The use of an acetoacetyl-CoA synthase in place of a -ketothiolase enhances poly-3-hydroxybutyrate production in sugarcane mesophyll cells

Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C-4 plants for the production of poly[(R)-3-hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield-potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing -ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl-CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB-producing sugarcane containing the -ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2x10(6)Da. These results are a major step forward in engineering a high biomass C-4 grass for the commercial production of PHB.

Sugarcane smut after three years: a policy retrospective.

The detection of sugarcane smut disease (Ustilago scitaminea) in the Bundaberg-Childers region of eastern Australia in 2006 triggered a comprehensive and united response from BSES Limited, Queensland Government and CANEGROWERS. The response to sugarcane smut in the Bundaberg-Childers area was the first test for the Emergency Plant Pest Response Deed, an agreement between Australian governments and plant industries to facilitate a response to a plant pest incursion. As part of this response and the subsequent inquiry, economic models of the likely pattern of spread and cost of the smut epidemic were prepared. This paper reviews the predictions of those models in the light of the subsequent three years' experience. It examines reasons for divergence from the modelled outcomes, some of which were good approximations of actual experience.

Interactions between rotation breaks, tillage and N management on sugarcane grown at Bundaberg and Ingham.

The impact of cropping histories (sugarcane, maize and soybean), tillage practices (conventional tillage and direct drill) and fertiliser N in the plant and 1st ratoon (1R) crops of sugarcane were examined in field trials at Bundaberg and Ingham. Average yields at Ingham (Q200) and Bundaberg (Q151) were quite similar in both the plant crop (83 t/ha and 80 t/ha, respectively) and the 1R (89 t/ha v 94 t/ha, respectively), with only minor treatment effects on CCS at each site. Cane yield responses to tillage, break history and N fertiliser varied significantly between sites. There was a 27% yield increase in the plant crop from the soybean fallow at Ingham, with soybeans producing a yield advantage over continuous cane, but there were no clear break effects at Bundaberg - possibly due to a complex of pathogenic nematodes that responded differently to soybeans and maize breaks. There was no carryover benefit of the soybean break into the 1R crop at Ingham, while at Bundaberg the maize break produced a 15% yield advantage over soybeans and continuous cane. The Ingham site recorded positive responses to N fertiliser addition in both the plant (20% yield increase) and 1R (34% yield increase) crops, but there was negligible carryover benefit from plant crop N in the 1R crop, or of a reduced N response after a soybean rotation. By contrast, the Bundaberg site showed no N response in any history in the plant crop, and only a small (5%) yield increase with N applied in the 1R crop. There was again no evidence of a reduced N response in the 1R crop after a soybean fallow. There were no significant effects of tillage on cane yields at either site, although there were some minor interactions between tillage, breaks and N management in the 1R crop at both sites. Crop N contents at Bundaberg were more than 3 times those recorded at Ingham in both the plant and 1R crops, with N concentrations in millable stalk at Ingham suggesting N deficiencies in all treatments. There was negligible additional N recovered in crop biomass from N fertiliser application or soybean residues at the Ingham site. There was additional N recovered in crop biomass in response to N fertiliser and soybean breaks at Bundaberg, but effects were small and fertiliser use efficiencies poor. Loss pathways could not be quantified, but denitrification or losses in runoff were the likely causes at Ingham while leaching predominated at Bundaberg. Results highlight the complexity involved in developing sustainable farming systems for contrasting soil types and climatic conditions. A better understanding of key sugarcane pathogens and their host range, as well as improved capacity to predict in-crop N mineralisation, will be key factors in future improvements to sugarcane farming systems.

Impact of tillage and residues from rotation crops on the nematode community in soil and surface mulch during the following sugarcane crop.

The impact of three cropping histories (sugarcane, maize and soybean) and two tillage practices (conventional tillage and direct drill) on plant-parasitic and free-living nematodes in the following sugarcane crop was examined in a field trial at Bundaberg. Soybean reduced populations of lesion nematode (Pratylenchus zeae) and root-knot nematode (Meloidogyne javanica) in comparison to previous crops of sugarcane or maize but increased populations of spiral nematode (Helicotylenchus dihystera) and maintained populations of dagger nematode (Xiphinema elongatum). However the effect of soybean on P zeae and M. javanica was no longer apparent 15 weeks after planting sugarcane, while later in the season, populations of these nematodes following soybean were as high as or higher than maize or sugarcane. Populations of P zeae were initially reduced by cultivation but due to strong resurgence tended to be higher in conventionally tilled than direct drill plots at the end of the plant crop. Even greater tillage effects were observed with M. javanica and X. elongatum, as nematode populations were significantly higher in conventionally tilled than direct drill plots late in the season. Populations of free-living nematodes in the upper 10 cm of soil were initially highest following soybean, but after 15, 35 and 59 weeks were lower than after sugarcane and contained fewer omnivorous and predatory nematodes. Conventional tillage increased populations of free-living nematodes in soil in comparison to direct drill and was also detrimental to omnivorous and predatory nematodes. These results suggest that crop rotation and tillage not only affect plant-parasitic nematodes directly, but also have indirect effects by impacting on natural enemies that regulate nematode populations. More than 2 million nematodes/m(2) were often present in crop residues on the surface of direct drill plots. Bacterial-feeding nematodes were predominant in residues early in the decomposition process but fungal-feeding nematodes predominated after 15 weeks. This indicates that fungi become an increasingly important component of the detritus food web as decomposition proceeds, and that that the rate of nutrient cycling decreases with time. Correlations between total numbers of free-living nematodes and mineral N concentrations in crop residues and surface soil suggested that the free-living nematode community may provide an indication of the rate of mineralisation of N from organic matter.

Baker's yeast expressing the Japanese encephalitis virus envelope protein on its cell surface: induction of an antigen-specific but non-neutralizing antibody response

Live recombinant Saccharomyces cerevisiae yeast expressing the envelope antigen of Japanese encephalitis virus (JEV) on the outer mannoprotein layer of the cell wall were examined for their ability to induce antigen-specific antibody responses in mice. When used as a modelantigen, parenteral immunization of mice with surface-expressing GFP yeast induced a strong anti-GFP antibody response in the absence of adjuvants. This antigen delivery approach was then used for a more stringent system, such as the envelope protein of JEV, which is a neurotropic virus requiring neutralizing antibodies for protection.Although 70% of cells were detected to express the total envelope protein on the surface by antibodies raised to the bacterially expressed protein, polyclonal anti-JEV antibodies failed to react with them. In marked contrast, yeast expressing the envelope fragments 238-398, 373-399 and 373-500 in front of a Gly-Ser linker were detected by anti-JEV antibodies as well as a monoclonal antibody but not by antibodies raised to the bacterially expressed protein. Immunization of mice with these surface-expressing recombinants resulted in a strong antibody response. However, the antibodies failed to neutralize the virus, although the fragments were selected based on neutralizing determinants.

DAQ00129 Improving the Integration of Legumes in Grain and Sugarcane Farming Systems in Southern Queensland

The farming systems and agribusinesses of the inland Burnett and southern coastal cropping regions of Queensland are becoming increasingly interlinked as grain legume crops, a key component of dryland cropping systems, become more firmly entrenched in the coastal sugarcane cropping areas. Soybeans, peanuts and possibly winter cereals like barley have a real and demonstrated role in sugarcane rotations, and assistance with the integration of those crops into viable and sustainable cropping systems with sugarcane will be critical to the futuer development of these industries.

Cropping solutions for the sugarcane farming systems of the Burdekin

The successful inclusion of break crops into the Burdekin sugar farming system will allow growers to diversify and capitalise on alternate crop income sources, particularly during cyclical downturns in sugar price. Secondly if cane productivity is improved through the inclusion of break crops, millers and growers stand to gain additional economic benefit compared to the current sugarcane monoculture.

A case study on the use of legume crop rotations in sugarcane

Growing legume fallow crops has proven to be an important factor in reducing the yield decline effect in sugarcane production. Legumes can also provide a direct economic benefit to sugarcane farmers by providing a source of nitrogen. Further, in some instances, income can flow from the sale, of grain or seed. The following case study provides an insight into the changes made by Russell Young, a sugarcane farmer situated in the Rita Island area of the Burdekin district. The case study focuses on the economics of the old farming system versus a new farming system. The old farming system is based on the conventional farming practices previously used by the Young family in 2002 compared to the 2006 farming system which involves a reduction in tillage practices and use of a Soybean rotational crop for seed production. A whole-of-farm was used to assess the impact of the new farming system on farm profitability. A whole-of-farm economic analysis looks at the impact of a change in farming practice across the whole business, rather than focusing on one single component. This case study is specific to an individual grower’s situation and is not representative of all situations. When evaluating a farming system change, it is important to have a detailed plan.

Economic Analysis of Sugarcane Farming Systems for Water Quality Improvement in the Great Barrier Reef Catchments.

In this report we analyse the private financial-economic impacts of transitioning to improved sugarcane management in the National Resource Management regions of the Wet Tropics, Burdekin Dry Tropics and Mackay Whitsundays. In order to do so, we: 1) compare farm GMs; 2) present information on capital investment associated with the transition; 3) perform a net present value analysis of the investments and; 4) undertake a risk analysis for cane and legume yields and prices. It must be noted that transaction costs are not captured within this project.

Nitrogen leaching from the root zone of sugarcane and bananas in the humid tropics of Australia

Loss of nitrogen in deep drainage from agriculture is an important issue for environmental and economic reasons, but limited field data is available for tropical crops. In this study, nitrogen (N) loads leaving the root zone of two major humid tropical crops in Australia, sugarcane and bananas, were measured. The two field sites, 57 km apart, had a similar soil type (a well drained Dermosol) and rainfall (∼2700 mm year -1) but contrasting crops and management. A sugarcane crop in a commercial field received 136-148 kg N ha -1 year -1 applied in one application each year and was monitored for 3 years (first to third ratoon crops). N treatments of 0-600 kg ha -1 year -1 were applied to a plant and following ratoon crop of bananas. N was applied as urea throughout the growing season in irrigation water through mini-sprinklers. Low-suction lysimeters were installed at a depth of 1 m under both crops to monitor loads of N in deep drainage. Drainage at 1 m depth in the sugarcane crops was 22-37% of rainfall. Under bananas, drainage in the row was 65% of rainfall plus irrigation for the plant crop, and 37% for the ratoon. Nitrogen leaching loads were low under sugarcane (<1-9 kg ha -1 year -1) possibly reflecting the N fertiliser applications being reasonably matched to crop requirements and at least 26 days between fertiliser application and deep drainage. Under bananas, there were large loads of N in deep drainage when N application rates were in excess of plant demand, even when applied fortnightly. The deep drainage loss of N attributable to N fertiliser, calculated by subtracting the loss from unfertilised plots, was 246 and 641 kg ha -1 over 2 crop cycles, which was equivalent to 37 and 63% of the fertiliser application for treatments receiving 710 and 1065 kg ha -1, respectively. Those rates of fertiliser application resulted in soil acidification to a depth of 0.6 m by as much as 0.6 of a unit at 0.1-0.2 m depth. The higher leaching losses from bananas indicated that they should be a priority for improved N management. Crown Copyright © 2012.

Reducing the risk of herbicide runoff in sugarcane farming through controlled traffic and early-banded application

The off-site transport of agricultural chemicals, such as herbicides, into freshwater and marine ecosystems is a world-wide concern. The adoption of farm management practices that minimise herbicide transport in rainfall-runoff is a priority for the Australian sugarcane industry, particularly in the coastal catchments draining into the World Heritage listed Great Barrier Reef (GBR) lagoon. In this study, residual herbicide runoff and infiltration were measured using a rainfall simulator in a replicated trial on a brown Chromosol with 90–100% cane trash blanket cover in the Mackay Whitsunday region, Queensland. Management treatments included conventional 1.5 m spaced sugarcane beds with a single row of sugarcane (CONV) and 2 m spaced, controlled traffic sugarcane beds with dual sugarcane rows (0.8 m apart) (2mCT). The aim was to simulate the first rainfall event after the application of the photosynthesis inhibiting (PSII) herbicides ametryn, atrazine, diuron and hexazinone, by broadcast (100% coverage, on bed and furrow) and banding (50–60% coverage, on bed only) methods. These events included heavy rainfall 1 day after herbicide application, considered a worst case scenario, or rainfall 21 days after application. The 2mCT rows had significantly (P < 0.05) less runoff (38%) and lower peak runoff rates (43%) than CONV rows for a rainfall average of 93 mm at 100 mm h−1 (1:20 yr Average Return Interval). Additionally, final infiltration rates were higher in 2mCT rows than CONV rows, with 72 and 52 mm h−1 respectively. This resulted in load reductions of 60, 55, 47, and 48% for ametryn, atrazine, diuron and hexazinone from 2mCT rows, respectively. Herbicide losses in runoff were also reduced by 32–42% when applications were banded rather than broadcast. When rainfall was experienced 1 day after application, a large percentage of herbicides were washed off the cane trash. However, by day 21, concentrations of herbicide residues on cane trash were lower and more resistant to washoff, resulting in lower losses in runoff. Consequently, ametryn and atrazine event mean concentrations in runoff were approximately 8 fold lower at day 21 compared with day 1, whilst diuron and hexazinone were only 1.6–1.9 fold lower, suggesting longer persistence of these chemicals. Runoff collected at the end of the paddock in natural rainfall events indicated consistent though smaller treatment differences to the rainfall simulation study. Overall, it was the combination of early application, banding and controlled traffic that was most effective in reducing herbicide losses in runoff. Crown copyright © 2012

L-rhamnose-1-dehydrogenase gene and L-rhamnose catabolism in the yeast Pichia stipitis

The purpose of this work was to identify some of the genes of the catabolic route of L-rhamnose in the yeast Pichia stipitis. There are at least two distinctly different pathways for L-rhamnose catabolism. The one described in bacteria has phosphorylated intermediates and the enzymes and the genes of this route have been described. The pathway described in yeast does not have phosphorylated intermediates. The intermediates and the enzymes of this pathway are known but none of the genes have been identified. The work was started by purifying the L-rhamnose dehydrogenase, which oxidates L-rhamnose to rhamnonic acid-gamma-lactone. NAD is used as a cofactor in this reaction. A DEAE ion exchange column was used for purification. The active fraction was further purified using a non-denaturing PAGE and the active protein identified by zymogram staining. In the last step the protein was separated in a SDS-PAGE, the protein band trypsinated and analysed by MALDI-TOF MS. This resulted in the identification of the corresponding gene, RHA1, which was then, after a codon change, expressed in Saccharomyces cerevisiae. Also C- or N-terminal histidine tags were added but as the activity of the enzyme was lost or strongly reduced these were not used. The kinetic properties of the protein were analysed in the cell extract. Substrate specifity was tested with different sugars; L-rhamnose, L-lyxose and L-mannose were oxidated by the enzyme. Vmax values were 180 nkat/mg, 160 nkat/mg and 72 nkat/mg, respectively. The highest affinity was towards L-rhamnose, the Km value being 0.9 mM. Lower affinities were obtained with L-lyxose, Km 4.3 mM, and L-mannose Km 25 mM. Northern analysis was done to study the transcription of RHA1 with different carbon sources. Transcription was observed only on L-rhamnose suggesting that RHA1 expression is L-rhamnose induced. A RHA1 deletion cassette for P. stipitis was constructed but the cassette had integrated randomly and not targeted to delete the RHA1 gene. Enzyme assays for L-lactaldehyde dehydrogenase were done similarly to L-rhamnose dehydrogenase assays. NAD is used as a cofactor also in this reaction where L-lactaldehyde is oxidised to L-lactate. The observed enzyme activities were very low and the activity was lost during the purification procedures.

Resistance gene analogues in sugarcane and sorghum and their association with quantitative trait loci for rust resistance

Fifty-four different sugarcane resistance gene analogue (RGA) sequences were isolated, characterized, and used to identify molecular markers linked to major disease-resistance loci in sugarcane. Ten RGAs were identified from a sugarcane stem expressed sequence tag (EST) library; the remaining 44 were isolated from sugarcane stem, leaf, and root tissue using primers designed to conserved RGA motifs. The map location of 31 of the RGAs was determined in sugarcane and compared with the location of quantitative trait loci (QTL) for brown rust resistance. After 2 years of phenotyping, 3 RGAs were shown to generate markers that were significantly associated with resistance to this disease. To assist in the understanding of the complex genetic structure of sugarcane, 17 of the 31 RGAs were also mapped in sorghum. Comparative mapping between sugarcane and sorghum revealed syntenic localization of several RGA clusters. The 3 brown rust associated RGAs were shown to map to the same linkage group (LG) in sorghum with 2 mapping to one region and the third to a region previously shown to contain a major rust-resistance QTL in sorghum. These results illustrate the value of using RGAs for the identification of markers linked to disease resistance loci and the value of simultaneous mapping in sugarcane and sorghum.

Triacylglycerol lipases of yeast and plants: more than just hydrolases

In the yeast, mobilization of triacylglycerols (TAG) is facilitated by TGL3, TGL4 and TGL5 gene products. Interestingly, experiments using [32P] orthophosphate as a precursor for complex glycerophospholipids revealed that tgl mutants had a lower steady-state level of these membrane lipids. To understand a possible link between TAG lipolysis and phospholipid metabolism, we performed overexpression studies with Tgl3p and Tgl5p which clearly demonstrated that these two enzymes enhanced the level of phospholipids. Domains and motifs search analyses indicated that yeast TAG hydrolases posses a GXSXG lipase motif but also a HX4D acyltransferase motif. Purified Tgl3p and Tgl5p did not only exhibit TAG lipase activity but also catalyzed acyl-CoA dependent acylation of lyso-phosphatidylethanolamine and lyso-phosphatidic acid (LPA), respectively. Search for lipase/hydrolase homologues in the Arabidopsis thaliana genome led to the identification of At4g24160 which possess three motifs that are conserved across the plant species such as GXSXG motif, a HX4D motif and a probable lipid binding motif V(X)3HGF. Characterization of At4g24160 expressed in bacteria revealed that the presence of an acyl-CoA dependent LPA acyltransferase activity. In addition, the purified recombinant At4g24160 protein hydrolyzed both TAG and phosphatidylcholine. We hypothesize that the plant enzyme may be involved in membrane repair. In summary, our results indicate that these TAG lipases play a dual role and thereby contribute to both anabolic and catabolic processes in yeast and plants.