**3. Gibberellins‐mediated control of seed dormancy release and germination**

A high level of gibberellins (GA) is needed for the counteraction of ABA activity in seeds. GA promotes seed dormancy release and radical protrusion during seed germination. The acti‐ vation of GA‐responsive genes induces cell wall–remodeling enzymes, such as endo‐β‐man‐ nase, xyloglucan endotransglycolase, expansin, and β‐1,3‐mannase. Their activity leads to the weakening of the embryo‐surrounding layers. Additionally, GA ensures the high‐growth potential of the embryo [68].

## **3.1. Role of GA metabolism in seed dormancy break**

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

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

mechanism of seed dormancy induction is similar in dicots and monocots.

**pathway**

ABI5 is detained (**Figure 1**) [52].

**2.3. Environmental cues and epigenetic modifications in the regulation of the ABA** 

The expression of Arabidopsis ABA metabolism and signaling genes is regulated through environmental factors. The red (R) light pulse irradiation applied to the far‐red (FR) light pulse pretreated, dark‐imbibed seeds inhibits and induces the expression of *NCED6* and *CYP707A2,* respectively. It suggests that the ABA metabolism genes are under the control of PHYB (phytochrome B), which regulates germination in response to FR and R pulse light [60]. On the other hand, the blue light has a negative impact on the germination of dormant grains in cereals. The blue light–associated secondary dormancy induces *HvNCED1* and *HvNCED2* and weakly reduces *HvABA8'OH‐1* expression in grains [61]. The activation of *HvNCED1* is under the regulation of phytochrome photoreceptor, cryptochrome 1 (HvCRY1). It indi‐ cates that ABA biosynthesis and catabolism take part in blue light–dependent regulation of seed dormancy [62, 63]. The temperature and NO also exert an impact on ABA pathway in Arabidopsis seeds. The high temperature promotes the expression of ABA biosynthesis genes in imbibed seeds, whereas NO positively regulates ABA signaling during seed dormancy breaking [52, 64]. NO action may be associated with N‐end rule pathway, leading to degrada‐ tion of proteins with destabilizing amino acid residues. NO and oxygen are sensed by N‐end rule pathway with the participation of many protein regulators [65]. The components of N‐ end rule pathway, proteolysis 6 (PRT6) and arginyl‐tRNA:protein arginyltransferase (ATE), regulate after‐ripening, inhibit ABA signaling, and finally promote seed germination. PRT6 is E3 ligase promoting protein degradation via 26S proteasome. Some PRT6 substrates belong to the ABA pathway. As a result, ABA signaling is inhibited, and the activation of ABI3 and GA biosynthesis takes place mainly in the radicle of the embryo, which in turn ensures ger‐ mination progression [69]. Arabidopsis seed germination is associated with the regulation of GA metabolism genes. The highest expression of GA‐biosynthesis genes, *gibberellin 3‐oxidase 1* (*GA3ox1*), *GA20ox3*, and *ENT‐kaurene oxidase 1* (*KO1*), was shown during the first 8 hours of imbibition [68]. The crucial role of GA in the breaking of seed dormancy was presented using a *ga requiring 1* (*ga1*) mutant in GA biosynthesis gene, *CPP synthase* (*CPS*). Interestingly, *ga1* capacity to germinate was renewed after removing testa and endosperm, without exogenous GA application. It was concluded that dormancy release and germination promotion was dependent on GA‐ABA balance in the embryo and the embryo‐surrounding layers of the seed [70]. The environmental factors, such as light and temperature, interact with GA bio‐ synthesis and signaling, which in turn promotes seed germination. The expression of *GA3ox1* is activated by red light and cold. Additionally, the low temperature determines the *GA3ox1* expression localization in the embryonic axis and the aleurone layer [71, 72]. Contrarily, low temperature represses the expression of GA catabolism gene, *GA2ox2* [71]. Two bHLH (basic helix‐loop‐helix) transcription factors, spatula (SPT) and phytochrome interacting factor 3‐ like 5 (PIL5), regulate seed germination after cold stratification, including GA biosynthesis pathway. SPT represses germination before stratification, whereas PIL5 also acts as an inhibi‐ tor of germination, but after cold stratification in darkness. Both, SPT and PIL5, act through negative regulation of *GA3ox1* and *GA3ox2* [73]. Another transcription factor, DOF affect‐ ing germination1 (DAG1) was found to mediate PIL5 negative regulation of *GA3ox1*. PIL5 promotes the expression of *DAG1* in darkness. Furthermore, DAG1 protein binds directly to *GA3ox1* promoter, inhibits its expression, and blocks germination [74]. Contrary to cold, high temperature represses the expression of *GA20ox1*, *GA20ox2*, *GA20ox3*, *GA3ox1*, and *GA3ox2* during seed imbibition and blocks germination [64]. Similar to ABA‐related genes, the expres‐ sion of GA metabolism genes is regulated seasonally. *GA3ox2* activation is associated with summer, whereas *GA2ox2* is expressed in winter [10].

In barley and wheat, the expression of GA biosynthesis genes occurs during imbibition of nondormant seeds [31, 72]. The rapid increase in *HvGA3ox2* involved in GA biosynthesis was observed in the after‐ripened grains during imbibition. The high expression level of *HvGA3ox2* is associated with *HvGA2ox2* activation [31]. The hypoxia‐related secondary dor‐ mancy in barley is associated with the modulation of the GA pathway. Low oxygen con‐ centration causes induction of *HvGA2ox3* and repression of *HvGA3ox1* and *HvGA20ox1* in dormant grains. The activity of GA‐responsive gene, *HvEXPANSIN11* (*HvEXPA11*), is also repressed [29]. Similar reaction was observed during seed dormancy imposed by blue light. The negative regulation of the GA pathway occurred through the promotion of *HvGA2ox3* and *HvGA2ox5* and the repression of *HvGA3ox2* [61]. The relationship between the expres‐ sion of GA metabolism genes and the induction of secondary dormancy at 30°C was also shown; however, the particular expression pattern depended on the embryo water content in barley. The embryo with high‐water content (1.60–1.87 g H2 O g−1 DW) shows the higher expression of GA catabolism and signaling genes than the embryo with lower water content (0.45 g H2 O g−1 DW) [75].

GA metabolism genes are involved in seed dormancy regulation in other monocot spe‐ cies. In wheat, after‐ripening causes induction of *GA20ox1* and *GA3ox2* [72]. The regulation of expression of GA synthesis and catabolism genes is more complex in rice. *OsGA20ox1*, *OsGA2ox5*, and *OsGA2ox6* expression pattern showed higher variability in a nondormant than in a dormant variety during seed development. Furthermore, the dormant variety accumulated less‐active GA in seeds in comparison to the nondormant variety. It resulted in appropriate dormancy phenotype of analyzed cultivars [24]. Similar analysis was con‐ ducted in terms of immature grains of sorghum inbred lines with contrasting dormancy level. Higher expression of *SbGA20ox1* and *SbGA20ox3* was observed for a less‐dormant line, whereas a strong induction of *SbGA2ox1* and *SbGA2ox3* was found in the line with higher dormancy [76]. To summarize, a proper regulation of GA biosynthesis and catabolism genes ensures the regulation of seed dormancy dependent on environmental conditions, both in dicot and monocot plants.

#### **3.2. Action of GA signaling components in seed dormancy regulation**

In Arabidopsis, GA signaling is mediated by GA insensitive dwarf1 (GID1) receptor. Overexpression of *GID1* promotes the release of seed dormancy. The impact of cold strat‐ ification and after ripening on *GID1* expression showed that imbibition at 4°C promoted expression of three *GID1* transcript forms: *GID1a*, *GID1b*, and *GID1c,* while after‐ripening storage induced only *GID1b*. Thus, both mechanisms of seed dormancy loss seem to be regu‐ lated differently [77]. In sorghum, exogenous GA represses *SbGID1* in immature grains. It suggests the role of SbGID1 in negative feedback regulation of the GA pathway [76]. The sleepy1 (SLY1) is a F‐box protein which enables 26S proteasome‐mediated degradation of DELLA proteins in the presence of active GA [78]. DELLA proteins act as repressors of GA signaling. *sly1* mutant shows reduced germination, even after the application of exogenous GA. It indicates that SLY1 is the crucial regulator of seed germination [79]. Another mutant related to GA signaling, *cts* (*comatose*), maintains seed dormancy even after stratification or after ripening. CTS functions as a peroxisomal ABC transporter and seems to be crucial for seed dormancy release [80]. The proper regulation of DELLA proteins is crucial for seed germination. Simultaneous deactivation of *repressor of GA* (*RGA*), *RGA‐like 1* (*RGL1*), *RGL2*, and *gibberellic acid insensitive* (*GAI*) results in insensitivity to GA and light during germina‐ tion. It indicates that DELLA proteins integrate environmental cues into GA signaling [81]. Among them, RGL2 seems to play a more important role in seed germination than other DELLAs. Thermoinhibition of seed germination demands activity of *RGL2,* which suggests its crucial role in the regulation of GA signaling in seeds [64]. Moreover, *GID1* transcripts are under control of RGL2 during cold stratification and after ripening. The RGL2 can promote or inhibit *GID1* expression according to a particular *GID1* transcript form and surround‐ ing conditions during dormancy loss [77]. *RGL2* activity associates with the regulation of shallow dormancy. Its expression is promoted during summer time [10]. Another negative regulator of GA signaling is *spindly* (*SPY*). The *spy* mutant demands the lower amount of GA to break seed dormancy and continue germination. *SPY* encodes *O*‐linked *N*‐acetylglu‐ cosamine (*O*‐GlcNAc) transferase which probably glycosylates components of GA signal‐ ing. SPY acts upstream of RGA through the modulation of its activity through *O*‐GlcNAc modification [82].

## **3.3. The role of essential seed dormancy regulator, DOG1, in GA pathway regulation**

*Delay of germination 1* (*DOG1*) is considered as the crucial, positive regulator of seed dor‐ mancy with unknown function. Expression of *DOG1* is seed specific, and *dog1* mutant shows disturbed seed dormancy in Arabidopsis [83]. Similar to ABA‐related genes, *DOG1* is under negative epigenetic regulation mediated by KYP/SUVH4, LDL1, and LDL2, which as a result reduces primary dormancy [66, 67]. *DOG1* expression is related to deep dormancy during winter season [10]. Recently, the role of DOG1 in temperature‐dependent coat dormancy through GA metabolism regulation was shown. DOG1 differently regulates the expression of GA biosynthesis genes, such as *GA3ox1* and *GA20ox*, at 18 and 24°C. This leads to the inhibi‐ tion of genes encoding cell wall remodeling enzymes: *expansin 2* (*EXPA2*), *EXPA9*, *xyloglucan endo‐transglycosylase 19* (*XTH19*) but only at 24°C. Therefore, DOG1 regulates the appropriate time of germination according to environment temperature [84].
