Plastid Signals Research Articles

EXECUTER1- and EXECUTER2-dependent transfer of stress-related signals from the plastid to the nucleus of Arabidopsis thaliana.

Shortly after the release of singlet oxygen ((1)O2), drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of (1)O2 generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of (1)O2-induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of (1)O2-dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid. Inactivation of both EXECUTER proteins in the ex1/ex2/flu triple mutant is sufficient to suppress the up-regulation of almost all (1)O2-responsive genes. Retrograde control of (1)O2-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment.

Inhibition of lycopene cyclase results in accumulation of chlorophyll precursors

Free porphyrins and their magnesium complexes, including chlorophylls, are potent photo-sensitizers. Plants usually accumulate these compounds bound to proteins together with protective compounds like carotenoids. Besides their protective role, carotenoids can play a structural role in these complexes. To analyze the effect of impaired carotenogenesis on plastid membranes we applied to barley seedlings the bleaching herbicide 2-(4-chlorophenylthio)triethylamine (CPTA) as a specific inhibitor for the cyclization of lycopene. To avoid interference with photo-oxidation, the essential experiments were performed on seedlings grown in darkness. While the amount of total carotenoids decreased, we found accumulation of more 6-carotene than lycopene in darkness clearly showing that CPTA inhibits the lycopene beta-cyclase more effectively than the lycopene epsilon-cyclase. The CPTA treatment resulted in accumulation of non-photoactive protochlorophyllide a; the amount of photoactive protochlorophyllide and NADPH:protochlorophyllide oxidoreductase remained constant. Further, the level of Mg protophorphyrin and its monomethyl ester increased to an extent similar to that obtained by application of 5-aminolevulinic acid (ALA). The perturbation of the ultrastructure of etioplast inner membranes, observed after CPTA-treatment, was not found after ALA-treatment; this excluded the accumulated tetrapyrroles as responsible for the perturbation. By contrast, the down-regulation of Lhcb and RbcS genes found after CPTA-treatment was compatible with the presumed role of Mg protophorphyrin as "plastid signal" for regulation of nuclear gene expression. Possible mechanisms for enhancement of tetrapyrrole accumulation by non-cyclic carotenoids are discussed.

Light and plastid signals regulate the expression of the pea plastocyanin gene through a common region at the 5' end of the coding region.

Expression of the pea plastocyanin gene (PetE) is regulated by light and plastid signals. Previous work indicated that light and plastid regulation of pea PetE operates post-transcriptionally in transgenic tobacco, and requires the correct 5' terminus of the PetE transcript and the PetE-coding region. The post-transcriptional light and plastid regulation of pea PetE has now been demonstrated to operate in transgenic Arabidopsis, where in contrast the endogenous PETE gene is regulated transcriptionally. Transgenic tobacco seedlings containing constructs with progressive 3' deletions of the PetE-coding region fused to the luciferase (Luc) reporter gene demonstrate that the first 60 nucleotides of the coding region are sufficient for regulated accumulation of Luc transcripts by light and plastid signalling pathways affected by treatment with norflurazon and lincomycin. PetE constructs containing premature stop codons were generated to investigate whether translation has a role in light or plastid regulation. Insertion of a stop codon in place of the second codon of the PetE-coding region diminished both light and plastid regulation of PetE transcripts, whereas stop codons inserted later in the transcript had no effect on light or plastid regulation. These experiments indicate that the 5' end of the plastocyanin-coding region contains sequences important for regulation by light and plastid signals.

2つのプラスチドシグナル伝達系：テトラピロール、非テトラピロール系

2つのプラスチドシグナル伝達系：テトラピロール、非テトラピロール系

Arabidopsis cue mutants with defective plastids are impaired primarily in the photocontrol of expression of photosynthesis-associated nuclear genes

Plant photoreceptors detect light cues and initiate responses ranging from chloroplast differentiation to the control of morphogenesis and flowering. The photocontrol of photosynthesis-related nuclear genes appears closely related to 'retrograde plastid signals' by which the status of the organelle controls the expression of nuclear genes. However, what specific role, if any, plastid-originated signals play in light responses is poorly understood: it has in the past been proposed that plastid signals play a role in all responses to 'high fluence' far-red light perceived by the light-labile phytochrome A, irrespective of whether they involve photosynthesis-related genes. To explore this further, we have re-examined the phenotype of three cue (cab-underexpressed) Arabidopsis mutants, defective in chloroplast development. The mutants have underdeveloped etioplasts, with increasing impairments in cue6, cue8 and cue3. The mutants show only small defects in photocontrol of hypocotyl elongation and cotyledon opening under prolonged far-red or red light, and normal photocontrol under blue. On the other hand, the expression of photosynthesis-associated nuclear genes is much more impaired in the mutants in the dark and following red or far-red light short treatments or continuous light, than that of those phytochrome-dependent genes tested which are not associated with photosynthesis. Furthermore, red/far-red photoreversible responses involving photosynthesis-related genes (induction of Lhcb1-cab promoter activity, and photoreversible extent of greening) mediated by phytochrome B and other photo-stable phytochromes, both show a reduction in the cue mutants, which correlates with the etioplast defect. Our evidence demonstrates that plastid-derived signals need to be operational in order for the phytochrome control of photosynthetic nuclear genes to occur.

The FLP proteins act as regulators of chlorophyll synthesis in response to light and plastid signals in Chlamydomonas

In photosynthetic organisms the accumulation of harmful photodynamic chlorophyll precursors is prevented because of the tight regulation of the tetrapyrrole pathway. FLU is one of the regulatory factors involved in this process in land plants. We have examined the function of a Flu-like gene (FLP) from Chlamydomonas that gives rise to two FLP transcripts through alternative splicing. These transcripts are translated into a short and a long protein that differ by only 12 amino acids but that interact differently with glutamyl-tRNA reductase, an enzyme involved in an early step of the chlorophyll biosynthetic pathway. Expression of FLPs is light-regulated at the level of RNA accumulation and splicing and is altered by mutations affecting the pathway. The relative levels of the long and short forms of FLP can be correlated with the accumulation of specific porphyrin intermediates, some of which have been implicated in a signaling chain from the chloroplast to the nucleus. Reciprocally, reduction of the FLP proteins by RNA interference leads to the accumulation of several porphyrin intermediates and to photobleaching when cells are transferred from the dark to the light. Thus the FLP proteins act as regulators of chlorophyll synthesis, and their expression is controlled by light and plastid signals.

Involvement of tetrapyrroles in inter-organellar signaling in plants and algae

For the assembly of a functional chloroplast, the coordinated expression of genes distributed between nucleus and chloroplasts is a prerequisite. While the nucleus plays an undisputed dominant role in controling biogenesis and functioning of chloroplasts, plastidic signals appear to control the expression of a subset of nuclear genes; the majority of which encodes chloroplast constituents. Tetrapyrrole biosynthesis intermediates are attractive candidates for one type of plastidic signal ever since an involvement of Mg-porphyrins in signaling from chloroplast to nucleus was first demonstrated in Chlamydomonas reinhardtii. Since then, Mg-protoporphyrin IX has been shown to exert a regulatory function on nuclear genes in higher plants as well. Here we review evidence for the role played by tetrapyrroles in inter-organellar communication. We also report on a screening for nuclear genes that may be subject to regulation by tetrapyrroles. This revealed that (i) >HEMA, the gene encoding the first enzyme specific for porphyrin biosynthesis is induced by Mg-protoporphyrin IX, (ii) several nuclear HSP70 genes are regulated by tetrapyrroles. Members of the gene family induced by the feeding of Mg-rotoporphyrin IX encode chaperones located in either the chloroplast or the cytosol. These results point to an important role of Mg-tetrapyrroles as plastidic signal in controling the initial step of porphyrin biosynthesis, and the synthesis of chaperones involved in protein folding in cytosol/stroma, protein transport into organelles, and the stress response.

The nuclear genes Lhcb and HEMA1 are differentially sensitive to plastid signals and suggest distinct roles for the GUN1 and GUN5 plastid‐signalling pathways during de‐etiolation

Feedback mechanisms are critical to the regulation of chloroplast development and signals from functional plastids are required to maintain nuclear gene expression of chloroplast proteins. To understand the role of these signals in de-etiolating Arabidopsis thaliana L. seedlings, we followed the expression of three nuclear genes, Lhcb, HEMA1 and GSA, under a variety of treatments (Norflurazon, lincomycin and a far-red light pre-treatment) leading to plastid damage in white light and in a range of genetic backgrounds known to modulate plastid signalling: the genomes uncoupled mutants, gun1, gun4, gun5 and the gun1,5 double mutant, and in a transgenic line over-expressing NADPH:protochlorophyllide oxidoreductase. The three nuclear genes were differentially sensitive to changes in plastid signalling, with Lhcb the most strongly repressed and GSA insensitive to all but the most severe treatments. Analysis of plastid morphology in seedlings grown under identical conditions demonstrated that these responses corresponded closely to the degree of plastid damage. Furthermore, although Lhcb and HEMA1 were responsive to both GUN1 and GUN5 signals, the relative inputs from these pathways differed for each transcript with GUN1 being dominant for HEMA1 regulation. Further analysis of HEMA1 expression in gun1 seedlings under non-photobleaching conditions indicates that GUN1 is an important suppressor of HEMA1 expression in the dark and under saturating white light. These results are consistent with plastid signals functioning in a feedback regulatory mechanism during chloroplast biogenesis, and suggest a key role for GUN1 during the early stages of chloroplast development.

Plastid-to-nucleus signalling

The function of the eukaryotic cell depends on the reciprocal interaction between its different compartments. Plastids emit signals that regulate nuclear gene expression to ensure the stoichiometric assembly of plastid protein complexes and to initiate macromolecular reorganisation in response to environmental cues. It is now clear that several different plastid processes produce signals that influence the expression of photosynthetic genes in the nucleus. The genome uncoupled (gun) mutants recently revealed one of the plastid signals, the chlorophyll intermediate Mg-protoporphyrinIX.

Knock-out of the genes coding for the Rieske protein and the ATP-synthase delta-subunit of Arabidopsis. Effects on photosynthesis, thylakoid protein composition, and nuclear chloroplast gene expression.

In Arabidopsis, the nuclear genes PetC and AtpD code for the Rieske protein of the cytochrome b(6)/f (cyt b(6)/f) complex and the delta-subunit of the chloroplast ATP synthase (cpATPase), respectively. Knock-out alleles for each of these loci have been identified. Greenhouse-grown petc-2 and atpd-1 mutants are seedling lethal, whereas heterotrophically propagated plants display a high-chlorophyll (Chl)-fluorescence phenotype, indicating that the products of PetC and AtpD are essential for photosynthesis. Additional effects of the mutations in axenic culture include altered leaf coloration and increased photosensitivity. Lack of the Rieske protein affects the stability of cyt b(6)/f and influences the level of other thylakoid proteins, particularly those of photosystem II. In petc-2, linear electron flow is blocked, leading to an altered redox state of both the primary quinone acceptor Q(A) in photosystem II and the reaction center Chl P700 in photosystem I. Absence of cpATPase-delta destabilizes the entire cpATPase complex, whereas residual accumulation of cyt b(6)/f and of the photosystems still allows linear electron flow. In atpd-1, the increase in non-photochemical quenching of Chl fluorescence and a higher de-epoxidation state of xanthophyll cycle pigments under low light is compatible with a slower dissipation of the transthylakoid proton gradient. Further and clear differences between the two mutations are evident when mRNA expression profiles of nucleus-encoded chloroplast proteins are considered, suggesting that the physiological states conditioned by the two mutations trigger different modes of plastid signaling and nuclear response.

Pathways of intracellular communication: tetrapyrroles and plastid-to-nucleus signaling.

Retrograde plastid-to-nucleus signaling plays a central role in coordinating nuclear and plastid gene expression. The gun (genomes uncoupled) mutants of Arabidopsis have been used to demonstrate that Mg-protoporphyrin (Mg-Proto) acts as a plastid signal to repress the transcription of nuclear photosynthesis genes (1). It is unclear how Mg-Proto triggers repression, but several components of this pathway have been recently identified. These include the products of GUN4 and GUN5. GUN5 is the ChlH subunit of Mg-chelatase, which produces Mg-Proto, and GUN4 is a regulator of ChlH activity (2). GUN4 might also play a role in photoprotection and in the trafficking of Mg-Proto.

Covariations in the nuclear chloroplast transcriptome reveal a regulatory master‐switch

The evolution of the endosymbiotic progenitor into the chloroplast organelle was associated with the transfer of numerous chloroplast genes into the nucleus. Hence, inter-organellar signalling, and the co-ordinated expression of sets of nuclear genes, was set up to control the metabolic and developmental status of the chloroplast. Here, we show by the differential-expression analysis of 3,292 genes, that most of the 35 environmental and genetic conditions tested, including plastid signalling mutations, elicit only three main classes of response from the nuclear chloroplast transcriptome. Two classes, probably involving GUN (genomes uncoupled)-type plastid signalling, are characterized by alterations, in opposite directions, in the expression of largely overlapping sets of genes.

GUN4, a Regulator of Chlorophyll Synthesis and Intracellular Signaling

Nuclear genes control plastid differentiation in response to developmental signals, environmental signals, and retrograde signals from plastids themselves. In return, plastids emit signals that are essential for proper expression of many nuclear photosynthetic genes. Accumulation of magnesium-protoporphyrin IX (Mg-Proto), an intermediate in chlorophyll biosynthesis, is a plastid signal that represses nuclear transcription through a signaling pathway that, in Arabidopsis, requires the GUN4 gene. GUN4 binds the product and substrate of Mg- chelatase, an enzyme that produces Mg-Proto, and activates Mg-chelatase. Thus, GUN4 participates in plastid-to-nucleus signaling by regulating Mg-Proto synthesis or trafficking.

Chloroplast redox control of nuclear gene expression--a new class of plastid signals in interorganellar communication.

Chloroplasts are genetically semiautonomous organelles that contain their own subset of 100-120 genes coding for chloroplast proteins, tRNAs, and rRNAs. However, the great majority of the chloroplast proteins are encoded in the nucleus and must be imported into the organelle after their translation in the cytosol. This arrangement requires a high degree of coordination between the gene expression machineries in chloroplasts and nucleus, which is achieved by a permanent exchange of information between both compartments. The existence of such coordinating signals has long been known; however, the underlying molecular mechanisms and signaling routes are not understood. The present data indicate that the expression of nuclear-encoded chloroplast proteins is coupled to the functional state of the chloroplasts. Photosynthesis, which is the major function of chloroplasts, plays a crucial role in this context. Changes in the reduction/oxidation (redox) state of components of the photosynthetic machinery act as signals, which regulate the expression of chloroplast proteins in both chloroplasts and nucleus and help to coordinate the expression both in compartments. Recent advances in understanding chloroplast redox regulation of nuclear gene expression are summarized, and the importance for intracellular signaling is discussed.

Coordination of plastid and nuclear gene expression.

The coordinated expression of genes distributed between the nuclear and plastid genomes is essential for the assembly of functional chloroplasts. Although the nucleus has a pre-eminent role in controlling chloroplast biogenesis, there is considerable evidence that the expression of nuclear genes encoding photosynthesis-related proteins is regulated by signals from plastids. Perturbation of several plastid-located processes, by inhibitors or in mutants, leads to decreased transcription of a set of nuclear photosynthesis-related genes. Characterization of arabidopsis gun (genomes uncoupled) mutants, which express nuclear genes in the presence of norflurazon or lincomycin, has provided evidence for two separate signalling pathways, one involving tetrapyrrole biosynthesis intermediates and the other requiring plastid protein synthesis. In addition, perturbation of photosynthetic electron transfer produces at least two different redox signals, as part of the acclimation to altered light conditions. The recognition of multiple plastid signals requires a reconsideration of the mechanisms of regulation of transcription of nuclear genes encoding photosynthesis-related proteins.

Multiple plastid signals regulate the expression of the pea plastocyanin gene in pea and transgenic tobacco plants.

The expression of nuclear genes encoding photosynthesis-related proteins is regulated by signals from plastids. To investigate how the pea PetE gene encoding plastocyanin is regulated by plastid signals, the effects of norflurazon, lincomycin and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), specific inhibitors of plastid-located processes generating plastid signals, have been examined. RNA-gel blot analysis of 7-day-old pea and tobacco seedlings containing the pea PetE gene showed that treatment with norflurazon and lincomycin, but not DCMU, decreased the accumulation of transcripts of pea PetE and endogenous Lhcb1 genes. Analysis of chimeric PetE gene constructs in tobacco seedlings showed that an intact PetE mRNA 5' terminus and elements within the PetE coding region were required to confer sensitivity to norflurazon and lincomycin, suggesting post-transcriptional regulation. Analysis of 4-week-old tobacco plants containing chimeric PetE constructs showed that DCMU treatment decreased the accumulation of pea PetE and Lhcb1 transcripts, but had opposite effects on the transcription of the genes in nuclear run-on assays. DCMU upregulated transcription from the pea PetE promoter whereas transcription of tobacco Lhcb1 genes was decreased. These experiments provide evidence for multiple plastid signals operating at different developmental stages and affecting transcriptional and post-transcriptional processes regulating expression of the pea PetE gene.

Loss of nuclear gene expression during the phytochrome A-mediated far-red block of greening response.

We have examined the expression of the HEMA1 gene, which encodes the key chlorophyll synthesis enzyme glutamyl-tRNA reductase, during the phytochrome A-mediated far-red light (FR) block of greening response in Arabidopsis. Our results demonstrate that the FR block of greening comprises two separate responses: a white light (WL) intensity-independent response that requires 3 d of FR and is associated with a loss of expression of the nuclear genes HEMA1 and Lhcb following the transfer to WL (transcriptionally coupled response) and a WL intensity-dependent response that is induced by 1 d of FR and is transcriptionally uncoupled. Both responses required phytochrome A. The transcriptionally uncoupled response correlated with a deregulation of tetrapyrrole synthesis and potential photooxidative damage and was inhibited by cytokinin. The transcriptionally coupled FR response was additive with the loss of expression following Norflurazon-induced photobleaching and was absent in the presence of sucrose or after lower fluence rate (1 micromol m(-2) s(-1)) FR treatments. Both pathways leading to the loss of nuclear gene expression were inhibited by overexpression of NADPH:protochlorophyllide oxidoreductase, indicating a role for plastid signaling in the FR-mediated pathway. The significance of identifying a distinct phytochrome A-mediated plastid signaling pathway is discussed.

Tissue-specific, light-regulated and plastid-regulated expression of the single-copy nuclear gene encoding the chloroplast Rieske FeS protein of Arabidopsis thaliana.

The single-copy PetC gene encoding the chloroplast Rieske FeS protein of Arabidopsis thaliana consists of five exons interrupted by four introns and encodes a protein of 229 amino acid residues with extensive sequence similarity to the chloroplast Rieske proteins of other higher plants. The N-terminal 50 amino acid residues constitute a presequence for targeting to the chloroplast and the remaining 179 amino acid residues make up the mature protein. Three of the introns are in identical positions in the PetC gene of Chlamydomonas reinhardtii, suggesting that they are of ancient origin. RNA-blot hybridisation showed that the gene was expressed in shoots, but not roots, and was light regulated and repressed by sucrose. The expression of chimeric genes consisting of PetC promoter fragments fused to the beta-glucuronidase (GUS) reporter gene was examined in A. thaliana and tobacco. In A. thaliana, GUS activity was detected in leaves, stems, flowers and siliques, but not in roots, and showed a strong correlation with the presence of chloroplasts. In transgenic tobacco, low levels of GUS activity were also detected in light-exposed roots. GUS activity in transgenic tobacco seedlings was light regulated and was decreased by norflurazon in the light suggesting regulation of PetC expression by plastid signals.

Functional properties and regulatory complexity of a minimal RBCS light-responsive unit activated by phytochrome, cryptochrome, and plastid signals.

Light-inducible promoters are able to respond to a wide spectrum of light through multiple photoreceptor systems. Several cis-acting elements have been identified as components of light-responsive promoter elements; however, none of these regulatory elements by itself appears to be sufficient to confer light responsiveness; rather, the combination of at least two elements seems to be required. Using phylogenetic structural analysis, we have identified conserved DNA modular arrays (CMAs) associated with light-responsive promoter regions that have been conserved throughout the evolutionary radiation of angiosperms. Here, we report the functional characterization of CMA5, a native 52-bp fragment of the Nicotiana plumbaginifolia rbcS 8B promoter, which contains an I- and a G-box cis-element. CMA5 behaves as a light-responsive minimal unit capable of activating a heterologous minimal promoter in a phytochrome-, cryptochrome-, and plastid-dependent manner. We also show that CMA5 light induction requires HY5 and that downstream negative regulators COP (constitutive photomorphogenic)/DET (de-etiolated) regulate its activity. Our results show that the simplest light-responsive promoter element from photosynthesis-associated genes described to date is the common target for different signals involved in light regulation. The possible mechanism involved in light-transcriptional regulation and tissue specificity of combinatorial elements units is discussed.

Research note: Characterization of a cDNA encoding glutamine synthetase II from Gelidium crinale (Rhodophyta)

SUMMARY A cDNA encoding glutamine synthetase (GS) was characterized from the red alga, Gelidium crinale (Turner) Gaillon, using reverse-transcriptase polymerase chain reaction and the 5′- and 3′-rapid amplification of cDNA ends. Sequence analysis of a 1231-bp GS cDNA transcript included both 5′ and 3′ untranslated regions and a 1056-bp open reading frame encoding a 352 amino acid polypeptide. Comparison with GS sequences from other organisms revealed that the G. crinale cDNA encodes a type-II GS, and the absence of a N-terminal plastid signal sequence suggests that it is a cytosolic isoenzyme. Phylogenetic analyses of GSII amino acid sequences supports the multiple origin of cytosolic and plastid isoenzymes during eukaryotic evolution.