Overexpression of AtruLEA1 from Acer truncatum Bunge Enhanced Arabidopsis Drought and Salt Tolerance by Improving ROS-Scavenging Capability

Abstract

Late embryonic developmental abundant (LEA) genes play a crucial role in the response to abiotic stress and are important target genes for research on plant stress tolerance mechanisms. Acer truncatum Bunge is a promising candidate tree species for investigating the tolerance mechanism of woody plants against abiotic stress. In our previous study, AtruLEA1 was identified as being associated with seed drought tolerance. In this study, LEA1 was cloned from A. truncatum Bunge and functionally characterized. AtruLEA1 encodes an LEA protein and is located in the nucleus. Phylogenetic tree analysis revealed a recent affinity of the AtruLEA1 protein to AT3G15760.1. Overexpression of AtruLEA1 resulted in enhanced tolerance of Arabidopsis thaliana to drought and salt stress and heightened the ABA sensitivity. Compared to wild-type (WT) plants, plants with overexpressed AtruLEA1 exhibited increased activities of antioxidant enzymes under drought stress. Meanwhile, the ROS level of transgenic Arabidopsis was significantly less than that of the WT. Additionally, the stoma density and stoma openness of AtruLEA1 Arabidopsis were higher compared to those in the WT Arabidopsis under salt and drought stress conditions, which ensures that the biomass and relative water content of transgenic Arabidopsis are significantly better than those of the WT. These results indicated that AtruLEA1 was involved in salt and drought stress tolerances by maintaining ROS homeostasis, and its expression was positively regulated by abiotic stress. These results indicate a positive role of AtruLEA1 in drought and salt stress and provide theoretical evidence in the direction of cultivating resistant plants.

Keywords: ABA; Acer truncatum Bunge; LEA1; drought stress; salt stress.

Figure 1

The phylogenetic analysis and conserved motif patterns of AtruLEA1. (A) The phylogenetic relationship between AtruLEA1 and LEAs from Arabidopsis. (B) Conserved motifs were identified by using the MEME tools. The five predicted motifs are represented by distinct colored boxes, and the gray lines indicate non-conserved regions for AtruLEA1.

Figure 2

Protein domain prediction. (A) This image shows a graphical summary of the conserved domains identified on the query sequence. The domains are color-coded according to the superfamilies to which they have been assigned. Hits with scores that pass domain-specific thresholds (specific hits) are drawn in bright colors; others (non-specific hits) and superfamily placeholders are drawn in pastel colors. (B) The AtruLEA1 protein’s conserved domain. (C) The domain not shown in the diagram.

Figure 3

The protein structure analysis of AtruLEA1. (A,B) The secondary and tertiary structures of the AtruLEA1 protein.

Figure 4

The transmembrane structure and the signal peptide. (A) AtruLEA1’s transmembrane structure. The inner part represents the intracellular region and the outer part the extracellular zone; the transmembrane represents the transmembrane region; and the larger the value of the transmembrane, the greater the possibility that this amino acid is in the transmembrane region. (B) The signal peptide: The abscissa is the protein sequence, while the ordinate is the probability.

Figure 5

Bioinformatic analysis of AtruLEA1 promoter. The promoter sequence information of the genes was obtained from figShare websites and analyzed by using Plant Care online tools. Multiple drought-related cis-acting elements were found in the promoter region of AtruLEA1. (A) The sequence of the AtruLEA1 promoter and the cis-acting elements. (B) Different colors correspond to different cis-acting elements.

Figure 5

Bioinformatic analysis of AtruLEA1 promoter. The promoter sequence information of the genes was obtained from figShare websites and analyzed by using Plant Care online tools. Multiple drought-related cis-acting elements were found in the promoter region of AtruLEA1. (A) The sequence of the AtruLEA1 promoter and the cis-acting elements. (B) Different colors correspond to different cis-acting elements.

Figure 6

Drought and salt tolerance analyses of AtruLEA1 transgenic seedlings under panel growth conditions. (A) The expression levels in AtruLEA1 transgenic and WT seedlings. (B) An image showing 35S::AtruEA1 (OE-3 and OE-10) and WT seedlings under normal conditions, NaCl treatment and drought treatment in a plate. (C,D) The fresh weights and root lengths of both WT and AtruLEA1-overexpressing seedlings were measured under normal conditions and under salt and drought stress treatments. The significance of differences among the lines was tested by using Student’s t-test (* p < 0.05 and ** p < 0.01).

Figure 7

Transgenic AtruLEA1 Arabidopsis seedling resistance to salt and drought stress in soil. (A) AtruLEA1-overexpressing and WT seedlings were cultivated in soil under normal growth conditions for two weeks. Following water supply withholding for 10 days, we rehydrated the plants to examine their ability to resist drought stress. (B) For salt treatment, the seedlings were irrigated with 50 mL of 200 mM NaCl every 3 days for 2 weeks. Photographs were taken after each course of treatment. (C) The survival rates of WT and AtruLEA1-overexpressing seedlings under control, salt stress and drought stress conditions. (D,E) The relative water content (RWC) and relative electrical conductivity (REC) of WT and AtruLEA1-overexpressing seedlings under control, salt stress and drought stress conditions. The values presented herein are the result of an average calculation based on three separate replicates. Student’s t-test was used to determine significant differences (* p < 0.05 and ** p < 0.01).

Figure 8

Early-seedling experiment under ABA treatment. (A) Seedlings in 1/2 MS solid medium and 1/2 MS solid medium with ABA (50 μM) for 5 days. (B,C) Effects of ABA treatment on root length and fresh weight. A photo was taken before measuring the root length and the fresh weight of the seedlings subjected to the different treatments. (D,E) The expression of the ABA synthesis-related genes (NCED9 and NCED5). The vertical bars represent the means ± standard deviations. Student’s t-test was used to determine significant differences (* p < 0.05 and ** p < 0.01).

Figure 9

(A) Stomatal density in OE-10, OE-3 and wild-type plants under drought and salt stress conditions. (B) Stomatal density is shown as the average number of stomata per square millimeter. The number of and means number of stomata in Arabidopsis, means the normal stomata, means the stomata tended to close. All the images in the figure were acquired at 40× magnification under a microscope. Student’s t-test was used to determine significant differences (* p < 0.05 and ** p < 0.01).

Figure 10

AtruLEA1 mediates ROS scavenging ability. (A,B) The NBT and DAB staining of leaves from 35S::AtruLEA1 (OE-3 and OE-10) and the WT under normal and stress treatments. (C–E) CAT, (C), POD (D) and SOD (E) activity in the WT and AtruLEA1-overexpressing seedlings under normal and stress treatments. Significance was tested by using Student’s t-test (* p < 0.05 and ** p < 0.01).