Error, Bias, and Long-Branch Attraction in Data for Two Chloroplast Photosystem Genes in Seed Plants

Sequences of two chloroplast photosystem genes, psaA and psbB, together comprising about 3,500 bp, were obtained for all five major groups of extant seed plants and several outgroups among other vascular plants. Strongly supported, but significantly conflicting, phylogenetic signals were obtained in parsimony analyses from partitions of the data into first and second codon positions versus third positions. In the former, both genes agreed on a monophyletic gymnosperms, with Gnetales closely related to certain conifers. In the latter, Gnetales are inferred to be the sister group of all other seed plants, with gymnosperms paraphyletic. None of the data supported the modern ‘‘anthophyte hypothesis,’’ which places Gnetales as the sister group of flowering plants. A series of simulation studies were undertaken to examine the error rate for parsimony inference. Three kinds of errors were examined: random error, systematic bias (both properties of finite data sets), and statistical inconsistency owing to long-branch attraction (an asymptotic property). Parsimony reconstructions were extremely biased for third-position data for psbB. Regardless of the true underlying tree, a tree in which Gnetales are sister to all other seed plants was likely to be reconstructed for these data. None of the combinations of genes or partitions permits the anthophyte tree to be reconstructed with high probability. Simulations of progressively larger data sets indicate the existence of long-branch attraction (statistical inconsistency) for third-position psbB data if either the anthophyte tree or the gymnosperm tree is correct. This is also true for the anthophyte tree using either psaA third positions or psbB first and second positions. A factor contributing to bias and inconsistency is extremely short branches at the base of the seed plant radiation, coupled with extremely high rates in Gnetales and nonseed plant outgroups. M. J. Sanderson,* M. F. Wojciechowski,*† J.-M. Hu,* T. Sher Khan,* and S. G. Brady

Investigation of highly regulated promoters for the production of recombinant proteins in plants

Plants have been identified as promising expression systems for the commercial production of recombinant proteins. Plant-based protein production or “biofarming” offers a number of advantages over traditional expression systems in terms of scale of production, the capacity for post-translation processing, providing a product free of contaminants and cost effectiveness. A number of pharmaceutically important and commercially valuable proteins, such as antibodies, biopharmaceuticals and industrial enzymes are currently being produced in plant expression systems. However, several challenges still remain to improve recombinant protein yield with no ill effect on the host plant. The ability for transgenic plants to produce foreign proteins at commercially viable levels can be directly related to the level and cell specificity of the selected promoter driving the transgene. The accumulation of recombinant proteins may be controlled by a tissue-specific, developmentally-regulated or chemically-inducible promoter such that expression of recombinant proteins can be spatially- or temporally- controlled. The strict control of gene expression is particularly useful for proteins that are considered toxic and whose expression is likely to have a detrimental effect on plant growth. To date, the most commonly used promoter in plant biotechnology is the cauliflower mosaic virus (CaMV) 35S promoter which is used to drive strong, constitutive transgene expression in most organs of transgenic plants. Of particular interest to researchers in the Centre for Tropical Crops and Biocommodities at QUT are tissue-specific promoters for the accumulation of foreign proteins in the roots, seeds and fruit of various plant species, including tobacco, banana and sugarcane. Therefore this Masters project aimed to isolate and characterise root- and seed-specific promoters for the control of genes encoding recombinant proteins in plant-based expression systems. Additionally, the effects of matching cognate terminators with their respective gene promoters were assessed. The Arabidopsis root promoters ARSK1 and EIR1 were selected from the literature based on their reported limited root expression profiles. Both promoters were analysed using the PlantCARE database to identify putative motifs or cis-acting elements that may be associated with this activity. A number of motifs were identified in the ARSK1 promoter region including, WUN (wound-inducible), MBS (MYB binding site), Skn-1, and a RY core element (seed-specific) and in the EIR1 promoter region including, Skn-1 (seed-specific), Box-W1 (fungal elicitor), Aux-RR core (auxin response) and ABRE (ABA response). However, no previously reported root-specific cis-acting elements were observed in either promoter region. To confirm root specificity, both promoters, and truncated versions, were fused to the GUS reporter gene and the expression cassette introduced into Arabidopsis via Agrobacterium-mediated transformation. Despite the reported tissue-specific nature of these promoters, both upstream regulatory regions directed constitutive GUS expression in all transgenic plants. Further, similar levels of GUS expression from the ARSK1 promoter were directed by the control CaMV 35S promoter. The truncated version of the EIR1 promoter (1.2 Kb) showed some differences in the level of GUS expression compared to the 2.2 Kb promoter. Therefore, this suggests an enhancer element is contained in the 2.2 Kb upstream region that increases transgene expression. The Arabidopsis seed-specific genes ATS1 and ATS3 were selected from the literature based on their seed-specific expression profiles and gene expression confirmed in this study as seed-specific by RT-PCR analysis. The selected promoter regions were analysed using the PlantCARE database in order to identify any putative cis elements. The seed-specific motifs GCN4 and Skn-1 were identified in both promoter regions that are associated with elevated expression levels in the endosperm. Additionaly, the seed-specific RY element and the ABRE were located in the ATS1 promoter. Both promoters were fused to the GUS reporter gene and used to transform Arabidopsis plants. GUS expression from the putative promoters was consitutive in all transgenic Arabidopsis tissue tested. Importantly, the positive control FAE1 seed-specific promoter also directed constitutive GUS expression throughout transgenic Arabidopsis plants. The constitutive nature seen in all of the promoters used in this study was not anticipated. While variations in promoter activity can be caused by a number of influencing factors, the variation in promoter activity observed here would imply a major contributing factor common to all plant expression cassettes tested. All promoter constructs generated in this study were based on the binary vector pCAMBIA2300. This vector contains the plant selection gene (NPTII) under the transcriptional control of the duplicated CaMV 35S promoter. This CaMV 35S promoter contains two enhancer domains that confer strong, constitutive expression of the selection gene and is located immediately upstream of the promoter-GUS fusion. During the course of this project, Yoo et al. (2005) reported that transgene expression is significantly affected when the expression cassette is located on the same T-DNA as the 35S enhancer. It was concluded, the trans-acting effects of the enhancer activate and control transgene expression causing irregular expression patterns. This phenomenon seems the most plausible reason for the constitutive expression profiles observed with the root- and seed-specific promoters assessed in this study. The expression from some promoters can be influenced by their cognate terminator sequences. Therefore, the Arabidopsis ARSK1, EIR1, ATS1 and ATS3 terminator sequences were isolated and incorporated into expression cassettes containing the GUS reporter gene under the control of their cognate promoters. Again, unrestricted GUS activity was displayed throughout transgenic plants transformed with these reporter gene fusions. As previously discussed constitutive GUS expression was most likely due to the trans-acting effect of the upstream CaMV 35S promoter in the selection cassette located on the same T-DNA. The results obtained in this study make it impossible to assess the influence matching terminators with their cognate promoters have on transgene expression profiles. The obvious future direction of research continuing from this study would be to transform pBIN-based promoter-GUS fusions (ie. constructs containing no CaMV 35S promoter driving the plant selection gene) into Arabidopsis in order to determine the true tissue specificity of these promoters and evaluate the effects of their cognate 3’ terminator sequences. Further, promoter truncations based around the cis-elements identified here may assist in determining whether these motifs are in fact involved in the overall activity of the promoter.

Development of descriptor tools for the characterization of Australian sugar mill evaporator scale

Cleaning of sugar mill evaporators is an expensive exercise. Identifying the scale components assists in determining which chemical cleaning agents would result in effective evaporator cleaning. The current methods (based on x-ray diffraction techniques, ion exchange/high performance liquid chromatography and thermogravimetry/differential thermal analysis) used for scale characterisation are difficult, time consuming and expensive, and cannot be performed in a conventional analytical laboratory or by mill staff. The present study has examined the use of simple descriptor tests for the characterisation of Australian sugar mill evaporator scales. Scale samples were obtained from seven Australian sugar mill evaporators by mechanical means. The appearance, texture and colour of the scale were noted before the samples were characterised using x-ray fluorescence and x-ray powder diffraction to determine the compounds present. A number of commercial analytical test kits were used to determine the phosphate and calcium contents of scale samples. Dissolution experiments were carried out on the scale samples with selected cleaning agents to provide relevant information about the effect the cleaning agents have on different evaporator scales. Results have shown that by simply identifying the colour and the appearance of the scale, the elemental composition and knowing from which effect the scale originates, a prediction of the scale composition can be made. These descriptors and dissolution experiments on scale samples can be used to provide factory staff with an on-site rapid process to predict the most effective chemicals for chemical cleaning of the evaporators.

Membrane filtration of clarified juice

A membrane filtration plant using suitable micro or ultra-filtration membranes has the potential to significantly increase pan stage capacity and improve sugar quality. Previous investigations by SRI and others have shown that membranes will remove polysaccharides, turbidity and colloidal impurities and result in lower viscosity syrups and molasses. However, the conclusion from those investigations was that membrane filtration was not economically viable. A comprehensive assessment of current generation membrane technology was undertaken by SRI. With the aid of two pilot plants provided by Applexion and Koch Membrane Systems, extensive trials were conducted at an Australian factory using clarified juice at 80–98°C as feed to each pilot plant. Conditions were varied during the trials to examine the effect of a range of operating parameters on the filtering characteristics of each of the membranes. These parameters included feed temperature and pressure, flow velocity, soluble solids and impurity concentrations. The data were then combined to develop models to predict the filtration rate (or flux) that could be expected for nominated operating conditions. The models demonstrated very good agreement with the data collected during the trials. The trials also identified those membranes that provided the highest flux levels per unit area of membrane surface for a nominated set of conditions. Cleaning procedures were developed that ensured the water flux level was recovered following a clean-in-place process. Bulk samples of clarified juice and membrane filtered juice from each pilot were evaporated to syrup to quantify the gain in pan stage productivity that results from the removal of high molecular weight impurities by membrane filtration. The results are in general agreement with those published by other research groups.

Fouling of tubular ceramic membranes during processing of cane sugar juice

This paper examines the fouling characteristics of four tubular ceramic membranes with pore sizes 300 kDa, 0.1 μm and 0.45 μm installed in a pilot plant at a sugar factory for processing clarified cane sugar juices. All the membranes, except the one with a pore size of 0.45 μm, generally gave reproducible results through the trials, were easy to clean and could handle operation at high volumetric concentration factors. Analysis of fouled and cleaned ceramic membranes revealed that polysaccharides, lipids and to a lesser extent, polyphenols, as well as other colloidal particles cause fouling of the membranes. Electrostatic and hydrophobic forces cause strong aggregation of the polymeric components with one another and with colloidal particles. To combat irreversible fouling of the membranes, treatment options that result in the removal of particles having a size range of 0.2–0.5 μm and in addition remove polymeric impurities, need to be identified. Chemical and microscopic evaluations of the juices and the structural characterisation of individual particles and aggregates identified options to mitigate the fouling of membranes. These include conditioning the feed prior to membrane filtration to break up the network structure formed between the polymers and particles in the feed and the use of surfactants to prevent the aggregation of polymers and particles.

Green cane impact on sugar processing : ISSCT process workshop 2008

The ISSCT Process Section workshop held in Réunion 20–23 October 2008 was attended by 51 delegates from 10 countries. The theme was Green cane impact on sugar processing. The workshop provided a valuable and timely opportunity to review and discuss the impact on factory operations and performance from a green cane supply that could include significant levels of trash. It was particularly relevant to those mills that were considering options to boost their biomass intake for increased co-generation capacity. Several of the speakers related their experiences with processing ‘whole of crop’ cane supplies through the factory. Speakers detailed the problems and increased losses that were incurred when processing cane with high trash levels. The consensus of the delegates was that the best scenario would involve a cane-cleaning plant at the factory so that only clean cane would be processed through the factory. The forum recommended that more research was required to address the issues of increased impurities in the process streams associated with high trash levels. Site visits to the two factories and a cane-delivery station were arranged as part of the workshop.

Formation of thermodynamically unstable calcium oxalate dihydrate in sugar mill evaporators

Calcium oxalate (CaOX) is the most intractable scale component to remove in sugar mill evaporators by either mechanical or chemical means. The operating conditions of sugar mill evaporators should preferentially favour the formation of the thermodynamically stable calcium oxalate monohydrate (COM), yet analysis of scale deposit from different sugar factories have shown that calcium oxalate dihydrate (COD) is usually the predominant phase, and in some cases is the only hydrate formed. The effects of trans-aconitic, succinic and acetic acids, all of which are present in sugarcane juice, and ethylenediamine tetraacetic acid disodium salt (EDTA) on the growth of CaOX crystals have been examined by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray powder diffraction (XRD) and thermogravimetric analysis (TGA). trans-Aconitic acid, which constitutes two-thirds of the organic acid component in sugarcane juice, in the presence of sugar resulted in the formation of COD and COM in a 3:1 ratio. EDTA was the most effective acid to promote the formation of COD followed by trans-aconitic acid, then acetic acid and lastly succinic acid.