Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol82.Issue3-4.Dec1999:

Short communication

Cucumber yield and nitrogen metabolism in

response to nitrogen supply

Juan Manuel Ruiz

*

, Luis Romero

Department of Plant Biology, Faculty of Sciences, University of Granada, 18071 Granada, Spain

Accepted 22 March 1999

Abstract

Cucumber plants (Cucumis sativusL. cv. Brunex) were grown under controlled conditions in an experimental greenhouse and treated with four rates of N in the form of KNO3(N1, 5 g/m2;

N2, 10 g/m2_{; N3, 20 g/m}2_{; and N4, 40 g/m}2_{). The intermediate N rates (N2 and N3) gave higher}

utilization of NO3ÿ in the leaves (highest NR activities) than treatment N1 (inadequate) and

N4 (excessive). This latter rate (N4) appears to result in excessive foliar assimilation of NO3

ÿ

, thereby increasing amino acids and proteins and inhibiting or reducing NR activity. N2 and, especially, N3 treatments strengthened the translocation of organic nitrogenous compounds (amino acids) towards the fruit, which enhanced the commercial yield.#1999 Elsevier Science B.V. All rights reserved.

Keywords: Cucumis sativus; Improvement; N fertilization; Nitrogen assimilation; Yield

1. Introduction

Plant growth is dependent on an adequate nitrogen (N) supply in order to form amino acids, proteins, nucleic acids and other cell constituents. It has long been recognized that the rate-limiting step for N assimilation, the reduction of NO3

ÿ -N to nitrite (NO2

ÿ

-N) catalyzed by nitrate reductase (NR), is highly regulated (Sivasankar and Oaks, 1996).

* Corresponding author. Tel.: +34-58-24-32-55; fax: +34-58-24-32-54

E-mail address:lromero@goliat.ugr.es (J.M. Ruiz)

Appropriate levels of NO3-N derived from proper N fertilization increase the

amount and activity of NR; this leads to a corresponding increase in the potential for NO3-N reduction and confers a greater capacity for general amino-acid

synthesis, protein synthesis or total N assimilation (Barneix and Causin, 1996). In addition, it has been shown that plant growth and yield rates are often dependent on N supply (Mattson et al., 1991). Nevertheless, excessive N fertilization can be harmful, and NO3-N accumulation is a problem in most crops (Quillere et al.,

1994). In addition, nitrogen fertilizers can contribute to groundwater and surface-water pollution through leaching and soil erosion (Sisson et al., 1991).

In the present work, our aim was to determine the relationship of different N rates with nitrogen metabolism in the leaves and fruits of the cucumber and with commercial yield in this crop grown under greenhouse conditions.

2. Materials and methods

2.1. Crop design

Cucumbers (Cucumis sativusL. c.v. Brunex) were seeded in cell flats (cell size 3_{310 cm) filled with peat: perlite mixture, placed on benches under the}

greenhouse conditions described below, for a period of eight weeks. Seedlings were then transplanted (October 1996) to soil and grown under controlled conditions in an experimental greenhouse at Centro de InvestigacioÂn y Desarrollo HortõÂcola, El Ejido, AlmerõÂa, Spain. The experiment was conducted from 1996 to 1997. In this area, the climate is semiarid and the land is intensively used for agriculture. The soil used was loamy sand with the following characteristics: sand (37.3%), silt (48.6%) and clay (10.1%), CaCO3 equivalent (26.82%), CaCO3

active (14.35%), total N (3.5 g kgÿ1), total organic C (36.1 g kgÿ1), PO (890 mg kgÿ1), K‡ (5.34 g kgÿ1), pH (H2O, 8.45; KCl; 8.01), electrical

conductivity (E.C.ˆ_{4.63 dS m}ÿ1_{). In the greenhouse, the relative humidity}

was 60±80% and the temperature range 24_{48C, with extremes of 158 and}

308C. The experimental design was a randomized complete block with four treatments. Container-grown cucumbers were transplanted into two rows 100 cm apart and drip-irrigated. Each treatment had four replicates in four individual plots of 4_{2 m}2_{wide (16 plots). Each plot contained eight plants. The irrigation}

water had the following properties: pH, 8.05; E.C. 2.03 dS mÿ1; Clÿ 483.9 mg lÿ1; Na‡ 305.76 mg lÿ1; K‡ 10.16 mg lÿ1; HCO 278.15 mg lÿ1.

The different treatments consisted of applying increasing rates of N in the following manner: N in the form of KNO3(N1, 5 g/m2; N2, 10 g/m2; N3, 20 g/

m2; and N4, 40 g/m2). P (8 g/m2) in the form of H3PO4, calcium (11 g/m2) and

magnesium (3 g/m2) were supplied as sulphates. The rate of each nutrient was applied gradually with the irrigation water over the entire experimental period.

Fertilization±irrigation was complemented with the following micronutrients: Fe, 0.5 mg lÿ1; B, 0.1 mg lÿ1; Mn, 0.1 mg lÿ1; Zn, 0.075 mg lÿ1; Cu, 0.075 mg lÿ1; and Mo, 0.05 mg lÿ1

. The pH values of the solution oscillated between 5 and 6; Fe was applied as FeEDDHA, B as H3BO3 and the remaining micronutrients as

sulphates.

2.2. Plant sampling

Leaf samples were taken only from plants with fully expanded leaves of the same 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 thrice in distilled water after disinfecting with 1% non-ionic detergent (Wolf, 1982), then blotted on filter paper. At each sampling, fresh matter (leaf and fruit pulp) was used for the NR assay (only in leaves), amino acids and proteins; a subsample was then dried in a forced-air oven at 708C for 24 h, ground in a Wiley mill and then stored in plastic bags for further analyses (NO3

ÿ

, organic N). Dry weight was recorded and expressed as (g DW/leaf).

2.3. Plant analysis

2.3.1. NO3 ÿ

determination

NO3

ÿ

-N levels were analysed from an aqueous extraction of 0.2 g of dried and ground material in 10 ml of Millipore-filtered water. The NO3

ÿ

-N concentration was measured by spectrophotometry by the method of Cataldo et al. (1975). The results were expressed as mg gÿ1dry weight (DW).

2.3.2. Detection of in vivo NR activity

The basic method was an adaptation of the in vivo NR assay by MaurinÄo et al. (1986). The resulting NO2

ÿ

was measured by the method of Snell and Snell (1949), and the NR activity was expressed as mmol NO2

ÿ

gÿ1 fresh weight (FW) hÿ1

.

2.3.3. Amino-acid and soluble-protein determination

Fresh leaf and fruit samples (0.5 g) were crushed with cold phosphate buffer (50 mM KH2PO4, pH 7.0) and centrifuged at 12 000g for 15 min. The

resulting supernatant was used for the determination of total amino acids by the ninhydrin method as described by Yemm and Cocking (1955), total free amino acids were expressed as mg glycine gÿ1

2.3.4. Organic N determination

A 0.1 g DW sub-sample was digested with sulphuric acid and H2O2 (Wolf,

1982). Organic N was measured by spectrophotometry according to Baethgen and Alley (1989). The results were expressed as mg gÿ1 DW.

2.3.5. Yield

Plant yield was expressed as the mean of fruit weight per plant. Cucumbers collected from each plant were weighed at sampling. Commercial yield (kg/plant) represents fruits with acceptable colour and size.

2.4. Statistical analysis

Standard analysis of variance techniques were used to assess the significance of treatment means. Analysis of regression (linear or quadratic) was performed and its significance was used, with each variable being analyzed as a function of N application rate. Levels of significance are represented by (*) atp< 0.05, (**) at

p< 0.01, (***) atp< 0.001, and NS, not significant.

3. Results and discussion

3.1. Effect of N fertilization on N metabolism in leaves

Most of the parameters of N metabolism analyzed in cucumber leaves showed a direct influence of the N application rate. One of the major and limiting stages of NO3

ÿ

assimilation is NR activity (Sivasankar and Oaks, 1996). Table 1 shows the values obtained in the in vivo assay of NR activity in leaves, with the highest activities at the 10 g/m2(N2) and 20 g/m2(N3) rates, with an increase in activity of 130% over values recorded at 5 g/m2 (N1) rate (p< 0.001). In contrast, the NO3

ÿ

levels (Table 1) showed a trend differing from that of the NR activity, with the highest concentration at 40 g/m2(N4), and the lowest at N1 (p< 0.001). One of the principal factors regulating both, the increase in de novo NR synthesis and its activity is the presence of NO3

ÿ

(Crawford, 1995). Our results showed that the treatments N2 and N3 facilitated uptake and transport of NO3

ÿ

towards the aerial part of the plant, thereby stimulating NR synthesis and activity and reducing NO3

ÿ

. The behaviour of the plants treated with N4 indicates that the heavy N fertilization apparently caused the foliar NO3

ÿ

concentration to exceed the assimilation capacity of these plants due to the partial inhibition or reduction of the activity of NR (Table 1) (Quillere et al., 1994). The N1 treated plants showed the lowest concentrations of NO3

ÿ

(Table 1), possibly owing to a reduced uptake or transport in these plants towards the aerial part, causing a low stimulation of the NR and, therefore, a decline in NO3

ÿ

assimilation.

Table 1

Actiivity of in vivo NR, acumulation of various N fractions and leaf dry weight as a function of N application rates

Treatments Nitrate reductase activity

Nitrate Amino acids Proteins Organic N L e a f d r y weight (g/m2_{) (}

mmol NO2

ÿ

gÿ1_{FW h}ÿ1_{) (mg g}ÿ1_{DW) (mg g}ÿ1_{FW) (mg g}ÿ1_{DW) (g)}

5 1.03 8.34 2.57 13.78 38.41 1.42

10 2.05 12.06 2.91 16.82 43.34 2.12

20 2.48 12.53 2.77 14.17 41.30 2.56

40 1.48 16.38 4.22 18.90 52.11 3.48

Significance Q*** Q*** Q*** Q** L*** L***

Note: Regression equations are for leaves: treatments, nitrate reductase activity, Yˆ0.24‡0.2Xÿ0.0043X2, R2ˆ0.94; treatments, nitrate,

Yˆ7.42‡0.35Xÿ0.0034X2, R2ˆ0.77; treatments, amino acids, Yˆ2.79ÿ0.026X‡0.0015X2, R2ˆ0.95; treatments, proteins,

Yˆ15.32ÿ0.099X‡0.0046X2, R2ˆ0.45; treatments, organic N, Yˆ37.22‡0.35X, R2ˆ0.72; and treatments, leaf dry weight,

Yˆ1.37‡0.054X,R2ˆ0.95.

Ruiz,

L.

Romer

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309±316

Nitrogenous compounds of low and high molecular weight, such as amino acids and proteins, are the principal products of NO3

ÿ

assimilation (Barneix and Causin, 1996; Ruiz et al., 1998), and, as might be expected, the least assimilation of NO3

ÿ

at N1 led to the lowest concentrations, both of amino acids (p< 0.001) and of proteins (p< 0.001) (Table 1). Conversely, the highest concentrations of these compounds were registered at N4 (Table 1). This suggests that initially the foliar assimilation of NO3

ÿ

in the plants treated with N4 was greater than that of the other treatments, thus significantly increasing the synthesis and accumulation of amino acids as well as proteins (Table 1). Nevertheless, the accumulation of nitrogenous compounds, together with the foliar excess of NO3

ÿ

in N4 (Table 1), could inhibit or diminish NR activity, as reported in several works (Barneix and Causin, 1996). This appears to account for the fact that N4 presented the highest concentrations of NO3

ÿ

, amino acids and proteins (Table 1) but an NR activity lower than in treatments N2 and N3 (Table 1). Finally, organic N is another N parameter that increased with intensified NO3

ÿ

reduction (Vincentz et al., 1933). This parameter showed a trend similar to that discussed above for the amino acids and proteins (p< 0.001) (Table 1).

The N treatments gave a significant increase in dry weight per leaf with the highest at N4, and the lowest at N1 (p< 0.001) (Table 1). A possible cause of this highest value at N4 may be the greatest concentrations of amino acids, proteins and organic N reached by the plants receiving this treatment (Table 1), as it is well known that dry weight per leaf, or biomass per leaf depends on N metabolism (McDonald et al., 1996). The relationship between these parameters in our experiment was significant and positive (amino acid-dry weight per leaf,

rˆ_0.87***_{; protein-dry weight per leaf, r}ˆ_0.73**_{; organic N-dry weight per}

leaf,rˆ_0.93***_).

3.2. Effect of N fertilization on N metabolism in fruit

Initially, the NO3

ÿ

levels in the fruits were low in all the treatments (Table 2), since this anion is scarcely transported through the phloem towards the fruit (Marschner et al., 1996). Nevertheless, when the highest NO3

ÿ

dosage was applied, the concentration of this anion in the fruit increased significantly, as found in N4 (p< 0.01). In contrast, the principal N compounds which are transported through the phloem towards the fruit are amino acids (Marschner et al., 1996). In our experiment, the highest levels of amino acids (Table 2) were found in plants treated with N2 and N3, and the lowest at N1 and N4 (p< 0.001). Proteins (p< 0.001) and organic N (p< 0.001) (Table 2) showed a trend similar to that described for the amino acids.

Finally, we studied the relationship between N fertilization and commercial yield. Table 2 shows the higher yields of the N2 and N3 plants compared to N1

and N4 plants, N2 giving the greatest commercial yield. For fruit growth, the mobility of inorganic nutrients and of the organic compounds such as amino acids through the phloem are fundamental (Ho, 1996). Amino acids are the principal nitrogenous compounds that are transported through the phloem, and originate both, from NO3ÿassimilation and from the hydrolysis of proteins and organic N

(Marschner et al., 1996). The greater translocation of amino acids from the leaves towards the fruit in treatments N2 and N3 could significantly influence commercial yield in these treatments (amino acids yield, rˆ_0.77**_{). On the}

other hand, the increased concentration of organic N in the fruits of the N2 and N3 plants also could have caused the differences (organic N commercial yield,

rˆ_0.87**_{), given that N is closely related to yield in most crops (McDonald}

et al., 1996; LoÂpez-Cantarero et al., 1997). The low production of N1 could be due to the lowest foliar assimilation of NO3

ÿ

presented by plants treated at this N level, which was evidently inadequate to stimulate growth and yield in cucumber plants. At N4, the excess N applied may have increased vegetative growth (primarily foliar dry weight) but simultaneously decreasing fruit yield (Davenport, 1996). In fact, N4 presented the highest foliar dry weight (Table 1), the lower yield being consistent with this result. It has been demonstrated experimentally in other cultivated plants that both, inadequate and, especially, excessive N fertilization increases non-commercial yield, but decreases commercial yield (LoÂpez-Cantarero et al., 1997).

Acknowledgements

We are indebted to Dr. Jose Maria Ramos and Dr. Joaquin HernaÂndez for their help in carrying out the statistical analyses.

Table 2

Acumulation of various N fractions and fruit yield as a function of N application rates

Treatments Nitrate Amino acids Proteins Organic N Fruit yield (g/m2) (mg gÿ1

DW) (mg gÿ1

FW) (mg gÿ1

DW) (kg/plant)

5 1.12 1.45 3.26 22.63 0.82

10 1.22 2.23 4.42 27.21 1.85

20 1.24 2.48 4.97 28.68 1.65

40 2.38 1.48 3.44 24.07 1.23

Significance Q** _Q*** _Q*** _Q*** _Q***

Note: Regression equations are for fruits: treatments, nitrate, Yˆ1.26ÿ0.026X‡0.0013X2,

R2ˆ_{0.44; treatments, amino acids,}Yˆ_0.85‡_0.15Xÿ_0.0035X2,R2ˆ_{0.94; treatments, proteins,}

Yˆ2.51‡0.025Xÿ0.0055X2, R2ˆ0.64; treatments, organic N, Yˆ19.35‡0.87Xÿ0.02X2,

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