Project description:Retrograde signaling from the chloroplast to the nucleus is necessary to regulate the chloroplast proteome during development and fluctuating environmental conditions. Although the specific chloroplast process(es) that must occur and the nature of the signal(s) that exits the chloroplast are not well understood, previous studies using drug inhibitors of chloroplast biogenesis have revealed that normal chloroplast development is required to express Photosynthesis Associated Nuclear Genes (PhANGs). In an attempt to determine which specific steps in chloroplast development are involved in retrograde signaling, we analyzed Arabidopsis mutants defective in the six genes encoding sigma factor (Sig) proteins that are utilized by the plastid-encoded RNA polymerase to transcribe specific sets of plastid genes. Here, we demonstrate that both Sig2 and Sig6 have partially redundant roles in not only plastid transcription, but also tetrapyrrole synthesis and retrograde signaling to control PhANG expression. Normal PhANG expression can be partly restored in the sig2 mutant by increasing heme synthesis. Furthermore, there is a genetic interaction between Sig and GUN (genomes uncoupled) genes to generate chloroplast-retrograde signals. These results demonstrate that defective plastid transcription is the source of at least two retrograde signals to the nucleus; one involving tetrapyrrole synthesis and the other involving the accumulation of an unknown plastid transcript. We also propose that the study of sig mutants (with defects in the expression of specific plastid genes) provides a new genetic system, which avoids the use of harsh inhibitors and their potential side effects, to monitor developmental retrograde signaling and to elucidate its mechanisms.
Project description:Plants depend on photosynthesis as their primary means to generate energy and building blocks, and adverse environmental conditions can stress the photosynthetic apparatus leading to the production of toxic byproducts. In the long term, stress experienced by the chloroplast must be communicated to the nucleus to adjust the expression of genes providing robust abiotic stress resilience in a process called retrograde signaling. We propose a retrograde signaling mechanism that starts with the accumulation of phosphatidic acid at the outer chloroplast membrane. A mutant of Arabidopsis thaliana, lppγ lppɛ1, disrupted in two chloroplast envelope membrane-located phosphatidic acid phosphatases shows reduced growth and activation of abscisic acid-mediated abiotic stress response pathways, among other changes, as determined by RNA-Seq analysis. The mutant is more resistant to freezing and osmotic stress. To identify components of the proposed retrograde signaling pathway, we conducted a suppressor screen in the lppγ lppɛ1 mutant and identified a mutation that causes the loss of MED16, which is a component of Mediator, a transcriptional complex in the nucleus affecting the expression of genes involved in freezing tolerance, among others. Based on these findings we are proposing a lipid-based, retrograde signaling mechanism in response to abiotic stresses such as freezing.
Project description:In the present study we investigated at the system level the proteome obtained from seedlings of Arabidopsis thaliana wild-type Col-0 and gun1-101 and gun1-102 mutant plants treated or not with lincomycin (Lin), a chloroplast translation inhibitor widely used to inhibit chloroplast biogenesis and to trigger the plastid-to-nucleus retrograde communication. Specifically, we attempted a description of cellular responses to Lincomycin in wild-type genetic background and in retrograde signaling-defective mutant gun1. Hypotheses and key players emerged by our holistic view were validated at the experimental level. Our findings suggest that, in the presence of Lincomycin, cell responses rely on the plastid compartment in a GUN1-dependent manner, while in the gun1-mutant background, the activity of extra-plastid compartments prevails. Moreover, results obtained by complementary approaches depict the Oxygen Evolving Complex subunit PsbO as an atypical Photosynthesis-related protein by accumulating in non-photosynthetic plastids and having a superhub role in modulating chloroplast dismantling upon impairment of plastid functions.
Project description:Photosystem I (PSI) is a critical component of the photosynthetic machinery in plants. Under conditions of environmental stress, PSI becomes photoinhibited, leading to redox imbalance in the chloroplast. PSI photoinhibition is caused by an increase in electron pressure within PSI, which damages the iron-sulfur centers. In this study, we investigated the effect of PSI electron acceptors on the susceptibility of PSI to photoinhibition at different CO2 concentrations in the plant environment. We also analyzed the global gene expression in plants exposed to PSI photoinhibition. PSI was photoinhibited using a specific illumination technique that inhibited PSI with minimal effect on PSII. CO2 levels neither increased nor decreased the likelihood of PSI photodamage. PSI photoinhibition, independent of CO2 levels, upregulated the genes involved in the response to iron excess in plants and downregulated the genes involved in iron deficiency. It also induced the genes of photosynthetic proteins that act as electron acceptors for PSI. We propose that PSI photoinhibition causes a release of iron from iron-sulfur centers, which initiates a retrograde signal from the chloroplast to the nucleus to modify gene expression. In addition, deprivation of CO2 from the air initiated a signal that induced flavonoid biosynthesis genes, probably via jasmonate production.
Project description:Inter-organellar communication has emerged as a critical factor in maintaining cellular homeostasis under stress conditions. Chloroplasts function not only as central organelles for energy production but are also increasingly recognized as stress sensors and signal integrators. SIGMA FACTOR-BINDING PROTEIN 1 (SIB1), encoded by a nuclear gene, has been identified as a positive regulator of plant immunity, with localization in both chloroplasts and the nucleus. In this study, we identified Arabidopsis SEC23A, a COPII complex component known to mediate membrane trafficking between the endoplasmic reticulum (ER) and the Golgi apparatus, as a novel interactor of SIB1. Our findings reveal that under normal conditions, both SIB1 and SEC23A localize to the ER, while SIB1 is also localized in the nucleus. Upon ER stress and/or treatment with the immunity inducer salicylate, both proteins are relocated from the ER to the chloroplasts. Notably, SEC23A, similar to SIB1, also functions as a positive regulator of disease resistance. In response to pathogen infection, SIB1 and SEC23A downregulate expression of chloroplast- and nucleus-encoded genes associated with photosynthesis while enhancing expression of defense-related genes. Together, our findings reveal a previously uncharacterized pathway of ER–chloroplast communication mediated by SIB1 and SEC23A during plant stress responses and immunity, providing novel insights into the intricate regulatory networks that govern inter-organellar communication under stress in plants.
Project description:Shortly after the release of singlet oxygen (1O2), 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 1O2 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 1O2-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 1O2-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 1O2-responsive genes. Retrograde control of 1O2-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment. Keywords: biotic and abiotic stress response, nuclear gene expression, plastid-derived signal, Col-0 ecotype, continuous light and then dark-incubated plants
Project description:Aim: To identify regulatory factors that control: (1) chloroplast protein importand (2) chloroplast-to-nucleus signalling. This project is a joint proposal from the Jarvis lab which is interested in chloroplast protein import [1] and the Moller lab which is interested in plastid-to-nucleus signalling [2]. Background: The majority of chloroplast proteins are encoded in the nucleus and imported post-translationally into chloroplasts. The abundance of chloroplast proteins may therefore be regulated at multiple levels. It is well documented that the nuclear gene expression is responsive to (largely unknown) signals from the chloroplast [23] and evidence is now emerging that protein import is also a regulated process [1]. Protein import into chloroplasts is mediated by protein complexes in the outer and inner envelope membranes called Toc and Tic respectively. Biochemical studies of pea chloroplasts identified several Toc/Tic components. These proteins are mechanistic or structural components of the import apparatus. Arabidopsis homologues of the pea Toc/Tic proteins were identified by the AGI. Pea Toc34 is represented in Arabidopsis by two genes "Toc33 and Toc34" and pea Toc75 is represented by three genes. These different Tocs have different expression patterns and are proposed to have different precursor protein recognition specificities. The factors that regulate Toc expression in concert with the needs of plastids in developmentally different cells are unknown. Proposal: Two Arabidopsis mutants will be analysed. The ppi1 mutant is null for the putative precursor protein receptor Toc33 [1]and the ppi3 mutant is null for a putative component of the protein import channel Toc75-IV (on chromosome IV). ppi1 plants are yellow-green in appearance but remarkably healthy and grow only slightly more slowly than wild type. By contrast ppi3 plants are indistinguishable from wild type by eye although analysis of the mutant's chloroplast proteome is beginning to reveal some differences (K. Lilley personal communication). Gene expression changes in ppi1 are likely to be quite extensive. Retardation of chloroplast development in ppi1 will activate retrograde signalling pathways so that many nuclear photosynthetic genes are down-regulated. Changes in the expression of photosynthetic genes and of the genes responsible for mediating these responses may therefore be observed. Any regulatory and signalling genes identified will be of interest to the Moller lab. The expression of factors that regulate Toc/Tic gene expression may also be altered in ppi1. It should be possible to distinguish these factors from those involved in the general control of chloroplast gene expression by comparing the results from the two mutants. Genes affected in both mutants are more likely to be involved in regulating chloroplast import since it is unlikely that widespread changes in gene expression will be observed in ppi3. Changes in the expression of factors that regulate import post-translationallyand of the Toc/Tic genes themselves (many are on the RNA) may also be observed. References: 1. Jarvis P. et al. (1998) Science 282: 100-103. 2. Moller S.G. et al. (2001) Genes Dev. 15:90-103. 3. Jarvis P. (2001) Curr. Biol. 11: R307-R310.
Project description:For establishing the photosynthetic apparatus plant cells must orchestrate the expression of genes encoded in both nucleus and chloroplast. Therefore a crosstalk between the two compartments is necessary. We employed a trivalent gene expression profiling approach in order to elucidate the changes in gene expression that occur during the early steps of light-induced chloroplast biogenesis.
Project description:To analyze the impact of photosynthetic redox signals, light sources with spectral qualities that preferentially excite either Photosystem I (PSI light) or Photosystem II (PSII light) were used. The light sources have been described in (Wagner et al, Planta 2008). Strong reduction signals were induced by light shifts from PSI to PSII light (PSI-II). In order to find primary regulated genes the acclimation responses were monitored at 30 min and 60 min after a light shift. The control was continuous Psi light at the same time. We used stn7 (a thylakoid redox regulated kinase) to specifically block transduction of photosynthetic redox signal in order to compare “real” redox regulated with that of other light acclimation pathways. Keywords: photosynthesis, redox regulation, light acclimation, retrograde signalling, long term response
Project description:Chloroplasts engaged in oxygenic photosynthesis frequently overproduce reactive oxygen species (ROS) under stress conditions, with singlet oxygen (1O2) being particularly harmful due to its high reactivity and short lifespan. The nuclear-encoded chloroplast protein EXECUTER1 (EX1) senses elevated 1O2 levels through Trp643 oxidation and undergoes proteolysis, a process essential for activating 1O2-induced EX1-mediated chloroplast-to-nucleus retrograde signaling (1O2-EX1 signaling). However, the link between EX1 proteolysis and subsequent nuclear transcriptome changes remains unclear. In this study, we isolated SOF1 (suppressor of flu 1) through a forward genetic screen using EMS-mutagenized flu mutant seeds of Arabidopsis thaliana harboring Flag-fused EX1 driven by its native promoter (referred to as fluEX1). Like flu, fluEX1 plants conditionally produce 1O2 in chloroplasts upon a dark-to-light shift. In the fluEX1 sof1, all 1O2-induced stress responses are largely suppressed, despite 1O2 levels being similar to those in the fluEX1. SOF1 encodes the chloroplast outer envelope-anchored preprotein import receptor TOC33. While TOC33 loss does not affect EX1 import, abundance, localization, and 1O2-induced proteolysis in the chloroplast, it blocks 1O2-induced chloroplast-to-nucleus retrograde signaling. TOC33 interacts with the UVR domain of EX1 (EX1-UVR) in the chloroplast envelope, facilitating 1O2-induced decrease of the chloroplast EX1-UVR and increase of the nucleus EX1-UVR. Moreover, ectopic expression of EX1-UVR outside of the chloroplast bypasses the restrictive barrier imposed by the chloroplast envelope, activating 1O2 signaling and inducing stress responses. Our findings demonstrate that SOF1/TOC33 mediates 1O2-EX1 signaling from the chloroplast to the nucleus and that the EX1-UVR domain can substitute for full-length EX1 in this signaling pathway.