AN ATTEMPT TO INTEGRATE PROTEOME AND TRANSCRIPTOME DATA: APPLICATION TO CARBOHYDRATE METABOLISM OF WHEAT GRAIN DEVELOPMENT

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Starchy endosperm

The inner endosperm is referred to as mealy or starchy endosperm and as the name indicates, contains the totality of energy-yielding starch of the grain, which is used by embryo as the major source of nutrition for development (Belderok 2000). In contrast to its high concentration of carbohydrates, this tissue is poor in proteins, fats, dietary fibers and minerals as compared to embryo and AL (Table 4). According to Popineau and Pineau 1988, the distribution of proteins and weight is not equal among different grain tissues, as can be seen in the Table 8 on page 25. In starchy endosperm three types of cell are reported, differing according to their size, shape and sites of occurrence (Greer et al 1951). i) Peripheral or sub-aleurone cells are adjacent to the aleurone cells. They are of similar size to these cells with about 60 μm of diameter. They are rich in AX and lenticular starch granules of A type (diameter >10 μm). ii) Prismatic cells radiate in columns from their junction with subaleurone cells. These cells are 128-200 μm long with 40-60 μm wide. iii) Central cells, are generally round or polygonal and have a length of 72-144 μm and a width of 69-120 μm (Bradbury et al. 1956b, Evers and Millar 2002). All three types of starch granules (A, B, C) are present in these two types of cells (prismatic and central), which differ in size and shape. The large one is lens shaped (A type) and the small one is near-spherical (B and C type).

Development of Wheat Plant and Grain

Winter wheats, like many other cereal grains, require vernalization (Dennis and Peacock 2009). They tolerate low temperatures for a specific required length of time to become competent to flower (plants convert from vegetative to reproductive growth). This process prevents the damage of the cold-sensitive flowering meristem during the winter. There is no requirement of vernalization in spring wheat varieties (Acevedo et al 2006). VNR1 gene is critical in division of polyploidy wheat varieties into the winter and spring categories (Tranquilli and Dubcovsky 2000, Law and Giorgi 1975). Vernalization may occur at three stages of the growing cycle of the wheat plant: during germination, during vegetative plant growth and during seed formation in the mother plant (Flood and Halloran 1986).
Five different scales are used worldwide for wheat growth stages. This classification varies from simple division in three phases of development to construction of detailed scales which covers all visual steps of development. Commonly used scales are Zadoks and Feekes-Large scales. The comparison between the two scales is not too easy but some key stages of development allow us to align these two scales (Table 5). In Feekes scale developmental stages are designated on a scale of 1 (seedling growth) to 11 (ripening) (Large 1954). The Zadoks scale is much more descriptive. Globally it goes from primary stage 00 (dry seed) to 90 (ripening) (i:e 10 primary stages, each of which is divided into 10 secondary stages = total of 100 stages) (Zadoks et al.

Germination to the End of vegetative growth

Germination is a complex process during which the imbibed mature seed must quickly shift from a maturation to a germination driven program of development and prepare for seedling growth (Nonogaki et al. 2010). Phytohormones such as abscisic acid (ABA), gibberellins (GAs), ethylene, brassinosteroids, salicylic acid, cytokinin, auxin, jasmonic acid and oxylipins are one the important factors influencing this phenomenon by forming an interlocked signaling network (Rajjou et al. 2012). ABA and gibberellins have antagonistic effects, where ABA is present at later stage of grain development to inhibit germination and thereafter grain imbibition its level is reduced, while GAs are essential germination activators. ABA/GA ratio regulates the metabolic transition required for germination process (Liu et al. 2010).
The mature grain contains enough stored reserves in the endosperm that are sufficient for the growth of embryo wheat plant. Minimum water content required in the grain for wheat germination is 35 to 45% by weight (Evans et al. 1975) with temperature range of 4-37°C being optimal from 12-25°C. Wheat crop emergence occurs at 3-4 leaf primordial stage of seed embryo and almost half of the leaf primordia already initiated (Baker and Gallagher 1983a, 1983b, Hay, and Kirby 1991). The first roots that appear are the seminal roots followed by coleoptiles which protect the emergence of the first leaf. Germination process lasts around 0-42 DAS (days after sowing of seeds, Table 5) and vegetative stage may vary from 60 to 150 days. With the emergence of the first leaf seedling, growth begins and continues until the next stage (tillering). Not all tillers produce spikes in wheat and many of them abort before anthesis (Gallagher and Biscoe 1978). Next is the stem elongation (jointing) phase which occurs as a result of internodes elongation. This phase completes with the emergence of last leaf from the whorl (flag leaf) (Krumm et al. 1990). Shortly after flag leaf emergence, the flag leaf sheath and the peduncle pushed up the developing head through the flag leaf sheath which obtains a swollen appearance to form a boot. Boot stage is rather short and coincides with meiosis which starts in the middle of the spike continuing later, above and below this zone, in wheat and barley plant growth (Zadoks et al. 1974).

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Non Protein Contents

The wheat grain (Triticum aestivum) is composed of 12-18% water, 63-74.5% glucids (starch and sugar), 8-12% proteins, 1.5-2% of lipids, 2.5-3% cellulose and 1.5-2% minerals. Wheat kernel components, particularly storage proteins and starch, have received a great deal of attention in the last two decades. Distribution of these contents is presented in (Table 2, 3 and 4). In this section, wheat proteins will be presented in detail.

Protein Contents

Traditionally wheat proteins have been classified into four types according to their solubility (Figure 11), soluble; metabolic proteins (albumins and globulins) and insoluble, gluten or storage proteins (gliadins and glutenins) (Osborne 1907, Table 7).

Metabolic Proteins (Non-prolamins or Soluble)

Albumins and globulins of flour are also known as soluble, non-prolamins or metabolic proteins. They are not specific to the endosperm; they can easily be extracted from any kernel tissue (endosperm, aleurone layer, and embryo) (Table 8).They are composed of thousands of enzymes and proteins needed for cell survival (most albumins) and cell structure (many globulins). They represent 15-20% of total wheat proteins (Shewry et al. 1986). Some high molecular weight albumins and globulins of triticins can also serve as storage proteins (Gupta et al. 1991, Singh and Skerritt 2001). Genes of globulins were localized mainly on chromosomes 4D, 5D, 6D, 7B and 7D, while characterization of products of these genes by IEF revealed that majority of them have pI that vary from 5.50 to 7.89 (Singh and Skerritt 2001). More information regarding this category of proteins is provided in Chapter 3.

Storage Proteins (Prolamins or non-soluble)

Prolamins are defined as proteins extractable in aqueous ethanol. Storage proteins (gliadins and glutenins) because they are very rich in proline and glutamine are also called as prolamins. As in addition to gliadins, the low molecular weight glutenins are also soluble in aqueous ethanol after reduction. They are evolutionarily closely related to the rest of the wheat prolamins (Shewry and Tatham 1999).
The storage proteins of wheat are unique because they are also functional proteins. They do not have enzymatic activity, but they are the only cereal proteins to form strong, cohesive dough. Gluten, which is simply obtained by water-washing of flour, is widely known to be responsible for its mixing properties. This water-insoluble network is a complex physico-chemical system composed of polymeric glutenins and monomeric gliadins (Bietz and Wall 1972).

Table of contents :

CHAPTER 1: INTRODUCTION
1 WHEAT
1.1 ORIGIN
1.2 EVOLUTIONARY HISTORY:
1.3 WHEAT GENOME
1.4 WHEAT CLASSIFICATION
1.5 WHEAT PLANT ANATOMY
1.6 WHEAT GRAIN STRUCTURE
1.6.1 Peripheral Layers (PL)
1.6.2 Endosperm (ESM)
1.6.3 Embryo
2 DEVELOPMENT OF WHEAT PLANT AND GRAIN
2.1 GERMINATION TO THE END OF VEGETATIVE GROWTH
2.2 FLOWERING AND FERTILIZATION
2.3 GRAIN DEVELOPMENT
2.3.1 Grain Growth
2.3.2 Grain Filling
2.3.3 Grain dehydration-maturation stage
3 WHEAT GRAIN CONTENTS (COMPOSITION)
3.1 NON PROTEIN CONTENTS
3.2 PROTEIN CONTENTS
3.2.1 Metabolic Proteins (Non-prolamins or Soluble)
3.2.2 Storage Proteins (Prolamins or non-soluble)
3.3 IMPACT OF WHEAT PROTEINS ON ITS QUALITY
4 PROTEOMICS TO STUDY EXPRESSION OF CANDIDATE GENES
4.1 PROTEOMICS
4.2 QUANTITATIVE PROTEOMICS
4.2.1 Relative quantification
4.2.2 Absolute Quantification
4.3 MASS SPECTROMETRY
4.3.1 MALDI-TOF (Matrix-Assisted Laser Desorption -Time of flight)
4.3.2 LC-MS (Liquid chromatography mass spectrometry)
4.3.3 Tandem mass spectrometry (MSMS or MS²)
5 TRANSCRIPTOMICS TO STUDY EXPRESSION OF CANDIDATE GENE
5.1 METHODS TO STUDY TRANSCRIPTIONAL EXPRESSION
5.1.1 OPEN SYSTEMS
5.1.2 CLOSED SYSTEMS
5.2 WHEAT TRANSCRIPTOMICS STUDIES
6 INTEGRATION OF PROTEOMICS AND TRANSCRIPTOMICS DATA
CHAPTER 2 (ARTICLE 1): PROTEOMIC ANALYSIS OF PERIPHERAL LAYERS DURING WHEAT (TRITICUM AESTIVUM L.) GRAIN DEVELOPMENT
CHAPTER 3 (ARTICLE 2): EXPRESSION PROFILING OF STARCHY ENDOSPERM METABOLIC PROTEINS AT TWENTY-ONE STAGES OF WHEAT GRAIN DEVELOPMENT
CHAPTER 4 (ARTICLE 3): AN ATTEMPT TO INTEGRATE PROTEOME AND TRANSCRIPTOME DATA: APPLICATION TO CARBOHYDRATE METABOLISM OF WHEAT GRAIN DEVELOPMENT
CHAPTER 5: COMPARATIVE PROTEOME STUDY OF PERIPHERAL LAYERS AND STARCHY ENDOSPERM DURING GRAIN DEVELOPMENT
1METABOLIC PROTEINS IDENTIFIED IN GRAIN TISSUES
2 EXPRESSION PROFILES OF PL AND ESM PROTEINS
3 FUNCTIONAL PROFILES OF PL AND ESM
3.1 METABOLISM
3.2 GENETIC INFORMATION AND PROCESSING
3.3 ENVIRONMENTAL INFORMATION AND PROCESSING
3.4 STORAGE PROTEINS (SP)
3.5 STRESS/ DEFENSE (SD)
3.6 REDOX HOMEOSTASIS
CONCLUSION AND PERSPECTIVES
REFERENCES

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