**4.2. Dormancy induction**

element motif. The RY motif also regulates the expression of genes encoding seed storage proteins (*SSP*) such as 2S albumins and 12S globulins. Four genes, such as *abi3* (ABA insensi‐ tive), *lec1*, *lec2* and *fus3*, are responsible for the conditional dormancy of embryos and are

Higher expressions of cell wall-remodeling enzymes (CWRE) is associated with the radicle protrusion. Endo-β-mannanase, β-1,3-glucanase, expansin, xyloglucan endo-transglycosylase, pectin methylesterase, polygalacturonase, and galactanase are the notable major enzymes involved in the cell-wall modification [12]. In *Arabidopsis*, the application of GA induced the expression of the *extension-like gene 1* (*epr1*). The *epr1* is involved in the strengthening of micropylar endosperm cell wall to elongate the radicle of seeds [69]. Cell-wall-associated gene expression under the imbibition is preferentially linked with ABA and GAs in the endosperm weakening. During the rehydration, increased oil bodies and protein storage vacuole packed in cells start to mobilize from the endosperm cells to radicle tip. Penfeild et al. reported that lipids and proteins produced in the endosperm following the imbibition are higher than those produced in the embryo [70]. Consequently, the proteolytic activities are also at higher rates. The GA suppresses the activation of DELLA proteins to enhance the process of seed germi‐ nation [66, 67, 71]. The E3 ubiquitin ligase SCF (skp1-cullin-F-box-Rbx) complex was reported to be involved in the degradation of DELLA proteins via the ubiquitin-26S proteasome pathway [72]. Perception of GA and its signal transduction determines various other mecha‐

**Figure 2.** Simplified framework of the dormancy release mediated by GA in concordant with the ROS. Interactions be‐

regulated by the ABA for proper maturation [68].

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

nisms in the plant along with the seed germination (**Figure 2**).

tween the genes are described in the text.

Enhanced dormancy occurred when the ABA content was increased in the *Arabidopsis* seeds due to the overexpression of ABA biosynthesis genes [73]. *Abscisic acid biosynthesis gene* (*aba2*), *9-cis-epoxycarotenoid dioxygenase* (*NCED*s), *Glc-insensitive1* (*GIN1*), and *short-chain dehydrogen‐ ase/reductase1* (*SDR1*) are the major genes regulating the synthesis of ABA [74]. Increase in the seed dormancy is also associated with the expression levels of the gene *delay of germination1 (DOG1*). Among the four regulatory genes, *abi3* and *fus3* are able to form the feedback loops. The *Lec1* and *lec2* mutants failed to express the *abi3* and *fus3*, suggesting the strong underlying molecular network between the regulators [75].

The ABA was involved in the vacuolation process to inhibit the endosperm loosening rather than the lipid breakdown [70]. Recently, it was found that the loss of function of the gene coding for E3 ligase ABI3-interacting protein 2 (AIP2) and ABI3-binding factor protein (AFP) leads to the ABA insensitivity and the nondormant phenotype even in the presence of ABA. Major receptors of the ABA are pyrabactin resistance1 (PYR1), PYR1-like protein (PYL), and regulatory components of ABA receptors (RCAR). Those loci encoding the main players in the ABA metabolism are also associated with the RNA translation and metabolism, proteindegradation pathways, and phosphatase components of the signaling pathways [69–75].

## **4.3. DELLA proteins**

The DELLA proteins [GA insensitive (GAI), repressor of ga1-3 (RGA), and RGA-like proteins (RGL 1-3)] belong to the subfamily of plant-specific Glycyl-TRNA synthetase (GRAS) proteins. The name of GRAS proteins was derived from the initially identified members such as *G*AI, *R*G*A*, and SCARECROW (*S*CR) [76]. Potential of GA was repressed by the DELLA-domain containing members such as GAI, RGA, and RGL 1-3. Among them RGL2 has the major influence on seed dormancy. Most of DELLA proteins act as the repressors of the GA biosyn‐ thesis [55]. Seeds of DELLA mutants show the symptoms of irregular shape and proportion, especially in the protruded radicle [77].

Recent research found that the GA signal inactivated the functional domain of DELLA protein. The GA induced repression of the RGA through protein degradation rather than the blocking of translational process [71]. The deletion of the conserved motif VHYNP present in the DELLA region or the region between the VHYNP-DELLA, releases the dormancy by enhancing GA metabolism. Additionally, GA-dependent degradation of proteins is also associated with *SLENDER RICE 1* (*SLR1*), especially S/T/V, a regulatory region. However, in the wild type the function of the *SLR1* was not clearly distinct from the mutant. On the other hand, phosphor‐ ylation of SCF-E3 ligase and the ubiquitin-26S leads to the proteasome pathway of *SLEEPY1* (*SLY1*) and *GA-INSENSITIVE DWARF2* (*GID2*). Bioactive GA mainly focuses on the inactiva‐ tion of RGA, GAI, and RGL 1-3 during the seed germination process [78, 79].

## **4.4.** *DWARF***,** *SLEEPY***,** *PICKLE***,** *SPY***, and** *SECRET AGENT*

The presence of GA at higher levels makes the plant thin and watery. Contrastingly, lack of GA-biosynthetic genes or lesser amount of endogenous GA produces the thicker leaves with 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 *GID2* and *SLY1* [82].

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].

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

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 [57–59, 82] (**Figure 3**).

Signaling Patterns of Reactive Oxygen Species and Phytohormones During Transition Period of Quiescent Seeds into Metabolically Active Organisms http://dx.doi.org/10.5772/64789 87

**Figure 3.** Heuristic networks on the dormancy induction by ABA. Interactions between the genes are described in the text. Arrows represent induction, barred lines represent inhibition, boxes represent sharing similar functions, question marks represent not clear, and crosses represent blocked or reduced synthesis. ABA, abscisic acid; Ca2+, calcium; CAT, catalase; *GAI*, *GA insensitive*; *RGA*, *repressor of ga1-3*; *RGL*, *RGA-like protein*; *GID, gibberellin-insensitive dwarf*; *SPY*, *spind‐ ly*; *OGT*, *O-linked N-acetylglucosamine trasnferase* and CHD3 functions as chromatin remodeling factor.
