Protein Hydrolysates and Mo-Biofortiﬁcation Interactively Modulate Plant Performance and Quality of ‘Canasta’ Lettuce Grown in a Protected Environment

: Since the use of protein hydrolysates (PHs) enhances overall plant performance and quality of vegetables, they might be considered as a toll to face a number of concerns essentially associated to the growing request of premium quality foodstuff realized in agreement with eco-friendly agriculture practices. Molybdenum (Mo) is considered a fundamental trace element for human body. Thus, its shortage determines several disorders mainly related to neurological lesion and esophageal cancer. Biofortiﬁcation of fruiting and leafy vegetables is a promising tool to prevent Mo deﬁciency in the human diet. The current study was carried out to assess the interactive effect of plant-derived PHs and Mo dosage (0.0, 0.5, 3.0, and 6.0 µ mol L − 1 ) on yield, morphology, nutritional and functional features, and nitrogen indices of ‘Canasta’ lettuce. Head fresh weight (HFW), head height (HH), ascorbic acid, K, Mg, total chlorophyll, as well as nitrogen use efﬁciency (NUE) index were positively correlated to PHs application. Furthermore, ascorbic acid and total chlorophyll were also improved by Mo supply. A great improvement in terms of soluble solid content (SSC), total sugars, total phenolic, carotenoids, Mo and N concentrations, nitrogen uptake efﬁciency (UE), and nitrogen physiological efﬁciency (PUE) indices was recorded when PHs application was combined with the highest Mo dosage (6.0 µ mol L − 1 ). Consequently, our results suggest that Mo-biofortiﬁcation and PHs application can positively modulate ‘Canasta’ lettuce plant performance and quality.


Introduction
Nowadays, the agriculture production sector must face both the multiple challenges to serve an increasing global population, and the pressing need to improve food quality and reduce the impact of cultivation on human health and environment [1]. In this scenario, the use of plant biostimulants is a technology with innovative application potentiality [2]. Several authors [3][4][5][6][7][8][9][10][11] have appraised that plant biostimulants are an eco-friendly means to enhance plant production and quality under favourable or unfavourable growth conditions. In this regard, the protein hydrolysates (PHs), represent a promising group of plant biostimulants [12]. Furthermore, PHs, when derived from enzymatic hydrolysis, are suitable plant biostimulants for an organic farm management [13]. PHs are either applied by foliar spraying and/or by root fertigation. As reported by Fernández and Eichert [14], when furnished via foliar spray, PHs are absorbed by (i) cuticle, (ii) epidermal cells and (iii) stomata, and finally reach the foliar mesophyll. Several studies [15][16][17][18] have confirmed that is imperative to appraise methods and doses. Based on the aforesaid evidence, the aim of our study was to assess the interactive influence of PHs application and Mo supply dose on yield, morphology, as well as the nutritional and functional features of an established lettuce variety in a typical cultivation area.

Plant Genetic Material and Experimental Site
The study was performed at the Department of Agricultural, Food and Forestry Sciences of the University of Palermo (SAAF), sited in Palermo (latitude 38°12′ N, longitude 13°36′ E, altitude 65 m). The trial was carried out in a tunnel 30.0 × 5.0 × 2.0 m 3 , length × width × height, respectively. The tunnel was covered with a transparent polyethylene film and equipped with a drip irrigation system. A data logger placed inside the tunnel collected daily minimum and maximum temperatures ( Figure 1). On 5 January 2020, 360 plug plants of 'Canasta' lettuce (Lactuca sativa L.) (Syngenta Seed, Basel, Switzerland), at the stage of four-five true leaves, were transplanted with 0.25 m and 0.20 m inter-rows and intra-rows spacing, respectively, making a density of 20 plants m −2 . During the entire cultivation period, all lettuce plant needs were assured in adherence to all the recommended cultivation practices [48]. The soil was basically sandy clay loam at pH 7.2, composed by 1.6% of total nitrogen and 3.2% of organic matter. All lettuce plants were harvested 55 days after transplant.

Plant-Derived Protein Hydrolysates Application and Molybdenum Biofortification
The protein hydrolysates (PHs) treatments were performed using TYSON ® (Mugavero fertilizers, Palermo, Italy), an organic biostimulant composed by 5% of total nitrogen (4.5% organic nitrogen), 25% of organic carbon and 31% of amino acids, obtained through enzymatic hydrolysis of soy plants at low temperature. PHs treatment was started one week after transplant, and it was accomplished weekly. Two doses of PHs were applied, 0 (control) or 3 mL L −1 (standard dose) and distributed via foliar spray. Four different doses of molybdenum (Mo) [0.0 (control), 0.5, 3.0 and 6.0 µmol Mo L −1 ], in form of sodium molybdate (Na2MoO4), were supplied via foliar spray. Mo fertilizations started one week after transplant and were applied every ten days. On 5 January 2020, 360 plug plants of 'Canasta' lettuce (Lactuca sativa L.) (Syngenta Seed, Basel, Switzerland), at the stage of four-five true leaves, were transplanted with 0.25 m and 0.20 m inter-rows and intra-rows spacing, respectively, making a density of 20 plants m −2 . During the entire cultivation period, all lettuce plant needs were assured in adherence to all the recommended cultivation practices [48]. The soil was basically sandy clay loam at pH 7.2, composed by 1.6% of total nitrogen and 3.2% of organic matter. All lettuce plants were harvested 55 days after transplant.

Plant-Derived Protein Hydrolysates Application and Molybdenum Biofortification
The protein hydrolysates (PHs) treatments were performed using TYSON ® (Mugavero fertilizers, Palermo, Italy), an organic biostimulant composed by 5% of total nitrogen (4.5% organic nitrogen), 25% of organic carbon and 31% of amino acids, obtained through enzymatic hydrolysis of soy plants at low temperature. PHs treatment was started one week after transplant, and it was accomplished weekly. Two doses of PHs were applied, 0 (control) or 3 mL L −1 (standard dose) and distributed via foliar spray. Four different doses of molybdenum (Mo) [0.0 (control), 0.5, 3.0 and 6.0 µmol Mo L −1 ], in form of sodium molybdate (Na 2 MoO 4 ), were supplied via foliar spray. Mo fertilizations started one week after transplant and were applied every ten days.

Yield, Nutritional and Bioactive Attributes of Lettuce Plants
All the analyzes on the agronomic and functional features of the 'Canasta' lettuce plants were carried out on five plant samples, randomly selected from each replicate. Measurements of head fresh weight (HFW), head height (HH), stem diameter (SD), and number of leaves (NL) were taken immediately after plant harvest. To assess soluble solids content (SSC), 120 g of leaf samples were juiced and clarified; the measure of SSC was Agronomy 2021, 11, 1023 4 of 18 acquired by a refractometer (MTD-045 nD, Three-In-One Enterprises Co. Ltd. New Taipei, Taiwan). SSC was expressed as Brix • . To determine the head dry matter (HDM) percentage, the samples were placed in a thermo-ventilated oven at 105 • C till reaching steady weight.
The HDM values were expressed as percentage.
Immediately after plant harvest, color coordinates were recorded through a colorimeter (Chroma-meter CR-400, Minolta corporation Ltd., Osaka, Japan) on five undamaged leaves arbitrarily designated from each replicate. CIELab colour coordinates a*, b* and L * indicate, respectively, the red-green axis, the yellow-blue axis and the lightness.
Total phenolic concentration was assessed following the Folin-Ciocalteu method [49]. Briefly, the leaf sample was mixed with deionized water, sodium carbonate and Folin-Ciocalteau reagent, then the solution was incubated at ambient temperature for 30 min. The 750 nm wavelength was used to measure the mixture's absorbance with the means of a spectrophotometer. After a conversion, total phenolic concentration was expressed as µg g −1 fresh weight.
Sugar concentration in lettuce plants was evaluated as reported by Serna et al. [50]. Briefly, three grams of the plant sample were centrifuged at 15,000× g for 20 min with 10 mL of distilled water. Then, a 10 µL aliquot was analyzed by high-performance liquid chromatography (HPLC). Results were shown as a percentage of fresh weight.

Plant Pigments and Mineral Profile
Total chlorophyll and carotenoid concentrations in leaf tissues were evaluated as reported by Costache et al. [51]. Briefly, 1 g of fresh leaf sample was mixed with methanol and homogenized, then the measure was accomplished with a spectrophotometer. Total chlorophyll was expressed as mg 100 g −1 fresh weight, whereas carotenoid concentration was shown as µg g −1 fresh weight.
Potassium (K), calcium (Ca), and magnesium (Mg) concentrations were determined following the method of Morand and Gullo [52]. The phosphorous (P) concentration in leaf tissues was measured as described by Fogg and Wilkinson [53]. Total nitrogen (N) concentration was assessed following the Kjeldahl method. Molybdenum (Mo) concentration was appraised by inductively coupled plasma-mass spectrometry (ICP-MS) (Plasma Quant MS Elite, Jena, Germany), as detailed by Sabatino et al. [41]. All the mineral concentrations in leaf tissues were expressed as mg g −1 dry weight.

Statistics and Experimental Design
A randomized complete block design was adopted for this experiment, with each block replicated three times. Two doses of PHs (0.0 or 3.0 mL L −1 ) were combined with four doses of Mo (0.0, 0.5, 3.0 or 6.0 µmol Mo L −1 ) in a two factorial trial displaying eight treatments in total (2 PHs doses × 4 Mo doses). Each treatment included 15 plants, rendering a total of 360 plants. All data were analyzed by implementing the SPSS software v.20 (StatSoft, Inc., Chicago, IL, USA) package through a two-way analysis of variance (ANOVA), setting PHs and Mo doses as fix factors. A Tukey honestly significant difference (HSD) test (p < 0.05) was used to separate the means. Percentage data were converted via arcsin transformation prior ANOVA analysis (Ø = arcsin(p/100) 1/2 ).
To summarize all plant traits, a heat map was also provided through the online program package clustvis (https://biit.cs.ut.ee/clustvis/ (accessed on 15 January 2021).) with Euclidean distance as the similarity measure and hierarchical clustering with complete linkage.

Crop Yield and Biometric Features
ANOVA for HFW, HH, SD and NL showed no significant interaction between PHs and Mo treatments (Table 1). Irrespective of the Mo treatments, PHs significantly increased HFW. Regardless of the PHs application, Mo dosage did not significantly affect HFW of lettuce plants. Data on HH supported the trend described for HFW (Table 1). Disregarding Mo fertilization, PHs application did not significantly influence SD values. Conversely, regardless of the PHs treatment, SD was significantly higher in non-biofortified plants than in plants supplied with Mo (Table 1). ANOVA for NL did not display a significant influence of the main factors and of their interaction (Table 1).

Mineral Profile in Leaf Tissues
ANOVA analysis for P, K, Ca, and Mg did not reveal a significant interaction PHs × Mo (Table 2).
PHs application and Mo concentration did not significantly affect P and Ca concentration in plant tissues ( Table 2). Irrespective of the PHs treatments, Mo dosage did not influence leaf K concentration. Conversely, regardless of the Mo dosage, plants treated with PHs showed a higher K concentration than non-treated ones ( Table 2). Data on Mg concentration sustained the tendency described for K concentration ( Table 2).
Plants not treated with PHs and without Mo supply had the highest leaf nitrogen concentration followed by those treated with PHs and without Mo supply which in turn revealed a higher N concentration than those supplied with 0.5 µmol Mo L −1 and not treated with PHs. The lowest N concentration was assessed in plants from the combination PHs × 6.0 µmol Mo L −1 (Figure 2). Table 2. Effect of PHs treatment and Mo-biofortification on phosphorous (P), potassium (K), calcium (Ca) and magnesium (Mg) concentrations of 'Canasta' lettuce leaves grown in a protected environment. ANOVA and mean separation for plant N concentration showed a significant interaction PHs × Mo ( Figure 2). PHs application and Mo concentration did not significantly affect P and Ca concentration in plant tissues ( Table 2). Irrespective of the PHs treatments, Mo dosage did not influence leaf K concentration. Conversely, regardless of the Mo dosage, plants treated with PHs showed a higher K concentration than non-treated ones ( Table 2). Data on Mg concentration sustained the tendency described for K concentration ( Table 2).
ANOVA and mean separation for plant N concentration showed a significant interaction PHs × Mo ( Figure 2).

Nutritional and Bioactive Compounds
Statistical analysis for ascorbic acid and CIELab parameters displayed no significant interaction PHs × Mo (Table 3). Table 3. Effect of PHs treatment and Mo-biofortification on ascorbic acid and CIELab parameters of 'Canasta' lettuce grown in a protected environment.

Treatments
Ascorbic Acid Disregarding the Mo concentration, PHs treated plants showed a higher ascorbic acid concentration than untreated ones. Irrespective of PHs treatments, our outcomes revealed that augmenting the Mo concentration resulted in a significant increase in ascorbic acid leaf content (Table 3). Results on a* colour parameter showed that, irrespective of the PHs

Nutritional and Bioactive Compounds
Statistical analysis for ascorbic acid and CIELab parameters displayed no significant interaction PHs × Mo (Table 3). Table 3. Effect of PHs treatment and Mo-biofortification on ascorbic acid and CIELab parameters of 'Canasta' lettuce grown in a protected environment.

Treatments
Ascorbic Acid (mg g −1 fw) a* b* L* Values within a column followed by different letters are significantly different at p ≤ 0.05. NS, *** non-significant or significant at 0.001, respectively. a*: red-green axis; b*: yellow-blue axis; L*: lightness.
Disregarding the Mo concentration, PHs treated plants showed a higher ascorbic acid concentration than untreated ones. Irrespective of PHs treatments, our outcomes revealed that augmenting the Mo concentration resulted in a significant increase in ascorbic acid leaf content (Table 3). Results on a* colour parameter showed that, irrespective of the PHs treatment, Mo biofortification did not significantly influence a* coordinate (Table 3). Contrariwise, PHs significantly decreased a* coordinate (Table 3). ANOVA and means separation revealed that both PHs application and Mo concentration did not significantly affect b* and L* CIELab parameters. (Table 3).
The highest total sugars concentration was exhibited in plants from the combination PHs × 6.0 Mo (Table 3). Meanwhile, the lowest total sugars values were observed in plants not treated with PHs and not supplied with Mo and in those treated with PHs and without Mo supply (Table 4).
Plants from the combination PHs × 6.0 revealed the highest total phenolic content, followed by those not treated with PHs and supplied with the highest dosage of Mo. The lowest total phenolic concentration was registered in plants not treated with PHs and not supplied with Mo and in PHs treated plants and without Mo supply (Table 4).
Results on leaf total chlorophyll showed no interaction between PHs and Mo treatments ( Figure 4B). Irrespective of the Mo treatments, total chlorophyll concentration was higher in plants treated with PHs. Averaged over PHs treatment, total chlorophyll concentration increased with the increase of the Mo concentration ( Figure 4B).  Results on leaf total chlorophyll showed no interaction between PHs and Mo tr ments ( Figure 4B). Irrespective of the Mo treatments, total chlorophyll concentration higher in plants treated with PHs. Averaged over PHs treatment, total chlorophyll centration increased with the increase of the Mo concentration ( Figure 4B).

Nitrogen Indices
Statistical analysis for NUE did not display a significant interaction PHs × Mo ( Fig  5A). Regardless of the Mo treatments, plants supplied with PHs revealed a higher N values than non-treated ones. Averaged over PHs, Mo application did not influence NUE ( Figure 5A).

Nitrogen Indices
Statistical analysis for NUE did not display a significant interaction PHs × Mo ( Figure 5A). Regardless of the Mo treatments, plants supplied with PHs revealed a higher NUE values than non-treated ones. Averaged over PHs, Mo application did not influence the NUE ( Figure 5A). Agronomy 2021, 11, x FOR PEER REVIEW 10 of 18 Statistics on UE underlined a significant interaction PHs × Mo ( Figure 5B); the highest UE index was obtained in plants supplied with PHs and not treated with Mo, followed by those recorded in plants supplied with PHs and treated with a dosage of 0.5 µmol Mo L −1 . Inversely, the combination PHs × 6.0 µmol Mo L −1 showed the lowest UE values ( Figure 5B).
ANOVA for PUE highlighted a significant interaction between PHs and Mo ( Figure 5C). The highest PUE values were recorded in the PHs × 6.0 µmol Mo L −1 combination, followed by those collected in the PHs × 3.0 µmol Mo L −1 combination. However, plants not treated with PHs and supplied with 0.5 µmol Mo L −1 did not significantly differ in terms of PUE neither from plants non treated with PHs and supplied with 3.0 or 6.0 µmol Mo L −1 , nor from those treated with PHs and supplied with 3.0 µmol Mo L −1 . The lowest PUE values were observed in PHs treated plants and not supplied with Mo ( Figure 5C).

Heat Map Analysis of All Recorded Plant Traits
A heat map analysis of all plant traits was carried out to reveal a chromatic assessment of the PHs and Mo treatments on plant traits. The heat map displayed two dendrograms, one situated on the top and called Dendrogram 1, and another placed on the left side, named Dendrogram 2 ( Figure 6). distinct letters significantly differ at p ≤ 0.05. NS, *** non-significant or significant at or 0.001, respectively.
Statistics on UE underlined a significant interaction PHs × Mo ( Figure 5B); the highest UE index was obtained in plants supplied with PHs and not treated with Mo, followed by those recorded in plants supplied with PHs and treated with a dosage of 0.5 µmol Mo L −1 . Inversely, the combination PHs × 6.0 µmol Mo L −1 showed the lowest UE values ( Figure  5B).
ANOVA for PUE highlighted a significant interaction between PHs and Mo ( Figure  5C). The highest PUE values were recorded in the PHs × 6.0 µmol Mo L −1 combination, followed by those collected in the PHs × 3.0 µmol Mo L −1 combination. However, plants not treated with PHs and supplied with 0.5 µmol Mo L −1 did not significantly differ in terms of PUE neither from plants non treated with PHs and supplied with 3.0 or 6.0 µmol Mo L −1 , nor from those treated with PHs and supplied with 3.0 µmol Mo L −1 . The lowest PUE values were observed in PHs treated plants and not supplied with Mo ( Figure 5C).

Heat Map Analysis of All Recorded Plant Traits
A heat map analysis of all plant traits was carried out to reveal a chromatic assessment of the PHs and Mo treatments on plant traits. The heat map displayed two dendrograms, one situated on the top and called Dendrogram 1, and another placed on the left side, named Dendrogram 2 ( Figure 6).  phenolic, SSC, ascorbic acid, carotenoids, total sugars, and total chlorophyll. The group on the left comprised untreated × 0.0 and non-treated × 0.5 combination. Within this cluster, PHs untreated × 0.0 µmol Mo L −1 combination is clearly separated by higher SD, N, HDM, UE, Mg, HFW, and NUE and lower a*, P, HH, Ca, PUE, Mo, total phenolic, SSC, ascorbic acid, carotenoids, total sugars, and total chlorophyll. Meanwhile, the grouping on the right contained the PHs untreated × 3.0 µmol Mo L −1 and the PHs untreated × 6.0 µmol Mo L −1 combinations. Within this cluster, the PHs untreated × 3.0 µmol Mo L −1 combination was parted by higher SD, N, b*, a*, L*, NL, P, Mg, and Mo and lower HDM, HFW, NUE, Ca, total phenolic, carotenoids, total sugars, and total chlorophyll.
On the right side of the Dendrogram 1 two clusters were identified, the first on the left comprised PHs treated × 0.0 µmol Mo L −1 and PHs treated × 0.5 µmol Mo L −1 combinations separately from the PHs treated × 3.0 µmol Mo L −1 and PHs treated × 6.0 µmol Mo L −1 combination, which had, particularly, higher SD, N, b*, HDM, UE and L*, but lower NL, HFW, NUE, PUE, Mo, total phenolic, SSC, ascorbic acid, carotenoids, total sugars, and total chlorophyll. The cluster on the left included the PHs treated × 0.0 µmol Mo L −1 combination. In this cluster, the PHs treated × 0.0 µmol Mo L −1 combination is noticeably separated by lower NL, P, HH, Ca, PUE, Mo, total phenolic, SSC, ascorbic acid, carotenoids, total sugars, and total chlorophyll, whereas the cluster on the right includes PHs treated × 3.0 µmol Mo L −1 and PHs treated × 6.0 µmol Mo L −1 combinations. Inside this cluster, the PHs treated × 3.0 µmol Mo L −1 combination is separated by lower L*, P, HH, PUE, total phenolic, SSC, ascorbic acid, carotenoids, total sugars, and total chlorophyll, and higher SD, N, b*, HDM, UE, a*, NL, K, Mg, HFW, NUE, and Ca. The clusters in Dendrogram 2 evidently point out the differential effects of PHs application and Mo supply.

Discussion
In recent years, PHs have acquired importance as plant biostimulants owing to their capacity to enhance the seed germination, yield, and quality of vegetables [2]. Concomitantly, plant mineral nutrition organization, including the supply of trace elements such as molybdenum, is a key pre-harvest aspect for yield and quality improving many vegetable crops. In the present research, the possible use of PHs and Mo biofortification, alone or combined, to improve yield and quality of lettuce was apprised. The study revealed that PHs significantly increased HFW and HH traits. These results are coherent with those listed by Di Mola et al. [54] who, by studying the influence of plant-based biostimulants on agronomical, physiological, and qualitative responses of baby rocket leaves under varied nitrogen levels, found that legume-derived PHs positively affected yield trait. Our outcomes are also in line with those indicated by Consentino et al. [10] in celery. There are also reports that PHs have a biostimulant role via the variation of plant molecular and physiological mechanisms that elicit growth and plant productivity [4,55]. PHs effects on plants comprise carbon and nitrogen metabolism stimulation. Furthermore, as reported by a number of authors [3,13,20], PHs have a significant role in the regulation of plant nitrogen uptake, via key enzymes included in the nitrogen assimilation mechanism, and in the regulation of the activity of three enzymes involved in the tricarboxylic acid cycle. Moreover, as specified by Colla et al. [17] and Colla et al. [20], bioactive peptides included in PHs could also interact with plant hormonal actions. Numerous researches have revealed that PHs can stimulate hormone-like activities (gibberellins and auxin), encouraging plant organ growth, and consequently crop yield [15][16][17][18]. In this study, the improved yield of plants treated with PHs could be also attributed to the certainty that PHs TYSON ® contains tryptophan (forerunner of indole−3-acetic acid) which determines shoot and root expansion in plants. Data indicated that although Mo biofortification did not affect HFW, HH, and NL, but it significantly influenced SD. These findings are consistent with those of Moncada et al. [40] on lettuce and with those of Biacs et al. [56] and Vieira et al. [57] on carrot and common bean.
Plant N concentration indicated that the PHs application reduced N accumulation in leaf tissue. These findings concur with those reported by Liu and Lee [58], who stated that amino acids mix treatments significantly reduced nitrate accumulation in several leafy vegetables. Moreover, data presented are in line with that reported by Tsouvaltzis et al. [59] who found that lettuce nitrate build-up reduces as PHs dose increases. PHs action in limiting the relevant accumulation of nitrogen in plants might be related to the huge regulation attitude of many metabolic pathways involved in the metabolism of nitrogen [2]. Additionally, other authors [20] pointed out that PHs with a high level of free amino acids guide to stout phloem loaded with amino acids which, accordingly, diminish root nitrate absorption and accumulation. Thus, as suggested by Consentino et al. [10] in celery, the reduction of N concentration in plants treated with PHs could be attributed to the use of their own nitrogen deposits without supplemental nitrogen uptake. Generally, the imperative function of amino acids is the modulation of a number of mechanisms and metabolic pathways related to the nitrogen metabolism in plants [55,58]. The present study revealed that N leaf concentration decreased as Mo dosage increased. These findings are coherent with those obtained by Moncada et al. [40], who report that a higher Mo dosage is effective in N leaf concentration reduction in lettuce, escarole and curly endive plants. Our results support those of Zhen et al. [60] who found that Mo supply reduces N content in lettuce plant tissues. There are reports of a negative relation between Mo dosage and N concentration in spinach and poinsettia plants [61][62][63]. As stated by Schwarz [64], molybdenum cofactors (Moco) contribute to the active site of nitrate reductase which has an imperative function in nitrate absorption and may enhance the nitrogen use efficiency.
PHs did not significantly affect P and Ca leaf concentration. On the contrary, PHs application resulted an effective tool to enhance K and Mg concentration. This is in accordance with the findings of Rouphael et al. [5], who indicated that plant-derived PHs meaningfully augment K and Mg concentrations in tomato fruits, and it also concurs with the results of Consentino et al. [10] who found that plant or animal-derived PHs significantly improve K and Mg concentrations in celery, although they do not affect P and Ca concentration. Furthermore, data on mineral profile revealed that Mo-biofortification did not significantly affect P, K, Ca and Mg concentration in lettuce.
PHs application enhanced Mo concentration in plant tissues. This output is in line with other studies [2,15,20] which report that PHs enhance plant macro-and micronutrient uptake. Additionally, while PHs non-treated plants showed a decrease in Mo concentration when Mo concentration in the nutrient solution was higher than 3.0 µmol L −1 , PHs treated plants revealed a positive relation between Mo supply dosage (up to 6.0 µmol Mo L −1 ) and Mo concentration in leaf tissues. Thus, we may hypothesize that PHs applications improves plant Mo tolerance.
PHs significantly increased ascorbic acid concentration. This is in accordance with other studies, which found that foliar application of plant-derived PHs enhances ascorbic acid in tomato and celery [2,5,10]. The ascorbic acid concentration could be linked to the improved mineral uptake of plants treated with PHs, which could in turn enhance the synthesis of some amino acids, like tyrosine and phenylalanine [5]. In our study, Mo supply positively affected ascorbic acid content. This is consistent with the report of Moncada et al. [40] on lettuce, escarole, and curly endive. As described by Valenciano et al. [65], ascorbic acid concentration can be influenced by Mo supply. In this regard, the intensification of enzymes responsible for ascorbic acid oxidation (phenol oxidase and peroxidase) is associated with Mo plant concentration. Contrariwise, Sabatino et al. [41] studying the interactive effect of genotype and Mo supply on tomato yield and fruit quality found a decrease in ascorbic acid concentration with an increase in Mo level. This contrasting result might be related to the different Mo plant organ distributions among plant species [66].
Colour of vegetables is a fundamental visual quality feature that often influences consumer's choice. Colour parameters revealed that PHs application increased leaf greenish, whereas it did not significantly affect b* and L* colour traits. This is consistent with the findings of Consentino et al. [10]. These results are partially coherent with those of other authors [67,68] who reported that PHs treatment does not significantly influence colour traits in perennial wall rocket. In this study, Mo biofortification did not affect colour traits. Our results partially differ from those of Moncada et al. [40], who found that Mo-enrichment does not affect L* coordinate, but significantly influences Chroma and Hue angle.
HDM indicated that PHs application combined with Mo biofortification (up to 3.0 µmol L −1 ), enhanced HDM. In contrast, PHs application combined with the highest Mo dosage (6.0 µmol L −1 ) negatively affected HDM parameter. This is in accordance with the findings of Sabatino et al. [41] who revealed that Mo supply decreases fruit water content in tomato. Moreover, the results presented are consistent with those of Moncada et al. [40], who stated that Mo-biofortification significantly increases head dry matter in lettuce, escarole and curly endive. Similar results are also reported by Boertje [69], Valenciano et al. [65] and Randall et al. [70] on lettuce, chickpea and grain, respectively. However, it can be hypothesized that the higher mineral uptake efficiency of PHs treated plants combined with the highest Mo level caused a HDM reduction, which in turn can be considered a plant toxicity signal. PHs and Mo supply significantly interacted enhancing total sugars. In particular, plants from the PHs × 6.0 µmol Mo L −1 combination had the highest sugar content. This is in accordance with the finding of Carillo et al. [71] who, by investigating the effect of PHs biostimulant and nitrogen fertilization level on morphological and physiological traits of spinach grown in a greenhouse, found that irrespective of the nitrogen fertilization level, PHs significantly increased sucrose concentration in spinach. Furthermore, the study underlined that Mo supply improved total sugars in 'Canasta' lettuce up to the highest dosage (6.0 µmol Mo L −1 ). Concurrently, the best results were observed in plants treated with PHs and supplied with the highest Mo dosage. This is partially consistent with the reported of Liu et al. [72] who, studying the effect of Mo-biofortification on nutrition, quality and volatile compounds of strawberry fruits, found that total sugars increased when Mo supply ranged from 0 to 135 g ha −1 , but decreased at the highest dosages. Thus, considering that a dose of 6.0 µmol Mo L −1 is not harmful for several plant physiological activities in lettuce and since Xi et al. [73] found that carbon dioxide supplementation could enhance total sugars concentration with a higher net photosynthesis, we may assume that Mo and carbon dioxide could increase total sugars concentration via the modulation of photosynthesis.
In addition, the study demonstrated that Mo-biofortification increased total phenolics in lettuce. However, the highest concentration of total phenolics was recorded in PHs treated plants enriched with 6.0 µmol Mo L −1 . There are reports that PHs plant or animal derived, increase total phenolic content in celery [10]. Our outcomes also agree with those of Colla et al. [2] who stated that both amino acids and peptides increase phenolic concentration in banana plants and they are also coherent with those reported by Sabatino et al. [41], who stated that Mo-biofortification increases polyphenolic content in tomato. A similar increase was also reported by Bergmann [74] and Gupta [75] in tomato and cauliflower. Thus, it seems that both PHs and Mo supply have a synergistic effect on total phenolics synthesis and accumulation.
PHs application and Mo supply improved carotenoids concentration. Similar results were observed in another study on the response of baby rocket to a legume-derived PHs [54]. We also highlighted that Mo-enrichment improved carotenoid concentration in lettuce.
PHs treatment increased total chlorophyll. There are reports that PHs determine an increase in total chlorophyll in baby rocket and lettuce [54,76]. This is perhaps due to the high amino acid increase measured in plants subjected to plant-derived PHs, which in turn supports the increase in total chlorophyll concentration and net photosynthesis rate [77]. Increasing Mo level corresponded a total chlorophyll upsurge. This is in line with the finding of Liu et al. [72], who report a chlorophyll increase with a Mo increase up to 135 g ha −1 . This suggest that, in 'Canasta' lettuce, a Mo dose of 6.0 µmol L −1 did not negatively affect the physiological mechanisms involved in chlorophyll synthesis. These results are also coherent with those of Yu et al. [78] and Zhang et al. [79] who declared a positive relation between Mo dose and chlorophyll concentration in winter wheat and Chinese cabbage.
PHs application positively affected NUE indices. This is in line with the results by Di Mola et al. [80] who investigated the effect of plant-based biostimulant on NUE indices and crop performance of lamb's lettuce and baby spinach. The current results also underlined that Mo-biofortification enhanced the physiological use efficiency of 'Canasta' lettuce. Based on our acquaintance, this is the first investigation on the influence of Mobiofortification on nitrogen indices in a vegetable crop.

Conclusions
In the current research, the tested PHs significantly improved yield and yield-related features, nutritional and functional traits, as well as nitrogen indices. Simultaneously, Mobiofortification considerably enhanced nutritional and bioactive parameters, like ascorbic acid and SSC. Combining plant-derived PHs with 3.0 or 6.0 µmol Mo L −1 meaningfully improved plant nutritional and functional features, such as SSC, total sugars, total phenolics, carotenoids, plant Mo and N concentrations, and nitrogen indices as compared with untreated plants. We may also hypothesize the highest dosage of PHs had a buffer effect on plant Mo toxicity.