# Proteomic analysis of dynamic processes taking place in inner pericarp during wheat grain development

Isabelle Nadaud, Claire Diogon, Brigitte Gaillard-Martinie, Gerard Branlard

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Isabelle Nadaud, Claire Diogon, Brigitte Gaillard-Martinie, Gerard Branlard. Proteomic analysis of dynamic processes taking place in inner pericarp during wheat grain development. COST meeting 2011, May 2011, DIJON, France. hal-05290682

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Submitted on 30 Sep 2025

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Distributed under a Creative Commons Attribution 4.0 International License
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Isabelle NADAUD<fnref n="1" />, Claire DIOGON<fnref n="1" />, Brigitte GAILLARD-MARTINIE<fnref n="2" /> and Gérard BRANLARD<fnref n="1" />

## Introduction

Wheat is the second largest cereal crop after rice used as a source of protein and starch for human consumption, animal feed, industrial raw materials and biofuels. Better understanding of the mechanisms taking place in wheat grain development and wheat grain composition will lead to produce high quality wheat. These last years, different tissues (endosperm, peripheral layers) were investigated by proteomic and transcriptomic approaches. From maternal origin, the knowledge of the peripheral layers (PL) (crossed and tubular cells, aleurone layer, testa, hyaline) is important for several points: nutritional value, grain size and responses to pathogens attacks. Recent proteomics studies were previously carried out on PL [1]. Here, we present the first proteomic attempt on tubular and crossed cells of inner pericarp from 150 to 850°Cday. Kinetics profiles of proteins expression were described on grain developmental phases.

## 1. Material and methods

**Material:** Grains of hexaploid wheat variety Récital were collected every 50°Cday (cumulative day temperature), from fertilization to maturation and frozen at -80°C. Six stages of development were studied: 152, 306, 459, 558, 709 and 847°Cday (figure 1).

**Manual dissection of tissues:** Under binocular embryo, external pericarp then endosperm were removed from peripheral layers. Aleurone layer then hyaline were carefully separated from PL to obtain the crossed and tubular cells (CTC) from inner pericarp (figures 2a, 2b).

**Extraction:** The method was performed for grain tissues. Samples of CTC were crushed under liquid nitrogen and the proteins were extracted using solution of glacial acetone containing 0.07% β-mercaptoethanol and 3.4% proteases Inhibitor.

**Solubilisation:** Total proteins of CTC were solubilised for 1H30 in CHAPS 4%, urea 7M, thiourea 2M, destreak reagent 1.2%, IPG buffer 1% then sonicated in ice. Complete solubilisation is reached after 30 min. Proteins were quantified at 595 nm by Bradford reagent.

**Two dimensional electrophoresis (2-DE):** Four replicates of 100 µg proteins were separated using under pH 3-10 gradient IPG (24cm) x SDS-PAGE at each stage of development. The gels (T=14%, C=2.1%) were stained during 24 hours by Blue Silver method performed on wheat tissues [2] [3].

**Image analysis:** SameSpots Nonlinear software was used to analyse quantitative and qualitative variation of protein spots by Anova test, PCA (Principal component analysis) and power analysis. HCA (Hierarchical Clustering Analysis) of normalised volume values of spots was used to group proteins with similar expression profiles (figure 3).

<figure><img src="image_3.png" /><figcaption>**Figure 3:** 2-DE of Master gel containing proteins extracted from stages 152, 558 and 709°Cday. HCA of proteins spots revealed six main groups, each color corresponds to spots belonging to a same type of profile.</figcaption></figure>

<caption>**Table 1:** identification of proteins by gels image comparison between PL and CTC</caption>

1: Germin like protein 2: HSP70 3,5. RubisCO large SU 4: Putative transketolase 6: Chlorophyll a/b binding protein of PSII 7: Xylanase inhibitor 8: Class II chitinase 9: Thiol specific antioxidant protein 10: Cytochrome b6 complex iron/sulfur SU 11: alpha amylase/substansin inhibitor 12: RubisCO small SU 13: Light harvesting complex 1 14: Choroplast oxygen evolving enhancer protein 1

<figure><img src="image_4.png" /><figcaption>Profile 2 (100 spots)</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Profile 3 (6 spots)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Profile 4 (43 spots)</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Profile 5 (33 spots)</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Profile 6 (91 spots)</figcaption></figure>

<figure><img src="image_9.png" /></figure>

Redox system, disponibility of carbone

Defense proteins

Stress proteins, lignification

<figure><img src="image_10.png" /><figcaption>**Figure 1:** Six grain developmental stages of soft wheat c.v Récital</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>Structure of wheat grain</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**Figure 2b:** Scanning electron microscopy of CTC at 459°C fully separated from the peripheral layers. Scale: 0.1 mm</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>**Figure 2a:** isolation of tubular and crossed cells (CTC) under binocular</figcaption></figure>

## 2. Results and Discussion

**Image analysis** revealed 670 spots detected in total over 6 stages of development analysed. Variance analysis revealed that 301 spots out of 670 were significantly varying.

**Power analysis** showed that 95.7% of our data were correctly aligned and that four replicates were sufficient for this analysis.

**Principal Component Analysis** showed that the technical replicates are well grouped and explained 66.55% of the total variance (PC1: 45.68%, PC2: 15.87%). Spots were divided in two majors groups: the first group with young and intermediate stages (152 to 558°Cd) and the second group with mature stages (709 and 847°Cd).

**Hierarchical Clustering Analysis** assembles the 301 spots in six profiles P1 to P6 (figure 3). Clustered spots were visualized by colour on master gel.

The mean of percentage of spot volume for spots belonging to the same type of profile was calculated and reported in µg of protein per grain [4].

**Comparison of gels images:** gels image of CTC were compared to gels image of peripheral layers (CTC, aleurone layer, testa, hyaline) previously reported in [1] for the same stages of development where 211 proteins were identified. Some specific spots of PL mapped and identified by MS were also recognized in our study on CTC. and know their identifications (N°ID spot on figure 3, table 1). So, we can give hypothesis for the nature of proteins involved for each kinetic profile, figure 4

**Profile 1** (9.3% of spots) showed proteins highly expressed at early stage 152°Cd like ATP synthases, kinases, phosphatases, elongation and transcription factors and proteins involved in arabinoxylans, major constituent of walls cells.

Proteins of **profile 2** (33.2% of spots) were associated to photosynthetic system which is important at young stages (152-306°C, spots 3, 5, 6, 10, 12, 13, 14) and to carbohydrate metabolism.

Their kinetics are directly correlated to energy production with proteins of **profile 3** and **4** (16.3%) strongly expressed at 306°Cd. We noticed a quantitative increase of some proteins between 459 and 558°Cd on **profile 4**. This stage corresponds to grain colour changing (green to yellow) coming with a change of redox system state (spot 9) and degradation of proteins in amino acids.

The remobilization of carbon allows the synthesis of new proteins involved in lignification of cells during maturation with a peak expression at 706°Cd (**profile 5**, 11% of proteins) with proteins associated to stress (spots 1, 2), and proteins of defense for grain protection like xylanases and alpha amylase inhibitors (spots 7, 8, 11, **profile 6**, 30.2% of proteins).

## 3. Conclusion

Tubular and crossed cells of wheat kernel development were analysed by proteomic approach. This study allowed to describe kinetic evolution of 301 proteins in six expression profiles. Proteins will be subjected to LC-MS/MS for identification to further investigate on precise the role of these cells in size determination of wheat grain, the processes of lignification, defense and resistance to pathogens.

## References

[1] Tasleem-Tahir, A., Nadaud, I., Girousse, C., Chambon, C., Bouzidi, M.F., Martre, P., Branlard, G., 2011, Proteomics, 11, 1-9

[2] Cardiano, G., Bruschi, M., Musante, L., Santucci, L., Ghigeri, G.M., and at, 2004, Electrophoresis, 25, 1327-1333

[3] Rabloud, T. (Ed), Springer, Germany 2000, pp. 107-126

[4] Nadaud, I., Girousse, C., Debton, C., Martre, P., Marion, D., Branlard, G., 2010, Proteomics, 10, 2901-2910

<footnote n="1">¹ UMR1095 INRA - UBP Unité GDEC, 234 avenue du brézet 63100 Clermont-Ferrand, France</footnote>

<footnote n="2">² INRA Plateau technique de microscopie, site de Their 63122 Saint-Genès-Champanelle, France</footnote>