Effect of Temperature on Gibberellin (GA) Responsiveness and on Endogenous GA(1) Content of Tall and Dwarf Wheat Genotypes

Near-isogenic wheat (Triticum aestivum L.) lines differing in height-reducing (Rht) alleles were used to investigate the effects of temperature on endogenous gibberellin (GA) levels and seedling growth response to applied GA₃. Sheath and lamina lengths of the first leaf were measured in GA treated and control seedlings, grown at 11, 18, and 25°C, of six Rht genotypes in each of two varietal backgrounds, cv Maris Huntsman and cv April Bearded. Endogenous GA₁ levels in the leaf extension zone of untreated seedlings were determined by gas chromatography-mass spectrometry with a deuterated internal standard in the six Maris Huntsman Rht lines grown at 10 and 25°C. Higher temperature increased leaf length considerably in the tall genotype, less so in the Rht1 and Rht2 genotypes, and had no consistent effect on the Rht1+2, Rht3 and Rht2+3 genotypes. In all genotypes, endogenous GA₁ was higher at 25°C than at 10°C. At 10°C the endogenous GA₁ was at a similar level in all the genotypes (except Rht2+3). At 25°C it increased 1.6-fold in the tall genotype, 3-fold in Rht1 and Rht2, 6-fold in Rht3, and 9-fold in Rht1+2. Likewise, the genotypic differences in leaf length were very conspicuous at 25°C, but were only slight and often unsignificant at 11°C. The response of leaf length to applied GA₃ in the Rht1, Rht2, and Rht1+2 genotypes increased significantly with lowering of temperature. These results suggest the possibility that the temperature effect on leaf elongation is mediated through its effect on the level of endogenous GA₁ and that leaf elongation response to endogenous or applied GAs is restricted by the upper limits set by the different Rht alleles.     

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

Influence of substrate curvature on osteoblast orientation and extracellular matrix deposition

Background

The effects of microchannel diameter in hydroxyapatite (HAp) substrates on osteoblast behavior were investigated in this study. Microchannels of 100, 250 and 500 μm diameter were created on hydroxyapatite disks. The changes in osteoblast precursor growth, differentiation, extra cellular matrix (ECM) secretion and cell attachment/orientation were investigated as a function of microchannel diameter.

Results

Curvature did not impact cellular differentiation, however organized cellular orientation was achieved within the 100 and 250 μm microchannels (mc) after 6 days compared to the 12 days it took for the 500mc group, while the flat substrate remained disorganized. Moreover, the 100, 250 and 500mc groups expressed a specific shift in orientation of 17.45°, 9.05°, and 22.86° respectively in 24 days. The secreted/mineralized ECM showed the 100 and 250mc groups to have higher modulus (E) and hardness (h) (E?=?42.6GPa; h?=?1.6GPa) than human bone (E?=?13.4-25.7GPa; h?=?0.47-0.74GPa), which was significantly greater than the 500mc and control groups (p?<?0.05). It was determined that substrate curvature affects the cell orientation, the time required for initial response, and the shift in orientation with time.

Conclusions

These findings demonstrate the ability of osteoblasts to organize and mineralize differentially in microchannels similar to those found in the osteons of compact bone. These investigations could lead to the development of osteon-like scaffolds to support the regeneration of organized bone.

     

A second gene at the tomato Cf-4 locus confers resistance to cladosporium fulvum through recognition of a novel avirulence determinant

The tomato Cf-4 and Cf-9 genes confer resistance to the leaf mould pathogen Cladosporium fulvum and map at a complex locus on the short arm of chromosome 1. It was previously shown that the gene encoding Cf-4, which recognizes the Avr4 avirulence determinant, is one of five tandemly duplicated homologous genes (Hcr9-4s) at this locus. Cf-4 was identified by molecular analysis of rare Cf-4/Cf-9 disease-sensitive recombinants and by complementation analysis. The analysis did not exclude the possibility that an additional gene(s) located distal to Cf-4 may also confer resistance to C. fulvum. We demonstrate that a number of Dissociation-tagged Cf-4 mutants, identified on the basis of their insensitivity to Avr4, are still resistant to infection by C. fulvum race 5. Molecular analysis of 16 Cf-4 mutants, most of which have small chromosomal deletions in this region, suggested the additional resistance specificity is encoded by Hcr9-4E. Hcr9-4E recognizes a novel C. fulvum avirulence determinant that we have designated Avr4E.     

The cover of living and dead corals from airborne remote sensing

Trends in coral cover are widely used to indicate the health of coral reefs but are costly to obtain from field survey over large areas. In situ studies of reflected spectra at the coral surface show that living and recently dead colonies can be distinguished. Here, we investigate whether such spectral differences can be detected using an airborne remote sensing instrument. The Compact Airborne Spectrographic Imager (Itres Research Ltd, Canada) was flown in two configurations: 10 spectral bands with 1-m² pixels and 6 spectral bands with 0.25-m² pixels. First, we show that an instrument with 10 spectral bands possesses adequate spectral resolution to distinguish living Porites, living Pocillopora spp., partially dead Porites, recently dead Porites (total colony mortality within 6 months), old dead (>6 months) Porites, Halimeda spp., and coralline red algae when there is no water column to confuse spectra. All substrata were distinguished using fourth-order spectral derivatives around 538 nm and 562 nm. Then, at a shallow site (Tivaru) at Rangiroa Atoll, Tuamotu Archipelago (French Polynesia), we show that live and dead coral can be distinguished from the air to a depth of at least 4 m using first- and fourth-order spectral derivatives between 562–580 nm. However, partially dead and recently dead Porites colonies could not be distinguished from an airborne platform. Spectral differences among substrata are then exploited to predict the cover of reef substrata in ten 25-m² plots at nearby Motu Nuhi (max depth 8 m). The actual cover in these plots was determined in situ using quadrats with a 0.01-m² grid. Considerable disparity occurred between field and image-based measures of substrate cover within individual 25-m² quadrats. At this small scale, disparity, measured as the absolute difference in cover between field and remote-sensing methods, reached 25% in some substrata but was always less than 10% for living coral (99% of which consisted of Porites spp.). At the scale of the reef (all ten 25-m² quadrats), however, disparities in percent cover between imagery and field data were less than 10% for all substrata and extremely low for some classes (e.g. <3% for living Porites, recently dead Porites and Halimeda). The least accurately estimated substrata were sand and coralline red algae, which were overestimated by absolute values 7.9% and 6.6%, respectively. The precision of sampling was similar for field and remote-sensing methods: field methods required 19 plots to detect a 10% difference in coral cover among three reefs with a statistical power of 95%. Remote-sensing methods required 21 plots. However, it took 1 h to acquire imagery over 92,500 m² of reef, which represents 3,700 plots of 25 m² each, compared with 3 days to survey 10 such plots underwater. There were no significant differences in accuracy between 1-m² and 0.25-m² image resolutions, suggesting that the advantage of using smaller pixels is offset by reduced spectral information and an increase in noise (noise was observed to be 1.6–1.8 times greater in 0.25-m² pixels). We show that airborne remote sensing can be used to monitor coral and algal cover over large areas, providing that water is shallow and clear, and that brown fleshy macroalgae are scarce, that depth is known independently (e.g. from sonar survey).