June 11, 2018

Proteomics as a tool in sorghum and cereal quality and abiotic stress

tolerance breeding

Maryke LabuschagneDept. of Plant Sciences, University of the Free StateBloemfontein, South AfricaEmail: labuscm@ufs.ac.za

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Prot

eom

ics 1994 is considered the birth

year of proteomics

Is the efficient and high-throughput identification of proteins in tissues and cells

First papers on the proteomics of plants only

appeared in late 1990’s

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Prot

eom

ics Comes from PROTEins

expressed by a genOME

Bioinformatics and computational science are connecting proteomics to

other “omics”

Genomics and proteomics are leading in discovery of

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What is “omics”?

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Chromatographic methods largely been used for analysis of cereal proteins until recently

Difficulties to separate proteins with these methods, so 2D electrophoresis has become method of choice for cereal protein identification

2DE is followed by mass spectrometry for specific protein identificationDO N

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Spots are excised, digested with trypsin or other proteases

Analysis with mass spectrometry

Spectra are then matched to theoretic data generated from a database of protein sequencesDO N

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The structure and composition of storage proteins in cereals determine their quality traits

Proteomics provides a tool to identify variation in protein composition in cereals

Which allows linking of proteomic profiles to phenotypic traits to develop markers for improved qualityDO N

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Sorghum proteomics

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June 11, 2018

The combination of Sorghum bicolor’s (sorghum) natural stress tolerance

And the recent completion of its genome sequencing

Makes it ideal for proteomics and genomics researchDO N

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The major storage proteins in sorghum are kafirins

Commercial value of sorghum often determined by endosperm composition

This includes composition and interaction of protein and starchDO N

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Kafirins grouped according to molecular weight, solubility, structure and amino acid composition

α-kafirin: most abundantβ-kafirinγ-kafirinδ- kafirin

Extensive sequence homology and similarity between maize and sorghum storage proteinDO N

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But sorghum protein bodies highly cross linked, reducing protein digestibility

Proteomics identified range of redox active proteins affecting sorghum storage protein chemistry (Cremer, 2014)

A range of putative uncharacterized proteins were found regulating folding and disulphide pairing of storage proteinsDO N

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α and β kafirin was correlated with protein digestibility

Some papers reported on proteomics as a tool in sorghum biofuel research (Ndimba et al., 2013, Salvato et al., 2017)

Proteomics is a way to understand biology behind biofuel production and manipulate this for increased biofuel productionDO N

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Salvato et al. (2017) reported on proteomics for identifying and characterizing proteins expressed in bioenergy production

Swami et al. (2011) identified 21 salt stress proteins from sorghum leaves

Ngara et al. (2012) divided sorghum leaf proteins into 6 categories and found carbohydrate metabolism proteins the most abundantDO N

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Wheat proteomics

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Proteomics has in the last years been successfully used to:

• Catalogue major proteins in wheat grains• Determine changes in the grain proteome during

germination and grain development• Determine responses of the proteome to biotic and

abiotic stresses

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Protein cataloguing

Many papers

reported on presence

and types of proteins

HMW-GS, LMW-GS, α, ω and γgliadins

Various methods used for

extraction and analysis

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Developmental studies

Accumulation profiles of proteins

throughout development in

endosperm

Provides insights into

molecular processes

taking place during grain

development

Establishes detailed picture of flour protein

composition critical for

understanding flour quality

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Major abiotic stresses

Drought Heat Combined heat and drought

Fertilizer

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Changes in relative amounts of protein spots

Up and down regulation of proteins due to stress conditions

Some authors extracted only a specific fraction for analysis, such as gliadins, albumins/globulins, HMW or LMW-GSDO N

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Visioli et al., 2016 (PLOS One)

Case study:

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Case studies:

• Hurkman et al. (2013) compared the effects of high temperature and fertilizer singly and in combination on the entire complement of gliadins and glutenins in flour from mature grain

• 122 gluten protein spots representing 19 gluten protein gene sequences responded significantly to fertilizer: five HMW-GS, six α-gliadins, and four ω-gliadins increased and three LMW-GS and one γ-gliadin decreased

• Zörb et al. (2010) reported that high N supply with low or high sulphur supply changed 41 and 66 of gliadin and glutenin proteins

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Wheat allergies

• Coeliac disease is the most common inflammatory condition in developed countries and affects about 1% of the population

• Is an immune-mediated enteropathy due to abnormal immune response to gluten in genetically susceptible people carrying HLA-DQ2 or DQ8 haplotype (Martinez-Estosa et al., 2016)

• 98.6% of coeliac toxic proteins have a minimum of 9 amino acids, which are likely to elicit toxic response

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• α gliadins have consistently high content of coeliac toxic motifs

• γ and ω gliadins also have toxic motifs• Proteomics can be used to target breeding for presence

of low numbers of toxic motifs• Proteomics to be used to test gluten free products for

absence of toxic motifs (Bromilow et al., 2017)

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Other

• Gobaa et al. (2008) showed 1BL.1RS translocation to cause large changes in endosperm proteome

• Fernando et al. (2015) reported decreases in HMW-GS spots with subsequent reduction in loaf volume under elevated CO2 conditions

• Studies on pre-harvest sprouting in wheat (Kamal et al., 2009) showed an effect on some glutenin and gliadin spots, among other proteins

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Other

• Einkorn, emmer and spelt wheat were analysed and peptides with immunogenic and toxic sequences were found in all samples (Prandi et al., 2017)

• There was higher quantity of peptides with immunogenic and toxic sequences in “old” wheat than modern wheat

• De Santis et al. (2017) reported reduced ω gliadins in modern wheat, and similar amounts of other immunotoxic gliadins in “old” and modern wheat

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Barley proteomics

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Early reports (Ostergaard et al., 2002, Shmelik et al., 2002) identified beta amylases and hordeins from grain

Guo et al. (2016) compared proteins in feed and malting barley

41 protein spots were differentially expressed between types of barley from which 23 proteins were identifiedDO N

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Barley proteomics: Comparison of a feed and malting barley cultlivar (Guo et al., 2016)DO NOT C

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Differentially expressed proteins were linked to malting quality and grain protein content

Various studies followed changes in cereal proteins during seed development (Ge et al., 2012, Guillon et al., 2012, Dong et al., 2014, Jiang et al., 2014)

This contributed significant knowledge to the molecular basis of grain filling and seed maturation in barley and other cerealsDO N

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The future

• Proteomics is still a relatively new science, which is developing rapidly

• It has the potential to contribute significantly to marker-assisted selection in breeding

• In terms of gluten proteins it can be used to select for good flour quality and against allergen sequences

• In barley it can be used to select for good malting quality

• In sorghum it can be used to select for good end product quality

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The future

• Not much has been reported so far in other cereals, but the field is developing all the time

• Technology is improving consistently• Databases are developing and are being expanded• It will further contribute to studies on effects of biotic

and abiotic stresses in the wheat proteome

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June 11, 2018

THANKS FOR YOUR ATTENTION!

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