Browsing by Author "Ngara, Rudo"

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    Identification and profiling of salinity stress-responsive proteins in Sorghum bicolor seedlings
    (Elsevier, 2012) Ngara, Rudo; Ndimba, Roya; Borch-Jensen, Jonas; Jensen, Ole Nørregaard; Ndimba, Bongani K.
    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.
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    Mapping and characterisation of the sorghum cell suspension culture secretome
    (Academic Journals, 2011) Ngara, Rudo; Ndimba, Bongani K.
    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.
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    A proteomic analysis of drought and salt stress responsive proteins of different sorghum varieties
    (University of the Western Cape, 2009) Ngara, Rudo; Rees, Jasper
    This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotic stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI-TOF and MALDI-TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germin proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism.
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    A proteomic analysis of drought and salt stress responsive proteins of different sorghum varieties
    (University of the Western Cape, 2009) Ngara, Rudo; Ndimba, Bongani K.
    Sorghum (Sorghum bicolorï, 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. This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotie stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI- TOF and MALDI- TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germ in proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism. Following 200 mM NaCl and 400 mM sorbitol stress treatments, the expression/abundance of a protein spot similar to a rice wall-associated protein kinase was upregulated in the sorghum secretome in response to both stresses. Amino acid sequence alignment of the matching peptides between these two proteins showed that the sorghum CF spot possesses a protein kinase domain. Therefore, this protein could possibly participate in cell signalling functions, which link the external environment with the cell's cytoplasm. Using whole plant systems, a comparative study of leaf protein expression between two sorghum varieties, AS6 (salt sensitive) and MN1618 (salt tolerant) was conducted. Forty well resolved spots of varying abundances were picked for MS analysis. Of these, 28 were positively identified, representing proteins with functions in carbohydrate metabolism (60.7%), proton transport (17.9%), protein synthesis (7.1%), hydrolytic functions (7.1%), nucleotide metabolism (3.6%) and detoxification (3.6%). Using PDQuest™ Advanced 2D Analysis Software version 8.0.1 (BIO-RAD), a comparative analysis of leaf proteome expression patterns between the two sorghum varieties was conducted. The results indicated proteins with similar expression patterns as well as qualitative and quantitative differences between the two leaf proteomes. The effect of 100 mM NaCI on leaf proteome expression between the two sorghum varieties was also studied. Western blotting analysis of leaf, sheath and root tissues using Hsp70 antibodies showed that this treatment induced Hsp70 expression, a known stress protein, in both varieties. Thereafter, the partially annotated leaf proteome map was used to landmark other salt responsive proteins. Examples of differential expression patterns included glutathione S transferase and hydroxynitrile lyase proteins whose abundances were upregulated in both varieties, while the large subunit of RuBisCo was downregulated in AS6 but upregulated in MN1618. Qualitative spot expression differences in response to salt stress were also observed between the two sorghum varieties but these remained unidentified after both MALDI-TOF and MALDI-TOF-TOF MS, possibly indicating novel and previously uncharacterised sorghum proteins. The results of this study can be used as reference tools by proteomics researchers worldwide as well as a foundation for future studies.