**5. The emerging role of auxin, jasmonates, brassinosteroids, and ethylene in seed dormancy regulation**

ABA and GA are not the only phytohormonal regulators of seed dormancy establishment and release. Their action is modulated by other phytohormones, such as auxin, jasmonates (JA), brassinosteroids (BR), and ethylene.

#### **5.1. Action of auxin pathway components in seeds**

Auxin promotes seed dormancy release and germination. Constitutive induction of auxin biosynthesis in *iaaM‐OX* line inhibits precocious germination in Arabidopsis. Contrarily, the switched off activity of *auxin response factor 10* (*ARF10*) and *ARF16*, auxin‐dependent tran‐ scription factors, in *arf10/arf16* double mutant, causes faster precocious germination than in the wild type. The role of auxin in the control of seed dormancy includes the action of *ABI3*. The double mutants, *abi3‐1/iaaM‐OX* and *abi3‐1/99999mARF16* (line resistant to miR160), show the reduced dormancy phenotype. Therefore, the activation of auxin signaling pro‐ motes *ARF10* and *ARF16*, which in turn induces *ABI3* and seed dormancy (**Table 1**) [93]. Analysis of after‐ripened wheat grains showed increased expression of *TaIAA‐alanine resis‐ tant 3* (*TaIAR3*) encoding hydrolase releasing IAA from conjugates. It was observed in paral‐ lel with the higher IAA level in seeds during imbibition. Probably, seed dormancy release may be associated with the increased auxin content in seeds of monocot plants. Furthermore, *TaAuxin‐resistant 1* (*TaAXR1*), *TaUbiquitin‐related protein 1* (*TaRUB1*), and *TaARF2* were also upregulated in after‐ripened wheat grains. TaAXR1 is associated with AUX/IAA proteasome‐ mediated degradation, whereas TaRUB1 is related to ubiquitin action. The higher expression of *TaAXR1* and *TaRUB1* can exert a negative impact on auxin signaling (**Table 1**) [72].

#### **5.2. Dual role of jasmonic acid in seed dormancy regulation**

The role of JA (Jasmonic Acid) in seed dormancy is ambiguous. The increased JA content was detected in nondormant Arabidopsis seeds. Probably, the decrease of JA content during imbi‐ bition in nondormant seeds is associated with germination promotion [94]. Application of JA precursor, 12‐oxo‐phytodienoic acid (OPDA) promotes the expression of *ABA1*, *ABI5*, and *RGL2* in after‐ripened seeds and inhibits seed germination. OPDA also exerts a regulatory action on the crucial seed dormancy component, *MFT* [95]. The opposite effect of JA on seed Seed Dormancy: The Complex Process Regulated by Abscisic Acid, Gibberellins, and Other Phytohormones... http://dx.doi.org/10.5772/intechopen.68735 89

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

ABA and GA are not the only phytohormonal regulators of seed dormancy establishment and release. Their action is modulated by other phytohormones, such as auxin, jasmonates (JA),

Auxin promotes seed dormancy release and germination. Constitutive induction of auxin biosynthesis in *iaaM‐OX* line inhibits precocious germination in Arabidopsis. Contrarily, the switched off activity of *auxin response factor 10* (*ARF10*) and *ARF16*, auxin‐dependent tran‐ scription factors, in *arf10/arf16* double mutant, causes faster precocious germination than in the wild type. The role of auxin in the control of seed dormancy includes the action of *ABI3*. The double mutants, *abi3‐1/iaaM‐OX* and *abi3‐1/99999mARF16* (line resistant to miR160), show the reduced dormancy phenotype. Therefore, the activation of auxin signaling pro‐ motes *ARF10* and *ARF16*, which in turn induces *ABI3* and seed dormancy (**Table 1**) [93]. Analysis of after‐ripened wheat grains showed increased expression of *TaIAA‐alanine resis‐ tant 3* (*TaIAR3*) encoding hydrolase releasing IAA from conjugates. It was observed in paral‐ lel with the higher IAA level in seeds during imbibition. Probably, seed dormancy release may be associated with the increased auxin content in seeds of monocot plants. Furthermore, *TaAuxin‐resistant 1* (*TaAXR1*), *TaUbiquitin‐related protein 1* (*TaRUB1*), and *TaARF2* were also upregulated in after‐ripened wheat grains. TaAXR1 is associated with AUX/IAA proteasome‐ mediated degradation, whereas TaRUB1 is related to ubiquitin action. The higher expression

of *TaAXR1* and *TaRUB1* can exert a negative impact on auxin signaling (**Table 1**) [72].

The role of JA (Jasmonic Acid) in seed dormancy is ambiguous. The increased JA content was detected in nondormant Arabidopsis seeds. Probably, the decrease of JA content during imbi‐ bition in nondormant seeds is associated with germination promotion [94]. Application of JA precursor, 12‐oxo‐phytodienoic acid (OPDA) promotes the expression of *ABA1*, *ABI5*, and *RGL2* in after‐ripened seeds and inhibits seed germination. OPDA also exerts a regulatory action on the crucial seed dormancy component, *MFT* [95]. The opposite effect of JA on seed

**5.2. Dual role of jasmonic acid in seed dormancy regulation**

**5. The emerging role of auxin, jasmonates, brassinosteroids, and**

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

**ethylene in seed dormancy regulation**

**5.1. Action of auxin pathway components in seeds**

brassinosteroids (BR), and ethylene.

(**Figure 2**) [54].

**Table 1.** Regulators of auxin, jasmonic acid, brassinosteroid, and ethylene pathways in seed dormancy promotion or release.

dormancy exists in wheat. JA was shown to reduce the promoting effect of blue light on seed dormancy in a nitrate‐dependent way [96]. Additionally, after ripening promotes expression of JA biosynthesis genes: *TaAllene oxide synthase* (*TaAOS*), *Ta3‐ketoacyl coenzyme A* (*TaKAT3*) and *TaLipoxygenase 5* (*TaLOX5*) in wheat grains. However, the level of JA decreases during imbibition (**Table 1**) [72]. The cold‐induced release of seed is associated with the increase in JA endogenous content. Cold stratification process promotes the expression of *TaAOS* and *TaAllene oxide cyclase* (*TaAOC*). Furthermore, JA positively regulates *TaNCED1* and *TaNCED2* activity and thus enables seed germination through ABA biosynthesis repression in wheat (**Table 1**) [96, 97].

#### **5.3. Brassinosteroids promote seed germination via repression of ABA signaling**

Brassinosteroids (BR) act opposite to ABA signaling in the regulation of seed dormancy and germination. In Arabidopsis, the crucial regulator of seed dormancy, *MFT*, is under BR regu‐ lation in seeds. Therefore, MFT acts as a mediator of ABA and BR pathways in seeds [98]. Brassinosteroid insensitive 2 (BIN2) is a GSK3‐like kinase playing a negative role in BR sig‐ naling, and furthermore, it ensures the communication with ABA signaling. BIN2 interacts with ABI5 and phosphorylates it, which in turn promotes ABI5 activity during seed germina‐ tion [99]. *TaBIN2* activity is downregulated in the after‐ripened wheat seeds (**Table 1**) [100]. Expression analysis also showed the induction of genes encoding the positive components of BR pathway: *TaDE‐etiolated 2* (*TaDET2*), *TaDWARF 4* (*TaDWF4*), and *TaBR signaling kinase 2* (*TaBSK2*) in wheat after‐ripened grains. *TaDET2* and *TaDWF4* encode crucial enzymes for BR biosynthesis, whereas *TaBSK2* promotes BR signaling (**Table 1**) [100].

#### **5.4. Ethylene represses ABA accumulation and promotes seed dormancy release**

Ethylene (ET) is positively related to seed dormancy release and germination promotion. In Arabidopsis, the expression of ET biosynthesis gene, *1‐aminocyclopropane‐1‐carboxylic acid oxi‐ dase* (*ACO*), is associated with imbibition; however, cold stratification reduces its expression (**Table 1**) [101]. Ethylene receptors, ethylene triple response 1 (ETR1) and ethylene insensitive 2 (EIN2) play a role in seed dormancy regulation. *etr1* and *ein2* mutants show the increased level of r seed dormancy associated with the increased level of seed ABA content [102, 103]. The higher expression of *NCED3* and lower activation of *CYP707A2* were observed in *ein2* and *etr1* mutants, respectively, compared to the wild type. It suggests a negative role of ethylene in the modulation of ABA pathway in seeds (**Table 1**) [104]. In wheat, after‐ripened grains express *TaACO* at a higher level than in dormant grains. Thus, the increased ET content in seeds is associated with dormancy loss also in wheat [100]. The role of ethylene in seed dor‐ mancy regulation includes regulation at epigenetic level. *SIN3‐like 1* (SNL1) and SNL2 reduce acetylation level of histone 3 lysine 9/18 and histone 3 lysine 14. The double mutant *snl1 snl2* shows reduced seed dormancy together with the increased expression of ethylene biosynthe‐ sis genes (*ACO1*, *ACO4*) and ABA catabolism genes (*CYP707A1*, *CYP707A2*). Therefore, SNL1 and SNL2 promote seed dormancy through simultaneous modulation of ethylene and ABA content in seeds [105].

## **6. Conclusions**

Proper regulation of seed dormancy is crucial for appropriate timing of germination. Many environmental factors, including light and temperature, exert action on switch from dormancy to germination stage. Their action is mediated by phytohormones: ABA and GA. ABA is a master player for the entrance to and the establishment of seed dormancy. Many ABA‐related genes are necessary for the quiescent stage of seeds. Contrary to ABA, GA‐mediated pathway promotes germination under favorable conditions. Similar mechanism of seed dormancy regu‐ lation exists in monocot plants. The seed response is dependent on the ABA and GA balance. The ABA‐GA crosstalk ensures the precise seed response according to developmental stage, environmental factors, and seasons. Many components of the ABA and GA pathway, for exam‐ ple ABI3, ABI4, ABI5, RGL2, MFT, and DOG1, are responsible for the proper regulation of seed dormancy. Additionally, auxin, jasmonic acid, brassinosteroids, and ethylene modulate the ABA pathway in seeds. Furthermore, epigenetic control of dormancy‐related components also occurs. Therefore, seed dormancy regulation appears to be a very elaborate process. In monocot plants, a part of the seed dormancy regulatory mechanism acts in a different manner. Action of MFT and JA pathway seems to be reverse in comparison to dicot plants. A better understand‐ ing of precise phytohormonal regulation of seed response of cereals can help in obtaining new varieties with the appropriate seed dormancy level.


2.1.5. Logistic Regression (LR)

Logistic regression is a supervised machine learning algorithm designed to solve classification problems where the target variable is categorical. The primary objective of logistic regression is to establish a mapping function from the dataset's features to the target. This allows the algorithm to predict the probability of a new data point belonging to a particular class [48]. As shown in Figure 1D, the input space is divided into two regions, which are separated by a boundary. Logistic regression is a widely used algorithm in many fields, such as marketing, healthcare, and finance, as it can help identify patterns and relationships between variables that can assist in making accurate predictions and decisions [49].


**1. Introduction**

Proteins under biological conditions exhibit marginal structural stability [1], and they unfold and refold repeatably in vivo [2]. Consequently, many of the biological processes that are facilitated by protein macromolecules are modulated by the properties and energetic character of the denatured state. Indeed, numerous efforts have shown that denatured state effects, such as residual structure [3], excluded volume [4], and intrinsic conformational propensities [5], have key roles in molecular recognition [6], allosteric signaling [7], folding [8,9], and stability [10]. A molecular-level understanding of how proteins are utilized for biological work thus requires characterization of the native, as well as the myriad of non-native, conformational states that exist in solution for a protein, the latter of which is referred to as its denatured state ensemble (DSE).

Despite its importance in understanding protein function, the probability and structural character of the full spectrum of states sampled by proteins are not known. Numerous studies have used short peptides as experimental models from which to probe the characteristics of the DSE [11–13]. The use of short peptides is advantageous because, being too short to fold, they offer access to unfolded states under otherwise folding conditions. Moreover, in the absence of folding, conformational preferences are simplified and locally driven by factors such as hydration [14] and steric hindrance [15]. These studies find that peptides

**Citation:** Paiz, E.A.; Lewis, K.A.; Whitten, S.T. Structural and Energetic Characterization of the Denatured State from the Perspectives of Peptides, the Coil Library, and Intrinsically Disordered Proteins. *Molecules* **2021**, *26*, 634. https:// doi.org/10.3390/molecules26030634

Academic Editor: Marilisa Leone Received: 1 January 2021 Accepted: 23 January 2021 Published: 26 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

have strong preferences for the polyproline II (PPII) backbone conformation, even at nonproline positions [12,16,17], suggesting that PPII structures are dominant components of the DSE. The PPII conformation is characterized by an extended left-handed helical turn with the amide hydrogen and the carboxyl oxygen of each peptide backbone projecting into solution, presumably making favorable contact with the solvent [18–20]. In addition, the PPII conformation appears to facilitate favorable intrachain n→π\* interactions, which should be a stabilizing factor [21]. Short peptides also exhibit conformational preferences for other backbone structures. At cold temperatures, alanine residues have intrinsic αhelix-forming tendencies (i.e., even in the absence of favorable side chain interactions) that are stabilized predominantly by peptide hydrogen bonds [22]. Elevated temperatures have been observed to promote low levels of β-strand [16] or β-turn [23], though the amino acid preferences for forming strand [24] or reverse turn structures [25,26] are thought to be highly context-dependent.

The protein coil library [27] also has been used as a structural model for the DSE [28–30]. These libraries are constructed from the segments of protein structure in the Protein Data Bank (PDB) that are found outside the α-helix and β-strand domains. Some libraries further omit additional conformationally restricted positions, such as those in reverse turns, or preceding prolines, or immediately flanking a region of secondary structure [29]. The underlying assumption when using a coil library as a DSE model is that site-specific effects on the intrinsic conformational preferences of the amino acids are minimized by averaging over many environments, and also by removing the regular and repetitive interactions associated with folded structures. Overall, coil libraries exhibit structural trends that are in good agreement with the results from peptide structural studies [29,31]. For example, chemical shifts and three-bond *J* couplings (3 *JHNα*) measured in peptides by NMR spectroscopy can be reproduced from structural models made from the protein coil library [32–34]. Notably, and similar to the results obtained from peptides, strong preferences for PPII that vary by amino acid type are found in structural surveys of the protein coil library [28–30].

Intrinsically disordered proteins (IDPs) offer another experimental system from which to assess structural preferences in unfolded states under nondenaturing conditions [35]. While chemically denatured proteins are known to adopt macromolecular sizes that depend weakly on sequence details other than chain length [36], IDPs in water exhibit strong sequence-dependent influences on structural size [37]. Computer simulations show that steric effects on disordered structure cannot account for the hydrodynamic size dependence on sequence observed in IDPs [38]. Additionally, temperature changes are found to induce large shifts in the hydrodynamic size for disordered proteins in water [39–41] that can exceed the change in size associated with the heat denaturation of folded proteins of the same chain length [42]. The implication of these findings, albeit expected, is that monomeric disordered protein structure is both under thermodynamic control and highly sensitive to the primary sequence.

In this review, we show that the sequence dependence of IDP hydrodynamic size can be described from the amino acid-specific biases for PPII in the denatured state. Because PPII-rich structures are extended [43], the magnitude of a PPII preference in the denatured state can affect its mean hydrodynamic size [44,45]. Specifically, experiments that evaluate how IDP hydrodynamic size changes with compositional changes in the protein give an independent measure of PPII bias, and further reveal amino acid-specific preferences for PPII that are in good quantitative agreement with PPII bias determined experimentally in peptides [37]. Good agreement is also found when the IDP results are compared to PPII bias in the protein coil library. Moreover, the analysis of heat effects on IDP hydrodynamic size indicates the PPII bias is driven by a significant and favorable enthalpy, and is partially offset by an unfavorable entropy [37], which, again, agrees quantitatively with the peptide results [46]. Across these three models (i.e., peptides, the coil library, and IDPs), the data indicate that the structural and energetic character of the DSE at normal temperatures follows the predictions of a PPII-dominant ensemble. At cold temperatures, both peptides

and IDPs reveal the DSE can shift in population toward the α-helix backbone conformation. To demonstrate these conclusions, the following sections review results obtained from numerous spectroscopic and calorimetric studies on short peptides [11–13,16,17,46], surveys of structures in the protein coil library [28–30], and the more recently acquired sequenceand temperature-based analysis of IDP hydrodynamic sizes [37], showing that these three experimental systems used for characterizing unfolded proteins under folding conditions convey a surprisingly consistent structural and energetic view of the DSE.

α