Production of Coumaroylserotonin and Feruloylserotonin in Transgenic Rice Expressing Pepper Hydroxycinnamoyl-Coenzyme A:Serotonin N-(Hydroxycinnamoyl)transferase

Abstract

Transgenic rice (Oryza sativa) plants were engineered to express a N-(hydroxycinnamoyl)transferase from pepper (Capsicum annuum), which has been shown to have hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase activity, a key enzyme in the synthesis of hydroxycinnamic acid amides, under the control of constitutive maize (Zea mays) ubiquitin promoter. The transgenic rice plants require foliar application of amines to support synthesis of hydroxycinnamic acid amides, suggestive of limiting amine substrates in rice shoots. In addition, when T 2 homozygous transgenic rice plants were grown in the presence of amines or phenolic acids, two novel compounds were exclusively identified in the leaves of the transgenic plants. These compounds eluted earlier than p-coumaroyltyramine and feruloyltyramine during HPLC chromatography and were identified as p-coumaroylserotonin and feruloylserotonin by liquid chromatography/mass spectrometry and other methods. To test whether the unpredicted production of serotonin derivatives is associated with the pepper N-(hydroxycinnamoyl)transferase, the substrate specificity of the pepper enzyme was analyzed again. Purified recombinant pepper N-(hydroxycinnamoyl)transferase exhibited a serotonin N-hydroxycinnamoyltransferase (SHT) activity, synthesized p-coumaroylserotonin and feruloylserotonin in vitro, and demonstrated a low K m for serotonin. SHT activity was inhibited by 10 to 50 mM tyramine. In addition, SHT activity was predominantly found in the root tissues of wild-type rice in parallel with the synthesis of serotonin derivatives, suggesting that serotonin derivatives are synthesized in the root of rice. This is the first report of SHT activity and the first demonstration, to our knowledge, that serotonin derivatives can be overproduced in vivo in transgenic rice plants that express serotonin N-(hydroxycinnamoyl)transferase.Hydroxycinnamic acid amides (HCAA) are thought to play a role in plant defense against pathogen attacks (Clarke, 1982;Hahlbrock and Scheel, 1989;Von Roepenack-Lahaye et al., 2003). Consistent with this idea, HCAA are synthesized in the cytosol and transported into the cell wall, where they undergo peroxidative polymerization (Negrel and Martin, 1984;Negrel and Jeandet, 1987;Negrel and Lherminier, 1987;Negrel et al., 1993). The accumulation of HCAA in the cell wall creates a resilient barrier against pathogens by reducing cell wall digestibility and/or by directly inhibiting the growth of fungal hyphae (Grandmaison et al., 1993). Accordingly, the synthesis of HCAA is induced in response to various stresses, including physical injury, pathogen infection, and elicitor treatment. These responses have been observed in Solanaceae plants such as potato (Solanum tuberosum), tobacco (Nicotiana tabacum), tomato (Lycopersicon esculentum), and grass plants such as maize (Zea mays;Clarke, 1982;Negrel and Martin, 1984;Miyagawa et al., 1998;Pearce et al., 1998;Ishihara et al., 2000).Hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110) is...

Functional Analysis of the Amine Substrate Specificity Domain of Pepper Tyramine and Serotonin N-Hydroxycinnamoyltransferases

“…7C). The K m values in the SHT-Y149F for two acyl donors, p-coumaroyl-CoA and feruloyl-CoA, were 25 and 5 mM, respectively, which are comparable to those measured in wild-type SHT (Jang et al, 2004). This indicates that the mutation in Tyr-149 into Phe-149 does not alter acyl donor substrate binding or catalysis.…”

“…The two highly similar THT isogenes identified have been demonstrated to encode two different enzymes, THT and SHT. In addition to its presence in the Solanaceae, THT activity has been observed in other plant families, such as the Papaveraceae (Yu and Facchini, 1999) and Gramineae (Ishihara et al, 2000;Jang et al, 2004), but no gene structure data have been reported as yet.…”

“…The enzyme has a 16-fold lower K m for serotonin (73 mM) than for tyramine (1,165 mM) with feruloyl-CoA as the acyl donor, revealing serotonin N-hydroxycinnamoyltransferase activity (SHT). The subsequent ectopic expression of pepper SHT in rice (Oryza sativa) induced the production of large amounts of HCAAs of serotonin, such as feruloylserotonin and p-coumaroylserotonin, in transgenic rice, verifying in vivo that SHT is a true serotonin N-hydroxycinnamoyltransferase (Jang et al, 2004;Kang et al, 2005). In spite of considerable progress in the study of N-hydroxycinnamoyltransferases, at both the molecular and substrate specificity analysis levels, a more detailed study of the residues and domains required for substrate binding and enzyme activity has not been conducted.…”

“…THT enzymes isolated from potato, tobacco, and tomato show the highest affinity for tyramine and octopamine (b-hydroxytyramine). Recently, a closely related N-hydroxycinnamoyltransferase gene was isolated from pepper (Capsicum annuum; Jang et al, 2004). This N-hydroxycinnamoyltransferase fused with a His tag was expressed in Escherichia coli and purified to homogeneity.…”

Evolutionarily Distinct BAHD N-acyltransferases are Responsible for Natural Variation of Aromatic Amine Conjugates in Rice

“…While BAHD proteins are responsible for the acylation of aliphatic and arylmonoamine acceptors, GNAT enzymes are active on arylamines (Bassard et al, 2010). Although they are from two distinct protein families that show low sequence identity, the BAHD N-acyltransferases, Os-THTs and Os-TBTs, identified in our study demonstrate that proteins from both families can act on aryldiamines (Tables 2 and 3; Jang et al, 2004;Kang et al, 2006). Comparing the specificity of the enzymes between the two families showed that whereas GNAT members are highly specific for tyramine, the BAHD enzymes may be regarded as bifunctional tryptamine/tyramine N-acyltransferases.…”