Abstract
Calcium (Ca) is necessary for plant growth and stress resistance, which are essential for the successful cultivation of Panax quinquefolium L. (American ginseng). However, information about the physiology of Ca nutrition in this species is limited. Therefore, the objective of this study was to determine the effect of Ca on the growth and physiological performance of American ginseng. Two-year-old American ginseng plants were supplemented with the following Ca concentrations [Ca2+] in a hydroponic system: 0, 160.17, 320.34, 640.68, and 961.02 mg⋅L−1. Measurements included growth biomass accumulation, chlorophyll (Chl) content and fluorescence, photosynthetic parameters, antioxidant enzyme activity, root activity, and malondialdehyde content. Biomass, stem height, leaf area, maximum photochemical efficiency, and superoxide dismutase activity peaked at [Ca2+] of 640.68 mg⋅L−1. Actual photochemical efficiency, minimum saturating irradiance, photosynthetic rate, catalase and peroxidase activities, and root activity reached their maximum at [Ca2+] of 320.34 mg⋅L−1. Stem diameter and regulated thermal energy dissipation increased with [Ca2+]. The sum of nonregulated heat dissipation and fluorescence emission and malondialdehyde content decreased to a minimum at [Ca2+] of 320.34 mg⋅L−1. The Chl content reached a maximum at [Ca2+] of 160.17 mg⋅L−1, but the Chl a/b ratio increased with [Ca2+]; the actual photochemical efficiency and photosynthetic rate reached their maximum level at Chl a/b ratios of 2.04 and [Ca2+] of 320.34 mg⋅L−1. Therefore, the optimal [Ca2+] for American ginseng growth was 320.34 mg⋅L−1. Furthermore, an appropriate increase [Ca2+] in the growth medium may improve biomass accumulation, light energy utilization efficiency, and stress resistance in American ginseng.
Panax quinquefolium L. (American ginseng) is an herb native to North America that is now cultivated in East Asia as well. Its active compounds, ginsenosides, have been documented to exert a wide range of biological activities resulting in hypoglycemic, anti-inflammatory, cardioprotective, and antitumor effects, and the plant has been postulated to have therapeutic applications for chronic obstructive pulmonary and menopause symptoms, among others (Szczuka et al., 2019). Currently, the American ginseng supply relies mainly on intensive field cultivation under artificial shade structures (Nadeau et al., 2003). However, this system commonly causes various diseases because of high planting density. Therefore, enhancing the health of American ginseng has become the focus of research.
Ca is the third most abundant element in plants, is necessary for plant growth, and has a role in resistance to abiotic and biotic stresses (Pathak et al., 2020). Ca in American ginseng cultivation has received attention since the 1980s. The absence of Ca was more restrictive to root growth and resulted in earlier foliar deficiency symptoms than withholding nitrogen, phosphorus, or magnesium from the nutrient solution in sand culture (Stoltz, 1982). Ca deficiency also led to a lower root/shoot ratio and relative root growth rate than iron or boron deficiency in the nutrient solution of hydroponic plants (Clippard et al., 2020). An analysis of soil samples (0–20 cm depth, within 15 cm adjacent to the root) collected from 54 wild and wild-simulated ginseng sites showed that American ginseng grows in a wide range of soil pH (4.2–7.1), but especially in Ca-rich soils with an average soil Ca content of 3500 kg⋅ha−1 [using the Mehlich 3 (ICP) method] (Burkhart, 2013). During research trials simulating wild habitat conditions, the survival and growth of American ginseng were improved with liming (Slak, 2005). Therefore, for example, dolomitic lime increased the germination, leaf area, and height and root weights of American ginseng in field cultivation (Konsler and Shelton, 1990; Thyroff, 2015). However, the soil pH and Ca concentration ([Ca2+]) depend on the type of Ca applied, which has different influences on American ginseng (Peever, 2004). For instance, excess gypsum (>2 Mt⋅ha−1) application decreased shoot and root growth and reduced the contents of total ginsenosides (Lee and Mudge, 2013). Therefore, maintaining a certain soil Ca content is one of the key elements for the successful cultivation of American ginseng.
However, information about the suitable [Ca2+] range and Ca nutrition physiology of American ginseng remains limited. Therefore, the objective of this study was to determine the adequate [Ca2+] for optimum American ginseng growth and physiological indices, namely, biomass, size, Chl content, Chl fluorescence, photosynthetic parameters, antioxidant enzyme activity, root activity, and malondialdehyde (MDA) content. Moreover, using the observed physiological function of Ca in American ginseng, we discussed why Ca promotes growth and enhances stress resistance of American ginseng.
Materials and Methods
Plant materials and treatments.
One-year-old American ginseng roots were selected as test materials in Nov. 2020; the average root weight was 1.62 ± 0.09 g. After the roots were carefully washed, the buds were immersed in a gibberellic acid solution (250 mg⋅L−1) for 2 h. Then, the roots were cultured in an artificial climate chamber at 26.5 °C with 58.9% relative humidity, 44.38 μmol⋅m−2⋅s−1 photosynthetically active radiation, and a 16-h/8-h light/dark regime.
The first year’s dormant buds began to germinate after 2 d (similar to the germination after natural dormancy, they were considered 2-year-old American ginseng). On day 7, when the bending stem began to straighten and the curly leaves began to unfold, plantlets were transplanted to hydroponic tanks containing Hoagland’s nutrient solution with the following five different [Ca2+] obtained by the addition of Ca(NO3)2: 0, 160.17, 320.34, 640.68, and 961.02 mg⋅L−1. Each [Ca2+] treatment was applied to 45 plantlets divided into three replicates of 15 plantlets each; all culture media were changed every 3 d and circulated by a pump. On day 20, when leaves appeared in different colors among the treatments, samples were collected and the growth parameters and physiological indices were measured.
Measurement of growth parameters.
Stem height was measured from the top of the rhizome to the base of the petiole. Stem diameter was measured at 1.5 cm below the base of the petiole. In addition, root, stem, and leaf weights were measured. The middle leaflet of one prong from each plant was selected as the sample, and the leaf area was calculated using ImageJ 1.52v (National Institutes of Health, Bethesda, MA) after scanning the leaves at 300 dpi. For all measured parameters, the average value for 15 plants was regarded as a replicate.
Measurement of Chl fluorescence.
The maximum photochemical efficiency of photosystem II (PS II; Fv/Fm), actual photochemical efficiency of PS II [Y(II)], regulated thermal energy dissipation [Y(NPQ)], and the sum of nonregulated heat dissipation and fluorescence emission [Y(NO)] (Ralph and Gademann, 2005) were measured with IMAG-MIN/B (Heinz Walz GmbH, Effeltrich, Germany). The actinic light source was a blue (450 nm) light-emitting diode with a saturation pulse light of 22 μmol⋅m−2⋅s−1 at 20-s intervals. The measure light was 0.05 μmol⋅m−2⋅s−1, with the basic fluorescence intensity kept between 0.03 and 0.1. The relative electron transfer rate for PS II was measured with a set of light pulses (0, 40, 80, 225, 325, 405, 495, and 625 μmol⋅m−2⋅s−1) at 20-s intervals. The rapid light curve and initial slope were fitted and calculated using Origin Pro 9.7 (OriginLab, Northampton, MA). Minimum saturating irradiance (Ek) was calculated according to a previously described method (Klughammer and Schreiber, 2008). Measurements were performed in a dark artificial climate chamber with a 20-min dark adaptation time.
Measurement of photosynthetic parameters.
The transpiration rate E, vapor pressure deficit (VPD), stomatal conductance (GH2O), photosynthetic rate (Pn), and intercellular CO2 concentration (Ci) were measured with a GFS-3000 portable photosynthesis measurement system (Heinz Walz GmbH). The gas flow rate was 750 μmol⋅m−2⋅s−1, the fan speed of the gas mixer comprised seven stages, and the leaf chamber area was 3 cm2. The measurements were performed in an artificial climate chamber, and the light was 44.38 μmol⋅m−2⋅s−1.
Determination of enzyme activity, root activity, Chl, and MDA content.
The activity of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) and the contents of dehydrogenase (DHA), Chl, and MDA were determined using the corresponding assay kits (Cominbio, Suzhou, China) according to the instructions provided by the manufacturer. For each [Ca2+] treatment, leaves or lateral roots (used for extracting DHA) of 15 plants were mixed as a replicate (0.1 g per sample). Leaf samples were stored at −80 °C until use. An equal weight of CaCO3 powder was added to each leaf sample for Chl extraction before grinding under liquid nitrogen, and the ground samples were extracted by adding 1 mL precooled sodium phosphate buffer (pH 7.0). The homogenates were extracted in an ice bath, followed by centrifugation at 8000 gn for 10 min at 4 °C; the supernatants were used for spectrophotometric determination. All spectrophotometric analyses were conducted using a SpectraMax iD3 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA).
Statistical analysis.
Each [Ca2+] treatment was analyzed in three replicates, and the data shown are the mean ± sd of the three replicates. An analysis of variance was used to estimate significant effects, followed by Fisher’s least significant difference test (P = 0.05) to separate the means. All analyses were conducted using SAS 8.0 (SAS Institute Inc., Cary, NC).
Results
Growth parameters of American ginseng.
Root weight of American ginseng decreased during sprouting, but root, stem, and leaf weights all increased after the leaves were fully unfolded. Ca had a significant effect on the total biomass of American ginseng, which increased and peaked at [Ca2+] of 640.68 mg⋅L−1; then, it decreased with further increases in [Ca2+] (Fig. 1A). Compared with the Ca deficiency treatment (0 mg⋅L−1 control), the maximum root weight and maximum sum of stem and leaf weights increased significantly by 45.56% and 58.82%, respectively (Table 1).
Effects of calcium concentrations ([Ca2+]) on the growth state, leaf area, and color of American ginseng.
Citation: HortScience 57, 1; 10.21273/HORTSCI16129-21
Effects of calcium concentrations ([Ca2+]) on the growth parameters of American ginseng.
Additionally, Ca had a significant effect on stem height, leaf area, and stem diameter. Stem height and leaf area first increased and then decreased with [Ca2+], reaching their maxima at [Ca2+] of 640.68 mg⋅L−1 and then decreasing with further [Ca2+] increases, whereas stem diameter increased continuously over the tested [Ca2+] range. Compared with Ca deficiency, the maximum stem height and leaf area increased significantly by 30.19% and 62.55%, respectively (Table 1).
Chl content and Chl a/b ratio of American ginseng.
Leaves deficient in Ca were light green in color, but at [Ca2+] >640.68 mg⋅L−1, yellow patches with an irregular shape appeared among the leaf veins and increased with increasing [Ca2+] (Fig. 1B). Moreover, [Ca2+] had a significant effect on the Chl content and Chl a/b ratio. Chl (a + b) and Chl a content increased and then decreased with increasing [Ca2+]; in contrast, Chl b decreased with increasing [Ca2+]. The Chl (a + b) content peaked at [Ca2+] of 160.17 mg⋅L−1, whereas Chl a peaked at [Ca2+] of 320.34 mg⋅L−1. The Chl a/b ratio increased from 1.81 to 2.22 (Table 2).
Effects of calcium concentrations ([Ca2+]) on the chlorophyll (Chl) content and Chl a/b ratio of American ginseng.
Chl fluorescence of American ginseng.
Ca had a significant concentration-dependent effect on the Chl fluorescence parameters of American ginseng (Fig. 2). Specifically, Fv/Fm and Y(II) first increased and then decreased with [Ca2+]; Fv/Fm reached a maximum value at [Ca2+] of 640.68 mg⋅L−1 (Fig. 2 A); however, Y(II) decreased before the Fv/Fm reached its peak and reached its maximum value at [Ca2+] of 320.34 mg⋅L−1 (Fig. 2B).
Effects of calcium concentrations ([Ca2+]) on the chlorophyll fluorescence parameters of American ginseng. Fv/Fm, maximum photochemical efficiency of photosystem II (PS II); Y(II), actual photochemical efficiency of PS II; Y(NPQ), regulated thermal energy dissipation; Y(NO), the sum of nonregulated heat dissipation and fluorescence emission; Ek, minimum saturating irradiance. Different lowercase letters indicate significant difference at P ≤ 0.05 according to Fisher’s least significant difference test.
Citation: HortScience 57, 1; 10.21273/HORTSCI16129-21
The values for Y(NPQ) increased with [Ca2+] (Fig. 2C); however, the Y(NO) value first decreased to a minimum at [Ca2+] of 320.34 and 640.68 mg⋅L−1; then, it increased at [Ca2+] of 961.02 mg⋅L−1 (Fig. 2D). With the Y(NPQ) increase and Y(NO) decrease, the Y(II) reached a peak at [Ca2+] of 320.34 mg⋅L−1 (Fig. 2 B). The Ek increased with [Ca2+] and reached the saturation state (73.60 μmol⋅m−2⋅s−1) at [Ca2+] of 320.34 mg⋅L−1 (Fig. 2E).
Photosynthetic parameters of American ginseng.
The Ca treatment had significant effects on E, GH2O, and Pn, all of which showed an increasing pattern, followed by a decrease with [Ca2+] (Table 3). Pn was similar at [Ca2+] of 160.17 and 320.34 mg⋅L−1, but it was significantly higher than Pn with other [Ca2+] treatments. The Ca concentrations of <160.17 mg⋅L−1 or >640.68 mg⋅L−1 significantly reduced E, GH2O, and Pn. However, the Ci increased with increasing [Ca2+] and could be divided into three different stages. At [Ca2+] <160.17 mg⋅L−1, Ca deficiency decreased the GH2O, thus limiting CO2 availability and ultimately reducing Pn. At [Ca2+] ranging from 160.17 to 640.68 mg⋅L−1, the stomata maintained an open state, and there was enough CO2 available for photosynthesis. However, when [Ca2+] was >640.68 mg⋅L−1, the GH2O and Pn decreased while the respiration increased, thus leading to Ci accumulation.
Effects of calcium concentrations ([Ca2+]) on the photosynthetic parameters of American ginseng.
Antioxidant enzyme activity, root activity, and MDA content of American ginseng.
The Ca treatment had a significant effect on the antioxidant enzyme activity and MDA content of American ginseng (Fig. 3). As expected, SOD, POD, and CAT activities increased and then decreased with [Ca2+]. The activity of SOD, which is the most important antioxidant enzyme, intensified as [Ca2+] increased to 640.68 mg⋅L−1 (Fig. 3A). The POD and CAT activities were more sensitive to increasing [Ca2+] (Fig. 3B and C), reaching a maximum level at [Ca2+] of 320.34 mg⋅L−1 and then decreasing before the inflection point for SOD activity.
Effects of calcium concentrations ([Ca2+]) on the antioxidant enzyme activity, root activity, and malondialdehyde (MDA) content of American ginseng. SOD, superoxide dismutase; CAT, catalase; POD, peroxidase; DHA, dehydrogenase. Different lowercase letters indicate significant difference at P ≤ 0.05 according to Fisher’s least significant difference test.
Citation: HortScience 57, 1; 10.21273/HORTSCI16129-21
Root activity influences water and mineral absorption; therefore, we chose DHA activity as an index of root activity to estimate the growth condition and vigor of the root system. Our data showed that DHA activity followed a similar change pattern with [Ca2+] as POD and CAT activities, suggesting that 320.34 mg⋅L−1 was the suitable [Ca2+] for root growth of American ginseng (Fig. 3D).
MDA is most commonly used to indicate the extent of damage caused by stress in plants; MDA first decreased and then increased with [Ca2+] (Fig. 3E). Furthermore, the MDA content did not differ between [Ca2+] of 160.17 and 320.34 mg⋅L−1 treatments and was significantly lower at these than at other [Ca2+] treatments tested herein, thus implying that both Ca deficiency and its excess cause severe damage to plant tissues.
Discussion
During the experiments summarized here, the Chl (a + b) and Chl a content increased and then decreased with [Ca2+], whereas that of Chl b decreased. Similar results were observed for passion fruit, except for the Chl a/b ratio (Bizarre et al., 2019), which has been associated with plant species and growth environment (Liang et al., 2009). For American ginseng, when [Ca2+] was 320.34 mg⋅L−1, the Chl a/b ratio was 2.04, at which point Pn, Y(II), and Ek reached maximum values.
The excitation energy absorbed in PS II is distributed among Y(II), Y(NPQ), and Y(NO). Under a given environmental condition, successful regulation of such partitioning is aimed at maximal values of Y(II), with a maximal ratio of Y(NPQ)/Y(NO). High values of Y(NPQ) are indicative of a high photoprotective capacity, whereas high values of Y(NO), mainly caused by closed PS II reaction centers, reflect the inability of a plant to protect itself against damage by excess illumination (Bizarre et al., 2019; Klughammer and Schreiber, 2008). Y(NPQ) is linked to the xanthophyll cycle, which involves nonphotochemical quenching of excess light energy in PS II (Jahns and Holzwarth, 2012). Exogenous Ca2+ can facilitate the xanthophyll cycle through the Ca2+/CaM-mediated expression of the violaxanthin de-epoxidase gene and increase in the antheraxanthin and zeaxanthin ratio (Yang et al., 2013). D1 protein constitutes the binding site of the oxygen-evolving complex and Mn4Ca cluster; it is highly susceptible to excitation energy and reactive oxygen species. As the primary site of photoinhibitory damage, the D1 protein has a high turnover rate when repairing the light-induced inactivation and functional closure of reaction centers (Townsend et al., 2018). The turnover of D1 protein consists of four processes: de novo synthesis, phosphorylation, dephosphorylation, and degradation (Li et al., 2017). The transcription and translation of the psbA gene (encoding D1 protein) are specifically inactivated by hydrogen peroxide and singlet oxygen (Kojima et al., 2007). However, exogenous Ca upregulates the antioxidant gene transcript levels and the expression of the psbA gene, resulting in an increase of antioxidant enzyme activity and upregulation of the D1 protein (Erinle et al., 2016). At [Ca2+] between 0 and 320.34 mg⋅L−1, the value of Y(NPQ) and SOD, CAT, and POD activities of American ginseng increased, whereas the value of Y(NO) decreased, and Y(NPQ)/Y(NO) reached a maximum value at [Ca2+] of 320.34 mg⋅L−1, at which point the Y(II) peaked.
The value for Ek, which reflects the adaptation of plants to light intensity, is determined by finding the maximum photosynthetic rate in the light-limited regions of rapid light curves (Ralph and Gademann, 2005; Sakshaug et al., 1997). Ek is related to quenching such that photochemical fluorescence quenching is dominant below and nonphotochemical quenching is dominant above the Ek value (Henley, 1993). The Ek value of American ginseng increased with [Ca2+] and reached the saturation state at [Ca2+] of 320 mg⋅L−1, indicating that the application of an appropriate amount of Ca improves American ginseng tolerance to high light intensity stress.
Furthermore, plant stomata are the main channel for gas exchange during photosynthesis and are coregulated by a signal network that includes the Ca2+ signal transduction pathway, the plant hormone pathway, and the reactive oxygen species signaling pathway (Wang et al., 2019). Reactive oxygen species comprise a key signal for stomatal regulation (Song et al., 2014). SOD catalyzes the dismutation of O2− to H2O2 and O2, whereas CAT mediates H2O2 cleavage to produce H2O and O2, and POD reduces H2O2 to H2O and prevents H2O2-mediated inhibition of amylase activity (Sparta et al., 2006). Additionally, amylase can regulate the water content of guard cells through starch degradation into soluble sugars, thus regulating stomatal movement (Leshem and Levine, 2013). At [Ca2+] between 160.17 and 320.34 mg⋅L−1, SOD, CAT, and POD activities increased, thereby reducing H2O2 content and relieving the inhibitory effect of H2O2 on amylase activity. Finally, Ca upregulated the water content of guard cells and kept the stoma open, thus enhancing gas exchange in American ginseng plants.
The Ca ion is a cofactor in the formation of activation sites on PS II (Popelkova et al., 2011), and it maintains the stability of the PS II reaction center components through the Ca/calmodulinsignal transduction pathway (Yang et al., 2015) and regulates the activities of sedoheptulose-1,7-bisphosphatase, fructose-l,6-bisphosphatase, and transketolase. However, high concentrations of exogenous Ca2+ inhibit carbon assimilation (Hochmal et al., 2015). Therefore, Ca can influence the stability of PS II and carbon assimilation-related enzyme activities, thus influencing the photosynthetic rate. The photosynthetic rate of American ginseng reached the maximum at [Ca2+] ranging from 160.17 to 320.34 mg⋅L−1. Therefore, this range is suitable for American ginseng to maximize CO2 assimilation.
Exogenous Ca2+ treatment not only affects membrane structure but is also involved in oxidative signal transduction (Agarwal et al., 2005), concomitant with the upregulation of antioxidant enzyme activity aimed at reducing stress-induced MDA accumulation (Gong et al., 1997). Our results show that the antioxidant enzyme activity of American ginseng increased and then decreased with [Ca2+], with the MDA content decreasing to its lowest levels at [Ca2+] of 160.17 mg⋅L−1 and 320.34 mg⋅L−1, which means that an appropriate amount of Ca applied effectively enhances membrane integrity. Moreover, root activity reached the maximum rate at [Ca2+] of 320.34 mg⋅L−1, further confirming that Ca improved the tolerance of American ginseng to waterlogging and hypoxia stress.
Nonetheless, excess Ca2+ released into the cytosol and sustained high cytosolic Ca2+ concentrations might be cytotoxic (White and Martin, 2003). Therefore, when [Ca2+] exceeded 320.34 mg⋅L−1, the Chl content, Y(II), Pn, and activities of CAT, POD, and root activity decreased significantly in American ginseng. Intriguingly, at [Ca2+] of 320.34 mg⋅L−1, the physiological parameters were most favorable for the growth of American ginseng, although the biomass did not reach its maximum; instead, biomass accumulation peaked at [Ca2+] of 640.68 mg⋅L−1, despite the leaves showing obvious stress symptoms, specifically, yellow patches.
Conclusion
The results of this study clearly showed that increasing [Ca2+] from 0 to 320.34 mg⋅L−1 effectively promoted a robust, healthy growth of American ginseng plants and increased plant resistance to high light intensity stress and hypoxia stress. However, [Ca2+] exceeding 320.34 mg⋅L−1 was toxic to American ginseng, and the toxicity increased with increasing [Ca2+]. Therefore, suitable [Ca2+] for growth promotion in American ginseng was 320.34 mg⋅L−1. Finally, although these results were obtained from American ginseng plantlets that were only 2 years old and grown in a hydroponic system, which might differ greatly from common [Ca2+] conditions in soil environments, our findings clearly indicated that growth and stress resistance of American ginseng might be effectively promoted by regulating the Ca supply.
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