Project description:Membrane contact sites (MCS) are fundamental for intracellular communication, but their role in intercellular communication remains unexplored. We show that in plants, plasmodesmata communication bridges function as atypical endoplasmic reticulum (ER)-plasma membrane (PM) tubular MCS, operating at cell-cell interfaces. Similar to other MCS, ER-PM apposition is controlled by a protein-lipid tethering complex, but uniquely, this serves intercellular communication. Combining high-resolution microscopy, molecular dynamics, pharmacological and genetic approaches, we show that cell-cell trafficking is modulated through the combined action of Multiple C2 domains and transmembrane domain proteins (MCTP) 3, 4, and 6 ER-PM tethers, and phosphatidylinositol-4-phosphate (PI4P) lipid. Graded PI4P amounts regulate MCTP docking to the PM, their plasmodesmata localization and cell-cell permeability. SAC7, an ER-localized PI4P-phosphatase, regulates MCTP4 accumulation at plasmodesmata and modulates cell-cell trafficking capacity in a cell-type specific manner. Our findings expand MCS's functions in information transmission, from intracellular to intercellular cellular activities.
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:We use Arabidopsis thaliana embryogenesis as a model system for studying intercellular transport via plasmodesmata (PD). A forward genetic screen for altered PD transport identified ise1 and ise2 mutants with increased intercellular transport of fluorescent 10-kDa tracers. Both ise1 and ise2 exhibit increased formation of twinned and branched PD. ISE1 encodes a mitochondrial DEAD-box RNA helicase, while ISE2 encodes a DEVH-type RNA helicase. Here we show that ISE2 foci are localized to the chloroplast stroma. Surprisingly, plastid development is defective in both ise1 and ise2 mutant embryos. In an effort to understand how RNA helicases that localize to different organelles have similar impacts on plastid and PD development/function we performed whole genome expression analyses. The most significantly affected class of transcripts in both mutants encodes products that target to and enable plastid function. These results reinforce the importance of plastid-mitochondria-nucleus crosstalk, add PD as a critical player in the plant cell communication network, and thereby illuminate a new signaling pathway, dubbed organelle-nucleus-plasmodesmata signaling (ONPS). Several genes with roles in cell wall synthesis and modification are also differentially expressed in both mutants, providing new targets for investigating PD development and function. Three biological replicates each of either ise1 or ise2 mutant seeds vs. sister wild-type controls
Project description:We use Arabidopsis thaliana embryogenesis as a model system for studying intercellular transport via plasmodesmata (PD). A forward genetic screen for altered PD transport identified ise1 and ise2 mutants with increased intercellular transport of fluorescent 10-kDa tracers. Both ise1 and ise2 exhibit increased formation of twinned and branched PD. ISE1 encodes a mitochondrial DEAD-box RNA helicase, while ISE2 encodes a DEVH-type RNA helicase. Here we show that ISE2 foci are localized to the chloroplast stroma. Surprisingly, plastid development is defective in both ise1 and ise2 mutant embryos. In an effort to understand how RNA helicases that localize to different organelles have similar impacts on plastid and PD development/function we performed whole genome expression analyses. The most significantly affected class of transcripts in both mutants encodes products that target to and enable plastid function. These results reinforce the importance of plastid-mitochondria-nucleus crosstalk, add PD as a critical player in the plant cell communication network, and thereby illuminate a new signaling pathway, dubbed organelle-nucleus-plasmodesmata signaling (ONPS). Several genes with roles in cell wall synthesis and modification are also differentially expressed in both mutants, providing new targets for investigating PD development and function.
Project description:In eukaryotes, membrane contact sites (MCS) allow direct communication between organelles. Plants have evolved unique MCS, the plasmodesmata intercellular pores, which combine organelle tethering with regulation of cell-to-cell signalling, the molecular mechanisms of which remains unknown. Here, we identify Multiple C2 domains and Transmembrane region Proteins (MCTPs) as tethers that link the endoplasmic reticulum (ER) to the plasma membrane (PM) within plasmodesmata. We report that MCTPs, including MCTP3 and 4 recently identified as modulators of SHOOTMERISTEMLESS trafficking, are ER-anchored proteins that cluster at plasmodesmata. MCTPs insert into the ER via their transmembrane region whilst their C2 domains dock to the PM through interaction with anionic phospholipids. A mctp3/4 loss-of function mutant induces plant developmental defects while MCTP4 expression in a yeast .tether mutant partially restores ER-PM tethering. Our data suggest that MCTPs are unique membrane tethers controlling both ER-PM contacts and cell-cell signalling.
Project description:Retrograde signals emanate from the DNA-containing cell organelles (plastids and mitochondria) and control the expression of a large number of nuclear genes in response to environmental and developmental cues. GENOMES UNCOUPLED1 (GUN1) participating in multiple retrograde signaling pathways that collectively regulate the nuclear transcriptome. We used microarrays to further investigate the regulation of nuclear gene expression by PGE retrograde signals mediated by GUN1.
Project description:Canonical retrograde signalling comprises information transmission from organelles to the nucleus and in particular controls gene expression for organellar proteins. The need to re-assess this paradigm was suggested by discrepancies between de novo protein synthesis and transcript abundance in response to excess light. Here we uncover major components of a translation-dependent retrograde signalling pathway that first impacts translation and then transcription. The response realization depends on the kinases Mitogen-activated protein kinase 6 (MPK6) and Sucrose non-fermenting 1-related kinase (SnRK1) subunit, AKIN10. Global ribosome foot-printing revealed differential ribosome association of 951 transcripts within 10 min after transfer from low to high light. Despite predominant translational repression, 15 % of transcripts were increased in translation and enriched for chloroplast-localized photosynthetic proteins. About one third of these transcripts, including Stress associated proteins (SAP) 2 and 3, share regulatory motifs in their 5`-UTR that act as binding sites for glyceraldehyde-3-phosphate dehydrogenase (GAPC) and light responsive RNA binding proteins (RBPs). SAP2 and 3 are both translationally regulated and interact with the calcium sensor Calmodulin-like 49 (CML49), which promotes relocation to the nucleus inducing a translation-dependent nuclear stress response. Thus, translation-dependent retrograde signalling bifurcates to directly regulate a translational circuit of chloroplast proteins and simultaneously initiate a nuclear circuit synchronizing retrograde and anterograde response pathways, serving as a rapid mechanism for functional acclimation of the chloroplast.
Project description:Canonical retrograde signalling comprises information transmission from organelles to the nucleus and in particular controls gene expression for organellar proteins. The need to re-assess this paradigm was suggested by discrepancies between de novo protein synthesis and transcript abundance in response to excess light. Here we uncover major components of a translation-dependent retrograde signalling pathway that first impacts translation and then transcription. The response realization depends on the kinases Mitogen-activated protein kinase 6 (MPK6) and Sucrose non-fermenting 1-related kinase (SnRK1) subunit, AKIN10. Global ribosome foot-printing revealed differential ribosome association of 951 transcripts within 10 min after transfer from low to high light. Despite predominant translational repression, 15 % of transcripts were increased in translation and enriched for chloroplast-localized photosynthetic proteins. About one third of these transcripts, including Stress associated proteins (SAP) 2 and 3, share regulatory motifs in their 5`-UTR that act as binding sites for glyceraldehyde-3-phosphate dehydrogenase (GAPC) and light responsive RNA binding proteins (RBPs). SAP2 and 3 are both translationally regulated and interact with the calcium sensor Calmodulin-like 49 (CML49), which promotes relocation to the nucleus inducing a translation-dependent nuclear stress response. Thus, translation-dependent retrograde signalling bifurcates to directly regulate a translational circuit of chloroplast proteins and simultaneously initiate a nuclear circuit synchronizing retrograde and anterograde response pathways, serving as a rapid mechanism for functional acclimation of the chloroplast CML49 KO and SAP3 KO 0' and 60' of low light (8µE) to high light (800µE) transfer in comparison to Col-0
Project description:GUN1 integrates retrograde signals in the chloroplast but the underlying mechanism is elusive. FUG1, a chloroplast translation initiation factor, and GUN1 are co-expressed at the transcript level, and FUG1 co-immunoprecipitates with GUN1. We used mutants of GUN1 (gun1-103) and FUG1 (fug1-3) to analyse their functional relationship at the physiological and systems-wide level, the latter including transcriptome and proteome analyses. Absence of GUN1 aggravates the effects of decreased FUG1 levels on chloroplast protein translation, resulting in transient additive phenotypes with respect to photosynthesis, leaf coloration, growth and cold acclimation. Variegation of the var2 mutant is enhanced by gun1-103 in terms of increasing the fraction of white sectors, in contrast to fug1-3 that acts as suppressor. The transcriptomes of fug1-3 and gun1-103 are very similar, but absence of GUN1 alone has almost no effects on protein levels, whereas chloroplast protein accumulation is markedly decreased in fug1-3. In gun1 fug1 double mutants, effects on transcriptomes and particularly proteomes are enhanced. Our results show that GUN1 function becomes critical when chloroplast proteostasis is perturbed by decreased translation (fug1) or degradation (var2) of chloroplast proteins. The functions of FUG1 and GUN1 appear to be related, corroborating the view that GUN1 operates in chloroplast proteostasis.
Project description:Chloroplast-nuclear retrograde signaling is viewed as a mechanism for inter-organelle communication. Here we show the SAL1-PAP (3′-phosphoadenosine 5′- phosphate) retrograde pathway functions more broadly in guard cells, interacting with abscisic acid (ABA) signaling at least in part via exoribonucleases. Unexpectedly, PAP bypasses the canonical signaling components ABA Insensitive 1 (ABI1) and Open Stomata 1 (OST1) by priming an alternative pathway that restores ABA-responsive gene expression, ROS bursts, ion channel function and stomatal closure in ost1-2. This alternative pathway up-regulates lowly expressed Calcium Dependent Protein Kinases (CDPKs) which have the capacity to activate the key slow anion channel SLAC1 in response to ABA-mediated and ost1-2 independent calcium release. The role of PAP in priming an alternative pathway to bypass components previously considered essential for stomatal closure demonstrates how a chloroplast signal can have broader roles as a secondary messenger to directly intersect with and tune hormone signaling.