NITROGEN PHOSPHORUS POTASSIUM EFFECTS ON FORMS OF SULFUR IN LEAVES AND FRUITS OF CUCUMBER

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1 JOURNAL OF PLANT NUTRITION Vol. 25, No. 10, pp , 2002 NITROGEN PHOSPHORUS POTASSIUM EFFECTS ON FORMS OF SULFUR IN LEAVES AND FRUITS OF CUCUMBER Luis R. López-Lefebre, Juan M. Ruiz, and Luis Romero* Department of Plant Biology, Faculty of Science, University of Granada, Av. Fuentenueva s=n, Granada E-18071, Spain ABSTRACT The impact that nitrogen (N), phosphorus (P), and potassium (K) application rates on the sulfur (S) fractions in leaves and fruits of greenhouse-grown cucumbers plants (Cucumis sativus L. cv. Brunex) are presented. The treatments were as follows: N (N1 ¼ 5g NO 3 NH =m 2, N2¼ 10 g NO 3 NH =m 2, N3¼ 20 g NO 3 NH =m 2,N¼0 g NO 3 NH =m 2 ), two levels of P (P1 ¼ 8g H 3 PO =m 2 and P2 ¼ 16 g H 3 PO =m 2 ), and two levels of K (K1 ¼ 20 g K 2 SO =m 2 and K2 ¼ 0 g K 2 SO =m 2 ). The foliar and fruit contents were determined for total S, organic S and sulfate. The influence of the N treatments on the total S (St: organic S þ sulfate) concentration, proved significant, showing a progressive increase in the leaf and fruit concentrations. In the leaves, the P slightly diminished the St concentration but values in the fruits did not appreciably differ from control. The K dosage did *Corresponding author. Fax: þ ; lromero@ugr.es 2151 DOI: =PLN Copyright # 2002 by Marcel Dekker, Inc (Print); (Online)

2 2152 LÓPEZ-LEFEBRE, RUIZ, AND ROMERO not cause the St concentration to differ from that of P, although in the fruit a slightly lower St concentration appeared in the K2 treatment. The response of the organic-s concentration in the leaves resembled that of St, and thus organic S should not be used as a diagnostic method for S status. In the relationship SO 2 =St, the SO2 concentration proved more influential than did the St form, providing a more accurate representation of the potential status of this nutrient in the plant. INTRODUCTION Sulfur (S) has long been known to play an important role in plant metabolism. Inorganic sulfate (SO 2 ) is the most common form of sulfur taken up by plants roots and is relatively abundant in the environment. It is actively transported into roots by plasmamembrane-localized H þ =SO 2 co-transporters. Reduction and most of assimilation of sulfate takes place into plastids. [1] In addition, sulfur forms part of the amino acids cysteine and methionine and also participates in the synthesis of many secondary compounds in plants. The amount and kind of these compounds, as well as proteins rich in sulfurcontaining amino acids that a plant is able to synthesize and store can influence the quality of a crop and its nutritional value for humans and animals. [2] To boost crop productivity, fertilizers are generally applied to correct assumed nutrient deficiencies. [3] Fertilization worldwide is dominated by the application of the macronutrients nitrogen (N), phosphorus (P), and potassium (K), which promote plant development as well as nutrient uptake by the plant. [] Nevertheless, little is known about the response of S to the application of NPK. The most widely applied fertilizer is N, given that its deficiency is the most common. Meanwhile, P is indispensable for the synthesis of phospholipids, ATP and nucleic acids, thus being essential to plant nutrition and therefore occasionally becoming limiting for development and growth. [5] The second most abundant mineral in the plant, after N, is K, which is essential for plant growth. [6] Deficiency of this cation inhibits growth and protein synthesis, [7] and thus an adequate supply of K favors crop growth and fruit production. [8] The deficiency or toxic excess of an element can affect other essential nutrients. It has been demonstrated that the nutritional status, primarily of N, P, and K, can determine the dry-matter production and distribution among the different organs of a plant. [9] The aim of the present work was to determine the response of S and several of its fractions in leaves and fruits of cucumber plants to N, P, and K treatments. The goal is to achieve more accurate estimations of the nutritional status of this crop.

3 SULFUR IN CUCUMBER 2153 MATERIAL AND METHODS Crop Design Cucumber plants (Cucumis sativus L. cv. Brunex) were grown in southeastern Spain under controlled conditions in a greenhouse equipped with a fertigation system. The experiment was conducted under the following conditions: 25 C=18 Cday=night temperature and 60%=80% day=night relative humidity. The experimental design consisted of 27 plots of eight N P K treatments and control with three replications each. All plots contained 1 plants each. The N P K treatments were applied as follows: four doses of N (N1 ¼ 5g NO 3 NH =m 2,N2¼ 10 g NO 3 NH =m 2,N3¼ 20 g NO 3 NH =m 2 and N ¼ 0 g NO 3 NH =m 2 ), two levels of P (P1 ¼ 8g H 3 PO =m 2 and P2 ¼ 16 g H 3 PO =m 2 ), and two levels of K (K1 ¼ 20 g K 2 SO =m 2 and K2 ¼ 0 g K 2 SO =m 2 ). Control plants were treated with N ¼ 2.5 g NO 3 NH =m 2,P¼ gh 3 PO =m 2 and K ¼ 10 g K 2 SO =m 2. Calcium (11 g Ca=m 2 ) and magnesium (3 g Mg=m 2 ) were applied as sulfates. All the treated plants received sulfur applied as K 2 SO, CaSO, MgSO, and H 2 SO,at55gS=m 2, to ensure equals levels of this macronutrient in the growing media. All fertilizers were applied throughout the growth cycle of the plants. The experimental plots were covered with micronutrient solutions as their sulfates, Mn (2 ppm), Zn (1 ppm), Cu (0.25 ppm), and Mo (0.05 ppm), except for Fe, which was applied as Fe-EDDHA (5 ppm), and B, applied as H 3 BO 3 (0.5 ppm). Plant Analysis Leaves samples were taken only from plants with fully expanded leaves of similar size. Leaves and fruits were removed from a zone at about two-thirds of the total height of the plant, when the fruits reached maturity (95 days after transplanting, January 1997). Leaves and fruits were rinsed three times in distilled water after decontaminating with 1% non-ionic detergent and then blotted on filter paper. At each sampling, tissue was dried in a forced-air oven 70 C for 2 h, ground in a Wiley mill. Dried plant material ( g D.M.) was digested with concentrated sulphuric acid and 30% hydrogen peroxide for determinations of organic N. [10] Dried plant material was subjected to nitric-perchloric (v=v) mineralization; in the product, S was measured by turbidimetry of the BaSO maintained in suspension by means of tensioactive agent (gum arabic), according to Novozamsky and van Eck. [11] SO 2 was determined following Cataldo. [12] From the aqueous extract obtained an aliquot was taken to which BaCl 2 -gum arabic was added, the SO 2 being measured by turbidimetry against a pattern curve. [11]

4 215 LÓPEZ-LEFEBRE, RUIZ, AND ROMERO Statistical Analysis Standard analysis of variance techniques were used to assess the significance of treatment means. The data shown are mean values S.E. Mean separation within columns by Duncan s Multiple Range Test, 5% level. Levels of significance are represented by * at P < 0.05, ** at P < 0.01, *** at P < 0.001, and ns: not significant. RESULTS AND DISCUSSION The concentration of total S (St: organic S þ sulphate) present in an organ is specific to each plant species and related to the concentration in the root zone. [13] In our experiment, the influence of the N treatments proved significant (P < 0.001), causing a progressive increase in the leaf and fruit St concentrations, with N3 registering the highest concentration, and N0 the lowest in both organs analyzed (Table 1). In the leaves, P slightly diminished the St concentration between P1 and P2, with values exceeding control by 12% in the P1 dosage and 9% in P2. In the fruit, the two P dosages presented highly similar St concentrations and did not strongly differ from control. Table 1. Effect of N Treatments on S Fractions in Leaves and Fruits of Cucumber Plants St S Org. SO 2 Treatments mg=g p.s. SO 2 =St Nt=St Leaves N0 10.0c 9.1b 0.90a 0.09a 6.77c N b 10.07a 0.92a 0.08a 7.27b N b 10.07a 0.9a 0.08a 7.58b N3 11.2a 10.11a 0.9a 0.08a 7.9a N 11.01b 10.07a 0.93a 0.08a 8.01a Fruits N0 9.02a 8.2a 0.60b 0.06a 2.26a N1 9.06a 7.61b 0.69a 0.07a 1.32c N2 9.09a 8.39a 0.70a 0.07a 1.33c N3 9.11a 8.2a 0.70a 0.07a 1.0b N 9.09a 8.0a 0.69a 0.07a 1.6b St: total sulfur; S org.: organic sulfur; SO 2 : sulfate; Nt: total nitrogen.

5 SULFUR IN CUCUMBER 2155 The K did not differ markedly from P, again slightly increasing, although not significantly (P > 0.05), the leaf St concentration, K2 and K1 presenting values of 10 and 9%, respectively, higher than control. Contrary to the results for the leaves, in the fruits a slight decline in the St concentration was found for the last K2 treatment, although this was not significant (P > 0.05). The behavior of the organic S in the leaves resembled that of St, and thus the former should not be used to diagnose the degree of S present in plant tissue. For the concentration of organic S, was the same as for St in the leaves and in the fruits for the N treatments (Table 1). Therefore, the N application showed a positive effect on organic S. These results differ from those of López- Cantarero, [1] who reported a decline in the leaf concentration of organic S, since this suggests a dilution effect with high amount of N in the medium. In our case, this dilution effect was not clearly manifested and a slight decline in the organic S concentration appeared only in N (0 g=m 2 ; Table 1). However, in the fruit, the behavior of organic S varied with respect to the leaf, since the highest concentration of the organic S in the fruit was recorded for the N0 treatment, some 11% stronger in concentration than for N1. This difference could be explained by a possible interference of the NO 3 that accumulated in this organ due to the application of the N treatments. Increased P fertilization negatively affected the leaf concentration of organic S, since a decline appeared between the P1 and P2 dosages. In addition, in fruit, P2 registered a concentration very similar to that of control (Table 2). The organic S in the leaves or fruits, plotted against the K dosage, proved identical to that described above for P (Table 3). Therefore, we conclude that the higher dosages of P and K did not have a positive effect on the leaf concentration Table 2. Effect of P Treatments on S Fractions in Leaves and Fruits of Cucumber Plants St S Org. SO 2 Treatments mg=g p.s. SO 2 =St Nt=St Leaves P0 10.0b 9.1b 0.90a 0.09a 6.77b P a 10.09a 0.93a 0.08a 7.72a P a 10.07a 0.9a 0.08a 7.68a Fruits P0 9.03a 8.2a 0.60a 0.06a 2.26a P1 9.09a 8.02a 0.69a 0.07a 1.35b P2 9.10a 8.0a 0.70a 0.08a 1.1b St: total sulfur; S org.: organic sulfur; SO 2 : sulfate; Nt: total nitrogen.

6 2156 LÓPEZ-LEFEBRE, RUIZ, AND ROMERO Table 3. Effect of K Treatments on S Fractions in Leaves and Fruits of Cucumber Plants St S Org. SO 2 Treatments mg=g p.s. SO 2 =St Nt=St Leaves K0 10.0b 9.1b 0.90a 0.09a 6.77b K a 10.09a 0.92a 0.08a 7.75a K a 10.07a 0.9a 0.08a 7.65a Fruits K0 9.02a 8.2a 0.60a 0.06a 2.26a K1 9.09a 8.01a 0.70a 0.07a 1.35b K2 9.09a 8.0a 0.68a 0.07a 1.0b St: total sulfur; S org.: organic sulfur; SO 2 : sulfate; Nt: total nitrogen. of organic S, this being apparently due to the increase in the other anions that may interfere with the S. [15] In plants with a low SO 2 content, this compound is usually closely related to St and in our experiment the dynamic, plotted against the N dosage of the leaf and fruit concentration of the SO 2, was in fact identical to that followed by St (Table 1). The highest concentration in both organs appeared in the N3 treatment and the lowest in N0. The N fertilization itself exerted no clear influence over the SO 2 concentration in the fruits (Table 1). However, concentration values for the N3 dosages proved 16% higher than in control. The fact that the SO 2 concentration in the fruits, contrary to that of organic S, did not accumulate in this organ might be explained by the fact that organic S was more mobile and a strong accumulation was mobilized from the leaves. [16] The higher P dosage did not increase the SO 2 concentration either in the leaves or the fruits, and thus there were no significant differences between the two P treatments (Table 2). The K treatments were the only ones that increased the SO 2 leaf concentration, giving a difference of 5% between K2 and control. The response of SO 2 in fruits to the K dosage proved significant (P < 0.05), but negative, given that the highest K dosage was found to interfere with the mobilization of these compounds towards the fruit, since they accumulated preferentially in the leaves (Table 3). As an approximate but reliable value of the S forms, the relationship SO 2 =St is used as an indicator of the nutritional state of S.[13] Tables 1 3 indicate that this relationship did not vary significantly in the leaves, control

7 SULFUR IN CUCUMBER 2157 registering the highest value for this parameter. From these results, we deduce that the control leaves concentrated S primarily in the form of SO 2 =St that is, without integrating it into the corresponding molecules, apparently by a lower rate of SO 2 reduction and assimilation than accumulation in the leaves. On the contrary, in the fruit, the N and P treatments applied had a more positive effect on this S fraction, and the dynamic was consistently the same with all the N and K dosages the values of this relationship increased to reach the highest in N3 and P2, with values 15% higher than control in both cases (Tables 1 and 2). On the other hand, as commented above, the K negatively influenced SO 2 accumulation in the fruit; this could on the one hand interfere with mobilization from the leaves, or, on the other hand, favor integration into organic molecules. Consequently, the SO 2 =St relationship was lower in the K2 than in K1 treatment (Table 3). It can be concluded that for the relationship SO 2 =St, the SO2 concentration was more influential than was the St form, providing a better representation of the potential state of this nutrient in the mineral metabolism of the plant. [17] Neither in herbaceous plants nor in perennials has the relationship total nitrogen=total sulfur (Nt=St) yet been demonstrated to be a good tool to analyze the nutritional status of S in these plants. [18] In this case, the relationship Nt=St was found to be favored by the N application, with the N dosage registering the highest value in the leaves, some 18% higher than the lowest value of control (Table 1). In fruit, this relationship was positively affected by the N treatments, but, despite this, the highest value was registered in control, the N1 values being 72% and the N 5% lower (Table 1). From these data, we deduce that control fruits suffered severe S deficiency. On the other hand, the P1 dosage positively affected this relationship, with values reaching 1% higher than in control. However, in P2, less difference was evident in the Nt=St relationship (Table 2). In fruit, as opposed to the situation in the leaves, P2 values showed less difference with respect to control than in P1 (Table 2). The dynamic of the Nt=St relationship in the leaves and fruits in response to the K treatments proved identical to that of P (Table 3). Despite the above, however, this Nt=St relationship should be used with caution, as more information is still needed to establish a solid relationship between this quotient and the nutritional status of S. In fact, Andrew [19] did not recommend the use of the Nt=St. In conclusion, given our experimental results, we propose that the SO 2 fraction is appropriate to be used as a criterion of the S status in agricultural plants, although further research is needed to remove doubts concerning the role of S and its fractions in plants.

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