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Annual Variation in Soil Enzyme Activity in a Paddy Field: Soil Temperature and Nutrient Availability Are Important for Controlling Enzyme Activities.

1. Introduction

Rice cultivation in paddy soils is a vital component of global agriculture [1, 2]; rice is the main staple food for more than half of the world's population and it provides about 20% of the world's dietary energy supply [3, 4]. Paddy soils experience temporal variation in redox conditions: paddy fields are kept flooded during the rice growing season, whereas they are drained during the nongrowing season. Variations in redox conditions cause complicated biogeochemical processes in paddy soils, mostly mediated by microorganisms [5-8], and lead to fluctuations in the soil nutrient availability [2]. The fluctuations in nutrient availability influence microbial activities in paddy soils, and vice versa.

Microorganisms produce various enzymes that are involved in organic matter degradation [9] and therefore influence nutrient cycling and soil fertility [10]. In spite of the significant roles of soil enzymes in nutrient cycling in paddy fields, limited information is available about the annual dynamics of enzyme activities and the factors controlling them in paddy soils. In the present study, we hypothesized that enzyme activities involved in carbon (C), nitrogen (N), phosphorus (P), and sulfur (S) cycling would vary according to the fluctuations in the soil nutrient availability caused by flooding and draining in paddy fields. The annual variations in enzyme activities and soil physicochemical properties were determined and compared to reveal possible links between them.

2. Materials and Methods

2.1. Soil Sampling. Soil samples were collected 18 times from November 2007 to October 2008 from a paddy field (1400 [m.sup.2]) in Matsumoto, Nagano Prefecture, Japan. The mean annual temperature was 11.8[degrees]C, and mean annual precipitation was 1,031 mm. The soil was classified as Gray Lowland soil in the Japanese system, which corresponds to Gleyic Fluvisol in the World Reference Base classification. The paddy field received cow manure (1.43 kg x [m.sup.-2]), compost (0.17 kg x [m.sup.-2]), and bone meal (0.04 kg x [m.sup.-2]) on May 1, 2008. The field was flooded on May 6, and rice seedlings were transplanted on May 17. The field remained flooded until late August and was then drained. The rice plants were harvested on September 25.

On each sampling date, soil sampling was conducted at around 2 p.m., and air and soil temperatures were measured in the paddy field. Soil samples were collected from five plots to 15 cm depth on each sampling occasion. After putting each sample into a polyethylene bag, air and water were removed from the bag, and then the samples were transported to the laboratory within 30 min after collection. Soil redox potential (Eh) and pH were measured for three soil samples as soon as possible; for the other measurements, the same amount of five soil samples were pooled and mixed together as a composite sample, and then they were stored at -80[degrees]C in bottles purged with [N.sub.2] gas until analysis.

2.2. Soil Physicochemical Analyses. Eh was measured as described in Onikura and Goto [11]. In brief, deoxygenated water was gently added to the wet soil, and then a Pt electrode was inserted into the soil. After leaving it to stand for 1 h under [N.sub.2] gas, Eh was determined using an Eh meter (PRN-41, Fujiwara Scientific Company, Tokyo, Japan) with a Pt-AgCl combination reference electrode. Soil pH was measured with a glass electrode in the water just above the soil. We did not correct the Eh for pH in this study, because there is a wide range of reported changes in Eh per unit of pH [12].

Acid-volatile sulfide (AVS) was measured by the coldacid purge and trap technique described by Allen et al. [13]. AVS is defined as the reactive fraction of sulfide in soil that releases [H.sub.2]S when exposed to cold HCl; it includes dissolved sulfide species and metastable iron sulfide minerals [14]. Briefly, the wet soil was treated with deoxygenated HCl, adjusted to a final concentration of 1 M, for 30 min in the reactor under a continuous flow of [N.sub.2] gas, and the evolved [H.sub.2]S was trapped in 0.5 M NaOH. The sulfide content in the trap was measured by the methylene blue spectrophotometric technique. The recovery of sulfide added as [Na.sub.2]S x 9[H.sub.2]O to the reactor was 116.5 [+ or -] 19.6% (n = 4). Exchangeable manganese (Mn) was extracted from the wet soil with deoxygenated 0.05 M Ca[Cl.sub.2] for 24 h [15, 16] under [N.sub.2] gas and then determined with an atomic absorption spectrometer (Perkin Elmer 5100 PC, Tokyo, Japan). Mn was selected as a metal sensitive to soil reduction [17]. Modified Bray-2P was extracted from the wet soil using deoxygenated dilute acid fluoride (0.03 M N[H.sub.4]F, 0.1 M HCl) at a 1: 20 soil: solution ratio (w/v) [18]. Concentrations of inorganic P (Pi) in the extracts were determined by the ammonium molybdate-ascorbic acid method [19]. In the colorimetric determination of Pi for the extract of the modified Bray-2 test, fluoride can interfere with color formation, and thus boric acid was added to form noninterfering fluoroborate [18]. For total C and N measurements, air-dried soil samples were used; the amounts were determined using an NC analyzer (Thermo Finnigan Flash EA1112, Thermo Fisher Scientific, Waltham, MA, USA). These analyses were performed in triplicate for each sample, and the mean values are expressed on a dry weight basis.

2.3. Soil Enzyme Analyses. We measured soil enzyme activities involved in C, N, P, and S cycling in soils stored at -80[degrees]C. [beta]-D-glucosidase activity was chosen as a representative C-acquiring enzyme because this enzyme is involved in hydrolysis of cellobiose, the main product in hydrolysis of cellulose by cellulases [20], and it was measured with p-nitrophenyl-[beta]-D-glucopyranoside as the substrate in a modified universal buffer (MUB) (tris(hydroxymethyl) aminomethane, 2.42 g; maleic acid, 2.32 g; citric acid, 2.8 g; boric acid, 1.26 g; and distilled water, 1 L) at pH 6.0 [20]. L-asparaginase activity was measured with L-asparagine as the substrate in a phosphate buffer at pH 7.6 [21]. L-asparaginase was selected as the N-acquiring enzyme, because it was reported that among several N-acquiring enzymes, L-asparaginase was the most representative of the N availability in soils [22]. Acid phosphatase activity was determined as the P-acquiring enzyme with p-nitrophenyl phosphate as the substrate in a MUB at pH 6.5 [20], because this enzyme plays an important role in P cycling in soils (e.g., [23, 24]). Arylsulfatase activity, which is partly responsible for S cycling in soils [20], was chosen as a representative S-acquiring enzyme and was determined with p-nitrophenyl sulfate as the substrate in an acetate buffer at pH 5.8 [20]. These assays were conducted in triplicate at the field soil temperature when the soil was collected. A standard curve was prepared on each occasion of measurement. The activities are expressed on a dry weight basis.

3. Results and Discussion

3.1. Annual Variations in Soil Physicochemical Properties. The lowest soil temperature (2.2[degrees]C) was observed on February 24, and the highest (28.3[degrees]C) was recorded on August 8 (Figure 1). Soil total C and N contents were slightly elevated during June and July. This increase might reflect a large input of organic matter through rice plants [25] and algae [26] in this period. Some soil physicochemical properties were drastically changed by water management practices. Soil Eh began to decrease after flooding on May 6 because of the oxygen depletion and stayed stable (-11 [+ or -] 31 mV to 53 [+ or -] 34 mV) under the flooded condition. Simultaneously with the decline in Eh, exchangeable Mn and Bray-2P concentrations and pH increased in the soil. The increase in pH with reduction in soil Eh is a well-known phenomenon (e.g., [16, 27]), which is caused by proton consumption through the formation of various reductants [28]: Ox + [ne.sup.-] + [mH.sup.+] = Red + (m/2)[H.sub.2]O, where Ox is the oxidized species and Red is the reduced species, and m and n are stoichiometric factors. The increase in exchangeable Mn concentration with reduction in soil Eh results from the reduction of Mn(IV) oxides to Mn(II) primarily by microorganisms [5, 29]. An increase in Bray-P concentration by soil flooding was also reported in other studies [30, 31, 32]. Such an enhancement of P availability has long been known in flooded paddy soils (e.g., [33, 34], primarily caused by the reduction of Fe(III) compounds holding P on their surfaces and by reduction of insoluble ferric phosphate to more soluble ferrous phosphate [2, 35]).

On September 28, a steep rise in Eh concurrent with soil draining caused a rapid drop in Bray-2P concentration from 119 [+ or -] 5 to 68 [+ or -] 1 [micro]g x P x [g.sup.-1] (Figure 1). In contrast, exchangeable Mn concentration declined gradually with soil oxidation, and an increased concentration of exchangeable Mn was still observed on October 9. Gotoh and Yamashita [36] and Phillips [37] also observed the slow reoxidation of Mn(II) formed in the reduced soil. These results indicate that Mn(IV) oxides are rapidly reduced by flooding, but the reoxidation of Mn(II) seems to be slow during the oxidation of anoxic soils. In stark contrast, the AVS concentration increased very slowly after flooding (beginning a month later than that of exchangeable Mn and Bray-2P) and decreased sharply from 66.4 [+ or -] 2.8 to 9.2 [+ or -] 3.3 [micro]g x [g.sup.-1] on September 9, more than a month ahead of the decline of exchangeable Mn. This behavior of AVS can be explained by the low Eh (<0 mV) at which sulfide is formed through microbial respiratory sulfate reduction [5] and by the rapid oxidation of sulfide via some oxidizable substances [13].

3.2. Soil Enzyme Activities. In this study, enzyme activities were determined at the field soil temperature when the soil was collected to reflect the annual changes of in situ activity in the paddy field. All the activities of four enzymes were high in summer (Figure 2(a)) and increased exponentially with soil temperature (Figure 3). Because soil temperature correlated positively with total C, total N, Bray-2P, and pH and negatively with Eh, most of these variables also correlated with enzyme activities (Table 1). However, these correlations were weaker than those between soil temperature and enzyme activities. These results might imply that soil temperature had a larger influence on in situ enzyme activities than flooding.

The effects of season and flooding/draining condition on the enzyme pool might be small in the paddy field, because the exponential relationship between enzyme activities and soil temperature (Figure 3) could be explained by temperature sensitivity of the structure and catalytic activity of the enzyme itself [38], which is described from first principles of thermodynamics. Inconsistent results have been reported regarding the effects of flooding and season on enzyme activities among soils and among enzymes in laboratory soil incubation studies [39, 40, 41] and field studies [42, 43]. Ishida and Shiraishi [42] found elevated levels of saccharase but similar levels of amylase activities under flooded conditions in summer compared with those under drained conditions in winter in a paddy field, whereas Kanazawa [43] reported a slight decrease in [beta]-D-glucosidase and N-acetyl-[beta]-glucosaminidase activities during the flooded period when compared with other periods in a paddy field (in both studies, enzyme activities were measured at a single reference temperature, not at the field soil temperature). High enzyme activity during the flooded period might be the result of the large input of organic matter from rice plants and algae as mentioned above. The decreased activity associated with flooding would not be related to the disappearance of [O.sub.2] because of the insensitivity of hydrolase to [O.sub.2] levels [44] but might be caused by the accumulation of phenolic compounds in the flooded paddy soil [44-46]. Alternatively, the decreases in the enzyme activities under flooded conditions might be a consequence of decreased microbial biomass size [39]. Further studies are necessary to understand the influence of flooding and season on enzyme activities and pools in paddy fields.

Variation in the stoichiometry of enzyme activities associated with nutrient acquisition can be explained by the resource allocation model for extracellular enzyme synthesis [47, 48] in which microorganisms will preferentially allocate their resources to enzymes for acquiring an element limiting their productivity. In aerobic agricultural and forest soils, the ratio of phosphatase to [beta]-D-glucosidase activity and the ratio of L-asparaginase to [beta]-D-glucosidase activity reflect P [49, 50] and N [22, 50] bioavailability, respectively. Thus, in the present study, we determined these enzyme ratios to evaluate annual variation in bioavailability of nutrients in the paddy field. The ratios of L-asparaginase to [beta]-D-glucosidase activity and arylsulfatase to [beta]-D-glucosidase activity exhibited similar temporal patterns: these ratios were generally higher in winter than in summer (Figure 2). In contrast, annual variation in the ratio of acid phosphatase to [beta]-D-glucosidase activity was small. In agreement with the results of aerobic agricultural and forest soils [22, 49, 50], significant negative correlations were observed between Bray-2P concentration and the ratio of acid phosphatase to [beta]-D-glucosidase activity (Spearman r = -0.631, p = 0.005) and between total N and the ratio of L-asparaginase to [beta]-D-glucosidase activity (r = -0.612, p = 0.007; Figure 4), which were comparable to those in previous studies. Therefore, it is suggested that acid phosphatase and L-asparaginase were synthesized by microorganisms depending on the temporal changes in soil P and N availability, although the microbial community, in particular the active microbial community, is known to vary substantially with redox conditions in paddy soils [5, 7, 51].

4. Conclusions

Exchangeable Mn, Bray-2P, and AVS concentrations and pH increased as the soil became more reduced under flooded conditions, and vice versa under drained conditions in the paddy field. Contrasting behavior was observed between exchangeable Mn and AVS: the exchangeable Mn concentration increased simultaneously with the decline in Eh after flooding and declined gradually with soil oxidation after draining, whereas the AVS concentration increased very slowly after flooding and decreased sharply after draining. Enzyme activities determined at the field soil temperature were high in summer, with the activity increasing exponentially with soil temperature. The ratio of acid phosphatase to [beta]-D-glucosidase activity increased with a decrease in Bray-2P concentration and the ratio of L-asparaginase to [beta]-D-glucosidase activity increased with a decrease in total N, indicating that acid phosphatase and L-asparaginase were synthesized by microorganisms depending on the temporal changes in soil P and N availabilities, although the microbial community is known to vary substantially with redox conditions in paddy soils. These results suggest the significance of soil temperature in controlling in situ enzyme activities in the paddy soil and also that the stoichiometry of extracellular enzyme activity associated with C, N, and P acquisition reflects the soil nutrient availability. Further studies are needed to confirm the usefulness of the stoichiometry of extracellular enzyme activity as an index of nutrient availability in paddy soils.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.


This study was supported by the Management Expenses Grant from Shinshu University.


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Takashi Kunito (iD), (1) Takashi Shiroma, (1) Hitoshi Moro, (1) and Hirotaka Sumi (2)

(1) Department of Environmental Sciences, Faculty of Science, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Japan

(2) Department of Biological Chemistry, College of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi 487-8501, Japan

Correspondence should be addressed to Takashi Kunito;

Received 5 June 2018; Revised 22 August 2018; Accepted 19 September 2018; Published 10 October 2018

Academic Editor: Rafael Clemente

Caption: Figure 1: Annual variations in soil physicochemical properties in a paddy field. (a) Air and soil temperatures. Filled circle, soil temperature; open circle, air temperature; (b) soil Eh; (c) exchangeable Mn; (d) Bray-2P; (e) total C and N. Filled circle, total C; open circle, total N; (f) soil pH; (g) acid-volatile sulfide. Data points represent means, and the error bars represent standard deviations.

Caption: Figure 2: Annual variations in enzyme activities involved in C, N, P, and S cycling (a) and the ratios of N-, P-, and S-acquiring enzymes to [beta]-D-glucosidase activity (b). (a) Filled triangle, arylsulfatase ([micro]mol p-nitrophenol [g.sup.-1] x [h.sup.- 1]); open circle, L-asparaginase ([micro]mol N[H.sub.4.sub.+]-N [g.sup.-1] x [h.sup.-1]); filled circle, acid phosphatase ([micro]mol p-nitrophenol [g.sup.-1] x [h.sup.-1]); open triangle, [beta]-D-glucosidase ([micro]mol p-nitrophenol [g.sup.-1] x [h.sup.-1]). (b) Open circle, ratio of L-asparaginase to [beta]-D-glucosidase activity; filled triangle, ratio of arylsulfatase to [beta]-D- glucosidase activity; filled circle, ratio of acid phosphatase to [beta]-D-glucosidase activity. Data points represent means, and the error bars represent standard deviations.

Caption: Figure 3: Relationships between soil temperature and enzyme activity in paddy soil. (a) Acid phosphatase; (b) L-asparaginase; (c) arylsulfatase; (d) [beta]-D-glucosidase.

Caption: Figure 4: Relationships between Bray-2P and ratio of acid phosphatase to [beta]-D-glucosidase activity (a) and between total N and ratio of L-asparaginase to [beta]-D-glucosidase activity (b) in paddy soil.
Table 1: Spearman rank correlation coefficients between soil
properties and enzyme activities.

                          Soil         Total N        Total C

Total N                 0.666 **
Total C                 0.482 *        0.820 **
Bray-2P                 0.856 **       0.564 *         0.416
Eh                     -0.723 **        -0.216         -0.206
pH                      0.808 **       0.472 *         0.453
Arylsulfatase           0.880 **       0.631 **        0.439
Acid phosphatase        0.893 **       0.649 **        0.414
[beta]-Glucosidase      0.874 **       0.608 **        0.414
L-asparaginase          0.839 **        0.265          0.273

                        Bray-2P           Eh             pH

Total N
Total C
Eh                     -0.757 **
pH                      0.868 **      -0.794 **
Arylsulfatase           0.740 **      -0.659 **       0.825 **
Acid phosphatase        0.643 **       -0.534 *       0.610 **
[beta]-Glucosidase      0.876 **      -0.748 **       0.856 **
L-asparaginase          0.717 **      -0.757 **       0.759 **

                      Arylsulfatase       Acid         [beta]-
                                      phosphatase    Glucosidase

Total N
Total C
Acid phosphatase        0.897 **
[beta]-Glucosidase      0.827 **        0.701 **
L-asparaginase          0.713 **        0.695 **       0.709 **

*, p < 0.05; **, p < 0.01.
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Title Annotation:Research Article
Author:Kunito, Takashi; Shiroma, Takashi; Moro, Hitoshi; Sumi, Hirotaka
Publication:Applied and Environmental Soil Science
Article Type:Report
Geographic Code:9JAPA
Date:Jan 1, 2018
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