Phytoalexins are inducible secondary metabolites possessing antimicrobial activity against phytopathogens. Rice produces a wide array of phytoalexins in response to pathogen attacks and environmental stresses. With few exceptions, most phytoalexins identified in rice are diterpenoid compounds. Until very recently, flavonoid sakuranetin was the only known phenolic phytoalexin in rice. However, recent studies have shown that phenylamides are involved in defense against pathogen attacks in rice. Phenylamides are amine-conjugated phenolic acids that are induced by pathogen infections and abiotic stresses including ultra violet (UV) radiation in rice. Stress-induced phenylamides, such as N-trans-cinnamoyltryptamine, N-p-coumaroylserotonin and N-cinnamoyltyramine, have been reported to possess antimicrobial activities against rice bacterial and fungal pathogens, an indication of their direct inhibitory roles against invading pathogens. This finding suggests that phenylamides act as phytoalexins in rice and belong to phenolic phytoalexins along with sakuranetin. Phenylamides also have been implicated in cell wall reinforcement for disease resistance and allelopathy of rice. Synthesis of phenolic phytoalexins is stimulated by phytopathogen attacks and abiotic challenges including UV radiation. Accumulating evidence has demonstrated that biosynthetic pathways including the shikimate, phenylpropanoid and arylmonoamine pathways are coordinately activated for phenolic phytoalexin synthesis, and related genes are induced by biotic and abiotic stresses in rice.

Cinnamyl alcohol dehydrogenase (CAD) is involved in the final step of the phenylpropanod pathway, catalyzing the NADPH-dependent reduction of hydroxy-cinnamaldehydes into the corresponding alcohols. The rice genome contains twelve CAD and CAD-like genes, collectively called OsCADs. To elucidate the biochemical function of the OsCADs, OsCAD1, 2, 6, and 7, which are highly expressed in rice, were cloned from rice tissues. The cloned OsCADs were heterologously expressed in Escherichia coli as His-tag fusion proteins. The activity assay of the recombinant OsCADs showed that OsCAD2, 6, and 7 have CAD activity toward hydroxycinnamaldehydes, but OsCAD1 has no detectable catalytic activity. The kinetic parameters of the enzyme reactions demonstrated that OsCAD2 has the highest catalytic activity among the examined enzymes. This result agrees well with the finding that the Zn binding and NADPH binding motifs and the residues constituting the substrate binding pocket in bona fide plant CADs were fully conserved in OsCAD2. Although they have large variations in the residue for the substrate binding pocket, OsCAD6 and 7 catalyzed the reduction of hydroxycinnamaldehydes with a similar efficiency. Alignment of amino acid sequences showed that OsCAD1 lacks the GxxxxP motif for NADPH binding and has mismatches in residues important in the reduction process, which could be responsible for the loss of catalytic activity. OsCAD2 belongs to CAD Class I with bona fide CADs from other plant species and is constitutively expressed throughout the developmental stages of rice, with preferential expression in actively lignifying tissues such as the root, stem, and panicle, suggesting that it is mainly involved in developmental lignification in rice. The expression of OsCAD2 was also induced by biotic and abiotic stresses such as Xanthomonas oryzae pv. oryzae (Xoo) infection and UV-irradiation, suggesting that it plays a role in the defense response of rice, in addition to a bona fide role in developmental lignification. OsCAD6 and 7 belong in CAD Class II. Their expression is relatively lower than that of OsCAD2 and is confined to certain tissues, such as the leaf sheath, stem, and panicle. The expression of OsCAD6 was stimulated by Xoo infection and UV-irradiation. Thus OsCAD6 appears to be an inducible OsCAD that is likely involved in the defense response of rice against biotic and abiotic stresses.

Cinnamoyl-CoA reductase (CCR) is the first committed enzyme in the monolignol pathway for lignin biosynthesis and catalyzes the conversion of hydroxycinnamoyl-CoAs into hydroxycinnamaldehydes. In the rice genome, 33 genes are annotated as CCR and CCR-like genes, collectively called OsCCRs. To elucidate the functions of OsCCRs, their phylogenetic relationships, expression patterns at the transcription levels and biochemical characteristics were thoroughly analyzed. Of the 33 OsCCRs, 24 of them encoded polypeptides of lengths similar to those of previously identified plant CCRs. The other nine OsCCRs had much shorter peptide lengths. Phylogenetic tree and sequence similarities suggested OsCCR4, 5, 17, 18, 19, 20, and 21 as likely candidates for functional CCRs in rice. To elucidate biochemical functions, OsCCR1, 5, 17, 19, 20, 21, and 26 were heterologously expressed in Escherichia coli and the resulting recombinant OsCCRs were purified to apparent homogeneity. Activity assays of the recombinant OsCCRs with hydroxycinnamoyl-CoAs revealed that OsCCR17, 19, 20, and 21 were biochemically active CCRs, in which the NAD(P)-binding and NADP-specificity motifs as well as the CCR signature motif were fully conserved. The kinetic parameters of enzyme reactions revealed that feruloyl-CoA, a precursor for the guaiacyl (G)-unit of lignin, is the most preferred substrate of OsCCR20 and 21. This result is consistent with a high content (about 70%) of G-units in rice lignins. Phylogenetic analysis revealed that OsCCR19 and 20 were grouped with other plant CCRs involved in developmental lignification, whereas OsCCR17 and 21 were closely related to stress-responsible CCRs identified from other plant species. In agreement with the phylogenetic analysis, expression analysis demonstrated that OsCCR20 was constitutively expressed throughout the developmental stages of rice, showing particularly high expression levels in actively lignifying tissues, such as roots and stems. These results suggest that OsCCR20 is primarily involved in developmental deposition of lignins in secondary cell walls. As expected, the expressions of OsCCR17 and 21 were induced in response to biotic and abiotic stresses, such as Magnaporthe grisea and Xanthomonas oryzae pv. oryzae (Xoo) infections, UV-irradiation and high salinity, suggesting that these genes play a role in defense-related processes in rice.

Rice produces a wide array of phytoalexins in response to pathogen attacks and UV-irradiation. Except for the flavonoid sakuranetin, most phytoalexins identified in rice are diterpenoid compounds. Analysis of phenolic-enriched fractions from UV-treated rice leaves showed that several phenolic compounds in addition to sakuranetin accumulated remarkably in rice leaves. We isolated two compounds from UV-treated rice leaves using silica gel column chromatography and preparative HPLC. The isolated phenolic compounds were identified as phenylamide compounds: N-trans-cinnamoyltryptamine and N-p-coumaroylserotonin. Expression analysis of biosynthetic genes demonstrated that genes for arylamine biosynthesis were upregulated by UV irradiation. This result suggested that phenylamide biosynthetic pathways are activated in rice leaves by UV treatment. To unravel the role of UV-induced phenylamides as phytoalexins, we examined their antimicrobial activity against rice fungal and bacterial pathogens. N-trans-Cinnamoyltryptamine inhibited the growth of rice brown spot fungus (Bipolaris oryzae). In addition to the known antifungal activity to the blast fungus, sakuranetin had antimicrobial activity toward B. oryzae and Rhizoctonia solani (rice sheath blight fungus). UV-induced phenylamides and sakuranetin also had antimicrobial activity against rice bacterial pathogens for grain rot (Burkholderia glumae), blight (Xanthomonas oryzae pv. oryzae) and leaf streak (X. oryzae pv. oryzicola) diseases. These findings suggested that the UV-induced phenylamides in rice are phytoalexins against a diverse array of pathogens.

Chalcone synthase (CHS) is a key enzyme in the flavonoid pathway, participating in the production of phenolic phytoalexins. The rice genome contains 31 CHS family genes (OsCHSs). The molecular characterization of OsCHSs suggests that OsCHS8 and OsCHS24 belong in the bona fide CHSs, while the other members are categorized in the non-CHS group of type III polyketide synthases (PKSs). Biochemical analyses of recombinant OsCHSs also showed that OsCHS24 and OsCHS8 catalyze the formation of naringenin chalcone from p-coumaroyl-CoA and malonyl-CoA, while the other OsCHSs had no detectable CHS activity. OsCHS24 is kinetically more efficient than OsCHS8. Of the OsCHSs, OsCHS24 also showed the highest expression levels in different tissues and developmental stages, suggesting that it is the major CHS isoform in rice. In oschs24 mutant leaves, sakuranetin content decreased to 64.6% and 80.2% of those in wild-type leaves at 2 and 4 days after UV irradiation, respectively, even though OsCHS24 expression was mostly suppressed. Instead, the OsCHS8 expression was markedly increased in the oschs24 mutant under UV stress conditions compared to that in the wild-type, which likely supports the UV-induced production of sakuranetin in oschs24. These results suggest that OsCHS24 acts as the main CHS isozyme and OsCHS8 redundantly contributes to the UV-induced production of sakuranetin in rice leaves.

Chalcone isomerase (CHI) is a key enzyme in flavonoid biosynthesis. In plants, CHIs occur in multigene families, and they are divided into four types, types I–IV. Type I and II CHIs are bona fide CHIs with CHI activity, and type III and IV CHIs are non-catalytic members with different functions. Rice contains seven CHI family genes (OsCHIs). Molecular analysis suggested that OsCHI3 is a type I CHI, and the other OsCHIs were classified into types III and IV. To elucidate their biochemical functions, OsCHI1, OsCHI3, OsCHI6, and OsCHI7 were expressed in Escherichia coli, and the recombinant OsCHI proteins were purified. An activity assay of recombinant OsCHIs showed that OsCHI3 catalyzed the isomerization of naringenin chalcone and isoliquiritigenin, whereas the other recombinant OsCHIs had no CHI activity. OsCHI3 also exhibited a strong preference to naringenin chalcone compared to isoliquiritigenin, which agrees well with the catalytic properties of type I CHIs. These results ascertain OsCHI3 to be a bona fide CHI in rice. OsCHI3 and the other OsCHIs were expressed constitutively throughout the rice growth period and different tissues. OsCHI3 expression was induced immediately in response to ultra-violet (UV) stress, suggesting its involvement in the biosynthesis of sakuranetin, a flavonoid phytoalexin in rice.