*Science-Based Approaches to Respond to COVID and Other Public Health Threats*

#### **Figure 1.**

*Status of clinical trials and stage: tocilizumab.*

**Table 2.**

*Comparing tocilizumab with other drugs employed for COVID-19 treatment.*

#### **Table 3.**

*Prominent publications reporting the treatment of COVID-19 using Tocilizumab.*


**4. Conclusion**

182 Etiology and Pathophysiology of Parkinson's Disease

finally, to be degraded by lysosomes (H. J. Lee et al., 2008). However, these results were obtained by using very high concentrations of recombinant α-synuclein and cationic

Importantly, recent data using cell-secreted α-synuclein have verified its impact on neuronal survival. Application of conditioned medium containing cell-secreted αsynuclein to neuronal cells induced cell death to the recipient cells (Emmanouilidou, Melachroinou et al., 2010). This toxic effect was concentration-dependent and was conferred synergistically by both oligomeric and monomeric α-synuclein species present in the conditioned medium. In this study, however, there was evidence of very low, if any, α-synuclein uptake by neuronal cells (Emmanouilidou, Melachroinou et al., 2010). Similarly, apoptotic death of neurons, both *in vitro* and *in vivo*, was observed upon their exposure to cell-derived extracellular α-synuclein (Desplats et al., 2009). Secreted αsynuclein, that was readily endocytosed by neurons, was transmitted from one cell to another thereby supporting the idea of a mechanism of pathological propagation in PD (Desplats et al., 2009). Cell-to-cell transfer of α-synuclein was also demonstrated using coculture systems (Hansen et al., 2010). In fact, this transfer did not require cell contact and was independent of the aggregation state of the protein. Fluorescently-labeled recombinant α-synuclein was uptaken by neuronal cells *in vitro* and *in vivo* via an endocytic mechanism. Altogether, these data demonstrated that endocytosed extracellular α-synuclein can be internalized by recipient cells, interact with the pool of intracellular α-

An alternative mechanism of neurodegeneration induced by extracellular α-synuclein may involve the initiation of neuroinflammatory responses. Microglia are resident immune cells that are sensitive to even minor disturbances in the homeostasis of the central nervous system (Soulet & Rivest, 2008). Activation of microglia results in a change in cell morphology (from a ramified to amoeboid shape) accompanied by alterations in surface receptor expression, production of reactive oxygen species (ROS) and release of chemokines and cytokines (Kim & Joh, 2006; Soulet & Rivest, 2008). There is increasing evidence suggesting that extracellularly added recombinant α-synuclein can trigger microglia activation which induces the production of various cytokines, such as IL1β and IL6, and inflammation-related enzymes (Su et al., 2008; Zhang et al., 2005). In fact, microglia activation has been shown to be one of the mechanisms by which α-synuclein induces dopaminergic neurodegeneration, rather than being an epiphenomenon following cell death (Zhang et al., 2007). Further dissection of the pathway of microglia activation, suggested that α-synuclein potentially binds to Mac-1 receptors which subsequently activate PHOX, a ROS-generating enzyme, to produce O2• ultimately leading to neurotoxicity. Importantly, microglia activation did not require internalization/phagocytosis of α-synuclein by microglial cells (Zhang et al., 2007).To this end, microglial prostaglandin E2 receptor subtype 2 (EP2) plays a critical role in αsynuclein-induced neurotoxicity partly by decreasing PHOX activation (Jin et al., 2007). Cell-produced α-synuclein also resulted in the activation of primary microglia, leading to the induction of inflammatory signaling pathways (E. J. Lee et al., 2010). It was suggested that α-synuclein-induced microglia activation involves the secretion of MMPs which in turn activate PAR-1 receptor (E. J. Lee et al.). Alternatively, recent data indicate that cellreleased α-synuclein can also be internalized by astrocytes thereby producing

liposomes to assist the uptake.

synuclein and seed aggregation (Hansen et al., 2010).

inflammatory responses both *in vitro* and *in vivo* (E. J. Lee et al., 2010).

α-Synuclein is genetically linked to PD. Maintenance of intracellular steady-state concentration of α-synuclein is considered to be a key challenge for neuronal homeostasis and total levels of the protein have been directly linked with PD pathogenesis. Importantly, Genome-Wide association Studies (GWAS) have provided a strong genetic link between αsynuclein and sporadic PD, and clearly point to α-synuclein as being one of the very few genetic loci consistently associated with disease progression. The physiological and aberrant functions of α-synuclein are still under investigation. However, cytoplasmic soluble oligomers/protofibrils of the protein appear to be one of the primary "suspects" in the pathogenesis of PD. Therefore, prevention of α-synuclein aggregation and intervention in the mechanisms of abnormal protein turnover appears to be a highly promising therapeutic target for the treatment of PD as well as other synucleinopathies.

From a therapeutic standpoint, it follows that enhancement of α-synuclein clearance via proteasomal or lysosomal degradation may represent a valid therapeutic intervention for PD. New evidence, suggests that α-synuclein is also physiologically secreted, and as such, it can exert as yet unknown paracrine effects in the brain. Still, the presence and exact levels of α-synuclein in the interstitial fluid in the brain remain to be clarified. Recent clinical observations have suggested that secreted α-synuclein may aggravate PD pathology via a mechanism that underlies cell-to-cell propagation of the protein. It is possible that a dynamic equilibrium between intracellular and extracellular α-synuclein exists, ensuring normal function of neuronal cells. In this respect, dysfunctions in the mechanism(s) regulating extracellular α-synuclein levels, such as mechanisms of secretion or extracellular clearance, may affect neuronal survival. Increases in extracellular α-synuclein may trigger the formation of toxic oligomers in neighbouring neurons and in the extracellular space, and result in inflammatory glia activation, utterly leading to a vicious cycle of neurodegeneration. Along these lines, compounds which block other signalling pathways switched on as a consequence of microglial activation which may ultimately lead to neuronal death- might also represent new targets for therapeutic intervention. Under this scope, manipulation of regulatory mechanisms that alleviate the extracellular α-synuclein "burden" represents a potential target for the development of novel treatment strategies for PD. It is obvious that α-synuclein can affect neuronal cell homeostasis in numerous ways and at multiple levels. The intrinsic complexity of the neuronal interface may suggest that its actions be considered within the context of non cell-autonomous models and thus be interpreted by taking into account that the nature of communication between brain cells is indeed very dynamic.


*Article* **Maturation and Post-Harvest Resting of Fruits Affect the Macronutrients and Protein Content in Sweet Pepper Seeds**

**Lidiane Fernandes Colombari 1, Larissa Chamma 1, Gustavo Ferreira da Silva 1,\*, Willian Aparecido Leoti Zanetti 2, Fernando Ferrari Putti 2 and Antonio Ismael Inácio Cardoso 1**

**Abstract:** There are few studies about the influence of fruit maturation and post-harvest resting on seed composition, which can be necessary for seedling development and future establishment. Thus, the objective of this study was to evaluate the effect of maturation and post-harvest resting of fruits on the macronutrient and protein content of sweet pepper seeds. The experimental design was a randomized block, with eight treatments, in a 4 × 2 factorial arrangement. The first factor was fruit maturation stages (35, 50, 65 and 80 days after anthesis), and the second, with and without post-harvest resting of the fruits for 7 days. The characteristics evaluated in seeds were the dry weight of one thousand seeds, macronutrient content, and content of albumin, globulin, prolamin and glutelin proteins. There were reductions in K, Ca and Mg content, and an increase in seed content of albumin, globulin and prolamins as a function of the fruit maturation stage. Post-harvest resting of the fruits provided higher Ca content and protein albumin in seeds. The decreasing order of macronutrients and protein content in seeds, independent of fruit maturation and resting stage of the fruits, was N > K > P > Mg > S > Ca, and albumin > globulin ≈ glutelin > prolamine, respectively.

**Keywords:** *Capsicum annuum* L.; maturation; nutritional quality; seed chemical composition

### **1. Introduction**

The main compounds that seeds store are carbohydrates, lipids, proteins, minerals, vitamins and plant hormones [1]. During plant development, nutrients are translocated to the fruits, and later, to seeds. The nutritional requirement of plants becomes intense in the reproductive phase, being more critical during seed formation, where the stored compounds will influence the formation of the embryo, and consequently, the metabolism, vigor and storage capacity of seeds [2,3]. Nitrogen is the most accumulated nutrient in seeds, followed by potassium and phosphorus, but it depends on the species [4,5]. As for protein reserves, two classes are mainly found in seeds: albumins and globulins, or prolamins [6].

Seed proteins are the main sources of nitrogen and sulfur, indispensable for the synthesis of new proteins, nucleic acids and secondary compounds [6–8]. Subsequently, the proteins that were synthesized and stored in the seeds will be broken down into amino acids for biosynthesis and energy generation, and, together with the other reserves, will be mobilized during germination for the development of the embryo, until the seedling manages to emerge above the ground and becomes photosynthetically active [6,9,10].

In the production of sweet pepper seeds, the fruits must be harvested at physiological maturity, when the maximum accumulation of dry matter occurs. If harvested before this point, the fruits must remain at rest after harvesting before extracting seeds for seven to ten days. This procedure allows early harvesting, reducing the time the fruits are exposed to unfavorable climatic conditions and the attack of pests and diseases [11,12]. During

**Citation:** Colombari, L.F.; Chamma, L.; da Silva, G.F.; Zanetti, W.A.L.; Putti, F.F.; Cardoso, A.I.I. Maturation and Post-Harvest Resting of Fruits Affect the Macronutrients and Protein Content in Sweet Pepper Seeds. *Plants* **2022**, *11*, 2084. https://doi.org/10.3390/ plants11162084

Academic Editors: Georgia Ouzounidou, Lord Abbey, Josephine Ampofo and Mason MacDonald

Received: 20 May 2022 Accepted: 26 July 2022 Published: 10 August 2022

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

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the post-harvest resting, the complete formation of the biochemical, morphological, and structural systems of seeds occurs [12,13].

During the maturation period and fruit post-harvest resting, the weight of seeds increases [11,12], probably because of translocation and accumulation of reserve compounds in seeds. However, there is no research showing the changes of this species in the chemical composition of seeds at each stage of fruit maturation and during fruit rest after harvest. Knowledge of these factors can help identify the best fertilization management approach, as it can indicate which nutrients are most important at each stage of plant development. Thus, the objective of this study was to evaluate the effect of maturation and post-harvest resting of fruits on the macronutrient and protein content of sweet pepper seeds.

### **2. Results**

### *2.1. Water Content, Dry Weight of One Thousand and Macronutrient Content of Seeds*

There was only a significant interaction between factors (maturation periods and post-harvest resting of the fruits) for nitrogen and sulfur content in the seeds. Therefore, for all other parameters, the factors were analyzed separately (Tables 1 and 2).

**Table 1.** Dry weight of one thousand seeds (DWTS), phosphorus (P), potassium (K), calcium (Ca) and magnesium (Mg) content in sweet pepper seeds as a function of post-harvest resting of fruits.

Means followed by the same letter do not differ from each other by the F-test at 5% probability.

**Table 2.** Nitrogen (N) and sulfur (S) content of sweet pepper seeds without and with post-harvest resting of fruits at each maturation stage.

Means followed by the same letter in each column and for each nutrient do not differ by the F-test at 5% probability.

Seed water content was adjusted to the decreasing linear model as a function of the maturation stage. The highest values were obtained at 35 DAA, and they reduced over time to a minimum of 56% and 53% at 80 DAA, without and with post-harvest fruit resting, respectively (Figure 1A).

A linear increase in dry weight of one thousand seeds (DWTS) was observed in fruit maturation, reaching a maximum value of 6.6 and 7.7 g at 80 DAA in seeds without and with post-harvest resting, respectively (Figure 1B).

There was a reduction of N content in seeds without fruit rest as a function of the maturation stage, with a minimum content of 28.5 g kg−1 of dry matter (DM) at 80 DAA (Figure 1C). The N content had a quadratic response with fruit rest, with a maximum estimated at 30.5 g kg−1 of DM, at 64 DAA.

Seed P contents were adjusted to the quadratic model, being estimated at a maximum of 5.1 g kg−1 of DM at 62 DAA, without rest, and 4.2 g kg−1 of DM at 74 DAA, with post-harvest resting of the fruits (Figure 1D).

**Figure 1.** Seed water content (**A**), dry weight of one thousand seeds (**B**), nitrogen—N (**C**), phosphorus—P (**D**), potassium—K (**E**), calcium—Ca (**F**), magnesium—Mg (**G**) and sulfur—S (**H**) content in sweet pepper seeds, as a function of maturation stage, without (•) and with (-) post-harvest resting of fruits. \* significant difference at 5% probability.

For K, Ca and Mg contents in the seeds, there were reductions during most periods of maturation, obtaining minimum contents of 9.6, 0.81 and 2.2 g kg−1 of DM at 74, 80 and 80 DAA, respectively, without post-harvest resting. For fruits with post-harvest resting, the minimum contents were 9.1, 0.95 and 2.1 g kg−1 of DM at 71, 70 and 80 DAA for K, Ca and Mg, respectively (Figure 1E–G).

S contents in seeds were also adjusted to the quadratic model; however, without fruit rest, there was an increase up to 50 DAA, with a reduction after this date. On the other hand, there was a reduction in contents up to 65 DAA with post-harvest resting, and a slight increase in values after this period (Figure 1H).

Comparing the factors post-harvest resting of the fruits, the presence of rest for seven days provided higher DWTS (Table 1). The post-harvest resting of the fruits also provided higher Ca content. Conversely, it was observed that the P content was higher without rest (Table 1).

Seed N content at 35 DAA was higher without fruit rest; however, at 65 and 80 DAA, higher contents were obtained after rest (Table 2). Without post-harvest resting, the N content in seeds reduced linearly with fruit maturation (Figure 1C); with rest, there was an increase until 64 DAA, and after this, a small decrease was observed.

The S content of seeds at 35 DAA was higher with fruit rest, but at 50 and 65 DAA, the contents were higher without fruit rest (Table 2).

Nutrient contents in the sweet pepper seeds followed the following decreasing order: N > K > P > Mg > S > Ca.

### *2.2. Seed Protein Content*

For proteins, the content of albumin and prolamine had quadratic responses, with maximum estimated values without fruit rest of 64.7 and 7.9 mg g−1 of DM, at 66 and 53 DAA, respectively. Post-harvest resting, the maximum albumin content was estimated at 65.9 mg g−1 of DM at 64 DAA, and prolamine was estimated at 7.1 mg g−1 of DM at 55 DAA (Figure 2A,C).

**Figure 2.** Albumin (**A**), globulin (**B**), prolamine (**C**) and glutelin (**D**) content in sweet pepper seeds, as a function of maturation stage, without (•) and with (-) post-harvest resting of fruits. \* significant difference at 5% probability.

Globulin protein had a linear increase, with maximum contents of 21.9 and 20.3 mg g−1 of DM, at 80 DAA, without and with fruit rest, respectively (Figure 2B).

Glutelin content adjusted to the quadratic model according to the maturation stage; however, there were reductions over the maturation stage, with the minimum without fruit rest estimated at 10.5 mg g−1 of DM at 72 DAA, and with rest estimated at 11.4 mg g−1 of DM at 68 DAA (Figure 2D).

It was observed that the post-harvest resting of the fruits enabled higher contents of albumin and glutelin. However, the prolamine protein content was higher without fruit resting (Table 3). Protein content in the sweet pepper seeds followed the following decreasing order: albumin > globulin ≈ glutelin > prolamine.

**Table 3.** Albumin, globulin, prolamine and glutelin content in sweet pepper seeds as a function of post-harvest resting of the fruits.

Means followed by the same letter do not differ from each other by the F-test at 5% probability.

### **3. Discussion**

### *3.1. Water Content, Dry Weight of One Thousand and Macronutrients Content of Seeds*

During maturation inside the fruits, seeds maintain a high water content (35 to 40%) and undergo dry matter accumulation. Water is considered the vehicle for photoassimilate translocation from the plant to the seeds, so it is necessary to synthesize their reserves [6,14]. However, the water content necessary is lower than that needed to initiate germination [11,12].

Studies conducted by refs. [13,14] determined the minimum water content in 'Magda' sweet pepper seeds to be 54% at 70 DAA, and 47% at 75 DAA in sweet yellow pepper, without fruit rest. The reduction in seed water content as a function of the fruit maturation stage was also reported in other species of the *Capsicum* genus [15,16].

An increase in the dry weight of one thousand seeds (DWTS) occurred because seeds tend to increase the dry weight until physiological maturity during the maturation process [17].

The beginning of seed development was characterized by the relatively slow accumulation of dry mass. This phase predominates the cell division and expansion, which are responsible for the constitution of the adequate structure to receive the substances transferred from the mother plant. Soon after, the replacement of water content with dry matter begins after the initial seed growth [17,18].

The stage of fruit harvest for better physiological seed quality can change according to species, cultivar and environmental conditions [13,19–22]. For the DWTS, higher values were obtained after post-harvest resting. Similar results were reported in other pepper seeds [23–25].

The post-harvest temporary storage of fruits before extraction allows the seeds to complete their physiological maturation [12,13,26,27]. Thus, the reserves continue to be metabolized and translocated to the seeds, allowing increases in weight and improving the physiological and nutritional quality of the seed.

Post-harvest resting of fruits is especially important in species with an indeterminate growth habit that produce fleshy fruits, such as pepper, cucumber, tomato and other species. The post-harvest resting in species with indeterminate growth habits is helpful to improve the uniformity generated by continuous flowering, reducing the number of harvests and the exposure of fruits and seeds to unfavorable field conditions [24].

An increase in the DWTS during the maturation stage is associated with the amount of reserve accumulated during seed maturation. Studies of the sweet pepper cultivar Amarela Comprida demonstrated increases in DWTS up to 75 DAA, with a maximum weight of 6.6 g [28]. However, in the pepper cultivar 'Malagueta', the maximum DWTS obtained was 3.2 g at 80 DAA, and in the 'Biquinho' pepper, it was 2.5 g at 70 DAA [25], thus showing that values can vary according to genotype.

The decreasing order of macronutrient content in the seeds was N > K > P > Mg > S > Ca. N was the most accumulated in sweet pepper seeds. Several studies reported that the N content in the seeds was always higher than the other nutrients [4,5,29,30]. Nitrogen occupies a prominent place in the plant metabolism system because all vital plant processes are associated with proteins, in which N is an essential constituent [31,32].

N is one of the most easily translocated nutrients from leaves to fruits [33]. However, there are no studies of this translocation from fruits at rest to seeds, as may have occurred

in this research, mainly in older fruits (more than 65 DAA). In younger fruits, with rest, there may have been a "dilution effect"; that is, the increase in dry seed weight with rest may have been greater than the translocation of N to seeds. In comparison, in older fruits, it is possible that the N translocation rate from fruit to seeds was more intense, favoring the increase of N content (Figure 1C and Table 2). "Nutrient dilution" is characterized when the DM growth rate is higher than the nutrient absorption rate. Similarly, it can occur in the seeds. With the advance of the maturation stage, together with the rest of the fruits, it allows the continuity of the seed maturation process and DM accumulation.

P was the third most accumulated nutrient in seeds. Phosphorous compounds are important in several reactions observed in seeds [34]. Furthermore, phosphorus is a constituent of the nucleic acid molecule, related to protein synthesis. Additionally, P is present in phospholipids and phosphate sugars, nucleotides and phytin, which is a salt with calcium and magnesium in seeds [30,35]. The requirement of this macronutrient for seeds may be associated with the fact that this nutrient provides faster initial root growth, improving initial seedling establishment. Seeds usually contain enough P to ensure maximum seedling growth for several weeks after germination [36].

The content of P in seeds is probably regulated by plants so that there is no deficiency of this nutrient during seed germination and the beginning of plant development. When there is a lack of P in the soil, it is translocated from the leaves to the fruits and seeds [37].

Regarding K, Ca and Mg content, the behavior was different from the dry matter accumulation in seeds. The content of these macronutrients had a reduction during most periods of the maturation stage (Figure 1E–G). According to [38], as the plant grows, nutrients are diluted, reducing the concentration in the tissues. The same probably happens in seeds as the maturation stages advance.

K was the second most accumulated nutrient in seeds, confirming the importance of this element in seed formation. The physiological role of K during fruit formation and maturation is mainly expressed in carbohydrate metabolism [39], which also makes it one of the most accumulated nutrients of sweet pepper seeds.

Ca was the nutrient with the lowest content in sweet pepper seeds, probably due to its low mobility in the phloem. According to ref. [29], Ca accumulation in seeds occurs only by absorption and transport during their maturation process, with no redistribution. Therefore, although fruit resting increases seed DM, it does not provide continuity of translocation of all macronutrients. This translocation occurs at a lower intensity than DM accumulation and is called the "dilution effect".

Mg in seeds is associated with proteins. However, its role is not yet fully understood, whether related to protein formation or whether this is a consequence of increased amino acid translocation from leaves to drains [40].

Without post-harvest resting, S content during maturation stages increased until 50 DAA, and decreased in older fruits (Figure 1H and Table 2), similarly to N, probably because of the "dilution effect", when the DM growth rate is higher than the nutrient absorption rate. With post-harvest resting, there is DM accumulation due to the maturation stage and to resting; as such, the S content decreases during all maturation stages, similarly to most nutrients. S regulates seed metabolism in terms of carbohydrates and storage proteins. S content in sweet pepper seeds was higher than Ca; S is responsible for carbohydrate regulation and storage of protein seed metabolism [41]. It is essential to mention that the S content in the seeds is low in most species, and that the mineral composition of the seeds can vary according to the species [42], emphasizing the importance of studying the mineral composition of seeds in different species. However, in cauliflower (*Brassica oleracea*), S was the second most accumulated nutrient in the seeds [30].

### *3.2. Seed Protein Content*

Although proteins are part of seed reserves, not all groups are found in the seeds of a determined species. Albumin and globulin are standard in dicotyledonous seeds [8]. They are probably translocated in greater proportions to sweet pepper seeds, favoring their

concentration, with albumin, the main storage protein, being present in greater quantity (Figure 2). The present study observed an increase in albumin content during maturation stages (Figure 2A), and, according to ref. [43], there are relationships between protein content, especially albumin, and seed physiological quality.

Glutelins are common proteins in cereals, and prolamins are common in grasses [8]; this may justify the lower prolamine content in sweet pepper seeds. Thus, plants accumulate reserves such as carbohydrates, oils and proteins during the maturation process, which are essential for germination and establishment [44]. As such, at the beginning of the germination process, the seeds are soaked in water, which begins the mobilization of a food reserve. The storage organs (cotyledons and endosperm) provide essential energy to nourish the seedling until stabilization [45]. Thus, the present result obtained in the study corroborates the literature. Since the reduction of storage proteins is degraded by the offending action and exopeptidases, proteolytic enzymes convert the storage proteins into soluble peptides that are further hydrolyzed into free amino acids, which are then mobilized to the embryonic axis to support growth [46,47].

### **4. Materials and Methods**

### *4.1. Site Description*

The experiment was conducted in a protected environment at the experimental area of São Paulo State University (UNESP), in São Manuel—SP (22◦46 S, 48◦34 W and altitude of 740 m). During the experiment, the average maximum daily temperature was 28.7 ◦C, the minimum was 22.6 ◦C, and the maximum and minimum relative humidity were 73 and 54%, respectively. The experiment was conducted in pots (13 L); the soil used in the pots was fertilized and corrected with limestone as is recommended by Bulletin 100 [44], and the top dressing by fertigation according to ref. [48].

The chemical characteristics of the soil used were: pH (CaCl2): 4.4; organic matter: 5 g dm−3; P(resin): 2 mg dm−3; H+Al: 26 mmolc dm−3; K: 1.1 mmolc dm−3; Ca: 33 mmolc dm−3; Mg: 4 mmolc dm−3; sum of bases: 39 mmolc dm−3; capacity of exchange cation: 64 mmolc dm−3; base saturation: 60%.

### *4.2. Experiment Conduction and Experimental Design*

Sowing was performed on 24th July 2017, and seedling transplantation at 47 days after sowing. An inbred line (SK 1730) from Sakata Seeds was used in this study. The management approach involved the withdrawal of sprouts until the appearance of the first flower, drip irrigation twice a day, and chemical pest and disease control when needed.

The experiment design was randomized in blocks, in a 4 × 2 factorial arrangement, and in four replications. The first factor comprised four maturation periods (35, 50, 65 and 80 days after anthesis (DAA)), and the second, the fruit post-harvest management (with and without the fruit rest for seven days after harvesting at laboratory conditions (25 ± 2 ◦C)). Ten plants were evaluated per plot, and all fruits fixed on the plants were harvested without thinning.

To determine the maturation period, all flowers were marked on the day of their anthesis. The harvests were performed when the fruits had the maturity stage corresponding to 35, 50, 65 and 80 DAA. Half of the fruits had their seeds extracted on the day of harvesting (without rest), and then half remained post-harvest, resting before seed extraction. At harvest time, the visual appearance of the fruits was: fully green fruits at 35 DAA; fruits with transient coloration from green to yellow at 50 DAA; fruits with 75% bright yellow color at 65 DAA; and at 80 DAA, the fruits were 100% yellow, but opaque and with less pulp firmness (Figure 3). Harvests were carried out manually, using scissors to separate the fruit from the mother plant.

**Figure 3.** Visual aspects of sweet pepper fruit at 35 (**A**), 50 (**B**), 65 (**C**) and 80 (**D**) days after anthesis.

### *4.3. Seed Analysis*

Seed water content was determined immediately after fruit extraction by the oven method at 105 ± 3 ◦C for 24 h, using 10 g of seeds [46]. After extraction, the seeds were put in a dry chamber (40% relative humidity and 20 ◦C) to reduce the seed water content to approximately 8% for storage.

The seed characteristics evaluated were the dry weight of one thousand seeds (DWTS), macronutrients (N, P, K, Ca, Mg and S) and protein (albumin, globulin, prolamine and glutelin) content.

To determine the dry weight of one thousand seeds (DWTS), seeds were dried in a forced-air oven at 65 ◦C until they reached a constant weight and weighed on a scale of 0.0001 g [49].

Sulfuric digestion was used to obtain the extract in order to determine the content of N, while P, K, Ca, Mg and S content were extracted by nitroperchloric acid digestion and determined by atomic absorption spectrophotometry, as described by AOAC [50]. To determine the contents of albumin, globulin, glutelin and prolamine proteins, the methodology proposed by ref. [51] was used. For all determinations, four replications were used.

### *4.4. Data Analysis*

The data were submitted for analysis of variance (F test) and, when significant, to compare the post-harvest resting periods of the fruits, the means were considered different by the F-test (*p* < 0.05). The effects of maturation periods were analyzed by regression analysis (*p* < 0.005).

### **5. Conclusions**

The determination of sweet pepper seed quality is affected by the stage of maturation and the presence of rest for 7 days. This is mainly due to the reduction of K, Ca and Mg, essential macronutrients for good germination. However, the reserve protein content (globulin and prolamine) increased due to the presence of maturation.

In this way, obtaining a good seed production with post-harvest rest helps with the accumulation of calcium, albumin and glutelin. Therefore, harvesting the sweet pepper fruit and resting is a management strategy that can improve the quality of seeds.

**Author Contributions:** Conceptualization, L.F.C. and A.I.I.C.; methodology, L.F.C., L.C., G.F.d.S. and W.A.L.Z.; formal analysis, L.F.C. and F.F.P.; investigation, L.F.C. and A.I.I.C.; writing—original draft preparation, L.F.C., G.F.d.S. and L.C.; writing—review and editing, G.F.d.S., L.C., F.F.P. and A.I.I.C.; supervision, A.I.I.C.; funding acquisition, L.F.C. and A.I.I.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Council for Scientific and Technological Development (CNPq), grant number 142044/2016-4.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to acknowledge the School of Agriculture (UNESP/ Botucatu) for supporting the development of this research.

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

### **References**

**Raphael Ofoe 1, Dengge Qin 1, Lokanadha R. Gunupuru 1, Raymond H. Thomas 2 and Lord Abbey 1,\***

**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** Pyroligneous acid (PA) is a reddish-brown liquid obtained through the condensation of smoke formed during biochar production. PA contains bioactive compounds that can be utilized in agriculture to improve plant productivity and quality of edible parts. In this study, we investigated the biostimulatory effect of varying concentrations of PA (i.e., 0%, 0.25%, 0.5%, 1%, and 2% PA/ddH2O (*v*/*v*)) application on tomato (*Solanum lycopersicum* 'Scotia') plant growth and fruit quality under greenhouse conditions. Plants treated with 0.25% PA exhibited a significantly (*p* < 0.001) higher sub-stomatal CO2 concentration and a comparable leaf transpiration rate and stomatal conductance. The total number of fruits was significantly (*p* < 0.005) increased by approximately 65.6% and 34.4% following the application of 0.5% and 0.25% PA, respectively, compared to the control. The 0.5% PA enhanced the total weight of fruits by approximately 25.5%, while the 0.25% PA increased the elemental composition of the fruits. However, the highest PA concentration of 2% significantly (*p* > 0.05) reduced plant growth and yield, but significantly (*p* < 0.001) enhanced tomato fruit juice Brix, electrical conductivity, total dissolved solids, and titratable acidity. Additionally, total phenolic and flavonoid contents were significantly (*p* < 0.001) increased by the 2% PA. However, the highest carotenoid content was obtained with the 0.5% and 1% PA treatments. Additionally, PA treatment of the tomato plants resulted in a significantly (*p* < 0.001) high total ascorbate content, but reduced fruit peroxidase activity compared to the control. These indicate that PA can potentially be used as a biostimulant for a higher yield and nutritional quality of tomato.

**Keywords:** *Solanum lycopersicum*; biostimulant; pyroligneous acid; vegetable production; post-harvest

## **1. Introduction**

Tomato (*Solanum lycopersicum*) is among the most cultivated greenhouse vegetable crops worldwide [1], and is known to be a rich source of health-promoting phytochemicals including carotenoids, phenolics, flavonoids, and ascorbic acid [2]. These phytochemicals exhibit antioxidant properties, which protect cells against oxidative stress by scavenging reactive oxygen species. Its antioxidant properties are known to induce anticancer, antiinflammatory, and chemo-preventive effects. Thus, contributing largely to the prevention of chronic diseases such as cardiovascular, cancer, atherosclerosis, and neurodegenerative disorders [2,3]. The flavor and dietary qualities of food, which strongly influence consumers preference, are usually associated with physical characteristics (e.g., chewability and texture) and chemical composition (pH, ◦Brix, elements, carotenoids, phenolics, and flavonoid) [4]. These properties can be influenced by growing conditions, environmental factors, and the genetic characteristics of the plant. As a result, current greenhouse producers seek alternative inputs which rely mostly on organic amendments to improve the yield and quality of tomato fruits. One such input is the use of pyroligneous acid (PA), which is a natural and environmentally friendly by-product of pyrolysis of plant biomass [5].

**Citation:** Ofoe, R.; Qin, D.; Gunupuru, L.R.; Thomas, R.H.; Abbey, L. Effect of Pyroligneous Acid on the Productivity and Nutritional Quality of Greenhouse Tomato. *Plants* **2022**, *11*, 1650. https:// doi.org/10.3390/plants11131650

Academic Editors: Tika Adhikari and Fermin Morales

Received: 24 May 2022 Accepted: 20 June 2022 Published: 22 June 2022

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

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

During pyrolysis, organic biomass is burnt at a high temperature under the presence of limited oxygen and the gaseous and smoke phase is condensed to produce a liquid smoke [6]. The condensed liquid smoke is stabilized by allowing it to stand for six months, which results in the formation of wood tar at the bottom, light oil at the top and condensed aqueous translucent PA. This aqueous translucent PA is also known as wood vinegar, biooil or liquid smoke [6]. PA has a smoky odor and the color may vary from light yellow to reddish-brown depending on the feedstock [7]. It is a complex mixture containing 80–90% water as a major component and over 200 water-soluble chemical compounds including nitrogen, phenolics, organic acids, sugar derivates, alcohols, and esters [6,8,9]. The chemical composition of PA mainly depends on the temperature, heating rate, feedstock, and residence time, and has been widely used in diverse areas including agriculture, food and medicine [6,10]. Evidence revealed that PA also contains a butanolide, a biologically active compound, that belongs to a new family of phytohormones known as karrikinolide or karrikins [11,12]. Interestingly, the signaling mechanism and mode of action of karrikins are analogous to that of known phytohormones [11,13,14], suggesting that PA at an appropriate concentration can positively influence plant growth and productivity. Furthermore, karrikins are thermal resistant, hydrophilic, and long lasting and can therefore remain highly potent at a wide range of concentrations. Several studies revealed that karrikins stimulate seed germination and regulate seedling photomorphogenesis by enhancing seedling sensitivity to light [11,12,15–18].

PA is commonly used as a biostimulant to improve plant growth and productivity [6]. Depending on the concentration, PA can be used as an antimicrobial agent [19,20], a herbicide [21], a soil enhancer [22], and an insect repellent [23] or promote root development [24,25] and microbial activities [26] when diluted. Recent studies reported that PA enhances seed germination rate, vegetative and reproductive growth of several plants species [6,24,25,27–29]. However, the concentration of PA applied to promote plant growth varied between studies. For instance, it was reported that the application of 1:500 (*v*/*v*) increased tomato yield but did not affect fruit nutritional quality, whereas according to Mungkunkamchao et al. [27], 1:800 PA enhanced the growth and yield of tomato. Similarly, soil drench with 20% PA increased the growth and yield of rockmelon (*Cucumis melo* var. *cantalupensis*) [30]. These suggest that the effectiveness of PA is dependent on its concentration, type of crop, and mode of application. Generally, the high acidity of PA necessitates its use at low concentrations for plant growth and productivity [6]. As such, an appropriate concentration can contain the right proportions of several bioactive compounds which induce beneficial effects on crop growth and quality [17]. Furthermore, phenolic compounds in PA induce high reactive oxygen species scavenging, reducing power activities and anti-lipid peroxidation capacity [8,31]. However, the chemical composition and individual chemical activities can be influenced by the pyrolytic temperature, as a high pyrolytic temperature between 311 and 550 ◦C was demonstrated to exhibit the strongest antioxidant activity [8]. It was amply demonstrated that a high PA concentration increases the availability of phenolics and organic acids that could adversely affect plant growth performance [32]. All these studies demonstrated the use of PA as a natural biostimulant with high efficacy for crop production but this was not extensively explored.

Accordingly, most studies on PA efficacy and use in crop production have focused on seed priming and foliar application. There is limited information on the efficacy of drench application on crop yield and especially on crop quality [6]. Additionally, agricultural use of PA in Canada and globally is in its infant stage due to limited studies on its efficacy for growth promotion and because recommended applications rate have not been clearly established. An understanding of how PA can regulate plant growth, yield and quality of tomato under greenhouse conditions is crucial not only to growers but also to consumers and researchers. In this study, we investigated the biostimulatory effect of varying concentrations of PA for production and increase in nutritional quality of tomato 'Scotia' under greenhouse conditions.

### **2. Results**

### *2.1. PA Chemical Composition*

The chemical composition of PA is presented in Supplementary Table S1. The most significant elements were nitrate, nitrite, calcium and potassium. Significant amounts of organic acids (i.e., salicylic acid, oxalic acid, propionic acid, and malic acid) and small amounts of shikimic acid and acylcarnithines were also present.

### *2.2. Morpho-Physiological Response*

PA application had no significant (*p* > 0.05) effect on plant height, stem diameter, and the number of branches and flowers (Table 1). Plant height non-significantly increased slightly with low PA concentrations, i.e., 0.25% and 0.5% PA, by *ca*. 5% compared to the control. The highest stem diameter was recorded with 0.25% PA followed by with 2% PA but was not statistically different from that of other treatments. Additionally, plants treated with 0.5% PA increased numbers of branches and flowers by *ca*. 13% and 8%, respectively, compared to that of the control although they were not statistically different (*p* > 0.05). Similarly, PA treatments had no significant (*p* > 0.05) effect on Fv/Fm, Fv/Fo, and chlorophyll content (Table 2). The effect of PA on Fv/Fm and Fv/Fo was comparable to the control. Likewise, PA had no significant (*p* > 0.05) effect on leaf intracellular CO2 and photosynthetic rate (Table 2). However, leaf transpiration rate, sub-stomatal CO2, and stomatal conductance were significantly (*p* < 0.001) reduced by PA compared to the control. Plants treated with 0.25% and 0.5% PA showed significant (*p* < 0.001) reductions in these physiological characteristics except for sub-stomatal CO2, which was increased by *ca*. 3% with 0.25% PA compared to the control. On the other hand, plants treated with 1% and 2% PA exhibited significant (*p* < 0.001) reductions in leaf transpiration rate, sub-stomatal CO2, and stomatal conductance compared to the other PA treatments.

**Table 1.** Morphological response of tomato 'Scotia' plants treated with pyroligneous acid (PA).

Values are the means ± SD of four replicates and different letters indicate significant (*p* < 0.05) difference according to Fisher's least significant difference (LSD) post hoc test.

A: photosynthetic rate; E: transpiration rate; gs: stomatal conductance; Ci: sub-stomatal CO2. Values are the means ± SD of four replicates and different letters indicate significant (*p* < 0.05) difference according to Fisher's least significant difference (LSD) post hoc test.

The application of 0.25% PA increased above-ground fresh weight but similar to the control (Figure 1A). However, tomato plants treated with 0.5% and 1% PA reduced the above-ground fresh weight by *ca*. 13% compared to the control. The above-ground dry weight of the tomato plant treated with 0.25% PA was significantly (*p* < 0.005) increased by *ca.* 11% compared to the control (Figure 1B). In contrast, the 0.5% and 1% PA reduced the

above-ground plant dry weight but was not significantly (*p* > 0.05) different from those of the control and the 2% PA treatment.

**Figure 1.** Pyroligneous acid effect on tomato plant above-ground biomass: (**A**) fresh weight and (**B**) dry weight. Values are the means of four replicates and different lowercase alphabetical letters indicate significant (*p* < 0.05) difference according to Fisher's least significant difference (LSD) post hoc test. Error bars show the standard deviations.

### *2.3. Fruit Yield and Quality*

The 0.5% PA treatment increased the total fruit weight by *ca*. 26% although not significantly different from that of 0.25% PA and the control (Figure 2A). However, 2% PA had a significant reduction in total fruit weight, which is not different from that of the 1% PA-treated plants. Similarly, the number of fruits was significantly (*p* < 0.005) increased by *ca*. 66% and *ca*. 34% by 0.5% and 0.25% PA, respectively, compared to the control (Figure 2B). Nevertheless, the application of 2% PA and e control reduced the number of fruits compared to the other PA treatments. Fruit morphological characteristics including polar (Figure 2C) and equatorial diameters (Figure 2D) were not significantly (*p* > 0.05) affected by PA treatment. Tomato fruit juice pH, ◦Brix, salinity, electric conductivity (EC), total dissolved solids (TSS) and titratable acidity (TA) were significantly (*p* < 0.001) affected by PA treatment (Table 3). Juice pH was significantly (*p* < 0.001) increased by *ca.* 3.3% and 1.3% following the application of 0.25% and 0.5% PA to the plants, respectively, compared to the control. An increase in PA concentration from 1% to 2% did not alter fruit juice pH. The ◦Brix content of the fruits was increased by *ca.* 13% following the application of 2% PA compared to the control (Table 3). However, ◦Brix content was significantly (*p* < 0.001) reduced by *ca.* 45% in fruits following the application of 0.25% PA compared to the control.

A significantly (*p* < 0.001) high fruit juice salinity was noticed with the 2% PA treatment compared to the control, while the 0.25% PA recorded the least salinity (Table 3). A considerable increase in fruit electrical conductivity was recorded with the 2% PA, while the least PA of 0.25% reduced fruit juice electrical conductivity. Likewise, the 2% PA recorded the highest fruit juice total dissolved solids (Table 3). Moreover, fruit titratable acidity was significantly (*p* < 0.001) increased by *ca*. 39% upon the application of 2% PA compared to the control (Table 3). Nevertheless, the 0.25% PA had a significant (*p* < 0.001) reduction on fruit TA, which was not different from those of 0.5% PA and 1% PA treatments.

**Figure 2.** Fruit yield of tomato 'Scotia' in response to pyroligneous acid treatment: (**A**) total fruit weight, (**B**) fruit number, (**C**) fruit polar diameter, and (**D**) fruit equatorial diameter. Values are the means of four replicates and different lowercase alphabetical letters indicate significant (*p* < 0.05) difference according to Fisher's least significant difference (LSD) post hoc test. Error bars show the standard deviations.

**Table 3.** Chemical quality of tomato 'Scotia' fruits from plants treated with pyroligneous acid (PA).

EC: electrical conductivity; TDS: total dissolved solids; TA: titratable acidity. Values are the means ± SD of four replicates and different letters indicate significant (*p* < 0.05) difference according to Fisher's least significant difference (LSD) post hoc test.

### *2.4. Fruit Biochemicals and Peroxidase Activities*

Carotenoid was significantly (*p* < 0.05) increased by the 0.5% PA and 1% PA by *ca*. 20% and 22%, respectively, compared to that of the control (Figure 3A). The carotenoid contents of the 0.5% and 1% PA fruits were not statistically (*p* > 0.05) different from that of the 2% PA, while the carotenoid content of the 0.25% PA fruits was low and comparable to the control. Tomato fruit total phenolics (Figure 3B) and flavonoid were significantly (*p* < 0.001) influenced with PA treatment (Figure 3C). The application of 2% PA exhibited a considerably higher fruit total phenolic compounds (*ca*. 23%) and flavonoid content (*ca*. 39%) compared to the control. The 0.5% PA reduced fruit TPC and flavonoid contents. Total ascorbate was increased by *ca*. 377%, *ca*. 177%, *ca*. 165% and *ca*. 129% following the application of 2%, 0.25%, 1% and 0.5% PA, respectively, compared to the control (Figure 3D). Although 0.5% PA had the highest impact on total fruit protein, it was not statistically (*p* > 0.05) different from those of the 0.25% PA and the control treatments (Figure 3E). However, the 2% PA significantly (*p* < 0.001) reduced total fruit protein content compared to the control. Furthermore, PA caused a significant (*p* < 0.001) reduction in total fruit sugar content (Figure 3F). The 1% PA-treated plants exhibited the least total fruit sugar content, while the 2% PA slightly increased total fruit sugar but was *ca*. 5% lower than that of the control. Furthermore, PA application exhibited a significant (*p* < 0.001) reduction in fruit peroxidase activity (Figure 4). The reduction in peroxidase activity was more obvious in the 0.25% PA fruits followed by the 1% PA and the 2% PA fruits.

**Figure 3.**  Values are the means of four replicates and different lowercase alphabetical letters indicate significant (*p* < 0.05) difference according to Fisher's least significant difference (LSD) post hoc test. Error bars show the standard deviations.

### *2.5. Fruit Elemental Composition*

Tomato 'Scotia' fruit N content was increased by *ca*. 10% upon plant application with 0.25% PA compared to the control but was reduced by the 0.5% PA (Table 4). Fruit Ca was markedly increased by *ca*. 29% upon plant treatment with the 1% PA, but was reduced by the 0.5% PA. Generally, PA had no effect on fruit K compared to the control. However, fruit Mg was increased by *ca*. 13% with the 0.25% PA but was reduced by *ca*. 12% with the 0.5% PA compared to the control. Fruit P content was increased slightly by the 2% PA, which was similar to the effect of the 0.25% PA but was reduced by the 0.5% PA treatment. Fruit Na content increased by *ca*. 59% following the application of 1% PA compared to the control, but was reduced by the 0.5% PA. Variation in PA concentration did not change fruit B content. Overall, Fe, Zn, Mn and Cu, contents were increased with the application of 0.25% PA by *ca*. 8%, *ca*. 8%, *ca*. 9% and *ca*. 15%, respectively, compared to the control. However, the 0.5% PA markedly reduced these four elements in the fruits.

**Figure 4.** Peroxidase activity of tomato 'Scotia' fruit in response to pyroligneous acid treatment. Values are the means of four replicates and different lowercase alphabetical letters indicate significant (*p* < 0.05) difference according to Fisher's least significant difference (LSD) post hoc test. Error bars show the standard deviations.

**Table 4.** Tomato 'Scotia' fruit elemental composition in response to pyroligneous acid (PA) treatments.

CV = coefficient of variation.

*2.6. Association between Morpho-Physiological Properties of Tomato Plants and Productivity and Quality in Response to PA Application*

Pearson's correlation coefficient (r) was used to further assess the association amongst the morpho-physiological, yield and quality of tomato plants in response to PA application (Table S2). The PCA biplot showed a projection of response variables in the factor spaces and explained *ca*. 69% of the total variations in the data set. The results revealed that the number of suckers had a significantly (*p* < 0.05) stronger positive correlation with the number of flowers (r = 0.903) and fruit K content (r = 0.914), while SPAD had a significantly (*p* < 0.05) stronger positive association with leaf intracellular CO2 content (r = 0.927) and a negative correlation with photosynthetic rate (r = −0.891). Similarly, leaf transpiration had a significantly strong positively correlated with sub-stomatal CO2

content (r = 0.888) and stomatal conductance (r = 0.996) and moderate association with photosynthetic rate (r = 0.608) and total fruit weight (r = 0.651) although this was not statistically significant. Total fruit weight exhibited a significant (*p* < 0.05) and strong positive correlation with plant height (r = 0.943) and fruit number (r = 0.887). However, it had a significantly (*p* < 0.05) strong negative interaction with total phenolics (r = −0.915) and flavonoid content (r = −0.953). Additionally, fruit number has a similar association with plant height (r = 0.915), total phenolics (r = −0.897) and flavonoid content (r = −0.906). Fruit salinity content showed a significantly strong positive correlation with EC (r = 0.998), TDS (r = 0.999) and Brix (r = 0.979), and a negative association with pH (r = −0.864).

### **3. Discussion**

Current crop production practices make use of natural products that can boost plant growth and the desirable dietary and nutritional quality without compromising the environment and agroecological systems. Therefore, the functional properties of various natural materials such as PA have recently attracted the interest of farmers and researchers. In this study, although the drench application of PA had no statistically significant effect on tomato 'Scotia' plant morphological parameters, they were slightly increased by 0.25% and 0.5% PA concentrations. These results agree with other studies where the foliar application of PA influenced the morphological growth of several plant species including tomato [27], soybean [33], rockmelon [30], and rapeseed [21]. The discovery of karrikins in PA has revolutionized its use in crop production because its signaling and biophysiological activities in plants mimic that of known phytohormones [11,12,15,16]. Moreover, karrikins have been demonstrated to stimulate seed germination and plant growth [12,18]. Hence, the increase in plant growth, although not significant, can be ascribed to the presence of karrikins. Compared to the other elements, N required for vegetative plant growth was considerably high in the PA used for this study. Therefore, the increase in plant growth with PA treatment was reflected in the above-ground fresh and dry weights, which can be attributed to increased nutrient uptake and promotion of cell division and elongation [27].

Stomatal conductance and transpiration rate play a pivotal role in thermoregulation and photosynthesis [34,35]. It was demonstrated that PA and other biostimulants affect stomatal conductance in plants under both stress and non-stress conditions [21,36]. We observed that lower concentrations of PA, i.e., 0.25% or 0.5% PA, exhibited a comparable stomatal conductance and leaf transpiration effect while higher PA concentrations, i.e., >1%, reduced these parameters drastically. A reduction in stomatal conductance is an adaptive strategy used by plants to minimize water loss during water-deficit and other related climatic stress conditions. This scenario adversely affects CO2 diffusion and net photosynthesis [37]. Although the photosynthesis rate in the present study was not affected by PA treatment, we surmised that the reduction in stomatal conductance with PA treatment could be due to adaptive thermoregulation of the photosynthesis system and stress mitigation mechanism [35], which will require further investigation.

Plant productivity (i.e., the total number of fruits and yield) increased with PA application as widely reported by many authors [18,21,27,30]. The composition of PA is complex and consists of numerous bioactive compounds including organic acids, phenolics, alcohol, alkane, and ester [18,21]. This suggests that plants with varying genotypic characteristics will respond differently to PA application. In the present study, an increase in the number of tomato fruits and fruit yield were observed with the application of 0.5% PA. The application of 0.5% and 0.25% PA may be considered less toxic to root systems and may promote root growth, thereby enhancing plant nutrient uptake and utilization [25]. Although data on trusses number were not considered, the increase in fruit number in plants treated with lower PA concentrations could suggest that fruit setting was higher in low-PA-treated plants compared to those treated with higher PA concentrations. This was reflected in the correlation analyses where total fruit weight had a strong association with fruit number. From the farmer's perspective, a slight increase in total fruit yield is considered significant improvement to the overall cashflow. Furthermore, the chemical

components of PA might have interacted with and stimulated the activities of various phytohormones including gibberellin, cytokinin, auxin, and various enzymes to enhance plant growth and development as previously reported [21].

Interestingly, determinants of fruit quality such as ◦Brix, titratable acidity, flavonoid, phenolics, and ascorbate were increased by the 2% PA. This suggests that PA could be used to enhance crop quality for human health and nutritional purposes. These results are inconsistent with the report by Kulkarni et al. [38]. The discrepancies may be due to differences in the tested concentration, time of application, and tomato variety. Generally, tomato fruits are considered an excellent source of phytochemicals including phenolics, flavonoids, and ascorbates, which exhibit high antioxidant properties by scavenging reactive oxygen species (ROS) radicals [2]. Studies demonstrated that higher PA concentration increases the availability of phenolics and organic acids that could affect plant growth [32]. Thus, the increased tomato fruits antioxidants in the present study was highly expected since previous studies have demonstrated that phenolic compounds in PA exhibited high ROS-scavenging activities, reducing power, and anti-lipid peroxidation capacity [8,31].

Accordingly, the present finding may be attributed to the increased phenolics and organic acids as reported in *Citrus limon* [39] and *Olea europaea* [40]. The ROS-scavenging abilities of these phytochemicals protect cells against oxidative stress, which are crucial for preventing chronic diseases including cancers, atherosclerosis, and inflammation disorders [2,3,41]. Moreover, fruit carotenoids are lipophilic pigments essential for human health [42]. Carotenoid content was higher in fruits harvested from plants that were treated with 0.5% and 1% PA compared to the control. This beneficial effect of PA can be attributed to the activation of pathways involved in N metabolism [43]. Furthermore, most plants adapt to stress conditions by accumulating these compounds, which ultimately enhances fruit dietary and nutritional quality. For instance, salinity stress increase TDS, sugar, and antioxidant compounds in tomato fruits [44,45]. Hence, it is plausible that although the 2% PA did not alter the growth of the tomato plants, it stimulated the plants to accumulate these phytochemicals in the fruits.

Mineral elements represent a minute fraction of the fruit dry matter content but constitute a vital component of the quality and nutritional profile of vegetables [46]. The present study demonstrated that the application of 0.25% PA enhanced tomato fruit N, Mg, P, and all the analyzed micronutrients except B. Additionally, the 1% PA increased Ca and Na in the tomato fruits. Some possible explanations could be (1) PA increased the uptake and translocation of mineral elements due to enhanced root growth and root functional activities [24]; (2) PA activated and promoted the expression of transporter genes in root cells for efficient nutrient element transport (not determined); and (3) some bioactive compounds in PA intensified the sink effect resulting in continuous flow and accumulation of these elements [21,47]. Therefore, it can be suggested that the optimal application rate of PA for enhancing tomato fruit elemental composition may range between 0.25% and 1% PA. Similar observations were made following the application of other biostimulants that enhanced the elemental composition of numerous crops including tomato [46,48] and eggplant [49]. Therefore, increased yield and dietary and nutrition quality of tomato can be obtained when the appropriate concentration of PA is applied in a greenhouse production system.

### **4. Materials and Methods**

### *4.1. Plant Material and Growing Condition*

This research was carried out in the greenhouse located in the Department of Plant, Food, and Environmental Sciences, Faculty of Agriculture, Dalhousie University between November 2020 and February 2021 and repeated in March (spring) and July (summer) 2021. Tomato (*Solanum lycopersicum*) cultivar 'Scotia' seeds were purchased from Halifax Seeds (Halifax, Canada). Seeds were sterilized with 10% sodium hypochlorite (NaClO) for 10 min, and thoroughly washed three times with sterile distilled water (ddH2O) followed by 70% ethanol sterilization for 5 min, and subsequently washed 5 times with sterile distilled water.

The sterilized seeds were germinated in a 32-cell pack containing Pro-Mix® BX (Premier Tech Horticulture, Québec, Canada) and grown for 30 days in a growth chamber with a day/night temperature regime of 25 ◦C, 16/8 h d−1 illumination, 300 μmol m−2·s−1 light intensity and 70% relative humidity. The seedlings were transplanted at the third to fourth true-leaf stage into 11.35 L-plastic pots containing approximately 1.5 kg of Pro-Mix® BX peat-based soilless medium. The plants were climate hardened for a week before the first treatment application under greenhouse conditions at 28 ◦C/20 ◦C (day/night cycle) temperature and 70% relative humidity with a 16 h photoperiod. Supplemental lighting was provided by a 600 W HS2000 high-pressure sodium lamp with NAH600.579 ballast (P.L. Light Systems, Beamsville, Canada) throughout the planting duration.

### *4.2. Experimental Treatment and Design*

The five experimental treatments were arranged in a completely randomized design with four replications. The experimental treatments consisted of 0.25%, 0.5%, 1%, and 2% PA, and distilled water was used as a negative control. The PA derived from white pine biomass was obtained from Proton Power Inc. (Lenoir City, TN, USA). The company (Proton Power Inc.) produces and sells graphene and biochar and not PA. The PA is a by-product to them. So, our study, which was funded by the federal agency, was to test this by-product for potential commercialization in the future by which time it will be available to purchase. At present, PA samples may be obtained from Proton Power for only research purposes before it can be available later for purchase. The chemical composition of the PA used in this study is listed in Table S1. All the treatments were applied biweekly as a soil drench to field capacity, and water-soluble compound fertilizer nitrogen-phosphoruspotassium (20:20:20) was applied at 20-day intervals. Pots were rearranged weekly on the bench to offset unpredictable occurrences due to variations in the environment. The entire study was repeated twice.

### *4.3. Plant Growth and Yield Components*

Plant growth parameters were measured at 50 days after transplanting (DAT). Plant height was measured from the stem collar to the highest leaf tip with a ruler and the stem girth (i.e., diameter of the main stem) was measured at 10 cm from the collar with Vernier calipers (Mastercraft®, Ontario, Canada). Total numbers of flowers and suckers (i.e., branching) were recorded for each treatment. Intracellular carbon dioxide concentration, net photosynthetic rate, and stomatal conductance were determined from the same four fully expanded leaves per plant using LC*i* portable photosynthesis system (ADC BioScientific Ltd., Hoddesdon, UK). Chlorophyll fluorescence indices including maximum quantum efficiency (Fv/Fm) and potential photosynthetic capacity (Fv/Fo) were measured on the same leaves using a Chlorophyll fluorometer (Optical Science, Hudson, NH, USA) [50]. Chlorophyll content was measured on the same leaves using a chlorophyll meter (SPAD 502-plus, Spectrum Technologies, Inc., Aurora, IL, USA). The total fresh weight of the above-ground tissues (i.e., leaves and shoot) was measured with a portable balance (Ohaus navigator®, ITM Instruments Inc., Sainte-Anne-de-Bellevue, QC, Canada) and subsequently oven-dried at 65 ◦C for 72 h for dry weight determination. Tomato fruit yield, determined as the total fresh weight of ripe fruits per plant, was recorded using the XT portable balance. The equatorial and polar diameters of the harvested fruits were measured with the digital Vernier caliper.

### *4.4. Fruit Quality and Phytochemical Analysis*

At harvest (75DAT), seven representative ripe fruits based on size and color were randomly selected and surface-sterilized with 70% ethanol. The pericarp (containing the epidermis) was excised from the longitudinal part of each fruit using a sterile scalpel blade. The pericarp was immediately frozen in liquid nitrogen and stored in a −80 ◦C freezer while the remaining fruits were frozen at −20 ◦C until further analysis. All frozen fruits were thawed at room temperature and fruit total soluble solids (TSS) were determined

using a handheld refractometer (Atago, Japan). Briefly, ripe fruits were cut, placed in a clear Ziploc bag and hand squashed. The juice was poured into a 50 mL beaker and 500 μL was used for TSS determination expressed as degree Brix (◦Brix). Fruit juice qualities including pH, salinity, total dissolved solids (TDS), and electrical conductivity (EC) were determined with a multi-purpose pH meter (EC 500 ExStik II S/N 252957, EXTECH Instrument, Nashua, New Hampshire, USA). For titratable acidity, 10 mL of juice from each treatment was diluted in 50 mL distilled water, and titratable acidity was determined at an endpoint of pH 8.1 with 0.1 N sodium hydroxide (NaOH). The mean titratable acidity was expressed in citric acid percentage [1]. The elemental composition of the fruits was determined at the Nova Scotia Department of Agriculture Laboratory Services, Truro, using inductively coupled plasma mass spectrometry (PerkinElmer 2100DV, Wellesley, Massachusetts, USA) [51].

### 4.4.1. Fruit Carotenoid Content

Fruit carotenoid content was determined as described by Lichtenthaler [52]. Briefly, 0.2 g of ground fruit pericarp was homogenized in 2 mL of 80% acetone. The homogenate was centrifuged at 15,000× *g* for 15 min and the absorbance of the supernatant was measured at 646.8, 663.2, and 470 nm using a UV–Vis spectrophotometer with 80% acetone alone as the blank. Total carotenoid content was expressed as μg g−1 fresh weight (FW) of the sample.

### 4.4.2. Total Ascorbate Content

Total ascorbate was measured following the method described by Ma et al. [53] with little modification. Approximately 0.2 g of ground fruit pericarp was homogenized in 1.5 mL ice-cold freshly prepared 5% trichloroacetic acid (TCA). The mixture was vortexed for 2 min and centrifuged at 12,000× *g* for 10 min at 4 ◦C. A volume of 100 μL of the supernatant was transferred into a new tube and 400 μL phosphate buffer (150 mM potassium dihydrogen phosphate (KH2PO4) (pH 7.4), 5 mM Ethylenediaminetetraacetic acid (EDTA)) was added. A volume of 100 μL of 10 mM Dithiothreitol (DTT) was added and vortexed for 30 s. A reaction mixture containing 400 μL of 10% (*w*/*v*) trichloroacetic acid (TCA), 400 μL of 44% orthophosphoric acid, 400 μL of 4% (*w*/*v*) α,α-dipyridyl in 70% ethanol and 200 μL of 30 g/L ferric chloride (FeCl3) was added to obtain color. The mixture was incubated at 40 ◦C for 60 min in a shaking incubator and the absorbance was measured at 525 nm. The total ascorbate content was determined using a standard L-ascorbic acid curve and expressed as μmol g−1 FW.

### 4.4.3. Soluble Sugar Content

The total sugar content of the tomato fruits was estimated following the phenolsulfuric acid method described by Dubois et al. [54]. An amount of 0.2 g of ground fruit pericarp was homogenized in 10 mL of 90% ethanol and the mixture was incubated in a water bath at 60 ◦C for 60 min. The final volume of the mixture was adjusted to 5 mL with 90% ethanol and centrifuged at 12,000 rpm for 3 min. An aliquot of 1 mL was transferred into a thick-walled glass test tube containing 1 mL of 5% phenol and mixed thoroughly. A volume of 5 mL of concentrated sulfuric acid was added to the reaction mixture, vortexed for 20 s, and incubated in the dark for 15 min. The mixture was cooled to room temperature and the absorbance was measured at 490 nm against a blank. Total sugar was calculated using a standard sugar curve and expressed as μg of glucose g−1 FW.

### 4.4.4. Total Phenolics Content

Total phenolics content (TPC) was determined by the Folin–Ciocalteu assay described by Ainsworth and Gillespie [55] with little modification. An amount of 0.2 g of ground fruit pericarp was homogenized in 1.5 mL of ice-cold 95% methanol and incubated in the dark at room temperature for 48 h. The mixture was centrifuged at 13,000× *g* for 5 min before mixing 100 μL of the supernatant to 200 μL of 10% (*v*/*v*) Folin–Ciocalteau reagent. The mixture was vortexed for 5 min, mixed with 800 μL 700 mM Na2CO3, and incubated

in the dark at room temperature for 2 h. The absorbance of the supernatant was measured at 765 nm against a blank. TPC was calculated using a gallic acid standard curve and expressed as mg gallic acid equivalents per g FW (mg GAE g−1 FW).

### 4.4.5. Total Flavonoid Content

Total flavonoid was estimated following the colorimetric method described by Chang et al. [56]. An amount of 0.2 g of ground fruit pericarp was homogenized in 1.5 mL of icecold 95% methanol followed by centrifugation at 15,000× *g* for 10 min. A volume of 500 μL of supernatant was added to a reaction mixture containing 1.5 mL of 95% methanol, 0.1 mL of 10% aluminum chloride (AlCl3), 0.1 mL of 1 M potassium acetate, and 2.8 mL distilled water. The mixture was incubated at room temperature for 30 min and the absorbance was measured at 415 nm against a blank lacking AlCl3. Total flavonoid content was estimated using quercetin equivalents and expressed as percentage flavonoid using the formula:

Total flavonoid = ([flavonoids](*μ*g/mL) × total volume of methanolic extract (mL)) mass of extract (g)

### 4.4.6. Protein Content and Peroxidase Activity

For protein content and antioxidant enzyme activity, approximately 0.2 g of ground sample was homogenized in 3 mL ice-cold extraction buffer (50 mM potassium phosphate buffer (pH 7.0), 1% polyvinylpyrrolidone, and 0.1 mM EDTA). The homogenate was centrifuged at 15,000× *g* for 20 min at 4 ◦C. The supernatant (crude enzyme extract) was transferred to a new microfuge tube on ice and the protein content was measured at 595 nm after 5 min of mixing with Bradford's reagent [57]. The protein content was estimated from a standard curve of bovine serum albumin (200–900 μg mL−1). Peroxidase (POD, EC <IP_ADDRESS>) activity was determined using Pyrogallol as substrate according to Chance and Maehly [58] with little modification. The reaction mixture consisted of 100 mM potassiumphosphate buffer (pH 6.0), 5% pyrogallol, 0.5 % H2O2 and 100 μL of crude enzyme extract. Following reaction mixture incubation at 25 ◦C for 5 min, 1 mL of 2.5 N H2SO4 was added to stop the reaction and the absorbance was read at 420 nm against a blank (ddH2O). One unit of POD forms 1 mg of purpurogallin from pyrogallol in 20 s at pH 6.0 at 20 ◦C.

### *4.5. Statistical Analysis*

All data obtained were subjected to one-way analysis of variance (ANOVA) with the averages of the two experiments using Minitab statistical software version 20 (Minitab Inc., State College, PA, USA). Treatment means were compared using Fisher's least significant difference (LSD) post hoc test at *p* ≤ 0.05. Pearson's correlation analysis was performed using XLSTAT version 19.1 (Addinsoft, New York, NY, USA).

### **5. Conclusions**

In conclusion, the drench application of low PA concentrations of 0.25% and 0.5% increases the morpho-physiological response of tomato plants. Overall, the application of 0.5% PA enhances the number of fruits and yield of tomato but reduces the quality of the fruits. Alternatively, the application of 0.25% PA will increase the elemental composition of tomato fruits. Additionally, the drench application of 2% PA can be considered stressful to tomato plants, but significantly enhanced fruit phytochemical contents including total phenolics and flavonoids and can be adopted to improve the nutritional and health benefits of tomato fruits. Hence, PA represents a novel natural product for improvement of plant growth, productivity, and nutritional content of tomato and other plants. However, further investigation is required to elucidate the molecular basis of the effect of PA on different plant species.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants11131650/s1, Table S1: Chemical composition of PA obtained from White pine; Table S2: Pearson's correlation between the morpho-physiological, yield and quality of tomato plants in response to PA application.

**Author Contributions:** Conceptualization, L.A. and R.O.; formal analysis, R.O. and L.R.G.; funding acquisition, L.A. and R.H.T.; investigation, R.O., L.R.G. and D.Q.; methodology, R.O., L.R.G. and L.A.; project administration, L.A. resources, L.A.; supervision, R.H.T. and L.A.; validation, R.O., L.R.G. and L.A.; writing—original draft, R.O.; writing—review and editing, R.O., L.R.G. and L.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), Grant #CRDPJ532183-18.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The lead author wishes to thank all her laboratory team for their unflinching support and suggestions during this study.

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

### **References**