Light regulation of succinate dehydrogenase expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> leaves

A potential mechanism of light regulation of the succinate dehydrogenase (SDH) expression in Arabidopsis thaliana leaves was studied. As was shown by dot-hybridization and polymerase chain reaction in real time (RT-PCR), the SDH mRNA level in wild-type Arabidopsis thaliana plants changed depending on light conditions. The level of SDH mRNA in darkness was higher than in the light. The analysis of Arabidopsis thaliana plants carrying the mutant genes of phytochromes A and B showed that phytochrome A was involved in the regulation of the SDH enzyme activity. The active form of phytochrome A suppressed the SDHI-2 gene expression, and that resulted in decreasing activity of SDH.     

The role of calcium cations in the mechanism of phytochrome-dependent regulation of the <Emphasis Type="Italic">sdh1-2</Emphasis> gene expression and succinate dehydrogenase activity in maize leaves

The regulation of succinate dehydrogenase (SDH) activity and its gene expression by the phytochrome system has been demonstrated: red light (660 nm) reduces the SDH activity and the level of expression of the sdh1-2 gene. At the same time, the content of calcium cations in the nuclear fraction increases; it seems to be one of the mechanisms of phytochrome signal transduction in plant cells. Far-red light (730 nm) had opposite effects, i.e., increased SDH activity and the level of expression of the sdh1-2 gene. The data suggest that the SDH activity can be regulated at the level of expression in the green leaves of Zea mays by the phytochrome system with the involvement of calcium ions as a signal transduction messenger.     

Ca is involved in phytochrome A-dependent regulation of the succinate dehydrogenase gene sdh1-2 in Arabidopsis

The mechanism of transduction of the phytochrome signal regulating the expression of succinate dehydrogenase in Arabidopsis has been investigated. Using the phytochrome mutants of Arabidopsis, it is demonstrated that the inhibition of succinate dehydrogenase in the light may result from the phytochrome A-dependent modulation of Ca²⁺ amount in the nuclear fraction of leaves. This leads to the activation of expression of the gene pif3 encoding the phytochrome-interacting factor PIF3, which binds to the promoter of the gene sdh1-2 encoding the SDHA subunit of succinate dehydrogenase and suppresses its expression. It is concluded that Ca²⁺ ions are involved in the phytochrome A-mediated inhibition of succinate dehydrogenase activity in the light.     

Three different genes encode the iron-sulfur subunit of succinate dehydrogenase in Arabidopsis thaliana

The iron-sulfur protein is an essential component of mitochondrial complex II (succinate dehydrogenase, SDH), which is a functional enzyme of both the citric acid cycle and the respiratory electron transport chain. This protein is encoded by a single-copy nuclear gene in mammals and fungi and by a mitochondrial gene in Rhodophyta and the protist Reclinomonas americana. In Arabidopsis thaliana, the homologous protein is now found to be encoded by three nuclear genes. Two genes (sdh2-1 andsdh2-2) likely arose from a relatively recent duplication event since they have similar structures, encode nearly identical proteins and show similar expression patterns. Both genes are interrupted by a single intron located at a conserved position. Expression was detected in all tissues analysed, with the highest steady-state mRNA levels found in flowers and inflorescences. In contrast, the third gene (sdh2-3) is interrupted by 4 introns, is expressed at a low level, and encodes a SDH2-3 protein which is only 67% similar to SDH2-1 and SDH2-2 and has a different N-terminal presequence. Interestingly, the proteins encoded by these three genes are probably functional because they are highly conserved compared with their homologues in other organisms. These proteins contain the cysteine motifs involved in binding the three iron-sulfur clusters essential for electron transport. Furthermore, the three polypeptides are found to be imported into isolated plant mitochondria.     

Altered Phytochrome Regulation of Greening in an aurea Mutant of Tomato

A brief pulse of red light accelerates chlorophyll accumulation upon subsequent transfer of dark-grown tomato (Lycopersicon esculentum) seedlings to continuous white light. Such potentiation of greening was compared in wild type and an aurea mutant W616. This mutant has been the subject of recent studies of phytochrome phototransduction; its dark-grown seedlings are deficient in phytochrome, and light-grown plants have yellow-green leaves. The rate of greening was slower in the mutant, but the extent (relative to the dark control) of potentiation by the red pulse was similar to that in the wild type. In the wild type, the fluence-response curve for potentiation of greening indicates substantial components in the VLF (very low fluence) and LF (low fluence) ranges. Far-red light could only partially reverse the effect of red. In the aurea mutant, only red light in the LF range was effective, and the effect of red was completely reversed by far-red light. When grown in total darkness, aurea seedlings are also deficient in photoconvertible PChl(ide). Upon transfer to white light, the aurea mutant was defective in both the abundance and light regulation of the light-harvesting chlorophyll a/b binding polypeptide(s) [LHC(II)]. The results are consistent with the VLF response in greening being mediated by phytochrome. Furthermore, the data support the hypothesis that light modulates LHC(II) levels through its control of the synthesis of both chlorophyll and its LHC(II) apoproteins. Some, but not all, aspects of the aurea phenotype can be accounted for by the deficiency in photoreception by phytochrome.     

Photophysiology of the Elongated Internode (ein) Mutant of Brassica rapa: ein Mutant Lacks a Detectable Phytochrome B-Like Polypeptide

Several phytochrome-controlled processes have been examined in etiolated and light-grown seedlings of a normal genotype and the elongated internode (ein/ein) mutant of rapid-cycling Brassica rapa. Although etiolated ein seedlings displayed normal sensitivity to prolonged far-red light with respect to inhibition of hypocotyl elongation, expansion of cotyledons, and synthesis of anthocyanin, they displayed reduced sensitivity to prolonged red light for all three of these deetiolation responses. In contrast to normal seedlings, light-grown ein seedlings did not show a growth promotion in response to end-of-day far-red irradiation. Additionally, whereas the first internode of light-grown normal seedlings showed a marked increase in elongation in response to reduced ratio of red to far-red light, ein seedlings showed only a small elongation response. When blots of protein extracts from etiolated and light-treated ein and normal seedlings were probed with monoclonal antibody to phytochrome A, an immunostaining band at about 120 kD was observed for both extracts. The immunostaining intensity of this band was substantially reduced for extracts of light-treated normal and ein seedlings. A mixture of three monoclonal antibodies directed against phytochrome B from Arabidopsis thaliana immunostained a band at about 120 kD for extracts of etiolated and light-treated normal seedlings. This band was undetectable in extracts of ein seedlings. We propose that ein is a photoreceptor mutant that is deficient in a light-stable phytochrome B-like species.     

The effect of light on the biosynthesis of the light-harvesting chlorophyll a/b protein

The effect of light on the biosynthesis of the light-harvesting chlorophyll a/b protein (LHCP) is investigated in wild-type barley (Hordeum vulgare L.) and in the chlorophyll b-less mutant chlorina f2. In dark-grown plants a short red light pulse triggers the appearance of mRNA activity for the LHCP. While the accumulation of this mRNA is controlled by phytochrome (Apel (1979) Eur. J. Biochem. 97, 183–188), the red light treatment is not sufficient to induce the appearance of the LHCP within the membrane. Thus, at least one of the subsequent steps in the biosynthetic pathway leading to the assembly of the LHCP is controlled by light. The red light-induced mRNA is taken up into the polysomes during the subsequent dark period and is translated in vitro in a cell-free protein synthesizing system. However, an accumulation of the freshly synthesized polypeptide within the plant is not observed. The apparent instability of the polypeptide might be explained by the deficiency of chlorophyll in the red light-treated plants. In the chlorophyll b-less barley mutant chlorina f2 an accumulation of the freshly synthesized apoprotein of the LHCP can be observed in the light. Thus, chlorophyll a formation seems to be a light-dependent step which is required for the stabilization of the LHCP.Abbreviations mRNA messenger RNA - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecylsulfate - LHCP light-harvesting chlorophyll a/b protein     

Identification of two loci involved in phytochrome expression in Nicotiana plumbaginifolia and lethality of the corresponding double mutant

Four Nicotiana plumbaginifolia mutants exhibiting long hypocotyls and chlorotic cotyledons under white light, have been isolated from M2 seeds following mutagenesis with ethyl methane sulphonate. In each of these mutants, this partly etiolated in white light (pew) phenotype is due to a recessive nuclear mutation at a single locus. Complementation analysis indicates that three mutants, dap5, ems28 and ems3-6-34, belong to a single complementation group called pew1, while dap1 defines the pew2 locus. The mutants at pew1 contain normal levels of immunochemically detectable apoprotein of the phytochrome that is relatively abundant in etiolated seedlings, but are deficient in spectrophotometrically detectable phytochrome, whether seedlings are grown in darkness or light. Moreover, biliverdin, a precursor of the phytochrome chromophore, restores light-regulated responses in pew1 mutants and increases their level of photoreversible phytochrome when grown in darkness. These results indicate that the pew1 locus may be involved in chromophore biosynthesis. The mutant at the pew2 locus displays no photoreversible phytochrome in etiolated seedlings, but does contain normal levels of photoreversible phytochrome when grown in the light. Biliverdin had little effect on light-regulated responses in this mutant. In addition, biliverdin did not alter the level of phytochrome in etiolated seedlings. These observations lead us to propose that this mutant could be affected in the phyA gene itself. We have also obtained the homozygous double mutant at the pew1 and pew2 loci. This double mutant is lethal at an early stage of development, consistent with a critical role for phytochrome in early development of higher plants.     

fhy1 defines a branch point in phytochrome A signal transduction pathways for gene expression

Physiological analysis of the fhy1 mutant of Arabidopsis has led to the proposal that the mutant is deficient in a downstream component of the phytochrome A signal transduction pathway. To define this lesion at the molecular level, we have examined the expression of a range of phytochrome-regulated genes in fhy1. In far-red light, the regulation of genes such as CHS and CHI is blocked in fhy1, whereas the induction of CAB and NR genes is affected minimally. In contrast, the induction of all genes tested is blocked in a phytochrome A-deficient mutant, confirming that gene expression in far-red light is regulated solely by phytochrome A. Thus, fhy1 defines a branch point in phytochrome A signal transduction pathways for gene expression. Contrary to the general opinion that responses to continuous red light are mediated by phytochrome B and other photostable phytochromes, we have shown also that red light-induction of CHS is mediated almost entirely by phytochrome A. Furthermore, phytochrome A-mediated induction of CHS by red light is blocked in fhy1. The induction of CHS by blue light, however, is normal in fhy1, suggesting that although FHY1 is a component of the phytochrome A signaling pathway, it is not a component of the blue-light signaling pathway for CHS expression.     

Roles of different phytochromes in Arabidopsis photomorphogenesis

The red/far-red light-absorbing phytochromes play fundamental roles in photoperception of the light environment and the subsequent adaptation of plant growth and development. Higher plants possess multiple, discrete phytochromes, the apoproteins of which are the products of a family of divergent (PHY) genes. Arabidopsis thaliana has at least five PHY genes, encoding the apoproteins of phytochromes A-E. Through the analysis of mutants that are deficient in phytochrome A or B and the corresponding double mutant, it is becoming clear that these phytochromes perform both discrete and overlapping roles throughout plant development. Through analysis of the phyA phyB double mutant, it has been possible to define several responses that are mediated by other members of the phytochrome family. This article reviews some of the recent progress in the study of phytochrome-deficient mutants of the model plant Arabidopsis thaliana.     

Photophysiology and phytochrome content of long-hypocotyl mutant and wild-type cucumber seedlings

Photomorphogenetic responses have been studied in a cucumber (Cucumis sativus L.) mutant (lh), which has long hypocotyls in white light (WL). While etiolated seedlings of this mutant have a similar phytochrome content and control of hypocotyl elongation as wild type, deetiolation is retarded and WL-grown seedlings show reduced phytochrome control. Spectrophotometric measurements exhibit that WL-grown tissues of the lh mutant (flower petals and Norflurazon-bleached leaves) contain 35 to 50% of the phytochrome level in the wild type. We propose that this is a consequence of a lack of light-stable phytochrome, in agreement with our hypothesis proposed on the basis of physiological experiments. The lh mutant lacks an end-of-day far-red light response of hypocotyl elongation. This enables the end-of-day far-red light response, clearly shown by the wild type, to be ascribed to the phytochrome, deficient in the lh mutant. Growth experiments in continuous blue light (BL) and continuous BL + red light (RL) show that when RL is added to BL, hypocotyl growth remains inhibited in the wild type, whereas the lh mutant exhibits significant growth promotion compared to BL alone. It is proposed that the hypocotyls fail to grow long in low fluence rate BL because photosynthesis is insufficient to sustain growth.     

Interactions of phytochromes A,B1 and B2 in light-induced competence for adventitious shoot formation in hypocotyl of tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.)

The interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phytochrome B2 (phyB2) in light-dependent shoot regeneration from the hypocotyl of tomato was analysed using all eight possible homozygous allelic combinations of the null mutants. The donor plants were pre-grown either in the dark or under red or far-red light for 8 days after sowing; thereafter hypocotyl segments (apical, middle and basal portions) were transferred onto hormone-free medium for culture under different light qualities. Etiolated apical segments cultured in vitro under white light showed a very high frequency of regeneration for all of the genotypes tested besides phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants. Evidence is provided of a specific interference of phyB2 with phyA-mediated HIR to far-red and blue light in etiolated explants. Pre-treatment of donor plants by growth under red light enhanced the competence of phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants for shoot regeneration, whereas pre-irradiation with far-red light enhanced the frequency of regeneration only in the phyAphyB1 mutant. Multiple phytochromes are involved in red light- and far-red light-dependent acquisition of competence for shoot regeneration. The position of the segments along the hypocotyl influenced the role of the various phytochromes and the interactions between them. The culture of competent hypocotyl segments under red, far-red or blue light reduced the frequency of explants forming shoots compared to those cultured under white light, with different genotypes having different response patterns.Abbreviations HIR: High irradiance response - LFR: Low fluence response - Pfr: Far-red absorbing form of phytochrome - phyA: Phytochrome A - phyB1: Phytochrome B1 - phyB2: Phytochrome B2 - phyA(B1, B2): Phytochrome mutant deficient in phyA (B1, B2) - phyAphyB1(B1B2,AB2): Double phytochrome mutant deficient in phyA and phyB1(B1, B2) - phyAphyB1phyB2: Triple mutant deficient in phyA, phyB1 and phyB2 - VLFR: Very low fluence response - WT: Wild-type tomato Communicated by R. Reski     

Role of differential expression of <Emphasis Type="Italic">sdh1-1</Emphasis> and <Emphasis Type="Italic">sdh1-2</Emphasis> genes in alteration of isoenzyme composition of succinate dehydrogenase in germinating maize seeds

A probable mechanism of alteration of the isoenzyme composition of succinate dehydrogenase (SDH) due to differential expression of genes encoding subunit A was considered. The alteration of SDH activity during maize seed germination was investigated, and its maximal activity on day 4-5 of germination was found. The alteration of the sdh1-1 and sdh1-2 gene expression level during maize seed germination was evaluated using the quantitative polymerase chain reaction method. The presence of four forms of the studied enzymes, providing multiple SDH functions was found in maize inflorescence using electrophoresis in polyacrylamide gel.     

Negative phototropic response of rhizoid cells in the fern Adiantum capillus-veneris

In general, phototropic responses in land plants are induced by blue light and mediated by blue light receptor phototropins. In many cryptogam plants including the fern Adiantum capillus-veneris, however, red as well as blue light effectively induces a positive phototropic response in protonemal cells. In A. capillus-veneris, the red light effect on the tropistic response is mediated by phytochrome 3 (phy3), a chimeric photoreceptor of phytochrome and full-length phototropin. Here, we report red and blue light-induced negative phototropism in A. capillus-veneris rhizoid cells. Mutants deficient for phy3 lacked red light-induced negative phototropism, indicating that under red light, phy3 mediates negative phototropism in rhizoid cells, contrasting with its role in regulating positive phototropism in protonemal cells. Mutants for phy3 were also partially deficient in rhizoid blue light-induced negative phototropism, suggesting that phy3, in conjunction with phototropins, redundantly mediates the blue light response.