**Managing the Product Quality of Vegetable Crops under Abiotic Stress**

Editor **Lilian Schmidt**

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This is a reprint of articles from the Special Issue published online in the open access journal *Horticulturae* (ISSN 2311-7524) (available at: https://www.mdpi.com/journal/horticulturae/special issues/Vegetable Crops Horticultural).

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## **Contents**

#### **Pinki Devi, Penelope Perkins-Veazie and Carol Miles**

## *Editorial* **Managing the Product Quality of Vegetable Crops under Abiotic Stress**

**Lilian Schmidt 1,2**

#### **1. Introduction**

Plants, as sessile organisms, are continuously exposed to varying environmental conditions and often face abiotic and biotic threats. Biotic stressors include pathogens such as fungi, bacteria or herbivores that can damage plant tissues locally and systemically. In addition to this, abiotic stress, such as sub- and supra-optimal temperatures, the availability of water, light conditions and nutrient supplies, may severely impact the performance of plants. This includes the effects on photosynthetic processes, growth and developmental alterations and modifications in metabolic pathways.

In consequence, plants have developed a wide range of strategies to cope with the challenging conditions, mainly the alteration of growth processes and the investment in defensive compounds [1]. These two strategies often represent a dilemma for plants as they require different resource allocation patterns.

When cultivating plants for use in human nutrition, farmers are faced with a similar dilemma: Besides a high crop yield, there is an increasing demand for products that are rich in health-promoting substances. These beneficial compounds are mostly secondary plant metabolites that are synthesized as a consequence of abiotic or biotic stresses. This is especially true for vegetable crops that are rich in secondary compounds such as polyphenols or carotenoids. In addition, vegetables can contain substances with undesired properties, e.g., oxalates and nitrate. It is thus of interest to cultivate vegetable crops with maximum contents of beneficial compounds and minimal contents of unfavorable compounds, as well as high yields.

Significant research is focusing on pre- and post-harvest procedures that can be used to achieve these goals. Abiotic stress is often applied by employing certain cultivation practices and storage conditions in order to maintain and potentially improve the internal and external properties of vegetable crops.

#### **2. Special Issue Overview**

This Special Issue compiles 11 manuscripts that deal with the effects of abiotic stress on the modulation of vegetable quality. It comprises 10 research articles and 1 review. The majority of the contributions deal with pre-harvest conditions and investigate whether cultivation practices can modulate the yield and nutritional value of vegetable crops and herbs under abiotic stresses. Among these publications, several deal with nutrient supply [2–5], salinity [6,7], water supply [2,3,8] or lighting conditions [6]. Two manuscripts describe either the effects of applying chemical substances such as gibberellic acid [9] or seaweed extract [10] on the yield and quality of vegetable crops. Grafting as a method to mitigate the impacts of abiotic stress in vegetables was elucidated by a research paper by Ellenberger et al. [3] and in a review by Devi et al. [11]. Elevated CO2 levels were either used as pre-harvest conditions in combination with different nitrogen forms [5] or as post-harvest treatment combined with different methods of packaging [12].

**Citation:** Schmidt, L. Managing the Product Quality of Vegetable Crops under Abiotic Stress. *Horticulturae* **2022**, *8*, 25. https://doi.org/ 10.3390/horticulturae8010025

Received: 1 December 2021 Accepted: 14 December 2021 Published: 26 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

In general, a large quantity of the contributions in this Special Issue present data on fruit vegetables such as watermelons, bitter gourd and tomatoes [2–4,9,11], with the latter being the focus of three publications. These crops were mostly grown in greenhouses [2–4], but some were grown in an open field [9] or under rain-out shelters [8]. Leafy vegetables and herbs—such as arugula [5], purslane [6] and basil [7]—were used in three experiments and were cultivated in growth cabinets [5,6] or in greenhouses [7]. The bulbs of onions were harvested from plants in a field trial [10], and the asparagus spears for a storage experiment were obtained from local farmers [12].

Altogether, a wide range of species, cultivation conditions and management practices (pre- and post-harvest) were used for elucidating the effect of abiotic stress on the quality of vegetable crops.

#### *2.1. Impacts of Reduced Water Supply*

The availability of water for the irrigation of vegetable crops will become of high relevance in the future; while heavy rainfall is predicted to occur more frequently, the opposite will be the case as well, with more and longer drought events as compared to the present conditions. One way to overcome these challenges is the use of vegetable varieties that are able to cope with these extreme conditions. Besides the breeding activities that aim at creating more resistant varieties, the use of traditional varieties that may be better adapted to specific situations is being increasingly suggested. In their study, Sica et al. [8] tested the local bean accessions of Veneto, Italy, concerning the yield potential and nutritional properties of the seeds as consequences of drought exposure during different growth stages. Interestingly, exposure to drought had no impact on the yield of dry seeds until the flowering phase. In general, most impacts on the nutraceutical properties of the bean seeds could be explained by variety, and rather little can be assigned to the drought treatments. One bean accession turned out to be most promising for cultivation in areas with increased risk of drought events.

#### *2.2. Effects of Alterations in Nutrient Supply in Combination with Other Abiotic Stresses*

Mineral nutrients are essential for plants. When applied in concentrations or forms that are not optimal for the crops, this may have consequences for the performance of plants and their products. Three contributions to this Special Issue deal with yield and quality aspects of tomatoes under different nutritional conditions that are regarded as "stressful" for the plants. Specifically, Ellenberger et al. [3] addressed the question of how grafting two commercial tomato varieties may affect the yield and fruit quality when exposed to a combination of nutrient deficiency and drought stress. They used the commercial rootstock 'Beaufort', two novel hybrid rootstocks of *S. pennellii* × *S. lycopersicum*, which are expected to be drought tolerant, and self-grafted plants. When exposed to a 50% reduction in fertigation, the 'Lyterno' variety grafted onto the novel rootstocks had higher fruit yields as compared to the plants grafted either on the commercial cultivar or the self-grafted plants. This was not observed for the tomato cultivar 'Tastery', which, in general, showed weak responses to the imposed stress. For all the combinations of grafting and fertigation supply, little effects in terms of fruit quality were assessed. The use of appropriate scion–rootstock combinations may thus provide potential for optimizing the stress resilience, the yield and the quality of tomatoes.

Moreover, de Luca et al. [2] combined potassium (K) supply and water shortage treatments for tomatoes, using a commercial cultivar and a transgenic line that may accumulate large quantities of K. The study aimed at testing whether different K supplies can alleviate the impact of periods of dehydration. Although it was obvious that the larger supply of K was able to increase the shoot growth and fruit yield under water stress, this did not result in yields comparable to those obtained for well-watered plants. Moreover, the transgenic tomato line with increased capacities to accumulate K turned out not to be beneficial under water stress in terms of fruit yield.

In another study on tomatoes, Schmidt and Zinkernagel [4] investigated the effects of fertilization with 50% less nitrogen (N) than recommended on the quality of three different varieties of cocktail tomatoes immediately after harvest and after eight days of storage at low temperatures. It was shown that the reduction in N had little effects on the yield, external quality and on the concentrations of valuable compounds in the tomato fruits. In addition, there were no impacts on the taste, as observed by a trained sensory panel, suggesting that the reduced N supply was not detrimental for the three varieties of tomato, at least under the experimental conditions.

One manuscript describes the effects of elevated atmospheric CO2 levels and N forms on the physiological processes, crop yield and nutritional quality of the leafy vegetable arugula [5]. Plants of two arugula varieties were grown in climate cabinets with either 400 or 800 ppm CO2 and received either pure nitrate or ammonium-dominated nitrogen fertilizer. Replacing 75% of nitrate-N with ammonium-N had little impact on photosynthetic CO2 assimilation, the nutritional quality (e.g., content of anti-oxidative compounds) or was disadvantageous for crop yield, regardless of the CO2 levels. Thus, ammonium-dominated N provides no benefits for arugula production under the elevated CO2 levels that are proposed for the future.

#### *2.3. Impact of Salinity*

Salinity is characterized by increased electrical conductivity due to an accumulation of mineral elements. Specifically, the enrichment with sodium (Na) and chloride (Cl) ions in the soil imposes a worldwide threat to plant production. However, as a mild stressor, saline conditions can be used to alter the quality of horticultural products.

This was conducted by Corrado et al. [7] by supplying sweet basil plants in hydroponics with three nutrient solutions with differing NaCl concentrations. Even low concentrations of NaCl (20 mM) resulted in reduced biomass development, while having positive effects on some nutritional quality parameters, such as antioxidant capacity and total polyphenols.

In a study by Giménez et al. [6], much higher salinity levels (80 mM of NaCl) were combined with different light conditions (fluorescent lamps, red–blue LED, red–blue–deep red LED). Purslane coped well with the salinity level under the two LED lighting regimes. The nutritional quality of this microgreen was improved as anti-nutritive compounds were lower when grown with LEDs and supply of NaCl. The study showed that it is possible to achieve high yields when growing purslane microgreens under saline conditions and an appropriate choice of light sources, although this was at the expense of some valuable plant pigments (chlorophylls, carotenoids, flavonoids).

#### *2.4. Effects of Application of Different Substances*

Stressful conditions can also be imposed by applying certain chemicals to plants. In particular, substances that mimic the action of plant hormones are the focus of attention. Gibberellic acid is known to regulate both vegetative and reproductive growth processes in plants. It thereby affects the quality of the product by enhancing its antioxidant status. Both the alteration of plant growth and developmental processes and the impact on the nutritional quality are very relevant for the tropical vegetable crop *Momordica charantia* (bitter gourd). Abbas et al. [9] conducted field trials with two varieties of bitter gourd exposed to five different concentrations of a commercial gibberellic acid product. They found that the exogenous application of the plant growth regulator at high concentrations enhances growth (e.g., increased length of petioles and internodes) and yield (increased fruit numbers, increased fruit weight) while decreasing the activity of several antioxidant enzymes at the same time.

In another contribution, Abbas et al. [10] investigated the effects of foliar spraying with seaweed extract on the productivity and quality of four onion cultivars. The bio-stimulant was applied at four concentrations, with the lowest concentration (0.5%) resulting in an increased yield, increased contents of some mineral nutrients and increased soluble solids of the bulbs. The highest concentration of the seaweed extract (3%) increased the concentrations of ascorbic acid in the bulbs as compared to those without the application of seaweed extract.

#### *2.5. Grafting as a Method to Reduce the Impact of Abiotic Stress*

Grafting is a commonly used method in overcoming abiotic and biotic stresses during the cultivation of fruit vegetable crops. The advantage of this cultivation technique is the combination of potential beneficial properties of two or more accessions in one plant.

Devi et al. [11] contributed a review dealing with the effects of grafting on the fruit maturity and quality of watermelons. The publication summarizes the results of research activities on the impact of grafting watermelon scions on different rootstocks, with a focus on the formation of hollow hearts and hard seeds, flesh firmness, total soluble solid content, pH, titratable acidity, lycopene content and concentration of the non-essential amino acid citrulline, which can be of relevance for the cardiovascular system.

#### *2.6. Modulation by Storage Conditions*

The impacts of post-harvest conditions on the quality of vegetables are represented as well. Wang et al. [12] combined elevated CO2 pre-treatments and different packaging methods in order to assess the effects on different quality characteristics of green asparagus in cold storage. The authors showed that continuous application of elevated CO2 reduced the decay of the asparagus spears to the greatest extent by inhibiting the microbial development. In general, elevated CO2 application, either continuously or as a three-day pre-treatment, did not only reduce the microbial load and the respiration rate but also improved the sensory and nutritional quality of the green asparagus. In combination with modified atmosphere packaging, the pre-treatment with elevated CO2 was able to preserve the visual quality and the fresh weight of the asparagus and reduce the development of an off-odor.

**Funding:** This research received no external funding.

**Acknowledgments:** I gratefully acknowledge all the authors that participated in this Special Issue.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**

## *Article* **Reassessing the Role of Potassium in Tomato Grown with Water Shortages**

**Anna De Luca 1,2, Mireia Corell 3, Mathilde Chivet 4, M. Angeles Parrado 1, José M. Pardo 2 and Eduardo O. Leidi 1,\***

**Abstract:** Potassium (K) is closely related to plant water uptake and use and affects key processes in assimilation and growth. The aim of this work was to find out to what extent K supply and enhanced compartmentation might improve water use and productivity when tomato plants suffered from periods of water stress. Yield, water traits, gas exchange, photosynthetic rate and biomass partition were determined. When plants suffered dehydration, increasing K supply was associated with reduction in stomatal conductance and increased water contents, but failed to protect photosynthetic rate. Potassium supplements increased shoot growth, fruit setting and yield under water stress. However, increasing the K supply could not counteract the great yield reduction under drought. A transgenic tomato line with enhanced K uptake into vacuoles and able to reach higher plant K contents, still showed poor yield performance under water stress and had lower K use efficiency than the control plants. With unlimited water supply (hydroponics), plants grown in low-K showed greater root hydraulic conductivity than at higher K availability and stomatal conductance was not associated with leaf K concentration. In conclusion, increasing K supply and tissue content improved some physiological features related to drought tolerance but did not overcome yield restrictions imposed by water stress.

**Keywords:** *Solanum lycopersicum*; drought potassium; vacuolar transporter

#### **1. Introduction**

Potassium (K) is the most abundant cation and plays important roles in plant growth and development where it contributes to charge balance, osmotic adjustment and enzyme catalysis [1]. Stored in vacuoles, K contributes to generating the osmotic pressure required for turgor and expansive growth. Potassium contributes to water uptake and the generation of turgor pressure in the stomatal guard cells and the phloem [1]. At low K availability, plants are more susceptible to wilting in drying soils and a mild K deficiency affects photosynthesis by reducing stomatal conductance and photosynthetic biochemical reactions [2]. High K availability might counteract the inhibitory effect of drought on CO2 assimilation by protecting photosynthetic capacity [3,4].

Plant K uptake is affected by available water as low soil water content reduces mass flow and diffusion of the nutrients to the rhizosphere, and simultaneously decreases root growth [5]. Potassium transport into aerial parts does not rely only on leaf transpiration, and other factors such as root pressure contribute to long distance transport [6]. However, greater water flow by active transpiration increases K flux to the shoot and K uptake by the roots when it is available [7]. In doing so, symplastic K transport depends on membrane transport proteins such as high-affinity KUP/HAK transporters and K-selective

**Citation:** De Luca, A.; Corell, M.; Chivet, M.; Parrado, M.A.; Pardo, J.M.; Leidi, E.O. Reassessing the Role of Potassium in Tomato Grown with Water Shortages. *Horticulturae* **2021**, *7*, 20. https://doi.org/10.3390/ horticulturae7020020

Received: 23 October 2020 Accepted: 25 January 2021 Published: 29 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

channels, which facilitate cation movement between plant cells and its final delivery into the xylem [8]. During drought, K fluxes into shoots become reduced by less transpired water (lower xylem water flow) and reduced expression of K-loading channels into the xylem [7].

Tomato requires high K supplements for high yields and optimum fruit quality [9] and low K affects flower development and fruit setting efficiency [10]. When K is scarce, tomato plants reduce sink activity (stems and fruits) prior to having any effect on photosynthesis [11,12]. Considering the great importance of K supply and water availability for fruit yield and quality, we aimed at studying how K availability and compartmentation into vacuoles might affect plant water use and productivity under water stress. The overexpression of a vacuolar K,Na/H antiporter (NHX1) contributes to salt-tolerance in tomato by increasing root K uptake and its storage plant vacuoles [8,13]. By including a transgenic line able to take up more of this nutrient, we tried to assess to what extent a greater plant K content might affect some physiological processes under stress. Specifically, the questions addressed were whether a greater K vacuolar accumulation might contribute to plant water acquisition and use for increasing productivity, and also if enhanced K capture might reduce water loss and provide greater protection of photosynthetic activity, resulting in increased water-use efficiency.

#### **2. Materials and Methods**

#### *2.1. Plant Material and Stress Treatments*

Two tomato lines, the cultivar MicroTom (henceforth, the wild-type, WT) [14] and a transgenic line (N367), were used. The line N367 overexpresses the vacuolar AtNHX1 cation/proton antiporter and is able to accumulate greater K quantities in vacuoles and tissues [13]. Seeds were pregerminated (28 ◦C, 4 days) and the seedlings were transplanted into quartz sand (0.5 L pots) and irrigated every three days with a modified Hewitt's nutrient solution supplemented with K at the concentrations required for each experiment. The basic nutrient solution contained (in mM): NO3 −, 4; H2PO4 −, 1; SO4 2−, 3; Ca2+, 2; Mg2+, 1; and (in μM): Fe, 50 (as FeEDDHA); Mn, 10; Cu, 1; Zn, 5; B, 30. Three different K concentrations were used as nutritional treatments by supplementing the basic solution with 0.1, 1 and 10 mM K (as K2SO4). When 0.1 mM K was added, 1 mM phosphate was provided as NaH2PO4. For addition of 10 mM K, Mg concentration in the solution was increased to 5 mM to avoid Mg deficiency [9]. Plants were grown in a greenhouse (mean temperature, 26 ◦C; mean relative humidity, 45.0%).

When plants reached the first flowering stage, water stress treatments were initiated by withholding irrigation during 2 weeks followed by one day with abundant watering (to flush pot sand) with nutrient solution supplemented with K as required. This treatment was repeated several times, depending on the experiment (Supplementary Materials Figure S1). When measuring yield, fruits were collected after six cycles of drying/watering, while control plants kept a regular watering regime every 3 days. To test the effect of acclimation, plants were treated with either one or four cycles of watering/drying. Plants submitted to a single water stress cycle were considered "nonacclimated", whereas plants that experienced four consecutive watering/drying cycles were judged as "acclimated". This preconditioning (priming) to stress allows osmotic adjustment and expression of drought-tolerance mechanisms [15,16]. Physiological measurements (leaf water status, photosynthesis, stomatal conductance) were taken at the end of the drying periods and before rewatering, as stated in the Results section. Control plants were irrigated normally every three days.

#### *2.2. Leaf Water Status*

Leaf water status was estimated by measuring relative water content (RWC) and leaf water content (LWCT) [17]. Leaf osmotic potentials were measured in leaf samples by using a thermocouple psychrometer (HR33 microvoltmeter and C-52 sample chamber, Wescor, Logan, UT, USA).

#### *2.3. Gas Exchange and Photochemical Reflectance Index*

Gas exchange measurements were performed in the youngest fully expanded leaves with an LC-Pro Photosynthesis System gas analyzer (ADC Bioscientific, Hoddesdon, UK). Leaf water-use efficiency (WUEi) was estimated as the ratio between photosynthesis (A) and stomatal conductance (gs). The photochemical reflectance index (PRI) was determined with a PlantPen PRI 200 (PSI, Drásov, Czech Republic).

#### *2.4. Hydroponic Culture and Root Hydraulic Conductivity*

For determining the effect of K supply on root hydraulic conductivity, a critical component of plant water uptake and transport, 1-week old seedlings of both MicroTom lines, WT and N367, were transferred into aerated nutrient solutions contained in 10-L plastic boxes as previously described [13] and cultivated hydroponically until flowering in the greenhouse.

Root hydraulic conductivity (L0) was estimated by measuring root exudation rates in detopped plants [18] grown hydroponically with nutrient solutions containing different K concentrations [13]. Xylem saps exuded from roots during 1 h were collected in Eppendorf tubes with micropipettes and frozen at −20 ◦C for further analysis of K concentration.

#### *2.5. Analysis of K Concentration*

Potassium was determined in leaves used for porometry and gas exchange, as well as in remaining leaves, stems, roots and fruits, or in xylem saps from root hydraulic conductivity measurements. Plant material (leaves, stems, roots and fruits) was weighed before and after drying (48 h, 70 ◦C), and ground to powder. Potassium was extracted with distilled water by heating (90 ◦C, 1 h) and sonication, and measured by flame photometry (PFP7, Jenway, Staffordshire, UK). Potassium in xylem saps was measured with the same equipment after appropriate dilutions.

#### *2.6. Harvest*

Fruits were harvested after plants had received six watering/drying cycles starting immediately after the onset of flowering. At harvest, shoot, fruit and root fresh and dry weights and fruit number were recorded. Sugar contents in dried leaves, stems and roots were estimated after hot water extraction with the phenol–sulphuric method using a sucrose standard [19]. Soluble solids in fruits were estimated by refractometry as Brix degrees (Atago Hand held refractometer). Harvest index was calculated as the ratio among fruit weight and total plant weight. Potassium use efficiency (KUE) was calculated by dividing the fruit mass by the total plant K uptake.

#### *2.7. Free Amino Acid Contents in Leaves and Flowers*

Considering the effect of available K on plant amino acids concentration [20,21] and the protection provided by compatible solutes against drought stress [22], we measured free amino acids in leaves and flowers of water-stressed tomato under two K regimes. At full flowering stage, and after two cycles of watering/drying, fully expanded leaves and flowers from plants grown at 0.1 and 10 mM K were frozen in liquid N2 and stored at −70 ◦C. Then, they were homogenized in Eppendorf tubes with plastic pestles and centrifuged (10,000 rpm, 5 min at 4 ◦C) and free amino acids in the supernatants were separated and quantified after derivatization with phenylisotiocyanate by reversed-phase high-performance liquid chromatography [23].

#### *2.8. Statistical Analysis*

The experimental design was a complete randomized design with a factorial 3 × 2 (3 K concentrations, 2 water treatments) with 3–4 plants per treatment or a factorial 3 × 2 (3 K concentrations, 2 tomato lines) with four plants per treatment. Analysis of variance (ANOVA) was applied in order to examine significant differences between treatments (K levels, watering) among variables (plant growth parameters, gas exchange measure-

ments, etc.). Normality of the data was checked by Shapiro–Wilks test. When differences were statistically significant, a post hoc test (Least Significant Difference, LSD) was applied for comparisons. A correlation analysis (Pearson) was done between the K concentration in leaves and gas exchange or PRI values, and between plant sugar and water content. Statistical analyses were performed with a standard package (Statistica, StatSoft Inc., Tulsa, OK, USA).

#### **3. Results**

To test the combined effect of K supply and water stress on the vegetative growth and yield, wild-type MicroTom plants were grown in sand and irrigated every 3 days with nutrient solution containing 0.1, 1 or 10 mM K. When plants reached the flowering stage, plants of each group were either irrigated normally or submitted to six consecutive cycles of drying and rewatering with nutrient solution (Supplementary Figure S1). Then, plants and fruits were harvested, and growth and yield parameters were measured. Results showed that water stress and the concentration of supplied K greatly affected fruit setting and therefore fruit yield (Table 1).

**Table 1.** Effect of K supply and watering treatment on plant growth and fruit production in tomato. Control plants (irrigated) were compared to plants submitted to six cycles of drying/watering after flower initiation (drought). Means followed by the same letter are not significantly different (LSD Test, *p* < 0.05). K and W in ANOVA represent potassium and water stress treatment factors, respectively.

Water stress after flowering significantly affected fruit yield but a greater K supply could partially counteract its negative impact on yield (Table 1). Plants growing in 0.1 K and submitted to water stress failed to produce fruits, and this could be prevented partially by increasing the K availability. However, when irrigation was restricted, the inhibition on fruit set had a much greater effect than K limitation. The same was true even when including line N367 (Table 2), which paradoxically showed a greater drought-induced reduction in fruit set and yield in comparison with the control line (WT) in spite of the higher K contents in the former (Table 3). Under regular irrigation, low K significantly reduced fruit set, and therefore, fruit yield. A surplus of K improved plant performance under drought by increasing shoot mass (greater photosynthetic area) while root mass was unaffected (Table 1).

**Table 2.** Effects of K supply on fruit yield, solute contents (Brix degrees) and fruit K concentration in two lines (wild-type (WT) and N367) differing in K uptake after six cycles of limited watering (see Section 2). Means followed by the same letter are not significantly different (LSD Test, *p* < 0.05). K and L in ANOVA represent potassium and line factors, respectively.

**Table 3.** Effects of K supply on plant growth and K concentration in different organs of two tomato lines (WT and N367) at harvest after receiving six cycles of limited watering. Means followed by the same letter are not significantly different (LSD Test, *p* < 0.05). K and L in ANOVA represent potassium and line factors, respectively. FW, fresh weight.

The NHX1-overexpressing line N367 failed to improve the growth of leaves, stems or roots at harvest when compared with the control line (WT) in spite of accumulating more K than the WT (Table 3). A significant effect of K on fruit yield was found when K was increased from 0.1 to 1 mM K whereas a further increase in K supply had a marginal effect (Table 2). Surprisingly, N367 with the highest plant K contents (Table 3) showed a tendency towards producing more fruits than the WT as the K availability increased, but had smaller fruits and lower yield than the WT (Table 2). The ANOVA showed a "line" factor (WT vs N367) only significant for fruit size and yield, whereas the "K" factor was critical in all fruit characters measured (Table 2).

Increasing K supply significantly favored fruit K and soluble solid (Brix) accumulation in both lines at harvest (Table 2). While no differences in harvest index were found between lines, a significant lower K use efficiency was recorded for line N367 (Table 4).

**Table 4.** Differences in harvest index and K use efficiency between two tomato lines (WT and N367) grown with different K availabilities and subjected to six cycles of drying/watering. In columns, means followed by the same letter are not significantly different (LSD Test, *p* < 0.05). K and L in ANOVA represent potassium and line factors, respectively.

Next, we determined key physiological parameters in wild-type and N367 grown with 1 mM K in the nutrient solution until flowering (to avoid severe effects of the lowest K supply on growth) and then submitted to different regimes of K availability (0.1, 1 and 10 mM K). At flowering, both lines were submitted as in the previous experiment to six rounds of 2-week drought and one day of rewatering with the corresponding nutrient solution. In drought-treated wild-type plants, shoot and root water contents were improved by a greater supply of K (Table 5). The same trend was found for lines WT and N367, which at harvest showed greater water content in leaves (0.1 vs. 1–10 mM K) or in roots (0.1–1 vs. 10 mM K) (Table 6). However, stem water contents remained similar in spite of significant increases in their K concentration by increasing K supply (see Table 6).

**Table 5.** Effect of K supply and watering on shoot and root water content (g H2O g dry weight−1) in tomato at harvest. Control plants (irrigated) were compared to plants submitted to six cycles of drying/watering after flower initiation (drought). Means followed by the same letter are not significantly different (LSD Test, *p* < 0.05). K and W in ANOVA represent potassium and water factors, respectively.

Plants receiving less K showed clear wilting symptoms at the end of each drought cycle in comparison with plants receiving a higher K supply (Figure 1).

At harvest, sugar accumulation in leaves was recorded in drought-treated WT and N367 tomato plants at the lowest K supply (Table 6) but no association was found between sugar accumulation and water content, with the exception of roots, which showed a significant correlation between their sugar and water contents (r = 0.61, *p* < 0.001, n = 24). The K concentration provided also had a significant effect on the water content of leaves, stems and roots (Table 6).

**Table 6.** Water and sugar contents in leaves, stems and roots of tomato WT and N367 lines at harvest when grown with different K concentrations and subjected to six cyclical drought periods. Means followed by the same letter are not significantly different (LSD Test, *p* < 0.05). K and L in ANOVA represent potassium and line factors, respectively.

**Figure 1.** Plants of tomato lines WT and N367 growing with 0.1 (**left**), 1 (**center**) or 10 mM K (**right**) after withholding irrigation for two weeks.

The potassium supply also affected the ability for water acquisition by modifying hydraulic conductivity (L0) in hydroponically grown plants. Root L0 values showed significant differences among K treatments (Table 7). In low 0.1 mM K, root L0 was greater than at higher K supplies. Greater L0 values correlated with greater sap flows (Jv) but lower K contents in the xylem sap, indicating that plants absorbed more water at low-K supply to satisfy nutritional requirements. Significant differences between lines were also recorded at 0.1 mM K (Table 7). N367 plants had a higher K content in their xylem sap compared to controls when grown in sufficient K (1 and 10 mM). They also showed reduced sap flows (Jv) and hydraulic conductivity (L0) at low 0.1 mM K.

Both water stress and K availability alter the concentration of amino acids in plants [19,20], some of which, like proline, have protective effects under drought stress [21]. Hence, we measured free amino acids in the leaves and flowers of water-stressed tomato plants submitted to two cycles of watering/drying under 0.1 and 10 mM K regimes. Indeed, nitrogen metabolism in MicroTom was also affected by K nutrition and water supply, as found in the variation in the free amino acids pool in leaves and flowers. At low K, the leaves from plants suffering water stress accumulated mostly arginine and proline (Supplementary Figure S2a) whereas at higher K supply, leaf proline content was significantly increased while arginine content remained rather unaffected. In flowers, glutamine, asparagine, proline and arginine were the main amino acids stored in both irrigated

and water-stressed plants and supplying more K increased the concentration of them all (Supplementary Figure S2b).

**Table 7.** Effect of available K on sap flow (Jv), root hydraulic conductivity (L0) and xylem sap K concentration in hydroponically grown tomato lines WT and N367 at flowering stage. Means followed by the same letter are not significantly different (LSD Test, *p* < 0.05). K and L in ANOVA represent potassium and line factors, respectively.

Units: Jv, sap flow, mg·g root fresh wt−1 h−1; *L*0, root hydraulic conductivity, mg·g root fresh wt−1 h−1 MPa−1; sap K, K concentration in the xylem sap, mM.

Priming plants with stress episodes improves plant adaptability to subsequent and repetitive stresses in a process known as acclimation, which enhances fitness and yield [15,16]. Hence, we tested how repetitive episodes of drying/watering (priming) affected the photosynthesis parameters, gas exchange and water-use efficiency of MicroTom plants at varying K availabilities. After reaching the flowering stage, plants growing with 0.1, 1 and 10 mM in the nutrient solution were submitted to either one cycle or four consecutive 2-week cycles of water withholding and rewatering (see Section 2). Plants experiencing only one water stress cycle were considered "nonacclimated", whereas primed plants that experienced three additional watering/drying cycles were deemed to be "acclimated". Physiological parameters were measured at the end of the one or four cycles of drying/watering and compared to the control plants that had been irrigated normally. The irrigation of control and treated plants was with complete nutrient solutions containing different K levels (0.1, 1 and 10 mM K). As expected, low water availability significantly reduced photosynthesis in acclimated and nonacclimated plants alike (Table 8). In plants suffering water stress, leaf photosynthetic and transpiration rates were limited by stomatal closure, and the increase in K supply did not prevent the drought-induced photosynthetic inhibition. However, increasing K availability to plants led to an improvement in intrinsic water-use efficiency (WUEi) in both irrigated and water-stressed plants (Table 8). Leaf K concentration was negatively associated with transpiration, stomatal conductance and photosynthesis (r = −0.54, r = −0.51, r = −0.46, *p* < 0.001, n = 36) but it was positively correlated with water-use efficiency (r = 0.50, *p* < 0.001, n = 36). The only positive association of leaf K contents was found in the photochemical reflectance index (PRI) (r = 0.61, *p* < 0.001, n = 44) at low K supply (see Supplementary Figure S3). At higher K supplies, leaf K contents were weakly and negatively associated with PRI (r = −0.27, *p* < 0.05, n = 80). When plants were grown with unlimited water supply, no association was found between K supply and stomatal conductance or transpiration rate (r = 0.06 and r = 0.09, n = 30). Priming plants led to an apparent improvement in the efficiency of C gain per mole of stomatal water lost, i.e., a greater increase in WUEi, in comparison with the irrigated controls (Table 8).

**Table 8.** Photosynthesis (*A*), transpiration rate (*E*), stomatal conductance (*gs*) and intrinsic water-use efficiency (WUE*i*) in plants that were watered every three days (irrigated), or received one cycle of drying/watering (nonacclimated) or four cycles (acclimated). Photosynthesis and gas exchange were measured after one or the four cycles of drying/watering, in comparison with plants that were irrigated normally. In the irrigation, plants had different K regimes (0.1, 1 and 10 mM K). Within each column, means (±standard deviation) followed by the same letter are not significantly different (LSD Test, *p* < 0.05). Units: *A*, μmol CO2 m−2 s−1; *E*, mmol H2O m−2 s−1; *gs*, mmol m−2 s−1; WUE*i*, μmol CO2·mmol−1. K and W in ANOVA represent potassium and watering regime, respectively.

#### **4. Discussion**

In this work we have reassessed the contribution of K supply towards key physiological parameters in tomato under water stress with the distinctive advantage of comparing two isogenic lines differing in their ability to take up and compartmentalize K into vacuoles [13]. We found that high K in the nutrient solution improved the water contents in wild-type plants, particularly under limited watering conditions (Tables 5 and 6). A significant beneficial effect of K supplementation was also found in fruit number and yield in MicroTom plants (Tables 1 and 2). The yield of well-irrigated wild-type plants doubled with K supplement in the 1–10 mM range compared to 0.1 mM, and the increase was five-fold in water-stressed plants. However, the yield was most severely impaired by drought and this could not be overcome by K supplements since the yield of stressed plants remained five- to ten-fold lower than that of control plants at 1 and 10 mM K (Table 1). The benefit of K supplements was more intense in the transgenic line N367 under limited watering, although the yield of N367 plants approached that of the wild-type only at 10 mM, the highest concentration tested (Table 2). This differential behavior of N367 plants is likely related to the larger proportion of cellular K that is compartmentalized inside vacuoles in the transgenic line at the expense of the cytosolic pool [13], so that a higher K supplement is needed to off-set the enhanced compartmentation. In other words, greater K compartmentation into vacuoles may be detrimental when low K availability and water stress are combined.

The enhanced accumulation of K in vacuoles, achieved by the overexpression of tonoplast- and endosomal-localized NHX antiporters, has been shown to the increase the salt tolerance of tomato plants by improving K homeostasis and reducing salt-induced K

loss [13,24,25]. However, we show that the tomato line N367 with greater K accumulation failed to demonstrate a better performance under water stress compared to wild-type when plants suffered from water restrictions in spite of the consistently higher K contents in N367 plants (Tables 2 and 3). In the line N367, K sequestration in stems and roots (see Table 3) probably limited the nutrients available for assimilate redistribution to developing sinks (i.e., fruits) thereby reducing its K use efficiency (Table 4). However, the line N367 accumulated a significant amount of sugars in the leaves and stems at low K (Table 6). Sugars may accumulate either in response to water stress [26] or as a symptom of K deficiency [27,28]. The line N367 accumulates sugars under salt-stress or limited K supply [13,21], and yet it shows greater sensitivity to K deficiency in spite of similar water contents compared to wild-type MicroTom plants (Table 6). Sugar accumulation in N367, which results in lower assimilate transport to sinks, might explain the reduced fruit set and size and fruit yield (Table 2).

The limitation in leaf transpiration imposed by water scarcity led to a significant improvement in WUE at optimum K nutrition, mostly in acclimated plants, by reducing more water loss than photosynthetic carbon gain. Increasing K also significantly affected water economy by favoring stomatal closure (reducing water loss) under water stress and by reducing root hydraulic conductivity (with no water restrictions, see Table 7). However, greater K supply did not protect the photosynthetic rate under water stress as previously shown in wheat [3,4]. Only when the external K concentration was low, the leaf K contents were associated with an increase in PRI, i.e., leaf K might have a protective effect on the photosynthetic apparatus under drought [29].

High K improved carbon gain under water stress by increasing photosynthetic area, i.e., promoting shoot growth and therefore leaf expansion (Tables 1 and 3) and probably through the increased synthesis of protective osmolytes like proline (Supplementary Figure S2a). In our controlled conditions, K improved the shoot/root ratio (Table 1) and leaf WUE (Table 8) in water-stressed plants but it led to marginal increase in fruit yield (Table 2). The improvement in shoot growth and water-use efficiency by increasing K has also been found in other species (albeit by using different approaches [30–32]). The role of K in the osmotic potential and turgor generation required for expansive growth [1,33] may explain the significant increase in shoot growth of either water-deprived plants or irrigated plants when K supply was increased (Table 3).

The greater root hydraulic conductivity found at low K in nutrient solutions (Table 7) might be explained by K-deficiency sensing, which causes root ABA accumulation, increases in radial water flow and facilitates root water uptake [34–37]. Potassium deficiency also causes ethylene release and therefore ethylene-induced defective stomatal closure cannot be discarded [38,39] in the observed higher stomatal conductance and transpiration rate at low K (Table 8). As K deficiency progresses, root water conductivity diminishes by inhibition of aquaporins and K-uptake channels [12] and the accumulation of ABA in leaves may limit the effects of ethylene on stomata [40] leading to stomatal closure. On the other hand, increasing K supply may reduce root hydraulic conductance (Table 7), which might be responsible, at least partially, for the decrease in stomatal conductance (Table 8), as was found for other species [18].

The increased leaf proline contents under high K (Supplementary Figure S2a) may have provided both osmotic adjustment and radical scavenging to protect metabolic processes under water stress [22,41], leading to increased fruit setting and growth (Table 3). In flowers, the small changes in amino acid concentrations recorded either under water or K shortages (Supplementary Figure S2b) show them as preferential sinks and therefore less affected by stress [42].

A strong association between plant water content and K uptake and use is evident from the experiments shown here and in the related literature [11,12]. Undoubtedly, K contributes to maintaining plant water status through its effects on the regulation of root water conductivity and stomatal movements. It improves photosynthetic carbon assimilation under water scarcity, and thereby fruit yield, by improving shoot growth, but had no effect on photosynthetic rates. However, the yield penalty caused by water limitation cannot be overcome by increasing the K supply. By using a tomato line (N367) able to accumulate higher K concentrations, the yield was not improved but in fact reduced by limiting K use efficiency (Table 4). The potassium stored in vegetative parts improved plant water contents but was a deterrent for getting better yields under drought as it was not available for the transport of assimilates into fruits.

When water and nutrient limitation affects different crops, the relationship between K availability and yield is not a simple one and the yield responses depend on several factors inherent to each species [43] and crop nutrient use efficiency [44]. In tomato, K may affect water acquisition and economy as well as C gain, and its partitioning in a close interplay and nutrient use efficiency is of paramount importance for fruit yield. A greater K accumulation (N367) was not translated into an improvement of plant growth or fruit yield when plants suffered water stress, but indeed proved to be a disadvantage by reducing plant growth and consequently fruit setting and size.

In conclusion, K supplementation had a protective effect on the yield of tomato plants under limited watering, but this benefit was insufficient to off-set the yield loss induced by water shortage. Increasing the plant K contents through enhanced compartmentation into vacuoles failed to protect yield under drought stress, even though it could improve some physiological traits.

**Supplementary Materials:** The following is available online at https://www.mdpi.com/2311-7524/ 7/2/20/s1. Figure S1: Scheme showing the different regimes of drying/rewatering used to measure the effect of K availability on the yield and acclimation of water-stressed plants. Figure S2: Free amino acids in (a) leaves and (b) flowers from tomato plants with or without water stress when grown at low (0.1 mM K) or high K (10 mM K). Figure S3: Effect of K and watering treatments on (a) leaf K concentration and (b) photochemical reflectance index in two tomato lines (WT and N367).

**Author Contributions:** E.O.L. designed and supervised the research; A.D.L., M.C. (Mireia Corell), M.C. (Mathilde Chivet) performed experiments and analyses; all authors analyzed and discussed the data; E.O.L. and J.M.P. wrote the manuscript and obtained the funding. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by grant RTI2018-094027-B-I00 from the Spanish Agencia Estatal de Investigación (AEI), of Ministerio de Ciencia, Innovación y Universidades (MCIU), and cofinanced by the European Regional Development Fund, to E.O.L. and J.M.P.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data supporting the present results are available on request to the corresponding author.

**Acknowledgments:** The authors thank R. Fernández, A. Cifarelli and D.M. Cabrera for their contributions to different experiments while in periods of training, and Francisco J. Quintero and Anna M. Lindahl for critical reading of the manuscript. F.J. Quintero produced the scheme in Supplementary Figure S1.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Novel** *S. pennellii × S. lycopersicum* **Hybrid Rootstocks for Tomato Production with Reduced Water and Nutrient Supply**

**Jan Ellenberger 1, Aylin Bulut 1, Philip Blömeke 1 and Simone Röhlen-Schmittgen 1,2,\***

**Abstract:** Drought stress and nutrient deficiency are limiting factors in vegetable production that will have a decisive role due to the challenges of climate change in the future. The negative effects of these stressors on yield can be mitigated by crop grafting. The increasing demands for resource-use efficiency in crop production, therefore, require the development and phenotyping of more resilient rootstocks, and the selection of appropriate scions. We tested the effect of combined drought stress and nutrient deficiency on yield and fruit quality of the two tomato cultivars 'Lyterno' and 'Tastery' in the greenhouse, grafted onto different rootstock genotypes. The use of four different rootstocks, including two novel *S. pennellii* × *S. lycopersicum* hybrids and the proven-effective use of 'Beaufort', as well as self-grafted plants, allowed conclusions to be drawn about the differential stress mitigation of the rootstocks used. The stress-induced yield reduction of the scion 'Lyterno' can be mitigated more significantly by the novel hybrid rootstocks than by the commercial rootstock 'Beaufort'. At the same time, however, the individual fruit weight and the lycopene content of the fruits were significantly reduced when grafted onto the hybrid rootstocks. In contrast, the cultivar 'Tastery' showed a weak stress response, so that a generally positive influence of the rootstocks independently of the scions could not be demonstrated. We conclude that, particularly for more sensitive cultivars, the selection of more resilient rootstocks offers the potential for sustainable and resource-efficient production not competing with the overall quality of tomatoes.

**Keywords:** grafting; water-use efficiency; nutrient use efficiency; vegetable production

#### **1. Introduction**

Among abiotic stresses, drought is the most critical threat to agricultural production. It is one of the most common limiting factors influencing plant growth and development [1]. Agronomists are already using a range of management tools to cope with dry conditions in order to harvest sufficient yields at reasonable water and nutrient input [2]. At the same time, both commercial and public organizations across the world continue to develop new crop varieties to cope with the changing environment. Now, as climate change continues, both the physical environment in which agriculture is practiced and the market environment, as well as consumer demands are changing. Customers in parts of Europe are increasingly demanding not only regional products, but also those with a low energy and resource footprint [3]. Modern crop breeding has to keep up with these changing demands. As a result, breeding techniques for resilience and resource-use efficiency are investigated [4]. Different wild crops were included to stabilize yield. Nowadays many tomatoes carry the genetic information of related wild plants [5]. There are many positive known aspects from wild plants to be used. *S. pennellii*, as a tomato plant example, consists of genes of arid-adapted plant species from South America. *S. pennellii* can be crossed with tomato [6], but as its fruits are not edible, using *S. pennellii*—based accessions as a stress-tolerant rootstock seems to be more promising. Although it is possible to use gene

**Citation:** Ellenberger, J.; Bulut, A.; Blömeke, P.; Röhlen-Schmittgen, S. Novel *S. pennellii* × *S. lycopersicum* Hybrid Rootstocks for Tomato Production with Reduced Water and Nutrient Supply. *Horticulturae* **2021**, *7*, 355. https://doi.org/10.3390/ horticulturae7100355

Academic Editor: Antonio Ferrante

Received: 27 August 2021 Accepted: 17 September 2021 Published: 2 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

editing to create de novo plants with beneficial root systems and edible fruits at the same time [7], the lack of public acceptance will likely prevent the commercial use of such plants in Europe in the coming years and perhaps decades [8,9]. Nevertheless, the grafting of horticultural crops remains a method to improve plant growth and yield production [10,11]. Indeed, grafting can be helpful in combining two advantageous characteristics (rootstock and scion) in a single plant. For example, the grafting method is used and researched in vegetable crops to increase yield, fruit quality, and nutrient uptake [12], and also to counteract pathogens and plant diseases [13,14].

For the tomato *(Solanum lycopersicum*), grafting is also seen as a promising technique to reduce water requirement [15]. The root system of grafted plants is reported to be stronger and more efficient in absorbing water and nutrients [16], which may indirectly improve yield and fruit quality [17]. As tomato production in Germany alone reached 108,000 tons in 2019 and tends to increase worldwide [18], the water and nutrient supply must be optimized and become more sustainable. In this regard, it is useful to resort to certain rootstock cultivars that are more stress-tolerant and to improve fruit quality. Grafting was shown to increase drought-stress tolerance in tomatoes, and drought-stress tolerance varies with the rootstock used [19]. The rootstock cultivar 'Beaufort' was proven to mitigate salt stress to some extent [20], and the water- and nitrogen-use efficiency of fieldgrown tomato 'Florida 47' was increased when grafted onto the rootstock 'Beaufort' [21]. Moreover, the uptake of macronutrients such as nitrogen, phosphorus, and calcium was significantly improved by grafting on 'Beaufort' [22–24]. Although 'Beaufort' is proving to be an efficient rootstock, newly developed cultivars are being hybridized as rootstock that may eventually exceed this performance. For example, novel rootstock cultivars have been obtained by crossing with the wild tomato species *Solanum pennellii* accession LA716, which is considered to be more tolerant in stress conditions than related tomato species *S. lycopersicum* [25].

In this study, we compare the yield and water- and nutrient-use efficiency of two commercial tomato lines grafted onto two novel interspecific hybrid rootstocks and the commercial rootstock 'Beaufort' in two fertigation regimes. Given the drought tolerance of *S. pennellii* described in the literature, we hypothesize that *S. pennellii* × *S. lycopersicum* hybrid rootstocks can limit the yield losses of commercial tomatoes under drought to a minimum. In order to check possible influences on fruit quality, the sugar content of the fruit (◦Brix), as well as flavonol and lycopene contents are also analyzed.

#### **2. Materials and Methods**

#### *2.1. Plant Material and Treatment*

The research was conducted in a greenhouse located in Meckenheim, Germany (latitude: 50◦37 27.7 N, longitude: 6◦59 20.1 E) in 2020. The two tomato (*S. lycopersicum*) F1 hybrids 'Lyterno RZ F1' (truss tomato) and the 'Tastery RZ F1' (cherry tomato) were self-grafted and grafted onto rootstock cultivars 'HUJ1', 'HUJ2' and 'Beaufort' (De-Ruiter, Bergshenhoek, NL), respectively. 'HUJ1' and 'HUJ2' are interspecific hybrids of an *S. lycopersicum* variety and the stress-tolerant wild tomato species *S. pennellii*, accession LA716 (described here [26]). In March 2020, one week prior to the scions, rootstock seeds were sown in rockwool substrate blocks (Grodan B.V.). After three weeks, right before grafting, rootstock seedlings were transferred to new rockwool blocks of size 8 cm × 8 cm × 10 cm. The grafting position was below the cotyledons of the rootstock seedlings and sealed with an appropriate clip.

Directly following this process grafted plants were put in a healing chamber for 72 h and covered with plastic bags to reduce light intensity and increase humidity to enhance the survival rate and then gradually accustomed to lower humidity levels. Thereafter, on May 10th, two plants of the same rootstock−scion combination each were planted in pots filled with *Miscanthus* × *giganteus* substrate. Twelve plants from each of the eight rootstock−scion combinations were prepared for the control and treated groups, respectively. The pots were placed in the greenhouse with an average temperature of

21.3 ◦C (±3.9 ◦C) and relative humidity of 77.9% (±16.2%). Fertigation was automatically supplied via drip irrigation. Until the third week in the greenhouse, the plants received a full supply of water and nutrients based on Groher et al. [27], to ensure the consistent growth of all plants. Subsequently, control plants received about 4 L of fertigation per day (589 L in 141 days), keeping the substrate at around 80% humidity by using a soil moisture sensor (SM150 kit, Delta T Devices Ltd., Cambridge, UK), while the treated group received about 2 L of fertigation per day (282 L in 141 days) (Figure 1). The reduced fertigation dose of technically 47.8% for treated plants is referred to as '50% reduction' from here on.

**Figure 1.** Daily amounts of fertigation for control (**A**) and drought stress (**B**). Occasional deviations from the desired 50% difference in fertigation between treatments occurred due to maintenance work.

#### *2.2. Determination of Total Yield per Plant, Single Fruit Weight and Water-Use Efficiency*

From the 20th week after sowing, ripe tomatoes were harvested once a week to determine the yield per plant and fruit quality in the greenhouse. Weekly harvesting was carried out until the 27th week. Harvested fruits per plant were categorized as marketable and nonmarketable fruits, based on visual assessment. The fruit number and weight of the harvested fruits of each plant were determined with a technical balance. Water-use efficiency (WUE) was calculated as the quotient of total yield per plant and fertigation per plant throughout greenhouse cultivation.

#### *2.3. Determination of Total Soluble Solids*

Total soluble solids (TTS) determination was performed on two ripe, marketable 'Lyterno' fruits (truss tomato) and five ripe, marketable 'Tastery' fruits (cocktail tomato), in order to achieve comparable amount of bulk material. Fully ripe fruits were selected from at least two and up to six plants of all rootstock−scion combinations of the control

and treatment group. A blender was used to mash the tomatoes to a homogeneous mass. The type PAL−1 refractometer (ATAGO CO., LTD., Tokyo, Japan) was used to determine the TSS in ◦Brix [%] of the tomato puree.

#### *2.4. Determination of Flavonoid and Lycopene Content*

For posterior analysis of lycopene and β-carotene, we used a protocol that had already been tested before [28]. In short, tomato puree was frozen, freeze-dried and ground for 1 min using a ball mill (MM200, Retsch GmbH, Haan, Germany). Freeze-dried and ground material (0.1 g) was homogenized and extracted with a 2 mL acetone–hexane mixture (4:6). The absorbance of the extracts was measured at 453 nm, 505 nm, 645 nm and 663 nm with a UV–visible spectrophotometer (Lambda 35 UV/VIS, spectrophotometer, Perkin Elmer, Waltham, MA, USA). Concentrations of pigments were calculated according to the following equations:

*lycopene*[mg/100mL] = 0.0458*A*663 + 0.204*A*645 + 0.372*A*505 − 0.0806*A*453

$$\beta-\acute{a}rotene[\text{mg/100mL}] = 0.216\_{A663} - 0.304\_{A505} + 0.452\_{A453}$$

The total flavonoid content was determined according to the slightly modified methodology described by Groher et al. [27]. Briefly, a methanolic extract (80% methanol plus 1.0% hydrochloric acid) was prepared from 0.05 g freeze-dried sample material. From an aliquot of the methanolic extract, 1 mL was mixed with 0.1 mL of 0.72 mol L−1 sodium nitrite solution, 0.1 mL of 0.75 mol L−1 aluminum chloride solution, and 0.1 mL of 1 mol L−1 sodium hydroxide at defined time intervals. After vortexing for 10 s, 1.7 mL water was added to adjust the sample to a final volume of 3.0 mL After 30 min of incubation, absorbance was measured at 510 nm using a UV–visible spectrophotometer (Lambda 35 UV/VIS, spectrophotometer, Perkin Elmer, USA). Quercetin was used as the standard for a linear curve between 0.1 and 1.0 mg mL−1, and the results were expressed as milligrams of quercetin equivalents in dry mass. Analyses were performed in triplicate, and the following equation was used to calculate flavonoid content:

$$Queretimequivalents = 4.5709\_{A510} - 0.06566$$

#### *2.5. Data Analysis*

As a preliminary step, the raw data was aggregated by the single fruit yield and total yield per plant and harvest date. For further analysis, only marketable fruits were taken into account. From those, the single fruit weight and total yield per plant were calculated. Data analysis and visualization was performed in R [29], figures were created using the R-package ggplot2 [30]. Tukey's HSD post hoc tests were used to identify differences in rootstock performance.

#### **3. Results**

As a result of the differences in fruit shape, Lyterno yields are generally higher than Tastery yields. Mean Lyterno yield per plant in optimal conditions in the seven weeks experimental period was 4906 g (±937 g), with no significant influence of the rootstock used (Figure 2A). For Tastery, the mean yield per plant was 2603 g (±250 g) (Figure 2C). When fertigation was reduced, Lyterno yields dropped by around 39% on average to 2961 g (±899 g) (Figure 2B), while the mean reduction in Tastery yields (24%) was a rather moderate 1978 g (±388 g) (Figure 2D). WUE is calculated as the quotient of yield per plant and fertigation per plant. The latter was held constant across rootstock−scion combinations within treated and control plants. The generally observed relative increase in WUE of stressed plants was higher in Tastery than in Lyterno (Tastery: 4.42 to 7.01 g/L; Lyterno: 8.33 to 10.50 g/L; Figure 2ii).

**Figure 2.** Average yield ((**i**), top) and water-use efficiency ((**ii**), bottom) per plant (n = 12) of scions from cultivar 'Lyterno' (**A**,**B**,**E**,**F**) and 'Tastery' (**C**,**D**,**G**,**H**) grafted onto different rootstocks and supplied with optimal (**A**,**C**,**E**,**G**) and reduced (**B**,**D**,**F**,**H**) fertigation. n.s.: not significant, *p*-value 0.05.

The large standard deviations in the Lyterno yields under reduced fertigation can partly be explained through the rootstock effect: the results of an analysis of variance and subsequent Tukey's HSD tests are presented in Table S1. Plants grafted onto Beaufort yields were significantly lower than those grafted onto HUJ 1 and HUJ 2. Self-grafted controls tended to yield lower than HUJ 1 and HUJ 2, but differences were not significant.

The results of fruit quality analyses of scion 'Lyterno' are presented in Figures 3 and 4. As no change in yield of scion 'Tastery' related to the different rootstock was found (Figure 2C,D), our further analyses focus on Lyterno. Tastery fruit quality parameters are presented in supplementary Figures S1 and S2.

**Figure 3.** Quality parameters of 'Lyterno' fruits grafted onto four different rootstocks and supplied with optimal (**A**,**C**,**E**) and reduced (**B**,**D**,**F**) fertigation: Individual fruit weight (g) (n = 49–84), (**A**,**B**); total soluble solids ('sugar') content of ripe fruits (◦Brix) (fruits of n = 2–6 plants), (**C**,**D**); fruit dry matter content [%]; (n = 2–6) (**E**,**F**). n.s.: not significant, *p*-value 0.05.

The individual weight of the Lyterno fruits drops by 11.2% from 83.1 g (±15.0 g) to 73.8 g (±13.1 g) when fertigation is reduced. Individual fruit weight of control plants grafted onto Beaufort is significantly higher than grafted onto both HUJ 1 and HUJ 2 (*p* < 0.01) (Figure 3A). In the reduced fertigation regime, self-grafted plants produce on average heavier fruits than scions grafted onto HUJ 1 and HUJ 2 (Figure 3B).

The pattern changes when looking at fruit sugar content. Sugar contents [◦Brix] are generally slightly increased when fertigation is reduced: From 5.05 ◦Brix (±0.29 ◦Brix) to 5.32 ◦Brix (±0.43 ◦Brix). Probably as a result of few marketable fruits from stressed plants grafted onto different rootstocks being available for laboratory analyses (n = 2–6), no significant differences in sugar content can be attributed to the different rootstocks. However, a tendency to higher sugar contents in stressed fruits grafted onto HUJ 1 and HUJ 2 is depicted in Figure 3D.

Under the influence of drought stress, the average dry matter content of Lyterno fruits increased from 12.66% (±0.51%) to 13.94% (±1.23%) (Figure 3E,F). While there were no rootstock dependent differences under optimal fertigation application, the dry matter content of self-grafted plants was higher under reduced fertigation application.

Only minor differences in the β-carotene, lycopene and flavonoid contents of the 'Lyterno' fruits grafted onto different rootstocks and supplied with optimal or reduced fertigation were detected (Figure 4).

As contents are presented in milligram per gram dry weight (Figure 4), and the dry matter of stressed fruits was on average higher than for control fruits (Figure 3E,F), the contents of β-carotene, lycopene and flavonoids per fresh matter were higher for stressed than for control fruits (not shown).

A comparison of the lycopene and β-carotene contents of the fruits indicates a higher responsiveness of β-carotene than lycopene to the applied changes in fertigation.

#### **4. Discussion**

A major objective of the present study was to exploit the potential of new genetic material for tomato grafting to target the necessary improvements for horticulture in the scope of climate-related challenges in future. We, therefore, verified to what extent novel hybrid rootstocks (*S. pennellii* × *S. lycopersicum*) could minimize tomato yield loss under limited irrigation conditions, as scarcity of water for irrigation will increase in the future [31]. Several studies have shown that remedial action can be provided by combining resilient rootstock and high yielding scions, while the physiological exchange of these plant parts significantly influences overall performance and resilience to environmental stressors. In addition, grafting can improve vegetable quality by enhancing biosynthesis of endogenous phytohormones, and by acquiring and transporting mineral nutrients [32,33]. Fruit flavor and nutritional value can be furthermore positively influenced by altered secondary metabolites, and by the concentration of health-promoting compounds, such as lycopene, carotenoids and amino acids [33,34]. Congruently, tomatoes grafted onto efficient rootstocks are also both nutritionally promising and at the same time more tolerant to abiotic and biotic stressors [35], which, besides the sustainable use of resources, in turn offers additional advantages in the case of more frequent and severe incidence of climate-related diseases.

#### *4.1. Impact of Different Rootstocks on Scion Performance*

Based on plant performance in yield and water-use efficiency, given the data presented (Figure 2i), we can conclude that the two novel rootstocks tested were indeed limiting yield losses of the truss-tomato cultivar Lyterno under drought stress conditions, as compared to the commercial rootstock 'Beaufort'. At the same time, underlining the potential of the new rootstock cultivars in optimal conditions, the yields of scions grafted onto the novel rootstocks were not different from those grafted onto 'Beaufort'.

While for the cocktail tomato Tastery, yields did not change depending on the rootstock used, either in optimal or drought conditions, where 5–10% losses were recorded.

The identical fertigation for plants with both scions apparently resulted in Lyterno being more stressed than Tastery, as can be seen from the greater yield reduction (Figure 2) and the more pronounced differences in fruit quality (Figure 3) for Lyterno.

This is in line with previous studies showing high-yielding performance under optimal conditions when grafted onto 'Beaufort' being advantageous for several scion cultivars [23,36]. Here, 'Beaufort' was ascertained as positive in greater stem height and diameter, as well as epigeous dry biomass [23]. Under stress conditions, for example, saline and drought treatment, 'Beaufort' was shown to be more tolerant than other genotypes, such as 'Body', 'Heman', 'Resistar', 'Spirit', 'Vigomax' and 'Vedi' [20], and 'Maxifort', 'Unifort', 'Vedi', 'Kemerit', 'King' 'Kong', 'Spirit', 'Resistar', '500292' and 'Toro' [19], respectively.

With a similarly promising performance, the novel rootstocks 'HUJ1' and 'HUJ2' that are based on the *S. pennellii* introgression lines (ILs), are considered to maintain stable growth when exposed to salt stress [25]. When stressed, the ILs performed significantly better at maintaining plant growth than the salt-tolerant wild species, as well as parental line LA716.

Nevertheless, for commercially relevant scions, we focused on two production varieties commonly used in central Europe and North America, namely the cocktail tomato 'Tastery' and the truss-tomato 'Lyterno' [37].

At this stage, it remains an open question whether Tastery is generally less responsive to different rootstocks, or whether the lack of rootstock-related differences is due to the relatively mild stress. The fact that yield and fruit weights were lower overall under drought stress was to be expected and was in line with other studies [38,39]. A surprising result was the consistently good performance of the self-grafted plants in our experiment, contradictory to other studies of different cultivars and scion−rootstock combinations [40,41]. The comparably good performance of our 'Tastery−Tastery' plants might be referred to the internal physiological communication between rootstock and scion, being identical for self-grafted plants. This essentially coordinated exchange of endogenous phytohormones, mineral elements, mRNA, and proteins [42], as well as relevant transporters (either influx or efflux carriers) might affect a smoother long-distance signaling of phytohormones through the plant vascular system [43] of genetically identical plants compared to different scion and rootstock genotypes.

#### *4.2. Effects of Rootstocks on Water-Use Efficiency during Cultivation Cycle*

In the scope of future challenges of water shortage, water-use efficiency (WUE) is a relevant option to determine plant performance. Reduced irrigation either by an improved WUE of (grafted) tomato genotypes [44] or by effective irrigation management during fruit developmental stages ensures sufficient yield and fruit quality [38].

However, under artificial conditions, the data on yield and WUE should be viewed with caution. Indeed, in order to assess validated data, the irrigation demands of every single plant would need to be considered. On the other hand, such an approach is not yet feasible for commercial-like conditions. Highly sophisticated greenhouse systems based on artificial intelligence might improve the current situation in the near future [45]. Nevertheless, in our experimental case, water demand and, thus, WUE would need to be explored under two separate irrigation regimes to adapt to the cultivar-specific needs of Tastery and Lyterno and/or rootstocks in more depth.

Furthermore, we focused on a rather short time period of productive cultivation cycles. The overall yield performance (Figure 2i) may therefore be used as an indicator for differences in yield among rootstocks, but the absolute values cannot be compared to commercial yields. While production cycles in commercial cultivation in Central European greenhouses last up to 10 months, our calculation of WUE only includes yields from a seven-week period. Moreover, we only started harvesting once all plants produced stable yields, rather than already picking the first tomatoes in the season. By the same token, WUE data presented in Figure 2ii may be used as an indicator for different WUE among rootstocks, but cannot be compared to WUE in commercial production: On the one hand, yield is not comparable to commercial production for the reasons above and on the other hand, water-use data is also different from commercial production, as, e.g., water consumption during plant germination, between grafting and planting out, and finally the water used to flood the *Miscanthus* substrate after transplanting to ensure plant stand establishment, are not included in our calculations in Figure 2ii. Since these factors were the same for all our experimental plants and rootstock−scion combinations, they can be neglected, but need to be considered when calculating the overall water use and thus, WUE of genotypes.

#### *4.3. Rootstock Influence on Fruit Quality Parameters*

Fruit quality, such as its nutritional value, taste and odor, is of great importance for consumers and therefore for producers [46]. Grafting has a significant effect on quality parameters improving health-promoting properties and flavor by the altered composition of secondary metabolites [33,34].

Looking at the characteristics of the fruits of plants grafted onto different rootstocks, some interesting results emerged. The two *S. pennellii*-based rootstocks seemed to favor the production of smaller fruits. Lyterno fruits from plants on these rootstocks were almost universally significantly lighter than the fruits on Beaufort or self-grafted Lyterno (Figure 3A,B). No significant differences were seen for Tastery, but again there is a slight trend toward smaller fruits on *S. pennellii*-based rootstocks (Figure S1A,B).

The fact that *S. pennellii* itself produces very small fruits with a fruit weight < 16 g [47] could be an indication that this trait has been transferred from the rootstock to the scion.

In contrast, we observed a fruit size-enhancing effect of 'Beaufort', resulting in the comparably high individual fruit weight of Lyterno, as was previously described for this type of vigorous interspecific rootstocks [10,11,36,48]. This effect on fruit size is significantly influenced by the grafting combination [41], and needs to be distinguished from yield performance that can also be attributed to an enhanced number of tomato fruits rather than an increased average fruit weight [49].

It is known that rootstock mediation affects fruit quality, due to altered water and nutrient uptake, and changes in the source−sink balance impacting ripening behavior [10,16]. Therefore, it can be concluded that some rootstocks favor early fruit set, as well as rapid fruit ripening [50]. In particular, the possible faster fruit ripening could have resulted in the

lower fruit weight of the Lyterno grafted onto 'HUJ1' and 'HUJ2'. Nevertheless, individual fruit weight was further reduced under 50% fertigation, implying that drought could also have accelerated tomato ripening [27].

Both overall increased dry matter and sugar content, and reduced fruit weight are a logical result of drought stress: with optimal water availability in the root zone, the high sugar concentration in the fruit ensures that water is transported from the root zone into the fruit by osmotic effects. When there is a lack of water, this 'sucking in' of water is not possible, and there is an increased content of soluble substances with a simultaneously reduced fruit weight [51].

The opposite trends observed in Lyterno for yield and fruit weight of *S. pennellii*-based rootstocks are noteworthy: total yield was relatively high when these rootstocks were used under drought stress (Figure 2B), but at the same time individual fruit weight was relatively low (Figure 3B and Figure S1). This can be explained by the fact that more but smaller fruits reached maturity [49].

These fruits, then, also tend to be sweeter (Figure 3D), which can be seen as another potential advantage provided by the novel rootstocks. Concerning taste and quality, this might be an approach to target current consumer needs.

Surprisingly, in regard to secondary metabolites in Lyterno fruits (Figure 4), we only found a significantly reduced lycopene content for the rootstock 'HUJ2' under optimal fertigation conditions. Here, 'Beaufort' showed the overall highest contents of β-carotene, lycopene and flavonoids. This is in contrast to previous findings, where 'Beaufort' resulted in a decrease of lycopene concentration of the scions 'Jeremy' and 'Jack' [52,53]. This might be attributed to the specific interaction of rootstock and scion [10,11]. However, under 50% fertigation, slightly reduced but comparably high metabolite contents were analyzed for Lyterno, when individual fruit weight and yield decreased. This is in agreement with previous field trials, where soluble solids, lycopene, and sugar content were increased in the tomato fruit, while fruit yield decreased between 1.9%–18.2% [54]. A similar increase in tomato fruit quality parameters was shown for a pot experiment with a two-thirds yield decrease under irrigation with 0.3% salt concentration in soil [55]. Based on our experiment, it could be argued that the reduction in fertigation also resulted in a reduced volume of 'Lyterno' tomatoes and thus both sugar content and metabolite contents increased in the fruit.

#### *4.4. Future Perspective of Renewable Substrate for Hydroponics*

Although there were no distinct patterns in the individual grafting combinations with lower fertigation dosages in our experiment, this project is still a sustainable approach for food production targeting resource-efficient tomato production by adjusting both plant performance (via grafting) and crop management. Eventually, a sustainable and more environmentally friendly substrate of *Miscanthus* × *giganteus* was used. In the study of Kraska et al. [56], it was shown that *Miscanthus* as shreds, chips and fibers does not differ much in comparison to rockwool as a substrate for the cumulative fruit yield (g/plant), which highlights in our experiment the fact, that the use of *Miscanthus* × *giganteus* should not negatively impact the yield measurement. Moreover, the yields and fruit quality of cucumber and tomatoes grown on *Miscanthus* as a potential alternative to rockwool as a growing medium for vegetables [57,58] and is overall seen as a promising alternative to rockwool as a growing medium for tomatoes in soilless cultivation [56].

To determine the optimal water and nutrient requirements in grafted 'Lyterno' and 'Tastery' tomato plants, further studies will need to be conducted in order to gain a more detailed insight into the interaction between rootstock and scions, as well as the potential to identify the genotype combinations being more tolerant towards drought stress and nutrient limitations. Ultimately, besides sustainability, it also makes financial sense to graft plants and thereby apply less water or nutrients for the same yield, than not grafting and thereby use up many limited resources, without losing fruit quality.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/horticulturae7100355/s1, Table S1: Tukey comparison of mean differences for plants with the scion Lyterno under stress conditions. Figure S1: Quality parameters of "Tastery" fruits grafted on four different rootstocks and supplied with optimal and reduced fertigation. Figure S2: Quality parameters of "Tastery" fruits grafted on four different rootstocks and supplied with optimal and reduced fertigation. Figure S3: Comparison of lycopene and β-carotene contents of ripe tomatoes from 8 rootstock \* scion – combinations supplied with optimal and reduced fertigation.

**Author Contributions:** Conceptualization, J.E., S.R.-S.; data analysis, J.E., A.B., P.B.; methodology, J.E.; writing—original draft preparation, J.E., A.B., P.B.; writing—review and editing, J.E., S.R.-S., A.B. and P.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** J.E. received funding from the European Union's Horizon 2020 research and innovation program under the Grant Agreement No. (727929) (TOMRES).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Raw data is available on request from the corresponding author.

**Acknowledgments:** The authors thank Dani Zamir, Hebrew University of Jerusalem, for providing *S. pennellii* × *S. lycopersicum* seeds. We also thank the technical staff of the Institute for Crop Science and Resource Conservation of Bonn University for their cooperation.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Opportunities of Reduced Nitrogen Supply for Productivity, Taste, Valuable Compounds and Storage Life of Cocktail Tomato**

**Lilian Schmidt 1,2,\* and Jana Zinkernagel 2**

**Abstract:** Vegetable production requires high nutrient input for ensuring high quality and high yield. As this is ecologically disadvantageous, it is necessary to determine if nitrogen (N) fertilization can be reduced without negative effects on productivity. For quality reasons, the effects of reduced N supply on taste, valuable compounds and storage life must be elucidated in parallel. This study examines whether reducing the N supply of cocktail tomatoes by 50% to recommendations affects the yield and quality of tomato fruits. Three varieties with different skin colors, yellow-orange ('Apresa'), red ('Delioso') and brown ('Bombonera'), were grown in soil in a greenhouse and harvested at the red-ripen stage. Quality parameters were assessed at harvest and after eight-day storage. Total yield decreased exclusively with 'Bombonera' due to reduced fruit weight. Firmness of the fruit pulp, concentrations of minerals, soluble solid contents, total acidity, total phenolics and liposoluble pigments of fruits were not influenced. However, storage affected chemical compositions positively, as shown by increased antioxidants. Descriptive sensory analyses revealed no impact of reduced N supply. From the perspective of the yield, quality and shelf life of fruits, reducing the N supply by 50% offers opportunities for the three cocktail tomato varieties in soil cultivation.

**Keywords:** tomato; product quality; nitrogen; shelf life; carotenoids; antioxidants; taste

#### **1. Introduction**

Nitrogen (N) is the most important mineral element for plants and, as a constituent of amino acids and proteins, often limiting for their growth processes [1]. In the context of plant production, N influences the appearance and internal quality of plant products, their yield and their shelf life. The supply of N is therefore decisive for the quality of food crops; the application of fertilizer in crop cultivation is essential for profitable plant production. In particular, the intensive cultivation of vegetable crops involves high amounts of N fertilizer (as well as other nutrients) and often results in an excessive N supply [2]. This implies that vegetable production often does not meet the requirements of environmental legislations for good ecological water status (e.g., the EU Nitrates Directive or the EU Water Framework Directive). Vegetable production is, like no other, faced with the tradeoff between ecological and economic demands. This adds to the importance for human health and global diet. Increasing the efficiency of nutrient supplies, especially nitrogen, is therefore essential for sustainable food production.

There is no general optimum for the nitrogen supply of vegetable crops; it depends on, among others, the production target (e.g., plant organ, quality). Consequently, the potential for a reduction in the N supply also varies among species and production systems. Generally, low N availability is known to decrease photosynthetic activity and thus plant growth [1]. Besides this, there are impacts on the secondary metabolism which can result

**Citation:** Schmidt, L.; Zinkernagel, J. Opportunities of Reduced Nitrogen Supply for Productivity, Taste, Valuable Compounds and Storage Life of Cocktail Tomato. *Horticulturae* **2021**, *7*, 48. https://doi.org/10.3390/ horticulturae7030048

Academic Editors: Wilfried Rozhon and Antonio Ferrante

Received: 29 January 2021 Accepted: 8 March 2021 Published: 11 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

in decreased synthesis of N-based compounds such as alkaloids and increased synthesis of C-based secondary metabolites [3]. A limited N supply may consequently increase crops' quality, especially in terms of phytonutrients that are potentially beneficial for human health. In fact, a low N supply of plants is one possible way to achieve enrichment of secondary metabolites, as shown for several vegetable crops such as lettuce [4], and for medicinal plants such as chamomile [5]. The enrichment of secondary metabolites while maintaining high yield and quality may be particularly important for crops that are consumed in large quantities. Tomato offers great potential in this regard, as it is the most widely grown and consumed vegetable in the world [6]. A serving of tomatoes (defined as around 200 g of fresh tomatoes) covers the recommended dietary allowances of several mineral elements and, to a larger extent, those of vitamin C [7]. Due to the high consumption and concentration of many valuable compounds such as carotenes and polyphenols, tomato fruits are important sources of further secondary plant metabolites and can improve the intake of antioxidants in the human diet [8]. Carotenes such as lycopene and β-carotene are strong antioxidants and may reduce the risks for some cancers and coronary heart diseases, as well as those for diabetes and osteoporosis [9–11]. Polyphenols (such as flavonoids) are known to have antioxidant effects as well. Several studies suggest that polyphenols may protect against coronary heart diseases, counteract cancer development and prevent neurodegenerative diseases [12]. Additionally, tomato fruits are relevant sources of other antioxidants, such as ascorbic acid and vitamin E. An increase in the concentration of these secondary compounds in tomato fruits is therefore favorable for human health.

The internal quality and the taste of tomato fruits can be improved by agronomic strategies such as moderate salinity stress or modulated mineral nutrition [13]. When subjected to repeated short-term nitrogen limitation, tomato leaves contain increased flavonol glycoside concentrations [14]. Evidence for the transferability from tomato leaves to the fruits is still pending. Reduced N supply is known to cause an increase in soluble sugars and a decrease in acid concentrations of tomato fruits [15]. This may improve tomatoes' taste as sugars and acids are the major constituents of the taste experience [16]. Moreover, the N supply may alter other flavor components such as the volatile organic compounds of tomato fruits [17].

In tomato seedlings grown under N deficiency, shikimate and soluble sugars increased in the xylem sap, which suggests that the biosynthesis of phenolic substances might be favored [18]. In fact, increased concentrations of phenolic compounds in fruits resulted from reduced N supply at transplant or anthesis of greenhouse-grown tomatoes, albeit there were little effects on the carotenoid concentration [19].

However, the positive effects of reduced N supply on the secondary metabolism have not been found reliably. Bénard et al. [15] showed little effects of reduced N supply on secondary metabolites such as phenolic compounds, ascorbic acid and carotenoids in tomato fruits. Studies with two lycopene-rich tomato varieties revealed no impacts of reduced N supply on yield, firmness, valuable compounds and sensory properties of the fruits [20]. In contrast, Wang et al. [17] observed close relationships between N application rates and flavor parameters such as titratable acidity, soluble sugars, soluble solids, volatile compounds and fruit firmness in cherry tomatoes. Nevertheless, studies on quality and sensory properties of tomatoes grown with reduced N supply are rare, especially with the focus on shelf life.

This study aims at closing this gap in the knowledge. In a one-year trial, it was tested whether a reduction in the N supply by 50% of the recommended amount [21] impacts the yield and quality of tomato fruits. Fertilizers' reduction was performed at the beginning of fruit ripening to minimize the impact on fruit yield while maximizing the impact on fruit quality. Three varieties of cocktail tomato, differing in skin color, were chosen for the experiments since small-fruited and highly aromatic tomatoes are increasingly demanded by consumers. Quality parameters of the tomatoes were assessed after harvest and after eight-day storage.

We hypothesized that the reduced N supply (i) has no impact on the yield, firmness and color of tomato fruits, (ii) results in better taste due to the altered sugar-to-acid ratio of the fruits and (iii) enhances the nutritional quality of the fruits by increasing their antioxidant content.

#### **2. Materials and Methods**

#### *2.1. Experimental Design and Plant Material*

Three one-factorial experiments with the factor "N supply" and two factor levels, control (con) and reduced (Nred) N supplies, were performed for three cocktail tomato varieties separately in a greenhouse in Geisenheim, Germany. The experiments were set up in a randomized block design with four repetitions. Each repetition (plot) included ten plants. Subsequently, a storage experiment was carried out with the harvested fruits. Quality parameters were measured immediately after harvest and after storage. Hypotheses were tested with two-factorial analysis with the factors "N supply" and "storage".

For the greenhouse experiment, seeds of the tomato varieties 'Delioso' (red color; Rijk Zwaan Welver GmbH, Welver, Germany), 'Apresa' (orange color; Hild Samen GmbH, Marbach am Neckar, Germany) and 'Bombonera' (green-brownish color; Uniseeds Select B.V., Bleiswijk, The Netherlands) were sown into the substrate (Floradur A; BayWa AG, München, Germany) at the beginning of April 2012. The seedlings were picked and transferred into Ø 10 cm pots containing "Einheitserde ED Koko 1+1" (Einheitserdewerke Werkverband e.V., Sinntal-Altengronau, Germany) 14 d later. The transplants were watered and fertilized (Ferty 2 Mega, Planta Düngemittel GmbH, Regenstauf, Germany) as required. At the beginning of May 2012, the transplants (30 days old, BBCH stage 103) were planted into the soil (sandy loam) in the greenhouse with 2.8 plants m−2. Plants of each tomato variety were planted onto eight randomly arranged plots (9.6 m2 each with 10 plants per plot).

#### *2.2. Cultivation and N Supply*

The "con" treatment received weekly N supplies of 3 g m−2, according to the recommendations for fertigated tomatoes [21]. For the "Nred" treatment, the weekly N supply was reduced by 50% at the onset of fruit ripening, yielding 1.5gNm−2 per week.

Until the differentiated N application, the fertilizer "Ferty 2 Mega" was applied (1 g N m−2 per week; Hauert HBG Dünger AG, Grossaffoltern, Switzerland). Starting mid-May 2012, the N dose was increased to2gm−2 per week until early June 2012. At the breaker stage, when the fruits started to change color (early June 2012), the N control plots received3gNm−2 per week (composed of Ferty Basis 1, calcium nitrate and ammonium sulphate; Planta Düngemittel GmbH). The reduced N treatments received 50% less N, yielding 1.5 g N m−2 per week (composed of Ferty Basis 1 and calcium nitrate; Beiselen GmbH, Ulm, Germany). In total, plants received 360 (con) and 210 (Nred) kg N ha−1 throughout the growing season. Albeit this represents a reduction in total N application of approximately 42%, this is further regarded as a 50% reduction.

The fertilizer was applied with the irrigation water as fertigation. Each plant was supplied by drip irrigation (two drippers per plant, with 0.85 L h−1), if soil tension fell below −9 kPa (measured at 20 cm soil depth by tensiometers). The application of water and fertilizer was controlled by the KLIWADU® software (developed by Michael Beck, Hochschule Weihenstephan-Triesdorf, Germany).

Pollination of the flowers was ensured by regular placement of bumblebees (re-natur GmbH, Stolpe, Germany) in the greenhouse. Plant protection was carried out by application of beneficial insects (*Amblyseius cucumeris* and *Encarsia formosa*, all from re-natur GmbH) when required.

The radiation inside the glass-covered greenhouse reached 724.7 kW m−2 during the cultivation period. The temperature regime throughout the cultivation season in the greenhouse is listed in Table 1.

**Table 1.** Temperature regime throughout the cultivation season.

#### *2.3. Harvest*

Mature tomato fruits were harvested at the fruit ripening stage "eating ripeness", which is chemometrically described as "red ripe" for red cultivars [22]. The different colored varieties were harvested analog to this. The harvest was carried out twice a week lasting from the end of June 2012 (week 26, corresponding to BBCH stage 801) to early September 2012 (week 36). The tomato fruits from each experimental plot were sorted into marketable and non-marketable fractions (broken, blossom end rot, green shoulder, soft, fissured, unfertilized, others). Fresh matter of tomato fruits was determined per plant and plot.

#### *2.4. Storage*

At least 12 marketable, similarly sized fruits from each experimental treatment were kept in a cold storage at 12 ◦C in the dark (CS 0300 T, Viessmann Kühlsysteme GmbH, Hof/Saale, Germany). The fruits were stored in plastic boxes in single layers and covered with plastic film to prevent water loss. After eight days, the fruits were removed from the cold storage for further analysis and sensory evaluations. During the course of the harvesting season, the storage experiment was repeated five times.

#### *2.5. Spectroscopic Measurements*

Non-invasive spectroscopic measurements were performed with the Multiplex® 3.6 device (Force-A, Orsay Cedex, France). Three marketable fruits from each plot were randomly chosen. Measurements were conducted in configuration 4 using red, blue, green and UV excitation at the bottom of the fruit. Parameters of interest were SFR\_G (indicating the chlorophyll content), FLAV (indicating the concentration of flavonoids) and ANTH\_RG (indicating the anthocyanin content) [23]. Averages of the indices of the fruits per plot were calculated for each date separately.

#### *2.6. Firmness of the Fruit Pulp*

For assessing the firmness of the fruit pulp, a single measurement was taken with the HHP-2001 device (Heinrich Bareiss Prüfgerätebau GmbH, Oberdischingen, Germany). A 0.25 cm2 test anvil was pressed against the bottom of the fruit and after one second, resistance readings were taken. Average values per plot and measuring date were calculated.

#### *2.7. Sampling and Chemical Analyses*

After the spectroscopic measurements and the determination of fruit pulp firmness, the fruits were weighed, cut and dried at 60 ◦C. After three days, the dry matter was determined. The dried tomato fruits were sampled for analysis of minerals. For other chemical analyses, an aliquot of approximately 1000 g of fruits per plot was sampled and stored at −20 ◦C until further analyses.

Dried tomato samples were ground to a fine powder. For determination of mineral concentrations, an aliquot of the sample was extracted according to the Kjeldahl procedure (TURBOTHERM, C. Gerhardt GmbH & Co. KG, Königswinter, Germany). The extracts were filtered, and the content of N, P K, Ca, Mg, Fe, Zn, Mn and Cu was determined by inductively coupled plasma optical emission spectrometry (ICP-OES; SPECTRO ARCOS, SPECTRO Analytical Instruments, Kleve, Germany).

For further analyses, the frozen fruit material was thawed at room temperature for 12 h and then homogenized with commercial kitchen blenders. Aliquots of this homogenate were taken for carotenoid analysis. The remaining homogenate was centrifuged at 4500 rpm for 10 min (ROTANTA 460 RS, Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany). The supernatant was filtered, and the resulting juice was kept deep-frozen at −20 ◦C until analysis of its composition.

The total content of liposoluble pigments (mainly carotenoids) from the tomato homogenate was extracted in acetone/hexane with addition of sodium sulphate and calcium carbonate (both of Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). The extracts were filtered (syringe filter ROTILABO PTFE 0.2 μm, Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and the concentration of the liposoluble pigments was determined spectrophotometrically at 450 nm as equivalents of β-carotene. Calibration curves were conducted using β-carotene (Merck KGAa, Darmstadt, Germany) as a standard.

The juice was thawed for 3 h at room temperature prior to analysis of its composition. The following parameters were assessed according to the standards of the International Fruit Juice Union (Paris): soluble solids (refractometric analysis), conductivity (conductometric analysis), pH (potentiometric analysis), total acidity (expressed as citric acid, potentiometric analysis after titration to pH 8.1 with 0.3 M NaOH; Titroline alpha, Schott, Mainz, Germany), concentration of ascorbic acid (automatic potentiometric titration with SCHOTT TitroLine alpha plus, SI Analytics, Mainz, Germany). The contents of total phenolics were estimated by the Folin–Ciocalteu assay, and the antioxidative capacity was estimated by a photometric assay (as Trolox equivalent antioxidative capacity, TEAC). The color space parameters a\* and b\* were assessed by reflection readings of the juice against a white standard (CR 200, Konica Minolta Sensing Europe B.V., Bremen, Germany) and the "chroma" value was calculated according to D'Souza et al. [24].

All chemical analyses were performed as duplicates.

#### *2.8. Sensory Evaluation*

The effects of reduced N supply and the variety on the sensory attributes taste (sweetness, sourness, typically tomato, foreign taste, watery), consistency (firmness of fruit pulp, firmness of the peel, mealiness), aftertaste (intensity, foreign) and overall liking were assessed by quantitative descriptive sensory evaluation. The intensity of the attributes was rated on a scale from 1 (non-existent) to 9 (very strong). For assessing possible differences between the treatments before and after storage, extended triangle tests (as forced choice tests) were used. An additional question was posed concerning the preference of one sample. All samples were coded with three-digit random numbers by the "FIZZ Data Aquisition und Calculation" software (Biosystèmes, Couternon, France). For sensory evaluations, the tomato fruits were cut into four parts. Each sample consisted of six quarters from six different tomato fruits per treatment. The samples were presented in random order to the panelists. The sensory panel comprised 15 to 17 trained people that were asked to take bits from each tomato piece to obtain sensory impressions of mixed samples. The panelists received water and matzo (Dr. Schär AG/SPA, Burgstall, Italy) to clear their palates between the sensory evaluations of two samples.

#### *2.9. Statistics*

The impact of N supply, cold storage and their interaction was assessed by two-way-ANOVA for each tomato variety separately, if the conditions of normal distribution (tested with the Shapiro–Wilk test) and variance homogeneity (tested with the Levené test) were met. In case of no interactions between the factors N supply and storage, differences between the treatments were analyzed by Tukey pairwise comparison. If the data were not normally distributed or homogeneity of variances was not observed, the Kruskal–Wallis test followed by Mann–Whitney pairwise comparison was performed.

Impacts of the N supply on fruit yield data and on the content of mineral elements in the fruits were assessed by a *t*-test. All statistics of yield and quality data were performed (with α = 0.05) using the PAST software (version 3.01, [25]).

The sensory data were analyzed by calculating averages for the single attributes and testing for significant differences with the Friedman test using the "FIZZ Data Aquisition und Calculation" software. When significant differences were observed, the means were separated by using least significant difference at α = 0.1. The triangle tests were analyzed by comparing the percentage of correct answers to the probability of the corresponding binomial law.

#### **3. Results**

#### *3.1. Yield*

The total yield (=marketable and non-marketable fruits) was not significantly impacted by the N supply for the varieties 'Apresa' and 'Delioso' (Table 2). In contrast, 'Bombonera' had lower total fruit yields when supplied with reduced N amounts. Nevertheless, there were tendencies of a larger proportion of marketable fruits in the Nred treatment of 'Bombonera' (*p* = 0.054). For 'Delioso' and 'Bombonera', the fruits of the control treatments were significantly heavier compared to the Nred treatment (Table 2). This was not observed for the variety 'Apresa'.

**Table 2.** Total yield, proportion of marketable fruits and weight of single fruits in three tomato varieties with normal (con) and reduced (Nred) N supplies. *N* = 4 ± SD. Asterisks in the "Nred" line indicate significant differences between the "con" and the "Nred" treatment for the respective tomato varieties (*t*-test at α = 0.05).

#### *3.2. External Quality Parameters*

The firmness of the fruit pulp did not differ between the control and the Nred treatments of the three tomato varieties, both before and after storage (Table 3). However, tendencies of differences in the fruit pulp firmness due to different N supplies were observed in 'Bombonera' immediately after harvest (*p* = 0.08). In all varieties and N supply treatments, the fruit pulp firmness decreased significantly after storage.

The spectroscopic parameters FLAV, ANTH\_RG and SFR\_G are indices for the contents of different pigments of the fruit skin. Immediately after harvest, exclusively 'Bombonera' showed differences due to the N supply, with lower ANTH\_RG indices in Nred compared to control fruits (Table 3). After storage, 'Apresa' fruits that were grown with reduced N supply had lower FLAV indices as compared to fruits grown under control conditions.

Fruits of the varieties 'Delioso' and 'Bombonera' did not show differences in the FLAV index between control and Nred treatments after storage. All indices were decreased after the storage in fruits of 'Delioso' grown under control and Nred conditions. The FLAV index was decreased after the storage in 'Apresa' control and Nred fruits but was not affected in 'Bombonera' fruits grown under both N supplies. In contrast, ANTH\_RG was increased after storage in both control and Nred fruits of 'Bombonera' but was not affected in 'Apresa'. The index SFR\_G was lowered after storage in 'Apresa' grown under reduced N supply, while there was no impact for 'Apresa' that was grown under control conditions. **Table 3.** External quality parameters assessed by non-invasive measurements of tomato fruits of three cocktail varieties grown with normal (con) and reduced (Nred) N supplies, immediately after harvest (before storage) and after 8 days of storage at 12 ◦C (after storage). Means of *N* = 22 to 36 for firmness, and *N* = 31 to 58 ± SD for FLAV, ANTH\_RG and SFR\_G. Different letters indicate significant differences due to storage within a variety and N supply. Asterisks indicate significant differences due to N supply within a variety and storage (two-way ANOVA with Tukey pairwise comparison, or Kruskal–Wallis test with Mann–Whitney pairwise comparison; at α = 0.05).

#### *3.3. Chemical Composition of Tomato Fruits*

The concentrations of the elements N, P, K, Mg, Fe, Zn, Mn and Cu differed between the varieties (data not shown) but were not influenced by N supply (Table 4).

**Table 4.** Mineral composition of tomato fruits at harvest. The concentrations of the mineral elements are expressed on a fresh weight base. Mean of *N* = 4 ± SD. No significant differences between the con and the Nred treatment for the respective tomato varieties were observed (*t*-test at α = 0.05).

The concentrations of total phenolics and total contents of liposoluble pigments were not affected by the N supply, both before and after storage, in all tomato varieties. Nevertheless, the eight-day storage of the fruits of 'Delioso' and 'Apresa' significantly increased their total content of liposoluble pigments (Table 5).

Ascorbic acid concentrations were higher in 'Delioso' fruits grown with reduced N supply both before and after storage. No changes in the ascorbic acid contents were observed in the fruits due to storage.

The antioxidant capacity (as TEAC) was higher in 'Delioso' fruits grown with reduced N supply before storage, but no differences between control and Nred fruits were observed after storage.

In general, the content of soluble solids, the pH and the total acidity of tomato fruits have impacts on their taste. The three parameters were not affected by the N supply before and after storage in fruits of all varieties here (Table 6). Storage had no effect on the content of soluble solids in all tested tomato samples but increased the pH in fruits of 'Delioso' and 'Apresa' grown under control and Nred conditions. There were no effects on the total acidity, except 'Delioso' under Nred conditions, where a decrease in total acidity was

observed after storage. The fruits of 'Bombonera' showed no changes in the content of soluble solids, total acidity and pH due to N supply and storage.

**Table 5.** Mean and standard deviation of the antioxidant status of fruits of three cocktail tomato varieties grown with normal (con) and reduced (Nred) N supplies, immediately after harvest (before storage) and after 8 days of storage at 12 ◦C (after storage). The antioxidant parameters are expressed on a fresh weight base. *N* = 3–4. Different letters indicate significant differences due to storage within a variety and N supply. Asterisks indicate significant differences due to N supply within a variety and storage (Two-Way ANOVA with Tukey pairwise comparison, or Kruskal-Wallis test with Mann-Whitney pairwise comparison; at α = 0.05).

**Table 6.** Composition and internal color (expressed as "chroma") of tomato fruits of three cocktail varieties grown with normal (con) and reduced (Nred) N supplies, immediately after harvest (before storage) and after 8 days of storage at 12 ◦C (after storage). Mean of *N* = 3–4 ± SD. Different letters indicate significant differences due to storage within a variety and N supply(two-way ANOVA with Tukey pairwise comparison, or Kruskal–Wallis test with Mann–Whitney pairwise comparison; at α = 0.05).

The color index "chroma" was not significantly affected by both the N and storage treatments in all varieties of cocktail tomatoes (Table 6). Exclusively, 'Bombonera' grown with reduced N tended to have higher "chroma" values after storage compared to the control (*p* = 0.067). Due to storage, 'Apresa' grown under controlled and reduced N supplies showed a significant decrease in "chroma".

#### *3.4. Sensory Evaluation*

Descriptive sensory evaluation revealed differences in the attributes sweetness, watery, foreign aftertaste, firmness of the fruit pulp and the peel, typically tomato and overall liking in the three varieties (Figure 1a, statistical data not shown). Differences due to N supply were not observed for all attributes.

In triangle tests, the sensory panel was not able to discriminate significantly between controls and Nred samples immediately after harvest (Figure 1b). However, there was a trend for sensory discrimination between control and Nred treatments in 'Bombonera' fruits (*p* = 0.08). After storage, a significant difference between controls and Nred fruits was detected for 'Bombonera'. However, there was no preference for either controls or Nred fruits.

**Figure 1.** Sensory attributes of three tomato varieties grown with normal (con) and reduced (Nred) N supplies (**a**) and results of triangle tests (**b**). In (**b**), the "correct answers" indicate the relative abundance of correct distinctions between Nred and control samples. Asterisks in (**b**) show significant sensory distinctions between controls and Nred samples. *N* = 16 for (**a**). *N* = 16 to 17 for (**b**).

#### **4. Discussion**

Reducing the nitrogen supply in vegetable production aims at lowering the nitrate leaching into the groundwater and thus negative impacts to the environment. However, a sub-optimal nitrogen supply is known to alter the metabolism of plants and thereby their allocation of resources. As a consequence of this abiotic stress, photosynthetic and growth processes might be impeded, which could lower the crop yield [1]. In order to achieve environmentally sound fertilization while maintaining yield and quality, this experiment examined for tomato crops, cultivated in soil in a greenhouse, whether this balance could be achieved by reducing the N supply by 50% at the onset of fruit ripening. In fact, the total yield of fruits of the cocktail tomato varieties 'Delioso' and 'Apresa' (Table 2) was not reduced, being in accordance with studies using similar N levels for soil-grown tomato plants to our experiment (210 vs. 360 kg N ha−1) [20,26–30].

The lower single fruit weight did not affect the total yield of 'Delioso', whereas this was not the case for 'Bombonera' (Table 2). Lower yields due to lower fruit weight with reduced N supply were reported by Qu et al. [31] and Wang et al. [32], but they occurred at lower N levels than in our experiment. It can be concluded that 'Bombonera' is more sensitive to reduced N availability under sub-optimal growing conditions as there were only around 37% marketable fruits in the control treatments (Table 2). Therefore, the reduction in the N supply was even beneficial for 'Bombonera' as the trend of increased fractions of marketable fruits resulted in comparable marketable yields of control and reduced N-grown fruits [33]. This positive effect of reduced N supply on the marketable fruit fraction was even significantly larger in 'Apresa' (Table 2) and was also observed in other studies, for example, due to lower numbers of rotten fruits [26,34].

Similarly to the marketable fruit yield, there was no impact of N reduction by 50% at the onset of fruit ripening on the firmness of the tomato fruits (Table 3). Albeit the

fruit firmness decreased after storage in all varieties and treatments (as reported in other studies [35–38]), which—as consequence of decreased turgor and water loss by transpiration [39]—is a commonly known phenomenon of fruit ripening and senescence, there was no effect of the reduced N supply. This is surprising as N limitation is known to enhance the senescence processes of plants [1], but this is probably not true for fruit softening, as several other studies also report little effects on the firmness of tomato fruits [15,20,40–42].

Besides degradation of the cell wall polysaccharides, the ripening of tomato fruits is characterized by breakdown of chlorophylls in the peel [35] and concomitant increases in pigments such as carotenoids. These pigments possess antioxidative properties and scavenge reactive oxygen species which occur increasingly at the onset of fruit ripening [43], thereby accelerating senescence and thus being detrimental for the shelf life of tomatoes [44]. The concentrations of carbon-based carotenoids and flavonoids are known to increase in plant tissues with low N availability due to accumulation of carbohydrates that could not be used for growth processes [3]. This is supported by several studies that observed increasing concentrations in phenolic substances (i) in vegetative tissue [14,45–47], (ii) in fruits as a result of strongly reduced N supply (e.g., [19]), (iii) in soilless cultivation systems such as hydroponics (e.g., [7]) and (iv) at earlier developmental stages (at transplant, see [19]).

However, N supplies of 210 and 360 kg ha−1 did not impact the concentration of soluble solids and acidity after harvest in our experiment (Table 6), which is in accordance with other studies [20,26,32,42,48]. The fact that sweetness and acidity are the major constituents of the tomato taste experience, along with volatile aroma compounds and texture parameters [17,49], explains the lack of significant differences in the strength of selected attributes assessed by the sensory panel (Figure 1a).

As expected from the similar soluble solid contents of fruits of the control and reduced N treatments, suggesting that excess carbohydrates did not occur in the Nred fruits, neither differences in the concentrations of total phenolics and total contents of liposoluble pigments in the whole tomato fruits (Table 5), in the inner color index "chroma" (Table 6), nor in the index FLAV were found between control and Nred tomato fruits. The lack of differences between the index SFR\_G of control and Nred fruits (Table 3) further suggests that the reduced N supply did not enhance chlorophyll breakdown and thus ripening and senescence processes. This is in accordance with other reports on the coloration of tomato fruits showing no effects of reduced N supply [15,41,50,51]. Similar to our findings, no impact of reduced N fertilization of tomato plants was observed on the content of the most important carotenoids in tomato fruits, lycopene, β-carotene and lutein [7,13,15,20,50,52].

In accordance with the very low effects of reduced N supply on phenolic substances (assessed by [19,20]), there were no impacts on the antioxidative capacity of water-soluble compounds (assessed as TEAC) of the fruits (Table 5), except 'Delioso' fruits. They showed higher TEAC values probably due to the higher contents of ascorbic acid (Table 5). Other studies showed that reductions in the N supply up to one third of the optimal level often had no effect on the vitamin C content of the tomato fruits [2,7,15,20,29,32]. Moreover, other results suggest that the antioxidative capacity of tomato fruits is little affected by the N supply [20,50,51].

Overall, there were very small effects of the reduced N supply on tomato fruit yield and quality. Even the N content of the fruits (Table 4) was not affected by the N supply, in the same way as the other mineral elements (Table 4), which is in accordance with other studies with a comparable or even more severe N reduction than in our experiment [26,29,40,42,53–55]. As proposed by Stefanelli et al. [3], reduced N contents in plants would be expected if the N supply to plants was low. However, the N content of the tomato leaves was not affected by the reduced N supply (data not shown), similarly to other reports of the N content in plants [40,53,54,56]. In other studies, there were no impacts of low N fertilizer levels on photosynthetic activity [57,58] and very little effects on the height and the aboveground biomass [27,40,53,56] of soil-grown tomato plants. This suggests that the reduced N supply in our experiment (210 kg N ha−1) was not a limiting factor for the tomato plants in soil cultivation.

In fact, Hartz and Bottoms [28] stated that a "seasonal N rate of 200 kg ha−1 appeared adequate to maximize fruit yield", which is supported by various studies of tomatoes [29,34,41,48,56,59]. Moreover, Zotarelli et al. [27] proposed that N application rates above 176 kg ha−1 are not beneficial for tomato fruit yields but increase the leaching of nitrate. According to the classification of Hartz and Bottoms [28], our control treatments (360 kg N ha−1) received excessive amounts of N and the tomato plants in our Nred treatments still had adequate N supplies, which might explain the lacking or minimal effects on tomato yield and quality.

These conclusions apply to tomato plants grown in soil, while soilless cultivation of tomato plants seems to increase their sensitivity towards reduced N supply as several studies report decreased photosynthetic activity [2], plant biomass [14,15,46,60] and fruit mineral contents [7]. However, it is possible to overcome N deficiency and ensure high fruit yields of soilless-grown tomato plants by supplying N levels larger than 4.5 mmol L−1 [2,15,19].

The very little effects of the N supply reduction by 50% at the onset of ripening were even less apparent after the storage. Exclusively, the higher ascorbic acid concentrations in 'Delioso', the lower FLAV index of 'Apresa' and the different taste observations in 'Bombonera' after the storage period could be ascribed to low N supply. All other storage effects, such as reduced firmness, decreased acidity and concomitant increase in pH and increases in liposoluble pigments such as carotenoids, are common phenomena that occur during ripening and senescence of tomato fruits [38]. However, in contrast to other observations [38,55,61], no increases in total soluble solid contents were observed in the three cocktail tomato varieties of our study.

Nevertheless, there was a trend (before storage) or even the significant perception (after storage) of different sensory properties between fruits of 'Bombonera' grown with either optimal or reduced N supply (Figure 1b). This sensory discrimination between the N supply levels can be less attributed to the soluble solid content and the total acidity than to the volatile organic compounds. It is possible that the concentration and composition of these compounds in tomato fruits were altered during the storage [62] and thus resulted in significantly different sensory properties of control and Nred-grown fruits of 'Bombonera' (Figure 1b).

Fruit storage at 12 ◦C for eight days had no effects on the content of ascorbic acid of all tomato varieties and treatments (Table 5) but increased the antioxidative capacity, significantly or in trend, in fruits of 'Apresa' and 'Delioso' (Table 5). This suggests that storage at 12 ◦C for eight days contributed to maintaining or even enhancing the potential nutritional quality of the tomato fruits, regardless of the N supply during their cultivation.

#### **5. Conclusions**

Reducing the N supply by 50% upon the onset of fruit ripening had little or no effects on the yield and external quality of three cocktail tomato varieties grown in soil. Contrary to our expectation, the products of secondary metabolism were not elevated, presumably due to the only moderately perceived stress. Reduced N supply thus did not result in increased nutritional quality and improved taste at harvest. However, fruit quality showed no deterioration with storage due to the lower N supply. The good shelf life was partly accompanied by increases in antioxidants. Consequently, the amount of nitrogen fertilizer can be reduced for several tomato varieties in soil cultivation while maintaining the yield, quality, taste and shelf life of tomato fruits.

**Author Contributions:** Conceptualization, L.S. and J.Z.; methodology, L.S.; investigation, L.S.; resources, J.Z.; data curation, L.S.; writing—original draft preparation, L.S. and J.Z.; writing—review and editing, L.S. and J.Z.; visualization, L.S.; supervision, J.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.

**Acknowledgments:** The authors thank Ralf Schweiggert, Ralph Lehnart, Anja Giehl, Anja Rheinberger and Petra Kürbel for help with the chemical analyses; and Mirjam Hey for help with the sensory evaluation. Seeds were kindly provided by Rijk Zwaan Welver GmbH, Hild Samen GmbH and Uniseeds Select B.V.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **For a Better Understanding of the Effect of N Form on Growth and Chemical Composition of C3 Vascular Plants under Elevated CO2—A Case Study with the Leafy Vegetable** *Eruca sativa*

**Lilian Schmidt 1,2 and Jana Zinkernagel 1,\***

**Abstract:** Plant responses to elevated atmospheric CO2 (eCO2) are well studied, but the interactions of the carbon and nitrogen metabolism in the process are still not fully revealed. This is especially true for the role of nitrogen forms and their assimilation by plants under eCO2. This study investigated the interacting metabolic processes of atmospheric CO2 levels and N form in the short-term crop arugula. The effects on physiological processes and their consequences for crop growth, yield and nutritional value were elucidated. Two varieties of arugula were grown in climate cabinets under 400 or 800 ppm CO2, respectively. The plants were fertilized with either pure nitrate or ammoniumdominated-N. Photosynthetic CO2 assimilation increased in response to eCO2 regardless of the N form. This did not affect the assimilation of nitrate and consequently had no impact on the biomass of the plants. The extra photosynthates were not invested into the antioxidative compounds but were probably diverted towards the leaf structural compounds, thereby increasing dry mass and "diluting" several mineral elements. The fertilization of arugula with ammonium-dominated N had little benefits in terms of crop yield and nutritional quality. It is therefore not recommended to use ammonium-dominated N for arugula production under future elevated CO2 levels.

**Keywords:** ammonium; climate change; food quality; photosynthesis; nitrogen source; nitrate; vegetable

#### **1. Introduction**

The atmospheric CO2 concentration is predicted to rise from 400 ppm up to 1142 ppm until the end of the 21st century [1]. Elevated CO2 concentrations (eCO2) promote growth for many plants as the water use efficiency is ameliorated and the carbon gain by photosynthetic activities is increased, at least in the short term, compared to ambient CO2 concentrations (aCO2) [2]). The beneficial effect of eCO2 in higher plants, however, is linked to certain conditions, such as (e.g., [3]): i. the plant's type of photosynthesis (e.g., C3 or C4 pathway), ii. the plant's growth period and therefore effect duration (short-, medium- or long-term), iii. the presence of other photosynthesis limiting factors (e.g., light, nutrients), and iv. the balance between source activity (net export of photo-assimilates by mature leaves) and sink activity (growth, storage). This complexity highlights that the finely-tuned mechanism of carbon assimilation is the prerequisite for better plant production under future increased carbon dioxide supply. From an agricultural perspective, the physiological processes involved that can be influenced by crop management are of particular interest. The supply of nitrogen and its assimilation in plants represents one such adjusting screw. Nitrogen (N) is incorporated in many organic compounds comprising all amino acids, thus being highly relevant for the biosynthesis of enzymes which are essential for metabolic

**Citation:** Schmidt, L.; Zinkernagel, J. For a Better Understanding of the Effect of N Form on Growth and Chemical Composition of C3 Vascular Plants under Elevated CO2—A Case Study with the Leafy Vegetable *Eruca sativa*. *Horticulturae* **2021**, *7*, 251. https://doi.org/10.3390/ horticulturae7080251

Academic Editor: Pietro Santamaria

Received: 8 July 2021 Accepted: 13 August 2021 Published: 17 August 2021

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