Rice Phytoalexins: Half a Century of Amazing Discoveries; Part I: Distribution, Biosynthesis, Chemical Synthesis, and Biological Activities

Abstract

Cultivated rice is a staple food for more than half of the world's population, providing approximately 20% of the world's food energy needs. A broad spectrum of pathogenic microorganisms causes rice diseases leading to huge yield losses worldwide. Wild and cultivated rice species are known to possess a wide variety of antimicrobial secondary metabolites, known as phytoalexins, which are part of their active defense mechanisms. These compounds are biosynthesized transiently by rice in response to pathogens and certain abiotic stresses. Rice phytoalexins have been intensively studied for over half a century, both for their biological role and their potential application in agronomic and pharmaceutical fields. In recent decades, the growing interest of the research community, combined with advances in chemical, biological, and biomolecular investigation methods, has led to a notable acceleration in the growth of knowledge on rice phytoalexins. This review provides an overview of the knowledge gained in recent decades on the diversity, distribution, biosynthesis, chemical synthesis, and bioactivity of rice phytoalexins, with particular attention to the most recent advances in this research field.

Keywords: Oryza; momilactones; oryzalexins; phenilammides; phytoalexins; phytocassanes; rice; sakuranetin.

Figure 5

Biosynthesis of IPP and DMAPP through MVA and MEP pathway in plants. Although IPP and DMAPP act as precursors for all terpenes, sesqui-, and triterpenes biosynthesis typically is fed by the cytosolic MEV pathway, while mono-, di-, and tetraterpenes biosynthesis typically is fed by the plastidial MEP pathway. AACT: acetoacetyl-CoA thiolase (EC 2.3.1.9); CMK: 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase (EC 2.7.1.148); DMAPP: dimethylallyl diphosphate; DPTS: diterpene synthase (E.C. 4.2.3.x); DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase (EC 1.1.1.267); DXS: 1-deoxy-D-xylulose 5-phosphate synthase (EC 2.2.1.7); FDP: farnesyl diphosphate; FPPS: farnesyl diphosphate synthase (EC 2.5.1.10); G3P: D-glyceraldehyde 3-phosphate; GPP: geranyl diphosphate; GPPS: geranyl diphosphate synthase (EC 2.5.1.29); GGPPS: geranylgeranyl diphosphate synthase (EC 2.5.1.29); HDR: (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase; HDS: (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (EC 1.17.1.2); HMGR: 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.17.1.2); HMGS: 3-hydroxy-3-methylglutaryl-CoA synthase (EC 2.3.3.10); IPPI: isopentenyl diphosphate Δ-isomerase (EC 5.3.3.2); IPP: isopentenyl diphosphate; MCT: 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (EC 2.7.7.60); MDD: diphosphomevalonate decarboxylase (EC 4.1.1.33); MDS: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (EC 4.6.1.12); MTPS: monoterpene synthase (EC:4.2.3.-); MVK: mevalonate kinase (EC 2.7.1.185); MVAP: mevalonate 5-phosphate; MVAPP: mevalonate diphosphate; PMK: phosphomevalonate kinase (EC 2.7.4.2); STPS: sesquiterpene synthase (EC 4.2.3.49; 4.2.3.47; 3.1.7.6); TPS: terpene synthase (EC 4.2.3.47); TTPS: triterpene synthase (EC 5.4.99.-) (adapted from [107]).

Figure 6

Proposed pathway for the biosynthesis of rice diterpenoid phytoalexins and gibberellins. The enzymes whose chloroplast localization is established are written in green. CPP: copalyl diphosphate; diTPS I: class I diterpene synthase; diTPS II: class II diterpene synthase; GGPP: geranylgeranyl diphosphate; OsCPS1: ent-copalyl diphosphate synthase (EC 5.5.1.13); OsCPS2 (OsCyc2): ent-copalyl diphosphate synthase (EC 5.5.1.13); OsCPS4 (OsCyc1): syn-copalyl diphosphate synthase (EC 5.5.1.14); OsKS1: ent-kaur-16-ene synthase (EC 4.2.3.19); OsKS4 (OsKSL4): syn-pimara-7,15-diene synthase (EC 4.2.3.35); OsKS7 (OsKSL7; OsDTC1): ent-cassa-12,15-diene synthase (EC 4.2.3.28); OsKS8 (OsKSL8; OsK8; OsDTC2): stemar-13-ene synthase (EC 4.2.3.33); OsKS10 (OsKSL10): ent-sandaracopimara-8(14),15-diene synthase (EC 4.2.3.29) (adapted from [110]).

Figure 1

Momilactones so far isolated from rice (A–E) and oryzalactone (F). The dual role of phytoalexins and allelochemicals has been demonstrated for momilactone A and B. Momilactones share a pimarane skeleton, while oryzalactone exhibits an abietane skeleton.

Figure 2

Phytocassanes so far isolated from rice.

Figure 3

Oryzalexins so far isolated from rice. Despite the name, oryzalexins (A–F) are distinguished from oryzalexin S both by the chemical structure (ent-sandaracopimaradiene- and stemarane-type, respectively) and by the metabolic intermediate from which they derive (ent-sandaracopimara-8(14),15 diene and syn-stemar-13-ene, respectively; vide infra).

Figure 4

Phenolic compounds that accumulate in rice after exposure to biotic and/or abiotic stresses. (A) is a flavanone compound, while (B–G) are phenylamides.

Figure 7

Schematic diagram of loci corresponding to genes involved in diterpene biosynthesis in rice. The biosynthetic gene clusters on chromosome 2 (c2BGC) and chromosome 4 (c4BGC) are located between LOC_Os02g35970 and LOC_Os02g36300 and between LOC_Os04g09800 and LOC_Os04g10240, respectively (adapted from [44,119,126]).

Figure 8

Proposed pathway for the biosynthesis of momilactones A and B. Chemical functions in blue indicate characterized reactions, while predicted functionalities are indicated in red. Dashed arrows indicate missing steps. Enzymes encoded by genes on chromosome 2, 4, and 6 (c2, c4, c6) are highlighted in green, yellow, and blue, respectively. GGPP: geranyl geranyl diphosphate; OsMAS: rice momilactone A synthase; OsKS4 (OsKSL4): syn-pimara-7,15-diene synthase (EC 4.2.3.35); OsCPS4 (OsCyc1): syn-copalyl diphosphate synthase (EC 5.5.1.14); CYP76M8: oryzalexin D synthase (EC 1.14.14.112; 1.14.14.123); CYP99A3: 9-beta-pimara-7,15-diene oxidase (EC 1.14.14.111); CYP99A2: cytochrome P450 99A2 (EC 1.14.-.-); CYP701A8: ent-sandaracopimaradiene 3-hydroxylase (EC 1.14.14.70) (adapted from [128]).

Figure 9

Proposed pathway for the biosynthesis of phytocassanes. The green highlight indicates enzymes encoded by genes belong to the biosynthetic gene cluster on chromosome 2 (c2BGC). Enzymes encoded by genes located on chromosomes 6 and in both 2 and 6 (c6 and c2–c6) are highlighted in blue and grey, respectively. GGPP: geranyl geranyl diphosphate; OsKS7 (OsKSL7; OsDTC1): ent-cassa-12,15-diene synthase (EC 4.2.3.28); OsCPS2 (OsCyc2): ent-copalyl diphosphate synthase (EC 5.5.1.13); CYP76M7: ent-cassadiene C11-alpha-hydroxylase 1 (EC 1.14.14.112); CYP76M8: oryzalexin D synthase (EC 1.14.14.112; 1.14.14.123); CYP701A8: ent-sandaracopimaradiene 3-hydroxylase (EC 1.14.14.70); C71Z7: ent-cassadiene hydroxylase (EC 1.14.14.69) (adapted from [138]).

Figure 10

Proposed pathway for the biosynthesis of oryzalexins A–E. Enzymes encoded by genes located on chromosomes 2, 4, 6, and 12 (c2, c4, c6, c12) are highlighted in green, yellow, blue, and pink, respectively. A, B, C: oryzalexin A, B, and C, respectively. CYP701A8: ent-sandaracopimaradiene 3-hydroxylase (EC 1.14.14.70); CYP76M6: oryzalexin E synthase (EC 1.14.14.122); CYP76M8: oryzalexin D synthase (EC 1.14.14.112; 1.14.14.123); GGPP: geranyl geranyl diphosphate; OsCPS4: syn-copalyl diphosphate synthase (EC 5.5.1.14); OsKS10: ent-sandaracopimara-8(14),15-diene synthase (EC 4.2.3.29) [123,138].

Figure 11

Biosynthesis of sakuranetin. ACC: acetyl-CoA carboxylase (EC 6.4.1.2); CHI: chalcone isomerase (EC 5.5.1.6); CHS: chalcone synthase (EC 2.3.1.74); OsNOMT: naringenin 7-O-methyltransferase (EC 2.1.1.232); SAH: adenosyl-L-homocysteine; SAM: S-adenosyl-L-methionine. Multiple arrows indicate multiple biosynthetic steps, while direct synthesis is indicated by single arrows.

Figure 12

Biosynthesis of phenylamides and sakuranetin. Multiple arrows indicate multiple enzymatic steps. E4P: erythrose 4-phosphate; PEP: phosphoenol pyruvate; T5H: tryptamine 5-hydroxylase (EC 1.14.-.-); TDC: L-tryptophan decarboxylase (aromatic-L-amino-acid decarboxylase, EC 4.1.1.28); TYDC: L-tyrosine decarboxylase (EC 4.1.1.25) (adapted from [21]).

Scheme 1

(−)-Phytocassane D chemical synthesis [144]. Reagents and conditions: (a) (i) 2,2-dimethyl-1,3-propandiol, BF₃.Et₂O; (ii) PhSH, aq. CH₂O, Et₃N, EtOH; (iii) Li, liq. NH₃, H₂O, THF, then MeI, THF; (iv) LiAlH₄, THF; (v) aq. HCl, THF; (vi) TBSCl, imidazole, DMF (40%, 6 steps). (b) (i) NaH, HCO₂Et, THF, PhMe; (ii) MeCOCH=CH₂, Et₃N; (iii) NaOMe, MeOH (78%, 3 steps). (c) (i) Li, liq. NH₃, EtOH, THF; (ii) PCC, 3-Å MS, CH₂Cl₂; (iii) KH, PhSO₂Me, THF; (iv) CaCO₃, PhMe, heat (77%, 4 steps). (d) (i) Me₂CuLi, Et₂O; (ii) diastereoisomer separation (47%). (e) (i) NaH, HCO₂Et, MeOH; (ii) NaBH₄, THF, MeOH; (iii) TBDPSCl, imidazole, DMF; (iv) PCC, 4-Å MS, CH₂Cl₂ (57%, 4 steps). (f) (i) TsNHNH₂, MgSO₄, PPTS, THF; (ii) excess LDA, THF then quenched with aq. NH₄Cl; (iii) TBAF, THF, room temp. (68%, 3 steps). (g) (i) MCPBA, NaHCO₃, CHCl₃; (ii) Dess–Martin periodinane, CH₂Cl₂; (iii) pyrrolidine, Et₂O (67%, 3 steps). (h) (i) Ph₃P=CH₂, THF; (ii) Ac₂O, DMAP, C₅H₅N; (iii) TBAF, THF, 50–60 °C; (iv) PCC, 4-Å MS, CH₂Cl₂ (59%, 4 steps). (i) (i) LiHMDS, TMSCl, THF; (ii) MCPBA, NaHCO₃, hexane; (iii) (CO₂H)₂, MeOH; (iv) Dess–Martin periodinane, CH₂Cl₂; (v) TBSCl, imidazole, DMF (25%, 5 steps). (l) LiAlH₄, THF, then aq. HCl (64%). (k) TPAP, 4-ÅMS, MeCN, CH₂Cl₂ (40%).

Scheme 2

Retrosynthetic scheme for (+)-oryzalexins A–B.

Scheme 3

(+)-Oryzalexins A–C chemical synthesis. Reagents and conditions: (a) (i) (CH₃O)₂SO₂, NaOH, Δ; (ii) Na, boiling alcohol (73%, 2 steps). (b) (i) pyrrolidine, C₆H₆, Δ; (ii) CH₃I, 1,4-dioxane, Δ; (iii) (C₂H₅)₂NCH₂CH₂COCH₃, CH₃I, C₆H₆, 0 °C (59% 3 steps). (c) KOC(CH₃)₃, CH₃I (75%). (d) (i) HOCOCH₃, Pd on C, H₂; (ii) NaI, CH₃CN, (CH₃)₃SiCl, room temp.; (iii) THF, Li, liquid NH₃, −60 °C; (iv) TsOH, CH₃OCOCH₃, Δ; (v) Raney Ni, H₂; (vi) Jones CrO₃ (34% 6 steps). (e) (i) NaOCH₃, HCOOCH₂CH₃, C₆H₆; (ii) TsOH, C₆H₆, BuSH, Δ; (iii) t-BuMe₂SiCl, DMF, imidazole, room temp.; NaBH₄, CH₃CH₂OH; (iv) CH₃CH₂OH, CdCO₃, HgCl₂, Δ (58%, 4 steps). (f) (i) C₆H₆, KOC(CH₃)₃, CH₃I, Δ; (ii) Ph₃P=CH₂, THF; (iii) HF, CH₃CN (40%, 3 steps). (g) (i) (−)-camphanoyl chloride, 0 °C, diastereoisomer separation by column chromatography; (ii) conc HCl, CH₃OH (40%, 2 steps). (h) (i) acetone, Jones CrO₃; (ii) C₆H₆, SeO₂, HOCOCH₃, H₂O (66% 2 steps). (i) (i) CH₃COOCOCH₃, pyridine; (ii) C₆H₆, SeO₂, HOCOCH₃, H₂O; (iii) (CH₃)₂SO, CH₃COOCOCH₃, (iv) K₂CO₃, CH₃OH (23%, 4 steps). (j) acetone, Jones CrO₃ (100%).

Scheme 4

(±)-Sakuranetin chemical synthesis. Reagents and conditions: (a) 2-methoxyethanol (79%). (b) H₃PO₄ (40%); (c) (i) H₂/Pd-C, EtOAc; (ii) BCl₃, CH₂Cl₂ (32% 2 steps).

Scheme 5

(±)-Sakuranetin chemical synthesis. Reagents and conditions: (a) LiAlH₄, MgBr₂ (45%). (b) DDQ (2 equiv), H₂O, CH₂Cl₂ (88%); (c) (i) ZnBr₂, CH₂Cl₂; (ii) K₂CO₃, MeI, acetone (36%, 2 steps).