Towards developing reliable models of leaf area on grapevines (Vitis vinifera L.)

A field study in three vineyards in southern Queensland (Australia) was carried out to develop predictive models for individual leaf area and shoot leaf area of two cultivars (Cabernet Sauvignon and Shiraz) of grapevines (Vitis Vinifera L.). Digital image analysis was used to measure leaf vein length and leaf area. Stepwise regressions of untransformed and transformed models consisting of up to six predictor variables for leaf area and three predictor variables for shoot leaf area were carried out to obtain the most efficient models. High correlation coefficients were found for log10 transformed individual leaf and shoot leaf area models. The significance of predictor variables in the models varied across vineyards and cultivars, demonstrating the discontinuous and heterogeneous nature of vineyards. The application of this work in a grapevine modeling environment and in a dynamic vineyard management context are discussed. Sample sizes for quantification of individual leaf areas and areas of leaves on shoots are proposed based on target margins of errors of sampled data.

Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels

Glutamate dehydrogenase (GDH; EC 1.4.1.2-1.4.1.4) catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate. In vascular plants the in vivo direction(s) of the GDH reaction and hence the physiological role(s) of this enzyme remain obscure. A phylogenetic analysis identified two clearly separated groups of higher-plant GDH genes encoding either the alpha- or beta-subunit of the GDH holoenzyme. To help clarify the physiological role(s) of GDH, tobacco (Nicotiana tabacum L.) was transformed with either an antisense or sense copy of a beta-subunit gene, and transgenic plants recovered with between 0.5- and 34-times normal leaf GDH activity. This large modulation of GDH activity (shown to be via alteration of beta-subunit levels) had little effect on leaf ammonium or the leaf free amino acid pool, except that a large increase in GDH activity was associated with a significant decrease in leaf Asp (similar to 51%, P=0.0045). Similarly, plant growth and development were not affected, suggesting that a large modulation of GDH beta-subunit titre does not affect plant viability under the ideal growing conditions employed. Reduction of GDH activity and protein levels in an antisense line was associated with a large increase in transcripts of a beta-subunit gene, suggesting that the reduction in beta-subunit levels might have been due to translational inhibition. In another experiment designed to detect post-translational up-regulation of GDH activity, GDH over-expressing plants were subjected to prolonged dark-stress. GDH activity increased, but this was found to be due more likely to resistance of the GDH protein to stress-induced proteolysis, rather than to post-translational up-regulation.

Developing pineapple fruit has a small transcriptome dominated by metallothionein

In a first step toward understanding the molecular basis of pineapple fruit development, a sequencing project was initiated to survey a range of expressed sequences from green unripe and yellow ripe fruit tissue. A highly abundant metallothionein transcript was identified during library construction, and was estimated to account for up to 50% of all EST library clones. Library clones with metallothionein subtracted were sequenced, and 408 unripe green and 1140 ripe yellow edited EST clone sequences were retrieved. Clone redundancy was high, with the combined 1548 clone sequences clustering into just 634 contigs comprising 191 consensus sequences and 443 singletons. Half of the EST clone sequences clustered within 13.5% and 9.3% of contigs from green unripe and yellow ripe libraries, respectively, indicating that a small subset of genes dominate the majority of the transcriptome. Furthermore, sequence cluster analysis, northern analysis, and functional classification revealed major differences between genes expressed in the unripe green and ripe yellow fruit tissues. Abundant genes identified from the green fruit include a fruit bromelain and a bromelain inhibitor. Abundant genes identified in the yellow fruit library include a MADS box gene, and several genes normally associated with protein synthesis, including homologues of ribosomal L10 and the translation factors SUI1 and eIF5A. Both the green unripe and yellow ripe libraries contained high proportions of clones associated with oxidative stress responses and the detoxification of free radicals.

Modelling the population dynamics of the Queensland fruit fly, Bactrocera (Dacus) tryoni: a cohort-based approach incorporating the effects of weather

Queensland fruit fly, Bactrocera (Dacus) tryoni (QFF) is arguably the most costly horticultural insect pest in Australia. Despite this, no model is available to describe its population dynamics and aid in its management. This paper describes a cohort-based model of the population dynamics of the Queensland fruit fly. The model is primarily driven by weather variables, and so can be used at any location where appropriate meteorological data are available. In the model, the life cycle is divided into a number of discreet stages to allow physiological processes to be defined as accurately as possible. Eggs develop and hatch into larvae, which develop into pupae, which emerge as either teneral females or males. Both females and males can enter reproductive and over-wintering life stages, and there is a trapped male life stage to allow model predictions to be compared with trap catch data. All development rates are temperature-dependent. Daily mortality rates are temperature-dependent, but may also be influenced by moisture, density of larvae in fruit, fruit suitability, and age. Eggs, larvae and pupae all have constant establishment mortalities, causing a defined proportion of individuals to die upon entering that life stage. Transfer from one immature stage to the next is based on physiological age. In the adult life stages, transfer between stages may require additional and/or alternative functions. Maximum fecundity is 1400 eggs per female per day, and maximum daily oviposition rate is 80 eggs/female per day. The actual number of eggs laid by a female on any given day is restricted by temperature, density of larva in fruit, suitability of fruit for oviposition, and female activity. Activity of reproductive females and males, which affects reproduction and trapping, decreases with rainfall. Trapping of reproductive males is determined by activity, temperature and the proportion of males in the active population. Limitations of the model are discussed. Despite these, the model provides a useful agreement with trap catch data, and allows key areas for future research to be identified. These critical gaps in the current state of knowledge exist despite over 50 years of research on this key pest. By explicitly attempting to model the population dynamics of this pest we have clearly identified the research areas that must be addressed before progress can be made in developing the model into an operational tool for the management of Queensland fruit fly. (C) 2003 Published by Elsevier B.V.

Fruit processing, seed viability and dormancy mechanisms of Persoonia sericea A. Cunn. ex R. Br. and P-virgata R. Br. (Proteaceae)

The morphology of the fruit and difficulties with fruit processing impose major limitations to germination of Persoonia sericea and P. virgata. The mesocarp must be removed without harming the embryo. Fermentation of fruit or manual removal of the mesocarp was effective but digestion in 32% hydrochloric acid (HCl) completely inhibited germination. The endocarp is extremely hard and therefore very difficult and time consuming to remove without damaging the seeds. The most efficient method was cracking the endocarp with pliers, followed by manual removal of seeds. Germination was completely inhibited unless at least half of the endocarp was removed. Microbial contamination of the fruit and seeds was controlled by disinfestation and germination of the seed under aseptic conditions. The results suggest that dormancy in these species is primarily due to physical restriction of the embryo by the hard endocarp.

Manipulating avocado fruit ripening with 1-methylcyclopropene

Previous investigations with 1-methylcyclopropene (1-MCP) on avocado (Persea americana Mill.) fruit have focussed mainly on improving storage life by reducing the severity of disorders causing discolouration of the flesh. Development of 1-MCP and ethylene treatments, which also help control the time to reach the eating ripe stage, may confer additional practical benefits. In this context, the current study investigated the potential of 1-MCP to accurately manipulate ripening of non-stored 'Hass' avocado fruit by treatment before or after ethylene and at different times during ripening. To investigate this, 500 nL L-1 1-MCP was applied within 1 day after harvest, followed by ethylene 0-14 days after 1-MCP. In addition, fruit were treated with ethylene, then 1-MCP 0-8 days after ethylene. Treatment of fruit with 500 nL L-1 1-MCP for 18 h at 20 degreesC provided the maximum effect by increasing the days from harvest to ripe (DTR) from 8 (with no 1-MCP) to 20. Fruit treated with 500 nL L-1 1-MCP for 18 h at 20 degreesC remained insensitive to 100 muL L-1 ethylene applied between 0 and 14 days after 1-MCP for 24 h at 20 degreesC. Ripening of fruit exposed to 100 muL L-1 ethylene for 24 h at 20 degreesC could be delayed by up to 3.3 days by applying 500 nL L-1 1-MCP for 18 h at 20 degreesC up to 2 days after ethylene treatment. However, once the fruit started to soften (sprung) there was little effect of 1-MCP on DTR, compared with no 1-MCP. 1-MCP treatment was associated with increased severity of body rots (caused mainly by Colletotrichum spp.) and stem-end rots (caused mainly by Dothiorella spp.), which was likely due to the increased DTR in these treatments. Significant differences in disease severity were found between orchards (replications), with replicates with low disease severity being less affected by 1-MCP treatment. These results indicate that 1-MCP can delay ripening, but careful sourcing of fruit is required to reduce the risk of diseases in ripe fruit. There is some capacity to delay ripening using 1-MCP after ethylene. There is little potential to control ripening using ethylene after treatment with 500 nL L-1 1-1-MCP, but lower concentrations may be more effective. (C) 2004 Elsevier B.V. All rights reserved.

Pax9 and Jagged1 act downstream of Gli3 in vertabrate limb development

From early in limb development the transcription factor Gli3 acts to define boundaries of gene expression along the anterior-posterior (AP) axis, establishing asymmetric patterns required to provide positional information. As limb development proceeds, posterior mesenchyme expression of Sonic hedgehog (Shh) regulates Gli3 transcription and post-translational processing to specify digit number and identity. The molecular cascades dependent on Gli3 at later stages of limb development, which link early patterning events with final digit morphogenesis, remain poorly characterised. By analysing the transcriptional consequences of loss of Gli3 in the anterior margin of the E11.5 and E12.5 limb bud in the polydactylous mouse mutant extra-toes (Gli3(Xt/Xt)), we have identified a number of known and novel transcripts dependent on Gli3 in the limb. In particular, we demonstrated that the genes encoding the paired box transcription factor Pax9, the Notch ligand Jagged1 and the cell surface receptor Cdo are dependent on Gli3 for correct expression in the anterior limb mesenchyme. Analysis of expression in compound Shh;Gli3 mutant mouse embryos and in both in vitro and in vivo Shh signaling assays, further defined the importance of Shh regulated processing of Gli3 in controlling gene expression. In particular Pax9 regulation by Shh and Gli3 was shown to be context dependent, with major differences between the limb and somite revealed by Shh bead implantation experiments in the chick. Jagged1 was shown to be induced by Shh in the chick limb and in a C3H10T1/2 cell based signaling assay, with Shh;Gli3 mutant analysis indicating that expression is dependent on Gli3 derepression. Our data have also revealed that perturbation of early patterning events within the Gli3(Xt/Xt), limb culminates in a specific delay of anterior chondrogenesis which is subsequently realised as extra digits. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Definition and spatial annotation of the dynamic secretome during early kidney development

The term secretome has been defined as a set of secreted proteins (Grimmond et al. [2003] Genome Res 13:1350-1359). The term secreted protein encompasses all proteins exported from the cell including growth factors, extracellular proteinases, morphogens, and extracellular matrix molecules. Defining the genes encoding secreted proteins that change in expression during organogenesis, the dynamic secretome, is likely to point to key drivers of morphogenesis. Such secreted proteins are involved in the reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (AM) that occur during organogenesis of the metanephros. Some key metanephric secreted proteins have been identified, but many remain to be determined. In this study, microarray expression profiling of E10.5, E11.5, and E13.5 kidney and consensus bioinformatic analysis were used to define a dynamic secretome of early metanephric development. In situ hybridisation was used to confirm microarray results and clarify spatial expression patterns for these genes. Forty-one secreted factors were dynamically expressed between the E10.5 and E13.5 timeframe profiled, and 25 of these factors had not previously been implicated in kidney development. A text-based anatomical ontology was used to spatially annotate the expression pattern of these genes in cultured metanephric explants.

Development of a DNA-based method for detection and identification of Phytophthora species

Phytophthora diseases cause major losses to agricultural and horticultural production in Australia and worldwide. Most Phytophthora diseases are soilborne and difficult to control, making disease prevention an important component of many disease management strategies. Detection and identification of the causal agent, therefore, is an essential part of effective disease management. This paper describes the development and validation of a DNA-based diagnostic assay that can detect and identify 27 different Phytophthora species. We have designed PCR primers that are specific to the genus Phytophthora. The resulting amplicon after PCR is subjected to digestion by restriction enzymes to yield a specific restriction pattern or fingerprint unique to each species. The restriction patterns are compared with a key comprising restriction patterns of type specimens or representative isolates of 27 different Phytophthora species. A number of fundamental issues, such as genetic diversity within and among species which underpin the development and validation of DNA-based diagnostic assays, are addressed in this paper.

Development of the Minimum Information Specification for in situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)

We describe the creation process of the Minimum Information Specification for In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE). Modeled after the existing minimum information specification for microarray data, we created a new specification for gene expression localization experiments, initially to facilitate data sharing within a consortium. After successful use within the consortium, the specification was circulated to members of the wider biomedical research community for comment and refinement. After a period of acquiring many new suggested requirements, it was necessary to enter a final phase of excluding those requirements that were deemed inappropriate as a minimum requirement for all experiments. The full specification will soon be published as a version 1.0 proposal to the community, upon which a more full discussion must take place so that the final specification may be achieved with the involvement of the whole community. This paper is part of the special issue of OMICS on data standards.

Expressed sequence tag analysis of genes expressed during development of the tropical abalone Haliotis asinina

The tropical abalone. Haliotis asinina. is,in ideal species to investigate the molecular mechanisms that control development. growth, reproduction and shell formation in all cultured haliotids. Here we describe the analysis of 232 expressed sequence tags (EST) obtained front a developmental H. asinina cDNA library intended for future microarray studies. From this data set we identified 183 unique gene Clusters. Of these, 90 clusters showed significant homology with sequences lodged in GenBank, ranging in function from general housekeeping to signal transduction, gene regulation and cell-cell communication. Seventy-one clusters possessed completely novel ORFs greater than 50 codons in length, highlighting the paucity of sequence data from molluscs and other lophotrochozoans. This study of developmental gene expression in H. asinina provides the foundation for further detailed analyses of abalone growth, development and reproduction.

Microsatellite markers for the pest fruit fly, Bactrocera papayae (Diptera : Tephritidae) and other Bactrocera species

The dorsalis complex contains some of the most economically important fruit fly pests of the Asia-Pacific regions, including Bactrocera dorsalis, Bactrocera papayae and Bactrocera carambolae. These species are morphologically indistinct and genetically very similar. We describe the development of 12 microsatellite markers isolated from a representative of the dorsalis complex, B. papayae. We show the potential utility of the B. papayae microsatellites and a set of microsatellites isolated from Bactrocera tryoni as population and species markers for the dorsalis complex.

Postharvest characteristics and handling of litchi fruit - an overview

Litchi ( Litchi chinensis Sonn.) is a tropical to subtropical crop that originated in South-East Asia. Litchi fruit are prized on the world market for their flavour, semi-translucent white aril and attractive red skin. Litchi is now grown commercially in many countries and production in Australia, China, Israel, South Africa and Thailand has expanded markedly in recent years. Increased production has made significant contributions to economic development in these countries, especially those in South-East Asia. Non-climacteric litchi fruit are harvested at their visual and organoleptic optimum. They are highly perishable and, consequently, have a short life that limits marketability and potential expansion of demand. Pericarp browning and pathological decay are common and important defects of harvested litchi fruit. Postharvest technologies have been developed to reduce these defects. These technologies involve cooling and heating the fruit, use of various packages and packaging materials and the application of fungicides and other chemicals. Through the use of fungicides and refrigeration, litchi fruit have a storage life of about 30 days. However, when they are removed from storage, their shelf life at ambient temperature is very short due to pericarp browning and fruit rotting. Low temperature acclimation or use of chitsoan as a coating can extend the shelf life. Sulfur dioxide fumigation effectively reduces pericarp browning, but approval from Europe, Australia and Japan for this chemical is likely to be withdrawn due to concerns over sulfur residues in fumigated fruit. Thus, sulfur-free postharvest treatments that maintain fruit skin colour are increasingly important. Alternatives to SO2 fumigation for control of pericarp browning and fruit rotting are pre-storage pathogen management, anoxia treatment, and dipping in 2% hydrogen chloride solution for 6-8 min following storage at 0 degrees C. Insect disinfestation has become increasingly important for the expansion of export markets because of quarantine issues associated with some fruit fly species. Thus, effective disinfestation protocols need to be developed. Heat treatment has shown promise as a quarantine technology, but it injures pericarp tissue and results in skin browning. However, heat treatment can be combined with an acid dip treatment that inhibits browning. Therefore, the primary aim of postharvest litchi research remains the achievement of highly coloured fruit which is free of pests and disease. Future research should focus on disease control before harvest, combined acid and heat treatments after harvest and careful temperature management during storage and transport.

The Pineapple EST sequencing and microarray project

We have initiated an EST sequencing project to survey a range of expressed sequences from green fruit, yellow fruit, roots, and root-knot nematode infected root/gall tissues. In total, 5681 edited EST sequences were retrieved. Clone redundancy was high in the fruit libraries, with the combined fruit 1548 clone sequences clustering into just 634 contigs comprising 191 consensus sequences and 443 singletons. Half of all fruit EST clone sequences clustered within approximately 14 and 9% of contigs from green unripe and yellow ripe libraries respectively, indicating that a small subset of genes dominates the majority of the transcriptome. The root and root/gall libraries had lower levels of redundancy than the fruit libraries. Half of the root/gall ESTs clustered within approximately 40% of all contigs, indicating the roots possess a more complex transcriptome. Contig assembly and cluster analysis revealed major differences in the abundant gene sequences expressed between the unripe green and the ripe yellow fruit tissues, or gene sequences expressed between the weeks 1-4 and weeks 5-10 nematode infected gall vascular cylinder libraries.