Induction of serotonin accumulation by feeding of rice striped stem borer in rice leaves

Tryptophan (Trp)-related secondary metabolism has been implicated in the defense against pathogen infection and insect feeding in various gramineous species. Recently, we also reported that rice plant accumulated serotonin and tryptamine as well as their amide compounds coupled with phenolic acids in response to the infection by fungal pathogen. These compounds were likely to play an important role in the formation of physical barrier to the invading pathogens. To extend our study to elucidate the defensive role of Trp-derived secondary metabolism in gramineous plants, we examined in this study whether it is activated in response to herbivore attack as well. Third leaves of rice plant were fed on by third instar larvae of rice striped stem borer for 24 h or 48 h. The analysis of four Trp-derived metabolites including tryptamine, serotonin feruloyltryptamine (FerTry) and p-coumaroylserotonin (CouSer) by liquid chromatography coupled with tandem mass spectrometry revealed that their contents clearly increased in response to the larvae feeding. The respective amounts of tryptamine, serotonin, FerTry and CouSer in the larvae-fed leaves were 12-, 3.5-, 33- and 140-fold larger than those in control leaves 48 h after the start of feeding.Key words: rice, Oryza sativa, Gramineae, serotonin, secondary metabolism, rice striped stem borer, Chilo suppressalisPlants defend themselves from environmental stresses by utilizing secondary metabolism. One of major biological stresses that plants have to cope with is attack by herbivorous insects. In the interactions with herbivorous insects, various secondary metabolites that are derived from tryptophan (Trp) pathway have been shown to play defensive roles in plants including gramineous species. For example, benzoxazinone glucosides in wheat (Triticum aestivum), rye (Secale sereale) and maize (Zea mays) express toxic and antifeeding effects on herbivorous insects.^1,2 Benzoxazinones are biosynthesized from indole-3-glycerol phosphate, an intermediate of Trp synthesis.^3,4 Another example of those compounds is gramine in barley (Hordeum vulgare). Gramine is a Trp-rerived indole amine,⁵ and has been received attention in the resistance mainly against aphids on the basis of its toxicity and deterrence.⁶We recently found that Trp-derived secondary metabolism is also involved in defense responses of rice (Oryza sativa) leaves to infection by brown spot fungus (Bipolaris oryzae).⁷ The infection of the fungus activates Trp biosynthesis and accumulation of serotonin and of smaller amounts of tryptamine, feruloyltryptamine (FerTry) and p-coumaroylserotonin (CouSer). In addition, the enhancement of serotonin peroxidase activity and incorporation of serotonin in the cell walls were detected. Thus, it is very likely that that serotonin-derived materials deposit in cell walls after oxidative polymerization to constitute a part of physical defense system of rice, which may be reminiscent of the wound sloughing in animals. These findings prompted us to investigate whether Trp-related secondary metabolism is also involved in the defense of rice plant against the attack by insects, as in the cases of other gramineous plants mentioned above. While the response of plants to pathogenic infection is generally different from that to insect herbivory, Trp-derived secondary metabolites have occasionally been implicated in both responses.^8–10 Here, we report the results of our study to examine the effects of herbivory by rice striped stem borer (Chilo suppressalis) on the Trp derived secondary metabolism in rice leaves.Rice (cv. Nipponbare) leaves were incubated with larvae of C. suppressalis in a feeding tube assembled according to Oikawa et al.,⁸ Aerial parts of two 12-day-old rice seedlings were excised, and their cutting ends were immersed in distilled water in a vial. Three third instar larvae of C. suppressalis were put on the leaves, and the leaves with larvae were covered by a plastic tube. For comparison, the control leaves were wounded by razor blade at the start of the incubation. After incubation for 24 h or 48 h with 16/8 h LD cycle at 28°C, the leaves were extracted with 10 volumes of 80% methanol, and analyzed by liquid chromatography coupled with tandem mass spectrometry in multiple reaction monitoring mode.As shown in Figure 1, the contents of tryptamine and serotonin increased along with time in the larvae-fed leaves. The respective contents of tryptamine and serotonin in the leaves were 12- and 3.5-fold larger than those in control leaves 48 h after the start of feeding. The accumulation of FerTry and CouSer was also observed after larvae feeding with the contents being 33- and 140-fold larger than those in control leaves, respectively. Their contents, however, were approximately 10-fold smaller than the corresponding amines.Open in a separate windowFigure 1Accumulation of Trp derived metabolites in the leaves attacked by rice striped stem borer. Chemical structures of analyzed compounds (A). The contents of tryptamine (B), serotonin (B), FerTry (C) and CouSer (D) were determined by LC-MS/MS analysis. The third leaves of 12-d-old rice seedlings were fed on by rice striped stem borer (brack bars) or wounded by razor blade as control (white bars). After incubation, the leaves were extracted by 80% methanol. The contents of metabolites at time 0 are represented as gray bars.In the interaction of rice plant with B. oryzae, serotonin was shown to be incorporated into cell walls as a part of physical defense system.⁷ In an analogous way, modification of cell walls by serotonin might function in sealing the sites injured by insect feeding to protect the leaves from desiccation, and opportunistic and insect-mediated infection by microorganisms. Indeed, at the cutting edge of the leaves, the formation of brown materials was observed. In addition, since serotonin is a neurotransmitter of insects and tryptamine has been indicated to be a neuroactive substance, their accumulation might directly affect behavior and physiology of some insects. High concentrations of tryptamine have been shown to express anti-oviposition activity toward Bemisia tabaci¹¹ and anti-feeding activities toward Malacosoma disstria and Manduca sexta.¹²The low levels of serotonin, tryptamine and their amides in the control leaves suggest that these compounds are induced in response to some components produced during the interaction between the plant and the herbivore. In this relation, it has been shown that elicitors are present in the saliva of some herbivous insects, which induce volatile emission from the plant to attracts their natural enemies.^13,14 Induction of Trp-derived secondary metabolites by the herbivore attack may likely be a result of recognition of some insect-derived molecules by rice leaves, similarly to the induction of volatile emission.The induced accumulation of indole amines and their hydroxycinnamic acid amides in the rice leaves attacked by C. suppressalis suggests that a common signaling pathway might be involved in the responses to pathogen infection and insect feeding. However, the composition of induced compounds was different between the responses to the two biological stresses. The content of tryptamine in the larvae-fed leaves was comparable to that reported in the B. oryzae-infected leaves, whereas the amount of serotonin (approximately 35 nmol/gFW) was much smaller than that in the infected leaves (approximately 250 nmol/gFW). This characteristic was similar to the response of rice leaves to methyl jasmonate (MeJA), which also induced accumulation of these Trp-derived secondary metabolites.⁷ The strong activation of the conversion of tryptamine to serotonin may require infection-specific signals.The serotonin accumulation in rice appears to be similar to the accumulation of gramine in barley in several aspects. Gramine accumulation has been demonstrated to be induced by either infection by pathogens⁹ or infestation by the aphid Schizaphis graminum.¹⁰ In addition, the gene encoding N-methyltransferase that catalyzes the final reaction in the gramine biosynthetic pathway is upregulated by MeJA, suggesting gramine synthesis is at least partly under the control of jasmonate signaling pathway.^15,16 The inducible serotonin production may be an archetypal form of the biosynthesis of more complicated indole amine in barley.     

WRKY33-mediated indolic glucosinolate metabolic pathway confers resistance against Alternaria brassicicola in Arabidopsis and Brassica crops

The tryptophan (Trp)-derived plant secondary metabolites, including camalexin, 4-hydroxy-indole-3-carbonylnitrile, and indolic glucosinolate (IGS), show broad-spectrum antifungal activity. However, the distinct regulations of these metabolic pathways among different plant species in response to fungus infection are rarely studied. In this study, our results revealed that WRKY33 directly regulates IGS biosynthesis, notably the production of 4-methoxyindole-3-ylmethyl glucosinolate (4MI3G), conferring resistance to Alternaria brassicicola, an important pathogen which causes black spot in Brassica crops. WRKY33 directly activates the expression of CYP81F2, IGMT1, and IGMT2 to drive side-chain modification of indole-3-ylmethyl glucosinolate (I3G) to 4MI3G, in both Arabidopsis and Chinese kale (Brassica oleracea var. alboglabra Bailey). However, Chinese kale showed a more severe symptom than Arabidopsis when infected by Alternaria brassicicola. Comparative analyses of the origin and evolution of Trp metabolism indicate that the loss of camalexin biosynthesis in Brassica crops during evolution might attenuate the resistance of crops to Alternaria brassicicola. As a result, the IGS metabolic pathway mediated by WRKY33 becomes essential for Chinese kale to deter Alternaria brassicicola. Our results highlight the differential regulation of Trp-derived camalexin and IGS biosynthetic pathways in plant immunity between Arabidopsis and Brassica crops.     

Metabolic changes in Arabidopsis thaliana expressing the feedback-resistant anthranilate synthase alpha subunit gene OASA1D

Anthranilate synthase (AS) is a key enzyme in tryptophan (Trp) biosynthesis. Metabolic changes in transgenic Arabidopsis plants expressing the feedback-resistant anthranilate synthase alpha subunit gene OASA1D were investigated with respect to Trp synthesis and effects on secondary metabolism. The Trp content varied depending on the transgenic line, with some lines showing an approximately 200-fold increase. The levels of AS activity in crude extracts from the transgenic lines were comparable to those in the wild type. On the other hand, the enzyme prepared from the lines accumulating high levels of Trp showed a relaxed feedback sensitivity. The AS activity, determined in the presence of 50 microM L-Trp, correlated well with the amount of free Trp in the transgenic lines, indicating the important role of feedback inhibition in control of Trp pool size. In Arabidopsis, Trp is a precursor of multiple secondary metabolites, including indole glucosinolates and camalexin. The amount of indol-3-ylmethyl glucosinolate (I3 M) in rosette leaves of the high-Trp accumulating lines was 1.5- to 2.1-fold greater than that in wild type. The treatment of the leaves with jasmonic acid resulted in a more pronounced accumulation of I3 M in the high-Trp accumulating lines than in wild type. The induction of camalexin formation after the inoculation of Alternaria brassicicola was not affected by the accumulation of a large amount of Trp. The accumulation of constitutive phenylpropanoids and flavonoids was suppressed in high-Trp accumulating lines, while the amounts of Phe and Tyr increased, thereby indicating an interaction between the Trp branch and the Phe and Tyr branch in the shikimate pathway.     

Leaf and root pectin methylesterase activity and 13C/12C stable isotopic ratio measurements of methanol emissions give insight into methanol production in Lycopersicon esculentum

Plant production of methanol (MeOH) is a poorly understood aspect of metabolism, and understanding MeOH production in plants is crucial for modeling MeOH emissions. Here, we have examined the source of MeOH emissions from mature and immature leaves and whether pectin methylesterase (PME) activity is a good predictor of MeOH emission. We also investigated the significance of below-ground MeOH production for mature leaf emissions. We present measurements of MeOH emission, PME activity, and MeOH concentration in mature and immature tissues of tomato (Lycopersicon esculentum). We also present stable carbon isotopic signatures of MeOH emission and the pectin methoxyl pool. Our results suggest that below-ground MeOH production was not the dominant contributor to daytime MeOH emissions from mature and immature leaves. Stable carbon isotopic signatures of mature and immature leaf MeOH were similar, suggesting that they were derived from the same pathway. Foliar PME activity was related to MeOH flux, but unexplained variance suggested PME activity could not predict emissions. The data show that MeOH production and emission are complex and cannot be predicted using PME activity alone. We hypothesize that substrate limitation of MeOH synthesis and MeOH catabolism may be important regulators of MeOH emission.     

Stimulation of ethylene production in detached rice leaves by vanadate

The effect of vanadate on ethylene biosynthesis in detached rice leaves was investigated. Vanadate at pH 5.0–7.0 effectively enhanced ethylene production within 3 h of its application. It promoted the conversion of ACC to ethylene. Treatment with vanadate did not decrease ACC level until late stage of incubation, i.e. at 12 h after incubation. Molybdate, an inhibitor of phosphatase had no or much less stimulatory effect on ethylene production than did vanadate at comparable concentrations. Azide, an inhibitor of F₁-ATPase, inhibited ethylene production in detached rice leaves. FC and vanadate were observed to be synergisticly increased ethylene production in detached rice leaves. In conclusion, plasma membrane H⁺-ATPase does not seem to be involved in ethylene biosynthesis in detached rice leaves.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - FC Fusicoccin     

Mutation of a rice gene encoding a phenylalanine biosynthetic enzyme results in accumulation of phenylalanine and tryptophan

Two distinct biosynthetic pathways for Phe in plants have been proposed: conversion of prephenate to Phe via phenylpyruvate or arogenate. The reactions catalyzed by prephenate dehydratase (PDT) and arogenate dehydratase (ADT) contribute to these respective pathways. The Mtr1 mutant of rice (Oryza sativa) manifests accumulation of Phe, Trp, and several phenylpropanoids, suggesting a link between the synthesis of Phe and Trp. Here, we show that the Mtr1 mutant gene (mtr1-D) encodes a form of rice PDT with a point mutation in the putative allosteric regulatory region of the protein. Transformed callus lines expressing mtr1-D exhibited all the characteristics of Mtr1 callus tissue. Biochemical analysis revealed that rice PDT possesses both PDT and ADT activities, with a preference for arogenate as substrate, suggesting that it functions primarily as an ADT. The wild-type enzyme is feedback regulated by Phe, whereas the mutant enzyme showed a reduced feedback sensitivity, resulting in Phe accumulation. In addition, these observations indicate that rice PDT is critical for regulating the size of the Phe pool in plant cells. Feeding external Phe to wild-type callus tissue and seedlings resulted in Trp accumulation, demonstrating a connection between Phe accumulation and Trp pool size.     

WRKY53 integrates classic brassinosteroid signaling and the mitogen-activated protein kinase pathway to regulate rice architecture and seed size

In rice (Oryza sativa) and other plants, plant architecture and seed size are closely related to yield. Brassinosteroid (BR) signaling and the mitogen-activated protein kinase (MAPK) pathway (MAPK kinase kinase 10 [MAPKKK10]–MAPK kinase 4 [MAPKK4]–MAPK6) are two major regulatory pathways that control rice architecture and seed size. However, their possible relationship and crosstalk remain elusive. Here, we show that WRKY53 mediated the crosstalk between BR signaling and the MAPK pathway. Biochemical and genetic assays demonstrated that glycogen synthase kinase-2 (GSK2) phosphorylates WRKY53 and lowers its stability, indicating that WRKY53 is a substrate of GSK2 in BR signaling. WRKY53 interacted with BRASSINAZOLE-RESISTANT 1(BZR1); they function synergistically to regulate BR-related developmental processes. We also provide genetic evidence showing that WRKY53 functions in a common pathway with the MAPKKK10–MAPKK4–MAPK6 cascade in leaf angle and seed size control, suggesting that WRKY53 is a direct substrate of this pathway. Moreover, GSK2 phosphorylated MAPKK4 to suppress MAPK6 activity, suggesting that GSK2-mediated BR signaling might also regulated MAPK pathway. Together, our results revealed a critical role for WRKY53 and uncovered sophisticated levels of interplay between BR signaling and the MAPK pathway in regulating rice architecture and seed size.

WRKY53 mediates crosstalk between BR and MAPK signaling to regulate rice architecture and seed size.     

Metabolic engineering and profiling of rice with increased lysine

Lysine (Lys) is the first limiting essential amino acid in rice, a stable food for half of the world population. Efforts, including genetic engineering, have not achieved a desirable level of Lys in rice. Here, we genetically engineered rice to increase Lys levels by expressing bacterial lysine feedback‐insensitive aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS) to enhance Lys biosynthesis; through RNA interference of rice lysine ketoglutaric acid reductase/saccharopine dehydropine dehydrogenase (LKR/SDH) to down‐regulate its catabolism; and by combined expression of AK and DHPS and interference of LKR/SDH to achieve both metabolic effects. In these transgenic plants, free Lys levels increased up to ~12‐fold in leaves and ~60‐fold in seeds, substantially greater than the 2.5‐fold increase in transgenic rice seeds reported by the only previous related study. To better understand the metabolic regulation of Lys accumulation in rice, metabolomic methods were employed to analyse the changes in metabolites of the Lys biosynthesis and catabolism pathways in leaves and seeds at different stages. Free Lys accumulation was mainly regulated by its biosynthesis in leaves and to a greater extent by catabolism in seeds. The transgenic plants did not show observable changes in plant growth and seed germination nor large changes in levels of asparagine (Asn) and glutamine (Gln) in leaves, which are the major amino acids transported into seeds. Although Lys was highly accumulated in leaves of certain transgenic lines, a corresponding higher Lys accumulation was not observed in seeds, suggesting that free Lys transport from leaves into seeds did not occur.     

A rice tryptophan deficient dwarf mutant, tdd1, contains a reduced level of indole acetic acid and develops abnormal flowers and organless embryos

Indole-3-acetic acid (IAA) plays a critical role in many aspects of plant growth and development; however, complete pathways of biosynthesis, localization and many aspects of functions of IAA in rice remain unclear. Here, we report the analysis of a rice tryptophan- (Trp-) and IAA-deficient mutant, tryptophan deficient dwarf1 ( tdd1 ) , which is embryonic lethal because of a failure to develop most organs during embryogenesis. Regenerated tdd1 plants showed pleiotropic phenotypes: dwarfing, narrow leaves, short roots and abnormal flowers. TDD1 encodes a protein homologous to anthranilate synthase β-subunit, which catalyses the first step of the Trp biosynthesis pathway and functions upstream of Trp-dependent IAA biosynthesis. TDD1-uidA and DR5-uidA expression overlapped at many sites in WT plants but was lacking in tdd1 , indicating that TDD1 is involved in auxin biosynthesis. Both Trp and IAA levels in flowers and embryos were much lower in tdd1 than in wild type (WT). Trp feeding completely rescued the mutant phenotypes and moderate expression of OsYUCCA1 , which encodes a key enzyme in Trp-dependent IAA biosynthesis, also rescued plant height and root length, indicating that the abnormal phenotypes of tdd1 are caused predominantly by Trp and IAA deficiency. In tdd1 embryos, the expression patterns of OSH1 and OsSCR , which mark the presumptive apical region and the L2 layer, respectively, are identical to those in WT, suggesting a possibility either that different IAA levels are required for basic pattern formation than for organ formation or that an orthologous gene compensates for TDD1 deficiency during pattern formation.     

Toxicity in leaves of rice exposed to cadmium is due to hydrogen peroxide accumulation

The production of H₂O₂ in detached rice leaves of Taichung Native 1 (TN1) caused by CdCl₂ was investigated. CdCl₂ treatment resulted in H₂O₂ production in detached rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase (NOX), prevented CdCl₂-induced H₂O₂ production, suggesting that NOX is a H₂O₂-genearating enzyme in CdCl₂-treated detached rice leaves. Phosphatidylinositol 3-kinase inhibitors wortmanin (WM) or LY294002 (LY) inhibited CdCl₂-inducted H₂O₂ production in detached rice leaves. Exogenous H₂O₂ reversed the inhibitory effect of WM or LY, suggesting that phosphatidylinositol 3-phosphate is required for Cd-induced H₂O₂ production in detached rice leaves. Nitric oxide donor sodium nitroprusside (SNP) was also effective in reducing CdCl₂-inducing accumulation of H₂O₂ in detached rice leaves. Cd toxicity was judged by the decrease in chlorophyll content. The results indicated that DPI, IMD, WM, LY, and SNP were able to reduce Cd-induced toxicity of detached rice leaves. Twelve-day-old TN1 and Tainung 67 (TNG67) rice seedlings were treated with or without CdCl₂. In terms of Cd toxicity (leaf chlorosis), it was observed that rice seedlings of cultivar TN1 are Cd-sensitive and those of cultivar TNG67 are Cd-tolerant. On treatment with CdCl₂, H₂O₂ accumulated in the leaves of TN1 seedlings but not in the leaves of TNG67. Prior exposure of TN1 seedlings to 45^oC for 3 h resulted in a reduction of H₂O₂ accumulation, as well as Cd tolerance of TN1 seedlings treated with CdCl₂. The results strongly suggest that Cd toxicity of detached leaves and leaves attached to rice seedlings are due to H₂O₂ accumulation.