A soil-free plant growth system to facilitate analysis of plant pathogen interactions in roots

Analysis of the interaction of pathogens with plant roots is often complicated by the growth of plants in a soil substrate. A soil-free plant growth system (SPS) was developed that removes the need for a substrate while supporting the growth of seedlings in a nutrient rich, oxygenated environment. The model legume Lupinus angustifolius was used to compare the growth of seedlings within soil and the SPS. Seedlings grown under both conditions were similar in morphology, anatomy and health (measured by leaf chlorophyll abundance) and importantly there was little difference in root growth and development although straighter and fuller root systems were achieved in the SPS. The ease of access to the root system proved efficient for the analysis of root and pathogen interactions with no interference from soil or adhering particulate matter. Following inoculation of L. angustifolius roots with Phytophthora cinnamomi the host/pathogen interaction was easily observed and tissues sampled undamaged.

Involvement of rootstocks and their hydraulic conductance in the drought resistance of grafted rubber trees

Improving drought resistance of rubber trees has become a pressing issue with the extension of rubber plantations and the prevalence of seasonal drought. Root system is vital to water and nutrients uptake of all plants, therefore, rootstocks could play decisive roles in drought resistance of grafted rubber trees on a specific scion clone. To investigate the responses of different clone rootstocks and their grafted trees to water stress and find applicable methods for selecting drought resistant rootstocks, seven related parameters and root hydraulic properties of both seeds originated and grafted saplings of PB86, PR107, RRIM600 and GT1 were measured to assess their drought resistance. It was shown that the rootstock drought resistance and root hydraulic conductance may improve the drought resistance of the grafted rubber trees. Among the four clone rootstocks, GT1, which demonstrated more resistant to drought and higher root hydraulic conductance, was comparatively resistant to drought both for the seed propagation seedlings and grafted saplings. In addition, studies on the grafted saplings with different root hydraulic conductance further validated the possibility of selecting drought resistant rootstocks on the basis of rootstock hydraulic conductance using a high-pressure flow meter.

Functional genomic analysis of systemic cell division regulation in legumes

Legumes develop root nodules from pluripotent stem cells in the rootpericycle in response to mitogenic activation by a decorated chitin-likenodulation factor synthesized in Rhizobium bacteria. The soybean genes encoding the receptor for such signals were cloned using map-based cloning approaches. Pluripotent cells in the root pericycle and the outer or inner cortex undergo repeated cell divisions to initiate a composite nodule primordium that develops to a functional nitrogen-fixing nodule. The process itself is autoregulated, leading to the characteristic nodulation of the upper root system. Autoregulation of nodulation (AON) in all legumes is controlled in part by a leucine-rich repeat receptor kinase gene (GmNARK). Mutations of GmNARK, and its other legume orthologues, result in abundant nodulation caused by the loss of a yet-undefined negative nodulation repressor system. AON receptor kinases are involved in perception of a long distance, root-derived signal, to negatively control nodule proliferation. GmNARK and LjHAR1 are expressed in phloem parenchyma. GmNARK kinase domain interacts with Kinase Associated Protein Phosphatase (KAPP). NARK gene expression did not mirror biological NARK activity in nodulation control, as q-RT-PCR in soybean revealed high NARK expression in roots, root tips, leaves, petioles, stems and hypocotyls, while shoot and root apical meristems were devoid of NARK RNA. High through-put transcript analysis in soybean leaf and root indicated that major genes involved in JA synthesis or response are preferentially down-regulated in leaf but not root of wild type, but not NARK mutants, suggesting that AON signaling may in part be controlled by events relating to hormone metabolism. Ethylene and abscisic acid insensitive mutants of L. japonicus are described. Nodulation in legumes has significance to global economies and ecologies, as the nitrogen input into the biosphere allows food, feed and biofuel production without the inherent costs associated with nitrogen fertilization [1]. Nodulation involves the production of a new organ capable of nitrogen fixation [2] and as such is an excellent system to study plant – microbe interaction, plant development, long distance signaling and functional genomics of stem cell proliferation [3, 4]. Concerted international effort over the last 20 years, using a combination of induced mutagenesis followed by gene discovery (forward genetics), and molecular/biochemical approaches revealed a complex developmental pathway that ‘loans’ genetic programs from various sources and orchestrates these into a novel contribution. We report our laboratory’s contribution to the present analysis in the field.

Role of P-glycoprotein in limiting the brain penetration of glabridin, an active isoflavan from the root of Glycyrrhiza glabra.

Purpose. Glabridin is a major active constituent of Glycyrrhiza glabra which is commonly used in the treatment of cardiovascular and central nervous system (CNS) diseases. Recently, we have found that glabridin is a substrate of P-glycoprotein (PgP/MDR1). This study aimed to investigate the role of PgP in glabridin penetration across the blood–brain barrier (BBB) using several in vitro and in vivo models.
Materials and Methods. Cultured primary rat brain microvascular endothelial cells (RBMVECs) were used in the uptake, efflux and transcellular transport studies. A rat bilateral in situ brain perfusion model was used to investigate the brain distribution of glabridin. The brain and tissue distribution of glabridin in rats with or without coadministered verapamil or quinidine were examined with correction for the tissue residual blood. In addition, the brain distribution of glabridin in mdr1a(-/-) mice was compared with the wild-type mice. Glabridin in various biological matrices was determined by a validated liquid chromatography mass spectrometric method.
Results. The uptake and efflux of glabridin in cultured RBMVECs were ATP-dependent and significantly altered in the presence of a PgP or multi-drug resistance protein (Mrp1/2) inhibitor (e.g. verapamil or MK-571). A polarized transport of glabridin was found in RBMVEC monolayers with
facilitated efflux from the abluminal (BL) to luminal (AP) side. Addition of a PgP or Mrp1/2 inhibitor in both luminal and abluminal sides attenuated the polarized transport across RBMVECs. In a bilateral in situ brain perfusion model, the uptake of glabridin into the cerebrum increased from 0.42 T 0.09% at 1 min to 9.27 T 1.69% (ml/100 g tissue) at 30 min and was significantly greater than that for sucrose. Coperfusion of a PgP or Mrp1/2 inhibitor significantly increased the brain distribution of glabridin by 33.6j142.9%. The rat brain levels of glabridin were only about 27% of plasma levels when corrected by tissue residual blood and it was increased to up to 44% when verapamil or quinidine was coadministered. The area under the brain concentration-time curve (AUC) of glabridin in mdr1a(-/-) mice was 6.0-fold higher than the wild-type mice.
Conclusions. These findings indicate that PgP limits the brain penetration of glabridin through the BBB and PgP may cause drug resistance to glabridin (licorice) therapy for CNS diseases and potential drugglabridin interactions. However, further studies are needed to explore the role of other drug transporters (e.g. Mrp1-4) in restricting the brain penetration of glabridin.

Investigation of plasmodiophora brassicae (clubroot disease) in vegetable brassica using arabidopsis thaliana as a model system

Clubroot, caused by Plasmodiophora brassicae, is the most devastating soil-borne disease of vegetable brassicas. It occurs all over the world and is responsible for crop losses of up to 10% every year. In Australia, the disease is being managed effectively with chemicals and cultural practices, but ideally control can be improved in the long term by the introduction of resistant cultivars. The life cycle ofP. brassicae and mode of action of plant resistance has not been fully elucidated because of the technical difficulties of working with an obligate, soil-borne plant pathogen. However, Arabidopsis thaliana, which is a host ofP. brassicae, has great potential as a model system for studying the life cycle, the infection process and development of resistance. We have developed a sand-liquid-culture system for growing Arabidopsis that allows easy observation of all life stages and, most importantly, the primary plasmodial stages within the root hair. The method was first optimised for observations of the lifecycle of the pathogen in a susceptible Arabidopsis ecotype (Col-3) where all stages of the lifecycle have now been observed and characterised. Further screening of Arabidopsis ecotypes for disease resistance has utilised one of the most virulent Australian pathotypes of brassica (ECD number 16/19/31). To date, Arabidopsis ecotype Ta-0 has shown a level of tolerance to the disease even though the roots get infected. It has been reported earlier that resistance toP. brassicae in Arabidopsis is due to one or a small number of genes. To examine changes in gene expression during the early, critical stages of infection, RNA was extracted from the susceptible and resistant ecotypes at two time points, 4 days and 17 days after inoculation. Microarray analysis will be used to investigate genome wide changes in gene expression during infection but also to identify candidate genes that may confer resistance to Australian isolates of the pathogen.

Development and use of a model system to monitor clubroot disease progression with an Australian field collection of Plasmodiophora brassicae

A modified sand–liquid culture method facilitated easy visualisation of the primary life cycle stages of Plasmodiophora brassicae within clean root hairs of the Arabidopsis host. Pathogen penetration occurred from day 4 onwards and then primary plasmodia developed within the host root. Several Arabidopsis ecotypes tested in varying growth conditions showed differences in disease expression. Defined growth cabinet conditions were found most suitable for studying disease progression in the ecotypes and for achieving uniform infection and disease development. Arabidopsis ecotypes Ta-0 and Tsu-0 known to be partially resistant to a German single-spore isolate of P. brassicae were susceptible to an Australian (Victorian) field population of P. brassicae. The European clubroot differential test was used to confirm virulence and describe the pathotype of the Victorian field population. Knowledge of the interaction of an Australian population of P. brassicae with its host will provide valuable information on a disease which is very difficult to control.

Spatial ecology of a root parasite : from pattern to process

Occurrence patterns of parasitic plants are constrained by the distribution of suitable hosts and movement patterns of seed vectors and, accordingly, represent a simplified system to study many aspects of spatial ecology and determinants of distribution. Previous work has focused on the aerially hemiparasitic mistletoes, and it is unclear whether root parasites are affected by similar factors. Here, we evaluate spatial patterns in the root parasitic Santalum lanceolatum in an arid shrubland in north-western New South Wales, central Australia. In this region, the principal host is a long-lived nitrogen fixing shrub Acacia tetragonophylla closely associated with ephemeral creek-lines. The location of 765 individuals of both species was mapped along a 250-m section of creek-line using a total survey station, and occurrence patterns of the root parasite related to host distribution and landscape context. We used Ripley's K-function and the O-ring statistic to determine whether the distribution of S. lanceolatum was random, aggregated or regular; the spatial scales at which these patterns occurred; and to quantify any spatial associations between the parasite and its host, A. tetragonophylla. While acacias were closely associated with the creek-line, S. lanceolatum plants were more tightly clustered, displaying significant clustering at two spatial scales (1.2 m and 8.8 m). We suggest that host quality may act as an important constraint, with only those acacias growing in or near the creek-line being physiologically capable of supporting a parasite to maturity. Insights gained from spatial analysis are used to guide ongoing research in this system, and highlight the utility of the O-ring statistic for understanding patterns of distribution affected by multiple processes operating at critical scales.

Study of the natriuretic peptide hormone system in plants

In this study, both physiological and cellular effects are elicited by natriuretic peptides (NPs), a novel type of plant hormone. It was found that rat ANP (rANP) influenced stomatal opening movement in Tradescantia sp., where a significant increase in stomatal opening was observed in the presence of 1 µM rANP. Furthermore, this effect is mediated by cGMP, a (putative) second messenger of NPs. Two inhibitors of guanylyl cyclase, LY 83583 and methylene blue, inhibited rANP-induced stomatal opening. In contrast, stomatal opening is induced in a concentration dependent manner by the cell permeant cGMP analogue 8-Br-cGMP. In addition it was found, that like in animals, the secondary structure of rANP is essential for rANP responses. Linearised rANP is biologically inactive. Since ANP elicit plant responses, an attempt was made to isolate NP analogues from plants. A protocol for partially purifying NP from plants was developed. It was found that two fractions eluted from an immunoaffinity chromatography column (0.5 M KCI eluted fraction and 0.75 M KCI eluted fraction) were biologically active. The level of cGMP in response to NPs was also tested. It is suggested that the receptor of NP is specific since only 0.75 M KCI eluted fractions increased cGMP levels in Zea mays root stele tissue. rANP did not elicit an effect on cGMP levels in this tissue and LY 83583 did not affect this response. It is therefore argued that a plant specific biologically active NP system is present in the stele and it is predicted that NPs modulate solute movement in this tissue. NPs also influence K⁺, Na⁺ and H⁺ fluxes in Zea mays root stele. Increase in both K⁺ and Na+ uptake were observed after 30 min., while H⁺ flux shifted immediately toward influx in the presence of both 0.5 and 0.75 KCI eluted fractions. Finally, a model is proposed for the effect of NPs on solute movement and its signalling system in plants.

Improve construction knowledge management system by using the soft system methodology

The construction industry has a poor record in the management of its knowledge and results into huge wastage of resources and detrimental effect to quality. Research has shown that data and information management system plus knowledge management are a critical part of today's project management practice for construction projects. Few people will deny that 'quality information' and 'useful knowledge' are extremely important to any decision-making. However, the current processes of handling information and knowledge in the construction industry and increasingly costly. One of the major reasons is the nature of this industry is not conducive to good knowledge management and the traditional data/information systems used in the industry has long been critisized. It is very common that information is often duplicated, inconsistent and not current. In turn, making knowledge becomes difficult to manage properly. Project managers have in the past found it very difficult to source and analyse data in order to make sound decisions. This paper is part of a doctoral research project which summarizes three exploratory surveys; namely ERP system, Partnering strategy and Leadership impact of a knowledge management system in a construction company. Those findings are described by using the Soft Systems Methodology (SSM) which later becomes the basis for actions research. SSM is useful to reveal complexities of the knowledge management situations that occur in construction industry. The first stage was to conduct interviews of the different practices in knowledge and reporting process. Then, the SSM rich picture was developed to present the problematic areas including difficulties in inputting data to enable the knowledge platform in place. The research then develops root definition and CATWOE, and a conceptual model was formed. Interviewees were conducted with structured questions to identify prioritized actions and activities that can be undertaken to improve and manage the knowledge platform.

A novel exhaust heat recovery system to reduce fuel consumption

Internal combustion engines release about 1/3 of the energy bound in the fuel as exhaust waste gas energy and another 1/3 energy is wasted through heat transfer into the ambient. On the other hand losses through friction are the third largest root cause for energy loss in internal combustion engines. During city driving frictional losses can be of the same size as the effective work, and during cold start these losses are even bigger. Therefore it is obvious to utilise wasted exhaust energy to warm up the engine oil directly. Frictional losses of any engine can be reduced during part load. Sensitivity analyses have been conducted for different concepts that utilise exhaust energy to reduce engine viscosity and friction. For a new system with an exhaust gas/oil heat exchanger the following benefits have been demonstrated:

• Fuel consumption reductions of over 7% measured as an average over 5 NEDC tests
compared to the standard system configuration.
• Significant reductions in exhaust emissions, mainly CO and NOx have been achieved
• Significantly higher oil temperatures during cold start indicate large potential to
reduce engine wear through reduced water condensation in the crankcase
• Fuel consumption reductions of further 3.3% to 4.6% compared to the 7% measured
over the NEDC test can be expected under real world customer usage conditions at
lower ambient temperatures.

Oil temperature measurements and analysis resulted in the idea of a novel system with further potential to reduce fuel consumption. This Oil Viscosity Energy Recovery System (OVER 7™) consists of 3 key features that add significant synergies if combined in a certain way: an oil warm up circuit/bypass, including oil pressure control and Exhaust Gas/Oil Heat Exchanger. The system separates the thermal inertias of the oil in the engine galleries and the oil pan, reduces hydraulic pumping losses, increases the heat transfer from the cylinder head to the oil, and utilises the exhaust heat to reduce oil friction.

The project demonstrated that sensitivity analysis is an important tool for the evaluation of different concepts. Especially for new concepts that include transient heat transfer such a qualitative approach in combination with accurate experiments and measurements can be faster and more efficient in leading to the desired improvements compared to time consuming detailed simulations.

Extending an industrial root controller : implementation and applications of a fast open sensor interface

An overview is given of the design and implementation of a platform for fast external sensor integration in an industrial robot system called ABB S4CPlus. As an application and motivating example, the implementation of force-controlled grinding and deburring within the AUTOFETT-project is discussed. Experiences from industrial usage of the fully developed prototype confirms the appropriateness of the design choices, thus also confirming the fact that control and software need to be tightly integrated. The new sensor can be used for the prototyping and development of a wide variety of new applications

Rhizophagus irregularis as an elicitor of rosmarinic acid and antioxidant production by transformed roots of Ocimum basilicum in an in vitro co-culture system

Arbuscular mycorrhiza is a symbiotic association formed between plant roots and soil borne fungi that alter and at times improve the production of secondary metabolites. Detailed information is available on mycorrhizal development and its influence on plants grown under various edapho-climatic conditions, however, very little is known about their influence on transformed roots that are rich reserves of secondary metabolites. This raises the question of how mycorrhizal colonization progresses in transformed roots grown in vitro and whether the mycorrhizal fungus presence influences the production of secondary metabolites. To fully understand mycorrhizal ontogenesis and its effect on root morphology, root biomass, total phenolics, rosmarinic acid, caffeic acid and antioxidant production under in vitro conditions, a co-culture was developed between three Agrobacterium rhizogenes-derived, elite-transformed root lines of Ocimum basilicum and Rhizophagus irregularis. We found that mycorrhizal ontogenesis in transformed roots was similar to mycorrhizal roots obtained from an in planta system. Mycorrhizal establishment was also found to be transformed root line-specific. Colonization of transformed roots increased the concentration of rosmarinic acid, caffeic acid and antioxidant production while no effect was observed on root morphological traits and biomass. Enhancement of total phenolics and rosmarinic acid in the three mycorrhizal transformed root lines was found to be transformed root line-specific and age dependent. We reveal the potential of R. irregularis as a biotic elicitor in vitro and propose its incorporation into commercial in vitro secondary metabolite production via transformed roots.