A cell suspension based uptake method to study high affinity glucosinolate transporters.

BACKGROUND: Glucosinolates are an important class of secondary metabolites characteristic to the order Brassicales. They are known to play a major role in plant defense and from the human perspective, can be anticarcinogenic or antinutritive. GTRs are plasma-membrane localized high affinity glucosinolate transporters, which are important components of the source (leaf) to sink (seed) translocation of intact glucosinolates in members of Brassicaceae family. GTRs are identified as major candidates for Brassica crop improvement, thus dictating a need for their functional characterization. However, currently there are limitations in availability of heterologous assay systems for functional characterization of plant secondary metabolite transporters. To date, the animal-based Xenopus oocyte system is the best established heterologous system for functional characterization of these transporters. Inherent biochemical and physiological attributes unique to the plant membranes necessitate the need for developing plant-based transporters assay systems as well. METHODS: In this study, Agrobacterium mediated transformation was used to develop GTR expressing cotton cell lines (CCL-1) for functional characterization of the Arabidopsis high affinity glucosinolate transporters, AtGTR1 and AtGTR2. Following sub-cellular localization of AtGTRs, we standardized the glucosinolate uptake assays using cell suspension cultures of AtGTR expressing CCL-1 its requirement of pH, salt, and time based glucosinolate uptake. Using the GTR expressing CCL-1, we subsequently performed kinetic analysis of AtGTR1 and AtGTR2 for different glucosinolate substrates, sinigrin, gluconapin and sinalbin. RESULTS: Several clones expressing each of AtGTR1 and AtGTR2 were obtained showing high level of GTR expression and were maintained through regular sub-culturing. Both AtGTR1 and AtGTR2 are predominantly plasma-localized proteins when overexpressed in CCL-1 cells. Uptake assays were standardized, suggesting that glucosinolate uptake of GTR expressing CCL-1 is robust within the physiological pH range 5-6, and at lower concentration of nitrate salts. GTR expressing CCL-1 cells show increasing glucosinolate accumulation in time course experiment. Kinetic studies over a wide glucosinolate concentrations (10-800 µM) revealed that our novel assay system displayed robust GTR-mediated uptake of different glucosinolates and unambiguously helps elucidate the saturable kinetics of GTRs. Our system confirms the high affinity of AtGTRs for both aliphatic and aromatic glucosinolates. CONCLUSION: The transporter assay system described in this study holds potential for studying sub-functionalization amongst GTR homologs present across Brassicaceae family. The fast growing CCL-1 cells, confer the benefits of an in vitro system for quick assays and is plant based thus enabling optimal expression without sequence modifications. The efficient functioning of the GTR transporters in the heterologous CCL-1 opens the possibility of using this plant cell suspension system for functional characterization of other metabolite transporters.

Molecular cloning and expression profiling of multiple Dof genes of Sorghum bicolor (L) Moench.

DNA binding with one finger (Dof) proteins represent a family of plant specific transcription factors associated with diverse biological processes, such as seed maturation and germination, phytohormone and light mediated regulation, and plant responses to biotic and abiotic stresses. In present study, a total of 21 Dof genes from Sorghum bicolor were cloned, sequenced and in silico characterized for homology search, revealing their identity to Dof like proteins. The expression profiling of SbDof genes using quantitative RT-PCR in different tissue types and also under drought and salt stresses was attempted. The SbDof genes displayed differential expression either in their transcript abundance or in their expression patterns under normal growth condition. Two of the SbDof genes namely SbDof8 and SbDof12 showed comparatively high level of transcript abundance in all the tissue types tested; whereas some of the SbDof genes showed a distinct tissue specific expression pattern. Further a total of 13 SbDof genes showed differential expression when subjected to either of the abiotic stress i.e. drought or salinity. Three of the SbDof genes namely SbDof12, SbDof19 and SbDof24 were found to be up-regulated in response to drought and salt stress. Comparative analysis of SbDof genes expression revealed existence of a complex transcriptional and functional diversity across plant growth and developmental stages.

Class-Specific Evolution and Transcriptional Differentiation of 14-3-3 Family Members in Mesohexaploid Brassica rapa.

14-3-3s are highly conserved, multigene family proteins that have been implicated in modulating various biological processes. The presence of inherent polyploidy and genome complexity has limited the identification and characterization of 14-3-3 proteins from globally important Brassica crops. Through data mining of Brassica rapa, the model Brassica genome, we identified 21 members encoding 14-3-3 proteins namely, BraA.GRF14.a to BraA.GRF14.u. Phylogenetic analysis indicated that B. rapa contains both ε (epsilon) and non-ε 14-3-3 isoforms, having distinct intron-exon structural organization patterns. The non-ε isoforms showed lower divergence rate (Ks < 0.45) compared to ε protein isoforms (Ks > 0.48), suggesting class-specific divergence pattern. Synteny analysis revealed that mesohexaploid B. rapa genome has retained 1-5 orthologs of each Arabidopsis 14-3-3 gene, interspersed across its three fragmented sub-genomes. qRT-PCR analysis showed that 14 of the 21 BraA.GRF14 were expressed, wherein a higher abundance of non-ε transcripts was observed compared to the ε genes, indicating class-specific transcriptional bias. The BraA.GRF14 genes showed distinct expression pattern during plant developmental stages and in response to abiotic stress, phytohormone treatments, and nutrient deprivation conditions. Together, the distinct expression pattern and differential regulation of BraA.GRF14 genes indicated the occurrence of functional divergence of B. rapa 14-3-3 proteins during plant development and stress responses.

Differential expression and interaction specificity of the heterotrimeric G-protein family in Brassica nigra reveal their developmental- and condition-specific roles.

Heterotrimeric G-proteins, comprised of α, ß and γ subunits, are important signal transducers across phyla. The G-proteins are well characterized in the model plants Arabidopsis and rice, and their inventories are possible from a few other plant species; however, information about the roles played by G-proteins in regulating various growth and developmental traits particularly from polyploid crops is still awaited. In this study, we have isolated one Gα (BniB.Gα1), three Gß (BniB.Gß1-BniB.Gß3) and four Gγ (BniB.Gγ1-BniB.Gγ4) coding sequences from the paleopolyploid Brassica nigra, a major condiment crop of the Brassicaceae family. Sequence and phylogenetic analysis revealed that whole-genome triplication events in the Brassica lineage had proportionally increased the inventory of the Gß subunit, but not of the Gα and Gγ subunits in B. nigra. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis showed that members of the G-protein subunit genes have distinct temporal and spatial expression patterns and were differentially altered in response to various stress and phytohormone treatments, thereby suggesting differential transcriptional regulation of G-protein genes in B. nigra. Interestingly, specific members of G-protein subunits were co-expressed across plant developmental stages, and in response to different elicitor treatments. Yeast-based interaction screens further predicted that the B. nigra G-protein subunits interacted in most of the possible combinations, although showing a high degree of interaction specificity between different G-protein subunits. Our data on physical interactions coupled with the co-expression pattern of the multiple G-protein subunit genes suggested that tissue- and condition-specific functional combinations of Gαßγ heterotrimers may exist in paleopolyploid B. nigra, to control diverse growth and development processes.

Evolution, expression differentiation and interaction specificity of heterotrimeric G-protein subunit gene family in the mesohexaploid Brassica rapa.

Heterotrimeric G-proteins, comprising of Gα, Gß, and Gγ subunits, are important signal transducers which regulate many aspects of fundamental growth and developmental processes in all eukaryotes. Initial studies in model plants Arabidopsis and rice suggest that the repertoire of plant G-protein is much simpler than that observed in metazoans. In order to assess the consequence of whole genome triplication events within Brassicaceae family, we investigated the multiplicity of G-protein subunit genes in mesohexaploid Brassica rapa, a globally important vegetable and oilseed crop. We identified one Gα (BraA.Gα1), three Gß (BraA.Gß1, BraA.Gß2, and BraA.Gß3), and five Gγ (BraA.Gγ1, BraA.Gγ2, BraA.Gγ3, BraA.Gγ4, and BraA.Gγ5) genes from B. rapa, with a possibility of 15 Gαßγ heterotrimer combinations. Our analysis suggested that the process of genome triplication coupled with gene-loss (gene-fractionation) phenomenon have shaped the quantitative and sequence diversity of G-protein subunit genes in the extant B. rapa genome. Detailed expression analysis using qRT-PCR assays revealed that the G-protein genes have retained ubiquitous but distinct expression profiles across plant development. The expression of multiple G-protein genes was differentially regulated during seed-maturation and germination stages, and in response to various phytohormone treatments and stress conditions. Yeast-based interaction analysis showed that G-protein subunits interacted in most of the possible combinations, with some degree of subunit-specific interaction specificity, to control the functional selectivity of G-protein heterotrimer in different cell and tissue-types or in response to different environmental conditions. Taken together, this research identifies a highly diverse G-protein signaling network known to date from B. rapa, and provides a clue about the possible complexity of G-protein signaling networks present across globally important Brassica species.