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The timing of flowering is a crucial factor for successful grain production at a wide range of latitudes. Domestication of rice (Oryza sativa) included selection for natural alleles of flowering-time genes that allow rice plants to adapt to broad geographic areas. Here, we describe the role of natural alleles of CIRCADIAN CLOCK ASSOCIATED1 (OsCCA1) in cultivated rice based on analysis of single-nucleotide polymorphisms deposited in the International Rice Genebank Collection Information System database. Rice varieties harboring japonica-type OsCCA1 alleles (OsCCA1a haplotype) flowered earlier than those harboring indica-type OsCCA1 alleles (OsCCA1d haplotype). In the japonica cultivar “Dongjin”, a T-DNA insertion in OsCCA1a resulted in late flowering under long-day and short-day conditions, indicating that OsCCA1 is a floral inducer. Reverse transcription quantitative PCR analysis showed that the loss of OsCCA1a function induces the expression of the floral repressors PSEUDO-RESPONSE REGULATOR 37 (OsPRR37) and Days to Heading 8 (DTH8), followed by repression of the Early heading date 1 (Ehd1)–Heading date 3a (Hd3a)–RICE FLOWERING LOCUS T 1 (RFT1) pathway. Binding affinity assays indicated that OsCCA1 binds to the promoter regions of OsPRR37 and DTH8. Naturally occurring OsCCA1 alleles are evolutionarily conserved in cultivated rice (O. sativa). Oryza rufipogon-I (Or-I) and Or-III type accessions, representing the ancestors of O. sativa indica and japonica, harbored indica- and japonica-type OsCCA1 alleles, respectively. Taken together, our results demonstrate that OsCCA1 is a likely domestication locus that has contributed to the geographic adaptation and expansion of cultivated rice.

Leaf angle shapes plant architecture, allowing for optimal light interception to maximize photosynthesis and yield, and therefore is a crucial agronomic trait. Here, we show that the rice (Oryza sativa L.) R2R3-type MYB transcription factor OsMYB7 determines leaf angle in a developmental stage-specific manner. OsMYB7-overexpressing lines produced wide-angled leaves and osmyb7 knockout mutants exhibited erect leaves. This phenotype was restricted to the lamina joints at the late developmental stage. In agreement with these observations, OsMYB7 was preferentially expressed in the lamina joints of post-mature leaves. Since OsMYB7 homologs are transcriptional repressors of lignin biosynthesis, we examined whether OsMYB7 might inhibit thickening of secondary cell walls. Although OsMYB7 repressed lignin biosynthesis, it enhanced thickening of sclerenchyma cell walls by elevating cellulose contents at the lamina joints. Furthermore, we found that OsMYB7 affects endogenous auxin levels in lamina joints, and the adaxial cells of lamina joints in OsMYB7-overexpressing lines and osmyb7 knockout mutants exhibited enhanced and reduced elongation, respectively, compared to the wild type. These results suggest that OsMYB7 promotes leaf inclination partially through decreasing free auxin levels and promoting cell elongation at the adaxial side of lamina joints.

Chloroplasts are essential organelles in plants that contain chlorophylls and facilitate photosynthesis for growth and development. As photosynthetic efficiency significantly impacts crop productivity, understanding the regulatory mechanisms of chloroplast development has been crucial in increasing grain and biomass production. This study demonstrates the involvement of OsGATA16, an ortholog of Arabidopsis GATA, NITRATE INDUCIBLE, CARBON-METABOLISM INVOLVED ( GNC), and GNC-LIKE/CYTOKININ-RESPONSIVE GATA FACTOR 1 ( GNL/CGA1), in chlorophyll biosynthesis and chloroplast development in rice ( Oryza sativa). The osgata16-1 knockdown mutants produced pale-green leaves, while OsGATA16-overexpressed plants ( OsGATA16-OE1) generated dark-green leaves, compared to their parental japonica rice. Reverse transcription and quantitative PCR analysis revealed downregulation of genes related to chloroplast division, chlorophyll biosynthesis, and photosynthesis in the leaves of osgata16-1 and upregulation in those of OsGATA16-OE1. Additionally, in vivo binding assays showed that OsGATA16 directly binds to the promoter regions of OsHEMA, OsCHLH, OsPORA, OsPORB, and OsFtsZ, and upregulates their expression. These findings indicate that OsGATA16 serves as a positive regulator controlling chlorophyll biosynthesis and chloroplast development in rice.

The cuticular wax layer on leaf surfaces limits non-stomatal water loss to the atmosphere and protects against pathogen invasion. Although many genes associated with wax biosynthesis and wax transport in plants have been identified, their regulatory mechanisms remain largely unknown. Here, we show that the MYB transcription factor OsMYB60 positively regulates cuticular wax biosynthesis and this helps rice (Oryza sativa) plants tolerate drought stress. Compared with the wild type (japonica cultivar 'Dongjin'), osmyb60 null mutants (osmyb60-1 and osmyb60-2) exhibited increased drought sensitivity, with more chlorophyll leaching and higher rates of water loss. Quantitative reverse-transcription PCR showed that the loss of function of OsMYB60 led to downregulation of wax biosynthesis genes, leading to reduced amounts of total wax components on leaf surfaces under normal conditions. Yeast one-hybrid, luciferase transient transcriptional activity, and chromatin immunoprecipitation assays revealed that OsMYB60 directly binds to the promoter of OsCER1 (a key gene involved in very-long-chain alkane biosynthesis) and upregulates its expression. Taken together, these results demonstrate that OsMYB60 enhances rice resilience to drought stress by promoting cuticular wax biosynthesis on leaf surfaces.

The vegetative-to-reproductive transition requires the complex, coordinated activities of many transcriptional regulators. Rice (Oryza sativa), a facultative short-day (SD) plant, flowers early under SD (≤10 h light/day) and late under long-day (LD; ≥14 h light/day) conditions. Here, we demonstrate that rice LATE FLOWERING SEMI-DWARF (LFS) encodes an APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) transcription factor that promotes flowering under non-inductive LD conditions. LFS showed diurnal expression peaking at dawn, and transcript levels increased gradually until heading. Mutation of LFS delayed flowering under LD but not SD conditions. Expression of the LD-specific floral repressor gene LEAFY COTYLEDON2 AND FUSCA3-LIKE 1 (OsLFL1) was upregulated in lfs knockout mutants, and LFS bound directly to the GCC-rich motif in the OsLFL1 promoter, repressing OsLFL1 expression. This suggests that increased LFS activity during vegetative growth gradually attenuates OsLFL1 activity. Subsequent increases in Early heading date 1, Heading date 3a, and RICE FLOWERING LOCUS T 1 expression result in flowering under non-inductive LD conditions. LFS did not affect the expression of other OsLFL1 regulators, including OsMADS50, OsMADS56, VERNALIZATION INSENSITIVE3-LIKE 2, and GERMINATION DEFECTIVE 1, or interact with them. Our results demonstrate the novel roles of LFS in inducing flowering under natural LD conditions.

During crop cultivation, water-deficit conditions retard growth, thus reducing crop productivity. Therefore, uncovering the mechanisms behind drought tolerance is a critical task for crop improvement. Here, we show that the rice (Oryza sativa) WRKY transcription factor OsWRKY5 negatively regulates drought tolerance. We determined that OsWRKY5 was mainly expressed in developing leaves at the seedling and heading stages, and that its expression was reduced by drought stress and by treatment with NaCl, mannitol, and abscisic acid (ABA). Notably, the genome-edited loss-of-function alleles oswrky5-2 and oswrky5-3 conferred enhanced drought tolerance, measured as plant growth under water-deficit conditions. Conversely, the overexpression of OsWRKY5 in the activation-tagged line oswrky5-D resulted in higher susceptibility under the same conditions. The loss of OsWRKY5 activity increased sensitivity to ABA, thus promoting ABA-dependent stomatal closure. Transcriptome deep sequencing and reverse transcription quantitative polymerase chain reaction analyses demonstrated that the expression of abiotic stress-related genes including rice MYB2 (OsMYB2) was upregulated in oswrky5 knockout mutants and downregulated in oswrky5-D mutants. Moreover, dual-luciferase, yeast one-hybrid, and chromatin immunoprecipitation assays showed that OsWRKY5 directly binds to the W-box sequences in the promoter region of OsMYB2 and represses OsMYB2 expression, thus downregulating genes downstream of OsMYB2 in the ABA signaling pathways. Our results demonstrate that OsWRKY5 functions as a negative regulator of ABA-induced drought stress tolerance, strongly suggesting that inactivation of OsWRKY5 or manipulation of key OsWRKY5 targets could be useful to improve drought tolerance in rice cultivars.

Leaf senescence is the final stage of leaf development and in cereal crops, the timing of senescence relative to grain filling has major effects on agronomic traits such as yield. Although many genetic factors are involved in the regulation of leaf senescence in cereals, the key regulators remain to be determined. Plant transcription factors with a conserved DOF (DNA-binding one zinc finger) domain play roles in multiple physiological processes. Here, we show a novel function for OsDOF24 as a repressor of leaf senescence in rice (Oryza sativa). In wild-type leaves, OsDOF24 expression rapidly decreased during natural senescence (NS) and dark-induced senescence (DIS). The gain-of-function mutant osdof24-D, which contains an enhancer-trap T-DNA in the OsDOF24 promoter, exhibited delayed leaf yellowing during NS and DIS. Transgenic plants overexpressing OsDOF24 showed the same phenotype during DIS. Reverse-transcription quantitative real-time PCR analysis revealed that senescence-associated genes (Osl85, Osl57 and OsNAP) and chlorophyll degradation genes (NYC1, NYC3 and SGR) were downregulated in the osdof24-D mutant during dark incubation. Among the phytohormones, only methyl jasmonate induced OsDOF24 expression. Furthermore, the reduced expression of jasmonate biosynthesis-related genes (OsLOX2, OsLOX8, OsHI-LOX, OsAOS1 and OsAOS2) in osdof24-D decreased endogenous jasmonate levels, resulting in delayed leaf senescence under DIS conditions. Yeast one-hybrid assays showed that OsDOF24 binds to the promoter region of OsAOS1. Taken together, our results demonstrate that OsDOF24 suppresses the induction of leaf senescence during vegetative growth by deactivating jasmonate biosynthetic pathways.

In higher plants, abscisic acid (ABA) biosynthesis and ABA signaling are associated with seed germination, leaf senescence, and abiotic stress tolerance. Here, we demonstrate that the MYB-related transcription factor Oryza sativa RADIALIS-LIKE3 (OsRL3) promotes dark-induced leaf senescence and reduces susceptibility to salt stress in rice. OsRL3 expression was upregulated in detached leaves under dark-induced senescence conditions and in seedlings under salt-stress conditions. In the dark, osrl3 null mutants exhibited a stay-green phenotype, with increased chlorophyll retention and photosynthetic capacity. RT-qPCR analysis demonstrated that, compared with wild type (WT), the expression levels of chlorophyll-degradation and senescence-associated genes were lower in osrl3 mutants; this was likely responsible for their stay-green phenotype. Under salt stress, proline levels were lower in osrl3 mutants than WT due to the reduced expression of proline biosynthesis genes, thereby leading to hypersensitivity to salt stress. Among phytohormones, OsRL3 expression was induced by only ABA, and osrl3 mutants were less sensitive to exogenous ABA treatment than WT. RT-qPCR analysis indicated that ABA signaling genes were downregulated in osrl3 mutants under both dark and salt-stress treatment. Taken together, these results indicate that OsRL3 promotes leaf senescence and delays the salt-stress response via ABA signaling pathways.

Soil degradation affects agriculture worldwide. Soils with high salt can result from local geological conditions or accumulation of salt from irrigation. Salt limits water uptake and reduces crop yields; therefore, salt tolerance is an important trait for crops grown in high-salt soils. Here, we show that the rice (Oryza sativa) phytochrome B (osphyB) mutant has greater tolerance to salt stress than its parent japonica rice (cv. Dongjin). We found that the osphyB mutant showed a higher survival rate, fresh weight, and levels of total chlorophylls and carotenoids, as well as enhanced membrane integrity under salt stress compared to the wild type. OsPHYB transcripts increased in tissues of the wild type after salt treatment; OsPHYB expression was much higher in the leaf blade than in the stem and root. The osphyB mutant accumulated less Na+ in the shoot and considerably more K+ in both the shoot and root, maintaining a significantly lower Na+ to K+ ratio, possibly due to a lower rate of Na+ uptake and a higher rate of K+ uptake. To elucidate the possible mechanism of salt tolerance in the osphyB mutant, we performed quantitative reverse transcription PCR analysis, which indicated that salt stress-associated genes, including transcription factors and high-affinity K+ transporters, are upregulated in the osphyB mutant under high-salinity conditions. Taken together, our findings show that the null mutation of OsPHYB contributes to a decrease in the Na+/K+ ratio and enhances cell membrane integrity through upregulation of salt stress-associated genes, resulting in improved tolerance to salt stress.

Exploring genetic methods to improve yield in grain crops such as rice (Oryza sativa) is essential to help meet the needs of the increasing population. Here, we report that rice ONAC096 affects grain yield by regulating leaf senescence and panicle number. ONAC096 expression increased rapidly in rice leaves upon the initiation of aging- and dark-induced senescence. Two independent T-DNA insertion mutants (onac096-1 and onac096-2) with downregulated ONAC096 expression retained their green leaf color during natural senescence in the field, thus extending their photosynthetic capacity. Reverse-transcription quantitative PCR analysis showed that ONAC096 upregulated genes controlling chlorophyll degradation and leaf senescence. Repressed OsCKX2 (encoding cytokinin oxidase/dehydrogenase) expression in the onac096 mutants led to a 15% increase in panicle number without affecting grain weight or fertility. ONAC096 mediates abscisic acid (ABA)-induced leaf senescence by upregulating the ABA signaling genes ABA INSENSITIVE5 and ENHANCED EM LEVEL. The onac096 mutants showed a 16% increase in grain yield, highlighting the potential for using this gene to increase grain production.

Lesion mimic mutants (LMMs) are usually controlled by single recessive mutations that cause the formation of necrotic lesions without pathogen invasion. These genetic defects are useful to reveal the regulatory mechanisms of defense-related programmed cell death in plants. Molecular evidence has been suggested that some of LMMs are closely associated with the regulation of leaf senescence in rice (Oryza sativa). Here, we characterized the mutation underlying spotted leaf4 (spl4), which results in lesion formation and also affects leaf senescence in rice. Map-based cloning revealed that the γ ray-induced spl4-1 mutant has a single base substitution in the splicing site of the SPL4 locus, resulting in a 13-bp deletion within the encoded microtubule-interacting-and-transport (MIT) spastin protein containing an AAA-type ATPase domain. The T-DNA insertion spl4-2 mutant exhibited spontaneous lesions similar to those of the spl4-1 mutant, confirming that SPL4 is responsible for the LMM phenotype. In addition, both spl4 mutants exhibited delayed leaf yellowing during dark-induced or natural senescence. Western blot analysis of spl4 mutant leaves suggested possible roles for SPL4 in the degradation of photosynthetic proteins. Punctate signals of SPL4-fused fluorescent proteins were detected in the cytoplasm, similar to the cellular localization of animal spastin. Based on these findings, we propose that SPL4 is a plant spastin that is involved in multiple aspects of leaf development, including senescence.

The plant-specific WUSCHEL-related homeobox (WOX) nuclear proteins have important roles in the transcriptional regulation of many developmental processes. Among the rice (Oryza sativa) WOX proteins, a loss of OsWOX3A function in narrow leaf2 (nal2) nal3 double mutants (termed nal2/3) causes pleiotropic effects, such as narrow and curly leaves, opened spikelets, narrow grains, more tillers, and fewer lateral roots, but almost normal plant height. To examine OsWOX3A function in more detail, transgenic rice overexpressing OsWOX3A (OsWOX3A-OX) were generated; unexpectedly, all of them consistently exhibited severe dwarfism with very short and wide leaves, a phenotype that resembles that of gibberellic acid (GA)-deficient or GA-insensitive mutants. Exogenous GA3 treatment fully rescued the developmental defects of OsWOX3A-OX plants, suggesting that constitutive overexpression of OsWOX3A downregulates GA biosynthesis. Quantitative analysis of GA intermediates revealed significantly reduced levels of GA20 and bioactive GA1 in OsWOX3A-OX, possibly due to downregulation of the expression of KAO, which encodes ent-kaurenoic acid oxidase, a GA biosynthetic enzyme. Yeast one-hybrid and electrophoretic mobility shift assays revealed that OsWOX3A directly interacts with the KAO promoter. OsWOX3A expression is drastically and temporarily upregulated by GA3 and downregulated by paclobutrazol, a blocker of GA biosynthesis. These data indicate that OsWOX3A is a GA-responsive gene and functions in the negative feedback regulation of the GA biosynthetic pathway for GA homeostasis to maintain the threshold levels of endogenous GA intermediates throughout development.

Light is an important factor influencing melatonin synthesis in response to cadmium treatment in rice. However, the effects of light quality on, and the involvement of phytochrome light receptors in, melatonin production have not been explored. In this study, we used light-emitting diodes (LEDs) to investigate the effect of light wavelength on melatonin synthesis, and the role of phytochromes in light-dependent melatonin induction in rice. Upon cadmium treatment, peak melatonin production was observed under combined red and blue (R + B) light, followed by red (R) and blue light (B). However, both far-red (FR) LED light and dark treatment (D) failed to induce melatonin production. Similarly, rice seedlings grown under the R + B treatment showed the highest melatonin synthesis, followed by those grown under B and R. These findings were consistent with the results of our cadmium treatment experiment. To further confirm the effects of light quality on melatonin synthesis, we employed rice photoreceptor mutants lacking functional phytochrome genes. Melatonin induction was most inhibited in the phytochrome A mutant (phyA) followed by the phyB mutant under R + B treatment, whereas phyB produced the least amount of melatonin under R treatment. These results indicate that PhyB is an R light receptor. Expression analyses of genes involved in melatonin biosynthesis clearly demonstrated that tryptophan decarboxylase (TDC) played a key role in phytochrome-mediated melatonin induction when rice seedlings were challenged with cadmium.

Chlorophyll breakdown is a vital catabolic process of leaf senescence as it allows the recycling of nitrogen and other nutrients. In the present study, we isolated rice senescence-induced receptor-like kinase (OsSRLK), whose transcription was upregulated in senescing rice leaves. The detached leaves of ossrlk mutant (ossrlk) contained more green pigment than those of the wild type (WT) during dark-induced senescence (DIS). HPLC and immunoblot assay revealed that degradation of chlorophyll and photosystem II proteins was repressed in ossrlk during DIS. Furthermore, ultrastructural analysis revealed that ossrlk leaves maintained the chloroplast structure with intact grana stacks during dark incubation, however, the retained green color and preserved chloroplast structures of ossrlk did not enhance the photosynthetic competence during age-dependent senescence in autumn. In ossrlk, the panicles per plant was increased and the spikelets per panicle were reduced, resulting in similar grain productivity between WT and ossrlk. By transcriptome analysis using RNA sequencing, genes related to phytohormone, senescence, and chlorophyll biogenesis were significantly altered in ossrlk compared to those in WT during DIS. Collectively, our findings indicate that OsSRLK may degrade chlorophyll by participating in a phytohormone-mediated pathway.

In rice (Oryza sativa), moderate leaf rolling increases photosynthetic competence and raises grain yield; therefore, this important agronomic trait has attracted much attention from plant biologists and breeders. However, the relevant molecular mechanism remains unclear. Here, we isolated and characterized Rolled Fine Striped (RFS), a key gene affecting rice leaf rolling, chloroplast development, and reactive oxygen species (ROS) scavenging. The rfs-1 gamma-ray allele and the rfs-2 T-DNA insertion allele of RFS failed to complement each other and their mutants had similar phenotypes, producing extremely incurved leaves due to defective development of vascular cells on the adaxial side. Map-based cloning showed that the rfs-1 mutant harbors a 9-bp deletion in a gene encoding a predicted CHD3/Mi-2 chromatin remodeling factor belonging to the SNF2-ATP-dependent chromatin remodeling family. RFS was expressed in various tissues and accumulated mainly in the vascular cells throughout leaf development. Furthermore, RFS deficiency resulted in a cell death phenotype that was caused by ROS accumulation in developing leaves. We found that expression of five ROS-scavenging genes [encoding catalase C, ascorbate peroxidase 8, a putative copper/zinc superoxide dismutase (SOD), a putative SOD, and peroxiredoxin IIE2] decreased in rfs-2 mutants. Western-blot and chromatin immunoprecipitation (ChIP) assays demonstrated that rfs-2 mutants have reduced H3K4me3 levels in ROS-related genes. Loss-of-function in RFS also led to multiple developmental defects, affecting pollen development, grain filling, and root development. Our results suggest that RFS is required for many aspects of plant development and its function is closely associated with epigenetic regulation of genes that modulate ROS homeostasis.

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.

During senescence of detached rice leaves, tryptophan (Trp) and Trp-derived secondary metabolites such as serotonin and 4-coumaroylserotonin accumulated in concert with methanol (MeOH) production. This senescence-induced MeOH induction was closely associated with levels of pectin methylesterase (PME)1 mRNA and PME enzyme activity. Exogenous challenge of detached rice leaves with 1% MeOH accelerated Trp and serotonin biosynthesis with induction of the corresponding genes. No other solvents, including ethanol, resulted in a Trp-inducing effect. This MeOH-induced Trp synthesis was positively regulated by abscisic acid but negatively regulated by cytokinin, suggesting hormonal involvement in the action of MeOH. Endogenous overproduction or suppression of MeOH either by PME1 overexpression or RNA interference (RNAi) gene silencing revealed that PME1 overexpressing lines produced twofold higher Trp levels with elevated Trp biosynthetic gene expression, whereas RNAi lines showed twofold reduction in Trp level in healthy control rice leaves, suggesting that MeOH acts as an endogenous elicitor to enhance Trp biosynthesis. Among many transcription factors induced following MeOH treatment, the WRKY family showed significant induction patterns, of which WRKY14 appeared to play a key regulatory role in MeOH-induced Trp and Trp-derived secondary metabolite biosynthesis.

N-acetylserotonin methyltransferase (ASMT), the last enzyme in the synthesis of melatonin, catalyzes N-acetylserotonin into melatonin. For the first time, we cloned ASMT from rice through the analysis of recombinant Escherichia coli harboring putative rice O-methyltransferase (OMT) cDNAs. In total, 18 full-length cDNAs, which show homology to wheat caffeic acid 3-O-methyltransferase, were expressed in E. coli and induced in the presence of N-acetylserotonin; we then analyzed the production of melatonin. Only recombinant E. coli line 15 showed melatonin synthesis; no other recombinant lines produced melatonin with the addition of N-acetylserotonin in E. coli culture. Line 15 clearly exhibited in vitro ASMT enzyme activity with 0.27 pkat/mg protein. ASMT enzyme activity was inhibited by various related compounds such as N-acetyltryptamine and N-acetyltyrosine. The open reading frame of ASMT consists of 364 amino acids possessing well-conserved motifs found in plant OMT such as S-adenosyl-L-methionine-binding and catalytic sites. Induction patterns of ASMT mRNA were well matched with the production of melatonin in rice leaves during senescence, as well as several stressors.

Transgenic rice plants overexpressing a rice tyrosine decarboxylase (TyDC) exhibited a dwarf phenotype with a high level of tyramine accumulation. The height of transgenic rice was reduced on average to 35% of the wild-type height, whereas the number of tillers increased to 190% that of wild type. When judged by cellular distribution of tyramine and tyramine derivatives, the level of tyramine in soluble and insoluble fractions was higher than that of tyramine derivatives such as 4-coumaroyltyramine (CT) in the transgenic rice plants, suggesting that tyramine rather than its derivatives was a causative compound triggering the dwarf phenotype. Microscopic observation revealed that cell size in the transgenic lines was maintained, with a slightly irregular arrangement in the leaf mesophyll cells. When wild-type rice seeds were grown in the presence of tyramine, rice seedlings also showed stunted phenotypes in a dose-dependent manner. When these stunted seedlings were employed to measure the degree of cellular proliferation by bromodeoxyuridine incorporation, only small numbers of cells were found to retain labeled nuclei in shoot tips compared with the untreated control. These results show that the dwarf phenotype associated with tyramine accumulation in transgenic rice plants is attributable to a reduction in cell number rather than cell size. In addition, our dwarf phenotype caused by tyramine was not closely associated with known dwarf genes such as D88.

Protoporphyrin IX is a photosensitizer and a causative agent of rice membrane lipid peroxidation in plant cells. Protoporphyrinogen IX oxidase (PPO) is the molecular target of PPO-inhibiting herbicides, which trigger a massive increase in protoporphyrin IX. Thus, any possible method to decrease the levels of protoporphyrin IX upon challenge with PPO-inhibiting herbicides could be employed to generate plants resistant to such herbicides. We generated transgenic rice plants overexpressing rice ferrochelatase isogenes encoding ferrochelatase enzymes, which convert protoporphyrin IX into protoheme, to see whether the transgenic plants have phenotypes resistant to PPO-inhibiting herbicides. The resulting transgenic rice plants were all susceptible to oxyfluorfen (a diphenyl-ether-type PPO-inhibiting herbicide), as judged by cellular damage with respect to cellular leakage, chlorophyll loss, and lipid peroxidation. In particular, the transgenic plants expressing rice ferrochelatase II without its plastid targeting sequence showed higher transgene expression and oxyfluorfen susceptibility than lines expressing the intact ferrochelatase II. Possible susceptibility mechanisms to oxyfluorfen herbicide in the transgenic rice plants are discussed.

Caffeoylserotonin (CaS) is a serotonin derivative that belongs to the family of phenylpropanoid amides, but has not previously been identified in plants. In this study, pepper fruits challenged with the anthracnose fungus, Colletotrichum gloeosporioides, were found to synthesize CaS, which accumulated at concentrations of up to 2.3 μg/g fresh weight. The induction of CaS was accompanied by the upregulation of both 4-coumarate-CoA ligase (4CL) and serotonin N-hydroxycinnamoyl transferase (SHT) in unripe pepper fruits. However, no induction of SHT and 4CL was observed in ripe pepper fruits consistent with the lower level of CaS production.

It has been suggested that serotonin biosynthesis is regulated by tryptophan decarboxylase (TDC) in plants. To determine if TDC plays a rate-limiting role in serotonin biosynthesis, two TDC genes, PepTDC1 and PepTDC2, were cloned from pepper (Capsicum annuum L.) fruits infected with anthracnose fungus and their expression was then examined in various organs, including fruit that had been treated with the fungus or various chemicals. PepTDC1 expression was highly induced in pepper fruits after treatment with fungus and ethylene, while PepTDC2 was constitutively expressed at low levels in all pepper tissues. Additionally, predominant induction of PepTDC1 mRNA and TDC enzyme activity was detected in the unripe-green fruit, but not in the ripe-red fruit upon pathogen infection. Higher expression of TDC in unripe-green fruit was closely associated with increased levels of tryptamine, serotonin, and serotonin derivatives. However, unlike the enhanced serotonin synthesis, tryptophan levels responded unchanged when challenged with the pathogen in both the unripe-green fruit and the ripe-red fruit. Expression of two key tryptophan biosynthetic genes, anthranilate synthase (ASalpha) and tryptophan synthase (TSbeta), remained unchanged in response to treatment. Also, anthranilate synthase enzyme activity remained steady regardless of pathogen infection. Taken together, these results suggest that the synthesis of serotonin was regulated by the induction of TDC without a simultaneous increase in tryptophan levels in pepper fruits.

Serotonin, which is well known as a pineal hormone in mammals, plays a key role in conditions such as mood, eating disorders, and alcoholism. In plants, although serotonin has been suggested to be involved in several physiological roles, including flowering, morphogenesis, and adaptation to environmental changes, its regulation and functional roles are as yet not characterized at the molecular level. In this study, we found that serotonin is greatly accumulated in rice (Oryza sativa) leaves undergoing senescence induced by either nutrient deprivation or detachment, and its synthesis is closely coupled with transcriptional and enzymatic induction of the tryptophan biosynthetic genes as well as tryptophan decarboxylase (TDC). Transgenic rice plants that overexpressed TDC accumulated higher levels of serotonin than the wild type and showed delayed senescence of rice leaves. However, transgenic rice plants, in which expression of TDC was suppressed through an RNA interference (RNAi) system, produced less serotonin and senesced faster than the wild type, suggesting that serotonin is involved in attenuating leaf senescence. The senescence-retarding activity of serotonin is associated with its high antioxidant activity compared to either tryptophan or chlorogenic acid. Results of TDC overexpression and TDC RNAi plants suggest that TDC plays a rate-limiting role for serotonin accumulation, but the synthesis of serotonin depends on an absolute amount of tryptophan accumulation by the coordinate induction of the tryptophan biosynthetic genes. In addition, immunolocalization analysis revealed that serotonin was abundant in the vascular parenchyma cells, including companion cells and xylem-parenchyma cells, suggestive of its involvement in maintaining the cellular integrity of these cells for facilitating efficient nutrient recycling from senescing leaves to sink tissues during senescence.

Tomato contains high levels of amines such as serotonin and tyramine and is a suitable host to enhance phenylpropanoid amides (PAs), an important class of nutraceuticals with strong antioxidant activity and chemotherapeutic effects, by ectopic expression of the corresponding gene, serotonin N-hydroxycinnamoyltransferase (SHT). To assess whether ectopic overexpression of SHT cDNA under the control of the CaMV 35S promoter would enhance levels of PAs, we generated transgenic tomato plants and analyzed the levels of PAs. Transgenic tomato plants exhibited increased synthesis of PAs such as feruloylserotonin (FS), 4-coumaroylserotonin (CS), feruloyltyramine (FT), 4-coumaroyltyramine (CT), and feruloyloctopamine (FO) in 1-month-old leaves compared to the wild type. The increase and relative levels of PAs were even more apparent in 3-month-old leaves of transgenic tomato. When tomato leaves were challenged by wounding, levels of PAs in the best transgenic line increased by 3- and 10-fold for CS + FS and CT + FT, respectively. In contrast to leaves, tomato fruit only showed enhanced synthesis of CT + FT, whereas CS + FS levels were not enhanced. Regarding amine content, the levels of tyramine were much higher than those of serotonin in tomato leaves and fruits. The high levels of tyramine may contribute to the preferential production of CT + FT rather than CS + FS, although SHT enzyme shows the highest substrate affinity toward serotonin rather than tyramine.

The plant-specific tyramine derivatives, feruloyltyramine (FT) and 4-coumaroyltyramine (CT), represent bioactive compounds found at low levels in many plant species. We generated transgenic rice seeds that produce high levels of CT (14 microg g(-1) seeds) and FT (2.7 microg g(-1) seeds) through the dual expression of tyramine N-hydroxycinnamoyltransferase and tyrosine decarboxylase, using the self-processing foot-and-mouth disease virus 2A sequence and the endosperm-specific prolamin promoter.

Tyramine, the decarboxylated product of tyrosine by catalysis via the tyrosine decarboxylase enzyme (TYDC), is employed as an acyl acceptor in the synthesis of hydroxycinnamic acid amides of tyramine, such as feruloyltyramine (FT) and 4-coumaroyltyramine (CT). Ectopic over-expression of TYDC cDNA through a maize ubiquitin promoter led to a massive production of tyramine in transgenic rice plants. Most transgenic rice plants exhibited stunted growth without seed production. From more than 120 transformants, we obtained several lines that were able to produce a few viable seeds, although they were dwarf morphs. Transgenic expression of TYDC was confirmed by Northern blot analysis and TYDC enzyme activity measurements. In parallel with high levels of tyramine, a fourfold increase in the levels of octopamine (β-hydroxytyramine) was observed in transgenic rice leaves relative to wild-type leaves. However, high levels of octopamine synthesis were not observed in transgenic rice seeds, even though the accumulation of tyramine in transgenic rice seeds was more than 80 times that in wild-type seeds. The unaltered levels of octopamine in transgenic seeds were attributed to the low levels of tyramine beta-hydroxylase (TβH) enzyme activity, which catalyzes tyramine to octopamine.

Plant-specific phenylpropanoid amides were produced in a recombinant Escherichia coli that expressed 4-coumarate:coenzyme A ligase from Arabidopsis and serotonin N-hydroxycinnamoyltransferase from pepper plants. Upon exogenous treatment with several precursors, high concentrations of the following phenylpropanoid amides were produced abundantly in the culture medium in a few hours: 4-coumaroylserotonin (215 mg/l), 4-coumaroyloctopamine (208 mg/l), and 4-coumaroyltyramine (187 mg/l).

Serotonin is a well-known pineal hormone that in mammals plays a key role in mood. In plants, serotonin is implicated in several physiological roles such as flowering, morphogenesis, and adaptation to environmental changes. However, its biosynthetic enzyme in plants has not been characterized. Therefore, we measured the serotonin content and enzyme activity responsible for serotonin biosynthesis in rice seedlings. Tryptamine 5-hydroxylase (T5H), which converts tryptamine into serotonin, was found as a soluble enzyme that had maximal activity in the roots. The maximal activity of T5H was closely associated with the enriched synthesis of serotonin in roots. Tetrahydropterine-dependent T5H activity was inhibited by tyramine, tryptophan, 5-OH-tryptophan, and octopamine, but remained unaltered by dopamine in vitro. The tissues of rice seedlings grown in the presence of tryptamine exhibited a dose-dependent increase in serotonin in parallel with enhanced T5H enzyme activity. However, no significant increase in serotonin was observed in rice tissues grown in the presence of tryptophan, suggesting that tryptamine is a bottleneck intermediate substrate for serotonin synthesis.

Protoporphyrinogen oxidase (PPO) is the target enzyme of peroxidizing herbicides. The overexpression of Myxococcus xanthus PPO ( Mx PPO) confers a high level of herbicide resistance in rice. Among the peroxidizing herbicides, butafenacil has an efficiency ∼1000-fold that of oxadiazon, as judged by calli susceptibility tests upon herbicide treatment. Butafenacil (0.1 μM) was used to select transgenic rice plants expressing Mx PPO under the control of the constitutive maize ubiquitin promoter. The ectopic expression of the Mx PPO transgene was investigated in the T 0 generation by Northern blot and Western blot analysis. The T 0 transgenic plants expressing the Mx PPO gene were resistant to butafenacil based on in vitro leaf disk and in vivo foliar spray tests.

Tyramine N -hydroxycinnamoyltransferase (THT) is a 28-kDa soluble protein that catalyzes the condensation of hydroxycinnamates and tyramine via the thioesters. The representative products synthesized by THT include feruloyltyramine (FT) and 4-coumaroyltyramine (CT). The THT gene of Capsicum annuum was introduced into the rice genome using Agrobacterium -mediated transformation. Eight independent transgenic rice plants were selected and characterized. The transgenic lines had a single- or two-copy insertion of the THT transgene. Abundant THT mRNA with high THT enzyme activity was detected in transgenic leaves, but not in wild-type leaves. In young leaves, the level of CT + FT was very low, but differed significantly between transgenics and the wild type. However, in old and senesced leaves, a high level of CT + FT was detected in both transgenics and the wild type, at 32 times higher in the best transgenic line than in the wild type. Tyramine applied exogenously to roots was directly coupled with the increased synthesis of CT + FT in young leaves in a dose-dependent manner. Our data suggest that CT and FT synthesis is developmentally regulated and closely associated with the presence of tyramine substrate in rice plants.

Pepper (Capsicum annuum) serotonin N-hydroxycinnamoyltransferase (SHT) catalyzes the synthesis of N-hydroxycinnamic acid amides of serotonin, including feruloylserotonin and p-coumaroylserotonin. To elucidate the domain or the key amino acid that determines the amine substrate specificity, we isolated a tyramine N-hydroxycinnamoyltransferase (THT) gene from pepper. Purified recombinant THT protein catalyzed the synthesis of N-hydroxycinnamic acid amides of tyramine, including feruloyltyramine and p-coumaroyltyramine, but did not accept serotonin as a substrate. Both the SHT and THT mRNAs were found to be expressed constitutively in all pepper organs. Pepper SHT and THT, which have primary sequences that are 78% identical, were used as models to investigate the structural determinants responsible for their distinct substrate specificities and other enzymatic properties. A series of chimeric genes was constructed by reciprocal exchange of DNA segments between the SHT and THT cDNAs. Functional characterization of the recombinant chimeric proteins revealed that the amino acid residues 129 to 165 of SHT and the corresponding residues 125 to 160 in THT are critical structural determinants for amine substrate specificity. Several amino acids are strongly implicated in the determination of amine substrate specificity, in which glycine-158 is involved in catalysis and amine substrate binding and tyrosine-149 plays a pivotal role in controlling amine substrate specificity between serotonin and tyramine in SHT. Furthermore, the indisputable role of tyrosine is corroborated by the THT-F145Y mutant that uses serotonin as the acyl acceptor. The results from the chimeras and the kinetic measurements will direct the creation of additional novel N-hydroxycinnamoyltransferases from the various N-hydroxycinnamoyltransferases found in nature.

Serotonin derivatives such as p-coumaroylserotonin and feruloylserotonin, a family of plant polyphenol compounds, have been implicated in an array of biological activities including antioxidative activity, but neither their production nor identification has been reported in crop plants. Here, we report the detection and enhanced synthesis of serotonin derivatives in rice seeds. We identified that wild-type rice seeds synthesized a basal levels of serotonin derivatives with around 30 ng g−1 seed weight. However, transgenic rice expressing the pepper hydroxycinnamoyl-CoA:serotonin N-(hydroxycinnamoyl)transferase (SHT) produced on average 274 ng g−1 seed weight which was nine-fold higher than wild-type. In addition, production of serotonin derivatives increased upon chemical treatments such as trans-cinnamic acid and tyramine in both wild-type and transgenic rice by two- to three-fold. The DPPH radical scavenging activities of transgenic rice were higher than that of wild-type and showed concentration dependent of serotonin derivatives. These results demonstrate that overexpression of SHT provides the feasibility of engineering increased neutraceutical serotonin derivative levels in plants using this gene.

Acylsugars are polyesters of short- to medium-length acyl chains on sucrose or glucose backbones that are produced in secretory glandular trichomes of many solanaceous plants, including cultivated tomato (Solanum lycopersicum). Despite their roles in biotic stress adaptation and their wide taxonomic distribution, there is relatively little information about the diversity of these compounds and the genes responsible for their biosynthesis. In this study, acylsugar diversity was assessed for 80 accessions of the wild tomato species Solanum habrochaites from throughout the Andes Mountains. Trichome metabolites were analyzed by liquid chromatography-time of flight-mass spectrometry, revealing the presence of at least 34 structurally diverse acylsucroses and two acylglucoses. Distinct phenotypic classes were discovered that varied based on the presence of glucose or sucrose, the numbers and lengths of acyl chains, and the relative total amounts of acylsugars. The presence or absence of an acetyl chain on the acylsucrose hexose ring caused clustering of the accessions into two main groups. Analysis of the Acyltransferase2 gene (the apparent ortholog of Solyc01g105580) revealed differences in enzyme activity and gene expression correlated with polymorphism in S. habrochaites accessions that varied in acylsucrose acetylation. These results are consistent with the hypothesis that glandular trichome acylsugar acetylation is under selective pressure in some populations of S. habrochaites and that the gene mutates to inactivity in the absence of selection.

N-acetylserotonin methyltransferase (ASMT), the last enzyme in the synthesis of melatonin, catalyzes N-acetylserotonin into melatonin. For the first time, we cloned ASMT from rice through the analysis of recombinant Escherichia coli harboring putative rice O-methyltransferase (OMT) cDNAs. In total, 18 full-length cDNAs, which show homology to wheat caffeic acid 3-O-methyltransferase, were expressed in E. coli and induced in the presence of N-acetylserotonin; we then analyzed the production of melatonin. Only recombinant E. coli line 15 showed melatonin synthesis; no other recombinant lines produced melatonin with the addition of N-acetylserotonin in E. coli culture. Line 15 clearly exhibited in vitro ASMT enzyme activity with 0.27 pkat/mg protein. ASMT enzyme activity was inhibited by various related compounds such as N-acetyltryptamine and N-acetyltyrosine. The open reading frame of ASMT consists of 364 amino acids possessing well-conserved motifs found in plant OMT such as S-adenosyl-L-methionine-binding and catalytic sites. Induction patterns of ASMT mRNA were well matched with the production of melatonin in rice leaves during senescence, as well as several stressors.

During senescence of detached rice leaves, tryptophan (Trp) and Trp-derived secondary metabolites such as serotonin and 4-coumaroylserotonin accumulated in concert with methanol (MeOH) production. This senescence-induced MeOH induction was closely associated with levels of pectin methylesterase (PME)1 mRNA and PME enzyme activity. Exogenous challenge of detached rice leaves with 1% MeOH accelerated Trp and serotonin biosynthesis with induction of the corresponding genes. No other solvents, including ethanol, resulted in a Trp-inducing effect. This MeOH-induced Trp synthesis was positively regulated by abscisic acid but negatively regulated by cytokinin, suggesting hormonal involvement in the action of MeOH. Endogenous overproduction or suppression of MeOH either by PME1 overexpression or RNA interference (RNAi) gene silencing revealed that PME1 overexpressing lines produced twofold higher Trp levels with elevated Trp biosynthetic gene expression, whereas RNAi lines showed twofold reduction in Trp level in healthy control rice leaves, suggesting that MeOH acts as an endogenous elicitor to enhance Trp biosynthesis. Among many transcription factors induced following MeOH treatment, the WRKY family showed significant induction patterns, of which WRKY14 appeared to play a key regulatory role in MeOH-induced Trp and Trp-derived secondary metabolite biosynthesis.

We examined to determine whether senescence-induced tryptophan levels are positively associated with levels of glutamine synthetase (GS1), the initial enzyme in tryptophan biosynthesis. We generated transgenic rice plants in which GS1 was suppressed by RNA interference technology. The transgenic line showed a dramatic decrease in GS1 protein and glutamine content, but the levels of tryptophan and mRNA of the key tryptophan biosynthetic genes upon senescence were comparable to those of the wild type.

A plant-specific biogenic amine, serotonin, was produced by heterologous expression of two key biosynthetic genes, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H), in Escherichia coli. The native T5H, a cytochrome P450 enzyme, was unable to be functionally expressed in E. coli. Through a series of N-terminal deletions or additions of tagging proteins, we generated a functional T5H enzyme construct (GST∆37T5H) in which glutathione S transferase (GST) was translationally fused with the N-terminal 37 amino acid deleted T5H. Dual expression of GST∆37T5H and TDC using a pCOLADuet-1 E. coli vector produced serotonin at concentrations of approximately 24 mg l⁻¹ in the culture medium and 4 mg l⁻¹ in the cells. An optimum temperature of approximately 20 °C was required to achieve peak serotonin production in E. coli because the low induction temperature gave rise to the highest soluble expression of GST∆37T5H.

Serotonin N-acetyltransferase (SNA), a rate-limiting enzyme in melatonin biosynthesis in vertebrates, is responsible for the production of N-acetylserotonin; this molecule is then converted to melatonin by hydroxyindole-O-methyltransferase. We generated transgenic rice plants via expression of the human SNA gene under the constitutive ubiquitin promoter using Agrobacterium-mediated gene transformation. We investigated the role of SNA in the biosynthesis of melatonin and the physiological role of melatonin in rice plants. The integration and expression of the transgene were confirmed in T(1) transgenic rice seedlings by Southern, Northern, and RT-PCR analyses. High SNA-specific enzyme activities were observed in the transgenic rice plants, whereas the wild type revealed a trace level of SNA enzyme activity. The functional expression of SNA protein was closely associated with the elevated synthesis of N-acetylserotonin and melatonin in the transgenic rice plants. Experiments using both exogenous treatment of serotonin and senescent detached leaves, which contain a pool of serotonin, significantly enhanced melatonin biosynthesis, indicating that endogenous serotonin levels play a bottleneck role in the pathway of melatonin biosynthesis. Finally, the transgenic rice seedlings with high levels of melatonin showed elevated chlorophyll synthesis during cold stress, suggesting a role for melatonin in cold-stress resistance.

Serotonin N-acetyltransferase (SNA), a rate-limiting enzyme in melatonin biosynthesis in vertebrates, is responsible for the production of N-acetylserotonin; this molecule is then converted to melatonin by hydroxyindole-O-methyltransferase. We generated transgenic rice plants via expression of the human SNA gene under the constitutive ubiquitin promoter using Agrobacterium-mediated gene transformation. We investigated the role of SNA in the biosynthesis of melatonin and the physiological role of melatonin in rice plants. The integration and expression of the transgene were confirmed in T(1) transgenic rice seedlings by Southern, Northern, and RT-PCR analyses. High SNA-specific enzyme activities were observed in the transgenic rice plants, whereas the wild type revealed a trace level of SNA enzyme activity. The functional expression of SNA protein was closely associated with the elevated synthesis of N-acetylserotonin and melatonin in the transgenic rice plants. Experiments using both exogenous treatment of serotonin and senescent detached leaves, which contain a pool of serotonin, significantly enhanced melatonin biosynthesis, indicating that endogenous serotonin levels play a bottleneck role in the pathway of melatonin biosynthesis. Finally, the transgenic rice seedlings with high levels of melatonin showed elevated chlorophyll synthesis during cold stress, suggesting a role for melatonin in cold-stress resistance.

Plant-specific bioactive compounds including feruloyltyramine (FT), 4-coumaroyltyramine (CT), and caffeoyltyramine (CaT) were simultaneously produced in Escherichia coli by heterologous expression of two biosynthetic genes encoding 4-coumarate:coenzyme A ligase and tyramine N-hydroxycinnamoyltransferase (THT) cloned from Arabidopsis thaliana and pepper, respectively. Simultaneous supplementation of substrates to the recombinant E. coli resulted in the secretion of multiple tyramine derivatives into the medium at high yield: CT (189 mg l(-1)), FT (135 mg l(-1)), CaT (40 mg l(-1)). In addition, the recombinant E. coli also produced, albeit at low concentration, a range of dopamine derivatives such as feruloyldopamine due to THT's ability to accept dopamine as a substrate.

Serotonin derivatives belong to a class of phenylpropanoid amides found at low levels in a wide range of plant species. Representative serotonin derivatives include feruloylserotonin (FS) and 4-coumaroylserotonin (CS). Since the first identification of serotonin derivatives in safflower seeds, their occurrence, biological significance, and pharmacological properties have been reported. Recently, serotonin N-hydroxycinnamoyl transferase (SHT), which is responsible for the synthesis of serotonin derivatives, was cloned from pepper (Capsicum annuum) and characterized in terms of its enzyme kinetics. Using the SHT gene, many attempts have been made to either increase the level of serotonin derivatives in transgenic plants or produce serotonin derivatives de novo in microbes by dual expression of key genes such as SHT and 4-coumarate-CoA ligase (4CL). Due to the strong antioxidant activity and other therapeutic properties of serotonin derivatives, these compounds may have high potential in treatment and prophylaxis, as cosmetic ingredients, and as major components of functional foods or feeds that have health-improving effects. This review examines the biosynthesis of serotonin derivatives, corresponding enzymes, heterologous production in plants or microbes, and their applications.

The L-tryptophan decarboxylase (TDC) gene of rice was heterologously expressed in various organisms. Transgenic rice overexpressing TDC showed accumulation of serotonin upon 5-hydroxytryptophan treatment, which was consistent with the in vitro 5-hydroxytryptophan decarboxylase enzyme activity of purified recombinant rice TDC in a pyridoxal phosphate-dependent manner. Recombinant yeast harboring TDC produced serotonin at the expense of the endogenous 5-hydroxytryptophan levels.

Biogenic monoamines such as serotonin, tryptamine, and tyramine function as neurotransmitters and mitogenic factors in animals and are involved in flowering, morphogenesis, and protection from and adaptation to environmental changes in plants. In plants, serotonin and tyramine are conjugated to form phenolic compounds via thioester linkages during the synthesis of hydroxycinnamic acid amides, including p-coumaroylserotonin (CS), feruloylserotonin (FS), p-coumaroyltyramine (CT), and feruloyltyramine (FT). In this study, we determined the amounts of the biogenic monoamines CS, FS, CT, and FT in commonly consumed vegetables using high-performance liquid chromatography. Serotonin, tryptamine, and tyramine were detected in all vegetables tested. The serotonin levels ranged from 1.8 to 294 microg/g of dry weight, the tryptamine levels ranged from 0.8 to 372 microg/g of dry weight, and the tyramine levels ranged from 1.4 to 286 microg/g of dry weight. The highest serotonin and tryptamine contents were found in tomato and cherry tomato (140.3-222 microg/g of dry weight), while paprika and green pepper had higher tyramine contents than the other vegetables (286 and 141.5 microg/g of dry weight, respectively). Overall, the levels of CS, FS, CT, and FT ranged from 0.03 to 13.8 microg/g of dry weight, with green onion possessing the highest levels of CS (0.69 microg/g of dry weight), FT (1.99 microg/g of dry weight), and CT (13.85 microg/g of dry weight).

The plant-specific serotonin derivatives feruloylserotonin (FS) and 4-coumaroylserotonin (CS) are synthesized by the enzymes 4-coumarate:coenzyme A ligase (4CL) and serotonin N-hydroxycinnamoyltransferase (SHT). To express these genes coordinately, SHT was fused in-frame with the self-processing FDMV 2A sequence followed by 4CL in a single open reading frame and introduced into Escherichia coli or Saccharomyces cerevisiae. The transgenes were abundantly expressed in both recombinant microbes, but functional expression was achieved only in yeast, with cleavage at the 2A sequence yielding monomeric SHT-2A and 4CL as judged by immunoblot and product analyses. In the presence of an exogenous supply of precursors such as serotonin and ferulic acid, the recombinant yeast synthesized 4.5 mg l(-1) FS in the medium while 0.02 mg l(-1) FS was produced in the cells. Time-course analysis indicated peak accumulation of FS at 24 h after induction, and this level was maintained until 96 h. The optimum precursor concentration was 2 mM. A series of serotonin derivatives was produced by adding various cinnamate derivative precursors with serotonin; 2.5 mg l(-1) caffeoylserotonin (CaS) and 1.4 mg l(-1) CS were produced, whereas no sinapoylserotonin or cinnamoylserotonin was yielded.

l-Tryptophan decarboxylase (TDC) and l-tyrosine decarboxylase (TYDC) belong to a family of aromatic l-amino acid decarboxylases and catalyze the conversion of tryptophan and tyrosine into tryptamine and tyramine, respectively. The rice genome has been shown to contain seven TDC or TYDC-like genes. Three of these genes for which cDNA clones were available were characterized to assign their functions using heterologous expression in Escherichia coli and rice (Oryza sativa cv. Dongjin). The purified products of two of the genes were expressed in E. coli and exhibited TDC activity, whereas the remaining gene could not be expressed in E. coli. The recombinant TDC protein with the greatest TDC activity showed a K m of 0.69 mM for tryptophan, and its activity was not inhibited by phenylalanine or tyrosine, indicating a high level of substrate specificity toward tryptophan. The ectopic expression of the three cDNA clones in rice led to the abundant production of the products of the encoded enzymes, tyramine and tryptamine. The overproduction of TYDC resulted in stunted growth and a lack of seed production due to tyramine accumulation, which increased as the plant aged. In contrast, transgenic plants that produced TDC showed a normal phenotype and contained 25-fold and 11-fold higher serotonin in the leaves and seeds, respectively, than the wild-type plants. The overproduction of either tyramine or serotonin was not strongly related to the enhanced synthesis of tyramine or serotonin derivatives, such as feruloyltyramine and feruloylserotonin, which are secondary metabolites that act as phytoalexins in plants.