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Crop adaptation, abiotic stress plant biochemistry

Abiotic Stress Proteomics of Crop Plants

Plants are continually challenged to recognize and respond to adverse changes in their environment to avoid detrimental effects on growth and development. Understanding the mechanisms that crop plants employ to resist and tolerate abiotic stress is of considerable interest for designing agriculture breeding strategies to ensure sustainable productivity.

The application of proteomics technologies to advance our knowledge in crop plant abiotic stress tolerance has increased dramatically in the past few years with the awareness that directly focusing on genes and their expression may not accurately portray conditions in the cell at a particular state and time during stress due to regulation at the RNA and protein level, including posttranslational regulation.

• Barkla, BJ (2018) Free Flow Zonal Electrophoresis for Fractionation of Plant Membrane Compartments Prior to Proteomic Analysis. Chapter in Methods in Molecular Biology. pp.1-12. DOI: 10.1007/978-1-4939-7411-5_1.
• Bravo-Adame ME, Vera-Estrella R, Barkla BJ, Martinez-Campos C, Flores-Alcantar A, Ocelotl-Oviedo JP, Pedraza-Alva G, Rosenstein Y (2017) An alternative mode of CD43 signal transduction activates pro-survival pathways of T lymphocytes. Immunology 150: 87-99.
• Nock CJ, Baten A, Barkla BJ, Furtado A, Henry RJ, King GJ. (2016) Genome and transcriptome sequencing characterises the gene space of Macadamia integrifolia (Proteaceae). BMC Genomics 17: 937.
• Barkla BJ, Rhodes T (2016) Use of infrared thermography for monitoring crassulacean acid metabolism. Functional Plant Biology 44: 46-51
• Ballindong J, Liu L, Ward RM, Barkla BJ, Waters DLE. (2016) Optimisation and standardisation of extraction and HPLC analysis of rice grain protein. Journal of Cereal Science 72:124-130, 10.1016/j.jcs.2016.10.005).
• Barkla BJ (2016) Identification of abiotic stress protein biomarkers by proteomic screening of crop cultivar diversity. Proteomes 4: 26.
• Carrasco Navarro U.; Vera Estrella R.; Barkla B.J.; Zúñiga León E.; Reyes Vivas H.; Fernández Perrino F.J.; Fierro F. (2016) Proteomic analysis of the signalling pathway mediated by the heterotrimeric Ga protein Pga1 of Penicillium chrysogenum. Microbial Cell Factories 15:173.
• Barkla BJ, Vera-Estrella R, Raymond C (2016) Single-cell-type quantitative proteomic and ionomic analysis of epidermal bladder cells from the halophyte model plant Mesembryanthemum crystallinum to identify salt-responsive proteins. BMC Plant Biology 16:110.
• Barkla, B. J., Castellanos-Cervantes, T., Diaz de León, J. L., Matros, A., Mock, H.-P., Perez-Alfocea, F., Salekdeh, G. H., Witzel, K. and Zörb, C. (2013), Elucidation of salt stress defence and tolerance mechanisms of crop plants using proteomics-Current achievements and perspectives. Proteomics, 13: 1885-1900. doi: 10.1002/pmic.201200399
• Barkla BJ, Vera-Estrella R. Pantoja O. (2013). Progress and challenges for abiotic stress proteomics of crop plants Proteomics, 3(12-13):1801-15. DOI: 10.1002/pic.201200401.

Focus on membrane proteins

On average, membrane proteins comprise approximately 30% of the eukaryotic proteome, and in Arabidopsis thaliana, close to 25% of the nuclear-encoded proteins are classified as membrane proteins. Membrane proteins would be expected to play an important role in abiotic stress tolerance and avoidance in crop plants, especially in those stresses involving changes in solute and water homeostasis, such as salinity, heavy metal, and water/drought stress and there are many reports in the literature which identify these proteins having a considerable role in stress tolerance.

However, proteomic reports in crop plants in which membrane proteins have been identified as stress responsive are scarce as most of the proteomic studies employ a single sample extraction approach and the majority of proteins identified in these studies are abundant proteins whose chemical properties facilitate extraction by the chosen method. If quantitative proteome analyses are to provide comprehensive information about cellular and regulatory processes that are stress responsive, and identify reliable stress biomarkers, they must be able to penetrate to the level of low abundant regulatory or transport proteins. To achieve this goal will require studies to include several approaches, including sample fractionation or enrichment studies to focus on particular sub proteomes or organelle proteomes.

One fractionation technique we have been exploiting is Free Flow Zonal Electrophoresis (FFZE). FFZE allows for the separation of specific membrane fractions from other cellular endomembranes based on net membrane surface charge during matrix free laminar flow through a thin aqueous layer. It is a highly versatile and reproducible fractionation technique and has been adapted to effectively separate cells, organelles, membranes and proteins from a wide variety of organisms and cell types; although only a few reports have appeared in which this technique has been utilized to separate plant membranes.

We have been adapting the FFE technique to separate highly purified tonoplast and more recently other membrane fractions, from several different plant species including Arabidopsis thaliana, Thellungiella halophila, Mesembryanthemum crystallinum, Ananis comosus (pineapple), barley and cowpea, as well as the green edaphic microalga, Neochloris oleoabundans. By manipulating the charge of specific membranes fractions through protein or chemical interactions we can control how the proteins are separated within the FFE chamber and this can be applied to all subcellular membrane compartments. Membrane fractions collected following FFZE are sealed and transport competent, allowing for their direct use in biochemical studies as well as providing high throughput starting material for downstream proteomics approaches.

• Calvert J, Baten A, Butler J, Barkla B, Shepherd M. (2017) Terpene synthase genes in Melaleuca alternifolia: comparative analysis of lineage-specific subfamily variation within Myrtaceae. Plant Systematics and Evolution DOI 10.1007/s00606-017-1454-3.
• Vera-Estrella R, Gomez-Mendez MF, Amezcua-Romero JC, Barkla BJ, Pantoja O. (2017) Cadmium and zinc activate mechanisms in Nicotiana tabacum similar to those observed in metal tolerant plants. Planta 246: 433–451.
• Garibay-Hernandez A, Barkla BJ, Vera-Estrella R, Martinez A, Pantoja O (2017) Membrane proteomics provides insights into the physiology and taxonomy of Ettlia oleoabundans. Plant Physiology 173: 390-416.
• Barkla BJ, Vera-Estrella R, Hernandez-Coronádo M, Pantoja O (2009) Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance. Plant Cell 41: 4044-4058.
• Barkla BJ, Vera-Estrella R, Pantoja O (2007) Enhanced separation of membranes during Free Flow Zonal Electrophoresis in plants. Analytical Chemistry 79: 5181-5187.

Using model plants to understand molecular mechanisms important for stress tolerance.

Mesembryanthemum crystallinum (common name ice plant) is a member of the Aizoaceae family in the order Caryophyllales of which sugar beet is the most economically valuable crop. Plants in this order are often found in marginal, stressful habitats and show a remarkable ability to tolerate diverse abiotic stress; particularly cold and salt stress. The ice plant is a highly salt tolerant species native to South Africa but now naturalized worldwide, predominantly in coastal areas, which show a Mediterranean climate, including Australia.

For most common crop plants, the presence of salt restricts the uptake of water and necessary nutrients, and once accumulated within the plant sodium can quickly reach toxic levels. Even small levels of salt in the soil can significantly reduce crop yield. The ice plant has developed adaptive mechanisms to enable the uptake of water under high salinity conditions and to actively accumulate the salt into the aerial parts of the plant.

The highest salt concentrations have been measured in the epidermal bladder cells (EBC) that line the leaf, stem and flower buds of M. crystallinum, and can reach 1.2 M sodium. These EBC's are modified single celled trichomes which remain compressed to the epidermal surface in unstressed plants but enlarge to comprise up to 25 % of the total aerial volume of the plant when plants are exposed to salinity.

Early work on the physiology of EBC in the ice plant concluded that these cells were predominantly involved in water storage during times of reduced water availability and more recently in sodium sequestration. However, by taking a proteomics approach we have been able to show that these cells are also metabolically active and unlike most epidermal cells, have functionally active chloroplasts (Barkla BJ et al., 2012).

Work in the lab is now underway to compare the transcriptome, proteome and metabolome of EBC from control grown plants to those grown in the presence of salt to identify novel stress responsive transcripts, proteins and metabolites that may help to shed a light on how sodium is sequestered, and what are other functions of these cells.

• Barkla B, Rhodes T, Tran K, Wijesinghege C, Larkin J, Dassanayake M (2018) Making epidermal bladder cells bigger: Developmental- and salinity-induced endopolyploidy in a model halophyte. Plant Physiology Vol 177 pp 615-632.
• Barkla BJ, Garibay‐Hernández A, Melzer M, Rupasinghe TWT, Roessner U. (2018) Single cell‐type analysis of cellular lipid remodelling in response to salinity in the epidermal bladder cells of the model halophyte Mesembryanthemum crystallinum. Plant Cell Environ. 2018;114.https://doi.org/10.1111/pce.13352
• Nock CJ, Baten A, Barkla BJ, Furtado A, Henry RJ, King GJ. (2016) Genome and transcriptome sequencing characterises the gene space of Macadamia integrifolia (Proteaceae). BMC Genomics 17: 937.
• Barkla BJ, Rhodes T (2016) Use of infrared thermography for monitoring crassulacean acid metabolism. Functional Plant Biology 44: 46-51
• Ballindong J, Liu L, Ward RM, Barkla BJ, Waters DLE. (2016) Optimisation and standardisation of extraction and HPLC analysis of rice grain protein. Journal of Cereal Science 72:124-130, 10.1016/j.jcs.2016.10.005).
• Barkla BJ, Vera-Estrella R, Raymond C (2016) Single-cell-type quantitative proteomic and ionomic analysis of epidermal bladder cells from the halophyte model plant Mesembryanthemum crystallinum to identify salt-responsive proteins. BMC Plant Biology 16:110.
• Barkla BJ (2016) Identification of abiotic stress protein biomarkers by proteomic screening of crop cultivar diversity. Proteomes 4: 26.
• Barkla BJ, Vera-Estrella R, Pantoja O. (2012) Protein profiling of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum. Proteomics 18: 2862-2865.
• Vera-Estrella R, Barkla BJ, Amezcua-Romero JC, Pantoja O (2011) Day/Night Regulation of Aquaporins During the CAM Cycle in Mesembryanthemum crystallinum. Plant Cell and Environment. 35: 485-501.

Manipulating seed storage proteins

• Rahman, M., Khatun, A., Liu, L., Barkla, B. (2018) Brassicaceae Mustards: Traditional and Agronomic Uses in Australia and New Zealand. Molecules 23 (1) 231; doi:10.3390/molecules23010231(Impact factor 2.861).