Soybean (Glycine max L. Merr.) is an important nitrogen-fixing crop that provides much of the world's protein and oil. However, the available tools for investigation of soybean gene function are ...limited. Nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as Targeting Induced Local Lesions IN Genomes (TILLING). We have applied TILLING to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate (EMS) and one with N-nitroso-N-methylurea (NMU).
We screened seven targets in each population and discovered a total of 116 induced mutations. The NMU-treated population and one EMS mutagenized population had similar mutation density (approximately 1/140 kb), while another EMS population had a mutation density of approximately 1/250 kb. The remaining population had a mutation density of approximately 1/550 kb. Because of soybean's polyploid history, PCR amplification of multiple targets could impede mutation discovery. Indeed, one set of primers tested in this study amplified more than a single target and produced low quality data. To address this problem, we removed an extraneous target by pretreating genomic DNA with a restriction enzyme. Digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template.
The development of four independent populations with considerable mutation density, together with an additional method for screening closely related targets, indicates that soybean is a suitable organism for high-throughput mutation discovery even with its extensively duplicated genome.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mitochondrial serine hydroxymethyltransferase (SHMT), combined with glycine decarboxylase, catalyzes an essential sequence of the photorespiratory C₂ cycle, namely, the conversion of two molecules of ...glycine into one molecule each of CO₂, NH₄⁺, and serine. The Arabidopsis (Arabidopsis thaliana) mutant shm (now designated shm1-1) is defective in mitochondrial SHMT activity and displays a lethal photorespiratory phenotype when grown at ambient CO₂, but is virtually unaffected at elevated CO₂. The Arabidopsis genome harbors seven putative SHM genes, two of which (SHM1 and SHM2) feature predicted mitochondrial targeting signals. We have mapped shm1-1 to the position of the SHM1 gene (At4g37930). The mutation is due to a G rightwards arrow A transition at the 5' splice site of intron 6 of SHM1, causing aberrant splicing and a premature termination of translation. A T-DNA insertion allele of SHM1, shm1-2, and the F₁ progeny of a genetic cross between shm1-1 and shm1-2 displayed the same conditional lethal phenotype as shm1-1. Expression of wild-type SHM1 under the control of either the cauliflower mosaic virus 35S or the SHM1 promoter in shm1-1 abrogated the photorespiratory phenotype of the shm mutant, whereas overexpression of SHM2 or expression of SHM1 under the control of the SHM2 promoter did not rescue the mutant phenotype. Promoter-beta-glucuronidase analyses revealed that SHM1 is predominantly expressed in leaves, whereas SHM2 is mainly transcribed in the shoot apical meristem and roots. Our findings establish SHM1 as the defective gene in the Arabidopsis shm1-1 mutant.
The dual affinity of ribulose-1,5-bisphosphate carboxylase/oxygenase for O₂ and CO₂ results in the net loss of fixed carbon and energy in a process termed photorespiration. The photorespiratory cycle ...is complex and occurs in three organelles, chloroplasts, peroxisomes, and mitochondria, which necessitates multiple steps to transport metabolic intermediates. Genetic analysis has identified a number of mutants exhibiting photorespiratory chlorosis at ambient CO₂, including several with defects in mitochondrial serine hydroxymethyltransferase (SHMT) activity. One class of mutants deficient in SHMT1 activity affects SHM1, which encodes the mitochondrial SHMT required for photorespiration. In this work, we describe a second class of SHMT1-deficient mutants defective in a distinct gene, GLU1, which encodes Ferredoxin-dependent Glutamate Synthase (Fd-GOGAT). Fd-GOGAT is a chloroplastic enzyme responsible for the reassimilation of photorespiratory ammonia as well as for primary nitrogen assimilation. We show that Fd-GOGAT is dual targeted to the mitochondria and the chloroplasts. In the mitochondria, Fd-GOGAT interacts physically with SHMT1, and this interaction is necessary for photorespiratory SHMT activity. The requirement of protein-protein interactions and complex formation for photorespiratory SHMT activity demonstrates more complicated regulation of this crucial high flux pathway than anticipated.
In chloroplasts of green plants and algae, CO₂ is assimilated into triose-phosphates (TPs); a large part of these TPs is exported to the cytosol by a TP/ phosphate translocator (TPT), whereas some is ...stored in the plastid as starch. Plastidial phosphate translocators have evolved from transport proteins of the host endomembrane system shortly after the origin of chloroplasts by endosymbiosis. The red microalga Galdieria sulphuraria shares three conserved putative orthologous transport proteins with the distantly related seed plants and green algae. However, red algae, in contrast to green plants, store starch in their cytosol, not inside plastids. Hence, due to the lack of a plastidic starch pool, a larger share of recently assimilated CO₂ needs to be exported to the cytosol. We thus hypothesized that red algal transporters have distinct substrate specificity in comparison to their green orthologs. This hypothesis was tested by expression of the red algal genes in yeast (Saccharomyces cerevisiae) and assessment of their substrate specificities and kinetic constants. Indeed, two of the three red algal phosphate translocator candidate orthologs have clearly distinct substrate specificities when compared to their green homologs. GsTPT (for G. sulphuraria TPT) displays very narrow substrate specificity and high affinity; in contrast to green plant TPTs, 3-phosphoglyceric acid is poorly transported and thus not able to serve as a TP/ 3-phosphoglyceric acid redox shuttle in vivo. Apparently, the specific features of red algal primary carbon metabolism promoted the evolution of a highly efficient export system with high affinities for its substrates. The low-affinity TPT of plants maintains TP levels sufficient for starch biosynthesis inside of chloroplasts, whereas the red algal TPT is optimized for efficient export of TP from the chloroplast.
Soybean (Glycine max (L.) Merr.) is an important crop that provides a sustainable source of protein and oil worldwide. Soybean cyst nematode (Heterodera glycines Ichinohe) is a microscopic roundworm ...that feeds on the roots of soybean and is a major constraint to soybean production. This nematode causes more than US$1 billion in yield losses annually in the United States alone, making it the most economically important pathogen on soybean. Although planting of resistant cultivars forms the core management strategy for this pathogen, nothing is known about the nature of resistance. Moreover, the increase in virulent populations of this parasite on most known resistance sources necessitates the development of novel approaches for control. Here we report the map-based cloning of a gene at the Rhg4 (for resistance to Heterodera glycines 4) locus, a major quantitative trait locus contributing to resistance to this pathogen. Mutation analysis, gene silencing and transgenic complementation confirm that the gene confers resistance. The gene encodes a serine hydroxymethyltransferase, an enzyme that is ubiquitous in nature and structurally conserved across kingdoms. The enzyme is responsible for interconversion of serine and glycine and is essential for cellular one-carbon metabolism. Alleles of Rhg4 conferring resistance or susceptibility differ by two genetic polymorphisms that alter a key regulatory property of the enzyme. Our discovery reveals an unprecedented plant resistance mechanism against a pathogen. The mechanistic knowledge of the resistance gene can be readily exploited to improve nematode resistance of soybean, an increasingly important global crop.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ustilago maydis, a basidiomycete, is a model organism among phytopathogenic fungi. A physical map of U. maydis strain 521 was developed from bacterial artificial chromosome (BAC) clones. BAC ...fingerprints used polyacrylamide gel electrophoresis to separate restriction fragments. Fragments were labeled at the HindIII site and codigested with HaeIII to reduce fragments to 50-750 bp. Contiguous overlapping sets of clones (contigs) were assembled at nine stringencies (from P less than or equal to 1 x 10(-6) to 1 x 10(-24)). Each assembly nucleated contigs with different percentages of bands overlapping between clones (from 20% to 97%). The number of clones per contig decreased linearly from 41 to 12 from P less than or equal to 1 x 10(-7) to 1 x 10(-12). The number of separate contigs increased from 56 to 150 over the same range. A hybridization-based physical map of the same BAC clones was compared with the fingerprint contigs built at P less than or equal to 1 x 10(-7). The two methods provided consistent physical maps that were largely validated by genome sequence. The combined hybridization and fingerprint physical map provided a minimum tile path composed of 258 BAC clones (18-20 Mbp) distributed among 28 merged contigs. The genome of U. maydis was estimated to be 20.5 Mbp by pulsed-field gel electrophoresis and 24 Mbp by BAC fingerprints. There were 23 separate chromosomes inferred by both pulsed-field gel electrophoresis and fingerprint contigs. Only 11 of the tile path BAC clones contained recognizable centromere, telomere, and subtelomere repeats (high-copy DNA), suggesting that repeats caused some false merges. There were 247 tile path BAC clones that encompassed about 17.5 Mbp of low-copy DNA sequence. BAC clones are available for repeat and unique gene cluster analysis including tDNA-mediated transformation. Program FingerPrint Contigs maps aligned with each chromosome can be viewed at http://www.siu.edu/~meksem/ustilago_maydis/.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
To test the function of candidate genes in soybean for resistance to the soybean cyst nematode (SCN), a large collection of EMS-mutants from the SCN-resistant soybean cultivar “Forrest” was developed ...for Targeting Induced Local Lesions IN Genomes (TILLING). Additionally, due to the complexity of the soybean genome, an integrated set of genomic and genetic analysis tools was employed to complement the TILLING approach. The efficiency of this integrated set of tools was tested using a candidate soybean gene for resistance to SCN, encoding a leucine-rich repeat receptor-like kinase (LRR-RLK) that was identified by map-based cloning at the Rhg4 locus. The Rhg4 locus is one of the major quantitative trait loci controlling soybean resistance against SCN race 3 (HG type 0) in cv. Forrest, but the gene(s) sequence for resistance remains to be determined. Using TILLING, a Forrest mutant containing a nonsense mutation in the LRR domain of the candidate resistance protein was identified and confirmed; however, the SCN-resistant phenotype of the mutant was not altered. Haplotyping and EcoTILLING of recombinant inbred lines along with complementation analysis corroborated the TILLING result and ruled out the possibility of functional redundancy by a second copy of the LRR-RLK gene identified in the soybean genome. This study validates the use of TILLING, in combination with an integrated set of genomic tools, as an efficient means of testing candidate genes for SCN resistance in soybean.
The transport of 14Cglycyl-glycine (Gly-Gly) has been characterized in leaf discs from mature exporting leaves of broad bean (Vicia faba L.). In terms of glycine (Gly) equivalents, the rate of ...transport of Gly-Gly was similar to that of Gly uptake. Uptake of Gly-Gly was localized mainly in the mesophyll cells, with little accumulation in the veins. It was optimal at pH 6.0, sensitive to thiol reagents and metabolic inhibitors, and exhibited a single saturable phase with an apparent Michaelis constant of 16 mM. Gly-Gly did not inhibit the uptake of labeled Gly. Addition of Gly-Gly induced a concentration-dependent pH rise in the medium, showing that peptide uptake is mediated with proton co-transport. Gly-Gly also induced a concentration-dependent transmembrane depolarization of mesophyll cells with an apparent Michaelis constant of 15 mM. This depolarization was followed by a transient hyperpolarization. When present at a 10-fold excess, various peptides and tripeptides were able to inhibit Gly-Gly uptake with the following decreasing order of efficiency: Gly-Gly-Gly = leucine-Gly > Gly-tyrosine > Gly-glutamine = Gly-glutamic acid > Gly-phenylalanine > Gly-threonine > Gly-aspartic acid = Gly-asparagine aspartic acid-Gly. Gly inhibited the uptake of Gly-Gly only slightly, whereas tetraGly and the tripeptide glutathione were not inhibitory. The dipeptides inhibiting Gly-Gly uptake also induced changes in the transmembrane potential difference of mesophyll cells and were able to affect in a complex way the response normally induced by Gly-Gly. Altogether, the data demonstrate the existence of a low-affinity, broad-specificity H+/peptide co-transporter at the plasma membrane of mesophyll cells. The physiological importance of this transporter for the exchange of nitrogenous compounds in mature leaves remains to be determined, as do the details of the electrophysiological events induced by the dipeptides.
The mature petiole of celery is an organ with versatile sink/source capacities where sucrose and mannitol are unloaded from and reloaded into the phloem cells. Plasma-membrane vesicles were purified ...by two-phase partitioning either from phloem strands isolated from mature petioles of celery (Apium graveolens L.) or from mature petioles devoid of vascular bundles. Both types of vesicle were comparable in purity (more than 86% of plasma-membrane origin), size (135 nm diameter) and orientation (72% right-side-out). Plasma-membrane vesicles from phloem tissues had a higher vanadate-sensitive ATPase activity than plasma-membrane vesicles from petioles. Plasma-membrane vesicles from phloem tissues accumulated mannitol and sucrose in response to an artificial proton-motive force, in agreement with the existence of proton/substrate carriers. Plasma-membrane vesicles from petioles devoid of vascular bundles accumulated only mannitol following application of an artificial proton-motive force. The data suggest the involvement of apoplasmic transport events. The pathway for sucrose uptake in storage parenchyma cells is discussed in the light of the available physiological data.
When we think of extremophiles, organisms adapted to extreme environments, prokaryotes come to mind first. However, the unicellular red micro-alga Galdieria sulphuraria (Cyanidiales) is a eukaryote ...that can represent up to 90% of the biomass in extreme habitats such as hot sulfur springs with pH values of 0-4 and temperatures of up to 56 degrees C. This red alga thrives autotrophically as well as heterotrophically on more than 50 different carbon sources, including a number of rare sugars and sugar alcohols. This biochemical versatility suggests a large repertoire of metabolic enzymes, rivaled by few organisms and a potentially rich source of thermo-stable enzymes for biotechnology. The temperatures under which this organism carries out photosynthesis are at the high end of the range for this process, making G. sulphuraria a valuable model for-physical studies on the photosynthetic apparatus. In addition, the gene sequences of this living fossil reveal much about the evolution of modern eukaryotes. Finally, the alga tolerates high concentrations of toxic metal ions such as cadmium, mercury, aluminum, and nickel, suggesting potential application in bioremediation. To begin to explore the unique biology of G. sulphuraria, 5270 expressed sequence tags from two different cDNA libraries have been sequenced and annotated. Particular emphasis has been placed on the reconstruction of metabolic pathways present in this organism. For example, we provide evidence for (i) a complete pathway for lipid A biosynthesis; (ii) export of triose-phosphates from rhodoplasts; (iii) and absence of eukaryotic hexokinases. Sequence data and additional information are available at http://genomics.msu.edu/galdieria.
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