Kathryn A. Schuller

Suppression of the Symbiotic Supernodulation Symptoms of Soybean

Summary Supernodulation mutants of soybean ( Glycine max L. Merr.) produce very high numbers of nodules and increased nodule mass compared to the parent cultivar Bragg in the absence or presence of nitrate. All (12 were tested) mutants also display a nitrate tolerant symbiotic (nts) as well as a supernodulation phenotype suggesting that nitrate inhibition of nodulation and endogenous autoregulation are at least in part jointly controlled by plant genes. Genetic analysis suggests that single recessive mendelian alleles at a single locus are involved. Supernodulation of nts mutants was suppressed by a variety of means. These include (a) suppression by low inoculum, (b) suppression by grafting, (c) suppression by another gene (epistatic), and (d) suppression by wild type vascular sap or methanol extract refeeding. These methods show that (1) nitrate tolerance of nodulation can be expressed even if supernodulation phenotype is not expressed, (ii) non-nodulation mutants epistatically suppress supernodulation, (iii) shoots of wild-type or non-nodulation mutant soybeans, or Glycine soja Sieb. and Zucc., suppress supernodulation and (iv) methanol extracts from inoculated wild type plants (but not uninoculated wild type or mutant nts382 plants) suppress supernodulation by 60 to 80 %. We are presently using these tools and the relevant material to investigate further the genetic basis of autoregulation of nodulation in legumes.

Monovalent Cation Activation of Plant Pyruvate Dehydrogenase Kinase

The pyruvate dehydrogenase kinase-catalyzed inactivation of the pyruvate dehydrogenase complex was studied using dialyzed, soluble proteins from mitochondria purified from green leaf tissue of Pisum sativum L. seedlings. At subsaturating ATP concentrations, K+ or NH4+, but not Na+, stimulated the pyruvate dehydrogenase kinase by lowering the Km(ATP). Micromolar concentrations of NH4+ were required to produce the same effect as millimolar concentrations of K+. This is apparent from the observations that the activation constant (Kact) for NH4+ was 0.1 mM, whereas the Kact(K+) was 0.7 mM. Maximal pyruvate dehydrogenase kinase velocities attained with NH4+ were higher than those with K+, and, therefore, NH4+ was able to stimulate PDH kinase further in the presence of saturating K+. This result supports our conclusion that photorespiratory NH4+ production in plant mitochondria may be involved in regulating the entry of carbon into the Krebs cycle by way of the pyruvate dehydrogenase complex.

Effect of Short-Term N2 Deficiency on Expression of the Ureide Pathway in Cowpea Root Nodules

Root systems of 28-d-old cowpea (Vigna unguiculata L. Walp cv Vita 3: Bradyrhizobiumsp. strain CB756) plants bearing nitrogen-fixing nodules in sand culture were exposed to an atmosphere of Ar:O2 (80:20, v/v) for 48 h and then returned to air. Root systems of control plants were maintained in air throughout. Nodules were harvested at the same times in control and Ar:O2-treated root systems. Activities of two enzymes of de novo purine synthesis, glycinamide ribonucleotide transformylase (GART; EC 2.1.2.2), aminoimidazole ribonucleotide synthetase (AIRS; EC 6.3.3.1), uricase (EC 1.7.3.3), and phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) were measured together with the protein level of each using immune-specific polyclonal antibodies. AIRS activity and protein both declined to very low levels within 6 h in Ar:O2 together with a decline in transcript level of pur5, the encoding gene. GART activity, protein, and transcript (pur3) levels were relatively stable. Uricase activity declined in Ar:O2 as rapidly as AIRS activity but the protein was stable. PEPC activity showed evidence of increased sensitivity to inhibition by malate but the protein level was stable. The data indicate that the flux of fixed N from bacteroids (N2-fixing nodule bacteria) is in some way associated with transcriptional control over pur5and possibly also catabolism of AIRS protein. In contrast, there is limited posttranslational control over GART and PEPC and close posttranslational control over uricase activity. The significance of these different levels of regulation is discussed in relation to the overall control of enhanced expression of plant enzymes in the cowpea symbiosis.

Plant Host Genetics of Nodulation Initiation in Soybean

Symbiotic nitrogen fixation as exemplified by the legume-Rhizobium (or Bradyrhizobium) root nodule interaction is a well researched phenomenon illustrating plant-microbe interaction. The functional nitrogen fixing symbiosis requires cooperation between the bacterium and the plant. The last decade has witnessed a rapid expansion of the definition of Rhizobium genes that are involved in the symbiosis. The plant’s contribution, although always recognised as being important, recently received more attention through two major developments. The first was the application of DNA technology to the analysis of gene expression of legume symbiotic genes (Verma et al 1985) and the second was the realisation that existant plant variability may be insufficient in many legumes to permit the isolation of symbiotically defective germplasm. For this reason, research with Pisum sativum (Feenstra and Jacobson, 1985) (LaRue et al, 1985), Cicer arietinum (Davies et al, 1985) and Glycine max (our laboratory) has concentrated on the isolation of symbiotic mutants after induced mutagenesis (Carroll et al, 1985a,b).