Insect cuticle is a first physical barrier to protect their body from multifarious environments. Cuticle tanning (sclerotization and pigmentation) is a complex process involves hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA), decarboxylation of DOPA to dopamine, N-acylation of dopamine to N-acetyldopamine (NADA) or N-β-alanyldopamine (NBAD), oxidation of NADA and NBAD to their corresponding quinones, and reactions between the quinones or quinone derivatives with cuticular proteins (CPs) resulting in protein cross-linking. N-acetyltransferase (NAT) catalyzes the conversion of dopamine to NADA whose covalent-linkage of CPs is correlated with colorless cuticle (β-sclerotization). In this study, we analyzed functions of TcNAT1 on cuticle tanning of adult Tribolium castaneum by RNAi. Injection of dsRNA for TcNAT1 (dsTcNAT1) had no affect on animal development and growth. However, some of the resulting adults (~70%) showed split elytra that could not cover their abdomen, resulting in improper folding of their hindwings. Interestingly, body color of the mature adults was darker than that of control dsTcVer-treated adults because probably due to the buildup of abnormally high levels of dopamine, which is used for dopamine eumelanin pigment synthesis (black pigment). On elytra and hindwings of these adults, darker pigments were observed around the sensory bristles located at the intervein regions, suggesting that NADA mediated β-sclerotization is occurred in these regions. Similarly, darker pigment was evident at veins of the hindwings of TcNAT1-deficient adults. These results suggest that TcNAT1 plays important roles in cuticle tanning of T. castaneum adult. To characterize enzymatic properties of TcNAT1, furthermore, recombinant TcNAT1 protein expressed in E. coli was purified by utilizing Ni-NTA affinity column chromatography. This work was supported by NRF (NRF-2012R1A2A1A01006467).

Insect cuticle/exoskeleton is a first physical barrier to protect their body from multifarious environments such as desiccation, natural enemies and entomopathogenic microorganisms. Cuticle tanning (sclerotization and pigmentation) is a vital procedure for generating suitable cuticle depending on body region by sclerotization and pigmentation in insects. Insect cuticle tanning is a complex process involves hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA), decarboxylation of DOPA to dopamine, N-acylation of dopamine to N-acetyldopamine (NADA) or N-β-alanyldopamine (NBAD), oxidation of NADA and NBAD to their corresponding quinones, and reactions between the quinones or quinone derivatives with cuticle protein (CP) side chains resulting in protein cross-linking. One type of pigmentation (quinone tanning) is associated with the covalent linkage of CPs to the ring component of NBAD. In contrast, linkage of CPs to the side chain of NADA (b-sclerotization) is correlated with colorless cuticle. N-acetyltransferase (NAT) catalyzes the conversion of dopamine to N-acetyl dopamine (NADA) in cuticle tanning pathway. In this study, we studied function of TcNAT1 on adult cuticle tanning by double stranded-RNA (dsRNA) mediated gene silencing. Injection of dsTcNAT1 had no affect on animal development, growth and molting such as larva to larva, larva to pupa and pupa to adult. However, some of the resulting adults (~70%) showed split elytra that could not cover their abdomen, resulting in improper folding of their hindwings. Interestingly, body color of the mature adults (older than 3 days) was darker than that of control dsTcVer treated adults because probably due to the buildup of abnormally high levels of dopamine, which is used for dopamine eumelanin pigment synthesis (black pigment) and dopamine quinone-mediated protein crosslinking. On elytra and hindwings of these adults, darker pigments were observed around the sensory bristles that are located in the intervein regions, suggesting that NADA mediated b-sclerotization is occurred at these regions. Similarly, darker pigment was evident at veins of the hindwings of the dsTcNAT1-mature adults. These results suggest that TcNAT1 have important roles in sclerotization and pigmentation of adult body and wings (elytron and hindwing). This work was supported by NRF (NRF-2012R1A2A1A01006467).

Serotonin N-acetyltransferase (SNAT), the penultimate enzyme in melatonin biosynthesis, catalyzes the conversion of serotonin into N-acetylserotonin. Plant SNAT is localized in chloroplasts. To test SNAT localization effects on melatonin synthesis, we generated transgenic rice plants overexpressing a sheep (Ovis aries) SNAT (OaSNAT) in their chloroplasts and compared melatonin biosynthesis with that of transgenic rice plants overexpressing OaSNAT in their cytoplasm. To localize the OaSNAT in chloroplasts, we used a chloroplast targeting sequence (CTS) from tobacco protoporphyrinogen IX oxidase (PPO), which expresses in chloroplasts. The purified recombinant CTS:OaSNAT fusion protein was enzymatically functional and localized in chloroplasts as confirmed by confocal microscopic analysis. The chloroplast-targeted CTS:OaSNAT lines and cytoplasmexpressed OaSNAT lines had similarly high SNAT enzyme activities. However, after cadmium and butafenacil treatments, melatonin production in rice leaves was severalfold lower in the CTS:OaSNAT lines than in the OaSNAT lines. Notably, enhanced SNAT enzyme activity was not directly proportional to the production of N-acetylserotonin, melatonin, or 2-hydroxymelatonin, suggesting that plant SNAT has a role in the homeostatic regulation of melatonin rather than in accelerating melatonin synthesis.

Ectopic overexpression of melatonin biosynthetic genes of animal origin has been used to generate melatonin-rich transgenic plants to examine the functional roles of melatonin in plants. However, the subcellular localization of these proteins expressed in the transgenic plants remains unknown. We studied the localization of sheep (Ovis aries) serotonin N-acetyltransferase (OaSNAT) and a translational fusion of a rice SNAT transit peptide to OaSNAT (TS:OaSNAT) in plants. Laser confocal microscopy analysis revealed that both OaSNAT and TS:OaSNAT proteins were localized to the cytoplasm even with the addition of the transit sequence to OaSNAT. Transgenic rice plants overexpressing the TS:OaSNAT fusion transgene exhibited high SNAT enzyme activity relative to untransformed wild-type plants, but lower activity than transgenic rice plants expressing the wild-type OaSNAT gene. Melatonin levels in both types of transgenic rice plant corresponded well with SNAT enzyme activity levels. The TS:OaSNAT transgenic lines exhibited increased seminal root growth relative to wild-type plants, but less than in the OaSNAT transgenic lines, confirming that melatonin promotes root growth. Seed-specific OaSNAT expression under the control of a rice prolamin promoter did not confer high levels of melatonin production in transgenic rice seeds compared to seeds from transgenic plants expressing OaSNAT under the control of the constitutive maize ubiquitin promoter.