# Source paper archive - Source title: Photosynthesis-derived carbohydrate supply sustains CuCl2-induced phytoalexin production in rice leaves - Author(s): Naoya Misawa, Yumi Oshima, Tomohiro Sagara, Natsumi Tate, Shuhei Yoshinaga, Tomoko Sakazawa, Emi Yumoto, Hisakazu Yamane, Koji Miyamoto - Source landing URL: https://www.biorxiv.org/content/10.64898/2026.10.29.761194v1 - PDF URL: https://www.biorxiv.org/content/10.64898/2026.10.29.761194v1.full.pdf - PDF SHA-256: 3ef23c9f0f66ad83bff76d50dda9f48649603fd509bfeea905a4c2d019f68cc6 - Converter: pymupdf4llm 0.0.27 - Limits: Automated PDF text extraction; reading order, equations, tables and formatting may be incomplete or incorrect. Images are omitted, and no OCR was performed. Markup and links are neutralized for safe downloading. Consult the original PDF for the authoritative version. --- [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1 **Title page** 2 3 **Photosynthesis-derived carbohydrate supply sustains CuCl** **2** **-induced phytoalexin production in rice** 4 **leaves** 5 Naoya Misawa [a,1], Yumi Oshima [b,1], Tomohiro Sagara [a], Natsumi Tate [a], Shuhei Yoshinaga [a], Tomoko 6 Sakazawa [a,c], Emi Yumoto [d], Hisakazu Yamane [a,b,d], Koji Miyamoto [a,b,c,d,] 7 8 a Department of Biosciences, Faculty of Science and Engineering, Teikyo University, 1-1 Toyosatodai, 9 Utsunomiya, Tochigi 320-8551, Japan 10 b Graduate School of Science and Engineering, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi 320 11 8551, Japan 12 c Department of Integrated Science and Engineering, Faculty of Science and Engineering, Teikyo University, 13 1-1 Toyosatodai, Utsunomiya, Tochigi 320-8551, Japan 14 d Advanced Instrumental Analysis Center, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi 320 15 8551, Japan 16 17 1 These authors contributed equally to this work. 18 - Corresponding author. 19 E-mail address: miyamoto.koji.dm@teikyo-u.ac.jp (K. Miyamoto). 20 1 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 21 **Abstract** 22 Phytoalexins are antimicrobial metabolites whose biosynthesis is triggered by signaling activated by 23 both biotic and abiotic elicitors, but the physiological conditions that sustain their production remain poorly 24 understood. Here, we examined how light and carbohydrate availability affect CuCl 2 -induced production of 25 diterpenoid (phytocassanes and momilactones) and flavonoid (sakuranetin) phytoalexins in rice ( _Oryza sativa_ 26 L.) leaf disks. Darkness strongly suppressed both the accumulation of all three phytoalexin groups and the 27 induction of their biosynthetic genes. In darkness, endogenous sucrose, glucose, and fructose declined to 3%– 28 8% of the levels in the light, whereas early accumulation of jasmonic acid and jasmonoyl- L -isoleucine (JA-Ile) 29 was not detectably reduced. Exogenous sucrose restored phytoalexin accumulation in darkness in a dose 30 dependent manner, together with the expression of the suppressed biosynthetic genes, whereas equimolar 31 mannitol had no effect. Glucose also restored sakuranetin accumulation in darkness but only partially restored 32 diterpenoid accumulation, whereas the glucose analog 2-deoxy- D -glucose did not, and it suppressed CuCl 2 33 induced accumulation in the light in a dose-dependent manner. In the light, 3-(3,4-dichlorophenyl)-1,1 34 dimethylurea (DCMU), an inhibitor of photosynthetic electron transport, depleted soluble sugars and 35 suppressed phytoalexin accumulation, and sucrose reversed this suppression. Sugars alone induced little 36 phytoalexin accumulation, indicating that carbohydrates act permissively rather than as elicitors. Diterpenoid 37 phytoalexins depended more strongly on light than sakuranetin, yet both classes depended on carbohydrate 38 availability. These results indicate that photosynthesis-derived carbohydrate supply sustains elicitor-induced 39 phytoalexin production in rice leaves and largely accounts for its light dependence. 40 **Keywords** 41 _Oryza sativa_ ; Phytoalexin; Specialized metabolism; Photosynthesis; Sugar; Darkness 42 2 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 43 **1. Introduction** 44 Phytoalexins are low-molecular-weight antimicrobial metabolites that plants newly synthesize when 45 exposed to pathogens or other stresses (Ahuja et al., 2012). Rice ( _Oryza sativa_ L.) produces structurally 46 diverse phytoalexins, including the diterpenoids phytocassanes and momilactones and the flavonoid 47 sakuranetin (Miyamoto, 2026; Valletta et al., 2023; Yamane, 2013). Upon infection by the blast fungus, 48 diterpenoid phytoalexins accumulate earlier and to higher levels in resistant than in susceptible plants 49 (Hasegawa et al., 2010). Genetic studies further support their role in disease resistance: knockout mutants of 50 _OsNOMT_, which are impaired in sakuranetin biosynthesis, and of _OsCPS2_, which have reduced phytocassane 51 levels, are more susceptible to the blast fungus (He et al., 2024; Lu et al., 2018), as is a knockdown mutant of 52 _OsCPS4_ with reduced momilactone levels (Toyomasu et al., 2014), although an _OsCPS4_ knockout did not 53 show increased susceptibility in another study (Lu et al., 2018). 54 The diterpenoid phytoalexins are derived from precursors supplied by the plastidic methylerythritol 55 phosphate (MEP) pathway, whose genes, including _OsDXS3_, are induced upon elicitation (Okada et al., 2007). 56 Their carbon skeletons are formed by OsCPS2 and OsKSL7 for phytocassanes and by OsCPS4 and OsKSL4 57 for momilactones (Yamane, 2013). In contrast, sakuranetin is derived from the flavonoid pathway and is 58 formed from naringenin by naringenin 7- _O_ -methyltransferase (OsNOMT) (Shimizu et al., 2012). 59 Rice phytoalexin production is induced by diverse biotic and abiotic elicitors. Among these, CuCl 2 is 60 widely used as a reproducible abiotic elicitor, and CuCl 2 treatment rapidly increases endogenous jasmonic acid 61 (JA) levels in rice leaves (Rakwal et al., 1996). The requirement for jasmonoyl- L -isoleucine (JA-Ile), a 62 biologically active jasmonate, however, differs among phytoalexin classes: JA-Ile is required for sakuranetin 63 production but not for the production of diterpenoid phytoalexins (Miyamoto et al., 2016; Shimizu et al., 64 2013). At the transcriptional level, Diterpenoid Phytoalexin Factor (DPF) and OsMYC2 regulate the 65 diterpenoid and sakuranetin pathways, respectively (Ishikawa et al., 2024; Ogawa et al., 2017; Yamamura et 66 al., 2015). Thus, the signaling and transcriptional networks that trigger phytoalexin biosynthesis have been 67 characterized in considerable detail. By contrast, the physiological conditions required to sustain these induced 68 responses, including the supply of carbon and energy for biosynthesis, have received much less attention. 69 Light is a major environmental factor affecting such conditions. Light influences plant defense 3 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 70 through multiple routes: photosynthesis supplies carbon skeletons, ATP, and reducing power, whereas 71 photoreceptors, chloroplast redox status, and reactive oxygen species also modulate defense signaling 72 (Kangasjärvi et al., 2012). In rice, phytochromes are required for JA- and salicylic acid-related responses and 73 for developmentally controlled resistance to the blast fungus (Xie et al., 2011). In _Arabidopsis thaliana_, 74 approximately 30% of strongly flg22-induced genes require light for their rapid induction, and inhibitors of 75 photosynthetic electron transport differ in their effects on this response (Sano et al., 2014). Because darkness 76 affects all of these processes simultaneously, a reduction in defense output in the dark cannot by itself be 77 attributed to carbon limitation. Combining light–dark comparisons with photosynthetic inhibition and sugar 78 supplementation therefore provides a means of evaluating the specific contribution of carbohydrate 79 availability. 80 In plant immunity, sugars act not only as sources of carbon and energy but also as signaling 81 molecules (Bolouri Moghaddam and Van den Ende, 2012). Carbohydrate availability can limit defense. 82 Repression of cell wall invertase impaired defense in tobacco source leaves (Essmann et al., 2008). Similarly, 83 starch-deficient _A. thaliana_ mutants showed reduced camalexin accumulation at late stages of _Colletotrichum_ 84 _higginsianum_ infection and increased susceptibility, although the consequences of carbon limitation depended 85 on the infection stage and pathogen lifestyle (Engelsdorf et al., 2013). Sugars also act in the regulation of 86 chemical defense. In _A. thaliana_, glucose-6-phosphate modulates defense signaling, and basal sugar uptake 87 through sugar transport proteins (STPs) is required for normal chitin-induced camalexin production (Yamada 88 and Mine, 2024). These findings connect carbohydrate status to both the execution and the regulation of 89 chemical defense. They also caution against interpreting the effects of sugar supplementation solely as the 90 provision of biosynthetic substrates. In rice, sucrose has been implicated in the priming of defense responses 91 and in enhanced resistance to the blast fungus (Gómez-Ariza et al., 2007). Sucrose also preferentially induces 92 the expression of _OsWRKY7_, which encodes a transcription factor that directly regulates the defense-related 93 gene _OsPR10a_ (Tun et al., 2023). Sugars applied exogenously to rice leaves induce phytoalexin accumulation 94 (Nishimura et al., 2021). Moreover, soluble sugars, jasmonates, and flavonoid phytoalexins increase together 95 in strigolactone-deficient plants, and spraying glucose or sucrose increases jasmonate accumulation in wild 96 type seedlings (Lahari et al., 2024). 4 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 97 These studies establish that sugars can promote defense responses in rice. However, it remains 98 unclear to what extent photosynthetically supplied carbohydrates contribute to the light dependence of elicitor 99 induced phytoalexin production. In particular, sugar-induced activation of defense needs to be distinguished 100 from the capacity of supplied sugars to sustain an elicitor-triggered response when photosynthetic carbon 101 supply is restricted. It is also unknown whether diterpenoid and flavonoid phytoalexins, which differ in 102 biosynthetic origin and JA-Ile requirement, share such a dependence on carbohydrate availability. Here, we 103 examined CuCl 2 -induced phytoalexin production in rice leaf disks under light and dark conditions. We 104 quantified phytocassanes, momilactones, and sakuranetin, together with transcripts of their biosynthetic genes, 105 endogenous soluble sugars, and early JA and JA-Ile levels. We then used exogenous sucrose and glucose, 106 mannitol, 2-deoxy- D -glucose (2-DG), and the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1 107 dimethylurea (DCMU). These treatments allowed us to distinguish the contribution of carbohydrate 108 availability from osmotic and other light-dependent effects. We show that darkness depleted soluble sugars 109 without detectably reducing early jasmonate accumulation. Sucrose and, at least partly, glucose, but not 110 mannitol or 2-DG, restored phytoalexin production in darkness. DCMU suppressed phytoalexin production in 111 the light in a sucrose-reversible manner. These results indicate that an adequate supply of metabolizable 112 carbohydrates, normally sustained by photosynthesis, is a major determinant of CuCl 2 -induced phytoalexin 113 production in rice leaves and that sugar depletion largely accounts for its suppression in darkness. 114 115 **2. Materials and methods** 116 **2.1. Plant materials and growth conditions** 117 We used _Oryza_ _sativa_ L. ‘Nipponbare’ as the wild type in the experiments for Figs. 5D–F and 6G–I, 118 and ‘Nihonmasari’ as the wild type in the other experiments. Rice plants were grown using a composite soil 119 (Bonsoru No. 2; Sumitomo Chemical, Tokyo, Japan), under natural daylight in a glasshouse at Teikyo 120 University (Utsunomiya, Tochigi, Japan; 36.60°N, 139.88°E) as described previously (Inagaki et al., 2023). 121 The temperature in the glasshouse was maintained at approximately 28 °C during the day and at 25 °C at night. 122 123 **2.2. Chemicals** 5 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 124 Copper(II) chloride dihydrate and D -(−)-fructose were purchased from Wako Pure Chemical Co. Ltd. 125 (Osaka, Japan). Sucrose, D -(+)-glucose, and D -(−)-mannitol were purchased from Kanto Chemical Co. Inc. 126 (Tokyo, Japan). 2-Deoxy- D -glucose (2-DG) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) were 127 purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). [ [13] C 12 ]-sucrose, [ [13] C 6 ]-glucose, and [ [13] C 6 ] 128 fructose were purchased from Omicron Biochemicals Inc. (South Bend, IN, USA). [ [2] H 2 ]-JA was purchased 129 from Tokyo Chemical Industry Co. Ltd. [ [13] C 6 ]-(−)-JA-Ile was synthesized from (±)-methyl jasmonate and 130 [ [13] C 6 ]-isoleucine and isolated by HPLC as described previously (Jikumaru et al., 2004; Riemann et al., 2013). 131 132 **2.3. Light and dark treatments with chemicals** 133 Leaf disks (6 mm in diameter) were punched from the youngest fully expanded leaves of 4-week-old 134 plants. For the quantification of JA and JA-Ile (Fig. 3D, E), leaf segments (approximately 1 cm long) were 135 prepared from the same leaves. Immediately after preparation, the leaf disks were floated on distilled water in 136 Petri dishes and incubated under continuous light (approximately 100 µmol m [−2] s [−1], white fluorescent lamps) 137 or in complete darkness at 25 °C for 24 h to allow recovery from wounding. The disks were then transferred to 138 Petri dishes containing the treatment solution (0 h) and incubated under their respective light or dark 139 conditions until sampling. Samples designated "Initial" were collected at the time of leaf-disk preparation, 140 before the light and dark treatments were started. Sampling times are given in the figure legends. Leaf disks 141 were prepared from 3–5 plants and randomly assigned to treatments. For phytoalexins and soluble sugars, each 142 biological replicate consisted of a single leaf disk; 6 disks per treatment were incubated together in one Petri 143 dish. 144 CuCl 2 was dissolved in distilled water at 1 M as a stock solution and diluted to 1 mM with distilled 145 water. Sugars and 2-DG were dissolved in distilled water or in 1 mM CuCl 2 solution and applied at 0 h at the 146 following concentrations: sucrose at 3, 30, or 300 mM (Fig. 4); sucrose and mannitol at 300 mM (Fig. 5A–C); 147 glucose and 2-DG at 300 mM in darkness (Fig. 5D–F) and at 3, 30, or 300 mM in the light (Fig. 6G–I). 148 DCMU was dissolved in ethanol at 10 mM as a stock solution and diluted 1000-fold with distilled 149 water to a final concentration of 10 µM (final ethanol concentration, 0.1% [v/v]); 0.1% (v/v) ethanol was used 150 as the control. For DCMU treatment (Fig. 6A–F), leaf disks were incubated in 10 µM DCMU or 0.1% ethanol 6 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 151 in Petri dishes under continuous white light during the 24 h preincubation period. The disks were then 152 transferred to Petri dishes containing the same concentration of DCMU or ethanol, with or without 1 mM 153 CuCl 2 and, where indicated, 300 mM sucrose, and incubated under white light for a further 24 h (soluble 154 sugars) or 72 h (phytoalexins). 155 156 **2.4. Quantification of phytoalexins and jasmonates** 157 Phytoalexins were quantified as described previously (Miyamoto et al., 2016) with minor 158 modifications. Each leaf disk was submerged in 1 mL of 80% (v/v) aqueous methanol and extracted at 4 °C for 159 at least 24 h. Aliquots (1 µL) of the extracts were analyzed without further purification by liquid 160 chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS; Agilent 6460 Triple 161 Quadrupole mass spectrometer with an Agilent 1200 separation module) equipped with a CAPCELL CORE 162 C18 column (50 mm long, 2.1 mm in diameter; Shiseido, Tokyo, Japan) in multiple reaction monitoring mode. 163 Phytocassanes A–E, momilactones A and B, and sakuranetin were quantified by comparison with the peak 164 areas of an authentic standard mixture (100 pg each). The amounts of phytocassanes and momilactones are 165 presented as the sums of the individual compounds. 166 For JA and JA-Ile, leaf segments (approximately 50 mg fresh weight per sample) were weighed and 167 homogenized in 80% (v/v) aqueous methanol containing 10 ng each of [ [2] H 2 ]-JA and [ [13] C 6 ]-JA-Ile as internal 168 standards. The extracts were purified on a Bond Elut C18 cartridge (100 mg, 3 mL; Agilent Technologies), 169 dried, dissolved in 0.5 mL of 50% (v/v) aqueous methanol, and analyzed by LC-ESI-MS/MS as described 170 previously (Miyamoto et al., 2016). 171 172 **2.5. Quantification of soluble sugars** 173 Each leaf disk was weighed on a precision balance (approximately 3 mg fresh weight) before 174 homogenization. The leaf disks were homogenized and suspended in 1 mL of 80% (v/v) aqueous methanol 175 containing 1 μg each of [ [13] C 12 ]-sucrose, [ [13] C 6 ]-glucose, and [ [13] C 6 ]-fructose as internal standards. The samples 176 were heated at 80 °C for 15 min. The supernatant was collected in a new tube, and then 1 mL of 80% (v/v) 177 aqueous methanol was added to the precipitate, followed by heating at 80 °C for 15 min. The second extract 7 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 178 was combined with the first supernatant from the previous step and centrifuged at 18,000 × _g_ for 5 min. The 179 supernatant was transferred to a new tube and evaporated to dryness under a stream of nitrogen. The residue 180 was dissolved in 1 mL of distilled water. The samples were loaded onto a Bond Elut C18 (100 mg, 3 mL; 181 Agilent Technologies, Santa Clara, CA, USA), which had been sequentially pre-washed with 3 mL each of 182 methanol and water, and eluted with 1 mL water. The flow-through and eluted fractions were combined, and 183 the sample was diluted 12.5-fold with 80% (v/v) aqueous acetonitrile. Sugars in the diluted samples were 184 quantified by LC-ESI-MS/MS using an Agilent 6460 Triple Quadrupole mass spectrometer coupled with an 185 Agilent 1200 separation module equipped with a Shodex HILICpak VG-50 2D column (150 mm long, 2.0 mm 186 in diameter; Resonac Holdings Co., Tokyo, Japan). The mobile phase consisted of water (solvent A) and 187 acetonitrile (solvent B). Elution was conducted at 80% of solvent B over 10 min at a flow rate of 0.3 mL/min. 188 The retention times of sucrose, glucose, and fructose were 5.9, 4.5, and 3.8 min, respectively. All sugars were 189 analyzed in negative ion mode with nitrogen as the collision gas. Each sugar was quantified by multiple 190 reaction monitoring. Precursor-to-product transitions monitored were _m_ / _z_ 341/89 for sucrose; _m_ / _z_ 353/92 for 191 [ [13] C 12 ]-sucrose; _m_ / _z_ 179/89 for glucose and fructose; _m_ / _z_ 185/92 for [ [13] C 6 ]-glucose and [ [13] C 6 ]-fructose. 192 193 **2.6. RNA extraction and quantitative RT-PCR** 194 Total RNA was extracted from leaf disks using Sepasol-RNA I Super G (Nacalai Tesque Inc., Japan) 195 and subjected to cDNA synthesis using a PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio Inc., 196 Japan). Quantitative RT-PCR (qRT-PCR) was performed as described previously (Inagaki et al., 2023) on an 197 ABI PRISM 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) using Fast SYBR 198 Green Master Mix (Applied Biosystems). Primer sequences are provided in Table S1 **.** Transcript levels were 199 normalized to that of the ubiquitin domain-containing protein gene _OsUBQ_ (Os10g0542200) and expressed as 200 relative values using the 2 [−ΔΔ] _[Ct]_ method. 201 202 **2.7. Statistical analysis** 203 All values are presented as means ± SE. The number of biological replicates is given in each legend. 204 Compounds that were below the limit of detection were treated as zero and are indicated as n.d. in the figures. 8 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 205 Comparisons between the light and dark treatments at individual time points (Figs. 1–3) were made using 206 Welch’s t-test with Bonferroni correction for the number of time points compared. Time points at which the 207 compound was not detected in both treatments were excluded from the analysis, and the number of 208 comparisons used for the correction was reduced accordingly. Comparisons among more than two treatments 209 (Figs. 4–6) were made using one-way ANOVA followed by Tukey’s HSD test on log(x + 1)-transformed 210 values. Treatments in which the compound was not detected were retained in the analysis as zero values and 211 are labeled n.d. in the figures. Analyses for Figs. 1–3 were performed with Microsoft Excel 2016 and those for 212 Figs. 4–6 with R version 4.3.1. Statistical analyses of transcript data were performed on Δ _Ct_ values ( _Ct_ of the 213 target gene minus _Ct_ of _OsUBQ_ ), which are on a log 2 scale, without further transformation. Differences were 214 considered significant at _p_ < 0.05. Results of all statistical tests are provided in Table S2. 215 216 **3. Results** 217 **3.1. Dark treatment suppressed CuCl** **2** **-induced phytoalexin production** 218 To investigate the effects of light conditions on CuCl 2 -induced phytoalexin production, we prepared 219 leaf disks from rice leaf blades and incubated them under white light or in the dark. After 24 h under these 220 conditions, the disks were treated with 1 mM CuCl 2, and phytoalexin accumulation was followed for a further 221 72 h (Fig. 1A). The diterpenoid-type phytoalexins, phytocassanes and momilactones, accumulated under light 222 conditions but showed almost no accumulation in the dark (Fig. 1B, C). The levels of the flavonoid-type 223 phytoalexin sakuranetin were slightly but significantly higher in the dark than in the light at 0 and 6 h after 224 CuCl 2 application. However, the amounts at these points were less than 2% of the level reached in the light at 225 72 h. From 24 h onward, under dark conditions, accumulation was significantly reduced to 11%–25% of the 226 level observed under light conditions (Fig. 1D). These results indicate that dark treatment suppresses CuCl 2 227 induced phytoalexin accumulation in rice leaves. 228 To examine whether the suppression of phytoalexin accumulation in the dark was accompanied by 229 reduced expression of the corresponding biosynthetic genes, we quantified transcript levels of four diterpenoid 230 pathway genes ( _OsCPS2_, _OsKSL7_, _OsCPS4_ and _OsKSL4_ ), the upstream MEP pathway gene _OsDXS3_, and the 231 sakuranetin biosynthetic gene _OsNOMT_ (Fig. 2). Under light conditions, all six genes were induced by CuCl 2, 9 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 232 although their kinetics differed: _OsKSL4_ and _OsDXS3_ peaked at 6 h and declined thereafter, _OsKSL7_ peaked at 233 12 h, whereas _OsCPS2_, _OsCPS4_ and _OsNOMT_ increased progressively until 24 h. _OsNOMT_ showed the 234 largest induction, reaching approximately 170,000-fold the initial level. At the time of CuCl 2 application, when 235 the disks had already been held under the respective light conditions for 24 h, transcript levels of all six genes 236 were below 10% of the maxima subsequently reached in the light, although small differences between the two 237 treatments were already detectable for four of them (Fig. 2A, C, D, F). Under dark conditions, the induction of 238 all six genes was markedly reduced. At 24 h, transcript levels in the dark corresponded to 0.8%–25% of those 239 in the light, and the difference was significant for every gene examined (Fig. 2A–F). At 6 h, the difference was 240 significant for five of the six genes, except for _OsCPS4_, whereas at 12 h it was significant only for _OsCPS2_ 241 and _OsNOMT_ ; _OsKSL7_ showed a similar difference that did not reach significance after Bonferroni correction 242 (Fig. 2B). These results indicate that dark treatment suppresses CuCl 2 -induced expression of the phytoalexin 243 biosynthetic genes, in parallel with the reduced accumulation of the phytoalexins themselves. 244 245 **3.2. Dark treatment decreased endogenous soluble sugars without a detectable effect on early CuCl** **2** **-** 246 **induced jasmonate accumulation** 247 To examine whether the dark treatment limited the supply of carbon available for phytoalexin 248 synthesis, we quantified endogenous soluble sugar levels (Fig. 3A–C). At 6, 12, and 24 h after CuCl 2 249 application, sucrose levels were significantly lower under dark than under light conditions. In particular, 12 250 and 24 h after CuCl 2 application, sucrose levels under dark conditions had decreased to approximately 5% of 251 those under light conditions (Fig. 3A). At 24 h after CuCl 2 application, glucose and fructose levels in dark 252 incubated leaf disks were reduced to 3%–8% of those under light conditions (Fig. 3B, C). These results show 253 that dark treatment markedly reduced endogenous soluble-sugar levels in rice leaf disks. 254 Jasmonic acid (JA) and its biologically active form, jasmonoyl- L -isoleucine (JA-Ile), are among the 255 signaling molecules that regulate stress-induced phytoalexin production. We investigated whether endogenous 256 JA and JA-Ile levels decrease under dark conditions. Since the accumulation of JA and JA-Ile is induced early 257 after stress treatment, we measured their endogenous levels 2 h after CuCl 2 application. At 2 h after CuCl 2 258 application, no significant differences in JA or JA-Ile accumulation were detected between light and dark 10 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 259 conditions (Fig. 3D, E). Thus, the suppression of phytoalexin accumulation in darkness was not accompanied 260 by a detectable reduction in the early jasmonate response at this time point. 261 262 **3.3. Sucrose application restored phytoalexin production under dark conditions** 263 To assess whether the decrease in endogenous soluble sugar levels suppresses phytoalexin 264 production, we examined whether exogenously applied sucrose could restore it. Phytoalexin accumulation 265 after CuCl 2 treatment in the dark was suppressed, similar to that shown in Fig. 1. When sucrose was applied 266 with CuCl 2 in the dark, phytoalexin accumulation increased in a dose-dependent manner after 72 h of treatment 267 (Fig. 4A–C). The addition of 3 mM sucrose did not increase phytoalexin accumulation, whereas 30 mM 268 sucrose partially restored it. When 300 mM sucrose was added, the accumulation levels of all three 269 phytoalexin groups were not significantly different from those induced by CuCl 2 under light conditions, 270 although the mean momilactone level reached only approximately one-third of that in the light. We then 271 analyzed the transcript levels of the corresponding biosynthetic genes, _OsCPS2_, _OsKSL7_, _OsCPS4_, _OsKSL4_, 272 _OsDXS3_, and _OsNOMT_ (Fig. 4D–I), 24 h after CuCl 2 treatment. Under light conditions, all six genes were 273 induced by CuCl 2 . In the absence of CuCl 2, transcript levels of the five diterpenoid-related genes were 274 approximately 4- to 25-fold higher in darkness than in the light, whereas that of _OsNOMT_ was lower. Under 275 dark conditions, the induction of _OsCPS2_, _OsKSL7_, _OsCPS4_, and _OsNOMT_ was reduced, whereas _OsKSL4_ 276 and _OsDXS3_ showed a similar tendency that did not reach statistical significance in this experiment. The 277 application of 300 mM sucrose together with CuCl 2 restored the expression of _OsCPS2_, _OsKSL7_, _OsCPS4_, and 278 _OsNOMT_ to levels comparable to those observed under light conditions. Treatment with sucrose alone had 279 little effect on inducing the expression of phytoalexin biosynthetic genes, except for _OsKSL7_ and _OsNOMT_, 280 which showed small but significant increases in the dark. However, these increases were less than 2% of the 281 level induced by CuCl 2 treatment under light conditions. These results indicate that exogenous sucrose restored 282 CuCl 2 -induced phytoalexin accumulation and substantially restored the expression of several phytoalexin 283 biosynthetic genes under dark conditions. 284 285 **3.4. Restoration was specific to metabolizable sugars** 11 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 286 We investigated whether the recovery of CuCl 2 -induced phytoalexin production in the dark 287 following the addition of sucrose was due to osmotic effects. Under dark conditions, 300 mM sucrose or 288 mannitol was applied together with CuCl 2, and phytoalexin accumulation was quantified. Phytoalexins were 289 not induced by the treatment of sucrose or mannitol alone (Fig. 5A–C). The addition of sucrose restored 290 CuCl 2 -induced phytoalexin accumulation under dark conditions, whereas mannitol at the same concentration 291 did not. These results indicate that the sucrose-mediated restoration was not due solely to an osmotic effect. 292 We further examined the effect of 2-deoxy- D -glucose (2-DG), a glucose analog that is 293 phosphorylated by hexokinase but is poorly metabolized beyond 2-deoxy- D -glucose-6-phosphate, thereby 294 interfering with glucose metabolism. In the dark, we applied 300 mM D -glucose (Glc) or 2-DG with CuCl 2 and 295 quantified phytoalexin accumulation after 72 h of treatment. Glc partially restored phytocassane and 296 momilactone accumulation under dark conditions (to 13% and 8% of the levels in the light, respectively), 297 whereas 2-DG did not; phytocassanes remained undetectable, and momilactone levels fell below those in leaf 298 disks treated with CuCl 2 alone in the dark (Fig. 5D, E). Glc restored sakuranetin accumulation to a level 299 comparable to that observed under light conditions, whereas 2-DG did not restore its accumulation (Fig. 5F). 300 The contrasting effects of glucose and 2-DG are consistent with a role for sugar metabolism in the restoration 301 of phytoalexin production, although inhibitory effects of 2-DG unrelated to glycolytic blockade cannot be 302 excluded. 303 304 **3.5. Inhibition of photosynthesis and treatment with 2-DG suppressed phytoalexin accumulation under** 305 **light conditions** 306 To investigate whether inhibiting photosynthesis can mimic the suppression of phytoalexin 307 production, we analyzed the effects of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of 308 photosynthetic electron transport, on phytoalexin accumulation. Treatment with 10 µM DCMU reduced 309 endogenous sucrose levels to 13% and 6% of the corresponding untreated controls in the absence and presence 310 of CuCl 2, respectively (Fig. 6A). Neither glucose nor fructose was detectable under the assay conditions 311 following DCMU treatment (Fig. 6B, C). Next, we quantified phytoalexin accumulation following the addition 312 of DCMU. The addition of DCMU suppressed the accumulation of all CuCl 2 -induced phytoalexins 12 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 313 (phytocassanes, momilactones, and sakuranetin) to 5% or less (Fig. 6D–F). When 300 mM sucrose was added 314 along with CuCl 2 and DCMU, phytoalexin production recovered to levels comparable to those in CuCl 2 315 treated leaf disks without DCMU (Fig. 6D–F). The restoration by sucrose suggests that reduced carbohydrate 316 availability contributes substantially to the suppression of phytoalexin production by DCMU under these 317 conditions. 318 We also examined the effect of 2-DG on phytoalexin accumulation under light conditions (Fig. 6G– 319 I). The addition of 2-DG dose-dependently inhibited CuCl 2 -induced phytoalexin accumulation under light 320 conditions. The addition of 3 mM 2-DG did not inhibit the accumulation of any phytoalexins. Instead, 3 mM 321 2-DG, like 3 mM glucose, significantly increased phytocassane accumulation (Fig. 6G). Treatment with 30 322 mM 2-DG significantly reduced the accumulation of all three phytoalexin groups. At 300 mM, 2-DG reduced 323 the accumulation of the three phytoalexin groups to 1%–14% of their respective controls. Under light 324 conditions, the addition of 300 mM glucose partially inhibited CuCl 2 -induced phytocassane accumulation but 325 significantly increased sakuranetin accumulation (Fig. 6G, I). Taken together, these results indicate that CuCl 2 326 induced phytoalexin production is supported by an adequate supply of metabolizable carbohydrates, which is 327 normally sustained by photosynthesis under light conditions, and that the depletion of endogenous soluble 328 sugars is a major contributing factor to its suppression under dark conditions. 329 330 **4. Discussion** 331 The present study shows that CuCl 2 -induced phytoalexin production in rice leaves depends strongly 332 on light and that this dependence is largely accounted for by the availability of metabolizable carbohydrates. In 333 darkness, endogenous sucrose, glucose and fructose declined to approximately 3%–8% of the levels in the light 334 by 24 h after CuCl 2 application (Fig. 3A–C), and both the induction of phytoalexin biosynthetic genes and the 335 accumulation of diterpenoid and flavonoid phytoalexins were strongly reduced (Figs. 1 and 2). Exogenous 336 sucrose restored phytoalexin accumulation in darkness in a dose-dependent manner and, at 300 mM, restored 337 the expression of the biosynthetic genes whose induction had been suppressed (Fig. 4). An equivalent 338 concentration of mannitol did not restore phytoalexin accumulation (Fig. 5A–C). Glucose, but not 2-DG, 339 restored sakuranetin accumulation and partially restored diterpenoid accumulation in darkness (Fig. 5D–F). In 13 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 340 the light, DCMU depleted soluble sugars and suppressed phytoalexin accumulation, and this suppression was 341 overcome by sucrose (Fig. 6A–F). Together, these results support a model in which light, through 342 photosynthesis, maintains a carbohydrate status that permits CuCl 2 -induced transcription of phytoalexin 343 biosynthetic genes and the subsequent accumulation of their products, and in which the suppression observed 344 in darkness results largely from carbohydrate depletion. 345 346 **4.1. Light sustains phytoalexin production mainly through carbohydrate supply** 347 Light affects plant defense not only through photosynthetic carbon supply but also through 348 photoreceptors, chloroplast redox status, and reactive oxygen species (Kangasjärvi et al., 2012). In rice, 349 phytochromes contribute to jasmonate- and salicylic acid-related responses and to resistance against the blast 350 fungus (Xie et al., 2011), and light-dependent accumulation of sakuranetin has been reported (Ueno et al., 351 2015). In this study, DCMU suppressed phytoalexin accumulation to 5% or less of the control under 352 continuous light (Fig. 6D–F). Because DCMU blocks electron transfer from photosystem II but does not 353 prevent light perception by photoreceptors, photoreceptor signaling in the light was not sufficient to sustain 354 phytoalexin production when photosynthetic carbon supply was restricted. Moreover, metabolizable sugars 355 restored phytoalexin production in darkness (Figs. 4 and 5), showing that light was dispensable when 356 carbohydrates were supplied exogenously. These two observations indicate that, in our system, the effect of 357 light on CuCl 2 -induced phytoalexin production is mediated largely through carbohydrate supply. However, 358 these observations do not exclude contributions from other light-dependent signals. DCMU also alters the 359 redox state of the photosynthetic electron transport chain, and in _A. thaliana_, inhibitors acting at different sites 360 of this chain differ in their effects on flg22-induced gene expression (Sano et al., 2014). In addition, 361 momilactone accumulation after sucrose supplementation in darkness reached approximately one-third of that 362 in the light, although the difference was not statistically significant (Figs. 4B and 5B). A minor contribution of 363 carbohydrate-independent light signals therefore cannot be ruled out, and photoreceptor mutants will be 364 needed to assess it directly. 365 366 **4.2. Carbohydrates act permissively, and their metabolism contributes to sustaining phytoalexin** 14 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 367 **production** 368 Exogenous sugars restored CuCl 2 -induced phytoalexin production but induced little on their own, 369 indicating that carbohydrates act permissively rather than as elicitors in this system. In darkness, neither 300 370 mM sucrose nor mannitol alone induced phytoalexin accumulation (Fig. 5A–C). Sucrose alone did not change 371 the transcript levels of four of the six biosynthetic genes, and the increases in _OsKSL7_ and _OsNOMT_ 372 transcripts were less than 2% of the levels induced by CuCl 2 in the light (Fig. 4D–I). This contrasts with a 373 report that glucose, sucrose, fructose and maltose at 10 mM induce phytoalexin accumulation in rice leaves 374 (Nishimura et al., 2021). The two studies differ in leaf age, tissue preparation, light conditions, sugar 375 concentration and treatment duration, and the present data do not allow us to attribute the difference to any one 376 of these factors. Our results add that, once phytoalexin production has been elicited, its magnitude depends on 377 carbohydrate availability. This view is compatible with the involvement of sucrose in the priming of defense 378 responses in rice (Gómez-Ariza et al., 2007). Similarly, in _A. thaliana_, glucose and sucrose alone did not 379 induce the salicylic acid biosynthetic gene _PBS3_ but enhanced its flg22-triggered expression, whereas mannitol 380 did not affect flg22-triggered defense gene expression (Yamada and Mine, 2024). 381 Whether carbohydrates act as substrates or as signals cannot be resolved from the present data. The 382 restoration of transcript levels by sucrose (Fig. 4D–I) shows that carbohydrate status affects the expression of 383 biosynthetic genes, but such restoration would also be expected if transcription were constrained by energy 384 supply. In darkness, 300 mM 2-DG did not substitute for glucose and reduced momilactone and sakuranetin 385 levels even below those in leaf disks treated with CuCl 2 alone (Fig. 5D–F). In the light, 2-DG suppressed 386 CuCl 2 -induced accumulation in a dose-dependent manner, whereas glucose at the same concentrations did not 387 reduce sakuranetin or momilactone accumulation (Fig. 6G–I). Because equimolar glucose, which imposes the 388 same osmotic load, did not reduce sakuranetin or momilactone accumulation, the suppression by 2-DG cannot 389 be attributed simply to osmotic stress, and its graded nature argues against an abrupt loss of tissue viability. 390 Since 2-DG is phosphorylated by hexokinase, these results suggest that hexokinase-dependent sugar signaling 391 alone is insufficient and that metabolism downstream of hexose phosphorylation contributes to sustaining 392 phytoalexin production. At the lowest concentration (3 mM), however, 2-DG increased phytocassane 393 accumulation, as did 3 mM glucose (Fig. 6G). In _A. thaliana_, 0.5 mM 2-DG induces camalexin accumulation, 15 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 394 and its induction of defense-related genes requires hexokinase and STPs (Yamada and Mine, 2024). 2-DG 395 should therefore not be regarded as an inert analog: at low concentrations it may act as a sugar signal, whereas 396 at high concentrations its inhibition of glucose metabolism appears to predominate. Energy- and nutrient 397 sensing pathways, such as those involving SnRK1, TOR and trehalose 6-phosphate (Figueroa and Lunn, 2016; 398 Margalha et al., 2019), are possible candidates for linking carbohydrate status to the expression of phytoalexin 399 biosynthetic genes. 400 401 **4.3. Diterpenoid phytoalexins depend more strongly on light than sakuranetin** 402 Diterpenoid phytoalexins were more strongly dependent on light than sakuranetin. In darkness, 403 phytocassanes and momilactones accumulated to less than 2% of the levels in the light at 48 and 72 h, whereas 404 sakuranetin still reached 11%–25% from 24 h onward (Fig. 1). The same tendency was observed in the 405 independent experiments shown in Figs. 4A–C and 5A–C. Several observations suggest that sakuranetin 406 production is partly buffered against carbohydrate limitation. Although _OsNOMT_ transcripts in darkness were 407 reduced to 0.8% of those in the light at 24 h (Fig. 2F), sakuranetin accumulation was reduced far less, and 408 during the first 6 h after CuCl 2 application sakuranetin levels were even slightly higher in the dark (Fig. 1D). 409 Moreover, within the same experiment, 300 mM glucose restored sakuranetin to the level in the light but 410 restored diterpenoids only partially (Fig. 5D–F). Both classes depend on plastid-derived precursors: the 411 diterpenoids on the MEP pathway (Okada et al., 2007) and sakuranetin on phenylalanine produced by the 412 plastidic shikimate pathway, together with cytosolic malonyl-CoA; sakuranetin is then formed from naringenin 413 by 7- _O_ -methylation (Shimizu et al., 2012). The MEP pathway, however, consumes glyceraldehyde 3 414 phosphate and pyruvate together with energy and reducing equivalents (Vranová et al., 2013), and in darkness 415 chloroplasts depend on an external supply of ATP (Weber and Linka, 2011). This demand may render 416 diterpenoid biosynthesis particularly sensitive to carbohydrate depletion. Notably, the two classes also differ in 417 their requirement for JA-Ile (Miyamoto et al., 2016; Shimizu et al., 2013), yet both depended on carbohydrate 418 availability. Carbohydrate supply may therefore represent a common constraint on phytoalexin production that 419 operates independently of differences in jasmonate signaling. 420 16 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 421 **4.4. Carbohydrate limitation was not accompanied by a detectable reduction in early jasmonate** 422 **accumulation** 423 Darkness did not detectably reduce JA or JA-Ile accumulation 2 h after CuCl 2 application (Fig. 3D, 424 E), even though the leaf disks had been kept in darkness for 24 h before elicitation. Because CuCl 2 rapidly 425 increases endogenous JA in rice leaves (Rakwal et al., 1996), this observation suggests that the early jasmonate 426 response is largely maintained in darkness and that carbohydrate status acts downstream of, or in parallel with, 427 jasmonate biosynthesis. Consistent with this interpretation, carbohydrate dependence was shared by 428 sakuranetin, whose production requires JA-Ile, and by the diterpenoid phytoalexins, whose production does not 429 (Miyamoto et al., 2016; Shimizu et al., 2013). Transcriptional regulators of the two pathways, such as DPF for 430 the diterpenoids and OsMYC2 for sakuranetin (Ishikawa et al., 2024; Ogawa et al., 2017; Yamamura et al., 431 2015), are possible points at which carbohydrate status could act. However, JA and JA-Ile were measured at a 432 single time point after elicitation with three biological replicates, and the mean JA level in the dark was 72% of 433 that in the light, a difference that this experiment could not resolve. Later jasmonate dynamics or differences in 434 jasmonate sensitivity therefore cannot be excluded. Spraying glucose or sucrose has been reported to increase 435 jasmonate accumulation in rice seedlings (Lahari et al., 2024). Our results do not contradict this; they indicate 436 that, under our conditions, the suppression of phytoalexin production in darkness was not accompanied by a 437 comparable reduction in early jasmonate accumulation. 438 439 **4.5. CuCl** **2** **elicitation is associated with depletion of soluble sugars** 440 CuCl 2 treatment itself reduced sucrose levels by approximately half, even in the light (Fig. 6A). 441 Whether this decrease reflects carbon consumption for defense or an inhibitory effect of CuCl 2 toxicity on 442 photosynthesis cannot be distinguished from the present data. In either case, elicited leaf tissues operate with a 443 diminished carbohydrate pool, and when photosynthetic supply is further restricted, this pool may become 444 insufficient to sustain phytoalexin production. This adds a physiological dimension to the concept that sugars 445 act as both nutrients and signals in plant immunity (Bolouri Moghaddam and Van den Ende, 2012), and to 446 observations that carbohydrate availability can limit defense in other species (Engelsdorf et al., 2013; Essmann 447 et al., 2008). In the field, leaf carbohydrate status fluctuates over the diurnal cycle (Smith and Stitt, 2007). Our 17 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 448 results raise the possibility that such fluctuations affect the capacity of rice leaves to produce phytoalexins 449 upon elicitation, a possibility that remains to be tested in intact plants and in pathogen interactions. 450 451 **4.6. Limitations and perspectives** 452 This study has several limitations. One concerns the generalizability of findings obtained using the 453 present experimental system. We used detached leaf disks, in which source–sink relationships are disrupted 454 and wounding may interact with elicitation. Whether the present findings extend to intact plants remains to be 455 determined. In addition, CuCl 2 is an abiotic elicitor, and it remains to be tested whether the same carbohydrate 456 dependence applies to responses induced by pathogens or by microbe-associated molecular patterns. 457 Continuous light and continuous darkness represent extreme conditions compared with natural diurnal cycles. 458 Prolonged darkness of detached leaf disks may induce starvation- and senescence-related responses, as 459 suggested by the elevated basal transcript levels of diterpenoid biosynthetic genes in darkness (Figs. 2 and 4). 460 The carbohydrate-manipulation experiments also require cautious interpretation. The 300 mM sugar 461 concentration used in the restoration experiments exceeds physiological levels. However, this concentration 462 has been used in studies of sucrose-induced defense priming in rice (Gómez-Ariza et al., 2007). Moreover, 463 equimolar mannitol did not reproduce the effect of sucrose (Fig. 5A–C), and sucrose restored phytoalexin 464 production in a dose-dependent manner over the range of 3–300 mM (Fig. 4A–C). Nevertheless, high sugar 465 concentrations may themselves influence phytoalexin production: 300 mM glucose moderately reduced 466 phytocassane accumulation in the light (Fig. 6G). Furthermore, endogenous sugar levels after supplementation 467 were not measured. DCMU and 2-DG may also have effects other than those intended; for example, 2-DG can 468 interfere with protein _N_ -glycosylation and sequester phosphate as 2-DG-6-phosphate. 469 Further constraints concern comparisons across experiments and parameters that were not measured. 470 The glucose and 2-DG experiments were performed with ‘Nipponbare’, whereas the remaining experiments 471 used ‘Nihonmasari’. Absolute phytoalexin levels also varied among independent experiments, potentially 472 reflecting differences in cultivar and plant growth conditions. We therefore restricted comparisons to 473 treatments and their corresponding controls within each experiment. We did not assess tissue viability or 474 copper uptake. In one experiment, CuCl 2 slightly reduced the already low basal levels of diterpenoid 18 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 475 phytoalexins in darkness (Fig. 5D, E), and light-dependent differences in copper toxicity cannot be excluded. 476 However, sucrose restored CuCl 2 -induced phytoalexin production in darkness, arguing against irreversible 477 tissue damage as the sole explanation. Gene expression was analyzed in the dark–sucrose system but not in the 478 DCMU experiments, in which the effects of DCMU were assessed only at the metabolite level. 479 Future studies using photoreceptor mutants and mutants affected in starch metabolism or sugar 480 transport, together with direct measurements of cellular energy status, will help clarify how carbohydrate 481 availability is translated into phytoalexin production. 482 483 **5. Conclusions** 484 CuCl 2 -induced phytoalexin production in rice leaves was strongly suppressed in darkness, and this 485 suppression was accompanied by depletion of soluble sugars but not by a detectable reduction in early 486 jasmonate accumulation. Metabolizable sugars, but not mannitol or 2-DG, restored phytoalexin production in 487 darkness, and inhibition of photosynthesis by DCMU reproduced the suppression in the light in a sucrose 488 reversible manner. These findings indicate that photosynthesis-derived carbohydrate supply sustains the 489 production of both diterpenoid and flavonoid phytoalexins in rice and is a major determinant of its light 490 dependence. 491 492 **CRediT authorship contribution statement** 493 Naoya Misawa: Formal analysis, Investigation, Visualization, Writing – review & editing. 494 Yumi Oshima: Formal analysis, Validation, Investigation, Visualization, Writing – review & editing. 495 Tomohiro Sagara: Investigation, Formal analysis. 496 Natsumi Tate: Investigation, Formal analysis. 497 Shuhei Yoshinaga: Investigation, Formal analysis. 498 Tomoko Sakazawa: Investigation. 499 Emi Yumoto: Investigation, Methodology, Formal analysis. 500 Hisakazu Yamane: Conceptualization, Resources, Supervision, Writing – review & editing, Funding 501 acquisition. 19 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 502 Koji Miyamoto: Conceptualization, Data curation, Methodology, Formal analysis, Resources, Visualization, 503 Writing – original draft, Writing – review & editing, Supervision, Project administration, Funding acquisition. 504 505 **Funding** 506 This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI 507 (JP18K14399 to K.M. and JP16K07722 to H.Y.); the Ministry of Education, Culture, Sports, Science and 508 Technology-Supported Program for the Strategic Research Foundation at Private Universities (S131052A01 to 509 H.Y.); the Advanced Comprehensive Research Organization (ACRO) Research Grants of Teikyo University 510 (TeTe20-01 to K.M.); and the ACRO Incubation Grants of Teikyo University (25-137 to K.M.). 511 512 **Declaration of competing interests** 513 The authors declare the following financial interests/personal relationships which may be considered as 514 potential competing interests: Koji Miyamoto reports financial support was provided by Japan Society for the 515 Promotion of Science. Hisakazu Yamane reports financial support was provided by Japan Society for the 516 Promotion of Science. Hisakazu Yamane reports financial support was provided by Government of Japan 517 Ministry of Education Culture Sports Science and Technology. If there are other authors, they declare that they 518 have no known competing financial interests or personal relationships that could have appeared to influence 519 the work reported in this paper. 520 521 **Acknowledgements** 522 We thank Dr. Tomoko Shinomura (formerly of Teikyo University) and Dr. Eiichi Minami (formerly 523 of the National Agriculture and Food Research Organization) for valuable advice on this study, Emeritus 524 Professor Moritoshi Iino (Osaka City University, now Osaka Metropolitan University) for providing seeds of 525 rice ‘Nihonmasari’, and Shota Abe (Teikyo University) for technical assistance. 526 527 **Declaration of generative AI and AI-assisted technologies in the manuscript preparation process** 528 During the preparation of this work, the authors used Claude (Anthropic), DeepL (DeepL SE), and 20 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 529 Perplexity (Perplexity AI) to draft, translate, and revise the English text and to assist with literature searches, 530 and Claude and Perplexity to generate R code for statistical analyses and to check the consistency of statistical 531 results. After using these tools, the authors reviewed and edited the content, verified the cited sources and the 532 analysis outputs, and take full responsibility for the content of the published article. 533 534 **Data availability** 535 The complete results of the statistical analyses are provided in Table S2. Raw data are available from 536 the corresponding author upon reasonable request. 537 21 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 538 **References** 539 Ahuja, I., Kissen, R., Bones, A.M., 2012. Phytoalexins in defense against pathogens. Trends Plant Sci. 17, 73– 540 90. https://doi.org/10.1016/j.tplants.2011.11.002 541 Bolouri Moghaddam, M.R., Van den Ende, W., 2012. Sugars and plant innate immunity. J. Exp. Bot. 63, 542 3989–3998. https://doi.org/10.1093/jxb/ers129 543 Engelsdorf, T., Horst, R.J., Pröls, R., Pröschel, M., Dietz, F., Hückelhoven, R., Voll, L.M., 2013. Reduced 544 carbohydrate availability enhances the susceptibility of Arabidopsis toward Colletotrichum 545 higginsianum. Plant Physiol. 162, 225–238. https://doi.org/10.1104/pp.112.209676 546 Essmann, J., Schmitz-Thom, I., Schön, H., Sonnewald, S., Weis, E., Scharte, J., 2008. RNA interference 547 mediated repression of cell wall invertase impairs defense in source leaves of tobacco. Plant Physiol. 548 147, 1288–1299. https://doi.org/10.1104/pp.108.121418 549 Figueroa, C.M., Lunn, J.E., 2016. A tale of two sugars: Trehalose 6-phosphate and sucrose. Plant Physiol. 172, 550 7–27. https://doi.org/10.1104/pp.16.00417 551 Gómez-Ariza, J., Campo, S., Rufat, M., Estopà, M., Messeguer, J., San Segundo, B., Coca, M., 2007. Sucrose 552 mediated priming of plant defense responses and broad-spectrum disease resistance by overexpression 553 of the maize pathogenesis-related PRms protein in rice plants. Mol. Plant Microbe Interact. 20, 832– 554 842. https://doi.org/10.1094/MPMI-20-7-0832 555 Hasegawa, M., Mitsuhara, I., Seo, S., Imai, T., Koga, J., Okada, K., Yamane, H., Ohashi, Y., 2010. 556 Phytoalexin accumulation in the interaction between rice and the blast fungus. Mol. Plant Microbe 557 Interact. 23, 1000–1011. https://doi.org/10.1094/MPMI-23-8-1000 558 He, Y., Zhao, Y., Hu, J., Wang, L., Li, L., Zhang, X., Zhou, Z., Chen, L., Wang, H., Wang, J., Hong, G., 2024. 559 The OsBZR1-OsSPX1/2 module fine-tunes the growth-immunity trade-off in adaptation to phosphate 560 availability in rice. Mol. Plant 17, 258–276. https://doi.org/10.1016/j.molp.2023.12.003 561 Inagaki, H., Hayashi, K., Takaoka, Y., Ito, H., Fukumoto, Y., Yajima-Nakagawa, A., Chen, X., Shimosato 562 Nonaka, M., Hassett, E., Hatakeyama, K., Hirakuri, Y., Ishitsuka, M., Yumoto, E., Sakazawa, T., 563 Asahina, M., Uchida, K., Okada, K., Yamane, H., Ueda, M., Miyamoto, K., 2023. Genome editing 564 reveals both the crucial role of OsCOI2 in jasmonate signaling and the functional diversity of COI1 22 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 565 homologs in rice. Plant Cell Physiol. 64, 405–421. https://doi.org/10.1093/pcp/pcac166 566 Ishikawa, K., Yamamura, C., Miyamoto, K., Kanda, Y., Inoue, H., Okada, K., Kamakura, T., Mori, M., 2024. 567 Rice transcription factor DPF regulates stress-induced biosynthesis of diterpenoid phytoalexins. 568 Biosci. Biotechnol. Biochem. 88, 1344–1348. https://doi.org/10.1093/bbb/zbae118 569 Jikumaru, Y., Asami, T., Seto, H., Yoshida, S., Yokoyama, T., Obara, N., Hasegawa, M., Kodama, O., 570 Nishiyama, M., Okada, K., Nojiri, H., Yamane, H., 2004. Preparation and biological activity of 571 molecular probes to identify and analyze jasmonic acid-binding proteins. Biosci. Biotechnol. 572 Biochem. 68, 1461–1466. https://doi.org/10.1271/bbb.68.1461 573 Kangasjärvi, S., Neukermans, J., Li, S., Aro, E.-M., Noctor, G., 2012. Photosynthesis, photorespiration, and 574 light signalling in defence responses. J. Exp. Bot. 63, 1619–1636. https://doi.org/10.1093/jxb/err402 575 Lahari, Z., van Boerdonk, S., Omoboye, O.O., Reichelt, M., Höfte, M., Gershenzon, J., Gheysen, G., Ullah, C., 576 2024. Strigolactone deficiency induces jasmonate, sugar and flavonoid phytoalexin accumulation 577 enhancing rice defense against the blast fungus Pyricularia oryzae. New Phytol. 241, 827–844. 578 https://doi.org/10.1111/nph.19354 579 Lu, X., Zhang, J., Brown, B., Li, R., Rodríguez-Romero, J., Berasategui, A., Liu, B., Xu, M., Luo, D., Pan, Z., 580 Baerson, S.R., Gershenzon, J., Li, Z., Sesma, A., Yang, B., Peters, R.J., 2018. Inferring roles in 581 defense from metabolic allocation of rice diterpenoids. Plant Cell 30, 1119–1131. 582 https://doi.org/10.1105/tpc.18.00205 583 Margalha, L., Confraria, A., Baena-González, E., 2019. SnRK1 and TOR: modulating growth-defense trade 584 offs in plant stress responses. J. Exp. Bot. 70, 2261–2274. https://doi.org/10.1093/jxb/erz066 585 Miyamoto, K., 2026. Regulatory mechanisms and evolutionary insights of phytoalexin biosynthesis in rice. 586 Biosci. Biotechnol. Biochem. 90, 158–165. https://doi.org/10.1093/bbb/zbaf166 587 Miyamoto, K., Enda, I., Okada, T., Sato, Y., Watanabe, K., Sakazawa, T., Yumoto, E., Shibata, K., Asahina, 588 M., Iino, M., Yokota, T., Okada, K., Yamane, H., 2016. Jasmonoyl-l-isoleucine is required for the 589 production of a flavonoid phytoalexin but not diterpenoid phytoalexins in ultraviolet-irradiated rice 590 leaves. Biosci. Biotechnol. Biochem. 80, 1934–1938. https://doi.org/10.1080/09168451.2016.1189319 591 Nishimura, A., Yoshioka, A., Kariya, K., Ube, N., Ueno, K., Tebayashi, S.-I., Osaki-Oka, K., Ishihara, A., 23 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 592 2021. Sugars in an aqueous extract of the spent substrate of the mushroom Hypsizygus marmoreus 593 induce defense responses in rice. Biosci. Biotechnol. Biochem. 85, 743–755. 594 https://doi.org/10.1093/bbb/zbaa122 595 Ogawa, S., Miyamoto, K., Nemoto, K., Sawasaki, T., Yamane, H., Nojiri, H., Okada, K., 2017. OsMYC2, an 596 essential factor for JA-inductive sakuranetin production in rice, interacts with MYC2-like proteins that 597 enhance its transactivation ability. Sci. Rep. 7, 40175. https://doi.org/10.1038/srep40175 598 Okada, A., Shimizu, T., Okada, K., Kuzuyama, T., Koga, J., Shibuya, N., Nojiri, H., Yamane, H., 2007. 599 Elicitor induced activation of the methylerythritol phosphate pathway toward phytoalexins 600 biosynthesis in rice. Plant Mol. Biol. 65, 177–187. https://doi.org/10.1007/s11103-007-9207-2 601 Rakwal, R., Tamogami, S., Kodama, O., 1996. Role of jasmonic acid as a signaling molecule in copper 602 chloride-elicited rice phytoalexin production. Biosci. Biotechnol. Biochem. 60, 1046–1048. 603 https://doi.org/10.1271/bbb.60.1046 604 Riemann, M., Haga, K., Shimizu, T., Okada, K., Ando, S., Mochizuki, S., Nishizawa, Y., Yamanouchi, U., 605 Nick, P., Yano, M., Minami, E., Takano, M., Yamane, H., Iino, M., 2013. Identification of rice Allene 606 Oxide Cyclase mutants and the function of jasmonate for defence against Magnaporthe oryzae. Plant 607 J. 74, 226–238. https://doi.org/10.1111/tpj.12115 608 Sano, S., Aoyama, M., Nakai, K., Shimotani, K., Yamasaki, K., Sato, M.H., Tojo, D., Suwastika, I.N., 609 Nomura, H., Shiina, T., 2014. Light-dependent expression of flg22-induced defense genes in 610 Arabidopsis. Front. Plant Sci. 5, 531. https://doi.org/10.3389/fpls.2014.00531 611 Shimizu, T., Lin, F., Hasegawa, M., Okada, K., Nojiri, H., Yamane, H., 2012. Purification and identification of 612 naringenin 7-O-methyltransferase, a key enzyme in biosynthesis of flavonoid phytoalexin sakuranetin 613 in rice. J. Biol. Chem. 287, 19315–19325. https://doi.org/10.1074/jbc.M112.351270 614 Shimizu, T., Miyamoto, K., Miyamoto, K., Minami, E., Nishizawa, Y., Iino, M., Nojiri, H., Yamane, H., 615 Okada, K., 2013. OsJAR1 contributes mainly to biosynthesis of the stress-induced jasmonoyl 616 isoleucine involved in defense responses in rice. Biosci. Biotechnol. Biochem. 77, 1556–1564. 617 https://doi.org/10.1271/bbb.130272 618 Smith, A.M., Stitt, M., 2007. Coordination of carbon supply and plant growth. Plant Cell Environ. 30, 1126– 24 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 619 1149. https://doi.org/10.1111/j.1365-3040.2007.01708.x 620 Toyomasu, T., Usui, M., Sugawara, C., Otomo, K., Hirose, Y., Miyao, A., Hirochika, H., Okada, K., Shimizu, 621 T., Koga, J., Hasegawa, M., Chuba, M., Kawana, Y., Kuroda, M., Minami, E., Mitsuhashi, W., 622 Yamane, H., 2014. Reverse-genetic approach to verify physiological roles of rice phytoalexins: 623 characterization of a knockdown mutant of OsCPS4 phytoalexin biosynthetic gene in rice. Physiol. 624 Plant. 150, 55–62. https://doi.org/10.1111/ppl.12066 625 Tun, W., Yoon, J., Vo, K.T.X., Cho, L.-H., Hoang, T.V., Peng, X., Kim, E.-J., Win, K.T.Y.S., Lee, S.-W., 626 Jung, K.-H., Jeon, J.-S., An, G., 2023. Sucrose preferentially promotes expression of OsWRKY7 and 627 OsPR10a to enhance defense response to blast fungus in rice. Front. Plant Sci. 14, 1117023. 628 https://doi.org/10.3389/fpls.2023.1117023 629 Ueno, M., Kihara, J., Arase, S., 2015. Tryptamine and sakuranetin accumulation in Sekiguchi lesions 630 associated with the light-enhanced resistance of the lesion mimic mutant of rice to Magnaporthe 631 oryzae. J. Gen. Plant Pathol. 81, 1–4. https://doi.org/10.1007/s10327-014-0560-0 632 Valletta, A., Iozia, L.M., Fattorini, L., Leonelli, F., 2023. Rice phytoalexins: Half a century of amazing 633 discoveries; Part I: Distribution, biosynthesis, chemical synthesis, and biological activities. Plants 12, 634 260. https://doi.org/10.3390/plants12020260 635 Vranová, E., Coman, D., Gruissem, W., 2013. Network analysis of the MVA and MEP pathways for 636 isoprenoid synthesis. Annu. Rev. Plant Biol. 64, 665–700. https://doi.org/10.1146/annurev-arplant 637 050312-120116 638 Weber, A.P.M., Linka, N., 2011. Connecting the plastid: transporters of the plastid envelope and their role in 639 linking plastidial with cytosolic metabolism. Annu. Rev. Plant Biol. 62, 53–77. 640 https://doi.org/10.1146/annurev-arplant-042110-103903 641 Xie, X.-Z., Xue, Y.-J., Zhou, J.-J., Zhang, B., Chang, H., Takano, M., 2011. Phytochromes regulate SA and JA 642 signaling pathways in rice and are required for developmentally controlled resistance to Magnaporthe 643 grisea. Mol. Plant 4, 688–696. https://doi.org/10.1093/mp/ssr005 644 Yamada, K., Mine, A., 2024. Sugar coordinates plant defense signaling. Sci. Adv. 10, eadk4131. 645 https://doi.org/10.1126/sciadv.adk4131 25 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 646 Yamamura, C., Mizutani, E., Okada, K., Nakagawa, H., Fukushima, S., Tanaka, A., Maeda, S., Kamakura, T., 647 Yamane, H., Takatsuji, H., Mori, M., 2015. Diterpenoid phytoalexin factor, a bHLH transcription 648 factor, plays a central role in the biosynthesis of diterpenoid phytoalexins in rice. Plant J. 84, 1100– 649 1113. https://doi.org/10.1111/tpj.13065 650 Yamane, H., 2013. Biosynthesis of phytoalexins and regulatory mechanisms of it in rice. Biosci. Biotechnol. 651 Biochem. 77, 1141–1148. https://doi.org/10.1271/bbb.130109 652 26 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 653 **Figure legends** 654 **Fig. 1.** CuCl 2 -induced phytoalexin accumulation was suppressed under dark conditions. 655 Leaf disks prepared from the leaf blades of rice ( _Oryza sativa_ L. ‘Nihonmasari’) were incubated under 656 continuous white light or in darkness from the time of preparation, and 1 mM CuCl 2 was applied 24 h later (0 657 h). Samples were collected at 0, 6, 24, 48, and 72 h after CuCl 2 application. "Initial" refers to the sample 658 collected at the time of leaf-disk preparation, before the light and dark treatments were started, and is common 659 to both treatments. (A) Experimental design. (B) Phytocassanes. (C) Momilactones. (D) Sakuranetin. *, _p_ < 660 0.05; Welch’s t-test with Bonferroni correction. Values are means ± SE ( _n_ = 6). 661 n.d., not detected; time points at which the compound was not detected in both treatments were excluded from 662 the analysis. 663 664 **Fig. 2.** CuCl 2 -induced expression of genes relevant to phytoalexin production was suppressed under dark 665 conditions. 666 Leaf disks prepared from the leaf blades of rice ( _O. sativa_ L. ‘Nihonmasari’) were incubated under white light 667 or in darkness from the time of preparation, and 1 mM CuCl 2 was applied 24 h later (0 h). Transcript levels 668 were determined by quantitative RT-PCR and normalized to _OsUBQ_ . (A) _OsCPS2_ . (B) _OsKSL7_ . (C) _OsCPS4_ . 669 (D) _OsKSL4_ . (E) _OsDXS3_ . (F) _OsNOMT_ . Samples were collected at 0, 6, 12, and 24 h after CuCl 2 application. 670 "Initial" indicates the sample collected at the time of leaf-disk preparation, before the light and dark treatments 671 were started, and is common to both treatments; all values are expressed relative to this sample (Initial = 1). 672 Note that the y axis in (F) is scaled by × 10 [4] . Values are means ± SE ( _n_ = 3). *, _p_ < 0.05; Welch’s t-test with 673 Bonferroni correction. 674 675 **Fig. 3.** Dark treatment decreased endogenous soluble sugars without a detectable decrease in early CuCl 2 676 induced jasmonate accumulation. 677 (A–C) Leaf disks prepared from the leaf blades of rice ( _O. sativa_ L. ‘Nihonmasari’) were incubated under 678 white light or in darkness from the time of preparation, and 1 mM CuCl 2 was applied 24 h later (0 h). Samples 679 were collected at 0, 6, 12, and 24 h after CuCl 2 application. "Initial" indicates the sample collected at the time 27 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 680 of leaf-disk preparation, before the light and dark treatments were started, and is common to both treatments. 681 (A) Sucrose. (B) Glucose. (C) Fructose. *, _p_ < 0.05; Welch’s t-test with Bonferroni correction. Values are 682 means ± SE ( _n_ = 3). 683 (D, E) Leaf segments (approximately 1 cm long) prepared from the leaf blades of rice ( _O. sativa_ L. 684 ‘Nihonmasari’) were incubated under white light or in darkness from the time of preparation, and 1 mM CuCl 2 685 was applied 24 h later (0 h). Samples were collected at 0 and 2 h after CuCl 2 application. (D) Jasmonic acid. 686 (E) Jasmonoyl- L -isoleucine. n.s., not significant (Welch’s t-test with Bonferroni correction). Values are means 687 ± SE ( _n_ = 3). 688 689 **Fig. 4.** Sucrose application restored CuCl 2 -induced phytoalexin production under dark conditions. 690 Leaf disks prepared from the leaf blades of rice ( _O. sativa_ L. ‘Nihonmasari’) were incubated under white light 691 or in darkness from the time of preparation, and 1 mM CuCl 2 was applied 24 h later, either alone or together 692 with sucrose (Suc; 3, 30, or 300 mM). (A–C) Phytoalexin levels 72 h after CuCl 2 application: (A) 693 phytocassanes, (B) momilactones, (C) sakuranetin. (D–I) Transcript levels 24 h after CuCl 2 application, 694 determined by quantitative RT-PCR, normalized to _OsUBQ_ and expressed relative to the mean of the light 695 control (Light Control = 1; note that Fig. 2 is expressed relative to the Initial sample): (D) _OsCPS2_ . (E) 696 _OsKSL7_ . (F) _OsCPS4_ . (G) _OsKSL4_ . (H) _OsDXS3_ . (I) _OsNOMT_ . Values are means ± SE ( _n_ = 6 for 697 phytoalexins and _n_ = 3 for transcripts). Different letters indicate significant differences ( _p_ < 0.05; one-way 698 ANOVA followed by Tukey’s HSD test on log(x + 1)-transformed values for phytoalexins and on Δ _Ct_ values 699 for transcripts). n.d., not detected; these treatments were included in the analysis as zero values. 700 701 **Fig. 5.** Mannitol did not substitute for sucrose in restoring CuCl 2 -induced phytoalexin production under dark 702 conditions (A–C), nor did 2-deoxy- D -glucose substitute for glucose (D–F), indicating that a metabolizable 703 sugar is required. 704 (A–C) Leaf disks prepared from the leaf blades of rice ( _O. sativa_ L. ‘Nihonmasari’) were incubated under 705 white light or in darkness from the time of preparation. In darkness, 300 mM sucrose (Suc) or mannitol (Man) 706 was applied 24 h later, with or without 1 mM CuCl 2, and phytoalexins were quantified 72 h thereafter. (A) 28 [bioRxiv preprint doi: https://doi.org/10.64898/2026.10.29.761194; this version posted November 2, 2026. The copyright holder for this](https://doi.org/10.64898/2026.10.29.761194) preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in [perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 707 Phytocassanes. (B) Momilactones. (C) Sakuranetin. 708 (D–F) Leaf disks prepared from rice ( _O. sativa_ L. ‘Nipponbare’) were treated as in (A–C), except that 300 mM 709 glucose (Glc) or 2-deoxy- D -glucose (2-DG) was applied with 1 mM CuCl 2 in darkness. (D) Phytocassanes. (E) 710 Momilactones. (F) Sakuranetin. (A–C) and (D–F) are independent experiments performed with different 711 cultivars; absolute values should not be compared across the two sets of panels. Values are means ± SE ( _n_ = 6). 712 Different letters indicate significant differences ( _p_ < 0.05; one-way ANOVA followed by Tukey’s HSD test on 713 log(x + 1)-transformed values). n.d., not detected; these treatments were included in the analysis as zero 714 values. 715 716 **Fig. 6. DCMU and high concentrations of 2-DG suppressed CuCl** **2** **-induced phytoalexin production in** 717 **the light.** 718 (A–C) Leaf disks prepared from the leaf blades of rice ( _O. sativa_ L. ‘Nihonmasari’) were preincubated with 719 0.1% ethanol (control) or 10 µM DCMU under white light for 24 h, and then incubated in the same solution 720 with or without 1 mM CuCl 2 for 24 h. Endogenous levels of (A) sucrose, (B) glucose, and (C) fructose. 721 (D–F) Leaf disks were preincubated as in (A–C) and then incubated with or without 1 mM CuCl 2, with or 722 without 300 mM sucrose, for 72 h. (D) Phytocassanes. (E) Momilactones. (F) Sakuranetin. (G–I) Leaf disks 723 prepared from rice ( _O. sativa_ L. ‘Nipponbare’) were preincubated under white light for 24 h and then 724 incubated with 1 mM CuCl 2 together with glucose (Glc) or 2-deoxy- D -glucose (2-DG) at 3, 30, or 300 mM for 725 72 h. (G) Phytocassanes. (H) Momilactones. (I) Sakuranetin. 726 Values are means ± SE ( _n_ = 3 for A–C and _n_ = 6 for D–I). Different letters indicate significant differences ( _p_ < 727 0.05; one-way ANOVA followed by Tukey’s HSD test on log(x + 1)-transformed values). n.d., not detected; 728 these treatments were included in the analysis as zero values. 729 730 **Supplementary material** 731 Table S1. Primers used for quantitative RT-PCR. 732 Table S2. Results of statistical analyses. 733 29 A Leaf disks preparation CuCl 2 application B C D 30 20 10 0 Initial 0 24 48 72 Time (h) after CuCl 2 application 150 100 50 0 Initial 0 24 48 72 Time (h) after CuCl 2 application 2000 Sakuranetin 1500 1000 500 0 Initial 0 24 48 72 Time (h) after CuCl 2 application Figure 1 A B _OsCPS2_ 50 40 30 20 10 0 _OsKSL7_ 15000 10000 5000 Initial 0 12 24 Time (h) after CuCl 2 application C D _OsCPS4_ 500 0 Initial 0 12 24 Time (h) after CuCl 2 application _OsKSL4_ 3000 400 300 200 100 0 Initial 0 12 24 Time (h) after CuCl 2 application E F _OsDXS3_ 20 2000 1000 0 Initial 0 12 24 Time (h) after CuCl 2 application _OsNOMT_ 20 15 10 5 0 15 10 5 0 Initial 0 12 24 Time (h) after CuCl 2 application Initial 0 12 24 Time (h) after CuCl 2 application Figure 2 A 50 Sucrose 5 Glucose B 40 30 20 10 0 Initial 0 12 24 Time (h) after CuCl 2 application C D 4 3 2 1 0 Initial 0 12 24 Time (h) after CuCl 2 application E Jasmonic acid Jasmonoyl-isoleucine n.s. 5 Fructose 4 3 2 1 0 1.0 0.8 0.6 0.4 0.2 0 Initial 0 12 24 Time (h) after CuCl 2 application 1.0 0.8 0.6 0.4 0.2 0 Figure 3 A B C 250 200 150 100 50 0 Phytocassanes Momilactones Sakuranetin 400 0 2500 2000 1500 1000 500 300 200 100 0 n.d. c c n.d. D E |Phytocassanes|Col2|Col3| |---|---|---| |bc<br>a<br>d<br>d<br>cd<br>b<br>a<br>n.d.|bc<br>a<br>d<br>d<br>cd<br>b<br>a<br>n.d.|bc<br>a<br>d<br>d<br>cd<br>b<br>a<br>n.d.| |0<br>0|0<br>1|0<br>0<br>3<br>30<br>300<br>0<br>1<br>1<br>1<br>1<br>Dark| |Light|Light|Light| |Momilactones|Col2|Col3| |---|---|---| |n.d.<br>b<br>a<br>c<br>c<br>c<br>b<br>a|n.d.<br>b<br>a<br>c<br>c<br>c<br>b<br>a|n.d.<br>b<br>a<br>c<br>c<br>c<br>b<br>a| |0<br>0|0<br>1|0<br>0<br>3<br>30<br>300<br>0<br>1<br>1<br>1<br>1<br>Dark| |Light|Light|Light| |Sakuranetin a|Col2|Col3| |---|---|---| |n.d.<br>c<br>a<br>d<br>b<br>b<br>a<br>a|n.d.<br>c<br>a<br>d<br>b<br>b<br>a<br>a|n.d.<br>c<br>a<br>d<br>b<br>b<br>a<br>a| |n.d.<br>c<br>a<br>d<br>b<br>b<br>a<br>a|n.d.<br>c<br>a<br>d<br>b<br>b<br>a<br>a|| |0<br>0|0<br>0<br>3<br>30<br>300<br>0<br>1<br>1<br>1<br>1<br>Dark|0<br>0<br>3<br>30<br>300<br>0<br>1<br>1<br>1<br>1<br>Dark| F 4000 3000 2000 3000 2000 1000 200 150 100 50 0 1000 0 0 |OsCPS2 a|Col2|Col3| |---|---|---| |d<br>a<br>c<br>b<br>c|d<br>a<br>c<br>b<br>c|d<br>a<br>c<br>b<br>c| |0<br>0|0<br>1|0<br>0<br>300<br>300<br>0<br>1<br>1<br>0<br>Dark| |Light|Light|Light| |OsKSL7 a|Col2|Col3| |---|---|---| |e<br>a<br>d<br>b<br>c<br>a|e<br>a<br>d<br>b<br>c<br>a|e<br>a<br>d<br>b<br>c<br>a| |0<br>0|0<br>1|0<br>0<br>300<br>300<br>0<br>1<br>1<br>0<br>Dark| |Light|Light|Light| |OsCPS4 a|Col2|Col3| |---|---|---| |d<br>ab<br>c<br>b<br>c<br>a|d<br>ab<br>c<br>b<br>c<br>a|d<br>ab<br>c<br>b<br>c<br>a| |0<br>0|0<br>1|0<br>0<br>300<br>300<br>0<br>1<br>1<br>0<br>Dark| |Light|Light|Light| G H I _OsKSL4_ _OsDXS3_ 1000 2500 2000 1500 1000 500 1500 1000 500 800 600 400 200 0 0 |OsDXS3 a|Col2|Col3| |---|---|---| |c<br>a<br>b<br>a<br>b<br>a|c<br>a<br>b<br>a<br>b<br>a|c<br>a<br>b<br>a<br>b<br>a| |0<br>0|0<br>1|0<br>0<br>300<br>300<br>0<br>1<br>1<br>0<br>Dark| |Light|Light|Light| Figure 4 0 |OsKSL4 a|Col2|Col3| |---|---|---| |c<br><br>b<br>a<br>b<br>a|c<br><br>b<br>a<br>b<br>a|c<br><br>b<br>a<br>b<br>a| |0<br>0|0<br>1|0<br>0<br>300<br>300<br>0<br>1<br>1<br>0<br>Dark| |Light|Light|Light| |OsNOMT a|Col2|Col3| |---|---|---| |d<br>a<br>e<br>b<br>c<br>a|d<br>a<br>e<br>b<br>c<br>a|d<br>a<br>e<br>b<br>c<br>a| |d<br>a<br>e<br>b<br>c<br>a||| |0<br>0<br>0<br>1<br>Light|0<br>1|0<br>0<br>300<br>300<br>0<br>1<br>1<br>0<br>Dark| 4000 3000 2000 0 A D |Phytocassanes a|Col2|Col3| |---|---|---| |bc<br>a<br>c<br>n.d.<br>n.d.<br>bc<br>c<br>bc<br>a<br>b|bc<br>a<br>c<br>n.d.<br>n.d.<br>bc<br>c<br>bc<br>a<br>b|bc<br>a<br>c<br>n.d.<br>n.d.<br>bc<br>c<br>bc<br>a<br>b| |-<br>0|-<br>1|-<br>Suc Man<br>-<br>Suc<br>0<br>0<br>1<br>0<br>1<br>Dark<br>Man<br>1| |Light|Light|Light| |Momilactones|Col2|Col3| |---|---|---| |bc<br>a<br>c<br>bc<br>c<br>b<br>a<br>b|bc<br>a<br>c<br>bc<br>c<br>b<br>a<br>b|bc<br>a<br>c<br>bc<br>c<br>b<br>a<br>b| |-<br>0|-<br>1|-<br>Suc Man<br>-<br>Suc<br>0<br>0<br>1<br>0<br>1<br>Dark<br>Man<br>1| |Light|Light|Light| |Sakuranetin a|Col2|Col3| |---|---|---| |n.d.<br>n.d.<br>c<br><br>c<br>c<br>c<br>b<br>a<br>b|n.d.<br>n.d.<br>c<br><br>c<br>c<br>c<br>b<br>a<br>b|n.d.<br>n.d.<br>c<br><br>c<br>c<br>c<br>b<br>a<br>b| |-<br>0|-<br>1|-<br>Suc Man<br>-<br>Suc<br>0<br>0<br>1<br>0<br>1<br>Dark<br>Man<br>1| |Light|Light|Light| 100 60 40 0 500 300 200 0 b c bc n.d.c bc b 100 bc c bc c b b 1000 c n.d. c n.d. 100 bc 500 300 200 0 CuCl 2 [(mM)] 1 CuCl 2 [(mM)] E F 1 CuCl 2 [(mM)] 400 3000 2000 1000 n.d.d c n.d.d b n.d.d 100 e c d b n.d.e 1000 n.d.e 300 200 100 n.d. c n.d. n.d. e c n.d. n.d. c d |Momilactones a|Col2|Col3| |---|---|---| |e<br>a<br>c<br>b<br>d<br>e<br>n.d.|e<br>a<br>c<br>b<br>d<br>e<br>n.d.|e<br>a<br>c<br>b<br>d<br>e<br>n.d.| |-<br>0|-<br>1|-<br>-<br>Glc<br>2-DG<br>0<br>1<br>1<br>1<br>Dark| |Light|Light|Light| Figure 5 0 0 CuCl 2 [(mM)] CuCl 2 [(mM)] CuCl [(mM)] 2 |Phytocassanes|Col2|Col3| |---|---|---| |n.d.<br>n.d.<br>d<br>a<br>c<br>b<br>d<br>d<br>n.d.|n.d.<br>n.d.<br>d<br>a<br>c<br>b<br>d<br>d<br>n.d.|n.d.<br>n.d.<br>d<br>a<br>c<br>b<br>d<br>d<br>n.d.| |-<br>0|-<br>1|-<br>-<br>Glc<br>2-DG<br>0<br>1<br>1<br>1<br>Dark| |Light|Light|Light| |Sakuranetin a|Col2|Col3| |---|---|---| |n.d.<br>e<br><br>c<br>a<br>b<br>d|n.d.<br>e<br><br>c<br>a<br>b<br>d|n.d.<br>e<br><br>c<br>a<br>b<br>d| |n.d.<br>e<br><br>c<br>a<br>b<br>d||| |-<br>0|-<br>1|-<br>-<br>Glc<br>2-DG<br>0<br>1<br>1<br>1<br>Dark| |Light|Light|Light| A B C D 100 [Phytocassanes] Sucrose 50 40 30 20 0 a 1 Glucose 1 a 0.8 0.4 0 CuCl 2 (mM) 0 1 0 1 80 60 40 20 Fructose 1 0.6 a 0.4 0 1 0 1 CuCl 2 (mM) 0 1 0 1 Control 10 μM DCMU Control 10 μM DCMU 0 1 0 1 CuCl 2 (mM) 0 1 0 1 Control 10 μM DCMU Control 10 µM DCMU Control 10 µM DCMU Suc (mM) 0 0 0 0 300 Control 10 µM DCMU 200 Phytocassanes Sakuranetin 2000 1600 1200 800 G 150 100 50 40 30 b 20 a n.d. n.d. 0 0 |Momilactones 0 1 0 1 Control 10 μM DCMU a a|Col2| |---|---| |n.d.<br>a<br>a<br>b<br>c<br>bc|n.d.<br>a<br>a<br>b<br>c<br>bc| |n.d.<br>a<br>a<br>b<br>c<br>bc|| ||| 0 1 0 1 1 0 1 0 1 1 0 2 (mM) 0 1 0 1 1 Suc (mM) 0 0 0 0 300 CuCl 2 (mM) 0 1 0 1 1 CuCl 2 (mM) 0 1 0 1 Control 1 mM CuCl 2 Sakuranetin Glc Glc Glc 2-DG 2-DG 2-DG 0 0 3 30 300 3 30 300 Glc Glc Glc 2-DG 2-DG 2-DG 3 30 300 3 30 300 (mM) Control 10 μM DCMU Suc (mM) 0 0 0 0 300 Control 10 μM DCMU H I Momilactones 100 ab a 80 60 40 20 0 ab ab a 1500 1000 500 0 Glc Glc Glc 2-DG 2-DG 2-DG 0 0 3 30 300 3 30 300 Glc Glc Glc 2-DG 2-DG 2-DG 3 30 300 3 30 300 (mM) Glc Glc Glc 2-DG 2-DG 2-DG 0 0 3 30 300 3 30 300 Glc Glc Glc 2-DG 2-DG 2-DG 3 30 300 3 30 300 (mM) Control 1 mM CuCl 2 Control 1 mM CuCl 2 Figure 6