The research was performed at the Panmugang Modern Forestry Demonstration Base of Zhejiang Agriculture and Forestry University, Zhejiang Province, China (119°58′ E, 30°29′ N). This area has a subtropical monsoon climate with an average annual temperature of 17.8°C, an average relative humidity of 70.3%, an annual precipitation of 1,454 mm, a frost-free period of 234 d, and 1,765 hours of sunshine per year. The relevant weather data is in Table S1 . The agricultural area has a hilly environment with hills that are typically less than 150 meters high. The soil is classified as a Ferralsol since it is largely originated from quaternary sandstone parent material. Natural precipitation and soil water storage are the primary sources of agricultural productivity (Xu et al., 2017).

The experimental P. praecox plot had a stand density of 15,000 plants per hectare, the average diameter at breast height of the bamboo culm was 3.89 cm, and the ratio of the number of bamboo culms in each year was year 1: year 2: year 3 = 1:1.89:0.58. The experimental area of the forest was split into twelve 50 m × 50 m plots, with the treatment arrangement being a full block with three replicates for each treatment. The treatments were (1) control; (2) mulching; (3) control + aeration (aeration) and (4) mulching + aeration (M+A). On December 17, 2020, the surface of the bamboo forest was mulched with organic material to increase the temperature and moisture content, and the hulled bran that had not decayed was removed on March 24, 2021. Once mulch has been removed, the shoot yield was recorded. The following was the mulching procedure: Initially, 4,500 kg·ha⁻¹ of chicken manure was spread to the soil surface. The chicken dung was subsequently covered with rice straw (3,750 kg·ha⁻¹). Finally, rice bran (412.5 t·ha⁻¹) was sprinkled on top to provide 15 cm of thickness. We randomly selected three plots that had been mulched for many years as aerated plots. The aeration measures were as follows: the holes were drilled in a straight line parallel below the ground at a depth of 50 cm (the bamboo rhizomes are mainly present in the 20-30 cm layer) in each plot at a spacing of 60 cm, and plastic ventilation pipes with an external diameter of 21 mm and a wall thickness of 1 mm were then inserted and connected in sequence, with small holes of 0.2 mm diameter every 30 cm in the wall for ventilation. An air pump was connected to the main pipe in each plot and the air was delivered by a compressor (AS7.5Hi, Quanzhou Jinba, China). The plots with aeration were aerated for the whole day. Samples were collected in March, June, September, and December 2021. In each sample plot, the bamboo root was sampled from a depth of 20-30 cm and taken to the laboratory for analysis. At the end of the experiment, the root system was scanned with the Winrhizo root analysis system and calculated for root length density, surface area density and volume density and record the diameter at breast height of the bamboo. Root samples were cleaned with distilled water, instantly dried, frozen in liquid nitrogen, and kept at -80°C until tested.

Using SPSS 20.0, all data were statistically assessed utilizing ANOVA and Duncan’s Multiple Range test (IBM Corp., Armonk, NY, USA). Correlation and redundancy analyses were carried out using R program v3.6.3. Origin v8.0 was used to create the figures (Origin Lab Corporation, Northampton, USA).

Compared to the control, shoot yield was significantly increased by 60.5%, 20.23% and 115.1% for mulching, aeration and M+A respectively, and by 34.0% for M+A compared to mulched ( Figure 1A ). As for diameter at breast height, the diameter at breast height in the aeration group was significantly increased compared to the mulched group ( Figure 1B ). Mulching significantly reduced root length density (67.9%), root surface area density (39.4%), and root volume density (73.0%), respectively, compared to the control ( Figures 1C–E ). But M+A significantly increased root length density (39.3%), root surface area density (22.7%), and root volume density (50.6%), respectively, compared to mulching. It showed that mulching combined with aeration techniques has a positive effect on the growth of bamboo.

When the bamboo mulched, soil oxygen concentration decreased rapidly, reaching its lowest level after two months ( Figure 2A ). Three months after mulching, soil oxygen concentration began to recover when the mulch was removed, but it was still lower than that of the control plots. There were no significant differences in oxygen concentration between the treatments of M+A and control in June, September and December. During the mulching period, soil temperature in the mulched treatments was significantly higher than the control, while aeration significantly reduced the soil temperature compared to the mulched ( Figure 2B ). When the mulch was removed in March, soil temperature increased and then decreased with time that was consistent with the air temperature. From June, there were no significant differences among all treatments. As indicated by Figure 2C , the mulching resulted in a lower root activity compared to the control, while aeration improved the activity significantly during the mulching peroid.

LDH, AlaAT, PDC, and ADH activities of mulching treatment were significantly elevated and the M+A treatment significantly lowered the activities of these anaerobic enzymes compared with mulching throughout the year ( Figures 3A–D ). There was no significant difference between the M+A and the control regarding anaerobic enzyme activity. Anaerobic enzyme activity of aeration group was significantly decreased compared to the control.

Mulching significantly improved the activities of ACO and ACS, aeration significantly reduced both activities ( Figures 3E, F ). For all four seasons, there were no statistically significant changes between the aeration treatment and the control. The activities of ACS and ACO were lower overall in June when compared to the other months of the year.

In March and September, mulching significantly increased ABA, GA, and ETH contents in the roots and decreased the IAA content compared to the control, while aeration significantly decreased the contents of ABA, GA, and ETH and increased the IAA content in the M+A group ( Figures 4A–D ). ABA, GA, and ETH contents were significantly reduced and IAA was enhanced in aeration treatment compared to the control.

Mulching significantly reduced the expression of Aux1, 2, 3, and 4 compared to the control throughout the year, while soil aeration significantly enhanced Aux gene expression in the M+A group ( Figures 5A–D ). Aux/IAAs gene expression of aeration was significantly increased compared to the control.

As shown in Figure 6 , the activities of ADH, PDC, LDH, and AlaAT all showed a linear correlation with soil oxygen concentration, and the activities of anaerobic enzyme increased as the oxygen concentration decreased.

Pearson’s correlation tests were also carried out to assess the correlations between each enzyme and hormones and genetic attribute under mulching and aeration ( Figure 7 ). The expression of Aux1, 2, 3, and 4 was substantially positively associated with IAA levels, and Aux/IAAs expression was significantly negatively associated with ETH, ABA and GA concentration (p< 0.05). Moreover, ETH was significantly positively related with the activities of PDC, ADH, LDH, AlaAT, and also with ABA and GA concentration. ABA concentration was significantly positively correlated with anaerobic enzyme activities and ETH and GA concentration, and significantly negatively correlated with Aux/IAAs expression and IAA concentration. As for anaerobic enzyme activity, LDH activity was significantly positively associated with the activities of PDC, AlaAT, and ADH, and the concentrations of ABA, ETH, and GA and significantly negatively correlated with IAA concentration and Aux/IAAs gene expression (p< 0.05). ADH, LDH and AlaAT activity was significantly positively associated with anaerobic respiration enzymes activities and ETH, GA, and ABA concentrations, and significantly negatively associated with IAA and Aux/IAAs gene expression (p< 0.05). The activity of PDC was significantly positively correlated with ETH, ABA and GA concentrations and the activities of anaerobic respiration enzymes, and it was significantly and negatively associated with Aux/IAAs gene expression (p< 0.05). The analysis showed that plant hormones have an important role in mulch-induced root hypoxia in P. praecox, influencing changes in Aux/IAAs expression and anaerobic enzymes activities.

We did redundancy analysis (RDA) to see whether there were any commonalities among the treatments in terms of enzyme and hormones (ADH, AlaAT, LDH, and PDC activities, hormone concentrations of ABA, IAA, GA, and ETH) and genetic characteristics (Aux gene expression) ( Figure 8 ). As a result, we found that enzyme and hormones and genetic characteristics interact with one another. The activities of ABA, PDC, and LDH, as well as the concentrations of ETH and IAA, had a significant influence on plant genetic composition (p< 0.05), with RDA1 and RDA2 exhibiting variances across all treatments, accounting for 56.15 and 35.88% of the variation, respectively ( Figure 8A ). Furthermore, Aux1, Aux2, Aux3, and Aux4 expression was strongly associated to plant enzyme and hormones parameters, with the first and second major axis accounting for 53.01 and 20.82% of the variance, respectively ( Figure 8B ).

In natural environments, soil hypoxia is caused by factors such as heavy rainfall, poor soil structure, and little drainage, which generate unfavorable porosity and ventilation interactions in the soil, restricting root growth and crop output considerably (Brookes et al., 1982; Weits et al., 2019; Strudley et al., 2008). Nevertheless, when bamboo forest grove is mulched, the organic material heats and ferments. The aerobic microorganisms consume oxygen directly from the soil, and the thick mulching material stops ambient oxygen from entering the soil, which causes a lack of oxygen at the root level, unlike flooding and soil slumping (Jiang et al., 2009; Qian et al., 2020). We discovered in the investigation that soil temperatures increased and soil oxygen concerntration decreased during the mulching period ( Figure 2A, B ; Table 2 ), which is in line with previous research findings (Qian et al., 2020). However, aeration significantly reduced soil temperature, increased oxygen content, diameter at breast height, shoot production and root growth, suggesting that soil aeration using buried pipes is effective in improving soil condition and bamboo growth. After removal of the mulch, soil oxygen concentrations recovered substantially but remained slightly lower than in the control. Also, soil temperatures did not differ significantly between the three treatments from June onwards. It is possible that mulching changed the soil microbial populations and soil structure, but after the organic material was removed, the soil repaired itself and gradually returned to the control level. Root growth is known to be limited by low soil oxygen availability (Christianson et al., 2010; Cruz et al., 2019). Hence, we found that mulching reduced root activity while aeration increased it. Also, it has stronger root activity in March than the other months ( Figure 2C ; Table 2 ). It is similar to previous research findings, Holthausen and Caldwell (1980) suggested that root system’s breathing capacity varies seasonally, meaning that respiratory capacity peaks in spring and a respiratory minimum occurs in late summer. This tendency might be attributed to environmental pretreatment together with an overall genetic-based program to extend the length of root activity and reduce the root system’s carbon requirement (Holthausen and Caldwell, 1980).

Pyruvate produced by glycolysis will undergo anaerobic respiration (also called fermentation) in plant cells once the oxygen content is low (Yazdani and Gonzalez, 2007). The reversible conversion of alanine and 2-ketoglutarate to pyruvate and glutamate is catalyzed by AlaAT (Ricoult et al., 2005). Fermentation may be classified into two types: lactic acid fermentation, in which LDH is responsible for catalyzing the transformation of lactate to pyruvate then back, with the end product being lactate; and alcoholic fermentation, in which ADH catalyzes the acetaldehyde-to-ethanol conversion. PDC catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and NADH, with carbon dioxide and ethanol as byproducts (Bailey-Serres and Chang, 2005; Arbona et al., 2010; Armstrong et al., 2019). Neither type of fermentation produces ATP molecules and both are detrimental to cell survival (Bailey-Serres and Chang, 2005). Our results showed that the activities of PDC, LDH, AlaAT and ADH were significantly increased of bamboo root during the mulching period ( Figure 3A-D ; Table 2 ). This discovery is in line with the findings of Qian et al. (2020). After the mulch was removed, anaerobic enzyme activity of mulching remained greater than in the control, but aeration drastically decreased anaerobic enzyme activity. It occurred because the mulching technique lowered soil oxygen concentration and enhanced anaerobic enzyme activity. There was a linear relationship between the activities of the anaerobic enzymes ADH, PDC, LDG, and AlaAT and soil oxygen concerntration ( Figure 6 ; Table 2 ). Extensive molecular and biochemical analyses revealed the mechanism behind these relationships. In hypoxic tissues in barley (Hordeum vulgare) and M. truncatula, AlaAT activity and gene expression are stimulated (Muench and Good, 1994; Bray et al., 2002; Ricoult et al., 2005). Previous studies have shown that flood-tolerant plants accumulate alanine by activating AlaAT, and that the alanine is carried via the xylem and becomes a transportable energy source (De Sousa and Sodek, 2003). AlaAT is essential for plant life not only in hypoxia, but also throughout the reoxygenation period following hypoxia (Nakamura and Noguchi, 2020). Additionally, when the oxygen content was inadequate in the root zone, the activities of LDH, PDC, and ADH, as well as the expression of the genes that encode these enzymes, were elevated in cucumber (Xu et al., 2014). Increased anaerobic enzyme activity may enhance plant tolerance to hypoxia (Kato-Noguchi, 2001). For example, plants of white clover with strong ADH activity, demonstrate better flood tolerance under flood stress than plants with weak ADH activity (Chan and Burton, 1992). Generally, subsurface buried pipe aeration reduced the anaerobic enzyme activity of bamboo roots caused by mulching with organic materials.

The levels of some phytohormones in P. praecox are highly susceptible to external environmental conditions, and the insulating effect of mulching disrupts the balance of endogenous hormones. Endogenous plant hormones, including IAA, GA, ABA, and ETH, are the “switches” that modulate and control plant growth (Davies, 2004). A previous study showed that lateral shoots at the base of bamboo plants had significantly higher IAA/ABA and ZT/ABA levels one year after mulching than did plants grown without mulching, thus promoting early differentiation of lateral shoots (Huang et al., 2002). However, this does not correspond with our experimental results, where we found significant increases in GA and ABA contents and a reduction in growth hormone content of roots treated with organic material mulch for consecutive years, and this was also found in degraded P. praecox stands ( Figure 4 ; Table 2 ). The ABA, GA, and cytokinin (CTK) contents of flowering bamboo in the degraded P. praecox forest were all higher than in unflowered bamboo, with the most significant increase being in ABA content (He et al., 2005). It might be because long-term mulching inhibited plant root growth, but the anoxic environment caused by mulching allowed the roots of bamboo to stretch towards soil surface to find more oxygen, thus increasing the contents of ABA, which promotes the formation of plant organ separation, and GA, which promotes cell elongation and division, ultimately leading to degradation of the bamboo forest (Davies, 2004). Previously, Xu et al. (2017) also found that long-term mulching caused roots to grow toward the ground in search of oxygen, which promoted root elongation. This phenomenon is also observed in rice. Deep-water rice leaves and internodes may stretch and grow above the water surface under flood circumstances to gather oxygen and prevent drowning (Ayano et al., 2015). We also found that the higher ABA contents in September and December and the higher IAA contents in March and June may be related to the growth habit of the plant ( Figure 4A, B ; Table 2 ), where the plant grows vigorously in spring and summer, while abscisic acid inhibits germination and promotes dormancy and plant organ separation in autumn and winter (Baktir et al., 2004; Davies, 2004). In addition, the actions of ABA and IAA are antagonistic, and one study showed that ABA may function as an inhibitor of GA and restrict root development, allowing the plant to survive during flooding (Wu and Hong, 2021). Our results suggest that there may be some antagonistic effects between ABA and IAA and GA in the hypoxic environment caused by organic material mulching ( Figure 7 ; Table 2 ). In addition to this, it has been shown that the fast buildup of ethylene in submerged tissues (through physical trapping and active synthesis) under anoxic circumstances, causes alterations in branch lengthening, glucose metabolism, and adventitious root development (Steffens and Sauter, 2006; Xu et al., 2006; Hattori et al., 2009). At the same time, the equilibrium of GA and ABA contents is likewise coordinated by ETH under anoxic conditions caused by submergence (Xu et al., 2006). In this investigation, we discovered significant increases in ETH content and the activities of enzymes involved in ethylene synthesis (ACO, ACS), and also a significant positive correlation between ETH and GA contents under organic material cover ( Figures 3E, F ; 4D ; 7 ; Table 2 ). It is due to the synergy established by the combination of ETH and GA. From this we can infer that ETH perception is essential for adventitious root development, and GA substantially promotes the ensuing ETH-induced adventitious root growth (Steffens and Sauter, 2006). Aeration from the buried pipes provided oxygen to alleviate soil hypoxia caused by mulching, thus changing the hormone contents in the bamboo roots by reducing the ABA, GA, and ETH contents, decreasing the activities of ACS and ACO, increasing the IAA content, and finally improving metabolic and physiological alterations in roots.

It is well known that auxin is the key regulator during plant growth (Wu et al., 2012; Eysholdt-Derzso and Sauter, 2017). IAA, a most abundant hormone in higher plants, is a weak acid, and growth hormone influx and efflux carriers promote its intercellular movement (Wu et al., 2012; Van and Licausi, 2015) Auxin transporters are necessary for the transfer of auxin into various cells. AUXIN1 (encoded by Aux1) is an auxin influx carrier. AUXIN1 is the major transporter for auxin uptake in root hairs and it controls root gravitropism, root hair formation, and leaf phyllotaxy (Ori, 2019). In this study, we found that mulching reduced the expression of Aux1 ( Figure 5A ), and that Aux1 expression was highly associated with growth hormone and unfavorably related to ETH concentrations (Li et al., 2015). Aux2 and Aux3 have been implicated in processes like as hypocotyl elongation and foliar growth in Arabidopsis and rice. Aux3 regulates lateral root growth and root hair production, whereas Aux4 regulates plant tiller height (Liscum and Reed, 2002; Overvoorde, 2005; Song and Xu, 2013). In the present study, mulching reduced gene expression of Aux genes, while aeration increased Aux gene expression ( Figure 5 ). Aux/IAAs expression was favorably linked with IAA and negatively associated with ethylene ( Figures 7 , 8 ). It is because ETH can control IAA synthesis by regulating Aux1 expression and growth hormone synthesis-related genes, which in turn regulate root development under adverse situations (Li et al., 2015).

Following aeration, the correlations between each enzyme and hormones indicator and gene expression were also investigated. Aux/IAAs gene expression was highly related to many enzyme and hormones factors, and the expression of Aux1, 2, 3, and 4 was closely associated with root development factors ( Figure 8 ). The results imply that Aux/IAAs genes are engaged in the management of hormone levels as well as the regulation of anaerobic enzymes and root respiration activity to keep proper root development, while Aux/IAAs contributing in this mechanism (Gao et al., 2022). Abiko et al. (2012) showed the expression of Aux/IAA in Zea nicaraguensis of hypoxic circumstances altered dramatically, and it may also govern the development of adventitious roots and the production of vented tissue. Mulching caused fast alterations in a number of critical enzyme and hormones markers in P. praecox. Here, LDH, PDC, ABA, IAA, and ETH all had significant impacts on the expression of Aux/IAAs ( Figure 8 ). It demonstrates that the overlay affects Aux/IAAs expression in plants, which in turn regulates changes in endogenous hormone levels that are involved in regulating anaerobic respiratory enzymes and ultimately improving ability of plant roots to cope with hypoxia caused by organic materials.

Overall, the findings of our investigation demonstrate that mulching with organic material degrades P. praecox forests ( Figure 9 ), which consistent with the phenomenon observed informally by local farmers (Xu et al., 2017). We are here for the first time to demonstrate the mechanism of underground pipeline aeration to mitigate the degradation of bamboo forest. We also explain for the first time that hormones crosstalk with Aux/IAAs and thus regulate changes in enzyme and hormones indicators under bamboo forest mulching. We therefore are of the opinion that our subsurface aeration strategy will help to mitigate soil hypoxia and, in turn, improve the growth of bamboo. However, the intensity and time of the aeration needs to be studied in detail in the future.

There are certain limitations to this study, namely that we only studied the effects of short-term aeration on plant biochemistry. Different aeration times may also have inconsistent effects on plant growth. Moreover, our experiment lasted for one year, which is a short period of time compared to a long-term bamboo mulch, and long-term monitoring of soil changes to determine physiological and biochemical changes in bamboo roots could be conducted in future studies. Due to the lack of research on the application of aeration systems to alleviate soil hypoxia caused by organic mulching, many scientific and technical problems remain. Our study could serve as a representative example of this research area and generate interest in further research on the role of post-mulching aeration in various cropping systems.