A novel small open reading frame gene, IbEGF, enhances drought tolerance in transgenic sweet potato

Abstract

Small open reading frames (sORFs) can encode functional polypeptides or act as cis-translational regulators in stress responses in eukaryotes. Their number and potential importance have only recently become clear in plants. In this study, we identified a novel sORF gene in sweet potato, IbEGF, which encoded the 83-amino acid polypeptide containing an EGF_CA domain. The expression of IbEGF was induced by PEG6000, H₂O₂, abscisic acid (ABA), methyl-jasmonate (MeJA) and brassinosteroid (BR). The IbEGF protein was localized to the nucleus and cell membrane. Under drought stress, overexpression of IbEGF enhanced drought tolerance, promoted the accumulation of ABA, MeJA, BR and proline and upregulated the genes encoding superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in transgenic sweet potato. The IbEGF protein was found to interact with IbCOP9-5α, a regulator in the phytohormone signalling pathways. These results suggest that IbEGF interacting with IbCOP9-5α enhances drought tolerance by regulating phytohormone signalling pathways, increasing proline accumulation and further activating reactive oxygen species (ROS) scavenging system in transgenic sweet potato.

Keywords: IbEGF; drought tolerance; phytohormone; sORF; sweet potato.

Figure 1

Expression analysis of IbEGF in in vitro-grown Xushu55-2 plants after different time points (h) in response to H₂O (control), 30% PEG6000, 100 mM H₂O₂,100 mM ABA, 100 μM MeJA and 100 nM BR, respectively. The expression level of IbEGF in the plant sampled at 0 h was set to 1. The data are presented as the means ± SEs (n = 3). ** indicates a significant difference from that of the untreated control (0 h) at P<0.01 according to Student’s t-test.

Figure 2

Subcellular localization of IbEGF in tobacco leaf hypodermal cells (A) and maize protoplasts (B). Confocal scanning microscopy images showing the localization of IbEGF-GFP to nucleus and cell membrane. The empty pSuper1300 vector (35S:GFP) was used as a control. Bars = 20 μm.

Figure 3

Responses of in vitro-grown transgenic sweet potato plants and WT cultured on MS medium without (control) or with 20% PEG6000 for 4 weeks. (A) Phenotypes. (B) Root length. (C) Fresh weight. The data are presented as the mean ± SEs (n = 3). * and ** indicate significant differences from that of WT at P<0.05 and P<0.01, respectively, according to Student’s t-test.

Figure 4

Responses of transgenic sweet potato plants and WT treated in Hoagland solution for 4 weeks (control) or in Hoagland solution with 20% PEG6000 for 2 weeks followed by 2 weeks of Hoagland solution. (A) Phenotypes. (B) Root number. (C) Fresh weight. * and ** indicate significant differences from that of WT at P<0.05 and P<0.01, respectively, according to Student’s t-test.

Figure 5

Responses of transgenic sweet potato plants and WT grown in transplanting boxes to drought stress. (A) Phenotypes of plants irrigated with Hoagland solution for 8 weeks (control) or stressed by an 8-week-long drought treatment. (B–H) ABA content, MeJA content, BR content, proline content, MDA content, SOD activity, and H₂O₂ content in the leaves of plants after 4 weeks of treatment, respectively. (I–M) Transcript levels of IbEGF, IbCOP9-5α, IbSOD, IbCAT, and IbPOD in the leaves of plants after 4 weeks of treatment, respectively. The transcript levels of the genes in WT under normal treatment were set to 1. * and ** indicate significant differences from that of WT at P<0.05 and P<0.01, respectively, according to Student’s t-test.

Figure 6

Stomatal aperture and leaf water loss rate of transgenic sweet potato plants and WT. (A, B) Stomatal aperture of greenhouse-grown plants under normal condition (-ABA) and treated with 20 μM ABA for 2 h. Bar = 5 μm. Data are presented as the means ± SD (n = 80). ** indicates significant difference from that of WT at P<0.01, according to Student’s t-test. (C) Leaf water loss rate of greenhouse-grown plants.

Figure 7

In vivo interaction between IbEGF and IbCOP9-5α. (A) IbEGF/IbCOP9-5α interaction in Y2H Gold cells by Y2H assay. (B) IbEGF/IbCOP9-5α interaction in the tobacco nucleus by BiFC assay. The yellow fluorescent protein (YFP) signals were predominantly localized in the nucleus. Bars = 20 μm. (C) IbEGF/IbCOP9-5α interaction by the co-IP assay. Total proteins from N. benthamiana leaf cells expressing Myc-IbEGF and IbCOP9-5α-GFP were extracted and incubated with anti-Myc magnetic beads. Proteins before (input) and after IP were detected with anti-Myc and anti-GFP antibodies.

Figure 8

Proposed working model of IbEGF in the transgenic sweet potato plants to drought stress.