Mapping and characterisation of the sorghum cell suspension culture secretome

Here we reported the first secretomic study of sorghum (Sorghum bicolor), a naturally drought tolerant cereal crop. In this study, we used a gel-based proteomic approach in combination with mass spectrometry to separate and identify proteins secreted into the culture medium of sorghum cell suspensions, a first step towards understanding their functions during plant growth and development. Proteins secreted into the culture medium of 10-day old sorghum cell suspension cultures termed culture filtrate (CF) proteins were separated by two-dimensional gel electrophoresis (2DE) and visualised using Coomassie brilliant blue (CBB) R-250 staining. Of the 25 visualised CBB stainable spots, 15 abundant, well-resolved and reproducible spots amongst the three biological replicates used were selected for identification using matrix assisted laser desorption/ionisation-time of flight/time of flight tandem mass spectrometry (MALDI-TOF-TOF MS). Of these spots, 14 were positively identified, representing four different protein classes: Peroxidases, germin proteins, oxalate oxidases and ?-galactosidases. All the identified proteins are known secretory proteins, with predicted signal peptides, which target proteins for the secretory pathway. The identified proteins have known functions in signalling processes, defence mechanisms and cell wall metabolism which is consistent with their location outside the cell. Western blotting analysis of the CF protein extracts using an antibody against ?-tubulin, a cytoplasmic protein, indicated that our CF protein preparations are free from any detectable amounts of this marker protein. Therefore, our sorghum cell culture system is ideal for use in the proteomic analysis of secreted proteins. The findings of this study are a step in the process of bridging the gap that currently exists in sorghum proteomics and also provides a foundation for future studies on understanding the roles played by secreted proteins during plant growth and development of the same crop

Mapping and characterisation of the sorghum cell suspension culture secretome

Here we reported the first secretomic study of sorghum (Sorghum bicolor), a naturally drought tolerant cereal crop. In this study, we used a gel-based proteomic approach in combination with mass spectrometry to separate and identify proteins secreted into the culture medium of sorghum cell suspensions, a first step towards understanding their functions during plant growth and development. Proteins secreted into the culture medium of 10-day old sorghum cell suspension cultures termed culture filtrate (CF) proteins were separated by two dimensional gel electrophoresis (2DE) and visualised using Coomassie brilliant blue (CBB) R-250 staining. Of the 25 visualised CBB stainable spots, 15 abundant, well resolved and reproducible spots amongst the three biological replicates used were selected for identification using matrix assisted laser desorption/ionisation-time of flight/time of flight tandem mass spectrometry (MALDI-TOF-TOF MS). Of these spots, 14 were positively identified, representing four different protein classes: Peroxidases, germin proteins, oxalate oxidases and α-galactosidases. All the identified proteins are known secretory proteins, with predicted signal peptides, which target proteins for the secretory pathway. The identified proteins have known functions in signalling processes, defence mechanisms and cell wall metabolism which is consistent with their location outside the cell. Western blotting analysis of the CF protein extracts using an antibody against β-tubulin, a cytoplasmic protein, indicated that our CF protein preparations are free from any detectable amounts of this marker protein. Therefore, our sorghum cell culture system is ideal for use in the proteomic analysis of secreted proteins. The findings of this study are a step in the process of bridging the gap that currently exists in sorghum proteomics and also provides a foundation for future studies on understanding the roles played by secreted proteins during plant growth and development of the same crop.Keywords: Sorghum, cell suspension cultures, culture filtrate, secreted proteins, proteomics analysis, two-dimensional gel electrophoresis, β-tubuli

Molecular adaptation mechanisms employed by ethanologenic bacteria in response to lignocellulose-derived inhibitory compounds

Current international interest in finding alternative sources of energy to the diminishing supplies of fossil fuels has encouraged research efforts in improving biofuel production technologies. In countries which lack sufficient food, the use of sustainable lignocellulosic feedstocks, for the production of bioethanol, is an attractive option. In the pre-treatment of lignocellulosic feedstocks for ethanol production, various chemicals and/or enzymatic processes are employed. These methods generally result in a range of fermentable sugars, which are subjected to microbial fermentation and distillation to produce bioethanol. However, these methods also produce compounds that are inhibitory to the microbial fermentation process. These compounds include products of sugar dehydration and lignin depolymerisation, such as organic acids, derivatised furaldehydes and phenolic acids. These compounds are known to have a severe negative impact on the ethanologenic microorganisms involved in the fermentation process by compromising the integrity of their cell membranes, inhibiting essential enzymes and negatively interact with their DNA/RNA. It is therefore important to understand the molecular mechanisms of these inhibitions, and the mechanisms by which these microorganisms show increased adaptation to such inhibitors. Presented here is a concise overview of the molecular adaptation mechanisms of ethanologenic bacteria in response to lignocellulose-derived inhibitory compounds. These include general stress response and tolerance mechanisms, which are typically those that maintain intracellular pH homeostasis and cell membrane integrity, activation/regulation of global stress responses and inhibitor substrate-specific degradation pathways. We anticipate that understanding these adaptation responses will be essential in the design of ‘intelligent’ metabolic engineering strategies for the generation of hyper-tolerant fermentation bacteria strains.IS

A Comparative Study of Selected Physical and Biochemical Traits of Wild-Type and Transgenic Sorghum to Reveal Differences Relevant to Grain Quality

Transgenic sorghum featuring RNAi suppression of certain kafirins was developed recently, to address the problem of poor protein digestibility in the grain. However, it was not firmly established if other important quality parameters were adversely affected by this genetic intervention. In the present study several quality parameters were investigated by surveying several important physical and biochemical grain traits. Important differences in grain weight, density and endosperm texture were found that serve to differentiate the transgenic grains from their wild-type counterpart. In addition, ultrastructural analysis of the protein bodies revealed a changed morphology that is indicative of the effect of suppressed kafirins. Importantly, lysine was found to be significantly increased in one of the transgenic lines in comparison to wild-type; while no significant changes in anti-nutritional factors could be detected. The results have been insightful for demonstrating some of the corollary changes in transgenic sorghum grain, that emerge from imposed kafirin suppression

Identification and profiling of salinity stress-responsive proteins in Sorghum bicolor seedlings

Sorghum bicolor, a drought tolerant cereal crop, is not only an important food source in the semi arid/arid regions but also a potential model for studying and gaining a better understanding of the molecular mechanisms of drought and salt stress tolerance in cereals. In this study, seeds of a sweet sorghumvariety, MN1618, were planted and grown on solid MS growth medium with or without 100mM NaCl. Heat shock protein expression immunoblotting assays demonstrated that this salt treatment induced stress within natural physiological parameters for our experimental material. 2D PAGE in combination with MS/MS proteomics techniques were used to separate, visualise and identify salinity stress responsive proteins in young sorghum leaves. Out of 281 Coomassie stainable spots, 118 showed statistically significant responses (p<0.05) to salt stress treatments. Of the 118 spots, 79 were selected for tandem mass spectrometric identification, owing to their good resolution and abundance levels, and of these, 55 were positively identified. Identified proteins were divided into six functional categories including both known and novel/putative stress responsive proteins. Molecular and physiological functions of some of our proteins of interest are currently under investigation via bioinformatic and molecular biology approaches.Web of Scienc

INPPO actions and recognition as a driving force for progress in plant proteomics: Change of guard, INPPO update, and upcoming activities

The International Plant Proteomics Organization (INPPO) is a non-profit organization whose members are scientists involved or interested in plant proteomics. Since the publication of the first INPPO highlights in 2012, continued progress on many of the organization’s mandates/goals has been achieved. Two major events are emphasized in this second INPPO highlights. First, the change of guard at the top, passing of the baton from Dominique Job, INPPO founding President to Ganesh Kumar Agrawal as the incoming President. Ganesh K. Agrawal, along with Dominique Job and Randeep Rakwal initiated the INPPO. Second, the most recent INPPO achievements and future targets, mainly the organization of first the INPPO World Congress in 2014, tentatively planned for Hamburg (Germany), are mentioned.Web of Scienc

Brown rot-type fungal decomposition of sorghum bagasse: variable success and mechanistic implications

Sweet sorghum is a promising crop for a warming, drying African climate, and basic information is lacking on conversion pathways for its lignocellulosic residues (bagasse). Brown rot wood-decomposer fungi use carbohydrate-selective pathways that, when assessed on sorghum, a grass substrate, can yield information relevant to both plant biomass conversion and fungal biology. In testing sorghum decomposition by brown rot fungi (Gloeophyllum trabeum, Serpula lacrymans), we found that G. trabeum readily degraded sorghum, removing xylan prior to removing glucan. Serpula lacrymans, conversely, caused little decomposition. Ergosterol (fungal biomarker) and protein levels were similar for both fungi, but S. lacrymans produced nearly 4x lower polysaccharide-degrading enzyme specific activity on sorghum than G. trabeum, perhaps a symptom of starvation. Linking this information to genome comparisons including other brown rot fungi known to have a similar issue regarding decomposing grasses (Postia placenta, Fomitopsis pinicola) suggested that a lack of CE 1 feruloyl esterases as well as low xylanase activity in S. lacrymans (3x lower than in G. trabeum) may hinder S. lacrymans, P. placenta, and F. pinicola when degrading grass substrates. These results indicate variability in brown rot mechanisms, which may stem from a differing ability to degrade certain lignincarbohydrate complexes

A comparison of faecal microbial populations of South African Windsnyer-type indigenous pigs (SAWIPs) and Large White × Landrace (LW × LR) crosses fed diets containing ensiled maize cobs

Faecal microbial communities in South African Windsnyer-type indigenous pigs (SAWIPs) and Large White × Landrace (LW × LR) crosses were investigated using high-throughput sequencing of the 16S rDNA genes. The faecal microbial communities in LW × LR crosses and SAWIPs fed control (CON) and high maize cob (HMC) diets were evaluated through parallel sequencing of 16S rDNA genes. Butrivibrio, Faecalibacterium and Desulfovibrio, although present in LW × LR pigs, were absent from the SAWIP microbial community. Bacteroides, Succiniclasticum, Peptococcus and Akkermansia were found in SAWIPs but not in LW × LR crosses. The ratios of Bacteroidia to Clostridia on the CON and HMC diets were similar (0.37 versus 0.39) in SAWIPs but different (0.24 versus 0.1) in LW × LR crosses. The faecal microbial profiles determined were different between the LW × LR and SAWIP breeds but not between pigs fed the CON and HMC diets. The composition of faecal bacterial communities in SAWIPs was determined for the first time. The differences in microbial communities detected may explain the enhanced ability of SAWIPs to digest fibrous diets compared with the LW × LR crosse

A comparative study of selected physical and biochemical traits of wild-type and transgenic sorghum to reveal differences relevant to grain quality

CITATION: Ndimba, R. J., et al. 2017. A comparative study of selected physical and biochemical traits of wild-type and transgenic sorghum to reveal differences relevant to grain quality. Frontiers in Plant Science, 8:952, doi:10.3389/fpls.2017.00952.The original publication is available at https://www.frontiersin.orgTransgenic sorghum featuring RNAi suppression of certain kafirins was developed recently, to address the problem of poor protein digestibility in the grain. However, it was not firmly established if other important quality parameters were adversely affected by this genetic intervention. In the present study several quality parameters were investigated by surveying several important physical and biochemical grain traits. Important differences in grain weight, density and endosperm texture were found that serve to differentiate the transgenic grains from their wild-type counterpart. In addition, ultrastructural analysis of the protein bodies revealed a changed morphology that is indicative of the effect of suppressed kafirins. Importantly, lysine was found to be significantly increased in one of the transgenic lines in comparison to wild-type; while no significant changes in anti-nutritional factors could be detected. The results have been insightful for demonstrating some of the corollary changes in transgenic sorghum grain, that emerge from imposed kafirin suppression.Publisher's versio

Correction: Cross-species multiple environmental stress responses: An integrated approach to identify candidate genes for multiple stress tolerance in sorghum (Sorghum bicolor (L.) Moench) and related model species.

[This corrects the article DOI: 10.1371/journal.pone.0192678.]