Cloning and characterization of a <i>Chlamydomonas reinhardtii</i> cDNA arylalkylamine <i>N</i>‐acetyltransferase and its use in the genetic engineering of melatonin content in the Micro‐Tom tomato

et al. 2012

164

234

Because of the absence of an arylalkylamine N-acetyltransferase (AANAT) homolog in the plant genome, the proposal was made that a GCN5-related N-acetyltransferase superfamily gene (GNAT) could be substituted for AANAT. To clone rice serotonin N-acetyltransferase (SNAT), we expressed 31 rice GNAT cDNAs in Escherichia coli and screened SNAT activity by measuring N-acetyltryptamine after application with 1 mm tryptamine. GNAT5 was shown to produce high levels of N-acetyltryptamine in E. coli, suggesting a possible rice SNAT. To confirm SNAT activity, the GNAT5 protein was purified through affinity purification from E. coli culture. The purified recombinant GNAT5 showed high SNAT enzyme activity catalyzing serotonin into N-acetylserotonin. The values for Km and Vmax were 385 μm and 282 pmol/min/mg protein, respectively. An in vitro enzyme assay of purified SNAT showed N-acetylserotonin formation to be proportional to enzyme concentration and time, with peak activity at pH 8.8. High substrate concentrations above 1 mm serotonin inhibited SNAT activity. Finally, the mRNA level of SNAT was higher in shoots than in roots, but it was expressed constitutively, unlike N-acetylserotonin methyltransferase (ASMT), the terminal enzyme in melatonin synthesis. These results suggest that ASMT rather than SNAT is the rate-limiting enzyme of melatonin biosynthesis in plants.

Section: Discussionmentioning

confidence: 99%

Molecular cloning of rice serotonin N‐acetyltransferase, the penultimate gene in plant melatonin biosynthesis

Kang

Lee

et al. 2012

164

234

“…Although the ectopic overexpression of NAT did not lead to the mass production of melatonin in the transgenic lines, our results indicate that ectopic overexpression of the NAT gene from other sources can produce high levels of melatonin stably through several generations. Similarly, an elevated melatonin level was achieved in transgenic tomato overexpressing Chlamydomonas reinhardtii NAT [4], suggesting the possible use of NAT for melatonin engineering in plants.…”

Section: Discussionmentioning

confidence: 99%

Melatonin promotes seminal root elongation and root growth in transgenic rice after germination

Back

2012

182

137

The effect of melatonin on root growth after germination was examined in transgenic rice seedlings expressing sheep serotonin N-acetyltransferase (NAT). Enhanced melatonin levels were found in T(3) homozygous seedlings because of the ectopic overexpression of sheep NAT, which is believed to be the rate-limiting enzyme in melatonin biosynthesis in animals. Compared with wild-type rice seeds, the transgenic rice seeds showed enhanced seminal root growth and an analogous number of adventitious roots 4 and 10 days after seeding on half-strength Murashige and Skoog medium. The enhanced initial seminal root growth in the transgenic seedlings matched their increased root biomass well. We also found that treatment with 0.5 and 1 μM melatonin promoted seminal root growth of the wild type under continuous light. These results indicate that melatonin plays an important role in regulating both seminal root length and root growth after germination in monocotyledonous rice plants. This is the first report on the effects of melatonin on root growth in gain-of-function mutant plants that produce high levels of melatonin.

“…Future studies and genetic analyses, together with the cloning of AANAT and analysis of melatonin levels in rice leaves, either during the natural senescence process or during a natural day/night cycle, will shed light on the regulation of melatonin biosynthesis by light and thus its possible functions [6, 7, 25, 28]. Additionally, the rate‐limiting step for light‐induced melatonin biosynthesis in rice plants remains unresolved.…”

Section: Discussionmentioning

confidence: 99%

Light‐regulated melatonin biosynthesis in rice during the senescence process in detached leaves

Byeon

Youngsoon

et al. 2012

109

103

The effect of light on melatonin biosynthesis was examined in detached rice (Oryza sativa cv. Asahi) leaves during the senescence process. The detached leaves were exposed to senescence treatment either in constant darkness or in constant light, and subjected to HPLC analysis for melatonin and its precursors. Higher melatonin levels were detected in rice leaves under constant light while very low levels were observed in constant darkness. Levels of the melatonin intermediates, tryptamine, serotonin, and N-acetylserotonin significantly decreased in the dark compared to those in the light. Furthermore, relative mRNA levels of melatonin biosynthetic genes and their corresponding proteins decreased accordingly in constant darkness. The most striking difference between constant light and dark was observed in levels of the protein tryptamine 5-hydroxylase. These results suggest that melatonin biosynthesis during senescence is dependent on light signals in rice leaves, contrary to the response found in animals.