**5. Signal transduction genes**

#### **5.1. 14-3-3 protein**

The members of the 14-3-3 protein family are dimeric proteins that are expressed in a wide range of organisms and tissues. They are involved in modulation of protein interactions through phosphorylation process. The other diverse functional roles include interaction with a large number of protein kinases, DNA, Raf kinase, and regulation of cell cycle progression [46]. In plants, 14-3-3 protein activates and regulates plasma membrane H+ -ATPase through fusicoccin responsive system [47]. Two Na+ /K+ -ATPase α -subunit forms were detected in the gill transcripts of crab *Pachygrapsus marmoratus*, which differed in presence of an 81-nucleotide sequence near the translation start site in the D form when compared to the C form. The extended D form was found to contain the binding motif for the regulatory protein 14-3-3, suggesting that Na+ /K+ -ATPase may be stimulated by this regulatory protein binding [48]. The two isoforms of 14-3-3 proteins identified from the *P. monodon* were shown to have varied gene expression profiles during salinity adaptation in response to hypo-osmotic (3 ppt) or hyper‐ osmotic (40 ppt) salinity stress conditions. Induction of 14-3-3B gene expression in gills of shrimp acclimated to low salinity water suggests that it is likely to be involved in controlling osmoregulation in *P*. *monodon* under hyperosmotic conditions [49]. The up-regulation of the 14-3-3 gene expression in the tissues of shrimp exposed to low (3 ppt) or high (55 ppt) salinity conditions suggests that *P. monodon* 14-3-3 gene may have a potential role in shrimp response to salinity stress [20-21].

#### **5.2. Calreticulin**

Calreticulin, a versatile lectin-like chaperone and important endoplasmic reticulum luminal resident protein, is involved in Ca2+ homeostasis and molecular chaperoning. Calreticulins, which are highly conserved in most of the eukaryotes, are involved in the synthesis of various molecules and in many other biological and physiological processes of an organism. The highest expression of calreticulin was detected in ovary of *F. chinensis.* The gene expression varied at different molting stages and could be induced by heat shock and WSSV challenge, indicating multifunctional role of calreticulin [50]. The *P. monodon* calreticulin showed changes in expression profile in response to high-temperature stress, indicating its potential as a biomarker for stress responses in shrimps [51]. Calreticulin also responds to salinity stress with significant increase in the gene expression levels in gills and muscle tissues of *P. monodon* [20].

#### **5.3. Innexins**

Innexins, which are members of large multigene families, are involved in formation of gap junctions for cell-to-cell communication [52]. In crustaceans, innexin expression has been associated with developing lobster stomatogastric nervous system [53]. In *P. monodon*, the transcripts of innexin-2-like protein showed increased expression in response to yellow head viral disease [54]. Innexin-2 was found to be more abundantly expressed in testes than ovaries of *P. monodon*, indicating functional role of innexin-2 in spermatogenesis but not in oogenesis [55]. The high gene expression observed for innexin 2 with 14.43-fold in muscle tissue of shrimp under low salinity stress of 3 ppt indicates gap junctions regulation during salinity stress in shrimps [20].

#### **6. Energy and metabolism genes**

#### **6.1. Arginine kinase**

Arginine kinase plays a major role in energy metabolism and is a phosphotransferase that catalyzes the reversible transfer of phosphate from phosphoguanidine to ADP, resulting in generation of ATP [56]. In shrimps, Penm2 allergen gene having conserved guanidino specificity region and showing very high sequence similarity with crustacean arginine kinase has been isolated and characterized from *P. monodon* [57]. In crustaceans, such as in blue crab *Callinectes sapidus*, the arginine kinase gene expression is associated with salinity changes. The arginine kinase flux reduced under hyperosmotic treatments and increased with the hypoosmotic treatments [58]. *M. japonicus* when subjected to severe hypoxic stress revealed upregulation of arginine kinase indicating metabolic response of arginine kinase under hypoxic stress [59]. Arginine kinase was found to be differentially expressed and up-regulated in WSSV-infected blue shrimp (*Penaeus stylirostris*) [60] and the gene expression could be induced against LPS immunostimulation in *L. vannamei* indicating its correlation with immune response in shrimps [61]. The differential expression of arginine kinase in gills, gut, and muscle tissues of shrimp exposed to low (3 ppt) salinity conditions indicates that arginine kinase plays an important role in metabolic process associated with salinity stress in crustaceans [20].

#### **6.2. Ferritin**

sequence near the translation start site in the D form when compared to the C form. The extended D form was found to contain the binding motif for the regulatory protein 14-3-3,

two isoforms of 14-3-3 proteins identified from the *P. monodon* were shown to have varied gene expression profiles during salinity adaptation in response to hypo-osmotic (3 ppt) or hyper‐ osmotic (40 ppt) salinity stress conditions. Induction of 14-3-3B gene expression in gills of shrimp acclimated to low salinity water suggests that it is likely to be involved in controlling osmoregulation in *P*. *monodon* under hyperosmotic conditions [49]. The up-regulation of the 14-3-3 gene expression in the tissues of shrimp exposed to low (3 ppt) or high (55 ppt) salinity conditions suggests that *P. monodon* 14-3-3 gene may have a potential role in shrimp response

Calreticulin, a versatile lectin-like chaperone and important endoplasmic reticulum luminal resident protein, is involved in Ca2+ homeostasis and molecular chaperoning. Calreticulins, which are highly conserved in most of the eukaryotes, are involved in the synthesis of various molecules and in many other biological and physiological processes of an organism. The highest expression of calreticulin was detected in ovary of *F. chinensis.* The gene expression varied at different molting stages and could be induced by heat shock and WSSV challenge, indicating multifunctional role of calreticulin [50]. The *P. monodon* calreticulin showed changes in expression profile in response to high-temperature stress, indicating its potential as a biomarker for stress responses in shrimps [51]. Calreticulin also responds to salinity stress with significant increase in the gene expression levels in gills and muscle tissues of *P. monodon* [20].

Innexins, which are members of large multigene families, are involved in formation of gap junctions for cell-to-cell communication [52]. In crustaceans, innexin expression has been associated with developing lobster stomatogastric nervous system [53]. In *P. monodon*, the transcripts of innexin-2-like protein showed increased expression in response to yellow head viral disease [54]. Innexin-2 was found to be more abundantly expressed in testes than ovaries of *P. monodon*, indicating functional role of innexin-2 in spermatogenesis but not in oogenesis [55]. The high gene expression observed for innexin 2 with 14.43-fold in muscle tissue of shrimp under low salinity stress of 3 ppt indicates gap junctions regulation during salinity stress in

Arginine kinase plays a major role in energy metabolism and is a phosphotransferase that catalyzes the reversible transfer of phosphate from phosphoguanidine to ADP, resulting in


suggesting that Na+

to salinity stress [20-21].

**5.2. Calreticulin**

**5.3. Innexins**

shrimps [20].

**6.1. Arginine kinase**

**6. Energy and metabolism genes**

/K+

108 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

Ferritin plays a functional role in iron storage and metabolism. In shrimps, this large multi‐ functional and multisubunit protein gene has been isolated and characterized from *L. vanna‐ mei*, which revealed that ferritin is expressed in most of the tissues of shrimp with major expression in hemocytes [62]. The administration of ferritin resulted in increased immune response in *L. vannamei* with enhanced survival rate in WSSV-challenged shrimps and maintained physiological homeostasis of shrimps [63]. In *M. rosenbergii*, the isolated ferritin gene showed conserved domain for the ferroxidase center and the administration of iron enhanced expression of ferritin gene in a tissue-specific manner [64]. The recombinant ferritin was shown to confer protection in *P. monodon* infected with *Vibrio harveyi* [65]. The expression of ferritin mRNA could be induced with heavy metal ions Cu2+ and Zn2+ and WSSV challenge in *F. chinensis* [66]. The gill tissues of *P. monodon* when subjected to low (3 ppt) salinity stress revealed significant increase in ferritin gene expression (8.79-fold), indicating its functional role in salinity stress in shrimps [20].

#### **6.3. Intracellular fatty-acid-binding proteins**

Intracellular fatty-acid-binding proteins (FABPs) are lipid-binding proteins that help in transport of fatty acids across extra- and intracellular membranes and are involved in various other biological processes such as modulation of signal transduction; gene transcription, especially of lipid metabolism; and cell growth and differentiation [67]. FABPs have been wellcharacterized in vertebrates as compared to that in invertebrates. In crustaceans, the FABP cDNA having fatty-acid-binding motifs has been cloned and characterized from the freshwater crayfish *Pacifastacus leniusculus* and *P. monodon* [68]. The activity of specific Na+ , K+ , Ca2+, and Cl ion channels are regulated by various fatty acids. These ion channel regulations may be carried out directly through fatty acid interactions [69]. The shrimp (*P. monodon*) gut tissue revealed highest gene expression level of FABP (14.05-fold) at high salinity stress conditions (55 ppt) and at low salinity stress conditions (3 ppt) with 13.30-fold; the osmoregulatory process may therefore involve the FABPs in shrimps [20-21].

### **6.4. Acyl-CoA binding protein**

Acyl-CoA binding protein (ACBP) is a highly conserved protein. In yeast it is involved in transportation of acyl-CoA esters from the fatty acid synthetase to acyl-CoA-consuming process [70]. The protein, which was first identified in mammals, acts as a neuropeptide that prevents binding of diazepam/endozepine to GABA receptor system [71-72] and is also involved in regulation of several acyl-CoA-dependent processes [73]. In addition, ACBP is involved in many other functions, which include regulating biosynthesis of fatty acid, functional regulation of enzymes and genes, intracellular acyl-CoA pool regulation, acyl-CoA esters donation required for β-oxidation and vesicular trafficking [74], and in regulation of mcalpain [75]. In plants *Arabidopsis thaliana*, different types of ACBPs are encoded [76], such as the ACBP1 and ACBP2, which are membrane-associated proteins [77-80]; ACBP3, which is the extracellularly targeted protein [81]; and ACBP4, ACBP5, and ACBP6, which are the cytosolic proteins [82-83]. ACBPs are involved in abiotic stress tolerance in plants. The ACBP2 in *Arabidopsis* was shown to be involved in heavy metal (Cd) tolerance [84-85]. ACBP6 and ACBP1 are functionally involved in increased freezing tolerance [78],[86]. In shrimp, ACBP functions in antibacterial [87] and antiviral response [88]. The ACBP gene in *P. monodon* was identified to be differentially expressed in the SSH libraries constructed from the gut tissues of both low (3ppt) and high (55ppt) salinity stressed shrimps. The complete cDNA sequence of ACBP consisted of 273 bp ORF coding for 90 amino and showed ligand-binding conserved domains similar to the other members of ACBP family. At 2 weeks post 3 ppt salinity stress conditions, a significant increase in the ACBP transcripts expression was observed in gills and muscle tissues with highest levels in the gut tissues (28.08-fold). Similar increase in the gene expression levels was observed in shrimps exposed to high salinity stress conditions of 55 ppt in gills and muscle tissues with gut tissues revealing high (11.95-fold) levels of gene expression [89]. These results suggest a functional role of ACBP gene during salinity stress in shrimps.

#### **6.5. Catechol-O-methyltransferase**

O-methyltransferase (OMT) is an enzyme found in a wide range of organisms such as microbes [90], where it is involved in antibiotic biosynthesis [91], and in fungi, where it is involved in biosynthesis of aflatoxins [92]. The OMT found in plants are well characterized for their functional role in O-methylation during biosynthesis of lignin, stress resistance, and disease tolerance [93]. In crustaceans, farnesoic acid O-methyltransferase (FAMeT) catalyzes farnesoic acid methylation resulting in production of isoprenoid methyl farnesoate, which is involved in metabolic and physiological regulation [94]. Catechol-O-methyltransferase (COMT), which is a type of O-methyltransferases, helps in catalyzing the transfer of methyl group to the hydroxyl group of catechol compounds from S-adenosyl-L-methionine. In higher animals, the COMT helps in catalysis of methylation of various macromolecules that are involved in different functional and regulatory purposes and is present in soluble and membrane-bound forms [95]. In shrimps, the construction of suppression subtractive hybridization (SSH) libraries from *P. monodon* gill tissues resulted in identification of COMT gene as one of the differentially expressed genes subjected to salinity stress. The COMT gene was differentially regulated in both the SSH libraries generated from low and high salinity conditions. The ORF of COMT gene of 666 bp size revealed the coding protein with 221 amino acids [96]. The *P. monodon* COMT showed the conserved domains present in superfamily of S-adenosylmethio‐ nine-dependent methyltransferases, which includes COMT, CCoAOMT family of indolethyl‐ amine N-methyltransferase from humans, and OMT from *Bacillus subtilis* [97]. The *P. monodon* COMT was found to be up-regulated in low and high salinity stress conditions at different time intervals in shrimp tissues (gills, guts, and muscles), suggesting a functional role of this gene in salinity stress tolerance in shrimps [96].
