Proteomic analysis reveals the differential proteins of endosperm between soft and hard wheat varieties

Feng Jia,Haicheng Yin,Qi Wang,Yu Chen,Jinshui Wang*

College of Biological Engineering,Henan University of Technology,Zhengzhou 450001,China

Keywords:

Soft and hard wheat

Proteome

Peptides surfaces

Endosperm proteins

ABSTRACT

In this study,we revealed the differential proteins from the wheat endosperms using proteomic analysis and investigated their surface properties.The pattern of the polypeptides obtained from the Yangmai-15 and Yangmai-16 wheat varieties were compared using two-dimensional polyacrylamide gels.In addition,we compared the characteristics of the grain such as grain hardness,protein content,wet gluten,dough development time,dough stability,gliadin and glutenin contents between Yangmai-15 and Yangmai-16,and the results were significantly different.Notably,216 and 197 protein spots were separated from Yangmai-15 and Yangmai-16,respectively.The isoelectric points of the identified proteins ranged from 4 to 10 and the molecular weights of proteins varied from 10 to 100 kDa.Further,21 and 8 specific differential protein spots were identified from the flour of Yangmai-15 and Yangmai-16,respectively.The surface properties of identified peptides consisted of hydrophobic or hydrophilic residues,as well as randomly scattered residues.The proteomic analysis of the wheat endosperms provides a novel insight into the biochemical basis for the differences in physicochemical properties between the soft and hard wheat varieties.

1.Introduction

Wheat(Triticum aestivum)is an important food crop that grows worldwide and is used as raw material for the manufacture of flour-derived food items in many countries[1,2].Based on the hardness of the grain,wheat can be divided into soft,hard,and durum wheat varieties[3].In China,the main varieties of wheat used are soft and hard wheat[4].Significant differences exist in physicochemical properties of the dough between the soft and hard wheat varieties,which depend largely on the amount and composition of the protein in them[5].

The protein components of wheat flour are represented by albumin,globulin,gliadin,and glutenin[6].The composition of the protein has an important effect on the processing and the end-use quality of the wheat flour[7].Additionally,the protein content has a positive effect on the volume of the bread loaf[8].Previous studies have shown that the volumes of bread were significantly affected by the amount and type of gluten in hard wheat[9].Moreover,the analysis of the roles of different protein fractions on dough quality demonstrated that gluten complexes can form polymers with viscoelastic properties[10,11].

Proteome analysis can be used both to visualize and compare complex protein mixtures,and in addition,it provides substantial information about the individual proteins involved in specific biological processes.Proteomic analysis of the wheat grain has already been done to identify the proteins in the germ and the endosperm[12,13],as well as to characterize the composition of the bran fractions and aleuronic layer[14,15].It has been reported that the biochemical composition varied among the wheat cultivars with different grain hardness[16].In recent years,researchers have revealed the changes in the protein composition during kernel development and in the mature kernel[17].Moreover,the proteomics results have also revealed that the number of protein spots identified in the Yangmai-15 variety were significantly higher than Yangmai-16 [18].Hence,the protein content of the wheat endosperm is an important factor for determining the yield and grain quality[19].

The aim of this study is to analyze the differences between the endosperm proteomes of Yangmai-15 and Yangmai-16.Our results elucidated the biochemical basis for the significant difference in the quality between soft and hard wheat from a proteomics perspective.This information can be potentially used for improving the flour quality by genetic engineering technology.

2.Materials and methods

2.1.Materials

Yangmai-15,a soft red winter wheat variety,and Yangmai-16,a hard red winter wheat variety,are commonly grown in the Yangtze-Huaihe Region of China.Both wheat varieties that have similar genotypes but different grain hardness were used in this study.The protein contents of these wheat varieties were 11.08%in the Yangmai-15 type and 13.5% in the Yangmai-16 type.

2.2.Analysis of the physicochemical properties of the wheat flour

To prepare the samples using the grains from the Yangmai-15 and the Yangmai-16 varieties,water was added to the grains and they were maintained at room temperature for 48 h to adjust the moisture content to 14%.The moisture content was increased by another 0.5%half an hour prior to milling.The grains were individually milled using a Chopin laboratory mill(Villeneuve la Garenne,France)into flour.The Yangmai-15 and Yangmai-16 flour samples were stored at 4°C and thawed for 3 h before analysis.The protein and wet gluten contents were determined according to the method described by Hegedus et al.[20].The time taken for developing the dough from these flours was decided based on the method described by Akhtar et al.[21].

2.3.Protein extraction

Mature and homogeneous wheat kernels were selected for the proteomic analysis.The protein fractions were extracted from the flour according to the method described by Zhang et al.[18].In brief,1 g flour was added to 1 mL extraction buffer solution (7 mol/L urea,2 mol/L thiourea,4%CHAPS,18 mmol/L Tris-HCl (pH 8),5.3 μg/mL DNase I,4.9 μg/mL RNase A and 1 mmol/L PMSF) to solubilize the crude protein.After 30 min of incubation at 4°C,dithiothreitol was added at a final concentration of 15 mmol/L and the flour samples were centrifuged for 10 min at 12,000 g at 4°C.The protein concentration of the supernatant was measured using the BCA Protein Assay Kit(Beyotime,Shanghai,China)using bovine serum albumin as the standard marker (Beyotime,Shanghai,China).The fractions were pooled,lyophilized,and stored at -80 °C prior to the two-dimensional electrophoresis(2-DE)experiments.

2.4.Two-dimensional electrophoresis and image analysis

The 2-DE experiments and image analysis were performed according to the method proposed by Zhang et al.[18].For this experiment,the gels were first processed with coomassie brilliant blue G-250,and scanned using an image scanner(GE Healthcare,Uppsala,Sweden)at 300 dpi and 16-bit grey scale level.After staining,the gels were scanned using the transmission mode and the scanning results were directly transferred to the computer.The scanned images were analyzed using the Melanie Viewer 7 software (http://www.expasy.org/proteomics).The identified proteins from the Yangmai-15 and Yangmai-16 varieties were matched with the proteins from the local database by the Mascot software (Matrix Science,London,UK,http://www.matrixscience.com).Each sample was separated by 2-DE in triplicate and all the results were analyzed digitally.

2.5.Trypsin digestion,characterization,and identification of proteins

Trypsin digestion,characterization,and identification of proteins by mass spectrometry(MS)were carried out following the protocol reported by Zhang et al.[18].The specific differential protein spots were excised from the replicate gels and pooled.400 mL ammonium bicarbonateacetonitrile(ABB)buffer(100 mmol/L)was added to destain the samples and then,5 mL of 10 ng/mL trypsin solution (Promega,Madison WI,USA) was added to digest the protein at 37°C for 20 min.The peptides were identified using a mass spectrometer(Voyager-DE Pro MALDI-TOF Applied Biosystems,Framingham,MA,USA).The monoisotopic peptide masses were used to search the protein database for matches using the Mascot software (Matrix Science,London,UK,http://www.matrixscience.com).The proteins were identified by MALDI-TOF if at least four nonredundant peptides matched with a single reference peptide from one of the databases.

2.6.Distribution of the hydrophobic and hydrophilic residues

The online Helical Wheel Projections software(http://rzlab.ucr.edu/scripts/wheel/wheel.cgi)was used to predict the distribution of the hydrophobic or hydrophilic residues in the amino acid sequences of the identified peptides.

2.7.Statistical analysis

The experimental data were analyzed using a one-way ANOVA model from the SPSS 17.0 software to assess the significance of the differences and the results of the Duncan's multiple comparative tests.

3.Results and discussion

3.1.Characteristics of the flour

Table 1 shows the characteristics of the flour obtained from Yangmai-15 and Yangmai-16.The characteristics of the grains such as grain hardness,protein content,wet gluten,dough development time,dough stability,gliadin and glutenin contents from Yangmai-15 and Yangmai-16 were compared,and the results were found to be significantly different.We observed that all the grain characteristics described above were significantly higher in Yangmai-16 than in Yangmai-15.However,the ratio of Glia/Glu in Yangmai-15(0.55)was significantly higher than Yangmai-16(0.47).The results of this study showed that PC of Yangmai-15 was significantly lower than that of Yangmai-16.Based on the hardness class,the hard varieties of wheat have higher average PC of 10.5%,compared to the soft wheat(9.5%)[22].Thus,the PC of soft wheat is lower than that of hard wheat[23].

3.2.Two-dimensional electrophoresis analysis of wheat endosperm proteins

The 2-DE analysis of the proteins from the endosperms of Yangmai-15 and Yangmai-16 identified 216 and 197 protein spots,respectively,and these proteins had different isoelectric points(4-10)and molecular weights(10-100 kDa)(Fig.1A and Fig.1B).Normalized spot volumes were used instead of raw spot volumes for the quantitative analysis of the protein expression,and the results were shown in Fig.1A’ and Fig.1B’.This was consistent with the report by Dziuba et al.[24],which indicated that the 82 gliadin spots identified from wheat cv.Sukces using 2-DE gels were likely to contain other proteins.Irar et al.[25]selected 18 spots from the 2-DE analysis of durum wheat embryo proteins that showed quantitative and qualitative variations compared that seen in the 2-DE gels of the two wheat varieties.On the other hand,20 differentially expressed proteins were identified by Wang et al.[26]from the regions of Westonia and Wyalkatchem that can be assigned to chromosomes 1D,3A,4A,4B,4D,6A,6B,7A,and 7B.

Table 1 Physicochemical,gluten,rheological and quality characteristics of wheat varieties.

Fig.1.Profile of the 2-DE analysis of the proteins from the endosperms of Yangmai-15(A)and Yangmai-16(B);A’and B’are three dimension patterns of A and B,respectively.

3.3.Analysis of the differential expression of proteins

Sixty-seven protein spots were identified from the 2-DE image using MS,out of which 21 and 8 protein spots corresponded to Yangmai-15 and Yangmai-16,respectively (Table 2).Overall,these 67 identified spots were classified based on comparative analysis.Of these,13 spots were found to be differentially expressed in Yangmai-15 and Yangmai-16(Fig.2),whereas the others could not be characterized using local database yet,which requires further enrichment of the protein database.These 13 identified spots of the endosperms from Yangmai-15 and Yangmai-16 matched with the proteins,which include 1-Cys peroxiredoxin PER1,aconitate hydratase,avenin-like,chromosome 3B,class I heat shock protein,fructose-bisphosphate aldolase,gliadin/avenin-like seed protein,and globulin-3A,respectively.The differential proteins identified were mainly globulin-3A(I6QQ39)and chromosome 3B(W5CWR9).The distribution of the structural and functional features of chromosome 3B revealed partitioning that correlated with meiotic recombination[27].The proportion of non-syntenic genes to the total gene count in wheat chromosome 3B was plotted in a sliding window of 10 Mb with a step of 1 Mb [28].Therefore,in this study,the identified protein spots mostly corresponded to globulin proteins.We speculate that the unidentified protein spots might correspond to glutenin and gliadin that determine the functional properties of dough and influence the food processing.

3.4.Amino acid distribution of identified proteins

The amino acid sequences of the identified proteins were analyzed using the Helical Wheel Projections software.The amino acid distributions of the proteins identified in Yangmai-15 and Yangmai-16 were predicted as shown in Fig.3.The surface of the peptide Q10464(Fig.3i)consisted of typical hydrophobic residues and the surfaces of four water soluble peptides,including P04724(Fig.3a),D2KFH1(Fig.3c),P0CZ05(Fig.3j)as well as D2KFG9(Fig.3l),contained hydrophilic residues.The amino acid sequences found on the surfaces of the remaining peptides had randomly scattered positive or negative charges.Hence,electrostatic interaction and hydrophobic interaction could play important roles in the α-helix formation[29].These results suggested that these peptides may have different protein characters.

4.Conclusion

Significant differences in the grain characteristics such as grain hardness,protein content,wet gluten,dough development time,dough stability,gliadin content,and glutenin content were detected between the Yangmai-15 and Yangmai-16 varieties.Using 2-DE,we obtained 216 and 197 protein spots from Yangmai-15 and Yangmai-16 varieties,respectively,and the isoelectric points and the molecular weights of the identified proteins ranged from 4 to 10 kDa and 10 to 100 kDa,respectively.Further,we identified 21 and 8 specific differential protein spots from the endosperms of Yangmai-15 and Yangmai-16,respectively,and the analysis of the surface properties of these peptides showed the presence of either hydrophobic or hydrophilic residues,as well as some randomly scattered residues.The proteomic analysis of the wheat endosperms provided a novel insight into the biochemical basis for the differences in physicochemical properties between the soft and hard wheat variety.

Declaration of Competing Interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work,there is no professional or other personal interest of any nature or kind in any product,service and/or company that could be construed as influencing the position presented in,or the review of,the manuscript entitled,“Proteomic analysis reveals the differential proteins of endosperm between soft and hard wheat variety”.

Acknowledgements

The authors thank for the financial support of the National Natural Science Foundation of China(No.31771897,31871852,and 31772023).

Table 2 Highly matched protein sequences using the local database analyzed using the Mascot software.

Fig.2.Protein class distribution of the identified proteins.

Fig.3.Amino acid distribution of the identified proteins.