This study focused on Pinus resinosa. This species was selected because it grows naturally in the City of Greater Sudbury, and it represents one of the main conifers used in the Sudbury regreening program ( Figure 1 ). Soil samples were collected from three locations in the City of Greater Sudbury (Canada). The GPS coordinates of the three sites are as follows: 46.467, −80.975 for site 1; 46.466, −80.973 for site 2; and 46.468, −80.971 for site 3. At each location, three red pine (Pinus resinosa) trees of 10 to 15 years were selected. For each tree, 10 subsamples were collected directly under the roots, representing the rhizosphere, and then mixed. Another group of 10 subsamples free from any root interactions representing the control were collected from the surrounding area, about 3 - 4 m away from each targeted tree. In total, 20 samples were collected per site. Plant materials, stones, and other residues were removed using a KimLab Economy Test Sieve with a 2 mm mesh size (#10). The soil samples were stored at 4˚C, and the enzyme activity was analyzed within 24 hours of collection.

Nine enzymes were measured and analyzed due to their relevance and importance in the soil ecological system ( Table 1 ). They include β-glucosidase (BG), cellobiohydrolase (CBH), β-N-acetylglucosaminidase (NAGase), aryl sulfatase (AS), acid phosphatase (AP), alkaline phosphatase (ALP), glycine aminopeptidase (GAP), leucine aminopeptidase (LAP), and peroxidase (PER). Specific substrate types were used to activate the enzyme activity. BG, CBH, NAGase, AS and AP were assayed using a p-nitrophenol (pNP) substrate. ALP and GAP were assayed using a p-nitroanilide substrate. The peroxidase was tested in two different forms due to the nature of its reaction. These consisted of the peroxidase and the buffer (POD), and the second form with POD and 10 μl of H₂O₂, turning it into polyphenol oxidase (PPO). These enzyme activities were assayed using the substrate L-DOPA (L-3, 4-dihydroxyphenylalanine). The original protocols for each assay can be found on the Environment RCN website ( http://enzymes.nrel.colostate.edu/ ).

† pNP represents 4-nitrophenyl.

To measure enzymatic activities, the soil samples were removed from the fridge, and 4 g of the soil was weighed out from each sample and mixed in its tube filled with 40 ml of 50 mM sodium acetate buffer (pH 5). Each tube was then vortexed for 1 min 30 secs, thus making our soil homogenate. For each sample, 10 tubes were filled with 500 μl of the respective substrate, and 500 μl of the soil homogenate, except the PPO, which had an additional 10 μl of 0.3% H₂O₂. For the control tube, each sample had two controls: 1 tube filled with 900 μl of buffer, and 900 μl of the soil homogenate. Control 2 had 500 μl of buffer and 500 μl of the soil homogenate, with an additional 10 μl of 0.3% H₂O₂. The tubes were then incubated for two hours to allow the reactions to fully occur. The pNP and p-nitroanilide type substrates, tubes and control 1 were incubated in the shaker at room temperature and 255 RMP. Then the PPO, POD and control 2 were attached to the rotating wheel that was placed in the fridge. Once the incubation was completed, the tubes were then centrifuged at 3400 RPM for 3 minutes and 30 seconds.

The enzyme assay starts with a 96-well plate, each tube had 100uL of its contents deposited into a well, and each tube was repeated in a triplicate, except for the control tubes, whose triplicate was repeated twice. Plate one, which contained pNP, p-nitroanilide tubes, and control one well, all had 5 uL of 1M NaOH added to them to stop the reaction, except for one of the buffer triplicates. For plate two, which contains the PPO, POD, and control 2, no NaOH was needed, as its reaction is typically very fast. Using the FLUOstar OPTIMA, plate one’s absorbance was read at 405 nm, and plate two at 450 nm.

Additionally, the dry weight of the soil was measured for calculations. Hence, for each sample, another 4 g sample was taken and weighed, and its weight boat was weighed separately. The soil was then placed in a chamber at 28˚C for one week to fully dehydrate the soil. Then all the samples were weighed again.

Statistical analyses were conducted using SPSS version 20 for Windows (IBM, NY, USA). A paired T-test was used to see if there was a significant difference in the enzyme activity between the Rhizosphere soil and the control for each site and across the sites.

The results of the analysis showed that a variety of enzymes had significantly higher activities in the rhizosphere compared to controls (P ≤ 0.05). In the first site, aryl sulfatase (AS), alkaline phosphatase (ALP) and glycine aminopeptidase (GAP) activities were significantly increased in the rhizosphere soil compared to the control (P ≤ 0.01) ( Figure 2 and Figure 3 ). However, Acid phosphatase and leucine aminopeptidase activity show the opposite effect, with significantly high levels in control soils compared to samples from the rhizosphere. Figure 2 and Figure 3 show the results of these LAP and AP activities. β-glucosidase (BG), cellobiohydrolase (CBH), β-N-acetylglucosaminidase (NAGase), and Peroxidase (PER) activities did not show any significant changes where samples from the rhizospheres and the controls were compared ( Figure 3 and Figure 4 ).

Site 2 shows some similarities with Site 1 as AP, ALP, AS, and GAP activities were significantly increased in the rhizosphere soil compared to the control (P ≤ 0.05) ( Figure 5 and Figure 6 ). At this site, BG, and NAGase activities were significantly higher in the rhizosphere soil compared to the control, differing from site 1 (P ≤ 0.05) ( Figure 5 and Figure 6 ). No significant differences were observed in CBH and LAP activities when the control and rhizosphere samples were compared ( Figure 6 and Figure 7 ).

Site 3 shows some similarities with the previous two sites. AP, ALP and AS showed highly significant activities in the rhizospheric soils compared to control samples (P ≤ 0.01) ( Figure 7 and Figure 8 ). BG and CBH activities were also significantly higher in rhizosphere soil compared to the control (P ≤ 0.05) ( Figure 8 ). No significant difference in NAGase, GAP, LAP and PER activities was observed when the two types of soils were compared ( Figure 9 and Figure 10 ).

While all three sites did not appear identical in terms of enzyme activity, the similarity of certain enzyme activities was observed when the three sites were compared. Aryl sulfatase (AS) and alkaline phosphatase (ALP) all showed significant increases in activity in the rhizosphere compared to the control, throughout all three sites, with site 1 and site 2 showing the highest level of significance (P ≤ 0.01). Activities of Acid phosphatase (AP) were significantly different in all three sites. The activities of this enzyme were higher in sites 2 and 3 in rhizospheres compared to the control, and the opposite trend was found in site 1. Glycine aminopeptidase (GAP) activity was 2× higher in the rhizosphere compared to the control in site 1, with the same trend observed in site 2 to a lesser degree; and no significant differences were observed between the two types of soil in site 3. The activities of β-glucosidase (BG) were 2× higher in the rhizosphere compared to controls in sites 2 and 3. The remaining enzymes cellobiohydrolase (CBH), β-N-acetylglucosaminidase (NAGase) and peroxidase (PER) were significantly higher in the rhizosphere compared to control samples only in one site. Leucine aminopeptidase was one of two enzymes whose activities were significantly higher in control compared to the rhizosphere, however, this only occurred in site 1.

β-glucosidase (BG) is known for its vital role in the degradation of cellulose, and it does this by catalyzing the hydrolysis reaction of cellobiose residues, eventually breaking it down into glucose [20] . BG has always been an indicator of soil health, given its importance to carbon cycling, where there is high BG activity, which means more decomposition of organic matter [21] . Given that it was significant in both sites 2 and 3, it can be concluded that these two sites had a similar amount of organic matter on the forest floor. It has been reported that the levels of pH play a major role in BG activity [21] . Studies have shown that as pH increases, the activity of BG enzymes decreases. This is supported by the activities of AP/ALP in sites 2 and 3, which show a similar pattern with both having much higher levels of AP activity than ALP, indicating that the soil was likely more acidic. In contrast, site 1 had a much higher level of ALP activities, meaning a more alkaline soil would have inhibited the BG enzyme activity, causing it to not be significantly different in either the control or rhizosphere in all 3 trees. It indicates that the BG enzyme could have significantly higher levels in the rhizosphere, assuming it has the optimal pH. This role of pH should be interpreted with caution since no clear differences in pH values were recorded in previous studies.

CBH is known for its vital role in degrading cellulose into cellobiose and ensuring its addition to the carbon cycle and nutrient cycling of the ecosystem [22] . It works on the microcrystalline structure of the cellulose, breaking away chains of cellulose and separating them [22] . Eventually, it breaks down into two products, glucose and fructose [23] . CBH is the beginning timeline of cellulose degradation, whereas BG is closer to the end of it in contrast. The results for CBH activities indicate no changes between the rhizosphere soil and the control soil, with only site 3 showing significant changes. That is, the site 3 increase could have been induced by factors such as the rain that occurred during the week and the sampling day that may have sped up the decomposition of large organic materials occurring in that area. It would be fair to say that CBH is a relatively consistent enzyme both with and away from the roots.

NAGase is known for its role in breaking down chitin, it also plays a key role in nutrient cycling. It breaks down the chitin by breaking down the chains and oxidizing its ends [24] . It then further breaks down the products by turning chitinase products into N-acetylglucosamine [25] . This breakdown of chitin products into amino sugars, has been shown to be a good indication of N mineralization in soil [24] . NAGase was only significantly higher in the rhizosphere only in site 2.

AP and ALP are both enzymes involved in phosphorus cycling [26] . They do this by catalyzing the hydrolysis of phosphomonoesters and phosphodiesters to release free phosphate into the soil for plant growth and soil health [26] . Its availability is influenced by the soil pH, as well as the already existing phosphorus that is available to the plant; if there is not enough available, more AP/ALP will be secreted to make up for it [21] . The activities of AP/ALP were found to be statistically significant in all three sites. They activities were significantly higher in rhizosphere compared to control in all the three sites with the exception of low activity of AP in site 1. Phosphorus plays a key role in plant life, as not only is it used in the plant’s metabolic processes, but it’s also involved in the management of abiotic stressors such as heavy metal toxicity and heat [27] . Given Sudbury’s unique environmental structure, it could explain the high levels of ALP found in general, but it’s safe to conclude that AP/ALP would be enzymes that would be consistently higher in the rhizosphere of the red pine.

Aryl sulfatase is an enzyme that helps with the sulphur cycling in soil. They catalyze the reaction that releases sulphur esters from sulphate, to make it available for plant use [28] . They are used most when the sulphur availability in soil is low, the microorganism secretes more AS to compensate [29] . Sulphur is a nutrient involved in plant growth, development and protection from oxidative damage [30] . These roles align with our findings, as AS was found to be significantly higher in the rhizosphere than in the control in all three sites. A community of fungi can typically be found around the root area, as they are part of what makes up the rhizosphere ecosystem, and promote plant sustainability [31] . Certain fungal and bacterial strains seem to have the capabilities to hydrolyze sulfate esters, with the help of AS [32] . One notable example is its partnership with Arbuscular Mycorrhiza Fungi (AMF), which is a fungus that attaches to the plant roots and helps with the upregulation of sulphates within the soil [30] . Together, they can help regulate the sulphur in the soil and make it bioavailable. This justifies the high activities of AS in the rhizosphere.

GAP and LAP are both aminopeptidases that target different amino acids. They hydrolyze their specific amino acid from the protein’s N-terminal [33] . They are highly involved in the nitrogen cycling in soil, as they help microbes absorb the nitrogen’s availability [33] . Leucine only showed a significant increase in the control samples of site 1. This may be explained by the fact that the area could be a protein hotspot [34] . When sampling, we may have hit an area that had a high soluble protein content, causing that large activity in leucine, whether it was an animal or insect that may have passed recently in that area [34] . GAP, on the other hand, was significantly higher in the rhizosphere in both sites 1 and 2. This implies that the rhizosphere is a richer source of Glycine than Leucine. Despite both being amino peptidase there’re a lot variations in their activities, most likely due to environmental factors, since in controlled environment their activities were similar [35] .

Peroxidases are enzymes that help break down polyphenol compounds like lignin with the help of hydrogen peroxide that oxidizes this reaction [36] . They tend to be environmentally unstable and are affected when they are exposed to minerals, which in the rhizosphere tend to be a lot of. However, their main role in soil is protecting against pests and pathogenic microbes by enhancing cell wall strength [37] . PER was only significantly different in the rhizosphere compared to the control in samples from site 2. This could be related to the increase observed with the NAGase on that same site. With NAGase breaking down chitin from insects, and PER protecting against pests, it seems likely that at the time of collection, the plant was battling some kind of herbivorous insect that was invading the red pines in that area.