Rice transcription factor OsMYB102 delays leaf senescence by down-regulating abscisic acid accumulation and signaling

Abstract

MYB-type transcription factors (TFs) play important roles in plant growth and development, and in the responses to several abiotic stresses. In rice (Oryza sativa), the roles of MYB-related TFs in leaf senescence are not well documented. Here, we examined rice MYB TF gene OsMYB102 and found that an OsMYB102 T-DNA activation-tagged line (termed osmyb102-D), which constitutively expresses OsMYB102 under the control of four tandem repeats of the 35S promoter, and OsMYB102-overexpressing transgenic lines (35S:OsMYB102 and 35S:GFP-OsMYB102) maintain green leaves much longer than the wild-type under natural, dark-induced, and abscisic acid (ABA)-induced senescence conditions. Moreover, an osmyb102 knockout mutant showed an accelerated senescence phenotype under dark-induced and ABA-induced leaf senescence conditions. Microarray analysis showed that a variety of senescence-associated genes (SAGs) were down-regulated in the osmyb102-D line. Further studies demonstrated that overexpression of OsMYB102 controls the expression of SAGs, including genes associated with ABA degradation and ABA signaling (OsABF4, OsNAP, and OsCYP707A6), under dark-induced senescence conditions. OsMYB102 inhibits ABA accumulation by directly activating the transcription of OsCYP707A6, which encodes the ABA catabolic enzyme ABSCISIC ACID 8'-HYDROXYLASE. OsMYB102 also indirectly represses ABA-responsive genes, such as OsABF4 and OsNAP. Collectively, these results demonstrate that OsMYB102 plays a critical role in leaf senescence by down-regulating ABA accumulation and ABA signaling responses.

Keywords: OsCYP707A6; OsMYB102; Abscisic acid; chlorophyll degradation; leaf senescence; rice; senescence-associated genes; transcriptional regulation.

Fig. 1.

osmyb102-D plants showed a delayed-senescence phenotype in natural paddy field conditions. (A, B) The relative transcript levels of OsMYB102 in senescing WT leaves (A) or detached leaf discs during DIS (B) were determined by RT-qPCR and normalized to the transcript levels of UBQ5. The mean and SD values were obtained from more than three biological samples. DDI, day(s) of dark incubation. (C) Gene structure of OsMYB102 and the T-DNA insertion site. (D) The relative transcript level of OsMYB102 in osmyb102-D was determined by RT-qPCR and normalized to the transcript level of UBQ5. (E, F) Phenotypes of the WT (the parental japonica rice cultivar ‘Dongjin’) and the osmyb102-D plants at 0, 50, and 70 DAH. (G, H) Changes of total Chl levels (G) and F_v/F_m ratios (H) in the WT and osmyb102-D plants during grain filling (0–50 DAH). The mean and SD values were obtained from more than five biological samples. These experiments were repeated twice with similar results. DAH, days after heading. Asterisks indicate significant difference compared with the expression level of OsMYB102 at 0 DDI (B), or difference between the WT and osmyb102-D (D, G, H) (Student’s t-test, *P<0.05, **P<0.01).

Fig. 2.

osmyb102-D plants showed a delayed senescence phenotype during dark-induced senescence. (A–D) Changes of detached leaf color (A), total Chl levels (B), photosystem protein levels (C), and ion leakage rates (D) in the leaf discs of the WT and osmyb102-D plants during dark incubation. Leaf segments were incubated on 3 mM MES (pH 5.8) buffer with the abaxial side up at 28 °C in darkness and sampled at the specified DDI for each experiment. (C) Antibodies against PSII antenna (Lhcb1 and Lhcb2), PSI antenna (Lhca1 and Lhca2), and PSII core (CP43) proteins were used for immunoblot analysis. RbcL was detected by Coomassie Brilliant Blue staining. (B, D) Black and white bars indicate the WT and osmyb102-D plants, respectively. The mean and SD values were obtained from more than five leaf samples. Asterisks indicate a significant difference between the WT and osmyb102-D plants (Student’s t-test, *P<0.05, **P<0.01). These experiments were repeated three times with similar results. DDI, day(s) of dark incubation.

Fig. 3.

OsMYB102-overexpressing transgenic rice plants show a stay-green phenotype during dark-induced leaf senescence. (A) The expression levels of OsMYB102 in leaf blades from 1-month-old WT, osmyb102-D, 35S:OsMYB102, and 35S:GFP-OsMYB102 plants. The transcript levels of OsMYB102 were determined by RT-qPCR and were normalized to the transcript levels of UBQ5. (B, C) The changes of leaf color (B) and total Chl content (C) in WT, osmyb102-D, 35S:OsMYB102, and 35S:GFP-OsMYB102 detached leaf discs during dark-induced leaf senescence. Asterisks indicate a significant difference compared with the WT (Student’s t-test, **P<0.01). The mean and SD values were obtained from more than five leaf samples.

Fig. 4.

Altered expression of SAGs in osmyb102-D plants during DIS. (A) Summary of the microarray analysis. A filter for Student’s t-test P-value of <0.05 was applied to the differentially expressed genes (DEGs). (B, C) Venn diagrams of the number of DEGs in osmyb102-D plants compared with the WT. The numbers of up-regulated (B) and down-regulated (C) genes in osmyb102-D plants at 0 and 4 DDI are shown. (D) The ratios of expression levels (osmyb102-D/WT) for known or putative SAGs at 0 and 4 DDI. The relative expression value was normalized to the WT expression level. DDI, day(s) of dark incubation.

Fig. 5.

Altered expression of SAGs and ABA-related genes in WT, osmyb102-D, 35S:OsMYB102, and 35S:GFP-OsMYB102 plants under DIS conditions. Plants grown for 1 month under LD (14 h light/day) conditions were transferred to darkness at 28 °C and incubated in darkness for 4 or 6 d. Relative transcript levels of OsCYP707A6 (A), OsABA2 (B), OsABA3 (C), OsABF1 (D), OsABF4 (E), OsNAP (F), OsSGR (G), OsNYC1 (H), OsEIN3 (I), OsLhcb1 (J), OsLhcb3 (K), and OsLhca1 (L) were determined by RT-qPCR and normalized to the transcript levels of UBQ5. Black, white, red, and blue bars indicate WT, osmyb102-D, 35S:OsMYB102, and 35S:GFP-OsMYB102 plants, respectively. The mean and SD values were obtained from more than three biological samples. Asterisks indicate a significant difference compared with the WT (Student’s t-test, *P<0.05, **P<0.01). These experiments were repeated twice with similar results. DDI, day(s) of dark incubation.

Fig. 6.

OsMYB102 directly activates the transcription of OsCYP707A6. (A) The positions of the AACXG binding motifs in the promoter of OsCYP707A6 and promoter fragments used for the ChIP assay (green horizontal lines), the transactivation assay, and Y1H assay (orange horizontal line). (B) Reporter and effector constructs used in the transactivation assay. Each construct also contains the NOS terminator (not shown). (C) The activation of the OsCYP707A6 promoter (–1516 to –1) by OsMYB102-MYC in the protoplast transient assay. The 35S promoter was used as a negative control. (D) OsMYB102 binding affinity to the promoter region of OsCYP707A6 in planta examined by ChIP assays. OsMYB102-Myc was transiently expressed in protoplasts isolated from 10-day-old WT seedlings. Fold-enrichment of the promoter fragments was measured by immunoprecipitation with an anti-Myc antibody (see Methods). SERINE/THREONINE PROTEIN PHOSPHATASE 2A (PP2A) was used as a negative control. (E) The binding activity of OsMYB102 to the promoter regions of OsCYP707A6 was examined by Y1H assays. Empty bait and prey plasmids (–) were used for the negative controls. The relative β-galactosidase activity was obtained by normalizing to the level of each negative control. The mean and SD values were obtained from more than five independent colonies. (F, G) The effects of disruption of the putative OsMYB102 binding sites in the OsCYP707A6 promoter in rice protoplasts. LUC was fused to the wild-type or mutated OsCYP707A6 promoter fragments, and the OsMYB102 overexpression vector or empty vector were co-transfected in the protoplasts. The 35S promoter was used as a negative control. The mean and SD values were obtained from more than four biological samples. (H) Relative ABA contents in WT, osmyb102-D, 35S:OsMYB102, and 35S:GFP-OsMYB102 plants at 0 (black bars), 2 (gray bars), and 4 d (white bars) after dark incubation. The mean and SD values were obtained from more than five biological samples. (C, D, E, G, H) Asterisks indicate a significant difference compared with the WT or negative control (Student’s t-test, *P<0.05, **P<0.01).

Fig. 7.

OsMYB102 is involved in down-regulation of OsNAP and OsABF4. (A) The change in the expression level of OsMYB102 during the treatment with 100 μM ABA. The transcript levels of OsMYB102 were determined by RT-qPCR and normalized to the transcript levels of UBQ5. The mean and SD values were obtained from more than three biological samples. (B) The positions of the AACXG binding motif in the promoters of OsNAP and OsABF4, and the promoter fragments used for the ChIP assay (green horizontal lines) and the transactivation assay (orange horizontal lines). (C) Reporter and effector constructs used in the transrepression assay. Each construct also contained the NOS terminator (not shown). (D) The repression of the promoters of OsNAP (–1502 to –1) and OsABF4 (–1516 to –1) by OsMYB102-MYC in the protoplast transient assay. The 35S promoter was used as a negative control. The mean and SD values were obtained from more than four biological samples. (E) OsMYB102 binding affinity to the promoter region of OsNAP and OsABF4 in planta examined by ChIP assays. OsMYB102-MYC was transiently expressed in protoplasts isolated from 10-day-old WT seedlings. Fold-enrichment of the promoter fragments was measured by immunoprecipitation with an anti-MYC antibody (see Methods). PP2A was used as a negative control. (F, G) The changes of leaf color and total Chl content in WT, osmyb102-D, 35S:OsMYB102, and 35S:GFP-OsMYB102 detached leaf discs during the treatment with 3 mM MES buffer (pH 5.8) containing 100 μM ABA. (A, D, E, G) Asterisks indicate a significant difference compared with the WT or negative control (Student’s t-test, *P<0.05, **P<0.01). The mean and SD values were obtained from more than five biological samples.

Fig. 8.

OsABF4 directly activates the transcription of OsSGR and OsNYC1. (A) The positions of the AACXG and ABRE binding sequences in the promoters of OsSGR and OsNYC1, and the promoter fragments used for the ChIP assay (green horizontal lines) and the transactivation assay (orange horizontal lines). (B) Reporter and effector constructs used in the transrepression and transactivation assays shown in (C) and (D). Each construct also contained the NOS terminator (not shown). (C) The repression of the promoters of OsSGR (–1514 to –1) and OsNYC1 (–1508 to –1) by OsMYB102-MYC in the protoplast transient assay. (D) The activation of the promoters of OsSGR and OsNYC1 by OsABF4-MYC and OsNAP-MYC in the protoplast transient assay. (C, D) The 35S promoter was used as a negative control. (E) OsABF4 binding affinity to the promoter regions of OsSGR and OsNYC1 in planta examined by ChIP assays. OsABF4-MYC was transiently expressed in protoplasts isolated from 10-day-old WT seedlings. Fold-enrichment of the promoter fragments was measured by immunoprecipitation with an anti-MYC antibody (see Methods). PP2A was used as a negative control. (C, D, E) The mean and SD values were obtained from more than three biological samples. Asterisks indicate a significant difference compared with the negative control (Student’s t-test, *P<0.05, **P<0.01).

Fig. 9.

Agronomic traits of osmyb102-D plants. Plant height (A), number of panicles per plant (B), panicle length (C), number of grains per panicle (D), fertility (E), 500-grain weight (F), grain yield per plant (G), images of the grains (top, fertile; bottom, sterile) (H), and panicle phenotype of main culm (I) were obtained from plants grown in the paddy field under natural LD conditions in 2017. The mean and SD values were obtained from at least 10 plant replicates. Asterisks indicate a significant difference between the WT and osmyb102-D plants (Student’s t-test, *P<0.05, **P<0.01).

Fig. 10.

Proposed model of the OsMYB102-mediated regulatory network of leaf senescence. OsMYB102 inhibits ABA biosynthesis by directly activating OsCYP707A6, which encodes an ABA catabolic enzyme. OsMYB102 indirectly represses the transcription of OsNAP and OsABF4, probably partially through the regulation of ABA biosynthesis. Then, OsNAP and OsABF4 directly activate genes encoding Chl catabolic enzymes, such as OsSGR and OsNYC1, leading to leaf yellowing.