Advancing energy cane cell wall digestibility screening by near-infrared spectroscopy.

Breeding energy cane for cellulosic biofuel production involves manipulating various traits. An important trait to optimize is cell wall degradability as defined by enzymatic hydrolysis. We investigated the feasibility of using near-infrared spectroscopy (NIRS) combined with multivariate calibration to predict energy cane cell wall digestibility based upon fiber samples from a range of sugarcane genotypes and related species. These samples produced digestibility values ranging between 6 and 31%. To preserve the practicality of the technique, spectra obtained from crudely prepared samples were used. Various spectral pre-processing methods were tested, with the best NIRS calibration obtained from second derivative, orthogonal signal-corrected spectra. Model performance was evaluated by cross-validation and independent validation. Large differences between the performance results from the two validation approaches indicated that the model was sensitive to the choice of test data. This may be remedied by using a larger calibration training set containing diverse sample types. The best result was obtained through independent validation which produced a R(2) value of 0.86, a root mean squared error of prediction (RMSEP) of 1.59, and a ratio of prediction to deviation (RPD) of 2.7. This study has demonstrated that it is feasible and practical to use NIRS to predict energy cane cell wall digestibility.

Co-ordinated synthesis of gentiobiitol and sorbitol, evidence of sorbitol glycosylation in transgenic sugarcane.

Sugarcane (a Saccharum spp. interspecific hybrid) was previously engineered to synthesize sorbitol (designated as sorbitolcane). Motivated by the atypical development of the leaves in some sorbitolcane, the polar metabolite profiles in the leaves of those plants were compared against a group of control sugarcane plants. Eighty-six polar metabolites were detected in leaf extracts by GC-MS. Principal component analysis of the metabolites indicated that three compounds were strongly associated with sorbitolcane. Two were identified as sorbitol and gentiobiose and the third was unknown. Gentiobiose and the unknown compound were positively correlated with sorbitol accumulation. The unknown compound was only abundant in sorbitolcane. This compound was structurally characterized and found to be a sorbitol-glucose conjugate. (13)C NMR analysis indicated that the glucopyranose and glucitol moieties were 1,6-linked. Ligand exchange chromatography confirmed that the compound was a beta-anomer, thus identifying the compound as 6-O-beta-d-glucopyranosyl-D-glucitol, or gentiobiitol.

Near-infrared spectroscopy for the prediction of disease ratings for Fiji leaf gall in sugarcane clones.

This paper demonstrates how inferential measurements or indirect methods using near-infrared (NIR) methodology and chemometrics can be used to predict sugarcane clonal performance. Fiji leaf gall resistance is used in this study as an example. Fiji leaf gall is one of Australia's most serious sugarcane diseases, representing a significant problem in almost half of the total area under production. Traditional rating of sugarcane clones for resistance/susceptibility is difficult and expensive because of the nature of field-based methods and variable infection levels of the trials. Thus, the aim of this work was to investigate the potential of NIR spectroscopy as an alternative means to rate clones from direct measurement of sugarcane leaf spectra and to examine its ability to successfully predict traditional resistance ratings using a calibration model based on a chemometrics method such as partial least squares (PLS). A scanning electron microscopy (SEM) study of the leaf substrate was undertaken to elucidate the nature of the NIR sample site. In addition, an NIR study of freeze-dried sugarcane leaf samples resolved the heavily overlapping O-H bands present in the NIR spectrum due to water/cellulose interaction. A significant decrease in the spectral intensity between 5205 and 5393 cm(-1) was observed and a similar decrease was noted in the OH stretching overtone (7114 cm(-1)) with an accompanying shift to lower wavenumbers. PLS modeling based on traditional ratings as the dependent variable and the corresponding NIR spectra showed satisfactory results with standard error of validation (SEV) and standard error of prediction (SEP) values being 0.98 (R(2) = 0.97) and 1.20 (R(2) = 0.88), respectively. This methodology has now been recommended for more extensive field trials.

Complex biopolymeric systems at stalk/epicuticular wax plant interfaces: a near infrared spectroscopy study of the sugarcane example.

Naturally occurring macromolecules present at the epicuticular wax/stalk tissue interface of sugarcane were investigated using near infrared spectroscopy (NIRS). Investigations of water, cellulose, and wax-cellulose interrelationships were possible using NIRS methods, where in the past many different techniques have been required. The sugarcane complex interface was used as an example of typical phenomena found at plant leaf/stalk interfaces. This detailed study showed that sugarcane cultivars exhibit spectral differences in the CH(n), water OH, and cellulose OH regions, reflecting the presence of epicuticular wax, epidermis, and ground tissue. Spectrally complex water bands (5276 cm(-1) and 7500-6000 cm(-1)) were investigated via freeze-drying experiments which revealed sequentially a complex band substructure (7500-6000 cm(-1)), a developing weak H-bonding system ( approximately 7301 cm(-1)), and strong H-bonding ( approximately 7062 cm(-1)) assigned to water-cellulose interactions. Principal component analysis techniques clarified complex band trends that developed during the desorption experiment. Bands from wax-free stalk were minimized in the 4327-4080 cm(-1) region (C--H(n) vibrational modes associated with long chain fatty compounds), while bands from the stalk tissue (particularly lignin and moisture) became more pronounced. This work is a comprehensive guide to similar studies by scientists involved in a variety of plant and fiber research fields. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 642-651, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.

A Paenibacillus sp. dextranase mutant pool with improved thermostability and activity.

Random mutagenesis was used to create a library of chimeric dextranase (dex1) genes. A plate-screening protocol was developed with improved thermostability as a selection criterion. The mutant library was screened for active dextranase variants by observing clearing zones on dextran-blue agar plates at 50 degrees C after exposure to 68 degrees C for 2 h, a temperature regime at which wild-type activity was abolished. A number of potentially improved variants were identified by this strategy, five of which were further characterised. DNA sequencing revealed ten nucleotide substitutions, ranging from one to four per variant. Thermal inactivation studies showed reduced (2.9-fold) thermostability for one variant and similar thermostability for a second variant, but confirmed improved thermostability for three mutants with 2.3- (28.9 min) to 6.9-fold (86.6 min) increases in half-lives at 62 degrees C compared to that of the wild-type enzyme (12.6 min). Using a 10-min assay, apparent temperature optima of the variants were similar to that of the wild type (T (opt) 60 degrees C). However, one of these variants had increased enzyme activity. Therefore, the first-generation dextranase mutant pool obtained in this study has sufficient molecular diversity for further improvements in both thermostability and activity through recombination (gene shuffling).

Growth and metabolism in sugarcane are altered by the creation of a new hexose-phosphate sink.

An efficient in planta sugarcane-based production system may be realized by coupling the synthesis of alternative products to the metabolic intermediates of sucrose metabolism, thus taking advantage of the sucrose-producing capability of the plant. This was evaluated by synthesizing sorbitol in sugarcane (Saccharum hybrids) using the Malus domestica sorbitol-6-phosphate dehydrogenase gene (mds6pdh). Mature transgenic sugarcane plants were compared with untransformed sugarcane variety Q117 by evaluation of the growth, metabolite levels and extractable activity of relevant enzymes. The average amounts of sorbitol detected in the most productive line were 120 mg/g dry weight (equivalent to 61% of the soluble sugars) in the leaf lamina and 10 mg/g dry weight in the stalk pith. The levels of enzymes involved in sucrose synthesis and cleavage were elevated in the leaves of plants accumulating sorbitol, but this did not affect sucrose accumulation in the culm. The activity of oxidative reactions in the pentose phosphate pathway and the non-reversible glyceraldehyde-3-phosphate dehydrogenase reaction were elevated to replenish the reducing power consumed by sorbitol synthesis. Sorbitol-producing sugarcane generated 30%-40% less aerial biomass and was 10%-30% shorter than control lines. Leaves developed necrosis in a pattern characteristic of early senescence, and the severity was related to the relative quantity of sorbitol accumulated. When the Zymomonas mobilis glucokinase (zmglk) gene was co-expressed with mds6pdh to increase the production of glucose-6-phosphate, the plants were again smaller, indicating that glucose-6-phosphate deficiency was not responsible for the reduced growth. In summary, sorbitol hyperaccumulation affected sugarcane growth and metabolism, but the outcome was not lethal for the plant. This work also demonstrated that impressive yields of alternative products can be generated from the intermediates of sucrose metabolism in Saccharum spp.

Design, development, and use of molecular primers and probes for the detection of Gluconacetobacter species in the pink sugarcane mealybug.

Molecular tools for the species-specific detection of Gluconacetobacter sacchari, Gluconacetobacter diazotrophicus, and Gluconacetobacter liquefaciens from the pink sugarcane mealybug (PSMB) Saccharicoccus sacchari Cockerell (Homiptera: Pseudococcidae) were developed and used in polymerase chain reactions (PCR) and in fluorescence in situ hybridizations (FISH) to better understand the microbial diversity and the numerical significance of the acetic acid bacteria in the PSMB microenvironment. The presence of these species in the PSMB occurred over a wide range of sites, but not in all sites in sugarcane-growing areas of Queensland, Australia, and was variable over time. Molecular probes for use in FISH were also designed for the three acetic acid bacterial species, and shown to be specific only for the target species. Use of these probes in FISH of "squashed" whole mealybugs indicated that these acetic acid bacteria species represent only a small proportion of the microbial population of the PSMB. Despite the detection of Glac. sacchari, Glac. diazotrophicus, and Glac. liquefaciens by PCR from different mealybugs isolated at various times and from various sugarcane-growing areas in Queensland, Australia, these bacteria do not appear to be significant commensals in the PSMB environment.

Diverse dextranase genes from Paenibacillus species.

Genes encoding dextranolytic enzymes were isolated from Paenibacillus strains Dex40-8 and Dex50-2. Single, similar but non-identical dex1 genes were isolated from each strain, and a more divergent dex2 gene was isolated from strain Dex50-2. The protein deduced from the Dex40-8 dex1 gene sequence had 716 amino acids, with a predicted M(r) of 80.8 kDa. The proteins deduced from the Dex50-2 dex1 and dex2 gene sequences had 905 and 596 amino acids, with predicted M(r) of 100.1 kDa and 68.3 kDa, respectively. The deduced amino acid sequences of all three dextranolytic proteins had similarity to family 66 glycosyl hydrolases and were predicted to possess cleavable N-terminal signal peptides. Homology searches suggest that the Dex40-8 and Dex50-2 Dex1 proteins have one and two copies, respectively, of a carbohydrate-binding module similar to CBM_4_9 (pfam02018.11). The Dex50-2 Dex2 deduced amino acid sequence had highest sequence similarity to thermotolerant dextranases from thermophilic Paenibacillus strains, while the Dex40-8 and Dex50-2 Dex1 deduced protein sequences formed a distinct sequence clade among the family 66 proteins. Examination of seven Paenibacillus strains, using a polymerase chain reaction-based assay, indicated that multiple family 66 genes are common within this genus. The three recombinant proteins expressed in Escherichia coli possessed dextranolytic activity and were able to convert ethanol-insoluble blue dextran into an ethanol-soluble product, indicating they are endodextranases (EC 3.2.1.11). The reaction catalysed by each enzyme had a distinct temperature and pH dependence.

Initial evaluation of sugarcane as a production platform for p-hydroxybenzoic acid.

Sugarcane (Saccharum hybrids) was evaluated as a production platform for p-hydroxybenzoic acid using two different bacterial proteins (a chloroplast-targeted version of Escherichia coli chorismate pyruvate-lyase and 4-hydroxycinnamoyl-CoA hydratase/lyase from Pseudomonas fluorescens) that both provide a one-enzyme pathway from a naturally occurring plant intermediate. The substrates for these enzymes are chorismate (a shikimate pathway intermediate that is synthesized in plastids) and 4-hydroxycinnamoyl-CoA (a cytosolic phenylpropanoid intermediate). Although both proteins have previously been shown to elevate p-hydroxybenzoic acid levels in plants, they have never been evaluated concurrently in the same laboratory. Nor are there any reports on their efficacy in stem tissue. After surveying two large populations of transgenic plants, it was concluded that the hydratase/lyase is the superior catalyst for leaf and stem tissue, and further studies focused on this pathway. p-Hydroxybenzoic acid was quantitatively converted to glucose conjugates by endogenous uridine diphosphate (UDP)-glucosyltransferases and presumably stored in the vacuole. The largest amounts detected in leaf and stem tissue were 7.3% and 1.5% dry weight (DW), respectively, yet there were no discernible phenotypic abnormalities. However, as a result of diverting carbon away from the phenylpropanoid pathway, there was a severe reduction in leaf chlorogenic acid, subtle changes in lignin composition, as revealed by phloroglucinol staining, and an apparent compensatory up-regulation of phenylalanine ammonia-lyase. Although product accumulation in the leaves at the highest level of gene expression obtained in the present study was clearly substrate-limited, additional experiments are necessary before this conclusion can be extended to the stalk.

Isolation and characterization of genes encoding thermoactive and thermostable dextranases from two thermotolerant soil bacteria.

Thermotolerant Paenibacillus strain Dex70-1B and unidentified strain Dex70-34 produce thermoactive dextran-degrading enzymes. Plasmid-based genomic DNA libraries constructed from mixed bacterial cultures containing Dex70-1B or Dex70-34 were screened for the ability to confer dextranolytic activity at 70 degrees C onto Escherichia coli. One gene, designated dex1, was isolated from each strain. The Dex70-1B and Dex70-34 dex1 gene sequences were non-identical, and encoded proteins containing 597 (M(r) 68.6 kDa) and 600 amino acids (M(r) 69.2 kDa), respectively. The Dex1 amino acid sequences were most similar to one another, and formed a new clade among the family 66 glycosyl hydrolase sequences. Expression of the Dex1 proteins in E. coli produced dextranolytic activity that converted ethanol-insoluble blue dextran into an ethanol-soluble form, suggestive of endodextranases (EC 3.2.1.11). Both enzymes were most active at about 60 degrees C and pH 5.5, and retained more than 70% maximal activity after incubation at 57 degrees C for 9.5 h in the absence of substrate.