Marielle Merlino

Proteomic analysis of amphiphilic proteins of hexaploid wheat kernels

Wheat proteins and specially gluten proteins have been well studied and are closely associated with baking products. Amphiphilic proteins (proteins that are soluble using nonionic detergent Triton X‐114 ) also play an important role in wheat quality. Some of them, like puroindolines, are lipid binding proteins, and are strongly linked to dough foaming properties and to fine crumb texture. However many amphiphilic proteins are still unknown and both their physiological and technological functions remain to be analysed. In order to explore these proteins, proteomic analysis was carried out using 81 F9 lines, progeny obtained from an interspecific cross “W7984”דOpata”, and already used to built a map of more than 2000 molecular markers (International Triticeae Mapping Initiative, ITMImap). Two‐dimensional electrophoresis (immobilized pH gradient (pH 6–11)×sodium dodecyl sulfate‐polyacrylamide gel electrophoresis) was performed on amphiphilic proteins with three to five replicates for each line. Silver stained gels were analysed using Melanie 3 software. Genetic determinism was carried out on 170 spots segregating between the two parental hexaploïd wheats. Many of these spots were mapped on different chromosomes of the ITMImap. Spots of interest were identified using matrix‐assisted laser desorption/ionization‐time of flight and some of them were partly sequenced using electrospray ionization‐tandem mass spectrometry. This proteomic approach provided some very useful information about some proteic components linked to bread wheat quality and particularly to kernel hardness.

Proteomes of hard and soft near-isogenic wheat lines reveal that kernel hardness is related to the amplification of a stress response during endosperm development

Wheat kernel texture, a major trait determining the end-use quality of wheat flour, is mainly influenced by puroindolines. These small basic proteins display in vitro lipid binding and antimicrobial properties, but their cellular functions during grain development remain unknown. To gain an insight into their biological function, a comparative proteome analysis of two near-isogenic lines (NILs) of bread wheat Triticum aestivum L. cv. Falcon differing in the presence or absence of the puroindoline-a gene (Pina) and kernel hardness, was performed. Proteomes of the two NILs were compared at four developmental stages of the grain for the metabolic albumin/globulin fraction and the Triton-extracted amphiphilic fraction. Proteome variations showed that, during grain development, folding proteins and stress-related proteins were more abundant in the hard line compared with the soft one. These results, taken together with ultrastructural observations showing that the formation of the protein matrix occurred earlier in the hard line, suggested that a stress response, possibly the unfolded protein response, is induced earlier in the hard NIL than in the soft one leading to earlier endosperm cell death. Quantification of the albumin/globulin fraction and amphiphilic proteins at each developmental stage strengthened this hypothesis as a plateau was revealed from the 500 °Cd stage in the hard NIL whereas synthesis continued in the soft one. These results open new avenues concerning the function of puroindolines which could be involved in the storage protein folding machinery, consequently affecting the development of wheat endosperm and the formation of the protein matrix.

A proteomics survey on wheat susceptibility to Fusarium head blight during grain development.

The mycotoxigenic fungal species Fusarium graminearum is able to attack several important cereal crops, such as wheat and barley. By causing Fusarium Head Blight (FHB) disease, F. graminearum induces yield and quality losses and poses a public health concern due to in planta mycotoxin production. The molecular and physiological plant responses to FHB, and the cellular biochemical pathways used by F. graminearum to complete its infectious process remain still unknown. In this study, a proteomics approach, combining 2D-gel approach and mass spectrometry, has been used to determine the specific protein patterns associated with the development of the fungal infection during grain growth on susceptible wheat. Our results reveal that F. graminearum infection does not deeply alter the grain proteome and does not significantly disturb the first steps of grain ontogeny but impacts molecular changes during the grain filling stage (impact on starch synthesis and storage proteins). The differentially regulated proteins identified were mainly involved in stress and defence mechanisms, primary metabolism, and main cellular processes such as signalling and transport. Our survey suggests that F. graminearum could take advantage of putative susceptibility factors closely related to grain development processes and thus provide new insights into key molecular events controlling the susceptible response to FHB in wheat grains.