Calcium Application Enhances Drought Stress Tolerance in Sugar Beet and Promotes Plant Biomass and Beetroot Sucrose Concentration

Abstract

Numerous studies have demonstrated the potential of sugar beet to lose the final sugar yield under water limiting regime. Ample evidences have revealed the important role of mineral nutrition in increasing plant tolerance to abiotic stresses. Despite the vital role of calcium (Ca²⁺) in plant growth and development, as well as in stress responses as an intracellular messenger, its role in alleviating drought stress in sugar beet has been rarely addressed. Here, an attempt was undertaken to investigate whether, and to what extent, foliar application of Ca²⁺ confers drought stress tolerance in sugar beet plants exposed to drought stress. To achieve this goal, sugar beet plants, which were grown in a high throughput phenotyping platform, were sprayed with Ca²⁺ and submitted to drought stress. The results showed that foliar application of Ca²⁺ increased the level of magnesium and silicon in the leaves, promoted plant growth, height, and leaf coverage area as well as chlorophyll level. Ca²⁺, in turn, increased the carbohydrate levels in leaves under drought condition and regulated transcriptionally the genes involved in sucrose transport (BvSUC3 and BvTST3). Subsequently, Ca²⁺ enhanced the root biomass and simultaneously led to induction of root (BvSUC3 and BvTST1) sucrose transporters which eventually supported the loading of more sucrose into beetroot under drought stress. Metabolite analysis revealed that the beneficial effect of Ca²⁺ in tolerance to drought induced-oxidative stress is most likely mediated by higher glutathione pools, increased levels of free polyamine putrescine (Put), and lower levels of amino acid gamma-aminobutyric acid (GABA). Taken together, this work demonstrates that foliar application of Ca²⁺ is a promising fertilization strategy to improve mineral nutrition efficiency, sugar metabolism, redox state, and thus, drought stress tolerance.

Keywords: carbohydrate synthesis; magnesium and silicon nutrition; metabolites; oxidative stress.

Figure 1

Influence of foliar application of Ca²⁺ on sugar beet root and shoot biomass and chlorophyll concentration under drought stress. (A) Beetroot dry weight; (B) beetroot diameter; (C) shoot dry weight; (D) chlorophyll concentration. Plants were grown in pots for a duration of 8 weeks. Five-week-old sugar beet plants were kept at 90% field capacity as control or exposed to drought stress (30% field capacity) for a duration of 3 weeks. Ca²⁺ was applied at BBCH14 and BBCH18 in concentration of 5 L ha⁻¹ corresponding to 540g Ca ha⁻¹. Leaves and beetroot were harvested at 60 days after sowing for biomass and chlorophyll analysis. Bars indicate means ± SD. Different letters denote significant differences according to ANOVA followed by SNK test (p < 0.05; n = 6).

Figure 2

Influence of foliar application of Ca²⁺ on sugar beet leaf coverage area shape and height under drought stress. (A) Representative image-based detection of leaf coverage area; (B) plant height estimation based on lateral imaging; and (C) leaf coverage area. Plants were grown in pots for a duration of 8 weeks. Five-week-old sugar beet plants were kept at 90% field capacity as control or exposed to drought stress (30% field capacity) for a duration of 3 weeks. Ca²⁺ was applied at BBCH14 and BBCH18 in concentration of 5 L ha⁻¹ corresponding to 540 g Ca ha⁻¹. Plants were imaged every four days from first Ca²⁺ application. Bars indicate means ± SD.

Figure 3

Influence of foliar application of Ca²⁺ on soluble sugar concentration in sugar beet exposed to drought stress. (A) Shoot glucose concentration; (B) shoot fructose concentration; (C) shoot sucrose concentration; (D) beetroot sucrose concentration; (E) correlation between beetroot sucrose content and shoot Mg content; and (F) correlation between beetroot sucrose content and shoot Si content. Plants were grown in pots for a duration of 8 weeks. Five-week-old sugar beet plants were kept at 90% field capacity as control or exposed to drought stress (30% field capacity) for a duration of 3 weeks. Ca²⁺ was applied at BBCH14 and BBCH18 in concentration of 5 L ha⁻¹ corresponding to 540 g Ca ha⁻¹. Leaves and beetroot were harvested at 60 days after sowing for soluble sugars analysis. Bars indicate means ± SEM. Different letters denote significant differences according to ANOVA followed by SNK test (p < 0.05; n = 6).

Figure 4

Influence of foliar application of Ca²⁺ on the expression levels of the genes involved in sugar transport in sugar beet plants exposed to drought stress. (A) Relative BvSUC3 mRNA levels in beetroot; (B) relative BvTST1 mRNA levels in beetroot; (C) relative BvSUT1 mRNA levels in beetroot; (D) relative BvSUC3 mRNA levels in leaves; (E) relative BvTST3 mRNA levels in leaves; and (F) relative BvSUT1 mRNA levels in leaves. Plants were grown in pots for a duration of 8 weeks. Five-week-old sugar beet plants were kept at 90% field capacity as control or exposed to drought stress (30% field capacity) for a duration of 3 weeks. Ca²⁺ was applied at BBCH14 and BBCH18 in concentration of 5 L ha⁻¹ corresponding to 540 g Ca ha⁻¹. Leaves and beetroot were harvested at 60 days after sowing for gene expression analysis. Bars indicate means ± SEM. Different letters denote significant differences according to ANOVA followed by SNK test (p < 0.05; n = 6).

Figure 5

Influence of foliar application of Ca²⁺ on shoot glutamate, GABA, and polyamine concentrations in sugar beet plants exposed to drought stress. (A) Shoot glutamate concentration; (B) shoot GABA concentration; (C) shoot putrescine concentration; (D) shoot spermidine concentration; and (E) shoot spermine concentration. Plants were grown in pots for a duration of 8 weeks. Five-week-old sugar beet plants were kept at 90% field capacity as control or exposed to drought stress (30% field capacity) for a duration of 3 weeks. Ca²⁺ was applied at BBCH14 and BBCH18 in concentration of 5 L ha⁻¹ corresponding to 540 g Ca ha⁻¹. Leaves and beetroot were harvested at 60 days after sowing for metabolite analysis. Bars indicate means ± SEM. Different letters denote significant differences according to ANOVA followed by SNK test (p < 0.05; n = 6).

Figure 6

Influence of foliar application of Ca²⁺ on shoot glutathione pool and on the expression of glutathione reductase in sugar beet exposed to drought stress. (A) Shoot GSH concentration; (B) shoot GSSG concentration; (C) shoot GSSH/GSH ratio; and (D) relative BvGR mRNA levels in leaves. Plants were grown in pots for a duration of 8 weeks. Five-week-old sugar beet plants were kept at 90% field capacity as control or exposed to drought stress (30% field capacity) for a duration of 3 weeks. Ca²⁺ was applied at BBCH14 and BBCH18 in concentration of 5 L ha⁻¹ corresponding to 540 g Ca ha⁻¹. Leaves and beetroot were harvested at 60 days after sowing for glutathione analysis. Bars indicate means ± SEM. Different letters denote significant differences according to ANOVA followed by SNK test (p < 0.05; n = 6).

Figure 7

Schematic representation of experimental design. After seed germination, the seedlings were grown for 8 weeks in the Roullier high-throughput plant phenotyping platform. Foliar application of Ca²⁺ was supplied twice at BBCH14 and BBCH18, each time with a dose of 5 L ha⁻¹ corresponding to 540 g Ca ha⁻¹. Drought stress (30% field capacity) was imposed 2 days after the second application of Ca²⁺ for a duration of 3 weeks, while control plants were kept in 90% of field capacity. From BBCH14 till end of the experiment, image-based phenotyping was applied every 4 days. All plants were harvested at 60 days after sowing for different physiological, biochemical, and molecular analyses.

Figure 8

Schematic model representing the regulatory role of Ca²⁺ on the response of sugar beet plants to drought stress. Ca²⁺ treated sugar beet plants effectively tolerated drought stress by regulating the concentration of Mg and Si in the leaves which consequently enhanced sugar metabolism both at the metabolic and transcriptional level. Ca²⁺ also increased the Na/K ratio in leaf displaying the possible role of Na in osmotic adjustment under drought stress. Shoots displayed increased leaf coverage area, higher biomass, and higher chlorophyll, along with increase in the beetroot diameter and beetroot biomass. Additionally, the lower GSSG/GSH ratio, higher putrescine levels, and reduced GABA level clearly showed that the Ca²⁺ treated plants were able to efficiently tolerate drought-induced oxidative stress. (Red arrows = increase/upregulation, blue arrows = decrease/downregulation).