Cellular Events During Germination

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Seed Development and Composition

Seed development in higher plants begins with a double fertilization process that occurs within the ovule and ends with a maturated seed primed to become the next plant generation (Goldberg et al., 1994). The major events that occur during seed development are shown in figure 1. Embryo development can be divided up into two phases, the first or “embryogenesis” involves cell divisions associated with morphogenetic events which form the basic cellular pattern for the development of the shoot-root organs and the primary tissue layers, and it also programs the regions of meristematic tissue formation. Following the cell division arrest at the end of the embryo growth phase, the seed enters the second phase, which is called “maturation phase”, this process involves cell growth and the storage of reserves, such as proteins, starch and oils, required as ‘food and energy supply’ during germination and seedling growth (Goldberg et al., 1994; Holdsworth et al., 2008). The seeds enter then, into a metabolically quiescent state related to dehydration after dry maturation, which represents the normal terminal event in the development process of orthodox seeds. Genetic studies in Arabidopsis have identified genes that provide new insight into molecular event underlying plant development (Le et al., 2010). Dormancy process is initiated during seed maturation and reached a maximum in harvest-ripe seeds (Karssen et al., 1983; Ooms et al., 1993; Raz et al., 2001). Seeds may remain in this dry dormant state from several days to many years and still retain their viability.
Fig. 1. A generalized overview of seed development and stages of the life cycle. OV, unfertilized ovule; Postfertilization, also called proembryo, this stage involved in terminal and basal cell differentiation, formation of suspensor and embryo proper; Globular-heart transition, differentiation of major tissue-type primordia at this stage, including establishment of radial (tissue-type) axis, visible appearance of shoot-root, hypocotyl-radicle development and differentiation of root meristem; Organ expansion and maturation, enlargement of cotyledons and axis by cell division and expansion before seed maturation, and following seed maturation storage reserves such as RNA, proteins and lipids are accumulated, and then after maturation involve in loss of water (dehydration) and dormancy status formed to inhibit precocious germination. Diagram adapted from Le et al. (2010), developmental events were modified from Goldberg et al. (1994).

Dormancy and Control of Germination

Baskin and Baskin (2004) have summarized the hierarchical system of classification for seed dormancy. The system has divided dormancy into five classes: (1) is physiological dormancy (PD), which contains three depth levels: deep, intermediate and non-deep. Depending on the PD depth level, dormancy can be released by different stratification treatments (imbibition at low or warm temperatures) or GA treatment; (2) morphological dormancy (MD), which caused by a delay of embryo development; (3) morphophysiological dormancy (MPD), it is a combinational dormancy (PD + MD); (4) physical dormancy (PY) is due to the existing of water-impermeable layers of palisade cells in the seed coat and it also can be released by mechanical or chemical scarification; and (5) combinational dormancy (PY + PD). However, inside all of these classifications, PD (non-deep level) is the most common kind of dormancy because it occurs in part of gymnosperms and in all major clades of angiosperms, and depending on the species, the dormancy alleviation correlated with stratification, a period of dry storage (after-ripening) or gibberellins (GAs) treatment. In the last decade, the works by Finch-Savage and Leubner-Metzger (2006) and Cadman et al. (2006) have provided insight into the molecular mechanisms of non-deep PD. Two terms of physiological seed dormancy are distinguished; the intrinsic molecular mechanisms determined by seed components, namely embryo and coat dormancy. The latter corresponds to the case when the intact seed is dormant but the isolated embryo can germinate normally, therefore, the seed coat enclosing the embryo exerts a constraint that the embryo cannot overcome. By contrast, embryo dormancy is characterized by the inability of the embryo itself to germinate normally after removal of the seed coat. Sunflower seeds are characterized by an embryo covered by seed-coats and a pericarp easily removable, this can help to deeply investigate the mechanism of embryo dormancy and eliminate the influence of coat dormancy.
Control of seed germination and growth is crucial to the survival of the next generation, there are several critical determinants for the transitions from dormancy to germination and from germination to growth (Fig. 2). At dispersal, the mature seed, when it encounters favourable environmental conditions, which can include light of a given wavelength, sufficient water availability, optimal temperatures, and adequate oxygen, releases dormancy and commences germination. Visible evidence of the completion of germination is usually radicle protrusion and elongation. Some seeds must be exposed to environmental cues, such as periods of warmdry conditions (after-ripening), moist chilling, or even smoke, to release dormancy (Stephen et al., 1986; Egerton-Warburton, 1998) (Fig. 2). The dormancy status reduces during afterripening, consequently, seeds are able to complete germination in wide range of environmental conditions. After-ripening largely depends on environmental conditions during seed storage and germination conditions (Donohue et al., 2005), while seed covering structures, moisture and temperature are the main factors to decide the speed of after-ripening (Manz et al., 2005). However, these primary dormant seeds may enter into a second stage of dormancy, called secondary dormancy, during imbibition if the environmental conditions are unfavourable for germination (Fig. 2, Bewley and Black, 1994; Kermode, 2005). Secondary dormancy is a safety mechanism that is implemented from the seed being exposed to adverse conditions once it falls from plant and function in imbibed seeds, it can be induced by anachronistic environmental conditions such as too low or high temperature, hypoxia and prolonged exposure to darkness or light (Khan and Karssen, 1980; Leymarie et al., 2008; Hoang et al., 2013 a, b, 2014).
Fig. 2. Relationship between dormancy and germination. The control of germination exists at the state of seed dormancy and the operation of environmental factors such as light, temperature and air humidity effect both dormancy and germination. The diagram is from Bewley and Black (1985).
A number of high-throughput, large scale-omics studies have been investigated to gain insight into molecular networks of seed dormancy and germination. Bentsink and Koornneef (2008) have presented the first high-throughput, large scale-omics studies investigating seed dormancy and germination, including transcriptomics, proteomics and metabolomics. Several transcriptomic analyses have provided gene expression profiles underlying dormancy-related differential gene expression (Gao et al., 2012; Dekkers et al., 2016). These new approaches and derived data sets provide an unprecedented level of detail concerning genome expression associated with germination potential, which suggest that RNA translation or post-translation are the major levels of control for germination completion, by contrast, transcriptome changes might reflect alteration in dormancy status or enhancement of germination vigor and effects on post-germination functions that relate to seedling growth. Indeed, Layat et al. (2014) and Basbouss-Serhal et al. (2015) have shown the importance of translational regulation in the control of this process. The absence of correlation between transcriptome and translatome reported suggests that translation regulation and consequently proteome change should be considered more than transcriptional regulation. Thus, proteomics is an increasingly important tool for the study of several plant functions because it allows the investigation of the underlying molecular processes in plant physiology (Trindade et al., 2018). Proteomic change investigated in dormant and non-dormant seeds pointed out the importance of metabolism, energy production, protein metabolism, cell growth and defense classes (Pawlowski and Staszak, 2016;
Xia et al., 2018a). Post-translational modifications (PTMs) represent another level of regulation in seeds especially phosphorylation, carbonylation and thiol oxidation (see below). Metabolomics represent the last branch of high throughput functional genomics that allowed measuring biochemical activity directly by monitoring the substrates and products converted by cell metabolism (Saito and Matsuda, 2010). Metabolomics is not yet actively investigated in seeds. During Arabidopsis seed stratification, Fait et al. (2006) have shown the importance of the groups of storage proteins, stress response and detoxification in dormant state and energy, amino acid metabolism, folding and mRNA and protein metabolism in non-dormant state. The comparison between two wheat cultivars with contrasting dormancy status revealed several raffinose family oligosaccharides as key markers (Das et al., 2017). Moreover, in sunflower seeds, Xia et al. (2018b) showed that among more than 100 metabolites quantified, only sugars present significant change between dormant and non-dormant seeds. These results highlight the importance of energy and metabolism regulation in non-dormant state. Beyond the importance of the level of regulation, the elements brought specially by the proteome and metabolome underlined the importance of central metabolism. Targeted enzyme activity can provide integrated information about gene expression and PTMs. Recently, Xia et al. (2018a) have highlighted PTMs regulation of enzymes involved in central metabolism in dormancy alleviation in sunflower by combining proteomic analysis and enzymatic profiling. In sum, multiple tevel of regulation has been shown in dormancy alleviation and germination processes.

Cellular Events During Germination

The dehydrated state of mature seed helps to withstand drought and extreme temperatures. Germination begins with water uptake by the dry seed during imbibition and ends with the embryonic axis or radicle elongation (Finch-Savage and Leubner-Metzger, 2006). During this process, a sequence of cellular events is initiated following seed water uptake which ultimately leads to emergence of the radicle and complete germination successfully. Metabolism commences in the seeds as soon as their cells are hydrated. Respiration and protein synthesis have been recorded within minutes of imbibition, using components conserved in the dry seed (Black et al., 2006; Galland et al., 2014). This is followed by synthesis of RNA, and DNA repair and synthesis. Numerous enzymes are either activated or de novo synthesized during germination, including lipases, proteinases, phosphatases, hydrolases, calmodulin, carboxypeptidases and others that appear to be particularly associated with this process (Mayer and Poljakoff-Mayber, 1982; Bewley and Black, 1985; Cocucci and Negrini, 1991; Washio and Ishikawa, 1994).

Seed Water Uptake

Water uptake by a mature dry seed has been defined as triphasic (Fig. 3). Phase I is a rapid initial uptake, imbibition is probably very fast into the peripheral cells of the seed and small tissues such as radicle. Metabolism can be activated from this phase within minutes of imbibition. The phase I is followed by a plateau (phase II), also called germination sensu stricto, both of dormant and non-dormant seeds are metabolically active during this phase, and for nondormant seeds, the major metabolic events that take place at this time is the preparation for radicle emergence. A further increase in water uptake in the phase III occurs only after radicle elongation. The dormant seeds are blocked in entering this phase because they cannot complete the germination process (Bewley, 1997a). The duration of each of these phases depends on several factors, including inherent properties of the seed such as seed size, seed coat permeability and genotypes, and also some environment factors such as temperature, and the moisture content and composition of medium (Bewley and Black, 1985).
Fig. 3. Triphasic pattern of water uptake and time course of major events involved in germination and subsequent post germination. The time for events to be completed varies from several hours to many weeks, depending on the plant species and germination conditions. Red dot line represents imbibition curve of seed that cannot complete germination and did not enter into Phase III. Adapted from Bewley (1997a) and Wang et al. (2015).
Before imbibition, dry seeds have a low moisture content, 5 to 12%, depending on the species. This low moisture content contributes to a remarkably low rate of metabolism. Marked changes in metabolism occur as soon as the seed imbibes (Copeland and McDonald, 2001). As the dry seed starts to take up water during phase I, influx of water into the cells lead to temporary structural perturbations, especially on membranes accompanied by a massive leakage of cellular solutes and low molecular weight metabolites including ions, sugars and amino acid into the surrounding imbibition solution (Powell and Matthews, 1978). This is a transition symptomatic of the membrane phospholipid components from the gel phase achieved by drying during seed maturation, to the normal and hydrated liquid-crystalline state (Crowe and Crowe, 1992). However, in order to deal with the damage imposed during dehydration, storage and rehydration, seeds activate a series of repair mechanisms during imbibition (Fig. 3), which include membrane repair and therefore the membranes return to more stable configuration and solute leakage is curtailed within a short time of rehydration. Dry seeds contain mRNAs stored during maturation, also called long-lived transcripts to indicate that they survived desiccation (Rajjou et al., 2004). Over 10 000 different stored mRNAs have been identified in transcriptome analyses of Arabidopsis (Nakabayashi et al., 2005; Kimura and Nambara, 2010; Okamoto et al., 2010). Similar numbers were found in barley and rice (Howell et al., 2009; Sreenivasulu et al., 2008). More transcripts (about 32 000) have been shown in sunflower (Meimoun et al., 2014). The dry seed transcriptome mirrors the process of seed maturation as well as prepares the seed for the following germination (Weitbrecht et al., 2011). As showed by Kimura and Nambara. (2010), the major portions of the dry seed transcriptomes are very similar between seeds of non-dormant Col and dormant Cvi Arabidopsis accession, and the majority of stored mRNAs are of the LEA (late embryogenesis abundant) group or transcripts of storage proteins. Meimoun et al. (2014) brought the evidence that there is no significant difference between D and ND at dry state in sunflower seeds. Hence, mature seeds contain ready to use mRNA for cell functioning upon imbibition, the difference between D and ND might be due to change mRNA and or protein quality and/or environment such as redox regulation that fine-tuned gene expression or protein activity.

Respiration

Upon imbibition, the rise in water content induces a parallel rise in the rate of metabolism including respiration and O2 consumption. One of the first changes upon imbibition is the resumption of respiratory activity, which can be detected within minutes. Three respiration pathways operate in a seed during germination: the glycolysis, the pentose phosphate pathway (PPP) and the tricarboxylic acid cycle (TCA cycle or Krebs’s cycle). They produce key intermediates in metabolism and energy in the form of adenosine triphosphate (ATP), and reducing power in the form of reduced pyridine nucleotides, the nicotine adenine dinucleotides (NADH and NADPH) (Côme and Corbineau, 1989; Black et al., 2006). Both of the glycolytic and PPP are restored during the phase I. Enzymes of the TCA cycle and the terminal oxidases are usually present in the dry seed and become activated or are resynthesized when oxygen is high enough in internal structures (Nicolas and Aldasoro, 1979; Salon et al., 1988). Germinating seeds frequently produce ethanol in many species (Morohashi and Shimokoriyama, 1972). This is often the result of an internal deficiency in oxygen that is caused by restrictions to gaseous diffusion by the structures that surround the seed and by the dense internal structure of most seeds. This oxygen deficiency may result in more pyruvate production than used for activities of the TCA cycle and electron transport chain. Application of inhibitors of respiration can break dormancy in several kinds of seeds including sunflower, lettuce, rice and barley (Côme and Corbineau, 1989; Bewley and Black, 1994). It has been demonstrated that cyanide, which can inhibit terminal oxidation and the TCA cycle in the mitochondria, the glycolysis inhibitor, fluoride, and electron acceptors such as nitrate, nitrite and methylene blue can break seed dormancy of sunflower and Arabidopsis (Oracz et al., 2007; Arc et al., 2013). Consequently, the concept of the pentose phosphate pathway playing a unique role in dormancy breaking arises largely from these studies. Thus, the system is established as dormant seeds are deficient in an alternative oxygen-requiring process essential for germination, which is depleted of oxygen because of its lower affinity for this gas than the cytochrome pathway of respiration, so inhibitors of glycolysis, TCA cycle and terminal oxidation reactions of the mitochondrial electron transport chain can broke dormancy by activating pentose phosphate pathway, suggested as the alternative oxygen-requiring process essential for germination (Fig. 4).
Fig. 4. The pentose phosphate pathway and dormancy breakage. The consumption of oxygen by conventional respiration is blocked by the inhibitors, and oxygen then become available for other processes, meanwhile, the pentose phosphate pathway requires oxygen for the oxidation of reduced NADP (NADPH). From Bewley and Black (1985).
In a recent enzymatic study, Xia et al. (2018a) have provided the evidence that temperature can also promote dormancy break by decreasing glycolysis and TCA cycle activities and increasing sucrose metabolism in sunflower seeds. Indeed, the activity of the respiratory enzymes related to TCA cycle (Aconitase, MDH) and glycolysis (Aldolase, enolase, PGK, GAPDH) decreased in phase I of germination sensu stricto and subsequently an increase in the enzyme related to sucrose metabolism (UGPase) was found. Consequently, one can hypothetize that such enzymes are the target of dormancy release regulatory events probably related to PTMs processes which remain however to be characterized in this process.

RNA and Protein Synthesis

Enzyme activation begins during Phases I and II of imbibition. Phase II which can vary widely in duration, is characterized by a stabilized water and oxygen uptake. The seed undergoes many processes essential for germination following Phase II of water uptake, including a sequential translation of mRNA related with antioxidant mechanisms, cell detoxification, protein fate, energy, and amino acids metabolism occurs (Copeland and McDonald, 2001; Galland and Rajjou, 2015). Proteomic approaches unveiled the main importance of protein synthesis during seed imbibition in order to meet the increasing demand for proteins for seedling growth (Rajjou et al., 2004; Kimura and Nambara, 2010, Galland et al., 2014). Dry mature seeds contain a large number of mRNA species, supposed to be ready for protein synthesis upon imbibition. Transcription inhibitor did not prevent seed germination suggesting that transcription is not strictly requested for seed germination (Rajjou et al., 2004). Previous studies have demonstrated that sunflower seed dormancy release by after-ripening is associated with oxidation of specific subsets of stored mRNAs and differential accumulation of polysome-associated mRNAs but not related to transcriptomic changes, the translatome differs between germinating and nongerminating sunflower embryos (Bazin et al., 2011; Meimoun et al., 2014; Layat et al., 2014). In agreement with this, Basbouss-Serhal et al. (2015) have shown that there is no correlation between transcriptome and translatome in Arabidopsis, and that germination regulation is translational implying a selective and dynamic recruitment of messenger RNAs to polysomes in both dormant and non-dormant seeds. Hence, translation machinery components are highly represented in transcriptomic and proteomic studies. By contrast, seed proteome appears quite unique and diverse when compared to other plant developmental stages. Seed proteins encompass several functional classes from primary and secondary metabolism to structural and antimicrobial defence. In a proteomic study on Arabidopsis thaliana seeds, Galland et al. (2014) have underlined the importance of neosynthesis of some specific proteins during seed germination sensu stricto by selective mRNA translation as a major regulatory mechanism for the sake of radicle protrusion and germination completion. They have shown that during phase I, stored mRNAs are transalted and a restart of the late seed developmental program as exemplified by the synthesis of seed storage proteins is in action (i.e. cruciferin and late embryogenesis abundant proteins). The transition from phase I to phase II was seen to be characterized by the action of important remodeling/repair (i.e. rotamase cyclophilin), antioxidant (i.e. monodehydroascorbate reductase), and detoxification mechanisms (i.e. mercaptopyruvate sulfurtransferase 1). As for phase II, the combined action of the proteasome (20S proteasome subunit) and peptidases (peptidase S8/S53, tripeptidyl peptidase II) is active for the degradation of seed storage and other proteins in order to fuel amino-acid-incorporating metabolism (glutamine synthetase 1.3). Entering to phase III of water uptake, proteins involved in seedling establishment are neosynthetized in preparation for the seedling growth (i.e. actin and tubulin). Therefore, it can be assumed that mRNA translation and protein post translational modifications constitute the main levels of control for germination completion. These processes are highly regulated in plants and represent rapid and efficient way to cope with environmental variations (Galland and Rajjou, 2015).

Cell Wall Modification and Cell Elongation

Cell growth which is regarded as an irreversible increase in cell volume can occur through two processes: by expansion (increase in cell size in two or three dimensions) or by elongation (expansion which is constrained preferentially to one dimension). During elongation or expansion, existing cell wall architecture must be modified to permit incorporation of new material, thus increasing the surface area of the cell and inducing water uptake by the protoplast (McCann and Roberts, 1994). Many conditions have to take part like the turgor pressure in the primary cell wall which is a prerequisite to drive cell expansion (Cosgrove, 1993), in addition to the extensibility of the cell architecture done by mechanisms operating for discrete biochemical loosening of the matrix to permit microfibril separation and insertion of newly synthesized polymers (McCann and Roberts, 1994; Hepler et al., 2013). The constraint on expansion needed for elongation rely in part on the presence of cellulose/xyloglucan network where possible rearrangements of microfibrils within and between layers require enzymes that act on the xyloglucans that cross-link them into the network. Small proteins, termed expansins, may displace xyloglucans from cellulose microfibrils by disrupting the hydrogen-bonding between the xyloglucan backbone and the microfibril (Cosgrove, 1993, 2005). Moreover, McCann and Roberts (1994) have explained that cellulose microfibrils undertake changes in orientation during cell growth. Hence, unidirectional microfibrils are inserted into the wall by apposition at the plasma membrane, then previously deposited randomly-oriented microfibrils will gradually move to the outer layers of the wall and become progressively ‘diluted’ by oriented microfibrils. Thus, either the old layers of microfibrils will be integrated into newer layers or the microfibrils within the old layers will become separated so far that they do not contribute significantly to wall thickness. Pectin takes as well a big part in this process. Elongation is correlated with increased esterification in pectin and the cessation of elongation with de-esterification in many species like carrots (McCann and Roberts, 1994) maize (Kim and Carpita, 1992), tobacco (McCann et al., 1994) and Arabidopsis (Derbyshire et al., 2007; Peaucelle et al., 2015). Indeed, Derbyshire et al. (2007) have shown that a minimum level of about 60% of degree of esterification is required for normal cell elongation in Arabidopsis hypocotyls.
Moreover, the pectic network in the wall is replaced by newly-synthesized highly-esterified pectins, and older un- or de-esterified pectins are contributing to the increase in surface area of the middle lamella region. Hepler et al. (2013) suggested that newly secreted pectin becomes inserted into the wall matrix where it loosens the bonds between the existing pectate linkages causing a reduction in bulk viscosity. Thus, the newly secreted pectin would compete away Ca2+ from the existing matrix, further weakening the wall and allowing turgor-driven expansion. From the above mentioned processes, one can conclude that one part of the cell wall extensibility might be the synthesis of highly esterified pectins that may alter the rheology of the pectic network. Another part might be the coordinated secretion or activation of xyloglucan endotransglycosylase (XET) and expansins during elongation to permit rearrangement of the cellulose/xyloglucan network as new microfibrils and xyloglucans are inserted. Furthermore, Peaucelle et al. (2011) have explained that increased cell wall hydration may in turn facilitate the sliding of wall polymers or the mobility of wall-modifying agents (expansins, Xyloglucan endotransglcosylase/hydrolase, XTH) thus increase extensibility. Besides, Barnes and Anderson et al. (2018) have shown that many enzymes are involved in the elongation process. For example, elongating Arabidopsis stems exhibit relatively high expression of polygalacturonases (PG), pectate-lyases (PL), XTH, ß-1,3-xylosidase, ß-D-xylosidase, α-L arabinofuranosidase, ß-D-glucuronidase, and ß-D-mannosidase genes (Minic et al., 2009). Little is known about the occurrence of these processes in seeds, especially during germination sensu stricto, but they may operate to prepare radicle protrusion. In fact, in seeds of some species, decreasing the endosperm mechanical resistance to such a level that radicle can protrude through the weakened tissues is a condition in proceeding for germination. Table 1 represents a general overview of cell wall related genes and proteins characterized in many seed species associated to seed dormancy and germination. Most cell wall modifying enzymes are represented suggesting their involvement in seed dormancy and germination. Thus, An and Lin (2011) have demonstrated that cell wall synthesis and modification pathway genes are preferentially up-regulated within the early germination phase, which may function to loosen cell walls for subsequent cell expansion and division. Hence, it has been demonstrated that cell wall and protein degradation pathways are in direct relation with plant hormones and ROS in the germination process (Müller et al., 2013; Miransari and Smith, 2014; Xiong et al., 2015).
For germination process, cell wall weakening is thought to be a prerequisite through the induction of cell wall hydrolases and the decrease in the force required for radicle protrusion.
Dynamic changes in the cytoskeleton and cell wall would assist cell expansion and reserve nutrition deposition which are essential for the germination process.

Table of contents :

Chapter 1 Introduction 
I. Seed Development and Composition
II. Dormancy and Control of Germination
III. Cellular Events During Germination
III.1. Seed Water Uptake
III.2. Respiration
III.3. RNA and Protein Synthesis
III.4. Cell Wall Modification and Cell Elongation
IV. Hormonal Regulation of Seed Dormancy and Germination
V. Reactive Oxygen Species Action in Seed Biology
V.1. ROS Production and Scavenging System
V.2. ROS in the Regulation of Dormancy Alleviation and Germination
V.3. ROS and Protein Oxidation
V.3.1. Carbonylation
V.3.2. Sulfur-centers oxidation
V.4. ROS in Cell Wall Loosening
VI. Objectives of the Thesis on Sunflower Seed Dormancy and Germination
Chapter 2 Cell wall loosening during dormancy alleviation in sunflower seeds
I. Introduction
II. Materials and Methods
II.1. Plant Material and Treatment
II.2. Germination Tests
II.3. Atomic Force Microscopy (AFM)
II.4. Cell Wall Monosaccharide Composition
II.5. Pectin Methyl-Esterase (PME) Activity
II.6. Immunofluorescence Studies and Histological Examination
II.7. Statistical Analysis
III. Results
III.1. Germination Assay
III.2. Wall Stiffness Assessment
III.3. Characterization of Cell Wall Monosaccharide Composition
III.4. PME Activity Measurements
III.5. In Situ Detection of Cell Wall Methylation
IV. Discussion
V. Conclusion
VI. Acknowledgements
VII. References
Supporting Information
Chapter 3 Proteomics of protein-bound methionine oxidation in sunflower seed 
I. Introduction
II. Materials and Methods
II.1. Seed Materials and Treatment Conditions
II.2. NAD(H), NADP(H), Glutathione and Ascorbate Pool Measurement
II.3. Protein Extraction
II.4. Protein Digestion
II.5. Diagonal Chromatography
II.6. LC-MS/MS Analysis
II.7. Data Processing
III. Results and Discussion
III.1. Germination Assay
III.2. Redox Metabolite Status
III.2.1. Redox treatments profile
III.2.2 Redox metabolites profile
III.3 Protein Identification and Expression Profiles among Different Treatments
III.4. Functions and Pathways Analysis of Differentially Expressed Proteins
III.4.1 Transcription and translation
III.4.2 Protein metabolism, binding and remodeling
III.4.3 Energy and metabolism
III.4.4 Stress response
III.4.5 Oxidation–reduction-related proteins
IV. Conclusion
V. Acknowlegments
Chapter 4 General conclusion 

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