Arabidopsis Protein Sequences Research Articles

Evolutionary Analysis and Functional Identification of Clock-Associated PSEUDO-RESPONSE REGULATOR (PRRs) Genes in the Flowering Regulation of Roses.

Pseudo-response regulators (PRRs) are the important genes for flowering in roses. In this work, clock PRRs were genome-wide identified using Arabidopsis protein sequences as queries, and their evolutionary analyses were deliberated intensively in Rosaceae in correspondence with angiosperms species. To draw a comparative network and flow of clock PRRs in roses, a co-expression network of flowering pathway genes was drawn using a string database, and their functional analysis was studied by silencing using VIGS and protein-to-protein interaction. We revealed that the clock PRRs were significantly expanded in Rosaceae and were divided into three major clades, i.e., PRR5/9 (clade 1), PRR3/7 (clade 2), and TOC1/PRR1 (clade 3), based on their phylogeny. Within the clades, five clock PRRs were identified in Rosa chinensis. Clock PRRs had conserved RR domain and shared similar features, suggesting the duplication occurred during evolution. Divergence analysis indicated the role of duplication events in the expansion of clock PRRs. The diverse cis elements and interaction of clock PRRs with miRNAs suggested their role in plant development. Co-expression network analysis showed that the clock PRRs from Rosa chinensis had a strong association with flowering controlling genes. Further silencing of RcPRR1b and RcPRR5 in Rosa chinensis using VIGS led to earlier flowering, confirming them as negative flowering regulators. The protein-to-protein interactions between RcPRR1a/RcPRR5 and RcCO suggested that RcPRR1a/RcPRR5 may suppress flowering by interfering with the binding of RcCO to the promoter of RcFT. Collectively, these results provided an understanding of the evolutionary profiles as well as the functional role of clock PRRs in controlling flowering in roses.

Different Roles of Heat Shock Proteins (70 kDa) During Abiotic Stresses in Barley (Hordeum vulgare) Genotypes.

In this work, the involvement of heat shock proteins (HSP70) in barley (Hordeum vulgare) has been studied in response to drought and salinity. Thus, 3 barley genotypes usually cultivated and/or selected in Italy, 3 Middle East/North Africa landraces and genotypes and 1 improved genotype from ICARDA have been studied to identify those varieties showing the best stress response. Preliminarily, a bioinformatic characterization of the HSP70s protein family in barley has been made by using annotated Arabidopsis protein sequences. This study identified 20 putative HSP70s orthologs in the barley genome. The construction of un-rooted phylogenetic trees showed the partition into four main branches, and multiple subcellular localizations. The enhanced HSP70s presence upon salt and drought stress was investigated by both immunoblotting and expression analyses. It is worth noting the Northern Africa landraces showed peculiar tolerance behavior versus drought and salt stresses. The drought and salinity conditions indicated the involvement of specific HSP70s to counteract abiotic stress. Particularly, the expression of cytosolic MLOC_67581, mitochondrial MLOC_50972, and encoding for HSP70 isoforms showed different expressions and occurrence upon stress. Therefore, genotypes originated in the semi-arid area of the Mediterranean area can represent an important genetic source for the improvement of commonly cultivated high-yielding varieties.

The cytosolic thiouridylase CTU2 of Arabidopsis thaliana is essential for posttranscriptional thiolation of tRNAs and influences root development.

BackgroundA large number of post-transcriptional modifications of transfer RNAs (tRNAs) have been described in prokaryotes and eukaryotes. They are known to influence their stability, turnover, and chemical/physical properties. A specific subset of tRNAs contains a thiolated uridine residue at the wobble position to improve the codon-anticodon interaction and translational accuracy. The proteins involved in tRNA thiolation are reminiscent of prokaryotic sulfur transfer reactions and of the ubiquitylation process in eukaryotes. In plants, some of the proteins involved in this process have been identified and show a high degree of homology to their non-plant equivalents. For other proteins, the identification of the plant homologs is much less clear, due to the low conservation in protein sequence.ResultsThis manuscript describes the identification of CTU2, the second CYTOPLASMIC THIOURIDYLASE protein of Arabidopsis thaliana. CTU2 is essential for tRNA thiolation and interacts with ROL5, the previously identified CTU1 homolog of Arabidopsis. CTU2 is ubiquitously expressed, yet its activity seems to be particularly important in root tissue. A ctu2 knock-out mutant shows an alteration in root development.ConclusionsThe analysis of CTU2 adds a new component to the so far characterized protein network involved in tRNA thiolation in Arabidopsis. CTU2 is essential for tRNA thiolation as a ctu2 mutant fails to perform this tRNA modification. The identified Arabidopsis CTU2 is the first CTU2-type protein from plants to be experimentally verified, which is important considering the limited conservation of these proteins between plant and non-plant species. Based on the Arabidopsis protein sequence, CTU2-type proteins of other plant species can now be readily identified.

Comparative Proteomics of Thellungiella halophila Leaves from Plants Subjected to Salinity Reveals the Importance of Chloroplastic Starch and Soluble Sugars in Halophyte Salt Tolerance

Thellungiella halophila, a close relative of Arabidopsis, is a model halophyte used to study plant salt tolerance. The proteomic/physiological/transcriptomic analyses of Thellungiella plant leaves subjected to different salinity levels, reported herein, indicate an extraordinary ability of Thellungiella to adapt to large concentrations of exogenous saline by compartmentalizing Na(+) into cell vacuoles and accumulating proline and soluble sugars as organic osmolytes. Salinity stress stimulated the accumulation of starch in chloroplasts, which resulted in a greatly increased content of starch and total sugars in leaves. Comparative proteomics of Thellungiella leaves identified 209 salt-responsive proteins. Among these, the sequences of 108 proteins were strongly homologous to Arabidopsis protein sequences, and 30 had previously been identified as Thellungiella proteins. Functional classification of these proteins into 16 categories indicated that the majority are involved in carbohydrate metabolism, followed by those involved in energy production and conversion, and then those involved in the transport of inorganic ions. Pathway analysis revealed that most of the proteins are involved in starch and sucrose metabolism, carbon fixation, photosynthesis, and glycolysis. Of these processes, the most affected were starch and sucrose metabolism, which might be pivotal for salt tolerance. The gene expression patterns of the 209 salt-responsive proteins revealed through hierarchical clustering of microarray data and the expression patterns of 29 Thellungiella genes evaluated via quantitative RT-PCR were similar to those deduced via proteomic analysis, which underscored the possibility that starch and sucrose metabolism might play pivotal roles in determining the salt tolerance ability of Thellungiella. Our observations enabled us to propose a schematic representation of the systematic salt-tolerance phenotype in Thellungiella and suggested that the increased accumulation of starch, soluble sugars, and proline, as well as subcellular compartmentalization of sodium, might collectively denote important mechanisms for halophyte salt tolerance.

Characterization and Prediction of Protein Phosphorylation Hotspots in Arabidopsis thaliana

The regulation of protein function by modulating the surface charge status via sequence-locally enriched phosphorylation sites (P-sites) in so called phosphorylation “hotspots” has gained increased attention in recent years. We set out to identify P-hotspots in the model plant Arabidopsis thaliana. We analyzed the spacing of experimentally detected P-sites within peptide-covered regions along Arabidopsis protein sequences as available from the PhosPhAt database. Confirming earlier reports (Schweiger and Linial, 2010), we found that, indeed, P-sites tend to cluster and that distributions between serine and threonine P-sites to their respected closest next P-site differ significantly from those for tyrosine P-sites. The ability to predict P-hotspots by applying available computational P-site prediction programs that focus on identifying single P-sites was observed to be severely compromised by the inevitable interference of nearby P-sites. We devised a new approach, named HotSPotter, for the prediction of phosphorylation hotspots. HotSPotter is based primarily on local amino acid compositional preferences rather than sequence position-specific motifs and uses support vector machines as the underlying classification engine. HotSPotter correctly identified experimentally determined phosphorylation hotspots in A. thaliana with high accuracy. Applied to the Arabidopsis proteome, HotSPotter-predicted 13,677 candidate P-hotspots in 9,599 proteins corresponding to 7,847 unique genes. Hotspot containing proteins are involved predominantly in signaling processes confirming the surmised modulating role of hotspots in signaling and interaction events. Our study provides new bioinformatics means to identify phosphorylation hotspots and lays the basis for further investigating novel candidate P-hotspots. All phosphorylation hotspot annotations and predictions have been made available as part of the PhosPhAt database at http://phosphat.mpimp-golm.mpg.de.

Rice CYP734A cytochrome P450s inactivate brassinosteroids in Arabidopsis

Endogenous brassinosteroid concentrations are an important target for optimizing the growth of crop plants because these hormones influence yield and stress tolerance. The CYP734A subfamily of cytochrome P450 enzymes has been shown to inactivate brassinosteroid hormones in Arabidopsis and tomato. Rice has three genes for CYP734A enzymes whose expression appears to be up-regulated by exogenous brassinolide. The amino acids predicted to be in the active site of the rice enzymes vary when compared with the Arabidopsis protein sequence, suggesting that there could be differences in their ability to inactivate the hormone. We have cloned three CYP734A rice genes and expressed them in Arabidopsis to assess their efficacy as brassinosteroid-inactivating enzymes. We found that incorrect transcript splicing can complicate the expression of monocot genomic clones in a eudicot. However, the Arabidopsis system allowed us to characterize an atypical splice variant in one of the rice genes. cDNA clones produced high levels of expression and conferred the brassinosteroid inactivation phenotype. This study shows that Arabidopsis is a useful heterologous system for testing plant genes predicted to act in biochemical pathways that are conserved between monocots and eudicots.

Identification, characterization and utilization of unigene derived microsatellite markers in tea (Camellia sinensis L.)

BackgroundDespite great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in tea (Camellia sinensis L.). The development of microsatellite markers will have a major impact on genetic analysis, gene mapping and marker assisted breeding. Unigene derived microsatellite (UGMS) markers identified from publicly available sequence database have the advantage of assaying variation in the expressed component of the genome with unique identity and position. Therefore, they can serve as efficient and cost effective alternative markers in such species.ResultsConsidering the multiple advantages of UGMS markers, 1,223 unigenes were predicted from 2,181 expressed sequence tags (ESTs) of tea (Camellia sinensis L.). A total of 109 (8.9%) unigenes containing 120 SSRs were identified. SSR abundance was one in every 3.55 kb of EST sequences. The microsatellites mainly comprised of di (50.8%), tri (30.8%), tetra (6.6%), penta (7.5%) and few hexa (4.1%) nucleotide repeats. Among the dinucleotide repeats, (GA)n.(TC)n were most abundant (83.6%). Ninety six primer pairs could be designed form 83.5% of SSR containing unigenes. Of these, 61 (63.5%) primer pairs were experimentally validated and used to investigate the genetic diversity among the 34 accessions of different Camellia spp. Fifty one primer pairs (83.6%) were successfully cross transferred to the related species at various levels. Functional annotation of the unigenes containing SSRs was done through gene ontology (GO) characterization. Thirty six (60%) of them revealed significant sequence similarity with the known/putative proteins of Arabidopsis thaliana. Polymorphism information content (PIC) ranged from 0.018 to 0.972 with a mean value of 0.497. The average heterozygosity expected (HE) and observed (Ho) obtained was 0.654 and 0.413 respectively, thereby suggesting highly heterogeneous nature of tea. Further, test for IAM and SMM models for the UGMS loci showed excess heterozygosity and did not show any bottleneck operating in the tea population.ConclusionUGMS markers identified and characterized in this study provided insight about the abundance and distribution of SSR in the expressed genome of C. sinensis. The identification and validation of 61 new UGMS markers will not only help in intra and inter specific genetic diversity assessment but also be enriching limited microsatellite markers resource in tea. Further, the use of these markers would reduce the cost and facilitate the gene mapping and marker-aided selection in tea. Since, 36 of these UGMS markers correspond to the Arabidopsis protein sequence data with known functions will offer the opportunity to investigate the consequences of SSR polymorphism on gene functions.

Comparative study of rice and Arabidopsis Actin-depolymerizing factors gene families

Actin-depolymerizing factors (ADF) is one of the small actin-binding proteins that regulate actin dynamics in cells. Analysis of the complete rice and Arabidopsis protein sequences revealed 12 ADF proteins, respectively. A further study on the similarities and differences between ADF throughout rice and Arabidopsis genome was carried out at the level of genomic organization and protein structure. The strict conservation of essential structural features suggested that the mode of action and physiological function of these proteins, as well as the expression pattern of their coding genes, might be very similar. The ADF proteins were divided into four groups based on the phylogenetic relationships of the amino acid sequences, and was comparable with previous studies.

Molecular Identification of a Functional Homologue of the Mammalian Fatty Acid Amide Hydrolase in Arabidopsis thaliana

N-Acylethanolamines (NAEs) are endogenous constituents of plant and animal tissues, and in vertebrates their hydrolysis terminates their participation as lipid mediators in the endocannabinoid signaling system. The membrane-bound enzyme responsible for NAE hydrolysis in mammals has been identified at the molecular level (designated fatty acid amide hydrolase, FAAH), and although an analogous enzyme activity was identified in microsomes of cotton seedlings, no molecular information is available for this enzyme in plants. Here we report the identification, the heterologous expression (in Escherichia coli), and the biochemical characterization of an Arabidopsis thaliana FAAH homologue. Candidate Arabidopsis DNA sequences containing a characteristic amidase signature sequence (PS00571) were identified in plant genome data bases, and a cDNA was isolated by reverse transcriptase-PCR using Arabidopsis genome sequences to develop appropriate oligonucleotide primers. The cDNA was sequenced and predicted to encode a protein of 607 amino acids with 37% identity to rat FAAH within the amidase signature domain (18% over the entire length). Residues determined to be important for FAAH catalysis were conserved between the Arabidopsis and rat protein sequences. In addition, a single transmembrane domain near the N terminus was predicted in the Arabidopsis protein sequence, similar to that of the rat FAAH protein. The putative plant FAAH cDNA was expressed as an epitope/His-tagged fusion protein in E. coli and solubilized from cell lysates in the nonionic detergent, dodecyl maltoside. Affinity-purified recombinant protein was indeed active in hydrolyzing a variety of naturally occurring N-acylethanolamine types. Kinetic parameters and inhibition data for the recombinant Arabidopsis protein were consistent with these properties of the enzyme activity characterized previously in plant and animal systems. Collectively these data now provide support at the molecular level for a conserved mechanism between plants and animals for the metabolism of NAEs.

A small GTP-binding protein from Arabidopsis thaliana functionally complements the yeast YPT6 null mutant.

A clone designated A.t.RAB6 encoding a small GTP-binding protein was isolated from a cDNA library of Arabidopsis thaliana leaf tissue. The predicted amino acid sequence was highly homologous to the mammalian and yeast counterparts, H.Rab6 and Ryh1/Ypt6, respectively. Lesser homology was found between the predicted Arabidopsis protein sequence and two small GTP-binding proteins isolated from plant species (44% homology to Zea mays Ypt1 and 43% homology to Nicotiana tabacum Rab5). Conserved stretches in the deduced amino acid sequence of A.t.Rab6 include four regions involved in GTP-binding, an effector region, and C-terminal cysteine residues required for prenylation and subsequent membrane attachment. Northern blot analysis demonstrated that A.t.Rab6 mRNA was expressed in root, leaf, stem, and flower tissues from A. thaliana with the highest levels present in roots. Escherichia coli produced histidine-tagged A.t.Rab6 protein-bound GTP, whereas a mutation in one of the guanine nucleotide-binding sites (asparagine122 to isoleucine) rendered it incapable of binding GTP. Functionally, the A.t.RAB6 gene was able to complement the temperature-sensitive phenotype of the YPT6 null mutant in yeast. The isolation of this gene will aid in the dissection of the machinery involved in soluble protein sorting at the trans-Golgi network of plants.