Auxin-binding-protein antibodies and peptides influence stomatal opening and alter cytoplasmic pH

Previous work has shown that stomatal opening induced by indole-3-acetic acid (IAA) in epidermal strips of the orchid Paphiopedilum tonsum L. is preceded by a reduction in cytoplasmic pH (pH_i) of the guard cells. We now report that Fab fragments of an auxin-agonist antibody (D16), directed against a putative auxin-binding domain of the auxin-binding protein ABP1, induce stomatal opening and decrease guard-cell pH_i, as monitored with the acetomethoxy ester of the ratiometric pH indicator Snarf-1. Similar activity was shown by a monoclonal antibody against the same domain. The C-terminal dodecapeptide, Pz152–163 of maize ABP1 (ABPzm1) induced guard-cell alkalinization and closed stomata, as did Fab fragments of a monoclonal antibody (MAC 256) recognising the C-terminal region of ABPzm1. By implicating, for the first time, an auxin-binding protein in mediation of an auxin-dependent physiological response, these findings strongly support an auxin-receptor role for ABP1. Received: 23 December 1997 / Accepted: 16 January 1998     

The topical niacin sensitivity test: an inter- and intra-rater reliability study in healthy controls

Topical application of nicotinic acid results in erythema, and in some cases oedema of the skin, supporting a strong relationship between niacin sensitivity and prostaglandin D2. The aim of this study was to examine the inter-rater and intra-rater reliability of a 12-min niacin sensitivity test in healthy adults. Three raters assessed the skin reaction of 12 volunteers, over 3-min intervals across four niacin concentrations (0.1, 0.01, 0.001, and 0.0001), and over six sessions. Inter-rater reliability estimates ranged from 0.85 to 0.97 for the total niacin sensitivity score. Similar inter-rater reliability estimates were found for niacin sensitivity ratings by concentration and time. Intra-rater reliability estimates ranged from 0.63 to 0.93 for the total niacin sensitivity score. These data indicate that the 12-min topical niacin sensitivity test has excellent reliability.     

Computer-assisted reconstruction of the male germ unit in pollen ofBrassica campestris

Summary In mature tricellular pollen of rapeseed,Brassica campestris L., the pair of sperm cells are held together within the common plasma membrane of the vegetative cell and are closely associated with the vegetative nucleus. Serial thin sections were cut of entire sperm cell associations of 7 pollen grains, and 3-dimensional coordinated information obtained by digitization. Precise lengths and placement of the sperm cells and vegetative nucleus in three dimensions were computed and stereoscopic images generated and confirmed by two manually constructed 3-dimensional models. The sperm cell most closely associated with the vegetative nucleus, possessed a long tail, > 10 m in length, that penetrated through a passage in the highly convoluted nucleus. This long tail contained a forked array of microtubules in all 7 grains examined. Arrays of microtubules occurred in the second sperm cell, aligned within plasma-membrane evaginations or ridges.     

Quantitative cytology of the sperm cells of Brassica campestris and B. oleracea

Pollen grains of Brassica campestris L. var. acephala DC and B. oleracea L. were serially sectioned and examined using transmission electron microscopy to determine the three-dimensional organization of sperm cells within the microgametophyte and the quantity of membrane-bound organelles occurring within each cell. Sperm cells occur in pairs within each pollen grain, but are dimorphic, differing in size, morphology and mitochondrial content. The larger of the two sperm cells (S_vn) is distinguished by the presence of a blunt evagination, which in B. oleracea wraps around and lies within shallow furrows on the vegetative nucleus and in B. campestris can penetrate through internal enclaves of the vegetative nucleus. This sperm cell contains more mitochondria in both species than the second sperm cell (S_ua). This latter cell is linked to the first by a common cell junction with the S _vn, but is not associated with the vegetative nucleus and lacks a cellular evagination. Such differences are indicative of a system of cytoplasmic heterospermy in which sperm cells possess significantly different quantities of mitochondria.Abbreviations mtDNA mitochondrial DNA - S_ua sperm cell unassociated with the vegetative nucleus - S_vn Sperm cell physically associated with the vegetative nucleus     

Ultrastructural analysis of the sperm cells of mature pollen of maize,Zea mays

Summary Ultrastructural analysis of the mature viable unhydrated pollen of maize,Zea mays from dehiscent anthers shows that the sperm cells are physically distant, each bounded by an envelope comprising their own plasma membrane and the inner plasma membrane of the vegetative cell. The chondriome is unusual in containing one or more filamentous complexes, up to 12m in length appressed to the side of the sperm nucleus. The extensions at each end of the elongate sperm cells contain longitudinally-oriented arrays of endoplasmic lamellae. In a three-dimensional reconstruction of serial thin sections, there is a long J-shaped sperm, c. 35 × 5m and up to 1m in thickness, sited within pointed evaginations of the vegetative nucleus and a second shorter sperm c. 20 × 5m and up to 3.5m in thickness.Abbreviations PA-TCH-SP periodic acid-thiocarbohydrazide-silver proteinate - DAPI 4,6-diamino-2-phenylindole - SC sperm cell - Sn sperm nucleus - Ua-Pb Uranyl acetate-lead citrate staining - ER endoplasmic reticulum     

An analysis of genetic variation in natural populations of Sticherus flabellatus [R. Br. (St John)] using amplified fragment length polymorphism (AFLP) markers

Amplified fragment length polymorphisms (AFLPs) were used to characterize the genetic diversity within and among natural populations of Sticherus flabellatus. Eight populations within the Sydney region of New South Wales, Australia were surveyed using 11 primer combinations. A total of 1108 reproducible bands were detected of which 469 (42%) were polymorphic. FST estimates averaged over all polymorphic loci indicated that significant genomic differentiation occurs among populations (average = 0.783). Genetic diversity within populations was assessed according to average heterozygosity (H) and percentage polymorphic loci (P) per population. Within-population diversity ranged from H = 0.12 and P = 33.69 to H = 0.04 and P = 15.99. Analysis of genetic similarity among populations suggested that the eight populations studied fall into two groups of four populations, based on population size and the condition of the habitat. Phenetic analysis (AMOVA) indicated that genetic variation is greater among populations (74.34%) than within populations (25.66%). These findings suggest that the breeding system of S. flabellatus is predominantly inbreeding, with genetic diversity maintained by occasional outcrossing in larger populations. The results presented in this study could provide evidence to support the proposal to protect natural stands of S. flabellatus, which has implications for the Australian horticulture industry.     

Studies ofNajas pollen tubes. Fine structure and the dependence of chlorotetracycline fluorescence on external free ions

Summary The distribution of membrane-associated calcium was investigated in pollen grains and tubes of the underwater pollinated angiospermNajas marina L. using chlorotetracycline (CTC). Tubes grown in distilled water (pH 6) showed the highest fluorescence in a subapical region that tapered basally into a fluorescent strand centrally located in the tube and extending back towards the pollen grain. The apical cap had low fluorescence as did the cytoplasm surrounding the fluorescent strand, the tube base and the pollen grain. Tubes grown in different pond waters (pH 8) revealed no intracellular CTC fluorescence. Instead there was an external fluorescence forming a distinct layer around the whole tube, frequently enhanced in a subapical region to form an external collar.Modification of the patterns of fluorescence could be induced by manipulation pH of the growth media and content of specific ions. For example tubes grown in distilled water with 10^–3 M Mg²⁺ salts showed a similar CTC fluorescence as those grown in pond water. In contrast, Ca²⁺ enrichment had no visible influence on the patterns of fluorescence. The pattern of fluorescence displayed by tubes grown in distilled water, could be reproduced in pond water if the pH was artificially reduced to pH 6.Ultrastructurally, there was no detectable difference in the markedly polar distribution of organelles between pollen tubes grown in the various growth media. The secretory vesicles found in the pollen grain prior to germination become distributed throughout the pollen tube but are least concentrated in regions that show highest internal CTC fluorescence. These regions appear to have large amounts of endoplasmic reticulum and include mitochondria.These results are discussed in relation to the significance of calcium gradients for tip growth and limitations in the use of CTC.Abbreviations CTC chlorotetracycline - SV secretory vesicle - ER endoplasmic reticulum - PIXE proton induced X-ray emissions     

Density‐dependent reproduction and pollen limitation in an invasive milkweed,Calotropis procera (Ait.) R. Br. (Apocynaceae)

Calotropis procera (Ait.) R.Br. (Apocynaceae), an invasive woody milkweed, has expanded its range in northern Australia affecting rangeland and pastoral productivity. While self‐compatibility should enhance the species range expansion, spread of C. procera is limited by the availability of larger wasp and bee species that are able to vector its solid pollinia. Pollination efficiency is thus likely dependent on both pollinator abundance and plant density. Calotropis procera flowers year round in Australia but fruiting is limited to the warm months of the year when pollinators are most abundant, indicating that seasonal regulation of reproduction may be due to pollinator limitation. We examine the propositions that C. procera reproduction is regulated by the interaction between plant population density and pollinator pressure and that low pollinator pressure causes low per capita plant fecundity. All pollinators belonged to Order Hymenoptera and pollinator composition was similar at six of the seven sites. Fruit production per plant (fecundity) was lower above and below intermediate densities (350–550 plants ha^?1) of flowering plants with evidence of a weak Allee effect at lower plant density. Pollinator visitation rates per plant were low at high and low plant densities, and greatest at intermediate densities, while pollen supplementation experiments showed that C. procera is pollen limited (Pollen Limitation Index_fruit = 0.9) even at intermediate densities. Pollen limitation caused by low pollinator pressure at low plant densities and pollinator satiation at high plant densities may account for these fruit production trends. Management should be conducted in the colder months when pollinator pressure is low and plants are not reproducing. In addition, where stand eradication cannot be achieved in one attempt, management should reduce flowering plants to below intermediate densities where the fecundity per plant is low.     

Effects of Exogenously Applied Jasmonates on Growth and Intracellular pH in Maize Coleoptile Segments

The effects of jasmonic acid (JA) on elongation growth of coleoptile segments from etiolated maize (Zea mays L.) were investigated in the presence and absence of auxin. When supplied alone, at physiological concentrations (10⁻⁹, 10⁻⁸, and 10⁻⁵ m), JA (or methyl-JA) inhibited growth. JA at a similar range of concentrations also inhibited auxin-induced elongation growth. To determine whether this effect on growth depended on endogenous abscisic acid (ABA), we grew maize coleoptiles in the presence of norflurazon (an inhibitor of carotenoid biosynthesis) that results in reduced endogenous ABA levels. Growth of etiolated coleoptile segments from these plants was inhibited by JA (or methyl-JA) in both the absence and presence of auxin. Previously, we have observed a correlation between elongation growth and cytosolic pH (pH_i), in which auxin lowers pH_i, and growth inhibitors such as ABA raise pH_i. We examined the effect of low concentrations of methyl-JA on pH_i with dual emission dye, carboxy seminaphthorhodafluor-1, and confocal microscopy. To confirm these studies, we also used in vivo ³¹P NMR spectrometry to ascertain the changes in pH_i after addition of jasmonate to maize coleoptiles. Coleoptiles grown in either the absence or presence of norflurazon responded to methyl-JA or JA by increases in pH_i of approximately 0.2 pH unit. This response occurs over a period of 15–20 min and appears to be independent of endogenous ABA. This alkalization induced by JA is likely to form a permissive environment for JA signal transduction pathway(s). Received February 5, 1999; accepted August 25, 1999