**3. Cell wall structure and composition**

Plant cell walls are complex and highly dynamic structures composed of a variety of polysac‐ charides, proteins, and aliphatic or aromatic compounds [28, 29]. They are continually being modified throughout development and in response to environmental stimuli [30, 31]. Primary cell walls of flowering plants can be classified in two main groups depending on its general architecture and composition, as well as their biosynthetic processes [32, 33]. Type I cell walls are the most common, present in dicotyledonous and the non‐commelinoid monocotyledo‐ nous plants (a more basal group of aroids, alismatids, and lilioids). Type II cell walls are found only in the commelinoid monocots that include the Poales (members of the families Poaceae, Bromeliaceae, and Cyperaceae) [32, 33].

#### **3.1. Primary cell wall polysaccharides**

Primary cell wall polysaccharides constitute the majority of the wall dry mass in land plants and can be grouped in three main classes: cellulose, hemicelluloses, and pectins [30]. Cellulose is a linear 1,4‐β‐D‐glucan that assembles into partially crystalline microfibrils, each of which contains about 36 parallel polysaccharide chains [34]. Cellulose is synthesized *in muro* by the cellulose synthase complex (CSC), embedded within the plasma membrane and formed by 6 rosette subunits that contain 6 cellulose synthase proteins (CESA) [35]. Aside from cellulose, all other cell wall polysaccharides are synthesized and processed for wall targeting in the trans‐ Golgi system [5].

Hemicelluloses are polymers whose backbones consist of β‐glucose, β‐xylose, or β‐mannose, with short side chains. In all vascular plants with type I walls, the most common hemicellu‐ loses are xyloglucans (XyG), whereas type II cell walls contain less XyG, being the most abundant glucuronoarabinoxylans (GAX) and β1,3:β1,4 mixed glucans [33]. Hemicellulose chains adhere to cellulose microfibrils, in a rope‐like manner, to restrain cell expansion [30, 34]. Also, in type I cell walls, hemicelluloses bind and cross‐link with pectin and form the hydrated matrix [30].

The group generally known as pectins comprises over 30% of the cell wall total mass in dicots [31, 36]. Pectins are acidic heteropolymers that form a hydrated gel, in which cellulose and other molecules are embedded in the plant cell wall. Their main defining feature is 1,4‐ linked α‐D‐galacturonic acid residues (GalA). Pectins interact covalently and non‐covalently with other pectin molecules or with hemicellulose xyloglucan or arabinogalactans [31]. Several studies support the hypothesis that the three major pectin classes, homoglacturonan (HG), rhamnogalacturonan I, and rhamnogalacturonan II, are covalently linked in the cell wall [29, 37], forming a hydrophilic macromolecular network. Pectin is deposited on the cell wall matrix in a highly methylesterified form [28]. The methyl group is removed by pectin methylester‐ ases (PMEs) in muro, providing an anionically charged matrix and changing the mechanic properties of the cell wall. Increasing evidence shows that the regulation of the degree of methylesterification of the pectic matrix plays a fundamental role in plant growth, develop‐ ment, morphogenesis, cell‐cell adhesion, cell expansion, seed hydration, and seed germina‐ tion [5, 16, 36, 38].

Other polysaccharides present in primary cell walls of various species are the mannans, arabinoxylans, and arabinogalactans. Mannans are formed by mannosyl residues linked by β‐ 1,4‐glycosidic linkages. This mannosyl backbone can contain glucose residues (glucomann‐ ans) or be further substituted by single galactose residues with α‐1,6‐linkages (galactomannans). Arabinoxylan consists of a (1,4)‐linked β‐D‐xylan backbone decorated with arabinose branches. Other residues, such as glucuronic acid and ferulic acid esters (FAE), are also attached in arabinoxylans that are particularly abundant in cereal grasses. Arabinogalac‐ tan and storage xyloglucans are used as reserves in cotyledons. The basic structure of storage xyloglucans differs from the primary wall xyloglucans in that it is not fucosylated [39].

#### **3.2. Cell wall proteins**

mutant seeds exhibit reduced growth rate phenotypes [25]. Also, GA enhances seed germina‐ tion by overcoming the mechanical restraint to radicle protrusion of the surrounding tissues. In *Solanum lycopersicum* (tomato), GA‐deficient embryos (unable to germinate unless incubated with exogenous GA) can grow into dwarf plants when the testa and the endosperm were removed mechanically [26]. This role of GAs in stimulating germination can be linked to the upregulation of several cell wall‐modifying proteins (CWMPs) detected in whole‐seed *Arabidopsis* transcriptomes of *ga1‐3* mutants treated with GA4 [24]. Jacobsen and Pressman [27] suggested that the embryo of celery (*Apium graveolens*) seeds does not secrete CWMPs but rather promote the activity of GA‐inducible CWMPs in the endosperm. The depletion of the endosperm in this species generates a space where the embryo cells can expand and eventually

158 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

An overrepresentation analysis (ORA) of gene ontologies showed that transcription regulation is enriched in both the endosperm and the embryo transcriptomes of *Arabidopsis* seeds. The ORA analysis also showed that in the endosperm, the main biological processes are associated with cell wall metabolism, cell death, response to biotic stimulus, and defense and response to ABA. The main biological processes in the embryo include phosphate metabolic process, protein amino acid phosphorylation, hormone metabolic process (particularly auxin synthesis and transport), cell division and cell cycle, post‐germination regulation of growth and organ

Plant cell walls are complex and highly dynamic structures composed of a variety of polysac‐ charides, proteins, and aliphatic or aromatic compounds [28, 29]. They are continually being modified throughout development and in response to environmental stimuli [30, 31]. Primary cell walls of flowering plants can be classified in two main groups depending on its general architecture and composition, as well as their biosynthetic processes [32, 33]. Type I cell walls are the most common, present in dicotyledonous and the non‐commelinoid monocotyledo‐ nous plants (a more basal group of aroids, alismatids, and lilioids). Type II cell walls are found only in the commelinoid monocots that include the Poales (members of the families Poaceae,

Primary cell wall polysaccharides constitute the majority of the wall dry mass in land plants and can be grouped in three main classes: cellulose, hemicelluloses, and pectins [30]. Cellulose is a linear 1,4‐β‐D‐glucan that assembles into partially crystalline microfibrils, each of which contains about 36 parallel polysaccharide chains [34]. Cellulose is synthesized *in muro* by the cellulose synthase complex (CSC), embedded within the plasma membrane and formed by 6 rosette subunits that contain 6 cellulose synthase proteins (CESA) [35]. Aside from cellulose, all other cell wall polysaccharides are synthesized and processed for wall targeting in the trans‐

penetrate the micropylar endosperm.

development, and signaling [17].

**3. Cell wall structure and composition**

Bromeliaceae, and Cyperaceae) [32, 33].

**3.1. Primary cell wall polysaccharides**

Golgi system [5].

Primary cell walls are mainly constituted by polysaccharides; however, proteins account for about 10% of the total dry mass of the wall [40]. Proteins that contain a secretion signal peptide, which targets them to the secretory pathway and in most cases is excised to allow activation or proper protein function, are commonly referred as classical cell wall proteins [40–42]. In *Arabidopsis*, about 17% (∼5000 genes) of the genome encodes for proteins targeted to the secretory pathway, and of this, about 1000–2000 genes could be cell wall proteins (CWPs). Cell wall proteins have several functions as structural, enzymatic, and defense and have been grouped in functional categories by different authors. The proteins with a structural func‐ tion or those acting on polysaccharides are the two main functional categories [30, 42]. These proteins are expressed in a tissue‐specific and process‐specific manner, contributing to the regulation of cell wall stabilization and rigidity [41].

#### *3.2.1. Structural proteins*

Structural proteins are usually classified by the predominant amino acids in their sequence, although some of them can belong to more than one category. The most common include the hydroxyproline‐rich glycoproteins (HRGPs), the glycine‐rich proteins (GRPs), the proline‐rich proteins (PRPs), and the arabinogalactan proteins (AGPs). These proteins vary greatly in abundance within plant species, cell tissues, and environmental conditions [4]. Arabinogalac‐ tan proteins, that are widely distributed among plant families and comprise about 2–10% of the total protein in the wall, are highly glycosylated. Also, AGPs are rich in hydroxyproline, serine, alanine, threonine, and glycine, and resistant to proteolysis in their native state [41]. Extensins are a family of HRGPs particularly abundant in dicots that have been involved in modification of wall extensibility in elongating tissues [43].

#### *3.2.2. Proteins acting on polysaccharides*

Within the CWPs acting on polysaccharides, there are a broad variety of activities. For instance, the group of glycosyl hydrolases (GHs) include glycosidases (β‐glucosidase, β‐galactosidase, β‐xylosidase, and α‐xylosidase, and exo‐polygalacturonases) and glycanases like β‐manna‐ nase, β‐xylanase, (1→4)‐β‐glucanase "cellulase", endo‐polygalacturonases, and xyloglucan endo‐hydrolase (XEH). The combined activity of these kinds of enzymes is theoretically capable of hydrolyzing most of the glycosidic bonds in the cell wall polysaccharides but do not imply that all enzymes are active at the same time or tissue. The glycosyltransferases (GTs) activity involves the formation of a glycosyl‐enzyme complex that is attacked by an acceptor substrate (another oligo/polysaccharide). This activity allows the integration of recently secreted polysaccharides into the matrix and the grafting of polysaccharides al‐ ready present in the wall matrix [44]. This category includes the xyloglucan endotransglyco‐ sylase (XET). Both XEH and XET proteins are commonly grouped within the xyloglucan endotransglucosylase/hydrolase family (XTH) due to some of their members (like β‐xyla‐ nase) that can have both GH and GT activities [30].

Polysaccharide lyases (PLs) promote cell separation by calcium‐dependent de‐polymeriza‐ tion of wall polygalacturonides. Plant pectate lyases are a group of enzymes that catalyze the cleavage of de‐methylesterified pectin. PL activity has being described in cell wall degrada‐ tion that occurs during fruit ripening [45], and Penfield et al. [7] report 34 pectate lyases that were downregulated in the endosperm of *Arabidopsis*imbibed seeds after treatment with ABA.

Carbohydrate esterases (CEs) include two enzyme families that have activity over pectins, the PMEs and pectin acetylesterases (PAEs), and the family of xylan acetylesterases. These enzymes cleave methyl or acetyl groups from the HG or Xyl backbone of polysaccharides [30].

PMEs catalyze the reaction by which methylesters are cleaved from a HG chain, producing a free carboxyl group and the release of a proton and methanol [46]. Plant PMEs are mainly alkaline isoforms bound to the wall matrix, while some isoforms are neutral and easily

solubilized or free apoplastic acidic isoforms. Alkaline isoforms seem to be the PMEs with most de‐methylesterification activity, but the kinetics of PME activity is affected by the ionic composition of the matrix, thus influencing PME activity and mobility [47]. PME activity can lead to two different cell wall fates: the first one would be the formation of a rigid, stable structure by Ca2+ interaction with de‐methylesterified GalA residues (>10) in the HG chains. The second fate of HG would be their degradation by polygalacturonases, where only small stretches or individual GalA residues are de‐methylesterified, thus leading to a more re‐ laxed matrix [28, 46]. Also, PME activity acidifies the cell wall; this acidification would allow expansin activity ("acid grow") [5]. PMEs are antagonistically regulated in the cell wall by proteinaceous PME inhibitors (PMEIs) meanwhile PGs by PG inhibitors (PGIPs) [28].

Expansins regulate cell wall loosening in a pH‐dependent manner by disruption of the hydrogen bonds between xyloglucans and cellulose. Sequence analysis indicates that expan‐ sins contain an N‐terminal domain slightly similar to the catalytic domain of the family‐45 endoglucanases; however no catalytic activity has been reported.

In *Arabidopsis*, about 10% of the total CWPs described in cell wall proteomes from different tissues and plants correspond to gene families with domains of unknown function (DUF) [42]. Mewalal et al. [48] have pointed out the relevance of these DUF families on cell wall dynam‐ ics. In particular, the two plant‐specific families DUF231 and DUF642 could be involved in pectin modification [49, 50].

#### **3.3. Cell wall modification**

proteins are expressed in a tissue‐specific and process‐specific manner, contributing to the

Structural proteins are usually classified by the predominant amino acids in their sequence, although some of them can belong to more than one category. The most common include the hydroxyproline‐rich glycoproteins (HRGPs), the glycine‐rich proteins (GRPs), the proline‐rich proteins (PRPs), and the arabinogalactan proteins (AGPs). These proteins vary greatly in abundance within plant species, cell tissues, and environmental conditions [4]. Arabinogalac‐ tan proteins, that are widely distributed among plant families and comprise about 2–10% of the total protein in the wall, are highly glycosylated. Also, AGPs are rich in hydroxyproline, serine, alanine, threonine, and glycine, and resistant to proteolysis in their native state [41]. Extensins are a family of HRGPs particularly abundant in dicots that have been involved in

Within the CWPs acting on polysaccharides, there are a broad variety of activities. For instance, the group of glycosyl hydrolases (GHs) include glycosidases (β‐glucosidase, β‐galactosidase, β‐xylosidase, and α‐xylosidase, and exo‐polygalacturonases) and glycanases like β‐manna‐ nase, β‐xylanase, (1→4)‐β‐glucanase "cellulase", endo‐polygalacturonases, and xyloglucan endo‐hydrolase (XEH). The combined activity of these kinds of enzymes is theoretically capable of hydrolyzing most of the glycosidic bonds in the cell wall polysaccharides but do not imply that all enzymes are active at the same time or tissue. The glycosyltransferases (GTs) activity involves the formation of a glycosyl‐enzyme complex that is attacked by an acceptor substrate (another oligo/polysaccharide). This activity allows the integration of recently secreted polysaccharides into the matrix and the grafting of polysaccharides al‐ ready present in the wall matrix [44]. This category includes the xyloglucan endotransglyco‐ sylase (XET). Both XEH and XET proteins are commonly grouped within the xyloglucan endotransglucosylase/hydrolase family (XTH) due to some of their members (like β‐xyla‐

Polysaccharide lyases (PLs) promote cell separation by calcium‐dependent de‐polymeriza‐ tion of wall polygalacturonides. Plant pectate lyases are a group of enzymes that catalyze the cleavage of de‐methylesterified pectin. PL activity has being described in cell wall degrada‐ tion that occurs during fruit ripening [45], and Penfield et al. [7] report 34 pectate lyases that were downregulated in the endosperm of *Arabidopsis*imbibed seeds after treatment with ABA.

Carbohydrate esterases (CEs) include two enzyme families that have activity over pectins, the PMEs and pectin acetylesterases (PAEs), and the family of xylan acetylesterases. These enzymes cleave methyl or acetyl groups from the HG or Xyl backbone of polysaccharides [30].

PMEs catalyze the reaction by which methylesters are cleaved from a HG chain, producing a free carboxyl group and the release of a proton and methanol [46]. Plant PMEs are mainly alkaline isoforms bound to the wall matrix, while some isoforms are neutral and easily

regulation of cell wall stabilization and rigidity [41].

160 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

modification of wall extensibility in elongating tissues [43].

nase) that can have both GH and GT activities [30].

*3.2.2. Proteins acting on polysaccharides*

*3.2.1. Structural proteins*

The molecular modification of the wall network can result in the relaxation of wall stress or "wall loosening" by the controlled rearrangement of cellulose/matrix polymers, which involve sliding of a cross‐link along a scaffold or the breakage of stress‐bearing cross‐links without substantial changes in wall dimensions. These rearrangements could include three processes: (a) the cleavage of the backbone of major matrix polymers, (b) the weakening of the non‐ covalent bonding between polysaccharides, and (c) the breakage of cross‐links [5]. Follow‐ ing cell wall loosening, there are three main types of outcomes: cell expansion, cell separation, and wall stiffening. Cell wall enlargement occurs secondarily as a result of water uptake and the reduction of turgor pressure resulting from wall loosening [44].

Reactive oxygen species (ROS) like hydrogen peroxide (H2O2), hydroxyl radical (·OH), and superoxide radical (O2 —) have been proposed to play a major role in germination by partici‐ pating in defense against pathogens, signaling, and promotion of cell wall loosening [2, 9]. ROS can negatively affect germination by reacting with almost all macromolecules stored in the seed, causing oxidative damage and cleavage of polysaccharide chains in the cell walls [5, 9]. The participation of ROS in cell wall loosening and promotion of germination might be indirect, through the ethylene signaling pathways that involve ROS production and down‐ stream activation of CWMPs [9]. Cosgrove [5] suggests the revision of ROS participation in the process of wall loosening, since in most studies reporting ROS‐mediated extensibility comprises only a small fraction (about 1% extensibility) and the assays with higher ROS concentrations provoke wall breakage.

#### **3.4. Role and regulation of cell wall enzymes and proteins during germination**

The study of plant cell wall structure and physiology has achieved a major progress from the input of "‐omics" technologies in the past two decades. These ‐omics technologies are able to capture the complexity of biological processes, like seed germination and cell wall modifica‐ tion, with high sensitivity and spatial‐temporal resolution. A tissue‐specific transcriptome analysis in *Arabidopsis* showed that both endosperm and embryo share gene expression patterns and biological processes during seed germination. About 10,800 transcripts (∼84% of total genes expressed) are present in both endosperm and embryo. Endosperm‐specific genes comprise about 415 genes that were highly expressed [17]. Of this gene set, 154 are cell wall‐ related genes, with most of them being expressed at the onset of testa rupture. Transcript abundance of several CWMPs shows a transient peak with a 6–24 h interval in tomato [51], *Arabidopsis* [17], and *Lepidium* seeds [16]. Although a change in transcript levels does not necessarily correlate to changes in protein abundance or enzymatic activity, it has been demonstrated that during *rice* germination, most cell wall‐related transcripts, as well as the resulting metabolites from cell wall modification, accumulate about 12–24 h after imbibition (HAI, although some metabolites can be detected by 3 HAI) [23].

Cell wall modification can occur at five different stages during seed germination: (a) during the cellular expansion process triggered by rehydration of tissues, (b) at the onset of testa rupture, (c) during endosperm weakening and rupture, (d) during cellular expansion related to radicle elongation, and (e) during wall degradation and mobilization of stored reserves in both living and nonliving storage tissues.

#### *3.4.1. Rehydration‐driven cellular expansion*

Seed imbibition is given by the difference in water potential between the seed and the environment. Nonviable seeds swell faster than viable seeds, as viable seeds develop turgor pressure that restricts further water uptake [2]. However, rapid imbibition can still occur and lead to solute leakage and damage of membranes. Gradual rehydration of seed tissues has been detected in legumes like peas and beans, where hydration starts in the tissues near to the micropyle. As water diffuses in the outermost tissues, a waterfront is formed between imbibed tissues and those about to be imbibed. The testa plays a significant role in modulating imbibition kinetics and the waterfront formation [2]. The seeds of mutants with altered testa structure or altered deposition of protecting substances (like flavonoids, cutin, suberin, and lignin) have increased permeability and lower longevity than the wild type [52]. Testa structure usually consists of several layers of highly compressed dead cells where protective substan‐ ces are deposited during seed development and maturation. Plant cell walls of living cells can also function as an interface that modulates water intake by changing wall porosity, thus allowing a gradual swelling of all tissues. This regulation could be achieved by rapid changes in wall extensibility as the ones generated by expansins. In support of this view, in whole unstratified *Arabidopsis* seeds, the upregulation of *EXPA1*, *EXPA2, EXPA3, EXPA8, EXPA9, EXPA15,* and *EXPA20* transcripts from 0 to 12 h has been reported. This induction was evident in seeds imbibed in the light and during moist cold stratification at 4°C in the dark [11]. Also, in whole‐seed transcriptomes, the expression of *AtXTH5*, *AtXTH6*, and *AtXTH33* transcripts within the first 6HAI and *AtXTH3* and *AtXTH*33 transcripts at 12HAI [24] was detected. The activity of XTHs from *chickpea* seeds has been detected in imbibed seeds from 1HAI until 24HAI, when the rate of radicle elongation slows down [53].

In many species from the Brassicaceae, Solanaceae, Linaceae, and Plantaginaceae, among others, the epidermis of the testa contains specialized cells that accumulate abundant pectins and heteroxylans, as well as some xyloglucans or arabinans during seed development. Upon imbibition these polysaccharides expand and burst out of the testa, generating a gel‐like structure. This phenomenon, known as myxospermy, has been used as a model to study several hydrolases and PME activity [54, 55]. Although there is still uncertainty about the actual role of myxospermy, the proposed roles include regulating hydration, preventing desicca‐ tion, being an oxygen barrier, or allowing the seed to attach to the substrate and animals [54– 56].

#### *3.4.2. Testa rupture*

**3.4. Role and regulation of cell wall enzymes and proteins during germination**

162 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

(HAI, although some metabolites can be detected by 3 HAI) [23].

both living and nonliving storage tissues.

*3.4.1. Rehydration‐driven cellular expansion*

The study of plant cell wall structure and physiology has achieved a major progress from the input of "‐omics" technologies in the past two decades. These ‐omics technologies are able to capture the complexity of biological processes, like seed germination and cell wall modifica‐ tion, with high sensitivity and spatial‐temporal resolution. A tissue‐specific transcriptome analysis in *Arabidopsis* showed that both endosperm and embryo share gene expression patterns and biological processes during seed germination. About 10,800 transcripts (∼84% of total genes expressed) are present in both endosperm and embryo. Endosperm‐specific genes comprise about 415 genes that were highly expressed [17]. Of this gene set, 154 are cell wall‐ related genes, with most of them being expressed at the onset of testa rupture. Transcript abundance of several CWMPs shows a transient peak with a 6–24 h interval in tomato [51], *Arabidopsis* [17], and *Lepidium* seeds [16]. Although a change in transcript levels does not necessarily correlate to changes in protein abundance or enzymatic activity, it has been demonstrated that during *rice* germination, most cell wall‐related transcripts, as well as the resulting metabolites from cell wall modification, accumulate about 12–24 h after imbibition

Cell wall modification can occur at five different stages during seed germination: (a) during the cellular expansion process triggered by rehydration of tissues, (b) at the onset of testa rupture, (c) during endosperm weakening and rupture, (d) during cellular expansion related to radicle elongation, and (e) during wall degradation and mobilization of stored reserves in

Seed imbibition is given by the difference in water potential between the seed and the environment. Nonviable seeds swell faster than viable seeds, as viable seeds develop turgor pressure that restricts further water uptake [2]. However, rapid imbibition can still occur and lead to solute leakage and damage of membranes. Gradual rehydration of seed tissues has been detected in legumes like peas and beans, where hydration starts in the tissues near to the micropyle. As water diffuses in the outermost tissues, a waterfront is formed between imbibed tissues and those about to be imbibed. The testa plays a significant role in modulating imbibition kinetics and the waterfront formation [2]. The seeds of mutants with altered testa structure or altered deposition of protecting substances (like flavonoids, cutin, suberin, and lignin) have increased permeability and lower longevity than the wild type [52]. Testa structure usually consists of several layers of highly compressed dead cells where protective substan‐ ces are deposited during seed development and maturation. Plant cell walls of living cells can also function as an interface that modulates water intake by changing wall porosity, thus allowing a gradual swelling of all tissues. This regulation could be achieved by rapid changes in wall extensibility as the ones generated by expansins. In support of this view, in whole unstratified *Arabidopsis* seeds, the upregulation of *EXPA1*, *EXPA2, EXPA3, EXPA8, EXPA9, EXPA15,* and *EXPA20* transcripts from 0 to 12 h has been reported. This induction was evident in seeds imbibed in the light and during moist cold stratification at 4°C in the dark [11]. Also, in whole‐seed transcriptomes, the expression of *AtXTH5*, *AtXTH6*, and *AtXTH33* transcripts

In many plant species, testa rupture starts at the micropylar seed end. In tobacco (*Nicotiana tabacum*) seeds, the testa ruptures at the micropyle and follows predetermined breaking points due to the presence of channel‐like structures underlying ridges in the testa [57]. In pea seeds the presence of xylogalacturonan in the inner walls of the testa was described, which coin‐ cides detached cells or with the junction sites between cells that are destined to detach, as described in other plant tissues [37]. In *Lepidium* seeds, HG composition shifts at the onset of testa rupture: while the seeds imbibe, non‐esterified HG is ubiquitously distributed in all tissues, but by the time of testa rupture, this non‐esterified HG is detected mostly on the endosperm and testa, meanwhile esterified HG is detected in the endosperm [38].

The seed testa is composed of several layers of nonliving cells, and thus the regulation and enzymes that facilitate cell separation in the testa must come from the living tissues under‐ neath. At the onset of testa rupture (∼25 HAI), it is possible to identify 90 cell wall‐related genes from the 501 upregulated genes (∼18%) in the micropylar endosperm and 58 from 282 genes (∼20%) upregulated in the radicle. Also, about 8 (∼8%) and 5 (∼4%) genes were downregulated in both tissues, respectively [17]. In *Lepidium* seeds, tissue‐specific transcript abundance patterns between the micropylar endosperm and the radicle accompany testa rupture, further supporting the view of this process as a decisive step in germination and in the regulation of cell wall‐related genes [38]. In **Table 1**, these transcripts and its predicted biological/biochemical function are enlisted.



The Dynamics of Plant Cell Wall *In Muro* Modifications and its Physiological Implications on Seed Germination http://dx.doi.org/10.5772/64085 165

**Endosperm (HAI) RA Endosperm (HAI) RA**

EXPA4 AT2G39700 No 16 β‐Gal AT4G26140 No 31 EXPA2 At5g05290 No 3 24 β‐Glu AT1G61820 EN Not specified EXPA8 At2g40610 No 3–12 24 β‐Glu AT1G70710 No 16 TR\* EXPA3 At2g37640 No 6–12 24 β‐Glu AT4G16260 EN Not specified EXPA9 AT5G02260 No 12 24 β‐Glu AT1G26560 EM Not specified

EXPB1 At2g20750 No 12 β‐Glu AT2G44450 No TR 31

EXLA1 AT3G45970 No 16 TR TR MAN5 AT4G28320 No Not specified EXLA2 AT4G38400 No 6–12\* TR TR MAN6 AT5G01930 EN Not specified EXT3 AT1G21310 EM Not specified MAN7 AT5G66460 No 6 24 EXT10 AT5G06640 EM Not specified GH AT5G49360 EN TR EXTL AT3G54590 EM Not specified GH AT5G57560 No 16 TR EXTL AT4G38770 EM Not specified GH AT5G08370 No 16c TR 31c EXTL AT2G27380 RA Not specified GH AT3G55430 No TR 31 24\* XTH5 AT5G13870 No 6 24 24 GH AT3G07320 No 24 31 24 XTH33 AT1G10550 No 12 16 TR TR GT AT3G10320 EN 31c XTH AT1G11545 No 16 TR GH AT3G13790 No 16 31 XTH AT1G32170 No TR GH AT5G64570 No 24 TR XTH17 AT1G65310 No TR GH‐DUF3357 AT1G12240 No 31 TR

XTH AT2G06850 No 16 TR 31 TR GH AT3G47010 EN 24 XTH AT2G36870 No 31 KOR2 AT1G65610 No 31 XTH AT3G23730 No TR TR CESA5 AT5G09870 No TR 31 24 XTH11 AT3G48580 No 16 24\* 31 CSLC AT4G07960 No TR TR XTH AT4G03210 No TR TR AGP AT3G11700 No TR TR

XTH AT4G14130 No TR TR AGP AT5G44130 EN TR XTH24 AT4G30270 EN 16 TR TR AGP AT1G28290 No 16 31 XTH18 AT4G30280 No 16 TR TR PRT AT3G54400 No 16 24 XTH AT4G30290 No 16 31 PRT AT3G61820 No TR\* 31 24 XTH AT4G37800 EM Not specified PRT AT4G16563 No TR TR XTH25 AT5G57550 No 6\* TR TR PL AT3G24670 EM 24 XTR8 AT3G44990 EN 16c 24 PL AT3G27400 No TR 24 XTR6 AT4G25810 No TR TR PL AT4G13710 EN Not specified

PL AT4G24780 No TR\* PMEI AT5G20740 No TR TR

EXPA20 AT4G38210 No 6–12 24 β‐Glu AT3G62750 No 3c

164 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

EXLA3 AT3G45960 No TR β‐Glu AT4G34480 No 12\*



The endosperm expression profiles are subdivided by hours after imbibition (HAI) and testa rupture (TR, ∼25HAI), whereas the radicle (Rad) expression only shows the moment of TR. *Abbreviations*: T‐S, tissue‐specific expression; EM, embryo; EN, endosperm; RA, radicle; MI, micropylar endosperm; EXPA, expansin; EXT, extensin; EXTL, extension‐like protein; XTH, xyloglucan‐transglycosylhydrolase; GH, glycosyl‐hydrolase; β‐Glu, β‐glucosidase; β‐Gal, β‐ galactosidase; AGP, arabinogalactan protein; CESA, cellulose synthase; PRT, protease; PL, pectate lyase; PX, peroxidase; CSLC, cellulose synthase‐like; LRR‐p, leucine‐rich repeat protein; DUF, domain of unknown function; PME, pectin methylesterase; PME2, PME with an inhibitory domain; PMEI, PME inhibitor; PTRI, protease inhibitor; GT, transglycanase; PG, polygalacturonase; PGIP, PG inhibitor. The \* means downregulation for that particular gene and time. The letter "c" beside a number means the expression was upregulated in the chalazal endosperm.

**Table 1.** Expression of some upregulated cell wall‐modifying genes in *Arabidopsis* tissue‐specific microarrays described by [17, 22] and expression profiles at http://bar.utoronto.ca/.

In *Arabidopsis* and *Lepidium* seeds, total PME activity increases gradually with imbibition time and peaks at the onset of testa rupture. ABA treatment does not affect testa rupture but endosperm rupture is delayed and PME activity fails to decrease following testa rupture [16, 38]. Two DUF642 genes, *BIIDXI* (*BDX*, *At4g32460*) and *At5g11420*, are expressed in the embryo and micropylar endosperm, respectively, during germination. In overexpression lines, testa and endosperm rupture of matrix‐primed seeds occurred earlier compared to wild type. The germination performance of overexpression seeds was accompanied by an increase of total PME activity, compared to the wild type [50].

In non‐endospermic seeds, testa rupture marks the end of germination. In this type of seeds, the testa rupture is accompanied by radicle elongation whose continued pressure in the inner face of the testa promotes cell separation [53].

#### *3.4.3. Endosperm weakening for radicle protrusion*

The endosperm functions as a barrier to control radicle protrusion as it can impose primary dormancy in many species like *Arabidopsis*, *Lepidium*, and yellow cedar, among others [2]. Endosperm structure of mature seeds varies greatly within species, where it can comprise one layer of cells as in *Arabidopsis*, *Lepidium*, and cucumber or to several layers as in tomato or tobacco [58, 59]. Structural studies in hard‐seeded species like fenugreek (*Trigonella foenum‐ graecum*) and coffee (*Coffea arabica*) have demonstrated that near the micropyle a zone of thin‐ walled cells that can be a low‐resistance area for radicle protrusion exists. Endosperm cell wall composition varies considerably among species: in the closely related species, *Arabidopsis* and *Lepidium* are rich in cellulose, non‐esterified HG, arabinans, and XG. However, these polysac‐ charides are not uniformly distributed in the endosperm: in *Lepidium*, an epitope for arabin‐ ans (LM13) was localized in the inner and outer walls of the cells, but absent from the traverse walls. The endosperm of tomato seed contains mannans that have been shown to contribute

to the control of radicle protrusion and general endosperm hardness rather than a storage function [39].

The hydrolytic activity of β‐glucanases or endo‐β‐mannanases can contribute to endosperm cell wall weakening in Brassicaceae and Solanaceae species, which have cell walls rich in mannans [59]. In *Arabidopsis* seeds the activity of *AtMAN5*, *AtMAN6*, and *AtMAN7* and regulation of their activity by the basic leucine‐zipper 44 transcription factor, *AtbZIP44*, whose knockout mutants have delayed germination have been described [60]. In hard‐seeded species, there was a negligible activity in the radicle and micropylar endosperm of endo‐β‐mannanas‐ es that could not be associated with endosperm weakening to allow radicle protrusion [61]. However it is still unexplored the activity of other CWMPs that could contribute to wall rearrangements during germination in these species. The upregulation of wall‐related genes in the endosperm has been reported; this includes α‐expansins (*AtEXPA2, AtEXPA8,* and *AtEXPA9*), β‐expansin (*AtEXPB1*), expansin‐like protein (*AtEXPL1*), cellulose synthase‐like proteins (*CSLA2* and *CSLC4*), xyloglucan endotransglycosylases (*AtXTH11*, *AtXTH17*, *AtXTH18, AtXTH33, AtXTH31, AtXTH23*, and *AtXTH24*), and mannanase (*AtMAN7*) [62].

Several reports indicate that endosperm weakening and rupture are inhibited or delayed by ABA, in a dose‐dependent manner, in some species of the Brassicaceae family [8, 16] and tobacco [63]. Microarray analysis of *Arabidopsis* seeds treated with exogenous ABA at the onset of radicle protrusion has shown a downregulation of several wall‐related transcripts in the endosperm, including PMEs, AGPs, and PLs [7]. In tobacco, ABA delays the accumulation and activity of β‐1,3‐glucanase in the micropyle before radicle protrusion [63]. PMEs contribute to seed germination in several species by modulating the degree of methylesterification of pectins in the endosperm [16, 38, 64, 65]. In yellow cedar a loss of the internal structure of the megagametophyte surrounding the radicle during germination was described. The resulting decrease in the mechanical strength of the megagametophyte would allow radicle protru‐ sion. There is a positive correlation between dormancy alleviation and PME activity, as well as with germination performance and PME activity in both the megagametophyte and the embryo. PME activity has also been demonstrated to positively correlate with germination performance in *Arabidopsis* [64]. The *LeXET4* gene transcripts, which are restricted to the endosperm cap, are detected in tomato seeds within 12 h of imbibition and reach a maxi‐ mum at 24 h [66]; they decline after radicle emergence despite a continued degradation of the lateral endosperm cell walls. PG *At2g43860* is expressed within the endosperm cells of the seed adjacent to the site of the emerging radicle [67]. In germinating tomato seeds, several reports indicate the expression of expansins [51], PGs (*LeXPG1*) [68], and XTHs (*LeXET4*), which accumulate in the endosperm region adjacent to the expanding radicle (∼40HAI). Tran‐ scripts are detected within 12HAI and generally peak by 24HAI, consistent with the endo‐ sperm weakening.

#### *3.4.4. Embryonic axis elongation*

**Endosperm (HAI) RA Endosperm (HAI) RA**

PMEI AT1G62770 RA Not specified GT AT1G08280 No 31 PMEI AT2G47670 EM 3‐12\* 24\* PGIP AT5G06860 No TR PMEI AT4G00080 No 31 TR PG AT2G43860 MI 24 31 PMEI AT4G12390 EM Not specified PG AT3G06770 No TR 24 The endosperm expression profiles are subdivided by hours after imbibition (HAI) and testa rupture (TR, ∼25HAI), whereas the radicle (Rad) expression only shows the moment of TR. *Abbreviations*: T‐S, tissue‐specific expression; EM, embryo; EN, endosperm; RA, radicle; MI, micropylar endosperm; EXPA, expansin; EXT, extensin; EXTL, extension‐like

166 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

protein; XTH, xyloglucan‐transglycosylhydrolase; GH, glycosyl‐hydrolase; β‐Glu, β‐glucosidase; β‐Gal, β‐ galactosidase; AGP, arabinogalactan protein; CESA, cellulose synthase; PRT, protease; PL, pectate lyase; PX, peroxidase; CSLC, cellulose synthase‐like; LRR‐p, leucine‐rich repeat protein; DUF, domain of unknown function; PME, pectin methylesterase; PME2, PME with an inhibitory domain; PMEI, PME inhibitor; PTRI, protease inhibitor; GT, transglycanase; PG, polygalacturonase; PGIP, PG inhibitor. The \* means downregulation for that particular gene and time. The letter "c" beside a number means the expression was upregulated in the chalazal endosperm.

by [17, 22] and expression profiles at http://bar.utoronto.ca/.

PME activity, compared to the wild type [50].

face of the testa promotes cell separation [53].

*3.4.3. Endosperm weakening for radicle protrusion*

**Table 1.** Expression of some upregulated cell wall‐modifying genes in *Arabidopsis* tissue‐specific microarrays described

In *Arabidopsis* and *Lepidium* seeds, total PME activity increases gradually with imbibition time and peaks at the onset of testa rupture. ABA treatment does not affect testa rupture but endosperm rupture is delayed and PME activity fails to decrease following testa rupture [16, 38]. Two DUF642 genes, *BIIDXI* (*BDX*, *At4g32460*) and *At5g11420*, are expressed in the embryo and micropylar endosperm, respectively, during germination. In overexpression lines, testa and endosperm rupture of matrix‐primed seeds occurred earlier compared to wild type. The germination performance of overexpression seeds was accompanied by an increase of total

In non‐endospermic seeds, testa rupture marks the end of germination. In this type of seeds, the testa rupture is accompanied by radicle elongation whose continued pressure in the inner

The endosperm functions as a barrier to control radicle protrusion as it can impose primary dormancy in many species like *Arabidopsis*, *Lepidium*, and yellow cedar, among others [2]. Endosperm structure of mature seeds varies greatly within species, where it can comprise one layer of cells as in *Arabidopsis*, *Lepidium*, and cucumber or to several layers as in tomato or tobacco [58, 59]. Structural studies in hard‐seeded species like fenugreek (*Trigonella foenum‐ graecum*) and coffee (*Coffea arabica*) have demonstrated that near the micropyle a zone of thin‐ walled cells that can be a low‐resistance area for radicle protrusion exists. Endosperm cell wall composition varies considerably among species: in the closely related species, *Arabidopsis* and *Lepidium* are rich in cellulose, non‐esterified HG, arabinans, and XG. However, these polysac‐ charides are not uniformly distributed in the endosperm: in *Lepidium*, an epitope for arabin‐ ans (LM13) was localized in the inner and outer walls of the cells, but absent from the traverse walls. The endosperm of tomato seed contains mannans that have been shown to contribute

Cell elongation, rather than cellular division, is the main process that drives embryo growth [14]. Cell division occurs after radicle protrusion and contributes to the rapid growth of the embryonic axis by generating new elongating cells [2]. Cell elongation that drives radicle protrusion occurs at the transition zone, which comprises the cells between the last proximal root hair cell in the radicle and the lower basal cells of the hypocotyl [69]. In *Arabidopsis* Col seeds that have been previously stratified, the radicle protrusion can initiate as early as 32 HAI. By this time, and immediately prior to the radicle emergence through the endosperm, the cells in the transition zone had incremented their size by 44% while the cells in the radicle 10% and in the hypocotyl 30%. By 40HAI, the radicle has already protruded and the elongated cells in the seedling have increased their size by 15% in the radicle, 52% in the hypocotyl, and 108% in the transition zone. Elongation is often accompanied by an increase in DNA content without subsequent mitosis (endoreduplication) [69].

In the micropylar endosperm of tomato seeds, an important mobilization of protein bodies occurs, but it seems that there is no cell degradation as the radicle protrudes. Instead, a process similar to cell separation to allow radical protrusion was suggested [70]. A similar process was observed in celery seeds, where the radicle tip also seems to penetrate the micropylar endosperm by separating the endosperm cells, but, since the embryo needs to grow before germination is completed, cell degradation for storage mobilization occurs in the endosperm adjacent to the embryo [27]. The expression of *LeEXP8* and *LePG1* in the embryo elongation zone of tomato seeds has been reported at the onset of radicle protrusion [51, 68].

#### *3.4.5. Cell wall participation in the mobilization of stored reserves*

Major reserve mobilization occurs once germination has concluded, and these reserves are utilized to feed the growing seedling rather than to fuel radicle protrusion. However, in cereal grains, the preparation for starch and oligosaccharide mobilization occurs within the first hours of germination [15]. In cereals, the endosperm is a nonliving storage tissue, and the endosperm cell walls protect its contents from enzymatic attack. Accordingly, the degrada‐ tion of cell walls is a limiting step in storage reserve mobilization that is induced by the GA produced by the embryo (at the scutellum) and secreted to the aleurone layer [2].

In most endospermic seeds this tissue is still living. Mannans in the endosperm cell walls of date palm (*Phoenix dactylifera*) and coffee are mobilized to support embryo development. It has been proposed that the mobilization of storage xyloglucans can be coupled to the growth rate of the seedling by transglycosylation. In legumes, endosperm galactomannans seems to function as reserves; they can constitute up to 30% of total seed dry weight. In fenugreek and *Schizolobium parahyba*, the cell walls of the endosperm are thickened with galactomannan and in some cases the cytoplasm is nonexistent. In *Tamarindus indica* and *Hymenaea courbaril*, reserve xyloglucans are stored between two primary walls and are degraded without hydrolyzing both walls [39]. During germination of celery seeds, the surrounding endosperm degrades leaving a small amount of un‐degraded polymers of the cell wall, except for the micropylar endosperm, in which only some protein bodies are mobilized and the rest of the cells persist until the radicle pushes through; once radicle protrusion has started, these micropylar cells are degraded [27].
