1. Introduction
The agricultural systems are primarily designed to increase yield and crop productivity, which might result in lower nutrient levels in crops and, consequently, deficient nutrient availability, which can lead to malnutrition in consumers [1]. However, more attention has being given to these aspects, and agriculture is currently shifting its focus from quantity to producing nutrient-rich food crops [1,2]. In this context, biofortification, namely, through agronomic management practices involving fertilization strategies, has emerged as a valuable method to enhance the nutritional status of crops [1,3,4].
Potato (Solanum tuberosum L.) is the third most popular consumed vegetable worldwide and ranked the fourth most produced one, after rice, maize, and wheat [5]. In Portugal, potato represents one of the most important horticultural crops, with its western region standing out as one of the major potato production areas. Potato is recognized for its health benefits and high nutritional value [6], and potato plays a crucial role in meeting the energy and nutritional needs of a significant part of the global population [7]. Potato is primarily recognized as a carbohydrate-rich food [8]. However, it also has the potential to address malnutrition, being a good source of essential minerals [9] and antioxidants [10], whereas it presents a high protein-to-calorie ratio, which makes this edible crop more efficient in supplying of energy and proteins compared to other major food crops [7]. Furthermore, potato can produce high yields, generating high dry matter accumulation in relatively short periods, and its adaptability to a variety of climatic conditions further contributes to its widespread cultivation, being also considered an ideal crop for mineral biofortification [11].
In recent decades, agronomic biofortification emerged as a promising strategy to enrich crops with essential nutrients, such as calcium (Ca), whose deficiency is associated with severe public health issues like osteopenia, osteoporosis, and rachitis [12,13], particularly in low- and middle-income countries and in more vulnerable age groups (children, adolescents, pregnant women, and older adults). Several agronomic strategies have been implemented worldwide to improve the mineral content in S. tuberosum L. plants, namely, of Ca [14,15,16,17], K [14,18], Fe [19,20,21,22,23], and Zn [24,25,26]. However, mineral accumulation in potato tubers is highly dependent on the cultivar, edapho-climatic conditions, and biofortification itinerary applied (including the chemical product that carries the applied mineral).
For instance, different studies indicated the different impacts on S. tuberosum L. plants of the foliar applications with Ca, i.e., an increase in plant height, number of stems, and plant area [14]; with CaCl2 solution, i.e., an increase in plant growth and tuber yield [15]; with either individual or combined applications of CaCl2 and Ca(NO3)2 [17], i.e., an increase in average tuber weight, leaf area, and chlorophyll content in leaves; and with Ca(NO3)2 solution, a decrease in number of tubers per plant [16]. As such, Liu et al. (2021) [27] and D’Imperio et al. (2016) [28] suggested that different calcium chemical forms can have varied effects on plant growth.
Foliar applications of Ca-EDTA have been studied in crops other than potato, such as sweet corn [29], mango [30], and pineapple [31], to increase the Ca content and/or to promote plant growth.
Research suggests that foliar applications are a more efficient agronomic biofortification approach than soil applications for enhancing the mineral content in the edible parts of crops [11,26]. However, this biofortification approach has limitations, including mineral accumulation and mobility, environmental conditions, and the stage of plant development when the biofortification is applied [1,11]. Moreover, the response variability among cultivars and local growing conditions emphasizes the importance of tailoring biofortification strategies to specific local/environmental/management contexts. While the literature has addressed the efficiency of Ca biofortification, focusing on the effects of its application on plant growth, increased mineral content, and its distribution in the tubers, a significant gap remains in understanding how applied calcium sources can influence the physiological aspects of plants, associated with the photosynthetic performance that is crucial for plant productivity.
In this context, we aim to evaluate the effectiveness of Ca biofortification (and Ca localization within the tuber) in Solanum tuberosum L., depending on cultivars (Picasso and Rossi) and calcium sources (calcium chloride and Ca-EDTA, 12 kg ha−1), together with the impact on photosynthetic functioning (as assessed through leaf gas exchanges and chlorophyll a fluorescence parameters), which is a determinant of plant productivity. These findings are crucial to optimize biofortification practices, in order to improve the potato nutritional quality while maintaining high yields. Moreover, we hypothesized that the foliar applications of CaCl2 and Ca-EDTA would enhance Ca accumulation and its redistribution within potato plants, particularly increasing the tuber Ca content, while also influencing photosynthetic performance, with potential differences between the two cultivars evaluated.
2. Materials and Methods
2.1. Location and Characteristics of the Potato Field
Two commercial S. tuberosum L. cvs. Rossi and Picasso were cultivated in Lourinhã (Figure 1), the western region of Portugal, in an area of about 50 m2. The soil used for both cvs. had a medium texture, a pH of 7.30–7.41, electrical conductivity ranging between 205 and 332 μS cm−1, 1.88–4.13% organic matter, 0.39–0.71% Ca content, 2.20–2.64% K content, and 0.19–0.23% P content. The application of fertilizer products before the planting was carried out with Plusmaster 8-12-12, soil set, and Pedrin.
The two cultivars are commonly grown in Portugal, being selected due to their agronomic relevance and popularity among farmers, particularly in the western region of Portugal. Both cvs. are widely appreciated for their yield stability and quality traits, making them a good choice for assessing calcium biofortification impacts under local production conditions.
2.2. Experimental Design
After the beginning of the tuberization of cvs. Picasso and Rossi (after 68 and 64 days after plantation, respectively), seven foliar applications were performed at intervals of 6–8 days, applying a single concentration (12 kg ha−1) of CaCl2 or Ca-EDTA (Table 1). The crop cycle duration for Picasso and Rossi were 132 and 148 days, respectively, and the farmers followed the standard technical itinerary commonly applied in potato production (phytopharmaceutical products against pin, infests, mildew, early blight, and scarab). Foliar applications were carried out by a professional using a handheld sprayer in order to guarantee the uniform distribution of the solution on the foliage. Moreover, the field trial was conducted with three independent replicates per treatment.
2.3. Climate Conditions
Climate conditions, such as temperature, air humidity, and precipitation, were obtained from the Weather Underground online platform for Lourinhã (Figure 2). During the crop cycle across the two fields, from the date of the 1st plantation (12 March) and the harvest (22 July for Picasso and 11 August for Rossi), the temperature ranged between 0 °C (15 April) and 39 °C (15 July) (Figure 2A), air humidity from 12% (15 April) to 99% (5 to 7 April) (Figure 2B), and precipitation from 0 mm to 19 mm (14 April and 10 May, respectively) (Figure 2C).
2.4. Monitoring of Crop During Biofortification Process
Plant development was monitored throughout the calcium biofortification process of S. tuberosum L. Regular photographic documentation was conducted to assess plant morphology and detect potential toxicity symptoms. The visual assessments focused on identifying any physiological or morphological changes that could indicate adverse effects of the applied biofortification products. For both cultivars (Rossi and Picasso), the first assessment was carried out six days after the 3rd foliar application, and the second and third assessments were conducted seven days following the 5th and the 7th foliar applications, respectively.
2.5. Photosynthetic Performance During Biofortification Process
Photosynthetic performance was monitored in both cultivars on 22 June and 30 July 2020, after the 5th and 7th spraying applications of 12 kg ha−1 of CaCl2 or Ca-EDTA solutions, i.e., by 96 days after plantation (for cvs. Picasso and Rossi) and 110 days (for cv. Rossi, considering that the cv. Picasso was already harvested on 22 of July), respectively. These evaluations were performed through the measurement of leaf gas exchanges and chlorophyll a fluorescence parameters briefly described below.
Leaf gas exchanges were evaluated under photosynthetic steady-state conditions after ca. 2–4 h of light exposure following Rodrigues et al. (2016) [32], using 5 random plants per treatment, and included net photosynthetic rate (Pn), stomatal conductance to water vapor (gs), and transpiration rate (E). For this purpose, a portable open-system infrared gas analyzer (Li-Cor 6400, LiCor, Lincoln, NE, USA) was used under environmental conditions, with a photosynthetic photon flux density (PPFD) within the range of 1200 to 1400 µmol m−2 s−1, air temperature of 33.8 ± 0.4 °C, and external air [CO2] set to ca. 400 ppm. The Pn-to-E ratio, representing the units of assimilated CO2 per unit of water lost through transpiration, allowed the calculation of leaf instantaneous water use efficiency (iWUE).
The determination of chlorophyll a fluorescence parameters was carried out using a PAM-2000 system (H. Walz, Effeltrich, Germany), as previously described in Daccak et al. (2024) [33], following the formulas discussed elsewhere [34,35,36]. Measurements were performed in 5 independent leaves from 5 different plants per treatment. The minimal fluorescence from the antennae (F0), maximal fluorescence (Fm), and maximal photochemical efficiency of photosystem II (PSII), represented by Fv/Fm, were measured on overnight dark-adapted leaves. F0 was assessed using a weak light (<0.5 μmol m−2 s−1) beam, while Fm was obtained using a saturation pulse of ca. 7500 μmol m−2 s−1 of actinic light for 0.8 s. Additional parameters were determined under photosynthetic steady-state conditions (after at least 2–4 h of light exposure), under natural irradiance (ca. 1200–1400 μmol m−2 s−1) and superimposed saturating flashes: Fv′/Fm′, qL, qN, Y(II), Y(NPQ), and Y(NO) [34,37,38]. F0′, needed for the quenching calculations, was measured in the dark, immediately after the actinic light was switched off, before the first fast phase of the fluorescence relaxation kinetics. Fv′/Fm′ expresses the PSII photochemical efficiency of energy conversion under light exposure. qL is the photochemical quenching, based on the concept of interconnected PSII antennae, and represents the proportion of energy captured by open PSII centers and driving photochemical events. Estimates of photosynthetic quantum yields of non-cyclic electron transfer (Y(II)), photoprotective regulated energy dissipation of PSII (Y(NPQ)), and non-regulated energy dissipation (heat and fluorescence) of PSII (Y(NO)) were also calculated, where Y(II) + Y(NPQ) + Y(NO) = 1.
2.6. Calcium Content in Tubers
Calcium content in the tubers of S. tuberosum L. was determined after harvest and at the end of the crop cycle. The samples were cut, dried at 60 °C until reaching a constant weight, and then ground. All samples were analyzed using an XRF analyzer (model XL3t 950 He GOLDD+, Thermo Fisher Scientific Inc., Waltham, MA, USA) following the methodology described by Mangueze et al. (2018) [39]. The analyses were performed in quadruplicate for each treatment. The detection limit of the equipment for Ca was 65 mg.kg−1.
2.7. Calcium Distribution in Tubers
Calcium distribution in the different tuber regions (relative to the equatorial area) of S. tuberosum L. after harvest was analyzed using the µ-EDXRF system (M4 Tornado™, Bruker, Berlin, Germany), as described by Marques et al. (2020) [40].
2.8. Yield, Caliber, and Tuber Weight
Total tuber yield, caliber (minimum and maximum diameters), and weight (average weight per tuber) were evaluated for both S. tuberosum L. cultivars under the different treatments. Caliber and weight were assessed in quadruplicate.
2.9. Statistical Analysis
Statistical analysis was performed using the IBM SPSS software (version 20, IBM Corp., Armonk, NY, USA). One-way ANOVA was applied to the photosynthetic performance parameters during the biofortification process for the cv. Picasso (due to the absence of plants in the second assessment) to assess differences between treatments (control, CaCl2, and Ca-EDTA). Additionally, one-way ANOVA was also applied to yield, caliber, and tuber weight for each cultivar (Picasso and Rossi) to assess differences between treatments. Two-way ANOVA was used to evaluate differences over time (22 June and 30 July) between all treatments for both cultivars. Also, the two-way ANOVA was applied to assess differences in Ca content in peeled and unpeeled tubers within the same treatment for each cultivar. Tukey’s test was used for mean comparison, and a 95% confidence level was adopted for all tests. The ANOVA model was applied with cultivar and calcium treatments as independent variables, and the normality of data was verified prior to analysis.
The PCA analysis was performed for all the analyzed parameters (photosynthetic performance parameters; Ca content in peeled and unpeeled tubers; yield; caliber; and tuber weight), and the data from the two main principal components (PC1 and PC2) were plotted, and the data from the loadings of the variables were assessed by considering PC1 and PC2.
3. Results
3.1. Monitoring of Culture During Biofortification Process
The monitoring of S. tuberosum L. plants subjected to foliar spraying with Ca solutions of CaCl2 or Ca-EDTA was performed in both experimental fields to assess potential stress occurrences or symptoms. The first assessment was carried out 6 days after the 3rd foliar application, the second assessment 7 days after the 5th foliar application, and the third assessment 7 days after the 6th foliar application (Figure 3).
The control plants of both cultivars under study showed normal development throughout their growth cycle (Figure 3). However, adverse effects were observed mostly for the Ca-EDTA-treated plants (greater in the Picasso cultivar) than in their CaCl2-treated counterparts. In fact, Ca-EDTA promoted a negative physiological impact associated with toxicity symptoms in both cvs., although the cv. Rossi showed a lower impact extent in all three assessments (Figure 3). Toxicity symptoms in the cv. Picasso become slightly evident shortly after the 3rd application (1st assessment), becoming markedly pronounced afterwards, with a clear loss/burn of the plant shoots. The cv. Rossi displayed less pronounced toxicity symptoms, visually detected following the 6th application of Ca-EDTA (2nd assessment). In sharp contrast, CaCl2 spraying induced only minor negative impacts following the 5th and 6th treatments (2nd and 3rd assessments, respectively), and only in Picasso plants.
3.2. Photosynthetic Performance During Biofortification Process
Leaf gas exchange evaluations to assess the potential impact of the biofortification process on the photosynthetic performance were performed on 22 June (6 days following the 5th foliar treatment) and 30 July (30 days after the 7th foliar treatment), close to the harvest time for Rossi (12 August) (Table 2). For the cv. Picasso, no data were collected on 30 July, as the plants were harvested by 22 July.
The use of CaCl2 did not result in significant changes in the photosynthetic rate (Pn) on 22 June, regardless of the cv. In sharp contrast, the Ca-EDTA treatment promoted a significant a 55% decline in Pn by 22 June in the Picasso plants, although no changes were observed in the Rossi plants, and it was noted that, on 22 June, no observable changes in photosynthetic rate (Pn) occurred in the Rossi cultivar. In contrast, the same treatment had a significant negative effect on the Picasso cultivar, leading to a 55.4% decrease in Pn (Table 2).
The significant impact on Pn promoted by in Ca-EDTA treatment in the Picasso cultivar was not associated with stomatal restrictions, since stomatal conductance (gs) and E exhibited similar values for the control plants by 22 June, consequently reducing iWUE to about half. On the other hand, in the cv. Rossi, the Ca-EDTA foliar applications led to significant increases in gs (1348%) and E (53%) values and a decline (23%) in iWUE by 22 June. In contrast, CaCl2-treated plants remained mostly unaffected, with regard to gs, E, and iWUE, since they exhibited similar values for the control plants, regardless of the evaluation date and the cv. (Table 2).
For a deeper analysis of the impact of the applied Ca solutions on the functioning of the photosynthetic apparatus, an additional analysis of chlorophyll a parameters was carried out (Table 3). The maximal photochemical efficiency of PSII exhibited values ranging from 0.77 to 0.83, indicating good photosynthetic efficiency and functional PSII, with the exception of the Picasso plants treated with Ca-EDTA (0.54) by 22 July, thus revealing a significant negative impact on PSII functioning by 22 June. Notably, even in the Rossi plants, measurements could no longer be taken due to the absence of photosynthetically active aerial parts by 30 July. In contrast, the plants of both cvs. exposed to CaCl2 spraying did not result in significant changes in these values on either date.
Globally, the other fluorescence parameters that reflect the functioning efficiency of the photosynthetic machinery showed patterns of variation similar to those of Fv/Fm, whereas those associated with energy dissipation processes showed an opposite pattern. In this context, in the Picasso plants, Ca-EDTA promoted the decline in the actual photochemical efficiency under light (Fv′/Fm′), the quantum yield of non-cyclic electron transport (Y(II)), and the photochemical quenching (qL), with the latter denoting the energy driving photochemical events. Furthermore, Ca-EDTA also decreased these parameters in the Rossi plants (significantly for Y(II) and qL), thus reflecting impairments in this cv. On the other hand, the plants of both cvs. treated with CaCl2 did not show any significant change.
Similarly, the quantum yield of regulated dissipation of energy in PSII (Y(NPQ)) and the non-photochemical quenching (qN)) were increased in the Picasso and Rossi plants sprayed with Ca-EDTA, thus reflecting a greater need of energy dissipation. Consistent with their better photochemical functioning, the plants with CaCl2 showed no impact on these energy dissipation-related parameters, regardless of the monitoring date and the cv. The quantum yield of unregulated dissipation (heat and fluorescence) dissipation of energy in PSII (Y(NO)) was mostly irresponsive, except in the cv. Rossi sprayed with CaCl2 by 30 July.
3.3. Calcium Content and Distribution in Tubers
Across cultivars, unpeeled tubers exhibited a higher Ca content compared to peeled tubers (Table 4) in controls, as well as with Ca treatments, except for CaCl2 treatment in the cv. Picasso, as assessed by the two-way ANOVA. This is justified by the fact that potato peel is a main localization site for Ca (Figure 4).
Considering the Ca distribution in the tuber regions, relative to the equatorial area (Figure 4), the µ-EDXRF analysis revealed a higher accumulation in the epidermal region, the outermost layer of the tubers, regardless of the treatment, in both cvs. The inner zones/regions of the tuber also showed substantial Ca accumulation, but this was less pronounced as compared to that in the epidermis, with no significant changes among treatments.
The unpeeled tubers from plants treated with Ca-EDTA exhibited a significantly higher Ca content as compared to their own controls, with increases of 88% and 79% in the Picasso and the Rossi cv., respectively. Notably, these tubers also showed significantly greater Ca levels than those in plants treated with CaCl2. In fact, the Ca levels from the latter tubers are similar to those of the control plants of both cvs.
In peeled tubers, both Ca treatments significantly increased Ca in the studied cvs., with greater increases in the Picasso cv. under the Ca-EDTA treatment (37%) and in the Rossi cv. with the CaCl2 treatment (30%).
3.4. Yield, Caliber, and Tuber Weight
A consistent pattern in tuber yield was observed for both cvs., with control plants always exhibiting the highest productivity, closely followed by that of plants treated with CaCl2. Finally, Ca-EDTA-treated plants showed a strong reduction of 43% (Picasso) and 72% (Rossi) when compared with their respective control yields (Table 5). In addition, tubers weight and diameters were not negatively impacted by the CaCl2 treatment in both cvs., but they exhibited lower values in Ca-EDTA treated plants. Notably, tubers from CaCl2-sprayed plants always showed greater values for these parameters than those from Ca-EDTA-treated plants, significantly for weight and minimum diameter in Picasso and maximal diameter in Rossi.
3.5. Principal Component Analysis
A principal component analysis (PCA) was performed for the different analyzed parameters to evaluate the similarity between variables and the correlation among the applied treatments and the loadings (Figure 5). PC1 accounts for the greater part of data variability (57.5%), while PC2 explains a smaller but still a significant part of the variation (21.7%). Together, PC1 and PC2 explain approximately 79.2% of the total variance, indicating a strong representation of the dataset.
PC1 is associated with gs, E, and iWUE (the positive part) and with Fv/Fm′, Y(II), and minimum and maximum diameters (the negative part), while PC2 is associated with the Ca content (unpeeled and peeled tubers) (the positive part) and with tuber weight and qL (the negative part). Given that these two components capture most of the variation in the data, the positioning of the treatments in the biplot reflects how they differ in terms of these variables. As such, treatments with positive values of PC1 are associated with higher gs, E, and iWUE, especially with the CaCl2 treatment in the cv. Rossi. On the other hand, treatments with negative PC1 values are associated with higher Fv/Fm′ and Y(II), as observed for the control treatments in both cvs. Additionally, Ca-EDTA (cv. Rossi) is more associated with the Ca content (unpeeled and peeled tubers) than with the lower yield (tuber weight)—located in the opposite side of Ca representation. Moreover, treatments associated with the negative values of PC2 are associated with a higher tuber weight, especially in the control treatments of both cvs.
Overall, CaCl2 treatments in both cultivars seem to create an equilibrium between photosynthetic parameters (Table 2 and Table 3) and tuber weight (Table 5), without affecting the Ca content (Table 4). However, the Ca-EDTA treatments in both cultivars are associated with a lower tuber weight (Table 5) but a higher Ca content (Table 4), especially for the cv. Rossi, and reveal a more pronounced effect on the characteristics associated with photosynthetic functioning (Table 2 and Table 3).
4. Discussion
Based on the monitoring of the crop during the biofortification process, toxicity symptoms were observed with Ca-EDTA applications as early as at the first assessment. These heavy symptoms led to the initiation of leaf necrosis and an extensive loss of plant shoots (stems and leaves) (Figure 3). This effect was observed in both cultivars, although with a much greater impact on the cv. Picasso from the 2nd assessment onwards. In sharp contrast, the application of CaCl2 did not promote such pronounced negative impacts in either cv. (Figure 3). These findings greatly aligned with the photosynthetic performance findings (Table 2 and Table 3). In fact, the Ca-EDTA treatments resulted in the lowest values for key physiological indicators associated with the photosynthetic performance in the Picasso plants (e.g., Pn, Y(II), qL), thus decreasing iWUE. Consistent with the visual assessment of plant health, the application of CaCl2 did not promote the negative effects on the photosynthetic performance of both cultivars. This supported the yield values similar to those of control plants, although with a lower Ca accumulation in the tubers than that promoted by the Ca-EDTA solution (Table 4). Still, the latter application showed a large yield reduction in both cvs., with a high potential risk for the economic sustainability of the crop and a high acceptance among farmers.
Interestingly, by 30 July, the cv. Rossi exhibited higher Pn values in treated plants compared to their control (Table 2), which seems to be a longer “stay green” result in these plants. Thus, this observation aligns with a previous study carried out in broccoli [41], which verified that foliar sprays containing CaCl2 improve the antioxidant system and delay chlorophyll degradation, which maintain the green color of the plant for more time.
The detrimental impacts caused by the Ca-EDTA treatment in S. tuberosum are in line with those reported in previous studies on tomato plants (Solanum lycopersicum L.), where Ca-EDTA was reported to promote blossom formation but induced toxicity when applied at high concentrations (namely, 5 g L−1), leading to chlorosis and necrosis in plants leaves [42], whereas it decreased chlorophyll levels in Vicia faba L. [43].
In sharp contrast, the global absence of adverse effects by the use of CaCl2 is in line with previous reports showing that exogenous Ca application enhanced photosynthetic efficiency in various crops, whereas the foliar application of CaCl2 solutions showed increases in chlorophyll content in potato plants [14].
Notably, the foliar application of commercial solutions with Ca chelate forms showed an increase in the Ca content in Fuji apples, as well as in improving the photosynthetic function of apple leaves and yield [44]. Additionally, exogenous Ca (CaCl2) has proved to reduce stress-induced damages in cucumber fruit, especially under hypoxic stress, and the applications of Ca revealed an enhanced Pn [45]. Moreover, the application of CaCl2 has been shown to enhance photosynthetic rates in tobacco plants [46] and tomatoes [47], when both are exposed to heat stress. These beneficial effects may be due to the vital role of Ca in regulating stomatal movement [48] and its involvement in various photosynthetic processes. Several proteins within the photosynthetic machinery bind directly to Ca2+, and Ca is also required for the activation and performance of C-assimilation [49] and PSII photochemical efficiency [50].
The increase in Ca content in the tubers (greater with Ca-EDTA spraying) was observed (Table 4), suggesting that Ca2+ mass flow through the xylem was complemented by the phloem-mediated redistribution of Ca resulting from the foliar applications [51,52,53,54,55], which is consistent with other reports [56,57,58]. Our data further showed that the pattern of Ca accumulation/localization in the tubers was maintained, with a prevailing accumulation in the epidermis region (Figure 4), in accordance with the findings of Subramanian et al. (2011) [55].
Moreover, the highest yield observed in the controls of both cvs. (Table 5) is probably related to the optimized management practices already adopted by farmers to maximize yield; consequently, the exogenous application of Ca through both products introduced a considerable shift in plant performance, resulting in a reduced yield but led to an increase in the tuber Ca content. Additionally, under the experimental conditions of this study, the soil Ca availability was not a limiting factor for plant growth and yield, with Ca application being the limiting factor, thus resulting in a decrease in yield.
The PCA (Figure 5) verified that CaCl2 has a similar effect on the analyzed variables when compared to the controls, showing an equilibrium between the photosynthetic parameter and tuber weight, without a substantial improvement in the Ca content of tubers. Moreover, considering the representation of both PC1 and PC2, the Ca-EDTA treatment showed a more pronounced effect on the parameters associated with photosynthetic functioning (as previously observed in Table 2 and Table 3).
Overall, although with a lower Ca accumulation in the tubers, CaCl2 proved to be the most effective option for Ca biofortification in peeled tubers (the form most commonly consumed by the general population). The application of this Ca solution, along with potato crop development, is an effective strategy to enhance its nutritional value, while maintaining yield and not promoting negative impacts on plant physiology, as evidenced by the normal functioning of the photosynthetic machinery, i.e., the basis of plant productivity. Furthermore, our analytical approach, based on the performance of the C-assimilation performance in the sprayed plants, together with the obtained yield and Ca content analysis, provides an accurate evaluation of the biofortification efficacy and efficiency for Solanum tuberosum.
Nevertheless, from a human health standpoint, increasing the dietary Ca intake through a widely consumed staple food crop, such as potato, may contribute to mitigating Ca deficiency across the world, based on the nutritional improvements observed in our study.
5. Conclusions
Among the applied solutions, Ca-EDTA, although with a greater Ca accumulation in the tubers, promoted strong negative impacts on the photosynthetic performance and a greater need for energy dissipation mechanisms (Y(NPQ) and qN), altogether contributing to the observed lower tuber size and total yield. The use of CaCl2 resulted in an increase in Ca content in peeled tubers of 17.1% in Picasso and 29.5% in Rossi. Furthermore, CaCl2 had no negative impact on the photosynthetic machinery of the plants, as judged by Pn and several parameters of chlorophyll a fluorescence that addressed the photosynthetic machinery functioning (such as, Fv/Fm, Fv′/Fm, Y(II), and qL). Our findings clearly showed that Ca-EDTA (for the applied concentration) should be avoided due to its severe negative implications on leaf photosynthesis-related parameters, tuber size, and total yield. In contrast, CaCl2, although with a moderate Ca accumulation in the tubers, did not affect the physiological and agricultural parameters, and, therefore, it can be used as an effective tuber Ca biofortification strategy for the Solanum tuberosum L. cvs. Picasso and Rossi, which can be implemented by farmers, thus providing a low-cost approach to improving the nutritional quality of potato in regions where these cultivars are already grown.
Conceptualization: F.C.L., A.R.F.C., J.C.R. and I.P.P.; methodology: A.R.F.C., M.G. and J.C.R.; investigation: A.R.F.C., A.P.R., I.L., A.C.M., C.C.P., D.D., M.G., J.C.R., A.P.R., J.N.S., M.M.S., M.S., P.S.-C. and P.L.; resources: M.G., A.P.R., J.N.S., I.L., A.C.M., C.C.P., D.D., F.C.L., M.S., M.M.S., P.L., P.S.-C. and F.H.R.; writing—original draft preparation: A.R.F.C., J.C.R. and I.P.P.; writing—review and editing: A.R.F.C., J.C.R. and I.P.P.; supervision: F.C.L. and J.C.R.; funding acquisition: F.C.L. and F.H.R. All authors have read and agreed to the published version of the manuscript.
Not applicable.
Not applicable.
The data are contained within the article.
The authors are grateful to Louricoop—Cooperativa de Apoio e Serviços do Concelho da Lourinhã—CRL—Portugal for its technical assistance in the field’s production.
The authors declare no conflicts of interest.
Footnotes
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Figure 1 National (red line) and regional geographic locations of the region of Lourinhã and the field location. Images were obtained through Google Earth (accessed on 21 February 2025).
Figure 2 Daily range of reported temperatures (A), air humidity (B), and precipitation (C) during the production cycle (12 March to 11 August 2020 in Lourinhã). The graphical projection was generated based on the data available from the Weather Underground online platform (
Figure 3 Overview of plants from Solanum tuberosum L. cvs. Picasso, and Rossi, treated with foliar spraying applications in control plants (with water only) or treated with 12 kg ha−1 of CaCl2 or Ca-EDTA solutions. The first monitoring corresponded to six days after the 3rd application, the 2nd was performed seven days following the 5th application, and the 3rd was performed seven days after the 6th application.
Figure 4 Calcium location in different regions of the equatorial area of the tubers of Solanum tu-berosum L. cvs. Picasso and Rossi at harvest, treated with foliar spraying applications in control plants (with water) or of 12 kg ha−1 of CaCl2 or Ca-EDTA solutions (yellow color indicates Ca content).
Figure 5 Principal component analysis (PCA) for PC1 and PC2 for each studied parameter (
Calcium biofortification itinerary for S. tuberosum L. cvs. Rossi and Picasso implemented in the field located in Lourinhã in 2020.
| Cultivar | Planting Date | Foliar Applications | Harvest Date | ||||||
|---|---|---|---|---|---|---|---|---|---|
| 1st | 2nd | 3rd | 4th | 5th | 6th | 7th | |||
| Picasso | 12/3 | 19/5 | 27/5 | 3/6 | 9/6 | 16/6 | 23/6 | 30/6 | 22/7 |
| Rossi | 16/3 | 19/5 | 27/5 | 3/6 | 9/6 | 16/6 | 23/6 | 30/6 | 11/8 |
Evaluation of net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (E), and instantaneous water use efficiency (iWUE) in the Solanum tuberosum L. cvs. Picasso and Rossi treated with foliar spraying applications in control plants (with water) or of 12 kg ha−1 of CaCl2 or Ca-EDTA solutions. Measurements were carried out on 22 June 2020 (96 days after plantation for the cvs. Picasso and Rossi, after five foliar applications) and 30 July 2020 (110 days after plantation for the cv. Rossi, close to harvest time, and 30 days after the 7th application, and the cv. Picasso was already harvested on 22 July 2020).
| Picasso | Rossi | ||||
|---|---|---|---|---|---|
| Parameter | Treatment | 22 June | 30 July | 22 June | 30 July |
| Pn | Control | 7.89 ± 0.54 a | - | 16.3 ± 0.80 aA | 7.90 ± 1.35 bB |
| CaCl2 | 8.85 ± 1.48 a | - | 17.7 ± 0.69 aA | 12.4 ± 0.71 aA | |
| Ca-EDTA | 3.52 ± 0.78 b | - | 17.5 ± 1.99 a | - | |
| gs | Control | 51.5 ± 1.95 a | - | 97.2 ± 10.8 bA | 77.1 ± 17.9 aA |
| CaCl2 | 61.0 ± 7.61 a | - | 118 ± 14.2 bA | 94.9 ± 4.39 aA | |
| Ca-EDTA | 44.8 ± 2.65 a | - | 227 ± 48.6 a | - | |
| E | Control | 1.54 ± 0.05 a | - | 1.92 ± 0.13 bA | 1.22 ± 0.23 aA |
| CaCl2 | 1.76 ± 0.15 a | - | 2.15 ± 0.20 abA | 1.56 ± 0.06 aA | |
| Ca-EDTA | 1.41 ± 0.08 a | - | 2.93 ± 0.36 a | - | |
| iWUE | Control | 4.37 ± 0.18 a | - | 7.21 ± 0.14 abA | 6.80 ± 0.32 aA |
| CaCl2 | 4.71 ± 0.47 a | - | 7.94 ± 0.19 aA | 7.87 ± 0.26 aA | |
| Ca-EDTA | 2.23 ± 0.15 b | - | 5.56 ± 0.57 b | - | |
Each value represents the mean ± SE (n = five plants). A two-way ANOVA analysis, p < 0.05, was performed for each parameter and separately for each cv. Different letters express significant differences between treatments in each date (a, b) or between dates within each treatment (A, B), with a and A representing the highest values. “-“ indicates that measurements were not carried out due to the absence of plants.
Evaluation of leaf chlorophyll a fluorescence parameters in the Solanum tuberosum L. cvs. Picasso and Rossi treated with foliar spraying applications in control plants (with water) or of 12 kg ha−1 of CaCl2 or Ca-EDTA solutions on the same days of leaf gas exchanges. Measurements were carried out on 22 June 2020 (96 days after plantation for the cvs. Picasso and Rossi, after five foliar applications) and 30 July 2020 (110 days after plantation for the cv. Rossi, close to harvest time, and 30 days after the 7th application, and the cv. Picasso was already harvested on 22 July 2020). The analyzed parameters included the maximal photochemical efficiency of PSII (Fv/Fm), the actual PSII photochemical efficiency of energy conversion under light-adapted conditions (Fv′/Fm′), the quantum yields of non-cyclic electron transport (Y(II)), as well as the regulated (Y(NPQ)) and non-regulated (Y(NO)) energy dissipation in PSII, and the photochemical (qL) and non-photochemical (qN) quenching coefficients.
| Picasso | Rossi | ||||
|---|---|---|---|---|---|
| Parameter | Treatment | 22 June | 30 July | 22 June | 30 July |
| Fv/Fm | Control | 0.825 ± 0.002 a | - | 0.797 ± 0.008 aA | 0.768 ± 0.010 aA |
| CaCl2 | 0.832 ± 0.003 a | - | 0.793 ± 0.015 aA | 0.773 ± 0.005 aA | |
| Ca-EDTA | 0.541 ± 0.041 b | - | 0.815 ± 0.003 a | - | |
| Fv’/Fm’ | Control | 0.458 ± 0.040 a | - | 0.478 ± 0.019 aA | 0.484 ± 0.022 aA |
| CaCl2 | 0.419 ± 0.055 a | - | 0.522 ± 0.028 aA | 0.471 ± 0.033 aB | |
| Ca-EDTA | 0.135 ± 0.018 b | - | 0.425 ± 0.048 a | - | |
| Y(II) | Control | 0.318 ± 0.033 a | - | 0.325 ± 0.017 aA | 0.297 ± 0.036 aA |
| CaCl2 | 0.282 ± 0.044 a | - | 0.374 ± 0.037 aA | 0.220 ± 0.034 aB | |
| Ca-EDTA | 0.044 ± 0.013 b | - | 0.235 ± 0.046 b | - | |
| Y(NPQ) | Control | 0.474 ± 0.030 b | - | 0.448 ± 0.018 bB | 0.504 ± 0.038 aA |
| CaCl2 | 0.537 ± 0.055 b | - | 0.398 ± 0.043 bB | 0.523 ± 0.033 aA | |
| Ca-EDTA | 0.756 ± 0.008 a | - | 0.565 ± 0.055 a | - | |
| Y(NO) | Control | 0.209 ± 0.009 a | - | 0.227 ± 0.008 aA | 0.199 ± 0.003 bB |
| CaCl2 | 0.181 ± 0.014 a | - | 0.228 ± 0.014 aA | 0.257 ± 0.017 aA | |
| Ca-EDTA | 0.200 ± 0.014 a | - | 0.200 ± 0.011 b | - | |
| qN | Control | 0.830 ± 0.018 b | - | 0.797 ± 0.016 bA | 0.818 ± 0.020 aA |
| CaCl2 | 0.858 ± 0.031 ab | - | 0.751 ± 0.043 bA | 0.795 ± 0.026 aA | |
| Ca-EDTA | 0.950 ± 0.005 a | - | 0.851 ± 0.033 a | - | |
| qL | Control | 0.556 ± 0.051 a | - | 0.531 ± 0.038 aA | 0.450 ± 0.047 aA |
| CaCl2 | 0.534 ± 0.033 a | - | 0.548 ± 0.050 aA | 0.329 ± 0.059 bB | |
| Ca-EDTA | 0.290 ± 0.061 b | - | 0.396 ± 0.042 b | - | |
Each value represents the mean ± SE (n = five plants). A two-way ANOVA analysis, p < 0.05, was performed for each parameter and separately for each cv. Different letters express significant differences between treatments for each date (a, b) or between dates within each treatment (A, B), with a and A representing the highest values. “-“ indicates that measurements were not carried out due to the absence of plants.
Calcium content (%) in peeled and unpeeled tubers (on a dry-weight basis) at harvest in the Solanum tuberosum L. cvs. Picasso and Rossi treated with foliar spraying applications in control plants (with water) or of 12 kg ha−1 of CaCl2 or Ca-EDTA solutions.
| Cultivar | Treatment | Peeled | Unpeeled |
|---|---|---|---|
| Picasso | Control | 0.041 ± 0.002 cB | 0.050 ± 0.000 bA |
| CaCl2 | 0.048 ± 0.001 bA | 0.046 ± 0.000 cA | |
| Ca-EDTA | 0.056 ± 0.001 aB | 0.094 ± 0.001 aA | |
| Rossi | Control | 0.044 ± 0.002 cB | 0.066 ± 0.001 bA |
| CaCl2 | 0.057 ± 0.001 aB | 0.064 ± 0.001 bA | |
| Ca-EDTA | 0.051 ± 0.001 bB | 0.118 ± 0.001 aA |
Each value represents the mean ± SE (n = 4), using the ANOVA analysis, p < 0.05. Different letters express significant differences among each cultivar and treatments among peeled or unpeeled tubers (a, b, c) or within each cultivar and treatment among peeled and unpeeled tubers (A, B), with letters a and A representing the highest value.
Evaluation of tubers, as regards their average weight (g), minimum diameter (cm), maximum diameter (cm), as well as yield (kg/total area) Solanum tuberosum L. cvs. Picasso at harvest, treated with foliar spraying applications in control plants (with water) or with 12 kg ha−1 of CaCl2 or Ca-EDTA solutions.
| Cultivar | Treatment | Tuber Weight | Minimum Diameter | Maximum Diameter | Yield |
|---|---|---|---|---|---|
| Picasso | Control | 205 ± 30.3 ab | 5.37 ± 0.273 ab | 9.40 ± 0.872 a | 105 |
| CaCl2 | 268 ± 25.3 a | 5.63 ± 0.376 a | 9.40 ± 0.208 a | 93.8 | |
| Ca-EDTA | 159 ± 23.3 b | 4.43 ± 0.176 b | 8.97 ± 0.578 a | 59.3 | |
| Rossi | Control | 227 ± 40.4 a | 6.70 ± 0.611 a | 19.3 ± 0.060 ab | 169 |
| CaCl2 | 195 ± 29.9 a | 5.87 ± 0.410 a | 11.5 ± 0.273 a | 160 | |
| Ca-EDTA | 186 ± 4.27 a | 5.32 ± 0.174 a | 8.73 ± 0.882 b | 46.9 |
Each value represents the mean ± SE (n = 4), using the ANOVA analysis, p < 0.05. Different letters express significant differences for each cultivar and treatments among parameters (a, b), with the letter a representing the highest value.
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; Pais, Isabel P 2
; Guerra, Mauro 3
; Rodrigues, Ana P 4
; Semedo, José N 2
; Inês, Luís 1
; Marques, Ana Coelho 1
; Pessoa, Cláudia C 1
; Daccak Diana 1
; Lidon, Fernando C 1 ; Simões Manuela 1
; Silva, Maria Manuela 1
; Legoinha Paulo 1
; Scotti-Campos, Paula 2
; Reboredo, Fernando H 1
; Ramalho, José C 5
1 Earth Sciences Department of NOVA School of Sciences and Technology, Campus de Caparica, 2829-516 Caparica, Portugal; [email protected] (I.L.); [email protected] (A.C.M.); [email protected] (C.C.P.); [email protected] (D.D.); [email protected] (F.C.L.); [email protected] (M.S.); [email protected] (P.L.); [email protected] (F.H.R.), GeoBioSciences, GeoTechnologies and GeoEngineering Unit (GeoBioTec), NOVA University Lisbon, 2829-516 Caparica, Portugal; [email protected] (I.P.P.); [email protected] (J.N.S.); [email protected] (P.S.-C.); [email protected] (J.C.R.)
2 GeoBioSciences, GeoTechnologies and GeoEngineering Unit (GeoBioTec), NOVA University Lisbon, 2829-516 Caparica, Portugal; [email protected] (I.P.P.); [email protected] (J.N.S.); [email protected] (P.S.-C.); [email protected] (J.C.R.), National Institute of Agrarian and Veterinary Research (INIAV), Quinta do Marquês, 2784-505 Oeiras, Portugal
3 Laboratory for Instrumentation, Biomedical Engineering and Radiation Physics (LIBPhys), Physics Department of NOVA School of Sciences and Technology, Campus daCaparica, 2829-516 Caparica, Portugal; [email protected]
4 PlantStress & Biodiversity Lab, Forest Research Center (CEF), Associate Laboratory TERRA, School of Agriculture (ISA), University of Lisbon (ULisboa), Quinta do Marquês, Av. República, 2784-505 Oeiras, Portugal; [email protected]
5 GeoBioSciences, GeoTechnologies and GeoEngineering Unit (GeoBioTec), NOVA University Lisbon, 2829-516 Caparica, Portugal; [email protected] (I.P.P.); [email protected] (J.N.S.); [email protected] (P.S.-C.); [email protected] (J.C.R.), PlantStress & Biodiversity Lab, Forest Research Center (CEF), Associate Laboratory TERRA, School of Agriculture (ISA), University of Lisbon (ULisboa), Quinta do Marquês, Av. República, 2784-505 Oeiras, Portugal; [email protected]