Irene S. Day

Coping with Stresses: Roles of Calcium- and Calcium/Calmodulin-Regulated Gene Expression

Abiotic and biotic stresses are major limiting factors of crop yields and cause billions of dollars of losses annually around the world. It is hoped that understanding at the molecular level how plants respond to adverse conditions and adapt to a changing environment will help in developing plants that can better cope with stresses. Acquisition of stress tolerance requires orchestration of a multitude of biochemical and physiological changes, and most of these depend on changes in gene expression. Research during the last two decades has established that different stresses cause signal-specific changes in cellular Ca2+ level, which functions as a messenger in modulating diverse physiological processes that are important for stress adaptation. In recent years, many Ca2+ and Ca2+/calmodulin (CaM) binding transcription factors (TFs) have been identified in plants. Functional analyses of some of these TFs indicate that they play key roles in stress signaling pathways. Here, we review recent progress in this area with emphasis on the roles of Ca2+- and Ca2+/CaM-regulated transcription in stress responses. We will discuss emerging paradigms in the field, highlight the areas that need further investigation, and present some promising novel high-throughput tools to address Ca2+-regulated transcriptional networks.

Analysis of EF-hand-containing proteins in Arabidopsis

BackgroundIn plants, calcium (Ca2+) has emerged as an important messenger mediating the action of many hormonal and environmental signals, including biotic and abiotic stresses. Many different signals raise cytosolic calcium concentration ([Ca2+]cyt), which in turn is thought to regulate cellular and developmental processes via Ca2+-binding proteins. Three out of the four classes of Ca2+-binding proteins in plants contain Ca2+-binding EF-hand motif(s). This motif is a conserved helix-loop-helix structure that can bind a single Ca2+ ion. To identify all EF-hand-containing proteins in Arabidopsis, we analyzed its completed genome sequence for genes encoding EF-hand-containing proteins.ResultsA maximum of 250 proteins possibly having EF-hands were identified. Diverse proteins, including enzymes, proteins involved in transcription and translation, protein- and nucleic-acid-binding proteins and a large number of unknown proteins, have one or more putative EF-hands. Phylogenetic analysis identified six major groups that contain some families of proteins.ConclusionsThe presence of EF-hand motif(s) in a diversity of proteins is consistent with the involvement of Ca2+ in regulating many cellular and developmental processes. Thus far, only 47 of the possible 250 EF-hand proteins have been reported in the literature. Various domains that we identified in many of the uncharacterized EF-hand-containing proteins should help in elucidating their cellular role(s). Our analyses suggest that the Ca2+ messenger system is widely used in plants and that EF-hand-containing proteins are likely to be the key transducers mediating Ca2+ action.

Kinesins in the Arabidopsis genome: A comparative analysis among eukaryotes

BackgroundKinesins constitute a superfamily of microtubule motor proteins that are found in eukaryotic organisms. Members of the kinesin superfamily perform many diverse cellular functions such as transport of vesicles and organelles, spindle formation and elongation, chromosome segregation, microtubule dynamics and morphogenesis. Only a few kinesins have been characterized in plants including Arabidopsis thaliana. Because of the diverse cellular functions in which kinesins are involved, the number, types and characteristics of kinesins present in the Arabidopsis genome would provide valuable information for many researchers.ResultsHere we have analyzed the recently completed Arabidopsis genome sequence and identified sixty-one kinesin genes in the Arabidopsis genome. Among the five completed eukaryotic genomes the Arabidopsis genome has the highest percentage of kinesin genes. Further analyses of the kinesin gene products have resulted in identification of several interesting domains in Arabidopsis kinesins that provide clues in understanding their functions. A phylogenetic analysis of all Arabidopsis kinesin motor domain sequences with 113 motor domain sequences from other organisms has revealed that Arabidopsis has seven of the nine recognized subfamilies of kinesins whereas some kinesins do not fall into any known family.ConclusionThere are groups of Arabidopsis kinesins that are not present in yeast, Caenorhabditis elegans and Drosophila melanogaster that may, therefore, represent new subfamilies specific to plants. The domains present in different kinesins may provide clues about their functions in cellular processes. The comparative analysis presented here provides a framework for future functional studies with Arabidopsis kinesins.

Analysis of the myosins encoded in the recently completed Arabidopsis thaliana genome sequence

BackgroundThree types of molecular motors play an important role in the organization, dynamics and transport processes associated with the cytoskeleton. The myosin family of molecular motors move cargo on actin filaments, whereas kinesin and dynein motors move cargo along microtubules. These motors have been highly characterized in non-plant systems and information is becoming available about plant motors. The actin cytoskeleton in plants has been shown to be involved in processes such as transportation, signaling, cell division, cytoplasmic streaming and morphogenesis. The role of myosin in these processes has been established in a few cases but many questions remain to be answered about the number, types and roles of myosins in plants.ResultsUsing the motor domain of an Arabidopsis myosin we identified 17 myosin sequences in the Arabidopsis genome. Phylogenetic analysis of the Arabidopsis myosins with non-plant and plant myosins revealed that all the Arabidopsis myosins and other plant myosins fall into two groups - class VIII and class XI. These groups contain exclusively plant or algal myosins with no animal or fungal myosins. Exon/intron data suggest that the myosins are highly conserved and that some may be a result of gene duplication.ConclusionsPlant myosins are unlike myosins from any other organisms except algae. As a percentage of the total gene number, the number of myosins is small overall in Arabidopsis compared with the other sequenced eukaryotic genomes. There are, however, a large number of class XI myosins. The function of each myosin has yet to be determined.

KIC, a novel Ca2+ binding protein with one EF-hand motif, interacts with a microtubule motor protein and regulates trichome morphogenesis

Kinesin-like calmodulin binding protein (KCBP) is a microtubule motor protein involved in the regulation of cell division and trichome morphogenesis. Genetic studies have shown that KCBP is likely to interact with several other proteins. To identify KCBP-interacting proteins, we used the C-terminal region of KCBP in a yeast two-hybrid screen. This screening resulted in the isolation of a novel KCBP-interacting Ca2+ binding protein (KIC). KIC, with its single EF-hand motif, bound Ca2+ at a physiological concentration. Coprecipitation with bacterially expressed protein and native KCBP, gel-mobility shift studies, and ATPase assays with the KCBP motor confirmed that KIC interacts with KCBP in a Ca2+-dependent manner. Interestingly, although both Ca2+-KIC and Ca2+-calmodulin were able to interact with KCBP and inhibit its microtubule binding activity, the concentration of Ca2+ required to inhibit the microtubule-stimulated ATPase activity of KCBP by KIC was threefold less than that required for calmodulin. Two KIC-related Ca2+ binding proteins and a centrin from Arabidopsis, which contain one and four EF-hand motifs, respectively, bound Ca2+ but did not affect microtubule binding and microtubule-stimulated ATPase activities of KCBP, indicating the specificity of Ca2+ sensors in regulating their targets. Overexpression of KIC in Arabidopsis resulted in trichomes with reduced branch number resembling the zwichel/kcbp phenotype. These results suggest that KIC modulates the activity of KCBP in response to changes in cytosolic Ca2+ and regulates trichome morphogenesis.

Localization and Dynamics of Nuclear Speckles in Plants

The generation of mature mRNAs from most genes (about 80%–90%) in autotrophic eukaryotes requires the removal of noncoding sequences (introns) and splicing of the coding regions (exons; [Labadorf et al., 2010][1]). During splicing in some precursor messenger RNAs (pre-mRNAs), the same splice sites

The role of the cytoskeleton and a molecular motor in trichome morphogenesis

We thank David Oppenheimer and Martin Hulskamp for providing seeds of zwichel mutants; Salah Abdel-Ghany for preparing scanning electron micrographs of trichomes. Work in our laboratory was supported by grants from the National Science Foundation and the National Aeronautics and Space Administration to A.S.N.R.

Localization and dynamics of plant splicing regulators

The generation of mature mRNAs from most genes (about 80%–90%) in autotrophic eukaryotes requires the removal of noncoding sequences (introns) and splicing of the coding regions (exons; [Labadorf et al., 2010][1]). During splicing in some precursor messenger RNAs (pre-mRNAs), the same splice sites

Isolation of a new mitotic-like cyclin from Arabidopsis: complementation of a yeast cyclin mutant with a plant cyclin

Cyclins, a large family of proteins, are the regulatory subunits of cyclin-dependent protein kinases that are essential activators of cell cycle progression in eukaryotes. Here we report isolation of a new cyclin cDNA (cyc1bAt) from Arabidopsis cDNA libraries using polymerase chain reaction amplified cyclin-box sequences as probes. The deduced amino acid sequence of the isolated cDNA showed the highest sequence similarity with mitotic cyclins. However, the nucleotide and predicted amino acid sequence of cyc1bAt is different from five other mitotic-like cyclins that have recently been isolated from the same system, indicating that it is a new mitotic-like cyclin. These results, together with previous reports, suggest that there are at least six different mitotic-like cyclins in Arabidopsis. Expression of cyc1bAt in yeast G1 cyclin-minus mutant (DL1) rescued the cyclin-minus phenotype, demonstrating that plant mitotic-like cyclin can complement cyclin function in yeast. Analysis of expression of cyc1bAt in different tissues by reverse transcription-polymerase chain reaction using primers corresponding to a unique region of the cDNA showed that cyc1bAt is differentially expressed in different tissues with highest expression in flowers and no detectable expression in leaves.

Origin and Evolution of Kinesin-Like Calmodulin-Binding Protein

Kinesin-like calmodulin-binding protein (KCBP), a member of the Kinesin-14 family, is a C-terminal microtubule motor with three unique domains including a myosin tail homology region 4 (MyTH4), a talin-like domain, and a calmodulin-binding domain (CBD). The MyTH4 and talin-like domains (found in some myosins) are not found in other reported kinesins. A calmodulin-binding kinesin called kinesin-C (SpKinC) isolated from sea urchin (Strongylocentrotus purpuratus) is the only reported kinesin with a CBD. Analysis of the completed genomes of Homo sapiens, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and a red alga (Cyanidioschyzon merolae 10D) did not reveal the presence of a KCBP. This prompted us to look at the origin of KCBP and its relationship to SpKinC. To address this, we isolated KCBP from a gymnosperm, Picea abies, and a green alga, Stichococcus bacillaris. In addition, database searches resulted in identification of KCBP in another green alga, Chlamydomonas reinhardtii, and several flowering plants. Gene tree analysis revealed that the motor domain of KCBPs belongs to a clade within the Kinesin-14 (C-terminal motors) family. Only land plants and green algae have a kinesin with the MyTH4 and talin-like domains of KCBP. Further, our analysis indicates that KCBP is highly conserved in green algae and land plants. SpKinC from sea urchin, which has the motor domain similar to KCBP and contains a CBD, lacks the MyTH4 and talin-like regions. Our analysis indicates that the KCBPs, SpKinC, and a subset of the kinesin-like proteins are all more closely related to one another than they are to any other kinesins, but that either KCBP gained the MyTH4 and talin-like domains or SpKinC lost them.