Methane uptake responses to heavy rainfalls co-regulated by seasonal timing and plant composition in a semiarid grassland

Abstract

Heavy rainfalls caused by global warming are increasing widespread in the future. As the second greenhouse gas, the biological processes of methane (CH4) uptake would be strongly affected by heavy rainfalls. However, how seasonal timing and plant composition affect CH4 uptake in response to heavy rainfalls is largely unknown. Here, we conducted a manipulative experiment to explore the effects of heavy rainfall imposed on middle and late growing season stage on CH4 uptake of constructed steppe communities including graminoid, shrub and their mixture in Inner Mongolia, China. The results of mixed effect model showed that both heavy rainfalls decreased CH4 uptake. Nevertheless, the effect magnitude and the pathways were varied with seasonal timing. Relatively, the late heavy rainfall had larger negative effects. Structural equation model suggested that late heavy rainfall decreased CH4 uptake through decreased diffusivity, pmoA abundance, and NH4⁺-N content, as products of high soil water content (SWC). However, middle heavy rainfall decreased CH4 uptake only by increasing SWC. Additionally, aboveground biomass (AGB) had negative effects on CH4 uptake under both heavy rainfalls. Additionally, plant composition not only affected CH4 uptake but also regulated CH4 uptake in response to heavy rainfalls. Late heavy rainfall had less negative effect on CH4 uptake in graminoid community than in other two communities, in coincidence with less reduction in NH4⁺-N content and less increase in SWC and AGB. In contrast, we did not observe obvious difference in effects of middle heavy rainfall on CH4 uptake across three communities. Our findings demonstrated that magnitude and pathways of heavy rainfall effects on CH4 uptake were strongly co-regulated by seasonal timing and plant composition.

Full text

Frontiers in Ecology and Evolution 01 frontiersin.org
Methane uptake responses to
heavy rainfalls co-regulated by
seasonal timing and plant
composition in a semiarid
grassland
ZhenzhenZheng
1, FuqiWen
1, CongjiaLi
1, ShuntianGuan
1,
YunqiXiong
1, YuanLiu
1, RuyanQian
1, MengboLv
1, ShaoruiXu
1,
XiaoyongCui
1, YanfenWang
2,3, YanbinHao
1,2 and LinfengLi
3*
1 College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China, 2 Beijing Yanshan
Earth Critical Zone National Research Station, University of Chinese Academy of Sciences, Beijing,
China, 3 College of Resources and Environment, University of Chinese Academy of Sciences, Beijing,
China
Heavy rainfalls caused by global warming are increasing widespread in the future.
As the second greenhouse gas, the biological processes of methane (CH4) uptake
would be strongly aected by heavy rainfalls. However, how seasonal timing
and plant composition aect CH4 uptake in response to heavy rainfalls is largely
unknown. Here, weconducted a manipulative experiment to explore the eects
of heavy rainfall imposed on middle and late growing season stage on CH4 uptake
of constructed steppe communities including graminoid, shrub and their mixture
in Inner Mongolia, China. The results of mixed eect model showed that both
heavy rainfalls decreased CH4 uptake. Nevertheless, the eect magnitude and
the pathways were varied with seasonal timing. Relatively, the late heavy rainfall
had larger negative eects. Structural equation model suggested that late heavy
rainfall decreased CH4 uptake through decreased diusivity, pmoA abundance,
and NH4+-N content, as products of high soil water content (SWC). However,
middle heavy rainfall decreased CH4 uptake only by increasing SWC. Additionally,
aboveground biomass (AGB) had negative eects on CH4 uptake under both
heavy rainfalls. Additionally, plant composition not only aected CH4 uptake but
also regulated CH4 uptake in response to heavy rainfalls. Late heavy rainfall had
less negative eect on CH4 uptake in graminoid community than in other two
communities, in coincidence with less reduction in NH4+-N content and less
increase in SWC and AGB. In contrast, wedid not observe obvious dierence in
eects of middle heavy rainfall on CH4 uptake across three communities. Our
findings demonstrated that magnitude and pathways of heavy rainfall eects on
CH4 uptake were strongly co-regulated by seasonal timing and plant composition.
KEYWORDS
CH4, climate extremes, greenhouse gasses, methanotrophs, community composition,
precipitation
OPEN ACCESS
EDITED BY
Kerou Zhang,
Chinese Academy of Forestry, China
REVIEWED BY
Gang Fu,
Institute of Geographic Sciences and Natural
Resources Research (CAS), China
Xiaolong Huang,
Nanjing Institute of Geography and Limnology
(CAS), China
*CORRESPONDENCE
Linfeng Li
lilinfeng@ucas.ac.cn
SPECIALTY SECTION
This article was submitted to
Population, Community, and Ecosystem
Dynamics,
a section of the journal
Frontiers in Ecology and Evolution
RECEIVED 22 January 2023
ACCEPTED 17 February 2023
PUBLISHED 08 March 2023
CITATION
Zheng Z, Wen F, Li C, Guan S, Xiong Y, Liu Y,
Qian R, Lv M, Xu S, Cui X, Wang Y, Hao Y and
Li L (2023) Methane uptake responses to heavy
rainfalls co-regulated by seasonal timing and
plant composition in a semiarid grassland.
Front. Ecol. Evol. 11:1149595.
doi: 10.3389/fevo.2023.1149595
COPYRIGHT
© 2023 Zheng, Wen, Li, Guan, Xiong, Liu, Qian,
Lv, Xu, Cui, Wang, Hao and Li. This is an open-
access 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 08 March 2023
DOI 10.3389/fevo.2023.1149595

Zheng et al. 10.3389/fevo.2023.1149595
Frontiers in Ecology and Evolution 02 frontiersin.org
Introduction
Methane (CH
4
) is a powerful greenhouse gas and strongly
contributes to global warming and resultant changes in precipitation,
as the global warming potential is 28–36 times than that of carbon
dioxide (CO
2
) at 100-y timescale (Jiang etal., 2012; Fischer and Knutti,
2016; Otto etal., 2018; IPCC, 2021). Aerobic soils are important CH4
sink, in which 9–47 Tg CH
4
year
−1
from the atmosphere was oxidated
by methanotroph through methane monooxygenase (MMO) (Elango
etal., 1997; Fest etal., 2015; Yue etal., 2019, 2022). e subunit genes
of MMOs, specically pmoA, are used as biomarker genes for the
presence and abundance of bacterial methanotrophs (Fest etal., 2015;
Tentori and Richardson, 2020). erefore, understanding eects of
changes in precipitation on CH
4
uptake in drylands and underlying
microbial mechanisms have great implications for prediction of future
carbon cycling and its feedback to climate changes.
It has been conrmed that precipitation changes are expected to
signicantly inuence the intensity of CH
4
sinks (Aronson etal., 2019;
Martins et al., 2021). For example, increased precipitation by 30%
signicantly increased CH
4
uptake in temperate deserts (Yue etal.,
2019). In contrast, CH
4
uptake was decreased and unchanged by
increased precipitation in alpine meadows and in degraded steppe
grasslands, respectively (Chen W. W. etal., 2013; Wu etal., 2020).
Meta-analysis studies suggested that increased precipitation can
decrease CH
4
uptake in terrestrial ecosystems at the global scale (Chen
H. etal., 2013; Yan etal., 2018). Although these results highlighted the
important role of increased precipitation in regulating CH4 uptake in
aerobic soils, to date, there is great uncertainty about the eects of
extreme precipitation with several days (e.g., heavy rainfall events),
rather than chronic increases in precipitation at seasonal timescale, on
CH4 uptake.
Eects of chronic increases in precipitation and heavy rainfall on
CH
4
uptake may belargely dierent. Soil moisture controlled CH
4
uptake through aecting the methanotroph community and altering
air-soil diusion (Wei etal., 2015). A bell-shaped relationship was
observed between soil moisture and CH
4
uptake with CH
4
uptake
reached the peak at intermediate soil moisture (Dijkstra etal., 2013;
Li etal., 2016; Zhang etal., 2021). Above the optimum soil moisture,
soil moisture would limit oxygen (O
2
) diusion in soils and depress
the activity of methanotroph communities, inhibiting CH
4
uptake
(Curry, 2007; Liptzin etal., 2011; Zhuang etal., 2013). Hence, slight
increases in precipitation may promote methanotroph community
and thereby increase CH
4
uptake while heavy rainfall caused
saturation soil moisture and thereby would reduce CH
4
uptake.
Additionally, CH
4
uptake is sensitive to soil ammonium (NH
4
+
-N)
and nitrate (NO
3
−
-N). NH
4
+
-N had an inhibiting eect on CH
4
uptake
mainly through replacing CH
4
to be oxidized by methanotroph
(Schnell and King, 1994; Yue et al., 2022), while NO
3
−
-N had an
inhibiting eect on CH
4
uptake through changing methanotroph
activity and composition or enhancing soil oxidation potential and
environment (Le Mer and Roger, 2001; Yue et al., 2022). Increased
precipitation is likely to enhance soil inorganic nitrogen by
accelerating mineralization (Cabrera and Kissel, 1988; Bai etal., 2012).
In contrast, soil inorganic nitrogen may decline through leaching and
runo under heavy rainfalls (Borken and Matzner, 2009; Cregger
etal., 2014). us, slight increases in precipitation and heavy rainfall
are likely to induce opposite impacts on CH
4
uptake through the
pathway of NH4 +-N and NO3
−-N content.
Furthermore, seasonal timing and plant composition potentially
modulate CH4 uptake in response to heavy rainfalls. Previous studies
suggested that seasonal timing strongly regulates eects of heavy
rainfall on multiple ecosystem attributes such as soil water, carbon,
and nitrogen availability, as well as plant biomass and phenology (Li
etal., 2019; Post and Knapp, 2020; Li etal., 2022). erefore, weexcept
that impacts of heavy rainfall on CH4 uptake may bealso regulated by
seasonal timing. Indeed, Zhao etal. (2017) found that CH4 uptake was
reduced by 62% and 45% during the period of middle and late heavy
rainfall, respectively. Besides, there were signicant dierences in the
composition and abundance of methanogens in soil with dierent
plant species, resulting in dierent potential of CH
4
uptake (Dai etal.,
2015). For example, CH
4
uptake capacity was stronger in soil of oat
than that in native vegetation (Hüppi etal., 2022). Moreover, plant
communities with higher-diversity were less negatively aected by
oods and mature plants can withstand ooding better than seedlings
(Gattringer etal., 2017; Wright etal., 2017). Although several studies
had reported that plant community composition and seasonal timing
could moderate heavy rainfall eects on CH
4
uptake capacity (Liebner
etal., 2015; Tong etal., 2017; Zhao etal., 2017; Yue etal., 2022), it is
unknown the interactions on CH4 uptake in the face of heavy rainfall.
To explore the individual and especially interactive eects of
heavy rainfall timing and species composition on CH
4
uptake in
response to heavy rainfall, weconducted a eld experiment in which
heavy rainfall occurring in middle and late growing season were
imposed on plots with three experimental plant communities of
graminoids, shrubs and their combination, respectively.
Wehypothesized that: (1) Heavy rainfalls would suppress CH4 uptake
due to reduced diusivity and methanotrophs activity, regardless of
seasonal timing, (2) Heavy rainfall occurring in middle growing
season with high air temperatures may cause less saturated soil
conditions, thus CH4 uptake is likely to beless decreased by middle
heavy rainfall than late heavy rainfall, and (3) Plant community
composition would adjust CH4 uptake in response to heavy rainfalls
though soil moisture, inorganic content, aboveground biomass and
methanotrophs activities.
Materials and methods
Study site
We carried out the study at the Research Station of Animal
Ecology (44°18′ N, 116°45′ E 1079 m.a.s.l) in a semiarid grassland of
Inner Mongolia Autonomous Region, China. e study site has a
temperate continental semi-arid climate, of which the mean annual
precipitation (1953 to 2017) is 281 mm and the mean annual
temperature is 2.5°C. e plant species in the study region is mainly
dominated by xeric rhizomatous grasses, needle grasses and perennial
forbs such as Leymus chinensis, Stipa grandis and Medicago falcata.
e soil type in this experimental region is classied as chestnut soil
consisting of 60% sand, 18% clay and 17% silt.
Experiment design
e experiment was began in 2012. In this study, wereported the
data measured in 2021. According to the statistical analysis of

Zheng et al. 10.3389/fevo.2023.1149595
Frontiers in Ecology and Evolution 03 frontiersin.org
∼60-year (1953–2012) historical meteorological data provided by e
Xilin Gol League Meteorological Administration, the longest
continuous rainfall period of daily precipitation (≥ 3 mm) was 20 days
during the growing season. e total eective precipitation was
calculated over all 20 days periods, which was 250 mm. us, heavy
rainfall was dened as 250 mm rainfall over 20 d in this study (12.5 mm
d
−1
) (Hao etal., 2017). We used a two-way split-plot experiment
design to study the eect of heavy rainfall on CH
4
uptake joint control
of seasonal timing and plant composition. e main treatment had 9
plots and each main plot was made up of 3 sub-plots, thus there were
total 27 sub-plots in heavy rainfall treatments experiment
(Supplementary Figure S1). Specically, three heavy rainfall
treatments were set up in the main plots with three replicates: ambient
control, mid-stage heavy rainfall (HR-mid, 15 July-5 August) and late-
stage heavy rainfall (HR-late, 15 August-5 September), respectively.
ree plant community compositions were set up in the sub-plots:
graminoid (Leymus chinensis and Stipa grandis), shrub (Caragana
microphylla and Artemisia frigida) and their mixture. Plant seeds of
dominant local species were cultivated at the start of the study in early
May 2012. e total coverage of graminoids, shrub and mixture
community were 70, 80, and 75%, respectively.
Twenty-seven 2 m × 2 m sub-plots were established with 1-m
intervals between sub-plots. e ambient control sub-plots remained
uncovered year-round. Heavy rainfall treatment sub-plots were
covered with 27 m
2
rainout shelters (4.5 m × 6 m, height 3 m) to
prevent natural rainfall and greenhouse eect during the treatment
periods. e rainout shelters were made of transparent polyester ber
material to ensure no signicant shading. Non-target plant seedlings
in each subplot were weekly removed to maintain xed plant
community composition during the entire growing season.
CH4 flux and soil water content
measurements
Soil water content (SWC) and CH
4
uxes were measured three
times monthly during the growing season in 2021. Time domain
reectometry (TDR 300 Soil Moisture Meter) with 20 cm probe was
used to record SWC. CH
4
uxes were measured by laser-based fast
greenhouse gas analyzer with an in-house closed chamber. e data
collection frequency of 1 Hz was utilized to measure CH4 uxes (Kang
etal., 2018). e volume of cube chamber is 1.25 × 10
5
cm
3
, which was
equipped with two electric fans in the center of the chamber ceiling to
mix air concentration. Laser-based fast greenhouse gas analyzer has
two 20-m rubber internal pipes, which were used to connect with the
closed chamber through two 2-cm diameter holes on top of the
chamber. Two pipes were used to transport gas from the greenhouse
gas analyzer to the chamber and return from the chamber to the
analyzer. Each subplot had a stainless frame (length × width ×
height = 50 cm × 50 cm × 10 cm) with 2 cm wide water groove. Each
frame was installed and inserted 7 cm deep in soil and retained 3 cm
above ground. Enough water should beput into the grooves of frames
to guarantee gas tightness before mounting the chamber on the frame.
Gas sampling area in each sub-plot was measured between 9:00 am
and 10:00 am local time. In each sampling area, the gas in chamber
was measured for 10 min, and the chamber should beopened for
2 min before the next measurement. Wecalculated CH4 ux from the
linear slope.
Fc dc
dt
M
V
P
P
T
T
H=××××
00
0
where F is the CH
4
ux rate [mg/(m
2
·h)];
dc dt/
is the cumulative
growth rate of CH
4
; M and P represent the molar mass of CH
4
(g/mol)
and the air pressure (Pa), respectively; V
0
and P
0
represent the standard
molar volume (22.41 m3/mol) and standard air pressure (101,325 Pa),
respectively; T and T
0
represent the absolute temperature inside the
chamber (oK) and absolute temperature (oK), respectively; and H is
the eective height of the chamber (m).
Above-ground biomass measurement
Harvest method was used to measure above-ground biomass
(AGB). We harvested all aboveground living plant tissues in a
50 cm × 50 cm quadrat of each sub-plot on September 21st, 2021. All
plant tissues of each quadrat were put in the oven and dried at 65°C
until they had constant weight.
Soil property measurement
ree soil cores were taken in 20 cm deep in each plot using an
auger (2.5 cm in diameter) on September 21st, 2021. Roots and stones
of soil were removed from three core sets and homogenized by 2 mm
sieves. e extract was a mixture solution of 10 g fresh soil sample and
40 ml 0.5 M K2SO4 solution, which were shaken for 30 min in shaker.
Aer mixing well, NH4 +-N and NO3
−-N concentration of soil sample
were detected by a continuous ow automatic ion analyzer (SEAL
Analytical GmbH, Norderstedt, Germany) (Wachendorf etal., 2008).
DNA extraction and qPCR
DNA were extracted from fresh soil of 0.5 g by Power Soil DNA
Isolation Kit (MOBIO Laboratories, UnitedStates) according to the
specication information. DNA quality of soil was assessed by
NanoDrop2000 UV–Vis spectrophotometer (ermo Scientic,
Wilmington, Delaware, USA). e methanotrophic pmoA gene
abundance was determined by quantitative polymerase chain
reaction (qPCR) using Eppendorf Masterpiece realplex sequence
detection system (Applied Biosystems 7500/7600). Standard curves
were created with plasmid DNA in ten-fold serial dilutions. e
primer sets were used for pmoA: 5′-GGNGACTGGGACTTCTGG-3′
and 5′-CCGGMGCAACGTCYTTACC-3′. e 20 μL qPCR
reaction consisted of 1 μL DNA template, 0.2 μL of front and back
primer, 10.4 μL mixture solution of ROX and Takara SYBR®Premix
Ex Taq ™ (Perfect RealTime) and 8.4 μL sterile water. Aer the
reaction solution has been thoroughly mixed, the hole in the
96-well plate was lled with 20 μL qPCR reaction solution.
Additionally, the contamination was detected by adding 19 μL
qPCR reaction solution into the hole of 96-well plate without DNA
template during the experiment. e sequential reaction conditions
for the pmoA gene were set as: an initial denaturation at 95°C for
30 s, followed by 40 cycles at 95°C for 30 s, 60°C for 45 s, and 68°C
for 45 s, with a nal extension at 80°C for 30 s.

Zheng et al. 10.3389/fevo.2023.1149595
Frontiers in Ecology and Evolution 04 frontiersin.org
Statistical analyses
We conducted Duncan’s multiple comparison to test
differences of heavy rainfall with seasonal timing, plant
composition and their interaction effects on variables including
CH
4
uptake, soil water content, aboveground biomass, pmoA
abundance, NH
4+
-N and NO
3
−
-N content. Mixed-effects models
were conducted using the NLME package in R v.3.4.4 (R Core
Team, 2018) to compare the effects of middle and late growing
season heavy rainfall, plant composition, and the interaction
effects of plant composition and heavy rainfall on the above
variables, respectively. Structural equation model (SEM) analyzes
were performed using the piecewise SEM package to explore
direct and indirect impacts of heavy rainfall on CH
4
uptake
(Domeignoz-Horta etal., 2020). The most variation can explain
by this model, including low Akaike Information Criterion (AIC),
a nonsignificant Chi-squared test (p > 0.05) and high Comparative
Fit Index (CFI > 0.9).
Result
Seasonal dynamics and response of soil
moisture content to heavy rainfalls
Total growing season precipitation (GSP) of control, HR-mid
and HR-late were 351 mm, 513.16 mm, and 559.02 mm,
respectively. Regardless of seasonal timing, SWC was significantly
increased by heavy rainfalls in all three communities (p = 0.01 and
<0.0001 for HR-mid and HR-late, respectively; Figure 1 and
Table1). Overall, SWC in HR-late seems slightly higher than that
in HR-mid (Figures 1E,F; Supplementary Figure S2). Plant
community composition had no significant effects on SWC
(p = 0.54 for composition, Table 1). However, the SWC in
graminoid community was slightly less increased by heavy
rainfalls compared with shrub and mixture communities
(Supplementary Figure S2).
Response of CH4 uptake to heavy rainfalls
Over the growing season, averaged CH
4
uptake signicantly
decreased by HR-mid (p = 0.03) and HR-late (p < 0.0001) in all three
communities (Figures2E,F). e reductions were mainly occurred
during the period of the heavy rainfalls. Relatively, HR-late had
larger negatively eects on CH
4
uptake than HR-mid. ere were
signicant dierences of CH
4
uptake among three communities
with the least CH4 uptake in shrub community (p < 0.0001, Table1;
Supplementary Figure S3). HR-late eects on CH4 uptake depended
on plant composition (p = 0.03 for HR-late × Composition), with
the least decreased CH4 uptake in graminoid community than that
in other two communities. ere was also a marginally signicant
interaction between HR-mid × Composition (p = 0.08) on CH
4
uptake, but the eects of HR-mid on CH
4
uptake were similar across
three communities. Collectively, negative eects of heavy rainfalls
on CH
4
uptake modulated by seasonal timing and plant
community composition.
Response of pmoA abundance, AGB,
NH4+-N and NO3
−-N to heavy rainfalls
Similarly, heavy rainfalls eects on soil inorganic N content and
pmoA abundance were changed with seasonal timing and plant
community composition. Both two heavy rainfalls signicantly
declined pmoA abundance for three communities but the eects were
larger in mixture community than in other two communities (p= 0.01
and 0.0001 for HR-mid × Composition and HR-late × Composition)
(Figure3A; Table1). Regardless of plant composition, HR-mid and
HR-late unchanged and signicantly increased AGB, respectively
(Figure3B; Table1), NH4+-N was signicantly increased by HR-mid
but signicantly declined by HR-late in three communities
(Figure 3C). Similarly, HR-mid signicantly increased NO
3
−
-N
content (Figure 3D; Table 1), mainly in graminoid and mixture
communities. In contrast, HR-late had little eects on NO
3
−
-N content.
The influence of abiotic and biotic factors
on CH4 uptake
Structural equation model showed that SWC had directly negative
impacts on CH
4
uptake and pmoA abundance under two heavy
rainfalls. Additionally, CH
4
uptake negatively correlated with AGB
under two heavy rainfalls. However, CH4 uptake positively correlated
with pmoA abundance in HR-late but not in HR-mid. SWC had
signicantly positive impact on NH4+ under HR-mid, while opposite
impact between SWC and NH
4+
was found in HR-late. Moreover,
NH
4+
positively correlated with pmoA abundance only in HR-late.
NO3
− had no signicant relationships with CH4 uptake (Figure4). In
short, heavy rainfalls with dierent seasonal timing decreased CH
4
uptake through dierent pathways.
Discussion
Understanding extreme precipitation scenario on CH
4
uptake has
important implications for predicting future global climate changes
and terrestrial C cycling. To explore how seasonal timing and plant
composition aected CH
4
uptake in response to heavy rainfalls,
weconducted a manipulative experiment in a semiarid grassland of
Inner Mongolia, China. In this study, we identied CH
4
uptake in
response to heavy rainfall are regulated by independent and especially
interactive eects of heavy rainfall timing and plant composition. Our
results demonstrate that seasonal timing strongly controls size and
pathway of negative eects of heavy rainfall on CH
4
uptake and
importantly the regulating eects of plant composition on CH
4
uptake
response to heavy rainfall via soil water content, pmoA abundance,
NH4+-N content and AGB.
Heavy rainfalls decrease CH4 uptake
CH
4
uptake was signicantly decreased by heavy rainfalls,
regardless of seasonal timing and plant community composition in
our study (Figure2). Previous study showed that soil moisture and
CH
4
uptake had a hump-shaped relationship, where the optimum

Zheng et al. 10.3389/fevo.2023.1149595
Frontiers in Ecology and Evolution 05 frontiersin.org
moisture is 10 % -12 % for highest CH4 uptake in a semiarid and arid
soils (Dijkstra etal., 2011; Li etal., 2016; Yue etal., 2022). Moderate
soil moisture could signicantly promote CH
4
uptake, which could
besignicantly inhibited by too- low or too- high soil moisture (Van
den Pol-van Dasselaar etal., 1998; Dijkstra etal., 2011). In our study,
SWC were above 12% in all treatments throughout the growing
season. High soil moisture induced by heavy rainfalls would cause
anaerobic soil conditions, low soil oxygen (O
2
) concentrations and
CH
4
diusion (Figure1). Additionally, pmoA abundance decreased in
both two heavy rainfalls (Figure3A). Taken together, these results
A
B
C
FIGURE1
Seasonal dynamic and mean of soil water content under heavy rainfall treatments in graminoid (A,D), shrub (B,E), and graminoid +shrub (C,F) plots.
Dierent letters above bars in d, e and f indicate significant dierence among treatments at p≤ 0.05. The orange and blue shaded regions in a–c
indicate the periods of the HR-mid (heavy rain imposed in middle of the growing season, orange line) and HR-late (heavy rainfall imposed late in the
growing season, blue line) treatments, respectively. Error bars show one standard error of the mean.
TABLE1 p-Value from mixed-eect model analyzes of HR-mid and HR-late, community composition and their interactions on soil water content
(SWC), aboveground biomass (AGB), CH4 uptake, abundance of pmoA, and content of NH4+-N and NO3
−-N.
Fixed eect DF SWC AGB CH4
uptake
pmoA NH4+-N NO3
−-N
Num Den
HR-mid 1 24 0.01 0.44 0.04 <0.0001 <0.0001 0.04
HR-late 1 24 <0.0001 0.05 <0.0001 <0.0001 0.004 0.77
Composition 2 24 0.27 <0.0001 <0.0001 0.001 0.03 0.21
HR-mid × Composition 2 24 0.85 0.85 0.08 0.01 0.22 0.10
HR-late × Composition 2 24 0.49 0.95 0.03 0.0001 0.58 0.96
p-Values in bold are statistically signicant to an alpha value of 0.05.

Zheng et al. 10.3389/fevo.2023.1149595
Frontiers in Ecology and Evolution 06 frontiersin.org
suggested that experimental heavy rainfalls continuously decreased
CH
4
diusivity and O
2
availability and thus inhibited the activity of
methanotrophs, supporting our rst hypothesis. As a result, SWC
showed negatively relationship with CH
4
uptake under two heavy
rainfalls in our study (Figure4).
Magnitude and pathways of heavy rainfall
eects on CH4 uptake depend on seasonal
timing
Although two heavy rainfalls had negative eects on CH4 uptake,
the eect magnitude varied with seasonal timing. Consistent with the
second hypothesis, CH
4
uptake is less decreased by HR-mid than
HR-late in all three communities (Figure2). is may be because
HR-mid received less precipitation than HR-late (513.16 mm vs.
559.02 mm, Figure1). In addition, higher air temperatures during the
period of HR-mid would induce larger evapotranspiration. As a result,
HR-mid caused less saturated soil conditions than HR-mid, which was
reected by slightly lower SWC in HR-mid than in HR-late
(Figure1F). As discussed above, SWC had negative impacts on CH
4
uptake in our study. us, lower SWC and corresponding less
saturated soil conditions under HR-mid induced less reduction in
CH4 uptake.
Structural equation model showed that SWC and resultant
anaerobic conditions were main controller of CH4 uptake (Wei etal.,
2015; Zhou etal., 2021). AGB also had direct negative eects on CH4
uptake under both heavy rainfalls (Figure 4B). Previous studies
showed similar trends that increased AGB may contribute to
increasing soil water-holding capacity, maintaining high soil moisture
and inhibiting soil substrate availability. As a result, methanotrophs
activities and CH
4
oxidation in soil were inhibited (Robson etal.,
2007; Zhang etal., 2012; Tang etal., 2018). Besides, high SWC directly
and indirectly inhibited pmoA abundance through decreasing NH4+-N
content, ultimately, suppressing CH
4
uptake in HR-late. CH
4
was
oxidated by methanotroph, thus, it is not surprising that low pmoA
A
B
C
FIGURE2
Seasonal dynamic and mean of CH4 uptake under heavy rainfall treatments in graminoid (A,D), shrub (B,E), and graminoid +shrub (C,F) plots. Dierent
letters above bars in d, e and f indicate significant dierence among treatments at p≤ 0.05. The orange and blue shaded regions in a–c indicate the
periods of the HR-mid (heavy rain imposed in middle of the growing season, orange line) and HR-late (heavy rainfall imposed late in the growing
season, blue line) treatments, respectively. Error bars show one standard error of the mean.

Zheng et al. 10.3389/fevo.2023.1149595
Frontiers in Ecology and Evolution 07 frontiersin.org
abundance would limit CH
4
uptake (Degelmann etal., 2010; Zhang
etal., 2019; Kaupper etal., 2021;). Previous studies have found that the
process of methanotrophs using CH
4
as both an energy and carbon
source generally requires soil NH
4+
-N as N source (Rigler and
Zechmeister-Boltenstern, 1999; Schimel and Weintraub, 2003;
Bürgmann, 2011), resulting in decreased soil NH
4+
-N content can
inhibit methanotrophs activities and pmoA abundance (Le Mer and
Roger, 2001; Xu and Inubushi, 2007; Yue etal., 2016, 2022). However,
some ndings of other studies suggest that decreased NH
4+
-N
availability in soils can promote CH4 oxidation as higher NH4+-N can
replace CH
4
to beoxidized by methanotroph (Song etal., 2020; Yue
et al., 2022). erefore, the net eects of NH
4+
-N on CH
4
uptake
depend on the relative size of the two processes. Nevertheless, the
mechanism was not suitable for HR-mid. Similar to HR-late, HR-mid
declined pmoA abundance, however, it had no signicant correlation
with CH
4
uptake. is may bebecause increased NH
4+
-N content
under HR-mid leaded to the oxidation of NH
4+
-N instead of CH
4
by
methanotrophs, thus resulting in the most decreased CH
4
uptake in
mixture community, although the largest reduction of pmoA
abundance were found in graminoid community. As NO
3
−
-N content
was little impacted by heavy rainfalls, it had no eects on CH4 uptake
in this study. Our results are consistent with the nding that soil
NO
3
−
-N concentrations and CH
4
uptake had no correlation in a
subtropical plantation forest ecosystem (Wang etal., 2014). Taken
together, our study proved that the pathways underlying CH4 uptake
in response to heavy rainfall depend on seasonal timing.
Plant composition regulates responses of
CH4 uptake to heavy rainfalls
Multiple lines of evidence proved that plant composition is a
controlling factor in regulation of soil CH
4
oxidation. CH
4
uptake
would increase with enhanced plant diversity as high plant biodiversity
promoted microbial activities (Altor and Mitsch, 2006; Bouchard
etal., 2007; Schultz et al., 2011; Hassan etal., 2019). Niklaus etal.
(2016) showed that the presence of legume plants inhibited soil CH
4
oxidation capacity due to decline in plant N acquisition. Likewise, CH4
AB
CD
FIGURE3
Responses of pmoA abundance (A), above-ground biomass (AGB) (B), NH4+-N (C) and NO3
−-N (D) to heavy rainfall treatments in three plant
communities. Dierent letters above bars indicate significant dierence among treatments at p ≤ 0.05.

Zheng et al. 10.3389/fevo.2023.1149595
Frontiers in Ecology and Evolution 08 frontiersin.org
uptake had signicant dierences among three communities, where
CH
4
uptake was less in shrub community than in graminoid and
mixture communities in our study (Supplementary Figure S3;
Table 1). It may be because shrubs had harmful eects on
methanotrophs activities and CH4 uptake through the chemistry of
root exudates and N competition among plants and microbes (Zak
etal., 2003; Hassan etal., 2019).
Importantly, plant composition regulated CH
4
uptake to heavy
rainfalls, reected by signicant interactions between plant
composition and heavy rainfalls (Table1). Negative eects of HR-late
on CH
4
uptake was the least in graminoid community than in other
two communities. e potential explanation may bethat HR-late had
larger positive eects on SWC and AGB and larger negative eects on
pmoA abundance and NH
4+
-N content in shrub and mixture
communities (Figures1F, 3A), although the interactions were only
signicant on SWC and pmoA abundance. is nding supports the
third hypothesis that plant composition would regulate CH
4
uptake in
response to heavy rainfalls though soil moisture, inorganic content,
aboveground biomass and methanotrophs activities. Although
previous studies proved that plant communities with higher-diversity
are less negatively aected by oods (Gattringer etal., 2017; Wright
etal., 2017), the least increase in SWC, AGB and the least decrease
NH
4+
-N content were observed in graminoid community under
HR-late. Previous studies have reported that precipitation inltration
and evaporation rate vary with plant species composition. Evaporation
and transpiration can remove water from shallow soil layers aer
rainfall and thus decrease soil moisture (Coughenour, 1984; Weltzin
etal., 2003; Springer etal., 2006; MacIvor and Lundholm, 2011; Moore
etal., 2022). Graminoids are shallower rooting with deeper and faster
inltration and faster evaporation rate, leading to lower soil moisture
and the duration of soil saturation in graminoid community (Springer
etal., 2006). erefore, HR-late had the least negative eect on CH
4
uptake in graminoid community under HR-late. Overall, SWC, pmoA
abundance and NH4+-N content had the least reduction in graminoid,
leading to the least decreased CH
4
uptake in HR-late. However,
HR-mid had similar negative eects on CH
4
uptake across three
communities although the interaction between HR-mid and plant
composition on CH
4
uptake was statistically signicant (p = 0.08).
erefore, weconcluded that CH
4
uptake in response to climate
extremes jointly controlled by interaction of seasonal timing and
plant composition.
Conclusion
Our results highlight the vital role of seasonal timing and plant
composition in regulating heavy rainfall eects on CH
4
uptake.
Specically, although both heavy rainfalls reduced CH
4
sink, late
heavy rainfall had larger negative eects than middle heavy rainfall.
is is because decreased NH4+-N induced by late heavy rainfall had
negative eects on pomA abundance and further suppressing CH
4
sink, in addition to directly negative eects of high soil moisture
induced by heavy rainfall. Besides, shrub community had lower CH4
uptake than graminoid and mixture communities. Moreover, late
heavy rainfall had the least negative eects on CH
4
uptake in
graminoid communities than in other two communities, indicating
that climate extremes-driven shis in dominant species would in turn
alter ecosystem feedbacks. erefore, to improve prediction accuracy
of terrestrial ecosystems feedbacks to climate changes, weencourage
future studies to further quantify the interactive eects between
seasonal timing and plant on regulating carbon cycling in response to
climate extremes.
AB
FIGURE4
Structural equation models analysis of the direct and indirect eects of soil, microbe and plant variables on CH4 uptakes under HR-mid (A) and HR-late
(B) treatment. SWC: soil water content; AGB: aboveground, biomass; NH4+ and NO3
−: soil ammonium and nitrate content. Solid and dashed lines
indicate significant (p ≤ 0.05) and nonsignificant (p > 0.05) relationships, respectively. Width of the line is proportional to the strength of path coecients
expressed by the numbers adjacent on lines. r2 values denote the proportion of variance explained for each variable.

Zheng et al. 10.3389/fevo.2023.1149595
Frontiers in Ecology and Evolution 09 frontiersin.org
Data availability statement
e original contributions presented in the study are included in
the article/Supplementary material, further inquiries can bedirected
to the corresponding author.
Author contributions
LL designed the experiments. ZZ and FW performed the
experiments. ZZ and LL analyzed the data. ZZ wrote the manuscript.
ZZ, FW, CL, SG, YX, YL, RQ, ML, SX, XC, YW, YH, and LL provided
the editorial advice. All authors contributed to the article and
approved the submitted version.
Funding
is project was funded by the funds for the National Natural
Science Foundation of China (42041005 and 32101313), China
Postdoctoral Science Foundation (2021M693138), and the Fundamental
Research Funds for the Central Universities (E1E40607 and E1E40511).
Conflict of interest
e authors declare that the research was conducted in the
absence of any commercial or nancial relationships that could
beconstrued as a potential conict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their aliated
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.
Supplementary material
e Supplementary material for this article can befound online
at: https://www.frontiersin.org/articles/10.3389/fevo.2023.1149595/
full#supplementary-material
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