Proteomic mapping of the sorghum bicolor (L.) moench cell suspension culture secretome and identification of its drought stress responsive proteins
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Date
2017-01
Authors
Ramulifho, Elelwani
Journal Title
Journal ISSN
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Publisher
University of the Free State (Qwaqwa Campus)
Abstract
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.
Description
Keywords
Sorghum -- Breeding, Droughts, Crops, Food security, Dissertation (M.Sc. (Plant Sciences))--University of the Free State (Qwaqwa Campus, 2017