POR structural domains important for the enzyme activity in R. capsulatus complementation system

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes hydrogen transfer from NADPH to protochlorophyllide (PChlide) in the course of chlorophyll biosynthesis in photosynthetic organisms and is involved in the regulation of the development of photosynthetic apparatus in higher plants, algae and cyanobacteria. To approach molecular factors determining the enzyme activity in a living cell, several mutants of POR from pea (Pisum sativum) with site-directed modifications in different parts of the enzyme were generated. The mutant enzymes were expressed in a R. capsulatus mutant deficient in BChl biosynthesis, and their catalytic activity and ability to integrate in bacterial metabolism were analyzed. Our results demonstrate that in heterologous bacterial cell system, higher plant POR is integrated in the porphyrin biosynthesis network and its activity leads to the formation of photosynthetic chlorophyll-proteins (CPs). The study of POR mutants in R. capsulatus reveals several POR domains important for the association of the enzyme with other subcellular components and for its catalytic activity, including identification of putative enzyme reaction center and substrate binding site. The study also demonstrated that an unknown structural factor is important for the formation of the enzyme photoactive complex in etiolated plants. Moreover, our findings suggest that POR might be directly involved in the regulation of the metabolism of other porphyrins. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant responses to fungal volatiles involve global posttranslational thiol redox proteome changes that affect photosynthesis

Microorganisms produce volatile compounds (VCs) that promote plant growth and photosynthesis through complex mechanisms involving cytokinin (CK) and abscisic acid (ABA). We hypothesized that plants' responses to microbial VCs involve posttranslational modifications of the thiol redox proteome through action of plastidial NADPH‐dependent thioredoxin reductase C (NTRC), which regulates chloroplast redox status via its functional relationship with 2‐Cys peroxiredoxins. To test this hypothesis, we analysed developmental, metabolic, hormonal, genetic, and redox proteomic responses of wild‐type (WT) plants and a NTRC knockout mutant (ntrc) to VCs emitted by the phytopathogen Alternaria alternata. Fungal VC‐promoted growth, changes in root architecture, shifts in expression of VC‐responsive CK‐ and ABA‐regulated genes, and increases in photosynthetic capacity were substantially weaker in ntrc plants than in WT plants. As in WT plants, fungal VCs strongly promoted growth, chlorophyll accumulation, and photosynthesis in ntrc–Δ2cp plants with reduced 2‐Cys peroxiredoxin expression. OxiTRAQ‐based quantitative and site‐specific redox proteomic analyses revealed that VCs promote global reduction of the thiol redox proteome (especially of photosynthesis‐related proteins) of WT leaves but its oxidation in ntrc leaves. Our findings show that NTRC is an important mediator of plant responses to microbial VCs through mechanisms involving global thiol redox proteome changes that affect photosynthesis.     

Chlorophyll Synthesis in Dark-Grown Pine Primary Needles

The pigment content of dark-grown primary needles of Pinus jeffreyi L. and Pinus sylvestris L. was determined by high-performance liquid chromatography. The state of protochlorophyllide a and of chlorophylls during dark growth were analyzed by in situ 77 K fluorescence spectroscopy. Both measurements unambiguously demonstrated that pine primary needles are able to synthesize chlorophyll in the dark. Norflurazon strongly inhibited both carotenoid and chlorophyll synthesis. Needles of plants treated with this inhibitor had low chlorophyll content, contained only traces of xanthophylls, and accumulated carotenoid precursors. The first form of chlorophyll detected in young pine needles grown in darkness had an emission maximum at 678 nm. Chlorophyll-protein complexes with in situ spectroscopic properties similar to those of fully green needles (685, 695, and 735 nm) later accumulated in untreated plants, whereas in norflurazon-treated plants the photosystem I emission at 735 nm was completely lacking. To better characterize the light-dependent chlorophyll biosynthetic pathway in pine needles, the 77 K fluorescence properties of in situ protochlorophyllide a spectral forms were studied. Photoactive and nonphotoactive protochlorophyllide a forms with emission properties similar to those reported for dark-grown angiosperms were found, but excitation spectra were substantially red shifted. Because of their lower chlorophyll content, norflurazon-treated plants were used to study the protochlorophyllide a photoreduction process triggered by one light flash. The first stable chlorophyllide photoproduct was a chlorophyllide a form emitting at 688 nm as in angiosperms. Further chlorophyllide a shifts usually observed in angiosperms were not detected. The rapid regeneration of photoactive protochlorophyllide a from nonphotoactive protochlorophyllide after one flash was demonstrated.     

Transgenic tobacco plants expressing the coat protein of cucumber mosaic virus show different virus resistance

Transgenic tobacco (Nicotiana tabacum cv. Xanthi-nc) plants were regenerated after cocultivation of leaf explants withAgrobacterium tumefaciens strain LBA4404 harboring a plasmid that contained the coat protein (CP) gene of cucumber mosaic virus (CMV-As). PCR and Southern blot analyses revealed that the CMV CP gene was successfully introduced into the genomic DNA of the transgenic tobacco plants. Transgenic plants (CP⁺) expressing CP were obtained and used for screening the virus resistance. They could be categorized into three types after inoculation with the virus: virus-resistant, delay of symptom development, and susceptible type. Most of the CP⁺ transgenic tobacco plants failed to develop symptoms or showed systemic symptom development delayed for 5 to 42 days as compared to those of nontransgenic control plants after challenged with the same virus. However, some CP⁺ transgenic plants were highly susceptible after inoculation with the virus. Our results suggest that the CP-mediated viral resistance is readily applicable to CMV disease in other crops.     

Photoregulation of Protochlorophyllide Oxidoreductase and Rubisco Large Subunit Accumulation in Phytochrome A-Deficient Transgenic Tobacco Plants

Some morphogenetic responses, induced by far red (FR) light in tobacco plants (Nicotiana tabacum L.), were studied. The inhibitory effect of FR irradiation on chlorophyll synthesis in transgenic plants with reduced phytochrome A content was almost absent. Phytochrome A-mediated repression of the por gene was demonstrated with the use of polyclonal antiserum against protochlorophyllide oxidoreductase. Continuous FR light induced the accumulation of Rubisco large subunits in wild-type but not in transgenic tobacco plants. Our data confirm the suggestion that phytochrome A mediates photoregulation of the synthesis of these proteins.     

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 ((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.     

An allele of ZmPORB2 encoding a protochlorophyllide oxidoreductase promotes tocopherol accumulation in both leaves and kernels of maize

Phytol is one of the key precursors for tocopherol synthesis in plants, however, the underlying mechanisms concerning the accumulation of tocopherol remain poorly understood. In this study, qVE5, a major QTL affecting tocopherol accumulation in maize kernels was identified via a positional cloning approach. qVE5 encodes a protochlorophyllide oxidoreductase (ZmPORB2), which localizes to the chloroplast. Overexpression of ZmPORB2 increased tocopherol content in both leaves and kernels. Candidate gene association analysis identified a 5/8‐bp insertion/deletion (InDel058) in the 5′ untranslated region (UTR) as the causal polymorphism in affecting ZmPORB2 expression and being highly associated with tocopherol content. We showed that higher expression of ZmPORB2 correlated with more chlorophyll metabolites in the leaf following pollination. RNA‐sequencing and metabolic analysis in near isogenic lines (NILs) support that ZmPORB2 participates in chlorophyll metabolism enabling the production of phytol, an important precursor of tocopherol. We also found that the tocopherol content in the kernel is mainly determined by the maternal genotype, a fact that was further confirmed by in vitro culture experiments. Finally, a PCR‐based marker based on Indel058 was developed in order to facilitate the high tocopherol (vitamin E) maize breeding.     

Structural and functional relationships in developing Pinus silvestris chloroplasts

The light dependent chloroplast development of dark grown seedlings of Pinus silvestris L. was followed by analyses of chlorophyll content, chlorophyll a/b ratios, chlorophyll/P700 ratios, chlorophyll-protein complexes and structural changes. Low-temperature fluorescence emission spectra of isolated chloroplasts and separation of sodium dodecyl sulphate solubilized chlorophyll-protein complexes by gel electrophoresis showed that the chlorophyll-protein complexes of photosystem 1 (P700-CPa), photosystem II (PS II-CPa) and the light-harvesting complex LH–CPa/b were present in dark grown seedlings. The low-temperature fuoorescence emission maxima of isolated P700–CPa and PS II–CPa shifted towards longer wavelengths during greening in light, indicating a light induced change of the chlorophyll organisation in the two photosystems. Illumination caused LH–CPa/b to increase relative to P700–CPa, whereas the ratio between LH–CPa/b and PS II–CPa remained essentially constant. Analyses of low-temperature fluorescence spectra with or without 0.01 M Mg²⁺ showed that the Mg²⁺ controlled distribution of excitation energy into PS I was activated upon illumination of the seedlings. The photosynthetic unit size, as defined by the chlorophyll/P700 ratio, did not change over a 96 h illumination period, although the chlorophyll content increased about 6–fold during that time. This result and the constant electron transport rate per unit chlorophyll and time during chlorophyll accumulation provided evidence for a sequential development of the photosynthetic units when illuminating dark grown pine cotyledons. Electron micrographs showed that exposure of dark grown seedlings to light for 2 h caused the prolamellar body to disappear and grana to form. These changes occurred prior to substantial accumulation of chlorophyll or change in the ratio between LH–CPa/b and P700–CPa. However, both the water-splitting system of photosystem II and the Mg²⁺ controlled redistribution of excitation energy was activated during this period.     

An analysis of precursors accumulated by several chlorophyll biosynthetic mutants of maize

Summary Several mutants of maize defective in chlorophyll synthesis are analysed. By feeding shoots of dark-grown seedlings -aminolevulinic acid, the regulatory step in chlorophyll biosynthesis is bypassed and chlorophyll precursors accumulate. In normal plants this results in a buildup of protoporphyrin IX and protochlorophyllide, while mutants accumulate precursors, depending on the site of the mutant-induced lesion. Mutants at three loci, l ^*-Blandy4, 113, and oy, are defective in conversion of protoporphyrin IX to Mg-protoporphyrin. Mutants at the oro and oro2 loci are defective in conversion of Mg-protoporphyrin monomethyl ester to protochlorophyllide. A dominant modifier gene, Orom, which allows oro seedlings to bypass their lesion is also described.Journal Paper No J-9076 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa Project No. 2035     

Light-dependent chlorophyll a biosynthesis upon chlL deletion in wild-type and photosystem I-less strains of the cyanobacterium Synechocystis sp. PCC 6803

Part of the chlL gene encoding a component involved in light-independent protochlorophyllide reduction was deleted in wild type and in a photosystem I-less strain of Synechocystis sp. PCC 6803. In resulting mutants, chlorophyll biosynthesis was fully light-dependent. When these mutants were propagated under light-activated heterotrophic growth conditions (in darkness except for 15 min of weak light a day) for several weeks, essentially no chlorophyll was detectable but protochlorophyllide accumulated. Upon return of the chlL ^- mutant cultures to continuous light, within the first 6 h chlorophyll was synthesized at the expense of protochlorophyllide at a rate independent of the presence of photosystem I. Chlorophyll biosynthesized during this time gave rise to a 685 nm fluorescence emission peak at 77 K in intact cells. This peak most likely originates from a component different from those known to be directly associated with photosystems II and I. Development of 695 and 725 nm peaks (indicative of intact photosystem II and photosystem I, respectively) required longer exposures to light. After 6 h of greening, the rate of chlorophyll synthesis slowed as protochlorophyllide was depleted. In the chlL ^- strain, greening occurred at the same rate at two different light intensities (5 and 50 E m^-2s^-1), indicating that also at low light intensity the amount of light is not rate-limiting for protochlorophyllide reduction. Thus, in this system the rate of chlorophyll biosynthesis is limited neither by biosynthesis of photosystems nor by the light-dependent protochlorophyllide reduction. We suggest the presence of a chlorophyll-binding chelator protein (with 77 K fluorescence emission at 685 nm) that binds newly synthesized chlorophyll and that provides chlorophyll for newly synthesized photosynthetic reaction centers and antennae.     

The Influence of Protochlorophyllide, Formed from Exogenous δ-Aminolevulinic Acid, on Chlorophyll Synthesis in Leaves during Flash Illumination

In the presence of large accumulations of protochlorophyllide, derived from exogenous δ-aminolevulinic acid, chlorophyll synthesis in excised leaves of two varieties of barley was less than in untreated leaves. In oat leaves the accumulated protochlorophyllide, from exogenous δ-laminolevulinic acid, stimulated chlorophyll synthesis to above the control level. — These relationships could only be demonstrated when phtodestruction of pigments was minimised by the use of flash illumination (2 milliseconds every 3 minutes). — These was no evidence from in vivo absorption spectra that the pigments in the barley leaves were different to those in leaves studied by other workers. However, the presence of the accumulated protochlorophyllide appeared to prevent the shift of the chlorophyll absorption maximum from 673 nm to 677 nm. — Possible mechanisms of inhibition are discussed.     

Pavel Siffel (1954–2003) or Life full of chlorophyll

Life and research results of Pavel Siffel, a talented but untimely deceased Czech scientist in photosynthesis, are reviewed. He studied biophysics and physiology of chlorophyll, its complexes with proteins, their absorption and fluorescence spectra, activities in mutants and transformants, dealt with chlorophyll biosynthesis and protochlorophyllide photoreduction, pigments in plants grown at CO₂ deficiency and under simulated acid rain, with changes accompanying leaf and plant development, photobleaching, etc. He participated in construction of specialised spectrofluorometers, finally he built the kinetic spectrophotometer SpeKin.     

Enhancements in sucrose biosynthesis capacity affect shoot branching in Arabidopsis

We previously demonstrated that transgenic tobacco plants expressing cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in the cytosol increased the number of lateral shoots and leaves at elevated CO₂ levels. These findings suggest that alterations in carbon partitioning affect the development of shoot branching. In order to elucidate the underlying mechanisms at the molecular level, we generated transgenic Arabidopsis plants overexpressing cyanobacterial fructose-1,6-bisphosphatase-II in the cytosol (AcF). At elevated CO₂ levels, the number of lateral shoots was significantly increased in AcF plants. Sucrose and hexose levels were also higher in AcF plants than in wild-type plants. The expression levels of MAX1, MAX4, YUCCA8, YUCCA9, and BRC1, which are involved in auxin or strigolactone biosynthesis and responses, were lower in AcF plants than in wild-type plants. These results suggest that alterations in sugar partitioning affect hormone metabolism and responses, resulting in enhanced shoot branching.     

The iron–sulfur cluster biosynthesis protein SUFB is required for chlorophyll synthesis,but not phytochrome signaling

Proteins that contain iron–sulfur (Fe–S) clusters play pivotal roles in various metabolic processes such as photosynthesis and redox metabolism. Among the proteins involved in the biosynthesis of Fe–S clusters in plants, the SUFB subunit of the SUFBCD complex appears to be unique because SUFB has been reported to be involved in chlorophyll metabolism and phytochrome‐mediated signaling. To gain insights into the function of the SUFB protein, we analyzed the phenotypes of two SUFB mutants, laf6 and hmc1, and RNA interference (RNAi) lines with reduced SUFB expression. When grown in the light, the laf6 and hmc1 mutants and the SUFB RNAi lines accumulated higher levels of the chlorophyll biosynthesis intermediate Mg‐protoporphyrin IX monomethylester (Mg‐proto MME), consistent with the impairment of Mg‐proto MME cyclase activity. Both SUFC‐ and SUFD‐deficient RNAi lines accumulated the same intermediate, suggesting that inhibition of Fe‐S cluster synthesis is the primary cause of this impairment. Dark‐grown laf6 seedlings also showed an increase in protoporphyrin IX (Proto IX), Mg‐proto, Mg‐proto MME and 3,8‐divinyl protochlorophyllide a (DV‐Pchlide) levels, but this was not observed in hmc1 or the SUFB RNAi lines, nor was it complemented by SUFB overexpression. In addition, the long hypocotyl in far‐red light phenotype of the laf6 mutant could not be rescued by SUFB overexpression and segregated from the pale‐green SUFB‐deficient phenotype, indicating it is not caused by mutation at the SUFB locus. These results demonstrate that biosynthesis of Fe–S clusters is important for chlorophyll biosynthesis, but that the laf6 phenotype is not due to a SUFB mutation.     

Arabidopsis miR171-Targeted Scarecrow-Like Proteins Bind to GT cis-Elements and Mediate Gibberellin-Regulated Chlorophyll Biosynthesis under Light Conditions

An extraordinarily precise regulation of chlorophyll biosynthesis is essential for plant growth and development. However, our knowledge on the complex regulatory mechanisms of chlorophyll biosynthesis is very limited. Previous studies have demonstrated that miR171-targeted scarecrow-like proteins (SCL6/22/27) negatively regulate chlorophyll biosynthesis via an unknown mechanism. Here we showed that SCLs inhibit the expression of the key gene encoding protochlorophyllide oxidoreductase (POR) in light-grown plants, but have no significant effect on protochlorophyllide biosynthesis in etiolated seedlings. Histochemical analysis of β-glucuronidase (GUS) activity in transgenic plants expressing pSCL27::rSCL27-GUS revealed that SCL27-GUS accumulates at high levels and suppresses chlorophyll biosynthesis at the leaf basal proliferation region during leaf development. Transient gene expression assays showed that the promoter activity of PORC is indeed regulated by SCL27. Consistently, chromatin immunoprecipitation and quantitative PCR assays showed that SCL27 binds to the promoter region of PORC in vivo. An electrophoretic mobility shift assay revealed that SCL27 is directly interacted with G(A/G)(A/T)AA(A/T)GT cis-elements of the PORC promoter. Furthermore, genetic analysis showed that gibberellin (GA)-regulated chlorophyll biosynthesis is mediated, at least in part, by SCLs. We demonstrated that SCL27 interacts with DELLA proteins in vitro and in vivo by yeast-two-hybrid and coimmunoprecipitation analysis and found that their interaction reduces the binding activity of SCL27 to the PORC promoter. Additionally, we showed that SCL27 activates MIR171 gene expression, forming a feedback regulatory loop. Taken together, our data suggest that the miR171-SCL module is critical for mediating GA-DELLA signaling in the coordinate regulation of chlorophyll biosynthesis and leaf growth in light.     

Pigment protein complexes and functional properties of tetratype resulting from crosses between CP1 and CP2 less Chlamydomonas mutants

A chlorophyll b-less mutant of Chlamydomonas reinhardtii (Pg ₂₇) was isolated after UV irradiation of the wild type cells. This photosynthetically competent mutant totally lacks chlorophyll b and the CP₂ chlorophyll-protein complex. However, SDS-PAGE, proteolytic digestions and immunodetections demonstrated that the 24–25 Kd apoproteins of the lacking CP₂ complex are still present in thylakoids of the Pg₂₇ mutant. It is concluded that this CP₂-less mutant is affected in the biosynthesis pathway of chlorophyll b.This CP₂-less mutant was crossed with a CP₁-less mutant (Fl₅) Fluorescence emission spectra and fluorescence inductions in the presence of DCMU were analysed in the resulting (cp ₂ ^– , cp ₁ ⁺ ), (cp ₂ ⁺ , cp ₁ ^– ), (cp ₂ ⁺ , cp ₁ ⁺ ), cp ₂ ^– , cp ₁ ^– )tetratype. Differences in PS 2 optical cross section and in the relative amplitude or localisation of fluorescence emission peaks fit well with a quadripartite model where PS1 and PS2 would each correspond to a reaction centre core complex (CP₁ and CP₂ respectively) associated to a light harvesting antenna (LHC1 and LHC2 respectively). The occurrence of energy transfers from PS1 peripheral antenna to PS2 in the Fl ₅ mutant shows that, in absence of CP₁, at least a part of its associated PS1 light harvesting antenna migrates in the PS2 containing appressed thylakoids.Abbreviations Chl Chlorophyll - LHC Light harvesting chl a/b complex - CP₂ Predominant form of LHC or SDS polyacrylamide gels - WT Wild type - DM Double mutant (cp ₁ ^– , cp ₂ ^– ) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - DOC-PAGE Deoxycholate polyacrylamide gel electrophoresis     

Differential effects of gabaculin and laevulinic acid on protochlorophyllide regeneration

Abstract The effects of gabaculin (3-amino 2,3-dihydrobenzoic acid) and laevulinic acid on the regeneration of protochlorophyllide from exogenous δ-aminolaevulinic acid in leaves of dark-grown barley (Hordeum vulgare) after a brief light treatment were compared. Gabaculin, a potent inhibitor of chlorophyll biosynthesis, did not inhibit this process showing that it affects the formation of δ-aminolaevulinic acid rather than its further metabolism. Laevulinic acid, which is an inhibitor of δ-aminolaevulinic acid dehydratase, prevented regeneration of protochlorophyllide provided pools of intermediates in the biosynthetic sequence were depleted. Formation of relatively large amounts of protochlorophyllide in some experiments suggests a lack of control in the utilization of δ-aminolaevulinic acid for protochlorophyllide synthesis.