**5. Role of Other Phytohormones in Seed Germination/Dormancy**

#### **5.1. Ethylene**

dwarf shoots. It was previously reported that GA-unresponsive dwarf phenotypes were observed in mutants such as *drwaf1* (*d1*) and *gid2* in rice, and *sly1* in *Arabidopsis* [80, 81]. The *GID1* and *SLY1* encoding for the F-box proteins are the subunit of the SCF complex belonging to the E3 ligase [78]. Genes (*SLR/RGA*) involved in the repression of GA were controlled by

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

The *PICKLE* (*PKL*), an important positive regulator involved in the control of radicle protru‐ sion, encodes a CHD3 chromatin remodeling factor. The *PKL* was reported to be actively involved in the later stage of seed germination, particularly on the root differentiation. Additionally, expression of the *PKL* was reported to be higher under an abiotic stress condition [83]. The gene *SPINDLY* (*SPY*) encodes the *O*-linked-*N*-acetyl glucosamine transferees (OGT), which negatively regulates the GA signaling (*GA1-3*) pathway [84]. The *SPY* decreases the GA effect by the suppression of the *SLR1* [85]. It is worthy mentioning that *GA1-3*, a precursor, is involved in the first step of the GA synthesis. Another gene, *SECRET AGENT* (*SEC*) which also encodes the *OGT* gene, found in *Arabidopsis*, did not show obvious phenotype alteration in the *sec* mutant (single mutant), whereas the mutant with both *spy* and *sec* (double mutant) showed the lethal effect in the gamete and also deeply affected the seed development. This double mutant could result from the alteration of not only the GA, but also the cytokine pathway [86]. The increased expression of tetratrico peptide repeats (TPRs) in *Arabidopsis* and *Petunia*, resulted with the repression of the *SPY*. The TRPs could either block directly by forming the

inactive heterodimers or indirectly via proteins interacting with the *SPY* [87].

The enzyme amylase plays an important role in the hydrolysis of endosperm starch into usable sugars. This provides the necessary energy for the emergence of radicle. Plants possess both alpha (α)- and beta (β)-amylases. The expression of α-amylase in the aleurone layer is induced by GA. Activation of the *α-amy1* gene is mediated by GA-responsive elements (GARE) along with the C/TCTTTT and TATCCAT [66, 78]. For the *α-amy2* gene, along with the factors required for *α-amy1*, the BOX1/O2S-like elements are required [88]. The KGM, a Ser-Thr kinase, could repress the *α-amy1* by blocking the expression of the *HyGAMYB* [89]. Translation and stability of the *GAMYB* plays a major role for GA signaling. Meanwhile, interaction of novel zinc finger protein HRT (*H ordeum ordeum* **r**epressor of **t**ranscription) with the *GARE* is able to repress the *α-amy2* gene expression [90]. After the inhibition for 12 h with GA4, the *Arabidopsis ga1-3* mutant showed that 138 genes were upregulated and 120 genes were downregulated. The 20% of the upregulated genes possessed the TAACAAA-like sequences, indicating the importance of GARE in the cleavage of endosperm [91]. The *LEAFY* genes in the shoot apex are linked with the *GAMYB*-like genes. The *GAMYB* gene is also present in the anthers and expressed on the epidermis, endothecium, middle layer, and tapetum in the initial stages of development [92]. The GA activates the Ca2+ signaling for the synthesis of hydrolases. Decrease in the suppression of the *SLENDER1* (*SLN1*) increased the cytosolic Ca2+ level. The ABA inhibits the hydrolase by blocking the *sln1*, which directly affects the α-amy. By increasing the Ca2+, GA activates the hydrolases via calmodulin signaling for successful emergence of the radicle

*GID2* and *SLY1* [82].

**4.5.** *AMYLASE***,** *GAMYB***, and** *LEAFY*

[57–59, 82] (**Figure 3**).

Ethylene was reported to be involved in various metabolisms such as dormancy breakage, root induction, defense against pathogens, and signaling [93]. Precisely, 1-aminocyclopropane-1 carboxylate oxidase (ACC oxidase), a precursor of ethylene synthesis, is required for higher synthesis of H2O2. Mutant lacking the GA synthesis gene (*gal-1*) possessed lower ethylene levels. Meanwhile, the exogenous supply of ethylene induced seed germination. The *ETR1* (ethylene receptor) was reported to be involved in the activation of serine/threonine kinase to overcome the dormancy in the *abi1-1* mutant [94]. In the straightway, mutants of *ein2* (*ethylene insensitive2*) and *etr1* (*ethylene receptor1*) showed higher degree of dormancy than the wild type. Especially, increase of dormancy ratio in the *ein2* mutant is directly proportional to the increased ABA content in the seeds. Meanwhile, decrease in the root ethylene content also decreased the ABA level [95].

#### **5.2. Brassinosteroids**

Brassinosteroids are well known for their functions in the cell elongation, cell cycle, and various other metabolisms. They are involved in the enhanced expression of *GA5*, a GA biosynthesis gene [96]. In the mutant of GA biosynthetic gene, *ga1*, and GA-insensitive mutant, *sly1,* application of brassinosteroids partially improved the seed germination under a lightdeprived condition [97]. Meanwhile, brassinosteroid receptor mutants such as *det2* (*deettiolated 2*) and *bri1* (*brassinosteroid insensitive*) were more sensitive to the ABA. Consequently, synthesis of GAs was also deeply affected [98]. This result signifies the importance of brassinosteriods in the GA synthesis for successful seed germination.

#### **5.3. Auxin**

Auxins are generally known for their roles in the root induction. Ogawa et al. reported upregulation of a number of auxin biosynthetic genes and genes encoding for auxin-carrying proteins in response to exogenous GA4 application [91]. The GA was well known to promote the auxin synthesis and the transportation of ethylene. Chiwocha et al. (2005) evidenced that the interaction of ethylene biosynthetic genes with the auxin signaling genes such as *axr1* and *axr2* was mediated by GA [99]. The BIG-gene, named due to its large size, encodes the calossin/ pushover protein involved in the efflux transportation of auxin [100]. Repressor of RGA proteins by the GA can be delayed by the attenuating auxin transportation or signaling [101]. Contrastingly, recent study in *Arabidopsis* by Lui et al. observed that the mutants of auxin receptors or biosynthesis genes showed the dramatic release of seed dormancy. This auxinmediated seed dormancy was coordinated with ABA signaling [102]. Both GA and ABA have strong influence on auxins during germination and dormancy, respectively. This kind of crosstalk between the hormones helps in the flexibility of the embryo/seeds in response to the environmental stimuli.

### **6. Conclusions**

During the developmental stage of embryos into the vigorous photoautrotropic organisms, numerous metabolic processes are activated and they include oxidation of proteins, cellular structural changes, and synthesis of macromolecules. The cascade of metabolic process ceases with the development of the radicle governed by the well-directed ROS accumulation. Interlinked relation between the GA and ABA aids in the proper development of the embryo, seed filling, desiccation tolerance, imbibition, hydrolysis, temporal and spatial distribution of ROS, proteolysis, and radicle protrusion. The recent evidences suggest that ABA-GA crosstalk with other phytohormones, such as ethylene, brassinosteroids and auxin, could play a vital role in the development of the seed. The important components other than the free radicals such as O2 - , H2O2, and •OH pertaining to the seed potential is the NO. Tapping of the NO linked with the GA-ABA and their responses to the light and temperature could be one of the interesting areas getting more attention on the seed research.

#### **Acknowledgements**

Prabhakaran Soundararajan and Abinaya Manivannan were supported by a scholarship from the BK21 Plus Program, the Ministry of Education, Republic of Korea.
