BackgroundSWEET (MtN3_saliva) domain proteins, a recently identified group of efflux transporters, play an indispensable role in sugar efflux, phloem loading, plant-pathogen interaction and reproductive tissue development. The SWEET gene family is predominantly studied in Arabidopsis and members of the family are being investigated in rice. To date, no transcriptome or genomics analysis of soybean SWEET genes has been reported.ResultsIn the present investigation, we explored the evolutionary aspect of the SWEET gene family in diverse plant species including primitive single cell algae to angiosperms with a major emphasis on Glycine max. Evolutionary features showed expansion and duplication of the SWEET gene family in land plants. Homology searches with BLAST tools and Hidden Markov Model-directed sequence alignments identified 52 SWEET genes that were mapped to 15 chromosomes in the soybean genome as tandem duplication events. Soybean SWEET (GmSWEET) genes showed a wide range of expression profiles in different tissues and developmental stages. Analysis of public transcriptome data and expression profiling using quantitative real time PCR (qRT-PCR) showed that a majority of the GmSWEET genes were confined to reproductive tissue development. Several natural genetic variants (non-synonymous SNPs, premature stop codons and haplotype) were identified in the GmSWEET genes using whole genome re-sequencing data analysis of 106 soybean genotypes. A significant association was observed between SNP-haplogroup and seed sucrose content in three gene clusters on chromosome 6.ConclusionPresent investigation utilized comparative genomics, transcriptome profiling and whole genome re-sequencing approaches and provided a systematic description of soybean SWEET genes and identified putative candidates with probable roles in the reproductive tissue development. Gene expression profiling at different developmental stages and genomic variation data will aid as an important resource for the soybean research community and can be extremely valuable for understanding sink unloading and enhancing carbohydrate delivery to developing seeds for improving yield.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1730-y) contains supplementary material, which is available to authorized users.
Senescence and reserve mobilization are integral components of plant development, are basic strategies in stress mitigation, and regulated at least in part by cytokinin. In the present study the effect of altered cytokinin metabolism caused by senescence-specific autoregulated expression of the Agrobacterium tumefaciens IPT gene under control of the P(SAG12) promoter (P(SAG12)-IPT) on seed germination and the response to a water-deficit stress was studied in tobacco (Nicotiana tabacum L.). Cytokinin levels, sugar content and composition of the leaf strata within the canopy of wild-type and P(SAG12)-IPT plants confirmed the reported altered source-sink relations. No measurable difference in sugar and pigment content of discs harvested from apical and basal leaves was evident 72 h after incubation with (+)-ABA or in darkness, indicating that expression of the transgene was not restricted to senescing leaves. No difference in quantum efficiency, photosynthetic activity, accumulation of ABA, and stomatal conductance was apparent in apical, middle and basal leaves of either wild-type or P(SAG12)-IPT plants after imposition of a mild water stress. However, compared to wild-type plants, P(SAG12)-IPT plants were slower to adjust biomass allocation. A stress-induced increase in root:shoot ratio and specific leaf area (SLA) occurred more rapidly in wild-type than in P(SAG12)-IPT plants reflecting delayed remobilization of leaf reserves to sink organs in the transformant. P(SAG12)-IPT seeds germinated more slowly even though abscisic acid (ABA) content was 50% that of the wild-type seeds confirming cytokinin-induced alterations in reserve remobilization. Thus, senescence is integral to plant growth and development and an increased endogenous cytokinin content impacts source-sink relations to delay ontogenic transitions wherein senescence in a necessary process.
A rapid methodology for the simultaneous analysis of a large number of cytokinins is presented. The cross-reactivity of a mixture of polyclonal antibodies against zeatin riboside and isopentenyladenosine was exploited in a protocol that can be used for immunoaffinity purification of 23 additional cytokinins. Ligands include the cytokinin bases zeatin, dihydrozeatin, isopentenyladenine, benzyl-adenine and kinetin, and their corresponding nucleoside, nucleoside-5'-monophosphate, and 9-glucoside derivatives, as well as cis-zeatin, cis-zeatin riboside, the 2-methylthiol derivatives of isopentenyladenosine and zeatin riboside, and benzyl-adenine-3-glucoside. Mixtures of cytokinins could be retained with high recoveries of all the components. Immunoaffinity purification of extracts of Arabidopsis thaliana (L.) Heynh. and Solarium tuberosum L. gave fractions clean enough, as verified by gas chromatographymass spectrometry (GC-MS), to allow analysis of endogenous cytokinins using a single high-performance liquid chromatography (HPLC) step with on-line UV-spectrum detection. The detection limit was 4-6 pmol. The procedure described forms a routine assaying technique that is faster and simpler, yet yields better qualitative and quantitative information than the commonly used procedure of immunoassaying of HPLC fractions.
The 3H-labeled nucleosides cytidine, deoxycytidine, and thymidine are rapidly incorporated into DNA via dCTP or dTTP pools. Between 30 and 60 minafter addition of tracer amounts of a labeled nucleoside to the medium ofrapidly growing 3T6 cells, dNTP pools attained a constant specific activity resulting from a steady-state equilibrium between incorporation of nucleoside, de novo synthesis, and linear incorporation of isotope into DNA. Removal oflabeled deoxycytidine or thymidine depleted the dNTP pools of isotope within a few minutes and incorporation into DNA stopped. When de novo synthesis of dTTP was blocked with amethopterin, the intracellular dTTP pool rapidly reached the specific activity of thymidine of' the medium and isotope-incorporation into DNA then measured absolute rates of DNA synthesis. In experiments with and without. amethopterin, we found no kinetic evidence for the existence ofmore than one dTTP pool and the decay of the pool suggested that all dTTP served as precursor of DNA. In contrast, experiments with deoxycytidine and cytidine suggested the presence of separate dCTP' pools with preferential DNA synthesis from the pool labeled from cytidine.Incorporation ofradioactive thymidine or, less frequently, other labeled nucleosides into DNA is often used to measure the rate of DNA synthesis. With thymidine and other deoxynucleosides, incorporation involves a series of phosphorylation steps leading directly to dNTPs; with ribonucleosides, reduction of the ribose moiety is required in addition. dNTPs are also formed de novo from small molecules. These interrelationships for the nucleosides thymidine, deoxycytidine, and cytidine are outlined below. Clearly, incorporation of a labeled nucleoside into DNA depends on other factors in addition to the rate of the final polymerization of dNTPs. In spite of this, incorporation of thymidine has been used widely, and often successfully, to monitor relative rates of DNA synthesis, neglecting possible errors introduced by changes in specific radioactivities ofdTTP. A safer, but more cumbersome, approach would be to measure the specific activity of the immediate precursor dNTP and relate incorporation of radioactivity into DNA to this value (1, 2). Such a procedure becomes imperative when one wishes to distinguish between effects of different manipulations-e. g., pharmacological interferenceon precursor synthesis and DNA replication. Knowing the specific activity of a dNTP under steady-state conditions may in addition permit determination of absolute rather than relative rates of DNA synthesis.The possibility of compartmentation of dNTP pools poses additionalicomplications (3). Fractionation of cells in nonaqueous media led to -the suggestion of separate cytoplasmic and. nuclear dNTP pools in Chinese hamster ovary cells (4). Kinetic experiment with HeLa cells were interpreted to indicate preferential incorporation of labeled thymidine into DNA via a dTTP pool that was not in equilibrium with dTTP formed de novo (5). The suggested channeling of DNA prec...
The tRNA of most organisms contain modified adenines called cytokinins. Situated next to the anticodon, they have been shown to influence translational fidelity and efficiency. The enzyme that synthesizes cytokinins on pre-tRNA, tRNA isopentenyltransferase (EC 2.5.1.8), has been studied in micro-organisms like Escherichia coli and SaccharomYces cerevisiae, and the corresponding genes have been cloned. We here report the first cloning and functional characterization of a homologous gene from a plant, Arabidopsis thaliana. Expression in S. cerevisiae showed that the gene can complement the anti-suppressor phenotype of a mutant that lacks MOD5, the intrinsic tRNA isopentenyltransferase gene. This was accompanied by the reintroduction of isopentenyladenosine in the tRNA. The Arabidopsis gene is constitutively expressed in seedling tissues.
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