1. Introduction
Cultivated lettuce (
Lactuca sativa L.; Asteraceae), which is usually consumed fresh, is one of the most important leafy vegetables in the world. Commercial lettuce varieties are classified based on head and leaf characteristics, and some of the most common horticultural types are romaine, iceberg (also named as crisphead; CHD), oak-leaf (i.e., green oak; GOAK, and red oak; ROAK), and butterhead. Breeding new lettuce cultivars involves manual pollination of genetically stable (i.e., pure) parent lines with agronomic traits of interest, followed by selection based on plant phenotyping and genotyping [
1,
2]. The availability of detailed genetic maps of cultivated lettuce [
3,
4,
5,
6,
7] has allowed significant progress for mapping agronomically-important traits and promoted the development of marker-assisted selection (MAS) and candidate gene identification in these species [
8]. Several studies have shown that most breeding target traits, such as disease resistance [
9], postharvest discoloration [
10], thermotolerance in seed germination [
7], or water and nitrate capture [
6], are complex traits and thus controlled by quantitative trait loci (QTL).
Spain is the third-largest producer of lettuce and chicory in the world after China and the USA, with a total of c.a. 1.1 million tons, with an area of 35,360 hectares dedicated for their cultivation [
11]. Most of the production is focused on the southeastern Mediterranean region, with a temperate climate that allows lettuce cultivation throughout the year, making Spain the world’s largest lettuce exporting country. However, water scarcity and soil availability are limiting factors for plant cultivation, and inadequate irrigation and fertilization management has increased the environmental impact of agricultural exploitation in this region [
12,
13]. Therefore, to contribute to sustainable lettuce production, there is a strong requirement for the development of new forms of farming to increase the crop’s resource use efficiency and the reduction of production costs. Floating systems or closed hydroponic methods have been successfully engaged for lettuce cultivation [
14,
15,
16]. This technique allows for the precise control of water and mineral nutrition, saves soil and labor costs, and provides shorter harvest cycles, high product quality, and good consumer acceptance [
16].
Tipburn is defined as the localized necrosis found on the distal margins of rapidly expanding leaves. It is a serious problem in controlled lettuce production, as it reduces the quality and shelf life of fresh lettuce, hence resulting in severe economic losses [
17,
18,
19]. Tipburn is influenced by many environmental factors, such as light intensity, air temperature, and soil conditions, and is considered a calcium deficiency-related physiological disorder, which is usually associated with rapidly growing tissues [
17,
18,
20,
21]. In addition, a strong variation for tipburn incidence among different lettuce cultivars has been reported [
19,
22], allowing for the development of tipburn-resistant varieties [
19,
23,
24]. The use of genomic tools has enabled the identification of QTL for tipburn incidence in several recombinant inbred line (RIL) populations and the development of linked molecular markers [
25,
26]. However, further research is needed to identify the underlying candidate genes for these QTL and the effect of their introgression into other lettuce cultivars. Also, only a few studies on tipburn incidence have been carried out in lettuce grown in hydroponics [
27,
28].
Within the framework of a company-based breeding program for lettuce, we devised a low-cost procedure for closed hydroponic cultivation and easy phenotyping of root and shoot attributes during early growth in three growing seasons (Fall, Winter, and Spring). A representative sample of lettuce varieties from different cultivars (CHD, GOAK and ROAK) were selected based on contrasting agronomically relevant traits such as tipburn tolerance. Our results allowed us to define genotype × environment (G × E) interactions for some of the studied traits, and to establish a strong correlation between leaf nutrient content and tipburn incidence, which may help to reduce leaf damage through adequate fertilization management.
4. Discussion
There is an increased demand for fresh, locally grown, and safe vegetables among the EU consumers [
38]. However, intensive agricultural exploitation might lead to water shortage and soil salinization, among other environmental damages [
39]. The greenhouse production of vegetables in closed hydroponic systems is a resource-efficient technique for the production of high-quality and high-yield crops [
40]. Here, we devised a low-cost hydroponic system (i.e., floating rafts) for lettuce cultivation, which was used to evaluate the early growth and quality parameters of 12 genotypes from different lettuce cultivars (CHD, GOAK, and ROAK) in three growing seasons (Fall, Winter, and Spring). These genotypes were selected based on agronomically-relevant traits.
Research on the role of root system architecture (RSA) traits that could enhance nutrient and water use efficiency has not received broad attention in lettuce breeding programs until quite recently [
6]. We found striking differences among the studied lettuce cultivars in regard to their root system (
Figure 2a,c,
Figure 3a,c, and
Figure 4a,c). The CHD genotypes showed deeper roots as compared to those from GOAK and ROAK. As it is known that deeper roots are crucial for improving drought resistance in plants [
41,
42], CHD cultivars may be more drought tolerant than GOAK and ROAK genotypes, although this hypothesis could not be directly tested in our hydroponics system. On the other hand, GOAK and ROAK root systems were heavier and more superficial than those in the CHD cultivar. Indeed, GOAK and ROAK are oak-leaf cultivars located on the same genetic clade [
43], which are mainly differentiated by their leaf anthocyanin content [
1]. The differences in the RSA of the CHD and GOAK/ROAK cultivars may thus account for the genotype-dependent behavior of cultivated lettuce in saline soils [
44] or in response to water and nutrient deficiency [
6]. Our experimental setup will allow evaluating growth responses under different soil stresses through the adjustment of the nutrient solution and/or the experimental conditions (i.e., temperature, aeration, etc.). In addition, the contrasting RSA phenotypes of the G3 and R5 genotypes (
Figure 3c and
Figure 4c), with a three-fold difference in their root fresh-weight during the Spring season (
Figure 5), may be used for the identification of the genetic determinants involved in RSA variation in the oak-leaf lettuce clade through the implementation of QTL mapping. We estimated the shoot growth rates of lettuce in hydroponic culture through dedicated image analysis (
Figure S2). Overall, shoot growth was much lower during Winter than in Spring or Fall, which is in agreement with previous studies where higher temperatures and high irradiance were found to be key factors, which affected growth product quality in these species [
16,
22]. However, we found an interesting G × E interaction for the estimated SA in some of the studied genotypes. On the one hand, the ROAK genotypes showed lower SA values in Spring than in Winter (
Figure 4b). On the other hand, SA values in C7 were highly affected by the growing season, as higher growth occurred during Spring for this genotype (
Figure 2b). However, the SA and RA values estimated from images were inaccurate descriptors of yield, as confirmed by the low correlations found between FW and DW values of the shoot system and the root system at the end of the experiment (
Figure S6). We found that FW was highly correlated with DW (both for root or shoot) for all the studied cultivars and during the different growing seasons (
Figure S6), and that their WC variation ranged from 92% to 98% (
Table S4). Interestingly, we found that WC (either in roots or shoots) was negatively correlated with root DW but not with shoot DW, which suggest that thinner roots may be more efficient in water uptake in lettuce plants grown in hydroponics, as compared to those plants grown in soil where root diameter may be directly related to the ability to penetrate the drying soil [
41]. In addition, the R:S ratio allowed us to identify genotypes with contrasting yield genotypes, such as C7 and R3 (
Table S4). While C7 had the lowest R:S values (and hence higher yield) in Fall, R3 showed the highest R:S ratio (thus lower yield) during Spring, indicating a G × E interaction for this trait, as well.
In Spain and Italy, two of the fifth largest lettuce and chicory producers in the world [
11], greenhouse lettuce production is often limited by the extent of tipburn and premature bolting. Tipburn is a physiological disorder characterized by necrotic lesions at the margins of the developing leaves, resulting from a localized Ca deficiency [
45]. Tipburn development in lettuce depends on environmental factors that promote growth [
46]. Ca translocation from the roots to the shoots occurs through the xylem due to transpiration, and Ca cannot be mobilized from older leaves to younger ones [
47]. Some of the climatic factors that characterize the Mediterranean region, specifically high temperature, high radiation, and long photoperiod, lead to the rapid shoot growth of lettuce, which cannot match Ca translocation from the roots. The lettuce genotypes studied in this work were selected based on their contrasting tipburn incidence when grown in soil. A previous study using a small number of lettuce cultivars grown in hydroponics showed that tipburn was not observed in the late Winter season, whereas it was severe during Spring [
48]. We found that tipburn incidence was higher during Spring but lower in Winter for most of the studied cultivars grown in hydroponics (
Figure 6a). And we also observed that the CHD cultivars showed a higher variation for tipburn incidence as compared to the studied oak-leaf types (GOAK and ROAK). These results were consistent with the greater genetic variability for tipburn responses found in the CHD cultivar as a result of earlier breeding efforts for tipburn tolerance in this cultivar [
19,
22,
24,
25]. In a recent study [
28], early bolting and tipburn behavior were studied on 18 genotypes from different lettuce cultivars grown in hydroponics at high temperature and extensive differences were also observed among them. Hence, the combined effect of high growth rates and high temperatures during Spring may lead to the reduced nutrient supply to the developing leaves, resulting in the observed enhancement of the tipburn severity during Spring. Only two of the studied genotypes, C3 and R2 showed severe tipburn symptoms in every season (
Figure 6a,c). Another two genotypes, C8 and G3 showed intermediate-to-severe tipburn symptoms only during Spring (
Figure 6a,c). On the other hand, C1, G1, and R5 showed tipburn tolerance when grown in hydroponics (
Figure 6a,c). These results perfectly matched the tipburn severity symptoms found in the studied genotypes when grown in soil (V.B., unpublished), which validates our experimental setup for the fast and high-throughput evaluation of tipburn responses in lettuce germplasm collections grown in hydroponics.
To investigate the nutritional causes of tipburn incidence during Spring in the studied genotypes, we measured the levels of several macro and micronutrients in leaves of different ages at the end of the experiment (45 dap;
Table S6). Ca and Na levels showed the highest M/J ratio, irrespectively of the cultivar type (
Table 2), which is consistent with the low Ca mobilization from mature tissues [
45] and the higher Na accumulation in older leaves [
49]. On the other hand, P displayed the lowest M/J values within the studied macronutrients (
Table 2), with lower P levels in the CHD than in the GOAK/ROAK genotypes. These latter results could be explained by the differences in RSA between the studied cultivars, as root responses to low phosphate favor the exploration of the shallower part of the soil, where phosphate tends to be more abundant [
50]. We noticed that the CHD genotypes contained a lower nutrient concentration than the GOAK and ROAK ones (
Table 4). Nutritional differences between lettuce cultivars have been described previously [
51]. The tipburn incidence and tipburn scores of the CHD genotypes perfectly matched their total nutrient content, hence, the genotypes with the lowest nutrient levels (C3 and C8) showed severe tipburn symptoms (
Figure 6a,b and
Table 5 and
Table 6). Our results suggest that tipburn in the studied CHD genotypes may be related to some nutrient imbalance, as has been proposed earlier in lettuce [
52]. The high growth rates observed during Spring for C3 and C8, combined with their contrasting R:S ratios, may result in decreased Ca concentrations in leaves and thus increased tipburn, as has been previously reported in other lettuce genotypes [
21,
45]. Because of the restricted Ca transport within the head-enclosed leaves of the CHD genotypes, Ca levels are much lower in the leaf margins, where tipburn symptoms arise early; high K levels in this region might also contribute to enhanced tipburn in CHD genotypes, as suggested previously [
45]. Overall, the studied ROAK and GOAK genotypes were less sensitive to tipburn, which was consistent with previous results which suggested a narrow genetic variation for this trait in oak-leaf type cultivars [
19]. R2 and G3, which displayed severe tipburn during the Spring season, were characterized by a strong decrease in K levels between mature and juvenile leaves, as compared with the tipburn tolerant G1 and R5 genotypes (
Figure 7). To assess whether tipburn in the studied lettuce genotypes is caused by an altered Ca/K homeostasis, we plan to evaluate tipburn susceptibility using an in vitro evaluation system [
24], with some modifications.
We also found striking differences in regard to the studied micronutrients (Fe, Mn, Zn, and Cu) depending on cultivar type and genotypes (
Table 3 and
Table 6). Alterations in micronutrient homeostasis (such as Fe and Mn) have commonly been associated with the appearance of shoot tip necrosis during pistachio in vitro culture [
53], which very much resembles the tipburn symptoms found in the ROAK and GOAK genotypes. Fe and Mn levels in leaves, as well as their M/J ratios, were much higher in the GOAK and ROAK genotypes than in the CHD ones (
Table 3). Consistent with previous results on K levels, Fe and Mn levels strongly decreased in the R2 and G3 genotypes from mature to juvenile leaves (
Figure 7), suggesting that a nutritional unbalance of some micronutrients (Fe and Mn) could explain tipburn in oak-leaf susceptible genotypes. Further experiments with additional ROAK and GOAK genotypes will allow us to confirm this hypothesis.