Anthocyanins are Key Regulators of Drought Stress Tolerance in Tobacco

Abstract

Abiotic stresses will be one of the major challenges for worldwide food supply in the near future. Therefore, it is important to understand the physiological mechanisms that mediate plant responses to abiotic stresses. When subjected to UV, salinity or drought stress, plants accumulate specialized metabolites that are often correlated with their ability to cope with the stress. Among them, anthocyanins are the most studied intermediates of the phenylpropanoid pathway. However, their role in plant response to abiotic stresses is still under discussion. To better understand the effects of anthocyanins on plant physiology and morphogenesis, and their implications on drought stress tolerance, we used transgenic tobacco plants (AN1), which over-accumulated anthocyanins in all tissues. AN1 plants showed an altered phenotype in terms of leaf gas exchanges, leaf morphology, anatomy and metabolic profile, which conferred them with a higher drought tolerance compared to the wild-type plants. These results provide important insights for understanding the functional reason for anthocyanin accumulation in plants under stress.

Keywords: leaf gas exchanges; leaf morphology and anatomy; metabolic profile; stomata.

Figure 1

A simplified representation of the anthocyanin biosynthetic pathway. CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; UFGT, flavonoid 3-O-glucosyltransferase. StAN1 is the transcription factor, which regulates anthocyanin biosynthesis in Solanum tuberosum.

Figure 2

StAN1 overexpression induces anthocyanin accumulation in tobacco plants. (A) Phenotype of a representative plant for wild-type (WT) and plants carrying p35S:StAN1 (AN1) at the end of the growth cycle; (B) Anthocyanin leaf content expressed as C3R equivalents.

Figure 3

Biometric parameters in AN1 and WT plants. (A) Leaves dry weight (DW); (B) Leaves fresh weight (FW); (C) Total leaf area. Asterisks indicate significant differences according to ANOVA (* p < 0.05).

Figure 4

Leaf gas exchanges in AN1 and WT plants. (A) CO₂ assimilation rate; (B) Stomatal conductance; (C) Transpiration rate. Asterisks indicate significant differences according to ANOVA (* p < 0.05; *** p < 0.001).

Figure 5

Leaf anatomical and morphological traits in AN1 and WT. (A) Leaf mass per area; (B) Pavement cells density; (C) Adaxial stomatal index; (D) Secondary vein density; (E) Succulence. Asterisks indicate significant differences according to ANOVA (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 6

Microscopy images of the adaxial side of (A) AN1 and (B) WT leaves (20×).

Figure 7

Lignin leaf content in AN1 and WT plants. Asterisks indicate significant differences according to ANOVA (*** p < 0.001).

Figure 8

Leaf carbohydrates content in AN1 and WT plants. (A) Glucose; (B) Fructose; (C) Sucrose; (D) Starch. Asterisks indicate significant differences according to ANOVA (* p < 0.05; ** p < 0.01).

Figure 9

Response of AN1 (grey boxes) and WT plants (white boxes) grown under control and drought condition. (A) CO₂ assimilation rate; (B) Shoot dry weight. Tukey post-hoc test was performed on significative differences according to two-way ANOVA (p < 0.05). Different letters indicate significant differences.

Figure 10

Schematic representation of the possible consequences of anthocyanin accumulation in tobacco leaves. The overexpression of StAN1 induces anthocyanin overproduction, which leads to a redirection of the phenylpropanoid pathway and/or a light screening effect mediated by anthocyanins. These events cause a full reshape of plant physiological, morphological and metabolic parameters, making StAN1 overexpressing plants more tolerant to drought stress.