CYP709B3, a cytochrome P450 monooxygenase gene involved in salt tolerance in Arabidopsis thaliana

Abstract

Background: Within the Arabidopsis genome, there are 272 cytochrome P450 monooxygenase (P450) genes. However, the biological functions of the majority of these P450s remain unknown. The CYP709B family of P450s includes three gene members, CYP709B1, CYP709B2 and CYP709B3, which have high amino acid sequence similarity and lack reports elucidating biological functions.

Results: We identified T-DNA insertion-based null mutants of the CYP709B subfamily of genes. No obvious morphological phenotypes were exhibited under normal growth conditions. When the responses to ABA and salt stress were studied in these mutants, only the cyp709b3 mutant showed sensitivity to ABA and salt during germination. Under moderate salt treatment (150 mM NaCl), cyp709b3 showed a higher percentage of damaged seedlings, indicating a lower tolerance to salt stress. CYP709B3 was highly expressed in all analyzed tissues and especially high in seedlings and leaves. In contrast, CYP709B1 and CYP709B2 were highly expressed in siliques, but were at very low levels in other tissues. Under salt stress condition, CYP709B3 gene expression was induced after 24 hr and remained at high expression level. Expression of the wild type CYP709B3 gene in the cyp709b3 mutant fully complemented the salt intolerant phenotype. Furthermore, metabolite profiling analysis revealed some differences between wild type and cyp709b3 mutant plants, supporting the salt intolerance phenotype of the cyp709b3 mutant.

Conclusions: These results suggest that CYP709B3 plays a role in ABA and salt stress response and provides evidence to support the functions of cytochrome P450 enzymes in plant stress response.

Figure 1

Identification of CYP709B subfamily mutants from T-DNA mutant collections. A. Structure of the CYP709B family genes showing the positions of the T-DNA insertions in mutants. B. Levels of transcripts in wild-type (WT) and mutants were determined by RT-PCR. WT: wild type; b1: cyp709b1; b2-1: cyp709b2-1; b2-2: cyp709b2-2; b3: cyp709b3. The ACTIN2 transcript level was used as a control (bottom panel).

Figure 2

Tissue-specific expression in wild typeArabidopsis. A:CYP709B1. B:CYP709B2.(C)CYP709B3. Se: 12-day-old seedlings; Inf: inflorescences; Rl: rosette leaves; FL: opening flowers; Silq: siliques (4–10 DAP). Real time PCR was performed to detect the level of transcripts in various organs of wild type. ACTIN2 was used as internal control. Values are the means ± SE of three replicates.

Figure 3

Phenotypic analysis ofcyp709b1,cyp709b2andcyp709b3germination. A. Germination of cyp709b3 is hypersensitive to ABA. Seeds were sown on wetted filter paper containing 0 and 1.5 μM ABA. After 2 days at 4°C, the plates were placed under continuous light. Germination (emergence of radicals) was scored at indicated times. B-C. Germination of cyp709b3 is sensitive to salt treatment. Seeds were sown on MS plates with 0, 100, 150 or 200 mM NaCl. Germination was scored at the indicated times after 2 days cold (4°C) treatment. B: Germination at day 3 under different NaCl conditions; C: Germination under 200 mM NaCl at indicated time. Error bars indicate SE (n = 3). Statistically different to wild type (p value < 0.05) is indicated using asterisks.

Figure 4

Thecyp709b3mutant plant is sensitive to salt stress. A. Rate of dead seedlings at day 5, 6 and 7 after transfer onto NaCl agar plates. Seeds were germinated on MS medium for 4 days and transferred onto MS agar plates supplemented with 0, 100, 150 or 200 mM NaCl. B. Growth of WT and cyp709b3 seedlings on MS medium (left) and medium supplemented with 150 mM NaCl (right) at day 5 after transfer. C.cyp709b1 and cyp709b2 mutant seedlings are not sensitive to salt stress. D. Growth of WT and cyp709b1, cyp709b2-1 and cyp709b3 mutant plants in soil. Seedlings were irrigated with water (left) or 150 mM NaCl (right) after 21 days. These pictures were taken at day 21 after treatment initiation. Error bars indicate SE (n = 3). Statistically different (p value < 0.05) is indicated using asterisks.

Figure 5

Expression of wild type CYP709B3 gene incyp709b3mutant can rescue salt intolerance phenotype. A-D. Expression pattern of Pro_CYP709B3:GUS in different organs. The results of GUS staining were observed in: seedling (A), leaf (B), silique (C) and flower (D). E. Expression of a wild type CYP709B3 gene can rescue the salt intolerance phenotype. Wild type and three independent Pro_CYP709B3:CYP709B3 transgenic homozygous lines (11–4, 18–8 and 40–2) in cyp709b3 mutant background were treated by 150 mM NaCl. The seedlings were counted at the indicated times. F. Analysis of the expression level of CYP709B3 in seedlings from wild type, cyp709b3 and independent transgenic homozygous lines (11–4, 18–8 and 40–2). ACTIN2 was used as internal control. Values are the means ± SE of three replicates. Statistically different (p value < 0.05) is indicated using asterisks.

Figure 6

Gene expression analysis in wild type and mutants under 150 mM NaCl treatment. Seeds were germinated on MS medium for 4 days and transferred to MS medium supplemented with 150 mM NaCl. Seedlings were harvested at indicated times. Gene expression was detected by quantitative real time PCR. A.CYP709B3 and CYP709B2 gene expression in wild type seedlings under 150 mM NaCl treatment. Expression of stress response genes: KIN2(B), RD29A(C), RD29B(D), DREB1A(E), ERD10(F) in seedlings from 150 mM NaCl-treated wild type and cyp709b3 mutant. ACTIN2 was used as internal control. Error bars indicate SE (n = 3). Statistically different (p value < 0.05) is indicated using asterisks.

Figure 7

The endogenous level of ABA in wild type and mutants. A. The change of endogenous ABA levels in wild type and cyp709b3 mutant during seed imbibition. The seeds were collected at 0, 12 and 24 h after imbibition. B. Endogenous ABA level in seedlings from 150 mM NaCl-treated seedlings from wild type and cyp709b3 at indicated times. Values are the means ± SE of three replicates. Statistically different (p value < 0.05) is indicated using asterisks.

Figure 8

Comparison of metabolite profiling between wild type andcyp709b3. Compounds related to membrane degradation (A-C), nucleic acid (D-F) and protein degradation (G-I) was increased in cyp709b3 mutants under salt stress compared to wild type (WT). Four-day-old seedlings were transferred onto 150 mM NaCl plates. Untreated and treated seedlings were collected at 2 days (2D) and 4 days (4D) after treatment. 100 mg of tissue was extracted and analyzed by LC/MS and GC/MS by Metabolon, Inc. Values are the means ± SD of four replicates. N: non-salt treatment; S: salt treatment. Y-axis: peak intensity.