**4. ABA and GA crosstalk during seed dormancy**

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

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

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

expression of GA catabolism and signaling genes than the embryo with lower water content

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

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

O g−1 DW) shows the higher

summer, whereas *GA2ox2* is expressed in winter [10].

in barley. The embryo with high‐water content (1.60–1.87 g H2

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

(0.45 g H2

O g−1 DW) [75].

dicot and monocot plants.

The seed dormancy maintenance or release and further promotion of the seed germination process are regulated by ABA and GA balance [1, 2, 12]. ABA‐mediated repression of GA biosynthesis enables the positive regulation of seed dormancy [60]. Many molecular interac‐ tions between ABA and GA pathways enable precise regulation of seed response according to environmental conditions.

#### **4.1. Activity of ABA and GA metabolism genes ensures the ABA‐GA interaction**

There is the relationship between ABA and GA biosynthesis in Arabidopsis. ABA‐deficient mutant, *aba2‐2*, shows the higher expression of *GA3ox1* and *GA3ox2* than the wild type [60, 64]. Interestingly, *AAO3* and *ABA2* expression were detected in a radicle, whereas *GA3ox2* in hypocotyl. It suggests that the places of ABA and GA biosynthesis are different in seeds [60]. *aba2‐2* shows also the reduced expression of *SPY* during seed imbibition in the presence of high temperature. Therefore, the negative regulator of GA signaling, *SPY*, is under the posi‐ tive action of ABA (**Figure 2**) [64]. NCED9 negatively influences GA biosynthesis. The appli‐ cation of paclobutrazol, GA biosynthesis inhibitor, causes better germination of *nced9* than the wild type. It is an evidence that ABA biosynthesis modulates GA pathway in seeds [85].

#### **4.2. ABA‐GA crosstalk depends on ABI transcription factors and DELLA proteins in seeds**

ABA and GA signaling components are involved in the ABA‐GA crosstalk in Arabidopsis seeds. ABI4 exerts action on GA biosynthesis genes. In *abi4* mutant, the expression of *GA3*,

**Figure 2.** Model for seed dormancy regulation by ABA‐GA crosstalk. ABA‐mediated promotion of seed dormancy and GA‐related release of seed dormancy are possible through ABA‐GA interactions. The seed dormancy regulator, *mother of FT and TFL 1* (*MFT1*), is promoted by ABA insensitive 5 (ABI5) and RGA‐like 2 (RGL2), but ABI3 downregulates its expression. ABI5 and RGL2 positively regulate reciprocal expression. RGL2 also promotes *XERICO* and ABA biosynthesis. Repressor of GA biosynthesis, delay of germination 1 (DOG1), activates *ABI3* and *ABI5*. GA biosynthesis is inhibited by spatula (SPT) and ABI4 via chotto1 (CHO1) activity. SPT also represses the expression of *ABI4* and *RGA repressor of GA (RGA)* but promotes *ABI5* and *RGL3*. *Spindly* (*SPY*), a negative regulator of GA signaling, is promoted by ABA. Modulation of ABA and GA responses also includes an epigenetic regulator, kryptonite/SU(VAR)3‐9 homolog 4 (KYP/SUVH4).

*GA3ox1*, *GA20ox1*, *GA20ox2*, *GA20ox3*, *ENT‐kaurenoic acid oxydase 1* (*KAO1*), and *KAO2* genes is upregulated in imbibed seeds. The *abi4* seeds also accumulate more GA [41]. *CHO1* acts downstream of *ABI4* in seed dormancy regulation, and its activity leads to the repression of GA biosynthesis genes (**Figure 2**) [49]. RGL2 seems to be one of the most important GA‐ related component acting in ABA‐GA crosstalk in seeds. The positive interaction between RGL2 and ABA biosynthesis through *XERICO* was described (**Figure 2**) [86]. Moreover, *RGL2* and *ABI5* positively regulate reciprocal expression during seed germination (**Figure 2**) [87]. Recently, the cooperation of NF‐YC transcription factor with RGL2 was identified during the regulation of *ABI5* expression in seeds [88].

**4.1. Activity of ABA and GA metabolism genes ensures the ABA‐GA interaction**

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

There is the relationship between ABA and GA biosynthesis in Arabidopsis. ABA‐deficient mutant, *aba2‐2*, shows the higher expression of *GA3ox1* and *GA3ox2* than the wild type [60, 64]. Interestingly, *AAO3* and *ABA2* expression were detected in a radicle, whereas *GA3ox2* in hypocotyl. It suggests that the places of ABA and GA biosynthesis are different in seeds [60]. *aba2‐2* shows also the reduced expression of *SPY* during seed imbibition in the presence of high temperature. Therefore, the negative regulator of GA signaling, *SPY*, is under the posi‐ tive action of ABA (**Figure 2**) [64]. NCED9 negatively influences GA biosynthesis. The appli‐ cation of paclobutrazol, GA biosynthesis inhibitor, causes better germination of *nced9* than the wild type. It is an evidence that ABA biosynthesis modulates GA pathway in seeds [85].

**4.2. ABA‐GA crosstalk depends on ABI transcription factors and DELLA proteins in seeds**

ABA and GA signaling components are involved in the ABA‐GA crosstalk in Arabidopsis seeds. ABI4 exerts action on GA biosynthesis genes. In *abi4* mutant, the expression of *GA3*,

**Figure 2.** Model for seed dormancy regulation by ABA‐GA crosstalk. ABA‐mediated promotion of seed dormancy and GA‐related release of seed dormancy are possible through ABA‐GA interactions. The seed dormancy regulator, *mother of FT and TFL 1* (*MFT1*), is promoted by ABA insensitive 5 (ABI5) and RGA‐like 2 (RGL2), but ABI3 downregulates its expression. ABI5 and RGL2 positively regulate reciprocal expression. RGL2 also promotes *XERICO* and ABA biosynthesis. Repressor of GA biosynthesis, delay of germination 1 (DOG1), activates *ABI3* and *ABI5*. GA biosynthesis is inhibited by spatula (SPT) and ABI4 via chotto1 (CHO1) activity. SPT also represses the expression of *ABI4* and *RGA repressor of GA (RGA)* but promotes *ABI5* and *RGL3*. *Spindly* (*SPY*), a negative regulator of GA signaling, is promoted by ABA. Modulation of ABA and GA responses also includes an epigenetic regulator, kryptonite/SU(VAR)3‐9 homolog 4

(KYP/SUVH4).

Coat‐mediated dormancy is also related to RGL2 action. RGL2 promotes ABA biosynthe‐ sis in endosperm, then coat‐derived ABA is released to the embryo, where it ensures the expression of *ABI5* and in consequence germination inhibition [40]. ABA and GA signaling genes are under the control of the negative regulator of GA biosynthesis, SPATULA (SPT): *ABI5* and *RGL3* are promoted, whereas *ABI4* and *RGA* are repressed by SPT. It suggests the universal role of SPT in seed dormancy induction and release through complex influ‐ ence on ABA and GA pathways (**Figure 2**) [89]. Induction of secondary dormancy through seed imbibition in darkness at 25°C is associated with changes in GA content and signal‐ ing. However, this process also includes positive action of RGL2 on *ABI5*. It suggests that ABA‐GA crosstalk is also important for entrance into secondary dormancy [90]. Epigenetic modifications are implicated in ABA‐GA interaction. *KYP/SUVH4* is promoted by GA and repressed by ABA. Regarding the role of KYP/SUVH4 in the regulation of *ABI3* and *DOG1* expression, this histone methylotransferase is also a part of ABA‐GA interaction (**Figure 2**) [66].

The interaction between ABI transcription factors and GA catabolism genes was described in monocot plants. In sorghum, SbABI4 and SbABI5 are able to bind with coupling element 1 (CE1) and ABA responsive element (ABRE), respectively, that are present in *SbGA2ox3* pro‐ moter and subsequently promote its expression. ABA‐dependent activation of GA catabolism can promote seed dormancy in grains [53].

## **4.3. Seed dormancy regulators, MFT and DOG1, are a part of the ABA‐GA crosstalk**

Mother of FT and TFL 1 (MFT) is one of the crucial regulators of seed dormancy enabling the interaction between ABA and GA signaling in Arabidopsis. MFT negatively regulates ABA signaling and seed dormancy, which in turn leads to germination. Its expression is repressed by ABI3 but promoted by RGL2. MFT also ensures a negative feedback loop in ABA sig‐ naling through the repression of *ABI5* transcription, whereas ABI5 induces *MFT* expression (**Figure 2**) [91]. However, the role of MFT is not completely clear. The wheat ortholog of MFT, TaMFT, acts in an opposite way in seed dormancy regulation. The increased expression of *TaMFT* is related to the lower germination index, and *TaMFT* overexpression causes inhibition of precocious germination of isolated embryos. Low temperature during seed development is associated with a higher level of dormancy. Under such environmental conditions, the activa‐ tion of *TaMFT* was observed during seed development [92]. Probably, the precise role of MFT in seed dormancy is different in dicots and monocots.

The role of DOG1, the GA‐related regulator of seed dormancy, was also described in ABA signaling in seeds. *ABI5* is positively promoted by DOG1, which in turn leads to the regulation of many *late embryogenesis abundant* (*LEA*) and *heat shock protein* (*HSP*) genes. Moreover, the double‐mutant *abi3‐1/dog1‐1* shows the lower sensitivity to ABA than *abi3‐1*, and in control condition, it produces mature dry green seeds. It suggests the positive relationship between *DOG1* and *ABI3; therefore,* DOG1 may be responsible for ABA‐GA interactions in seeds (**Figure 2**) [54].
