9- cis-Epoxycarotenoid Dioxygenase 3 Regulates Plant Growth and Enhances Multi-Abiotic Stress Tolerance in Rice

Abstract

Although abscisic acid (ABA) is an important hormone that regulates seed dormancy, stomatal closure, plant development, as well as responses to environmental stimuli, the physiological mechanisms of ABA response to multiple stress in rice remain poorly understood. In the ABA biosynthetic pathway, 9-cis-epoxycarotenoid dioxygenase (NCED) is the key rate-limiting enzyme. Here, we report important functions of OsNCED3 in multi-abiotic stress tolerance in rice. The OsNCED3 is constitutively expressed in various tissues under normal condition, Its expression is highly induced by NaCl, PEG, and H₂O₂ stress, suggesting the roles for OsNCED3 in response to the multi-abiotic stress tolerance in rice. Compared with wild-type plants, nced3 mutants had earlier seed germination, longer post-germination seedling growth, increased sensitivity to water stress and H₂O₂ stress and increased stomata aperture under water stress and delayed leaf senescence. Further analysis found that nced3 mutants contained lower ABA content compared with wild-type plants, overexpression of OsNCED3 in transgenic plants could enhance water stress tolerance, promote leaf senescence and increase ABA content. We conclude that OsNCED3 mediates seed dormancy, plant growth, abiotic stress tolerance, and leaf senescence by regulating ABA biosynthesis in rice; and may provide a new strategy for improving the quality of crop.

Keywords: CRISPR/Cas9 system; OsNCED3; abiotic stress; abscisic acid (ABA); growth; leaf senescence; seed germination.

Figure 1

Expression analysis and subcellular localization of OsNCED3. (A) Histochemical staining of Pro_NCED3-GUS transgenic plants, including mature seed (a), bud sheath (b), root (c), leaf (d), culm and node (e), flower (f), stigma (g), pollens(h). Bars = 20 μM. (B) Transcription level of OsNCED3 in different organs. E(embryo), R(root), L(leaf), C(culm), N(node), F(flower). (C) OsNCED3 expression in abiotic stress by qRT-PCR. The three-leaf stage seedlings were performed to 150 mM NaCl, 250 mM Mannitol, 25% PEG and 100 mM H₂O₂ treatment, respectively. The seedling samples were collected for OsNCED3 expression analysis. Data are the mean ± SD for three replicates.

Figure 2

Subcellular location of OsNCED3 proteins. OsNCED3-eGFP fusion protein transiently expressed in Arabidopsis chloroplast and visualized by confocal microscopy, Bars = 20 μm.

Figure 3

CRISPR/Cas9 induced mutation of OsNCED3 and the phenotype of seed germination and early seedling growth in nced3 mutants. (A) Targeted mutagenesis of the OsNCED3 gene. The OsNCED3 mutation site is shown on the gene structure. The sequence in the red box represents the OsNCED3 spacer, and PAM is labeled by underline. The indels are shown in red dashes or letters. (B) Germination rate of wild type and nced3 mutants. Data are the mean ± SD for three replicates (each replicate containing 100 seeds). (C) Growth state at post-germination 2 d, 3 d, and 4 d for wild type and nced3. (D) Root and (E) shoot length of plants post-germination 4 d. (F) Seedling characteristics in the three-leaf stage. (G) Length measurement of (E) seedlings. Data shown are ± SD (n = 14). Similar results in three individual experiments. Asterisks indicate statistically significant differences (Student's t-test; ^*P < 0.05, ^**P < 0.01).

Figure 4

The nced3 mutants showed decreased salt and PEG stress tolerance. (A) The phenotype of three-leaf stage seedlings of nced3 mutants and wild type under NaCl and PEG stress. (B,C) Statistics for survival rate after salt and PEG stress. The number of surviving plants as a proportion of the total plants is shown. Data shown are ± SD from three independent replicates. Asterisks indicate statistically significant differences (Student's t-test; ^**P < 0.01).

Figure 5

Analysis of H₂O₂ accumulation and oxidative enzymes under H₂O₂ stress condition. (A) Phenotype of nced3 and wild type leaves after 100 mM H₂O₂ treatment for 2 d. (B) DAB staining of H₂O₂ accumulation. Three independent replicates were performed. (C) SOD and CAT activities of nced3 and wild type three-leaf-stage seedlings under 100 mM H₂O₂ treatment for 24 h. Data shown are ± SD from three independent replicates. Asterisks indicate statistically significant differences (Student's t-test; ^*P < 0.05).

Figure 6

Water stress tolerance of nced3 seedlings. (A) Phenotype of nced3 in water stress. (B) Survival rate after drought stress. The number of surviving plants as a proportion of the total plants is shown. (C, D) Proline and relative electrolyte leakage determination after water treatment. Data shown are ± SD from three independent replicates. (E) Measurement of water loss rate in wild type, nced3-1, and nced3-2 seedlings. Values are the mean ± SD (n = 6 plants). Similar results were obtained from three independent replicates. Asterisks indicate statistically significant differences (Student's t-test; ^*P < 0.05, ^**P < 0.01).

Figure 7

Stomata closure of nced3 mutant under water stress. (A) Scanning electron microscopy images of three levels of stomatal apertures, Bars = 10 μm. (B) The percentage of three levels of stomatal apertures in nced3-2 mutant and wild type plants were calculated under normal and water stress condition (n = 105 stomata for wild type; n = 98 stomata for nced3-2 mutant). (C) Stomata numbers per mm² were counted in middle leaves from nced3-2 mutant and wild type plants, respectively. Data shown are ± SD from there independent replicate.

Figure 8

ABA accumulation and ABA-related gene expression in nced3 mutants. (A) The shoot and roots ABA content in normal condition, (B) The leaf ABA content under water stress, (C) The leaf ABA-related genes expression in wild type and nced3 mutants three-leaf stage seedlings under water stress for 5 d. Data shown are ± SD from there independent replicate. Asterisks indicate statistically significant differences (Student's t-test; ^*P < 0.05, ^**P < 0.01).

Figure 9

The phenotype of OsNCED3-overexpressing plants and ABA content and ABA-related genes transcript levels. (A) The phenotype of three-leaf stage seedlings of OsNCED3-overexpressing and wild type under 150 mM NaCl stress. (B) Statistics for survival rate after salt stress. The number of surviving plants as a proportion of the total plants is shown. (C) The phenotype of three-leaf stage seedlings of OsNCED3-overexpressing and wild type under water stress. (D) Statistics for survival rate after water stress. (E) ABA content and (F) ABA-related genes expression in OsNCED3-overexpressing and wild type three-leaf stage seedlings under water stress for 5 d. Data shown are ± SD from there independent replicate. Asterisks indicate statistically significant differences (Student's t-test; ^*P < 0.05, ^**P < 0.01).

Figure 10

Dark-induced leaf senescence phenotype of leaf sections in nced3 mutant and OsNCED3-overexpressing plants. (A) Quantitative real-time PCR analysis of OsNCED3 expression after 3-day dark treatment. Data are the mean ± SD for three replicates. (B) Leaf senescence phenotype of 8-week-old nced3-1,2 and OE-1,2 plants after 3-day dark treatment. (C) Measurement of chlorophyll content in the 8-week-old nced3-1,2 and OE-1,2 leaves after dark treatment. (D) DAB staining of detached nced3-1,2 and OE-1,2 leaves after dark treatment. (E) Relative expression of OsSGR, OsNAP, OsI85, and OsNAC2 genes after 3-day dark treatment in detached nced3-1,2 and OE-1,2 leaves. Data shown are ± SD from three independent replicates. Asterisks indicate statistically significant differences (Student's t-test; ^*P < 0.05, ^**P < 0.01).