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Study shows why trees won't benefit much from extra CO2 in the air

By Michael Irving
March 26, 2020
Study shows why trees won't benefit much from extra CO2 in the air
Trees growing under simulated heat and drought conditions reveal that the negatives of increased atmospheric CO2 levels outweigh the benefits for plants
Trees growing under simulated heat and drought conditions reveal that the negatives of increased atmospheric CO2 levels outweigh the benefits for plants
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Trees growing under simulated heat and drought conditions reveal that the negatives of increased atmospheric CO2 levels outweigh the benefits for plants
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Trees growing under simulated heat and drought conditions reveal that the negatives of increased atmospheric CO2 levels outweigh the benefits for plants

The amount of carbon dioxide in the atmosphere is increasing steadily, having recently reached the highest concentration in human history. While that’s undoubtedly bad news for the planet, one argued silver lining is that plants are better off due to more of their food being in the air. But a new study has dashed those hopes, finding that the more extreme heat and drought brought on by climate change would cancel out most of the benefits for trees.

Carbon dioxide is the main nutrient for plants, who pluck it out of the atmosphere and, with the help of sunlight, convert it into chemical energy so they can grow. So it follows that more CO2 would be a good thing for our photosynthesizing friends, right?

To an extent that’s true, but things are more complex than that. More CO2 means a warmer and drier planet, and these factors would also have an effect on plants. And just how much of an effect was the focus of a new study, conducted by researchers at Karlsruhe Institute of Technology (KIT), Ludwig-Maximilians-Universität München, the University of Vienna, and Weizmann Institute of Science.

The researchers grew a series of Aleppo pines from seeds under two different concentrations of CO2 – 421 parts per million (ppm), which is a little higher than the current atmospheric level, and an elevated level of 867 ppm.

When the trees were 18 months old, the team began testing them. For the first month, they watered one group well, while leaving others without, to simulate drought conditions. Then, they planted both groups in chambers where they could control the temperature. Over 10 days, the heat was gradually increased from a pleasant 25 °C (77 °F) to a sweltering 40 °C (104 °F), while the scientists measured the trees’ responses.

The team found that higher CO2 levels did help the trees use their water more efficiently, and lose less of it, as the heat rose. That was largely thanks to root proteins becoming more stable, and the trees closing their stomata – the pores in leaves that allow for gas exchange.

... the increasing CO2 concentration of the atmosphere cannot compensate the stress of the trees resulting from extreme climate conditions
Nadine Rühr

But that’s where the benefits end, unfortunately. Closed stomata meant the stressed-out trees took up significantly less carbon from the air, and the heat had a negative effect on their metabolism as well.

“Overall, the impact of the increased CO2 concentration on stress reactions of the trees was rather moderate,” says Nadine Rühr, lead author of the study. “With increasing heat and drought, it decreased considerably. From this, we conclude that the increasing CO2 concentration of the atmosphere cannot compensate the stress of the trees resulting from extreme climate conditions.”

Of course, this is just one brief study on one species of tree, so there’s no assurance that the results apply to all other species. But it does feed into what we already know about tree biology, and contributes to the complex picture of how plants are responding to climate change, and what effects that will have.

Other recent studies have found that more CO2 is making trees grow faster and die younger, so they return their captured carbon to the atmosphere earlier than usual. And even when more are growing, the types of trees and the environments they’re growing in are also factors. For instance, another study found that “woody” plants are on the rise, covering more of the world’s savannas and tundras, but these changing biomes could have unexpected negative consequences.

The new study was published in the journal New Phytologist.

Source: KIT

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EnvironmentTreeEnvironmentPlantsKarlsruhe Institute of TechnologyClimate CrisisPhotosynthesisCarbon DioxideWeizmann InstituteUniversity of ViennaLudwig-Maximilians-Universität MunichNew Atlas Audio
12 comments
Michael Irving
Michael Irving
Michael has always been fascinated by space, technology, dinosaurs, and the weirder mysteries of the universe. With a Bachelor of Arts in Professional Writing and several years experience under his belt, he joined New Atlas as a staff writer in 2016.

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12 comments
Anne Ominous
There is a problem with this scenario.

Global Warming is supposed to create a WETTER world overall, not a drier one.

The physics of this is exceedingly simple:

1) Higher temperatures mean more evaporation.

2) Since most of Earth's surface is water, there will be an overall increase in evaporation, not a decrease.

3) Increased evaporation = more clouds and precipitation.



Brian M
Anne Ominous makes a good point, global warming does not necessarily imply a dryer world, more energy in the weather systems would perhaps indicate a wetter world. The contra to that is how are the weather and ocean current patterns going to be effected. Suspect this might be a case of some areas dryer, some wetter. Plus of course plants themselves can help retain water in the environment, so a possible feedback loop there.

A few degrees is not going to make that much difference to the dryness of the overall climate but its effect on weather patterns might.
paul314
My impression of the models is that some places will be wetter, others drier. Fine for the people, animals and plants that can move to the more-salubrious environment, not so good for everyone/everything else.
Douglas Rogers
What about prehistoric times with much more CO2 while being much warmer and wetter? This seems like a forced outcome to me.
PeteKK
Agree with Anne, One must also consider that temperate zones will shift and while plants that can't tolerate the heat will die off, they will also be replaced by plants that flourish in the warmer climate.
Skip Robinson
The comments make more sense than the article. Good job folks. I saw a couple of mistakes in the article as well. Our planet has to be relatively self-regulating. Many say Co2 levels are not the highest in history and water evaporates more, the hotter it is, that which then blocks more sunlight and heat. I like golfing when it cloudy.
George Smathers
Anne's correct; warmer and wetter. Already there has been a 12% greening from 1990-2015 due (probably) to less need to open stomata.
BeinThayer
The actual study is interestingly seemingly at odds with its own conclusions:
"... New Phytologist Trust


New PhytologistEarly View
Full Paper Open Access
Hot drought reduces the effects of elevated CO2 on tree water‐use efficiency and carbon metabolism
Benjamin Birami Thomas Nägele Marielle Gattmann … See all authors
First published:03 February 2020
https://doi.org/10.1111/nph.16471
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Summary
Trees are increasingly exposed to hot droughts due to CO2‐induced climate change. However, the direct role of [CO2] in altering tree physiological responses to drought and heat stress remains ambiguous.
Pinus halepensis (Aleppo pine) trees were grown from seed under ambient (421 ppm) or elevated (867 ppm) [CO2]. The 1.5‐yr‐old trees, either well watered or drought treated for 1 month, were transferred to separate gas‐exchange chambers and the temperature gradually increased from 25°C to 40°C over a 10 d period. Continuous whole‐tree shoot and root gas‐exchange measurements were supplemented by primary metabolite analysis.
Elevated [CO2] reduced tree water loss, reflected in lower stomatal conductance, resulting in a higher water‐use efficiency throughout amplifying heat stress. Net carbon uptake declined strongly, driven by increases in respiration peaking earlier in the well‐watered (31–32°C) than drought (33–34°C) treatments unaffected by growth [CO2]. Further, drought altered the primary metabolome, whereas the metabolic response to [CO2] was subtle and mainly reflected in enhanced root protein stability.
The impact of elevated [CO2] on tree stress responses was modest and largely vanished with progressing heat and drought. We therefore conclude that increases in atmospheric [CO2] cannot counterbalance the impacts of hot drought extremes in Aleppo pine.
Introduction
Forests are exposed to a rapidly changing climate world‐wide, and extreme weather events such as heatwaves and drought spells are predicted to increase in frequency and severity as atmospheric [CO2] a[CO2]) is rising (Coumou & Rahmstorf, 2012; Baldwin et al., 2019; Pfleiderer et al., 2019). This has pronounced impacts on forest carbon (C) and water (H2O) cycling (Williams et al., 2013), particularly in already H2O‐limited ecosystems (Choat et al., 2018). Yet, the interacting effects of elevated [CO2] (e[CO2]) with extreme environmental conditions (such as drought, heat stress, and the combination of both) on tree stress resistance are far from clear.

Heatwaves during extended drought periods can be a main cause of forest decline (Anderegg et al., 2013). Hot droughts are particularly stressful because evaporative demand is high, while H2O availability is low and trees need to tightly regulate H2O loss (Ameye et al., 2012; Ruehr et al., 2016; Birami et al., 2018). This typically induces stomatal closure to maintain the integrity of the H2O transporting system (Tyree & Zimmermann, 2002). Simultaneously, C assimilation rates decline while C is needed to support osmoregulation and cellular maintenance (Hsiao, 1973; Huang et al., 2012; Hartmann & Trumbore, 2016). Therefore, a C imbalance can arise under progressing stress, which triggers a cascade of metabolic adjustments.

A driving force of metabolic activity in plants is respiration. Typically, c. 30–80% of the daily photosynthetic C gain is released back to the atmosphere (Atkin & Tjoelker, 2003). During stressful conditions, the amount of respiration to assimilation can change dramatically and trees can become a net source of CO2. It has been shown that the C loss in trees subjected to higher temperatures and increasing drought is larger and occurs earlier than under cooler conditions. This was due to respiration continuing at relatively high rates whereas assimilation started to decline earlier in drought‐treated trees grown under 35°C compared with 25°C (Zhao et al., 2013). Other work has shown that respiration can strongly increase under rapid warming, even in combination with drought, until rates drop at very high temperatures (Gauthier et al., 2014). By contrast, if trees are exposed to elevated growth temperatures, respiration typically acclimates, nearly offsetting the effect of the warming (Reich et al., 2016; Drake et al., 2019). Although much research has focused on the temperature relationship of respiration, we have little mechanistic understanding to predict how respiration will respond to day‐long heatwaves, let alone in combination with drought and/or changes in [CO2].

Increasing a[CO2] may affect tree stress responses through a variety of plant physiological processes. For instance, e[CO2] often suppresses photorespiration and dark respiration (Drake et al., 1999; Dusenge et al., 2019), whereas it stimulates C assimilation and productivity under nonstressful conditions (Ainsworth & Long, 2005; Ainsworth & Rogers, 2007; Ameye et al., 2012; Simón et al., 2018; Zinta et al., 2018). Alongside increases in C uptake, stomatal conductance gs typically declines (Eamus, 1991). This reduction in gs corresponds with a larger leaf‐intercellular [CO2] Ci, stimulated photosynthesis, and increased plant H2O‐use efficiency (WUE) – the ratio of C uptake via assimilation per unit H2O loss from transpiration (Lavergne et al., 2019). Increases in WUE under e[CO2] have been observed in many studies (Eamus, 1991), particularly in H2O‐limiting environments (Wullschleger et al., 2002). However, the combined effects of e[CO2] stress responses during extreme heat and/or drought stress have rarely been investigated, and results remain inconclusive. For instance, e[CO2] could not mitigate extreme drought stress (withholding H2O until mortality occurred) in Pinus radiata and Callitris rhomboidea (Duan et al., 2015) or in Eucalyptus globullus when +240 ppm CO2 was combined with a constant +4°C warming (Duan et al., 2014), whereas it alleviated extreme heat stress in Pinus taeda and Quercus rubra (Ameye et al., 2012; +320 ppm, +12°C heatwave) and Larrea tridentata (Hamerlynck et al., 2000; +340 ppm CO2, +8°C heatwave).

A more comprehensive picture on the interacting effects of e[CO2] on plant stress performance could be gained through a whole‐tree C perspective – integrating sink and source responses (Dusenge et al., 2019; Ryan & Asao, 2019). Moreover, investigating changes in the primary metabolism could allow identification of some of the underlying mechanisms (Xu et al., 2015; Mohanta et al., 2017). For instance, e[CO2] can increase sugar and starch concentrations, which might buffer plant C losses during drought via enhanced C supply and/or improved osmoregulation (Ainsworth & Long, 2005; Ainsworth & Rogers, 2007) as well as may reduce oxidative stress (Zinta et al., 2014). However, e[CO2] may also affect the C : nitrogen (N) stoichiometry and N dilution, as has been observed, resulting in decreased protein and amino acid concentrations (Poorter et al., 1997; Johnson & Pregitzer, 2007). A decrease in protein content may affect assimilation and respiration rates (Drake et al., 1999; Xu et al., 2015; Dusenge et al., 2019), could dampen stress‐induced upregulation of amino acids important for osmoregulation (Zinta et al., 2018), and may affect the abundance of heat‐shock proteins, and therefore plant thermotolerance (Coleman et al., 1991; Huang et al., 2012; Zhang et al., 2018). Hence, e[CO2] can trigger metabolic processes that may directly interact with tree drought and heat stress responses. Yet, results remain inconclusive because we miss an integrated understanding of the interactive effects of e[CO2] and stress on the C balance and primary metabolism of trees.

Here, we provide novel insights into the impacts of e[CO2] on whole‐tree shoot and root stress responses in Aleppo pine saplings originating from a semi‐arid forest at the arid timberline (Grünzweig et al., 2009). To elucidate the effects of [CO2] in combination with drought and heat stress on physiological responses, we combined measurements of whole‐tree C balance, WUE, and primary metabolites. More specifically, our hypotheses were as follows: first, that e[CO2] increases photosynthesis, which results in a larger net C uptake maintained during heat stress; second, that WUE increases proportionally with a[CO2] and that this increase can be maintained during heat stress but not during hot drought, when stomata are closed; and third, the tree metabolic response to temperature is suppressed under e[CO2], which is reflected in a concurrent change in respiratory activity and primary metabolites.

Materials and Methods
Plant material
Pinus halepensis (Miller) saplings were grown from seeds for 18 months under ambient CO2 (c. 420 ppm) or e[CO2] (c. 870 ppm, within the range of representative concentration pathway 8.5 for 2100; IPCC, 2014) in a scientific glasshouse facility in Garmisch‐Partenkirchen, Germany (732 m asl, 47°28′32.87ʺN, 11°3′44.03ʺE) with highly UV‐transmissive glass (70%). The origin of the seed material is a 50‐yr‐old Aleppo pine plantation in Israel (Yatir Forest). Cones of trees were sampled growing in close proximity to a meteorological station and flux tower (IL‐Yat, 650 m asl, 31°20′49.2ʺN, 35°03′07.2ʺE).

In the following, the experimental design of the study is explained in detail from the germination of the seedlings until the 18‐month‐old saplings were transferred into separate tree gas‐exchange chambers (see Fig. 1). Seeds germinated on vermiculite in two transparent growth chambers either under ambient a[CO2] or e[CO2]. About 10 wk after germination, in July 2016, the seedlings were transferred to pots (5 × 5 × 5 cm3, 0.125 l) containing a C‐free potting mixture of 1 : 1 : 0.5 quartz sand (0.7 mm and 1–2 mm), vermiculite (c. 3 mm), and quartz sand (Dorsolit 4–6 mm) with 1 cm of expanded clay (8–16 mm) as a drainage. Seedlings were fertilized with 2 g of slow‐release fertilizer (Osmocote® Exact 3‐4M 16‐9‐12 + 2MgO+TE; ICL Specialty Fertilizers, Heerlen, the Netherlands) supplemented by liquid fertilizer (Manna® Wuxal Super; Wilhelm Haug Gmbh & Co. KG, Ammerbuch, Germany). Placement of the seedlings within the two growth chambers was randomized every second week; and to overcome a possible chamber effect, the CO2 treatments were moved at monthly intervals between the chambers (Fig. 1). After the saplings were 7 months old they were placed in two glasshouse compartments referring either to a[CO2] and e[CO2] conditions and 10‐month‐old seedlings were individually transferred to larger pots (4.5 l) for a second time. The potting mixture was again a C‐free substrate of 1 : 1 : 2 vermiculite (3–6 mm), coarse (4–6 mm), and fine quartz sand (2–3 mm) with 1 cm of expanded clay (8–16 mm) as a drainage. Slow‐release fertilizer (5 g, Osmocote® Exact Standard 5‐6M 15‐9‐12+2MgO+TE; ICL Specialty Fertilizers) was added to the mixture and supplemented by liquid fertilizer, phosphate, and magnesium addition once. Incoming light from outside was supplemented with plant growth‐lamps (T‐agro 400 W; Philips, Hamburg, Germany) and the saplings were irrigated regularly to saturation. A possible effect of the placement within the glasshouse was again overcome by iterating the CO2 treatments between the two glasshouse bays four times before the start of the heat stress experiment in September 2017 (Fig. 1).

image
Fig. 1
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Experimental timeline from seedling (Pinus halepensis) germination until two heat experiments (each 10 d) were conducted 18 months later. The initiation of the CO2 (elevated [CO2] (e[CO2]): dark blue; atmospheric [CO2] (a[CO2]): light blue) and drought treatments (orange) is also shown. Seedlings of the four treatments (a[CO2]W, e[CO2]W, a[CO2]D, e[CO2]D; D, drought; W, well‐watered) were randomly selected and transferred to the gas‐exchange chambers where temperature was increased stepwise (25°C, 30°C, 35°C, 38°C, 40°C) and above and belowground gas‐exchange measured. The heat experiment was repeated with a new set of seedlings to increase number of replicates to eight per treatment. Note that one gas‐exchange chamber per treatment was left blank to serve as a quality control. The yellow dotted lines depict iteration of the CO2 treatments between two growth chambers or two glasshouse bays.
Atmospheric CO2 differed greatly between the glasshouse compartments (421 ± 105 ppm in a[CO2] and 867 ± 157 ppm in e[CO2] on average, increase in [CO2] of 106% during growth period), whereas all other growth conditions were kept similar (see Supporting Information Fig. S1). Moisture sensors (10HS; Decagon Devices Inc., Pullman, WA, USA; calibrated to potting substrate) and an automated drip irrigation system were installed (Rain Bird, Azusa, CA, USA) when seedlings were 15 months old. The irrigation was adapted to result in a relative substrate H2O content (RSWC) of 50% (Fig. S2) close to the soil H2O content in the Yatir Forest during spring conditions. RSWC was calculated as follows:

urn:x-wiley:0028646X:media:nph16471:nph16471-math-0001
(Eqn 1)
(SWCmax (g g−1), maximum amount of H2O held by the substrate (e.g. field capacity); SWCmin (g g−1), minimum amount of H2O held by the substrate, set to zero; SWCsample, measured substrate H2O content, derived from the calibrated moisture sensors).

When seedlings were about 17 months old, half of the seedlings from each CO2 treatment were randomly selected and assigned to a drought treatment (D). In the drought trees, irrigation was slowly reduced to maintain daily‐averaged RSWC at c. 10%, whereas RSWC in the well‐watered trees (W) was maintained at 50%, leading to a pronounced decrease in H2O potential. Two sets of seedlings from each of the four treatments (a[CO2]W, e[CO2]W, a[CO2]D, e[CO2]D) were randomly selected 40 d and 50 d after drought had been initiated (Fig. 1), transferred to custom‐built separate tree gas‐exchange chambers (see section Chamber system) and exposed to increasing heat stress (n = 4 per treatment) for a period of 10 d.

Tree gas‐exchange chambers
Chamber system
We developed a tree gas‐exchange system with 20 separate chambers divided into above and belowground compartments to continuously measure the exchange of H2O and CO2. Each of the 20 aboveground compartments were individually temperature‐controlled (Fig. 2). The aboveground and belowground compartments were separated and gas tightness between the above‐ and belowground compartment was ensured after enclosing the tree stem. For details on the set‐up and constant air supply of the tree gas‐exchange system, see S1.

image
Fig. 2
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Whole‐tree gas‐exchange system separated in an above and belowground compartment, shown exemplified for one chamber (n = 20 in total). The arrows indicate the direction of flow. The air supply to the chambers is given in black (Airsupply), and the sample air is given in green (Airsample). The Li‐840 measured absolute [CO2] and [H2O] connected to an Li‐7000 to measure differences between Airsupply and Airsample. Note that trees (Pinus halepensis) were potted in carbon‐free substrate and the belowground CO2 efflux is therefore interpreted as root respiration.
The chamber system was installed in the glasshouse and outside light was supplemented with plant growth lamps (T‐agro 400 W; Philips, Hamburg, Germany). Canopy light conditions inside each chamber were measured automatically with a photodiode (G1118; Hamamatsu Photonics, Hamamatsu, Japan), which had been cross‐calibrated with a high‐precision photosynthetic active radiation (PAR) sensor (PQS 1; Kipp & Zonen, Delft, the Netherlands). Root‐zone conditions were monitored with temperature sensors (TS 107; Campbell Scientific Inc., Logan, UT, USA) and moisture sensors (10HS; Decagon Devices Inc.). These data were logged half‐hourly (CR1000; Campbell Scientific Inc.).

Gas‐exchange measurements
The gas‐exchange chambers were constantly supplied with an air stream (Airsupply) of either 408 ppm or 896 ppm CO2. Sample air (Airsample) was drawn at a rate of 500 ml min−1, and each seedling was measured once every 80 min using differential gas analysis. We used two gas analyzers: the analyzer measuring absolute [CO2] and [H2O] (LI‐840; Li‐Cor, Lincoln, NE, USA) was connected to a differential gas analyzer (Li‐7000; Li‐Cor) quantifying [CO2] and [H2O] differences between Airsupply and Airsample. The data were logged at 10 s intervals. The gas analyzers were calibrated following the manufacturer's recommendations.

To eliminate any offset between Airsupply and Airsample not caused by plant gas‐exchange, empty aboveground and belowground compartments (n = 1 per treatment, four in total) containing C‐free potting substrate only, were measured and offsets (on average +0.33 ± 1.2 ppm CO2 and 0.02 ± 0.05 ppt H2O in the aboveground compartments) removed accordingly. Differences in CO2 were slightly larger in the belowground compartments (c. +2 ppm on average) and may be due to some microbial activity in the potting substrate.

Gas‐exchange fluxes of CO2 and H2O were calculated from the concentration differences between Airsupply and Airsample. Plant H2O loss via transpiration E (mol s−1) was calculated as follows:

urn:x-wiley:0028646X:media:nph16471:nph16471-math-0002
(Eqn 2)
(ṁ, air mass flow (mol s−1) into the chamber compartment; Wsample, H2O vapor concentration of Airsample (mol mol−1); Wsupply, H2O vapor concentration of Airsupply (mol mol−1)).
From daytime E and H2O vapor concentrations we determined stomatal conductance gs (mol s−1) as follows:

urn:x-wiley:0028646X:media:nph16471:nph16471-math-0003
(Eqn 3)
(Wleaf (mol mol−1), leaf H2O vapor concentration, derived from the ratio of saturation vapor pressure (kPa) at a given air temperature (°C) and atmospheric pressure). This approach, which neglects boundary‐layer conductance, is suitable under well‐coupled conditions, as confirmed by negligible temperature differences between chamber and tree canopy (< 1°C; see Table S1).
CO2 gas exchange (mol s−1) – that is, net photosynthesis ANet, shoot respiration Rshoot, and root respiration Rroot – was calculated as follows:

urn:x-wiley:0028646X:media:nph16471:nph16471-math-0004
(Eqn 4)
(Csample, [CO2] of Airsample (mol mol−1); Csupply, [CO2] of Airsupply (mol mol−1); E, used to correct for dilution through transpiration (mol s−1)). In the case of Rroot (where sample air was dried), the H2O vapor dilution term became negative. The daily net C uptake (mg) per tree was calculated based on daily‐averaged ANet and respiration as follows:
urn:x-wiley:0028646X:media:nph16471:nph16471-math-0005
(Eqn 5)
In order to determine changes in whole‐tree WUE, apparent WUE was derived as follows:

urn:x-wiley:0028646X:media:nph16471:nph16471-math-0006
(Eqn 6)
To allow comparison of tissue gas‐exchange activity between treatments and because root surface area was not available, we calculated gas‐exchange rates per shoot (i.e. needle and woody tissues) or root DW, if not stated otherwise. The percentage share of soluble C from tissue biomass was small (< 4%; Table S2), hence we refrained from taking normalization to soluble C into account. Tree biomass was determined at the end of the experiment and separated into needles, roots, and woody tissues before drying at 60°C for 48 h (Table 1).

Table 1. Needle, shoot, root, total tree biomass, needle area, and total soluble carbon (C, calculated as C equivalents of all measured metabolites) for 1.5‐yr‐old Pinus halepensis seedlings are given as treatment averages ± 1 SE (n = 16 per treatment) at the end of the experiment (post‐stress).
Treatment Biomass (g DW) Needle area (cm2) Soluble C (µmol g−1 DW)
Needle Root Wood Total
a[CO2]W 33.4 ± 1.0 A 51.1 ± 1.4 B 15.4 ± 0.6 A 99.9 ± 2.1 A 1296 ± 38 A 1090 ± 209 A
e[CO2]W 47.6 ± 1.5 B 66.7 ± 1.9 D 24.5 ± 1.2 B 138.8 ± 2.1 B 1925.3 ± 84 B 847 ± 79 A
a[CO2]D 33.3 ± 1.5 A 43.6 ± 1.2 A 15.8 ± 0.9 A 92.7 ± 2.9 A 1414.6 ± 60 A 2623 ± 454 B
e[CO2]D 47.5 ± 1.4 B 60.0 ± 2.2 C 24.2 ± 1.4 B 131.6 ± 4.5 B 2052.5 ± 65 B 2578 ± 349 B
D, drought; W, water treated; a, atmospheric; e, elevated.
Significant differences between treatments were derived from ANOVA followed by Tukey's honestly significant difference and are given in upper‐case letters (P < 0.05).
Heat stress experiment
Responses of shoot and root gas‐exchange with increasing temperature and evaporative demand were assessed continuously using the tree gas‐exchange system described in the Tree gas‐exchange chambers section and Notes S1. In brief, randomly selected seedlings were placed into separate gas‐exchange chambers (n = 4 per treatment, Fig. 1). The chambers containing one tree each were positioned next to each other in a randomized block design. The heat stress experiment was repeated in order to double the numbers of replicates per treatment. Each heat experiment lasted 10 d, and after the initial 2 d at 25°C (20°C nighttime) the temperature was increased stepwise every second day to the following daytime temperatures: 25, 35, 38, and 40°C (Fig. 3a). We refrained from temperatures above 40°C as tree mortality has been found to strongly increase in Aleppo pine seedlings above this threshold, particularly in combination with drought (Birami et al., 2018). As during a typical heatwave in the Yatir Forest (Tatarinov et al., 2016), vapor pressure deficit (VPD) increased alongside increasing temperature, and this increase was slightly greater in the drought‐treated saplings due to low E (Fig. 3c). PAR was kept relatively constant between gas‐exchange chambers, and daily averages were 456 ± 140 µmol m−2 s−1. To overcome some of the light limitations (saturating PAR for photosynthesis was at 1200 µmol m−2 s−1) daytime length was set to 16 h, well above the average summer day length in Yatir Forest. Irrigation was controlled to maintain the RSWC of well‐watered trees at c. 50% and of drought‐treated trees at c. 10% (Fig. 3). The irrigation amount did not differ between the [CO2] treatments (a[CO2]W and e[CO2]W: 300 ml d−1; a[CO2]D and e[CO2]D: 50 ml d−1) and drought‐treated seedlings reached a midday needle H2O potential ψmidday indicating stomatal closure (Fig. S3; Table 2).

image
Fig. 3
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Environmental drivers during the heat stress experiment (Pinus halepensis) given per treatment. (a) Air temperature TAir, (b) soil temperature TSoil, (c) vapor pressure deficit (VPD), and (d) relative substrate water content (RSWC) are shown. Data are treatment‐averages during daytime (lines including symbols) or nighttime (lines), and the shaded areas are ± 1 SD (n = 8). Daytime is defined as photosynthetic active radiation (PAR) > 100 and nighttime as PAR = 0. Note the temperature difference between day and nighttime was not constant due to technical limitations but was kept within 7–10°C.
Table 2. Gas‐exchange rates at 25°C expressed per tissue DW and tree net carbon (C) uptake for 1.5‐yr‐old Pinus halepensis seedlings given as treatment averages ± 1 SE (n = 8 per treatment).
Treatment
E

(µmol s−1 g−1)

g s

(mmol s−1 g−1)

A Net

(nmol s−1 g−1)

R dark

(nmol s−1 g−1)

C i

(µmol mol−1)

WUE

(µmol mmol−1)

Net C uptake/tree

(mg d−1)

ψ midday

(MPa)

a[CO2]W 3.29 ± 0.15 B 0.38 ± 0.04 B 15.3 ± 1.3 B 7.4 ± 0.3 B 263 ± 3 A 4 ± 0.3 A 165.1 ± 29.8 AC −1.49 ± 0.07 A
e[CO2]W 2.03 ± 0.28 D 0.23 ± 0.07 B 16.5 ± 2.2 B 6 ± 0.2 C 612 ± 5 C 7.2 ± 0.5 B 379.2 ± 68.8 C −1.15 ± 0.04 A
a[CO2]D 0.66 ± 0.17 A 0.08 ± 0.01 A 3.4 ± 1.1 A 3.9 ± 0.4 A 287 ± 11 A 3.4 ± 0.7 A −73.2 ± 18.3 B −2.68 ± 0.3 B
e[CO2]D 0.67 ± 0.16 A 0.04 ± 0.02 A 6.7 ± 1.2 C 4.2 ± 0.3 A 482 ± 14 B 9.4 ± 1.3 B 23.2 ± 38.7 AB −1.83 ± 0.2 A
D, drought; W, water treated; a, atmospheric; e, elevated.
E, transpiration; gs, stomatal conductance; ANet, net photosynthesis; Rdark, dark respiration; Ci, leaf‐intercellular [CO2]; WUE, water‐use efficiency; ψmidday, midday needle water potential.
Tree net C uptake is the sum of photosynthesis ANet minus respiration R. ψmidday is given and was measured at the time of tissue sampling for metabolite analysis. Significant differences between treatments were derived from ANOVA followed by Tukey's honestly significant difference and are given in upper‐case letters (P < 0.05).
Sample preparation
We sampled needle tissue for analysis of primary metabolites on the last day of the following temperature levels: 25, 35, 38 and 40°C. To avoid disturbance of belowground fluxes, root biomass was only sampled at 25°C (additional set of saplings, not used for the experiments) and at 40°C. Sampling took place in the afternoon between 15:00 h and 16:00 h; samples were immediately frozen in liquid N2 and then stored at −80°C until ground to fine powder in liquid N2 before freeze‐drying for 72 h with cooling aggregate at −80°C and sample temperature at −30°C (Alpha 24 LSC; Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). The freeze‐dried samples were stored in the dark in closed vials at room temperature, and analyses of primary metabolites were completed within 2 months (Fürtauer et al., 2019). For details on analysis of primary metabolites via gas chromatography coupled with time‐of‐flight mass spectrometry (Fürtauer et al., 2016; Weiszmann et al., 2018) and protein content via Bradford assay (Fürtauer et al., 2018), please see Notes S2.

Statistical data analysis
Data processing, analysis, and statistics were carried out using R v.3.5.2 (R Core Team, 2018). Gas‐exchange measurements of each chamber were carefully inspected before analyses, and day or nighttime fluxes outside 1.5 times the interquartile range (above the upper quartile and below the lower quartile) per temperature were considered outliers. This removed, on average, 3.8% of CO2 and 5.7% of H2O gas‐exchange data.

Primary metabolites were scaled to SD before treatment effects in needles and roots at 25°C were visualized by hierarchical clustering, utilizing R packages ggplot2 (Wickham, 2016) and cluster (Maechler et al., 2018). Further, the overall changes in the primary metabolome depending on tissue, treatment, and temperature were analyzed after centering of the scaled data via principal components analysis.

Treatment effects on biomass, gas‐exchange rates, and metabolites at specific temperature levels were tested using ANOVA, and differences between treatments were revealed by post hoc analysis (Tukey honestly significant difference (HSD)). Treatment and temperature dependencies of gas‐exchange fluxes and metabolites (fixed effects) were checked by implementing a linear mixed effects model (lmerTest; Kuznetsova et al., 2017). In order to account for temporal autocorrelation, tree was accounted as a random factor. Using the reduced sample size Akaike information criteria (Akaike, 1974; Giraud, 2015), the most parsimonious model was selected with or without tree as random factor, and the treatment and temperature effects included with and without interaction. We report a pseudo‐R2 (pR2) for the selected model (MuMIn; Barton, 2019). Homoscedasticity and normality of residuals were checked and, if applicable, loge transformation applied.

To analyze differences in the temperature relationship of ANet we applied an exponential decay function (y = e−bx); in the case of respiration R, we fitted a second‐order polynomial function following (Gauthier et al., 2014):

urn:x-wiley:0028646X:media:nph16471:nph16471-math-0007

The uncertainties of all fitted functions are given as 95% confidence intervals derived from first‐order Taylor expansion using the propagate package (Spiess, 2018).

Results
Tree biomass
Differences in above and belowground biomass were distinct after growing P. halepensis seedlings for more than 1 yr under a[CO2] of 421 ppm or e[CO2] of 867 ppm (Table 1). A doubling of atmospheric [CO2] increased total tree biomass by 35%. This increase was particularly pronounced in woody tissues (stem and twigs, +47%) and to a lesser extend in needles (+26%). The 1‐month drought period had no obvious effect on aboveground biomass, but reduced belowground biomass under ambient (−15%) and e[CO2] (−11%). The amount of nonstructural carbohydrates in total biomass varied between 1.5 and 3.5% and was slightly larger under drought with no clear trend of [CO2] ..."
ljaques
Bzzzt! Invalid testing methodology. // Man didn't evolve from ape in 10 days. They used 421ppm tree seeds. Grow the trees in 867ppm CO2, collect their seeds, and regrow in 867 several generations. NOW do your tests. // BUT, it matters now whether trees do better in higher CO2 levels. Those levels are coming no matter what man does. These scientists don't even know why the ocean sinks so much CO2, or when or how. There are far too many facets of this process which evade the minds of men. Leave Mother Nature alone, as she'll do her thing with or without us. As the Marines are wont to say "Innovate, adapt, overcome.", but don't forget to evolve during all this, too.
Kpar
Anne is on the money, so is Douglas. A wetter environment also come with a side effect- the more clouds, the higher the Earth's albedo, and more sunlight is reflected back into space, reducing the "global warming". Indeed, "weather" is the Earth's air conditioner- a negative feedback system that stabilizes temperatures. The "warm-mongers" choose to ignore this salient fact in order to push their agenda of control over humanity.
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