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

Annual variations in enzyme activities involved in carbon (C), nitrogen (N), phosphorus (P), and sulfur (S) cycling and soil physicochemical properties were examined in a Japanese paddy field. All the enzyme activities determined at the field soil temperature (range, 2.2°C–28.3°C) increased exponentially with soil temperature (p< 0.001). Significant negative correlations were found between Bray-2P concentration and the ratio of acid phosphatase to β-D-glucosidase activity (Spearman r�−0.631, p� 0.005) and between total N and the ratio of L-asparaginase to β-D-glucosidase activity (r�−0.612, p � 0.007), suggesting that in accordance with the resource allocation model, acid phosphatase and L-asparaginase were synthesized by microorganisms depending on the temporal changes in soil P and N availability. +ese results suggest the significance of soil temperature in controlling in situ enzyme activities in paddy soil and also that the stoichiometry of enzyme activities associated with C, N, and P acquisition reflects the soil nutrient availability.


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][6][7][8], and lead to fluctuations in the soil nutrient availability [2].e 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.
e annual variations in enzyme activities and soil physicochemical properties were determined and compared to reveal possible links between them.

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 2 ) in Matsumoto, Nagano Prefecture, Japan.e mean annual temperature was 11.8 °C, and mean annual precipitation was 1,031 mm. e soil was classified as Gray Lowland soil in the Japanese system, which corresponds to Gleyic Fluvisol in the World Reference Base classification.e paddy field received cow manure (1.43 kg•m −2 ), compost (0.17 kg•m −2 ), and bone meal (0.04 kg•m −2 ) on May 1, 2008.e field was flooded on May 6, and rice seedlings were transplanted on May 17. e field remained flooded until late August and was then drained.
e 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 °C in bottles purged with N 2 gas until analysis.

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 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 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 2 gas, and the evolved H 2 S was trapped in 0.5 M NaOH.e sulfide content in the trap was measured by the methylene blue spectrophotometric technique.
e recovery of sulfide added as Na 2 S•9H 2 O to the reactor was 116.5 ± 19.6% (n � 4).Exchangeable manganese (Mn) was extracted from the wet soil with deoxygenated 0.05 M CaCl 2 for 24 h [15,16] under N 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 NH 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 ( ermo Finnigan Flash EA1112, ermo Fisher Scientific, Waltham, MA, USA).ese analyses were performed in triplicate for each sample, and the mean values are expressed on a dry weight basis.

Soil Enzyme Analyses.
We measured soil enzyme activities involved in C, N, P, and S cycling in soils stored at −80 °C.β-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 pnitrophenyl-β-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 pnitrophenyl sulfate as the substrate in an acetate buffer at pH 5.8 [20].ese 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.e activities are expressed on a dry weight basis.

Annual Variations in Soil Physicochemical Properties.
e lowest soil temperature (2.2 °C) was observed on February 24, and the highest (28.3 °C) was recorded on August 8 (Figure 1).Soil total C and N contents were slightly elevated during June and July.
is 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 ± 31 mV to 53 ± 34 mV) under the flooded condition.Simultaneously with the decline in Eh, exchangeable Mn and Bray-2P concentrations and pH increased in the soil.e 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 − + mH + � Red + (m/2)H 2 O, where Ox is the oxidized species and Red is the reduced species, and m and n are stoichiometric factors.
e 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 2 Applied and Environmental Soil Science been known in ooded 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 ± 5 to 68 ± 1 µg•P•g −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.ese results indicate that Mn(IV) oxides are rapidly reduced by ooding, 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 ooding (beginning a month later than that of exchangeable Mn and Bray-2P) and decreased sharply from 66.4 ± 2.8 to 9.2 ± 3.3 µg•g −1 on September 9, more than a month ahead of the decline of exchangeable Mn. is behavior of AVS can be explained by the low Eh (<0 mV) at which sul de is formed through microbial respiratory sulfate reduction [5] and by the rapid oxidation of sul de via some oxidizable substances [13].

Soil Enzyme Activities.
In this study, enzyme activities were determined at the eld soil temperature when the soil was collected to re ect the annual changes of in situ activity in the paddy eld.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.ese results might imply that soil temperature had a larger in uence on in situ enzyme activities than ooding.e e ects of season and ooding/draining condition on the enzyme pool might be small in the paddy eld, 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 which is described from rst principles of thermodynamics.Inconsistent results have been reported regarding the e ects of ooding and season on enzyme activities among soils and among enzymes in laboratory soil incubation studies [39,40,41] and eld studies [42,43].Ishida and Shiraishi [42] found elevated levels of saccharase but similar levels of amylase activities under ooded conditions in summer compared with those under drained conditions in winter in a paddy eld, whereas Kanazawa [43] reported a slight decrease in β-D-glucosidase and N-acetylβ-glucosaminidase activities during the ooded period when compared with other periods in a paddy eld (in both studies, enzyme activities were measured at a single reference temperature, not at the eld soil temperature).High enzyme activity during the ooded period might be the result of the large input of organic matter from rice plants and algae as mentioned above.e decreased activity associated with ooding would not be related to the disappearance of O 2 because of the insensitivity of hydrolase to O 2 levels [44] but might be caused by the accumulation of phenolic compounds in the ooded paddy soil [44][45][46].Alternatively, the decreases in the enzyme activities under ooded conditions might be a consequence of decreased microbial biomass size [39].Further studies are necessary to understand the in uence of ooding and season on enzyme activities and pools in paddy elds.
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 β-D-glucosidase activity and the ratio of L-asparaginase to β-D-glucosidase activity re ect P [49,50] and N [22,50] bioavailability, respectively.us, in the present study, we determined these enzyme ratios to evaluate annual variation in bioavailability of nutrients in the paddy eld.
e ratios of L-asparaginase to β-Dglucosidase activity and arylsulfatase to β-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 β-D-glucosidase activity was small.In agreement with the results of aerobic agricultural and forest soils [22,49,50], signi cant negative correlations were observed between Bray-2P concentration and the ratio of acid phosphatase to β-D-glucosidase activity (Spearman r −0.631, p 0.005) and between total N and the ratio of L-asparaginase to β-Dglucosidase activity (r −0.612, p 0.007; Figure 4), which were comparable to those in previous studies.erefore, 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].

Conclusions
Exchangeable Mn, Bray-2P, and AVS concentrations and pH increased as the soil became more reduced under ooded conditions, and vice versa under drained conditions in the paddy eld.Contrasting behavior was observed between exchangeable Mn and AVS: the exchangeable Mn concentration increased simultaneously with the decline in Eh after ooding and declined gradually with soil oxidation after draining, AVS concentration increased very slowly after ooding and decreased sharply after draining.Enzyme activities determined at the eld soil temperature were high in summer, with the activity increasing exponentially with soil temperature.
e ratio of acid phosphatase to β-D-glucosidase activity increased with a decrease in Bray-2P concentration and the ratio of L-asparaginase to β-D-glucosidase activity increased with a decrease in total N,   Applied and Environmental Soil Science 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.ese 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 re ects the soil nutrient availability.Further studies are needed to con rm the usefulness of the stoichiometry of extracellular enzyme activity as an index of nutrient availability in paddy soils.

Figure 1 :
Figure 1: Annual variations in soil physicochemical properties in a paddy eld.(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 sul de.Data points represent means, and the error bars represent standard deviations.

Figure 4 :
Figure 4: Relationships between Bray-2P and ratio of acid phosphatase to β-D-glucosidase activity (a) and between total N and ratio of Lasparaginase to β-D-glucosidase activity (b) in paddy soil.

Table 1 :
Spearman rank correlation coe cients between soil properties and enzyme activities.