The battle of two ions: Ca2+ signalling against Na+ stress.

Soil salinity adversely affects plant growth, crop yield and the composition of ecosystems. Salinity stress impacts plants by combined effects of Na+ toxicity and osmotic perturbation. Plants have evolved elaborate mechanisms to counteract the detrimental consequences of salinity. Here we reflect on recent advances in our understanding of plant salt tolerance mechanisms. We discuss the embedding of the salt tolerance-mediating SOS pathway in plant hormonal and developmental adaptation. Moreover, we review newly accumulating evidence indicating a crucial role of a transpiration-dependent salinity tolerance pathway, that is centred around the function of the NADPH oxidase RBOHF and its role in endodermal and Casparian strip differentiation. Together, these data suggest a unifying and coordinating role for Ca2+ signalling in combating salinity stress at the cellular and organismal level.

Calcium Signaling during Salt Stress and in the Regulation of Ion Homeostasis.

Soil composition largely defines the living conditions of plants and represents one of their most relevant, dynamic and complex environmental cues. The effective concentrations of many either tolerated or essential ions and compounds in the soil usually differ from the optimum that would be most suitable for plants. In this regard, salinity - caused by excess of NaCl - represents a widespread adverse growth condition but also shortage of ions like K+, NO3- and Fe2+ restrains plant growth. During the past years many components and mechanisms that function in the sensing and establishment of ion homeostasis have been identified and characterized. Here, we reflect on recent insights that extended our understanding of components and mechanisms, which govern and fine-tune plant salt stress tolerance and ion homeostasis. We put special emphasis on mechanisms that allow for interconnection of the salt overly sensitivity pathway with plant development and discuss newly emerging functions of Ca2+ signaling in salinity tolerance. Moreover, we review and discuss accumulating evidence for a central and unifying role of Ca2+ signaling and Ca2+ dependent protein phosphorylation in regulating sensing, uptake, transport and storage processes of various ions. Finally, based on this cross-field inventory, we deduce emerging concepts and arising questions for future research.

Loss of the mitochondrial cox2 intron 1 in a family of monocotyledonous plants and utilization of mitochondrial intron sequences for the construction of a nuclear intron.

The intron content of plant organellar genes is a useful marker in molecular systematics and evolution. We have tested representatives of a wide range of monocotyledonous plant families for the presence of an intron (cox2 intron 1) in one of the most conservative mitochondrial genes, the cox2 locus. Almost all species analyzed were found to harbor a group II intron at a phylogenetically conserved position. The only exceptions were members of a single monocot family, the Ruscaceae: representatives of all genera in this family were found to lack cox2 intron 1, but instead harbor an intron in the 3' portion of the cox2 coding region (cox2 intron 2). The presence of cox2 intron 1 in families of monocotyledonous plants that are closely related to the Ruscaceae suggests that loss of the intron is specific to this family and may have accompanied the evolutionary appearance of the Ruscaceae. Interestingly, sequences that are highly homologous to cox2 intron 2 are found in a nuclear intron in a lineage of monocotyledonous plants, suggesting that the originally mitochondrial group II intron sequence was transferred to the nuclear genome and reused there to build a spliceosomal intron.

Interaction of the response regulator ARR4 with phytochrome B in modulating red light signaling.

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.

Molecular characterization of At5PTase1, an inositol phosphatase capable of terminating inositol trisphosphate signaling.

The inositol triphosphate (IP(3))-signaling pathway has been associated with several developmental and physiological processes in plants, but we currently know little about the regulation of this pathway. Inositol 5' phosphatases (5PTases) are enzymes that remove a 5' phosphate from several potential second messengers, including IP(3). In catalyzing the removal of a 5' phosphate from second messenger substrates, 5PTases can act to terminate signal transduction events. We describe the molecular analysis of At5PTase1, a 5PTase gene from Arabidopsis. When expressed transiently in Arabidopsis leaf tissue or ectopically in transgenic plants, At5PTase1 allowed for the increased hydrolysis of I(1,4,5)P(3) and I(1,3,4,5)P(4) substrates. At5PTase1 did not hydrolyze I(1)P, I(1,4)P(2), or PI(4,5)P(2) substrates. This substrate specificity was similar to that of the human Type I 5PTase. We identified 14 other potential At5PTase genes and constructed an unrooted phylogenetic tree containing putative Arabidopsis, human, and yeast 5PTase proteins. This analysis indicated that the Arabidopsis 5PTases were grouped in two separate branches of the tree. The multiplicity of At5PTases indicates that these enzymes may have different substrate specificities and play different roles in signal termination in Arabidopsis.

The response regulator ARR2: a pollen-specific transcription factor involved in the expression of nuclear genes for components of mitochondrial complex I in Arabidopsis.

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.

The NAF domain defines a novel protein-protein interaction module conserved in Ca2+-regulated kinases.

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.

Novel protein kinases associated with calcineurin B-like calcium sensors in Arabidopsis.

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.

RNA editing in an untranslated region of the Ginkgo chloroplast genome.

mRNAs in plant cell organelles can be subject to RNA editing, an RNA processing step altering the identity of single nucleotide residues. In higher plant chloroplasts, editing proceeds by C-to-U conversions at highly specific sites. All known plastid RNA editing sites are located in protein-coding regions and, typically, change the coding properties of the mRNA. To gain more insight into the evolution of editing, we have determined the molecular structure and RNA editing pattern of the psbE operon of the primitive seed plant Ginkgo biloba. We report here the identification of altogether four sites of C-to-U editing, two of which are unique to Ginkgo and have not been found in other species. Surprisingly, one of the sites is located in an intercistronic spacer, thus being the first chloroplast editing site detected outside a protein-coding region. This indicates that the plastid editing machinery can operate also in untranslated regions and without having apparent functional consequences.

Degrading chloroplast mRNA: the role of polyadenylation.

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.

Genes for calcineurin B-like proteins in Arabidopsis are differentially regulated by stress signals.

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.

The cox2 locus of the primitive angiosperm plant Acorus calamus: molecular structure, transcript processing and RNA editing.

Acorus calamus, or sweet flag, is a semiaquatic plant of uncertain taxonomic position. Molecular phylogenetic analysis using plastid rbcL sequences have suggested that Acorus calamus might be the most ancient surviving representative of the ancestral monocotyledonous plants. In order to provide molecular and phylogenetic data for the mitochondrial genetic system of Acorus, we have determined the structure of a mitochondrial locus, the cytochrome oxidase subunit II gene cox2. The Acorus cox2 gene harbors an unusually small group II intron, the smallest plant mitochondrial intron known to date. The transcript undergoes C-to-U RNA editing at eight sites. One of these sites is likely to play a dual functional role in both intron splicing and protein function. The 3' end of the mature transcript folds into a characteristic stem-loop structure that is presumably required for mitochondrial mRNA stability. Phylogenetic analysis of the cox2 sequence data, as well as the unusual intron structure, all support an evolutionarily isolated position for Acorus calamus.

Molecular characterization of a plant FKBP12 that does not mediate action of FK506 and rapamycin.

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.

Polyadenylation accelerates degradation of chloroplast mRNA.

The expression of chloroplast genes is regulated by several mechanisms, one of which is the modulation of RNA stability. To understand how this regulatory step is controlled during chloroplast development, we have begun to define the mechanism of plastid mRNA degradation. We show here that the degradation petD mRNA involves endonucleolytic cleavage at specific sites upstream of the 3' stem-loop structure. The endonucleolytic petD cleavage products can be polyadenylated in vitro, and similar polyadenylated RNA products are detectable in vivo. PCR analysis of the psbA and psaA-psaB-rps14 operons revealed other polyadenylated endonucleolytic cleavage products, indicating that poly(A) addition appears to be an integral modification during chloroplast mRNA degradation. Polyadenylation promotes efficient degradation of the cleaved petD RNAs by a 3'-5' exoribonuclease. Furthermore, polyadenylation also plays an important role in the degradation of the petD mRNA 3' end. Although the 3' end stem-loop is usually resistant to nucleases, adenylation renders the secondary structure susceptible to the 3'-5' exoribonuclease. Analysis of 3' ends confirms that polyadenylation occurs in vivo, and reveals that the extent of adenylation increases during the degradation of plastid mRNA in the dark. Based on these results, we propose a novel mechanism for polyadenylation in the regulation of plastid mRNA degradation.

Molecular characterization of a FKBP-type immunophilin from higher plants.

Immunophilins are intracellular receptors for the immunosuppressants cyclosporin A, FK506, and rapamycin. In addition to their use in organ transplantation, these natural products have been used to investigate signaling pathways in yeast, plant, and mammalian cells. We have recently described the identification of an immunosuppressant-sensitive signaling pathway in and the purification of several immunophilins from Vicia faba plants. We now report the molecular characterization of a 15 kDa FK506- and rapamycin-binding protein from V. faba (VfFKBP15). The amino acid sequence deduced from the cDNA starts with a signal peptide of 22 hydrophobic amino acids. The core region of VfFKBP15 is most similar to yeast and mammalian FKBP13 localized in the endoplasmic reticulum (ER). In addition, VfFKBP15 has a carboxyl-terminal sequence that is ended with SSEL, a putative ER retention signal. These findings suggest that VfFKBP15 is a functional homolog of FKBP13 from other organisms. Interestingly, two distinct cDNAs corresponding to two isoforms of FKBP15 have been cloned from Arabidopsis and also identified from rice data base, suggesting that pFKBP15 (plant FKBP15) is encoded by a small gene family in plants. This adds to the diversity of plant FKBP members even with the same subcellular localization and is in contrast with the situation in mammalian and yeast systems in which only one FKBP13 gene has been found. Like the mammalian and yeast FKBP13, the recombinant VfFKBP15 protein has rotamase activity that is inhibited by both FK506 and rapamycin with a Ki value of 30 nM and 0.9 nM, respectively, illustrating that VfFKBP15 binds rapamycin in preference over FK506. The mRNA of VfFKBP15 is ubiquitously expressed in various plant tissues including leaves, stems, and roots, consistent with the ER localization of the protein. Levels of VfFKBP15 mRNA are elevated by heat shock, suggesting a possible role for this FKBP member under stress conditions.

The molecular structure, chromosomal organization, and interspecies distribution of a family of tandemly repeated DNA sequences of Antirrhinum majus L.

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.

Chloroplast mRNA 3'-end processing by a high molecular weight protein complex is regulated by nuclear encoded RNA binding proteins.

In the absence of efficient transcription termination correct 3'-end processing is an essential step in the synthesis of stable chloroplast mRNAs in higher plants. We show here that 3'-end processing in vitro involves endonucleolytic cleavage downstream from the mature terminus, followed by exonucleolytic processing to a stem-loop within the 3'-untranslated region. These processing steps require a high molecular weight complex that contains both endoribonucleases and an exoribonuclease. In the presence of ancillary RNA binding proteins the complex correctly processes the 3'-end of precursor RNA. In the absence of these ancillary proteins 3'-end maturation is prevented and plastid mRNAs are degraded. Based on these results we propose a novel mechanism for the regulation of mRNA 3'-end processing and stability in chloroplasts.

Tissue- and stage-specific modulation of RNA editing of the psbF and psbL transcript from spinach plastids--a new regulatory mechanism?

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.

RNA editing in tobacco chloroplasts leads to the formation of a translatable psbL mRNA by a C to U substitution within the initiation codon.

The psbL gene which codes for a 38 amino acid peptide of photosystem II, together with the photosynthetic genes psbE and psbF, is contained in a conserved position of many species of higher plant plastomes. The alignment of the psbL nucleotide sequences from ten species shows strong conservation, which is indicative of a functional gene. The tobacco and spinach psbL genes have, however, an ACG codon instead of the initiator ATG codon observed in the homologous position of the other eight species. Evidence is presented that in tobacco chloroplasts a translatable psbL mRNA containing an AUG initiator codon is formed by a C to U editing of the ACG codon. This observation, following the previously reported editing of an rpl2 gene in maize chloroplasts, underlines a more widespread occurrence of this type of posttranscriptional mRNA modification and demonstrates its presence in a dicotyledon plant.