Zn²⁺ induces changes in activities of mitochondrial respiratory chain complexes and emissions of floral volatiles in Dendrobium huoshanense

The experimental plant material was a 3-year-old D. huoshanense, grown in the greenhouse (Figures 1A–1D) in the botanical garden of West Anhui University, Lu’an City, Anhui Province, China. The greenhouse was covered with the sunshade net in 70% light transmittance of natural light, on the roof, and was automatically controlled in 40%–60% of humidity when the diurnal temperature was from −5 °C to 40 °C The culture medium was pine barks, which were naturally fermented after being piled on the ground.

Figure 1

D. huoshanense plants at a full-bloom stage were randomly divided into four groups: three treatment groups and one control group. Each group contains 10 D. huoshanense plants. The plant's petals of three different treatment groups were sprayed with 50 mL ZnSO₄ solution in 2 mM, 4 mM and 8 mM concentrations, respectively. The control group's petals were treated with the same volume of distilled water. Each processing was set to three repetitions. A total of 600 mL of ZnSO₄ solution was applied, and 120 D. huoshanense plants were examined. The spraying of the ZnSO₄ solution did not cause obvious visual changes to the petals. At 6 h after the treatment, sufficient fresh petals were collected to prepare protoplast and analyse intracellular Zn²⁺ concentrations. Furthermore, at 6 h, 9 h and 12 h, petals of 5 g were collected from every experiment group and were cut into pieces to put into a 20-mL headspace (HS) bottle. Subsequently, 5 g of anhydrous sodium sulphate and 10 μL of 0.05 mg · mL⁻¹ 2-nonanone were added into each bottle as the internal standards for chromatographic analysis, and the bottle was sealed with an attached cap. At every abovementioned moment, a sufficient number of petals collected were instantly frozen in liquid nitrogen and stored at −80 °C for further use, including the analysis of respiratory chain complex activities and the precursor and ATP levels (at 6 h, 9 h and 12 h after the treatment).

To comprehensively assess the change of intracellular Zn²⁺ concentrations, we determined the intracellular fluorescence intensity of Zn²⁺ and zinc concentrations in petals. Then, 10 g of fresh petals were cut into 1-mm pieces. The petal pieces were used to prepare cell protoplast suspension by the enzymatic hydrolysis method (Guilley and Hahne, 1989). Then, 10 μL fluorescent dye ZnAF-2F DA (Chemodex Ltd., St. Gallen, Switzerland) was added to 10 mL prepared protoplast suspension and was incubated in a petri dish under room temperature and dark conditions for 30 min. After the incubation, a fluorescence confocal microscope (FluoView FV1200, Olympus, Tokyo, Japan) was used for the observation of fluorescence intensity. Representative photos (60×) were taken, and the fluorescence intensity was analysed with Image J software. Zn²⁺ fluorescence intensity value was the mean of nine fluorescence measurements of three photos/protoplasts. Fluorescence intensity measurement of each time was calculated using the ratio of “integrated density/area”. The “area” was the region occupied by a specific number of protoplasts measured in the image, while “integrated density” was the sum of fluorescence intensities in the region. As for zinc concentrations, sufficient petals were ground and passed through a No. 40 sieve. Next, 50 g of the test sample was taken and carbonised into powder. Then, the powder was heated in a muffle furnace until it turned ash grey. According to the method described by Ashok Kumar et al. (2013), zinc concentrations were determined by the atomic absorption spectrophotometer (AA-6000).

Thirty grams of petals were cut into pieces; then, 120 mL of the extraction medium was added and immediately ground in a mortar to homogenate under ice bath conditions. The above extraction medium consisted of 20 mM HEPES-Tris (pH 7.5), 0.3 M sucrose, 1 mM 1,4-dithioerythritol (DTE), 5 mM sodium-ethylenediaminetetraacetic acid (Na-EDTA), 0.3% (w/v) bovine serum albumin (BSA) and 0.6% (w/v) polyvinylpolypyrrolidone (PVPP). After homogenising, the mitochondrial components were sequentially extracted and purified by differential centrifugation and Percoll density gradient centrifugation according to the method of Petrussa et al. (2008). The final purified material was resuspended in suspension [20 mM HEPES-Tris, pH 7.5, 0.1% (w/v) defatted BSA, 0.3 M sucrose]. The concentration of mitochondrial protein was monitored by the coomassie brilliant blue method and adjusted to 0.5 mg · mL⁻¹, and the protein was then stored on ice.

The activity of complex I (NADH-ubiquinone oxidoreductase) was evaluated by measuring the NADH oxidation rate according to the method described by Estornell et al. (Estornell et al., 1993; Srivastava et al., 2017). The reaction was started by adding the appropriate amount of buffer to 50 μL of the protein sample. The buffer consisted of 100 mM coenzyme Q1, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 2 mM N-ethylmaleimide (NEM), 2 mM potassium cyanide (KCN) and 50 mM KCl. The absorbance change of the reaction solution was measured with a spectrophotometer (UV-5100) at 340 nm. The activities of complex II (succinate dehydrogenase), III (ubiquinol cytochrome c reduction) and IV (cytochrome c oxidase) were measured according to the method described by Srivastava et al. (Srivastava et al., 2017; Zamiri et al., 2017). Briefly, the activity of complex II was determined by reducing 2,6-dichlorophenolindophenol (DCPI) with phenazine methyl sulphate (PMS). A 50-μL protein sample was taken and added to a medium containing 100-μM PMS, 50-mM KH₂PO₄ (pH 7.5), 16-mM sodium succinate and 1.5-mM KCN. After 3-min incubation at 37°C, 100 μM of DCPI was added to measure the difference in absorbance at 600 nm. The activity of complex III was determined by measuring the absorbance of cytochrome C at 550 nm. Approximately, 50 μL of the sample was added to the reaction medium containing 35 mM KH₂PO₄ (pH 7.5), 125 mM oxidized cytochrome C, 10 μg · mL⁻¹ rotenone, 5 mM MgCl₂, 1.8 mM KCN and 2.5 mg · mL⁻¹ BSA. After 30 s of incubation at 37 °C, the reaction was initiated by adding 31.8 μM reduced ubiquinone 2. Absorbance with and without 10 μg · mL⁻¹ antimycin A was monitored. The activity of complex IV was determined by measuring the oxidation rate of cytochrome C. The reaction was performed using 50 μL of the protein sample in 50 mM phosphate buffer (7.4 pH). Absorbance was monitored at 550 nm with or without 2 mM KCN. Regarding complex V (ATP synthase), its activity was determined according to the report of Zamiri et al. (2017). Approximately, 50 μL of the protein sample was added to a medium containing 50 mM Tris (pH 8.0), 50 mM KCl, 20 mM MgCl₂, 15 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP), 10 mM phosphoenolpyruvate, 5 mM antimycin A, 2.5 mM ATP, 5 mg · mL⁻¹ BSA, 1 mM NADH, 20 U of pyruvate kinase and 20 U of lactate dehydrogenase. After 30 s of incubation at 37 °C, a reaction was performed in the presence and absence of 3 μM oligomycin. Absorbance was monitored at 340 nm.

Depending on the method described by Zhou et al. (2013), IPP and DMAPP contents were estimated by measuring the isoprene production rate. Briefly, fresh petals were ground and divided into two halves directly into 5 mL test tubes. Ice cold extraction buffer (100 mM NH₄HCO₃:isopropanol:acetonitrile = 1:1:3) was added to the tubes to extract the powder. The extracts were centrifuged at 4°C for 15 min. Supernatants were transferred and dried. Then, isoprene synthase and/or isopentenyl diphosphate isomerase was added to initiate the reaction. DMADP content was quantified by converting it to isoprene using isoprene synthase. The total content of IPP plus DMADP was measured by including isopentenyl diphosphate isomerase in the reaction mixtures. IPP content was determined according to the difference between the total measurement and the DMADP measurement. Isoprene emission from the reaction was measured with a fast isoprene sensor (LY500-C5H8). Phe content was established by referring to the method of Li et al. (2017). Petal samples were ground and extracted with 1.0 mL of extraction liquid (chloroform: methanol, 1:3, v:v). Subsequently, the extracts were centrifuged at 4 °C for 10 min. The supernatant was transferred into a glass vial and dried in a vacuum chamber at 30 °C. Then, samples were derivatised with N-trimethylsilyl-N-methyl trifluoroacetamide at 70 °C for 1 h, and the Phe content was determined using gas chromatography. The operating conditions of gas chromatography (ThermoFisher Trace) were as follows: (1) the carrier gas was helium, with a flow rate of 1 mL · min⁻¹, (2) the injector and detector temperatures were both set at 280 °C and (3) the column temperature was kept at 70 °C for the first 2 min and then increased to 300 °C at a rate of 10 °C min⁻¹ for the next 10 min. LA and LNA contents were determined according to the method of Glew et al. (2010). Before analysis, petals were ground to a powder with a mortar and pestle and dried to constant weight under vacuum. Then, 2 g of the sample was extracted using 40 mL of the extraction solution (methanol: chloroform, 1:1, v:v). The extracts were well mixed and filtered. The filtrate was concentrated using a rotary evaporator. Subsequently, at 80°C, the sample was methylated by adding a solution (four drops of benzene, 4 mL of 5% methanol/anhydrous hydrochloric acid), and the LA and LNA contents were assayed using gas chromatography. The following were the gas chromatographic operating settings: (1) H₂ served as the carrier gas, with a flow rate of 1 mL · min⁻¹, (2) the temperatures of the injector and detector were both set at 250 °C and (3) the initial temperature was 45 °C, which was held for 4 min. Then, the temperature was programmed at 13 °C · min⁻¹ to 175 °C, which was held for 27 min, and afterward programmed at 4 °C · min⁻¹ to 215 °C, which was held for 35 min. ATP content was established by an ATP determination kit (Sigma Chemical Co., St. Louis, USA) in strict conformity with the operational procedures described by the kit. Petal samples (0.5 g) were weighed and added to 5 mL of the lysis buffer from the ATP kit for homogenisation in an ice bath. The homogenate was centrifuged with 8,000 g of petals at 4 °C for 10 min. Next, the bioluminescence intensity of the supernatant was determined by a microplate photometer (Multiskan FC, USA). A linear regression equation and serial ATP concentrations were used to determine the ATP content.

Headspace SPME technology was in time used to extract the samples collected. The volatiles from samples were separated and identified by GC–MS analysis (GC–MS, ThermoFisher Trace ISQ-LT). For HS-SPME to fully extract the volatile components from the samples, the HS bottle was balanced in a water bath at 50 °C; the microextraction head (Supelco) pre-aged at 250 °C for 2 h in the GC injection port was inserted into the HS bottle 1 cm away from the top of the samples for 40 min. The chromatography conditions were as follows: the column was 5% phenyl methyl siloxane (HP-5MS) (0.25 mm × 30 m × 0.25 μm) and carrier gas, helium (99.999%), and column flow rates were 1 mL · min⁻¹. The column temperature was programmed: the initial temperature was maintained at 40 °C for 3 min, then ramped to 150 °C at 5 °C · min⁻¹ and then increased to 250 °C at 10 °C · min⁻¹ and held for 5 min. The MS parameters were: ion source temperature 250 °C; transmission line temperature 250 °C; ionization voltage 70 eV; mass range m/z 35–450; national institute of standards and technology (NIST) standard reference database number 69. To qualitatively identify floral volatile components, 0.1 μL of n-alkaline mixtures (c6–c20) was used as a standard sample for GC–MS analysis, and their retention indexes (RI) were calculated by a linear heating formula. The relative mass of volatile compounds was calculated by the internal standard method.

All experimental indicators were evaluated in triplicates, and the measurement data were expressed as the mean plus or minus the standard error (SE). SPSS 17.0 software was used for statistical analysis of the data. One-way analysis of variance (ANOVA) was applied to compare the difference in the mean of the data. Canonical correlation analysis was conducted to explore relationships among the activities of respiratory chain complexes, production rates of precursors and ATP and emissions of floral volatiles.

Zn²⁺ fluorescence intensity is an important indicator reflecting the Zn²⁺ concentrations in the cell. ZnAF-2F DA, as a Zn²⁺ fluorescent probe, can be hydrolysed by esterase in the protoplast into ZnAF-2F. The latter can selectively recognise intracellular free Zn²⁺. When there was a higher Zn²⁺ concentration in cells, the fluorescence intensity of the complex constituted by the combination of ZnAF-2F and Zn²⁺ was stronger and vice versa. The study indicated that ZnSO₄ treatments significantly increased Zn²⁺ fluorescence intensity (Figures 2A–2D, Figures 2a–2d; Table 1). Compared with the control group, the Zn²⁺ fluorescence intensity in 2–8 mM ZnSO₄ treatments was stronger (Figures 2A–2D, Figures 2a–2d). Quantitative analysis showed Zn²⁺ fluorescence intensity increased with the increasing ZnSo₄ concentration (from 2 mM to 8 mM). In the 8 mM ZnSo₄ treatments, both brightfield and darkfield fluorescence intensities reached their maximum values, which were ~54.59% (p < 0.01) and ~28.17% (p < 0.01), respectively (Table 1). Zn concentrations in petals also showed similar changes (Table 1). In 2–8 mM ZnSO₄ treatments, the Zn concentrations were increased by 15.07%, 29.80%, and 81.53%. The results implied that the ZnSO₄ application increased the intracellular Zn²⁺ concentrations.

Figure 2

To evaluate the response of the activities of mitochondrial respiratory chain complex I, II, III, IV and V to the elevated intracellular Zn²⁺ concentrations, we measured the activity values at 6 h, 9 h and 12 h after the ZnSO₄ application (Figures 3A–3E). In 2 mM or 4 mM ZnSO₄ treatments, the activity values of complex I, III and IV were not statistically significantly different from those of the control (Figures 3A–3D). However, the activity values of complex II and V were significantly increased, and the increase in activity values varied from 5.42% (p < 0.05) to 86.38% (p < 0.01) (Figures 3B and 3E). In 8 mM ZnSO₄ treatments, the activity values of complex I, II, III, IV and V were all significantly decreased (p < 0.01), and the reduction in activity values was at most ~55.39% (p < 0.01) (Figures 3A–3E). Elevated intracellular Zn²⁺ concentrations induced the different changes of mitochondrial respiratory chain complex activities.

Figure 3

Floral volatile emissions can be influenced by precursors and ATP production rates; hence, we measured precursor and ATP contents in D. huoshanense petals after the ZnSO₄ application (Figures 4A–4F). In 2 mM or 4 mM ZnSO₄ treatments, the content of precursors (DMAPP, IPP, Phe, LA and LNA) and ATP was significantly increased with elevated intracellular Zn²⁺ concentrations compared with the control group, and the increase in proportion varied from 3.76% (p < 0.01) to 26.15% (p < 0.01) and from 21.90% (p < 0.01) to 69.43% (p < 0.01) in 2 mM and 4 mM ZnSO₄ treatments, respectively. However, in 8 mM ZnSO₄ treatments, the content of precursors and ATP was significantly decreased, and the decrease in proportion varied from 7.37% (p < 0.01) to 27.69% (p < 0.01) (Figures 4A–4F). The content changes of precursors and ATP indicated that their production rates in D. huoshanense petals were promoted or inhibited by elevated intracellular Zn²⁺ concentrations after ZnSO₄ treatments.

Figure 4

The total ion chromatograms of volatile components were obtained by GC–MS analysis, including the different concentrations of ZnSO₄-treated samples at 6 h, 9 h and 12 h (Figures S1A–S1D, Figures S2A–S2D, Figures S3A–S3D). Based on the retrieved data of the MS library, floral volatile components were distinguished and are listed in Table 2. A total of 42 volatile compounds, with a total mass of 59.01 μg · g⁻¹ FW, were identified in the control group, including 17 terpenoids, 38.38 μg · g⁻¹ FW; 5 benzoids, 2.53 μg · g⁻¹ FW; and 21 fatty acid derivatives, 18.10 μg · g⁻¹ FW. Compared with the control group, 2 mM and 4 mM · L⁻¹ ZnSO₄ treatments promoted floral violate emissions; the number of floral volatile compounds increased by one (2 mM ZnSO₄ treatments) and three (4 mM ZnSO₄ treatments), and their total mass increased by 18.09% (p < 0.01), 66.88% (p < 0.01) respectively; in addition, the mass of terpenoids, benzoids and fatty acid derivatives all increased. However, 8 mM ZnSO₄ treatments significantly reduced floral violate emissions; there were only 39 compounds, losing 3 compounds compared with the control group; the total mass of volatile compounds decreased by 18.79% (p < 0.01), and the mass of terpenoids, benzoids and fatty acid derivatives all decreased (Figures 5A–5D; Table 2). The mass and number changes of floral volatile compounds indicated that their emissions in D. huoshanense petals were affected by elevated intracellular Zn²⁺ concentrations after ZnSO₄ treatments.

Figure 5

Canonical correlation analysis is a statistical method for studying the overall relevance between two sets of variables, which can more comprehensively reflect the internal relationship between variable sets. Thus, we performed canonical correlation analysis to understand the overall relationships among the activities of mitochondrial respiratory chain complexes (X group), productions of precursors and ATP (Y group) and emissions of floral volatiles (Z group) (Figures 6A–6C, Tables S1–S3). The diagram in Figure 6A shows that the canonical coefficient of this pair of variables (X group, Y group) is 0.999 and statistically significant at the 1% level, indicating that there is a very close positive correlation between the five indicators in the X group and the six indicators in the Y group. By analysing the internal structure of the X or Y group, it is found that a canonical load of complex II and V is relatively larger in the X group and a canonical load of ATP in the Y group too. Similar to Figure 6A, Figure 6B shows there are also positive correlations between X and Z groups. The diagram in Figure 6C shows that there are two canonical coefficients between the Y and Z group, and the two canonical coefficients are both statistically significant at the 1% level, indicating that there is a very close positive correlation between the six indicators in the Y group and the three indicators in the Z group. In the internal structure of the Y and Z group, the canonical load of each indicator affiliated to the first correlation coefficient is larger than corresponding to that affiliated to the second correlation coefficient. In conclusion, canonical correlation analysis of the above variable sets indicates that there are overall positive relations among mitochondrial respiratory chain complex activities, precursor and ATP productions and floral volatile emissions.

Figure 6

In the present study, intracellular Zn²⁺ concentrations treated with 2 mM, 4 mM and 8 mM exogenous Zn²⁺ are all increased significantly (Figures 2A–2D, Figures 2a–2d, Table 1). This indicates that the exogenous Zn²⁺ treatments have obvious effects on intracellular Zn²⁺ regulation in D. huoshanense. Under normal physiological conditions, the total Zn²⁺ level in plant cells is strictly regulated (Colvin et al., 2010). When cells are stimulated by a low concentration of Zn²⁺, intracellular Zn²⁺ concentrations can remain relatively stable because the intracellular Zn²⁺ concentration can be regulated by a cell wall barrier, absorption, and the mechanism of Zn²⁺ transfer into and out of cells. But when cells are stimulated by a high concentration of Zn²⁺, the Zn²⁺ concentration inside the cells may increase due to exceeding the self-regulating ability of intracellular Zn²⁺ (Eide, 2006; Colvin et al., 2010). Given exogenous Zn²⁺ treatments in this study exceeded the self-regulation ability of the cells, the intracellular Zn²⁺ concentrations were increased in petal cells of D. huoshanense. The elevated concentration of Zn²⁺ may be an intracellular signal to induce or directly participate in the regulation of physiological and biochemical activity in cells by complex-mediated pathways, affect enzyme activities and change material and energy metabolism. For example, Amiri et al. (2015) reported that a high concentration of exogenous Zn²⁺ treatment significantly increased the activity of superoxide dismutase in almond seedlings under salt stress.

Further, this study showed that elevated intracellular Zn²⁺ concentrations had diverse effects on the activities of mitochondrial respiratory chain complex I, II, III, IV and V (Figures 3A–3E). This may be attributed to the fact that Zn²⁺ can regulate protease activity in various ways. Zn²⁺ has a strong ability to accept electrons and can combine with some nitrogen, sulphur and other atoms in proteins through electrostatic attraction to form a tetrahedral structure that plays an important role in protease function display (Takahashi et al., 2003). Zn²⁺ can also act as an activator and regulator of some enzymes (Rezaeieh et al., 2016). Many studies have shown that there are one or more zinc-binding sites in respiratory chain complex I, III, IV and V (Sharpley and Hirst et al., 2006; Vygodina et al., 2008; Rose et al., 2011; Yi et al., 2017); due to differences in the zinc-binding rate and plasticity at discrete sites, their activities are variable under different Zn²⁺ concentration treatments. For complex II, although there are limited reports about the influence of Zn²⁺ as a structural function on its activity, Zn²⁺ can exert an effect through the activation and regulation function (Pan et al., 2013). All in all, intracellular Zn²⁺, in theory, can act on respiratory chain complexes (I, II, III, IV and V) through structural, activating or regulatory functions. Different Zn²⁺ concentrations have various effects on the activity of respiratory chain complexes because of the difference in the action mechanism of Zn²⁺ on respiratory chain complexes.

Mitochondria are semi-autonomous organelle. The signalling of mitochondrial to nucleus is termed as retrograde signalling, which can participate in the regulation of cellular material and energy metabolism (Carlsson et al., 2008). Studies have shown that mitochondrial alternative oxidase pathways can affect plant volatile emissions (Chen et al., 2020). But, so far, there are limited reports on the relationship between the activities of mitochondrial respiratory chain complexes and floral volatile emissions.

In theory, respiratory chain complex II is directly involved in the TCA cycle, while complex I, III, IV and V indirectly affect the TCA cycle and directly affect oxidative phosphorylation by electron transfer (Pan et al., 2013; Heidarvand et al., 2017). Changes in the mitochondrial respiratory chain and the TCA cycle can induce nuclear genes to regulate metabolic intermediate and ATP productions (Carlsson et al., 2008). Our research results directly confirmed that changes in the activity of respiratory chain complexes affected precursor and ATP productions in D. huoshanense. For example, in the 2 mM or 4 mM ZnSO₄ treatments, the activities of respiratory chain complex II and V significantly enhanced (Figures 3B and 3E), and the precursor and ATP productions increased (Figures 4A–4F). Canonical correlation analysis further confirmed that changes in respiratory chain complex activities were significantly correlated with the production of precursors and ATP. Moreover, within the respiratory chain complexes, complex II and V have relatively larger canonical loadings, indicating that they are the main factors causing changes in the production of precursors and ATP (Figure 6A). Other studies also have shown that complex II and V are closely linked to the TCA cycle, oxidative phosphorylation and production of precursors and ATP (Pan et al., 2013; Lee and Chen, 2014; Liang et al., 2019).

Floral volatile emissions are regulated by a precursor and ATP supply. Firstly, flower volatiles are mainly synthesised by the mevalonic acid (MEP)/the methylerythritol-phosphate (MVA) pathway, shikimic acid (SKA) pathway and LOX pathway (Gentner et al., 2014). In the MEP/MVA pathway, terpenoids are regulated by the supply of IPP and DMAPP; in the SKA pathway, benzoids are regulated by the supply of Phe; while in the SKA pathway, fatty acid derivatives are regulated by the supply of LA and LNA (van Schie et al., 2006; Gentner et al., 2014). Secondly, the emissions of floral volatiles require energy. It was reported that floral volatiles are synthesised in plant sub-organelles; their emission must at least cross the barrier of the cytosol, plasma membrane, hydrophilic cell wall and sometimes the cuticle (Adebesin et al., 2017). This was also confirmed by our study. After being treated with different concentrations of Zn²⁺, floral volatile emissions correspondingly increased or decreased with up or down of the precursor and ATP levels, and there is a significant positive correlation between the former and the latter (Figures 4A–4F, Figures 5A–5D, Figure 6C). Therefore, when the level of IPP and DMAPP was higher, the category and quantity of terpenoids are more. Similarly, the relationship between Phe and benzoids or between LA and LNA and fatty acid derivatives is similar. As for ATP, it provides energy for their emissions.

In summary, our research results confirmed that changes in the activity of respiratory chain complexes, especially complex II and V, affected volatile emissions by regulating precursor and ATP productions in D. huoshanense. However, it remains still unclear how the changes in mitochondrial complex activities trigger the mitochondrial-to-nucleus gene retrograde signalling to regulate precursor and ATP productions, as well as floral violate emissions. Further study needs to be focussed on this point.