SLocX: Predicting Subcellular Localization of Arabidopsis Proteins Leveraging Gene Expression Data

Despite the growing volume of experimentally validated knowledge about the subcellular localization of plant proteins, a well performing in silico prediction tool is still a necessity. Existing tools, which employ information derived from protein sequence alone, offer limited accuracy and/or rely on full sequence availability. We explored whether gene expression profiling data can be harnessed to enhance prediction performance. To achieve this, we trained several support vector machines to predict the subcellular localization of Arabidopsis thaliana proteins using sequence derived information, expression behavior, or a combination of these data and compared their predictive performance through a cross-validation test. We show that gene expression carries information about the subcellular localization not available in sequence information, yielding dramatic benefits for plastid localization prediction, and some notable improvements for other compartments such as the mitochondrion, the Golgi, and the plasma membrane. Based on these results, we constructed a novel subcellular localization prediction engine, SLocX, combining gene expression profiling data with protein sequence-based information. We then validated the results of this engine using an independent test set of annotated proteins and a transient expression of GFP fusion proteins. Here, we present the prediction framework and a website of predicted localizations for Arabidopsis. The relatively good accuracy of our prediction engine, even in cases where only partial protein sequence is available (e.g., in sequences lacking the N-terminal region), offers a promising opportunity for similar application to non-sequenced or poorly annotated plant species. Although the prediction scope of our method is currently limited by the availability of expression information on the ATH1 array, we believe that the advances in measuring gene expression technology will make our method applicable for all Arabidopsis proteins

Functional analysis of the Landsberg erecta allele of FRIGIDA

Abstract Background: Most of the natural variation in flowering time in Arabidopsis thaliana can be attributed to allelic variation at the gene FRIGIDA (FRI, AT4G00650), which activates expression of the floral repressor FLOWERING LOCUS C (FLC, AT5G10140). Usually, late-flowering accessions carry functional FRI alleles (FRI-wt), whereas early flowering accessions contain non-functional alleles. The two most frequent alleles found in early flowering accessions are the ones present in the commonly used lab strains Columbia (FRI-Col) and Landsberg erecta (FRI-Ler), which contain a premature stop codon and a deletion of the start codon respectively

Characterization of the Putative Acylated Cellulose Synthase Operon in Komagataeibacter xylinus E25

Bacterial cellulose is a natural polymer with an expanding array of applications. Because of this, the main cellulose producers of the Komagataeibacter genus have been extensively studied with the aim to increase its synthesis or to customize its physicochemical features. Up to now, the genetic studies in Komagataeibacter have focused on the first cellulose synthase operon (bcsI) encoding the main enzyme complex. However, the role of other accessory cellulose operons has been understudied. Here we aimed to fill this gap by performing a detailed analysis of the second cellulose synthase operon (bcsII), which is putatively linked with cellulose acylation. In this study we harnessed the genome sequence, gene expression and protein structure information of K. xylinus E25 and other Komagataeibacter species to discuss the probable features of bcsII and the biochemical function of its main protein products. The results of our study support the previous hypothesis that bcsII is involved in the synthesis of the acylated polymer and expand it by presenting the evidence that it may also function in the regulation of its attachment to the cell surface and to the crystalline cellulose fibers

Taxonomic review and microbial ecology in bacterial nanocellulose fermentation

Acetic acid bacteria (AAB) have a long history of use in several fermentation processes. Their exploitation gradually emerged in biotechnologic applications, especially in the biosynthesis of useful chemicals and processes for the manufacture of several fermented food products. Taxonomic studies, from traditional to polyphasic approaches, have gradually allowed the proper classification of several ABB into distinct genera and species, among them, the bacterial nanocellulose (BNC) producers, notably Komagataeibacter xylinus. Despite the advantages in using specific (isolated) strains for biotechnologic processes toward controlling the kinetics and process yield, mixed culture fermentations may provide an interesting approach to tailoring the properties of BNC and to increase the product yield when aiming at industrial scale. Microbial population dynamics may play a synergistic role in the coordinative substrate consumption and metabolites production, especially if using complex media (as is the case with low cost substrates, eg, residues from other processes). This chapter will first review the main historic steps involved in the taxonomic classification of AAB. It will then address the lying potential behind mixed microbial fermentations, from kombucha to nata de coco, both sharing in common, the contribution of cellulose-producing bacteria for the fermentation process.(undefined

Functional analysis of the Landsberg erecta

Background: Most of the natural variation in flowering time in Arabidopsis thaliana can be attributed to allelic variation at the gene FRIGIDA (FRI, AT4G00650), which activates expression of the floral repressor FLOWERING LOCUS C (FLC, AT5G10140). Usually, late-flowering accessions carry functional FRI alleles (FRI-wt), whereas early flowering accessions contain non-functional alleles. The two most frequent alleles found in early flowering accessions are the ones present in the commonly used lab strains Columbia (FRI-Col) and Landsberg erecta (FRI-Ler), which contain a premature stop codon and a deletion of the start codon respectively. Results: Analysis of flowering time data from various Arabidopsis natural accessions indicated that the FRI-Ler allele retains some functionality. We generated transgenic lines carrying the FRI-Col or FRI-Ler allele in order to compare their effect on flowering time, vernalization response and FLC expression in the same genetic background. We characterize their modes of regulation through allele-specific expression and their relevance in nature through re-analysis of published datasets. We demonstrate that the FRI-Ler allele induces FLC expression, delays flowering time and confers sensitivity to vernalization in contrast to the true null FRI-Col allele. Nevertheless, the FRI-Ler allele revealed a weaker effect when compared to the fully functional FRI-wt allele, mainly due to reduced expression. Conclusions: The present study defines for the first time the existence of a new class of Arabidopsis accessions with an intermediate phenotype between slow and rapid cycling types. Although using available data from a common garden experiment we cannot observe fitness differences between accessions carrying the FRI-Ler or the FRI-Col allele, the phenotypic changes observed in the lab suggest that variation in these alleles could play a role in adaptation to specific natural environments

Crystal structure and functional characterization of a glucosamine-6-phosphate N-acetyltransferase from Arabidopsis thaliana

GlcNAc (N-acetylglucosamine) is an essential part of the glycan chain in N-linked glycoproteins. It is a building block for polysaccharides such as chitin, and several glucosaminoglycans and proteins can be O-GlcNAcylated. The deacetylated form, glucosamine, is an integral part of GPI (glycosylphosphatidylinositol) anchors. Both are incorporated into polymers by glycosyltransferases that utilize UDP-GlcNAc. This UDP-sugar is synthesized in a short pathway comprising four steps starting from fructose 6-phosphate. GNA (glucosamine-6-phosphate N-acetyltransferase) catalyses the second of these four reactions in the de novo synthesis in eukaryotes. A phylogenetic analysis revealed that only one GNA isoform can be found in most of the species investigated and that the most likely Arabidopsis candidate is encoded by the gene At5g15770 (AtGNA). qPCR (quantitative PCR) revealed the ubiquitous expression of AtGNA in all organs of Arabidopsis plants. Heterologous expression of AtGNA showed that it is highly active between pH 7 and 8 and at temperatures of 30–40°C. It showed Km values of 231 μM for glucosamine 6-phosphate and 33 μM for acetyl-CoA respectively and a catalytic efficiency comparable with that of other GNAs characterized. The solved crystal structure of AtGNA at a resolution of 1.5 Å (1 Å=0.1 nm) revealed a very high structural similarity to crystallized GNA proteins from Homo sapiens and Saccharomyces cerevisiae despite less well conserved protein sequence identity.</jats:p

VvMATE1 and VvMATE2 encode putative proanthocyanidin transporters expressed during berry development in Vitis vinifera L.

Univ Talca, Inst Ciencias Biol, Talca, Chile; Perez-Diaz, R (Perez-Diaz, Ricardo); Perez-Diaz, J (Perez-Diaz, Jorge); Casaretto, JA (Casaretto, Jose A.); Gonzalez-Villanueva, E (Gonzalez-Villanueva, Enrique); Ruiz-Lara, S (Ruiz-Lara, Simon)VvMATE1 and VvMATE2 encode putative PA transporters expressed during seed development in grapevine. The subcellular localization of these MATE proteins suggests different routes for the intracellular transport of PAs. Proanthocyanidins (PAs), also called condensed tannins, protect plants against herbivores and are important quality components of many fruits. PAs biosynthesis is part of the flavonoid pathway that also produces anthocyanins and flavonols. In grape fruits, PAs are present in seeds and skin tissues. PAs are synthesized in the cytoplasm and accumulated into the vacuole and apoplast; however, little is known about the mechanisms involved in the transport of these compounds to such cellular compartments. A gene encoding a Multidrug And Toxic compound Extrusion (MATE) family protein suggested to transport anthocyanins-named VvMATE1-was used to identify a second gene of the MATE family, VvMATE2. Analysis of their deduced amino acid sequences and the phylogenetic relationship with other MATE-like proteins indicated that VvMATE1 and VvMATE2 encode putative PA transporters. Subcellular localization assays in Arabidopsis protoplasts transformed with VvMATE-GFP fusion constructs along with organelle-specific markers revealed that VvMATE1 is localized in the tonoplast whereas VvMATE2 is localized in the Golgi complex. Major expression of both genes occurs during the early stages of seed development concomitant with the accumulation of PAs. Both genes are poorly expressed in the skin of berries while VvMATE2 is also expressed in leaves. The presence of putative cis-acting elements in the promoters of VvMATE1 and VvMATE2 may explain the differential transcriptional regulation of these genes in grapevine. Altogether, these results suggest that these MATE proteins could mediate the transport and accumulation of PAs in grapevine through different routes and cellular compartments

Plant genome and transcriptome annotations: from misconceptions to simple solutions

Next-generation sequencing has triggered an explosion of available genomic and transcriptomic resources in the plant sciences. Although genome and transcriptome sequencing has become orders of magnitudes cheaper and more efficient, often the functional annotation process is lagging behind. This might be hampered by the lack of a comprehensive enumeration of simple-to-use tools available to the plant researcher. In this comprehensive review, we present (i) typical ontologies to be used in the plant sciences, (ii) useful databases and resources used for functional annotation, (iii) what to expect from an annotated plant genome, (iv) an automated annotation pipeline and (v) a recipe and reference chart outlining typical steps used to annotate plant genomes/transcriptomes using publicly available resources

Domestication selected for deceleration of the circadian clock in cultivated tomato

The circadian clock is a critical regulator of plant physiology and development, controlling key agricultural traits in crop plants1. In addition, natural variation in circadian rhythms is important for local adaptation2, 3, 4. However, quantitative modulation of circadian rhythms due to artificial selection has not yet been reported. Here we show that the circadian clock of cultivated tomato (Solanum lycopersicum) has slowed during domestication. Allelic variation of the tomato homolog of the Arabidopsis gene EID1 is responsible for a phase delay. Notably, the genomic region harboring EID1 shows signatures of a selective sweep. We find that the EID1 allele in cultivated tomatoes enhances plant performance specifically under long day photoperiods, suggesting that humans selected slower circadian rhythms to adapt the cultivated species to the long summer days it encountered as it was moved away from the equator