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Stimulus-specific accumulation of second messengers like reactive oxygen species (ROS) and Ca(2+) are central to many signaling and regulation processes in plants. However, mechanisms that govern the reciprocal interrelation of Ca(2+) and ROS signaling are only beginning to emerge. NADPH oxidases of the respiratory burst oxidase homolog (RBOH) family are critical components contributing to the generation of ROS while Calcineurin B-like (CBL) Ca(2+) sensor proteins together with their interacting kinases (CIPKs) have been shown to function in many Ca(2+)- signaling processes. In this study, we identify direct functional interactions between both signaling systems. We report that the CBL-interacting protein kinase CIPK26 specifically interacts with the N-terminal domain of RBOHF in yeast two-hybrid analyses and with the full-length RBOHF protein in plant cells. In addition, CIPK26 phosphorylates RBOHF in vitro and co-expression of either CBL1 or CBL9 with CIPK26 strongly enhances ROS production by RBOHF in HEK293T cells. Together, these findings identify a direct interconnection between CBL-CIPK-mediated Ca(2+) signaling and ROS signaling in plants and provide evidence for a synergistic activation of the NADPH oxidase RBOHF by direct Ca(2+)-binding to its EF-hands and Ca(2+)-induced phosphorylation by CBL1/9-CIPK26 complexes.

Cyclic photosynthetic electron flow (CEF) is crucial to photosynthesis because it participates in the control of chloroplast energy and redox metabolism, and it is particularly induced under adverse environmental conditions. Here we report that down-regulation of the chloroplast localized Ca(2+) sensor (CAS) protein by an RNAi approach in Chlamydomonas reinhardtii results in strong inhibition of CEF under anoxia. Importantly, this inhibition is rescued by an increase in the extracellular Ca(2+) concentration, inferring that CEF is Ca(2+)-dependent. Furthermore, we identified a protein, anaerobic response 1 (ANR1), that is also required for effective acclimation to anaerobiosis. Depletion of ANR1 by artificial microRNA expression mimics the CAS-depletion phenotype, and under anaerobic conditions the two proteins coexist within a large active photosystem I-cytochrome b(6)/f complex. Moreover, we provide evidence that CAS and ANR1 interact with each other as well as with PGR5-Like 1 (PGRL1) in vivo. Overall our data establish a Ca(2+)-dependent regulation of CEF via the combined function of ANR1, CAS, and PGRL1, associated with each other in a multiprotein complex.

Background: Calcium serves as a versatile messenger in many adaptation and developmental processes in plants. Ca2+ signals are represented by stimulus-specific spatially and temporally defined Ca2+ signatures. These Ca2+ signatures are detected, decoded and transmitted to downstream responses by a complex toolkit of Ca2+ binding proteins that function as Ca2+ sensors., Scope of Review: This review will reflect on advancements in monitoring Ca2+ dynamics in plants. Moreover, it will provide insights in the extensive and complex toolkit of plant Ca2+ sensor proteins that relay the information presented in the Ca2+ signatures into phosphorylation events, changes in protein-protein interaction or regulation of gene expression., Major Conclusions: Plants' response to signals is encoded by different Ca2+ signatures. The plant decoding Ca2+ toolkit encompasses different families of Ca2+ sensors like Calmodulins (CaM), Calmodulin-like proteins (CMLs), Ca2+-dependent protein kinases (CDPKs), Calcineurin B-like proteins (CBLs) and their interacting kinases (CIPKs). These Ca2+ sensors are encoded by complex gene families and form intricate signaling networks in plants that enable specific, robust and flexible information processing., General Significance: This review provides new insights about the biochemical regulation, physiological functions and of newly identified target proteins of the major plant Ca2+ sensor families. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling., (Copyright © 2011. Published by Elsevier B.V.)

Calcineurin B-like (CBL) proteins contribute to decoding calcium signals by interacting with CBL-interacting protein kinases (CIPKs). Currently, there is still very little information about the function and specific targeting mechanisms of CBL proteins that are localized at the vacuolar membrane. In this study, we focus on CBL2, an abundant vacuolar membrane-localized calcium sensor of unknown function from Arabidopsis thaliana. We show that vacuolar targeting of CBL2 is specifically brought about by S-acylation of three cysteine residues in its N-terminus and that CBL2 S-acylation and targeting occur by a Brefeldin A-insensitive pathway. Loss of CBL2 function renders plants hypersensitive to the phytohormone abscisic acid (ABA) during seed germination and only fully S-acylated and properly vacuolar-targeted CBL2 proteins can complement this mutant phenotype. These findings define an S-acylation-dependent vacuolar membrane targeting pathway for proteins and uncover a crucial role of vacuolar calcium sensors in ABA responses.

Calcineurin B-like proteins (CBLs) represent a family of calcium sensor proteins that interact with a group of serine/threonine kinases designated as CBL-interacting protein kinases (CIPKs). CBL-CIPK complexes are crucially involved in relaying plant responses to many environmental signals and in regulating ion fluxes. However, the biochemical characterization of CBL-CIPK complexes has so far been hampered by low activities of recombinant CIPKs. Here, we report on an efficient wheat germ extract-based in vitro transcription/translation protocol that yields active full-length wild-type CIPK proteins. We identified a conserved serine residue within the C terminus of CBLs as being phosphorylated by their interacting CIPKs. Remarkably, our studies revealed that CIPK-dependent CBL phosphorylation is strictly dependent on CBL-CIPK interaction via the CIPK NAF domain. The phosphorylation status of CBLs does not appear to influence the stability, localization, or CIPK interaction of these calcium sensor proteins in general. However, proper phosphorylation of CBL1 is absolutely required for the in vivo activation of the AKT1 K(+) channel by CBL1-CIPK23 and CBL9-CIPK23 complexes in oocytes. Moreover, we show that by combining CBL1, CIPK23, and AKT1, we can faithfully reconstitute CBL-dependent enhancement of phosphorylation of target proteins by CIPKs in vitro. In addition, we report that phosphorylation of CBL1 by CIPK23 is also required for the CBL1-dependent enhancement of CIPK23 activity toward its substrate. Together, these data identify a novel general regulatory mechanism of CBL-CIPK complexes in that CBL phosphorylation at their flexible C terminus likely provokes conformational changes that enhance specificity and activity of CBL-CIPK complexes toward their target proteins.

Temporally and spatially defined calcium signatures are integral parts of numerous signalling pathways. Monitoring calcium dynamics with high spatial and temporal resolution is therefore critically important to understand how this ubiquitous second messenger can control diverse cellular responses. Yellow cameleons (YCs) are fluorescence resonance energy transfer (FRET)-based genetically encoded Ca(2+) -sensors that provide a powerful tool to monitor the spatio-temporal dynamics of Ca(2+) fluxes. Here we present an advanced set of vectors and transgenic lines for live cell Ca(2+) imaging in plants. Transgene silencing mediated by the cauliflower mosaic virus (CaMV) 35S promoter has severely limited the application of nanosensors for ions and metabolites and we have thus used the UBQ10 promoter from Arabidopsis and show here that this results in constitutive and stable expression of YCs in transgenic plants. To improve the spatial resolution, our vector repertoire includes versions of YCs that can be targeted to defined locations. Using this toolkit, we identified temporally distinct responses to external ATP at the plasma membrane, in the cytosol and in the nucleus of neighbouring root cells. Moreover analysis of Ca(2+) dynamics in Lotus japonicus revealed distinct Nod factor induced Ca(2+) spiking patterns in the nucleus and the cytosol. Consequently, the constructs and transgenic lines introduced here enable a detailed analysis of Ca(2+) dynamics in different cellular compartments and in different plant species and will foster novel approaches to decipher the temporal and spatial characteristics of calcium signatures., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Ca(2+) is a crucial second messenger that is involved in mediating responses to various biotic and abiotic environmental cues and in the regulation of many developmental processes in plants. Intracellular Ca(2+) signals are realized by spatially and temporally defined changes in Ca(2+) concentration that represent stimulus-specific Ca(2+) signatures. These Ca(2+) signatures are sensed, decoded and transmitted to downstream responses by a complex tool kit of Ca(2+) binding proteins that function as Ca(2+) sensors. Plants possess an extensive and complex array of such Ca(2+) sensors that convey the information presented in the Ca(2+) signatures into phosphorylation events, changes in protein-protein interactions or regulation of gene expression. Prominent Ca(2+) sensors like, Calmodulins (CaM), Calmodulin-like proteins (CMLs), calcium dependent protein kinases (CDPKs), Calcineurin B-like proteins (CBLs) and their interacting kinases (CIPKs) exist in complex gene families and form intricate signaling networks in plants that are capable of robust and flexible information processing. In this review we reflect on the recently gained knowledge about the mechanistic principles of these Ca(2+) sensors, their biochemical properties, physiological functions and newly identified targets proteins. These aspects will be discussed in the context of emerging functional principles that govern the information processing via these signaling modules., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Dynamic protein-protein interactions are essential in all cellular and developmental processes. Protein-fragment complementation assays allow such protein-protein interactions to be investigated in vivo. In contrast to other protein-fragment complementation assays, the split-luciferase (split-LUC) complementation approach facilitates dynamic and quantitative in vivo analysis of protein interactions, as the restoration of luciferase activity upon protein-protein interaction of investigated proteins is reversible. Here, we describe the development of a floated-leaf luciferase complementation imaging (FLuCI) assay that enables rapid and quantitative in vivo analyses of protein interactions in leaf discs floating on a luciferin infiltration solution after transient expression of split-LUC-labelled interacting proteins in Nicotiana benthamiana. We generated a set of eight Gateway-compatible split-LUC destination vectors, enabling fast, and almost fail-safe cloning of candidate proteins to the LUC termini in all possible constellations. We demonstrate their functionality by visualizing the well-established homodimerization of the 14-3-3 regulator proteins. Quantitative interaction analyses of the molybdenum co-factor biosynthesis proteins CNX6 and CNX7 show that the luciferase-based protein-fragment complementation assay allows direct real-time monitoring of absolute values of protein complex assembly. Furthermore, the split-LUC assay is established as valuable tool to investigate the dynamics of protein interactions by monitoring the disassembly of actin filaments in planta. The new Gateway-compatible split-LUC destination vector system, in combination with the FLuCI assay, provides a useful means to facilitate quantitative analyses of interactions between large numbers of proteins constituting interaction networks in plant cells., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

The plant-specific calcium binding protein CAS (calcium sensor) has been localized in chloroplast thylakoid membranes of vascular plants and green algae. To elucidate the function of CAS in Chlamydomonas reinhardtii, we generated and analyzed eight independent CAS knockdown C. reinhardtii lines (cas-kd). Upon transfer to high-light (HL) growth conditions, cas-kd lines were unable to properly induce the expression of LHCSR3 protein that is crucial for nonphotochemical quenching. Prolonged exposure to HL revealed a severe light sensitivity of cas-kd lines and caused diminished activity and recovery of photosystem II (PSII). Remarkably, the induction of LHCSR3, the growth of cas-kd lines under HL, and the performance of PSII were fully rescued by increasing the calcium concentration in the growth media. Moreover, perturbing cellular Ca(2+) homeostasis by application of the calmodulin antagonist W7 or the G-protein activator mastoparan impaired the induction of LHCSR3 expression in a concentration-dependent manner. Our findings demonstrate that CAS and Ca(2+) are critically involved in the regulation of the HL response and particularly in the control of LHCSR3 expression.

Potassium (K(+)) channel function is fundamental to many physiological processes. However, components and mechanisms regulating the activity of plant K(+) channels remain poorly understood. Here, we show that the calcium (Ca(2+)) sensor CBL4 together with the interacting protein kinase CIPK6 modulates the activity and plasma membrane (PM) targeting of the K(+) channel AKT2 from Arabidopsis thaliana by mediating translocation of AKT2 to the PM in plant cells and enhancing AKT2 activity in oocytes. Accordingly, akt2, cbl4 and cipk6 mutants share similar developmental and delayed flowering phenotypes. Moreover, the isolated regulatory C-terminal domain of CIPK6 is sufficient for mediating CBL4- and Ca(2+)-dependent channel translocation from the endoplasmic reticulum membrane to the PM by a novel targeting pathway that is dependent on dual lipid modifications of CBL4 by myristoylation and palmitoylation. Thus, we describe a critical mechanism of ion-channel regulation where a Ca(2+) sensor modulates K(+) channel activity by promoting a kinase interaction-dependent but phosphorylation-independent translocation of the channel to the PM.

Ribonuclease E (RNase E) represents a key enzyme in bacterial RNA metabolism. It plays multifarious roles in RNA processing and also initiates degradation of mRNA by endonucleolytic cleavage. Plastids (chloroplasts) are derived from formerly free-living bacteria and have largely retained eubacterial gene expression mechanisms. Here we report the functional characterization of a chloroplast RNase E that is encoded by a single-copy nuclear gene in the model plant Arabidopsis thaliana. Analysis of knockout plants revealed that, unlike in bacteria, RNase E is not essential for survival. Absence of RNase E results in multiple defects in chloroplast RNA metabolism. Most importantly, polycistronic precursor transcripts overaccumulate in the knockout plants, while several mature monocistronic mRNAs are strongly reduced, suggesting an important function of RNase E in intercistronic processing of primary transcripts from chloroplast operons. We further show that disturbed maturation of a transcript encoding essential ribosomal proteins results in plastid ribosome deficiency and, therefore, provides a molecular explanation for the observed mutant phenotype., (© 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.)

Fluorescent tagging of proteins and confocal imaging techniques have become methods of choice in analysing the distributions and dynamic characteristics of proteins at the subcellular level. In common use are a number of strategies for transient expression that greatly reduce the preparation time in advance of imaging, but their applications are limited in success outside a few tractable species and tissues. We previously developed a simple method to transiently express fluorescently-tagged proteins in Arabidopsis root epidermis and root hairs. We describe here a set of Gateway-compatable vectors with fluorescent tags incorporating the ubiqutin-10 gene promoter (P(UBQ10) ) of Arabidopsis that gives prolonged expression of the fluorescently-tagged proteins, both in tobacco and Arabidopsis tissues, after transient transformation, and is equally useful in generating stably transformed lines. As a proof of principle, we carried out transformations with fluorescent markers for the integral plasma membrane protein SYP121, a member of the SNARE family of vesicle-trafficking proteins, and for DHAR1, a cytosolic protein that facilitates the scavenging of reactive oxygen species. We also carried out transformations with SYP121 and its interacting partner, the KC1 K(+) channel, to demonstrate the utility of the methods in bimolecular fluorescence complementation (BiFC). Transient transformations of Arabidopsis using Agrobacterium co-cultivation methods yielded expression in all epidermal cells, including root hairs and guard cells. Comparative studies showed that the P(UBQ10) promoter gives similar levels of expression to that driven by the native SYP121 promoter, faithfully reproducing the characteristics of protein distributions at the subcellular level. Unlike the 35S-driven construct, expression under the P(UBQ10) promoter remained elevated for periods in excess of 2 weeks after transient transformation. This toolbox of vectors and fluorescent tags promises significant advantages for the study of membrane dynamics and cellular development, as well as events associated with environmental stimuli in guard cells and nutrient acquisition in roots., (© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.)

Ca(2+) signals are core transducers and regulators in many adaptation and developmental processes of plants. Ca(2+) signals are represented by stimulus-specific signatures that result from the concerted action of channels, pumps, and carriers that shape temporally and spatially defined Ca(2+) elevations. Cellular Ca(2+) signals are decoded and transmitted by a toolkit of Ca(2+) binding proteins that relay this information into downstream responses. Major transduction routes of Ca(2+) signaling involve Ca(2+)-regulated kinases mediating phosphorylation events that orchestrate downstream responses or comprise regulation of gene expression via Ca(2+)-regulated transcription factors and Ca(2+)-responsive promoter elements. Here, we review some of the remarkable progress that has been made in recent years, especially in identifying critical components functioning in Ca(2+) signal transduction, both at the single-cell and multicellular level. Despite impressive progress in our understanding of the processing of Ca(2+) signals during the past years, the elucidation of the exact mechanistic principles that underlie the specific recognition and conversion of the cellular Ca(2+) currency into defined changes in protein-protein interaction, protein phosphorylation, and gene expression and thereby establish the specificity in stimulus response coupling remain to be explored.

During adaptation and developmental processes cells respond through nonlinear calcium-decoding signaling cascades, the principal components of which have been identified. However, the molecular mechanisms generating specificity of cellular responses remain poorly understood. Calcineurin B-like (CBL) proteins contribute to decoding calcium signals by specifically interacting with a group of CBL-interacting protein kinases (CIPKs). Here, we report the subcellular localization of all 10 CBL proteins from Arabidopsis and provide a cellular localization matrix of a plant calcium signaling network. Our findings suggest that individual CBL proteins decode calcium signals not only at the plasma membrane and the tonoplast, but also in the cytoplasm and nucleus. We found that distinct targeting signals located in the N-terminal domain of CBL proteins determine the spatially discrete localization of CBL/CIPK complexes by COPII-independent targeting pathways. Our findings establish the CBL/CIPK signaling network as a calcium decoding system that enables the simultaneous specific information processing of calcium signals emanating from different intra- and extracellular stores, and thereby provides a mechanism underlying the specificity of cellular responses.

Ca(2+) signals are a core regulator of plant cell physiology and cellular responses to the environment. The channels, pumps, and carriers that underlie Ca(2+) homeostasis provide the mechanistic basis for generation of Ca(2+) signals by regulating movement of Ca(2+) ions between subcellular compartments and between the cell and its extracellular environment. The information encoded within the Ca(2+) transients is decoded and transmitted by a toolkit of Ca(2+)-binding proteins that regulate transcription via Ca(2+)-responsive promoter elements and that regulate protein phosphorylation. Ca(2+)-signaling networks have architectural structures comparable to scale-free networks and bow tie networks in computing, and these similarities help explain such properties of Ca(2+)-signaling networks as robustness, evolvability, and the ability to process multiple signals simultaneously.

Calcium serves as a versatile messenger in many adaptation and developmental processes in plants. Cellular calcium signals are detected and transmitted by calcium-binding proteins functioning as sensor molecules. The family of calcineurin B-like (CBL) proteins represents a unique group of calcium sensors and contributes to the decoding of calcium transients by interacting with and regulating the family of CBL-interacting protein kinases (CIPKs). In higher plants, CBL proteins and CIPKs form a complex signaling network that allows for flexible but specific signal-response coupling during environmental adaptation reactions. This review presents novel findings concerning the evolution of this signaling network and key insights into the physiological function of CBL-CIPK complexes. These aspects will be presented and discussed in the context of emerging functional principles governing efficient and specific information processing in this signaling system.

Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluorescence complementation (BiFC) is a suitable technique to investigate the formation of protein complexes and the localization of protein-protein interactions in planta. However, the generation of large plasmid collections to facilitate the exploration of complex interaction networks is often limited by the need for conventional cloning techniques. Here, we report the implementation of a GATEWAY vector system enabling large-scale combination and investigation of candidate proteins in BiFC studies. We describe a set of 12 GATEWAY-compatible BiFC vectors that efficiently permit the combination of candidate protein pairs with every possible N- or C-terminal sub-fragment of S(CFP)3A or Venus, respectively, and enable the performance of multicolor BiFC (mcBiFC). We used proteins of the plant molybdenum metabolism, in that more than 20 potentially interacting proteins are assumed to form the cellular molybdenum network, as a case study to establish the functionality of the new vectors. Using these vectors, we report the formation of the molybdopterin synthase complex by interaction of Arabidopsis proteins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous formation of Cnx6/Cnx6 and Cnx6/Cnx7 complexes revealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector systems should significantly facilitate the large-scale investigation of complex regulatory networks in plant cells.

Plant growth and development is driven by osmotic processes. Potassium represents the major osmotically active cation in plants cells. The uptake of this inorganic osmolyte from the soil in Arabidopsis involves a root K(+) uptake module consisting of the two K(+) channel alpha-subunits, AKT1 and AtKC1. AKT1-mediated potassium absorption from K(+)-depleted soil was shown to depend on the calcium-sensing proteins CBL1/9 and their interacting kinase CIPK23. Here we show that upon activation by the CBL.CIPK complex in low external potassium homomeric AKT1 channels open at voltages positive of E(K), a condition resulting in cellular K(+) leakage. Although at submillimolar external potassium an intrinsic K(+) sensor reduces AKT1 channel cord conductance, loss of cytosolic potassium is not completely abolished under these conditions. Depending on channel activity and the actual potassium gradients, this channel-mediated K(+) loss results in impaired plant growth in the atkc1 mutant. Incorporation of the AtKC1 subunit into the channel complex, however, modulates the properties of the K(+) uptake module to prevent K(+) loss. Upon assembly of AKT1 and AtKC1, the activation threshold of the root inward rectifier voltage gate is shifted negative by approximately -70 mV. Additionally, the channel conductance gains a hypersensitive K(+) dependence. Together, these two processes appear to represent a safety strategy preventing K(+) loss through the uptake channels under physiological conditions. Similar growth retardation phenotypes of akt1 and atkc1 loss-of-function mutants in response to limiting K(+) supply further support such functional interdependence of AKT1 and AtKC1. Taken together, these findings suggest an essential role of AtKC1-like subunits for root K(+) uptake and K(+) homeostasis when plants experience conditions of K(+) limitation.

Calcium serves as a critical messenger in many adaptation and developmental processes. Cellular calcium signals are detected and transmitted by sensor molecules such as calcium-binding proteins. In plants, the calcineurin B-like protein (CBL) family represents a unique group of calcium sensors and plays a key role in decoding calcium transients by specifically interacting with and regulating a family of protein kinases (CIPKs). Several CBL proteins appear to be targeted to the plasma membrane by means of dual lipid modification by myristoylation and S-acylation. In addition, CBL/CIPK complexes have been identified in other cellular localizations, suggesting that this network may confer spatial specificity in Ca2+ signaling. Molecular genetics analyses of loss-of function mutants have implicated several CBL proteins and CIPKs as important components of abiotic stress responses, hormone reactions and ion transport processes. The occurrence of CBL and CIPK proteins appears not to be restricted to the plant kingdom raising the question about the function of these Ca2+ decoding components in non-plant species.

The specificity of intracellular signaling and developmental patterning in biological systems relies on selective interactions between different proteins in specific cellular compartments. The identification of such protein-protein interactions is essential for unraveling complex signaling and regulatory networks. Recently, bimolecular fluorescence complementation (BiFC) has emerged as a powerful technique for the efficient detection of protein interactions in their native subcellular localization. Here we report significant technical advances in the methodology of plant BiFC. We describe a series of versatile BiFC vector sets that are fully compatible with previously generated vectors. The new vectors enable the generation of both C-terminal and N-terminal fusion proteins and carry optimized fluorescent protein genes that considerably improve the sensitivity of BiFC. Using these vectors, we describe a multicolor BiFC (mcBiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. Application to a protein interaction network acting in calcium-mediated signal transduction revealed the concurrent interaction of the protein kinase CIPK24 with the calcium sensors CBL1 and CBL10 at the plasma membrane and tonoplast, respectively. We have also visualized by mcBiFC the simultaneous formation of CBL1/CIPK1 and CBL9/CIPK1 protein complexes at the plasma membrane. Thus, mcBiFC provides a useful new tool for exploring complex regulatory networks in plants.

Arabidopsis thaliana calcineurin B-like proteins (CBLs) interact specifically with a group of CBL-interacting protein kinases (CIPKs). CBL/CIPK complexes phosphorylate target proteins at the plasma membrane. Here, we report that dual lipid modification is required for CBL1 function and for localization of this calcium sensor at the plasma membrane. First, myristoylation targets CBL1 to the endoplasmic reticulum. Second, S-acylation is crucial for endoplasmic reticulum-to-plasma membrane trafficking via a novel cellular targeting pathway that is insensitive to brefeldin A. We found that a 12-amino acid peptide of CBL1 is sufficient to mediate dual lipid modification and to confer plasma membrane targeting. Moreover, the lipid modification status of the calcium sensor moiety determines the cellular localization of preassembled CBL/CIPK complexes. Our findings demonstrate the importance of S-acylation for regulating the spatial accuracy of Ca2+-decoding proteins and suggest a novel mechanism that enables the functional specificity of calcium sensor/kinase complexes.

INTRODUCTIONBimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein-protein interactions in living cells. This method has been successfully adapted to a variety of expression systems in different organisms. BiFC is based on the formation of a fluorescent complex by fragments of the enhanced yellow fluorescent protein (eYFP) when brought together by the interaction of two associating proteins fused to these fragments. Interaction of these proteins restores fluorescence and allows the visualization of spatial localization patterns of protein complexes. Absence of interaction prevents reassembly of the fluorescent protein and results only in background fluorescence. The specificity of bimolecular fluorescence complementation must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. This protocol describes the Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana leaf cells. This method exhibits a high transformation rate (up to 90% of the cells) and allows the simultaneous expression of multiple proteins in single cells. Therefore, this expression system enables colocalization analyses of fluorescently labeled proteins with the formation of BiFC complexes for determination of cellular complex localization. In addition, protein interaction assays in N. benthamiana leaves permit the investigation of protein interactions at different time points of expression, allow analysis of proteins that are normally toxic in protoplasts, and enable comparative protein interaction investigation in epidermal cells as well as in mesophyll protoplasts.

* Guard cell movements are regulated by environmental cues including, for example, elevations in extracellular Ca(2+) concentration. Here, the subcellular localization and physiological function of the Ca(2+)-sensing receptor (CAS) protein was investigated. * CAS protein localization was ascertained by microscopic analyses of green fluorescent protein (GFP) fusion proteins and biochemical fractionation assays. Comparative guard cell movement investigations were performed in wild-type and cas loss-of-function mutant lines of Arabidopsis thaliana. Cytoplasmic Ca(2+) dynamics were addressed in plants expressing the yellow cameleon reporter protein YC3.6. * This study identified CAS as a chloroplast-localized protein that is crucial for proper stomatal regulation in response to elevations of external Ca(2+). CAS fulfils this role through modulation of the cytoplasmic Ca(2+) concentration. * This work reveals a novel role of the chloroplast in cellular Ca(2+) signal transduction.

Calcium serves as a critical messenger in many adaptation and developmental processes. Cellular calcium signals are detected and transmitted by sensor molecules such as calcium-binding proteins. In plants, the calcineurin B-like protein (CBL) family represents a unique group of calcium sensors and plays a key role in decoding calcium transients by specifically interacting with and regulating a family of protein kinases (CIPKs). We report here that the CBL protein CBL10 functions as a crucial regulator of salt tolerance in Arabidopsis. Cbl10 mutant plants exhibited significant growth defects and showed hypersensitive cell death in leaf tissues under high-salt conditions. Interestingly, the Na(+) content of the cbl10 mutant, unlike other salt-sensitive mutants identified thus far, was significantly lower than in the wild type under either normal or high-salt conditions, suggesting that CBL10 mediates a novel Ca(2+)-signaling pathway for salt tolerance. Indeed, the CBL10 protein physically interacts with the salt-tolerance factor CIPK24 (SOS2), and the CBL10-CIPK24 (SOS2) complex is associated with the vacuolar compartments that are responsible for salt storage and detoxification in plant cells. These findings suggest that CBL10 and CIPK24 (SOS2) constitute a novel salt-tolerance pathway that regulates the sequestration/compartmentalization of Na(+) in plant cells. Because CIPK24 (SOS2) also interacts with CBL4 (SOS3) and regulates salt export across the plasma membrane, our study identifies CIPK24 (SOS2) as a multi-functional protein kinase that regulates different aspects of salt tolerance by interacting with distinct CBL calcium sensors.

Calcium signalling involves sensor proteins that decode temporal and spatial changes in cellular Ca2+ concentration. Calcineurin B-like proteins (CBLs) represent a unique family of plant calcium sensors that relay signals by interacting with a family of protein kinases, designated as CBL-interacting protein kinases (CIPKs). In a reverse genetic screen for altered drought tolerance, we identified a loss-of-function allele of CIPK23 as exhibiting a drought-tolerant phenotype. In the cipk23 mutant, reduced transpirational water loss from leaves coincides with enhanced ABA sensitivity of guard cells during opening as well as closing reactions, without noticeable alterations in ABA content in the plant. We identified the calcium sensors CBL1 and CBL9 as CIPK23-interacting proteins that targeted CIPK23 to the plasma membrane in vivo. Expression analysis of the CIPK23, CBL1 and CBL9 genes suggested that they may function together in diverse tissues, including guard cells and root hairs. In addition, expression of the CIPK23 gene was induced by low-potassium conditions, implicating a function of this gene product in potassium nutrition. Indeed, cipk23 mutants displayed severe growth impairment on media with low concentrations of potassium. This phenotype correlates with a reduced efficiency of K+ uptake into the roots. In support of the conclusion that CBL1 and CBL9 interact with and synergistically serve as upstream regulators of CIPK23, the cbl1 cbl9 double mutant, but not the cbl1 or cbl9 single mutants, exhibit altered phenotypes for stomatal responses and low-potassium sensitivity. Together with the recent identification of the potassium channel AKT1 as a target of CIPK23, these results imply that plasma membrane-localized CBL1- and CBL9-CIPK23 complexes simultaneously regulate K+ transport processes in roots and in stomatal guard cells.

The tolerance responses of plants to many abiotic stresses are conjectured to be controlled by complex gene networks. In the frame of the AtGenExpress project a comprehensive Arabidopsis thaliana genome transcript expression study was performed using the Affymetrix ATH1 microarray in order to understand these regulatory networks in detail. In contrast to earlier studies, we subjected, side-by-side and in a high-resolution kinetic series, Arabidopsis plants, of identical genotype grown under identical conditions, to different environmental stresses comprising heat, cold, drought, salt, high osmolarity, UV-B light and wounding. Furthermore, the harvesting of tissue and RNA isolation were performed in parallel at the same location using identical experimental protocols. Here we describe the technical performance of the experiments. We also present a general overview of environmental abiotic stress-induced gene expression patterns and the results of a model bioinformatics analysis of gene expression in response to UV-B light, drought and cold stress. Our results suggest that the initial transcriptional stress reaction of Arabidopsis might comprise a set of core environmental stress response genes which, by adjustment of the energy balance, could have a crucial function in various stress responses. In addition, there are indications that systemic signals generated by the tissue exposed to stress play a major role in the coordination and execution of stress responses. In summary, the information reported provides a prime reference point and source for the subsequent exploitation of this important resource for research into plant abiotic stress.

Intracellular release of calcium ions belongs to the earliest events in cellular stress perception. The molecular mechanisms integrating signals from different environmental cues and translating them into an optimized response are largely unknown. We report here the functional characterization of CIPK1, a protein kinase interacting strongly with the calcium sensors CBL1 and CBL9. Comparison of the expression patterns indicates that the three proteins execute their functions in the same tissues. Physical interaction of CIPK1 with CBL1 and CBL9 targets the kinase to the plasma membrane. We show that, similarly to loss of CBL9 function, mutation of either CBL1 or CIPK1 renders plants hypersensitive to osmotic stress. Remarkably, in contrast to the cbl1 mutant and similarly to the cbl9 mutant, loss of CIPK1 function impairs abscisic acid (ABA) responsiveness. We therefore suggest that, by alternative complex formation with either CBL1 or CBL9, the kinase CIPK1 represents a convergence point for ABA-dependent and ABA-independent stress responses. Based on our genetic, physiological and protein-protein interaction data, we propose a general model for information processing in calcium-regulated signalling networks.

Group II introns are widespread in plant cell organelles. In vivo, most if not all plant group II introns do not self-splice but require the assistance of proteinaceous splicing factors. In some cases, a splicing factor (also referred to as maturase) is encoded within the intronic sequence and produced by translation of the (excised) intron RNA. However, most present-day group II introns in plant organellar genomes do not contain open reading frames (ORFs) for splicing factors, and their excision may depend on proteins encoded by other organellar introns or splicing factors encoded in the nuclear genome. Whether or not the ancestors of all of these noncoding organellar introns originally contained ORFs for maturases is currently unknown. Here we show that a noncoding intron in the mitochondrial cox2 gene of seed plants is likely to be derived from an ancestral reverse transcriptase/maturase-encoding form. We detected remnants of maturase and reverse transcriptase sequences in the 2.7 kb cox2 intron of Ginkgo biloba, the only living species of an ancient gymnosperm lineage, suggesting that the intron originally harbored a splicing factor. This finding supports the earlier proposed hypothesis that the ancient group II introns that invaded organellar genomes were autonomous genetic entities in that they encoded the factor(s) required for their own excision and mobility.

Potassium channel function is fundamental to many physiological processes in plants. In this issue of Cell, identify a protein complex in Arabidopsis consisting of a potassium channel, a protein kinase, and a calcium sensor. This study reveals that plants cope with limited soil potassium by coupling potassium channel activation to a cellular calcium signaling network.

Dynamic networks of protein-protein interactions regulate numerous cellular processes and determine the ability to respond appropriately to environmental stimuli. However, the investigation of protein complex formation in living plant cells by methods such as fluorescence resonance energy transfer has remained experimentally difficult, time consuming and requires sophisticated technical equipment. Here, we report the implementation of a bimolecular fluorescence complementation (BiFC) technique for visualization of protein-protein interactions in plant cells. This approach relies on the formation of a fluorescent complex by two non-fluorescent fragments of the yellow fluorescent protein brought together by association of interacting proteins fused to these fragments (Hu et al., 2002). To enable BiFC analyses in plant cells, we generated different complementary sets of expression vectors, which enable protein interaction studies in transiently or stably transformed cells. These vectors were used to investigate and visualize homodimerization of the basic leucine zipper (bZIP) transcription factor bZIP63 and the zinc finger protein lesion simulating disease 1 (LSD1) from Arabidopsis as well as the dimer formation of the tobacco 14-3-3 protein T14-3c. The interaction analyses of these model proteins established the feasibility of BiFC analyses for efficient visualization of structurally distinct proteins in different cellular compartments. Our investigations revealed a remarkable signal fluorescence intensity of interacting protein complexes as well as a high reproducibility and technical simplicity of the method in different plant systems. Consequently, the BiFC approach should significantly facilitate the visualization of the subcellular sites of protein interactions under conditions that closely reflect the normal physiological environment.

Hormones are important regulators of plant growth and development. In Arabidopsis, perception of the phytohormones ethylene and cytokinin is accomplished by a family of sensor histidine kinases including ethylene-resistant (ETR) 1 and cytokinin-response (CRE) 1. We identified the Arabidopsis response regulator 2 (ARR2) as a signalling component functioning downstream of ETR1 in ethylene signal transduction. Analyses of loss-of-function and ARR2-overexpressing lines as well as functional assays in protoplasts indicate an important role of ARR2 in mediating ethylene responses. Additional investigations indicate that an ETR1-initiated phosphorelay regulates the transcription factor activity of ARR2. This mechanism may create a novel signal transfer from endoplasmic reticulum-associated ETR1 to the nucleus for the regulation of ethylene-response genes. Furthermore, global expression profiling revealed a complex ARR2-involving two-component network that interferes with a multitude of different signalling pathways and thereby contributes to the highly integrated signal processing machinery in higher plants.

Calcium plays a pivotal role in plant responses to several stimuli, including pathogens, abiotic stresses, and hormones. However, the molecular mechanisms underlying calcium functions are poorly understood. It is hypothesized that calcium serves as second messenger and, in many cases, requires intracellular protein sensors to transduce the signal further downstream in the pathways. The calcineurin B-like proteins (CBLs) represent a unique family of calcium sensors in plant cells. Here, we report our analysis of the CBL9 member of this gene family. Expression of CBL9 was inducible by multiple stress signals and abscisic acid (ABA) in young seedlings. When CBL9 gene function was disrupted in Arabidopsis thaliana plants, the responses to ABA were drastically altered. The mutant plants became hypersensitive to ABA in the early developmental stages, including seed germination and post-germination seedling growth. In addition, seed germination in the mutant also showed increased sensitivity to inhibition by osmotic stress conditions produced by high concentrations of salt and mannitol. Further analyses indicated that increased stress sensitivity in the mutant may be a result of both ABA hypersensitivity and increased accumulation of ABA under the stress conditions. The cbl9 mutant plants showed enhanced expression of genes involved in ABA signaling, such as ABA-INSENSITIVE 4 and 5. This study has identified a calcium sensor as a common element in the ABA signaling and stress-induced ABA biosynthesis pathways.

Calcium signals mediate a multitude of plant responses to external stimuli and regulate a wide range of physiological processes. Calcium-binding proteins, like calcineurin B-like (CBL) proteins, represent important relays in plant calcium signaling. These proteins form a complex network with their target kinases being the CBL-interacting protein kinases (CIPKs). Here, we present a comparative genomics analysis of the full complement of CBLs and CIPKs in Arabidopsis and rice (Oryza sativa). We confirm the expression and transcript composition of the 10 CBLs and 25 CIPKs encoded in the Arabidopsis genome. Our identification of 10 CBLs and 30 CIPKs from rice indicates a similar complexity of this signaling network in both species. An analysis of the genomic evolution suggests that the extant number of gene family members largely results from segmental duplications. A phylogenetic comparison of protein sequences and intron positions indicates an early diversification of separate branches within both gene families. These branches may represent proteins with different functions. Protein interaction analyses and expression studies of closely related family members suggest that even recently duplicated representatives may fulfill different functions. This work provides a basis for a defined further functional dissection of this important plant-specific signaling system.

Calcium ions represent both an integrative signal and an important convergence point of many disparate signaling pathways. Calcium-binding proteins, like calcineurin B-like (CBL) proteins, have been implicated as important relays in calcium signaling. Here, we report the in vivo study of CBL1 function in Arabidopsis. Analyses of loss-of-function as well as CBL1-overexpressing lines indicate a crucial function of this calcium sensor protein in abiotic stress responses. Mutation of CBL1 impairs plant responses to drought and salt stresses and affects gene expression of cold-regulated genes, but does not affect abscisic acid (ABA) responsiveness. Conversely, overexpression of CBL1 reduces transpirational water loss and induces the expression of early stress-responsive transcription factors and stress adaptation genes in non-stressed plants. Together, our data indicate that the calcium sensor protein CBL1 may constitute an integrative node in plant responses to abiotic stimuli and contributes to the regulation of early stress-related transcription factors of the C-Repeat-Binding Factor/dehydration-responsive element (CBF/DREB) type.

The CDPK-SnRK superfamily consists of seven types of serine-threonine protein kinases: calcium-dependent protein kinase (CDPKs), CDPK-related kinases (CRKs), phosphoenolpyruvate carboxylase kinases (PPCKs), PEP carboxylase kinase-related kinases (PEPRKs), calmodulin-dependent protein kinases (CaMKs), calcium and calmodulin-dependent protein kinases (CCaMKs), and SnRKs. Within this superfamily, individual isoforms and subfamilies contain distinct regulatory domains, subcellular targeting information, and substrate specificities. Our analysis of the Arabidopsis genome identified 34 CDPKs, eight CRKs, two PPCKs, two PEPRKs, and 38 SnRKs. No definitive examples were found for a CCaMK similar to those previously identified in lily (Lilium longiflorum) and tobacco (Nicotiana tabacum) or for a CaMK similar to those in animals or yeast. CDPKs are present in plants and a specific subgroup of protists, but CRKs, PPCKs, PEPRKs, and two of the SnRK subgroups have been found only in plants. CDPKs and at least one SnRK have been implicated in decoding calcium signals in Arabidopsis. Analysis of intron placements supports the hypothesis that CDPKs, CRKs, PPCKs and PEPRKs have a common evolutionary origin; however there are no conserved intron positions between these kinases and the SnRK subgroup. CDPKs and SnRKs are found on all five Arabidopsis chromosomes. The presence of closely related kinases in regions of the genome known to have arisen by genome duplication indicates that these kinases probably arose by divergence from common ancestors. The PlantsP database provides a resource of continuously updated information on protein kinases from Arabidopsis and other plants.

The exoribonuclease polynucleotide phosphorylase (PNPase) has been implicated in mRNA processing and degradation in bacteria as well as in chloroplasts of higher plants. Here, we report the first comprehensive in vivo study of chloroplast PNPase function. Modulation of PNPase activity in Arabidopsis chloroplasts by a reverse genetic approach revealed that, although this enzyme is essential for efficient 3'-end processing of mRNAs, it is insufficient to mediate transcript degradation. Surprisingly, we identified PNPase as also being indispensable for 3'-end maturation of 23S rRNA transcripts. Analysis of tRNA amounts in transgenic Arabidopsis plants suggests a direct correlation of PNPase activity and tRNA levels, indicating an additional function of this exoribo nuclease in tRNA decay. Moreover, the extent of polyadenylated mRNAs in chloroplasts is negatively correlated with PNPase activity. Together, our results attribute novel functions to PNPase in the metabolism of all major classes of plastid RNAs and suggest an unexpectedly complex role for PNPase in RNA processing and decay.

The Arabidopsis thaliana response regulator 4, expressed in response to phytochrome B action, specifically interacts with the extreme amino-terminus of the photoreceptor. The response regulator 4 stabilizes the active Pfr form of phytochrome B in yeast and in planta, thus elevates the level of the active photoreceptor in vivo. Accordingly, transgenic Arabidopsis plants overexpressing the response regulator 4 display hypersensitivity to red light but not to light of other wavelengths. We propose that the response regulator 4 acts as an output element of a two-component system that modulates red light signaling on the level of the phytochrome B photoreceptor.

The Arabidopsis calcineurin B-like calcium sensor proteins (AtCBLs) interact with a group of serine-threonine protein kinases (AtCIPKs) in a calcium-dependent manner. Here we identify a 24 amino acid domain (NAF domain) unique to these kinases as being required and sufficient for interaction with all known AtCBLs. Mutation of conserved residues either abolished or significantly diminished the affinity of AtCIPK1 for AtCBL2. Comprehensive two-hybrid screens with various AtCBLs identified 15 CIPKs as potential targets of CBL proteins. Database analyses revealed additional kinases from Arabidopsis and other plant species harbouring the NAF interaction module. Several of these kinases have been implicated in various signalling pathways mediating responses to stress, hormones and environmental cues. Full-length CIPKs show preferential interaction with distinct CBLs in yeast and in vitro assays. Our findings suggest differential interaction affinity as one of the mechanisms generating the temporal and spatial specificity of calcium signals within plant cells and that different combinations of CBL-CIPK proteins contribute to the complex network that connects various extracellular signals to defined cellular responses.

Two-component signal systems regulate a variety of cellular activities. They involve at least two common signalling molecules: a signal-sensing kinase and a response regulator that mediates the output response. Multistep systems also require proteins containing phosphotransfer domains. Here we report that the response regulator ARR2 from Arabidopsis is predominantly expressed in pollen and is localized in the nuclear compartment of the plant cell. Furthermore, ARR2 is transcriptionally active in yeast and binds to the promoters of nuclear genes for several components of mitochondrial respiratory chain complex I (nCI) from Arabidopsis. The nuclear nCI genes are up-regulated in pollen during spermatogenesis. The transcription factor functions of ARR2 are mediated by its C-terminal output domain. Our data identify ARR2 as the first eukaryotic response regulator which functions as a transcription factor at a known promoter sequence. Yeast two-hybrid analysis and in vitro interaction studies suggest that ARR2 very probably forms part of a multistep two-component signalling mechanism that includes HPt proteins like AHP1 or AHP2. These findings point to an as yet unidentified signal transduction system that may regulate aspects of floral and mitochondrial gene expression.

Members of the Arabidopsis calcineurin B-like Ca(2)+ binding protein (AtCBL) family are differentially regulated by stress conditions. One AtCBL plays a role in salt stress; another is implicated in response to other stress signals, including drought, cold, and wounding. In this study, we identified a group of novel protein kinases specifically associated with AtCBL-type Ca(2)+ sensors. In addition to a typical protein kinase domain, they all contain a unique C-terminal region that is both required and sufficient for interaction with the AtCBL-type but not calmodulin-type Ca(2)+ binding proteins from plants. Interactions between the kinases and AtCBLs require micromolar concentrations of Ca(2)+, suggesting that increases in cellular Ca(2)+ concentrations may trigger the formation of AtCBL-kinase complexes in vivo. Unlike most serine/threonine kinases, the AtCBL-interacting kinase efficiently uses Mn(2)+ to Mg(2)+ as a cofactor and may function as a Mn(2)+ binding protein in the cell. These findings link a new type of Ca(2)+ sensors to a group of novel protein kinases, providing the molecular basis for a unique Ca(2)+ signaling machinery in plant cells.

Chloroplast development involves changes in the stability of specific plastid mRNAs. To understand how the half-lives of these mRNAs are modified, several laboratories are investigating how plastid mRNAs are degraded. This has led to the isolation of a high-molecular-weight complex that contains an endoribonuclease and a 3'-5' exoribonuclease, and the discovery that efficient mRNA degradation requires polyadenylation. These findings are similar to recent discoveries in Escherichia coli. However, an important difference between the two systems is that chloroplast mRNA degradation involves nuclear-encoded proteins. Modification of these proteins could provide the mechanism for altering plastid-mRNA half-lives in response to developmental stimuli.

An important effector of Ca2+ signaling in animals and yeast is the Ca2+/calmodulin-dependent protein phosphatase calcineurin. However, the biochemical identity of plant calcineurin remained elusive. Here we report the molecular characterization of AtCBL (Arabidopsis thaliana calcineurin B-like protein) from Arabidopsis. The protein is most similar to mammalian calcineurin B, the regulatory subunit of the phosphatase. AtCBL also shows significant similarity with another Ca2+-binding protein, the neuronal calcium sensor in animals. It contains typical EF-hand motifs with Ca2+-binding capability, as confirmed by in vitro Ca2+-binding assays, and it interacts in vivo with rat calcineurin A in the yeast two-hybrid system. Interaction of AtCBL1 and rat calcineurin A complemented the salt-sensitive phenotype in a yeast calcineurin B mutant. Cloning of cDNAs revealed that AtCBL proteins are encoded by a family of at least six genes in Arabidopsis. Genes for three isoforms were identified in this study. AtCBL1 mRNA was preferentially expressed in stems and roots and its mRNA levels strongly increased in response to specific stress signals such as drought, cold, and wounding. In contrast, AtCBL2 and AtCBL3 are constitutively expressed under all conditions investigated. Our data suggest that AtCBL1 may act as a regulatory subunit of a plant calcineurin-like activity mediating calcium signaling under certain stress conditions.

Immuonosuppressive drugs FK506 and rapamycin block a number of signal transduction pathways in eukaryotic systems. The 12 kDa FK506 binding protein (FKBP12) mediates the action of both FK506 and rapamycin against their functional targets. In this report, we cloned, sequenced and characterized a gene encoding FKBP12 in Vicia faba (VfFKBP12). While VfFKBP12 is highly homologous to animal and yeast FKBP12, it does not mediate the action of FK506 and rapamycin. There are unique features in plant FKBP12 sequences that cause the variation in their function. One lies in the domain that is critical for interaction with calcineurin (CaN), the mammalian and yeast target of FKBP12-FK506 complex. Protein-protein interaction assays revealed a low-affinity and unstable VfFKBP12-FK506-CaN ternary complex. In the genetic assay, VfFKBP12 did not restore the sensitivity of yeast FKBP12 mutant to rapamycin or FK506, supporting that plant FKBP12-ligand complexes are unable to block the function of the drug target. Also unique to plant FKBP12 proteins, a pair of cysteines is spatially adjacent to potentially form disulfide linkage. Treatment of VfFKBP12 with reductant dithiothreitol (DTT) abolished the formation of VfFKBP12-FK506-CaN ternary complex. Site-directed mutagenesis to substitute one of the cysteines, Cys26, with Ser produced a similar effect as DTT treatment. These results indicate that an intramolecular disulfide bond is a novel structural feature required for the low-affinity interaction between plant FKBP12 and CaN. In conclusion, plant FKBP12 proteins have evolved structural changes that modify their protein-protein interacting domains and cause loss of function against the drug targets.

Monomers of a major family of tandemly repeated DNA sequences of Antirrhinum majus have been cloned and characterized. The repeats are 163-167 bp long, contain on average 60% A+T residues, and are organized in head-to-tail orientation. According to site-specific methylation differences two subsets of repeating units can be distinguished. Fluorescent in situ hybridization revealed that the repeats are localized at centromeric regions of six of the eight chromosome pairs of A. majus with substantial differences in array size. The monomeric unit shows no homologies to other plant satellite DNAs. The repeat exists in a similar copy number and conserved size in the genomes of six European species of the genus Antirrhinum. Tandemly repeated DNA sequences with homology to the cloned monomer were also found in the North American section Saerorhinum, indicating that this satellite DNA might be of ancient origin and was probably already present in the ancestral genome of both sections.

The psbE operon of spinach chloroplasts, which includes the genes psbE, psbF, psbL and psbJ, encodes two RNA editing sites. One site corresponds to the initiation codon of the psbL transcript, as has been described earlier for the homologous transcript from tobacco, while at a second editing site, newly reported here, an internal phenylalanine codon of the psbF transcript is restored. Both these sites were investigated with respect to the extent of editing in spinach plastids at various developmental stages. The apparent existence of only completely edited transcripts in etioplasts and chloroplasts, indicates that light-induced processes are not acting as determinants in eliciting the editing process. Reduced editing is, however, observed in the psbF and psbL transcript from seeds and roots. This finding suggests that the RNA editing process is differentially down-regulated in leucoplasts and proplastids and that editing may, therefore, function as a regulatory device in plastid gene expression.