Panacis quinquefolii radix is an important traditional Chinese medicine that comes from the dried
root of Panax quinquefolium L. (American ginseng);
a plant that belongs to the Araliaceae family (Rao et al. 2004; Proctor et al.
2010; Chinese Pharmacopoeia Commission 2015). Ginsenosides, which are secondary
metabolites of American ginseng, possess diverse pharmacological activities,
which include stimulating cell metabolism, enhancing immune system function,
protecting the cardiovascular system, and antioxidant and anticancer properties
(Attele et al. 1999; Liao et al. 2002; Xie et al. 2007; Yuan et al.
2010; Qi et al. 2011; Kim 2012; Wang et al. 2013; Kochan et al. 2014; Shkryl et al. 2018). Ginsenosides are classified into the following three groups
according to their structures: protopanaxadiols (Rb group: Ra1, Ra2,
Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rh2,
and others); protopanaxatriols (Rg group: Rg1, Rg2, Re,
Rf, Rh1, and others); and oleanolic acid derivatives (Ro group:
oleanolic acid) (Wang et al. 2013;
Kochan et al. 2014).
American ginseng is at risk of
extinction because of threats to its natural habitat and overexploitation (Shkryl et al. 2018). Hence, wild resources are rare and cannot
satisfy growing medicinal demand. Cultivation is an alternative way to
supply this plant with high medicinal substances (Wang and Wu 2003; Proctor et al. 2010). In China, American ginseng
is cultivated at 25 cm high and 1.5 m wide seedbeds (Hou 2017), whereas the
length and direction are determined by the situation of the cultivated land. Therefore, the long axis of the seedbeds can be oriented east-west
(E-W) or north-south (N-S). Because the seedbeds are not strictly flat,
the short-axis section is slightly mounded. Thus, the leaves of American ginseng growing on different sides of the
seedbeds have different orientations. Studies on other plants have
shown that the ridge direction alters the light intensity that reaches the
leaves, and thus affects the photosynthesis and yield of plants (Ma et al. 2015).
American ginseng is a shade perennial, and its growth and development are sensitive to light intensity
(Proctor et al. 2010; Cheng and Shen
2011; Proctor and Palmer 2017). Lower light intensity cannot maintain the
normal level of photosynthesis and reduces the yield, whereas higher light
intensity bleaches the leaves, inhibits photosynthesis, and causes severe death
of leaves and plants (Proctor and Palmer 2017). The optimum light intensity for
American ginseng is determined by age. Generally, the appropriate range of
light transmission rate (LTR) of 1 to two-year-old
plants is 18–20%, whereas for 3 to four-year-old plants, it is 25–30% (Li et al. 2004; Wang et al. 2010). Therefore, it is essential to
provide optimum light intensity for American ginseng during cultivation.
Methods for studying plant photosynthesis include determination of leaf
gas exchange parameters and fast chlorophyll a fluorescence
induction kinetics. Fast chlorophyll a fluorescence analysis
is a transient record of a plant or leaf or photosynthesizing organism exposed
to actinic radiation 1–2 s. This transient record has a typical polyphasic O-J-I-P
(OJIP curve) shape. O refers to the initial fluorescence level at 20 μs; J
and I refer to the fluorescence level at 2 and 30 ms, respectively; and P
refers to the maximum fluorescence level (Gao et al. 2018). Thus, fast chlorophyll a fluorescence analysis
is also called the JIP-test (Campos et
al. 2014; Yang et al. 2018). The
JIP-test provides information about the structure, activities, and
electron transport of PSⅡ. The method has the
advantages of measuring more than 50 parameters, being fast (1–2 s), and not
damaging the plants. Although the JIP-test is widely used to study plant
photosynthesis (Li et al. 2006; Cuchiara et al. 2013; Jedmowski et al. 2015), there are few
reports on the photosynthesis of American ginseng.
We hypothesize that the growing position
and LTR
could affect the activities of PSII in American ginseng.
Therefore, we examined the photosynthesis, chlorophyll concentration, OJIP
curve, parameters related to the JIP-test, and the ginsenoside contents of two-year-old
American ginseng grown on different sides of seedbeds with N-S or E-W
orientations and different LTRs.
Plant
material and growth conditions
The plants were grown in an experimental field of Wendeng
Agricultural Bureau, Wendeng, Shandong Province, China (37°12′N,
122°03′E). Wendeng is located in the North Temperate Zone and has a continental monsoon Table 1: Summary of conditions during the course of experiment
|
Group name |
Conditions |
|
ES-11%, ES-15%, ES-20% |
South side of the E-W seedbed, LTRs of 11%, 15%, and 20%, respectively. |
|
EN-11%, EN-15%, EN-20% |
North side of the E-W seedbed, LTRs of 11%, 15%, and 20%, respectively. |
|
NE-11%, NE-15%, NE-20% |
East side of the N-S seedbed, LTRs of 11%, 15%, and 20%, respectively. |
|
NW-11%, NW-15%, NW-20% |
West side of the N-S seedbed, LTRs of 11%, 15%, and 20%, respectively. |
|
N-11% |
Both sides of the N-S seedbed, LTR of 11% (i.e., NE-11% and NW-11%) |
|
N-15% |
Both sides of N-S the seedbed, LTR of 15% (i.e., NE-15% and NW-15%) |
|
N-20% |
Both sides of N-S the seedbed, LTR of 20% (i.e., NE-20% and NW-20%) |
|
E-11% |
Both sides of E-W the seedbed, LTR of 11% (i.e., ES-11% and EN-11%) |
|
E-15% |
Both sides of E-W the seedbed, LTR of 15% (i.e., ES-15% and EN-15%) |
|
E-20% |
Both sides of E-W the seedbed, LTR of 20% (i.e., ES-20% and EN-20%) |
climate. The average annual temperature is 11.1şC. The annual
precipitation is 816.7 mm, which mainly occurs in summer, accounting for 82.2%
of the total rainfall amount, whereas there is less precipitation in spring and
autumn, and drought often occurs in these two seasons. The annual sunshine is
2522 h and the frost-free period is 194 days.
The study was carried out from
March 16 to October 16, 2018. Six shading sheds (area of 50 m2 each
and height of 1.8 m) were constructed, and the LTRs of the sheds were 11, 15, and 20%. There
were two sheds for each LTR, one containing N-S seedbed and the other containing an E-W seedbed. The LTR in each experiment was controlled by the number of layers of black polyethylene net. The conditions in which the plants were cultivated were named according to the
orientation and side of the seedbed and the LTR (Table 1). For example, the
south side of the E-W seedbed with LTR of 15% is called ES-15%.
Two-year-old American ginseng plants were obtained from Wendeng Agricultural Bureau, and were transplanted into the seedbeds
(length × width × height, 10 × 1.5 × 0.25 m) at a density of 1 plant per 20 × 8
cm on March 16, 2018 (Hou 2017). The seedbed soil had a pH of 4.69, hydrolyzed
nitrogen of 52.9 mg·kg-1, available phosphorus of 140.9 mg·kg-1, available potassium of 65 mg·kg-1,
and organic matter of 1.03%. The field management measures of each treatment were
consistent.
Measurements of gas
exchange parameters
The net photosynthesis rates (Pn), stomatal conductance
(gs), and internal CO2 (Ci) of
the fully expanded two-year-old American ginseng leaves were determined by a
portable photosynthesis system
(CIRAS-3, PP Systems, Amesbury, MA, USA). Measurements were taken during the period
of fastest root growth (Liu et al.
1987), at 09:00–11:00 am on August 10, 2018 on a sunny day under light saturation (700
μmol·m-2·s-1), illumination, ambient carbon dioxide of 400 ppm, average, relative
humidity of 40%, and temperature of 30şC.
Determination of chlorophyll
content
The
chlorophyll contents of two-year-old American ginseng leaves were
measured with a chlorophyll meter (SPAD-502, Minolta, Tokyo, Japan) at the
center of the full expanded leaves, except for major veins.
JIP-test
Fast
chlorophyll a fluorescence transient tests of the fully expanded leaves
of two-year-old American ginseng were performed from 09:00 to 11:00 am on a
sunny day using a Plant Efficiency Analyzer (Handy PEA, Hansatech Instruments
Ltd., UK). After 30 min dark adaptation using dark
adaptation clips on the leaves, as described previously (Li et al. 2006), the middle leaflet was illuminated with
continuous red light (peak at 650 nm) for 1 s at 3000 μmol·m−2·s−1 to
generate a true maximum fluorescence intensity (Ma et al. 2017).
The OJIP curve was obtained from the fast chlorophyll a
fluorescence transient test. The meanings and analysis methods of each
parameter and the phases of the OJIP curve are described
in the literature (Strasser et al. 2000; Force et al. 2003; Strasser et al. 2004; Li et al. 2006; Stirbet and Govindjee 2011)
and methods of normalization
of the initial curve acquired from the JIP-test have
been described previously (Qiu et al.
2012). In this study, the two methods of normalization were as
follows. First is normalization with (FM - F0),
where the normalized
fluorescence signal data is relative variable fluorescence, Vt = (Ft - F0)/(FM - F0), and Ft is the fluorescence value at
time t, FM is the maximum fluorescence, and F0
is the initial fluorescence. In the OJIP curve plotted with Vt, the value of the O-phase is 0, whereas
that of the P-phase is 1. The second is normalization with (FJ - F0), where the normalized fluorescence
signal data is Wt = (Ft - F0)/(FJ - F0), and FJ is the
fluorescence at the J-step. In the OJIP curve plotted with Wt, the value of the O-phase is 0,
whereas that of the J-phase is 1.
Fluorescence parameters can be obtained by mathematical
analysis of the fluorescence kinetic curve (Strasser et al. 2000; Strasser et al.
2004). In this study, the radar plot was constructed from the fluorescence
parameters obtained for the NW-15% conditions with a
calculated value of 1, and the ratio of the fluorescence parameters obtained
for the other conditions to the control parameters.
High-performance
liquid chromatography analysis of ginsenosides
The roots of the 12 groups of
plants were collected on October 16, 2018. The roots were washed sequentially
with clean water and distilled water, and then the residual water was removed
with absorbent paper. The cleaned roots were cut into slices 1 cm thick, kept
initially at 38şC for 24 h, and then at 40şC until dried.
For each group of plants, 1 g dried powder from 20 dried American ginseng
plants was placed in a conical flask with a plug, extracted with 50 mL of
water-saturated n-butanol by refluxing for 1 h, cooled to room temperature,
and filtered. A 10 mL sample of the extracted solution was evaporated to
dryness. The residue was dissolved in 50% methanol, and adjusted to an exact
volume of 5 mL using a 5 mL volumetric flask. The extract was filtered through
a membrane filter with a pore size of 0.45 μm. The filtrate was used for high-performance liquid chromatography (HPLC) analysis
(Yu et al. 2019).
Standard ginsenoside components, such as ginsenosides Rg1, Re,
Rb1,
Rc, and Rd, were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai,
China). The HPLC separation was carried out on an HPLC system (U3000, Thermo
Fisher Scientific, Waltham, MA, USA) with a C18 column (4.6 × 250 mm, 5
μm), at a temperature of 40şC, a flow rate of 1 mL·min-1, and a
detection wavelength of 203 nm. The mobile phases were acetonitrile (Solvent A) and 0.05%
phosphoric acid (Solvent B) and the gradient was as follows: 19% A (0–35 min); 19–29%
A (35–70 min); 29–40% A (70–100 min). A one-point curve method was used for
ginsenoside quantification analysis (mg·g-1 DW) and was
performed by comparing retention times and peak areas of standards and samples
(Yu et al. 2019). The total content
of Rg1 and Re is the Rg group content, the total content of Rb1,
Rc, and Rd is the Rb group content, and the
ratio of the two values is Rg/Rb (Jang et al. 2015).
All measurements were repeated
six times. All statistical analyses were performed by using SAS 19.0 software
(SAS Institute, Cary, NC, USA). The statistical significance of the differences
was determined using Duncan’s multiple range test and one-way analysis of
variance (ANOVA), evaluating significant differences at P < 0.05. All
data were compared at the 5% significance level and were reported as mean ±
standard deviation.
Results
Effects of LTRs and
seedbed orientation on Pn, gs,
and Ci
%20343-351_files/image002.png)
Fig. 1: Pn (A), gs (B), and Ci
(C) of 2-year-old American ginseng grown on two
seedbeds with different LTRs
The vertical error bars represent the standard errors (n = 6). The same letters at the top
of each bar indicate that there is statistically no difference at P > 0.05 using Duncan’s multiple
range test
In seedbeds with the same orientation, Pn and gs
of American ginseng leaves grown on both sides of the seedbeds increased with
LTR significantly (P < 0.05) (Fig. 1). At
the same LTR, Pn and gs showed no
significant difference between the two sides of the same seedbed (P
> 0.05), except for the E-11% conditions (P < 0.05),
for which the values of Pn and gs were the
lowest. However, this difference was not observed for the N-11% conditions.
Otherwise, at the same LTRs, the parameters for the leaves grown on both sides
of the E-W seedbeds were lower than those for the N-S seedbeds (P
< 0.05). Ci was the
same for LTRs of 20% and 15% (P > 0.05) (Fig. 1). Ci
values for LTR of 11%, except for the NE-11% conditions, were the smallest
compared with those for LTRs of 20% and 15% (P > 0.05), and
the NE-11% conditions showed no differences from those with LTRs of 20% and 15%
(P > 0.05).
Effects of LTRs and seedbed
orientation on chlorophyll content
On
the south side of the E-W seedbeds, the chlorophyll content of the American ginseng leaves increased with increase in LTR; ES-11% had a
significantly lower content than ES-15% and ES-20% (P <
0.05), although there was no significant difference between ES-15% and ES-20% (Fig.
2). On the north side of the E-W seedbeds, the same trend was observed, and
significant differences were observed (P > 0.05) among the three LTRs (EN-11%, EN-15%,
and EN-20%). Otherwise, there were no significant differences between the sides
of the N-S seedbeds for the three LTRS (P > 0.05).
At LTR of 11%, the chlorophyll
content for both sides of the N-S
seedbeds were significantly higher than those for the E-W seedbeds (P
< 0.05), whereas the content was similar for both sides of seedbeds
with the same orientation (P > 0.05). For LTRs of 15%, NW-15% had the
highest content, followed by NE-15%, ES-15%, and EN-15%. EN-15% was the lowest, and
showed a significant (P < 0.05) difference from NW-15%
and NE-15%. ES-15% was higher than EN-15% (P > 0.05), and
lower than NW-15% (P < 0.05) and
NE-15% (P > 0.05). The
chlorophyll contents for groups with
LTR of 20% were greater than those of the other LTRs, and there were no
differences among the groups with LTR of 20% (P > 0.05).
%20343-351_files/image004.jpg)
Fig. 2: Chlorophyll contents of 2-yr-old American ginseng
grown on two seedbeds with different LTRs
The vertical error bars
represent the standard errors (n = 6). The same letters at the top of
each bar indicate that there is statistically no difference at P > 0.05 using Duncan’s multiple
range test
%20343-351_files/image006.png)
Fig. 3: Fast
chlorophyll fluorescence curves normalized
by Vt (A) and Wt (B) (n = 6)
Effects of LTRs and seedbed
orientation on chlorophyll a fluorescence transients
Chlorophyll a fluorescence is a sensitive
method for studying the performance of PSII. OJIP-curves normalized
by Vt and Wt of American ginseng grown in
seedbeds with two different orientations and different LTRs are shown in Fig.
3A–B. The OJIP chlorophyll a fluorescence-induced kinetics curves were
typical, indicating that the plants had a normal status. The fluorescence
intensity of the J-step of EN-11% was the biggest, followed
by ES-11%, and the values for the other groups were similar (Fig.
3A). The
fluorescence intensity of the I-step of EN-11% was the largest, followed by
NW-20% and NE-11%, and the values for NE-20% and ES-20% showed a dramatic
decrease (Fig. 3B). For the P-step, the fluorescence intensity of EN-11% was
the biggest, whereas the values for NE-20% and ES-20% were the lowest, the same
as for the I-step (Fig. 3B).
The heterogeneity of the oxidized plastoquinone (PQ)
pool is reflected by phases J-I and I-P. The PQ pool can
be divided into the rapidly reducible PQ pool (phase J-I) and slowly reducible
PQ pool (phase I-P). In the J-I phase, the fluorescence
intensity of EN-11% was the
highest, whereas that of NE-20% was the lowest (P < 0.05) (Fig. 3A).
The I-P phase was the same as the J-I phase, although the
fluorescence intensities of NE-20% and ES-20% were the lowest (P
<0.05) (Fig. 3B). Both
the rapidly and slowly reducible PQ pools of the
leaves in the NE- Table 2: Parameters of JIP-test in
dark-adapted leaves
|
LTR |
Fv/FM |
VJ |
VI |
Sm |
N |
RC/CSm |
PI total |
|
NW-20% |
0.7900±0.017b |
0.5254±0.008d |
0.8823±0.006f |
17.8733±0.009j |
28.5156±0.568m |
1671.81±134.227q |
0.5700±0.050s |
|
NE-20% |
0.7934±0.021b |
0.5038±0.003d |
0.8802±0.002f |
18.4434±2.114i |
30.2525±1.266l |
1694.6034±114.552q |
0.5873±0.040s |
|
ES-20% |
0.8052±0.002a |
0.4658±0.017e |
0.8632±0.046g |
23.0241±2.444h |
36.3403±0.954K |
3552.5556±150.553n |
0.8642±0.064s |
|
EN-20% |
0.7951±0.003b |
0.4719±0.009e |
0.8714±0.044g |
20.0975±0.521i |
33.6774±0.649l |
3451.6111±89.774p |
0.6852±0.017s |
|
NW-15% |
0.7873±0.003b |
0.462±0.013e |
0.8455±0.010g |
22.0076±3.663h |
35.5831±0.756k |
3352.2727±44.228p |
0.9100±0.065r |
|
NE-15% |
0.7767±0.012b |
0.4386±0.004e |
0.8325±0.004g |
23.6752±3.591h |
36.9804±1.031k |
3107.8571±164.014p |
0.9849±0.054r |
|
ES-15% |
0.7994±0.002a |
0.4285±0.017e |
0.866±0.014g |
19.5791±1.031i |
32.7989±1.276l |
3284.625±147.222p |
0.7825±0.048s |
|
EN-15% |
0.7956±0.024b |
0.4497±0.015e |
0.8625±0.041g |
18.8403±2.131i |
31.3094±1.243l |
3237.8182±123.996p |
0.7815±0.128s |
|
NW-11% |
0.7866±0.003b |
0.4930±0.007d |
0.8766±0.005f |
18.3296±1.423i |
29.5854±2.475l |
3439.0556±112.669p |
0.6437±0.110s |
|
NE-11% |
0.7991±0.005b |
0.4886±0.006d |
0.8750±0.046f |
19.6168±0.475i |
30.6032±0.753l |
3383.8889±130.653p |
0.7229±0.134s |
|
ES-11% |
0.743±0.004c |
0.5800±0.013d |
0.8501±0.006g |
26.5799±1.354h |
46.5620±9.765k |
3043.0000±109.271p |
0.5353±0.009t |
|
EN-11% |
0.7197±0.001c |
0.6593±0.054d |
0.8438±0.002g |
17.9446±0.429i |
33.8413±5.629k |
3092.0909±115.207p |
0.4637±0.029t |
Mean values ± standard error based on six independent determinations. The
same letters at the end of each value indicate that there is statistically no
difference at P > 0.05 using
Duncan’s multiple range test
%20343-351_files/image008.jpg)
Fig. 4: Radar plot of parameters derived from the JIP-test
Measurements were carried out in dark adapted
leaves of the plants. Average values are expressed in relation to the plants
under SW-15% conditions, for which the values were normalized to 1. Data are
mean values (n = 6)
20% and
ES-20% groups were the largest, whereas those in the EN-11% group
were the smallest.
In this study, the seven fluorescence parameters with the most fundamental
physiological significance are shown in Table 2 and Fig. 4. The maximum
quantum yields for PSII photochemistry, FV/FM,
were the lowest for EN-11% and ES-11% (P < 0.05), and there was no
significant (P > 0.05) difference between them (Table 2, Fig. 4). The
largest values of FV/FM were recorded for
ES-15% and ES-20%, and these values showed significant differences to the other
groups (P < 0.05), although there was no significant difference between them (P
> 0.05). In the E-W seedbeds, FV/FM
values for plants grown on the south side of the seedbed with the same LTR were
larger than those grown on the north side, and there were significant
differences (P < 0.05) between the two sides of the seedbeds for LTRs of 15
and 20% (E-15% and E-20%). In contrast, there was no significant (P
> 0.05) difference in values between the two sides of the E-W seedbed
with an LTR of 11% (E-11%). There was no difference (P > 0.05) between
the sides of the N-S seedbeds for the three LTRs (N-11%, N-15%, and N-20%). The
values of FV/FM for E-11% were the lowest,
whereas those for ES-20% were the highest.
The 12 groups of plants were divided into two groups according to the
significance of the relative variable fluorescence at 2 ms, VJ, and the relative variable
fluorescence at 30 ms, VI (Table 2; Fig. 4). For VJ, N-20% and the conditions
with LTR of 11% (N-11% and E-11%) were in the higher VJ group, whereas the other conditions
were in the lower VJ group, and the VJ values of the two groups were significantly
different from each other (P < 0.05). For VI, N-11% and N-20% were in the
higher VI group, whereas the other conditions were in the
lower VI group, and there was a significant difference
between the two groups (P
< 0.05).
Table 3: Ginsenoside
composition in roots
|
LTR |
Rg1 |
Re |
Rb1 |
Rc |
Rd |
|
NW-20% |
0.4392±0.0685b |
5.7303±0.5432d |
9.0580±1.4553g |
9.1476±0.9933m |
1.1353±0.3590x |
|
NE-20% |
0.4370±0.0543b |
5.6149±0.7765d |
9.0394±1.5642g |
9.1175±0.9694m |
1.1275±0.6543x |
|
ES-20% |
0.4364±0.0323b |
5.5081±0.8392d |
8.9873±1.7653g |
8.9536±0.8833m |
1.1620±0.3345x |
|
EN-20% |
0.4132±0.0453c |
5.3420±0.8372e |
8.9566±1.4421h |
8.6098±1.1124n |
1.6788±0.3821w |
|
NW-15% |
0.4012±0.0654c |
5.3002±0.5564e |
8.8397±1.2212h |
8.5893±1.0235n |
1.7345±0.5467w |
|
NE-15% |
0.4555±0.0699b |
5.2260±0.0222e |
8.7847±1.3424h |
8.5672±0.8839n |
1.6717±0.4321w |
|
ES-15% |
0.4373±0.0943b |
5.2020±0.0543e |
8.7768±1.6537h |
8.5691±0.6623n |
1.6596±0.2231w |
|
EN-15% |
0.4243±0.0754b |
5.2254±0.0768e |
8.8781±1.8724h |
8.5848±0.7653n |
1.7082±0.3234w |
|
NW-11% |
0.4620±0.1094a |
4.6141±0.0599f |
7.6815±1.5541k |
7.6241±0.9782p |
1.6134±0.6998w |
|
NE-11% |
0.4636±0.0884a |
4.5067±0.4998f |
7.6376±1.6241k |
7.1050±0.6136p |
1.1980±0.7742x |
|
ES-11% |
0.4558±0.0675a |
4.1868±0.0740f |
7.2704±1.2342k |
6.9075±0.1100p |
0.9237±0.2211y |
|
EN-11% |
0.4523±0.0433a |
4.1831±0.0042f |
7.2463±1.4456k |
6.8856±0.3345p |
0.9170±0.1021y |
Mean values ± standard error
based on six independent determinations. The same letters at the end of each
value indicate that there is statistically no difference at P > 0.05 using Duncan’s multiple
range test
The conditions were divided into three groups according to the significance
(P < 0.05) of total electron carriers per
reaction center (RC), Sm, and time-dependent turnover number
of primary quinone electron acceptor of PSII (QA), N (Table 2; Fig. 4). ES-20%,
N-15%, and ES-11% had the highest Sm values, the value of
NW-20% was the lowest (P
< 0.05), and the values of the other six conditions were intermediate. The values of N were
similar to those of Sm, except that the group with the
highest values consisted of ES-20%, E-11%, and N-15%, and the other conditions
had similar values.
The value
of RC/CSM [number of active PSII reaction centers
per unit area (t = tFM)] and PItotal
[performance index (potential) for energy conservation from photons absorbed by
PSII to the reduction of PSI end acceptors] for N-20% was the lowest (Table 2, Fig. 4). The value
of RC/CSM for
ES-20% was the biggest, and the value of PItotal for EN-20% and
ES-20% were the biggest (P < 0.05).
Effects of LTRs and seedbed
orientation on ginsenoside contents
The ginsenoside contents of Rg1,
Re, Rb1, Rc, and Rd are shown in Table 3. For the same LTRs, the
contents of Re, Rb1, and Rc decreased in the order of seedbed
orientation and side of NW, NE, ES, and EN, except for Rb1 and Rc
for EN-15%, although there were no significant differences (P
> 0.05). Maximum Re, Rb1, and Rc contents were observed for the
N-20% and ES-20% conditions, and showed significant differences compared with
the other conditions (P < 0.05). Re, Rb1, and Rc contents for LTR of 11% for both orientations (N-11% and E-11%) were
significantly lower than those for other conditions (P < 0.05), but there was no difference among the contents for N-11% and
E-11% (P
> 0.05).
The contents of Rg1
for LTR of 11% (N-11%
and E-11%) were higher than
those for the other LTRs (P < 0.05). For LTR of 20%, the Rg1 contents of the roots of the
plants grown on both sides of the N-S seedbeds (N-20%) were higher than those
of the E-W seedbeds (E-20%). For LTR
of 15%, the Rg1 content of NW-15% was the lowest, and showed a
significant difference from the other three groups with the same LTR (P
< 0.05) (Table 3).
The Rd content
increased, and then decreased as the LTR increased (Table 3). The conditions
were divided into three groups according to their significance (P
< 0.05). The group with the highest Rd contents consisted of EN-20%, E-15%,
N-15% and NW-11% (P < 0.05), and there were no significant
differences among the members of this group (P > 0.05).
N-20%, ES-20%, and NE-11% had the second highest Rd contents, and there was no
significant difference among the group (P > 0.05). E-11% had the lowest Rd,
and there was no significant difference between the two sides of the seedbed (P
> 0.05).
The total concentrations of the Rg and Rb groups and the Rg/Rb indices are
shown in Table 4. The values of the Rg groups for NE-20%, NW-20%, and ES-20%
showed no significant differences (P > 0.05), and were significantly
higher than the values for the other groups (P
< 0.05). ES-11% and EN-11% had the lowest values
that were significantly (P < 0.05) lower than those for the other groups,
although there was no significant (P > 0.05) difference between these two
groups. These values for the Rb groups followed a similar trend to the values
for Rb1. The values of Rb groups for NW-20%, NE-20%, and ES-20% were
significantly (P < 0.05) higher than those for the other groups,
whereas those for E-11% and N-11% differences were the lowest (P
< 0.05).
Table 4: Total
concentration of Rg and Rb groups and Rg/Rb index in roots
|
LTR |
Rg |
Rb |
Rg/Rb |
Total |
|
NW-20% |
6.1695±0.9933a |
19.3410±1.5632d |
0.3190±0.0112g |
25.5105±1.5412k |
|
NE-20% |
6.0520±0.8970a |
19.2844±2.0013d |
0.3138±0.0287g |
25.3363±4.2210k |
|
ES-20% |
5.9445±0.9782a |
19.1029±1.0093d |
0.3112±0.0491g |
25.0474±2.1240k |
|
EN-20% |
5.7552±0.9932b |
19.2452±1.1209e |
0.2990±0.0352i |
25.0004±2.6786m |
|
NW-15% |
5.7014±0.6352b |
19.1635±1.4325e |
0.2975±0.0333i |
24.8649±1.4432m |
|
NE-15% |
5.6815±0.8897b |
19.0236±1.6320e |
0.2987±0.0549i |
24.7051±1.0098m |
|
ES-15% |
5.6394±0.6722b |
19.0054±1.4424e |
0.2967±0.0093i |
24.6448±1.7849m |
|
EN-15% |
5.6497±0.5409b |
19.1711±1.6521e |
0.2947±0.2019i |
24.8208±1.3030m |
|
NW-11% |
5.0761±0.2095b |
16.9190±1.7732f |
0.3000±0.1129h |
21.9951±1.3950n |
|
NE-11% |
4.9704±0.1443b |
15.9406±1.5430f |
0.3118±0.0388h |
20.9110±1.3306n |
|
ES-11% |
4.6425±0.3320c |
15.1016±1.4352f |
0.3074±0.0408h |
19.7441±1.4509n |
|
EN-11% |
4.6354±0.4234c |
15.0488±1.6642f |
0.3080±0.0333h |
19.6842±1.8876n |
Rg = Rg1 + Re; Rb = Rb1 + Rc +
Rd. Mean values ± standard error based on six independent determinations.
The same letters at the end of each value indicate that there is statistically
no difference at P > 0.05 using
Duncan’s multiple range test
NW-20% had the highest Rg/Rb ratio, with 1.08-fold more ginsenosides than
EN-15%, which had the lowest ratio (P < 0.05). The ratios for LTR of 11% were not the
lowest, and were close to those for ES-20% (P
> 0.05). A total of five ginsenosides were
significantly up-regulated as the LTRs increased markedly (P
< 0.05), which was consistent with the trend for Pn
(Fig. 1A).
Discussion
Photosynthesis is the basis of plant
morphogenesis, growth, and development. Because
photosynthesis provides 90–95% of the plant’s dry weight, it is crucial to
the quality and yield of the plant (Cheng and Shen 2011). Light transmission and the planting position affects
the effective light intensity on leaves, especially for shade plants like American ginseng (Wang et al. 2010; Xu et al.
2011; Li et al. 2014; Song et al. 2017).
We investigated the
response of PSII in
two-year-old P. quinquefolium plants to different LTRs and seedbed orientations through gas
exchange, JIP-test, chlorophyll content determination, and ginsenoside
analysis. The seedbed orientation seemed to have no effect on the chlorophyll
content below LTR of 20%, and increasing the LTR increased the chlorophyll
content of the leaves of P. quinquefolium.
The quantum yields (FV/FM)
indicated that conditions E-11% (ES-11% and EN-11%) and ES-20% had the largest
and smallest effects on the light photochemical efficiencies of the
leaves, respectively (Li et al. 2006). The J-step
(the fluorescence intensity at 2 ms) corresponds to the first transient accumulation
of the primary quinone electron acceptor of PSII (QA), and reflects
the efficiency of the electron transfer and the degree of closure of active RCs
(Force et al. 2003; Li et al. 2006). When the LTR was 11%, 2 ms
after illumination, the electron reception on the acceptor side of PSII
(J-step) and the proportions of RCs were smallest for the
plants grown under E-11% conditions (Li et
al. 2006). That is, on the acceptor PSII side, the
transport of electrons from QA- to the secondary acceptor QB
was blocked, and the majority of the active RCs were shut down (Bordenave et al. 2019). Thus, electrons could not
be transferred to the dark reactions (Pan et
al. 2010), which down-regulated the assimilatory power and the rate of the
dark reaction during carbon assimilation and reduced the production of
photosynthates (Sun et al. 2009).
For LTR of 20%, the values of Pn
for N-20% and
ES-20% were the largest. For ES-20%, the value of the maximum quantum yield of
PSII (FV/FM), performance indexes (PItotal),
and number of active PSII (QA reducing) RCs per unit area (RC/CSM,
t = tFM) were the highest (Li et al. 2006). A highest rapidly
reducible PQ pool (J-I phase) and slowly reducible PQ pool (I-P phase) together
resulted in the highest time-dependent turnover number of QA (N), most electron
carriers per RC (SM), and the lower proportions of reaction
centers (RCs) closed at 2 ms (VJ) and 30 ms (VI).
A higher chlorophyll content of ES-20% also prompted the electron transfer
rate and the quantum yield of
electron transport beyond QA (Li et al.
2006; Qiu et al. 2012). In addition, total electron carriers per RC (Sm) and
time-dependent turnover number of QA (N) of NW-20% were the
smallest, and the proportions of reaction centers (RCs) closed at 2 ms (VJ)
and 30 ms (VI) of N-20% were the highest. The increase in VI
for N-20% indicated the accumulation of reduced QA, which could not
transfer electrons to the dark reactions (Pan et al. 2010). Thus, the activity of PSII in ES-20% was the highest.
Our results demonstrated that chlorophyll a fluorescence can provided a
relative accurate measure of PSII (Strasser et al. 2000; Strasser et al.
2004; Li et al. 2006).
NE-20%, NW-20%, and
ES-20% had the highest Rg group content and Rg/Rb ratio. This result indicated that
photooxidation occurred because Rg group are relatively weak antioxidants (Lü et al. 2012; Shkryl et al. 2018), whereas the highest value of Pn
indicated that photooxidation did not affect Pn. In
addition, the change in the total ginsenoside content was consistent with the change
in Pn (Fig. 1A; Table 4), and the results agree with earlier
works on Panax Ginseng L. (Jang et
al. 2015).
Data suggested that E-W seedbed orientation was better for growing
American ginseng than the N-S orientation for LTR of 20%. This is consistent
with earlier works on other plants (Bai et
al. 2005; Xu et al. 2011; Song
2017). For LTR of 15%, the total ginsenoside content, and the Rg and Rd groups were
similar for both orientations and sides of the seedbeds.
The analysis of Pn, chlorophyll a fluorescence,
and ginsenoside content showed that the lowest values
were for E-11%, confirming that the lower light intensity was the sole limit
source, subsequently affecting the ginsenoside contents (Xu et al. 2002). Pn and ginsenoside content increased with the
LTR. According to the gas exchange
parameters, the lowest Pn for LTR-11% was attributed to the
stomatal factor.
Conclusion
The changes in the ginsenoside contents were
consistent with the changes in LTRs and photosynthesis, whereas the lowest Pn
at LTR of 11% was attributed to
the stomatal factor. American ginseng plant grown on the E-W seedbeds with LTR of 20% had higher PSII
activity and ginsenoside contents than the N-S seedbeds, regardless of the side
of the seedbed. Moreover, the fluorescence data and ginsenoside contents showed
that light oxidation occurred in PSII of plant leaves grown in the N-20% seedbed, even though the Pn
was the highest. Therefore, future work should be focused on the long-term
effect of light oxidation in American ginseng by prolonging the cultivation
period for two or three years.
This study was supported financially by Shandong
Province Modern Agricultural Industry Technical System via the Chinese Herbal
Medicine Innovation Team Construction Project and the National Key Research and
Development Project (2017YFC1702702, 2017YFC1700705).
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