea leaves predarkened for 17 h resulted in a 1–2 h lag phase of magnesium chelatase activity with consequent strong stimulation. After 3 h of illumination it reached 200% compared with the enzyme activity immediately before illumination. Cycloheximide, if applied during the entire period of dark or light treatment, decreased the magnesium chelatase activity by 19% in the dark and by 39% in light, in comparison with the corresponding control variant on H 2O. The ADP and especially the ATP content increased in the leaves treated with cycloheximide both in darkness and in light. The photosynthetic activity, measured as O 2 evolution by leaf segments, did not change in the presence of cycloheximide. The results are discussed as an additional influence of light on the magnesium chelatase activity not only via photosynthetic supply with ATP but also through light induced synthesis of the enzyme molecules de novo.

Light-independent chlorophyll biosynthesis: involvement of the chloroplast gene chlL (frxC).

The Chlamydomonas reinhardtii chloroplast gene chlL (frxC) is shown to be involved in the light-independent conversion of protochlorophyllide to chlorophyllide. The polypeptide encoded by chlL contains a striking 53% amino acid sequence identity with the bacteriochlorophyll (bch) biosynthesis bchL gene product in the photosynthetic bacterium Rhodobacter capsulatus. In a previous analysis, we demonstrated that bchL was involved in light-independent protochlorophyllide reduction, thereby implicating chlL in light-independent protochlorophyllide reduction in photosynthetic eukaryotes. To perform a functional/mutational analysis of chlL, we utilized particle gun-mediated transformation to disrupt the structural sequence of chlL at its endogenous locus in the chloroplast genome of Chlamydomonas. Transformants for which the multicopy chloroplast genome was homoplasmic for the disrupted chlL allele exhibit a "yellow-in-the-dark" phenotype that we demonstrated to be a result of the dark accumulation of protochlorophyllide. The presence of a chlL homolog in distantly related bacteria and nonflowering land plants, which are thought to be capable of synthesizing chlorophyll in the dark, was also demonstrated by cross-hybridization analysis. In contrast, we observed no cross-hybridization of a probe of chlL to DNA samples from representative angiosperms that require light for chlorophyll synthesis, in support of our conclusion that chlL is involved in light-independent chlorophyll biosynthesis. The role of chlL in protochlorophyllide reduction as well as recent evidence that both light-independent and light-dependent protochlorophyllide reductases may be of bacterial origin are discussed.