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).     

Evidence for a SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in Arabidopsis

Compartmentation of the eukaryotic cell requires a complex set of subcellular messages, including multiple retrograde signals from the chloroplast and mitochondria to the nucleus, to regulate gene expression. Here, we propose that one such signal is a phosphonucleotide (3'-phosphoadenosine 5'-phosphate [PAP]), which accumulates in Arabidopsis thaliana in response to drought and high light (HL) stress and that the enzyme SAL1 regulates its levels by dephosphorylating PAP to AMP. SAL1 accumulates in chloroplasts and mitochondria but not in the cytosol. sal1 mutants accumulate 20-fold more PAP without a marked change in inositol phosphate levels, demonstrating that PAP is a primary in vivo substrate. Significantly, transgenic targeting of SAL1 to either the nucleus or chloroplast of sal1 mutants lowers the total PAP levels and expression of the HL-inducible ASCORBATE PEROXIDASE2 gene. This indicates that PAP must be able to move between cellular compartments. The mode of action for PAP could be inhibition of 5' to 3' exoribonucleases (XRNs), as SAL1 and the nuclear XRNs modulate the expression of a similar subset of HL and drought-inducible genes, sal1 mutants accumulate XRN substrates, and PAP can inhibit yeast (Saccharomyces cerevisiae) XRNs. We propose a SAL1-PAP retrograde pathway that can alter nuclear gene expression during HL and drought stress.     

The chloroplast division mutant caa33 of Arabidopsis thaliana reveals the crucial impact of chloroplast homeostasis on stress acclimation and retrograde plastid-to-nucleus signaling

Retrograde plastid-to-nucleus signaling tightly controls and coordinates the nuclear and plastid gene expression that is required for plastid biogenesis and chloroplast activity. As chloroplasts act as sensors of environmental changes, plastid-derived signaling also modulates stress responses of plants by transferring stress-related signals and altering nuclear gene expression. Various mutant screens have been undertaken to identify constituents of plastid signaling pathways. Almost all mutations identified in these screens target plastid-specific but not extraplastidic functions. They have been suggested to define either genuine constituents of retrograde signaling pathways or components required for the synthesis of plastid signals. Here we report the characterization of the constitutive activator of AAA-ATPase (caa33) mutant, which reveals another way of how mutations that affect plastid functions may modulate retrograde plastid signaling. caa33 disturbs a plastid-specific function by impeding plastid division, and thereby perturbing plastid homeostasis. This results in preconditioning plants by activating the expression of stress genes, enhancing pathogen resistance and attenuating the capacity of the plant to respond to plastid signals. Our study reveals an intimate link between chloroplast activity and the susceptibility of the plant to stress, and emphasizes the need to consider the possible impact of preconditioning on retrograde plastid-to-nucleus signaling.     

At3g53630 encodes a GUN1-interacting protein under norflurazon treatment

Chloroplasts are semi-autonomous organelles, with more than 95% of their proteins encoded by the nuclear genome. The chloroplast-to-nucleus retrograde signals are critical for the nucleus to coordinate its gene expression for optimizing or repairing chloroplast functions in response to changing environments. In chloroplasts, the pentatricopeptide-repeat protein GENOMES UNCOUPLED 1 (GUN1) is a master switch that senses aberrant physiological states, such as the photooxidative stress induced by norflurazon (NF) treatment, and represses the expression of photosynthesis-associated nuclear genes (PhANGs). However, it is largely unknown how the retrograde signal is transmitted beyond GUN1. In this study, a protein GUN1-INTERACTING PROTEIN 1 (GIP1), encoded by At3g53630, was identified to interact with GUN1 by different approaches. We demonstrated that GIP1 has both cytosol and chloroplast localizations, and its abundance in chloroplasts is enhanced by NF treatment with the presence of GUN1. Our results suggest that GIP1 and GUN1 may function antagonistically in the retrograde signaling pathway.

     

Light intensity-dependent retrograde signalling in higher plants

Plants are able to acclimate to highly fluctuating light environment and evolved a short- and long-term light acclimatory responses, that are dependent on chloroplasts retrograde signalling. In this review we summarise recent evidences suggesting that the chloroplasts act as key sensors of light intensity changes in a wide range (low, high and excess light conditions) as well as sensors of darkness. They also participate in transduction and synchronisation of systemic retrograde signalling in response to differential light exposure of distinct leaves. Regulation of intra- and inter-cellular chloroplast retrograde signalling is dependent on the developmental and functional stage of the plastids. Therefore, it is discussed in following subsections: firstly, chloroplast biogenic control of nuclear genes, for example, signals related to photosystems and pigment biogenesis during early plastid development; secondly, signals in the mature chloroplast induced by changes in photosynthetic electron transport, reactive oxygen species, hormones and metabolite biosynthesis; thirdly, chloroplast signalling during leaf senescence. Moreover, with a help of meta-analysis of multiple microarray experiments, we showed that the expression of the same set of genes is regulated specifically in particular types of signals and types of light conditions. Furthermore, we also highlight the alternative scenarios of the chloroplast retrograde signals transduction and coordination linked to the role of photo-electrochemical signalling.     

ROS and redox signalling in the response of plants to abiotic stress

The redox state of the chloroplast and mitochondria, the two main powerhouses of photosynthesizing eukaryotes, is maintained by a delicate balance between energy production and consumption, and affected by the need to avoid increased production of reactive oxygen species (ROS). These demands are especially critical during exposure to extreme environmental conditions, such as high light (HL) intensity, heat, drought or a combination of different environmental stresses. Under these conditions, ROS and redox cues, generated in the chloroplast and mitochondria, are essential for maintaining normal energy and metabolic fluxes, optimizing different cell functions, activating acclimation responses through retrograde signalling, and controlling whole-plant systemic signalling pathways. Regulation of the multiple redox and ROS signals in plants requires a high degree of coordination and balance between signalling and metabolic pathways in different cellular compartments. In this review, we provide an update on ROS and redox signalling in the context of abiotic stress responses, while addressing their role in retrograde regulation, systemic acquired acclimation and cellular coordination in plants.     

'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.     

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.     

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.     

Identification and differential accumulation of two isoforms of the CF1-beta subunit under high light stress in Brassica rapa.

The chloroplast ATP synthase coupling factor CF1 complex contains five nonidentical subunits, alpha, beta, gamma, delta, and epsilon, with a stoichiometry of 3:3:1:1:1. The beta subunit contains the catalytic site for ATP synthesis during photooxidative phosphorylation in the chloroplast. In this study, we have identified two isoforms of the CF1-beta subunit at 56 and 54 kDa in the chloroplast of Brassica rapa, through isolation/purification, proteolytic digestion and internal peptide sequencing. Examining their accumulation pattern demonstrates that both isoforms coexist during chloroplast biogenesis and in mature thylakoid membranes, but the 54 kDa isoform is more apparently upregulated by light or under light stress. LiDS-PAGE shows that the 56 kDa is a major isoform of the CF1-beta subunit under normal light conditions, and its amount was not influenced during high light or other light stress treatments. The 54 kDa isoform is a minor band at normal conditions; however, it significantly increased under excess light stresses, such as high or low light with drought and/or high temperature. Particularly, a ninefold increase was observed after 8-10 h of high light treatment with drought and high temperature. The results suggest that light stress induction of the 54 kDa CF1-beta isoform may present a positive response during chloroplast photoacclimation.     

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.