Rice ONAC039 Drives Leaf Senescence Through Direct Transcriptional Activation of Multiple Senescence-Associated Genes

doi:10.1016/j.rsci.2026.08.007

Abstract

Abstract:

Leaf senescence is a programmed cell death process that reallocates nutrients from aging leaves to developing parts of the plant, influencing crop yield and stress responses. NAC transcription factors play a crucial role in this transition, but the upstream signals and downstream targets in rice remain largely unclear. In this study, we identified ONAC039 as a positive regulator that connects abscisic acid (ABA) signaling to chlorophyll degradation. Transcripts of ONAC039 increased during age-dependent and dark-induced senescence, as well as following ABA treatment. CRISPR-Cas9-generated lines exhibited delayed yellowing and retained higher chlorophyll levels, whereas ONAC039 activation-tagged and overexpression lines showed accelerated senescence. ONAC039 upregulated key senescence-associated and chlorophyll catabolic genes, along with ABA signaling components. Yeast one-hybrid, chromatin immunoprecipitation, and dual-luciferase reporter assays demonstrated that ONAC039 directly bound to the promoters of three senescence-related genes and one ABA signaling gene, resulting in their transcriptional activation. Collectively, ONAC039 plays a crucial role in linking ABA signaling with senescence-associated genes in rice, providing a foundation for future studies aimed at modulating senescence timing.

Key words: abscisic acid, chlorophyll degradation, cis-regulatory element, leaf senescence, NAC transcription factor, ONAC039, rice, senescence-associated gene

Yehyun Yim, Kiyoon Kang, Suyeon Ko, Hee-Jin You, Sichul Lee, Nam-Chon Paek, Choon-Tak Kwon. Rice ONAC039 Drives Leaf Senescence Through Direct Transcriptional Activation of Multiple Senescence-Associated Genes[J]. Rice Science, 2026, 33(5): 687-700.

Figures/Tables 7

Fig. 1. Expression profiles of ONAC039. A, Spatial expression of ONAC039 in senescing flag leaves sampled 50 d after heading. Leaves were divided into three sections (base, middle, and tip) based on visible senescence, and transcript levels were quantified by qRT-PCR using OsUBQ5 as an internal control. Data are mean ± SD (n = 4). Different letters indicate statistically significant differences among groups (one-way ANOVA followed by Tukey’s test, P < 0.05). B, Temporal expression of ONAC039 during natural senescence of wild type (WT) leaves under natural long-day conditions. The red arrow indicates heading date. Data are mean ± SD (n = 3-6). C, Expression dynamics of ONAC039 during dark-induced senescence conditions in leaf discs sampled every 24 h. Representative images are shown for each time point. DDI, Days after dark incubation. Data are mean ± SD (n = 3-4). Different letters indicate statistically significant differences among groups (one-way ANOVA followed by Tukey’s test, P < 0.05). D, Tissue-specific expression of ONAC039 in WT plants. Samples were collected from leaf blade (LB), leaf sheath (LS), flag leaf (FL), flag leaf sheath (FLS), node (N), internode (IN), panicle (P), and roots (R). Relative transcript levels were determined by qRT-PCR and normalized to OsUBQ5. VS, Vegetative stage; RS, Reproductive stage. Data are mean ± SD (n = 4). The raw data underlying this figure are provided in Table S1.

Fig. 2. ONAC039 positively regulates leaf senescence in rice under natural long-day (NLD) conditions. A and B, Whole-plant phenotypes (A) and detached flag leaves (B) of wild type (WT), onac039-1, and onac039-2 plants at 0 and 50 d after heading (DAH) under NLD conditions. Scale bars, 9 cm. C and D, Whole-plant phenotypes (C) and detached flag leaves (D) of WT, ONAC039-D, and ONAC039-OX plants at 0 and 50 DAH under NLD conditions. Scale bars, 9 cm. E, Total chlorophyll content in flag leaves from WT, onac039-1, and onac039-2 plants after heading. Data are mean ± SD (n = 3-6). F, Maximum quantum efficiency of photosystem II (Fv/Fm ratio) in flag leaves of WT, onac039-1, and onac039-2 plants during natural senescence under NLD conditions. Data are mean ± SD (n = 5-29). G, Total chlorophyll content in flag leaves from WT, ONAC039-D, and ONAC039-OX plants after heading. Data are mean ± SD (n = 3-6). H, Fv/Fm ratio in flag leaves of WT, ONAC039-D, and ONAC039-OX plants during natural senescence under NLD conditions. Data are mean ± SD (n = 5-29). Statistical significance was assessed by two-way ANOVA with Geisser-Greenhouse correction and Dunnett’s multiple comparison test, using WT as the control (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, Not significant). The raw data underlying panels E-H are provided in Table S2.

Fig. 3. ONAC039 overexpression accelerates dark-induced senescence (DIS). A-C, Delayed leaf senescence of wild type (WT), onac039-1, and onac039-2 plants after 0 and 4 d of dark incubation (DDI). D-F, Accelerated leaf senescence in WT, ONAC039-D, and ONAC039-OX plants after 0 and 3 DDI. A and D, Representative leaf discs of WT, onac039-1, onac039-2 (A), ONAC039-D, and ONAC039-OX (D) plants. B and E, Total chlorophyll content of WT, onac039-1, onac039-2 (B), ONAC039-D, and ONAC039-OX (E) plants. C and F, Ion leakage rate of WT, onac039-1, onac039-2 (C), ONAC039-D, and ONAC039-OX (F) plants. Data are mean ± SD (n = 4). Statistical significance was assessed by two-way ANOVA followed by Dunnett’s multiple comparison test, using WT as the control (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, Not significant). The raw data underlying panels B, C, E, and F are provided in Table S2.

Fig. 4. ONAC039-dependent expression of senescence-associated and chlorophyll degradation genes during dark treatment. A-L, Relative transcript levels of OsNAP (A and B), OsNOL (C and D), OsNYC1 (E and F), OsNYC3 (G and H), OsSGR (I and J), and OsPAO (K and L) among WT, onac039-1, and onac039-2 after 0 and 4 d of dark incubation (DDI) (A, C, E, G, I, and K) and among WT, ONAC039-D, and ONAC039-OX after 0 and 3 DDI (B, D, F, H, J, and L). Data were normalized to OsUBQ5 and are shown as mean ± SD (n = 4). Statistical significance was assessed by two-way ANOVA followed by Dunnett’s multiple comparison test, using WT as the control (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, Not significant). The raw data underlying this figure are provided in Table S1.

Fig. 5. Exogenous abscisic acid (ABA) accelerates chlorophyll degradation in ONAC039-dependent leaf discs. A, Representative images of leaf discs from wild type (WT), onac039-1, and onac039-2 plants incubated on 1/2 Murashige and Skoog (MS) medium containing 0 or 2 μmol/L ABA for 0 or 3 d after treatment (DAT). B, Representative images of leaf discs from WT, ONAC039-D, and ONAC039-OX plants incubated for 0 or 3 DAT. C and D, Total chlorophyll content of samples in A (C) and B (D), measured at 0 and 3 DAT. Data are mean ± SD (n = 7-10). Statistical significance was assessed by two-way ANOVA followed by Dunnett’s multiple comparison test, using WT as the control (**, P < 0.01; ***, P < 0.001; ns, not significant). The raw data underlying panels C and D are provided in Table S2.

Fig. 6. ONAC039-dependent expression of abscisic acid (ABA) biosynthetic and signaling genes during ABA treatment. A-L, Relative transcript levels of OsABF2 (A and B), OsABF4 (C and D), OsABI5 (E and F), OsbZIP23 (G and H), OsEEL (I and J), and OsNCED1 (K and L) among wild type (WT), onac039-1, and onac039-2 after 0 and 3 d after treatment (DAT) (A, C, E, G, I, and K) and among WT, ONAC039-D, and ONAC039-OX after 0 and 3 DAT (B, D, F, H, J, and L). Data were normalized to OsUBQ5 are shown as mean ± SD (n = 4). Statistical significance was assessed by two-way ANOVA followed by Dunnett’s multiple comparison test, using WT as the control (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, Not significant). The raw data underlying this figure are provided in Table S1.

Fig. 7. ONAC039 directly regulates transcriptions of OsNAP, OsABI5, OsNYC1, and OsSGR. A, Schematic of the promoter regions of OsNAP, OsABI5, OsNYC1, and OsSGR. Green lines (a-d) indicate fragments used for yeast one hybrid assays. Yellow lines (P1-P4) mark regions used for chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR). Red boxes indicate putative ONAC039-binding motifs (C[C/A]CTCCTC). B, Yeast one-hybrid assays showing ONAC039 interaction with promoter fragments of OsNAP, OsABI5, OsNYC1, and OsSGR. Blue coloration indicates positive binding. C, ChIP-qPCR analysis confirming in vivo ONAC039 enrichment at the indicated promoters in ONAC039-OX plants. The actin promoter served as a negative control. Data are presented as fold enrichment and are shown as mean ± SD (n = 3). Different lowercase letters represent statistically significant differences among groups, as determined by one-way ANOVA followed by Tukey’s multiple comparison test (P < 0.05). D, Schematic representation of dual-luciferase reporter assay. Promoter regions of OsNAP, OsABI5, OsNYC1, and OsSGR were fused to the LUC reporter. 35S, 35S promoter; LUC, Firefly luciferase gene; NOS, Nopaline synthase transcription termination sequence; proUbi, Ubiquitin promoter. E, Co-transfection with proUbi::ONAC039-MYC significantly enhanced reporter activity in rice protoplasts compared with the control (proUbi::MYC). Relative LUC/REN ratios were normalized to the control (set as 1). Data are shown as mean ± SD (n = 5). Statistical significance was assessed using a two-tailed Student’s t-test (***, P < 0.001). The raw data underlying panels C and E are provided in Table S3.

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