Proteomic mapping of the sorghum bicolor (L.) moench cell suspension culture secretome and identification of its drought stress responsive proteins
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Drought (also known as osmotic stress), adversely effects crop productivity. With the projected increase in global surface temperatures, the frequency and intensity of drought is predicted to increase, worldwide. It is therefore important to develop crops that can withstand drought and thus alleviate food insecurity. However, the success of such breeding initiatives requires prior understanding of plant stress response mechanisms. Sorghum (Sorghum bicolor), a naturally drought tolerant cereal crop, is a potentially good model system for studying plant responses to drought stress. The objectives of this study were to establish a sorghum cell suspension culture system, map its secretome and identify the osmotic stress responsive proteins. In this study, seeds from eight sorghum genotypes, namely SA 1441, ICSV 210, ICSV 112, ICSV 213, ICSB 78, ICSB 338, Macia, and White sorghum, were used to establish callus and cell suspensions for use in secretome analysis. Murashige and Skoog Basal Salt with minimal organics medium supplemented with varying concentrations of plant growth hormones, 1- naphthaleneacetic acid (NAA) and 2,4-Dichlorophenoxyacetic acid (2,4-D) were used for callus induction. ICSB 338 and White sorghum produced large friable callus masses on medium supplemented with 2.5 mg/L NAA and 3 mg/L 2,4-D. These callus masses were subsequently used to establish cell suspension cultures, which were further characterised in terms of cell growth and viability patterns following sorbitol-induced osmotic stress. The cell growth plots conformed to a typical sigmoidal growth curve with distinct lag, exponential, and stationary phases. Osmotic stress experiments were carried out on ICSB 338 and White sorghum cell cultures using 400 mM sorbitol for 72 hr. Cell viability and microscopic analysis indicated a change in metabolic activity and structural changes of cells following osmotic stress treatment. Culture filtrate proteins (referred to as secreted proteins in this study), were extracted from both cell cultures. Differential protein expressions of the secreted proteins of the two cultures were observed on Coomassie Brilliant Blue-stained one-dimensional sodium dodecyl sulfatepolyacrylamide gels. The White sorghum secreted proteins after 48 hr of sorbitol treatment were further analysed by the isobaric tags for relative and absolute quantitation (iTRAQ) method. A total of 178 sorghum secreted proteins were positively identified, with some matching proteins from plant peroxidase, glycoside hydrolase, Expansin/Lol pl, germin, and peptidase C1A protein families. However, 78% of the 178 positively identified proteins were uncharacterised, possibly indicating novel sorghum proteins. SignalP 4.1 predicted signal peptides on 128 (72%) of the positively identified proteins, indicating that they are classically secreted into the extracellular matrix, while 50 (28%) were not. Out of the 178 positively identified secreted proteins, 152 were differentially expressed in response to osmotic stress with 148 (97%) and 4 (3%) being up-regulated and down-regulated, respectively. The osmotic stress responsive proteins were predicted to have putative functions in metabolism (33.5%), disease/defence (23%), protein destination and storage (13%), signal transduction (8%), energy (6.5%), cell growth/division (6%), cell structure (3%), intracellular traffic (1%), and secondary metabolism (1%); while 3% were unclassified and 2% unclear classifications, respectively. This study reports the first comprehensive sorghum cell suspension culture secretome map and its osmotic stress responsive proteins. The secretome mapping data reported in this study can be used as a reference for studies focusing on characterising sorghum secreted proteins in response to a wide range of biotic and abiotic stresses, thus further advancing existing knowledge on sorghum response networks.