'happy on norflurazon' (hon) mutations implicate perturbance of plastid homeostasis with activating stress acclimatization and changing nuclear gene expression in norflurazon-treated seedlings

Various mutant screens have been undertaken to identify constituents involved in the transmission of signals from the plastid to the nucleus. Many of these screens have been performed using carotenoid-deficient plants grown in the presence of norflurazon (NF), an inhibitor of phytoene desaturase. NF-treated plants are bleached and suppress the expression of nuclear genes encoding chloroplast proteins. Several genomes uncoupled (gun) mutants have been isolated that de-repress the expression of these nuclear genes. In the present study, a genetic screen has been established that circumvents severe photo-oxidative stress in NF-treated plants. Under these modified screening conditions, happy on norflurazon (hon) mutants have been identified that, like gun mutants, de-repress expression of the Lhcb gene, encoding a light-harvesting chlorophyll protein, but, in contrast to wild-type and gun mutants, are green in the presence of NF. hon mutations disturb plastid protein homeostasis, thereby activating plastid signaling and inducing stress acclimatization. Rather than defining constituents of a retrograde signaling pathway specifically associated with the NF-induced suppression of nuclear gene expression, as proposed for gun, hon mutations affect Lhcb expression more indirectly prior to initiation of plastid signaling in NF-treated seedlings. They pre-condition seedlings by inducing stress acclimatization, thereby attenuating the impact of a subsequent NF treatment.     

Plastid signals remodel light signaling networks and are essential for efficient chloroplast biogenesis in Arabidopsis

Plastid signals are among the most potent regulators of genes that encode proteins active in photosynthesis. Plastid signals help coordinate the expression of the nuclear and chloroplast genomes and the expression of genes with the functional state of the chloroplast. Here, we report the isolation of new cryptochrome1 (cry1) alleles from a screen for Arabidopsis thaliana genomes uncoupled mutants, which have defects in plastid-to-nucleus signaling. We also report genetic experiments showing that a previously unidentified plastid signal converts multiple light signaling pathways that perceive distinct qualities of light from positive to negative regulators of some but not all photosynthesis-associated nuclear genes (PhANGs) and change the fluence rate response of PhANGs. At least part of this remodeling of light signaling networks involves converting HY5, a positive regulator of PhANGs, into a negative regulator of PhANGs. We also observed that mutants with defects in both plastid-to-nucleus and cry1 signaling exhibited severe chlorophyll deficiencies. These data show that the remodeling of light signaling networks by plastid signals is a mechanism that plants use to integrate signals describing the functional and developmental state of plastids with signals describing particular light environments when regulating PhANG expression and performing chloroplast biogenesis.     

Heme synthesis by plastid ferrochelatase I regulates nuclear gene expression in plants

Chloroplast signals regulate hundreds of nuclear genes during development and in response to stress, but little is known of the signals or signal transduction mechanisms of plastid-to-nucleus (retrograde) signaling. In Arabidopsis thaliana, genetic studies using norflurazon (NF), an inhibitor of carotenoid biosynthesis, have identified five GUN (genomes uncoupled) genes, implicating the tetrapyrrole pathway as a source of a retrograde signal. Loss of function of any of these GUN genes leads to increased expression of photosynthesis-associated nuclear genes (PhANGs) when chloroplast development has been blocked by NF. Here we present a new Arabidopsis gain-of-function mutant, gun6-1D, with a similar phenotype. The gun6-1D mutant overexpresses the conserved plastid ferrochelatase 1 (FC1, heme synthase). Genetic and biochemical experiments demonstrate that increased flux through the heme branch of the plastid tetrapyrrole biosynthetic pathway increases PhANG expression. The second conserved plant ferrochelatase, FC2, colocalizes with FC1, but FC2 activity is unable to increase PhANG expression in undeveloped plastids. These data suggest a model in which heme, specifically produced by FC1, may be used as a retrograde signal to coordinate PhANG expression with chloroplast development.     

A new gun mutant of oilseed rape with a reduced porphyrin flux through Mg-chelatase

Plastid-to-nucleus retrograde signaling coordinates the expression of nuclear photosynthetic genes with the developmental and functional state of the plastid. These signals are essential not only for coordinating the expression of photosynthetic genes both in the plastome and nuclear genome, but also for plants to respond optimally to environmental stress. In the present study, we found that the expression of the nuclear genes that encode plastid and non-plastid photosynthesis-related proteins was still maintained or slightly higher in cr3529, a chlorophyll deficient mutant of oilseed rape that possesses an arrested development of chloroplasts, suggesting that the expression of photosynthesis-related nuclear genes was uncoupled from the normal dependence on the developmental state of the chloroplast in cr3529. When the development of the plastid in cr3529 and the wild type was completely inhibited by lincomycin, much higher expression of photosynthesis-related nuclear genes was observed in cr3529, suggesting that the genomes uncoupled (gun) phenotype of cr3529 is even more apparent than under normal growth conditions. Lincomycin treatment also derepressed the expression of plastid genes in cr3529. The determination of porphyrin flux through Mg-chelatase showed that the content of protoporphyrin IX and Mg-protoporphyrin decreased in cr3529. The obvious gun phenotype of cr3529 under normal growth conditions and the pattern of tetrapyrrole metabolism in cr3529 suggest that it is a new gun mutant that could be used to study the regulation of the expression of nuclear and plastid genes by plastid-to-nucleus retrograde signaling under more physiological conditions and the mechanism of plant stress responses mediated by plastid signals.     

Modulation of 1O2-mediated retrograde signaling by the PLEIOTROPIC RESPONSE LOCUS 1 (PRL1) protein, a central integrator of stress and energy signaling

Shortly after the release of singlet oxygen (¹O₂) in chloroplasts, 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. Extensive genetic screens aimed at identifying constituents involved in ¹O₂-mediated plastid-to-nucleus signaling have failed to identify extraplastidic signaling components. This finding suggests that ¹O₂-mediated signals are not translocated to the nucleus via a single linear pathway, but rather through a signaling network that is difficult to block by single mutations. The complexity of this signaling network has been tackled by mutagenizing a transgenic flu line expressing the luciferase reporter gene under the control of the promoter of a ¹O₂-responsive AAA-ATPase gene (At3g28580) and isolating second site mutants that constitutively express the reporter gene at a high level. One of the mutants was shown by map-based cloning and sequencing to contain a single amino acid change in the PLEIOTROPIC RESPONSE LOCUS 1 (PRL1) protein. PRL1 suppresses the expression of AAA-ATPase and other ¹O₂-responsive genes. PRL1 seems to play a major role in modulating responses of plants to environmental changes by interconnecting ¹O₂-mediated retrograde signaling with other signaling pathways.     

Mutations in CHLOROPLAST RNA BINDING provide evidence for the involvement of the chloroplast in the regulation of the circadian clock in Arabidopsis

The Arabidopsis circadian system regulates the expression of up to 36% of the nuclear genome, including many genes that encode photosynthetic proteins. The expression of nuclear-encoded photosynthesis genes is also regulated by signals from the chloroplasts, a process known as retrograde signaling. We have identified CHLOROPLAST RNA BINDING (CRB), a putative RNA-binding protein, and have shown that it is important for the proper functioning of the chloroplast. crb plants are smaller and paler than wild-type plants, and have altered chloroplast morphology and photosynthetic performance. Surprisingly, mutations in CRB also affect the circadian system, altering the expression of both oscillator and output genes. In order to determine whether the changes in circadian gene expression are specific to mutations in the CRB gene, or are more generally caused by the malfunctioning of the chloroplast, we also examined the circadian system in mutations affecting STN7, GUN1, and GUN5, unrelated nuclear-encoded chloroplast proteins known to be involved in retrograde signaling. Our results provide evidence that the functional state of the chloroplast may be an important factor that affects the circadian system.     

Influence of plastids on light signalling and development

In addition to their contribution to metabolism, chloroplasts emit signals that influence the expression of nuclear genes that contribute to numerous plastidic and extraplastidic processes. Plastid-to-nucleus signalling optimizes chloroplast function, regulates growth and development, and affects responses to environmental cues. An incomplete list of plastid signals is available and particular plastid-to-nucleus signalling mechanisms are partially understood. The plastid-to-nucleus signalling that depends on the GENOMES UNCOUPLED (GUN) genes couples the expression of nuclear genes to the functional state of the chloroplast. Analyses of gun mutants provided insight into the mechanisms and biological functions of plastid-to-nucleus signalling. GUN genes contribute to chloroplast biogenesis, the circadian rhythm, stress tolerance, light signalling and development. Some have criticized the gun mutant screen for employing inhibitors of chloroplast biogenesis and suggested that gun alleles do not disrupt significant plastid-to-nucleus signalling mechanisms. Here, I briefly review GUN-dependent plastid-to-nucleus signalling, explain the flaws in the major criticisms of the gun mutant screen and review the influence of plastids on light signalling and development.     

Implication of chlorophyll biosynthesis on chloroplast-to-nucleus retrograde signaling

The biogenesis and function of chloroplast are controlled both by anterograde mechanisms involving nuclear-encoded proteins targeted to chloroplast and by retrograde signals from plastid to nucleus contributing to regulation of nuclear gene expression. A number of experimental evidences support the implication of chlorophyll biosynthesis intermediates on the retrograde signaling, albeit an earlier-postulated direct link between accumulation of chlorophyll intermediates and changes in nuclear gene expression has recently been challenged. By characterization of Arabidopsis mutants lacking the chloroplast localized NADPH-thioredoxin reductase (NTRC) we have recently proposed that imbalanced activity of chlorophyll biosynthesis in developing cells modifies the chloroplast signals leading to alterations in nuclear gene expression. These signals appear to initiate from temporal perturbations in the flux through the pathway from protoporphyrin to protochlorophyllide rather than from the accumulation of a single intermediate of the tetrapyr-role pathway.Key words: chloroplast biogenesis, NADPH-thioredoxin reductase, porphyrins, ROS, signaling, tetrapyrrole, thioredoxinOrchestrated regulation of gene expression in the nucleus and plastids is crucial for the proper biogenesis of the organelle during the development and for the acclimation of plants to environmental cues. Multiple potential candidates for initiating plastidial signals have been recognized, including intermediates of the tetrapyrrole biosynthetic pathway, redox state of chloroplast electron transfer components and reactive oxygen species (ROS). These multiple signaling pathways are likely to interact with each others, resulting in a complex signaling network between plastid and nucleus (reviewed in ref. ¹).     

Photosynthetic redox control of nuclear gene expression

Chloroplasts contain 3000-4000 different proteins but only a small subset of them is encoded in the plastid genome while the majority is encoded in the nucleus. Expression of these genes therefore requires a high degree of co-ordination between nucleus and chloroplast. This is achieved by a bilateral information exchange between both compartments including nucleus-to-plastid (anterograde) and plastid-to-nucleus (retrograde) signals. The latter represent a functional feedback control which couples the expression of nuclear encoded plastid proteins to the actual functional state of the organelle. The efficiency of photosynthesis is a very important parameter in this context since it is influenced by many environmental conditions and therefore represents a sensor for the residing environment. Components of the photosynthetic electron transport chain exhibit significant changes in their reduction/oxidation (redox) state depending on the photosynthetic electron flow and therefore serve as signalling parameters which report environmental influences on photosynthesis. Such redox signals control chloroplast and nuclear gene expression events and play an important role in the co-ordination of both genetic compartments. It is discussed here which photosynthetic parameters are known to control nuclear gene expression, how these signals are transduced toward the nucleus, and how they interact with other plastid retrograde signals and cytosolic light perception systems.     

The discovery of plastid-to-nucleus retrograde signaling—a personal perspective

DNA and machinery for gene expression have been discovered in chloroplasts during the 1960s. It was soon evident that the chloroplast genome is relatively small, that most genes for chloroplast-localized proteins reside in the nucleus and that chloroplast membranes, ribosomes, and protein complexes are composed of proteins encoded in both the chloroplast and the nuclear genome. This situation has made the existence of mechanisms highly probable that coordinate the gene expression in plastids and nucleus. In the 1970s, the first evidence for plastid signals controlling nuclear gene expression was provided by studies on plastid ribosome deficient mutants with reduced amounts and/or activities of nuclear-encoded chloroplast proteins including the small subunit of Rubisco, ferredoxin NADP+ reductase, and enzymes of the Calvin cycle. This review describes first models of plastid-to-nucleus signaling and their discovery. Today, many plastid signals are known. They do not only balance gene expression in chloroplasts and nucleus during developmental processes but are also generated in response to environmental changes sensed by the organelles.     

Variegation mutants and mechanisms of chloroplast biogenesis

Variegated plants typically have green‐ and white‐sectored leaves. Cells in the green sectors contain normal‐appearing chloroplasts, whereas cells in the white sectors lack pigments and appear to be blocked at various stages of chloroplast biogenesis. Variegations can be caused by mutations in nuclear, chloroplast or mitochondrial genes. In some plants, the green and white sectors have different genotypes, but in others they have the same (mutant) genotype. One advantage of variegations is that they provide a means of studying genes for proteins that are important for chloroplast development, but for which mutant analysis is difficult, either because mutations in a gene of interest are lethal or because they do not show a readily distinguishable phenotype. This paper focuses on Arabidopsis variegations, for which the most information is available at the molecular level. Perhaps the most interesting of these are variegations caused by defective nuclear gene products in which the cells of the mutant have a uniform genotype. Two questions are of paramount interest: (1) What is the gene product and how does it function in chloroplast biogenesis? (2) What is the mechanism of variegation and why do green sectors arise in plants with a uniform (mutant) genotype? Two paradigms of variegation mechanism are described: immutans (im) and variegated2 (var2). Both mechanisms emphasize compensating activities and the notion of plastid autonomy, but redundant gene products are proposed to play a role in var2, but not in im. It is hypothesized that threshold levels of certain activities are necessary for normal chloroplast development.     

Signaling pathways from the chloroplast to the nucleus

Genetic and physiological studies have to-date revealed evidence for five signaling pathways by which the chloroplast exerts retrograde control over nuclear genes. One of these pathways is dependent on product(s) of plastid protein synthesis, for another the signal is singlet oxygen, a third employs chloroplast-generated hydrogen peroxide, a fourth is controlled by the redox state of the photosynthetic electron transport chain, and a fifth involves intermediates and possibly proteins of tetrapyrrole biosynthesis. These five pathways may be part of a complex signaling network that links the functional and physiological state of the chloroplast to the nucleus. Mutants defective in various steps of photosynthesis reveal a surprising diversity in nuclear responses suggesting the existence of a complex signaling network.     

In vivo visualization of Mg-protoporphyrin IX, a coordinator of photosynthetic gene expression in the nucleus and the chloroplast

The photosynthetic apparatus is composed of proteins encoded by genes from both the nucleus and the chloroplast. To ensure that the photosynthetic complexes are assembled stoichiometrically and to enable their rapid reorganization in response to a changing environment, the plastids emit signals that regulate nuclear gene expression to match the status of the plastids. One of the plastid signals, the chlorophyll intermediate Mg-ProtoporphyrinIX (Mg-ProtoIX) accumulates under stress conditions and acts as a negative regulator of photosynthetic gene expression. By taking advantage of the photoreactive property of tetrapyrroles, Mg-ProtoIX could be visualized in the cells using confocal laser scanning spectroscopy. Our results demonstrate that Mg-ProtoIX accumulated both in the chloroplast and in the cytosol during stress conditions. Thus, the signaling metabolite is exported from the chloroplast, transmitting the plastid signal to the cytosol. Our results from the Mg-ProtoIX over- and underaccumulating mutants copper response defect and genome uncoupled5, respectively, demonstrate that the expression of both nuclear- and plastid-encoded photosynthesis genes is regulated by the accumulation of Mg-ProtoIX. Thus, stress-induced accumulation of the signaling metabolite Mg-ProtoIX coordinates nuclear and plastidic photosynthetic gene expression.     

四吡咯代谢中间产物在植物质体向细胞核信号转导中的作用

在植物细胞内,除了顺向的信号转导通路,即核基因控制着质体基因的转录和翻译之外,还存在着逆向的信号转导通路,即质体的代谢状况作为一种信号去调控核基因的表达。过去对这条逆向的信号转导通路,亦称质体因子,研究得非常少。近几年来,随着对基因组解偶联突变体的深入研究,人们对这条通路的认识大大加深了。现着重介绍质体中的四吡咯代谢中间产物参与信号的产生,以及质体向细胞质搬运这些中间产物启动了对编码质体蛋白的核基因的表达调控。     

The Arabidopsis chloroplast ribosomal protein L21 is encoded by a nuclear gene of mitochondrial origin.

Many chloroplast genes of cyanobacterial origin have been transferred to the nucleus during evolution and their products are re-addressed to chloroplasts. The RPL21 gene encoding the plastid r-protein L21 has been lost in higher plant chloroplast genomes after the divergence from bryophytes. Based on phylogenetic analysis and intron conservation, we now provide evidence that in Arabidopsis a nuclear RPL21c gene of mitochondrial origin has replaced the chloroplast gene. The control of expression of this gene has been adapted to the needs of chloroplast development by the acquisition of plastid-specific regulatory promoter cis-elements.