Simultaneous Suppression of Two Distinct Serotonin N-Acetyltransferase Isogenes by RNA Interference Leads to Severe Decreases in Melatonin and Accelerated Seed Deterioration in Rice.

Ok Jin Hwang,Kyoungwhan Back

doi:10.3390/biom10010141

Abstract

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in the melatonin biosynthetic pathway, in which serotonin is converted into N-acetylserotonin (NAS) in plants. To date, two SNAT isogenes with low amino acid sequence homologies have been identified. Their single suppression in rice has been reported, but their double suppression in rice has not yet been attempted. Here, we generated double-suppression transgenic rice (snat1+2) using the RNA interference technique. The snat1+2 exhibited retarded seedling growths in conjunction with severe decreases in melatonin compared to wild-types and single-suppression rice plants (snat1 or snat2). The laminar angle was decreased in the snat1+2 rice compared to that of the wild-types and snat1, but was comparable to that of snat2. The reduced germination speed in the snat1+2 was comparable to that of snat2. Seed-aging testing revealed that snat1 was the most severely deteriorated, followed by snat1+2 and snat2, suggesting that melatonin is positively involved in seed longevity.

Highlights

Melatonin is a multifunctional regulator, serving as a signal molecule for various metabolic processes, and is a potent antioxidant in plants [1,2,3,4]
Tryptamine is further hydroxylated into serotonin by the action of tryptamine 5-hydroxylase (T5H), a P450 enzyme localized in the endoplasmic reticulum
The resulting 393-bp chimeric fragment was cloned into the T&A cloning vector (T&A:OsSNAT1+2; RBC Bioscience), and the antisense SNAT1+2 insert was obtained by SacI and SpeI double-digestion and ligated into the pTCK303 binary vector [18], which had been digested by the SacI and SpeI restriction enzymes

Summary

Introduction

Melatonin is a multifunctional regulator, serving as a signal molecule for various metabolic processes, and is a potent antioxidant in plants [1,2,3,4]. Downregulation of SNAT2 in rice (snat2) led to an enhanced tolerance to abiotic stress, a semidwarf phenotype with erect leaves, and a decrease in melatonin content [14,15]. The major difference between snat and snat rice was the level of brassinosteroid (BR), of which snat was more deficient than the wild-type, while snat had a BR content comparable to that of the wild-type Both snat and snat rice had decreased melatonin levels, but they had different phenotypes and abiotic stress responses, depending on their BR levels [15,16]. These contradictory results prompted us to generate a double-suppression rice mutant (snat1+2) to observe the effects of the simultaneous downregulation of both genes

Methods

Results

Conclusion