The “Oxygen Sink” of Bamboo Shoots Regulates and Guarantees the Oxygen Supply for Aerobic Respiration

Abstract

The amazingly rapid growth of bamboo shoots requires strong respiration and provides a large amount of energy and intermediate metabolites. Strong aerobic respiration requires a large amount of O₂. This raises the following question: What is the source and mechanism of O₂ supply to meet aerobic respiration? However, currently, this remains unknown. The underground buds (US), the 2-m-high overground buds (AS), and the 8-m-high growth arrest buds (HS) of bamboo (Phyllostachys prominens) were collected to represent their different stages of growth and development. The fifth bamboo shoot node at each stage was sealed by two membranes, and treated in a polyethylene zip-lock bag filled with air (21% O₂ + 79% N₂) and nitrogen (100% N₂) for 1.5 h. The concentrations of free O₂ and CO₂ in the shoot cavities in polyethylene zip-lock bags, and the ethanol content in the shoot body before and after treatment were determined. In addition, the photosynthetic rates of the fifth bamboo internodes of 1 y/o, 2 y/o and 3 y/o bamboo in the field were measured. The results indicated that: (1) When treated with air and nitrogen, US, AS and HS mainly exhibited aerobic respiration, and there was almost no anaerobic respiration; (2) When treated with air, 59.66%, 54.47% and 45.84% of the O₂ in the aerobic respiration of US, AS and HS came from the polyethylene zip-lock bag, 0.06%, 0.57% and 0.650% came from the shoot cavity, but 40.28%, 44.96% and 53.51% of the O₂ was of an unknown source; (3) Treated by nitrogen, 0.19%, 4.71% and 4.79% of O₂ in aerobic respiration of US, AS and HS came from shoot cavity, while the other 99.808%, 95.290% and 95.21% of O₂ came from unknown sources; and (4) The photosynthesis of the fifth internodes of 1 y/o, 2 y/o and 3 y/o bamboo generated little oxygen that could not absolutely meet the huge O₂ supply for aerobic respiration. It was concluded that the respiration of P. prominens shoots in its different growth and development stages was dominated by aerobic respiration. O₂ supply pathways were mainly via the sheath stomata; however, there was little absorption from dissolved O₂ in the soil water and little supply produced by shoot/stem photosynthesis. It was found that the large supply of oxygen in the aerobic respiration of bamboo shoots and young bamboo was of an unknown source under air treatment and nitrogen treatment, i.e., 40.28%–53.51% and 95.21%–99.81% of oxygen in the aerobic respiration of bamboo shoots and young bamboo was of unknown origin, respectively. Therefore, we proposed that bamboo shoots may exhibit the phenomenon of acting as an “oxygen sink”, which can provide a large amount of O₂ from unknown sources to ensure the rapid growth of bamboo shoots and young bamboo.

1. Introduction

Bamboo is one of the fastest growing plants on earth, of which, Moso bamboo (Phyllostachys edulis (Carriere) J. Houzeau) grows more than 100 cm per day in its growth period and completes its growth period in approximately 50 days to reach a height of approximately 20 m [1,2]. The mechanism underlying why it grows at such a rapid rate remains the subject of research and one of the mysteries in botany. Studies on bamboo structural anatomy have shown that bamboo consists of many internodes composed of meristematic tissue zones, elongation zones and maturation zones in an upward sequence; each internode actually functions as a “growth point”, and many “growth points” are superimposed systematically to accelerate its rapid growth [3,4]. Exogenous gibberellin (GA) treatment applied to Moso bamboo seedlings significantly increased internode length [5]. Chen et al. [3] found there was a high content of GA in both the cell division and elongation regions of the rapidly growing internodes of Moso, and the cell division and elongation rate of Moso bamboo seedlings could be increased by 2.23–3.89 times when treated with exogenous GA. Song et al. proposed that mature Moso bamboo plant supplied the assimilates from leaves, branches, stalks and roots to fast-growing bamboo shoots via its underground rhizomes [6]. However, Zhang et al. found that the starch supplied for the growth of Moso young shoots was only stored in the shoot body rather than in the rhizomes [7]. Cui et al. found that the protein abundance of Moso bamboo growing from a shoot to young culm was regulated temporally and, to some extent, spatially. The sequential development from the base to the apex of the bamboo culm was implemented by the temporal and spatial expression of enzymes; energy was mainly derived from sucrose degradation, and the regulation of anaerobic and aerobic modes of respiration appeared to play an important role in energy generation [8]. It was found that the internodal growth zone of Moso bamboo could elongate by up to 11.8 cm per day, produce approximately 570 million cells and deposit approximately 28 mg/g lignin (dry weight) and approximately 44 mg/g cellulose (dry weight); this exceeded, by far, the growth rates of other plants [3]. Consequently, such an amazing growth rate of bamboo should be supported by strong respiration to produce enough energy and the intermediate metabolites required for its growth and development. Of course, strong aerobic respiration should be supplied by a large amount of O₂. However, the following question still remains: What is the source and mechanism of O₂ supply to meet aerobic respiration? Unfortunately, no research has provided an answer to this question yet. Unearthing the mysterious mechanism behind the rapid growth of bamboo represents a fundamental scientific puzzle that should be solved. In response to this scientific problem, we hypothesized that there should be an oxygen supply mechanism in bamboo shoots, to meet the oxygen supply of aerobic respiration in a short timeframe in order to ensure the rapid growth and development of bamboo shoots.

To study such a mechanism and test this hypothesis, the fifth bamboo internodes of Phyllostachys. prominens at three different growth and development stages, i.e., underground shoots (US), 2-m-high aboveground shoots (AS), and 8-m-high height-growth-ceased shoot/young bamboo (HS), were treated for 1.5 h in a polyethylene zip-lock bag filled with air (21% O₂ + 79% N₂) and nitrogen (100% N₂). The respiration intensity of shoot internodes, the free O₂ and CO₂ concentrations inside the internode cavity and in the bag, and the ethanol content of shoot internodes before and after treatment with air and nitrogen were measured to reveal the source and mechanism of O₂ for satisfying aerobic respiration during the rapid growth of bamboo shoots.

2. Materials and Methods

2.1. Overview of the Test Site

The experimental site was located in a P. prominens bamboo forest (29°79′ N, 119°57′ E) in the Eshan Township, Tonglu County, Hangzhou City, Zhejiang Province. It has a subtropical monsoon climate with four distinct seasons, an annual average temperature of 16.5 °C, annual average rainfall of 1525 mm, sandy loam soil, and a bamboo forest density of 5000–6900 plants/hm².

2.2. Research Methods

2.2.1. Test Materials

On 3 June 2022, US, AS and HS without mechanical damage or insect attack (Figure 1) and of nearly the same sizes were selected and transported to the laboratory for further treatment and analysis within 2 h.

2.2.2. Treatment of the Materials

The 5th internode with both septa was taken as the test material. The bamboo shoots were rinsed in clean water, soaked in a 1% sodium hypochlorite solution for sterilization for 30 s, and then taken out and dried. They were randomly divided into 2 groups: Group 1 was placed in a closed 6.1 L polyethylene zip-lock bag at room temperature and filled with high-concentration nitrogen (100% N₂) for 1.5 h; Group 2 was placed in a closed 6.1 L polyethylene zip-lock bag at room temperature and filled with air (21% O₂ + 79% N₂) as the control. In each group, 8 shoots were used as 8 biological replicates.

2.2.3. Air Tightness Test of O₂ and CO₂ in the Bags

Two 6.1 L polyethylene zip-lock bags (thickness 0.12 mm, length × width = 42 cm × 35 cm) were filled with nitrogen (100% N₂) and air (21% O₂ + 79% N₂), respectively, until each was balanced with the outside air pressure, and then sealed. One oxygen electrode (Unisense OX-NP, Aarhus, Denmark) probe was inserted into each of the polyethylene zip-lock bags and stabilized for 20 s, to measure the oxygen content [9], and CO₂ concentration was determined using a GXH-3010E infrared carbon dioxide analyzer. The experiment was set at 4 time points, i.e., 0 h, 1.5 h, 3 h and 6 h.

2.2.4. Measurement of Respiration Rate

For the two experimental groups, the respiration rate of the internodes was measured using one infrared carbon dioxide analyzer GXH-3010E [10,11], with eight biological replicates; the respiration rate was expressed as mg CO₂ kg⁻¹h⁻¹.

2.2.5. Determination of O₂ and CO₂ Concentrations

O₂ and CO₂ concentrations were determined using a headspace analyzer (CheckMate 3 from Molcon, Boston, MA, USA) and a self-made gas extraction device. The gas analyzer was kept open and preheated for 10 min. The gas in the collection tube was put into the self-made gas extraction device, and the measuring system was closed under the condition of 25 °C. The intermittent automatic measurement mode was adopted until the volume fraction of CO₂ or O₂ did not change significantly for several consecutive measurements.

2.2.6. Determination of Ethanol Content in Bamboo Shoots

The ethanol content was determined by high-performance liquid chromatography (HPLC). A total of 2 g of the prepared sample was homogenized in a 20 mL plugged centrifuge tube, and 20.0 mL acetonitrile was added and centrifuged for 2 min at 4000 r·min⁻¹. Approximately 5 g sodium chloride was added, and the mixture was violently shaken for 1 min and centrifuged for 5 min at 4000 r·min⁻¹ to separate acetonitrile and water. A 10 mL acetonitrile layer was placed in a 50 mL pear-shaped bottle and concentrated to near dry by rotary evaporation at 40 °C. After the sample was blown dry, the dissolved residue was purified for determination by ultra-high-performance liquid chromatography. The chromatographic parameters were set as follows: mobile phase flow rate 0.5 mL·min⁻¹; fluorescence detection wavelength Ex = 280 nm, Em = 330 nm; column temperature 40 °C; and sample size 4 μL. Standard ethanol (purity > 99%) was used for quantitative determination.

2.2.7. Calculation of Anaerobic Respiration and Aerobic Respiration Ratio of Bamboo Shoots

The total yield of 1.5 h CO₂ was calculated according to the variation in CO₂ concentration, and the corresponding volume of the closed polyethylene zip-lock bag and bamboo shoot cavity. According to the ethanol content and the mass of bamboo, the ethanol mass of bamboo was calculated, and the CO₂ produced by anaerobic respiration was calculated by the ethanol mass. The proportion of anaerobic respiration was expressed as the ratio of the amount of anaerobic respiration to the total amount of CO₂. One-hundred percent minus the proportion of anaerobic respiration was equal to the proportion of aerobic respiration. The calculation formula is as follows:

$$\begin{gathered} a=c_1\times v_1+c_2\times v_2; \\ b=c_3\times m\times \frac{44}{46.07}; \\ d=\frac{b}{a}\times 100\%;e=100\%-d. \end{gathered}$$

where a is the total yield of 1.5 h CO₂ (mg); c₁ and c₂ are the changes in CO₂ concentration of the polyethylene zip-lock bag and the bamboo shoot cavity (mg·L⁻¹), respectively; v₁ and v₂ represent the gas volume of the polyethylene zip-lock bag (L) and the volume of the bamboo shoot cavity (L), respectively; b is the amount of CO₂ produced by anaerobic respiration(mg); c₃ is the concentration of ethanol (mg·kg⁻¹); m is the internode mass of shoots (kg); d is the proportion of anaerobic respiration; and e is the proportion of aerobic respiration.

2.2.8. Calculation of the O₂ Consumption Ratio of Bamboo Shoots from Different Sources

According to the variation in O₂ concentration and the corresponding volumes of the sealed polyethylene zip-lock bags and bamboo shoot cavities, the respective consumption of O₂ over 1.5 h was calculated. The amount of CO₂ corresponding to aerobic respiration equaled the total amount of CO₂ produced minus the amount of CO₂ produced by anaerobic respiration. The amount of CO₂ corresponding to aerobic respiration was calculated as the total O₂ demand for aerobic respiration. The ratio of O₂ consumption of the polyethylene zip-lock bag and the bamboo shoot cavity to O₂ demand of aerobic respiration represented the O₂ source ratio of the polyethylene zip-lock bag and the bamboo shoot cavity. One-hundred percent minus the O₂ source ratio of the polyethylene zip-lock bag and the bamboo shoot cavity equaled the O₂ source ratio of unknown sources.

$$\begin{gathered} f=c_4\times v_1;g=c_5\times v_2; \\ h=(a-b)\times \frac{32}{44}; \\ i=\frac{f}{h}\times 100\%;j=\frac{g}{h}\times 100\%;k=100\%-i-j. \end{gathered}$$

where f and g are the O₂ consumption (mg) of the polyethylene zip-lock bag and the bamboo shoot cavity, respectively; c₄ and c₅ are the changes in O₂ concentration of the polyethylene zip-lock bag and the bamboo shoot cavity, respectively (mg·L⁻¹); h is the total requirement of O₂ for aerobic respiration (mg); and i, j and k are the polyethylene zip-lock bag, bamboo shoot cavity and O₂ of unknown origin, respectively.

2.2.9. O₂ Consumption and CO₂ Production Per Unit Volume of Bamboo Shoots

The volume ratios of O₂ consumption and CO₂ production in the bamboo shoot cavity and the polyethylene zip-lock bag, total O₂ consumption, total CO₂ production and bamboo shoot segment were expressed, including O₂ consumption per unit volume and CO₂ production (eliminating the effects of different volumes).

2.2.10. Determination of the Photosynthetic Rate of Bamboo Culms

The photosynthetic rate of the bamboo stems of high-jointed bamboo shoots was measured as follows: (1) It was measured using a LIi-6400XT(LICOR Beijing Ecotek Technology Company) portable photosynthesizer and homemade sealed air chamber (Figure 2). The structure of the gas chamber was modified with reference to the gas chamber for the CO₂ emission flux from the tree trunk [12]. The device was made of transparent acrylic material with a radius of 7.5 cm and a height of 10 cm. (2) The operation and sealing condition of the equipment were checked before the CO₂ emission flux was measured. After it was confirmed that there was no problem, an air chamber was installed on the stalk to ensure that the stalk and the device were well fitted, and sealing mud was applied to ensure that the whole space was sealed. (3) After the measurement was taken, the transparent device was removed to make the internal air condition consistent with the outside. Furthermore, opaque tin foil was pasted on the side of the gas chamber to block the outside light, the gas chamber was installed at the same position in the same section, and the photosynthesizer was set to automatically record the concentration of CO₂ in the gas chamber every 10 s and measure for 120 s. (4) The date of measurement was June 2022. The CO₂ emission flux between the fifth nodes aged 1, 2 and 3 years were measured at 10:00 a.m. and 15:00 p.m. on a sunny day. (5) The gas exchange method was used to measure the photosynthetic rate of bamboo stems and the photosynthetic rate of cortex was equal to the reduction in CO₂ emission flux under light compared with that under shading [13,14]. (6) The photosynthetic rate was used to calculate the amount of O₂ produced per unit volume of bamboo stems. The calculation formula is as follows:

$$\begin{gathered} R=\frac{10V_1{P}_0\times (1-\frac{W}{1000})}{FS(T_0+273.15)}\times R_S; \\ A=\left|R_d-R_1\right|; \\ C=3.6\times {10}^{-4}\times \frac{32}{44}\times \frac{AST}{V_2} \end{gathered}$$

where R is the CO₂ emission flux (μmol·m⁻²s⁻¹); R_S is the CO₂ change slope of the sealing device; V is the gas volume of the sealing device; P₀ is the atmospheric pressure of the sealing device (KPa); W is the initial water vapor content (mmol mol⁻¹); F is the constant 8.314; S is the stem surface area (cm²); T₀ is the temperature in the sealing device (°C); A is the photosynthetic rate (μmol·m⁻²s⁻¹); R_d is the light-blocking CO₂ emission flux (μmol·m⁻²s⁻¹); R₁ is the light-blocking CO₂ emission flux (μmol·m⁻²s⁻¹); C is the yield of stem cortex O₂ over a period of time (mg·L⁻¹); AST is the time (h); V₂ is the stem volume in the sealing device (L); and 3.6 × 10⁻⁴ × 32/44 is the conversion factor.

2.2.11. Statistical Analysis

The data obtained are expressed as the mean ± standard error (SE). SPSS 25 statistical analysis software was used for data significance analysis, and one-way ANOVA was used for Tukey multiple comparison (p < 0.05). The Origin 2022 drawing software was also used.

3. Results

3.1. Gas-Tightness of the Polyethylene Zip-Lock Bag to O₂ and CO₂

In order to detect whether the bag was sealed in terms of O₂ and CO₂, the O₂ and CO₂ gas-tightness in the bags were measured and the results showed no significant changes in O₂ and CO₂ contents at the four experimental time points (Figure 3). It was shown that the bags were effectively gas-tight to O₂ and CO₂ and met the experimental gas-tightness requirements.

3.2. Changes in Respiration Rates of Bamboo Shoots Treated with Air and Nitrogen

As is shown in Figure 4, the respiration rates of shoots in different growth and development stages coincided with their growth features, i.e., the highest respiration rate of 350–360 mg CO₂·kg⁻¹h⁻¹ was observed in the 2-m-high bamboo shoots in the rapid growth period, and the lowest respiration rate of 170–230 mg CO₂·kg⁻¹h⁻¹ was observed in the HS; however, the respiration rates of shoots at the same stage (US, AS and HS) treated with air and nitrogen were not significantly different.

3.3. Changes in Ethanol Content in Bamboo Shoots Treated with Air and Nitrogen

The degree and level of anaerobic respiration can be represented by the amount of ethanol produced by bamboo shoots. Shoots at the same stages of US, AS and HS did not significantly differ in terms of ethanol content under the different treatments of air and nitrogen (Figure 5), indicating that there was no significant difference in the intensity of anaerobic respiration between the two treatments.

3.4. Ratios of Aerobic and Anaerobic Respiration of Bamboo Shoots

The ratios of aerobic respiration and anaerobic respiration at the three stages of US, AS and HS with two different treatments are shown in

Table 1. Ratio of aerobic respiration and anaerobic respiration of bamboo shoots in different growth and development stages.

Treatment	Stage	Aerobic Respiration Ratio (%)	Anaerobic Respiration Ratio (%)
Air	US	99.99	0.01
	AS	99.97	0.03
	HS	99.98	0.02
Nitrogen	US	99.99	0.01
	AS	99.97	0.03
	HS	99.98	0.02

. This shows that their aerobic respiration ratio was above 99.90%, indicating that during the rapid growth period of bamboo shoots, aerobic respiration was the main respiratory function, while anaerobic respiration was almost absent. Within 1.5 h of nitrogen treatment, i.e., without external O₂ supply, its respiration was still aerobic, indicating that the O₂ supply was sufficient.

3.5. Proportion of O₂ Consumption from Different Sources of Bamboo Shoots

In order to find out the source of O₂ consumption in aerobic respiration, the O₂ consumption of different treatments at different growth and development stages was calculated (

Table 2. Proportion of O₂ sources of aerobic respiration in two treatments of bamboo shoots at different growth and development stages.

Treatment	Stage	O2 Consumption Ratio of Shoot Cavities (%)	O2 Consumption Ratio of Polyethylene Zip-Lock Bags (%)	Proportion of O2 from Unknown Sources (%)
Air	US	0.06	59.66	40.28
	AS	0.57	54.47	44.96
	HS	0.65	45.84	53.51
Nitrogen	US	0.19	-	99.81
	AS	4.71	-	95.29
	HS	4.79	-	95.21

). Under the air treatment, with the growth of bamboo shoots, the proportion of O₂ consumed in the shoot cavity also increased, with a rate of 0.06%–0.65%. On the contrary, the proportion of O₂ consumed in the polyethylene zip-lock bags was 45.84%–59.66%. What is more interesting is that 40.28%–53.51% of the O₂ was from unknown sources. Under the nitrogen treatment, due to the absence of O₂ in the surrounding environment, the proportion of O₂ consumed by the shoot cavity was far greater than that with the air treatment, and 95.21%–99.81% of the O₂ was from unknown sources. The results showed that the proportion of O₂ consumed by the shoot cavity was not only related to the growth and development stage of the shoot, but was also affected by the external O₂ content. What are the unknown sources of O₂? This is a very interesting question.

3.6. O₂ Consumption and CO₂ Production Per Unit Volume of Bamboo Shoots

In order to compare the relationship between O₂ consumption and CO₂ production in bamboo shoots, O₂ consumption and CO₂ production were expressed as O₂ consumption and CO₂ production per unit volume of bamboo shoots. Under the air treatment, there was no significant difference in shoot cavity O₂ consumption between the US, AS and HS growth periods. Moreover, both O₂ consumption in the polyethylene zip-lock bag and total O₂ consumption were significantly higher in the US stage than in the other two stages. Under the nitrogen treatment, O₂ consumption in the shoot cavity and total O₂ consumption were significantly lower in the US stage than in the other two stages. (

Table 3. O₂ consumption of bamboo shoots in different growth and development stages (mean ± SE).

Treatment	Stage	O2 Consumption in Bamboo Shoot Cavities (mg·L−1)	O2 Consumption in Polyethylene Zip-Lock Bags (mg·L−1)	Total O2 Consumption (mg·L−1)
Air	US	4.69 ± 1.04 a	5663.07 ± 749.40 a	5667.760 ± 749.63 a
	AS	8.80 ± 2.41 a	828.34 ± 106.77 b	837.14 ± 108.03 b
	HS	9.05 ± 3.10 a	694.89 ± 108.32 b	703.94 ± 108.17 b
Nitrogen	US	15.65 ± 2.06 b	-	15.65 ± 2.06 b
	AS	55.77 ± 5.62 a	-	55.77 ± 5.62 a
	HS	53.67 ± 4.40 a	-	53.67 ± 4.40 a

Note: Different lowercase letters indicate significant differences at the level of p < 0.05 in different periods.

).

There was no significant difference in CO₂ production in the shoot cavity among the US, AS and HS stages treated with air and nitrogen. Furthermore, both CO₂ production in the polyethylene bag and total CO₂ production were significantly higher in the US stage than in the other two stages (

Table 4. CO₂ generation of bamboo shoots under two treatments at different growth and development stages (mean ± SE).

Treatment	Stage	CO2 Generation in Shoot Cavities (mg·L−1)	CO2 Generation in Polyethylene Zip-Lock Bags (mg·L−1)	Total CO2 Generation (mg·L−1)
Air	US	−0.38 ± 1.14 a	13,116.92 ± 1687.84 a	13,116.92 ± 1687.84 a
	AS	0.75 ± 2.35 a	2129.98 ± 299.31 b	2130.73 ± 299.63 b
	HS	−7.25 ± 5.05 a	2087.76 ± 253.46 b	2080.51 ± 252.47 b
Nitrogen	US	−3.65 ± 1.43 a	12,877.43 ± 2190.27 a	12,877.43 ± 2190.27 a
	AS	1.64 ± 2.53 a	1985.62 ± 397.41 b	1987.25 ± 396.10 b
	HS	−4.650 ± 4.12 a	1980.4 ± 346.57 b	1975.80 ± 343.41 b

Note: Different lowercase letters indicate significant differences at the level of p < 0.05 in different periods.

). Under the air treatment, both O₂ consumption and CO₂ production showed a downward trend from the US–HS period, indicating that the smaller bamboo shoots had a more vigorous metabolism, higher O₂ consumption and CO₂ production, and higher total CO₂ production than O₂ consumption. After nitrogen treatment, only a small amount of O₂ was provided by the bamboo shoot cavity, and the total amount of CO₂ generated was much higher than the O₂ consumption.

3.7. O₂ produced by Photosynthesis of Bamboo Culms

The CO₂ emission flux of bamboo culms was measured to calculate the photosynthetic rate of bamboo culms and the amount of O₂ produced by photosynthesis of bamboo culms in 1.5 h. The results showed that there was no significant difference between the photosynthetic rates of bamboo culms at the same age between the morning and afternoon, and the maximum photosynthetic rate was only 0.4 μmol·m⁻²·s⁻¹ (Figure 6a). The amount of O₂ produced by photosynthesis in 1.5 h was not significantly different between bamboo culms at the same age in the morning and afternoon, and was only 0.01–0.13 mg·L⁻¹ (Figure 6b), which was much lower than the amount of O₂ required for aerobic respiration. The amount of O₂ required for aerobic respiration was much lower than the amount of O₂ required for anaerobic respiration, so the amount of O₂ provided by bamboo culm photosynthesis for aerobic respiration was negligible.

4. Discussion

4.1. Respiratory Alternation of Bamboo Shoots

Higher plants usually undergo a short period of anaerobic respiration in anoxic environments, e.g., when seeds absorb water for germination, or when the plant is flooded; anaerobic respiration also usually occurs in the internal tissues of fertile stems and leaves, and large fleshy fruits and tubers where the anoxic environments are formed due to difficulties in diffusing O₂ into the internal tissues [15]. This study showed that the proportion of aerobic respiration in P. prominens shoots was up to 99.90% at all three different growth and development stages, either with the air or nitrogen treatment, indicating that the bamboo shoots were dominated by aerobic respiration, with anaerobic respiration being almost absent.

It was found in our previous study, where Moso bamboo shoots were treated with air (21% O₂ + 79% N₂) and hyperoxia (90% O₂ + 10% N₂), that harvested Moso bamboo shoots had a very high respiratory intensity, and its respiration type or approach was subject to the ambient O₂ contents: Under air treatment, the bamboo shoots mainly underwent anaerobic respiration, supplemented by aerobic respiration; however, under high oxygen treatment, the shoots mainly underwent aerobic respiration, supplemented by anaerobic respiration [16]. By carrying out a proteomic study on Moso bamboo shoots in different growth and development stages, Cui et al. [3] found that a lot of enzymes such as superoxide dismutase, peroxide dismutase and catalase existed in its earlier growth and development stage; enzymes related to aerobic respiration such as fructose diphosphate aldolase, phosphoglycerate kinase, malate dehydrogenase and succinate dehydrogenase were observed in its later stage. Therefore, it was supposed that earlier bamboo shoots were subjected to an anaerobic environment by being covered by shoot sheaths, which made bamboo shoots proceed mainly with anaerobic respiration. In the later stage, increased shoot sheath shedding made it easier for the shoots to access external O₂, which caused them to be dominated by aerobic respiration, gradually decreasing anaerobic respiration. Therefore, whether bamboo shoots undergo aerobic or anaerobic respiration depends on the bamboo species and the availability of O₂ in the environment.

4.2. The Sources of O₂ Supply for the Aerobic Respiration of Bamboo Shoots

O₂ supply for the aerobic respiration of plants has several sources. It is traditionally recognized that there are mainly three sources: First, O₂ enters the cell interstices by diffusion and convection through plant stomata and stem lenticels from the outside atmosphere. Second, O₂ is absorbed by the root system from soil crevices and dissolved O₂ in aqueous soil solutions, and transported with water over a long distance to oxygen-demanding organs, as driven by root pressure and transpiration. Third, under light, O₂ is produced via photosynthesis of the cells in the green parts of the plant.

Each bamboo shoot sheath is borne at each corresponding node and inter-wrapped tightly by each other. A lot of stomata are distributed on both sheath sides and they have a much higher stomatal conductance than that of mature leaves [17,18,19]. The shoot sheath structure mainly consists of air cavities and thin-walled cells that create large cellular spaces [20]. Ding and Liese [21] found that “phloem valves” distributed at the shoot nodes functioned mainly to promote the distributary transport of substances. Atmospheric O₂ enters into the shoot body either by the culm sheath stomata via diffusion and convection, or by “phloem valves” in a “stoma-cavity system”. Consumption of O₂ driven by aerobic respiration creates a favorable gradient of O₂ concentration between the air, shoot sheath and shoot body to promote O₂ entry. In the bag filled with air, the proportion of O₂ supply for shoot aerobic respiration was up to 45.84%–59.66% from the air inside the bag, but less than 1% from the shoot cavity. Consequently, the following question arises: Where does the rest of the O₂ come from? In the bag filled with nitrogen, the proportion of O₂ supply for shoot aerobic respiration reached up to 99.99%–99.98% from the bag but only 0.19%–4.79% from the shoot cavity. Consequently, further questions arise: Where does this large amount of O₂ come from? What other O₂ supply pathway remains so mysterious?

Yang et al. [22] found, through the use of heat dissipation probes, that the root pressure mainly drove soil water transport to replenish bamboo shoots at night, and mainly took the water stored for guttation by sheaths at night and transpiration during the day. Therefore, water stored in the shoots should be limited to a certain degree and not continue to take on water from the soil. The average guttation produced by Moso shoots was only 0.342–0.514 L at night [23], and the maximum transpiration rate of Bambusa blumeana was 1.4 mm·d⁻¹ [24]; the generally dissolved O₂ in water was only 5.18–10.6 mg·L⁻¹ [25]. Therefore, regardless of whether it occurs via water transpiration during the day or water spitting at night, the uptake of dissolved O₂ in water by bamboo shoots is quite limited. This is “a drop in the bucket” compared with the large amount of O₂ required for bamboo shoot’ respiration. O₂ produced by stem photosynthesis is important for maintaining aerobic respiration in stems. However, the results of this study showed that the photosynthetic rate in 1-, 2- and 3-year-old bamboo culms was very little, with a maximum of only 0.4 μmol·m⁻²·s⁻¹. The O₂ produced by photosynthesis in 1.5 h was only 0.01–0.13 mg·L⁻¹, which was far lower than the O₂ requirement for aerobic respiration. Culms at 1-, 2- and 3-years-old, for which their sheaths were shedding, has a significantly increased chlorophyll content and were able to receive direct sunlight for photosynthesis; however, they had very low photosynthetic rates. The O₂ generated by photosynthesis of the bamboo shoots in the US, AS and HS growth stages was wrapped tightly by the sheaths. However, it was negligible for shoot aerobic respiration, since their chlorophyll was underdeveloped. In summary, the O₂ for aerobic respiration in bamboo shoots was supplied largely from the outside air through the stomata on shoot sheaths, and a little was supplied through soil water uptake and photosynthesis; however, the source of a large amount of O₂ is still unknown.

4.3. Bamboo Shoot “Oxygen Sink” Phenomenon

In this study, it was found that the supply of O₂ ranging from 40.28 to 53.51% for the aerobic respiration of P. prominens shoots during its different growth and development stages had an unknown source. There was no significant difference in the respiration rate and shoot ethanol content in shoots in the three different growth and development stages treated with both air and nitrogen. More interestingly, in the nitrogen treatment group, the percentage of the aerobic respiration of shoots was still higher than 99.90% in the three different growth and development stages. In addition, the total CO₂ emission was much higher than the total O₂ consumption, providing no external O₂ supply or only a small amount of O₂ supply available from the shoot cavity. In the air treatment group, the total CO₂ emission was approximately twice that of the O₂ consumption. This is inconsistent with common sense or principle; the total O₂ consumption and total CO₂ emission by aerobic respiration should be equal [17]. If this unknown source of O₂ does not exist, then the bamboo shoots should be in a state of O₂ deficiency; however, the rapid growth rate of bamboo shoots indicates that bamboo shoots are not O₂ deficient. The content of free O₂ in the flesh of bamboo shoots was very low, and the content of O₂ in the flesh of bamboo shoots was less than 1 mg·L⁻¹. Therefore, our data support the existence of an “oxygen sink” during the rapid growth and development of bamboo shoots and young bamboo: The bamboo shoots absorb O₂ via different methods during the growth and development process, forming combined O₂ “storage”. Under the condition of insufficient external O₂ supply, combined O₂ will be released to meet the need of O₂ for aerobic respiration in a short timeframe to ensure the rapid growth of bamboo shoots. The phenomenon of an “oxygen sink” may be the result of the long-term evolution and adaptation of bamboo plants to the environment. This would explain the amazingly rapid growth of bamboo requiring a large amount of O₂ to be supplied immediately despite the difficulty in obtaining a sufficient O₂ supply from the outside due to the tight sheath. Therefore, this theory is helpful in explaining the mechanism behind the rapid growth of bamboo shoots.

5. Conclusions

The respiration of P. prominens shoots in their different growth and development stages were dominated by aerobic respiration. Their O₂ supply pathways were mainly via the sheath stomata; however, there was little absorption from dissolved O₂ in the soil water and little supply from shoot/stem photosynthesis. The unknown supply of oxygen for the aerobic respiration of bamboo shoots and young bamboos treated with air and nitrogen reached 40.28%–53.51% and 95.21%–99.81%, respectively. Therefore, we proposed that bamboo shoots may have access to the phenomenon of an “oxygen sink”, which can provide a large amount of O₂ from unknown sources to ensure the rapid growth of bamboo shoots and young bamboo.