Angela C. Delves

Extended target-site specificity for a hammerhead ribozyme.

In vitro mutagenesis has been used to systematically mutate the GUC target site cleaved by a synthetic ribozyme based on the catalytic domain of the satellite RNA of tobacco ringspot virus. Amongst the spectrum of changes, it is found that GUC, UUC, CUC, GUA and GUU targets show equivalent rates of cleavage. An AUC target does not cleave, in contrast to observations from other studies. For a GUG target site, the normal ribozyme cannot induce cleavage, but an alteration of the stem-loop in the catalytic domain leads to the formation of a weakly active ribozyme. Certain double mutations, not previously studied, showed slow but discernable cleavage. This mutational approach shows that general rules for cleavage at NUY triplets for the target site of hammerhead ribozymes should be modified. Not all NUY targets cleave under all circumstances, and there are some targets with nucleotides other than U in the centre position which show significant, discernable cleavage.

Plant Genetic Approaches to Symbiotic Nodulation and Nitrogen Fixation in Legumes

We happily accepted the invitation to write this chapter because we have experienced the development of new approaches to research into the genetics, microbiology and biochemistry of symbiotic nitrogen fixation. We were confronted with a broad range of advances in the biochemical genetics of Rhizobium, especially relating to the genetic elements controlling the key symbiotic phenotypes: nitrogen fixation (the nif and fix genes) and nodulation (controlled by the nod genes). Likewise there has been a major application of plant molecular biology to the analysis of nodule-specific or nodule-enhanced plant gene products (Lee et al., 1983; Fuller et al., 1983; Govers et al., 1985; and Kaninakis and Verma, 1985). This major class of proteins or nodulins allows the molecular visualisation of the plant genome’s contribution to the symbiosis (Verma et al., 1985).

Biochemistry and physiology of esterases in organophosphate-susceptible and -resistant strains of the Australian sheep blowfly, Lucilia cuprina

Abstract Eight esterases identified using native polyacrylamide gel electrophoresis were characterized in a susceptible and an organophosphate-resistant strain of Lucilia cuprina. Developmental profiles and tissue distributions were determined for all eight esterases and substrate and inhibitor specificities were also determined for six of them. Esterases E1 and E2 were isozymes of acetylcholinesterase, with E1 present throughout development and E2 largely confined to adults. Esterases E8, which was mainly confined to the neuromuscular and digestive tissues of early larvae, and E7, which was only recovered from 2- to 3-day-old adults, were not abundant enough for biochemical analysis. E4 and E13, classified as carboxylesterases, were only moderately sensitive to inhibition by organophosphates and found mainly in hemolymph. E3 and E9 were also identified as carboxylesterases but were highly sensitive to inhibition by organophosphates. They were the only enzymes found to differ between the two strains, both being detected only in the organophosphate-susceptible strain. In this strain they were both abundant in pupae, and E3 was also abundant in larvae and sexually mature adults, localized mainly in digestive tissues and the adult reproductive tract. The inhibitor, developmental, and tissue specificities of E3 all support genetic data reported previously that E3 is encoded by the major organophosphate resistance locus, ROP-1. While genetic data are not available for E9, its concentration in pupae suggests that it is unlikely to confer major gene resistance in feeding stages of L. cuprina.

Asymmetric somatic hybrid plants between Medicago sativa L. (alfalfa, lucerne) and Onobrychis viciifolia Scop. (sainfoin)

This paper reports on the production of intergeneric somatic hybrid plants between two sexually incompatible legume species. Medicago sativa (alfalfa, lucerne) leaf protoplasts were inactivated by lethal doses of iodoacetamide. Onobrychis viciifolia (sainfoin) suspension-cell protoplasts were gamma-irradiated at lethal doses. Following electrofusion under optimized conditions about 50,000 viable heterokaryons were produced in each test. The fusion products were cultured with the help of alfalfa nurse protoplasts. Functional complementation permitted only the heterokaryons to survive. A total of 706 putative heterokaryon-derived plantlets were regenerated and 570 survived transplantation to soil. Experimentation was aimed at the introduction of proanthocyanidins (condensed tannins) from sainfoin, a bloat-safe plant, to alfalfa, a bloat-causing forage crop; however, no tannin-positive regenerant plants were detected. Most regenerant plants have shown morphological differences from the fusion parents, although, as expected, all resembled the “recipient” parent, alfalfa. Southern analysis using an improved total-genomic probing technique has shown low levels of sainfoin-specific DNA in 43 out of 158 tested regenerants. Cytogenetic analysis of these asymmetric hybrids has confirmed the existence of euploid (2n=32; 17%) as well as aneuploid (2n=30, 33–78; 83%) plants. Pollen germination tests have indicated that the majority of the hybrids were fertile, while 35% had either reduced fertility or were completely sterile.

Genetic analysis and complementation studies on a number of mutant supernodulating soybean lines

Genetic analysis was done on a number of nitrate tolerant supernodulating (nts) mutant soybean lines. These lines are altered in the autoregulation response, and each was isolated as a separate mutational event following chemical mutagenesis. Crosses were made betweennts lines on a diallel pattern, and each was also crossed usingnts lines as female parent, to wild-type nodulation cultivars. F1 and F2 data were analysed from each cross for nodulation type and number. No complementation was noted wherents lines were intercrossed, suggesting that in each line the same gene was affected. Wherents lines were crossed with wild-type cultivars all the F1 progeny were wild-type, confirming that thenls gene is recessive and, with one exception,nts 1116, all of the F2 progeny segregated into a 3:1 wild-type to supernodulating phenotype, indicating that a single gene is involved. The hypernodulating linents 1116 gave a 1:1 ratio in its F2 progeny when crossed with othernts lines. This line behaved as a dominant in the latter crosses. No wild-type segregants were recovered, therefore again no complementation look place. This line may be a leaky mutant with partial autoregulation as its segregation ratios do not fall into any of the obvious patterns.

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.

Supernodulation in Interspecific Grafts between Glycinemax (Soybean) and Glycine soja

Summary Nitrate tolerant symbiosis (nts) soybean Glycine max (L.) Merr. mutants, altered in theirautoregulation response to allow extremely high (supernodulating) levels of nodulation were used in grafting experiments with the normal wild-type variety Bragg and a closely related species Glycine soja Sieb. and Zucc. Where the nts mutant was used as the shoot, supernodulation levels of nodule numbers were seen on both G. soja and soybean roots. Where either G. soja or the wild-type Bragg was used as the shoot, normally autoregulated, lower levels of nodulation were seen on all root types. These data confirm the shoot control of supernodulation, and that this phenomenon can be induced on another legume species by a mutant Glycine max shoot. This may suggest a similar method of chemical signalling to induce autoregulation amongst legumes.

Plant Host Genetics of Nodulation and Symbiotic Nitrogen Fixation in Pea and Soybean

The need to study the plant functions controlling nodulation and symbiotic nitrogen fixation has received progressive recognition and attention. Comprehensive reviews of recent advances can be found in LaRue et al (1985) as well as Miflin and Cullimore (1984) and Verma and Nadler (1984).

Inheritance of supernodulation in soybean and estimation of the genetically effective cell number

SummaryProvided the nature of inheritance is known, the frequency of homozygous mutant plants in individual M2 families (derived from M1 seed) can be used to estimate the genetically effective cell number (GECN). Segregation ratios in M3 families derived from M2 wild-type plants indicated that the supernodulation characters nts382, nts1007 and nts183 are inherited as Mendelian recessives. The nature of inheritance was also known or confirmed to be recessive by crossing the wild type to these and several other mutants derived from the same population of M2 families. Subsequently, using the frequency of mutant plants in individual M2 families, the GECN for soybean was calculated to be approximately two.

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).