**2. Seed germination**

Seed germination is a physiological process initiated with water uptake and culminating with the emergence of the embryo through its protective tissues, which might include the testa, endosperm, perisperm, or pericarp [2]. The testa and the endosperm rupture must be coordi‐ nated with environmental seasonality to facilitate germination in the most favorable conditions [1, 6]. Several mechanisms have evolved to ensure proper synchronization of germination with environmental cues; among these is the interplay of hormonal signaling pathways via abscisic acid (ABA), gibberellins (GA), ethylene, and jasmonates [7–10]. These hormones exert their regulation on germination through different pathways including cell wall remodeling [7, 11].

In the classical model of seed germination described by Bewley et al. [12], the process of germination is divided into three phases, distinguished by the rate of water absorption by the seed tissues. The phase I, or imbibition phase, is characterized by a rapid water uptake rate driven by the difference in water potential between the seed and the environment. In this phase also the reactivation of primary metabolism and DNA repair pathways starts. Next, in phase II or activation phase, the imbibition rate decreases, water content remains stable, and major changes in the metabolic pathways and activation of other cellular processes take place. In this phase the integration of environmental cues with the internal status of the seed that will determine whether or not the seed will enter into the next phase occurs. Finally, in phase III there is another rapid water uptake driven by radicle protrusion and is mainly related to seedling growth. Germination is completed once the radicle has emerged at the onset of phase III. This triphasic model of imbibition can be applied to all seeds analyzed thus far [12, 13]. The imbibition time needed for completion of germination is highly variable among species and even within seed lots, and it depends on several factors like seed history and environmental conditions experienced by the mother plant at the moment of seed dispersion and during the after‐ripening period [12, 14, 15].

(testa) that is derived from the maternal tissues and in some species by one or more layers of storage tissue (endosperm) [2]. Seeds can function as resistance structures. Several mecha‐ nisms have evolved, in tight relation with the environment, to ensure the survival of the quiescent embryo [3]. Part of these mechanisms includes the modification of the structure and composi‐

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

One characteristic feature of plant cells is that they are enclosed in a polysaccharide and protein matrix, denominated as cell wall [4]. Plant cells can have two different types of wall. Primary walls, produced during cytokinesis, are flexible structures that regulate cell growth and shape. The secondary walls are deposited after the cell has achieved its final size and shape, by the inclusion of lignin and other phenolic compounds, thus making the cell wall rigid and usually impermeable. Cell walls have several functions that include the regulation of cell‐cell adhesion and abscission, apoplastic transport, mechanical support and maintenance of turgor pressure, and defense against pathogens [2, 5]. In seeds, cell walls are modified in order to generate hard, and in some cases impermeable, coats that protect the embryo from the environmental conditions. Also, seed cell walls can store energy that can be mobilized to feed embryo growth and development. Finally, cell walls regulate the timing of seed germination by fine‐tuning the processes of matrix polysaccharide loosening/breakage, as well as the integration of environmental cues with the hormonal and physiological status of the embryo [4, 6]. In this chapter we will focus only on primary cell walls and their importance on seed germination.

Seed germination is a physiological process initiated with water uptake and culminating with the emergence of the embryo through its protective tissues, which might include the testa, endosperm, perisperm, or pericarp [2]. The testa and the endosperm rupture must be coordi‐ nated with environmental seasonality to facilitate germination in the most favorable conditions [1, 6]. Several mechanisms have evolved to ensure proper synchronization of germination with environmental cues; among these is the interplay of hormonal signaling pathways via abscisic acid (ABA), gibberellins (GA), ethylene, and jasmonates [7–10]. These hormones exert their regulation on germination through different pathways including cell wall remodeling [7, 11].

In the classical model of seed germination described by Bewley et al. [12], the process of germination is divided into three phases, distinguished by the rate of water absorption by the seed tissues. The phase I, or imbibition phase, is characterized by a rapid water uptake rate driven by the difference in water potential between the seed and the environment. In this phase also the reactivation of primary metabolism and DNA repair pathways starts. Next, in phase II or activation phase, the imbibition rate decreases, water content remains stable, and major changes in the metabolic pathways and activation of other cellular processes take place. In this phase the integration of environmental cues with the internal status of the seed that will determine whether or not the seed will enter into the next phase occurs. Finally, in phase III there is another rapid water uptake driven by radicle protrusion and is mainly related to seedling growth. Germination is completed once the radicle has emerged at the onset of phase

tion of plant cell walls.

**2. Seed germination**

It is now generally accepted that radicle protrusion occurs by two nonexclusive processes [2, 13]. The first process involves a decrease in the mechanical resistance of the enclosing tissues, especially in the micropylar region of the testa and endosperm [2, 10]. The second process deals with an increasing growth potential of the embryo, driven by turgor pressure and cellular expansion in the embryonic axis [2, 13]. Most of the knowledge generated about the regulation of radicle protrusion comes from endospermic seeds, where testa and endosperm rupture can occur in two easily distinguishable stages (*Arabidopsis thaliana –Arabidopsis–*, *Chenopodium album*, *Lepidium* sp., *Nicotiana* sp., to mention a few) [2].

In recent years, with the advent of whole genome/transcriptome analysis, it has been possible to study the process of germination with high spatial‐temporal resolution. Transcriptomic analysis allows a comprehensive view of seed germination by dissecting "early" or "late" germination processes, the first being the initial response to water and the second correspond‐ ing to the interval from the imbibed seed to the radicle protrusion [14, 15]. Also, in endospermic seeds, an important landmark is the distinction between the processes that occur prior to testa rupture and after it that leads to endosperm rupture [8, 16–18].

Several studies demonstrate that the main transcripts, enzymes, and other proteins accumu‐ lated in dry seeds participate in primary metabolism, starch and storage protein mobilization, reactive oxygen species (ROS) scavenging, and cell wall synthesis [14, 15]. Aside from providing building blocks to sustain protein production and cell growth, the reactivation of primary metabolism in the early stages of seed germination plays a major role in the generation of the proper redox state to promote the activity of different enzymes and produce energy to support processes essential for radicle protrusion [14, 19].

In *Arabidopsis*, the seed development and maturation programs are regulated by the LAFL transcription factor network (LEAFY COTYLEDON 1 (LEC1) and LEC1‐LIKE (L1L), ABA INSENSITIVE 3 (ABI3), FUSCA 3 (FUS3), and LEC2), which activates other downstream transcription factor networks in concerted action of hormone, sugar, and light signalization pathways. Some target genes are involved in ABA, GA, ethylene, brassinoesteroids (BR), auxin, jasmonic acid (JA), and cytokinin (CK) signalization pathways [20]. The ABA signalization pathway participates in the regulatory networks of seed maturation, reserve accumulation, and desiccation tolerance acquisition [21]. GA blocks the LAFL and ABA networks during germination. The degradation of transcripts and enzymes related to seed maturation, which accumulated in the dry seed, has been described to occur in the first 6–12 h of seed imbibition in *Arabidopsis* [22] and within the first 24 h in *rice* and *barley* [14, 23].

Gibberellins play a major role in promoting a myriad of developmental programs, and its antagonistic role in ABA‐mediated block of germination has been described [24]. GA stimu‐ lates seed germination by enhancing embryo growth; embryos of *Arabidopsis* GA‐deficient 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 penetrate the micropylar endosperm.

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 development, and signaling [17].
