Enzymatic Activity Responses to Transport and Low-Temperature Storage: Implications for Plant Nitrogen Metabolism Studies

Abstract

Understanding how transport and storage conditions affect enzymatic activity is essential for accurate biomonitoring of nitrogen metabolism in plants. This study evaluated the effects of transport conditions and low-temperature storage on the enzymatic activities of nitrate reductase (NR), glutamine synthetase (GS), and phosphomonoesterase (PME) for Chloris gayana, Fraxinus uhdei, and Trifolium repens. Enzymatic activities were measured for leaf samples immediately after collection, after 18 h at room temperature, or after 18 h on ice. Additionally, samples were stored at −16 °C or −45 °C for up to 28 days. NR activity decreased to near-zero levels under all storage conditions, indicating that this enzyme is unsuitable for delayed analysis. In contrast, GS and PME activities showed species-dependent responses to storage, with increased activity observed for T. repens and C. gayana, potentially reflecting tissue degradation processes. F. uhdei exhibited greater stability in enzyme activities, suggesting a higher resilience to storage. These findings highlight the importance of minimizing storage time to preserve enzymatic integrity, particularly for NR, while providing insight into the potential for delayed analysis of GS and PME in specific species. This work offers practical recommendations for future biomonitoring efforts in nitrogen deposition studies.

1. Introduction

Human impacts on planetary systems have intensified over the past several decades, one of the most affected being the nitrogen biogeochemical cycle [1]. The rate at which reactive nitrogen species, removed from the N₂ atmospheric sink, are introduced into the biosphere has doubled since the mid-20th century, with high-biodiversity tropical regions being particularly vulnerable [2,3].

Many temperate forest species evolved in nutrient-rich soils, in contrast to plants from high-biodiversity tropical regions, which typically originated in oligotrophic soils [4]. As a result, their responses to the increasing availability of anthropogenic reactive nitrogen species are determined by their particular nitrogen requirements. Atmospheric nitrogen deposition has thus become a key driver of floristic change, favoring the proliferation of nitrophilous species while threatening those that are unable to acclimate to the increasingly high levels of reactive nitrogen [5,6].

Understanding how plant species respond to elevated nitrogen availability is, thus, crucial for projecting biodiversity changes in the Anthropocene. However, while plant responses to climate change can be estimated probabilistically through ecological niche modeling and downscaling of increasingly accurate climate change models [7,8], predicting potential plant responses to increased nitrogen deposition still requires direct physiological assessments.

Quantifying enzyme activities provides a direct method for assessing plant responses to increased nitrogen availability in this changing environment [9,10]. In particular, nitrate reductase is the first enzyme of plant nitrogen metabolism, catalyzing the reduction of nitrate (NO₃⁻) to nitrite (NO₂⁻) [11,12]. In turn, glutamine synthetase plays a subsequent role by integrating ammonium (NH₄⁺) into glutamate, thereby converting inorganic nitrogen into organic forms [12]. While phosphomonoesterase is not directly involved in nitrogen metabolism, its activity is enhanced by nitrogen availability, as it releases phosphate from organic molecules, making it a useful indicator in nutrient-cycling studies [13,14,15].

Protocols for analyzing enzyme activity generally recommend immediate processing of plant tissue samples in order to ensure enzymatic integrity [16,17]. However, studies conducted in remote areas or those involving the collection of large sample sizes may not have access to laboratories or their immediate processing may be logistically unfeasible. In this respect, physiological activity is reduced by low temperatures, especially below 0 °C, as enzymatic reactions slow and cellular metabolism is inhibited. However, while freezing can preserve tissue integrity, it also induces osmotic stress and pH changes during ice formation, which can denature proteins, including enzymes, or reduce their activity [16,17]. For instance, nitrate reductase can become inactivated during prolonged storage at freezing temperatures, given conformational changes [16]. This contrasts the case of phosphomonoesterase, whose activity can remain stable after low-temperature storage but is sensitive to changes in cell phosphorus status during freezing [13]. Despite such potential limitations, freezing remains a practical option for preserving tissue samples collected in remote locations, thus requiring an explicit assessment of whether enzymatic integrity can be maintained during transport and storage.

This study aims to evaluate whether the enzymatic activities of nitrate reductase, glutamine synthetase, and phosphomonoesterase can be preserved during transport and after different periods of low-temperature storage. The study focuses on Chloris gayana, Fraxinus uhdei, and Trifolium repens—three plants of different life forms with broad distribution, considered invasive in certain regions and appearing to be tolerant to high nitrogen levels, as suggested by their presence in disturbed environments.

2. Materials and Methods

2.1. Plant Material

Plant material was collected from the gardens of the Universidad Nacional Autónoma de México, Morelia Campus, located in a periurban area of a mid-sized city in west-central Mexico. The spontaneous flora at this location has increasingly acquired exotic species over the past two decades as urbanization has extended [18,19].

Three plant species that have potential for utilization as biomonitors of atmospheric nitrogen deposition were utilized in the present study. In particular, Chloris gayana Kunth. (Poaceae) is an annual C₄ African grass that is tolerant of high nitrogen levels and has become a cosmopolitan element of ruderal and urban floras [20,21,22,23]. In turn, Fraxinus uhdei (Wenz.) Lingelsh (Oleaceae) is a Mexican dioecious tree that grows in humid environments and is tolerant to shade; it is cultivated for erosion control and to vegetate urban parks and has been introduced in India and Hawaii, an archipelago where it has become invasive [21,24,25]. The individuals utilized in the present study were planted ca. 2010 as part of the landscaping of a university campus that was undergoing construction [19]. Finally, Trifolium repens L. (Fabaceae) is a perennial nitrogen-fixing legume from Europe, northern Africa, and eastern Asia, which is utilized as forage, green manure, and cover crop and has become a cosmopolitan element of ruderal and urban floras [21,26,27].

2.2. Experimental Manipulations

2.2.1. Transport

Three common transport and handling conditions were evaluated for mature leaves from 5 individuals of each study species that were collected between 11:00 a.m. and 12:15 p.m. on 11–13 April 2023. This sample size was informed by previous works [9,23], which successfully detected enzymatic responses to experimental manipulations. The leaves were placed in resealable polypropylene bags (16.4 × 8.2 cm) and stored in a polystyrene cooler (volume of 21 L) that was kept in the shade until use. Enzymatic activities were determined for samples that were either processed immediately (within 2 h after collection), after 18 h for samples kept at room temperature (20–22 °C), or after 18 h for samples kept on ice (0–2 °C).

2.2.2. Storage

To evaluate the effect of storage on enzymatic activities, mature leaves from 5 individuals of each species were collected as described above and transported to the laboratory. A total of 35 tissue samples per individual were separated into resealable polypropylene bags and randomly assigned to experimental treatments of storage time and temperature (n = 5 per treatment). Specifically, enzymatic activities were determined for fresh samples (within 2 h after collection) and after 7, 14, and 28 days of storage at either −16 °C (in an Acros AS7818A deep freezer, Whirlpool, Celaya, Gto., Mexico) or at −45 °C (in a Thermo Scientific ULT2586-6-A41 ultra-freezer, Thermo Electron Corporation, Asheville, NC, USA).

2.3. Enzymatic Activities

2.3.1. Nitrate Reductase (NR)

The nitrate reductase (EC 1.7.1.1) activity was quantified based on the Greis-Ilosuay reaction [14]. Tissue samples were incubated for dark induction for 12 h in 5 mL KNO₃ 3 mM. The samples were infiltrated under vacuum (−58 cmHg) for 3 min with 5 mL of buffer (50 mM KH₂PO₄, 100 mM KNO₃, 100 mM potassium acetate, and 1.5% v:v propanol-1-ol, pH 7.5 adjusted 0.1 M NaOH), and placed in a dark water bath at 30 °C for 30 min. Nitrite (NO₂⁻) production was quantified by mixing 1 mL of the incubation supernatant with 1 mL of a 1% (w:v) sulphanilic acid solution in 1.5 M HCl and 1 mL of a 0.02% (w:v) n-(1-naphtyl ethylendiamine-HCl) solution. The aliquots were incubated in the dark for 40 min before measuring their absorbance at 540 nm with a Genesis 20 spectrophotometer (Thermo Fisher Scientific Inc., Madison, WI, USA). The tissue samples were dried at 45 °C for 48 h and weighed (resolution of 0.01 mg). A standard curve was made with NaNO_2, and the NR activity was reported as NO₂ produced per gram of dry weight per min (nM NO₂⁻ g⁻¹ dw m⁻¹).

2.3.2. Glutamine Synthetase (GS)

The glutamine synthetase (EC 6.3.1.2) activity was quantified based on the production of g-glutamyl hydroxamate [28]. Foliar tissue was weighed (resolution of 0.01 mg) and incubated in a water bath at 30 °C for 30 min in 300 µL of buffer (50 mM Tris-HCl, 2 mM mercaptoethanol and 1 mM EDTA; pH 7.5 adjusted with 0.1 M NaOH), 500 µL of 200 mM Tris-HCl, 200 mL of 50 mM ATP, 500 µL of 500 mM glutamic acid, 100 mL of 1 M MgSO₄, 300 µL of 100 mM hydroxylamine, and 100 µL of 100 mM cysteine. After a 1:1 dilution of the supernatant with a solution of 200 mM FeCl₃, 500 mM TCA, and 330 mM HCl, the absorbance was measured at 540 nm. A standard curve was made with g-glutamyl hydroxamate, and the GS activity was reported as g-glutamyl hydroxamate produced per gram of fresh weight per second (nM g-glutamyl hydroxamate g⁻¹ fw s⁻¹).

2.3.3. Phosphomonoesterase (PME)

The phosphomonoesterase (EC 3.1.3.2) activity was quantified based on the release of p-nitrophenol (p-NP) from p-nitrophenyl phosphate [13]. Tissue samples were placed in glass vials with 4 mL of 4 mM p-NPP in a buffer (0.1 M citric acid/0.1 M NaOH, adjusted to pH 5 with 0.1 M NaOH) and incubated for 120 min in a shaking water bath at 37 °C. Aliquots of 200 µL of the reaction supernatant were mixed with 3 mL of a terminating solution (0.1 M Tris adjusted to pH 12 with 0.1 M NaOH), and their absorbance was measured immediately at 410 nm. The leaf samples were dried at 45 °C for 48 h and weighed (resolution of 0.01 mg). A standard curve was made with p-NP standards, and the PME activity was expressed as p-NP released per gram of dry weight per second (nmol p-NP g⁻¹ dw s⁻¹).

2.4. Statistical Analyses

Data analyses were performed using RStudio 4.2.2 (RStudio Team, Boston, MA, USA). The effects of transport on enzymatic activities were analyzed using Friedman repeated measures tests for each species, followed by Nemenyi post hoc tests [29]. The effects of storage on enzymatic activities were assessed with generalized linear mixed models (GLMM) for each species, considering two levels of storage temperature and four levels for time under storage [30]. Data for the GLMM data were transformed using the natural logarithm, specifying either a Gaussian or Gamma distribution and a quadratic term was included when appropriate. Pairwise post hoc comparisons were conducted with Tukey tests [31]. A significance level of p < 0.05 was assumed for all analyses.

3. Results

3.1. Transport

The nitrate reductase activity did not respond to transport for Chloris gayana nor Fraxinus uhdei (Figure 1A,B;

Table 1. Friedman repeated measures test for the effect of transport to the laboratory on enzyme activities for samples of Chloris gayana, Fraxinus uhdei, and Trifolium repens. Numbers in bold indicate statistical differences.

Enzyme	Species	d.f.	X2	p
Nitrate reductase
	Chloris gayana	2	0.4	0.82
	Fraxinus uhdei	2	1.2	0.55
	Trifolium repens	2	6.4	0.04
Glutamine synthetase
	Chloris gayana	2	8.4	0.01
	Fraxinus uhdei	2	7.6	0.02
	Trifolium repens	2	3.3	0.20
Phosphomonoesterase
	Chloris gayana	2	2.8	0.25
	Fraxinus uhdei	2	0.4	0.82
	Trifolium repens	2	1.2	0.55

and Table S1). For Trifolium repens, NR activity did not change 18 h after collection compared with those that were processed immediately, but the NR activity for the samples kept at room temperature was significantly lower than for those that were kept on ice (Figure 1C; Table 1 and Table S1).

Transport conditions also affected the glutamine synthetase activity for C. gayana and F. uhdei (Figure 1D,E; Table 1 and Table S1). In particular, for C. gayana, GS activity decreased by 51% for samples kept on ice for 18 h relative to those that were processed immediately after collection (Figure 1D). Similarly, for F. uhdei, GS activity decreased by 27% for samples kept on ice for 18 h compared to those processed immediately (Figure 1E). In turn, the GS activity for T. repens did not respond to the transport treatment (Figure 1F).

The phosphomonoesterase activity did not respond to transport for any of the species considered in the study (Figure 1G–I; Table 1 and Table S1).

3.2. Cold Storage

Nitrate reductase activity decreased to near-zero values for all species after all storage times, regardless of the storage temperature (Figure 2A–C;

Table 2. Generalized linear mixed models for the effect of time under cold storage on enzymatic activities for samples of Chloris gayana, Fraxinus uhdei, and Trifolium repens. Numbers in bold indicate statistical differences.

Enzyme	Species	Variable	t	p
Nitrate reductase
	Chloris gayana	Time	−6.62	<0.05
		Storage	−0.15	0.82
		Time: Storage	−0.13	0.86
	Fraxinus uhdei	Time	5.43	<0.05
		Storage	0.28	0.78
		Time: Storage	−0.89	0.38
	Trifolium repens	Time	−3.61	<0.05
		Storage	0.30	0.77
		Time: Storage	0.13	0.89
Glutamine synthetase
	Chloris gayana	Time	3.85	<0.05
		Storage	−0.77	0.45
		Time: Storage	0.39	0.70
	Fraxinus uhdei	Time	0.99	0.33
		Storage	0.50	0.62
		Time: Storage	−1.01	0.32
	Trifolium repens	Time	2.18	<0.05
		Storage	1.00	0.32
		Time: Storage	0.24	0.81
Phosphomonoesterase
	Chloris gayana	Time	2.58	<0.05
		Storage	−0.97	0.34
		Time: Storage	0.85	0.40
	Fraxinus uhdei	Time	−1.75	0.09
		Storage	−1.12	0.23
		Time: Storage	0.34	0.74
	Trifolium repens	Time	5.07	<0.05
		Storage	0.65	0.52
		Time: Storage	−0.27	0.79

and Table S2).

Glutamine synthase activity responded to storage time, regardless of storage temperature, for C. gayana and T. repens (Figure 2D,F; Table 2 and Table S2). For F. uhdei, the GS activity of stored samples did not differ significantly from those of fresh tissue (Figure 2E).

Phosphomonoesterase activity was affected by time under cold storage for C.gayana and T. repens (Figure 2G,I; Table 2 and Table S2). Specifically, for C. gayana, PME activity increased by 125% at 28 days (Figure 2G). For F. uhdei, the PME activity of stored samples did not differ from that of fresh tissue (Figure 2H). For T. repens, PME activity increased significantly under both storage temperatures (Figure 2I).

4. Discussion

4.1. Transport and Storage

The activities of the three enzymes studied here—nitrate reductase (NR), glutamine synthetase (GS), and phosphomonoesterase (PME)—were affected by both transport conditions and storage time. Indeed, natural degradation processes begin upon harvesting plant material [16]. Moreover, low temperatures, such as those occurring when plant samples are transported on ice, may cause cellular damage, while proteolytic activity and a decrease in antioxidant activity have been observed at temperatures below −25 °C [16,32]. In turn, proteins, including enzymes, can also be affected by the increased concentration of salts and changes in pH that occur as water freezes [16]. Rapid freezing below −25 °C is thus recommended to reduce such damage, for instance, by utilizing liquid nitrogen for those samples that will not be processed fresh [33].

Once the samples were frozen, the storage temperature appeared to be less consequential on enzymatic activity. For example, no significant differences were observed between the samples stored at −16 °C and those stored at −45 °C.

4.2. Enzymatic Activities

Nitrate reductase activity was the most susceptible enzyme to both storage and transport conditions. The enzyme showed minimal activity after freezing, regardless of the species or temperature, reflecting that it becomes inactivated over time when stored, making it unsuitable for delayed analysis [34,35]. This was particularly evident for T. repens, which displayed the lowest NR activity among the three species considered, likely due to its reliance on nitrogen-fixing symbiosis [12,36]. In contrast, C. gayana had the highest NR activity, consistent with observations for fast-growing ruderal species that are often more responsive to nitrogen availability [37].

Glutamine synthetase activity exhibited a species-dependent response to storage time. For both C. gayana and T. repens, the enzyme’s activity increased after a week of storage, suggesting the onset of tissue degradation and the activation of GS isoforms that mobilize nitrogen during senescence [38]. Such an increase in GS activity over time indicates that delaying analysis beyond a species-specific threshold can lead to the misinterpretation of metabolic processes. In contrast, F. uhdei showed minimal changes in GS and PME activities under either storage condition, suggesting that woody species may be more resilient to storage-related degradation.

Phosphomonoesterase activity also showed species-dependent responses. In particular, PME activity increased significantly after 28 days of freezing for C. gayana, which may indicate a response to phosphorus remobilization under nutrient stress [39]. Similarly, T. repens showed increased PME activity over time, reinforcing the idea of the onset of tissue degradation following tissue collection.

4.3. Plant Responses

The distinct responses observed across the three study species can be attributed to their differing life forms and ecophysiological strategies. Trifolium repens, a nitrogen-fixing legume, exhibited unique behavior when compared with the non-leguminous species studied. As a legume, T. repens benefits from its symbiotic relationship with nitrogen-fixing bacteria, allowing it to access atmospheric nitrogen directly. This biological advantage likely explains its lower nitrate reductase activity, as the plant relies less on soil-derived nitrate and more on ammonium produced via symbiotic nitrogen fixation [12,36]. The increase in both GS and PME activities for T. repens during storage may be tied to its nitrogen assimilation pathway, where excess ammonium is rapidly integrated into organic compounds through glutamine synthetase, preventing toxicity [40]. Such a heightened metabolic response during storage might also reflect early tissue senescence, wherein nitrogen and phosphorus remobilization are activated [38,39,40,41,42]. In turn, Chloris gayana, a fast-growing C₄ grass, and Fraxinus uhdei, a shade-tolerant tree, showed contrasting enzymatic responses. The higher initial NR rate for the grass was probably a result of its ability to become established and develop under high nitrogen availability [23]. For its part, the relatively stable responses that were observed for the tree probably reflect its relatively slow growth rate, as well as its ability to thrive under an ample range of nitrogen availability [23,43,44]. These life form-specific patterns highlight the need for species-specific considerations when interpreting enzymatic data in biomonitoring contexts.

5. Conclusions

This study reaffirms the need for immediate analysis of nitrate reductase (NR) activity, as its enzymatic integrity could not be preserved during freezing, regardless of temperature or species. However, our findings also highlight the resilience of glutamine synthetase (GS) and phosphomonoesterase (PME) activities under the storage conditions utilized. Both enzymes demonstrated potential for delayed analysis, provided that species-specific responses are carefully considered. This finding is particularly valuable for large-scale biomonitoring studies where immediate processing is logistically challenging. Notably, Trifolium repens exhibited an unexpected increase in both GS and PME activities during storage, likely linked to its nitrogen-fixing symbiosis and unique metabolic pathways. These results suggest that while freezing may affect enzyme activity, certain species or life forms are better suited to handling delayed processing. Thus, while NR should be analyzed for fresh tissue, GS and PME activities can be reliably measured from frozen samples, offering practical flexibility for biomonitoring projects conducted over long distances or extended timeframes.

As anthropogenic nitrogen deposition continues to drive changes in ecosystems, particularly in high-biodiversity regions, accurate monitoring of plant nitrogen metabolism remains critical for understanding species’ responses to these environmental changes. This study underscores the utility of nitrogen metabolism enzymes for biomonitoring in large-scale or remote field studies, emphasizing the importance of enzyme-specific storage protocols. While immediate or minimally delayed analysis is indispensable for NR, GS and PME activities can provide reliable data after storage, enabling broader application of biomonitoring techniques. Future research should prioritize the development of strategies to further preserve enzymatic integrity during storage, particularly in scenarios where immediate processing is not impractical.