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. 2018 Apr 12;9(4):209.
doi: 10.3390/genes9040209.

A Novel G-Protein-Coupled Receptors Gene from Upland Cotton Enhances Salt Stress Tolerance in Transgenic Arabidopsis

Pu Lu  1 , Richard Odongo Magwanga  2   3 , Hejun Lu  4 , Joy Nyangasi Kirungu  5 , Yangyang Wei  6 , Qi Dong  7 , Xingxing Wang  8 , Xiaoyan Cai  9 , Zhongli Zhou  10 , Kunbo Wang  11 , Fang Liu  12
Affiliations

A Novel G-Protein-Coupled Receptors Gene from Upland Cotton Enhances Salt Stress Tolerance in Transgenic Arabidopsis

Pu Lu et al. Genes (Basel). .

Abstract

Plants have developed a number of survival strategies which are significant for enhancing their adaptation to various biotic and abiotic stress factors. At the transcriptome level, G-protein-coupled receptors (GPCRs) are of great significance, enabling the plants to detect a wide range of endogenous and exogenous signals which are employed by the plants in regulating various responses in development and adaptation. In this research work, we carried out genome-wide analysis of target of Myb1 (TOM1), a member of the GPCR gene family. The functional role of TOM1 in salt stress tolerance was studied using a transgenic Arabidopsis plants over-expressing the gene. By the use of the functional domain PF06454, we obtained 16 TOM genes members in Gossypium hirsutum, 9 in Gossypium arboreum, and 11 in Gossypium raimondii. The genes had varying physiochemical properties, and it is significant to note that all the grand average of hydropathy (GRAVY) values were less than one, indicating that all are hydrophobic in nature. In all the genes analysed here, both the exonic and intronic regions were found. The expression level of Gh_A07G0747 (GhTOM) was significantly high in the transgenic lines as compared to the wild type; a similar trend in expression was observed in all the salt-related genes tested in this study. The study in epidermal cells confirmed the localization of the protein coded by the gene TOM1 in the plasma membrane. Analysis of anti-oxidant enzymes showed higher concentrations of antioxidants in transgenic lines and relatively lower levels of oxidant substances such as H₂O₂. The low malondialdehyde (MDA) level in transgenic lines indicated that the transgenic lines had relatively low level of oxidative damage compared to the wild types. The results obtained indicate that Gh_A07G0747 (GhTOM) can be a putative target gene for enhancing salt stress tolerance in plants and could be exploited in the future for the development of salt stress-tolerant cotton cultivars.

Keywords: G-protein-coupled receptors; antioxidant; gene; salt tolerance; transgenic; wild type.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Phylogenetic tree, gene structure, and motif of all the cotton TOM genes. Red colour indicates the exons, blue indicates the up/downstream, and the grey lines are the introns. The motifs are numbered 1 to 20 and are illustrated by differently-coloured motifs unique to cotton TOM genes; (B) Common motifs which define the cotton-specific TOM gene.
Figure 2
Figure 2
Multiple sequence alignment of Gh_A07G0747 (GhTOM) protein and phylogenetic tree analysis. (A) Gh_A07G0747 (GhTOM) proteins tree analysis built using MEGA 7.0 program together with proteins from other plants as illustrated in the key. (B) Amino acids alignment of Gh_A07G0747 (GhTOM) with other TOM-GPCRs from other plants: Cotton_A_24028.1; Gorai.001G092500.1; Thecc1EG002003; Potri.005G203600.1; Potri.002G058500.1; AT3G59090.2; Thecc1EG029520; Gorai.011G042100.1; Gh_D10G0373.1; Cotton_A_17563.1; Gh_A10G0365.1 and LOC_Os01g54784.1.
Figure 3
Figure 3
Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the expression of the transformed gene Gh_A07G0747 (GhTOM). (A) Total RNA isolated from two-week-old cotton plant under normal conditions; (B) Total RNA extracted from salt-stressed cotton seedlings; (C) Polymerase chain reaction (PCR) analysis performed to check 978 bp coding sequence (CDS) integration in transformed T0 generation, number 1–13 transgenic lines, 14 positive control (pWM101-Gh_A07G0747 (GhTOM)), and 15 is the negative control (wild-type, WT). Expression of the transformed genes in transgenic; (D) the transcripts levels of the Gh_A07G0747 (GhTOM) of T2 transgenic lines analysed through qRT-PCR.
Figure 4
Figure 4
Changes of malondialdehyde (MDA) and H2O2 in Gh_A07G0747 (GhTOM) transgenic lines under salt stress. (A) Determination of MDA accumulation in leaves of wild-type (WT) and both overexpressed (OE) lines (OE-3, OE-7, and OE-9) after 8-day salt stress; (B) Quantitative determination of H2O2 accumulation in leaves of WT and both OE lines (OE-3, OE-7, and OE-9) after 8-day salt stress. In (A,B), each experiment was repeated three times. Bar indicates standard error (SE). Different letters indicate significant differences between wild-type and OE lines (ANOVA; p < 0.05). CK: normal conditions.
Figure 5
Figure 5
Activities of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) in WT and Gh_A07G0747 (GhTOM)—transgenic lines. Four weeks old WT and transgenic plants were salt-stressed for 8 days and then the leaf samples were taken and used to detect activities of CAT, SOD and POD. (A) CAT activity; (B) SOD activity; (C) POD activity. Data are means ± SE calculated from three replicates. Different letters indicate a significant difference between the WT and both transgenic lines (ANOVA; p < 0.05). Three independent experiments were carried out and the results were similar.
Figure 6
Figure 6
Comparative analysis of Gh_A07G0747 (GhTOM) transgenic and WT plant lines under salt stress conditions. (A) Gh_A07G0747 (GhTOM) overexpressing and WT plants were grown vertically in 0.5 Murashige and Skoog (MS) medium supplemented with 0, 150, and 200 mM NaCl and incubated for 6 days. (B) Survival rate of transgenic and wild type; (C) Comparison of root length between transgenic lines and WT seedlings grown on 0.5 MS medium containing different concentrations of NaCl; (D) Comparison of survival rate between transgenic lines and WT seedlings grown on 0.5 MS medium containing different concentrations of NaCl after 4 days of salt stress. Each value is the mean ± SD (n = 3), the letters “a” and “b” indicate that there is significant difference between the Gh_A07G0747 (GhTOM) transgenic and the wild-type seeds, which were determined through t-tests (* p = 0.05).
Figure 7
Figure 7
Expression levels of salt stress-responsive genes (CBL1, ABF4, RD29A, and SOS2) in wild-type and transgenic lines. Arabidopsis ACTIN2 was used as the reference gene, mean values with ± SD. * p < 0.05 as calculated by Student’s t-test.
Figure 8
Figure 8
Phenotypes of wild-type (WT) and Gh_A07G0747 (GhTOM)-transgenic Arabidopsis plants in response to salt stress. (A) Salt tolerance of potted plants of wild-type and Gh_A07G0747 (GhTOM)—OE Arabidopsis. Four-week-old WT and transgenic OE (OE-3, OE-7, and OE-9) plants were grown in soil in pots for 8 and 16 days under salt stress; (B) Chlorophyll determination between the wild-type and the transgenic lines after 8 days of salt stress. Values are mean ± SE, n = 12, and significant differences between wild-type and OE lines are indicated by different letters.
Figure 9
Figure 9
Localization of Gh_A07G0747 (GhTOM) in onion epidermal cells. (A–C) Onion epidermal cells transformed with 35S::GFP; (D–F) Onion epidermal cells transformed with 35S::GFP–Gh_A07G0747 (GhTOM). (A,D) Light field with magnification of X400 to display morphology. (B,E) Dark field images for the detection of green fluorescent protein (GFP) fluorescence. (C,F) Superimposed light and dark field images.
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