A C2-Domain Abscisic Acid-Related Gene, IbCAR1, Positively Enhances Salt Tolerance in Sweet Potato (Ipomoea batatas (L.) Lam.)

Abstract

Plant C2-domain abscisic acid-related (CAR) protein family plays an important role in plant growth, abiotic stress responses, and defense regulation. In this study, we cloned the IbCAR1 by homologous cloning method from the transcriptomic data of Xuzishu8, which is a sweet potato cultivar with dark-purple flesh. This gene was expressed in all tissues of sweet potato, with the highest expression level in leaf tissue, and it could be induced by NaCl and ABA. Subcellular localization analyses indicated that IbCAR1 was localized in the nucleus and plasma membrane. The PI staining experiment revealed the distinctive root cell membrane integrity of overexpressed transgenic lines upon salt stress. Salt stress significantly increased the contents of proline, ABA, and the activity of superoxide dismutase (SOD), whereas the content of malondialdehyde (MDA) was decreased in overexpressed lines. On the contrary, RNA interference plants showed sensitivity to salt stress. Overexpression of IbCAR1 in sweet potatoes could improve the salt tolerance of plants, while the RNAi of IbCAR1 significantly increased sensitivity to salt stress in sweet potatoes. Meanwhile, the genes involved in ABA biosynthesis, stress response, and reactive oxygen species (ROS)-scavenging system were upregulated in overexpressed lines under salt stress. Taken together, these results demonstrated that IbCAR1 plays a positive role in salt tolerance by relying on the ABA signal transduction pathway, activating the ROS-scavenging system in sweet potatoes.

Keywords: IbCAR1 gene; abscisic acid; salt stress resistance; sweet potato.

Figure 1

Sequence analysis of IbCAR1. (A) Features of IbCAR1 protein. (B) Sequences alignment of IbCAR1 with its closest homologs from other species. The C2 domain is represented with red lines. (C) Analysis of IbCAR1 protein homology tree in different species. (D) The structure diagrams of IbCAR1. Exons are represented by ellipses, and introns are represented by lines. The blue boxes represent the 5′ and 3′ untranslated regions (UTR).

Figure 2

Expression analysis of IbCAR1. (A) Expression analysis of IbCAR1 in storage root, pencil root, fibrous root, leaf, stem, and petiole tissues of Xuzishu8. Data are presented as means ± SE (n = 3). Different lowercase letters indicate a significant difference at p < 0.05 based on Student’s t-test. (B) Expression analysis of IbCAR1 in whole plants of Xuzishu8 after different times (h) in response to 200 mM NaCl and 100 mM ABA, respectively. The sweet potato b-actin gene was used as an internal control. Data are presented as means ± SE (n = 3). ** indicates a significant difference compared with 0 h (p < 0.01) based on Student’s t-test. * indicates a significant difference compared with 0 h (p < 0.05) based on Student’s t-test.

Figure 3

Subcellular localization of IbCAR1. OsMADS53 and AtSYP122 were used as nuclear and membrane markers, respectively. The n stands for nucleus. The cm stands for cytoplasmic membrane. Confocal scanning microscopic images showed that the IbCAR1-GFP fusion expression protein localized on the nucleus and membrane. GFP as the control. Bars = 25 μm.

Figure 4

Effects of NaCl stress on the root cell membrane integrity. The plasma membrane integrity in root cells was checked by using propidium iodide (PI) staining. The PI staining experiment revealed the distinctive root cell membrane integrity of overexpressed transgenic lines upon salt stress. The PI staining image of the root elongation zone (three independent experiments) for each treatment. Bars = 200 μm.

Figure 5

Analysis of the function of sweet potato IbCAR1 under normal and salt stress. (A) Phenotypic analysis of WT and transgenic sweet potato under salt stress. (B–E) The SOD (B), Proline (C), MDA (D), and ABA (E) contents of WT and overexpressed plant leaves under normal and salt stress. (F–I) The SOD (F), proline (G), MDA (H), and ABA (I) contents of WT and RNAi plant leaves under normal and salt stress. Data are presented as means ± SE (n = 3). ** and * indicate significant differences between the transgenic lines and WT at p < 0.01 and p < 0.05 levels, respectively.

Figure 6

Expression levels of stress-responsive genes in transgenic and WT sweet potato plants. (A) Expression patterns of stress-related genes in the WT and the overexpressed transgenic lines L1, L5, and L16. (B) Expression patterns of stress-related genes in the WT and the RNAi transgenic lines #10, #11. Plants grown in the transplanting boxes were sampled for analysis after treating with no stress (normal) for 12 h and salt stress for 12 h. Data are presented as means ± SE (n = 3). ** and * indicate significant differences between the transgenic lines and WT at p < 0.01 and p < 0.05 levels, respectively.

Figure 7

A proposed model for regulation of IbCAR1 in salt stress tolerance in transgenic sweet potato. Red arrows indicate up-regulation of gene coding these enzymes.