Table 1.
Characteristics of genes encoding citrus GolS proteins.
Fig 1.
Phylogeny and expression of C. sinensis GolS (CsGolS) genes.
(A) Phylogenetic relationships of CsGolS amino acid sequences with those of Arabidopsis thaliana (At). (B) RNA-Seq data of CsGolS expression in the different C. sinensis tissues. RPKM: reads per kilobase per million mapped. (C-G) Expression analysis of CsGolS genes in response to drought (C, D) and salt (E, F) stresses and ‘Ca. L. asiaticus’ infection (G). Ratios (log2) of relative mRNA levels in leaves and roots between stressed and control plants of sweet orange (C-F) and in leaves between infected and control plants of rough lemon (RL) and sweet orange (SW) at 0, 7, 17, and 34 WAI (G), as measured by qPCR. GAPC2 was used as an endogenous control. Data are means ± SE from three biological replicates. *,**,***Significantly different from control treatment (C-F) or sweet orange at the respective WAI (G) by the Student’s t test at P ≤ 0.05, P ≤ 0.01 or P ≤ 0.001, respectively.
Fig 2.
In vitro stress tolerance assay of CsGolS6-overexpressing transgenic tobacco plants.
(A) Representative phenotypes of WT and CsGolS6-overexpressing transgenic (L3-12) lines grown under control, salt and PEG treatments for 20 days. (B) Seedling biomass of WT and transgenic lines under control, salt and PEG treatments for 20 days. The data are means ± SE of three technical replicates composed of fifteen seedlings for each line. *,**,***Significantly different from WT at the respective treatment by the Student’s t test at P ≤ 0.05, P ≤ 0.01 or P ≤ 0.001, respectively.
Fig 3.
Physiological analysis of WT and CsGolS6-overexpressing transgenic tobacco plants under different water regimes in greenhouse conditions.
WT and transgenic (L3-L12) plants were exposed to three water regimes: (i) control (leaf water potential at -0.3 a -0.5 MPa), (ii) drought (leaf water potential at -1.5 a -2.0 MPa) and (iii) rehydration (leaf water potential at -0.3 a -0.5 MPa after a cycle of drought stress). The data are means ± SE of ten biological replicates. *,**Significantly different from WT at the respective water treatment by the Dunnett test at P ≤ 0.05 or P ≤ 0.01, respectively.
Fig 4.
Growth performance of WT and CsGolS6-overexpressing transgenic tobacco under control (irrigated) and drought-stress conditions.
WT and transgenic (L3-L12) plants grown under greenhouse conditions were exposed to a progressive soil water deficit (drought) until reach leaf water potentials of ~-2.0 MPa, or maintained at leaf water potentials of -0.3 to -0.5 MPa as the control treatment. The data are means ± SE of ten biological replicates. *,**Significantly different from WT at the respective water treatment by the Dunnett test at P ≤ 0.05 or P ≤ 0.01, respectively.
Fig 5.
Anatomical analysis of leaf cross-sections of WT and CsGolS6-overexpressing transgenic plants under control (irrigated) and drought stress conditions.
(A) Leaf cross-section of WT and CsGolS6-overexpressing transgenic (L3, L6 and L10) plants subjected to drought stress, as observed under photonic microscope. The mesophyll region is shown. Magnification bars represent 100 μm. Adaxial side (adx), abaxial side (adx), palisade parenchyma (pp), spongy parenchyma (pe), glandular trichomes (tg), stomata (*), vascular tissue (arrow) and idioblasts (i). (B) Quantitative analysis of the leaf anatomy of WT and transgenic (L3, L6, L10 and L12) plants subjected to control (irrigated) and drought stress treatments. The data are means ± SE of three technical replicates, each containing three slides composed for each plant line. *Statistically significant differences between WT and transgenic plants by the Dunnett test at P ≤ 0.05.
Fig 6.
Primary metabolite profiles of WT and CsGolS6-overexpressing transgenic tobacco under control (C) and drought (D) conditions.
Green and red colors represent, respectively, increase and decrease of metabolites using a false-color scale. Values are the means of three biological replicates.