**Moderation of Inulin and Polyphenolics Contents in Three Cultivars of** *Helianthus tuberosus* **L. by Potassium Fertilization**

#### **Anna Michalska-Ciechanowska 1, Aneta Wojdyło 1, Bo ˙zena Bogucka 2,\* and Bogdan Dubis <sup>2</sup>**


Received: 30 October 2019; Accepted: 10 December 2019; Published: 13 December 2019

**Abstract:** Jerusalem artichoke, a widely consumed edible, is an excellent source of inulin and selected phytochemicals. However, the improvement of its chemical composition by potassium fertilization has not yet been studied. Thus, the aim of the study was to evaluate the effect of different potassium (K) fertilization levels (K2O 150 kg ha<sup>−</sup>1, 250 kg ha−1, 350 kg ha−1) on the content of inulin; profile and changes in polyphenolic compounds; and the antioxidant capacity, including on-line ABTS antioxidant profiles of freeze-dried tubers originated from Violette de Rennes, Topstar, and Waldspindel cultivars. Inulin content was highest in the early maturing *cv*. Topstar. The application of 350 kg ha−<sup>1</sup> of K fertilizer rates during the growth of *cv*. Topstar increased the inulin content of tubers by 13.2% relative to the lowest K fertilizer rate of 150 kg ha<sup>−</sup>1. In *cv*. Violette de Rennes, inulin accumulation increased in response to the fertilizer rate of 250 kg ha<sup>−</sup>1. A further increase in K fertilizer rates had no effect on inulin content. The inulin content of *cv*. Waldspindel was not modified by any of the tested K fertilizer rates. Thus, the accumulation of the inulin was cultivar-dependent. In the cultivars analyzed, 11 polyphenolic compounds were identified and polyphenolic compound content was affected by the applied rate of potassium fertilizer, which was dependent on the cultivar. Chlorogenic acid was the predominant phenolic acid in all cultivars, and it accounted for around 66.4% of the identified polyphenolic compounds in *cv*. Violette de Rennes and for around 77% of polyphenolic compounds in *cv*. Waldspindel and Topstar.

**Keywords:** traditional crop varieties; Jerusalem artichoke; inulin; fertilization; polyphenols; antioxidant capacity

#### **1. Introduction**

Jerusalem artichoke (*Helianthus tuberosus* L.) is a plant with a long history of cultivation that has been making a revival as an edible vegetable in recent years. Jerusalem artichoke is a minor crop, which is why Food and Agriculture Organization (FAO) and EUROSTAT statistics for its cultivated area are not available. Jerusalem artichoke tubers contain inulin, which increases the concentration of cell fluids and confers resistance to very low temperatures (−30 ◦C). Inulin is a fructan with hypoglycemic properties, and it is used as a dietary supplement in diabetes management on account of its low energy value. According to the literature, Jerusalem artichoke exerts therapeutic effects by stabilizing sugar blood levels; reducing cholesterol levels; regulating blood pressure; protecting the liver and kidneys; enhancing the absorption of calcium, magnesium, and iron; and preventing osteoporosis [1–3]. The discussed plant promotes the elimination of toxic metabolites, boosts immunity, relieves stress, and improves concentration [4].

Roberfroid et al. [5] analyzed chicory inulin and found that all fructans are well fermented by gut bifidobacteria, which contributes to their anticarcinogenic properties. Inulin induces a 10-fold increase in the Lactobacillus population, which is why it suppresses appetite, regulates the passage rate of digesta, and stimulates the immune system [3,6]. Inulin consumed in a daily dose of approximately 20 g does not produce adverse side effects (such as bloating). Inulin consumption is estimated at 3–11 g in Europe and 1–4 g in the United States [7]. Jerusalem artichoke contains Si, Zn, Mn, Se, K, and Mg, and when consumed regularly, it stimulates pancreatic cells to produce insulin. The discussed plant is also characterized by unique antiparasitic activity in the treatment of giardiasis.

Jerusalem artichoke tubers are a rich source of phytochemicals. Tubers contain 221 mg of phenolic compounds in 100 g of fresh matter, and the content of phenolic acids with antioxidant properties is estimated at 16.6% on a dry matter basis. According to Petkova et al. [8], Jerusalem artichoke flour delivers health benefits due to its high total polyphenolic content. Tchoné et al. [9] identified 22 phenolic compounds in Jerusalem artichoke tubers, with a predominance of chlorogenic acid. According to Cie´slik and Filipiak-Florkiewicz [10] and Florkiewicz et al. [11], the nutritional composition of Jerusalem artichoke renders it highly suitable for the production of functional foods. Similar to potatoes, Jerusalem artichokes are consumed cooked, roasted, or fried. The plant can be processed into flour, chips, salads, and additives for the production of desserts, ice cream, and fruit preserves [10–13]. Jerusalem artichoke has a high yield potential of approximately 90 t ha−<sup>1</sup> tubers [10,14]. Research has demonstrated that the yield and biological value of Jerusalem artichoke tubers are influenced by cultivar, production technology, and harvest date [15,16]. Most studies of Jerusalem artichoke cultivation have focused on the effect of nitrogen fertilization [17–20]. Sawicka [17], who investigated three Jerusalem artichoke cultivars and four nitrogen fertilization levels, observed differences in the chemical composition of tubers depending on cultivar. Fertilizer rates higher than 100 kg N ha−<sup>1</sup> decreased the biological value of tubers. Another study by Sawicka et al. [19] revealed that rational mineral fertilization, in particular with nitrogen, contributes to the high nutritional value of Jerusalem artichoke. The highest content of macronutrients in the aboveground parts of plants was noted in plots fertilized with 50 kg N ha<sup>−</sup>1. Gao et al. [20] also reported that the tuber and biomass yield of *H. tuberosus* was highest at a fertilizer rate of 50 kg N ha−1. Praznik et al. [16] and Matias et al. [18] demonstrated that the agronomic performance of Jerusalem artichoke was affected by harvest date rather than by different levels of NPK fertilization. Research shows that the yield and biological value of Jerusalem artichoke tubers are determined by cultivar, cultivation technology, and harvest date [15,18]. However, the influence of potassium (K) fertilization on the inulin content and the composition of polyphenolic compounds in different Jerusalem artichoke cultivars has not been investigated to date. In view of the above, the aim of this study was to determine the effect of different K fertilizer rates (150 kg ha<sup>−</sup>1, 250 kg ha−1, and 350 kg ha−1; K2O) on the content of inulin and polyphenolic compounds, and the antioxidant capacity of Jerusalem artichoke cultivars Topstar, Violette de Rennes, and Waldspindel in order to improve the nutrition value of such widely consumed edibles.

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

#### *2.1. Materials*

A field experiment was conducted at the Agricultural Experiment Station in Tomaszkowo (53◦ 42 N, 20◦ 26 E, Poland). The experiment had a two-level factorial design with randomized blocks. The first experimental factor was cultivar. The following German cultivars were investigated: Topstar, an early maturing, edible cultivar with yellow-brown tubers and high yields; Violette de Rennes, a medium-late maturing, edible cultivar with red tubers; and Waldspindel, a medium-late maturing cultivar with red tubers, used in the production of herbal supplements and in the distilling industry (all cultivars were obtained from Topinambur Manufaktur, an organic farm in Germany). The second experimental factor was the rate of mineral K fertilizer applied to soil: (I) K2O 150 kg ha−1, (II) K2O 250 kg ha−1, or (III) K2O 350 kg ha−<sup>1</sup> (50% potassium sulfate). Nitrogen and phosphorus fertilizers

were applied once before planting (80 kg N ha<sup>−</sup>1; urea (46%), 70 kg P2O5 ha<sup>−</sup>1; triple superphosphate (46%), CaO 90 kg ha<sup>−</sup>1). Organic fertilizers were not applied. Jerusalem artichoke was planted in the last ten days of April 2016 at a depth of 6–8 cm, 30 cm apart, with row spacing of 75 cm. Tubers were harvested in the last ten days of October 2016. Fertilizer rates were determined based on the results of an experiment conducted in Germany during 1994–2001 [21].

The experiment was established on Haplic Luvisol loamy sand [22]. Composite soil samples were obtained from each plot at a depth of 20 cm to determine the chemical properties of soil. The soil pH was 4.12, and soil nutrient levels were determined at 81.5 mg kg−<sup>1</sup> P (Egner–Riehm method), 107 mg kg−<sup>1</sup> K (Egner–Riehm method), and 31 mg kg−<sup>1</sup> Mg (Atomic Absorption Spectrophotometry-ASS) [23,24]. Pesticides were not used during the experiment.

#### 2.1.1. Sample Preparation

Jerusalem artichoke (*Helianthus tuberosus* L.) tubers (12 tubers from each fertilization treatment) were washed, peeled, and cut into 0.5 cm cubes. The cubes were freeze-dried in the Alpha 1-2LD Plus freeze-drier (Martin Christ GmbH, Osterode am Harz, Germany) (50 h; −72 ◦C) [25,26]. After freeze-drying, the samples were pulverized in a laboratory grinder, vacuum-packed, and stored (−20 ◦C) until analyses. The dry matter of the samples was on average 24.8%.

#### 2.1.2. Identification of Inulin

Inulin content was determined by the methods proposed by Megazyme [26] and Topolska et al. [27]. The results were expressed in grams per 100 g of freeze-dried samples (*n* = 2).

#### 2.1.3. Identification and Quantification of Polyphenolic Compounds

Polyphenolic compounds were identified in extracts prepared according to the method proposed by Wojdyło et al. [28]. Identification was performed in the Acquity Ultraperformance Liquid Chromatograpy system [29] equipped with a photodiode sensor (PDA, UPLC) (Waters Corp., Milford, MA, USA) and G2 QToF Micromass spectrometer (Waters, Manchester, UK) with electrospray ionization (ESI). Polyphenols were separated on an UPLC BEH column (1.7 μL, 2.1 × 100 mm; Waters Corp., Milford, MA, USA) at a temperature of 30 ◦C.

Polyphenols were quantified in the Acquity Ultraperformance LC system [29]. The retention times (*tR*) of polyphenolic compounds in tubers were compared against commercial standards (Table 1). Standard curves for chlorogenic, neochlorogenic, cryptochlorogenic, and ferulic acids were developed over a concentration range of 0.05 to 5 mg mL−<sup>1</sup> (*r2* = 0.9998). Polyphenol concentrations were determined in a series of replicates and expressed in mg kg−<sup>1</sup> on a dry matter (DM) basis.


**Table 1.** Polyphenols identified in Jerusalem artichoke tubers by LC/MS QToF (*tR*: retention time).

#### 2.1.4. Antioxidant Capacity

The antioxidant capacity of Jerusalem artichokes was determined in water–methanol extracts of freeze-dried tubers (1:4; v/v) according to the method described by Wojdyło et al. [28]. Antioxidant capacity was measured in the ABTS radical scavenging activity assay (TEAC ABTS) [30] and the Ferric Reducing Antioxidant Potential (FRAP) assay [31]. The results were expressed in Trolox equivalents per 100 g DM (*n* = 3).

#### 2.1.5. Antioxidant Profiling by On-Line HPLC-PDA with Post-Column Derivatization with ABTS

The antioxidant activity of polyphenolic compounds in Jerusalem artichokes was determined by on-line HPLC-PDA coupled with post-column derivatization with ABTS according to the method proposed by Kusznierewicz et al. [32] and described by Tkacz et al. [33].

#### *2.2. Statistical Analysis*

The results were processed statistically by one-way analysis of variance (ANOVA) (Statistical 10.0, StatSoft, Tulsa, OK, USA). Significant differences (*p* ≤ 0.05) between samples were determined by Tukey's test.

#### **3. Results and Discussion**

#### *3.1. Inulin*

Jerusalem artichoke tubers are most abundant in inulin between mid-October and December. In successive months, polyfructose content decreases and the concentration of simple sugar increases. The accumulation of oligofructans in tubers is influenced by weather conditions [34].

The inulin content of the evaluated cultivars ranged from 45.94 to 60.85 g 100 g−<sup>1</sup> of freeze-dried samples (Table 2). Similar results were noted by Cie´slik et al. [35] and Florkiewicz et al. [11] in Polish cultivars Albik and Rubik (41.4–50.4 g·100 g<sup>−</sup>1). The coefficient of variation did not exceed 20% in the tested cultivars, which indicates that inulin content is a stable trait. Inulin concentration was most stable in *cv*. Waldspindel.

**Table 2.** The effect of cultivar and potassium fertilizer rate on the inulin content of Jerusalem artichoke tubers (g·100g−<sup>1</sup> freeze-dried sample).


Note: a, b, c, d = values in columns marked with the same letters do not differ significantly at *p* ≤ 0.05 (Tukey's test, analysis of variance (ANOVA)).

An analysis of K fertilization levels revealed that inulin content was highest in response to the highest fertilizer rate (350 kg K2O ha−1). The analyzed cultivars responded differently to higher rates of K fertilizer. According to Sawicka [17,36], Jerusalem artichoke tubers are characterized by cultivar-dependent variations in nutrient levels. Matias et al. [18] found no differences in inulin content in response to higher rates of NPK fertilizers (Level 1: 54, 108, 162; Level 2: 108, 216, 324) and concluded that unlike harvest date, fertilization has a minor effect on tuber yields.

Inulin accumulation was significantly higher in the early-maturing *cv*. Topstar relative to medium-late maturing cultivars. Higher rates of K fertilizer exerted the greatest influence on the inulin content of Jerusalem artichoke *cv*. Topstar. The inulin content of freeze-dried tubers increased by 8.02 g ·100 g−<sup>1</sup> when the fertilizer rate was increased by 200 kg K2O ha<sup>−</sup>1.

Violette de Rennes was characterized by the lowest inulin content and the smallest variations in insulin levels. In this cultivar, the inulin content of freeze-dried tubers increased by 4.57 g·100g−<sup>1</sup> when the fertilizer rate was increased from 150 kg K2O ha<sup>−</sup><sup>1</sup> to 250 kg K2O ha<sup>−</sup>1. The inulin content of the medium-late *cv*. Waldspindel, which is used in the production of herbal supplements and in the distilling industry, did not change in response to higher rates of K fertilizer.

#### *3.2. Polyphenols*

The polyphenols content of Jerusalem artichoke tubers is influenced by cultivar [37], harvest date, and storage conditions [38]. According to Terzi´c et al. [39] and Kapusta et al. [37], differences in the concentrations of polyphenolic compounds are also genetically conditioned. The effect of various rates of K fertilizer on the content of polyphenolic compounds in Jerusalem artichoke tubers has not been investigated to date. In the present study, the total content of the polyphenolic compounds identified in the analyzed cultivars of Jerusalem artichoke ranged from 1477 to 1801 mg kg−<sup>1</sup> DM. Similar results were reported by Kapusta et al. [37] (Table 3).

In treatments fertilized with 150 kg K ha<sup>−</sup>1, polyphenol levels were lowest in *cv*. Violette de Rennes and highest in *cv*. Topstar. In *cv.* Violette de Rennes, total polyphenolic content increased by 5% and 13% when the rate of K fertilizer was increased from 150 kg ha−<sup>1</sup> to 250 and 350 kg ha<sup>−</sup>1, respectively. Similar observations were made by Kavalcova et al. [40] in onions, where polyphenol levels measured spectrophotometrically increased with a rise in K fertilizer rate. In this regard, it was concluded that the content of polyphenolic compounds could be a varietal trait in Jerusalem artichoke. In *cv*. Waldspindel and Topstar, polyphenol levels decreased by 6.4% and 1.5% on average, respectively, when K fertilizer rate was increased by 100 and 200 kg ha−1. These findings suggest that the potassium-stimulated synthesis of polyphenolic compounds is affected by the unique chemical composition of different cultivars of Jerusalem artichoke.

Chlorogenic acid was the predominant polyphenolic compound in Jerusalem artichoke tubers and leaves [41]. The average content of chlorogenic acid was estimated at 66.4% in *cv*. Violette de Rennes and at 77% in *cv*. Waldspindel and Topstar, regardless of K fertilizer rate. The content of chlorogenic acid was lowest in *cv*. Violette de Rennes and highest in *cv*. Topstar, regardless of K fertilizer rate. In *cv*. Violette de Rennes, the content of chlorogenic acid increased by 5% and 15% in response to K fertilizer rates of 250 kg ha−<sup>1</sup> and 350 kg ha<sup>−</sup>1, respectively. In *cv*. Waldspindel and Topstar, the content of chlorogenic acid decreased by 6% and 10% on average when the K fertilizer rate was increased to 250 and 350 ha−1, respectively. The above findings indicate that cultivar and chemical composition significantly affect the potassium-induced synthesis of chlorogenic acid.



 dm).

Four isomers of dicaffeoylquinic acid (1,5-, 3,4-, 3,5-, and 4,5-dicaffeoylquinic acids) were identified in Jerusalem artichoke tubers in this study, which is consistent with the results reported by Kapusta et al. [37]. Here, 1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, and 3,5-dicaffeoylquinic acid accounted for approximately 25% of the identified polyphenolic compounds in *cv*. Violette de Rennes and for 16.5% of the identified polyphenolic compounds in *cv*. Waldspindel and Topstar, regardless of K fertilizer rate. In *cv*. Violette de Rennes, the content of 1,5-dicaffeoylquinic acid and 3,4-dicaffeoylquinic acid increased with a rise in K fertilization levels and was highest in response to the rate of 350 kg K ha<sup>−</sup>1. In *cv*. Waldspindel, the concentrations of the above compounds did not change considerably in response to increasing rates of K fertilizer. In *cv*. Topstar, the content of above components decreased when the K fertilizer rate was increased from 150 kg ha−<sup>1</sup> to 250 and 350 kg ha−1. The analyzed cultivars also contained neochlorogenic acid, which accounted for 3.5% of all polyphenolic compounds on average. The variations in the concentration of neochlorogenic acid across fertilizer treatments were cultivar-dependent, and they differed from those observed in the content of dicaffeoylquinic acids. Cryptochlorogenic acid was also identified in the analyzed Jerusalem artichoke cultivars. On average, cryptochlorogenic acid accounted for 1.2% of the identified polyphenolic compounds regardless of cultivar and K fertilizer rate. The content of cryptochlorogenic acid was highest in *cv*. Topstar and it more than doubled in response to a K fertilizer rate of 250 kg ha−1. The content of caffeoyl-glucoside acid and caffeic acid increased in all cultivars in response to a K fertilizer rate of 350 kg ha<sup>−</sup>1. The concentration of *p*-coumaroyl-quinic acid was highest in *cv.* Violette de Rennes, and it increased with a rise in K fertilizer rate. A reverse trend was observed in the remaining cultivars, which indicates that polyphenol synthesis is a varietal trait in Jerusalem artichoke.

#### *3.3. Antioxidant Capacity*

Antioxidant capacity ranged from 0.87 to 3.28 μmol Trolox kg−<sup>1</sup> DM in the ABTS radical scavenging activity assay (Table 4).


**Table 4.** The effect of potassium fertilizer rates on the antioxidant capacity of three Jerusalem artichoke cultivars (μmol Trolox kg<sup>−</sup>1).

Note: average ± standard deviation; a, b, c, d, e: values in columns marked with the same letters do not differ significantly at *p* ≤ 0.05 (Tukey's test, ANOVA).

Antioxidant capacity measured by TEAC ABTS assay was the highest in *cv*. Violette de Rennes and the lowest in *cv*. Waldspindel. Considerable differences in the antioxidant capacity of different Jerusalem artichoke cultivars were also reported by Catana et al. [42]. The antioxidant capacity of *cvs*. Violette de Rennes, Waldspindel, and Topstar increased by approximately 53%, 24%, and 44%, respectively, when K fertilizer rate was increased from 150 kg ha−<sup>1</sup> to 350 kg ha−1. The content of polyphenolic compounds was correlated with antioxidant capacity measured in the ABTS assay only in *cv*. Violette de Rennes (*r* = 0.998).

The chromatographic profile of phenolic components in Jerusalem artichoke before and after derivatization of the negative control (ABTS reagent) is presented in Figure 1. Chlorogenic acid exerted the strongest effect on the antioxidant potential of Jerusalem artichokes, followed by 3,5-caffeoylquinic acid, 3,4-caffeoylquinic acid, 1,5-caffeoylquinic acid, and neochlorogenic acid.

**Figure 1.** Standard UV chromatograms (blue line) and on-line ABTS antioxidant profiles (black line) of Jerusalem artichoke. Peaks: **1**: neochlorogenic acids; **2**: chlorogenic acid; **3**: 1,5-dicaffeoylquinic acid; **4**: 3,4-dicaffeoylquinic acid; **5**: 3,5-dicaffeoylquinic acid.

In summary, no significant correlations were found between the concentrations of individual polyphenolic compounds in Jerusalem artichoke tubers and the antioxidant capacity of the extracts determined spectrophotometrically in the ABTS radical scavenging assay.

On the other hand, the FRAP values indicated that the highest antioxidant capacity was noted for *cv*. Topstar in the treatment supplied with K fertilizer at 150 kg ha−1. The increase of the K content during fertilization led to the decrease in antioxidant capacity measured by FRAP. Thus, the ability of compounds able to reduce the Fe ion in Jerusalem artichoke can be moderated by the K levels.

#### **4. Conclusions**

Based on the results obtained, it was concluded that among *cv*. Violette de Rennes, Waldspindel, and Topstar, the inulin accumulation was significantly higher in the early-maturing *cv*. Topstar. Higher rates of K fertilizer exerted the greatest influence on the inulin content of Jerusalem artichoke in this cultivar. This led to the increase of inulin content of 4.4 g 100 g−<sup>1</sup> when the K fertilizer rate was increased from 150 kg K2O ha<sup>−</sup><sup>1</sup> to 350 kg K2O ha<sup>−</sup>1.

Eleven polyphenolic compounds were identified in 3 cultivars of Jerusalem artichoke. The content of polyphenolic compounds ranged from 1.5 to 1.8 g kg−<sup>1</sup> DM of tuber samples, and it was influenced by the rate of K fertilizer. Chlorogenic acid was the predominant phenolic acid in all cultivars, and it accounted for around 66.4% of the identified polyphenolic compounds in *cv*. Violette de Rennes and for around 77% of polyphenolic compounds in *cv*. Waldspindel and Topstar. Four isomers of dicaffeoylquinic acid were also identified in the evaluated tubers, and 1,5-dicaffeoylquinic acid was the predominant isomer. The content of the remaining compounds varied across cultivars and K fertilization treatments. Chlorogenic acid, 3,5-, 3,4-, 1,5-caffeoylquinic acids, and neochlorogenic acid had the strongest influence on antioxidant potential measured by the ABTS on-line profiling method. Taking the above into consideration, fertilization with selected microelements of edibles, including Jerusalem artichoke, could be a new strategy for the improvement of the nutritional value of such plants. Nevertheless, the polyphenolic compounds are stress metabolites, and their content in the plants can be modified by the type of the fertilizer as well as the quantity applied; thus, numerous aspects should be considered in order to provide a thorough recommendation for a single polyphenolic component [43].

**Author Contributions:** Conceptualization, B.B.; methodology, B.B., A.M.-C., and A.W.; formal analysis, B.B., A.M.-C., A.W.; writing—original draft preparation, B.B., A.M.-C., A.W., and B.D.

**Funding:** This research was co-funded by Ministry of Science and Higher Education in the frame of the program entitled 'Regional Initiative of Excellence' for the years 2019-2022, Project No. 010/RID/2018/19, amount of funding 12.000.000 PLN.

**Acknowledgments:** The publication is the result of the research group activity: 'Plants4food'.

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

#### **References**


© 2019 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 (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Growing Conditions A**ff**ect the Phytochemical Composition of Edible Wall Rocket (***Diplotaxis erucoides***)**

**Carla Guijarro-Real 1, Ana M. Adalid-Martínez 1, Katherine Aguirre 2, Jaime Prohens 1, Adrián Rodríguez-Burruezo <sup>1</sup> and Ana Fita 1,\***


Received: 20 November 2019; Accepted: 6 December 2019; Published: 7 December 2019

**Abstract:** Wall rocket (*Diplotaxis erucoides*) is a wild vegetable with the potential to become a crop of high antioxidant quality. The main bioactive compounds include ascorbic acid (AA), sinigrin, and a high content of total phenolic compounds (TP). It also accumulates nitrates. Since these compounds are affected by environmental conditions, adequate crop management may enhance its quality. Eleven accessions of wall rocket were evaluated under field and greenhouse conditions during two cycles (winter and spring) and compared to *Eruca sativa* and *Diplotaxis tenuifolia* crops. The three species did not differ greatly. As an exception, sinigrin was only identified in wall rocket. For the within-species analysis, the results revealed a high effect of the growing system, but this was low among accessions. The highest contents of AA and TP were obtained under field conditions. In addition, the levels of nitrates were lower in this system. A negative correlation between nitrates and antioxidants was determined. As a counterpart, cultivation in the field–winter environment significantly decreased the percentage of humidity (87%). These results are of relevance for the adaptation of wall rocket to different growing conditions and suggest that the field system enhances its quality. The low genotypic differences suggest that intra-species selections in breeding programs may consider other aspects with greater variation.

**Keywords:** ascorbic acid; *Diplotaxis erucoides*; field; greenhouse; new crops; nitrates; sinigrin

#### **1. Introduction**

Modern societies have become increasingly aware of the importance of diet as part of a healthy lifestyle. Thus, many consumers look for additional health benefits to be obtained from specific foods, which are known as functional foods [1,2]. On the other hand, some consumers are demanding products with new and differentiated aromas and tastes to enrich daily dishes and increase the culinary experience [3]. These demands offer an opportunity for the enhancement of wild edible plants (WEPs). In fact, several WEPs have high bioactive properties and may be considered as potential functional foods [4,5], while they have also differentiated organoleptic characteristics that are highly appreciated [6]. Apart from the direct harvest from the wild, a promising strategy for such revalorization could be domestication and adaptation into cultivation systems; this is an alternative that offers several advantages such as better yields, uniformity and accessibility [3].

Mediterranean cultures have a rich ethnobotanic knowledge and tradition in the consumption of WEPs, as has been compiled in many works (e.g., [7–9]). These reports show a great diversity of WEPs that have potential as new crops, including the edible *Diplotaxis erucoides* (L.) DC. (wall rocket). Wall rocket is an annual plant from the *Brassicaceae* family, broadly distributed along the Mediterranean areas of Europe and Africa to the Middle East [7]. Considered as a weed for many crops, the species is also appreciated as a wild vegetable for its tender leaves, with a characteristic pungent flavor, and also for its flowers as decorative elements. Wall rocket is eaten fresh or cooked, added to salads, soups, pasta dishes or even fried in omelettes [8,9]. One commercial variety of wall rocket is currently available (var. Wasabi, Shamrock Seed Company, Inc.), but as far as we know, its cultivation is negligible.

Wall rocket is taxonomically related to the popular rocket crops *Eruca sativa* Mill. (salad rocket) and *D. tenuifolia* (L.) DC. (wild rocket). These crops accumulate large amounts of phytochemicals including vitamin C, phenolic compounds and glucosinolates [10] and could therefore be considered for in vivo models and clinical assays addressed to test their potential bioactive properties. In fact, both vitamin C and phenolic compounds are potent antioxidants against plant oxidative stress, and such compounds could be also involved in reducing the risk of different illnesses such as cardiovascular diseases, hepathotoxicity and general inflammation risks [11–14]; in addition, vitamin C is an essential microelement with antiscorbutic activity [15]. Glucosinolates (GSLs) are secondary metabolites from *Brassicaceae* and other families within the *Brassicales* order [16]. The enzymatic hydrolysis of GSLs releases volatile compounds that are responsible of the bitter and pungent flavor of *Brassicaceae* species [17]. In addition, different compounds in this class have been analyzed in terms of their potential health benefits using in vivo models, as reviewed by Dinkova-Kostova and Kostov [18]. Together with the bioactive compounds, rocket crops also accumulate high amounts of nitrates [19,20], considered to be antinutrients with potential health risks [21]. Thus, maximum levels are established for the commercial production of rocket crops (*E. sativa* and *Diplotaxis* sp.) and other vegetables in Europe [22].

Rocket crops are cultivated under field and greenhouse conditions [23] and can be grown in the Mediterranean regions for most of the year. As a result, these crops are subjected to growth under variable agronomic and environmental factors that include temperature, length and incidence of sunlight, irrigation, soil type, or time of harvest, among others. These environmental changes can affect the accumulation of bioactive compounds [24]. For instance, an increase of light intensity and photoperiod can decrease the content of nitrates [25]. Stresses such as heat shock, chilling or high light conditions activate the accumulation of protective phytochemicals such as ascorbic acid or phenolic compounds [26]. Abiotic stresses such as growing under non-optimal temperature conditions can increase the content of glucosinolates as well [27].

Although these are general behaviors, information related to the effect of cultivation on wall rocket is scarce. Ceccanti et al. [4] suggested that, as part of the breeding programs of wild edible plants into new crops, it is important to study the proper cultivation practices to allow large-scale, high-yield production and, at the same time, ensure a good-quality product including nutritional quality. In this sense, testing different growing environments may lead to identifying the most adequate conditions for the development of wall rocket as a crop. In addition, the use of a local germplasm may help to establish the crop in the Mediterranean regions, as these materials would have been naturally selected for its adaptation to these conditions [28]. Thus, the current study aimed to analyze the effect of growing systems (greenhouse and field) and cycles (winter and spring) on selected compounds of relevance including ascorbic acid, sinigrin and nitrates, as well as the content of total phenolics, for pre-selected accessions of wall rocket derived from local germplasm. In addition, accessions of *D. tenuifolia* and *E. sativa* were used as reference materials with the aim of contextualizing the values obtained for wall rocket, as the acceptance of a new, differentiated crop will also depend on its recognisable differences from other crops. Overall, the study allows us to gain a general insight of the behaviour of wall rocket as a crop. Moreover, the current study can be useful for establishing a basis for the future exploitation of this emerging crop, which may have a high added value due to its content of bioactive compounds.

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

#### *2.1. Plant Material and Cultivation*

Ten pre-selected accessions of wall rocket and four commercial cultivars of rocket species were evaluated in the experiment. The pre-selected accessions corresponded to the second generation seedlings from wild populations collected in the Valencian Community (Spain) (Table S1). Seeds are conserved at the Universitat Politècnica de València (UPV, Valencia, Spain), where a domestication program is being developed. The commercial cultivars (from Shamrock Seed Co., Salinas, CA, USA) included the species *D. tenuifolia* (var. SSC2402 and var. Wild Rocket), *E. Sativa* (var. S. Rocket SSC2965), and the only commercial variety of *D. erucoides* that, to our knowledge, is currently available (var. Wasabi).

The experiments were performed at the UPV following the same experimental design as described in Guijarro-Real et al. [29]. Thus, two independent growing cycles were evaluated: the late autumn–winter season (hereafter called the winter season) and late winter–early spring season (hereafter called the spring season). In each cycle, assays were simultaneously carried out in two cultivation systems: a heated glasshouse (39◦29 0" N, 0◦20 26" W) and an experimental field under an anti-pest mesh (39◦28 56" N, 0◦20 11" W).

First of all, seeds were treated with a pre-germinative treatment in order to break the possibly secondary dormancy and increase the germination uniformity [30]. Thus, seeds were treated with commercial sodium hypochlorite 2.5% (v/v) for 5 min plus gibberellic acid 100 ppm (Duchefa Biochemie, Haarlem, The Netherlands) for 24 h. Treated seeds were sown in commercial Neuhaus Humin-substrat N3 substrate (Klasmann-Deilmann Gmbh, Geeste, Germany) and placed in a growing chamber with long day conditions (16/8 h, 25 ◦C) for two days. For materials used in the greenhouse system, sowing was directly performed in 40 <sup>×</sup> 25 cm<sup>2</sup> trays, in which plants remained for the entire experiment; plants used for the field system were instead sown in seedling trays.

Two days after being sown, trays were moved to a greenhouse. Trays used for the greenhouse system remained in these conditions during the entire experiment. In contrast, plants used in the field system were allowed to grow in the greenhouse until the appearance of the second true leaf and then were transplanted to the field until the end of the experiment. In both the greenhouse and field systems, the same experimental design was followed: a complete randomized block design with five blocks, with each block including one replicate of 30 plants per accession. This totals 8400 plants used for the experiments performed.

#### *2.2. Preparation of Samples*

All plants in each replicate were harvested together as a pool, except for plants with visible growing damages (e.g., a very small size compared to the average of the block) that were discarded. Samples were processed on the same day as harvesting. One fresh sub-sample was used for the analysis of ascorbic acid, and the rest were frozen at −80 ◦C and then lyophilized. The difference between the weight before and after lyophilization was used to calculate the percentage of moisture. The lyophilized material was powdered with a commercial grinder and stored in darkness until being analyzed for total phenolics, sinigrin and nitrates. All results were expressed as contents per each 100 g of fresh weight (FW) using the percentage of moisture for conversion, as this result provides a more appropriate value considering that the product is eaten raw.

#### *2.3. Traits Measured*

The content of ascorbic acid (AA) was measured according to Cano and Bermejo [31] with slight modifications. Briefly, 1.0 g of fresh material was homogenized with 5 mL of cold meta-phosphoric acid 3.0% (v/v) for 1 min using a mortar. The aqueous phase was filtered through a 0.22 μm PVDF filter and analyzed on a HPLC 1220 Infinity LC System (Agilent Technologies; Santa Clara, CA, USA) using a BRISA C18 column (150 mm × 4.6 mm i.d., 3 μm particle size; Teknokroma; Barcelona, Spain). The mobile phase consisted of methanol: 1% acetic acid (5:95) for 15 min at a flow rate of 1 mL min<sup>−</sup>1. The injection volume was 5 μL, and quantification was performed at 254 nm using an external standard calibration of *L*-ascorbic acid (Sigma-Aldrich, Saint Louis, MO, USA).

The content of sinigrin (SIN) was determined as described by Grosser and van Dam [32] with slight modifications. Firstly, 0.1 g of powdered samples was heated for 2 min at 75 ◦C using a Termoblock TD150 P2 (Falc Instruments, Treviglio, Italy) for myrosinase inactivation [33]. Extraction was then performed using 1 mL of methanol 70% (v/v) for 15 min at 75 ◦C. After centrifugation, the supernatant was collected. The extraction step was repeated with 1 mL of methanol 70% (v/v) for another 15 min at 75 ◦C. Both supernatants were mixed and injected into an SPE column containing a DEAE Sephadex anion exchanger (A-25, Sigma-Aldrich, Saint Louis, MO, USA) activated with 20 mM sodium acetate buffer (pH 5.5) and incubated with 20 μL of diluted sulfatase (Sigma-Aldrich, Saint Louis, MO, USA) overnight. Desulphonated sinigrin was eluted with 500 μL plus 500 μL of milliQ water and analyzed using the same HPLC apparatus as for AA analysis and a Luna® Omega C18 column (150 mm <sup>×</sup> 4.6 mm i.d., 3 μm particle size; Phenomenex, Torrance, CA, USA). The mobile phases consisted of acetonitrile (A) and water (B), with the following gradient: from 98% A to 65% A in 35 min, then equilibrated for 5 min to the initial conditions. The injection volume was 10 μL and the flow rate was 0.75 mL min<sup>−</sup>1. Quantification was performed at 229 nm using desulphoned sinigrin hydrate (PhytoPlan, Heidelberg, Germany) as an external standard.

The content of total phenolics (TP) was determined according to the Folin–Ciocalteu procedure [34] as in Guijarro-Real et al. [35]. For that, 0.125 g of lyophilised material was extracted with 5 mL of acetone 70% (v/v) containing acetic acid 0.5% (v/v) for 24 h under continuous stirring. Aliquots of 65 μL were incubated with 500 μL of diluted Folin-Ciocalteu (1:10; Scharlab S.L., Sentmenat, Spain) for 5 min; then, 500 μL of sodium carbonate 60 g L−<sup>1</sup> was added and incubated for other 90 min. Quantification was performed at 765 nm in a iMarkTM Microplate Reader spectrophotometer (Bio-Rad, Hercules, CA, USA). Chlorogenic acid (Sigma-Aldrich) was used as an external standard and the results were expressed as mg of chlorogenic acid equivalents (mg CAE 100 g−<sup>1</sup> FW).

Finally, the content of nitrates was determined using a nitrate-selective ion (Crison Instruments S.A., Alella, Barcelona, Spain), with an extraction protocol adapted from Egea-Gilabert et al. [36]. Nitrates from 0.1 g were extracted with 50 mL of distilled water for 15 min under continuous stirring and stabilized with 1 mL of 2 M diammonium sulfate ((NH4)2 SO4) buffer at the moment of measurement using the nitrate-selective ion.

#### *2.4. Data Analysis*

Data were subjected to a fixed effects model analysis of variance [37] using the Statgraphics Centurion XVII v.17.2 (Statpoint Technologies, Inc., Warrenton, VA, USA). Two different analyses were performed: (1) a comparison among materials from different species, and (2) a comparison among accessions of wall rocket. For the analysis of species, the average values for accession considering the five replicates per environment were used as data. Data were then submitted to a multivariate analysis of variance (ANOVA) and the effects of species (S, corresponding to three levels: wall rocket, wild rocket, salad rocket), environment (E, four levels: greenhouse in winter, field in winter, greenhouse in spring, field in spring) and the S × E interaction were tested. The linear model applied was

$$\chi\_{\rm ijk} = \mu + \mathbf{S}\_{\rm i} + \mathbf{E}\_{\rm j} + (\mathbf{S} \times \mathbf{E})\_{\rm ij} + \mathbf{e}\_{\rm ij(k)}$$

where Xijk is the value for accession k of species i and environment j, μ is the general mean, Si is the effect of the species i, Ej is the effect of the environment j, (S × E)ij is the effect of the interaction between species i and environment j, and eij(k) is the residual error of the accession k. Mean values and standard error were obtained for the three species and significant differences determined using the Student–Newman–Keuls multiple range test (*p* = 0.05).

The analysis of wall rocket aimed to study the presence of differences among accessions and/or among systems, considering each growing cycle independently [29]. Thus, individual data were submitted to a multivariate analysis of variance (ANOVA) and the effects of accession (A, eleven accessions), growing system (GS, two levels: greenhouse, field) and the A × GS interaction were tested. The linear model applied was

$$\mathbf{X}\_{\mathrm{ijkl}} = \boldsymbol{\mu} + \mathbf{A}\_{\mathrm{i}} + \mathbf{B}\_{\mathrm{j}(\mathrm{ik})} + \mathbf{G} \mathbf{S}\_{\mathrm{j}} + (\mathbf{A} \times \mathbf{G} \mathbf{S})\_{\mathrm{ik}} + \mathbf{e}\_{\mathrm{i}\mathbf{j}k(\mathrm{l})}$$

where Xijkl is the value for replicate l of accession i in block j and growing system k, μ is the general mean, Ai is the effect of the genotype i, Bj(ik) is the effect of block j for accession i and system k, GSj is the effect of the growing system j, (A × GS)ik is the effect of the interaction between accession i and system k, and eijk(l) is the residual error of the replicate l. Mean values and standard errors were obtained, and significant differences among environments were determined according to the LSD test (*p* = 0.05). Accessions were ranked for their average values of AA, TP, SIN and NO3 − within each environment, where high levels of AA, TP and SIN and low levels of NO3 − were a positive trait. These ranks were then used to obtain a global ranking table for the eleven accessions of wall rocket. Finally, the Spearman rank coefficients of correlation (ρ) were calculated for phenotypic (*n* = 44) and environmental (*n* = 213) correlations.

#### **3. Results**

#### *3.1. Di*ff*erences among Materials of Di*ff*erent Species*

Wall rocket was compared to the reference materials including two accessions of *D. tenuifolia* and one accession of *E. sativa* in terms of the percentage of moisture and contents of AA, TP and NO3 −. Differences in the contents of SIN were not analyzed because this compound was only present in wall rocket. A significant effect of the environment was determined in the four traits evaluated (Table 1). This factor was the main contributor to the total sum of squares in all cases, with values ranging between 52.8% (NO3 −) and 72.6% (TP). On the contrary, the species factor was only significant for the percentage in moisture and the content in AA. Their contribution was, in any case, lower than 10.5%. Finally, a significant S × E interaction was determined for all traits except for the content of AA, and the effect of this interaction accounted for up to 17.7% of the total sum of squares (Table 1).

**Table 1.** Sum of squares (in percentage, %) and degrees of freedom (*d.f*) for the effects of species (S) with three levels: wall rocket (*n* = 44), wild rocket (*n* = 8) and salad rocket (*n* = 4); environment (E) with four levels: greenhouse–winter, field–winter, greenhouse–spring, and field–spring; S × E interaction and residuals for the percentage in moisture and the content in ascorbic acid (AA), total phenolics (TP) and nitrates (NO3 −).


ns, \*, \*\* and \*\*\* indicate no significant or significant at *p* < 0.05, 0.01 and 0.001, respectively.

The average values for each trait are represented in Figure 1. The percentage of moisture was close to 90.0% for the three species, with salad rocket displaying the highest values on average. Both wild rocket and wall rocket significantly decreased the moisture of leaves under the field–winter environment, while the percentage in salad rocket was stable for the environments tested. Regarding the content of AA, wall rocket accumulated the highest value on average (70.02 mg AA 100 g−1), at approximately 30% greater than wild rocket. The effect of the environment was similar in the three species, with the greenhouse environments providing the lowest values. As an exception, the accumulation of AA in salad rocket was not affected by the growing system (field or greenhouse) during the spring cycle (Figure 1).

**Figure 1.** Mean values ± SE for the traits determined in each environment tested (greenhouse–winter, G-W; field–winter, F-W; greenhouse–spring, G-S; field–spring, F-S) for wall rocket (*n* = 11), wild rocket (*n* = 2) and salad rocket (*n* = 1), and global average values: (**A**) Percentage of moisture (%); (**B**) Content of ascorbic acid (AA, expressed as mg in 100 g−<sup>1</sup> FW); (**C**) Content of total phenolics (TP, expressed as mg of chlorogenic acid equivalents, CAE, in 100 g−<sup>1</sup> FW); (**D**) Content of nitrates (NO3 −, expressed as mg in 100 g−<sup>1</sup> FW).

Although no significant differences were established among species for the contents of TP and NO3 <sup>−</sup>, both traits were affected by the S × E interaction (Table 1). The content of TP was not significantly affected by the environment for salad rocket, while the field environments increased the estimated TP in the other species (Figure 1). Finally, none of the three species showed total average values above 600 mg NO3 <sup>−</sup> 100 g−1. However, the spring environments significantly increased the accumulation of these ions, with the maximum value obtained for wall rocket growing in the greenhouse–spring environment.

#### *3.2. Variation among Wall Rocket Accessions*

#### 3.2.1. Effects of Accession, Growing System and Interaction

The effects of accession (A), growing system (GS) and A × GS interaction were independently analyzed for each growing cycle (Table 2). The winter cycle was highly affected by the growing system for all traits except for the content of NO3 −. The contribution of this factor to the total sum of squares ranged between 16.5% (NO3 −) and 81.1% (TP); moreover, this factor was the greatest contributor to the percentage of moisture, AA and TP (>50%). On the contrary, the contribution of the growing system to the total sum of squares was lower during the spring cycle, with percentages significantly decreasing for all traits (Table 2). Moreover, during this cycle, its effect was only significant for the contents of AA and TP. As in the winter cycle, it remained the main contributor to the total sum of squares for both AA and TP, accounting for 52.8% and 57.3%, respectively.


**Table 2.** Sum of squares (in percentage, %) and degrees of freedom (*d.f*) for the effects of accession (A, *n* = 11), growing system (GS, field or greenhouse), A × GS interaction, block and residuals for the percentage in moisture, ascorbic acid (AA), total phenolics (TP), sinigrin (SIN) and nitrates (NO3 −) evaluated in the eleven accessions of wall rocket during the winter and spring cycles.

ns, \*, \*\* and \*\*\* indicate no significance or significance at *p* < 0.05, 0.01 and 0.001, respectively.

On the other hand, the effect of accession was not significant for most traits in any of the cycles (Table 2). In fact, this factor only affected the contents of AA and SIN during the winter cycle, accounting for 3.8% and 10.0% of the total sum of squares, respectively. In a similar way, the A × GS interaction effects were mostly non-significant (Table 2). As an exception, an interaction effect was determined during the spring cycle for the contents in AA and TP.

#### 3.2.2. Effects of Accession, Growing System and Interaction

The average values and dispersion for the different traits evaluated in each environment are summarized in Figure 2. Results were compared between systems for each cycle, while the indirect effect of the growing period was also evaluated by comparing within systems.

**Figure 2.** Box and whisker plot reflecting the average values (marked as +) and distribution for the traits evaluated in the accessions of wall rocket (*n* = 11) growing under greenhouse or field systems, during the winter and spring cycles. (**A**) Percentage of moisture (%). (**B**) Content of ascorbic acid (AA, expressed as mg in 100 g−<sup>1</sup> FW). (**C**) Content of sinigrin (SIN, expressed as mg in 100 g−<sup>1</sup> FW). (**D**) Content of total phenolics (TP, expressed as mg of chlorogenic acid equivalents, CAE, in 100 g−<sup>1</sup> FW). (**E**) Content of nitrates (NO3 <sup>−</sup>, expressed as mg in 100 g−<sup>1</sup> FW). \* indicates significant differences between systems within cycles according to the LSD test (*p* = 0.05). Different letters indicate significant differences between cycles within systems according to the LSD test (*p* = 0.05).

The percentage of moisture was only affected by the field–winter cycle. Plants growing under these conditions reduced the accumulation of water in leaf tissues in approximately 4% in comparison with the other environments tested, where the percentage in moisture was around 90.7%. On the contrary, the contents of AA, TP and SIN significantly increased when plants grew in the field-winter environment. Values under these conditions were more than two-fold greater with respect to the greenhouse system (Figure 2). A similar performance was found during the spring cycle for the contents of AA and TP but not for the content of SIN. Thus, the accumulation of AA and TP also increased for plants growing in the field system in the second cycle, but differences among systems were lower in this case.

An indirect effect of the growing period was also found (Figure 2). Plants growing in the greenhouse displayed the least differences between cycles. In this system, the cycle only affected the levels of SIN and NO3 −, with plants growing in spring displaying the highest contents. Thus, the accumulation of NO3 − displayed a 2.6-fold increase with respect to the winter cycle (Figure 2). In fact, this environment provided the maximum levels of NO3 − considering the four environments (974 mg 100 g−<sup>1</sup> FW). Regarding the field system, the results indicated that all traits were influenced by the growing cycle (Figure 2). Plants growing during the winter cycle had higher contents of AA, TP and SIN. The greatest increase was found for the levels of SIN, corresponding to a two-fold increase (35.4 mg 100 g−<sup>1</sup> vs. 73.0 mg 100 g−<sup>1</sup> FW). On the contrary, the winter cycle resulted in a reduction of the levels in NO3 - . In fact, plants growing in this environment displayed the lowest accumulation (164 mg NO3 <sup>−</sup> 100 g−<sup>1</sup> FW) (Figure 2).

The accessions were ranked considering their bioactive properties as well as the levels of NO3 − along the four environments tested (Table 3). The high content of bioactive compounds was considered as a positive trait, while the accumulation of NO3 − was considered as negative. The commercial cv. Wasabi ranked second together with accession DER055-1. The first in rank was DER001-1, although it had a low rank position for the levels of NO3 −. DER006-1was also very close to the commercial cultivar. On the contrary, accessions DER064-1 and DER085-1 had the lowest scores.


**Table 3.** Average values and coefficient of variation (CV, %) for the contents of ascorbic acid (AA), sinigrin (SIN), total phenolics (TP) and nitrates (NO3 −) evaluated in the 11 accessions of wall rocket across the four environments tested, and overall rank. Contents are expressed as mg 100 g−<sup>1</sup> FW, and the levels of total phenolics are expressed as equivalents of chlorogenic acid (CAE). *n* = 4.

#### 3.2.3. Correlation between Nutritional Traits

All Spearman rank phenotypic correlations among traits were highly significant (Table 4). A positive correlation was found between the percentage of moisture and the levels of NO3 − in the tissue (ρ = 0.498). On the contrary, both traits were negatively correlated with the content of bioactive compounds. Among them, the highest correlation coefficients were obtained among moisture and

bioactive compounds, while the correlations with the content in NO3 <sup>−</sup> were ρ < −0.56. On the contrary, positive correlations were found among bioactive compounds, with the content of AA–TP having the highest coefficient (ρ = 0.921) (Table 4).

**Table 4.** Phenotypic (above the symmetry axis, *n* = 44) and environmental (below the symmetry axis, *n* = 213) Spearman rank correlations (ρ) between the percentage in moisture, ascorbic acid (AA), total phenolics (TP), sinigrin (SIN), and nitrates (NO3 −) determined in the accessions of wall rocket.


ns, \*, \*\* and \*\*\* indicate no significant or significant at *p* < 0.05, 0.01 and 0.001, respectively.

Similar results were obtained for the analysis of environmental correlations, with lower coefficients being generally found in this case (Table 4). The greatest decrease was found for the moisture –NO3 − correlation (ρ = 0.237). A high reduction in the ρ coefficient comparing environmental vs. phenotypic correlations was also determined for the AA–NO3 <sup>−</sup> content (−0.215 vs. −0.555, respectively). Finally, a moderate environmental correlation was found for the AA–TP (ρ = 0.649) (Table 4).

#### **4. Discussion**

Wall rocket is a common weed in the Mediterranean regions. However, it is also appreciated as a wild edible vegetable [38] and therefore has the potential to become a new crop. The present work aimed at studying the effect of different cultivation environments on the nutritional quality of pre-selected accessions of wall rocket. Due to the close phylogenetic relationships and similarities in terms of growth and commercial use, wall rocket may be potentially cultivated in similar environments as the already established rocket crops. Thus, it might be produced in field or greenhouse systems, although soil-less systems may also be available [23,39]. The greenhouse and field environments differ in several factors such as temperature, light intensity, air humidity or the effect of rains, among others [40]. These factors can also differ between growing cycles. Environmental factors have been proven to affect the leaf morphology of wall rocket [29]. In addition, environmental factors have been proven to influence the accumulation of secondary metabolites and nitrates in different crops [41–43]. Thus, the quality of wall rocket in terms of bioactive compounds and nitrates may be affected as well.

As phytochemicals, the contents of AA, TP, SIN and NO3 − were evaluated. Only the reduced form of vitamin C was evaluated since we previously concluded that the AA form represented around 90% of the total vitamin C in wall rocket materials [44]. Differences between wall rocket and the reference materials were moderate and only significant for the content of AA and the percentage of moisture. Thus, the traits analyzed in the present work were not useful enough to clearly separate among them. These results are in contrast to our previous work evaluating the leaf morphology in the three species, where materials were clearly differentiated by the shape and size of leaves [29]. In consequence, the exploitation of distinctiveness in wall rocket with a commercial purpose could focus on other traits such as visual traits. As exception, wall rocket had as a distinctive trait the accumulation of SIN as main glucosinolate, since this compound was neither determined in *E. sativa* nor *D. tenuifolia* materials. These findings are in agreement with previous works comparing the glucosinolate profile of the three species [23,45]. Nevertheless, different profiles in other wall rocket materials, characterized for the absence of SIN, have been identified as well [45,46]. Discrepancies may correspond to differences related to the origin of materials, inter-subspecies differences—i.e., the analysis of *D. erucoides* subsp. *erucoides* or subsp. *longisiliqua* materials—as suggested by D'Antuono et al. [45], or they may even correspond to inter-specific crosses.

In a second analysis, the 11 accessions of wall rocket were compared among systems under two different growing periods. It has been previously observed that the growing period in a short-cycle species such as wall rocket can affect its morphology [29,47]. Moreover, the accumulation of different compounds such as nitrates can be affected by the growing period as well, as Bonasia et al. [48] described for wild rocket. Our results showed a low contribution of the accession effect to the total sum of squares, together with a general absence of significance. This result was an indicator of low nutritional variation among the accessions analyzed. The lack of variation may be due to the close geographic origin of materials, as original populations were collected in a relatively small territory. On the other hand, the low variation found may be due in part to a high intra-population variability considering that no homogenization efforts have been addressed, as suggested by the residual effect. However, a commercial cv. was included, which is assumed to be obtained from different populations, presumably with a different origin, and to show a high degree of uniformity. Thus, these low differences may also correspond to the low variation of wall rocket as a species in terms of bioactive compound contents and NO3 − accumulation capacity. Nevertheless, some accessions could be considered in new programs as promising materials, including, for example, accessions DER055-1 and DER006-1. The latter was, in fact, also selected by its morphology as a promising material [29].

A comparison of different environments demonstrated a high effect on the final phytochemical composition of the product. Plants growing in the field during the winter cycle experienced the most extreme environment, both considering the two systems (greenhouse vs. field during the winter cycle) and the different growing periods (winter vs. spring in the field system). High adverse conditions took place during this growing cycle with remarkable low temperatures, so plants were subjected to high abiotic stresses. Abiotic stress increases the levels of reactive oxygen species, causing oxidative stress in plants [49]. As part of the defense response to this possible oxidative damage in plant tissues, the content of secondary metabolites such as AA and phenolic compounds can increase as well. Oh et al. [26] found that plants of lettuce exposed to cold stress increased the accumulation of protective metabolites by the activation of genes involved in their biosynthesis. In the same way, it has been observed among *Brassicaceae* that plants accumulate a greater content of glucosinolates when they grow under non-optimal temperatures [27], as our results suggest. In particular, it has been observed that a decrease in temperature can increase the accumulation of glucosinolates [50,51]. By contraposition, the filed-winter environment resulted in the accumulation of the lowest content in NO3 −, which is also of interest for a commercial purpose. Light intensity has been positively correlated to nitrate reductase activity and a consequent lower accumulation of NO3 − [48]. This season-dependence explains the different maximum limits established for lettuce and rocket crops in the European Union [22]. However, our experiment was conducted in two consecutive cycles with similar light exposure, and therefore light differences may be not be high enough to affect the reductase activity. Thus, other physiological processes may be related to this different accumulation.

The field–spring environment also provided a product accumulating high levels of pytochemicals of interest (AA, SIN, TP) and low levels of NO3 −. The content of AA was significantly higher than previously described by Salvatore et al. [52], who found that mature, wild plants of wall rocket on average accumulated 13.9 mg AA 100 g−1. Values were also comparable or even greater than levels of vitamin C (VC) previously described for wild rocket by Spadafora et al. [10] (22 mg 100 g−<sup>1</sup> FW) or Durazzo et al. [24] (21–81 mg 100 g−<sup>1</sup> FW). The accumulation of SIN, however, did not reach the levels previously described for wall rocket by Di Gioia et al. [23], with an average level of 11.6 mg g−<sup>1</sup> DW. In addition, the content of NO3 −, although greater in this cycle, was below the maximum limit of 7000 mg kg−<sup>1</sup> established for the commercialization of rocket crops harvested before April [22]. Comparable or slightly lower levels have been previously found in cultivated rocket crops [43], usually ranging between 3500–4500 mg kg−<sup>1</sup> but reaching 7349 mg kg<sup>−</sup>1. Levels described for non-cultivated wall rocket would be between 2000–2500 mg kg−<sup>1</sup> [53,54], suggesting that the species tends to increase this accumulation under cultivated conditions. Finally, the increased percentage of moisture was reflected in a greater visual appearance and less coriaceous aspect—traits that are essential for consumer acceptance.

In contrast, the greenhouse system may not be adequate for the commercial production of wall rocket according to our results. Heated greenhouses are used to provide a more appropriate and stable temperature for plant growth compared to field conditions, but also affect other factors such as wind, air humidity, solar radiation, the effect of rains and storms or crop management [40]. Thus, our results suggest that growing wall rocket under greenhouse conditions would enhance the homogenization of most of the traits evaluated but provide a product of lower quality, especially in terms of AA and TP contents. Moreover, plants in this system accumulated high levels of NO3 −, which in the spring cycle exceeded the limits established [22] and made the product obtained not commercially acceptable.

Finally, phenotypic coefficients of correlations were greater than the environmental ones. These results indicated that the different factors evaluated had a similar effect on materials on average; however, the residuals among those traits had a lower correlation. The high correlation between AA and TP may be explained by their antioxidant function in the plants and their accumulation in response to environmental stresses [55]. However, both AA and TP had lower correlations with the content of SIN. As with AA and phenolic phytochemicals, the accumulation of glucosinolates in plant tissues is part of the plant response mechanism against abiotic stress conditions and can therefore be affected by environmental factors such as light intensity, season or fertilization [16,27,41], but it is also highly related to the biotic stress by pests and pathogens [16,27]. The combined effect of both biotic and abiotic stress could be responsible for this lower correlation. On the other hand, a negative correlation between the antioxidant phytochemicals and the percentage of moisture was found. This may correspond to a concentration effect in the tissues [41], although it could be also related to the plant behavior and defense system against cold stress. Król et al. [56] found that leaves of grapevine developed under cold stress reduce their percentage of moisture, although the total phenolics were also decreased; on the contrary, Oh et al. [26] found that the exposure of lettuce to chilling conditions increased the total phenolics against oxidative damage. Moreover, the negative correlation with these compounds and the content of NO3 − has been previously observed [48], in agreement with our results. Finally, a positive correlation between the percentage of moisture and the levels of NO3 − was found, as extensively observed in many species [57]. This positive correlation between both traits is related to the osmotic effect of NO3 − ions, meaning that its accumulation increases the capacity of tissues to retain water [20,57].

#### **5. Conclusions**

This work aimed to evaluate the most adequate conditions for the establishment of wall rocket as a new crop. Our results indicated that growing this vegetable under field conditions would enhance the accumulation of AA and TP in the final product. Moreover, the accumulation of NO3 − was reduced in this environment compared to the greenhouse system. Among all environments, the field–winter system resulted in the lowest content in NO3 −, which is a trait of high interest for a commercial purpose, but also the lowest percentage of moisture, with this reducing the visual quality and presumably consumer acceptance. Thus, our results suggest that stressful conditions such as low temperatures in winter may not be adequate for commercial production in an unprotected field. In this sense, the use of crop thermal blankets may reduce such stress.

The low variability of the phytochemicals among accessions of wall rocket may reflect the low genotypic differences among the selected materials or at a species level. Moreover, the levels found in wall rocket did not clearly differ from the reference crops. As an exception, wall rocket had, as a distinctive trait, the presence of sinigrin as its main glucosinolate unlike the other species, as previously described [23,45]. These results increase the information available for the species and are of relevance for breeding programs and future commercial strategies, suggesting that the promotion of the distinctiveness of this new crop among the other rocket crops should focus on other aspects such as visual quality or flavor instead of bioactive traits.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4395/9/12/858/s1, Table S1: Geographical location of the original ten wild populations of wall rocket.

**Author Contributions:** Conceptualization, A.F., A.R.-B. and J.P.; data curation, A.M.A.-M., C.G.-R. and K.A.; formal analysis, A.F., C.G.-R. and J.P.; investigation, A.M.A.-M., C.G.-R. and K.A.; methodology, A.F., C.G.-R. and J.P.; project administration, A.F., C.G.-R., and J.P.; resources, A.F., A.R.-B. and J.P.; supervision, A.F., A.R.-B. and J.P.; validation A.M.A.-M., and C.G.-R.; visualization, A.F., A.R.-B., C.G.-R., and J.P..; writing—original draft preparation, A.F., C.G.-R. and J.P.; writing—review and editing, A.F., A.R.-B. and J.P.

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

**Acknowledgments:** C.G. is grateful to the Ministerio de Educación, Cultura y Deporte of Spain (MECD) for the financial support by means of a predoctoral FPU grant (FPU14-06798). The authors also thank Ms. E. Moreno and Ms. M.D. Lerma for their help in the field tasks.

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

#### **References**


© 2019 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 (http://creativecommons.org/licenses/by/4.0/).

*Article*
