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Rational metabolic-flow switching is an effective strategy that we proposed for producing exogenous high-value natural products using transformed plant cells. In an earlier proof-of-concept study, we generated bamboo ( Phyllostachys nigra ; Pn) cells expressing the 4-hydroxycinnamoyl-CoA hydratase/lyase gene of Pseudomonas putida KT2440 ( PpHCHL ). The encoded enzyme catalyzes the formation of 4-hydroxybenzaldehyde and vanillin from p -coumaroyl-CoA and feruloyl-CoA, respectively. The PpHCHL-transformed Pn cells accumulated mono-glucose conjugates (glucoside and glucose ester) of 4-hydroxybenzoic acid and vanillic acid, indicating that the products (aldehydes) of the PpHCHL-catalyzed reaction were oxidized by endogenous enzyme(s) in Pn cells. In this study, we re-examined the extracts of PpHCHL-transformed Pn cells to screen for additional 4-hydroxybenzoic acid derivatives. An unidentified compound was detected exclusively in the PpHCHL-transformed Pn cells. This compound was purified via column chromatography and then identified as a di-glucose conjugate of 4-hydroxybenzoic acid (i.e., β-D-glucopyranosyl 4- O -β-D-glucopyranosylbenzoate), implying that some of the mono-glucose conjugates of 4-hydroxybenzoic acid were converted to the di-glucose conjugate by endogenous enzyme(s) in Pn cells. The maximum production titer of this di-glucose conjugate in the suspension-cultured cells was 0.38 g l -1 , which was the second highest titer among the four glucose conjugates produced by the PpHCHL-transformed Pn cells. The study findings further support the utility of PpHCHL-transformed Pn cells for the bioproduction of 4-hydroxybenzoic acid and its derivatives., Competing Interests: Conflict of interestThe authors have no conflicts of interest to declare., (© 2024 Japanese Society for Plant Biotechnology.)

Plants produce dimerized phenolic compounds as secondary metabolites. Hordatine A (HA), a dehydrodimer of p-coumaroylagmatine (pCA), is an antifungal compound accumulated at high levels in young barley (Hordeum vulgare) seedlings. The enzyme responsible for the oxidative dimerization of pCA, which is the final step of the hordatine biosynthetic pathway, has not been identified. In this study, we first verified the presence of this enzyme activity in the crude extract of barley seedlings. Because the enzyme activity was not dependent on H 2 O 2 , the responsible enzyme was not peroxidase, which was previously implicated in HA biosynthesis. The analysis of the dissection lines of wheat (Triticum aestivum) carrying aberrant barley 2H chromosomes detected HA in the wheat lines carrying the distal part of the 2H short arm. This chromosomal region contains two laccase genes (HvLAC1 and HvLAC2) that are highly expressed at the seedling stage and may encode enzymes that oxidize pCA during the formation of HA. Changes in the HvLAC transcript levels coincided with the changes in the HA biosynthesis-related enzyme activities in the crude extract and the HA content in barley seedlings. Moreover, HvLAC genes were heterologously expressed in Nicotiana benthamiana leaves and in bamboo (Phyllostachys nigra) suspension cells and HA biosynthetic activities were detected in the crude extract of transformed N. benthamiana leaves and bamboo suspension cells. The HA formed by the enzymatic reaction had the same stereo-configuration as the naturally occurring HA. These results demonstrate that HvLAC enzymes mediate the oxidative coupling of pCA during HA biosynthesis., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

Most commercially circulating mushrooms are produced via cultivation using artificially produced mushroom substrates. However, after mushroom harvesting, the disposal of spent mushroom substrates (SMSs) is a serious problem for the mushroom industry owing to the need for a disposal site and the cost involved. Thus, in view of the possibility of recycling SMSs as a soil modifier, we examined the effect of soil mixed with SMSs on the infection of Arabidopsis leaves by Alternaria brassicicola, the causal agent of cabbage leaf spot. The mixing of SMSs used for Hypsizygus marmoreus, Pholiota microspora, Lyophyllum decastes, and Auricularia polytricha into culture soil suppressed the lesion formation caused by A. brassicicola. The defense responses of Arabidopsis were not induced by the culturing of these seedlings in soils containing SMSs. Suppressed lesion formation was observed after the seedlings were treated with volatiles emitted from SMSs that were incubated with soil for 7 days and used for H. marmoreus, P. microspora, L. decastes, A. polytricha, Lentinula edodes, and Cyclocybe cylindracea. The volatiles from the SMSs reduced the elongation of A. brassicicola hyphae. GC-MS analyses of extracts from the SMS containing soils led to the detection of various volatile compounds; among these, skatole, 2,4-di-tert-butylphenol, γ-dodecalactone, butyric acid, guaiacol, 6-amyl-2-pyrone, and 1-octen-3-ol were examined for inhibitory activity on A. brassicicola and found to suppress hyphae elongation. These findings indicate that the antifungal volatile compounds emitted by the SMSs suppress A. brassicicola infection., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Phytoalexins play a pivotal role in plant-pathogen interactions. Whereas leaves of rice (Oryza sativa) cultivar Nipponbare predominantly accumulated the phytoalexin sakuranetin after jasmonic acid induction, only very low amounts accumulated in the Kasalath cultivar. Sakuranetin is synthesized from naringenin by naringenin 7-O-methyltransferase (NOMT). Analysis of chromosome segment substitution lines and backcrossed inbred lines suggested that NOMT is the underlying cause of differential phytoalexin accumulation between Nipponbare and Kasalath. Indeed, both NOMT expression and NOMT enzymatic activity are lower in Kasalath than in Nipponbare. We identified a proline to threonine substitution in Kasalath relative to Nipponbare NOMT as the main cause of the lower enzymatic activity. Expanding this analysis to rice cultivars with varying amounts of sakuranetin collected from around the world showed that NOMT induction is correlated with sakuranetin accumulation. In bioassays with Pyricularia oryzae, Gibberella fujikuroi, Bipolaris oryzae, Burkholderia glumae, Xanthomonas oryzae, Erwinia chrysanthemi, Pseudomonas syringae, and Acidovorax avenae, naringenin was more effective against bacterial pathogens and sakuranetin was more effective against fungal pathogens. Therefore, the relative amounts of naringenin and sakuranetin may provide protection against specific pathogen profiles in different rice-growing environments. In a dendrogram of NOMT genes, those from low-sakuranetin-accumulating cultivars formed at least two clusters, only one of which involves the proline to threonine mutation, suggesting that the low sakuranetin chemotype was acquired more than once in cultivated rice. Strains of the wild rice species Oryza rufipogon also exhibited differential sakuranetin accumulation, indicating that this metabolic diversity predates rice domestication., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Phytoalexins are inducible antimicrobial metabolites in plants, and have been indicated to be important for the rejection of microbial infection. HPLC analysis detected the induced accumulation of three compounds 1 - 3 in barley ( Hordeum vulgare ) roots infected by Fusarium culmorum , the causal agent of Fusarium root rot. Compounds 1 - 3 were identified as cinnamic acid amides of 9-hydroxy-8-oxotryptamine, 8-oxotryptamine, and (1 H -indol-3-yl)methylamine, respectively, by spectroscopic analysis. Compounds 1 and 2 had been previously reported from wheat, whereas 3 was an undescribed compound. We named 1 - 3 as triticamides A-C, respectively, because they were isolated from barley and wheat, which belong to the Triticeae tribe. These compounds showed antimicrobial activities, indicating that triticamides function as phytoalexins in barley. The administration of deuterium-labeled N -cinnamoyl tryptamine (CinTry) to barley roots resulted in the effective incorporation of CinTry into 1 and 2 , which suggested that they were synthesized through the oxidation of CinTry. Nine putative tryptamine hydroxycinnamoyl transferase (THT)-encoding genes ( HvTHT1 - HvTHT9 ) were identified by database search on the basis of homology to known THT gene sequences from rice. Since HvTHT7 and HvTHT8 had the same sequences except one base, we measured their expression levels in total by RT-qPCR. HvTHT7 / 8 were markedly upregulated in response to infection by F. culmorum . The HvTHT7 and HvTHT8 enzymes preferred cinnamoyl- and feruloyl-CoAs as acyl donors and tryptamine as an acyl acceptor, and (1 H -indol-3-yl)methylamine was also accepted as an acyl acceptor. These findings suggested that HvTHT7/8 are responsible for the induced accumulation of triticamides in barley.

We investigated the effect of treatment with hot water extracts from the spent mushroom substrates (SMSs) of Lentinula edodes and Hypsizygus marmoreus on the resistance of rice leaves to Pyricularia oryzae infection. The spraying of the SMS extracts clearly suppressed the development of lesions caused by Py. oryzae infection. The accumulation of phytoalexins momilactones A and B, oryzalexin A, and sakuranetin was markedly induced by the spraying of extracts. The enhanced expression of defense related genes PR1b and PBZ was also found in leaves sprayed with the extracts. Treatments with the extracts also affected phytohormone levels. The levels of N 6 -(Δ 2 -isopentenyl)adenine and trans -zeatin markedly increased in response to treatment, whereas the levels of salicylic and jasmonic acids were largely unchanged.

The ethyl acetate extract of the culture filtrate of Phlebia tremellosa promoted elongation of the lateral roots of lettuce seedlings at 250 µg/mL. We purified two compounds that promote root elongation by using activity-guided chromatographic fractionation. On the basis of spectroscopic analyses, these compounds were identified to be isolactarane sesquiterpenes derived from the dehydrogenation of merulactone, which was previously isolated from the same species. We named the purified compounds phlelactones A and B. Phlelactones A and B promoted primary root elongation at 100-300 and 10-30 µg/mL and the elongation and formation of lateral roots at 300-1000 and 30-100 µg/mL, respectively.

The inducible metabolites in rice leaves treated with 1 mM jasmonic acid (JA) were analyzed using HPLC. We detected an increase in the levels of two compounds, 1 and 2 . Based on the comparison with mass spectra and chromatographic behavior with authentic compounds, 1 and 2 were identified as 13-oxooctadeca-9,11-dienoic acid (13-KODE) and 9-oxooctadeca-10,12-dienoic acid (9-KODE), respectively, which have not been detected in rice to date. The accumulation of these compounds was also induced by an infection by Bipolaris oryzae . Treatment of rice leaves with KODEs induced the accumulation of defensive secondary metabolites, sakuranetin, naringenin, and serotonin, suggesting that KODEs may play a role in the elicitation of defense responses. The compounds that have an α, β-unsaturated carbonyl group similar to KODEs did not reproduce the response of accumulation of defensive secondary metabolites, suggesting that additional structural factors such as long hydrophobic carbon chain are needed to elicit defense responses.

In view of the possibility that spent mushroom substrates (SMSs) may be used as agricultural materials to prevent crop diseases, we examined the effect of treatment with a hot water extract from the SMS of Lentinula edodes on plant resistance to pathogenic infection. The extract of Le. edodes SMS was sprayed onto the leaves of rice plants, followed by inoculation of the leaves with the conidia of rice blast fungus. The development of lesions was suppressed by treatment with the SMS extract. The extract markedly inhibited the germination of Pyricularia oryzae conidia. We purified compounds 1 , 2 , and 3 , which showed inhibitory effects on conidial germination, from the Le. edodes SMS extract of by successive solvent extraction, column chromatography, and preparative HPLC. Spectroscopic analyses revealed that 1 , 2 , and 3 were phenolic acids with two carboxyl groups in common.