[Show abstract][Hide abstract] ABSTRACT: Environmental stresses profoundly altered accumulation of nonsense mRNAs including intron retaining (IR) transcripts in Arabidopsis. Temporal patterns of stress-induced IR mRNAs were dissected using both oscillating and non-oscillating transcripts. Broad range thermal cycles triggered a sharp increase in the long intron retaining CCA1 isoforms and altered their phasing to different times of day. Both abiotic and biotic stresses such as drought or P. syringae infection induced similar increase. Thermal stress induced a time delay in accumulation of CCA1 I4Rb transcripts whereas functional mRNA showed steady oscillations. Our data favor a hypothesis that stress-induced instabilities of the central oscillator can be in part compensated through fluctuations in abundance and out of phase oscillations of CCA1 IR transcripts. Altogether, our results support a concept that mRNA abundance can be modulated through altering ratios between functional and nonsense/IR transcripts. SR45 protein specifically bound to the retained CCA1 intron in vitro, suggesting that this splicing factor could be involved in regulation of intron retention. Transcriptomes of NMD-impaired and heat-stressed plants shared a set of retained introns associated with stress- and defense-inducible transcripts. Constitutive activation of certain stress response networks in an NMD mutant could be linked to disequilibrium between functional and nonsense mRNAs.
[Show abstract][Hide abstract] ABSTRACT: The Arabidopsis Ca(2+)/calmodulin-binding transcription factor SIGNAL RESPONSIVE1 (AtSR1/CAMTA3) was previously identified as a key negative regulator of plant immune responses (Galon et al., 2008, Du et al., 2009). Here, we report a new role for AtSR1 as a critical component of plant defense against insect herbivory. Loss of AtSR1 function impairs tolerance to feeding by the generalist herbivore Trichoplusia ni as well as wound-induced jasmonate (JA) accumulation. The susceptibility of the atsr1 mutant is associated with decreased total glucosinolate (GS) levels. The two key herbivory deterrents, indol-3-ylmethyl (I3M) and 4-methylsulfinylbutyl (4MSOB), showed the most significant reductions in atsr1 plants. Further, changes in AtSR1 transcript levels led to altered expression of several genes involved in GS metabolism including IQD1, MYB51, and AtST5a. Overall, our results establish AtSR1 as an important component of plant resistance to insect herbivory as well as one of only three described proteins involved in Ca(2+)/calmodulin(CaM)-dependent signaling to function in the regulation of GS metabolism, providing a novel avenue for future investigations of plant-insect interactions.
[Show abstract][Hide abstract] ABSTRACT: Calcium signaling depends on proteins at three nodes: generation of Ca2+ signature, sensing of changes in cellular Ca2+ level, and transduction of a Ca2+ signal. Plant cells have multiple mechanisms for generating increases in [Ca2+]cyt suggesting the capacity to produce complex spatiotemporal patterns of [Ca2+] cyt elevation. A large number of Ca2+ sensors have been identified experimentally or have been predicted on the basis of sequence similarity to known Ca2+-binding proteins or the presence of Ca2+-binding domains. The number of target proteins is expected to be large, as a given sensor can interact with multiple proteins.
Proteins involved in Ca2+signaling in plants have been identified using Ca2+-binding, protein–protein interaction, yeast two-hybrid, and coprecipitation screens. With the completion of genomic sequencing
of several plants, researchers have identified many Ca2+ sensors and target proteins on a global scale. In Arabidopsis, about 3–4% of the proteome appears to participate in Ca2+ signaling. The challenge now is the elucidation of the function of each verified/predicted protein involved in Ca2+ signaling on a local and global scale.
[Show abstract][Hide abstract] ABSTRACT: Ca(2+), a universal messenger in eukaryotes, plays a major role in signaling pathways that control many growth and developmental processes in plants as well as their responses to various biotic and abiotic stresses. Cellular changes in Ca(2+) in response to diverse signals are recognized by protein sensors that either have their activity modulated or that interact with other proteins and modulate their activity. Calmodulins (CaMs) and CaM-like proteins (CMLs) are Ca(2+) sensors that have no enzymatic activity of their own but upon binding Ca(2+) interact and modulate the activity of other proteins involved in a large number of plant processes. Protein-protein interactions play a key role in Ca(2+)/CaM-mediated in signaling pathways. In this review, using CaM as an example, we discuss various experimental approaches and computational tools to identify protein-protein interactions. During the last two decades hundreds of CaM-binding proteins in plants have been identified using a variety of approaches ranging from simple screening of expression libraries with labeled CaM to high-throughput screens using protein chips. However, the high-throughput methods have not been applied to the entire proteome of any plant system. Nevertheless, the data provided by these screens allows the development of computational tools to predict CaM-interacting proteins. Using all known binding sites of CaM, we developed a computational method that predicted over 700 high confidence CaM interactors in the Arabidopsis proteome. Most (>600) of these are not known to bind calmodulin, suggesting that there are likely many more CaM targets than previously known. Functional analyses of some of the experimentally identified Ca(2+) sensor target proteins have uncovered their precise role in Ca(2+)-mediated processes. Further studies on identifying novel targets of CaM and CMLs and generating their interaction network - "calcium sensor interactome" - will help us in understanding how Ca(2+) regulates a myriad of cellular and physiological processes.
[Show abstract][Hide abstract] ABSTRACT: Calmodulin (CaM) is a calcium-binding protein that is involved in a variety of cellular processes, interacting with many proteins. Since many CaM interactions are calcium-dependent, they are difficult to detect using high-throughput methods like yeast-two-hybrid. Furthermore, detection of CaM binding sites requires a significant experimental effort. Using a collection of CaM binding sites extracted from the Calmodulin Target Database we trained SVM-based classifiers to detect CaM binding sites using a variety of sequence features; our best classifier achieved an area under the ROC curve of 0.89 for detecting binding site locations at the amino acid level. We apply our classifiers to the problem of detecting CaM binding proteins in Arabidopsis; at a false-positive level of 0.05 we detected 638 novel putative CaM binding proteins. These proteins share overrepresented Gene Ontology terms associated with the functions of known CaM binders.
[Show abstract][Hide abstract] ABSTRACT: Kinesins and dyneins are two superfamilies of microtubule motor proteins that regulate many diverse fundamental cellular and
developmental processes including cell shape, cell division and intracellular transport as well as spatial and temporal organization
of molecules and organelles within the eukaryotic cells. These motor proteins use chemical energy from ATP to move unidirectionally
using microtubules as roadways or to regulate microtubule dynamics. This review focuses on a comparative analysis of kinesins
in eukaryotes, especially in the green lineage and their roles in plants. Comprehensive comparative analysis of kinesins among
completed genome sequences of animal and several photosynthetic eukaryotes ranging from algae to monocots revealed considerable
expansion of kinesins in flowering plants. Much of this expansion is due to an increase in members of two families (Kinesin-7
and Kinesin-14). Of the fourteen recognized families of kinesins in eukaryotes, members of four families are not found in
flowering plants. However, a group of plant-specific kinesins does not fall into any of the recognized families, and some
plant kinesins form plant-specific clades inside of their respective families. Some known domains are found exclusively either
in plant and animal lineages, suggesting their functional specialization. Arabidopsis has the highest number of kinesins of
any known multicellular eukaryotes, including humans, with a total of 61 kinesins. Although the processes regulated by many
plant kinesins are yet to be discovered, functions of some kinesins have been elucidated in recent years using cell biological,
molecular and genetic approaches and these are discussed briefly here. In addition, insights into regulatory mechanisms of
a unique plant Ca2+/CaM-interacting motor called kinesin-like calmodulin-binding protein (KCBP) obtained through biochemical assays and crystal
structure studies of its motor domain alone and as a complex with a calcium-binding protein are presented.
[Show abstract][Hide abstract] ABSTRACT: Recently we reported that CRLK1, a novel calcium/calmodulin-regulated receptor-like kinase plays an important role in regulating plant cold tolerance. Calcium/calmodulin binds to CRLK1 and upregulates its activity. Gene knockout and complementation studies revealed that CRLK1 is a positive regulator of plant response to chilling and freezing temperatures. Here we show that MEKK1, a member of MAP kinase kinase kinase family, interacts with CRLK1 both in vitro and in planta. The cold triggered MAP kinase activation in wild-type plants was abolished in crlk1 knockout mutants. Similarly, the cold induced expression levels of genes involved in MAP kinase signaling are also altered in crlk1 mutants. These results suggest that calcium/calmodulin-regulated CRLK1 modulates cold acclimation through MAP kinase cascade in plants.
[Show abstract][Hide abstract] ABSTRACT: Referee: Dr. Gabor Lazar, Chief, Dept. of Molecular Biology, Massachsetts General Hospital, Harvard Medical School, Wellman Eleven, Boston, MA 02114 The coding regions of about 80% of plant nuclear genes contain one or more noncoding intervening sequences (introns). The transcription of these genes results in a precursor mRNA (pre-mRNA) with coding sequences (exons) and introns. The noncoding intervening sequences are then accurately removed and the coding regions are joined in the nucleus to generate functional mRNAs by a process called pre-mRNA splicing. In addition to basic/constitutive splicing, many plant pre-mRNAs, like metazoan pre-mRNAs, undergo alternative splicing, thereby contributing to proteomic complexity. The splicing of pre-mRNAs takes place in a large RNA-protein complex named the spliceosome, which is made up of several small nuclear ribonucleoprotein (snRNP) particles and other associated proteins. Until recently, it was thought that there is only one type of spliceosome in all eukaryotes. However, it is now established that most metazoans have a second (minor) type of spliceosome that is compositionally different from the widely studied major spliceosome and functions in splicing of some rare introns. Based on the conservation of many components of major and minor spliceosomes in plants, it is likely that plants contain both types of spliceosomes and the basic mechanisms involved in spliceosome formation and intron removal are likely to be similar between plants and animals. However, a number of reports using in vivo splicing assays have shown that the cis-elements that are necessary for intron recognition and proper splicing of plant introns differ considerably from yeast and animals. These studies suggest that the mechanisms of intron recognition in plants are likely to differ from yeast and animals and involve novel proteins that recognize the plant-specific cis-elements. In recent years, several proteins that are implicated in plant pre-mRNA splicing have been characterized, including some novel ones that are not present in metazoans. The recent completion of the Arabidopsis and other eukaryotic genomes sequence should facilitate the identification of plant orthologs of various animal spliceosomal proteins in the near future. However, identification and functional analysis of the splicing proteins that are specific to plants will demand novel approaches.
[Show abstract][Hide abstract] ABSTRACT: Intracellular calcium transients during plant-pathogen interactions are necessary early events leading to local and systemic acquired resistance. Salicylic acid, a critical messenger, is also required for both of these responses, but whether and how salicylic acid level is regulated by Ca(2+) signalling during plant-pathogen interaction is unclear. Here we report a mechanism connecting Ca(2+) signal to salicylic-acid-mediated immune response through calmodulin, AtSR1 (also known as CAMTA3), a Ca(2+)/calmodulin-binding transcription factor, and EDS1, an established regulator of salicylic acid level. Constitutive disease resistance and elevated levels of salicylic acid in loss-of-function alleles of Arabidopsis AtSR1 suggest that AtSR1 is a negative regulator of plant immunity. This was confirmed by epistasis analysis with mutants of compromised salicylic acid accumulation and disease resistance. We show that AtSR1 interacts with the promoter of EDS1 and represses its expression. Furthermore, Ca(2+)/calmodulin-binding to AtSR1 is required for suppression of plant defence, indicating a direct role for Ca(2+)/calmodulin in regulating the function of AtSR1. These results reveal a previously unknown regulatory mechanism linking Ca(2+) signalling to salicylic acid level.
[Show abstract][Hide abstract] ABSTRACT: U1-70K, a U1 snRNP-specific protein, and serine/arginine-rich (SR) proteins are components of the spliceosome and play critical roles in both constitutive and alternative pre-mRNA splicing. However, the mobility properties of U1-70K, its in vivo interaction with SR proteins, and the mobility of the U1-70K-SR protein complex have not been studied in any system. Here, we studied the in vivo interaction of U1-70K with an SR protein (SR45) and the mobility of the U1-70K/SR protein complex using bimolecular fluorescence complementation (BiFC) and fluorescence recovery after photobleaching (FRAP). Our results show that U1-70K exchanges between speckles and the nucleoplasmic pool very rapidly and that this exchange is sensitive to ongoing transcription and phosphorylation. BiFC analyses showed that U1-70K and SR45 interacted primarily in speckles and that this interaction is mediated by the RS1 or RS2 domain of SR45. FRAP analyses showed considerably slower recovery of the SR45/U1-70K complex than either protein alone indicating that SR45/U1-70K complexes remain in the speckles for a longer duration. Furthermore, FRAP analyses with SR45/U1-70K complex in the presence of inhibitors of phosphorylation did not reveal any significant change compared to control cells, suggesting that the mobility of the complex is not affected by the status of protein phosphorylation. These results indicate that U1-70K, like SR splicing factors, moves rapidly in the nucleus ensuring its availability at various sites of splicing. Furthermore, although it appears that U1-70K moves by diffusion its mobility is regulated by phosphorylation and transcription.
PLoS ONE 02/2008; 3(4):e1953. DOI:10.1371/journal.pone.0001953 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The general organization ofeukaryotic nuclei, including plant nuclei, into functional domains is now widely recognized. Conventional immunocytochemistry and visualization of proteins fused to fluorescent proteins (FP) have revealed that in plants, RNA and protein components of pre-mRNA splicing are spatially organized depending on the stage of cell cycle, development, and the cell's physiological state. Application of some of the latest microscopy techniques, which reveal biophysical properties such as diffusion and interaction properties of proteins, has begun to provide important insights into the functional organization of spliceosomal proteins in plants. Although some progress has been made in understanding the spatial and temporal organization of splicing machinery in plants, the mechanisms that regulate this organization and its functional consequences remain unresolved.
Current topics in microbiology and immunology 01/2008; 326:103-18. DOI:10.1007/978-3-540-76776-3_6 · 4.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A substantial fraction (approximately 30%) of plant genes is alternatively spliced, but how alternative splicing is regulated remains unknown. Many plant genes undergo alternative splicing in response to a variety of stresses. Large-scale computational analyses and experimental approaches focused on select genes are beginning to reveal that alternative splicing constitutes an integral part of gene regulation in stress responses. Based on the studies discussed in this chapter, it appears that alternative splicing generates transcriptome/proteome complexity that is likely to be important for stress adaptation. However, the signaling pathways that relay stress conditions to splicing machinery and if and how the alternative spliced products confer adaptive advantages to plants are poorly understood.
Current topics in microbiology and immunology 01/2008; 326:257-75. DOI:10.1007/978-3-540-76776-3_14 · 4.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Arabidopsis Flagellin sensitive2 (FLS2) is a transmembrane leucine-rich repeat receptor-like kinase, which recognizes a conserved 22 amino acid peptide (flg22) of bacterial flagellin and activates downstream defense signaling pathways resulting in enhanced resistance against plant pathogens. The underlying mechanisms for the activation of FLS2 in the cell membrane, however, are not fully understood. Using fluorescence recovery after photobleaching (FRAP), we demonstrate that approximately 75% of the FLS2 in the plasma membrane diffuses laterally with a diffusion coefficient of 0.34 microm(2) s(-1), indicating that it moves rapidly. Further, we show that FLS2 is less mobile in the presence of flg22, suggesting its ligand-dependent confinement to microdomains or transient interaction with other less mobile membrane proteins. Using an in vivo bimolecular fluorescence complementation (BiFC) system and fluorescence resonance energy transfer (FRET), which reveals in vivo protein-protein interactions, we show that FLS2 does not homodimerize either constitutively or in the presence of flg22. Our data suggest that the reduced mobility of FLS2 after binding flg22 and its existence in monomeric form are important mechanistic features of FLS2 early signaling.
[Show abstract][Hide abstract] ABSTRACT: Serine-arginine-rich (SR) proteins, a family of spliceosomal proteins, function at multiple steps in the assembly of the spliceosome in non-plant systems. Limited studies with metazoan SR splicing factors (ASF/SF2 and SC35) indicated that their mobility is not dependent on ATP and phosphorylation. In addition, inhibition of transcription slightly increased their mobility. Here, we analyzed the mobility of SR45, a plant-specific SR protein with unique domain organization, and SR1/SRp34, a plant homolog of metazoan ASF/SF2, using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). Our results show that, in contrast to metazoan SR splicing factors, the movement of the plant SR proteins is dependent on ATP, phosphorylation and transcription. To understand the underlying mechanism for these observations, we carried out mobility analyses with the domain-deletion mutants of SR45 in ATP-depleted cells and in the presence of inhibitors of transcription or phosphorylation. Our results show that the sensitivity of SR45 to these inhibitors is conferred by an RNA-recognition motif (RRM) and the serine-arginine-rich (RS) domain 2. These results provide important insights into the mechanisms of plant SR protein movement and suggest fundamental differences in the regulation of the mobility of plant and animal SR splicing factors.
[Show abstract][Hide abstract] ABSTRACT: Functional studies with ZWICHEL ( ZWI ), which encodes a Ca(2+)-calmodulin-regulated kinesin, have shown its involvement in trichome morphogenesis and cell division. To identify regulatory regions that control the ZWI expression pattern, we generated transgenic Arabidopsis plants with a GUS reporter driven by different lengths of the ZWI gene 5' region alone or 5' and 3' regions together. The 5' fusions contain varying lengths of the coding and non-coding regions of beta - HYDROXYISOBUTYRYL-CoA HYDROLASE 1 ( CHY1 ), which is upstream of ZWI, and a 162 bp intergenic region. In transgenic plants with 5' 460::GUS, GUS activity was observed primarily in the root hairs whereas transgenic plants with an additional 5' 266 bp region from the CHY1 gene (5' 726::GUS) showed strong GUS accumulation in the entire root including root hairs and root tip, calli and at various developmental stages in trichomes and pollen. However, very little GUS accumulation was detected in roots of dark-grown or root tips of cold-treated seedlings with 5' ZWI constructs. These results were further confirmed by quantifying GUS enzyme activity and transcripts in these seedlings. Calli and pollen transformed with the 5' distal 268 bp fused in antisense orientation to the proximal 460 bp did not show GUS expression. Further, IAA-treated dark-grown seedlings with 726::GUS, but not with 460::GUS, showed high GUS expression in specific regions (outer layer 2a cells) at the base of the lateral roots. The ZWI 3' region (3 kb) did not influence the GUS expression pattern driven by the 5' 726 bp. The absence of CHY1 transcripts in the chy1-2 mutant did not alter either ZWI expression or ZWI-mediated trichome morphogenesis. Thus, our data suggest that the 3' part of the CHY1 gene contains regulatory elements that control ZWI gene expression in dividing cells and other cells that exhibit polarized growth such as root hairs, pollen and trichomes. This is the first evidence that the regulatory regions conferring developmental and cell-specific expression of a gene reside in the introns and exons of its upstream protein-coding gene.
[Show abstract][Hide abstract] ABSTRACT: Ca2+ and calmodulin (CaM), a key Ca2+ sensor in all eukaryotes, have been implicated in defense responses in plants. To elucidate the role of Ca2+ and CaM in defense signaling, we used 35S-labeled CaM to screen expression libraries prepared from tissues that were either treated with an elicitor derived from Phytophthora megasperma or infected with Pseudomonas syringae pv. tabaci. Nineteen cDNAs that encode the same protein, pathogen-induced CaM-binding protein (PICBP), were isolated. The PICBP fusion proteins bound 35S-CaM, horseradish peroxidase-labeled CaM and CaM-Sepharose in the presence of Ca2+ whereas EGTA, a Ca2+ chelator, abolished binding, confirming that PICBP binds CaM in a Ca2+-dependent manner. Using a series of bacterially expressed truncated versions of PICBP, four CaM-binding domains, with a potential CaM-binding consensus sequence of WSNLKKVILLKRFVKSL, were identified. The deduced PICBP protein sequence is rich in leucine residues and contains three classes of repeats. The PICBP gene is differentially expressed in tissues with the highest expression in stem. The expression of PICBP in Arabidopsis was induced in response to avirulent Pseudomonas syringae pv. tomato carrying avrRpm1. Furthermore, PICBP is constitutively expressed in the Arabidopsis accelerated cell death2-2 mutant. The expression of PICBP in bean leaves was also induced after inoculation with avirulent and non-pathogenic bacterial strains. In addition, the hrp1 mutant of Pseudomonas syringae pv. tabaci and inducers of plant defense such as salicylic acid, hydrogen peroxide and a fungal elicitor induced PICBP expression in bean. Our data suggest a role for PICBP in Ca2+-mediated defense signaling and cell-death. Furthermore, PICBP is the first identified CBP in eukaryotes with four Ca2+-dependent CaM-binding domains.
[Show abstract][Hide abstract] ABSTRACT: Calmodulin (CaM) plays an important role in sensing and transducing changes in cellular Ca2+ concentration in response to several biotic and abiotic stresses. Although CaM is implicated in plant-pathogen interactions, its molecular targets and their role in defense signaling pathway(s) are poorly understood. To elucidate the signaling pathways that link CaM to defense responses, we screened a cDNA library constructed from bean leaves undergoing a hypersensitive response (HR) with radiolabeled CaM isoforms. A total of 26 putative CBPs were identified. Sequencing of the cDNAs revealed that they represent 8 different genes. They are homologues of previously identified CaM-binding proteins (CBPs) in other systems. However, some CBPs are novel members of known CBP families. The proteins encoded by these clones bound CaM in a Ca2+-dependent manner. To determine if these CBPs are involved in plant defense responses, we analyzed their expression in bean leaves inoculated with compatible, incompatible and nonpathogenic bacterial strains. Expression of three CBPs including an isoform of cyclic nucleotide-gated channels (PvCNGC-A) and two hypothetical proteins (PvCBP60-C and PvCBP60-D) was induced whereas the expression of two other isoforms of CNGCs (PvCNGC-B and PvCNGC-C) was repressed in response to incompatible pathogens. The expression of the rest, a small auxin up RNA (PvSAUR1) and two hypothetical proteins (PvCBP60-A and PvCBP60-B), was not changed. The expression of most of the pathogen-regulated genes was also affected by salicylic acid, jasmonic acid, hydrogen peroxide and a fungal elicitor, which are known to induce defense responses. Our results strongly suggest that at least five bean CBPs are involved in plant defense responses.