**2. Role of ABA metabolism and signaling in maintaining seed dormancy**

ABA is considered as a crucial phytohormone for seed dormancy establishment and main‐ tenance. Many of the ABA metabolism‐ and signaling‐related genes play a crucial role in the control of seed dormancy.

## **2.1. ABA biosynthesis and catabolism activity in the regulation of seed dormancy**

**1. Introduction**

Seed dormancy is defined as the inability of seeds to germinate under favorable conditions. The quiescent stage of seeds enables their survival during the adverse period for further seedling development. The high level of seed dormancy is considered as a negative trait due to germination retardation and reduction in the length of the growing season. On the other hand, low level of seed dormancy leads to preharvest sprouting (PHS) and yield loss. Thus, the varieties with medium value of seed dormancy are the most desirable [1–4]. Seed dor‐ mancy is considered as a quantitative trait under the control of the genetic and environmen‐ tal signals. The primary dormancy is induced during seed maturation, and its expression occurs mainly in freshly harvested seeds in order to prevent precocious seed germination. After‐ripening, which is dry seeds' storage at room temperature, can reduce primary seed dormancy [1]. The secondary dormancy can be induced in the presence of unfavorable con‐ ditions even in initially nondormant seeds [5–7]. Environmental conditions such as cold or heat temperature (stratification), light, nitrate (NO3−), and nitric oxide (NO) can break the dormancy stage [1, 3, 6, 8, 9]. The level of seed dormancy depends on the season of a year. Deep dormancy is associated with sensing slow seasonal changes in winter. Shallow dor‐

78 Phytohormones - Signaling Mechanisms and Crosstalk in Plant Development and Stress Responses

Induction and release of seed dormancy is mainly under the control of abscisic acid (ABA) and gibberellic acid (GA). ABA promotes seed dormancy and germination inhibition. Action of ABA is counteracted by GA, which promotes seed germination at appropriate time. The balance between ABA and GA is regulated by environmental conditions (light, temperature) and endog‐ enous signals [4, 6, 7, 11]. Other phytohormones, such as auxin, brassinosteroids, and ethylene, modulate the interaction between ABA and GA in the regulation of seed dormancy [2, 4, 12].

Seed dormancy in cereals is established during seed development; however, the time of seed dormancy release can be different. Some varieties loose dormancy when the harvest maturity is reached. There are also varieties ready for germination after seed physiological maturity (fully developed, but not dried seeds). In cereals, such as barley (*Hordeum vulgare*), wheat (*Triticum aestivum*), and sorghum (*Sorghum bicolor*), the switch between physiological and har‐

Here, we discuss the genetic and molecular bases of seed dormancy entrance and breaking in Arabidopsis and monocot plants, considering the action of components belonging to ABA, GA, and other phytohormone pathways. Additionally, the influence of environmental cues

ABA is considered as a crucial phytohormone for seed dormancy establishment and main‐ tenance. Many of the ABA metabolism‐ and signaling‐related genes play a crucial role in the

**2. Role of ABA metabolism and signaling in maintaining seed** 

mancy senses rapid condition changes in summer [10].

vest maturity is related to ABA decrease [7].

on ABA‐ and GA‐related genes is described.

**dormancy**

control of seed dormancy.

ABA produced in the embryo is fundamental for the promotion of seed dormancy. ABA syn‐ thesized in maternal tissues or ABA applied externally is not able to induce seed dormancy [13]. However, Kanno et al. [14] showed that ABA produced by maternal tissues can be trans‐ ported to the embryo in order to take part in seed dormancy induction. ABA biosynthesis is catalyzed in several steps, and the rate‐limiting reaction is mediated by carotenoid cleavage dioxygenase (NCED) [15, 16].

Many ABA biosynthesis genes are implicated in the regulation of seed dormancy in Arabidopsis. *NCED6* and *NCED9* are considered as the key ABA biosynthesis genes for induction of seed dormancy. They are expressed specifically during seed development. Double mutant *nced6/nced9* shows reduced seed dormancy [17]. Additionally, overexpres‐ sion of *NCED6* results in an increase in the ABA content in seeds and in the inhibition of pre‐ cocious germination [18]. *NCED5* is also described as a seed dormancy regulator (**Figure 1**) [19]. Other enzymes necessary for ABA biosynthesis and seed dormancy are encoded by *ABA deficient 2* (*ABA2*) and *abscisic aldehyde oxidase 3* (*AAO3*). *aba2‐2* and *aao3* mutants show a reduced ABA content and similar disorders in seed dormancy as *nced6/nced9* (**Figure 1**) [14, 20]. ABA level in seeds depends also on degradation process. The catabolism of ABA is mediated by ABA8'hydroxylase encoded by *cytochrome P450* (*CYP707A*) genes [15, 16]. The activity of *CYP707A* genes is related to the loss of seed dormancy. *CYP707a* mutants show higher level of seed dormancy than the wild type (WT), especially the *CYP707A2*. The expression of *CYP707A2* is induced in seeds during imbibition. Furthermore, *CYP707A2* activity and after‐ripening show a positive relationship. Therefore, *CYP707A2* is proposed to be responsible mainly for ABA degradation during release of seed dormancy and the ger‐ mination process (**Figure 1**) [21, 22]. The other *CYP707A* genes, *CYP707A1* and *CYP707A3*, also take part in ABA catabolism in seeds; however, their role in breaking dormancy is minor [22, 23].

The regulation of ABA metabolism genes plays also a very important role in seed dor‐ mancy of monocot plants. In rice (*Oryza sativa*), the expression of *OsNCED2* is activated at the early or the late stage of seed development, in dormant and nondormant cultivars, respectively. The different times of ABA biosynthesis in seeds could result in a high or low dormancy level [24]. In barley, the expression pattern of *HvNCED* genes in developing grains shows the higher level of *HvNCED2* transcript in comparison to *HvNCED1* [25–27]. Moreover, *HvNCED2* activation in the field is independent of weather conditions, in con‐ trary to *HvNCED1* and *HvABA8'OH1/HvCYP707A1*. On the other hand, the induction of *HvABA8'OH1* expression occurs in after‐ripened seeds, but not in the dormant seeds during imbibition. Thus *HvNCED2* seems to play a more significant role in ABA biosynthesis and in the preventing of preharvest sprouting than *HvNCED1*. Furthermore, *HvABA8'OH1* activity mediates dormancy breaking [25].

Barley seed dormancy is associated with the presence of glumellae (lemma and palea). It was shown that dehulled grains have no induction of *HvNCED1*, *HvNCED2*, and *HvABA8'OH1* genes. The contrary reaction was observed in whole, dormant grains [28]. The induction of secondary dormancy in barley is also dependent on ABA metabolism genes. While *HvNCED1*

**Figure 1.** Probable function of ABA‐related genes in seed dormancy promotion. Seed dormancy is positively regulated by ABA biosynthesis genes: *carotenoid cleavage dioxygenase 2 (NCED2*), *NCED3*, *NCED5*, *NCED6*, *NCED9*, *ABA2 ABA deficient 2* (*ABA2*), and *abscisic aldehyde oxidase 3* (*AAO3*). Genes encoding ABA‐related transcription factors, *ABA insensitive 3* (*ABI3*), *ABI4*, and *ABI5*, also promote seed dormancy. *HONSU* (*HON*) is a positive regulator of seed dormancy, but it represses ABA signaling. ABA catabolism genes*, cytochrome P450* (*CYP707A1*) and *CYP707A2*, are responsible for seed dormancy release. Other regulators like, *lysinespecific demethylase 1* (*LDL1*), *LDL2, kryptonite/SU(VAR)3‐9 homolog 4* (*KYP/ SUVH4*), *proteolysis6 (PRT6)* negatively regulates the ABA pathway and seed dormancy. ABI4 modulates the expression of ABA biosynthesis genes (*NCED2*, *NCED3*) probably via *CHOTTO1* (*CHO1*) and ABA catabolism genes (*CYP707A1*, *CYP707A2*).

shows higher activation at 30°C, the transfer of grains to 20°C is associated with *HvNCED2* and *HvABA8'OH1* induction. Probably, the expression of secondary dormancy depends mostly on *HvNCED2*, whereas the promotion of *HvABA8'OH1* may be a response to immediate increase in the ABA level in seeds [26]. *HvNCED2* is also mostly expressed during hypoxia‐related seed dormancy [29]. The after‐ripening process is associated with the increased expression of *HvABA8'OH1* in coleorhizae [30–32]. The barley lines with silenced *HvABA8'OH1* expres‐ sion show the increased ABA accumulation and seed dormancy level [31]. In *Brachypodium*  *distachyon*, the higher expression of *BdNCED1* was observed in dormant grains in comparison to after‐ripened grains. Contrarily, after ripening promoted the induction of *BdABA8'OH‐1* at the second day of imbibition. Probably, *BdABA8'OH‐1* plays a prominent role in the after‐rip‐ ening process [33].

### **2.2. Regulation of seed dormancy via ABA signaling components**

The core ABA signaling is mediated by pyrabactin resistance proteins/PYR‐like proteins/regu‐ latory components of ABA receptor (PYR/PYL/RCAR), phosphatase 2C (PP2C), SNF1‐related protein kinase 2 (SnRK2), and abscisic acid responsive elements‐binding factor (AREB) basic leucine zipper (bZIP) transcription factors [34–36]. In Arabidopsis, ABA signaling genes are also implicated in seed dormancy regulation. *ABA insensitive 1* (*ABI1*) encodes PP2C phos‐ phatase, which acts as the negative regulator of ABA signaling [37]. *abi1* was described as the mutant with decreased seed dormancy level and better germination in the presence of ABA [38]. The other PP2C phosphatase, HONSU (HON), also represses ABA signaling, specifically in seeds. However, its role in seed dormancy is inconclusive. *HON* expression is associated with both, dormancy establishment and release (**Figure 1**) [39]. *ABI* genes, such as *ABI3*, *ABI4*, and *ABI5* encode crucial ABA‐dependent transcription factors expressed in seeds. Expression of *ABI3, ABI4*, and *ABI5* is higher in dormant seeds than in seeds with reduced seed dormancy level (**Figure 1**) [40–42]. Among *ABI* genes, *ABI3* is the most substantial for seed dormancy establishment. *ABI3* is expressed in developing seeds. It regulates the accumulation of chlo‐ rophyll, anthocyanins, and storage proteins together with two other seed‐related regulators, *FUSCA3* (*FUS3*) and *leafy cotyledon 1* (*LEC1*) [43, 44]. *abi3* mutant shows no seed dormancy, and immature seeds are able to germinate [45]. ABI3 is under direct regulation of WRKY DNA‐binding protein 41 (WRKY41) during the establishment of primary seed dormancy. WRKY41 binds directly to *ABI3* promoter and induces its expression [46].

ABI4 is another ABA‐activated transcription factor with APETALA 2 (AP2) domain, expressed in seeds. It takes part in the regulation of abiotic stress responses and different aspects of plant development [47]. *abi4* mutant germinates faster than the wild type without stratification. The expression analysis showed decreased activation of *NCED2* and *NCED3* in *abi4* seeds. Moreover, ABI4 binds to *CYP707A1* and *CYP707A2* promoters and represses their expression. It indicates the important role of ABI4 in seed dormancy maintenance (**Figure 1**) [41]. It is worth noting that *ABI4*, *NCED2*, and *NCED6* are under positive regulation of a common ABA‐dependent regulator, myeloblastosis 96 (MYB96). The activation of *NCED2* and *NCED6* ensures ABA biosynthesis and seed dormancy promotion, whereas *ABI4* induc‐ tion inhibits lipid breakdown and further seed germination [48]. One of the downstream tar‐ get of ABI4 is *CHO1* (*CHOTTO 1*), encoding a transcription factor with double AP2 domain. *CHO1* acts also as a positive regulator of primary seed dormancy (**Figure 1**) [49, 50]. ABI5 is a bZIP transcription factor regulating ABA signaling in seeds [42]. The role of *ABI5* in seed dormancy regulation is not clear. *abi5* mutant shows a normal dormancy level [51]. However, many studies described below showed a distinct relationship between *ABI5* and seed dor‐ mancy [40, 52–54].

shows higher activation at 30°C, the transfer of grains to 20°C is associated with *HvNCED2* and *HvABA8'OH1* induction. Probably, the expression of secondary dormancy depends mostly on *HvNCED2*, whereas the promotion of *HvABA8'OH1* may be a response to immediate increase in the ABA level in seeds [26]. *HvNCED2* is also mostly expressed during hypoxia‐related seed dormancy [29]. The after‐ripening process is associated with the increased expression of *HvABA8'OH1* in coleorhizae [30–32]. The barley lines with silenced *HvABA8'OH1* expres‐ sion show the increased ABA accumulation and seed dormancy level [31]. In *Brachypodium* 

*CYP707A2*).

**Figure 1.** Probable function of ABA‐related genes in seed dormancy promotion. Seed dormancy is positively regulated by ABA biosynthesis genes: *carotenoid cleavage dioxygenase 2 (NCED2*), *NCED3*, *NCED5*, *NCED6*, *NCED9*, *ABA2 ABA deficient 2* (*ABA2*), and *abscisic aldehyde oxidase 3* (*AAO3*). Genes encoding ABA‐related transcription factors, *ABA insensitive 3* (*ABI3*), *ABI4*, and *ABI5*, also promote seed dormancy. *HONSU* (*HON*) is a positive regulator of seed dormancy, but it represses ABA signaling. ABA catabolism genes*, cytochrome P450* (*CYP707A1*) and *CYP707A2*, are responsible for seed dormancy release. Other regulators like, *lysinespecific demethylase 1* (*LDL1*), *LDL2, kryptonite/SU(VAR)3‐9 homolog 4* (*KYP/ SUVH4*), *proteolysis6 (PRT6)* negatively regulates the ABA pathway and seed dormancy. ABI4 modulates the expression of ABA biosynthesis genes (*NCED2*, *NCED3*) probably via *CHOTTO1* (*CHO1*) and ABA catabolism genes (*CYP707A1*,

80 Phytohormones - Signaling Mechanisms and Crosstalk in Plant Development and Stress Responses

In monocot plants, the activation of ABA signaling is also associated with seed dormancy. The maize (*Zea mays*) ortholog of *ABI3*, the *viviparous 1* (*VP1*), is a crucial regulator of seed dormancy. *vp1* mutant shows premature embryo germination (vivipary) and reduced ABA sensitivity [55]. The overexpression of maize *VP1* in wheat induces increased seed dormancy and prevents pre‐ harvest sprouting [56]. Some rice varieties produce truncated versions of *OsVP1* transcript. There is a relation between incorrect transcripts' amount and preharvest sprouting. This phenomenon is associated with developmental stage: immature embryos accumulate a higher number of truncated transcripts than mature embryos [57]. Another gene, the *seed dormancy 4* (*SDR4*) is a rice quantitative trait locus (QTL) responsible for seed dormancy promotion in ABA‐dependent manner. The *japonica* varieties have reduced dormancy and possess only SDR4‐n allele, whereas more dormant varieties of *indica* type include SDR4‐n and SDR4‐k alleles. OsVP1 was shown to positively promote *SDR4* expression [58]. Other ABA‐related genes also take part in seed dormancy maintenance. Expression analysis of sorghum grains with various dormancy level identified a set of differentially regulated ABA signaling genes. A dormant inbred line of sor‐ ghum showed increased expression of *SbABA‐responsive protein kinase* (*SbPKABA1.1*), *SbABI1*, *SbVP1*, *SbABI4*, and *SbABI5* during grain imbibition. However, no induction of these genes in a nondormant inbred line was observed [59]. In barley, dormancy expression is associated with increased induction of *HvPKABA*, *HvVP1*, and *HvABI5* [28]. Probably, the general ABA‐related mechanism of seed dormancy induction is similar in dicots and monocots.
