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. 2016 Jun;171(2):1259-76.
doi: 10.1104/pp.16.00059. Epub 2016 May 2.

The De-Etiolated 1 Homolog of Arabidopsis Modulates the ABA Signaling Pathway and ABA Biosynthesis in Rice

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The De-Etiolated 1 Homolog of Arabidopsis Modulates the ABA Signaling Pathway and ABA Biosynthesis in Rice

Guangchao Zang et al. Plant Physiol. .
Free PMC article

Abstract

DEETIOLATED1 (DET1) plays a critical role in developmental and environmental responses in many plants. To date, the functions of OsDET1 in rice (Oryza sativa) have been largely unknown. OsDET1 is an ortholog of Arabidopsis (Arabidopsis thaliana) DET1 Here, we found that OsDET1 is essential for maintaining normal rice development. The repression of OsDET1 had detrimental effects on plant development, and leaded to contradictory phenotypes related to abscisic acid (ABA) in OsDET1 interference (RNAi) plants. We found that OsDET1 is involved in modulating ABA signaling in rice. OsDET1 RNAi plants exhibited an ABA hypersensitivity phenotype. Using yeast two-hybrid (Y2H) and bimolecular fluorescence complementation assays, we determined that OsDET1 interacts physically with DAMAGED-SPECIFIC DNA-BINDING PROTEIN1 (OsDDB1) and CONSTITUTIVE PHOTOMORPHOGENIC10 (COP10); DET1- and DDB1-ASSOCIATED1 binds to the ABA receptors OsPYL5 and OsDDB1. We found that the degradation of OsPYL5 was delayed in OsDET1 RNAi plants. These findings suggest that OsDET1 deficiency disturbs the COP10-DET1-DDB1 complex, which is responsible for ABA receptor (OsPYL) degradation, eventually leading to ABA sensitivity in rice. Additionally, OsDET1 also modulated ABA biosynthesis, as ABA biosynthesis was inhibited in OsDET1 RNAi plants and promoted in OsDET1-overexpressing transgenic plants. In conclusion, our data suggest that OsDET1 plays an important role in maintaining normal development in rice and mediates the cross talk between ABA biosynthesis and ABA signaling pathways in rice.

Figures

Figure 1.
Figure 1.
Analysis of OsDET1 expression. A, Histochemical staining of POsDET1::GUS transgenic plants. Transgenic plants were tested during protoplast culture and the flowering stage. Images are as follows: callus (a), leaf sheaths (b), leaves (c), roots, stems, and internodes (d), branches (e), panicle spikelets (f), and pistils and ovaries (g). B, Effects of phytohormones and abiotic stresses on OsDET1 expression. Two-week-old cv Nipponbare plants were used for the experiment. GA, Gibberellic acid; IAA, indole-3-acetic acid; KT, 6-furfurylaminopurine; PEG, polyethylene glycol; SA, salicylic acid. **, P ≤ 0.01 (Student’s t test). C, Kinetic analysis by qRT-PCR of OsDET1 expression after treatment with 50 μm ABA.
Figure 2.
Figure 2.
OsDET1 deficiency accelerates dark-induced leaf senescence in OsDET1 RNAi plants. A, qRT-PCR analysis of the expression of OsDET1 in OsDET1 RNAi lines and the wild type (WT). B, Suppression of OsDET1 promoted dark-induced leaf senescence. C, Changes with time of chlorophyll content in OsDET1 RNAi lines and wild-type plants during dark incubation. Values are means ± sd of nine measurements. The penultimate leaves were detached and incubated with water in darkness at the tillering stage. FW, Fresh weight. D, Changes with time of Fv/Fm values in OsDET1 RNAi lines and wild-type plants during dark incubation. Values are means ± sd of nine measurements. E, Changes with time of membrane ion leakage in OsDET1 RNAi lines and wild-type plants during dark incubation. Values are means ± sd of nine measurements. F to H, Expression of NOL, SGR, and OsNAP in the wild type and OsDET1 RNAi lines during dark incubation. **, P ≤ 0.01 (Student’s t test).
Figure 3.
Figure 3.
OsDET1 deficiency effects on ABA-induced leaf senescence. A, OsDET1 deficiency accelerates ABA-induced leaf senescence. The penultimate leaves were detached and incubated with water containing 50 μm ABA in light, with no added ABA as a control. B, Changes with time of chlorophyll contents in OsDET1 RNAi lines and wild-type (WT) plants after treatment with 50 μm ABA. Values are means ± sd of nine measurements. FW, Fresh weight. C, Changes with time of membrane ion leakage in OsDET1 RNAi lines and wild-type plants after treatment with 50 μm ABA. Values are means ± sd of nine measurements. D, Expression of CDGs (PAO, SGR, NYC1, NYC3, RCCR, PPH, and NOL) and SAGs (OsNAP and OsI57) in wild-type plants and OsDET1 RNAi plants after treatment with 50 μm ABA. *, P ≤ 0.05 and **, P ≤ 0.01 (Student’s t test).
Figure 4.
Figure 4.
OsDET1 influences rice ABA synthesis and inactivation. A, ABA content in wild-type (WT) and transgenic plants. The penultimate leaves were detected at the tillering stage. Values are means ± sd of six measurements. **, P ≤ 0.01 (Student’s t test). FW, Fresh weight. B to D, Expression of ABA inactivation genes (OsABA8ox1, OsABA8ox2, and OsABA8ox3) in the wild type and transgenic lines. *, P ≤ 0.05 and **, P ≤ 0.01 (Student’s t test). E to H, Expression of ABA biosynthesis genes (OsNCED1, OsNCED3, OsNCED4, and OsZEP) in the wild type and transgenic lines. *, P ≤ 0.05 and **, P ≤ 0.01 (Student’s t test).
Figure 5.
Figure 5.
OsDET1 influences seed germination and seedling growth. A, Overexpression or suppression of OsDET1 inhibited seed germination in transgenic plants. B, Germination time courses on one-half-strength MS medium. C, Overexpression or suppression of OsDET1 inhibited seed germination in transgenic plants during ABA treatment. D, Germination time courses on one-half-strength MS medium containing 2 µm ABA. E, Overexpression or suppression of OsDET1 inhibited shoot growth and promoted root growth. F, Overexpression or suppression of OsDET1 inhibited the growth of shoot and root during ABA treatment. G and H, Root and shoot growth rates of the wild type (WT) and OsDET1 RNAi and OE-OsDET1 plants. Three independent experiments were carried out with similar results. Representative graphs are shown (n = 30 seeds in each experiment). *, P ≤ 0.05 and **, P ≤ 0.01 (Student’s t test).
Figure 6.
Figure 6.
Interaction of OsDET1, OsDDB1, OsCOP10, OsDDA1, and OsPYL5 in vivo. A, OsDET1 interacts with OsDDB1 and OsCOP10 in yeast cells. The coding range of OsDET1 was inserted into pGBKT7 as bait, and the coding ranges of OsDDB1 and OsCOP10 were inserted into pGADT7 as prey. B, OsDDA1 interacts with OsDDB1 and OsPYL5 in yeast cells. The coding range of OsDDA1was inserted into pGBKT7 as bait, and the full-length coding sequence of OsDDB1 and a truncated version of the ABA receptor OsPYL5 (amino acids 90–209) were fused separately to pGADT7 as prey. AbA, Aureobasidin A; 3-AT, 3-amino-1,2,4-triazole; SD, synthetic dextrose. C, OsDET1 interacts with OsDDB1 and OsCOP10 in vivo. YFPN-OsDET1/YFPC-OsDDB1 and YFPN-OsDET1/YFPC-OsCOP10 were transformed into onion epidermal cells by Agrobacterium tumefaciens-mediated transformation to test their interactions. After 48 h of incubation in the dark, YFP signal was detected by confocal microscopy. Bright field was used to indicate the localization of nuclei. Empty vectors were used as negative controls. D, OsDDA1 interacts with OsDDB1 and OsPYL5 in vivo. YFPN-OsDDA1/YFPC-OsDDB1 and YFPN-OsDDA1/YFPC-OsPYL5 were transformed into onion epidermal cells by A. tumefaciens-mediated transformation to test their interactions.
Figure 7.
Figure 7.
OsDET1 influences the degradation of OsPYL5 and activates the expression of ABA mark genes. Fourteen-day-old seedlings were treated with ABA solution (one-half-strength MS medium with 5 μm ABA) for 12 h. Shoots were detected by qRT-PCR. A to D, Expression of OsPYL5, OsRAB16, OsLIP9, and OsLEA3 in the wild type (WT) and OsDET1 RNAi lines during ABA treatment. **, P ≤ 0.01 (Student’s t test). E, Western-blot analysis of OsPYL5 from detached OsDET1-GFP leaves at the tillering stage after 12 h of ABA treatment. F, Western-blot analysis of OsPYL5 from detached OE-OsDET1 leaves at the tillering stage after 12 h of ABA treatment. G, Western-blot analysis of OsPYL5 from detached OsDET1 RNAi leaves after 12 h of ABA treatment.
Figure 8.
Figure 8.
OsDET1 deficiency affects the pollination and fertilization processes in flower. A, Spikelets from the wild type (WT) and OsDET1 RNAi lines showed reductions in the number of filled seeds in the transgenic lines. B and C, Pollen grain morphology analysis showing the pollen grains of OsDET1 RNAi plants under ABA stress. Pollen grains were isolated from the wild type and OsDET1 RNAi plants and tested with a microscope. Three photographs were used for statistical analysis. Representative graphs are shown. **, P ≤ 0.01 (Student’s t test). Bars = 200 μm.
Figure 9.
Figure 9.
OsDET1 deficiency leads to contradictory ABA-related phenotypes in leaf. A, Stomatal aperture and wax crystallization of OsDET1 RNAi plants and wild-type plants (WT) observed by SEM. B, Frequency of open stomata. Values are means ± sd (n = 6). **, P ≤ 0.01 (Student’s t test). C, Water loss assay of leaves from OsDET1 RNAi plants and wild-type plants.
Figure 10.
Figure 10.
Transcriptome comparison between leaves of the wild type and DRI-16 using RNA-SEQ. A, Volcano plot showing DEGs of the wild type and DRI-16. Biological significance (log2 fold change) is depicted on the x axis and statistical significance (log10) is depicted on the y axis. Statistical significance was corrected at P < 0.05. B, Scatterplot of KEGG pathway enrichment statistics from wild-type and DRI-16 leaves. Rich factor is the ratio of the number of DEGs to the number of background genes in a KEGG pathway. The top 20 enriched KEGG pathways are listed.
Figure 11.
Figure 11.
Model of how OsDET1 deficiency leads to contradictory phenotypes related to ABA in OsDET1 RNAi plants. OsDDB1 interacts with OsCOP10, OsDET1, and OsDDA1 to form the CDDD complex. The reduction of CDDD complex function hinders the ubiquitination of OsPYLs and causes the accumulation of OsPYLs. The accumulation of PYLs and ABA promotes the stability of the PP2C-ABA-PYL ternary complex (Irigoyen et al., 2014). However, OsDET1 deficiency also leads to a decline in the content of ABA in normal conditions. Thus, due to these opposing factors, OsDET1 deficiency only partly causes the ABA hypersensitivity phenotype, such as closed stomatal pores and changes of pollen grain morphology, although it is unable to induce leaf senescence in normal development. The modulation of cuticular wax biosynthesis by the ABA signaling pathway is distinct from the governing stomatal regulation, and the accumulation of cuticular waxes is reduced in OsDET1 RNAi plants. During dark treatment, ABA is induced by continuous dark, which further enhances the ABA response and finally leads to increased leaf senescence. The leaf senescence in turn promotes the synthesis of ABA. Finally, OsDET1 RNAi plants exhibit a significantly accelerated leaf senescence phenotype.

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