Proteomic mapping of the sorghum bicolor (L.) moench cell suspension culture secretome and identification of its drought stress responsive proteins

[摘要] Drought (also known as osmotic stress), adversely effects crop productivity. With theprojected increase in global surface temperatures, the frequency and intensity of droughtis predicted to increase, worldwide. It is therefore important to develop crops that canwithstand drought and thus alleviate food insecurity. However, the success of suchbreeding initiatives requires prior understanding of plant stress response mechanisms.Sorghum (Sorghum bicolor), a naturally drought tolerant cereal crop, is a potentially goodmodel system for studying plant responses to drought stress. The objectives of this studywere to establish a sorghum cell suspension culture system, map its secretome andidentify the osmotic stress responsive proteins. In this study, seeds from eight sorghumgenotypes, 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 insecretome analysis. Murashige and Skoog Basal Salt with minimal organics mediumsupplemented with varying concentrations of plant growth hormones, 1-naphthaleneacetic acid (NAA) and 2,4-Dichlorophenoxyacetic acid (2,4-D) were used forcallus induction. ICSB 338 and White sorghum produced large friable callus masses onmedium supplemented with 2.5 mg/L NAA and 3 mg/L 2,4-D. These callus masses weresubsequently used to establish cell suspension cultures, which were furthercharacterised in terms of cell growth and viability patterns following sorbitol-inducedosmotic stress. The cell growth plots conformed to a typical sigmoidal growth curve withdistinct lag, exponential, and stationary phases. Osmotic stress experiments werecarried out on ICSB 338 and White sorghum cell cultures using 400 mM sorbitol for 72hr. Cell viability and microscopic analysis indicated a change in metabolic activity andstructural 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 wereobserved on Coomassie Brilliant Blue-stained one-dimensional sodium dodecyl sulfatepolyacrylamidegels. The White sorghum secreted proteins after 48 hr of sorbitoltreatment were further analysed by the isobaric tags for relative and absolutequantitation (iTRAQ) method. A total of 178 sorghum secreted proteins were positivelyidentified, with some matching proteins from plant peroxidase, glycoside hydrolase,Expansin/Lol pl, germin, and peptidase C1A protein families. However, 78% of the 178positively identified proteins were uncharacterised, possibly indicating novel sorghumproteins. SignalP 4.1 predicted signal peptides on 128 (72%) of the positively identifiedproteins, indicating that they are classically secreted into the extracellular matrix, while50 (28%) were not. Out of the 178 positively identified secreted proteins, 152 weredifferentially expressed in response to osmotic stress with 148 (97%) and 4 (3%) beingup-regulated and down-regulated, respectively. The osmotic stress responsive proteinswere 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 secondarymetabolism (1%); while 3% were unclassified and 2% unclear classifications,respectively. This study reports the first comprehensive sorghum cell suspension culturesecretome map and its osmotic stress responsive proteins. The secretome mapping datareported in this study can be used as a reference for studies focusing on characterisingsorghum secreted proteins in response to a wide range of biotic and abiotic stresses,thus further advancing existing knowledge on sorghum response networks.

[发布机构] University of the Free State