Stress Responses in Alfalfa (Medicago sativa L.) : V. Constitutive and Elicitor-Induced Accumulation of Isoflavonoid Conjugates in Cell Suspension Cultures

The isoflavonoid conjugates medicarpin-3-O-glucoside-6″-O-malonate (MGM), afrormosin-7-O-glucoside (AG), and afrormosin-7-O-glucoside-6″-O-malonate (AGM) were isolated and characterized from cell suspension cultures of alfalfa (Medicago sativa L.), where they were the major constitutive secondary metabolites. They were also found in alfalfa roots but not in other parts of the plant. The phytoalexin medicarpin accumulated rapidly in suspension cultured cells treated with elicitor from Colletotrichum lindemuthianum, and this was subsequently accompanied by an increase in the levels of MGM. In contrast, net accumulation of afrormosin conjugates was not affected by elicitor treatment. Labeling studies with [¹⁴C]phenylalanine indicated that afrormosin conjugates were the major de novo synthesized isoflavonoid products in unelicited cells. During elicitation, [¹⁴C]phenylalanine was incorporated predominantly into medicarpin, although a significant proportion of the newly synthesized medicarpin was also conjugated. Treatment of ¹⁴C-labeled, elicited cells with l-α-aminooxy-β-phenylpropionic acid, a potent inhibitor of PAL activity in vivo, resulted in the initial appearance of labeled medicarpin of very low specific activity, suggesting that the phytoalexin could be released from a preformed conjugate under these conditions. Our data draw attention to the involvement of isoflavone hydroxylases during the constitutive and elicitor-induced accumulation of isoflavonoids and their conjugates in alfalfa cell cultures.     

Ultrastructural features of egg development in oviparae of the vetch aphid, Megoura viciae buckton

In the ovaries of the oviparous morph of the aphid, Megoura viciae, resting oocytes are located in the basal region of each germarium. During previtellogenic egg development, electron-dense spheres appear in the ooplasm. During vitellogenesis a brush border develops at the oolemma, and numerous protein and lipid-like spheres accumulate in the egg cytoplasm. Follicle cells are of two morphologically distinct types, termed 'type 1' and 'type 2' follicle cells. Unlike the more numerous 'type 1' cell, 'type 2' cells do not become patent. The acellular tunica propria exterior to follicle cell apices remains intact throughout egg development. During late vitellogenesis symbiont invasion of eggs takes place via 'receptor' cells encircling the pedicel at the posterior egg pole. These cells shrink and/or degenerate to create intercellular spaces that facilitate symbiont transmission. The end of vitellogenesis is marked by vitelline membrane formation and secretion of the chorionic layers, at which time the next egg in the ovariole undergoes final stages of previtellogenic growth and enters vitellogenesis.     

Genetic and radiation hybrid mapping of the hyperekplexia region on chromosome 5q.

Hyperekplexia, or startle disease (STHE), is an autosomal dominant neurologic disorder characterized by muscular rigidity of central nervous system origin, particularly in the neonatal period, and by an exaggerated startle response to sudden, unexpected acoustic or tactile stimuli. STHE responds dramatically to the benzodiazepine drug clonazepam, which acts at gamma-aminobutyric acid type A (GABA-A) receptors. The STHE locus (STHE) was recently assigned to chromosome 5q, on the basis of tight linkage to the colony-stimulating factor 1-receptor (CSF1-R) locus in a single large family. We performed linkage analysis in the original and three additional STHE pedigrees with eight chromosome 5q microsatellite markers and placed several of the most closely linked markers on an existing radiation hybrid (RH) map of the region. The results provide strong evidence for genetic locus homogeneity and assign STHE to a 5.9-cM interval defined by CSF1-R and D5S379, which are separated by an RH map distance of 74 centirays (roughly 2.2-3.7 Mb). Two polymorphic markers (D5S119 and D5S209) lie within this region, but they could not be ordered with respect to STHE. RH mapping eliminated the candidate genes GABRA1 and GABRG2, which encode GABA-A receptor components, by showing that they are telomeric to the target region.     

Conserved histidine residues in soybean lipoxygenase: functional consequences of their replacement.

Sequences of 13 lipoxygenases from various plant and mammalian species, thus far reported, display a motif of 38 amino acid residues which includes 5 conserved histidines and a 6th histidine about 160 residues downstream. These residues occur at positions 494, 499, 504, 522, 531, and 690 in soybean lipoxygenase isozyme L-1. Since the participation of iron in the lipoxygenase reaction has been established and existing evidence based on M?ssbauer and EXAFS spectroscopy suggests that histidines may be involved in iron binding, the effect of the above residues has been examined in soybean lipoxygenase L-1. Six singly mutated lipoxygenases have been produced in which each of the His residues has been replaced with glutamine. Two additional mutants have been constructed wherein the codons for His-494 and His-504 have been replaced by serine codons. All of the mutant lipoxygenases, which were obtained by expression in Escherichia coli, have mobilities identical to that of the wild-type enzyme on denaturing gel electrophoresis and respond to lipoxygenase antibodies. The mutated proteins H499Q, H504Q, H504S, and H690Q are virtually inactive, while H522Q has about 1% of the wild-type activity. H494Q, H494S, and H531Q are about 37%, 8%, and 20% as active as the wild type, respectively. His-517 is conserved in the several lipoxygenase isozymes but not in the animal isozymes. The mutant H517Q has about 33% of the wild-type activity. The inactive mutants, H499Q, H504Q, H504S, and H690Q, become insoluble when heated for 3 min at 65 degrees C, as does H522Q. The other mutants and the wild-type are stable under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for two Ca2(+)-mobilizing purinoceptors on rat hepatocytes.

Aequorin measurements of cytosolic free Ca2+ in single rat hepatocytes show that ADP and ATP, thought to act through the same P2Y purinoceptor, elicited very different responses in the majority of cells tested. ADP invariably induced transients of short duration (approx. 9 s), whereas ATP induced either similar transients or transients with a much longer duration (approx. 49 s). We explain this variability in terms of two separate purinoceptors on rat hepatocytes, one of which responds to either ATP or ADP to generate free-Ca2+ transients of short duration, and the other responds to ATP only, with transients of longer duration.     

Small-scale environmental heterogeneity and the analysis of species distributions along gradients

Abstract. The observed distribution of a species along an environmental gradient is strongly affected by environmental variability within a quadrat. Because a quadrat does not represent a point along an environmental gradient, but rather a range of conditions, it is likely to contain species not typically associated with the mean conditions in the quadrat. Systematic relationships exist between a species' true distribution, the observed distribution as a function of mean quadrat environment, and the frequency distribution of the environment within that quadrat. The observed species habitat breadth increases and the observed maximum abundance decreases as within-quadrat environmental heterogeneity increases. If species distributions or beta diversities are to be compared among species or coenoclines, they should be correctedforintra-quadratheterogeneity.Wederive simple corrections for environmental heterogeneity. The distributions of hardwood forest understory species along a soil acidity gradient in the North Carolina piedmont are presented as an example.     

Ventilatory effects of substance P, vasoactive intestinal peptide, and nitroprusside in humans.

Animal studies suggest that the neuropeptides, substance P and vasoactive intestinal peptide (VIP), may influence carotid body chemoreceptor activity and that substance P may take part in the carotid body response to hypoxia. The effects of these peptides on resting ventilation and on ventilatory responses to hypoxia and to hypercapnia have been investigated in six normal humans. Infusions of substance P (1 pmol.kg-1.min-1) and of VIP (6 pmol.kg-1.min-1) were compared with placebo and with nitroprusside (5 micrograms.kg-1.min-1) as a control for the hypotensive action of the peptides. Both peptides caused significantly less hypotension than nitroprusside. Substance P and nitroprusside caused significantly greater increases in ventilation and in the hypoxic ventilatory response than VIP. No changes were seen in hypercapnic sensitivity. The stimulation of ventilation and the differential effects on ventilatory chemosensitivity that accompanied hypotension are consistent either with stimulation of carotid body chemoreceptor activity or with an interaction with peripheral chemoreceptor input to the respiratory center, as is seen in animals. The similar cardiovascular but different ventilatory effects of the peptides suggest that substance P may also stimulate the carotid body in a manner independent of the effect of hypotension. This is consistent with a role of substance P in the hypoxic ventilatory response in humans.     

Kinetic disposition of an aqueous formulation of alphacypermethrin applied to the dorsal mid-line of sheep with long wool and its effect on lice

A group of 5 adult Merino sheep with fleeces about 70 mm long (7-months growth of wool) was treated with a topical formulation of the synthetic pyrethroid insecticide, alphacypermethrin, applied to the dorsal mid-line. Insecticide concentrations at the tip, middle and base of wool staples collected from meridians along the back, upper and lower flanks were measured at intervals from 1 to 98 days after treatment. Some movement of the alphacypermethrin from the back to the lower body occurred within 24h after treatment, but despite careful application of the insecticide there was wide variation in the concentration between and within meridians. The majority of the alphacypermethrin remained close to the dorsal mid-line and near the tip of the staple. There were significant differences in the concentration between the tip, middle and base segments of the staples in the back and lower flank meridians (P < 0.05). Despite exposure of the sheep to normal weathering, there was no significant difference in the concentration of alphacypermethrin between samples collected at day 1 or day 98 after treatment (P > 0.05). Numbers of pyrethroid-susceptible lice surviving exposure in vitro for 20 h differed significantly between samples collected at different times after treatment (P < 0.05). The numbers of lice surviving in samples collected within 28 days after treatment tended to be lower than in those collected from 28 to 98 days but, in some samples, regardless of time after treatment, lice survived for 20 h in wool taken from parts of the fleece that contained high concentrations of alphacypermethrin.     

Functional dissection of a bean chalcone synthase gene promoter in transgenic tobacco plants reveals sequence motifs essential for floral expression

Expression of chalcone synthase (CHS), the first enzyme in the flavonoid branch of the phenylpropanoid biosynthetic pathway in plants, is induced by developmental cues and environmental stimuli. We used plant transformation technology to delineate the functional structure of the French bean CHS15 gene promoter during plant development. In the absence of an efficient transformation procedure for bean, Nicotiana tabacum was used as the model plant. CHS15 promoter activity, evaluated by measurements of -d-glucuronidase (GUS) activity, revealed a tissue-specific pattern of expression similar to that reported for CHS genes in bean. GUS activity was observed in flowers and root tips. Floral expression was confined to the pigmented part of petals and was induced in a transient fashion. Fine mapping of promoter cis-elements was accomplished using a set of promoter mutants generated by unidirectional deletions or by site-directed mutagenesis. Maximal floral and root-specific expression was found to require sequence elements located on both sides of the TATA-box. Two adjacent sequence motifs, the G-box (CACGTG) and H-box (CCTACC(N)₇CT) located near the TATA-box, were both essential for floral expression, and were also found to be important for root-specific expression. The CHS15 promoter is regulated by a complex interplay between different cis-elements and their cognate factors. The conservation of both the G-box and H-box in different CHS promoters emphasizes their importance as regulatory motifs.