Fig 1.
Spatial and temporal pattern of SlGLK2 transcript abundance.
Histogram representation of SlGLK2 relative mRNA levels in wild-type (SlGLK2), Slglk2 and au-SlGLK2 mutant genotypes. Values represent means ± SE of at least three biological replicates normalised against the basal IG3 sample from SlGLK2 genotype. Statistically significant differences between the pedicellar and basal samples are indicated by asterisks (P < 0.05). Within the pedicellar samples from each genotype, statistically significant differences to the respective IG3 sample are indicated by triangles (P < 0.05). Within the pedicellar samples of each stage, statistically significant differences to SlGLK2 genotype are indicated by plus signal (P < 0.05). ND: Not detected. The entire set of data is presented in S2 Table.
Fig 2.
Interplay between SlGLK2 and auxin and cytokinin production and signalling.
(A) Auxin and cytokinin responsiveness in genotypes encoding functional (SlGLK2) and truncated (Slglk2) SlGLK2 proteins. Responsiveness was addressed by evaluating the activity of the GUS reporter, whose expression was controlled by the auxin- or cytokinin-responsive promoter DR5 and ARR5, respectively. Different letters indicate statistically significant differences between stages within each genotype (P < 0.05). Asterisks denote statistically significant differences between genotypes within the same fruit stage (P < 0.05). Values represent mean ± SE of at least three biological replicates. (B) Relative transcript value of SlGLK2 along fruit development and ripening in wild-type (SlGLK2), auxin insensitive (dgt) and low-cytokinin (35S::CKX2) genotypes. Different letters indicate statistically significant differences between stages within each genotype (P < 0.05). Asterisks denote statistically significant differences between genotypes within the same fruit stage in comparison to the SlGLK2 sample (P < 0.05). Values represent mean ± SE of at least three biological replicates.
Fig 3.
Phenotype, chlorophyll and tocopherol content of fruits.
Phenotype of fruits at IG5 stage from SlGLK2, au-SlGLK2 and Slglk2 genotypes. Scale bar = 1 cm (A). Chlorophyll (B) and total tocopherol (C) content in the pedicellar portion of fruits of wild-type (SlGLK2), Slglk2 and au-SlGLK2 mutant genotypes. Different letters indicate statistically significant values between genotypes within each stage (P < 0.05). Values represent mean ± SE of at least three biological replicates.
Fig 4.
Transcript profile of tocopherol-related encoding genes.
Heatmap representation of the mRNA abundance of tocopherol-related encoding genes in the pedicellar portion of immature green (IG5) and ripe (Br+5) fruits of wild-type (SlGLK2), truncated SlGLK2-encoding (Slglk2) and au-SlGLK2 mutant genotypes. Coloured squares represent statistically significant differences in relation to the respective wild-type (SlGLK2) sample (P < 0.05). Values represent mean of at least three biological replicates. Relative transcript values are detailed in S4 Table. Methylerythritol phosphate pathway (orange), shikimate pathway (red), chlorophyll metabolism (green), phytol recycling (purple) and tocopherol biosynthesis (blue). Dotted lines indicate that intermediate steps were omitted. Enzymes: DXS: 1-DEOXY-D-XYLULOSE-5-P SYNTHASE; GGDR: GERANYLGERANYL DIPHOSPHATE REDUCTASE; VTE1: TOCOPHEROL CYCLASE; VTE2: HOMOGENTISATE PHYTYL TRANSFERASE; VTE3: 2,3-DIMETHYL-5-PHYTYLQUINOL METHYL TRANSFERASE; VTE4: TOCOPHEROL γ-METHYL TRANSFERASE; VTE5: PHYTOL KINASE; VTE6: PHYTYL-PHOSPHATE KINASE. Metabolites: G3P: glyceraldehyde 3-phosphate; DXP: 1-deoxy-D-xylulose-5P; GGDP: geranylgeranyl-2P; PDP: phytyl diphosphate; HGA: homogentisate; HPP: hydroxyphenylpyruvate; PEP: phosphoenolpyruvate; E4P: erythrose 4-phosphate; MPBQ: 2-methyl-6-phytylquinol; DMBQ: 2,3-dimethyl-5-phytylquinol; PP: phytyl phosphate.
Fig 5.
Characterisation of SlGLK2 overexpressing lines in au-Slglk2 background.
(A) SlGLK2 relative transcript ratio in the pedicellar region of IG5 fruits from control genotypes (SlGLK2 and au-Slglk2) and SlGLK2-overexpressing lines (L2, L7 and L8). Letters indicate statistically significant differences among the genotypes (P < 0.05). Values are normalised against au-Slglk2 sample and represent mean ± SE of at least three biological replicates. (B) Detection of SlGLK2 protein (34.4 KDa) by Western blot in the pedicellar portion of IG5 fruits from control genotypes (SlGLK2 and au-Slglk2) and SlGLK2-overexpressing lines (L2, L7 and L8). (C) Fruit phenotypes from control genotypes (SlGLK2 and au-Slglk2) and SlGLK2-overexpressing lines (L2, L7 and L8). (D) Chlorophyll content in the pedicellar portion of IG5 fruits from control genotypes (SlGLK2 and au-Slglk2) and SlGLK2-overexpressing lines (L2, L7 and L8). Letters indicate statistically significant differences among the genotypes (P < 0.05). Values represent mean ± SE of at least three biological replicates.
Fig 6.
Chloroplast number and structure in SlGLK2 overexpressing lines.
(A) and (B) Number and area of chloroplasts at the pedicellar portion of IG5 fruits of untransformed genotypes (SlGLK2 and au-Slglk2) and transformed au-Slglk2 SlGLK2-overexpressing L2 line. Letters indicate statistically significant differences among the genotypes (P < 0.05). Values represent mean ± SE of at least 10 biological replicates. (C) Chloroplast ultrastructure of IG5 fruits of untransformed genotypes (SlGLK2 and au-Slglk2) and transformed au-Slglk2 SlGLK2-overexpressing L2 line. Scale bar = 0.5 μM (above) or 0.2 μM (below).
Fig 7.
Sugar and tocopherol content in ripe fruits from SlGLK2 overexpressing line.
(A) Total soluble solids (°BRIX) in Br+5 ripe fruits from untransformed genotypes (SlGLK2 and au-Slglk2) and SlGLK2-overexpressing L2 line. Letters indicate statistically significant differences among the genotypes (P < 0.05). Values represent mean ± SE of at least 12 biological replicates. Soluble sugars (glucose, fructose and sucrose) (B) and tocopherol content (C) in the pedicellar portion in Br+5 ripe fruits from untransformed genotypes (SlGLK2 and au-Slglk2) and SlGLK2-overexpressing L2 line. Letters indicate statistically significant differences among the genotypes (P < 0.05). Values represent mean ± SE of at least three biological replicates.
Fig 8.
Transcript profile of tocopherol-related encoding genes in fruits from SlGLK2 overexpressing line.
Heatmap representation of the mRNA abundance of tocopherol-related encoding genes in the pedicellar portion of immature green (IG5) and ripe (Br+5) fruits of untransformed genotypes (SlGLK2 and au-Slglk2) and transformed au-Slglk2 SlGLK2-overexpressing L2 line. Values are normalised against the respective au-Slglk2 sample. Coloured squares indicate statistically significant differences compared to au-Slglk2 (P < 0.05). Values represent mean of at least three biological replicates. Relative transcript values are described in S6 Table. Methylerythritol phosphate pathway (orange), shikimate pathway (red), chlorophyll metabolism (green), phytol recycling (purple) and tocopherol biosynthesis (blue). Dotted lines indicate that intermediate steps were omitted. Enzymes: DXS: 1-DEOXY-D-XYLULOSE-5-P SYNTHASE; HPPD2: 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE (2); GGDR: GERANYLGERANYL DIPHOSPHATE REDUCTASE; VTE1: TOCOPHEROL CYCLASE; VTE2: HOMOGENTISATE PHYTYL TRANSFERASE; VTE3: 2,3-DIMETHYL-5-PHYTYLQUINOL METHYL TRANSFERASE; VTE4: TOCOPHEROL Γ -METHYL TRANSFERASE; VTE5: PHYTOL KINASE; VTE6: PHYTYL-PHOSPHATE KINASE; PPH: PHEOPHYTINASE; PPHL1: PHEOPHYTINASE LIKE-1; CHLG: CHLOROPHYLL SYNTHASE. Metabolites: G3P: glyceraldehyde 3-phosphate; DXP: 1-deoxy-D-xylulose-5P; GGDP: geranylgeranyl-2P; PDP: phytyl diphosphate; HGA: homogentisate; HPP: hydroxyphenylpyruvate; PEP: phosphoenolpyruvate; E4P: erythrose 4-phosphate; MPBQ: 2-methyl-6-phytylquinol; DMBQ: 2,3-dimethyl-5-phytylquinol; PP: phytyl phosphate.
Fig 9.
Phytochromes (PHY) acts as positive regulators of auxins [39], which, in turn, repress SlGLK2 expression either directly or indirectly via SlAFR4, a negative regulator of auxin signalling [6]. In dgt background, an auxin response impaired genotype [33], the negative regulation of auxin over SlGLK2 gene expression is disrupted. Cytokinin response is upregulated in the presence of SlGLK2. The positive effect of cytokinins over AtGLK2 expression, described by [3] (dashed arrow), was not verified in tomato fruits. SlGLK2 is a master transcription factor that promotes the differentiation of proplastids into chloroplasts with the corresponding chlorophyll accumulation in green fruits, which is directly proportional to VTE content in ripe fruits. SlARF4 inhibits AGPase expression and enzyme activity [6]. Moreover, auxin is known to inhibit AMYLASE activity, which is associated with ripening-inducing starch degradation for soluble sugar accumulation in climacteric ripe fruits [56], probably mediated by SlGLK2. The balance between AGPase and AMYLASE enzyme activities determines the starch and, in part, soluble sugar content in green and ripe stages of fruits development.