Reduction of inositol (1,4,5)-trisphosphate affects the overall phosphoinositol pathway and leads to modifications in light signalling and secondary metabolism in tomato plants

Abstract

The phosphoinositol pathway is one of the major eukaryotic signalling pathways. The metabolite of the phosphoinositol pathway, inositol- (1,4,5) trisphosphate (InsP(3)), is a regulator of plant responses to a wide variety of stresses, including light, drought, cold, and salinity. It was found that the expression of InsP 5-ptase, the enzyme that hydrolyses InsP(3), also dramatically affects the levels of inositol phosphate metabolites and the secondary metabolites in transgenic tomato plants. Tomato plants expressing InsP 5-ptase exhibited a reduction in the levels of several important inositol phosphates, including InsP(1), InsP(2), InsP(3), and InsP(4). Reduced levels of inositol phosphates accompanied an increase in the accumulation of phenylpropanoids (rutin, chlorogenic acid) and ascorbic acid (vitamin C) in the transgenic fruits of tomato plants. The enhanced accumulation of these metabolites in transgenic tomato plants was in direct correspondence with the observed up-regulation of the genes that express the key enzymes of ascorbic acid metabolism (myo-inositol oxygenase, MIOX; L-galactono-γ-lactone dehydrogenase, GLDH) and phenylpropanoid metabolism (chalcone synthase, CHS1; cinnamoyl-CoA shikimate/quinate transferase, HCT). To understand the molecular links between the activation of different branches of plant metabolism and InsP(3) reduction in tomato fruits, the expression of transcription factors known to be involved in light signalling was analysed by real-time RT-PCR. The expression of LeHY5, SIMYB12, and LeELIP was found to be higher in fruits expressing InsP 5-ptase. These results suggest possible interconnections between phosphoinositol metabolism, light signalling, and secondary metabolism in plants. Our study also revealed the biotechnological potential for the genetic improvement of crop plants by the manipulation of the phosphoinositol pathway.

Fig. 1.

Levels of basic inositol phosphates (InsP₁, InsP₂, InsP₃, InsP₄) in leaves of transgenic tomato lines expressing the InsP 5-ptase gene (L6, L7) and control lines (WT, EV). Levels of ³H-labelled inositol phosphates (InsP₁, InsP₂, InsP₃, InsP₄) expressed as counts per minute (CPM) per mg fresh weight of transgenic tomato seedlings lines expressing the InsP 5-ptase gene (L6, L7) and control lines (WT, EV). (A) Inositol monophosphate (InsP₁); (B) inositol bisphosphate (InsP₂); (C) inositol trisphosphate (InsP₃); and (D) inositol tetrakisphosphate (InsP₄). Vertical bars indicate ±SE of three biological replicates.

Fig. 2.

Effect of expression of InsP 5-ptase gene (B) on expression of LeCHS1, tomato chalcone synthase gene (A), expression of HCT, cinnamoyl-CoA shikimate/quinate transferase gene (C) in tomato green fruits and production of chlorogenic acid and rutin (D) in red fruits. Vertical bars (C and D) indicate ±SE of three biological replicates. Analysis of gene expression was performed by RT-PCR (A, B) and real-time PCR (C).

Fig. 3.

Light signalling is modified in transgenic tomato plants expressing the InsP 5-ptase gene. (A) The phenotype of wild-type and InsP 5-ptase-expressing tomato plants cultivated under high-light conditions (800 μmol m⁻² s⁻¹ with 16 h light (25 °C) and 8 h dark (22 °C) for 2 months on incubation in vitro. (B) Expression of genes involved in light signalling in tomato green fruits expressing InsP 5-ptase (L6 and L7) and control lines (WT and EV) analyzed by real-time PCR. Expression levels are given relative to WT for each gene as separate assays. Vertical bars indicate ±SE of three biological replicates. (C) Hypothetical model of suggested network between InsP₃, light signalling factors, phenylpropanoids, and protection against light that may occur in transgenic tomato plants expressing InsP 5-ptase.

Fig. 4.

Simplified, schematic representation of cross-talk between general phosphoinositol pathway and biosynthetic pathways of ascorbic acid (vitamin C).

Fig. 5.

Effect of genetic reduction of InsP₃ in tomato plants on the expression of genes encoding key enzymes of ascorbic acid pathway (A) and the accumulation of ascorbic acid (B) in transgenic and control tomato fruits. Expression analysis (real-time PCR) was performed in green tomato fruits. Expression levels are given relative to WT for each gene as separate assays. Analysis of accumulation of ascorbic acid was performed in green and mature (red) fruits. Vertical bars indicate ±SE of three biological replicates.