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doi: 10.1111/gcb.14293
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DR. VICTOR ALEXANDRE HARDT FERREIRA SANTOS (Orcid ID : 0000-0002-3510-5035)
Article type : Primary Research Articles
Causes of reduced leaf-level photosynthesis during strong El Niño drought in a Central
Amazon forest
Amazon photosynthesis resilience to drought
Victor Alexandre Hardt Ferreira dos Santos1; Marciel José Ferreira2*; João Victor Figueiredo
Cardoso Rodrigues3; Maquelle Neves Garcia1; João Vitor Barbosa Ceron1; Bruce Walker
Nelson1; Scott Reid Saleska4
1 Environmental Dynamics Department, Brazil´s National Institute for Amazon Research,
Manaus, Amazonas, Brazil. (vichardt@hotmail.com; maquelleneves@gmail.com;
joaovitorceron@gmail.com; bnelsonbr@gmail.com)
2Department of Forest Sciences, Federal University of Amazonas, Manaus, Amazonas,
Brazil. (mjf.ufam@gmail.com)
3Center for Distance Education, Federal University of Amazonas, Manaus, Amazonas, Brazil.
(joao.ufam@gmail.com)
4Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona,
USA. (saleska@email.arizona.edu)
*(corresponding author): Telephone: +55 (92) 33054042; Email: mjf.ufam@gmail.com
Keywords: Tropical forest, warming, drought stress, stomatal conductance, climate change,
chlorophyll fluorescence
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Primary Research Article
Abstract
Sustained drought and concomitant high temperature may reduce photosynthesis and cause
tree mortality. Possible causes of reduced photosynthesis include stomatal closure and
biochemical inhibition, but their relative roles are unknown in Amazon trees during strong
drought events. We assessed the effects of the recent (2015) strong El Niño drought on leaf-
level photosynthesis of Central Amazon trees via these two mechanisms. Through four
seasons of 2015, we measured leaf gas exchange, chlorophyll a fluorescence parameters,
chlorophyll concentration and nutrient content in leaves of 57 upper canopy and understory
trees of a lowland terra firme forest on well-drained infertile oxisol. Photosynthesis
decreased 28% in the upper canopy and 17% in understory trees during the extreme dry
season of 2015, relative to other 2015 seasons and was also lower than the climatically
normal dry season of the following non-El Niño year. Photosynthesis reduction under
extreme drought and high temperature in the 2015 dry season was related only to stomatal
closure in both upper canopy and understory trees, and not to chlorophyll a fluorescence
parameters, chlorophyll, or leaf nutrient concentration. The distinction is important because
stomatal closure is a transient regulatory response that can reverse when water becomes
available, whereas the other responses reflect more permanent changes or damage to the
photosynthetic apparatus. Photosynthesis decrease due to stomatal closure during the 2015
extreme dry season was followed two months later by an increase in photosynthesis as rains
returned, indicating a margin of resilience to one-off extreme climatic events in Amazonian
forests.
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Keywords: Tropical forest, warming, drought stress, stomatal conductance, climate change,
chlorophyll fluorescence
Introduction
Amazon forests significantly influence the global carbon cycle, storing ~120 PgC in
biomass and contributing 14% of the net annual carbon fixed by terrestrial photosynthesis
(Malhi et al., 2006; Malhi et al., 2008; Zhao & Running, 2010; Fauset et al., 2015). However,
this function is at risk due to drought events in the region, linked to global climate change
(Aragão et al., 2014; Brienen et al., 2015). Reduction in ecosystem scale photosynthesis and
widespread tree mortality during and after drought have been reported, suggesting that these
forests may be increasingly at the risk of shift from a carbon sink to a source (Bonal et al.,
2008; Phillips et al., 2009; Aragão et al., 2014; Gatti et al., 2014; Brienen et al., 2015;
Doughty et al., 2015; Bonal et al., 2016; Cavaleri et al., 2017).
Concerning seasonal drought, evidence suggests that gross ecosystem productivity of
Central Amazon forests is not limited by rainfall during a typical dry season, but instead
increases due to coordinated dry season leaf-flush and litterfall that shifts the composition of
the canopy towards younger leaves with greater photosynthetic capacity (Kitajima et al.,
1997; Doughty & Goulden, 2008; Restrepo-Coupe et al., 2013; Wagner et al., 2016; Wu et
al., 2016; Wagner et al., 2017). However, important remaining questions include how in situ
leaves of a given age respond to drought or heat stress, and how they respond under more
extreme or long-term droughts like those occurring during dry phases of El Niño and the
Atlantic Multidecadal Oscillation (Davidson et al., 2012; Bonal et al., 2016).
The effects of normal seasonality and of extreme drought events on leaf-level
photosynthetic processes of mature leaves are much debated. Some reports indicate no
seasonal change in photosynthesis at the leaf level during normal but prolonged (5 mo)
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seasonal drought (Domingues et al., 2014) and maintenance of growth and photosynthetic
capacity (despite increased mortality by xylem embolism) during 15y of a through-fall
exclusion experiment (Rowland et al., 2015a; Rowland et al., 2015b) in the eastern Amazon.
Tree growth rates over a 2 y period that included the extreme 2005 drought were no different
from other multi-year intervals that did not include droughts (Phillips et al., 2009). On the
other hand, leaf-level photosynthesis reduction has been reported during the normal
prolonged seasonal drought typical of southern Amazonia (Miranda et al., 2005), during the
extreme 2010 drought in a Bolivian forest (Doughty et al., 2015) and at Paracou, French
Guiana, during a pronounced dry season in 2008 (Stahl et al., 2013a). The variability between
individual trees responses (both inter and intra-specific) to drought also adds to the difficulty
of estimating ecosystem-scale drought effects when examining leaf-level photosynthetic
processes in tropical forests (Miranda et al., 2005; Stahl et al., 2013a). Critical to resolving
questions about the sensitivity of leaf-level photosynthesis to drought and to accurately
representing tropical forest response to climate change in Earth System models is a more
detailed understanding of the mechanisms involved in the leaf-level response to drought and
to high temperatures (Corlett, 2016).
The mechanism of leaf-level photosynthesis reduction during drought stress might be
stomatal closure (which limits CO2 assimilation), non-stomatal limitations to photosynthetic
function, or a combination of the two (Flexas & Medrano, 2002; Lloyd & Farquhar, 2008).
Under increased vapor pressure deficit from low humidity and/or high temperature, plants
close their stomata (Cunningham, 2004; Slot & Winter, 2017). This maintains leaf turgor and
water column integrity in xylem vessels at least up to a critical leaf water potential despite
low availability of soil water and high atmospheric evaporative demand (Bonal et al., 2000;
Flexas & Medrano, 2002; Chastain et al., 2014; Sperlich et al., 2015). Stomatal closure
protects against hydraulic failure but reduces photosynthesis, limits CO2 diffusion to the
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substomatal cavity and reduces leaf cooling under the high irradiance and high temperatures
concurrent with natural drought (Teskey et al., 2015).
During the stomatal closure, reduced use of energy for CO2 assimilation can
overexcite chlorophyll. Together with high leaf temperature from reduced evaporative
cooling, this may damage the integrity and functionality of photochemical processes in the
chloroplast membranes (Oukarroum et al., 2009; Desotgiu et al., 2012, Campos et al., 2014).
The distinction between reductions in photosynthesis due to stomatal closure alone, versus
reductions that follow damage to leaf biochemistry, are critically important in terms of their
implications for long-term drought response of the forest. Stomatal closure is transient and
may be reversed once the drought is past, but damage to leaf photosynthetic infrastructure
may be longer-lived, and require valuable carbon and water resources to repair or replace
damaged leaves (Flexas & Medrano, 2002; Lloyd & Farquhar, 2008).
In 2015, a widespread warming and extreme drought occurred over the Amazon forest
due to a strong El Niño phase in the El Niño-Southern Oscillation (ENSO) (Jiménez-Muñoz
et al., 2016). We used this natural event to assess the effects of drought on leaf-level
photosynthesis and on the mechanisms which control it and to thereby improve our
understanding of climate change impacts on tropical forests.
We hypothesize that drought reduces photosynthesis in mature leaves. We endeavor to
confirm this general hypothesis and test further key hypotheses about the mechanisms
regulating photosynthesis under drought and high maximum temperatures in a Central
Amazon forest. In particular, we test whether changes in photosynthesis are due only to
changes in stomatal conductance (brought on by drought and/or temperature-induced increase
in vapor pressure deficit), or also due to other changes in photosynthetic function (as
indicated by photosynthetic capacity, chlorophyll fluorescence, chlorophyll concentration and
nutrient content).
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We examined 57 trees cohabiting the forest upper canopy and the understory during
four consecutive seasons in 2015 to assess the effects of the strong El Niño on leaf-level
photosynthesis and its regulation. The El Niño drought was strong in only the 2015 dry
season. However, to be sure that we were not detecting typical seasonal differences between
the 2015 dry season and the other three seasons of 2015, we made a second set of dry season
measurements in 2016, which had normal seasonal rainfall and temperature.
Materials and Methods
Study site
The study site is a Central Amazon forest at the LBA (Large Scale Biosphere-
Atmosphere Experiment in Amazonia) K-34 tower (2.6° S, 60.2° W). The region is
characterized by low seasonal variation in air temperature (24 - 27°C monthly average), high
humidity (75% - 92% daily average) and moderately high precipitation (2200 mm annually)
(Araújo et al., 2002). The terrain is undulating with altitude ranging from 60 m in valleys to
120 m a.s.l. on plateaus. Plateau soil is predominantly well-drained clay oxisol while valleys
and lower slopes have poorly drained sandy spodosol (Luizão et al., 2004). The forest
structure is characterized by high tree density (626 trees ha-1), tree basal area (28-30 m2 ha-1)
and tree aboveground biomass (360 Mg ha-1) (Chambers et al., 2004; Vieira et al., 2004).
We measured leaf traits in 19 trees inhabiting the upper canopy layer and 38 trees in
the understory (Table S1), during four field campaigns in the wet, wet/dry transition, dry and
dry/wet transition seasons of 2015. Campaign mid-dates in 2015 were May 7, July 7,
September 13 and November 26, respectively. Extreme drought occurred prior to and during
the September campaign. To compare the 2015 extreme dry season to a normal dry season,
gas exchange measurements were also made in the dry season of 2016, with a mid-date on
Oct 17, 2016.
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The trees are all located on a plateau in the footprint of K-34 LBA tower. The upper
canopy layer was accessed from the tower and two nearby canopy walkways, each 30m long
and 25-30m above the ground. In the understory, the leaves were accessible from the ground.
Environmental conditions
Daily and monthly precipitation for 1998-2016 were obtained from products 3B42v7
and 3B43v7, respectively of the Tropical Rainfall Measuring Mission database (NASA,
2016). We used the daily precipitation to calculate a 30 d running sum prior to each day.
From this we obtained the Accumulated Daily Water Deficit as of the mid-date of each field
campaign. This is a modified version of the Maximum Climatological Water Deficit, or
MCWD, often used as a metric of drought intensity across Amazonia (Malhi et al., 2009;
Phillips et al., 2009; Lewis et al., 2011; Aragão et al., 2014; see Text S5). In addition to our
modified MCWD, we used the soil moisture from six depths (10, 20, 30, 40, 60 and 100 cm)
to calculate the Relative Extractable Water index (REW) (Granier et al., 1999; Wagner et al.,
2011; Stahl et al., 2013a).
The maximum monthly air temperature was from the Brazilian National Institute of
Meteorology (INMET Manaus station #81730, 3.13° S; 59.95° W). Anomalies for monthly
rainfall and maximum air temperature were calculated according to Saleska et al. (2007). To
address rainfall and temperature associations with ENSO intensity, these monthly anomalies
were correlated to the Multivariate ENSO Index (MEI) (Wolter & Timlin, 1998;
http://www.cdc.noaa.gov/people/klaus.wolter/MEI/table.html). Air temperature and relative
humidity for the four 2015 field campaigns were obtained from a sensor (HMP45C, Vaisala
Oyj, Finland) installed above the canopy at 51 m height on the K-34 tower. A data gap for
relative humidity occurred in July. Photosynthetic photon flux density sensors were installed
near the ground (1.3 m) and above the canopy (51 m) during 5-7 days in each campaign
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(MQS-B PAR sensor and ULM-500 logger, Heinz Walz, Germany). Soil moisture was
determined at the six depths with a profile probe (PR1, Delta-T Devices, UK). Soil moisture,
air temperature and relative humidity also were recorded during the 2016 dry season.
Leaf traits
For all leaf traits, we selected samples from mature, healthy, fully expanded leaves
with no signs of aging or senescence. To control for leaf age effects, in a parallel study of leaf
demography, we monitored the leaf age of our upper canopy trees with a precision of +/- 15
days. This allowed us to confidently select only mature-stage leaves in each season and each
year of the present study. We measured one leaf per tree in each season for gas exchange
parameters and three leaves per tree for the other traits. Gas-exchange and chlorophyll a
fluorescence parameters were obtained in vivo from attached leaves and branches. The same
leaves were then harvested for the other measurements.
Light-saturated photosynthetic rate (Asat), dark respiration (Rd), transpiration (E) and
stomatal conductance (gs) were measured between 08:00h and 13:00h using a LI-6400
portable photosynthesis system (LICOR, USA). The LI-6400 chamber was adjusted to a fixed
CO2 concentration (400 µmol mol-1), leaf temperature (31°C) and H2O vapor fraction (21
mmol mol-1), consistent with optimal conditions for photosynthesis in tropical trees (Araújo
et al., 2002; Santos Junior et al., 2006; Doughty & Goulden, 2008; Lloyd & Farquhar, 2008;
Domingues et al., 2014). Asat, E and gs were measured at a photosynthetic photon flux density
(PPFD) of 2000 µmol m-2 s-1 and dark respiration (Rd) at a PPFD of 0 µmol m-2 s-1. Gas
exchange rates stabilized after five minutes of maintaining leaves in the saturated light or
dark inside the IRGA chamber, but we left leaves in the chamber for 30 minutes. While we
could use a model to adjust our measurements to the 25°C convention, we prefer to report the
standard temperature that was in fact used. All measurements were made at a leaf
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temperature of 31°C, which is close to optimal for net photosynthesis (Tribuzy, 2005; Lloyd
& Farquhar, 2008; Slot & Winter, 2017; Tan et al., 2017). We report both photosynthesis and
dark respiration at this leaf temperature. High PPFD guaranteed steady state saturated
photosynthesis in both canopy leaves and in understory trees. We did not detect evidence for
damage to the leaves from this PPFD, i.e. no reduction in photosynthesis with increase up to
2000 µmol m-2 s-1 (Fig. S8). Water use efficiency (WUE) and intrinsic water use efficiency
(WUEi) were calculated as the ratio of light-saturated photosynthesis to transpiration and to
stomatal conductance, respectively. The maximum carboxylation rate of ribulose-1,5-
bisphosphate carboxylase/oxygenase (Vcmax) was estimated for 17 upper canopy trees during
the four 2015 seasons according to the Farquhar et al. model (Farquhar et al., 1980) and the
Sharkey et al. (2007) A/Ci curve fitting utility, version 1.0. For Vcmax, the LI-6400 chamber
was adjusted to a photosynthetic photon flux density of 1000 µmol m-2 s-1, leaf temperature
of 31°C, H2O vapor fraction of 21 mmol mol-1 and successive CO2 concentrations of 400,
300, 250, 200, 150, 100, 50, 400, 400, 450, 500, 600, 700, 800, 1000, 1200 µmol mol-1.
Polyphasic transient of chlorophyll a fluorescence was recorded in three leaves per
tree between 08:00h and 10:00h. Leaves were dark adapted for 30 min, then exposed to a
saturated light pulse of 3000 µmol m-2 s-1 with a wavelength of 650 nm during 1 s using a
portable fluorimeter (PEA, MK2 9600 Hansatech, Norfolk, UK). Chlorophyll a
fluorescence parameters (Maximum quantum yield of photosystem II - Fv/Fm, Performance
index - PIABS, and Total performance index - PItotal) were calculated by the JIP-test (Strasser
et al., 1995; Strasser et al., 2010; Santos Junior et al., 2015). For detailed information about
the fluorescence parameters used in this paper see Strasser et al. (1995); Strasser et al.
(2010); Stirbet & Govindjee (2011) and Stirbet et al. (2018). In summary, during the first
second of exposure to light of a dark-acclimated leaf sample the chlorophyll fluorescence
rises in a polyphasic shape. Four phases or steps of this transient fluorescence signal (O-J-I-
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P) are used to derive the parameter Fv/Fm and the performance indices (PIABS and PItotal). The
Fv/Fm ratio is the maximum quantum yield of the primary Photosystem II photochemistry.
The ratio is derived from the minimum (Fo or O) and the maximum (Fm or P) fluorescence
(Fm-Fo/Fm). The two performance indices are more sensitive but also more complex, being
the product of three parameters from the polyphasic transient curve that define PIABS, plus a
fourth parameter to define PItotal. The PIABS is formed by the ratio of the total number of
active PSII reaction centers (RC) per absorption flux (ABS), Fv/Fm and the probability that an
electron moves further than reduced quinone A [(Fm FJ)/FV)]. Finally, PItotal is calculated
multiplying PIABS by the probability with which an electron from the intersystem electron
carriers is transferred to reduce end electron acceptors at the PSI acceptor side [(Fm FI)/ (Fm
FJ)]. Therefore, PIABS is the potential for energy conservation from photons absorbed by
PSII to the reduction of intersystem electron acceptors and PItotal is the potential for energy
conservation from photons absorbed by PSII to the reduction of PSI end acceptors.
Chloroplast pigment concentrations were determined using a spectrophotometric
method modified from Lichtenthaler & Wellburn (1983), following acetone-filtered
extraction. We used Hendry and Price (1993) equations for chlorophyll (a,b) concentrations.
Leaf nitrogen was determined by the Kjeldahl method with distillation and titration
ate
method (Murphy & Riley, 1962) and K was determined by atomic absorption spectrometry
(1100B; PerkinElmer, Ueberlingen, Germany).
Additionally, predawn (5:30h - 6:30h) and midday (12:00h 13:00h) leaf water
potentials were measured in 11 upper canopy trees using a pressure pump 3005-1422 (Soil
Moisture Equipment Corp, USA) (Scholander et al., 1964; Turner, 1981) during four months
(May, July, September and October) of the abnormally dry year of 2015. We measured three
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leaves per tree and the canopy walkway allowed each measurement to be completed in about
three minutes.
Statistical analyses
Leaf trait data were subjected to Lilliefors and Mauchly tests to check assumptions of
normality and sphericity, respectively. Seasonal effects were analyzed by one-way repeated
measures ANOVA in each forest layer (upper canopy and understory) and then the seasons
(three 2015 non-drought seasons vs 2015 extreme dry season) were compared by planned
comparison with univariate test of significance (Zar, 1999). Being a repeated-measures test
we included only trees sampled in all seasons for each type of test. We obtained the
difference in photosynthesis between the 2015 dry season and the 2015 annual average
excluding that dry season ( Dry/ Annual)-1) and correlated this difference with leaf traits.
Photosynthesis and stomatal conductance during dry seasons of the 2015 El Niño and the
2016 non-El Niño years were compared by a two-tailed paired sample t-test. All analyses
were performed using Statistica 9.0 software (StatSoft Inc., 2010 East 14th Street, Tulsa, OK,
USA).
Results
Environmental conditions
Starting in August 2015, the El Niño altered the normal Central Amazon rainfall and
air temperature patterns (Fig. 1a, c). However, only the September 2015 field campaign was
preceded by strong drought (Figs. S6, S7). The drought indices ADWD and REW both show
intense water deficits prior to and during the 2015 dry season. ADWD reached -117 mm at
the mid-date of the 2015 dry season field campaign and REW fell to zero. Before and during
the following campaign (dry-to-wet transition), soil water was not yet fully replenished, but
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REW exceeded 0.4 and ADWD oscillated near the deficit (Fig. S6). Additionally,
predawn leaf water potential -- an indicator of plant-available soil water -- reached its most
negative values during dry months of September and October in 2015 (Fig. S9),
corroborating the two drought indices. In August, September and October of 2015, maximum
monthly air temperatures reached their highest values for the entire 19 y period of record
(Fig. 1c). Rainfall and maximum air temperature were correlated with the Multivariate ENSO
index in a negative and positive way, respectively.
Among the four field campaigns of 2015, the dry season (September) had the highest
mid-day extremes of irradiance (in both upper canopy and understory), highest air
temperature, highest vapor pressure deficit and lowest relative humidity (Fig. 2). Soil
moisture was also lowest in the 2015 dry season at all depths from 10-100 cm (Fig. 2f). In
contrast, the 2016 dry season was not extreme for air temperature, relative humidity or vapor
pressure deficit, having values similar to the dry-to-wet transition campaign of 2015 (Fig. 2c,
d, e). Soil moisture below the surface in the 2016 dry season was similar to that of the pre-
drought wet season of 2015 (Fig. 2f).
Gas exchange
The light-saturated photosynthetic rate varied between the 2015 seasons, reaching a
minimum during the extreme drought of September (Fig. 3a). In the upper canopy and
understory, 93% and 79% of the trees, respectively, reduced their Asat values in this extreme
dry season relative to the mean of the other three seasons in 2015. Across all trees, the dry
season decrease in photosynthesis relative to the other season average was 28% for upper
canopy (F = 10.59; P < 0.01; Fig. 3a) and 17% for understory (F = 11.54; P < 0.01; Fig. 3a).
Two months later, during the dry/wet transition season, in both upper canopy (F = 0.94; P =
0.35) and understory (F = 0.01; P = 0.9), photosynthesis recovered to the pre-drought mean
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value. During the normal dry season of 2016 a non-El Niño year Asat and stomatal
conductance were much higher in the upper canopy and understory when compared with the
2015 El Niño dry season (Fig 4).
Following the trends in Asat, a high percentage of trees reduced their stomatal
conductance (gs) during the dry season. Seventy-three percent of upper canopy trees reduced
gs in the wet-to-dry transition season and 88% kept low values through the extreme dry
season of 2015. In the understory, a high percentage of trees with reduced gs (83%) were seen
only during the extreme dry season. Means of gs and of E across all understory trees declined
in the extreme dry season of 2015 relative to the three other seasons (gs F = 32.89; P <
0.0001; Fig. 3b; E F = 23.31; P < 0.0001; Fig. 3d). In the upper canopy, however, these two
variables were lower in both the wet-to-dry transition season and the extreme dry season,
when compared to the other season average (gs F = 20.86; P < 0.001; E F = 26.59; P < 0.001).
In the post-drought dry-to-wet transition season, gs recovered to wet season values in both
forest layers (Understory - F = 2.93; P = 0.10; Upper Canopy - F = 4.56; P = 0.05). However,
E recovered to wet season values only in the understory (Understory - F = 0.82; P = 0.37;
Canopy - F = 5.17; P = 0.04). For upper canopy trees, the WUE and WUEi increased in the
wet-to-dry transition season and maintained high efficiency through the extreme dry and dry-
to-wet seasons of 2015 (Fig. 3e,f). In understory, the trees increased their WUE and WUEi
only during the extreme dry season, but the magnitude of increase was similar to upper
canopy trees (Fig. 3e,f).
Rd remained unchanged through all four seasons of 2015 in both upper canopy and
understory (Fig. 3c). Similarly, no significant seasonal changes in Vcmax were observed in
upper canopy trees species (Fig. S4).
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Chlorophyll concentration and fluorescence
The maximum quantum yield of photosystem II (Fv/Fm) ranged from 0.73 to 0.85 and
the upper canopy mean quantum yield did not differ among the four 2015 seasons (Fig. 5b).
On the other hand, mean Fv/Fm in the understory was different between the year 2015 dry-to-
wet and the extreme dry season (F = 35.79; P < 0.001). PIABS, PItotal and leaf chlorophyll
concentration did not change with the seasons (Fig. 5a, c, d).
Leaf nutrients concentration
In both upper canopy and understory leaves, mean leaf N concentration did not reduce
during the 2015 extreme dry season (Fig. 6a). P concentration was lowered during wet and
dry-to-wet seasons, but only in the understory (F = 6.39; P < 0.01; Fig. 6b). In the understory,
during the extreme 2015 dry season, mean leaf K concentration was slightly higher compared
with all other seasons (F = 2.58; P = 0.08; Fig. 6c).
Relationships between reduction in Asat and leaf traits
Asat reduction during the 2015 dry season was correlated only with a reduced stomatal
conductance. This was true in both upper canopy and understory (Table S3; Fig. 7). Seasonal
changes in photochemical performance parameters (Fv/Fm, PIABS, PItotal), chlorophyll
concentration and leaf nutrient content were not correlated with Asat reduction (Table S3).
Discussion
El Niño drought inhibits leaf level photosynthesis in Central Amazon trees
We have shown that a strong El Niño drought in the dry season of 2015 inhibited leaf
level photosynthesis (Asat) in mature leaves of a Central Amazon forest. This was not a
normal seasonal pattern; Asat reduction did not occur during the climatically normal dry
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season of the following year. Extreme drought and/or concomitant high air temperature
induced a diffusive restriction to CO2 assimilation by forcing stomatal closure (Fig. 2 and
Fig. 3). This is consistent with previous reports of photosynthesis reduction during extreme
droughts (Stahl et al., 2013a; Gatti et al., 2014, Aragão et al., 2014; Hilker et al., 2014;
Doughty et al., 2015; Inoue et al., 2017) or warming (Doughty, 2011; Cavaleri et al., 2015).
We found no evidence that photosynthesis reduction could be attributed to causes beyond
stomatal closure, i.e., declines in photosynthetic infrastructure such as reduced photosynthetic
capacity, photochemical performance, chlorophyll concentration or leaf nutrient content
(Table S2).
This is an important finding: even during
(Fig. 1; Jiménez-Muñoz et al., 2016), the only detectable mechanism of photosynthetic
reduction was stomatal closure, a transient regulatory response that can potentially reverse
when the drought ends.
Stomatal conductance as the main driver of photosynthesis reduction during drought
Stomatal closure-induced reductions in leaf level photosynthesis were sensitive to
canopy layer (Fig. 3a). Reduced photosynthesis, stomatal conductance and transpiration were
more pronounced in upper canopy than in understory trees (Fig. 3c and Fig. 3d). Both upper
canopy and understory trees could have deep roots allowing access to deep soil water stores
(Nepstad et al., 1994, Stahl et al., 2013b). However, the typical microclimate conditions in
the upper canopy layer are low air humidity, high air temperature and high irradiance
(Chazdon & Fetcher, 1984; Kumagai et al., 2001; Kamakura et al., 2015), all of which are
aggravated during the dry season, especially in an El Niño year (Fig. 2). Thus, the more upper
canopy leaves are exposed to demanding atmospheric conditions, the more likely they are to
exhibit a strong reduction of stomatal conductance and photosynthesis during drought,
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compared to understory trees (Sperlich et al., 2015). Moreover, taller trees must cope with a
longer water-transport path from the soil to their leaves. Higher atmospheric evaporative
demand increases the risk of embolism and hydraulic failure (Bennett et al., 2015). In sum,
our results provide support for more pronounced drought effects on leaf gas exchange in tall
upper canopy trees, exposed to drier and hotter atmosphere, than in understory trees.
Our focus on individual mature leaves means that several important attributes of
whole canopy photosynthesis were outside the scope of this study. We do not here examine,
for example, whether drought influences canopy phenology (leaf growth, mortality, or leaf
age-composition). In a normal (non-El Niño) year, dry season leaf flush drives a dry season
increase in whole-canopy photosynthesis at this site (Restrepo-Coupe et al., 2013; Wu et al.,
2016). Newly flushed leaves -- if supplied sufficient water during this 2015 El Niño dry
season (as in more typical dry seasons) -- would counteract some of the leaf-level suppression
of photosynthetic Asat, potentially allowing canopy photosynthesis to increase despite a
drought.
An important result of our study is that it shows, for the first time, that photosynthetic
reduction at the leaf-level during an El Niño drought was caused exclusively by stomatal
closure. However, the relative importance of drought and of high temperature on
photosynthesis of Amazon trees are still not fully elucidated. In parts of the Eastern Amazon
normal seasonal dry periods last five months but do not limit photosynthesis (Domingues et
al., 2014). In a 15y long through-fall exclusion experiment, where soil water was artificially
reduced but upper canopy air temperature was unaffected, only vulnerable trees reduced
photosynthesis in association with a drop in stomatal conductance (Rowland et al., 2015a).
Trees died by embolism without any pre-death reduction of growth rate or of non-structural
carbohydrate stores (Rowland et al., 2015b). By contrast, during a single year of extreme
drought in 2010 in western Amazonia, photosynthesis was suppressed at leaf and plot levels
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with an estimated assimilation reduction of 0.38 petagrams of carbon across the Amazon
Basin (Doughty et al., 2015).
Non-stomatal processes were not impaired during extreme drought in Central Amazon trees
The cause of stomatal closure during the dry season was presumably to maintain leaf
water potential under low available soil water and high vapor pressure deficit, as observed in
11 canopy trees in this survey and previously reported for tropical tree species (Fig S9; Reich
& Borchert 1988; Bonal et al., 2000; Cao, 2000; Miranda et al., 2005). With increasing
severity and duration of the drought, other processes related to photosynthesis can be
affected, such as photochemistry in the thylakoid membranes (Flexas & Medrano 2002;
Flexas et al., 2006; Atkin & Macherel 2009). For example, in evergreen and semi-deciduous
trees of a monsoonal tropical dry forest the electron transport rate (ETR) decreased in
response to drought stress during the dry season (Ishida et al., 2014). Further, Hung et al.
(2013) demonstrated an inhibition of both linear electron flow and non-photochemical
quenching under prolonged drought for tropical trees growing on limestone. However,
according to JIP-test parameters, the electron transport performance was not impaired during
the extreme 2015 dry season (Fig. 5a, b, d). The JIP-test has been used in several studies that
analyzed the effects of drought stress on the integrity and functionality of electron transport
(Strasser et al., 2010; Redillas et al., 2011; Desotgiu et al., 2012; Campos et al., 2014). PItotal
integrates the energy conservation from photons absorbed by PSII through to the reductions
of PSI end acceptors and is among the more sensitive JIP-test parameters to biotic and abiotic
stresses (Strasser et al., 2010; Stirbet & Govindjee 2011; Stirbet et al., 2018). The uncoupling
between electron transport and photosynthesis during the dry season suggests that another
process, such as photorespiration, was contributing to energy and power reduction (ATP,
NADPH, Fd-) utilization generated in the electron transport chain (Martinez-Ferii et al., 2000,
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Haupt-Herting & Fock, 2002; Lawlor & Tezara, 2009). Moreover, this uncoupling can add
more complexity for obtaining landscape-level photosynthesis estimates based on the
chlorophyll fluorescence signal (Osuna et al., 2015; Yang et al., 2017).
In conclusion, our results have important implications for improving models of the
sensitivity of tropical forests to climate anomalies by providing insight into the effects of
extreme climatic events (e.g. El Niño) on leaf-level photosynthesis. This work also adds some
early evidence about the consequences of future climate change on the current carbon cycle
of Amazonia. Because stomatal conductance best explained changes in photosynthesis as
affected by drought stress, including this variable in models may lead to improved model
performance under drought stress conditions for Amazon forest.
Acknowledgments
We thank the Federal University of Amazonas (UFAM), Brazil´s National Institute
for Amazon Research (INPA) and the Large-Scale Biosphere-Atmosphere Experiment in
Amazonia Program (LBA) for logistic support; the GOAmazon project, funded jointly by the
U.S. Department of Energy (DOE, # DE. SC0008383), by the Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP), and by the Fundação de Amparo à Pesquisa do
Estado do Amazonas (FAPEAM, # 062.00570/2014). The authors declare no conflict of
interest.
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Fig. 1 Monthly average rainfall (a) and maximum monthly air temperature (c) in 2015 (bars).
Monthly averages for 19 y (1998-2016) are solid lines; the range for each month´s average
rainfall and for each month´s maximum air temperature are dashed lines. Anomalies for 19 y
of trimester average precipitation (b) and trimester maximum air temperature (d) are plotted
against trimester anomalies for Multivariate ENSO Index MEI. The four 2015 trimesters
are solid black symbols (JFM - inverted triangle; AMJ - triangle; JAS - square and OND -
circle).
Fig. 2 Diurnal variation in photosynthetic photon flux density above the canopy (PPFD; a)
and in the understory (PPFD; b); air temperature (Tair; c), relative humidity (RH; d) and
vapor pressure deficit (VPD; e) above the canopy; and soil moisture (f) of a Central Amazon
forest during the four field seasons of the 2015 El Niño year and dry season of the 2016 non-
El Niño year. Symbols with black outlines at each time of day are the mean for that time of
day for each of the five field campaigns. Light grey symbols are daily values.
Fig. 3 Box plots of light-saturated photosynthetic rate (Asat; a), stomatal conductance (gs; b),
leaf dark respiration (Rd; c); transpiration (E; d), intrinsic water use efficiency (WUEi, e) and
water use efficiency (WUE; f) in upper canopy and understory trees of a Central Amazon
forest during the four field seasons of the 2015 El Niño year. Solid line inside the box is the
median, dashed line is the mean, box-plot range is 50% of the data, error bars are 5th and 95th
percentiles and points are outliers. Asterisk p-values are n.s.(p>0.05), *(p<0.05), **(p<0.01),
***(p<0.001) for a one-way repeated-measures ANOVA, i.e., testing for a difference
between any pair of seasons (Degrees of freedom are shown in Table S2). Comparisons
between dry season and the other three seasons grouped together are reported in the text.
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Fig 4 Box plots of light-saturated photosynthetic rate (Asat; a) and stomatal conductance (gs;
b) during the 2015 (El Niño year) and the 2016 (non-El Niño year) dry seasons for the upper
canopy and understory trees. For box interpretation see Fig 3. The P-values are for two-tailed
paired sample t-tests (nunderstory = 21; ncanopy = 14).
Fig. 5 Box plots of chlorophyll concentration (Chl; a), maximum quantum yield of
photosystem II (Fv/Fm; b), photochemical performance index (PIABS; c), and total
photochemical performance index (PItotal; d) in upper canopy and understory trees of a
Central Amazon forest during the four field seasons of the 2015 El Niño year. Boxplot and
ANOVA details are as described in Fig. 3.
Fig. 6 Box plots of leaf nitrogen (N; a), phosphorus (P; b) and potassium (K; c) concentration
in upper canopy and understory trees of a Central Amazon forest during the four field seasons
of the 2015 El Niño year. Boxplot and ANOVA details are as described in Fig. 3.
Fig. 7 Correlations between light saturated photosynthesis (Asat) and stomatal conductance
(gs), using their respective relative differences () between the extreme dry season of 2015
( Dry) and the mean of the other three seasons ( Annual), where Dry/ Annual)-1. (ncanopy =
15; nunderstory = 24).
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Supporting Information captions
Table S1. Canopy and understory species list
Table S2. Univariate test for repeated measure (season)
Figure S4. Box plot of maximum carboxylation rate of ribulose-1, 5-bisphosphate
carboxylase/oxygenase (Vcmax) seasonality
Supplementary Text S5. Accumulated Daily Water Deficit (ADWD) and Relative Extractable
Water (REW)
Figure S6. Two years of Accumulated Daily Water Deficit, Daily Rainfall, and Relative
Extractable Water
Figure S7. Nineteen years of Accumulated Daily Water Deficits
Figure S8. A_PPFD response curve
Figure S9. Box plot of predawn and mid-day leaf water potential
Supplementary Text S10. Repeated-measures ANOVA to detect effects of season and of
plant family
Table S11. Repeated Measures Analysis of Variance for understory trees
Figure S12. Photosynthesis seasonality in the understory for four tree families
Table S13. Repeated Measures Analysis of Variance for upper canopy trees
Figure S14. Photosynthesis seasonality in the upper canopy for four tree families.
Supplementary Text S15 Repeated-measures ANOVA to detect effects of season and of light
environment
Table S16. Repeated Measures Analysis of Variance
Figure S17. Photosynthesis seasonality for the four light levels from Dawkins index.
Excel worksheet S18. Data
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