Paclobutrazol induces tolerance in tomato to deficit irrigation through diversified effects on plant morphology, physiology and metabolism

Abstract

Dwindling water resources combined with meeting the demands for food security require maximizing water use efficiency (WUE) both in rainfed and irrigated agriculture. In this regard, deficit irrigation (DI), defined as the administration of water below full crop-water requirements (evapotranspiration), is a valuable practice to contain irrigation water use. In this study, the mechanism of paclobutrazol (Pbz)-mediated improvement in tolerance to water deficit in tomato was thoroughly investigated. Tomato plants were subjected to normal irrigated and deficit irrigated conditions plus Pbz application (0.8 and 1.6 ppm). A comprehensive morpho-physiological, metabolomics and molecular analysis was undertaken. Findings revealed that Pbz application reduced plant height, improved stem diameter and leaf number, altered root architecture, enhanced photosynthetic rates and WUE of tomato plants under deficit irrigation. Pbz differentially induced expression of genes and accumulation of metabolites of the tricarboxylic acid (TCA) cycle, γ-aminobutyric acid (GABA-shunt pathway), glutathione ascorbate (GSH-ASC)-cycle, cell wall and sugar metabolism, abscisic acid (ABA), spermidine (Spd) content and expression of an aquaporin (AP) protein under deficit irrigation. Our results suggest that Pbz application could significantly improve tolerance in tomato plants under limited water availability through selective changes in morpho-physiology and induction of stress-related molecular processes.

Figure 1. Pbz induced morphological adaptations under deficit irrigation.

Effects of PBZ application (0, 0.8 and 1.6 ppm) in irrigated and deficit-irrigated tomato plants grown over a period of 105-days on the morphological parameters including (A) stem thickness, (B) leaf number, (C) plant height, (D) leaf area, (E) root area, (F) leaf area(LA)/root area (RA) ratio, (G) root dry weight (RDW), (H) shoot dry weight (ShDW) and (I) RDW/ShDW ratio.

Figure 2. Pbz application modulates endogenous abscisic acid and spermidine contents.

Effects of PBZ application (0, 0.8 and 1.6 ppm) in irrigated (I) and deficit-irrigated (DI) tomato plants on the endogenous content of (A) abscisic acid (ABA) and expression of ABA biosynthesis genes (B,C) SlZEP, SlNCED and SlAAO1, and relative content of (D) spermidine (Spd) and expression of (E) spermidine synthase gene (SlSPDS) in the leaf tissue of 105-days old tomato plants. Letters (A,B and a,b,c) indicate significant differences (one way ANOVA, p < 0.05) from the control in irrigated and deficit-irrigated conditions, respectively.

Figure 3. Pbz induced tricarboxylic acid cycle (TCA) adaptations confer tolerance to deficit irrigation.

Effects of Pbz application (0, 0.8 and 1.6 ppm) in irrigated (I) and deficit- irrigated (DI) tomato plants on the relative content of the citric/tricarboxylic acid cycle (TCA cycle) intermediates, sugar metabolism and GABA content and the gene expression of enzymes implicated in their metabolism in the leaf tissue of 105-days-old tomato plants as shown in fold change in Log-scale (−1 to +1.5) under irrigated (I) and deficit- irrigated (DI) conditions.

Figure 4. Pbz improved deficit irrigation tolerance by inducing anti-oxidant adaptation via modulation of the Glutathione-Ascorbate cycle.

Effects of Pbz application (0, 0.8 and 1.6 ppm) in irrigated (I) and deficit-irrigated (DI) tomato plants on the relative content of (A) ascorbic acid, ASC and dehydroascorbate, DHA, (B,C,F) expressions analysis of genes related to GSH and ASA biosynthesis (SlGLDH, SlPGbeta, SlAPX, SlMDHAR and SlGR) and (D,E) glutathione (GSH and GSSG-ascorbate cycle components) in the leaf tissue of 105-days old tomato plants. Letters (A,B and a, b) indicate significant differences from the control in irrigated and deficit-irrigated conditions, respectively (one way ANOVA, p < 0.05).

Figure 5. Pbz contributes in tolerance to deficit irrigation by modulating cell wall metabolism.

Effects of PBZ application (0, 0.8 and 1.6 ppm) in irrigated (I) and deficit-irrigated (DI) conditions on cell wall metabolites conferring cell wall stability (A) fucose, (C) xylose and itaconate, (E) galactouronic acid and ferrulic acid; and expression analysis of genes related to cell wall organization (B,D) SlPME, SlEXP1 and SlXTH5. Capital letters (A,B) indicate significant differences from the PBZ untreated control in the irrigated plants. Small italicized letters (a, b) indicate significant differences from the PBZ untreated control in the deficit-irrigated plants (one way ANOVA, p < 0.05).

Figure 6. A schematic model describing mechanism of Pbz mediated water deficit tolerance in tomato.

Drought stress in plants is accompanied by secondary oxidative or osmotic stress leading to reduction in water potential, disruption of ionic and osmotic homeostasis and damage to proteins and membranes, ultimately resulting in reduced photosynthetic efficiency and overall growth of the plant. Such a signal, perceived in the nucleus leads to activation of stress responsive genes. Similarly, anti-oxidant genes, genes responsible for ABA biosynthesis as well as genes governing primary metabolism (sugar synthesis, glucose, fructose, sucrose) and constituents of cell wall permeability are induced imparting osmoprotection, re-establishment of cellular homeostasis and tolerance to water deficit. Water deficit stress when applied in conjunction with Pbz engenders modulation of central metabolism through enhanced TCA cycle activity and regulation of gene expression associated with GABA shunt signaling. Generally, external stimuli like drought stress or deficit irrigation described here lead to increase in endogenous GABA levels permitting its adherence to cell-surface binding sites, enabling an interim increase in Ca²⁺ pools and its import into cells through high affinity GABA transporters (e.g., GAT174). Considerably, GAD is activated through a Ca²⁺/CaM complex. Thereafter, increased intracellular GABA activates various signaling cascades and genes of primary metabolism (SlHK and SlPGM) while inhibiting some, like genes responsible for cell wall-modifications. Additionally, subject to outside environment, a substantial portion of cytosolic GABA makes way into mitochondria via the GABA permease, At GABP, for catabolism by GABA-T and SSADH, producing succinate for feeding into the Calvin Cycle. Enzyme represent oval grey boxes, their reactions represented by black arrows. Brown lines indicate a regulatory effect. Blue spheres denote GABA, red crescents denote GABA receptors. Abbreviations: Pbz, Paclobutrazol; GABA, γ-aminobutric acid; GAT1, GABA transporter 1 SlNCED, 9-cis-epoxy-carotenoid dioxygenase; SlTIP2, tonoplast intrinsic protein 2; SlHK, hexokinase; SlPGM, phosphoglucomutase; SlPME, Pectin methyl esterase; SlXTH5, Xyloglucan endotransglycosylase; SlEXP1, expansin1; SlGDH, Glutamate dehydrogenase; CaM, calmodulin; GAD, glutamate decarboxylase; GABA-T, GABA transaminase; SSADH, succinic semialdehyde dehydrogenase; respectively.