Divergent Impacts of Moderate and Severe Drought on the Antioxidant Response of Calendula officinalis L. Leaves and Flowers ^†

Abstract

We studied the impacts of moderate and severe drought on different pot marigold (Calendula officinalis L.) genotypes, evaluating the antioxidant performance of leaves and flowers concerning the levels of proline and malondialdehyde, the activity of antioxidant enzymes (catalase, peroxidase, and ascorbate peroxidase), as well as impacts on flower production. Overall, we found high resilience to moderate drought. However, the severe drought significantly affected flower production, despite the high level of antioxidants, proline, and malondialdehyde. Results also indicate significant variation in drought tolerance among pot marigolds, providing an opportunity to identify valuable tolerance traits.

1. Introduction

Drought stress is a major abiotic stress, limiting crop production and yield [1,2]. Drought decreases photosynthesis and chlorophyll synthesis, and alters nutrient metabolism, ion uptake, and translocation, ultimately limiting plant vegetative growth. Drought also has an impact on flower number and size, affecting the viability and durability of flowers [3]. The abortion of reproductive organs is a major limiting factor for flower production under water stress. Thus, understanding its impacts has become a major aim for the sustainability of the floricultural industry.

Drought triggers the overproduction of reactive oxygen species (ROS), which are toxic and cause damage to proteins, lipids, carbohydrates, and DNA, leading to oxidative stress and, ultimately, plant death [4,5]. To cope with the effects of drought, plants activate the antioxidant system that includes a variety of ROS scavengers, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POX), and catalase (CAT), as well as non-enzymatic metabolites such as carotenoids, flavonoids, and proline [6]. Malondialdehyde (MDA) produced by membrane lipids in response to ROS, is often used as a drought indicator to evaluate the degree of membrane damage and the level of drought tolerance since plants with low amounts of MDA are generally considered to be more tolerant to drought [7]. Nevertheless, the antioxidant response is highly dependent on the level and duration of drought and the species, and even varies within genotypes of the same species [8,9,10].

Calendula officinalis L. (pot marigold or calendula), from the plant family Asteraceae, is a widely used plant in ornamental horticulture, as well as in traditional healing treatments, due to its wide range of secondary metabolites, flavonoids, and carotenoids content [11]. It has recently been proposed as an oilseed crop since the oil from its seeds has high amounts of fatty acids [11]. Nevertheless, it is highly affected by drought, although some genotypes show some tolerance to water scarcity [12]. For instance, drought caused a significant decline in the number of leaves and leaf area in Calendula cv. Orange King [13]. In measurements performed under field conditions, drought increased the activity of CAT, POX, and APX, as well as the levels of MDA and leaf and root proline while reducing seed yield in several pot marigold genotypes [12]. Nevertheless, high variation in responses was found between genotypes [12]. Drought can be a significant constraint in this species, but studies performed under controlled conditions remain scarce. Consequently, this study aims to understand the impacts of moderate and severe drought in four different pot marigold genotypes under a controlled environment. Specifically, we aim to understand the response of the pot marigold antioxidant machinery to drought, namely, the levels of proline and malondialdehyde, and the enzymatic activity of catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX) in the leaves and flowers. We also aim to detect the impact of drought on the production of flowers.

2. Material and Methods

2.1. Plant Experimental Design

The antioxidant performance of Calendula officinalis was tested in four different genotypes: cv. Indian Prince, Golden Emperor, Orange Prince, and Sun Glow. Seeds were grown in 2 L capacity pots in a controlled environmental chamber under a long-day photoperiod (16 h light), at a temperature of 23/1 °C (light/dark period) and relative humidity of 72–76%. Plants were watered every two days using the Hoagland nutritive solution. One-month-old Calendula plants were subjected to different water treatments (control, 100% field capacity (FC); moderate drought, 60% FC; and severe drought, 35% FC) and followed for three weeks [12]. Each treatment consisted of 10 biological replications per cultivar.

2.2. Non-Enzymatic Activities in Leaves and Flowers

Proline was determined according to acid–ninhydrin and toluene methods [14]. Absorbance was determined at 520 nm. Results are expressed in micrograms of proline per gram of dry weight. Lipid peroxidation was quantified according to [15] and measured in terms of malondialdehyde content (MDA). Absorbance was measured at 532 nm. Results are expressed as nmol of MDA per gram of dry weight.

2.3. Antioxidative Enzyme Activities in Leaves and Flowers

The activity of catalase (CAT) was determined in a 1.5 mL reaction mixture with 50 mM K-phosphate buffer (pH 7.0), 10 mM H₂O₂, and the enzyme following [16], and measured at 240 nm. The results were expressed in CAT mg⁻¹ of protein. Peroxidase activity (POX) was determined as described by [17], measuring the absorbance at 430 nm. Results are expressed in units mg⁻¹ of protein. Ascorbate peroxidase (APX) was determined according to [18], measured at 290 nm. Results are expressed in APX mg⁻¹ of protein. Data are expressed in dry weight (DW).

2.4. Impacts of Drought on the Production of Flowers

For each genotype and experimental treatment, we quantified the total number of flowers produced. The dry matter content was acquired by drying samples to a constant weight using a thermo-ventilated oven at 65 °C.

2.5. Statistical Analysis

Mean values (±SE) were calculated from 10 replicates per cultivar using IBM SPSS v.22. To analyze the effects of salinity, we used a multivariate ANOVA or a t-test after checking the homogeneity of variance using Levene’s Test for Equality of Variances. Significant differences between means were also subjected to Tukey’s test for post hoc comparisons (at the 1% significance level).

3. Results and Discussion

3.1. Non-Enzymatic Activities in Leaves and Flowers

Moderate drought had no significant effects on the levels of proline in the leaves of the Calendula genotypes (F_2,11 = 0.341, p = 0.871), while some slight significant increases were already reported in the flowers of Golden Emperor and Sun Glow (respectively, F_2,11 = 11.872, p < 0.05; F_2,11 = 10.201, p < 0.05; Figure 1A). In contrast, severe drought significantly increased the levels of proline in all genotypes, either considering leaves or flowers (Figure 1A). The accumulation of proline under drought stress has been reported in many plant species [19], including Calendula [12]. Proline is mainly synthesized in the leaves and transported to other areas to balance osmotic pressure and scavenge ROS, allowing plants to cope with drought [20]. Under stress, proline also maintains cell turgor or osmotic balance, stabilizes membranes, and prevents oxidative bursts in plants. Thus, the increase found in this study suggests its role in protecting Calendula cultivars from the effects of severe drought.

The levels of MDA showed no significant differences between the control conditions and moderate drought when recorded in the leaves (F_2,11 = 0.121, p < 0.05; Figure 1B). However, slight increases were already felt in the flowers of the Golden Emperor and Sun Glow genotypes (Figure 1B). The highest increase in MDA was recorded under severe drought, in all genotypes, especially in the Golden Emperor and Sun Glow genotypes (Figure 1B). ROS induces lipid peroxidation, giving rise to MDA, an indicator of membrane damage, especially during stress. Overall, the more the plant is stressed, the higher its MDA content. Thus, this stress marker indicates that the flowers of some Calendula genotypes were already affected by moderate drought, while severe drought had strong negative effects on lipid peroxidation in the leaves and flowers of all genotypes. The level of MDA can be used in future studies to evaluate the degree of plasma membrane damage and the ability of Calendula plants to tolerate drought stress.

3.2. Antioxidative Enzyme Activities in Leaves and Flowers

Under moderate drought, the enzymatic activities of catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX) measured in the leaves showed no significant differences from control conditions in all genotypes (

Table 1. Enzyme activities of catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX), expressed as mg of protein/DW under control water conditions, moderate drought (MD), and severe drought (SD) in four Calendula officinalis genotypes. Results indicate measurements in leaves/flowers. Results are expressed as means ± SE (n = 10). Different superscripts indicate significant differences between water levels for the same species (ANOVA followed by a Tukey test at p < 0.001) considering only leaves or flowers.

	Control	MD	SD
CAT
Indian Prince	11.23 ± 2.21 a/8.14 ± 1.17 a	11.66 ± 3.44 a/8.11 ± 1.19 a	28.37 ± 4.22 b/10.14 ± 1.29 a
Golden Emperor	14.25 ± 2.74 a/7.89 ± 1.15 a	14.23 ± 3.01 a/8.55 ± 2.20 b	21.99 ± 4.05 b/10.31 ± 2.17 c
Orange Prince	12.07 ± 2.22 a/7.99 ± 1.11 a	12.03 ± 2.66 a/9.01 ± 1.25 b	16.28 ± 3.31 b/14.20 ± 1.99 c
Sun Glow	10.03 ± 2.00 a/6.45 ± 1.11 a	10.20 ± 2.11 a/7.25 ± 1.19 b	17.25 ± 3.07 b/9.29 ± 2.33 c
POX
Indian Prince	1.24 ± 0.22 a/0.33 ± 0.07 a	1.51 ± 0.44 a/1.03 ± 0.98 b	3.25 ± 0.22 b/2.07 ± 0.01 c
Golden Emperor	0.98 ± 0.21 a/0.41 ± 0.11 a	1.03 ± 0.11 a/0.48 ± 0.67 a	2.71 ± 0.56 b/1.06 ± 0.23 b
Orange Prince	1.08 ± 0.22 a/0.37 ± 0.13 a	1.13 ± 0.66 a/0.41 ± 0.21 a	2.08 ± 0.71 b/1.03 ± 0.01 b
Sun Glow	1.12 ± 0.19 a/0.56 ± 0.212 a	1.18 ± 0.18 a/0.23 ± 2.21 a	2.01 ± 0.98 b/1.20 ± 0.21 b
APX
Indian Prince	9.44 ± 2.01 a/4.55 ± 1.04 a	9.51 ± 2.03 a/4.23 ± 2.01 a	18.23 ± 3.05 b/5.20 ± 2.24 b
Golden Emperor	10.21 ± 2.33 a/3.03 ± 1.89 a	10.25 ± 2.26 a/2.99 ± 2.28 a	16.39 ± 2.56 b/4.56 ± 1.98 b
Orange Prince	11.23 ± 2.04 a/6.21 ± 2.23 a	10.99 ± 3.01 a/6.15 ± 1.99 a	14.99 ± 2.27 b/8.05 ± 2.01 b
Sun Glow	8.05 ± 2.31 a/4.02 ± 1.89 a	8.12 ± 2.01 a/4.11 ± 2.17 a	11.13 ± 2.27 b/6.73 ± 2.35 b

). Nevertheless, the flowers of some genotypes already showed a significant increase in the activity of CAT (Golden Emperor, Orange Prince, and Sun Glow) and POX (Indian Prince) under moderate conditions (Table 1).

The activities of all enzymes increased significantly under severe drought in all genotypes and considering both leaves and flowers (Table 1). However, significant variation in enzyme activities was found between genotypes (always p > 0.05). The highest enzymatic activities in the leaves were reported in the genotype Indian Prince under severe drought, while the highest enzymatic values were recorded in the flowers of the genotype Orange Prince, also under severe drought (Table 1). An increase in the activity of antioxidant enzymes was also found in other Calendula genotypes subjected to drought, although high variation was found between genotypes [12]. Together with the increase in the content of proline, the higher antioxidant enzyme activities found under drought suggest a good antioxidant mechanism to cope with drought. Altogether, these enzymatic and non-enzymatic components help to reduce the oxidative stress in Calendula triggered by drought, as reported in other plants [18,21]. Calendula plants can keep ROS under control through this efficient and versatile scavenging system. This would help to protect cellular structures and functions as well as to maintain water balance and the efficiency of physiological processes.

3.3. Impacts of Drought on the Production of Flowers

Although flowers are crucial for the floriculture industry, studies on understanding the impacts of abiotic stresses, especially concerning Calendula species, are remarkably scarce. In this study, flower production decreased by 5.01% in Indian Prince, 4.39% in Golden Emperor, 6.99% in Orange Prince, and 6.81% in Sun Glow under moderate drought. However, a harsh effect was felt under severe drought, decreasing the production of flowers by 23.67% in Indian Prince, 25.66% in Golden Emperor, 37.53% in Orange Prince, and 39.88% in Sun Glow. In general, drought decreases flower production in many species [22] since flower development and the related reproductive processes are very sensitive to stress, and also because resources are allocated to plant survival under stress conditions. Therefore, it is not surprising to find that drought also has a strong impact on Calendula plants. However, as impacts vary between genotypes, it is crucial to conduct further tests on drought tolerance by screening additional Calendula plants. Understanding the effects of stress on flower development or abortion would help to develop high-yield cultivars that can cope with environmental changes using traditional and molecular breeding approaches.

4. Conclusions

Calendula plants showed high resilience to moderate drought, in contrast with severe drought, which had a harsh impact on most genotypes. The levels of proline and MDA can be used in future studies as stress markers to understand the impacts of drought on these plants. The antioxidant machinery studied here increased under the harsh drought effect but did not prevent negative effects on flower production, which was significantly affected by drought. As drought showed a negative impact on flower production, future studies should focus on understanding its effects on flower development and fertility. Additionally, high variation was found between pot marigold genotypes, suggesting differences in drought tolerance, which can be used to screen useful tolerance traits. Apart from adjustments in the antioxidant system, it should be noted that drought tolerance depends on additional plant features, which should be measured to characterize the severity of drought. Useful traits for future studies should include, for example, net photosynthesis, the abundance of osmoprotectants, ABA content, and membrane integrity. It would also be useful to identify genes that respond to abiotic stresses in Calendula plants.