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Article

Triacontanol Reverses Abscisic Acid Effects on Stomatal Regulation in Solanum lycopersicum L. under Drought Stress Conditions

by
María Asunción Bravo-Díaz
1,
Emilia Ramos-Zambrano
1,
Tomás Ernesto Juárez-Yáñez
1,
María de Jesús Perea-Flores
2 and
Alma Leticia Martínez-Ayala
1,*
1
Centro de Desarrollo de Productos Bióticos, Instituto Politécnico Nacional, Carretera Yautepec-Jojutla, Col. San Isidro, Km. 6, Calle CEPROBI No. 8, Yautepec 62731, Mexico
2
Unidad Profesional Adolfo López Mateos, Centro de Nanociencias y Micro y Nanotecnologías, Instituto Politécnico Nacional (IPN), Av. Luis Enrique Erro S/N, Zacatenco, Delegación Gustavo A. Madero, Mexico City 07738, Mexico
*
Author to whom correspondence should be addressed.
Horticulturae 2024, 10(9), 985; https://doi.org/10.3390/horticulturae10090985
Submission received: 20 August 2024 / Revised: 10 September 2024 / Accepted: 14 September 2024 / Published: 18 September 2024
(This article belongs to the Section Biotic and Abiotic Stress)
Browse Figures
Versions Notes

Abstract

:
When applied under abiotic stress conditions, triacontanol (TRIA) is effective in regulating the physicochemical processes in plants through mechanisms of defence such as abscisic acid (ABA) signalling. However, TRIA’s role in relation to ABA and stomatal opening is unclear. Therefore, the objective of this study was to evaluate the effects of TRIA and ABA and their combinations on different variables related to stomatal regulation in Solanum lycopersicum, which is subjected to drought stress, and on the leaf epidermis. The negative effects of stress and responses triggered by ABA were reversed in plants treated with TRIA. TRIA increased stomatal conductance and photosynthetic activity in the early hours, and it was determined that TRIA produced larger stomata than did the other treatments. Moreover, the chloroplasts of plants treated with TRIA were significantly smaller and more numerous than those of the control, which could improve CO2 diffusion efficiency and may be related to the regulation of stomatal opening and photosynthesis. Finally, the abaxial epidermis tests reaffirmed the inhibitory effects of TRIA on ABA on stomatal opening. These results confirm the important role of TRIA in regulating various processes in plants and processes triggered by ABA, such as those related to stomatal regulation.

Graphical Abstract

1. Introduction

Drought is one of the most serious environmental factors affecting plant productivity. Indeed, the biomass of a fresh plant body contains 80–95% water, which plays a crucial role in several physiological processes [1]. Therefore, drought is considered one of the most notable limiting factors for sustainable agriculture, which results in a more than 50% reduction in crop yields each year [2]. Different degrees of intensity and durations of drought stress can occur, producing a wide range of effects on plant growth and development. These differences depend on plant species, age, and growth stage [3]. Solanum lycopersicum is one of the most important vegetable crops worldwide but is susceptible to biotic and abiotic stress, with drought stress representing the most prevalent example [4]. It is also important as a research study model in the responses to abiotic and biotic stresses [5,6,7].
Plants have different response mechanisms under drought stress conditions, which can occur rapidly. These responses include stomatal closure and long-term effects such as decreased leaf area, stomatal frequency, and compatible solute accumulation [8]. Stomatal closure not only affects the transpiration process but also influences limited CO2 absorption by leaves, which is closely related to the stomatal control of water loss.
Consequently, there is a reduction in the net carbon assimilation rate/photosynthesis and a decrease in the intercellular CO2 concentration, which are considered early symptoms of drought stress [9]. During the stress process, there is a hormonal response in which plants accumulate phytohormones such as abscisic acid (ABA), salicylic acid, and methyl jasmonate. Among these hormones, ABA is considered an essential and versatile compound in plant development, which leads to a series of events, including stomatal closure—essential for the plant to conserve its water. At the same time, ABA regulates genes that promote osmotic adjustment in leaves [10], regulate transpiration and photosynthesis processes, inhibit the growth of aerial parts, and regulate seed dormancy [11]. It was also demonstrated that drought stress causes other series of events. Ristic and Cass [12] reported that drought stress affected some characteristics of chloroplasts, while Głowacka et al. [13] reported that chloroplast size affected photosynthetic efficiency, indicating that smaller chloroplasts had a proportionally greater surface area, which could improve CO2 diffusion. Zhang et al. [14] reported that chloroplasts in plants under severe drought stress moved closer to the cell centre, which caused chloroplasts to become deformed and assume a blurred shape.
Biostimulants are employed to combat the effects of drought stress in plants and play an important role in agricultural development. One of these biostimulants is triacontanol (TRIA), which is a long-chain fatty alcohol composed of 30 carbon atoms (CH3(CH2)28CH2OH) and is considered a non-toxic compound due to its natural origins. Indeed, TRIA can be obtained from plant and insect waxes such as beeswax, sugar cane wax, tea, and wheat germ [15,16,17]. It was previously demonstrated that the foliar application of TRIA at low concentrations in plants increases growth, internal CO2 concentrations, stomatal conductance, photosynthetic pigments, photosynthesis rate, compatible osmolytes, nitrate reductase and carbonic anhydrase activities, membrane stability, and genetic regulation, thereby improving the yields of various crops [18,19], including tomatoes [20,21]. A recent study carried out by Manai et al. [21] determined that TRIA had no effects on the growth of S. lycopersicum L., var. Minibel, but did exert an influence at the biochemical level as a proteomics analysis of the leaves demonstrated that TRIA increased the abundance of proteins related to development and responses to abiotic stress. It was also shown that TRIA can improve plant tolerance to abiotic stress because it reverses the negative effects caused by salinity stress, heat stress, high irradiation, heavy metals, and drought stress [20,22,23,24,25]. Ramos-Zambrano et al. [20] reported that applying TRIA to Solanum lycopersicum under light stress (600 μmol m−2 s−1) reversed the associated negative effects, increasing photochemical responses and photosynthetic irradiation parameters. The addition of TRIA also stimulated the chlorophyll content, improved stomatal conductance, and affected the size and morphology of the stomata and chloroplasts, resulting in larger stomata and smaller chloroplasts. Under drought stress, it was reported that TRIA improves plant water conditions through osmotic adjustment, increasing the accumulation of some osmolytes, such as proline, free amino acids, and total soluble ones, helping to reduce the osmotic potential of cells and, therefore, reducing water loss under these stress conditions [26].
It is generally known that TRIA biostimulant activity triggers an ABA-mediated defence response; however, these signals are not entirely clear. In some reports, TRIA reduced ABA levels [27,28] in plants under normal conditions, but in others, TRIA increased ABA levels under normal [21] and drought stress conditions [21,26]. These results may be contradictory because TRIA stimulates ABA concentrations but, at the same time, stimulates stomatal opening, which should be inhibited in the presence of ABA. Therefore, in this work, we analysed the effects of TRIA and ABA in relation to stomatal regulation under conditions of water stress in tomato plants. In this way, we evaluated the effects of TRIA, ABA, and TRIA + ABA treatments on stomatal regulation and the operating efficiency of PSII photochemistry behaviour throughout the day. The chlorophyll content, stomatal density, stomatal area and conductance, and chloroplast volume were also evaluated. Finally, bioassays were carried out to evaluate the effect of TRIA, ABA, and TRIA +ABA on stomatal opening on the epidermis of tomato leaves.

2. Materials and Methods

2.1. Plant Material

An indeterminate tomato variety of the Saladette type, “sun 7705”, was established under greenhouse conditions at the Centro de Desarrollo de Productos Bióticos (CEPROBI-IPN) located in Yautepec, Morelos, Mexico, at latitude 18°49′44.278″ N, longitude 99°5′34.296″ W, and an altitude of 1064 m.a.s.l. Solanum lycopersicum seeds were disinfected with a 1% sodium hypochlorite solution for 5 min, incubated for 6 h in distilled water, and placed on moist paper in a container in the dark for 2 days. Once the seeds germinated, they were transferred to polystyrene seed trays with coconut fibre and agrolite (50:50 v/v) as a substrate. While the seedlings were in the seedbed, they were irrigated every third day with 100% nutrient solution (SN) containing 0.1 g/L calcium nitrate (Ca (NO3)2), 0.2 g/L monoammonium phosphate (MAP), 0.1 g/L monopotassium phosphate (MKP), and 0.015 g/L of a commercial micronutrient mixture (Ultrasolmicro), with a final pH of 6.5. After 30 days in the seedbed (early growth), the plants were transplanted into 4 L bags with agrolite and coconut fibre (50:50 v/v) as a substrate. Under these conditions, the plants were irrigated through automatic fertigation with the nutrient solution previously used on the seedlings in the seedbed [20] (Figure 1b). Crop cycling was carried out in November and December, reaching 600 μmol m−2 s−1 maximum irradiance, temperatures of 23–31 °C during the day, and a relative humidity of 35%. All the plants grew uniformly and were used for treatment application.

2.2. Application of Treatments in a Greenhouse Tomato Crop

In the results obtained by Ramos-Zambrano et al. [20], TRIA influenced some physiological variables in S. lycopersicum under stress conditions when applied to the plants 30 and 45 days after early growth. Consequently, we employed a completely randomised design (Figure 1a) in the present study, with treatments applied foliarly to the entire plant for each treatment 30 and 45 days after early growth (Figure 1b). The design consisted of two factors with 3 repetitions per treatment, as shown in Table 1.

2.3. Drought Stress Conditions

According to Flores et al. [29], the water requirement of a tomato plant is 0.2–1.5 L, depending on its physiological stage. In this study, over the last 15 d of the tomato culture, the water flow required per plant was 214.3 mL/day (December). Therefore, the irrigation amount was reduced to 117.1 mL/day of water to induce drought stress. The chlorophyll content, stomatal density, area and conductance, chloroplast volume, and photosystem II efficiency were measured in December, with 3 repetitions per treatment (Figure 2).

2.4. Measurement of Chlorophyll Content

The chlorophyll content was determined using a Minolta SPAD 502 Plus Chlorophyll Meter (Spectrum Technologies Inc., Aurora, IL, USA) in the last fully expanded plant leaf. Measurements were carried out 15 days after application of the second treatment. Each measurement was performed in triplicate.

2.5. Measurement of the Operating Efficiency of PSII Photochemistry at Different Times of the Day

The operating efficiency of PSII photochemistry, Fq′/Fm′, was measured using a FluorPen FP 100 portable fluorimeter (Photon Systems Instruments, Drasov, Czech Republic) [30]. Measurements were carried out from 9:00 to 10:00 a.m., 12:00 to 1:00 p.m., and 4:00 to 5:00 p.m. Each measurement was performed in triplicate.

2.6. Measurement of Stomatal Conductance at Different Times of the Day

Stomatal conductance was determined using a SC-1 Leaf Porometer (Decagon Devices, Inc., Washington, USA) in the first fully extended leaf from the plant apex [20]. The stomatal conductance and temperature were measured from 6:00 to 8:00 a.m., 9:00 to 10:00 a.m., 12:00 to 1:00 p.m., and 4:00 to 5:00 p.m. Each measurement was performed in triplicate.

2.7. Morphometric Characterisation of Stomata

Each measurement was performed on the first leaf fully extended from the apex of the plant. Transparent varnish was applied to the leaves to obtain an impression of the stomatal shape and density. After 30 min, the layer formed with the varnish was removed and placed on a slide. The samples were analysed on an optical microscope (Nikon eclipse 80i, Tokyo, Japan) at 10× objective magnification to analyse the density and at 20× to measure the length and width to determine the aspect ratio and stomatal area [31].
The stomatal density was calculated by determining the number of stomata per visual field of the objective. In this case, the visual field was determined by calculating the image area, and the result was extrapolated to 1.0 mm2.
The aspect ratio of the stomata was calculated according to the following equation reported by Khanna et al. [32]:
Aspect ratio: L/W
where L is the length and W is the width.
We used Fiji ImageJ software version 1.54f (National Institutes of Health, Bethesda, MD, USA) to estimate the size of the stomata. For this purpose, the length and width of the guard cells were measured in the micrographs of the varnish impression. To determine the stomatal area, we assumed that the stomata had an oval shape, and the following equation was used:
A = abπ
where a is the largest radius, b is the smallest radius, and π is 3.1416. The largest radius was half the length of the guard cell, and the smallest radius was half the width of the guard cell.

2.8. Morphometric Characterisation of Chloroplasts

Each measurement was carried out on 3 × 3 mm segments of S. lycopersicum leaves and performed in triplicate. Leaf segments were fixed in 3.5% (v/v) glutaraldehyde solution for 1 h in the dark and subsequently transferred to a 0.1 M Na2 EDTA solution (pH 9). Samples were stored at 4 °C until micrographs were obtained. Samples were observed on an LSM 710 NLO confocal–multiphoton microscope (Carl Zeiss, Baden-Wurttemberg, Germany) with a 640 nm laser (red fluorescence). Micrographs with a resolution of 1130 × 1130 pixels were stored in the TIFF format. Each 0.58 μm slice was imaged to a depth of 0–20 μm approximately from the beginning of the parenchymal palisading tissue of the leaf through the epidermal layer. Image analysis was carried out with sample micrographs using the Fiji ImageJ software version 1.54f (National Institutes of Health, Bethesda, MD, USA) [33] with the ImageJ 3D suite employed to manipulate and analyse the 3D images [34]. Images were imported for stack rearrangement, and the scale and depth of each layer (slice) were adjusted. Pre-processing was performed by applying contrast improvements in a normalised way, with a saturation value of 0.3%. For segmentation, the “3D Simple Segmentation” tool with a threshold value of 85 was used, limiting the particle size to >500 voxels. The volume of the segmented zones (chloroplasts) was measured with the “3D Geometrical Measure” tool. The percentage of volume occupied by chloroplasts (%VolChlor) was calculated using the following equation:
TotalClorVol (%) = (Chloroplast Vol.)/(Vol. Total) × 100
The unit volume of chloroplasts was calculated using the following equation reported by Li et al. [35]:
Vchl = (4/3) × π × a × b2
where a is Lchl/2 (chloroplast length), b is Dchl/2 (chloroplast thickness), and π is 3.1416.
The number of chloroplasts was calculated according to the following equation:
Chloroplast number = TotalVolClor/Vchl

2.9. TRIA Effect on ABA in Epidermal Strips

We also evaluated the effect that TRIA has on stomatal closure with ABA. Epidermal strips were floated in a buffer (30 mM KCl, 10 mM MES, pH 6.15) and exposed to light (250 mol m−2 s−1 PPFD) for 2 h to open the stomata. Once the stomata were open, the epidermal strips were transferred to the same buffer plus ABA (10 μM), ABA (10 μM) + TRIA (0.15 mM), and TRIA (0.15 mM). Samples were observed on a Zeiss LSM800 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany). The autofluorescence emission signal was captured in the range of 410–595 nm (green fluorescence) using a 20×/0.5 EC lens. Images 2048 × 2048 pixels in size were obtained and stored in the TIFF format, and all stomata were analysed in triplicate [20].

2.10. Statistical Analysis

Statistical analysis was performed using a bifactor analysis of variance (ANOVA), which was based on a completely randomised design. Significant differences between plants exposed to treatments were examined using Fisher’s LSD with the MINITAB 17 software. All data were expressed as the means ± standard error, calculated from three biological replicates (n = 3). However, for the chloroplast analysis, 10 images per replicate were processed, which was equivalent to 30 images per treatment. Significant differences were examined using Fisher’s LSD method. Graphs were constructed using the MATLAB software version 24.1.0.2622093 (R2024a) (The MathWorks Inc., Natick, MA, USA).
The BioRender platform “BioRender.com (accessed on 24 July 2024)” was used to design some illustrative figures.

3. Results

Plants were exposed to different treatments, enabling us to evaluate certain variables that elucidate the effects of TRIA on photosynthesis, the morphometric characteristics of the stomata and chloroplasts, and ABA applied exogenously to the plants under drought stress. We also evaluated the chlorophyll content as an indicator of drought stress. Subsequently, the operating efficiency of PSII photochemistry, the stomatal conductance, and the morphometric characteristics of the stomata and chloroplasts, such as their size, density, and volume, were analysed.

3.1. Chlorophyll Content

Treatments were the predominant factor, and the chlorophyll content decreased by 7.0% in control plants under drought stress compared to plants under normal conditions. However, when TRIA was applied to the plants, the chlorophyll content increased by 11.69% under normal conditions, and the negative effects on chlorophyll reduction under drought stress were reversed. Moreover, the chlorophyll content decreased when ABA was applied under normal conditions, but this effect was reversed with increases of 16.92 and 9.63% when TRIA + ABA treatment was applied to plants under normal and drought stress conditions, respectively (Figure 3).

3.2. The Operating Efficiency of PSII Photochemistry, Fq′/Fm′

In the operating efficiency of PSII photochemistry, Fq′/Fm′ (ϕPSII) water flow was the predominant factor at 9–10 a.m. and 4–5 p.m., while treatments were the predominant factor at 12–1 p.m. and decreased significantly by 12.32% from 9 to 10 a.m., 8.45% from 12 to 1 p.m., and 10.38% from 4 to 5 p.m. in control plants under drought stress compared to those under normal conditions (Figure 4). This decrease in ϕPSII values was reversed when TRIA was applied to the plants. The values increased by 5.73, 12.32, and 8.25% compared to the control values under drought stress from 9 to 10 a.m., 12 to 1 p.m., and 4 to 5 p.m., respectively. For the plants that received TRIA under drought stress, the ϕPSII values did not present significant differences from the control under normal conditions, as shown by the readings from 12 to 1 p.m. and from 4 to 5 p.m. This outcome reflects a positive result because the plants indicated a return to their photosystem II efficiency from a state of stress to normal conditions (Figure 4).
When ABA was added to plants under normal conditions, the ϕPSII values decreased by 6.57% at 12 p.m., possibly because of stomatal closure. However, this effect was reversed when TRIA + ABA was applied to the plants, after which the ϕPSII values increased by 0.91, 1.88, and 0.87% from 9 to 10 a.m., 12 to 1 p.m., and 4 to 5 p.m., respectively. However, when the plants were treated with ABA under drought stress conditions, the ϕPSII values did not present significant differences from the control plants under the same water flow conditions. This effect could be related to high ABA concentrations because, in addition to its applications, ABA can be synthesised in plants due to drought stress.
During the evaluated time periods, TRIA improved the operating efficiency of PSII photochemistry in plants under drought stress conditions. From 9 to 10 a.m., the ϕPSII values indicated that plants treated with TRIA began photosynthesis earlier than the control plants under drought stress conditions. Subsequently, this efficiency increased after 12–1 p.m. in plants treated with TRIA.

3.3. Stomatal Conductance

Stomatal conductance was measured in mmol m−2 s−1 at different times during the day (6–8 a.m., 9–10 a.m., 12–1 p.m., and 4–5 p.m.) to evaluate the effects of treatments under drought stress conditions. We observed that stomatal conductance was dependent on leaf temperature: the higher the temperature, the greater the stomatal conductance. The leaf temperature was 9.88 ± 0.63 °C from 6 to 8 a.m., 23.88 ± 1.15 °C from 9 to 10 a.m., 31.26 ± 0.21 °C from 12 to 1 p.m., and 29.50 ± 0.49 °C from 4 to 5 p.m. (Figure 5).
Water flow was the predominant factor at 6–8 a.m., 9–10 a.m., and 12–1 p.m., while treatments were the predominant factor at 4–5 p.m. Starting at 6–8 a.m., the plants exposed to TRIA exhibited increased stomatal conductance compared to the control plants under normal conditions, providing an opportunity for the plants to open their stomata earlier than the other plants. Consequently, the photosynthetic activity of these plants also began earlier, as shown by the values of ϕPSII observed in this study. Moreover, TRIA significantly increased stomatal conductance compared to the control, by 37.81% under normal conditions and by 30.64% under drought conditions from 9 to 10 a.m. (Figure 5b). However, this increase was minimal compared to the stomatal conductance readings from 12 to 1 p.m., for which the increase was 85.64% under normal conditions and 88.46% under drought conditions (Figure 5c).
When ABA was added to plants under normal conditions, stomatal conductance did not show significant differences from the control plants. Under drought stress conditions, stomatal conductance decreased from 9 to 10 a.m., but this effect was reversed when TRIA + ABA was applied. This treatment significantly increased stomatal conductance in plants under normal conditions and drought stress (Figure 5b).

3.4. Stomata Morphometric Characteristics

Stomatal density (Figure 6b) did not present significant differences in most treatments under both water flow conditions, except when TRIA was applied to plants under stress conditions, which yielded a significant increase in stomatal density, so treatments were the predominant factor in stomatal characteristics. On the other hand, the stomatal area and the stomatal aspect ratio (Figure 6a,c,d) showed significant differences between TRIA and TRIA + ABA treatments compared to the control and ABA treatments. Specifically, TRIA treatment increased the stomatal area by 28.87% (369.30 ± 21.22 µm2) and 57.14% (380.14 ± 27.54 µm2) compared to the control (286.56 ± 19.84 and 241.91 ± 17.19 µm2), respectively, under normal conditions and drought stress.
When ABA was applied, the stomatal area was 261.39 ± 20.69 µm2 and 224.97 ± 15.33 µm2, respectively, under normal and drought stress conditions, while TRIA + ABA treatment increased the stomatal area by 36.27% (356.21 ± 24.05 µm2) and 57.37% (354.04 ± 28.53 µm2), respectively, under normal conditions and drought stress (Figure 6a,c). The stomatal area increased significantly when TRIA was applied to plants under normal and drought stress conditions and reversed the effects of ABA. In addition, the aspect ratio showed greater stomatal opening in plants treated with TRIA under normal and drought stress conditions (Figure 6a,d).

3.5. Chloroplast Morphometric Characteristics

The interaction of two factors was predominant in the chloroplast characteristics. TRIA enhanced the volume of chloroplasts as compared to the other treatments in plants under normal conditions and drought stress (Figure 7a). These results were corroborated by the statistical analysis, in which TRIA significantly increased the total volume (%) of chloroplasts compared to the control by 51.23% under normal conditions and 21.22% in plants under normal conditions and drought stress. When ABA was applied, the chloroplast volume decreased by 35.88% and 53.08%, compared to that of the control under normal conditions and under drought stress, respectively.
Although the volume of the chloroplasts was significantly greater in plants under TRIA treatment, their size was smaller than that of the chloroplasts in the control plants. Chloroplasts exposed to ABA exhibited a significantly reduced size when compared to those in the control and TRIA treatments under normal and drought stress conditions (Figure 7b). Due to the size of the chloroplasts, the number of chloroplasts was significantly greater under TRIA than under the other treatments (Figure 7c).

3.6. TRIA’s Effect on ABA in Epidermal Strips

Other results in this study indicated that TRIA reversed the effects of ABA on the variables that were evaluated. For this reason, we evaluated the effect that TRIA has on ABA in epidermal strips. We observed that ABA decreased the stomatal opening under light conditions. However, when TRIA was added together with ABA, the stomatal opening regained the conditions observed in the control (Figure 8).

4. Discussion

The operating efficiency of PSII photochemistry and stomatal conductance and the characteristics of the stomata and chloroplasts were affected by drought stress in this study. However, when TRIA was applied, the negative effects on these physiological parameters were reversed, and all analysed variables depended on each other. Larger stomata, greater stomatal conductance, and smaller chloroplasts resulted in more efficient photosynthesis (Figure 9).
Even when S. lycopersicum plants were placed under drought stress in this study, their stomatal size was larger under TRIA than under the other treatments. Specifically, greater stomatal conductance was observed in plants treated with TRIA under drought stress conditions at 12 p.m., which simultaneously yielded a significant increase in the operating efficiency of PSII photochemistry. However, not only the stomatal conductance but also the characteristics of the chloroplast influenced the operating efficiency of PSII photochemistry, with the chloroplasts being smaller than those of the control plants. In addition, the chlorophyll content (SPAD) increased when TRIA was applied. Overall, we observed that the stomatal density may or may not be related to the results obtained for the stomatal conductance of plants under drought stress conditions.
The results obtained in this work agree with those of previous studies in which TRIA increased stomatal conductance in plants under either normal [25,36] or stress conditions [20,37]. Ramos-Zambrano et al. [20] reported that stomatal conductance was correlated with the size of stomata and found that TRIA had no effect on stomatal density but did affect stomatal size and morphology. This result reflects a previous study indicating that gas diffusion through the stomata is mainly determined by their size, depth, opening, and density [38], which can affect the operating efficiency of PSII photochemistry [36,39]. Related to the chloroplast size obtained in this work, Xiong et al. [40] determined that small and large populations of chloroplasts were more efficient in CO2 diffusion. Ivanov and Angelov [41] found that TRIA modified the membrane fluidity of chloroplasts. The results obtained in this work agree with those of Ramos-Zambrano et al. [20], who evaluated the effects of TRIA in S. lycopersicum plants under light stress (600 μmol m−2 s−1) and identified that the chloroplasts were smaller than those in the control plants. The chlorophyll content (chlorophyll a and chlorophyll b) represents an important pigment in the primary reaction of photosynthesis [42]. In this work, the chlorophyll content (SPAD) decreased when plants were placed under drought stress and increased when TRIA was applied, which agrees with previous reports on plants under normal [43] and stress conditions, such as salinity stress [44] and drought stress [26].
Finally, we observed throughout this study that ABA decreased the values of the operating efficiency of PSII photochemistry, stomatal conductance, and the characteristics of the stomata and chloroplasts in plants. This decrease could be related to the high concentration of this hormone, which was applied exogenously and possibly synthesised by the plants due to drought stress conditions. In this regard, some reports indicate that the use of exogenous ABA reduces stomatal size and stomatal conductance [45,46] and that elevated ABA concentrations are generally associated with water deficits in plants [47]. In this study, even though ABA negatively affected the variables evaluated, TRIA inhibited the effects of ABA in the physiological variables and epidermal bioassays.
Currently, information on the mechanism of TRIA is limited to physiological effects; therefore, there are few reports that demonstrate the expression of genes or interactions with proteins involved in stomatal movement. Lesniak et al. [48] and Morré et al. [49] reported that TRIA could favour CO2 fixation and stimulate the activity of ATPase and NADP oxidase, resulting in higher concentrations of ATP and NADH that increase photosynthetic activity. The results obtained in this work could be related to those reported by Lesniak et al. [48] and Morré et al. [49], in which greater stomatal conductance was observed in plants with TRIA.
On the other hand, TRIA is a mixture of policosanol [50], which is involved in lowering cholesterol and serum lipids through AMPK activity (adenosine 5′monophosphate-activated protein kinase) in humans [51]. SnRK1/Snf1/AMPK are conserved heterotrimeric kinase complexes activated under energy limitations to induce metabolic reprogramming and achieve energy homeostasis in plants, yeast, and mammals. In plants, SnRK1 signalling regulates growth, development, and stress adaptations through metabolic reprogramming [52]. SnRK2s can form a complex with SnRK1 under normal conditions, thus preventing the interaction between SnRK1 and the TARGET OF RAPAMYCIN (TOR) complex and activating the TOR complex to promote plant growth. Under drought stress, SnRK1 is released from sequestration by the SnRK1–PP2C–SnRK2 complex and inhibits the TOR complex, triggering stress responses and delaying growth [53] because TOR promotes guard cell starch degradation and induces stomatal opening; conversely, the inactivation of TOR impairs guard cell starch degradation and stomatal opening [54]. According to Romero-Martínez et al. [55], TRIA may interact with the SnrK1 protein. Therefore, if SnRK1 interacts directly with TRIA, then the SnRK1–TOR complex would not form, enabling the plant to continue its development as though it were under normal conditions. In addition, Carianopol et al. [52] identified that the SnRK1 subunits interact with ABA signalling and response proteins, including positive and negative regulators, suggesting that SnRK1 modulates the ABA response pathway at multiple levels. In another study, Pang et al. [16] found that TRIA promoted the synthesis of ABA but inhibited its signal transduction through enhanced activities of the negative regulators “protein phosphatase 2C” (PP2C) and serine/threonine protein kinase SRK2 (SNRK2) under normal conditions, in addition to other ABA-triggered transduction signals.
The inhibitory effects of TRIA on exogenously applied ABA were also demonstrated in this study, and most of the evaluated variables were found to be reversed. It was reported that TRIA increased gene expression related to photosynthetic activity at the genetic level in plants and, in turn, decreased the expression of genes related to abiotic stress and ABA responses [56]. However, more recent studies have determined that TRIA can increase ABA levels, among other phytohormones [27,28,43]. Moreover, proteomic studies observed that TRIA increases the levels of proteins related to defence against abiotic stress [21]. For this reason, TRIA is considered to have an impact on different physiological aspects of the plant [21]. This differential response could explain the effects that increased TRIA has on ABA concentrations alongside the inhibition of certain ABA-triggered responses, such as a decrease in the expression of the AF039573 gene [57], which encodes the ASR1 protein (ABA-induced Serine-rich Repressor 1) closely related to responses to drought stress [57]. This protein causes stomatal closure in response to abiotic stress and ABA [58], which is how TRIA partially inhibited the effects of ABA, among other responses triggered by this phytohormone that have not yet been confirmed.
This regulatory effect of TRIA can also be observed by decreasing the gene expression that codes for a wounding-inducible protein (WIP1), which, according to Chen et al. [56], is very similar to Bowman–Birk proteinase inhibitors. In a study by Manai et al. [21] on the abundance of proteins modified by the application of TRIA, the authors observed an increase in the quantity of Proteinase inhibitor II. Likewise, another study carried out by Ramanarayan et al. [59] found that TRIA negatively modulated jasmonic acid stimulated by proteinase inhibitors in Lycopersicon sculetum. Jasmonic acid is another phytohormone involved in the stomatal closure processes triggered by stress. Proteinase inhibitors have a wide variety of functions in plant responses to biotic and abiotic stress [60,61,62]. The differential effects of TRIA on these proteins would also cause plants to increase their responses to stress but, at the same time, protect against effects on key processes such as stomatal opening and photosynthesis in order to help plants withstand the negative effects of abiotic stress.

5. Conclusions

TRIA reversed the negative effects of drought stress on the physiological variables evaluated, including the chlorophyll content, operating efficiency of PSII photochemistry, stomatal conductance, and morphometric characteristics of stomata and chloroplasts. The size of the stomata was larger than in other treatments; consequently, greater stomatal conductance was identified. It can be concluded that TRIA leads to greater stomatal opening at an earlier age, resulting in an earlier start for photosynthesis, and thus offers advantages, compared to no TRIA treatment. These results suggest that plants have higher photosynthetic activity but also a longer photosynthesis time per day when treated with TRIA. Although the chloroplasts of plants exposed to TRIA were smaller than those of the control under both water flow conditions, this difference was related to improvements in photosystem II efficiency. In addition, TRIA reversed the negative effect of ABA by increasing the stomatal conductance, operating efficiency of PSII photochemistry, stomatal size, and chloroplast volume. Based on the results, it can be assumed that TRIA plays an important role in regulating stomatal opening and in the development of stomata and chloroplasts. These results also confirm TRIA’s selective role in regulating different pathways in plant metabolism, in response to stress, by inhibiting certain ABA-mediated responses.

Author Contributions

M.A.B.-D.: conceptualisation, methodology, validation, formal analysis, investigation, and writing—original draft; E.R.-Z.: conceptualisation, methodology, validation, formal analysis, and investigation; T.E.J.-Y.: methodology, validation, and investigation; M.d.J.P.-F.: resources, writing—review and editing, and supervision; A.L.M.-A.: conceptualisation, resources, writing—review and editing, supervision, project administration, funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Instituto Politécnico Nacional (project number SIP20240634).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article; further inquiries can be directed to the corresponding author.

Acknowledgments

We acknowledge the Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT) and the Instituto Politécnico Nacional (IPN) for the scholarships given to María Asunción Bravo-Díaz and Tomás Ernesto Juárez-Yáñez during the course of this investigation; Program: “Estancias Postdoctorales por Mexico”, CONAHCYT for the scholarship awarded to Emilia Ramos Zambrano; and the IPN for financial support.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Experimental design of treatment application during the cultivation of Solanum lycopersicum. (a) Graphic description of design, in which each color indicates the treatment applied in normal and drought stress in plants. (b) treatments applied during the cultivation of Solanum lycopersicum in two stages of plant growth: 30 d and 45 d after germination.
Figure 1. Experimental design of treatment application during the cultivation of Solanum lycopersicum. (a) Graphic description of design, in which each color indicates the treatment applied in normal and drought stress in plants. (b) treatments applied during the cultivation of Solanum lycopersicum in two stages of plant growth: 30 d and 45 d after germination.
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Figure 2. Graphic description of treatments applied during cultivation of Solanum lycopersicum. Stages of plant growth and application of treatments at 30 d and 45 d after germination; readings of chlorophyll content, photosystem II efficiency, morphometric characterisation of stomata, and chloroplasts at 60 d after germination.
Figure 2. Graphic description of treatments applied during cultivation of Solanum lycopersicum. Stages of plant growth and application of treatments at 30 d and 45 d after germination; readings of chlorophyll content, photosystem II efficiency, morphometric characterisation of stomata, and chloroplasts at 60 d after germination.
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Figure 3. Chlorophyll content [SPAD] in plants exposed to two water flows (normal conditions and drought stress) and different treatments (Control, TRIA, ABA, and TRIA + ABA). Values represent the mean ± SE of at least three replicates. Different letters indicate significant differences, Fisher’s LSD, p < 0.05, in which treatments were the predominant factor.
Figure 3. Chlorophyll content [SPAD] in plants exposed to two water flows (normal conditions and drought stress) and different treatments (Control, TRIA, ABA, and TRIA + ABA). Values represent the mean ± SE of at least three replicates. Different letters indicate significant differences, Fisher’s LSD, p < 0.05, in which treatments were the predominant factor.
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Figure 4. Operating efficiency of PSII photochemistry, Fq′/Fm′ (ϕPSII), in plants exposed to drought stress and different treatments: (a) ϕPSII at 9–10 a.m.; (b) ϕPSII at 12–1 p.m.; (c) ϕPSII at 4–5 p.m. Values represent the mean ± SE of at least three replicates. Different letters indicate significant differences, Fisher’s LSD, p < 0.05, in which water flow was the predominant factor at 9–10 a.m. and 4–5 p.m., while treatments were the predominant factor at 12–1 p.m.
Figure 4. Operating efficiency of PSII photochemistry, Fq′/Fm′ (ϕPSII), in plants exposed to drought stress and different treatments: (a) ϕPSII at 9–10 a.m.; (b) ϕPSII at 12–1 p.m.; (c) ϕPSII at 4–5 p.m. Values represent the mean ± SE of at least three replicates. Different letters indicate significant differences, Fisher’s LSD, p < 0.05, in which water flow was the predominant factor at 9–10 a.m. and 4–5 p.m., while treatments were the predominant factor at 12–1 p.m.
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Figure 5. Stomatal conductance in mmol m−2 s−1 in plants exposed to drought stress and different treatments: (a) 6–8 a.m.; (b) 9–10 a.m.; (c) 12–1 p.m.; (d) 4–5 p.m. Values represent the mean ± SE of at least three replicates. Different letters indicate significant differences, Fisher’s LSD, p < 0.05, in which water flow was the predominant factor at 6–8 a.m., 9–10 a.m., and 12–1 p.m., while treatments were the predominant factor at 4–5 p.m.
Figure 5. Stomatal conductance in mmol m−2 s−1 in plants exposed to drought stress and different treatments: (a) 6–8 a.m.; (b) 9–10 a.m.; (c) 12–1 p.m.; (d) 4–5 p.m. Values represent the mean ± SE of at least three replicates. Different letters indicate significant differences, Fisher’s LSD, p < 0.05, in which water flow was the predominant factor at 6–8 a.m., 9–10 a.m., and 12–1 p.m., while treatments were the predominant factor at 4–5 p.m.
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Figure 6. Morphometric characteristics of stomata from plants exposed to drought stress and different treatments: (a) Micrographs of stomata obtained using an optical microscope. Reference bar: 50 µm. (b) Stomatal density/mm2 area. (c) Area calculated with the equation A = abπ. (d) Stomatal aspect ratio. Values represent the mean ± SE of at least three replicates. Different letters indicate significant differences, Fisher’s LSD, p < 0.05, in which treatments were the predominant factor in stomatal characteristics.
Figure 6. Morphometric characteristics of stomata from plants exposed to drought stress and different treatments: (a) Micrographs of stomata obtained using an optical microscope. Reference bar: 50 µm. (b) Stomatal density/mm2 area. (c) Area calculated with the equation A = abπ. (d) Stomatal aspect ratio. Values represent the mean ± SE of at least three replicates. Different letters indicate significant differences, Fisher’s LSD, p < 0.05, in which treatments were the predominant factor in stomatal characteristics.
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Figure 7. Micrographs and morphometric characteristics of chloroplasts from plants exposed to drought stress and different treatments: (a) Micrographs obtained using a confocal laser scanning microscope, reference bar: 20 µm. (b) Chloroplast size in µm3. (c) Chloroplast number per 10,000 µm3. Values represent the mean ± SE of at least three replicates. Different letters indicate significant differences, Fisher’s LSD, p < 0.05, in which the interaction of two factors was predominant in the chloroplast characteristics.
Figure 7. Micrographs and morphometric characteristics of chloroplasts from plants exposed to drought stress and different treatments: (a) Micrographs obtained using a confocal laser scanning microscope, reference bar: 20 µm. (b) Chloroplast size in µm3. (c) Chloroplast number per 10,000 µm3. Values represent the mean ± SE of at least three replicates. Different letters indicate significant differences, Fisher’s LSD, p < 0.05, in which the interaction of two factors was predominant in the chloroplast characteristics.
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Figure 8. Effect of TRIA on stomatal opening under light conditions and its effects together with ABA in epidermal strips. Values represent the mean ± SE of at least three replicates. Different letters indicate significant differences, Fisher’s LSD, p < 0.05.
Figure 8. Effect of TRIA on stomatal opening under light conditions and its effects together with ABA in epidermal strips. Values represent the mean ± SE of at least three replicates. Different letters indicate significant differences, Fisher’s LSD, p < 0.05.
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Figure 9. Correlation of results obtained under TRIA application in Solanum lycopersicum plants under drought stress conditions compared to the control. Up arrows indicate an increase, down arrows indicate a decrease, and the symbol = represents no significant differences.
Figure 9. Correlation of results obtained under TRIA application in Solanum lycopersicum plants under drought stress conditions compared to the control. Up arrows indicate an increase, down arrows indicate a decrease, and the symbol = represents no significant differences.
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Table 1. Description of treatments applied to Solanum lycopersicum cultivation.
Table 1. Description of treatments applied to Solanum lycopersicum cultivation.
FactorLevelsDescriptionReference
Treatment applicationControlSurfactant (0.1% (v/v) Tween 20 solution)[20]
TRIA1 mg/L of TRIA dissolved in surfactant
ABA40 µM of ABA dissolved in surfactant[11]
TRIA + ABA1 mg/L of TRIA + 40 µM of ABA dissolved in surfactant
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Bravo-Díaz, M.A.; Ramos-Zambrano, E.; Juárez-Yáñez, T.E.; Perea-Flores, M.d.J.; Martínez-Ayala, A.L. Triacontanol Reverses Abscisic Acid Effects on Stomatal Regulation in Solanum lycopersicum L. under Drought Stress Conditions. Horticulturae 2024, 10, 985. https://doi.org/10.3390/horticulturae10090985

AMA Style

Bravo-Díaz MA, Ramos-Zambrano E, Juárez-Yáñez TE, Perea-Flores MdJ, Martínez-Ayala AL. Triacontanol Reverses Abscisic Acid Effects on Stomatal Regulation in Solanum lycopersicum L. under Drought Stress Conditions. Horticulturae. 2024; 10(9):985. https://doi.org/10.3390/horticulturae10090985

Chicago/Turabian Style

Bravo-Díaz, María Asunción, Emilia Ramos-Zambrano, Tomás Ernesto Juárez-Yáñez, María de Jesús Perea-Flores, and Alma Leticia Martínez-Ayala. 2024. "Triacontanol Reverses Abscisic Acid Effects on Stomatal Regulation in Solanum lycopersicum L. under Drought Stress Conditions" Horticulturae 10, no. 9: 985. https://doi.org/10.3390/horticulturae10090985

APA Style

Bravo-Díaz, M. A., Ramos-Zambrano, E., Juárez-Yáñez, T. E., Perea-Flores, M. d. J., & Martínez-Ayala, A. L. (2024). Triacontanol Reverses Abscisic Acid Effects on Stomatal Regulation in Solanum lycopersicum L. under Drought Stress Conditions. Horticulturae, 10(9), 985. https://doi.org/10.3390/horticulturae10090985

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