Kinetic Basis for the Conjugation of Auxin by a GH3 Family Indole-acetic Acid-Amido Synthetase

The GH3 family of acyl-acid-amido synthetases catalyze the ATP-dependent formation of amino acid conjugates to modulate levels of active plant hormones, including auxins and jasmonates. Initial biochemical studies of various GH3s show that these enzymes group into three families based on sequence relationships and acyl-acid substrate preference (I, jasmonate-conjugating; II, auxin- and salicylic acid-conjugating; III, benzoate-conjugating); however, little is known about the kinetic and chemical mechanisms of these enzymes. Here we use GH3-8 from Oryza sativa (rice; OsGH3-8), which functions as an indole-acetic acid (IAA)-amido synthetase, for detailed mechanistic studies. Steady-state kinetic analysis shows that the OsGH3-8 requires either Mg²⁺ or Mn²⁺ for maximal activity and is specific for aspartate but accepts asparagine as a substrate with a 45-fold decrease in catalytic efficiency and accepts other auxin analogs, including phenyl-acetic acid, indole butyric acid, and naphthalene-acetic acid, as acyl-acid substrates with 1.4–9-fold reductions in k_cat/K_m relative to IAA. Initial velocity and product inhibition studies indicate that the enzyme uses a Bi Uni Uni Bi Ping Pong reaction sequence. In the first half-reaction, ATP binds first followed by IAA. Next, formation of an adenylated IAA intermediate results in release of pyrophosphate. The second half-reaction begins with binding of aspartate, which reacts with the adenylated intermediate to release IAA-Asp and AMP. Formation of a catalytically competent adenylated-IAA reaction intermediate was confirmed by mass spectrometry. These mechanistic studies provide insight on the reaction catalyzed by the GH3 family of enzymes to modulate plant hormone action.     

Path analysis of natural selection on plant chemistry: the xylem resin of ponderosa pine

We analyzed the pattern of correlations among fitness components, herbivory, and resin characteristics in a natural all-aged stand of ponderosa pine, to infer the strength and mechanism of natural selection on plant chemistry. Male and female cone production were monitored yearly for 15 years, and levels of herbivory for 9 years in 165 trees. Resin flow rate and monoterpene composition were determined for these same trees. Multiple regression of fitness components on resin characteristics showed significant associations consistent with directional selection for increased resin flow rates and increased proportions of α- and β-pinene, myrcene and terpinolene. However, negative correlations among monoterpene fractions of the resin constrained the overall selection. Selective herbivory by aphids approached statistical significance and monoterpenes showed some (non-significant) effect as deterrents against deer browse. Resin characteristics were not correlated with attack by cone insects or porcupines. However, the association between resin characteristics and fitness is significantly different from that predicted by the path coefficients involving herbivores. Therefore the hypothesis that these herbivores mediate selection on the resin is not supported by the observed pattern of correlations among resin characteristics, herbivory, growth and fecundity. In this population, most of the association between resin characteristics and fitness appears to be mediated by some other factor independent of attack by herbivore species present. Received: 18 March 1996/Accepted: 18 July 1996     

The nuclear pores of early meiotic prophase nuclei of plants

Summary Pollen mother cells at early meiotic prophase fromFritillaria lanceolata, F. mutica, Tulbaghia violacea, the lily “Formobel”,Triticum aegilopoides, T. dicoccoides, T. aestivum and synaptic and asynaptic forms ofT. durum were studied in thin sections with the electron microscope (a) in relation to distribution of nuclear pores (b) in respect of fine structure of the pore complex in those of the first four. The pores were distributed in random clusters during leptotene to pachytene in all plants, except in the two forms ofT. durum where there were either no pores or so few that they were not detectable. Probably correlated with this, the two membranes of the nuclear envelope were often widely separated and frequently sacculated. No pores were seen at leptotene in the part of the envelope to which, in theFritillarias and lily, the nucleolus was adpressed at this time. Evidence supporting a recent model which proposes that annuli are composed of three rings of eight granular subunits was obtained. These subunits as well as a dense central element, observed in most pores, were composed of filaments about 3 nm in diameter and evidently protein in character. There was evidence of a continuity between filaments in the central element and those in the rings of subunits which encircle the pore aperture at both the nuclear and cytoplasmic sides of the pore. In profiles of pores knobbed filaments were sometimes seen extending laterally from the pore wall into the perinuclear space at two sides. Questions concerning the role of the annulus are discussed. The author wish to thank Mr. R. F. Scott for construction to the model.     

Senescence in cut carnation flowers: Temporal and physiological relationships among water status, ethylene, abscisic acid and membrane permeability

Changes in water status, membrane permeability, ethylene production and levels of abscisic acid (ABA) were measured during senescence of cut carnation flowers ( Dianthus caryophyllus L. cv. White Sim) in order to clarify the temporal sequence of physiological events during this post-harvest period. Ethylene production and ABA content of the petal tissue rose essentially in parallel during natural senescence and after treatment of young flowers with exogenous ethylene, indicating that their syntheses are not widely separated in time. However, solute leakage, reflecting membrane deterioration, was apparent well before the natural rise in ethylene and ABA had begun. In addition, there were marked changes in water status of the tissue, including losses in water potential (ψ_w), and turgor (ψ_p), that preceded the rise in ABA and ethylene. As senescence progressed, ψ_w continued to decline, but ψ_p returned to normal levels. These temporal relationships were less well resolved when senescence of young flowers was induced by treatment with ethylene, presumably because the time-scale had been shortened. Thus changes in membrane permeability and an associated water stress in petal tissue appear to be earlier symptoms of flower senescence than the rises in ABA or ethylene. These observations support the contention that the climacteric-like rise in ethylene production is not the initial or primary event of senescence and that the rise in ABA titre may simply be a response to changes in water status.     

Consumer pressure, seed versus safe-site limitation, and plant population dynamics

Plants often suffer reductions in fecundity due to insect herbivory. Whether this loss of seeds has population-level consequences is much debated and often unknown. For many plants, particularly those with long-lived seedbanks, it is frequently asserted that herbivores have minimal impacts on plant abundance because safe-site availability rather than absolute seed number determines the magnitude of future plant recruitment and hence population abundance. However, empirical tests of this assertion are generally lacking and the interplay between herbivory, spatio-temporal variability in seed- or safe-site-limited recruitment, and seedbank dynamics is likely to be complex. Here we use a stochastic simulation model to explore how changes in the spatial and temporal frequency of seed-limited recruitment, the strength of density-dependent seedling survival, and longevity of seeds in the soil influence the population response to herbivory. Model output reveals several surprising results. First, given a seedbank, herbivores can have substantial effects on mean population abundance even if recruitment is primarily safe-site-limited in either time or space. Second, increasing seedbank longevity increases the population effects of herbivory, because annual reductions in seed input due to herbivory are accumulated in the seedbank. Third, population impacts of herbivory are robust even in the face of moderately strong density-dependent seedling mortality. These results imply that the conditions under which herbivores influence plant population dynamics may be more widespread than heretofore expected. Experiments are now needed to test these predictions. Received: 3 November 1999 / Accepted: 15 February 2000     

Suppression of pecan and peach pathogens on different substrates using Xenorhabdus bovienii and Photorhabdus luminescens

Prior research indicated the ability of concentrated metabolites from Xenorhabdus spp. and Photorhabdus spp. to suppress a variety of peach and pecan diseases in vitro, and on detached pecan leaves or terminals. In the current study, our objectives were to (1) determine if bacterial broths (in addition to concentrated metabolites tested previously) have suppressive ability and (2) determine if metabolites or bacterial broths are active in a soil medium. In laboratory studies, two pathogens of pecan (Fusicladium effusum and Phytophthora cactorum) and one peach pathogen (Armillaria tabescens) were tested for susceptibility to Xenorhabdus bovienii (SN) and Photorhabdus luminescens (VS) bacterial broths or concentrated metabolites on three different substrates. Treatments were applied to lesions of F. effusum on terminals to ascertain any suppressive effect on sporulation, to A. tabescens in soil to determine effect on survival of mycelia, and to lesions caused by P. cactorum on pecan leaf surfaces to assess any reduction in lesion development. Acetone (the metabolite solvent), un-inoculated media (tryptic soy broth) and water were included as controls. The X. bovienii metabolite treatment was as efficacious as a commercial fungicide (fenbuconazole) in reducing sporulation of F. effusum on pecan terminals. The P. luminescens metabolite treatment also caused reduced sporulation relative to water and acetone controls but bacterial broths had no effect. In contrast, all bacterial broth and metabolite treatments suppressed lesion growth caused by P. cactorum (measured on detached leaves maintained on agar). However, in soil, only the P. luminescens metabolite treatment was suppressive to A. tabescens (this is the first report of Photorhabdus or Xenorhabdus toxicity to Armillaria spp.). This study provides a basis for further research on the use of Xenorhabdus and Photorhabdus metabolites or bacterial broth for suppression of pecan and peach diseases.     

Life history of Japanese Stypocaulon durum (Sphacelariales, Phaeophyceae)

Phenology, morphology, life history and responses to different temperature and photoperiod conditions were studied in Japanese Stypocaulon durum (Ruprecht) Okamura. Erect thalli of the species were collected year-round, but the mature thalli forming either uniloc-ular sporangia or two different types of plurilocular structures (evidently gametangia) on separate thalli were found only in winter. ln culture, an isomorphic life history is suggested for the species, alternating between a sporophyte forming unilocular sporangia and gam-etophytes forming plurilocular macro- (female) and micro- (male) gametangia. Contents of unilocular sporangia were not released, but germinated in situ, developing into erect thalli forming plurilocular gametangia. Macrogametangia released aplanogametes (oospores), but male gametangia appeared to be non-functional, although flagellated cells were once formed in the loc-uli. This is the first report of plurilocular gametangia in the species. Although the species grew well and matured under considerably lower temperature conditions than European Stypocaulon scoparium (L.) Sauvageau, its temperature requirements showed similarity to northwestern Atlantic Stypocaulon species. This supports the notion that northwestern Atlantic Stypocaulon is conspecific with S. durum.     

A critical rebuttal of the proposed division of the genus Arcobacter into six genera using comparative genomic,phylogenetic, and phenotypic criteria

The proposal to restructure the genus Arcobacter into six distinct genera was critically examined using: comparative analyses of up to 80 Epsilonproteobacterial genome sequences (including 26 arcobacters); phylogenetic analyses of three housekeeping genes and also 342 core genes; and phenotypic criteria. Genome sequences were analysed with tools to calculate Percentage of Conserved Proteins, Average Amino-acid Identity, BLAST-based Average Nucleotide Identity, in silico DNA–DNA hybridisation values, genome-wide Average Nucleotide Identity, Alignment Fractions and G + C percentages. Genome analyses revealed the genus Arcobacter sensu lato to be relatively homogenous, and phylogenetic analyses clearly distinguished the group from other Epsilonproteobacteria. Genomic distinction of the genera proposed by Pérez-Cataluña et al. [2018] was not supported by any of the measures used and a subsequent risk of strain misidentification clearly identified. Similarly, phenotypic analyses supported the delineation of Arcobacter sensu lato but did not justify the position of the proposed novel genera. The present polyphasic taxonomic study strongly supports the continuance of the classification of “aerotolerant campylobacters” as Arcobacter and refutes the proposed genus-level subdivision of Pérez-Cataluña et al. [2018].