Meta-analysis of the responses of tree and herb to elevated CO2 in Brazil

The CO2 concentration has increased in the atmosphere due to fossil fuel consumption, deforestation, and land-use changes. Brazil represents one of the primary sources of food on the planet and is also the world's largest tropical rainforest, one of the hot spots of biodiversity in the world. In this work, a meta-analysis was conducted to compare several CO2 Brazilian experiments displaying the diversity of plant responses according to life habits, such as trees (79% natives and 21% cultivated) and herbs (33% natives and 67% cultivated). We found that trees and herbs display different responses. The young trees tend to allocate carbon from increased photosynthetic rates and lower respiration in the dark—to organ development, increasing leaves, roots, and stem biomasses. In addition, more starch is accumulated in the young trees, denoting a fine control of carbon metabolism through carbohydrate storage. Herbs increased drastically in water use efficiency, controlled by stomatal conductance, with more soluble sugars, probably with a transient accumulation of carbon primarily stored in seeds as a response to elevated CO2.


Results
Photosynthetic parameters, biomass, and starch increased in leaves of tropical plants under elevated CO 2 .The eCO 2 increased plants' assimilation rate by 44% (Fig. 1; Table 1).Overall, trees + herbs responses in biomass showed an average increase of 20% in leaves, 41% in stems, and 43% in roots (Fig. 2; Table 1).The results in non-structural carbohydrates composed of glucose, fructose, sucrose, and starch present in the leaves of trees and herbs under CO 2 are shown in Fig. 3.However, only total soluble sugars and starch content showed an increase of 7% and 47%, respectively (Fig. 3; Table 1).
Elevated CO 2 effect in trees and herbs according to life habits.The life habits were essential to distinguish responses in total biomass, stomatal conductance (gs), transpiration foliar (E), water use efficiency(WUE), and maximum rate of electron transport (J max ) (Table 2).The biomass increase is different per organ between trees and herbs under eCO 2 .The biomass increased more in trees than in the herbs category, being higher on leaves (194%), stems (245%), and roots (250%) (Fig. 2; Table 1).In herbs, the biomass increased by 28% and 77% in stems and roots, respectively.Furthermore, changes in the biomass of leaves were not significant in herbs (Fig. 2; Table 1).The grain biomasses were only measured in herbs, which had no alteration in plants cultivated under eCO 2 (Fig. 2).Starch increased by 61% in trees, while in the herbs, the fructose, sucrose, and soluble sugars increased by 13%, 15%, and 14%, respectively (Fig. 3; Table 1).When trees and herbs were analyzed separately, the assimilation increased by 39% and 52%, respectively (Fig. 3; Table 1).Stomatal conductance negatively affected herbs (p = 0.001; Table 2; Fig. 1).The reduction of gs (39%) in herbs increased WUE (117%) (Fig. 3).Thus, the photosynthesis parameters WUE, E, and J max differed among herbs and trees at eCO 2 (Fig. 1; Table 2).These results may reflect a tendency for the opposite effects of these variables in trees and herbs (Fig. 1; Table 1).On the other hand, the trees displayed no significant effect in gs, WUE, and E at eCO 2 (Fig. 1; Table 1).Under eCO 2 , trees significantly reduced dark respiration (17%).Furthermore, Ci/Ca, Jmax, Vcmax, Fv/Fm, and total Chl in trees and herbs under eCO 2 did not change under eCO 2 (Figs. 1, 4).The lack of effect could reflect the small number of observations in those variables (Fig. 5), which calls for more studies to provide consistent analysis for these variables.
Heterogeneity and publication bias analysis.Heterogeneity (I 2 ) analysis in the analytical models was used to evaluate the variation in results among observations.The high heterogeneity indicates variation in the effect of eCO 2 among observations.The heterogeneity was high (I 2 > 75) for total biomass, A, gs, Rd, E, WUE, Ci/ Ca, J max , total chl, starch, and proteins (Table 2).High heterogeneity shows that external factors may influence the variation of the estimated effects among observations.The Vc max showed moderate heterogeneity, and the Fv/Fm had low heterogeneity (Table 2).These results demonstrate less variation among observations in Vc max and Fv/Fm variables.
No publication bias was found for net CO 2 assimilation, dark respiration, foliar transpiration, water use efficiency, intercellular/ambient CO 2 rate, maximum carboxylation rate, the potential quantum efficiency of PSII, total chlorophyll, and total soluble sugars (Table 2).However, the Egger test identified publication bias for biomass, gs, J max , and starch (Table 2).

Discussion
Plants can be used to capture carbon to delay the effects of climate change through photosynthesis, which assimilates carbon in the form of CO 2 and accumulates it into the plant's biomass.Thus, higher carbon availability is expected to generate changes in these processes and intensify plant growth 15,47 .In the meta-analysis presented in this work, data from species planted as crops and native species to the neotropics were examined.We confirmed previous literature observations regarding the physiology of temperate species, showing that several neotropical ones alter their photosynthesis parameters, biomass accumulation, and sugars (biochemicals) under eCO 2 (Fig. 6).The elevated CO 2 in plants through photosynthesis is directly connected to their growth and productivity 48 .In addition, elevated CO 2 stimulated photosynthetic assimilation in neotropical herbs, improving WUE due to stomata closure and conductance reduction (Fig. 1).This behavior corroborates evidence reported in other meta-analyses 12,28 , except that neotropical trees did not alter the stomatal conductance responses as happens in temperate trees 13 .
Stomatal conductance (gs) and assimilation rates control the intercellular/ambient CO 2 ratio, which dictates the internal carbon allocation in plants 49 .Elevated CO 2 increases the concentration of intracellular CO 2 in leaves 38 , but to continue the assimilation, the mesophyll CO 2 needs to display lower concentrations than the atmospheric partial pressure of CO 2 50 .This regulation is performed by the closure and opening of the stomata, which leads to a decrease in stomatal conductance 38,51 .
It has been reported that European forests grown in eCO 2 decreased J max and Vc max by 10% 52 .The authors attributed this decrease to the limiting levels of nitrogen in leaves.The Neotropical species examined in the present work did not decrease J max and Vc max changes (Fig. 1), possibly indicating that the leaf nitrogen status in the experiments used for this meta-analysis was not limited.According to Bonan et al. 53 , the Vc max parameter displays relevant implications for large-scale modeling.Carbon flux models show that simulated photosynthetic rates are particularly susceptible to Vc max and J max , with the former being pointed out by Bonan et al. 53 as a model-dependent parameter.Therefore, accuracy in these parameters is critical for a more effective prediction and modeling by the global panels.
The sugars produced during photosynthesis can be metabolized for maintenance and developmental processes.Catabolism of sugars leads to the consumption of ATP by respiration, which may increase or decrease, depending on the species, when plants are exposed to unfavorable conditions 54 .When neotropical plant species were subjected to elevated CO 2 during growth, they displayed a decrease in dark respiration (Rd) (see Overall in Fig. 1), which is expected to increase the efficiency of the net productivity of carbon gain 55,56 .Thus, the efficiency of the carbon metabolism increases under eCO 2 .The decrease in Rd may be associated with the higher   1).The same pattern of reduction of Rd was observed for temperate trees 12 .However, no meta-analysis has been performed considering sugar metabolism and photosynthesis, so temperate and neotropical species could not be directly compared via meta-analysis.
An explanation for the higher accumulation of starch in leaves of neotropical species growing under eCO 2 is that the photosynthetic assimilation rate can exceed the growth capacity, leading to the accumulation of non-structural carbohydrates 19,57,58 .We found that starch increase (47%) represents the primary non-structural carbohydrate in plant leaves under eCO 2 (Fig. 2).
The increased starch levels in eCO 2 are usually the main element responsible for increasing the content of total non-structural carbohydrates 59 .Starch is composed of insoluble and long-term storage polysaccharides (amylose and amylopectin) that are not readily available to participate in plant metabolic processes 60 but can be used to increase biomass in leaves, stems, and roots, as observed in this meta-analysis (Fig. 2).The carbohydrates synthesized in leaves from extra CO 2 supply were translocated into tree stems (Fig. 2), suggesting that the reserve biomass is driven to this organ, boosting secondary growth 61 .Furthermore, stimulation of photosynthesis with eCO 2 had a response in the biomass increase different in the development of organs and plant seed mass 62 .Li et al. 63 synthesized 71 tree species and data of a more significant increase in starch than soluble sugars in leaves under eCO 2 .The results obtained in this work show that the responses of neotropical plant species to eCO 2 are consistent with those on the global scale (temperate climates mainly), suggesting that the predictions made by models of climate change would answer similarly to temperate and neotropical species 13,47,52 .However, in Brazil, relatively few experiments were carried out with eCO 2 in plants from the biomes Pantanal, Caatinga, Cerrado, Amazon, and the Pampas, the latter in a temperate region (Table 3).Thus, more profound exploration should provide relevant information on how different biomes could answer to eCO 2 and climate change 64,65 .Also, establishing long-term experiments to test the effect of eCO 2 on plants over time in Brazil is needed once a significant portion of the neotropical plants is located there.This would allow an understanding of the physiological responses to climate change 66 .
Native plants in neotropical regions have evolved to adapt to their specific environmental conditions, including CO 2 levels.Elevated CO 2 can positively affect native plants by increasing photosynthesis, promoting plant growth, increasing carbon sequestration, and potentially acting as a CO 2 sink 16 .In contrast, plants grown in neotropical regions are often grown for agricultural purposes.They may have different responses to eCO 2 compared to native plants, although this hypothesis needs to be checked in further studies with more species.Cultivated plants can exhibit increased photosynthetic rates and grow under elevated CO 2 35 .This can benefit crop productivity and potentially increase carbon sequestration in farming systems 67 .However, the response of cultivated plants to elevated CO 2 may vary depending on factors such as plant species, nutrient availability, management Table 2. Meta-analyses result in different variables according to life habits: Trees and Herbs, publication bias, and heterogeneity.Bold letters represent significant differences between Trees and Herbs p < 0.05.For data from the column in publication bias, the p-value < 0.05 does not indicate publication bias.For heterogeneity, analyses were considered I 2 ≤ 25 low, I 2 > 25 to 75 moderate, and I 2 > 75 high heterogeneity.practices, and genetic improvement techniques 68 .Therefore, it is important to note that the potential of native and cultivated plants to act as CO 2 sources or sinks is influenced by several factors.These include the specific plant species, their physiological characteristics, duration of exposure to elevated CO 2 , and general ecosystem  Figure 6 summarizes the responses of the neotropical species analyzed in this work.Temperate and neotropical species respond similarly to eCO 2 , which is likely to reflect directly in the consistency of modeling regarding the adjustment of parameters.Trees and herbs display different responses.The trees studied are primarily young and, therefore, rapidly growing.As they are not yet at the reproductive stage, young trees tend to allocate carbon-from increased photosynthetic rates and lower respiration in the dark-to organ development, significantly increasing leaves, roots, and stem biomasses.As growth rates are limited in comparison with the growth capacity of most herbs, more starch is accumulated in trees, denoting a tight control of carbon metabolism through carbohydrate storage.Herbs, mainly crop plants, reached reproductive maturity during the experiments.Their strategy to respond to eCO 2 involved a drastic increase in water use efficiency, controlled by stomatal conductance.In addition, the plants tend to display more soluble sugars, probably with a transient accumulation of carbon primarily stored in seeds.

Conclusion
The responses of species native or cultivated in the neotropics to eCO 2 can be attributed to contrasting growth strategies and physiological features of trees and herbs.Trees display greater carbon sink capacity and can allocate more resources for growth and storage.The higher rates of photosynthesis in response to eCO 2 (39%) led to greater starch storage (61%) and a more significant biomass accumulation in tree organs (Table 1).This behavior may be attributed to the tree's long lifespan and ability to allocate resources for growth and storage.
In contrast, herbs, which display shorter lifespans, prioritize rapid growth and reproduction and tend to allocate resources that would support higher water use efficiency (117%) due to decreased stomatal conductance (− 39%) under conditions of eCO 2 .Herbs responded differently, increasing net CO 2 assimilation (52%) and soluble sugars such as sucrose and fructose (14%, 15%, and 13%).Understanding these responses would be crucial to predicting the impacts of increased CO 2 levels on different types of plants in the face of eCO 2 increases.
Finally, it is essential to note that eCO 2 alone does not represent the complete response of plants to climate change.Combinations of eCO 2 with stresses of temperature and water will be necessary to assess the systemic response of plants to global climate change.Thus, more experiments are needed using these parameters that, together with modeling work, could help understand how the neotropics, with their rather large proportion of world biodiversity, will respond to climate change in this century.

Data collection.
For data collection, a systematic review was performed.A systematic review is a technique that selects primary studies on a given subject 69 .For the elaboration of the systematic review, it is necessary to identify and describe the steps taken to study selection and data extraction.These steps must follow a protocol that can be consulted and reproducible 69 .The flowchart with steps for data collection is shown in Supplementary Fig. 1.Literature search for the data collection on the effect of the elevated CO 2 on plants was performed in three databases: Web of Science, Scielo, and Brazilian Digital Library of Theses and Dissertations (https:// bdtd.ibict.br) [70][71][72] .For each database, a combination of keywords was used (Supplementary Table 1) that recovered 2096 works on the eCO 2 .In addition, 35 studies were manually included from leading Brazilian researchers by Lattes search (https:// lattes.cnpq.br) 72 .Lattes is a Brazilian platform for integrating Curriculum, Research Groups, and  www.nature.com/scientificreports/Institution databases into a single information system 72 .The search resulted in a total of 2127 analyzed works in the systematic review (Supplementary Fig. 1).A database was assembled with 68 studies published before October 1st, 2021 (see Table 3; Supplementary Fig. 1).The included works were: (a) studies on Brazilian manipulative experimentation, reporting results from both the treatment groups (eCO 2 ) and the control groups (ambient CO 2 = aCO 2 ); (b) studies on trees or herbs; and (c) studies with the mean, sample size, and standard deviation of error of the selected variables.The data from articles were grouped as trees and herbs on 28 and 16 species, respectively (Table 3).The collected data were extracted in three theoretical categories: growth (biomass  www.nature.com/scientificreports/data were collected from total biomass or biomass per plant organ.Each biomass result per organ was considered a biomass observation.Each soluble sugar (glucose, fructose, sucrose, raffinose, and myoinositol) was considered an observation for the biochemical category.A dataset contemplated a total of 437 observations.In general, the duration of the studies was 90 days.The average high CO 2 concentration was from ~ 400 to ~ 800 ppm.Fifty studies were performed in Open Top Chambers (OTC), 13 in Free Air CO 2 Enrichment (FACE), and 7 in Glasshouse (GC).The most frequently studied species among trees was Coffea arabica, with 12 different studies.On the other hand, among herbs was Panicum maximum with six different studies.Fourteen variables were analyzed [A, gs, E, WUE, Rd, Ci/Ca, biomass, total soluble sugars, starch, proteins, Fv/Fm, total Chl, Vc max , J max ] (Fig. 5).The most frequent variables were biomass (79), with 46 observations for trees and 33 for herbs (Fig. 5).From the total species analyzed, 30% represent cultivated ones.Among the trees, 21% are cultivated, and 79% are native species.Among herbs, 33% are native, and 67% are cultivated.The experiments were considered unstressed unless the author had identified some stress factor.In the case of stress treatments, data from the control treatments were used.Most of the works had an average duration of experimentation of 90 days.The plants were grown in pots.
Plants that received fertilizer treatment were not included in this analysis.The plants were watered regularly and exposed to natural light.
Observations of each study at the end of the experiment were grouped, and there was no categorization by experiment period.There was also the group for the elevated CO 2 levels of the different studies.Curtis and Wang 13 examines each subgroup for categorical divisions such as pot size and exposure time.However, a meta-analysis by these authors did not find significant differences among the groups by pot size and experiment time.This is an example that, throughout all studies, suggests significant differences in the response of plants under the CO 2 environment and, however, not among those grown in different pot sizes or experiment duration.
Mean values, standard deviation/error, and sample size under eCO 2 and aCO 2 were collected for each observation.WebPlotDigitizer v4.1 73 was used to obtain the numerical data from the figures.For works that showed only the standard error value, the following equation was used: (SD = SE × √n) (n is the sample size, SE is the standard error, and SD is the standard deviation) 74 .Data from temporal experiments were considered only the last harvest to represent the maximum exposure of these plants to eCO 2 cultivation.

Meta-analysis.
Meta-analysis assessed plant responses to eCO 2 in growth, biochemical composition, and photosynthesis categories.To evaluate the relative changes of these responses between treatment (eCO 2 ) versus control (aCO 2 ), it was applied the logarithmic response ratio ln (RR), calculated as the size effect, where X̅ t is the mean of the experimental/treatment group, and X̅ c is the mean of the control group 68 .The natural log of the response ratio (lnRR = X̅ t/X̅ c) was used and is reported as the mean percentage change [(lnRR − 1) × 100] 75 .Values of lnRR higher than zero indicate that the eCO 2 effect increases, while negative values indicate that the eCO 2 effect decreases concerning aCO 2 .A hierarchical mixed-effects model was used to estimate the mean and 95% confidence interval (CI) of the lnRR for each type of response variable.If the 95% CI of a response variable overlaps zero, the lnRR of the treatment is not significantly different from the control 76 .The effect was reported as a percentage change from the control: ((e lnRR − 1) × 100).In addition, life habits were used as a fixed predictor variable while the study and species were considered random variables to control for the lack of independence of observations from the same study or/and carried out with the same plant species 77,78 .Furthermore, heterogeneity (I 2 ) was tested to verify the variation in results between studies 77,79 .The Egger regression test was used to identify publication bias 80,81 .Bias analyses for the multilevel models were conducted with meta-analytic residuals 77 .Analyzes were performed using the package "metafor" 82 , and the graphics were generated using the package "ggplot2" 78 , both in R version program 3.6.0 83.

Figure 1 .
Figure 1.Responses of photosynthetic variables: Net CO 2 assimilation (A), stomatal conductance (gs), dark respiration (Rd), foliar transpiration (E), water use efficiency (WUE), intercellular/ambient CO 2 rate (Ci/Ca), maximum electron transport rate (J max ), and maximum Rubisco carboxylation rate (Vc max ) according to life habits: Trees (a), Herbs (b), and Overall (c) in plants grown in elevated CO 2 .The circles represent the percentage changes in elevated CO 2 .Error bars represent 95% confidence intervals.Study numbers for each variable are shown in parentheses.

Figure 2 .
Figure 2. Biomass responses in each plant organ (leaf, stem, root, grain, and total) in plants grown into elevated CO 2 according to life habits: Trees (a), Herbs (b), and Overall (c).The circles represent the percentage changes in elevated CO 2 .Error bars represent 95% confidence intervals.Study numbers for each variable are shown in parentheses.

Figure 3 .
Figure 3. Responses of non-structural carbohydrates (glucose, fructose, sucrose, total soluble sugars, and starch) in plants grown to elevated CO 2 , according to life habits: Tree (a), Herbs (b), and Overall (c).The circles represent the percentage changes to elevated CO 2 .Error bars represent 95% confidence intervals.Study numbers for each variable are shown in parentheses.

Figure 4 .
Figure 4. Responses of potential quantum efficiency of photosystem II (Fv/Fm), total chlorophyll content, and proteins in plants grown in elevated CO 2 , according to life habits: Trees (white), Herbs (gray), and Overall (black).The circles represent the percentage change in elevated CO 2 .Error bars represent 95% confidence intervals.Study numbers for each variable are shown in parentheses.

Figure 5 .
Figure 5.Observation numbers from the literature extracted were divided into biomass, biochemical, and photosynthesis components according to life habits: Trees (black) and Herbs (gray) in experiments with elevated CO 2 .The variables correspond to total biomass, total soluble sugars, starch, proteins, net CO 2 assimilation (A), stomatal conductance (gs), foliar transpiration foliar (E), water use efficiency (WUE), dark respiration (Rd), intercellular/ambient CO 2 ratio (Ci/Ca), the potential quantum efficiency of PSII (Fv/Fm), total chlorophyll (total Chl) maximum Rubisco carboxylation rate (Vc max ), and maximum electron transport rate (J max ).

Figure 6 .
Figure 6.Tropical climate trees and herbs responses to elevated CO 2 .

Table 1 .
Meta-analysis with the percentage change of the biomass, photosynthesis, and biochemical variables measured in Trees and Herbs under elevated CO 2 .Observation numbers (k).The effect size values are represented as Log response rate (LnRR) and percentage.Average estimates with lower and upper Confidence Intervals (CI).Bold letters represent significant differences (p < 0.05).

Table 3 .
Species found in a literature search with plants grown at different CO 2 atmospheric concentrations (ambient CO 2 = aCO 2 and elevated CO 2 = eCO 2 ), classified according to life habits: Tree and Herbs.OTC Open top chambers, FACE Free Air Carbon Enrichment, and GC Glasshouse, ppm parts per million.