1. Introduction
Global climate change is without a doubt the greatest challenge for humanity nowadays. This phenomenon causes global temperatures to gradually increase throughout the planet, most of the time accompanied by a reduction in precipitation, which diminishes water availability and increases the occurrence of extreme weather events [
1]. Given these challenging environmental conditions, agricultural systems face great difficulties in maintaining optimal crop production despite higher temperatures and water scarcity. Indeed, the combined stresses have been shown to adversely affect plant growth, as proved by the 21 and 40% yield reductions observed in wheat (
Triticum aestivum L.) and maize (
Zea mays L.) [
2], as well as the 28% yield reduction accounted for in tomato [
3]. Therefore, the development of new crop varieties better adapted to these extreme conditions has become crucial for enhancing crop productivity and ensuring access to food.
Tomato (
Solanum lycopersicum L.) is a major crop with a global production of 186,107,972.48 million tons in 2022, according to FAO, and one of the most widely consumed as part of the Mediterranean diet [
4], where its major production areas in Europe are located (
https://www.fao.org/statistics/, accessed on 16 May 2024). It is also one of the most appreciated vegetables due to its nutritional qualities, like a higher content of antioxidants such as polyphenols, β-carotene, and lycopene, as well as essential minerals like manganese and zinc [
5]. Tomatoes are grown in a variety of climate zones and are frequently subjected to extreme temperature stress, either in greenhouses or when grown outdoors [
6]. Temperatures ranging from 25 to 30 °C during the day and 20 °C at night have been reported to be ideal for tomato growth [
7]. Nevertheless, even a small increase in any of these values can have a severe impact, since fruit setting has been reported to be interrupted when day and night temperatures exceed 26 and 20 °C, respectively [
8]. This sensitivity is reflected in alterations in male gametophyte development such as inadequate anther dehiscence and abnormal tapetum development in the early phases of pollen formation, ultimately causing male sterility, flower loss, and a significant decrease in yield [
9]. Also, the impact of drought is quite significant in this crop since flower bud abscission and a reduction in photosynthetic parameters have been reported under reduced watering conditions [
10].
Over the past few decades, huge efforts have been made to genetically improve this species, although breeding activities have been focused mainly on fruit quality traits [
11]. Thus far, tomato heat tolerance breeding programs have been scarce and mostly carried out in the same production regions, thus not considering climate-adverse conditions [
12]. Furthermore, these programs have outlined the limited genetic basis of cultivated tomatoes for heat tolerance, which has sparked interest in exploiting tomato wild relatives, frequently used as sources for abiotic and biotic stresses [
13]. The challenges posed by climate change make it necessary to develop tomato varieties suitable for production under this agronomic scenario. One appropriate solution to improve tomato performance under heat-stress conditions and mitigate production losses is the exploration of new sources of tolerance. Natural germplasm is revealed to be a useful material for broadening the genetic basis of tomato abiotic stress tolerance by means of different breeding strategies. However, it may not be sufficient. The key lies in combining the exploration of natural variation with the implementation of large-scale mutagenesis programs, enabling the generation of new tolerance alleles and thereby increasing genetic diversity.
With the aim of identifying new sources of resilience to climate change impact, we evaluated germplasm collection under combined heat and drought stress conditions. This collection includes an ethyl methanesulfonate (EMS) mutagenized population [
14] and natural germplasm accessions represented by wild and cultivated tomato species. The tolerant variants identified provide valuable insights into understanding the genetic basis of tomato thermotolerance and, simultaneously, serve as suitable materials for breeding programs aimed at increasing tomato resilience to adverse climate conditions.
3. Results
3.1. Screening for Combined Heat and Drought Stress Tolerance in Nursery Conditions
With the aim of identifying new sources of tolerance to abiotic stress in tomatoes for their introduction into breeding programs, two trials were performed under combined heat and drought stress conditions in the summer cycles of 2018 and 2019. As part of these trials, we screened the tomato EMS mutant collection developed in the Moneymaker (MM) genetic background [
14] and a natural germplasm collection. The cultivar MM was used as a control and grown alongside the other plants in each trial.
Trials were conducted under nursery conditions to detect phenotypic alterations caused by combined heat and drought stress in the early vegetative developmental stages. A total of 7769 M2 families and 395 natural accessions were sown for screening under these conditions. In the initial step, the germination rate was assessed, and only families and accessions with five or more plants (a germination rate higher than 40%) were evaluated. Thus, 72.6% and 91.7% of the total sown M2 families and natural accessions, respectively, were assessed. Concretely, 3459 M2 families were screened in the summer2018 cycle and 2180 M2 families and 362 natural accessions in the summer2019 cycle.
At the initial stages of each trial, which took place in early summer, plants were cultivated until they developed a proper root system and 4–6 fully expanded leaves. At this point, stress was implemented by reducing water availability and increasing temperatures. Vegetative traits such as decreased growth rate, reduced leaf development and expansion, leaf senescence and chlorosis, leaf necrosis, and damage to the shoot apex were assessed. In addition, any other visual symptoms of hypersensitivity or tolerance responses were also noted. An overview of the combined heat and drought stress treatment effects, including phenotype features before and after stress condition implementation, can be observed in
Figure 1.
After completing the stress period, which lasted 48 days in the summer2018 trial and 53 days in the summer2019 trial, most M2 families showed a hypersensitive response to the stress treatment since they showed premature death, extreme chlorosis, necrosis, and/or scarce leaf formation. However, 161 EMS M2 families (2.9% of the total assessed) were identified as including at least one tolerant plant among their members. This tolerance response was primarily characterized by either the presence of green and turgid shoot apices, the absence of senescence, or reduced chlorosis symptoms in basal leaves. Some of the tolerant lines exhibited compact vegetative growth, while others were also able to develop new axillary shoots after the necrosis of the main shoot apex (
Figure 1D). To obtain M3 progenies, tolerant plants were transplanted and grown to maturity under organic greenhouse conditions. Among these lines, 152 (94.4%) displayed a tolerant phenotype fitting a monogenic recessive inheritance pattern, while the remaining ones (5.6%) showed a phenotype segregation consistent with a single dominant mutation. Regarding the natural germplasm collection, we identified 24 tolerant accessions (6.1%) from the wild species
S. peruvianum,
S. corneliomuelleri,
S. pimpinellifolium, and
S. huaylasense, as well as from
S. lycorpesicum var.
cerasiforme and from the cultivated species
S. lycopersicum. Among them, seven lines exhibited consistent and uniform tolerant behavior (29.2%). On the contrary, a heterogeneous stress response, from absolute susceptibility to great tolerance, was observed within the remaining 17 lines. The tolerant lines identified with both the EMS collection and the natural germplasm, along with the inheritance pattern and the number of detected tolerant plants, are displayed in
Supplementary Tables S3 and S4.
3.2. Stress Tolerance of Selected Germplasm under Climatic Chamber Conditions
To further assess the stress tolerance phenotype of a set of 20 M2 families selected during the nursery trials, a new trial under controlled heat and drought conditions was set up. With this aim, 15 plants from each M2 family, along with control plants, were grown in a climatic chamber where growth parameters, i.e., photoperiod, temperature, water availability, and humidity, were precisely determined. When plants had developed four true leaves, a stress treatment consisting of a combination of high temperature and drought stresses was applied (for details, see the Material and Methods section). Daily phenotyping was carried out by assessing parameters such as leaf darkening, loss of turgidity, and symptoms of wilting and necrosis. Furthermore, leaf temperature, which is directly related to water loss by transpiration, was measured daily using an infrared thermography (IT) camera, a non-invasive method that allows the early identification of stress.
A representation of leaf temperature as recorded by the IT camera is shown in
Figure 2A, which includes temperatures before the implementation of stress treatment (0 DAS) and those records corresponding to one to three days after stress treatment was applied (1–3 DAS). As a result of this chamber trial, a tolerant response was corroborated for the BT20250, BT20450, and BT20520 families. Whereas the leaf temperatures of sensitive plants continuously increased, the tolerant ones exhibited a thermographic profile characterized by lower temperature values (
Figure 2B) and a reduced water evaporation rate (
Supplementary Figure S2), even though stress symptoms were not yet evident in sensitive plants. After seven days of stress treatment, tolerant plants exhibited leaflets that remained bright green, showing no signs of wilting or other stress-related symptoms (
Figure 2C). In contrast, control and sensitive plants displayed severe wilting and notable chlorophyll degradation, as evidenced by the dark brown color of the leaves and the entire plant (
Figure 2C). Conclusive results were challenging to obtain for 11 M2 lines due to the unequal vegetative development exhibited by the plants at the time of assessment. Indeed, tolerance was only detected in the smaller plants, likely due to lower water evaporation. Finally, the stress-tolerant phenotype was uncertain for the remaining six M2 lines, as the plants with thermographic profiles showing lower temperature values ultimately exhibited sensitive symptoms at the end of the stress treatment.
3.3. Characterization of Stress Tolerance Response under Greenhouse Conditions
Given that reproductive traits such as flower number, pollen viability, and fruit setting have a direct impact on crop productivity, we conducted new trials under greenhouse conditions to assess the effects of combined heat and drought stresses on these reproductive traits. In addition, plant height and stem thickness were also assessed to determine the impact of stress on vegetative development. Field trials were carried out during the summer2020 and summer2021 cycles using a random two-block design of eight M3 plants each. In both trials, M3 progenies, derived from 14 M2 plants identified as tolerant during the previous nursery screenings, were evaluated along with control plants. Temperature and humidity were constantly monitored to ensure that both day and night temperatures exceeded the established threshold values (25 °C during the day and 20 °C at night) [
7].
After three weeks of growth under stress conditions, control plants began to display leaf chlorosis and reduced growth compared to tolerant lines (
Figure 3A). Other stress-related features observed in control plants included senescence of the shoot apex, a burnt leaf appearance, and curly leaflets. In contrast, leaves of tolerant lines such as BT20330, BT20230, BT21400, BT20220, and BT21210 remained green, fully expanded, and lacked senescence symptoms (
Figure 3B,C).
To ensure that observed phenotypic differences in plant height and stem thickness were solely attributed to the stress effect, these vegetative traits were measured before and after the implementation of stress conditions. No significant differences were found between the control and mutant genotypes before the stress conditions were implemented (
Supplementary Figure S3). After the stress treatment, significant differences in plant height were observed in the BT20100 and BT20330 lines compared to control plants in the summer2020 trial, while no significant differences were observed for this trait during the summer2021 trial between EMS lines and control MM plants (
Figure 3D;
Supplementary Tables S5 and S6). It is worth mentioning that the latest trial was affected by an infestation of the
Tuta absoluta pest, which could be responsible for some of the differences observed between the two trials. Additionally, significant differences in stem thickness were observed in the summer2020 trial for the lines BT20040, BT20090, BT20230, BT20330, and BT21140, which showed higher values than control plants under stress conditions. Interestingly, similar differences were reproducible in the summer2021 trial for the BT20230, BT20330, and BT21140 lines. (
Supplementary Figure S4; Supplementary Tables S5 and S6).
Regarding the assessment of reproductive traits, we focused on fruit setting, as well as the presence of flower and inflorescence damages such as burning and abortion. Three weeks after the onset of stress, unlike the observed response in tolerant lines like BT20230 (
Figure 4B), some flowers of control plants began to show burning and inflorescence abortion (
Figure 4A). Furthermore, to assess the impact of stress on pollen viability, a tetrazolium staining assay was performed on pollen grains harvested from the first inflorescence produced under these conditions (3rd truss). This analysis not only confirmed that heat and drought conditions have a severe effect on pollen viability (
Figure 4C,D), but more importantly, it revealed that tolerant EMS lines identified in this study yielded an increased ratio of viable pollen compared to control plants (
Figure 4E,F). As a consequence of the higher pollen viability, enhanced fruit setting was observed in tolerant plants such as BT20220, BT20300, BT20330, BT21210, BT21230, and BT21400 in the summer2020 trial. Despite the presence of the
T. absoluta pest, significant differences in fruit setting were also noted for a few lines (BT20100, BT20300, and BT21400) during the summer2021 cycle (
Figure 4G).
3.4. TILLING and Eco-TILLING Analysis for Mutant Variant Detection
TILLING and Eco-TILLING analyses were applied as proof-of-concept to isolate new genetic variants that can be used for functional analysis of regulatory genes and as sources of tolerance to abiotic stress in tomato breeding programs. For this purpose, we screened both the EMS mutant and natural germplasm collections for polymorphisms in the CALCINEURIN B-LIKE PROTEIN 10 (SlCBL10) gene (Solyc08g065330). The loss-of-function mutant of this gene shows a hypersensitive phenotype when exposed to salt stress conditions, resulting in severe inhibition of vegetative development and collapse of the shoot apical meristem [
17]. Furthermore, the pivotal role of SlCBL10 in ion homeostasis and its function as a positive regulator of the abiotic stress response in tomatoes have been demonstrated in recent years [
18,
19]. Therefore, the identification of new alleles of SlCBL10 through TILLING analysis could facilitate further research into its biological function and serve as genetic tools for the development of new sustainable tomato varieties.
Specific primers covering most of the coding regions of the
SlCBL10 (
Solyc08g065330) gene were designed (
Figure 5A;
Supplementary Table S2). A workflow depicting the setup of the TILLING and EcoTILLING platforms, as well as the HRM analysis, is shown in
Supplementary Figure S5. Our analysis revealed 11 putative mutations, with five identified in the natural germplasm accessions and six in the EMS collection. Subsequently, Sanger amplicon sequencing confirmed the presence of seven of these single-nucleotide mutations at the base-pair level in individual plants. Three of these mutations were located in the coding exons of the
SlCBL10 gene, resulting in one synonymous and two non-synonymous mutations (
Figure 5B;
Supplementary Table S7). Hence, our findings confirm that these reverse genetics strategies are an effective method for detecting mutations in tomato genes related to stress tolerance, and the populations used in this study have the potential to serve as a comprehensive platform for future reverse genetic studies in tomato.
4. Discussion
As assessed to date by several public and private institutions, global temperature data show a rising trend that makes heat stress a critical issue that must be addressed to ensure global crop yield and productivity [
20]. This is particularly serious for tomatoes, a major vegetable crop whose proper growth is severely hampered by high temperatures [
9]. Understanding the genetic and physiological responses to heat stress is crucial for improving their agronomic performance under the climate change scenario. With this aim, we characterized a natural germplasm collection, including a set of tomato wild relative accessions, and an EMS-mutagenized population, the latter developed from the commercial cv. Moneymaker, to identify new sources of genetic tolerance to high temperatures.
Some of the tolerant lines identified as part of our study displayed a compact phenotype. Previous works have reported this trait’s occurrence, probably as a resource to reduce water evaporation to cope with abiotic stress conditions [
21]. This is the case of the EMS heat-tolerant mutant line HT7, obtained from the ornamental Micro-Tom genetic background, which shows a compact phenotype as described by Pham et al. [
22]. HT7 plants developed a narrow canopy and had less stomatal density than WT plants under stress conditions, which could contribute to a decreasing transpiration rate. Furthermore, HT7 plants produced a high number of viable pollen grains under long-term heat stress, resulting in high fruit-setting rates and increased fruit yield [
22]. In our mutant collection, some tolerant lines, such as BT20090 (
Figure 1D), exhibited compact vegetative growth when subjected to combined heat and drought stresses in nursery greenhouse conditions, which supports the idea that this strategy is effective for mitigating adverse climatic conditions.
The impact of heat stress on male gametophyte fertility has been broadly described to date [
23,
24,
25]. In our greenhouse conditions, thermal stress induced several alterations in the reproductive traits of control and hypersensitive lines, including pollen number and viability. In contrast, tolerant lines showed higher pollen viability rates than control plants, where pollen grains were both fewer and non-viable. Previous studies have highlighted the particular sensitivity of flower buds to heat stress, especially during the 7 to 15 days before anthesis, a critical period that encompasses the meiosis phase [
9,
26]. Thus, when pollen mother cells are subjected to heat stress, the quality and quantity of pollen are significantly reduced [
9]. In addition to reduced pollen viability, control and hypersensitive genotypes showed severe burning of entire inflorescences (
Figure 4), which ultimately compromised their yield. Thus, the tolerant plants identified in our screenings demonstrated superior agronomic performance, particularly in terms of higher fruit setting when compared to control ones. Furthermore, under nursery conditions, we identified seven tolerant accessions from wild species such as
S. peruvianum,
S. corneliomuelleri,
S. pimpinellifolium, and
S. huaylasense. Despite their small fruit size and lack of commercial value, these accessions exhibited remarkable tolerance behavior and serve as valuable genetic resources for broadening the genetic base to improve abiotic stress tolerance in cultivated tomatoes through backcross breeding strategies.
A comprehensive study of the genetic basis of abiotic stress tolerance is essential for developing new resilient varieties. Given the polygenic nature of this trait, advanced QTL analyses have been conducted to dissect the genetic architecture of traits related to stress tolerance in tomatoes [
27]. QTLs associated with several reproductive traits affected by temperature, such as flower number, fruit number per truss and fruit set percentage, stigma exertion, and pollen viability, have been identified [
28]. Moreover, QTLs have been detected for traits that exhibit high plasticity under high temperatures, such as flowering time, fruit weight, and yield [
29], underscoring the ability of tolerant accessions to display different phenotypic features depending on the environmental conditions. Notably, our greenhouse trials have revealed significant variability in the behavior of tolerant accessions from one season to another. This variability is likely attributed to the complexity of heat tolerance, a polygenic trait often associated with plasticity effects in response to changing environmental conditions. Therefore, gaining knowledge of the genomic regions and genes associated with the tolerant phenotype displayed by the accessions identified in our study, which requires forward genetics analyses involving the development of mapping populations and whole-genome sequencing approaches, will contribute to a deeper understanding of heat and drought stress tolerance in tomatoes.
Furthermore, reverse genetics methodologies may be applied to isolate new drought and heat tolerance alleles. To achieve this goal, we combined extensive phenotyping with TILLING analysis to identify mutations in target genes previously involved in regulating the heat tolerance response in other plant model species. This is the case of the
SlCBL10 gene [
17]. The TILLING strategy has proven useful in the past for identifying new mutant lines carrying SNVs in genes controlling tomato thermotolerance [
30]. Through this approach, we identified seven new variants of the
SlCBL10 gene among both natural germplasm and EMS mutant collections. While this work focuses on this gene, our TILLING analysis has been extended to include other 14 genes, with the hope of identifying new tolerant variants of genes of interest. Therefore, our work proves that the combination of extensive phenotyping under controlled greenhouse conditions and the use of molecular biology techniques, such as TILLING, enables the identification of novel alleles associated with abiotic stress tolerance in tomatoes. These variants hold a great value for future breeding programs aimed at enhancing tomato resilience to adverse climate conditions.