Overexpression of a Gene Encoding Trigonelline Synthase from Areca catechu L. Promotes Drought Resilience in Transgenic Arabidopsis

Abstract

Areca catechu L. is a commercially important palm tree widely cultured in tropical and subtropical areas. Its growth and production are severely hindered by the increasing threat of drought. In the present study, we investigated the physiological responses of areca seedlings to drought stress. The results showed that prolonged drought-induced yellowing on the overall area of most leaves significantly altered the chlorophyll fluorescence parameters, including maximum chemical efficiency (Fv/Fm), photochemical efficiency of PSII (Y(II)), photochemical chlorophyll fluorescence quenching (qP) and non-photochemical chlorophyll fluorescence quenching (NPQ). On the 10th day of drought treatment, the contents of proline in the areca leaves and roots increased, respectively, by 12.2 times and 8.4 times compared to normal watering. The trigonelline levels in the leaves rose from 695.35 µg/g to 1125.21 µg/g under 10 days of water shortage, while no significant changes were detected in the content of trigonelline in the roots. We determined the gene encoding areca trigonelline synthase (AcTS) by conducting a bioinformatic search of the areca genome database. Sequence analysis revealed that AcTS is highly homologous to the trigonelline synthases in Coffea arabica (CaTS 1 and CaTS 2) and all possess a conserved S-adenosyl- L-methionine binding motif. The overexpression of AcTS in Arabidopsis thaliana demonstrated that AcTS is responsible for the generation of trigonelline in transgenic Arabidopsis, which in turn improves the drought resilience of transgenic Arabidopsis. This finding enriches our understanding of the molecular regulatory mechanism of the response of areca to water shortage and provides a foundation for improving the drought tolerance of areca seedlings.

Keywords: areca seedling; drought resilience; gene expression; trigonelline synthase.

Figure 1

Growth of the areca seedlings under treatment (n = 3). (A) Phenotype of the areca seedlings. Scale bar = 10 cm. (B) The value of Fv/Fm. (C) The value of Y(II). (D) The value of qP. (E) The value of NPQ. Data are expressed as the means ± standard errors of the means. * p value < 0.05; ** p < 0.01.

Figure 2

The content of trigonelline and proline in the areca leaves and roots (n = 3). (A) The content of trigonelline in the areca leaves. (B) The content of trigonelline in the areca roots. (C) The content of proline in the areca leaves. (D) The content of proline in the areca roots. DW: dry weight. Data are expressed as the means ± standard errors of the means. * p value < 0.05; ** p < 0.01.

Figure 3

Sequence analysis of trigonelline synthases from different plant species. (A) Phylogenetic tree and conserved motifs of AcTS, CaTS1, CaTS2, AtTS, GmTS and OsTS. Different motifs are represented by different boxes. The location of each motif can be estimated using the scale at the bottom. (B) Alignment of amino acid sequences of AcTS, CaTS1, CaTS2, AtTS, GmTS and OsTS. Three proposed SAM regions are shown in the open boxes. AcTS: Areca catechu trigonelline synthase; AtTS: Arabidopsis thaliana trigonelline synthase; OsTS: Oryza sativa trigonelline synthase; CaTS: Coffea arabica trigonelline synthase; GmTS: Glycine max trigonelline synthase.

Figure 4

The transcript abundance of AcTS in areca leaves and roots under treatment (n = 3). (A) Transcript abundance of AcTS in acrea leaves under treatment. (B) Transcript abundance of AcTS in acrea roots under treatment. Data are expressed as the means ± standard errors of the means. * p-value < 0.05; ** p < 0.01.

Figure 5

The potential role of AcTS in response to drought. (A) The content of trigonelline in Col-0 and transgenic Arabidopsis overexpressing AcTS (OxAcTS14 and OxAcTS14). (B) The phenotype of Col-0 and OxAcTS under drought and rehydration treatments. (C) The phenotype of Col-0 and OxAcTS on half-strength MS medium and half-strength MS medium supplemented with mannitol. (D) The root length of Col-0 and OxAcTS on half-strength MS medium and half-strength MS medium supplemented with mannitol. (E) The phenotype of Col-0 on half-strength MS medium, half-strength MS medium supplemented with trigonelline, half-strength MS medium supplemented with mannitol and half-strength MS medium supplemented with trigonelline and mannitol. A minus (−) sign indicates that the growth medium lacked the corresponding component, and a plus (+) sign indicates that the growth medium was supplemented with the corresponding ingredients. (F) The root length of Col-0 on half-strength MS medium, half-strength MS medium supplemented with trigonelline, half-strength MS medium supplemented with mannitol and half-strength MS medium supplemented with trigonelline and mannitol. Data are expressed as the means ± standard errors of the means. * p value < 0.05; ** p < 0.01.