SOX9 enhances aggrecan gene promoter/enhancer activity and is up-regulated by retinoic acid in a cartilage-derived cell line, TC6

SOX9 is a transcription factor that plays a key role in chondrogenesis, Aggrecan is one of the major structural components in cartilage; however, the molecular mechanism of aggrecan gene regulation has not yet been fully elucidated, TC6 is a clonal chondrocytic cell line derived from articular cartilage, The purpose of this study was to examine whether SOX9 modulates aggrecan gene expression and to further identify molecules that regulate Sox9 expression in TC6 cells. SOX9 overexpression in TC6 cells enhanced by similar to 3-fold the transcriptional activity of the AgCAT-8 construct containing S-kilobase (kb) promoter/first exon/first intron fragments of the aggrecan gene. SOX9 enhancement of aggrecan promoter activity was lost when we deleted a 4.5-kb fragment from the 3'-end of the 8-kb fragment corresponding to the region including the first intron, In TC6 cells, SOX9 enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence >10-fold. SOX9 enhancement of aggrecan gene promoter activity and SOX9 transactivation through the Sry/Sox consensus sequence were not observed in osteoblastic osteosarcoma cells (ROS17/2.8), indicating the dependence on the cellular background. Northern blot analysis indicated that TC6 cells constitutively express Sox9 mRNA at relatively low levels. To examine regulation of Sox9 gene expression, we investigated the effects of calciotropic hormones and cytokines, Among these, retinoic acid (RA) specifically enhanced Sox9 mRNA expression in TC6 cells. The basal levels of Sox9 expression and its enhancement by RA were observed similarly at both permissive (33 degrees C) and nonpermissive (39 degrees C) temperatures. Furthermore, RA treatment enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence in TC6 cells. Moreover, RA treatment also enhanced the transcriptional activity of another reporter construct containing the enhancer region of the type II procollagen gene in TC6 cells. These observations indicate that SOX9 enhances aggrecan promoter activity and that its expression is up-regulated by RA in TC6 cells.

Canopy carbon and oxygen isotope composition of 9-year-old hoop pine families in relation to seedling carbon isotope composition, growth, field growth performance, and canopy nitrogen concentration

Carbon isotope composition (delta C-13), oxygen isotope composition (delta O-18), and nitrogen concentration (N-mass) of branchlet tissue at two canopy positions were assessed for glasshouse seedlings and 9-year-old hoop pine (Araucaria cunninghamii Ait. ex D. Don) trees from 22 open-pollinated families grown in 5 blocks of a progeny test at a water-limited and nitrogen-deficient site in southeastern Queensland, Australia. Significant variations in canopy delta C-13, delta O-18, and N-mass existed among the 9-year-old hoop pine families, with a heritability estimate of 0.72 for branchlet delta C-13 from the upper inner canopy position. There was significant variation in canopy delta C-13 of glasshouse seedlings between canopy positions and among the families, with a heritability estimate of 0.66. The canopy delta C-13 was positively related to canopy N-mass only for the upper outer crown in the field (R = 0.62, p < 0.001). Phenotypic correlations existed between tree height and canopy delta C-13 (R = 0.37-0.41, p < 0.001). Strong correlations were found between family canopy delta C-13 at this site and those at a wetter site and between field canopy delta C-13 and glasshouse seedling delta C-13. The mechanisms of the variation in canopy delta C-13 are discussed in relation to canopy photosynthetic capacity as reflected in the N-mass and stomatal conductance as indexed by canopy delta O-18.

Denitrification, leaching and immobilisation of N-15-labelled nitrate in winter under windrowed harvesting residues in hoop pine plantations of 1-3 years old in subtropical Australia

A field study was carried out to investigate the impacts of windrowed harvesting residues on denitrification, immobilisation and leaching of N-15-labelled nitrate applied at 20 kg N ha(-1) to microplots in second-rotation hoop pine (Araucaria cunninghamii) plantations of 1-3 years old in southeast Queensland, Australia. The PVC microplots were 235 mm in diameter and 150 mm. long, and driven into the 100 mm soil. There were three replications of such microplots for each of the six treatments which were areas just under and between 1-, 2- and 3-year-old windrows of harvesting residues. Based on gaseous N losses estimated by the difference between the recoveries of bromide (Br) applied at 100 kg Br ha(-1) and N-15-labelled nitrate, denitrification was highest (23% based on N-15 loss) in the areas just under the 1-year-old windrows 25 days after a simulated 75 mm rainfall and following several natural rainfall events. There was no significant difference in N-15 losses (14-17%) among the other treatments. The N-15 immobilisation rate was highest for microplots in the areas between the 1-year-old windrows and generally higher for microplots in the areas just under the windrows (30-39%) than that (26-30%) between the windrows. Direct measurement of N-15 gas emissions (N-15(2) + (N2O)-N-15) confirmed that the highest denitrification rate occurred in the microplots under the 1-year-old windrows although the gaseous N-15 loss calculated by gas emission was only about one-quarter that estimated by the N-15 mass balance method. A significant, positive linear relationship (P < 0.05) existed between the gaseous N-15 losses measured by the two methods used. The research indicates that considerable mineral N could be lost via denitrification during the critical inter-rotation period and early phase of the second rotation. However, the impacts of windrowed harvesting residues on N losses via denitrification might only last for a period of about 2 years. Published by Elsevier Science B.V.

Effects of moist chilling and solid matrix priming on germination of loblolly pine (Pinus taeda L.) seeds

Loblolly pine ( Pinus taeda L.) seeds from sources with a mild climate under maritime influence (North Carolina) required shorter moist chilling to achieve maximum germination vigor than seeds from sources with a harsher continental climate (Oklahoma). Solid matrix priming (SMP) for 6 d achieved as much as 60 d of moist chilling to improve rapidity, synchrony and completeness of germination for three of the four families studied. SMP after moist chilling increased the rapidity, synchrony and completeness of germination. The benefit of SMP was greatest for non-stratified seeds and the benefit decreased with length of moist chilling. In general, delaying planting for one week after SMP had minor effects on germination when seeds were kept in the SMP matrix at 4 degreesC. Delayed planting after SMP can increase germination rapidity and synchrony of seeds that have received long moist chilling and reduce the benefit of SMP in non-moist-chilled seeds.

Global distribution and genetic discontinuities of mangroves - emerging patterns in the evolution of Rhizophora

Mangroves are often described as a group of plants with common features and common origins based mostly on their broad distributional patterns, together with an erroneous view of comparable abilities in long-distance dispersal. However, whilst mangroves have common needs to adapt to rigorous environmental constraints associated with regular seawater inundation, individual taxa have developed different strategies and characteristics. Since mangroves are a genetically diverse group of mostly flowering plants, they may also have evolved at quite different geological periods, dispersed at different rates from different locations and developed different adaptive strategies. Current distributions of individual taxa show numerous instances of unusual extant distribution which demonstrate finite dispersal limitations, especially across open water. Our preliminary assessment of broad distribution and discontinuities reveals important patterns. Discontinuities, in the absence of current dispersal barriers, may be explained by persistent past barriers. As we learn more about discontinuities, we are beginning to appreciate their immense implications and what they might tell us about past geological conditions and how these might have influenced the distribution and evolution of mangroves. In this article, we describe emerging patterns in genetic relationships and distributions based on both current knowledge and preliminary results of our studies of molecular and morphometric characteristics of Rhizophora species in the Indo West Pacific region.

Inhibition of adventitious rooting in Backhousia citriodora F. Muell cuttings correlate with the concentration of essential oil

Backhousia citriodora is typical of the many commercially valuable woody Australian Myrtaceae species that are recalcitrant in forming adventitious roots from cuttings after maturation. A series of experiments were conducted to identify an endogenous rooting inhibitor in line with established criteria. Endogenous levels of citral were correlated with the rooting capacities of juvenile versus mature, and easy- versus difficult-to-root genotypes of B. citriodora, in both winter and summer. The biological activity of citral was confirmed in bioassays on mung beans and easy-to-root B. citriodora seedlings. Evidence of a common mechanism of root inhibition with other species in the Myrtaceae and the role of action of citral are discussed.

Towards the clonal propagation of coconut

Past studies from our laboratory have shown that whole immature, or mature sliced, zygotic embryos are a very good starting explant for coconut somatic embryogenesis. The highest rate of somatic embryogenesis was obtained when certain polyamines were added into the culture medium as well as activated charcoal (AC) to absorb unwanted phenolics. These past studies also showed that the development and maturation of the somatic embryos produced could be improved by the addition of abscisic acid (ABA), alone or with one of several osmotically active agents, into the culture medium. In the present study this well characterised somatic embryogenic system for zygotic tissues is being modified and applied to somatic tissues. This recent approach should be a better method for the rapid production of clonal, true-to-type coconut palms. The present research approach is focused on young leaf section explants which have been found to be very responsive to callus production. Young leaf sections produced optimum callus when cultured on media containing 2,4-D (150 μM) and the amount produced could be increased by soaking the sections in sterile water (15 to 60 minutes) or ascorbic acid (15 to 30 minutes) prior to culturing. Further improvement in callus production, as well as a reduction in the time taken for callogenesis was obtained when casein hydrolysate and/or certain polyamines were added to the callus induction medium. The development of the somatic embryos was improved by using ABA and polyethylene glycol (PEG) in the maturation medium. Despite these initial successes in improving coconut somatic embryogenesis, further studies are now being considered to shorten the time to achieve somatic embryogenesis, to better germinate somatic embryos and to improve the rate of somatic seedling conversion into plantlets.

Competition and resource availability in an annual plant community dominated by an invasive species, Carrichtera annua (L. Aschers.), in South Australia

The last decade has seen spirited debates about how resource availability affect the intensity of competition. This paper examines the effect that a dominant introduced species, Carrichtera annua, has upon the winter annual community in the arid chenopod shrublands of South Australia. Manipulative field experiments were conducted to assess plant community response to changing below-ground resource levels and to the manipulation of the density of C. annua. Changes in the density of C. annua had little effect on the abundance of all other species in the guild. Nutrient addition produced an increase in the biomass of the most abundant native species, Crassula colorata. An analysis of the root distribution of the main species suggested that the areas of soil resource capture of C. annua and C. colorata are largely segregated. Our results suggest that intraspecific competition may be stronger than interspecific competition, controlling the species responses to increased resource availability. The results are consistent with a two-phase resource dynamics systems, with pulses of high resource availability triggering growth, followed by pulses of stress. Smaller plants were nutrient limited under natural field conditions, suggesting that stress experienced during long interpulse phases may override competitive effects after short pulse phases. The observed differences in root system structure will determine when plants of a different species are experiencing a pulse or an interpulse phase. We suggest that the limitations to plant recruitment and growth are the product of a complex interplay between the length and intensity of the pulse of resource availability, the duration and severity of the interpulse periods, and biological characters of the species.

Dry matter production and allocation in Eucalyptus cloeziana and Eucalyptus argophloia seedlings in response to soil water deficits

Effects of soil water availability on seedling growth, dry matter production and allocation were determined for Gympie ( humid coastal) and Hungry Hills ( dry inland) provenances of Eucalyptus cloeziana F. Muell. and for E. argophloia Blakely ( dry inland) species. Seven-month-old seedlings were subjected to well-watered (100% field capacity, FC), moderate (70% FC) and severe (50% FC) soil water regimes in a glasshouse environment for 14 wk. There were significant differences in seedling growth, biomass production and allocation patterns between species. E. argophloia produced twice as much biomass at 100% FC, and more than three times as much at 70% and 50% FC than did either E. cloeziana provenance. Although the humid provenance of E. cloeziana had a greater leaf area at 100% FC conditions than did the dry provenance, total biomass production did not differ significantly. Both E. cloeziana provenances were highly sensitive to water deficits. E. argophloia allocated 10% more biomass to roots than did E. cloeziana. Allometric analyses indicated that relative biomass allocation patterns were significantly affected by genotype but not by soil water availability. These results have implications for taxon selection for cultivation in humid and subhumid regions.

Leaf water relations of Eucalyptus cloeziana and Eucalyptus argophloia in response to water deficit

Leaf water relations responses to limited water supply were determined in 7-month-old plants of a dry inland provenance of Eucalyptus argophloia Blakely and in a humid coastal provenance (Gympie) and a dry inland provenance (Hungry Hills) of Eucalyptus cloeziana F. Muell. Each provenance of E. cloeziana exhibited a lower relative water content at the turgor loss point, a lower apoplastic water content, a smaller ratio of dry mass to turgid mass and a lower bulk modulus of elasticity than the single provenance of E. argophloia. Osmotic potential at full turgor and water potential at the turgor loss point were significantly lower in E. argophloia and the inland provenance of E. cloeziana than in the coastal provenance of E. cloeziana. There was limited osmotic adjustment in response to soil drying in E. cloeziana, but not in E. argophloia. Between-species differences in water relations parameters were larger than those between the E. cloeziana provenances. Both E. cloeziana provenances maintained turgor under moderate water stress through a combination of osmotic and elastic adjustments. Eucalyptus argophloia had more rigid cell walls and reached lower water potentials with less reduction in relative water content than either of the E. cloeziana provenances, thereby enabling it to extract water from dryer soils.

Growth hormone regulates osteogenic marker mRNA expression in human periodontal fibroblasts and alveolar bone-derived cells

Background: Growth hormone (GH) is a potent regulator of bone formation. The proposed mechanism of GH action is through the stimulation of osteogenic precursor Cell proliferation and, following clonal expansion of these cells. promotion of differentiation along the osteogenic lineage. Objectives: We tested this hypothesis by studying the effects of GH on primary cell populations of human periodontal ligament cells (PLC) and alveolar bone cells (ABC), which contain a spectrum of osteogenic precursors. Method: The cell populations were assessed for mineralization potential after long-term culture in media containing beta-glycerophosphate and ascorbic acid, by the demonstration of mineral deposition by Von Kossa staining. The proliferative response of the cells to GH was determined over a 48-h period using a crystal violet dye-binding assay. The profile of the cells in terms of osteogcnic marker expression was established using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for alkaline phosphatase (ALP), osteopontin. osteocalcin, bone sialoprotein (BSP), as well as the bone morphogenetic proteins BMP-2, BMP-4 and BMP-7. Results: As expected, a variety of responses were observed ranging from no mineralization in the PLC populations to dense mineralized deposition observed in one GH-treated ABC population. Over a 48-h period GH was found to be non-mitogenic for all cell populations. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) BSP mRNA expression correlated well with mineralizing potential of the cells. The change in the mRNA expression of the osteogenic markers was determined following GH treatment of the cells over a 48-h period. GH caused an increase in ALP in most cell populations, and also in BMP expression in some cell populations. However a decrease in BSP. osteocalcin and osteopontin expression in the more highly differentiated cell populations was observed in response to GH. Conclusion: The response of the cells indicates that while long-term treatment with GH may promote mineralization, short-term treatment does not promote proliferation of osteoblast precursors nor induce expression of late osteogenic markers.

Rootstocks influence 'Hass' avocado fruit quality and fruit minerals

To determine the potential for rootstocks to improve avocado quality, 'Hass' fruit from trees on seedling 'Velvick' (SV), clonal 'Velvick' (CV) or clonal 'Duke 7' (CD) rootstocks were harvested in 1999 and 2000, ripened at 20 C immediately after harvest (non-stored) or stored at 5 C for four weeks (stored), and then fruit quality and mineral concentrations determined. In 2000, non-stored CV fruit had lower severity of body rots (5% of flesh volume affected, caused mainly by Colletotrichum gloeosporioides) than CD fruit (20%), while body rots severity in stored CV fruit was 20% compared with 38% in stored CD fruit. Stored CV fruit had less severe diffuse discoloration (3% and 9% in 1999 and 2000, respectively) than CD fruit (7% and 20%, respectively). Stored CV fruit also had less severe vascular browning (19%) than CD fruit (33%) in 2000. In both seasons, CV fruit had 15-19% higher flesh calcium concentrations, 17-22% higher flesh boron concentrations, and 14-15% lower flesh nitrogen concentrations than CD fruit. In 2000, CV fruit also had 27% more skin calcium and 14% less skin nitrogen than CD fruit. There were no effects of rootstock on yield, canopy volume, measured length of non-suberized roots, average fruit weight, fruit length: width ratio, skin thickness, dry matter, proportion of the fruit skin purple-black when ripe, or seed mass: fruit weight ratio. These results suggest that there is potential to improve 'Hass' avocado quality through rootstock selection, and that tree and fruit mineral concentrations have a role in this relationship.

Composition and quality of harvest residues and soil organic matter under windrow residue management in young hoop pine plantations as revealed by solid-state C-13 NMR spectroscopy

Solid-state C-13 nuclear magnetic resonance (NMR) with cross-polarisation (CP) and magic-angle-spinning (MAS) was used to: (a) examine the changes in carbon (C) composition of windrowed harvest residues during the first 3 years of hoop pine plantations in subtropical Australia; (b) assess the impacts of windrowed harvest residues on soil organic matter (SOM) composition and quality in the 0-10 cm soil layer. Harvest residues were collected from 0-, 1-, 2- and 3-year-old windrows of ca. 2.5 m width (15 m apart for 0-, 1- and 2-year-old sites and 10 m apart for 3-year-old site). Soils from the 0 to 10 cm soil layer were collected from the 1-, 2- and 3-year-old sites. The 13C NMR spectra of the harvest residues indicated the presence of lignin in the hoop pine wood, foliage and newly incorporated organic matter (NIOM). Condensed tannin structures were found in the decay-resistant bark, small wood and foliage, but were absent in other residue components and SOM. The NMR spectra of small wood samples contained condensed tannin structures because the outer layer of bark was not removed. NIOM showed a shift from foliage-like structures (celluloses) to lignin-type structures, indicating an incorporation of woody residues from the decomposing harvest residues. Suberins were also present in the small wood, foliage and bark. The 13C CP NMR spectra of SOM indicated that in areas where windrows were present, SOM did not show compositional changes. However, an increase in SOM quality under the windrows in the second year after their formation as characterised by the alkyl C/O-alkyl C (A/O-A) ratio was mainly due to inputs from the decomposition of the labile, readily available components of the windrowed harvest residues. (C) 2002 Published by Elsevier Science B.V.