Cyanoglucoside biosynthesis in white clover (Trifolium repens)

l-valine-(U-¹⁴C), isobutyraldoxime-(U-¹⁴C) and 2-hydroxyisobutyraldoxime-(1,3-¹⁴C) were fed to white clover shoots which contained or lacked cyanoglucoside. Labelling results indicated that plants which were unable to synthesize linamarin from these precursors lacked the ability to glucosylate 2-hydroxyisobutyronitrile. When l-valine-(U-¹⁴C) was fed to cyanoglucoside-producing plants in the presence of cold isobutyraldoxime, radioactivity could be trapped in this compound. No radioactivity was trapped in isobutyraldoxime fed to non-cyanoglucoside-producing shoots demonstrating that this phenotype also lacks the ability to convert valine into the aldoxime. This suggests that the non-cyanoglucoside-producing plants have at least two steps in the biosynthesis of linamarin missing. Differences between the linamarin biosynthetic enzymes of white clover and flax were demonstrated using the inhibitor O-methylthreonine and by the failure of cyanoglucoside-producing white clover shoots to synthesize linamarin from fed 2-hydroxyisobutyraldoxime.     

In vitro characterization of the Ac locus in white clover (Trifolium repens L.)

Microsomal fractions from developing shoots of adult white clover plants (of genotype AcAc) and cotyledons of dark germinated clover seedlings can synthesize 2-hydroxy-2-methylpropanenitrile and 2-hydroxy-2-methylbutanenitrile, the aglycone precursors of the cyanogenic glucosides, linamarin and lotaustralin, from various precursors in the presence of NADPH. l-Valine, 2-methylpropanal oxime, and 2-methylpropanenitrile are converted to 2-hydroxy-2-methylpropanenitrile and are detected as cyanide and acetone. l-Isoleucine and 2-methylbutanal oxime are converted to 2-hydroxy-2-methylbutanenitrile and are detected as cyanide and 2-butanone. At least two steps in these conversions are missing in microsomes from plants of genotype acac.     

The response to selection for increased nodule tissue in white clover ( Trifolium repens L.)

Summary S. 100 white clover was selected over three generations for increased total nodule volume. In S₂, large plants which had a small nodule volume were also selected. A third selection line was made by crossing the S₂ selections for large and small nodule volume, this being referred to as thelarge×small selection. Progeny assessment in test tube culture and in sterile sand in open cartons gave similar results. Selection for increased nodule volume was successful for two generations but regressed in the third. Selection for small nodule volume in S₂ restored nodule volume to its S₁ level, whereas thelarge×small crosses were almost identical to the S₃ large selection. The increase in total volume was due to an increased number of nodules, which were smaller on average than in the original population, and symbiotic efficiency generally declined with selection. The results indicate that the size of the individual nodule unit is relevant to the efficiency of the symbiosis and this is discussed in relation to breeding of an improved variety of white clover.     

Multivariate analysis of intraspecific responses to UV-B radiation in white clover (Trifolium repens L.)

White clover (Trifolium repens L.) is experiencing increased levels of ultraviolet‐B (UV‐B) radiation in temperate pastures due to the depletion of the stratospheric ozone layer. Based on 17 morphological, morphogenetic and physiological attributes, this study analysed the consequences of enhanced UV‐B on 26 white clover populations using principal components analysis (PCA). After 18 d of exposure to 13·3 kJ m ^{? 2} d ^{? 1} UV‐B in controlled environments, UV‐B significantly decreased above‐ground and below‐ground plant growth attributes, epidermal cell surface area and maximum quantum efficiency of photosystem II photochemistry (F_v/F_m). Aspects of cell division and cell expansion both were negatively affected by UV‐B. Stomatal density, specific leaf mass, root‐to‐shoot ratio and levels of UV‐B‐absorbing compounds increased in response to UV‐B. In the multivariate analysis, the main dimension of UV‐B sensitivity was characterized by changes in plant growth attributes. Alterations in partitioning within and between plant organs constituted a secondary tier of UV‐B responsiveness. Plant characteristics related to UV‐B tolerance included lower growth rate, smaller epidermal cell surface area and higher UV‐B‐induced levels of UV‐B‐absorbing compounds. The results suggest overall UV‐B tolerance for slower‐growing populations from less productive habitats with higher natural UV‐B irradiance.     

Flowering and gibberellins in a mutant red clover (Trifolium pratense L.)

Relationships between gibberellins and floral initiation were investigated in a conditional non-flowering mutant of red clover, Trifolium pratense. Untreated mutant plants will not flower under long-days, but will do so when certain GAs are applied. Gibberellins, A₃, A₁, A₇, and A₅ all resulted in both stem elongation and flowering whilst GA₄ produced the elongation only. Applications of GA₂₀, GA₈ and GA₁₃ under long-days had no detectable effect. Thus, by combining the use of the mutant with the application of different GAs, the correlation between the processes of stem elongation and floral initiation, which is normally strongly expressed in this species, was broken. Endogenous gibberellins shown to be present in normal plants were also found in the mutant genotype. Gibberellins alone were not sufficient to initiate floral development in the mutant, there being an essential element of interaction with long-days. These results are discussed in relation to the nature of the lesion in the mutant and the signal provided by the applied gibberellin.     

Molecular analysis of a null mutant for pea (Pisum sativum L.) seed lipoxygenase-2

A mutant line of Pisum fulvum was identified that lacked seed lipoxygenase-2 (LOX-2). The mutant phenotype was introgressed into a standard Pisum sativum cv. Birte to provide near-isogenic lines with or without seed LOX-2. Genetic analyses showed the mutation to behave as a single, recessive Mendelian gene. Northern and dot-blot analyses showed a large reduction in LOX-2 mRNA from developing seeds of the LOX-2-null mutant. A restriction fragment length polymorphism associated with the 5 end of the LOX-2 gene(s) co-segregated with the null phenotype, indicating that the reduction of LOX-2 mRNA was neither a consequence of deletion of the LOX genes nor a consequence of the action of a genetically distant regulatory gene. Analysis of the 5-flanking sequences of LOX-2 genes from Birte and the near-isogenic LOX-2-null mutant revealed a number of insertions, deletions and substitutions within the promoter from the LOX-2-null mutant that could be responsible for the null phenotype. Incubation of crude seed LOX preparations from Birte and the LOX-2-null mutant showed that the latter generated relatively less 13-hydroperoxides and also produced relatively more hydroxy- and ketoacid compounds that have implications for the fresh-frozen pea industry.     

Inheritance of phosphorus response in white clover (Trifolium repens L.)

Genotypes of white clover that exhibited divergent responses to P were identified in a glasshouse pot trial. Six high P-responding genotypes were selected from previously identified high P-responding cultivars and 5 low P-responding genotypes were selected from previously identified low P-responding cultivars. These were crossed in a full diallel design without selfing and reciprocals were kept separate. The P-response of progeny lines was compared with parents. High P-response was dominant over low P-response with progeny from crosses between high and low P-response genotypes being similar to the high P-response parent. Reciprocal effects were not significant. The general combining abilities of high P-response genotypes were generally greater than that of the low P-response genotypes, although there were significant specific combining abilities. Narrow sense heritabilities for P response were moderate, 0.46 based on the linear coefficient and 0.33 based on the quadratic coefficient of the fitted response curves.The mode of inheritance, feasibility of manipulating differences in P response by breeding and future directions of this work are discussed.Deceased.Deceased.     

Near-full length sequencing of 16S rDNA and RFLP indicates that Rhizobium etli is the dominant species nodulating Egyptian winter Berseem clover (Trifolium alexandrinum L.)

Egyptian winter Berseem clover (EWBC) is one of the main important forage legume crops in Egypt that is used for animal feeding in winter and it occupies about 2.5 million feddans (Feddan = 4200 m²) in winter agricultural rotation systems. Forty-eight rhizobial isolates that nodulated this legume host from different geographical regions within Egypt were isolated. RFLP analyses of 16S rDNA (1.5 kb) and whole ribosomal DNA (5 kb), the sequencing of 16S rDNA, and the sequencing of nodC, nifH and house keeping genes were used to identify these isolates. The RFLP analysis of 16S rDNA (1.5 kb) among 15 representative strains with three enzymes generated two genotypes. The largest genotype was similar to Rhizobium etli CFN42T (93.33%) except for strain 902 that failed to re-nodulate EWBC. RFLP analysis of complete ribosomal DNA (5 kb) produced five genotypes. The majority of tested strains shared the genotype with R. etli CFN42T (53.33%). Only one strain (1002) shared the genotype with Rhizobium leguminosarum sv. trifolii 3023. The other four strains were comprised of two unique genotypes. Phylogenetic analysis of 16S rDNA sequences revealed that seven representative strains could be divided into two genetic clusters sharing the ancestral clad with R. etli CFN42T. A phylogenetic tree based on nodC gene sequence confirmed that all the examined strains shared the genetic lineage with R. leguminosarum sv. trifolii WSM1325. The phylogenetic trees of house keeping genes are supported strongly the identification of majority of strains as a novel symbiovar of R. etli with new lineages.     

Detection of selection in populations of white clover (Trifolium repens L.)

Seed populations of white clover polymorphic for the presence/absence of both ovariogenic glucosides and the hydrolysing enzyme linamarase, were introduced into three natural populations. Over the first six months of life a significant increase in the frequency of linamarase containing individuals occurred. Estimated selection coefficients against plants lacking linamarase were in the region of 0.3. This result may have been due to selection at the enzyme locus alone, or to selection favouring cyanogenic individuals which possess both cyanogenic glucosides and enzyme.     

Inheritance of white, black and brown coat colours in alpaca (Vicuna pacos L.)

An experimental trial of the segregation of white vs. pigmented and black vs. brown colours in alpacas was conducted at the Peruvian INIA Quimsachata Experimental Station. One hundred and forty five offspring were born from the following matings: 4 white sires × 36 white dams, 4 white sires × 39 pigmented dams, and 9 pigmented sires × 70 pigmented dams. Among these last matings were, 4 black sires × 25 black dams, 2 black sires × 20 brown dams, and 3 brown sires × 25 brown dams. Statistical tests validate that the inheritance of white is due to a single gene which is dominant over pigmentation, without any modifying effect and independent of segregation of black and brown patterns. However, the evidence does not support a simple dominant inheritance of the black vs. brown.