Overexpression of the Glutathione Peroxidase 5 (RcGPX5) Gene From Rhodiola crenulata Increases Drought Tolerance in Salvia miltiorrhiza

Abstract

Excessive cellular accumulation of reactive oxygen species (ROS) due to environmental stresses can critically disrupt plant development and negatively affect productivity. Plant glutathione peroxidases (GPXs) play an important role in ROS scavenging by catalyzing the reduction of H₂O₂ and other organic hydroperoxides to protect plant cells from oxidative stress damage. RcGPX5, a member of the GPX gene family, was isolated from a traditional medicinal plant Rhodiola crenulata and constitutively expressed in Salvia miltiorrhiza under control of the CaMV 35S promoter. Transgenic plants showed increased tolerance to oxidative stress caused by application of H₂O₂ and drought, and had reduced production of malondialdehyde (MDA) compared with the wild type. Under drought stress, seedlings of the transgenic lines wilted later than the wild type and recovered growth 1 day after re-watering. In addition, the reduced glutathione (GSH) and total glutathione (T-GSH) contents were higher in the transgenic lines, with increased enzyme activities including glutathione reductase (GR), ascorbate peroxidase (APX), and GPX. These changes prevent H₂O₂ and O₂ ^- accumulation in cells of the transgenic lines compared with wild type. Overexpression of RcGPX5 alters the relative expression levels of multiple endogenous genes in S. miltiorrhiza, including transcription factor genes and genes in the ROS and ABA pathways. In particular, RcGPX5 expression increases the mass of S. miltiorrhiza roots while reducing the concentration of the active ingredients. These results show that heterologous expression of RcGPX5 in S. miltiorrhiza can affect the regulation of multiple biochemical pathways to confer tolerance to drought stress, and RcGPX5 might act as a competitor with secondary metabolites in the S. miltiorrhiza response to environmental stimuli.

Keywords: Rhodiola crenulata; Salvia miltiorrhiza; drought; glutathione peroxidase; secondary metabolites.

FIGURE 1

Identification and characterization of transgenic Salvia miltiorrhiza plants expressing RcGPX5. (A) The identities of wild type and transgenic lines are abbreviated as follows: WT, wild type; L1/L4/L5/L8, four individual plants from positive transgenic lines. (B) RT-PCR analysis of primary transformants using several primer combinations. Using SmACTIN as control, PCR amplification assays were conducted using cDNA from WT and putative transgenic lines produced by transformation with the plasmid vector p35SS::RcGPX5. L1–L8 indicate the putative transgenic lines. V is the plasmid vector; M is the DNA marker. 35SF is the forward primer that anneals to the 35S promoter and RcGPX5R is the reverse primer that anneals to a site in RcGPX5. The primer pair RcGPX5F/R was used to amplify the entire coding region of RcGPX5. The primer pair RcGPXqF/R was used for semi-quantitative real-time PCR to analyze the relative expression levels of RcGPX5 in the different transgenic lines. (C) RT-PCR analysis to quantify the tissue-specific expression of RcGPX in the L1 transgenic plantlets. The bottom was amplification production of Smactin primer and the top was qRcGPX5.

FIGURE 2

Effect of exogenous H₂O₂ on leaf disks and whole leaves of WT and three transgenic lines of S. miltiorrhiza. (A) Leaf squares after H₂O₂ treatment. The leaf pieces from WT and the transgenic lines (L1, L4, and L8) were divided into similar size groups. The squares were immersed in 0.5X liquid MS media containing 50 μmol/L H₂O₂ and 0.1% Tween 20. The top panel shows the control condition and the below shows the treatment condition. (B) Whole leaves after H₂O₂ treatment. Leaves were treated with 10 mmol/L H₂O₂. (C) Whole leaves were from the WT and transgenic lines were stained with diaminobenzidine (DAB). The leaves were treated with H₂O₂ prior to DAB staining. The brown staining shows sites of H₂O₂ accumulation and indicates the relative degree of leaf damage.

FIGURE 3

Effects of H₂O₂ stress on malondialdehyde (MDA) and pigment contents in WT and three transgenic lines of S. miltiorrhiza. Contents of (A) MDA, (B) xanthophylls, (C) chlorophyll a, (D) chlorophyll b, (E) total chlorophyll. The values in (E) represent the sum of chlorophyll a and b. Bars represent the mean ± SE of three independent experiments. ^∗∗ indicate significant differences at p < 0.01 compared with WT.

FIGURE 4

Morphological changes in plants of WT and four transgenic lines of S. miltiorrhiza in response to drought stress. D, Drought; R, Re-watered; W, well-watered; WT, wild type; L1/L4/L5/L8, transgenic lines that express RcGPX5.

FIGURE 5

Effects of drought stress on metabolite contents and enzyme activities in WT and three transgenic lines of S. miltiorrhiza. Leaves from WT and transgenic plants were used for the physiological measurements after 11 days of drought stress. (A) H₂O₂ accumulation; (B) O₂^- production rate; (C) superoxide dismutase (SOD) enzyme activity; (D) glutathione peroxidase (GPX) enzyme activity; (E) glutathione reductase (GR) enzyme activity; (F) L-ascorbate peroxidase (APX) enzyme activity; (G) reduce glutathione (GSH) content; (H) total glutathione (T-GSH) content; (I) GSH/GSSG ratio. The oxidized glutathione (GSSG) content was calculated using formula [GSSG = 1/2(Total GSH-GSH)]; (J) ascorbate (ASA) content; (K) malondialdehyde (MDA) content. The physiological measurements were made from leaves of WT and the transgenic lines under well-watered conditions. Bars represented the mean ± SE of three independent experiments. ^∗ and ^∗∗ represent significant differences at p < 0.05 and p < 0.01 compared to WT.

FIGURE 6

Relative expression levels of genes in the ROS pathways. qRT-PCR primers were designed from gene sequences in the S. miltiorrhiza transcriptome. Genes were annotated as encoding catalase (CAT), c43537; glutathione synthetase (GSH II), c50060; L-ascorbate peroxidase (APX), c14053; monodehydroascorbate reductase (MDHAR), c40572; and glutathione reductase (GR), c34040. mRNA levels were normalized with respect to SmACTIN. Data represent the means ± SE of at least three replicates.

FIGURE 7

Expression levels of genes related to drought stress. The materials were used with leaves of WT and transgenic lines under control and drought stress. Primers were designed by the sequences from transcriptome. c16091, c18179, c28876, c36053, c43595, and c47803 were annotated as encoding proteins with functions in energy production and conversion or photosystem pathways; c17368 was annotated as a gene for transcription factor dehydration-responsive element-binding protein 1s (DREB1s)/C-repeat-binding factor (CBF); c45322 was annotated as a protein phosphatase 2C ABI2 gene; c18143, c40726, c42086, and c42905 were annotated as genes with functions related to the stress response. mRNA levels were normalized with respect to SmACTIN. Data represent the means ± SE of at least three replicates.

FIGURE 8

Appearance of representative whole plants (top) and roots (bottom) of WT and three transgenic lines of S. miltiorrhiza. The plantlets were transferred to the experimental field and grown for 5 months. Scale bar = 10 cm.

FIGURE 9

Physiological indices measured in roots of WT and two transgenic lines of S. miltiorrhiza. (A) Root weights after natural drying; (B–E) several indices of the glutathione cycle were measured in fresh roots. (B) Glutathione peroxidase (GPX) enzyme activity; (C) reduced glutathione (GSH) content; (D) total glutathione (T-GSH) content; (E) GSH/GSSG ratio. (F–I) The active ingredient contents were measured in dried roots. (F) cryptotanshinone; (G) Tanshinone I; (H) Tanshinone IIA; (I) Salvianolic acid B. Bars represent the mean ± SE of three independent experiments. ^∗∗ represents significant differences at p < 0.01 compared with WT.

FIGURE 10

Expression of the salvianolic acid biosynthesis pathway genes in roots of WT and RcGPX5 transgenic S. miltiorrhiza plants that were grown in the field for 5 months.

FIGURE 11

Expression of terpenoid biosynthesis pathway genes in roots of WT and RcGPX5 transgenic S. miltiorrhiza plants.