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The reduced growth due to
elevated CO
2
concentration
hinders the sexual reproduction
of mature Northern pipevine
(Aristolochia contorta Bunge)
Si-Hyun Park
1
and Jae Geun Kim
1,2
*
1
Department of Biology Education, Seoul National University, Seoul, Republic of Korea,
2
Center for Education Research, Seoul National University, Seoul, Republic of Korea
The phenology has gained considerably more attention in recent times of climate
change. The transition from vegetative to reproductive phases is a critical process
in the life history of plants, closely tied to phenology. In an era of climate change,
understanding how environmental factors affect this transition is of paramount
importance. This study consisted of field surveys and a greenhouse experiment
on the reproductive biology of Northern pipevine (Aristolochia contorta Bunge).
During field surveys, we investigated the environmental factors and growth
characteristics of mature A. contorta, with a focus on both its vegetative and
reproductive phases. In its successful flowering during the reproductive phase, A.
contorta grew under the conditions of 40% relative light intensity and 24% soil
moisture content, and had a vertical rhizome. In the greenhouse experiments, we
examined the impact of increased CO
2
concentrationonthegrowthand
development of 10-year-old A. contorta, considering the effect of rhizome
direction. Planted with a vertical rhizome direction, A. contorta exhibited
sufficient growth for flowering under ambient CO
2
concentrations. In contrast,
when planted with a horizontal rhizome direction, it was noted to significantly
impede successful growth and flowering under elevated CO
2
concentrations.
This hindered the process of flowering, highlighting the pivotal role of substantial
vegetative growth in achieving successful flowering. Furthermore, we observed a
higher number of underground buds and shoots under the conditions of
elevated CO
2
concentration and a horizontal rhizome direction instead of
flowering. Elevated CO
2
concentrations also exhibited diverse effects on
mature A. contorta’sflower traits, resulting in smaller flower size, shorter
longevity, and reduced stigma receptivity, and pollen viability. The study shed
light on elevated CO
2
concentrations can hinder growth, potentially obstructing
sexual reproduction and diminishing genetic diversity.
KEYWORDS
climate change, CO
2
concentration, growth inhibition, phenology, reproduction,
rhizome direction
Frontiers in Plant Science frontiersin.org01
OPEN ACCESS
EDITED BY
Huanjiong Wang,
Institute of Geographic Sciences and Natural
Resources Research (CAS), China
REVIEWED BY
Yachen Liu,
Xi’an University, China
Xiangjin Shen,
Chinese Academy of Sciences (CAS), China
*CORRESPONDENCE
Jae Geun Kim
jaegkim@snu.ac.kr
RECEIVED 22 December 2023
ACCEPTED 04 March 2024
PUBLISHED 20 March 2024
CITATION
Park S-H and Kim JG (2024) The
reduced growth due to elevated CO
2
concentration hinders the sexual
reproduction of mature Northern
pipevine (Aristolochia contorta Bunge).
Front. Plant Sci. 15:1359783.
doi: 10.3389/fpls.2024.1359783
COPYRIGHT
© 2024 Park and Kim. This is an open-acc ess
article distributed under the terms of the
Creative Commons Attribution License (CC BY).
The use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
TYPE Original Research
PUBLISHED 20 March 2024
DOI 10.3389/fpls.2024.1359783
1 Introduction
Climate change is a critical global issue with far-reaching
consequences for the natural environment (Kumar et al., 2020).
Climatechangeposesnumerouschallenges to plant species,
influencing their biology, including their growth and reproductive
strategies (Piao et al., 2019;Pareek et al., 2020). Phenology has
garnered significant interest in recent years (Piao et al., 2019;Shen
et al., 2022). Phenological shifts, which involve the timing of key
events in plant life cycles such as flowering, fruiting, leafing out, and
senescence, represent some of the most visible ecological effects of
globalclimatechange(CaraDonna et al., 2014). Among these
phenological events, flowering naturally serves as a significant
indicator of the transitions between vegetative and reproductive
phases and is important in maintaining ecological balance
(Schwartz, 2003). Climate change crucially modifies flowering
time, which is a vital adaptation for ensuring successful
reproduction in response to shifting environmental conditions
(Cortes-Flores et al., 2017;König et al., 2017). There are various
types of evidence indicating that phenological traits can be changed
rapidly when there is strong natural selection which is influenced by
environmental factors (Schwartz, 2003;Elzinga et al., 2007;Chuine,
2010). For example, elevated CO
2
concentrations may offer certain
benefits as they enhance photosynthesis, increase the number of
flowers and fruits, and promote blooming (Chaudhry and Sidhu,
2022). However, other plants’changes in CO
2
concentration can
disrupt the delicate balance of plant reproductive timing, potentially
affecting reduced seed production (Boyle and Bronstein, 2012;Nam
et al., 2020).
In response to the challenges posed by climate change, certain
plant species have displayed adaptations in their flowering times,
with certain species flowering earlier or later than usual to align
with the changing climate (Inouye, 2020;Rafferty et al., 2020).
These shifts in flowering patterns enable them to optimize their
reproductive success in response to altered seasonal cues and
environmental conditions (Rafferty et al., 2020;Rosbakh et al.,
2021). Furthermore, some species exhibit modified flowering
patterns, adjusting the timing of flowering to coincide with
periods of increased pollinator availability (Inouye, 2020;Martins
et al., 2021;Faust and Iler, 2022). Many other plants have evolved
specialized underground structures that exhibit a remarkable ability
to adjust their asexual reproductive structure in response to specific
characteristics of the environment (Karlova et al., 2021;Huang
et al., 2022). This adaptation is particularly advantageous when the
ability to sprout and establish new growth from the rhizomes
becomes a crucial strategy for plant resilience (Ziska et al., 2004;
McPeek and Wang, 2007). Understanding how plants respond and
adapt to environmental factors is vital for predicting their long-term
survival, maintaining ecosystem stability, and contributing to
overall biodiversity (Cleland et al., 2007;Sun and Frelich, 2011;
Davies et al., 2013). Moreover, identifying the specific factors that
drive the reproduction and investigating how selection by these
factors impacts the most advantageous are crucial aspects of
studying the evolution of an organism’s life history (Raza et al.,
2020;Pesendorfer et al., 2021).
Northern pipevine (Aristolochia contorta Bunge)has garnered
attention for research on climate change impacts. This perennial
herbaceous vine plant is found in fragmented natural populations
along forest edges and riverbanks in East Asia (Nam et al., 2020). It
employs both sexual and asexual reproductive mechanisms (Lee,
2012). During sexual reproduction, this plant typically produces
flowers after three or more years of growth (Park et al., 2019). The
unique features of its flowers include a straight tubular perianth,
enclosing fused styles, stigma, and anthers within a chamber known
as the utricle, which forms a gynostemium (Park and Kim, 2023).
During asexual reproduction, the plant stores reserves in the root or
rhizome and undergoes seasonal changes, shedding its aboveground
parts in winter and regrowing new stems from underground buds
annually (Lee, 2012). Additionally, the vulnerable butterfly,
Sericinus montela, whose larvae feed solely on A. contorta,
underscores the importance of conservation studies (Park
et al., 2023).
Previous studies of A. contorta have mainly focused on its
functional aspects, such as plant’s secondary metabolites and its
optimal habitat conditions (Hashimoto et al., 1999;Cheung et al.,
2006;Heinrich et al., 2009;Nakonechnaya et al., 2013;Park et al.,
2019). Studies have shown that the genetic variation indices of A.
contorta are low, similar to those of other rare plants
(Nakonechnaya et al., 2012;Nam et al., 2020). Elevated CO
2
concentrations inhibit the growth of 1- or 2-year-old A. contorta,
decrease photosynthesis, and increase plant resistance, negatively
impacting specialist herbivores (Park et al., 2021). The effect of
climate change on the interaction between A. contorta and its
specialist and generalist herbivores vary depending on the
ontogenetic stage of the plant (Park et al., 2022). However,
previous studies have focused only on A. contorta growth during
the vegetative phase, and no research has been conducted on its
reproductive phase. Furthermore, there is a lack of research on the
reproductive biology of this plant under climate change.
Toaddressthisgap,weinvestigatedthegrowthand
reproductive characteristics of mature A. contorta under different
CO
2
concentrations while also examining trade-off patterns in its
reproductive strategies. The field survey provided valuable insights
into the conditions for flowering through a comparison of the
vegetative and reproductive phases, and the greenhouse experiment
was guided by the findings from the field survey to identify the
factors hindering or triggering flowering. We hypothesized that (1)
the elevated CO
2
concentration will impede the growth of mature A.
contorta, which in turn will hinder flowering and lead to a transition
from sexual reproduction to asexual reproduction, and (2) the
impact of elevated CO
2
concentration will be more pronounced
when the rhizome direction of mature A. contorta is horizontal.
This research underscores the novel contributions of our study
within the broader context of the impacts of climate change on
plant species, with a particular focus on A. contorta. Delving into
the relatively unexplored domain of its reproductive biology under
varying CO
2
conditions, our investigation provides critical insights
into the adaptive responses of mature A. contorta to climate change
and its reproductive behavior. Such knowledge is pivotal in guiding
conservation efforts, aiding in mitigating the impact of climate
Park and Kim 10.3389/fpls.2024.1359783
Frontiers in Plant Science frontiersin.org02
change on A. contorta populations, preserving their genetic
diversity, and ensuring their long-term survival.
2 Materials and methods
2.1 Vegetative and reproductive
phases comparison
2.1.1 Environmental factor analysis
The research was conducted in Anyang, Gyeonggi Province,
South Korea (37°24’2.58”N, 126°58’18.3”E) during the flowering
period from May to August in, 2023, documented a mean
temperature of 24.82 ± 0.29°C and a mean precipitation of 16.07
± 3.23 mm. For the region, the 30-year annual mean temperature
from, 1993 to, 2022 was 12.60 ± 0.10°C, and the annual mean
precipitation was, 1337.01 ± 52.86 mm (Korea Meteorological
Administration, 2023). The site was situated in a riparian area
within the native distribution range of A. contorta. Relative light
intensity (RLI) was measured by comparing the light intensity
(µmol m
−2
s
−1
)recordedatthetopofA. contorta with that
measured at the same height in an open space at the same time
(Lee and Cho, 2000). We measured soil properties, including
water content, pH, EC, PO
4
–P, NH
4
–N, NO
3
–N, Ca
2+
,K
+
,Na
+
,
and Mg
2+
. Analyses of soil properties were performed by obtaining
soil samples from a depth of 15 - 25 cm near the rhizomes of each
individual A. contorta. To preserve moisture, the samples were
sealed in plastic bags and transported to the laboratory. The soil
samples were then sieved through a 2 mm mesh, and a mixture of
deionized water was added at a ratio of soil 1: water 5. The resulting
solution was filtered using Whatman filter paper No. 42 (Sigma-
Aldrich, St. Louis, MO, USA) and used for analyses of soil
environmental characteristics. The pH level was determined using
a pH meter (model AP 63; Fisher Scientific, Pittsburgh, PA, USA),
and the electrical conductivity (EC) was measured using a
conductivity meter (Corning Checkmate model 311; Corning
Incorporated, Tewksbury, MA, USA). For soil nutrients, we
determined the contents of PO
4
–P, NH
4
–N and NO
3
–N using the
methods of hydrazine (Kamphake et al., 1967), indo-phenol
(Murphy and Riley, 1962), and ascorbic acid reduction
(Solorzano, 1969), respectively. Exchangeable cations (Ca
2+
,K
+
,
Na
+
and Mg
2+
) were measured using an atomic absorption
spectrometer (model AA240FS; Varian Medical Systems, Palo
Alto, CA, USA) after extraction with 1 M ammonium acetate
solution. Additionally, soil water content was determined by
drying the fresh soil samples at 105°C for more than 48 hours
(Kim et al., 2004), while soil organic matter contents were analyzed
using loss on ignition at 450°C (John, 2004).
2.1.2 Growth trait analysis
During the transition from vegetative phase to reproductive
phase (when the flower buds began to appear), 20 individuals (older
than 4 years; approximately 2 m stem length, the length of the onset
of flowering according to observations in field survey) were taken
into account for both the flowering group, consisting of
10 individuals spaced more than 10 meters apart, and the non-
flowering group of 10 individuals. The measured growth factors
were stem thickness at ground level, internode length, number of
branches, number of leaves, single leaf area, total leaf area, rhizome
thickness, rhizome length, direction of rhizome growth, chlorophyll
content, fresh and dry weights (stems, leaves, rhizomes with roots,
and flowers), and C/N ratios of each part (stem, leaves, and
rhizome). Stem and rhizome thickness were measured with a
vernier calipers (Mitutoyo, Kanagawa, Japan; resolution,
0.01 mm). The first internode length above the ground and
number of leaves were recorded for each individual. To calculate
the total leaf area, we first determined the leaf area for each
individual by measuring the average area of ten leaves using
ImageJ (Schneider et al., 2012). Subsequently, the average leaf
area was applied to the total number of leaves. For assessing
rhizome direction, we excavated the soil to a depth of
approximately 20 cm. We utilized an angle gauge to measure the
angle between the rhizomes and the horizontal plane, providing a
clear indication of their orientation. In order to gauge the
chlorophyll content of leaves, we employed a chlorophyll meter
(SPAD-502, Konica Minolta, Tokyo; Rodriguez and Miller, 2000).
Moreover, the dry weights of stems, leaves, and rhizomes with roots
were measured, and belowground/aboveground ratio was calculated
by dividing the dry weight of the rhizome with roots by the dry
weight of the aboveground shoot components of a plant. In order to
investigate the distribution of carbon and nitrogen resources in
various plant parts, we performed stoichiometric analyses of the
rhizomes, stems, leaves, flowers, and fruits. The plant parts were
dried in a dry oven at 60°C and then ground using a ball mill
(Pulverisette 23; Fritsch, Germany) to ensure uniform mixtures for
the analysis. C/N ratio of each part was measured using an
elemental analyzer (Flash EA, 1112, Thermo Electron, USA) at
the National Instrumentation Center for Environmental
Management (NICEM) at Seoul National University.
2.2 Effects of elevated CO
2
on growth and
reproduction in different
rhizome directions
2.2.1 Experimental design
To investigate how individuals with different inclinations respond
to elevated CO
2
concentrations, we conducted greenhouse experiments
to elucidate the changes in the growth and reproduction of A. contorta
under different CO
2
concentrations. For this purpose, we purchased
10-year-old A. contorta rhizomes in March 23, 2023.
We observed that growth and reproductive characteristics
differed based on whether the rhizomes were oriented vertically
or horizontally in the field surveys. To replicate the natural
conditions closely, we planted a total of 40 rhizomes of these
plants individually, both horizontally and vertically, in 5 liters of
soil each (Superlative soil, Gumok, Pohang-si, Republic of Korea;
Park et al., 2019). The greenhouse, located at Seoul National
University, Seoul, Republic of Korea, provided a relative light
intensity of 37.9% (Park et al., 2019). We installed hexagonal
Park and Kim 10.3389/fpls.2024.1359783
Frontiers in Plant Science frontiersin.org03
Open Top Chambers (OTC; Park et al., 2021) in the greenhouse to
manipulate the carbon dioxide (CO
2
) concentration, simulating two
scenarios (Thomson et al., 2011): 1) Representative Concentration
Pathways 4.5 (RCP 4.5) climate change scenario with a CO
2
concentration of 540 ppm, and 2) current ambient conditions
with a CO
2
concentration of 400 ppm. Each OTC had its own
CO
2
control system to regulate elevated CO
2
concentration. The
control system consisted of a sensor-transmitter coupled with a
CO
2
controller (SH-MVG260, Soha-tech, Korea), capable of
maintaining CO
2
concentrations within the range of 0 –2000
ppm. Additionally, a solenoid valve and individual CO
2
gas tanks
(40 L, 99.999% purity) were used in the setup (Park et al., 2021).
Therefore, we conducted a greenhouse experiment using a total of
40 plants, with 10 plants for each specific condition. The experiment
involved two CO
2
concentrations (ambient 400 ppm and elevated
540 ppm), and two rhizome directions planting (horizontal rhizome
planting, H; and vertical rhizome planting, V), resulting in four
experimental treatments (400ppmCO
2
/H, 400ppmCO
2
/V,
540ppmCO
2
/V, 540ppmCO
2
/H) arranged in a factorial design.
Temperature and relative humidity sensors (HOBO Pro v2,
Onset, Bourne, MA, USA) were installed in each chamber, and
these variables were ensured to remain consistent across all
chambers throughout the experimental period. The mean air
temperature and humidity recorded in the OTCs during the
experimental period were as follows: 24.3°C and 69.5% in
ambient CO
2
, 24.1°C and 68.4% in elevated CO
2
.
Pots were set on 50 mm thickness plates to minimize external
wind effects from the bottom of the chamber induced by the
ventilation system. To measure the amount of nutrients absorbed
by each plant, bottom watering was used. A rope wick was inserted
into the base of the pots to facilitate water absorption from the tray
below the pot. During harvesting, excess water at the bottom was
directed into the soil to reduce nutrient loss. To compare the
nutrient absorption efficiency based on the treatment conditions,
we evaluated the differences in soil nutrient levels between before
and after the experiment, including NO
3
–N, NH
4
–N, PO
4
–P, as
well as cations (K
+
,Na
+
,Ca
2+
, and Mg
2+
), along with the C/N ratio.
The calculation of nutrient uptake by plants and nutrient losses
(efforts were made to minimize nutrient loss during irrigation, and
while some leaching was expected, the uniform water supply likely
resulted in consistent nutrient loss) involved subtracting the
nutrient content in the soil after the experiment from that before
the experiment.
2.2.2 Analysis of growth and reproductive traits
To assess the growth and reproductive (sexual and asexual)
traits of A. contorta under two CO
2
concentrations and two
rhizome direction plantings, we harvested all plants in the
greenhouse at the initial signs of aboveground senescence. We
separated the collected plants into stems, leaves, and flowers and
measured the growth traits of stem length, internode length, stem
thickness, number of branches, total branch length, number of
leaves, single leaf area, total leaf area, chlorophyll content, changes
in fresh and dry weight of each component (stem, leaf, rhizome and
roots), and C/N ratios of each part (stem, leaves, rhizome and root).
We assessed variations in reproductive traits related to sexual
reproduction, including the first flowering day (FFD), number of
flowers, flowering duration, flower longevity, perianth size,
diameter of the utricle, pollen grain size, stigmatic receptivity,
pollen viability, and C/N ratio of the perianth and ovary (the
breeding process of A. contorta heavily relied on pollinators. Only
one fruit was produced and we opted to measure the ovary instead).
Flowering duration refers to the period from the FFD until the last
flower wilts or fades, while flower longevity denotes the duration
from the budding of a single flower until it withers. The sizes of the
perianth, utricle, fruit, and seed were measured using a digital
vernier calipers. Ten flowers were assessed under each condition,
with ten pollen grain diameters measured from each individual
flower. The sizes of pollen grains were measured using an optical
microscope (DE/Axio Inager A1 microscope, Carl Zeiss, Germany)
and ImageJ. For stigmatic receptivity assessment, we collected ten
samples from each flowering individual on the first day of flower
opening. Subsequently, we conducted separate applications of a 3%
hydrogen peroxide (H
2
O
2
) solution to the stigmas of both the
female and male phases (Dafni and Maues, 1998;Serrano and
Olmedilla, 2012). The stigmas were observed for a duration of 3
minutes under a stereoscopic microscope (GB-742, Global4U,
Republic of Korea), and the presence of bubbles served as a
reliable indicator to assess their receptivity. Stigmas that displayed
a substantial number of bubbles were classified as highly reactive to
the compound, while those with minimal or no bubble formation
were categorized as having low reactivity. It was assessed using the
approach adapted from Dafni and Maues (1998), involving the
assignment of scores based on the number of bubbles. These scores
included no reaction (0), a weak positive reaction (1), a strong
positive reaction (2), and a very strong positive reaction (3). Pollen
viability was assessed using the 1% neutral red staining method
(Georgieva and Kruleva, 1993). Three samples were collected from
each flowering individual and prepared. Viability was calculated by
counting stained (viable or semi viable) and unstained (non-viable)
grains from ten samples from each flowering individual on the third
day (after 48 h of opening, male phase) of flower opening. The
number of underground buds and shoots were recorded as
reproductive organs related to asexual reproduction. To explore
the variations in the distribution of carbon and nitrogen resources
under the two different CO
2
concentrations, we conducted
stoichiometric analyses on the rhizomes, stems, leaves, flowers,
and fruits. The experimental methodology was consistent with the
previously described procedure.
2.3 Statistical analyses
We employed an analysis of variance (ANOVA) after the
homogeneity of variance test and post-hoc tests (Duncan’s test) to
determine the significance of the observed variations in growth
and reproductive parameters between the control and treatment
groups. Canonical correlation analysis (CCA) was employed to
confirm the relationships between environmental factors and
growth traits (including the number of flowers) during the field
Park and Kim 10.3389/fpls.2024.1359783
Frontiers in Plant Science frontiersin.org04
survey, as well as between growth and reproductive traits in the
greenhouse experiment. This analysis was performed using
PC-ORD for Windows version 5 (B. McCune and MJ Mefford,
MjM Software, Gleneden Beach, OR, USA). To comprehend the
impact and interaction between rhizome direction and CO
2
concentration, we conducted a multivariate analysis of variance
(MANOVA). We utilized SPSS software version 23.0 (SPSS, Inc.,
Chicago, IL, USA) for statistical analysis, with the significance
level set at p<0.05.
3 Results
3.1 Vegetative and reproductive
phases comparison
3.1.1 Environmental factor analysis
Among the environmental factors, relative light intensity and
soil water content showed statistically significant differences
between the vegetative and reproductive phases (Figure 1), and
BC
DE F
GH I
JKL
A
FIGURE 1
Comparison of environmental variables between vegetative and reproductive phases. (A) relative light intensity, (B) soil water content, (C) soil
organic matter content, (D) pH, (E) EC, (F) PO
4
–P, (G) NH
4
–N, (H) NO
3
–N, (I) Ca
2+
,(J) K
+
,(K) Na
+
,(L) Mg
2+
. Bars indicate standard errors. *p< 0.05;
***p< 0.001.
Park and Kim 10.3389/fpls.2024.1359783
Frontiers in Plant Science frontiersin.org05
there were no significant differences in other environmental factors.
The vegetative phase had a higher RLI (85.91%) than the
reproductive phase (39.74%, Figure 1A).
3.1.2 Growth traits analysis
The growth traits of A. contorta during the vegetative and
reproductive phases were significantly different (Figure 2). Stems
were thicker in the reproductive phase (2.90 ± 0.21 mm) than in the
vegetative phase (1.69 ± 0.17 mm). Internode length was longer in
the reproductive phase (26.4 ± 1.2 cm) than in the vegetative phase
(13.8 ± 1.2 cm). Branch and leaf numbers per quadrat were more in
the reproductive phase (8.0 ± 0.5 branches, 106.4 ± 9.1 leaves) than
in the vegetative phase (3.7 ± 0.7 branches, 59.0 ± 13.8 leaves).
Single leaf area (34.46 ± 1.35 cm
2
) and total leaf area (3690.80 ±
267.46 cm
2
) in reproductive phase were greater than those in
vegetative phase (single leaf area, 26.25 ± 1.67 cm
2
; total leaf area,
1542.42 ± 460.30 cm
2
). There were broad and horizontal leaves in
reproductive phase and small leaves pasted vertically on shorter
internodes in vegetative phase. Rhizome thickness (8.71 ± 0.66 mm)
and length (57.4 ± 5.9 cm) were greater at reproductive phase than
BC
DE
F
GHI
JK
A
FIGURE 2
Growth traits of A.contorta in vegetative and reproductive phases. (A) stem thickness, (B) length of the first internode, (C) number of branches,
(D) number of leaves, (E) single leaf area, (F) total leaf area, (G) rhizome thickness, (H) rhizome length, (I) direction of rhizome, (J) chlorophyll
content, (K) dry weight of stem, leaves, rhizome and root. Bars indicate standard errors. **p< 0.01; ***p< 0.001.
Park and Kim 10.3389/fpls.2024.1359783
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(rhizome thickness, 2.46 ± 0.11 mm; root length, 29.1 ± 3.4 cm).
Direction of root growth in vegetative phase was 33.5 ± 5.9°, and
those in reproductive phase was 71.0 ± 5.6°. Chlorophyll contents in
vegetative phase (33.16 ± 0.91 mg/m
2
) and reproductive phase
(40.27 ± 1.63 mg/m
2
) were different. Dry weight of each part in
reproductive phase (stem, 5.70 ± 0.33 g; leaves, 3.17 ± 0.11 g;
rhizome and root, 4.59 ± 0.29 g) were significantly larger than those
in vegetative phase (stem, 3.15 ± 0.27 g; leaves, 1.81 ± 0.11 g;
rhizome and root, 2.99 ± 0.28 g; Figure 2). The belowground/
aboveground ratio showed a ratio of 0.56 in vegetative phase and
0.51 in reproductive phase (p=0.070). C/N ratios were significantly
different each part of the vegetative and reproductive phases. Each
part in vegetative phase were: a stem, 28.34 ± 0.74; leaves, 12.55 ±
0.46; a rhizome, 17.23 ± 2.27; and those at reproductive phase were:
a stem, 41.89 ± 1.97; leaves, 14.18 ± 0.45; a rhizome, 32.84 ± 1.73
(Figure 3). The environmental variables were correlated with
internode length, single leaf area, rhizome length, direction of
rhizome, dry leaf weight, dry rhizome weight, dry flower weight,
and number of flowers (Figure 4). Axes 1 and 2 accounted for 46.1%
and 29.3% of the explained variance, respectively.
3.2 Effects of elevated CO
2
on growth and
reproduction in different
rhizome directions
3.2.1 Nutrient uptake of A. contorta and soil
environmental conditions
In the greenhouse experiment, there were no significant
differences in nutrient uptake for any nutrient except for potassium
(Figure 5). Absorbed potassium was significantly largest in the
540ppmCO
2
/H (770.85 ± 33.17 mg·kg
-1
), followed by 540ppmCO
2
/
V (766.13 ± 30.13 mg·kg
-1
), 400ppmCO
2
/V (569.14 ± 40.99 mg·kg
-1
),
and 400ppmCO
2
/H (386.17 ± 36.60 mg·kg
-1
).
In the case of soil C/N ratio, the significantly highest value was
observed in 400ppmCO
2
/V (37.66 ± 0.09), followed by
540ppmCO
2
/H (33.84 ± 0.37), 400ppmCO
2
/H (33.40 ± 0.99), and
540ppmCO
2
/H (32.73 ± 0.47).
3.2.2 Elevated CO
2
and rhizome direction effects
on growth and reproductive traits
Stems, internodes, and branches of A. contorta were significantly
longer at 400ppmCO
2
than at 540ppmCO
2
(Figure 6). In addition, A.
contorta produced a greater number of branches, and larger leaves at
400ppmCO
2
than at 540ppmCO
2
. However, chlorophyll content was
significantly higher at 540ppmCO
2
than at 400ppmCO
2
.A. contorta
with rhizome planted vertically showed longer stem, internode, and
branches, thicker stem, higher number of branches and leaves, and
larger leaves than individuals with rhizome planted horizontally
(Figure 6). Stem length was longest in the 400ppmCO
2
/V (614.1 ±
67.5 cm), followed by 540ppmCO
2
/V (445.3 ± 40.5 cm),
400ppmCO
2
/H (326.8 ± 46.1 cm), and 540ppmCO
2
/H (216.2 ±
29.4 cm). Internode length was also longest in the 400ppmCO
2
/V
(15.6 ± 1.2 cm), followed by 540ppmCO
2
/V (10.4 ± 0.8 cm),
400ppmCO
2
/H (10.4 ± 0.6 cm), and 540ppmCO
2
/H (7.5 ± 0.5
cm). Stem thickness was largest in the 400ppmCO
2
/V (2.83 ± 0.18
mm), followed by 540ppmCO
2
/V (2.64 ± 0.12 mm), 540ppmCO
2
/H
(2.07 ± 0.23 mm), and 400ppmCO
2
/H (2.02 ± 0.16 mm). Rhizome
direction influenced the number of branches and total branch length,
with the largest values observed in the 400ppmCO
2
/V treatment
(branches, 10.4 ± 0.9 cm; total branch length, 536.1 ± 39.2 cm),
followed by the 540ppmCO
2
/V treatment (branches, 4.2 ± 0.4; total
branch length, 220.8 ± 43.7 cm), the 400ppmCO
2
/H treatment
(branches, 3.1 ± 0.5; total branch length, 135.7 ± 33.7 cm), and the
540ppmCO
2
/H treatment (branches, 2.8 ± 0.6; total branch length,
98.1 ± 19.1 cm). Number of leaves was largest in the 400ppmCO
2
/V
(225.2 ± 22.4), followed by 540ppmCO
2
/V (170.1 ± 23.5),
400ppmCO
2
/H (102.1 ± 20.9), and 540ppmCO
2
/H (72.7 ± 9.7).
Dried stem weight differed significantly under different CO
2
concentrations (400ppmCO
2
, 5.35 ± 0.76 g; 540ppmCO
2
,3.44±
FIGURE 3
C/N ratio of each part in the vegetative phase and reproductive phase.
Bars indicate standard errors. *p< 0.05; **p< 0.01; ***p< 0.001.
FIGURE 4
Canonical correlation analysis (CCA) plots to determine the
relationships among the environmental factors and growth traits.
The percentage (%) of each axis represents the explained variance.
Dotted curves indicate groups of individuals in the vegetative and
reproductive phases, which are represented by pink triangles. The
arrows are strongly correlated with the axis.
Park and Kim 10.3389/fpls.2024.1359783
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0.45 g) and dried stem and leaf weights differed significantly under
different rhizome directions (stem: H, 2.69 ± 0.33 g; V, 6.11 ± 0.68 g,
leaves: H, 2.71 ± 0.76 g; V, 5.24 ± 0.53 g) (Figures 7A–C). The
belowground/aboveground ratio was highest in the 540ppmCO
2
/H
(0.61), followed by 400ppmCO
2
/H (0.39), 540ppmCO
2
/V (0.27), and
400ppmCO
2
/V (0.16) (Figure 7D). There was a 26-day difference in
the FFD between conditions with 400ppmCO
2
(occurring on June
13th) and conditions with 540ppmCO
2
(occurring on July 9th).
Under 400ppmCO
2
,flower longevity was extended, accompanied
by an increased number of flowers (Figure 8) and larger flower size
(perianth size and diameter of utricle) compared to 540ppmCO
2
(Figure 9). Additionally, flowers exhibited earliest flowering, with a
greater number of flowers, greater flower longevity, and larger
perianth size (diameter of utricle) in vertical rhizome as opposed to
horizontal rhizome. On the other hand, in terms of asexual
reproductive traits, the number of underground buds and shoots
was higher at 540ppmCO
2
compared to 400ppmCO
2
.
All sexual reproductive traits were responsive to rhizome
direction in both 400ppmCO
2
and 540ppmCO
2
. Flower longevity
was longest in 400ppmCO
2
/H(16.0±0.6days),followedby
B
CD
EF
GH
IJ
A
FIGURE 5
Sum of nutrient uptake by A.contorta and nutrient losses under two CO
2
concentrations (400 ppm, 540 ppm), and two rhizome directions
(horizontal rhizome planting, H; and vertical rhizome planting, V). (A) PO
4
–P, (B) NH
4
–N, (C) NO
3
–N, (D) Ca
2+
,(E) K
+
,(F) Na
+
,(G) Mg
2+
,(H) pH,
(I) EC, (J) soil C/N ratio. Letters on the graph indicate significant differences at the 5% level, based on Duncan’s test. Bars indicate standard errors.
Park and Kim 10.3389/fpls.2024.1359783
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400ppmCO
2
/V (15.3 ± 0.2 days), and 540ppmCO
2
/V (12.3 ± 0.2
days). Perianth size was largest in 400ppmCO
2
/H (28.8 ± 1.4 mm),
followed by 400ppmCO
2
/V (28.2 ± 0.6 mm), and 540ppmCO
2
/V
(25.2 ± 0.5 mm). Diameter of utricle was largest in 400ppmCO
2
/V
(5.11 ± 0.08 mm), followed by 400ppmCO
2
/H (4.76 ± 0.12 mm),
and 540ppmCO
2
/V (3.78 ± 0.18 mm).
Stem C/N ratio was highest in 400ppmCO
2
/V (19.06 ± 1.97),
followed by 540ppmCO
2
/V (17.87 ± 0.37), 400ppmCO
2
/H (16.89
± 0.76), and 540ppmCO
2
/H (12.3 ± 0.33) (Figure 10). The highest
value of leaf C/N ratio was observed in 540ppmCO
2
/V (14.92 ±
0.69), followed by 540ppmCO
2
/H (14.24 ± 1.27), 400ppmCO
2
/V
(12.78 ± 0.15), and 400ppmCO
2
/H (11.21 ± 0.66). The highest
ratio of rhizome C/N ratio was found in 400ppmCO
2
/V (18.39 ±
0.17), followed by 540ppmCO
2
/V (18.04 ± 0.35), 540ppmCO
2
/H
(14.70 ± 0.51), and 400ppmCO
2
/H (14.67 ± 0.16). Flower C/N
ratio was highest in 400ppmCO
2
/H (9.76 ± 0.01), followed by
400ppmCO
2
/V (9.65 ± 0.14), and 540ppmCO
2
/V (7.09 ± 0.01).
For ovary C/N ratio was highest in 400ppmCO
2
/V 12.04 ± 0.01),
followed by 400ppmCO
2
/H (10.78 ± 0.01), and 540ppmCO
2
/V
(10.02 ± 0.02).
B
CD
EF
GH
I
A
FIGURE 6
The growth traits of A.contorta under two CO
2
concentrations (400 ppm, 540 ppm), and two rhizome directions (horizontal rhizome planting, H;
and vertical rhizome planting, V). (A) stem length, (B) length of the first internode, (C) stem thickness, (D) number of branches, (E) total branch
length, (F) number of leaves, (G) single leaf area, (H) total leaf area, (I) chlorophyll content. Letters on the graph indicate significant differences at the
5% level, based on Duncan’s test. Bars indicate standard errors.
Park and Kim 10.3389/fpls.2024.1359783
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The stigma reactions were the most intense at the 400ppmCO
2
/V
and there were numerous bubbles at the 400ppmCO
2
/H (Table 1).
The reactions of stigmas were not intense on at the 540ppmCO
2
/V
with only a few bubbles on the stigma. Pollen viability rates were
highest at 400ppmCO
2
/V (the viability range between 90.0 ± 1.2 -
97.0 ± 0.8%, reaching up to 100%), followed by 400ppmCO
2
/H
(88.3 ± 1.7 - 93.3 ± 1.7%), and lowest at 540ppmCO
2
/V (71.7 ± 6.0 -
85.0 ± 2.9%, reaching up to 60%) in the observations conducted
under an optical microscope (Table 2).
3.2.3 Interactive effects of elevated CO
2
concentrations and rhizome direction on growth
and reproductive traits
Morphological and reproductive traits responded to differences
in CO
2
concentrations and rhizome directions (Table 3). For
instance, number of shoots was only affected by CO
2
concentrations, and stem thickness and dry leaf weight were only
affected by rhizome directions. There were no interactive effects on
dry rhizome weight. Morphological differences according to the
CO
2
concentrations were more apparent when the rhizome
direction was different. We detected a significant interaction of
CO
2
concentrations and rhizome direction on number of branches,
total branch length, single leaf area, total leaf area, dry stem weight,
and sexual reproductive traits (number of flowers, flower longevity,
perianth size, diameter of utricle, pollen grain size, stigmatic
receptivity, and pollen viability).
Growth traits at 400ppmCO
2
exhibited strong correlations with
reproductive characteristics, including the number of flowers,
flower longevity, perianth size, diameter of the utricle, pollen
grain size, stigmatic receptivity, and pollen viability (Figure 11).
Growth traits at 540ppmCO
2
displayed strong correlations with the
number of buds and shoots. Axes 1 and 2 accounted for 55.1% and
59.7% of the explained variance, respectively.
4 Discussion
4.1 Environmental factors and growth traits
in A. contorta
We specifically focused on environmental factors and
established a standard for assessing their significant influence on
B
CD
A
FIGURE 7
Dry weight of each part of A.contorta and allocation of dry weight under two CO
2
concentrations (400 ppm, 540 ppm), and two rhizome directions
(horizontal rhizome planting, H; and vertical rhizome planting, V). (A) stem weight, (B) leaves weight, (C) rhizome and root weight, (D) allocation of
dry weight. Letters on the graph indicate significant differences at the 5% level, based on Duncan’s test. Bars indicate standard errors.
Park and Kim 10.3389/fpls.2024.1359783
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optimal flowering in mature A. contorta. During the reproductive
phase, RLI was significantly lower (approximately 40%) than that
during the vegetative phase, and the soil water content was notably
higher than that during the vegetative phase (Figures 1A,B).
Appropriate light intensity has been recognized as a significant
driver of growth in mature A. contorta. Adequate light during the
vegetative phase can enhance photosynthesis and establish a
resource base for future reproductive efforts (Wimalasekera,
2019). However, excessive light such as RLI 100% can disrupt
growing and flowering of mature A. contorta (Park et al., 2019;
Wimalasekera, 2019). This inhibition can be attributed to various
factors based on prior research, including photoinhibition, altered
hormone regulation, and increased oxidative stress in plants
exposed to excessively intense light conditions (Huang et al.,
2019;Maai et al., 2020;Bassi and Dall'Osto, 2021;Roeber
et al., 2021).
There was a negative correlation between RLI and soil water
content (-0.512, p< 0.05) in both the vegetative and reproductive
phase, with high light intensity identified as a factor contributing to
a decrease in soil water content. Due to the higher RLI, A. contorta
grown in arid soil conditions of the vegetative phase group
exhibited inferior growth compared to the reproductive phase
group and did not flower sufficiently. Adequate soil water is
essential for nutrient uptake, photosynthesis, and overall plant
health (Zainul et al., 2020;Chtouki et al., 2022). Without
sufficient water, plants struggle to maintain their physiological
processes and reach the reproductive phase, which is vital for
successful flowering and reproductive success (Jomo et al., 2015;
Chauhan et al., 2019).
The field survey also explored the role of soil nutrients,
specifically aiming to identify the precise soil nutrient levels that
are essential for optimal growth. While no significant differences in
soil nutrient levels were observed between the vegetative and
reproductive phase during the field survey (Figure 1), this
suggests that the soil may contain an appropriate abundance of
the necessary nutrients to support sufficient growth and flowering
(Uchida, 2000;Pestana et al., 2005;Fathi, 2022). Prior research has
consistently demonstrated that insufficient or excessive soil nutrient
levels can significantly hinder growth and flowering processes
(Uchida, 2000), highlighting the profound influence of nutrient
availability on plant reproduction. While these vegetative and
reproductive phases contain similar soil nutrient contents, the
discrepancy in flowering can be attributed to differences in RLI
and soil water content. These environmental factors were found to
have a strong correlation with the growth characteristics and
number of flowers of A. contorta (Figure 4). This implies that
under suitable environmental conditions, A. contorta exhibits
optimal growth, which is essential for flowering.
In addition to environmental factors from the field surveys, the
results of the greenhouse experiment also demonstrated that
elevated CO
2
concentrations had a hindering effect on the growth
of mature A. contorta. There was a significant decrease in stem
length, internode length, leaf size, and branch development under
540ppmCO
2
compared to 400ppmCO
2
(Figure 6). Some plant
responses, such as increased leaf thickness and mesophyll size,
may enhance photosynthesis and growth (Prior et al., 2011;Zhang
et al., 2013;Broughton et al., 2016;Choi et al., 2017;Chavan et al.,
2019;Thruppoyil and Ksiksi, 2020). However, in other plants,
specific changes in stomatal conductance and biochemical
composition can counteract these positive effects, resulting in
variable outcomes for growth and photosynthesis under elevated
CO
2
conditions (Poorter and Perez-Soba, 2002;Wang et al., 2012;
Zheng et al., 2019). Elevated CO
2
levels reduce stomatal
conductance. This reduction decreases water loss but also limits
CO
2
diffusion into the leaf, affecting photosynthesis (Poorter and
Perez-Soba, 2002;Wang et al., 2012). This reduction can affect the
overall photosynthetic efficiency and, subsequently, growth.
Interestingly, in our experiment at 540ppmCO
2
,eventhough
there was a higher leaf chlorophyll content, the aboveground
growth was not as favorable. This is because it is speculated that
FIGURE 8
Number of flowers of A. contorta under two CO
2
concentrations and two rhizome directions.
Park and Kim 10.3389/fpls.2024.1359783
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this additional resource allocation is directed towards other parts,
such as rhizomes, root buds, and shoots. Such resource allocation
can indicate trade-offs between biological growth and reproductive
structures (Ziska et al., 2004;McPeek and Wang, 2007;Qin et al.,
2022). Over time, plants exposed to elevated CO
2
may undergo
photosynthetic acclimation, where the initial increase in
photosynthetic rate plateaus or decreases due to downregulation
of Rubisco activity and other photosynthetic enzymes (Zhang et al.,
2013;Broughton et al., 2016). This acclimation can limit growth
benefits from increased CO
2
.
It exhibited higher C/N ratios in stem, rhizome, flower and
ovary parts except for the leaves at the 400ppmCO
2
condition
(Figure 10). The different C/N ratios in leaves compared to other
plant parts result from their influence on carbon and nitrogen
levels. Carbon levels are regulated through leaves by affecting
photosynthetic capabilities and carbohydrate export, while
nitrogen quantities are modulated by their transport from stems
to reproductive tissues (Yasumura, 2009). Plants possess the ability
to independently govern carbon and nitrogen, a crucial trait in
natural environments where external factors can readily disrupt the
delicate carbon-nitrogen balance (Yasumura, 2009). In addition,
elevated CO
2
can affect nitrogen assimilation and utilization,
leading to lower nitrogen content in tissues (Choi et al., 2017;
Chavan et al., 2019). Since nitrogen is vital for chlorophyll and
B
CD
A
FE
FIGURE 9
Reproductive (sexual and asexual) traits of A.contorta under two CO
2
concentrations and two rhizome directions. (A) flower longevity, (B) perianth
size, (C) diameter of utricle, (D) pollen diameter, (E) number of underground buds, (F) number of shoots. Letters on the graph indicate significant
differences at the 5% level, based on Duncan’s test. Bars indicate standard errors.
Park and Kim 10.3389/fpls.2024.1359783
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protein synthesis, its reduced availability can limit growth despite
the increased photosynthetic potential.
Our study also revealed a noticeable difference in potassium
absorption under elevated CO
2
conditions (Figure 5D). Potassium
serves as a catalyst for a range of enzymes and plays a crucial role in
regulating the intracellular osmotic balance and facilitating the
transport of membrane proteins (Ye et al., 2019). Additionally, it
has a significant impact on carbohydrate transportation in plants,
contributing to their overall metabolism and resilience to stress
factors (Wang and Wu, 2013;Nieves-Cordones et al., 2019). At
540ppmCO
2
, the enhanced potassium absorption, likely
attributable to its involvement in photosynthesis, may not have
been the predominant factor affecting the flowering of A. contorta.
These results underscore the intricate interplay between CO
2
concentrations and various physiological and morphological traits
in A. contorta.
4.2 Growth traits and their impact on
reproductive traits in A. contorta
Our study revealed variations in the growth traits of A. contorta,
such as thicker stems, longer internodes, increased leaf area, and
more flowers in the reproductive phase (Figure 2). Before plants
reach the flowering and reproduction stages, most plants go
through a period of vegetative growth (Sola and Ehrlen, 2007).
B
CD
E
A
FIGURE 10
C/N ratio of each part of A.contorta under two CO
2
concentrations and two rhizome directions. (A) stem C/N ratio, (B) leaf C/N ratio, (C) rhizome
C/N ratio, (D) flower C/N ratio, (E) ovary C/N ratio.
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Vegetative and reproductive phases can be viewed as developmental
phases where new organs continuously develop, each exhibiting
distinct morphological traits such as internode length, leaf area, and
cell size (Poethig, 2003;Huijser and Schmid, 2011;Wang et al.,
2011). During this period, plants typically experience a rapid
increase in their ability to perform photosynthesis and their
overall size and mass (Sola and Ehrlen, 2007). Generally, plants
develop reproductive organs only during the adult vegetative phase
(Araki, 2001). This also aligns with the findings of Park et al. (2019),
indicating that when an appropriate level of RLI is provided, A.
contorta growth becomes more vigorous and has flowers. These
observations underscore the importance of achieving a minimum
level of growth during the transition from the vegetative to the
reproductive phase, which is essential for successful flowering
(Araki, 2001). Furthermore, the observed higher C/N ratio in the
reproductive phase compared to the vegetative phase (Figure 3)
carries substantial implications for nutrient availability and
allocation (Uchida, 2000). As documented in earlier research
(Zhang et al., 2022), plants may allocate resources toward
nitrogen uptake when soil nitrogen levels exceed optimal ranges,
potentially delaying or inhibiting the initiation of the flowering
process. This phenomenon is often observed when plants adapt
their resource allocation strategies to optimize their survival and
reproduction under varying environmental conditions (Körner,
2015). Resource allocation in A. contorta refers to the distribution
of limited resources among different physiological processes,
including growth and reproduction (Hartmann et al., 2020).
When an appropriate level of resources is available, as indicated
by factors such as proper RLI and soil nutrient levels, plants allocate
more resources to growth (Tandon et al., 2020). This allocation
leads to thicker stems, longer internodes, and increased leaf areas,
all of which are associated with a more vigorous growth phase
(Tandon et al., 2020).
In greenhouse experiments, as in the field survey, plant growth
in response to CO
2
concentration contributed to the transition from
the vegetative to the reproductive phase. Adequate vegetative
growth, as observed at 400ppmCO
2
, characterized by long and
thick stems and abundant leaves, was associated with a higher
number of flowers (Figures 6,10). On the other side, at
540ppmCO
2
, insufficient growth was observed, which hindered
flowering (Figures 6,10). In addition, at 400ppmCO
2
,flowering
occurred earlier than under other conditions, while at 540ppmCO
2
,
flowering was hindered. This may be associated with a greater
allocation of resources to aboveground structures rather than
belowground structures (Figure 7D). Elevated CO
2
concentration
also had an additional effect on plant flowering. These findings are
TABLE 1 Stigmatic receptivity of A. contorta under two CO
2
concentrations and two rhizome directions.
400ppmCO
2
540ppmCO
2
Horizontal rhizome
3.00 ± 0.00 (+++) /
3.00 ± 0.00 (+++) /
2.67 ± 0.33 (+++) /
2.33 ± 0.33 (++) /
2.33 ± 0.33 (++) /
2.00 ± 0.00 (++) /
2.33 ± 0.33 (++) /
2.67 ± 0.33 (+++) /
2.33 ± 0.33(++) /
2.67 ± 0.33 (+++) /
Vertical rhizome
3.00 ± 0.00 (+++) 1.67 ± 0.33 (++)
2.80 ± 0.13 (+++) 2.00 ± 0.00 (++)
2.90 ± 0.10 (+++) 2.67 ± 0.33 (+++)
3.00 ± 0.00 (+++) 2.00 ± 0.00 (++)
3.00 ± 0.00 (+++) 1.33 ± 0.33 (+)
2.80 ± 0.13 (+++) 1.00 ± 0.00 (+)
2.90 ± 0.10 (+++) 1.33 ± 0.33 (+)
2.50 ± 0.17 (++) 1.00 ± 0.00 (+)
2.90 ± 0.10 (+++) 1.67 ± 0.33 (++)
2.80 ± 0.13 (+++) 1.33 ± 0.33 (+)
+++, strong receptivity; ++, moderate receptivity; +, weak receptivity; /, no flower.
TABLE 2 Proportion of the stained pollen grains at A. contorta
individuals (%).
400ppmCO
2
540ppmCO
2
Horizontal rhizome
91.67 ± 1.67 /
89.33 ± 0.67 /
93.33 ± 3.33 /
91.67 ± 1.67 /
88.33 ± 1.67 /
91.67 ± 1.67 /
93.33 ± 1.67 /
90.00 ± 2.89 /
91.67 ± 1.67 /
91.67 ± 1.67 /
Vertical rhizome
90.00 ± 1.18 81.67 ± 4.41
97.00 ± 0.82 85.00 ± 2.89
94.70 ± 1.58 71.67 ± 6.01
93.50 ± 1.45 74.00 ± 2.08
93.60 ± 1.48 80.00 ± 2.89
96.00 ± 0.67 70.00 ± 2.89
95.30 ± 1.52 75.00 ± 2.89
95.50 ± 0.90 74.33 ± 2.33
95.60 ± 1.54 85.00 ± 2.89
94.50 ± 1.17 77.67 ± 1.45
n=30.
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in agreement with those of previous research (Jablonski et al., 2002),
which also highlighted the influence of CO
2
concentration on the
reproductive traits of various plant species.
Our study also revealed significant reductions in flower
longevity, decreased flower abundance, and smaller flower size
under 540ppmCO
2
(Figure 9). In the same context, several plant
species (Trifolium pratense, Capsicum annuum,andCucurbita
pepo)exhibitedareducednumberofflowers and shortened
flower longevity under elevated CO
2
conditions (Rusterholz and
Erhardt, 1998;Lopez-Cubillos and Hughes, 2016). Conversely,
different plant species (Phalaenopsis Queen Beer, Lotus
corniculatus,Gerbera jamesonii, and Vitis vinifera L.) produce a
greater number of larger flowers and experience extended flower
longevity in response to elevated CO
2
conditions (Rusterholz and
Erhardt, 1998;Lopez-Cubillos and Hughes, 2016;Arrizabalaga-
Arriazu et al., 2020). This highlights the divergent responses of plant
species to elevated CO
2
, indicating that the impact varies depending
on the specific plant type. Remarkably, traits like stigma receptivity
and pollen viability also demonstrated increased vitality at the
400ppmCO
2
condition, which may contribute to the reproductive
success of A. contorta. This parallels a study on maize crop
production, where elevated CO
2
concentrations were found to
have adverse effects not only on stigma receptivity and pollen
viability but also on reproductive processes and yield (Prasad
et al., 2006;Bokshi et al., 2021).
Moreover, it’s important to highlight that 540ppmCO
2
appeared to be less favorable for overall growth, thereby
promoting asexual reproduction which were the number of
underground buds and shoots (Figures 9E,F). In the elevated
CO
2
, the absence of flowering suggests that plants may have
adopted an asexual reproduction strategy due to the challenging
conditions for sexual reproduction. This trade-off highlights the
flexibility of the reproductive strategies of mature A. contorta,
suggesting dynamic resource allocation in response to different
environmental cues to maximize overall reproductive fitness. This
phenomenon aligns with observations in other plant species, such
as Cirsium arvense and Taraxacum officinale (Ziska et al., 2004;
McPeek and Wang, 2007). This shift towards increased asexual
reproduction at 540ppmCO
2
could potentially lead to reduced
genetic diversity. Such a change in reproductive strategy has
implications for genetic variation, since it may limit the
introduction of new genetic variations typically associated with
TABLE 3 Two-way analysis of variance results for traits of A. contorta in the greenhouse experiments; Fstatistics are shown.
Traits of A. contorta CO
2
concentrations Rhizome directions
CO
2
concentrations
×
Rhizome directions
Growth traits
Stem length 8.483* 28.980*** 0.368
Internode length 24.155*** 24.418*** 2.001
Stem thickness 0.181 15.232*** 0.454
Number of branches 17.564*** 14.471*** 31.465***
Total branch length 15.189*** 33.373*** 9.405**
Number of leaves 4.515* 30.746*** 0.418
Single leaf area 59.127*** 20.065*** 5.421*
Total leaf area 22.367*** 42.636*** 1.509
Chlorophyll content 5.491* 7.086* 0.038
Dry stem weight 7.445* 28.980*** 0.448
Dry leaf weight 0.338 7.300* 1.354
Dry rhizome weight 0.137 0.196 0.078
Sexual reproductive traits
Number of flowers 13.51** 17.581*** 10.603**
Flower longevity 37.136*** 7.631** 10.089**
Perianth size 22.862*** 7.366* 8.214**
Diameter of utricle 34.269*** 9.295** 5.930*
Pollen grain size 228.499*** 233.732* 214.661***
Stigmatic receptivity 180.523*** 46.536*** 20.401***
Pollen viability. 1306.355*** 724.843*** 609.611***
Asexual reproductive traits
Number of buds 70.248*** 46.035*** 0.637
Number of shoots 5.126* 3.821 1.263
The two treatments were CO
2
concentrations (400ppmCO
2
and 540ppmCO
2
) and rhizome directions (horizontal and vertical). df=1, 39 for traits. Significant effects are shown in boldface (*, p<
0.05; **, p< 0.01; ***, p< 0.001).
Park and Kim 10.3389/fpls.2024.1359783
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sexual reproduction (Nakonechnaya et al., 2012;Nam et al., 2020;
Yu et al., 2021).
Therefore, based on vigorous growth, mature A. contorta (e.g.
400ppm/V) may strategically allocate resources to maximize its
sexual reproductive success; otherwise, it (eg. 540ppm/H) may
invest in asexual reproduction (Figure 11). In response to elevated
CO
2
concentrations, these findings suggest potential shifts in
growth dynamics and reproductive patterns, while also shedding
light on the broader ecological and evolutionary implications of
resource allocation strategies in plant reproduction under the global
environmental changes.
4.3 Interactive effects of elevated CO
2
and
rhizome direction on A. contorta
All morphological differences were influenced by either CO
2
concentrations or the direction of the rhizomes (Table 3). A direct
influence of CO
2
concentration on the number of shoots was
observed, indicating that the production of new shoots from the
rhizome system was stimulated by elevated CO
2
concentration. This
finding aligns with previous research highlighting the impact of
elevated CO
2
on increased asexual reproduction (Ziska et al., 2004;
McPeek and Wang, 2007).
Rhizome direction also has emerged as a key determinant
influencing various aspects of plant morphology, particularly stem
thickness and the weight of dry leaves (Table 3). This suggests that the
rhizome direction plays a pivotal role in shaping the structural
characteristics of the aboveground parts of the plant, potentially by
affecting water, nutrient uptake and resource allocation (Silva et al.,
2019). In situations where environmental conditions undergo rapid
and unpredictable changes, the adaptability conferred by horizontal
rhizomes becomes particularly advantageous (Berntson and
Woodward, 1992). They are well-suited for scenarios where
securing water resources swiftly is crucial, especially in arid regions.
The distribution of rhizomes is recognized as critical for a plant’s
ability to acquire essential water and nutrients (Miao et al., 1992).
In contrast, vertical rhizomes, which had more flowers than
horizontal rhizome (Figure 7), can contribute to genetic stability
and the promotion of evolutionary processes by facilitating the
reliable transfer of genetic information from one generation to the
next (Kobayashi, 2019). This is achieved through sexual
reproduction, making them potentially more suited for stable and
consistent environments (Liu et al., 2021). In the context of
evolution, the adaptability of rhizome direction becomes a crucial
factor (Berntson and Woodward, 1992). The documented
enhancements in reproductive success associated with rhizome
systems further support the idea that belowground factors play a
significant role in influencing aboveground reproductive traits.
The influence of hormonal signaling pathways, such as auxin in
the case of vertical rhizomes (Scott, 1972), in regulating growth
responses to environmental cues further underscores the complex
interplay between rhizome direction and environmental
adaptability. Conversely, horizontal rhizomes may obstruct
hormonal movement, leading to suboptimal growth conditions
(Mellor et al., 2020). Therefore, under the same CO
2
concentration, horizontal rhizomes had a more detrimental effect
on growth and development than vertical rhizomes.
The most remarkable outcomes stemmed from the interaction
between CO
2
concentrations and rhizome directions (Table 3). This
FIGURE 11
Canonical correlation analysis (CCA) plots to determine the relationships between growth and reproductive traits. The percentage (%) of each axis
represents the explained variance. The dotted curves indicate the groups of individuals in the treatment groups, which are represented by the blue
triangles. The arrows are strongly correlated with the axis.
Park and Kim 10.3389/fpls.2024.1359783
Frontiers in Plant Science frontiersin.org16
interaction led to substantial variations in a range of morphological
and reproductive traits, such as branch number, total branch length,
single leaf area, and total leaf area (Figure 11,Table 3). These changes
signify an impact in the plant’s growth and structural characteristics
when these factors are considered simultaneously. Furthermore, the
interaction of CO
2
concentrations and rhizome direction significantly
influenced sexual reproductive traits (Table 3; the number of flowers,
flower longevity, perianth size, diameter of utricle, pollen grain size,
stigmatic receptivity, and pollen viability). These changes in
reproductive traits have important implications for the plant’s
overall reproductive success, suggesting that environmental
variations can have far-reaching effects on its life cycle and
ecological role. Therefore, our findings emphasize the intricate
nature of plant responses to changes in CO
2
concentrations and
rhizome directions, providing valuable insights into the adaptability
of A. contorta. Moreover, understanding the ecological implications
of these responses is crucial, especially in the context of ongoing
global environmental changes.
4.4 Limitations and challenges
Our research provides valuable insights into how environmental
factors affect A. contorta’s growth and reproduction, yet it’s
conducted in controlled settings that may not fully reflect natural
ecosystem complexities. The study’s duration may also fall short of
capturing A. contorta’s long-term adaptations to increased CO
2
,
indicating the necessity for longer observation in future research.
The variability in plant responses to elevated CO
2
across different
species, genotypes, and individuals highlights the challenge of
generalizing findings and necessitates a broader spectrum of
studies. Interactions with other environmental factors such as
temperature, water availability, and nutrient levels further
complicate the isolation of CO
2
effects. Additionally, the detailed
physiological and molecular mechanisms underlying CO
2
’sinfluence
on plant growth remain partially understood, emphasizing the need
for advanced, interdisciplinary approaches to unravel
these complexities.
5 Conclusion
Our study provided a comprehensive understanding of the various
factors that influence the probability of flowering in mature A. contorta
(Figure 12). The research encompassed field surveys and greenhouse
treatments, revealing the intricate interplay of environmental and
physiological elements in shaping plant reproductive patterns. Field
surveys have underscored the pivotal roles of light intensity, soil water
content, and rhizome direction in influencing growth and flowering.
Greenhouse experiments have revealed the interactive effects of CO
2
concentration and rhizome conditions on flowering. CO
2
concentration and rhizome direction also influenced growth traits,
emphasizing the significance of substantial vegetative growth for
successful flowering. Additionally, elevated CO
2
concentrations
exhibited diverse negative effects on the mature A. contorta’s
reproductive traits, impacting flower size, longevity, stigma
receptivity, and pollen viability, showing the complex interplay
between environmental conditions and reproductive outcomes.
Remarkably, our findings highlighted how environmental factors can
inhibit growth and, in turn, hinder the sexual reproduction of mature
A. contorta. Moreover, it triggers a shift towards increased asexual
reproduction at elevated CO
2
concentrations, potentially leading to
reduced genetic diversity. These findings provide valuable insights into
the adaptability and resource allocation strategies of mature A. contorta
in response to ever-changing environmental cues. Moreover, our study
sheds light on the broader ecological and evolutionary implications of
these interactions, emphasizing the crucial role of environmental
influences in shaping the reproductive patterns of mature A. contorta.
FIGURE 12
Comprehensive understanding of various factors that influence the asexual and sexual reproductions in mature A. contorta.
Park and Kim 10.3389/fpls.2024.1359783
Frontiers in Plant Science frontiersin.org17
Data availability statement
The original contributions presented in the study are included
in the article/supplementary material. Further inquiries can be
directed to the corresponding author.
Author contributions
S-HP: Conceptualization, Data curation, Formal analysis,
Investigation, Methodology, Visualization, Writing –original
draft. JK: Conceptualization, Funding acquisition, Methodology,
Project administration, Supervision, Writing –review & editing.
Funding
The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. This work
was supported by Basic Science Research Program through the
National Research Foundation of Korea (NRF) funded by the
Ministry of Education (NRF-2021R1I1A2041895) and by Korea
Environment Industry & Technology Institute (KEITI) through
‘Wetland Ecosystem Value Evaluation and Carbon Absorption
Value Promotion Technology Development Project’, funded by
Korea Ministry of Environment (MOE)(RS-2022-KE002025).
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
The author(s) declared that they were an editorial board
member of Frontiers, at the time of submission. This had no
impact on the peer review process and the final decision.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
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