Overexpression of SoCYP85A1, a Spinach Cytochrome p450 Gene in Transgenic Tobacco Enhances Root Development and Drought Stress Tolerance

Abstract

Brassinosteroids (BRs) play an essential role in plant growth, development, and responses to diverse abiotic stresses. However, previous studies mainly analyzed how exogenous BRs influenced plant physiological reactions to drought stress, therefore, genetic evidences for the endogenous BRs-mediated regulation of plant responses still remain elusive. In this study, a key BRs biosynthetic gene, SoCYP85A1 was cloned from Spinacia oleracea, which has a complete open reading frame of 1,392 bp encoding a 464 amino acid peptide and shares high sequence similarities with CYP85A1 from other plants. The expression of SoCYP85A1 which was higher in leaf compared with root and stem, was induced by treatments of PEG6000, abscisic acid (ABA), low temperature and high salt. Increases in both SoCYP85A1 transcripts and endogenous BRs in transgenic tobacco which resulted in longer primary root and more lateral roots enhanced drought tolerance compared with wild types. The transgenic tobacco accumulated much lower levels of reactive oxygen species and malondialdehyde (MDA) than wild types did, accompanied by significantly higher content of proline and notably enhanced activities of antioxidant enzymes. Besides, transcriptional expressions of six stress-responsive genes were regulated to higher levels in transgenic lines under drought stress. Taken together, our results demonstrated that SoCYP85A1 involves in response to drought stress by promoting root development, scavenging ROS, and regulating expressions of stress-responsive genes.

Keywords: ROS; SoCYP85A1; brassinosteroids; drought stress tolerance; stress-responsive genes; transgenic tobacco.

FIGURE 1

Organ expression assay of SoCYP85A1 and expression profiles of SoCYP85A1 under treatments with PEG, ABA, 4°C and NaCl. (A) Organ expression assay of SoCYP85A1 in spinach. The organs (stem, leaf, and root) are represented by S, L, and R, respectively. (B) 20% PEG6000; (C) 100 μM ABA; (D) 4°C; (E) 200 mM NaCl. The expression level of stem for organ expression assay and for stress assay at time point 0 were defined as 1. Error bars represent standard deviations (SD) for three independent replicates with different letters at P < 0.05 significant level.

FIGURE 2

Molecular identifications of transgenic tobacco lines. (A) PCR analysis of genomic DNA with the specific primers for the SoCYP85A1 gene. PCR amplification of a 1,392 bp fragment of the SoCYP85A1 gene in transgenic lines. (B) Expressions of SoCYP85A1 in transgenic lines. M, molecular marker; P, plasmid; N, negative control; W, water; WT, wild types; 2 and 8, transgenic lines.

FIGURE 3

Effect of overexpression of SoCYP85A1 on root development before and after drought stress. Phenotypes of seedlings at 6-week-old under the normal conditions (A) and water loss for 10 d (B). Primary root length (C) and lateral root number (D) of seedlings at 6-week-old with and without water. Error bars represent SD and values with different letters are significant at P < 0.05.

FIGURE 4

BRs biosynthesis capacity in WT and overexpressing lines (L2 and L8). (A) Transcriptional accumulations of the SoCYP85A1 gene in WT, L2 and L8 before and after 10-day drought stress. The expression level of L2 and L8 at time point 0 was defined as 1, respectively. (B) Detection of CS content. (C) Detection of BL content. CS, castasterone; BL, brassinolide; FW, fresh weight. The leaves from tobacco plants at 6-week-old stage were sampled for BRs analysis before and after 10-day water loss. Data are the means ± SD of three replicates and values with different letters are significant at P < 0.05.

FIGURE 5

The endogenous contents of ABA in WT and transgenic lines under control and drought treatment. Error bars represent SD and values with different letters are significant at P < 0.05.

FIGURE 6

Analysis of the physiological indices in WT and transgenic lines (L2 and L8) under drought stress. Six-week-old seedlings were withheld water for 10 days to measure the value of RWC (A), water loss rate (B), proline content (C) and MDA (D). Error bars represent SD and values with different letters are significant at P < 0.05.

FIGURE 7

Analysis of three antioxidant enzymes’ activities and H₂O₂ accumulation in WT and transgenic lines (L2 and L8) under drought stress. Six-week-old seedlings were withheld water for 10 days to assess SOD (A), POD (B), CAT (C), and H₂O₂ (D). Error bars represent SD and values with different letters are significant at P < 0.05.

FIGURE 8

Expression levels of stress-relative genes in WT and transgenic lines (L2 and L8) under drought stress. Six-week-old seedlings were withheld water for 10 days. The tobacco leaves were sampled to extract the total RNA to synthesize cDNA. Expression levels of nine genes were detected. NtActin was used as the internal control. Error bars represent SD and values with different letters are significant at P < 0.05.