Multigeneic QTL: the laccase encoded within the soybean Rfs2/rhg1 locus inferred to underlie part of the dual resistance to cyst nematode and sudden death syndrome.

Multigeneic QTL present significant problems to analysis. Resistance to soybean (Glycine max (L) Merr.) sudden death syndrome (SDS) caused by Fusarium virguliforme was partly underlain by QRfs2 that was clustered with, or pleiotropic to, the multigeneic rhg1 locus providing resistance to soybean cyst nematode (SCN; Heterodera glycines). A group of five genes were found between the two markers that delimited the Rfs2/rhg1 locus. One of the five genes was predicted to encode an unusual diphenol oxidase (laccase; EC 1.10.3.2). The aim of this study was to characterize this member of the soybean laccase gene-family and explore its involvement in SDS resistance. A genomic clone and a full length cDNA was isolated from resistant cultivar 'Forrest' that were different among susceptible cultivars 'Asgrow 3244' and 'Williams 82' at four residues R/H168, I/M271, R/H330, E/K470. Additional differences were found in six of the seven introns and the promoter region. Transcript abundance (TA) among genotypes that varied for resistance to SDS or SCN did not differ significantly. Therefore the protein activity was inferred to underlie resistance. Protein expressed in yeast pYES2/NTB had weak enzyme activity with common substrates but good activity with root phenolics. The Forrest isoform may underlie both QRfs2 and rhg1.

Genomic analysis of the rhg1 locus: candidate genes that underlie soybean resistance to the cyst nematode.

The rhg1 gene or genes lie at a recessive or co-dominant locus, necessary for resistance to all Hg types of the soybean (Glycine max (L.) Merr.) cyst nematode (Heterodera glycines I.). The aim here was to identify nucleotide changes within a candidate gene found at the rhg1 locus that were capable of altering resistance to Hg types 0 (race 3). A 1.5 +/- 0.25 cM region of chromosome 18 (linkage group G) was shown to encompass rhg1 using recombination events from four near isogenic line populations and nine DNA markers. The DNA markers anchored two bacterial artificial chromosome (BAC) clones 21d9 and 73p6. A single receptor like kinase (RLK; leucine rich repeat-transmembrane-protein kinase) candidate resistance gene was amplified from both BACs using redundant primers. The DNA sequence showed nine alleles of the RLK at Rhg1 in the soybean germplasm. Markers designed to detect alleles showed perfect association between allele 1 and resistance to soybean cyst nematode Hg types 0 in three segregating populations, fifteen additional selected recombination events and twenty-two Plant Introductions. A quantitative trait nucleotide (QTN) [corrected] in the RLK at rhg1 was inferred that alters A87 to V87 in the context of H274 rather than N274. [corrected] Contiguous DNA sequence of 315 kbp of chromosome 18 (about 2 cM) contained additional gene candidates that may modulate resistance to other Hg-types including a variant laccase, a hydrogen-sodium ion antiport and two proteins of unknown function. A molecular basis for recessive and co-dominant resistance that involves interactions among paralagous disease-resistance genes was inferred that would improve methods for developing new nematode-resistant soybean cultivars.

Characterization of Glutathione Uptake in Broad Bean Leaf Protoplasts.

Transport of reduced glutathione (GSH) and oxidized glutathione (GSSG) was studied with broad bean (Vicia faba L.) leaf tissues and protoplasts. Protoplasts and leaf discs took up GSSG at a rate about twice the uptake rate of GSH. Detailed studies with protoplasts indicated that GSH and GSSG uptake exhibited the same sensitivity to the external pH and to various chemical reagents. GSH uptake was inhibited by GSSG and glutathione conjugates. GSSG uptake was inhibited by GSH and GS conjugates, and the uptake of metolachlor-GS was inhibited by GSSG. Various amino acids (L-glutamic acid, L-glutamine, L-cysteine, L-glycine, L-methionine) and peptides (glycine-glycine, glycine-glycine-glycine) affected neither the transport of GSH nor GSSG. Uptake kinetics indicate that GSH is taken up by a single saturable transporter, with an apparent Km of 0.4 mM, whereas GSSG uptake exhibits two saturable phases, with an apparent Km of 7 [mu]M and 3.7 mM. It is concluded that the plasma membrane of leaf cells contains a specific transport system for glutathione, which takes up GSSG and GS conjugates preferentially over GSH. Proton flux measurements and electrophysiological measurements indicate that GSH and GSSG are taken up with proton symport. However, a detailed analysis of these measurements suggests that the ion movements induced by GSSG differ from those induced by GSH.

Characterization of leucine-leucine transport in leaf tissues.

Uptake of the dipeptide [(3)H]Leu-Leu into leaf discs from mature broad bean (Vicia faba L.) was characterized. Uptake was maximal at pH 6.0 and appeared to be mediated by three systems with apparent K(m) values of 20 µM, 350 µM and 43 mM, respectively. Leu-Leu uptake was sensitive to N-ethylmaleimide, p-chloromercuribenzenesulphonic acid, diethylpyrocarbonate, and carbonyl-cyanide-m-chlorophenylhydrazone. Nitrate did not compete with peptide uptake, although the peptide transporter and the nitrate transporter have been reported to be homologous. The ability of leaf tissues to take up peptides strongly decreased with leaf age, and the phloem export of peptides as measured by exudation experiments was very low. It is concluded that the leaf tissues contain a peptide transporter that may take up some peptides with a high affinity, but that this transporter is not involved in the long-distance transport of nitrogen under the form of di- or tri-peptides.

Dipeptide transport in barley mesophyll vacuoles.

The transport of [14C]glycyl-glycine (Gly-Gly) across the tonoplast of isolated barley vacuoles has been characterized. Uptake of the dipeptide Gly-Gly into barley mesophyll vacuoles was strongly increased by the addition of ATP, while Mg2+ inhibited the ATP-dependent fluxes. Inhibition of the vacuolar proton pump by bafilomycin or dissipation of the delta pH had no effect on the ATP-dependent Gly-Gly uptake. Only the non-hydrolysable ATP analogue 5'-adenylylimidodiphosphate could partially substitute for ATP; ADP, GTP and UTP had no effect. Transport of Gly-Gly was saturable exhibiting an apparent Km of 49 +/- 5 mM. Uptake was inhibited by the sulphydryl reagents N-ethylmaleimide and p-chloromercuribenzenesulphonic acid. When present at 30 mM, various dipeptides as well as tri-glycine inhibited Gly-Gly (10 mM) uptake by 30-50%, whereas 5 mM phenylalanine was able to inhibit Gly-Gly uptake almost completely. Comparison with published data suggests that Gly-Gly is transferred across the tonoplast by the same system as several amino acids and inorganic ions.

Proton-Peptide Co-Transport in Broad Bean Leaf Tissues.

The transport of [14C]glycyl-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.