Environmental Pollution xxx (2014) 1e6

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Is guava phenolic metabolism influenced by elevated atmospheric CO2? Fernanda Mendes de Rezende*, Amanda Pereira de Souza, Marcos Silveira Buckeridge,
udia Maria Furlan Cla ~o Paulo, Rua do Mata ~o, 277, CEP 05508-090, Sa ~o Paulo, SP, Brazil Department of Botany, Institute of Bioscience, University of Sa

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 February 2014 Received in revised form 17 July 2014 Accepted 24 July 2014 Available online xxx

Seedlings of Psidium guajava cv. Pedro Sato were distributed into four open-top chambers: two with ambient CO2 (~390 ppm) and two with elevated CO2 (~780 ppm). Monthly, five individuals of each chamber were collected, separated into root, stem and leaves and immediately frozen in liquid nitrogen. Chemical parameters were analyzed to investigate how guava invests the surplus carbon. For all classes of phenolic compounds analyzed only tannins showed significant increase in plants at elevated CO2 after 90 days. There was no significant difference in dry biomass, but the leaves showed high accumulation of starch under elevated CO2. Results suggest that elevated CO2 seems to be favorable to seedlings of P. guajava, due to accumulation of starch and tannins, the latter being an important anti-herbivore substance. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Starch Flavonoids Tannins Open top chamber Elevated CO2

1. Introduction Besides the traditional polluting gases known, carbon dioxide (CO2) has been attracting the attention of many researchers due to its association with the greenhouse effect. It is estimated that the atmospheric concentration of carbon dioxide [CO2] increases about 1% per year, and that [CO2] can reach 600e750 ppm within 90 years (IPCC, 2013). In 2013, the Brazilian Panel on Climate Change (PBMC), created in 2009 by the Ministry of Environment (MMA) and Ministry of Science, Technology and Innovation (MCTI), released the first national assessment report (RAN1). This is the most complete diagnosis produced about main trends of future climate in the country. Forecasts suggest that rising in [CO2] will lead an increase in temperature of 3e6
C by 2100 in all Brazilian regions and changes in precipitation patterns (RAN1, 2013). Changes in [CO2] also are supposed to have some effect on the physiological behavior of plants as CO2 is important to photosynthesis and may influence the metabolic processes in plants. Secondary metabolites are important compounds in plant defense mechanism and affect all interactions of plants with their

* Corresponding author. E-mail addresses: [email protected], [email protected] (F. Mendes de Rezende).

biotic and abiotic environment, showing a high genetic plasticity and diversity that guarantees plant adaptations to the continuous alterations on their environment (Fernie, 2007; Hartmann, 2007; Edreva et al., 2008; Ibrahim and Jaafar, 2011). There are several publications reporting effects on physiology of plants that grow in an atmosphere enriched with CO2 (Griffin and Seemann, 1996; Ceulemans et al., 1999; Pritchard et al., 2001; Luo et al., 1999; Kerstiens, 2001; Ainsworth and Long, 2005; Malhi € rner, 2006; to name but a few). Also there and Phillips, 2004; Ko are many studies that discuss the relationship between increased levels of [CO2] and the synthesis of secondary metabolites (Lincoln et al., 1993; Lavola and Julken-Titto, 1994; Kinney et al., 1997; Hartley et al., 2000; Coviella et al., 2002; Chen et al., 2005; Peltonen et al., 2005; Braga et al., 2006; Koike et al., 2006; Kelly et al., 2010; Wu et al., 2011). However, these studies are mostly focused on temperate species, including agricultural crops, herbaceous and woody species (Iason et al., 2012). Here we report results of effects of elevated [CO2] on Psidium guajava L. (Myrtaceae), an important tropical crop. It is used for production of fruits in Brazil, the fourth major country on guava production (FAO, 2011). It is also used in popular medicine mainly
rrez et al., 2008). Tannins because of its antidiarrheal effect (Gutie and flavonoids (quercetin derivatives) have been described as the major secondary compounds produced by this species (Park et al., 2012; Mailoa et al., 2013; Yousaf et al., 2013), and the therapeutic activity of guava has been associated to the presence of these two

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classes of substances (Meckes et al., 1996; Shaheen et al., 2000;
rrez et al., 2008). Guitie Guava has also been described in the literature as a bioindicator of atmospheric pollutants both in a field experiment, as in a controlled experiment of fumigation (Pandey and Pandey, 1994; Moraes et al., 2002; Furlan et al., 2007; Pina et al., 2007; Pina and Moraes, 2010). Guava has an active production of phenolic compounds, in presence of ozone (O3) guava leaves showed visible symptoms, red tint, correlated with the presence of two classes of phenolic compounds: anthocyanins and tannins (Furlan et al., 2007; Rezende and Furlan, 2009). The cultivar Pedro Sato showed less intense symptoms, bronzed tint, and inversely correlated to contents of anthocyanins and tannins (Moraes et al., 2011). According to growthedifferentiation balance theory (GDB theory) the increase of resource availability first induces to a fast increase on gross primary production rate and on defense-related metabolism, while the growth-related metabolism shows a slow increase (Matyssek et al., 2012). The increase in C-based secondary metabolites frequently occurs when environmental conditions also promote an accumulation of non-structural carbohydrates in plants, since these carbohydrates can be used as building blocks for secondary metabolites (Ghasemzadeh et al., 2010). However, while the resource availability is high, the gross primary production rate and the growthrelated metabolism continue to increase, but the defense-related metabolism shows reduction, once growth and defense metabolism respond in complementary ways to each other (Matyssek et al., 2012). Given the economic importance as a tropical fruit species in Brazil, and the reported alterations in phenolic production in the presence of atmospheric pollutants such as O3 and fluorides, P. guajava became an interesting species to be evaluated under elevation of atmospheric [CO2]. This study aimed at evaluating how a species with high production of phenolic compounds will invest the surplus carbon provided by an elevated [CO2]. We hypothesized that investment will be on growth or accumulating non-structural carbohydrates and/or phenolic compounds (in terms of trade-off). In order to test this hypothesis we analyzed the contents of soluble carbohydrates and starch, carbon/nitrogen ratio, and phenolic compounds (flavonoids and tannins) in seedlings of P. guajava subjected to an elevated CO2 atmosphere.

extractions with 1.5 mL of 80% ethanol for 20 min at 80
C in a water bath. The supernatants were collected and dried under vacuum, taken up in 1 mL of ultrapure water plus 0.5 mL of dichloromethane (CH2Cl2) for the removal of soluble pigments, such as chlorophyll (De Souza et al., 2013). The quantification of soluble sugars (modified from Massuko et al., 2005) was performed with an ELISA-type microplate reader at 490 nm, in each well 30 ml of the diluted aqueous extract containing soluble sugars were mixed with 150 ml of concentrated sulfuric acid and 30 ml of 5% phenol (water was used as negative control). The plate was incubated at 75
C for 5 min and cooled in an ice bath for 5 min. The results obtained were compared to a standard curve using glucose as reference (23e700 mg mL1) and are expressed in mg g1 of dry weight (DW). In order to analyze starch, the precipitate was treated with a-amylase (120 U mL1) from Bacillus licheniformis and amyloglucosidase from Aspergillus niger. Content of starch was determined with an ELISA-type microplate reader at 490 nm with 5 ml of extract diluted in 45 mL of water and 250 mL of glucose PAP, the plate was incubated for 15 min at 37
C (Amaral et al., 2007). A standard curve was prepared using glucose solutions ranging from 20 to 300 mg mL1. Results are expressed in mg g1 of dry weight (DW). 2.3. C/N ratio Two milligrams of freeze-dried and powered plant material from root, stem and leaves were analyzed by a PerkineElmer 2400 CHN elemental Analyzer. The percentages of carbon and nitrogen were used to calculate the C/N ratio. 2.4. Phenolic compounds Hydroalcoholic extracts were prepared using 15 mL of 80% methanol and 100 mg of plant material from root, stem and leaves. After incubation for 1 h at 70
C in a dry bath, the extract was filtered and the volume was adjusted to 25 mL. This extract was used for analysis in UVevisible spectrophotometer to quantify: total tannins, proanthocyanidins (condensed tannins), total phenolics and total flavonoids. The following assays were used to quantify total tannins, proanthocyanidins, total phenolics and total flavonoids, respectively, according to Watanabe et al. (2011): protein precipitationeBSA, acid butanol, Folin-Ciocalteu and aluminum chloride assay. 2.5. Statistical analysis Analyses were performed using Statistica software (version 12) to test normality and homoscedasticity. A box-plot was constructed to identify outliers and extremes (n ¼ at least 8 for each treatment). ANOVA considering treatment (ambient or elevated CO2) and exposure time (in days) was performed followed by a post-hoc

2. Material and methods 2.1. Experimental designs Seedlings of P. guajava cv. Pedro Sato (~30 cm tall) were obtained from a Bra~o Paulo (southeast of Brazil). zilian producer, Sítio S~ ao Jo~ ao II, Taquaritinga, Sa ~o Paulo, Brazil in 10 L (120 cm height and Seedlings were planted at University of Sa 9 cm diameter) pots using standardized substrate (Plantmax Eucatex®) and vermiculite (2:1). They were kept for 30 days in an open top chamber (OTC) before exposure. The CO2 exposure was conducted for three months (August 29 to November 24, 2011). During the period of experiment mean air temperature, relative air humidity and CO2 concentration were monitored by a software RICS (RICSRemote Integrated Control System). During experimental period, water supply was provided to the plants on a daily basis and fertilizer on a monthly basis provided by addition of 50 mL of Hoagland solution (Epstein, 1975). Four polycarbonate OTC's (3.5 m height  1.5 m diameter; 90% of transmittance of visible light e up to 400 nm) were used: two were kept under elevated [CO2] (~780 ppm), while the other two chambers provided a current ambient [CO2] (~390 ppm). To avoid effects caused by micro environmental differences, the pots were weekly rotated into the chamber. Five plants of each chamber were harvested every 30 days, totaling ten plants for each treatment. One day before harvesting stem height and number of leaves were measured, and on the harvesting day was also measured root length. After harvesting, plant material was separated into leaves, stems and roots. The material was immediately weighed and frozen in liquid nitrogen. Subsequently, the material was lyophilized and weighed again to obtain dry biomass. Samples were powdered in a ball mill for all chemical analyses. 2.2. Carbohydrates For extraction of total soluble sugars and starch, 10 mg of the lyophilized and grounded material from roots, stems and leaves was subjected to four successive

Fig. 1. (A) Mean temperature (
C), (B) relative humidity (%) and (C) CO2 concentration (ppm) along the exposure period. -- elevated CO2 and C ambient CO2.

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F. Mendes de Rezende et al. / Environmental Pollution xxx (2014) 1e6

Fig. 2. Biomass (g DW) during the 90 days of exposure. Asterisk indicates significant difference (p < 0.05) between treatments: ambient CO2 (A) and elevated [CO2] (C); (n ¼ 10).

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saplings growing at elevated [CO2] showed a tendency to increment their total dry biomass (Fig. 2). Data on growth as stem height, number of leaves and root length (data not shown) showed no significant differences between treatments. Regarding biochemical assays, significant increase in starch was observed in leaves, but not in stems and roots. Soluble carbohydrates accumulation did not vary significantly in any organ (Fig. 3). A significant increase (p < 0.05) was observed in the C/N ratios in leaves and stems of guava seedlings growing at elevated [CO2] (Table 1). Contents of total phenolic compounds and flavonoids in root, stem and leaves showed no significant changes between treatments (Table 2). However, leaves of P. guajava at elevated [CO2] showed a significant accumulation of tannins after 90 days of exposure to elevated [CO2] (p < 0.05). Comparing the amounts of phenolic compounds on the different organs of P. guajava under high [CO2], it has been observed that the roots showed higher levels of total phenolic compounds (Table 2). 4. Discussion

Tukey test. We analyzed statistically the results also separately by chamber, and there was no significant difference between the chambers of the same treatment.

3. Results During the experimental period the average temperature and relative humidity were 22.7
C and 69.25%, respectively. The average of CO2 concentration was 780 ppm at the elevated [CO2] chambers and 390 ppm at the ambient [CO2] (Fig. 1). Visible injury symptoms were not observed on the surfaces of the leaves under elevated [CO2]. Dry biomass showed no significant differences between treatments. After 60 and 90 days of exposure,

A few studies examined the effect of elevated CO2 concentrations in tropical tree species relating to secondary metabolites production. The present study showed that surplus carbon leads to starch and tannins accumulation in leaves of P. guajava but not to a significant growth increment. Studying saplings of tropical trees and crop species, several authors observed that elevated [CO2] increase carbon assimilation, leaf area and total dry biomass (Aidar et al., 2002; Godoy et al., 2009; Rasineni et al., 2011; De Oliveira et al., 2012; Roy et al., 2012; Satapathy et al., 2014; Kumari and Agrawal, 2014). According to Rai et al. (2010) P. guajava presents a slow growth rate and a substantial variability in seedling populations, which

Fig. 3. Amounts of starch (g g1 DW) and soluble carbohydrates (g g1 DW) during 90 days of CO2 fumigation. Asterisk indicates significant difference (p < 0.05) between treatments: ambient CO2 ( ) and elevated [CO2] (-); (n ¼ 10).

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Table 1 Carbon and nitrogen percentages, and C/N ratio during 90 days of CO2 fumigation. Asterisk indicates significant difference (p < 0.05) between treatments: ambient CO2 and elevated CO2; (n ¼ 5). Days of exposure

Leaves Elevated CO2

Ambient CO2 C (%)

N (%)

C/N ratio

C (%)

N (%)

C/N ratio

0 30 90

53.31 ± 0.13 45.64 ± 0.39 45.19 ± 0.27

1.37 ± 0.24 1.05 ± 0.12 0.86 ± 0.19

33.74 ± 5.90 45.41 ± 3.62 47.79 ± 4.00

45.51 ± 0.51 44.92 ± 0.31 45.06 ± 0.54

1.14 ± 0.06 0.81 ± 0.11 0.70 ± 0.11

39.88 ± 2.09 *56.29 ± 8.26 *64.47 ± 9.78

Days of exposure

Stem Elevated CO2

Ambient CO2 C (%)

N (%)

C/N ratio

C (%)

N (%)

C/N ratio

0 30 90

43.65 ± 0.33 43.81 ± 0.78 43.76 ± 0.88

2.41 ± 1.16 1.16 ± 0.40 1.15 ± 0.50

30.32 ± 24.21 44.59 ± 18.81 44.93 ± 25.53

42.73 ± 0.77 43.76 ± 0.93 43.15 ± 0.31

0.68 ± 0.10 0.47 ± 0.11 0.43 ± 0.08

63.24 ± 9.81 *98.45 ± 26.48 *102.25 ± 20.7

Days of exposure

Roots Elevated CO2

Ambient CO2

0 30 90

C (%)

N (%)

C/N ratio

C (%)

N (%)

C/N ratio

43.28 ± 0.42 42.91 ± 0.93 42.45 ± 1.93

0.9 ± 0.11 0.61 ± 0.04 0.47 ± 0.06

48.66 ± 6.72 70.33 ± 7.26 90.05 ± 12.47

44.18 ± 0.64 44.23 ± 0.48 44.35 ± 0.69

0.78 ± 0.16 0.55 ± 0.03 0.53 ± 0.06

57.91 ± 12.69 79.89 ± 6.02 84.56 ± 11.7

species Hymenaea courbaril. The accumulation of non-structural carbohydrates in leaves of C3 plants due to the high [CO2] is a well-documented effect (Stitt, 1991; Cheng et al., 1998; Rogers et al., 2004; Ainsworth and Rogers, 2007; Leakey et al., 2009; Tausz et al., 2013). The hypothesis that there would be quantitative differences in phenolic compounds with higher availability of carbon was

affect guava production. These two characteristics allied to a short time exposure (90 days) may have reflected on the low biomass increment found, even under elevated [CO2], denoting the fact that P. guajava behaves as a late successional species. The extra carbon provided by the enhanced [CO2] was accumulated as starch and tannins in leaf tissues of guava. Similar results were observed by Costa (2004) studying another tropical tree

Table 2 Levels of total phenolic, tannins, flavonoids and proanthocyanidins (mg g1 dw; mean ± standard deviation) in different plant organs. Asterisk indicates significant difference (p < 0.05) between treatments: ambient CO2 and elevated CO2. Days of exposure

Total phenolic Roots

Stems

Ambient CO2 0 30 60 90 Days of exposure

143.59 123.14 111.79 137.97

± ± ± ±

13.64 13.95 18.88 27.67

Elevated CO2 218.86 115.36 109.24 121.37

± ± ± ±

82.19 21.72 18.04 16.02

2.75 2.54 2.32 2.06

± ± ± ±

0.54 0.32 0.28 1.48

Elevated CO2 1.86 2.79 2.80 3.25

± ± ± ±

1.54 0.85 0.68 0.87

15.69 9.75 8.31 22.39

Elevated CO2 50.86 39.86 51.88 58.67

± ± ± ±

7.94 12.35 7.34 8.55

Ambient CO2 110.63 122.06 115.97 103.79

± ± ± ±

11.99 19.59 15.18 26.73

Elevated CO2 115.35 98.82 108.64 128.89

± ± ± ±

15.83 12.81 19.67 21.03

Leaves

Ambient CO2 42.53 45.21 42.84 63.97

± ± ± ±

18.86 7.03 7.63 8.57

Elevated CO2 46.11 37.51 51.62 60.26

± ± ± ±

1.30 24.09 5.74 10.43

Ambient CO2

Elevated CO2

84.12 ± 13.78 105.36 ± 19.52 113.04 ± 13.17 *102.8 ± 11.68

90.67 92.89 122.06 129.23

± ± ± ±

16.16 19.28 10.60 11.27

Total flavonoids Roots

Stems

Ambient CO2 ± ± ± ±

0 30 60 90

1.53 1.75 1.45 2.71

Days of exposure

Proanthocyanidins

0.41 0.54 0.14 0.84

Elevated CO2 2.34 1.55 2.03 2.19

± ± ± ±

0.80 0.51 0.63 0.47

Roots

0.45 0.55 0.44 0.37

± ± ± ±

Leaves

Ambient CO2 3.13 2.73 2.19 2.38

± ± ± ±

1.53 0.45 0.77 1.28

Elevated CO2 3.49 2.17 1.38 3.21

± ± ± ±

1.40 1.09 0.20 0.31

Stems

Ambient CO2 0 30 60 90

± ± ± ±

Stems

Ambient CO2

Days of exposure

Ambient CO2 46.43 42.93 43.67 46.85

Total tannins Roots

0 30 60 90

Leaves

0.25 0.12 0.20 0.25

Elevated CO2 0.26 0.55 0.54 0.63

± ± ± ±

0.29 0.21 0.28 0.37

Ambient CO2 23.88 25.95 26.05 21.01

± ± ± ±

2.08 3.17 3.22 3.68

Elevated CO2 21.18 22.47 22.04 22.83

± ± ± ±

1.71 3.23 3.82 3.49

Leaves

Ambient CO2

Elevated CO2

Ambient CO2

Elevated CO2

± ± ± ±

3.68 ± 0.90 5.08 ± 3.46 12.01 ± 2.91 13.42 ± 2.57

36.11 ± 9.75 57.72 ± 12.71 65.01 ± 13.05 *55.36 ± 12.43

42.07 46.79 70.23 77.87

7.94 9.10 9.24 17.76

4.37 3.51 3.05 4.13

± ± ± ±

17.53 12.01 15.45 8.62

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confirmed. P. guajava cv. Pedro Sato showed high amounts of total tannins in elevated [CO2], these compounds being important as herbivore deterrence and contributing, along with flavonoids, to pharmacological effects of guava extracts. The mechanism by which elevated [CO2] affected the biosynthesis of phenolic compounds or why these compounds are accumulated or have reduced levels in plants has not been fully elucidated. Stutte et al. (2008), studying two Scutellaria species with high contents of flavonoids, after CO2 enrichment, suggested that this gas might affect enzyme activities. For example, the CO2 enrichment seems to activate a glucosyl-transferase responsible for glucosylation of bacalein into baicalin, one of the major flavonoids found on Scutellaria species. The authors also suggested that hydroxylases seem to have been activated with elevated [CO2], resulting in the accumulation of scutellarein, baicalin and apigenin. Ibrahim and Jaafar (2011) hypothesized that when the synthesis of protein is restricted by the high carbonenitrogen ratio availability, a lower demand for amino acids could determine stimulation on the synthesis of phenolic compounds. This hypothesis predicts high accumulation of carbon based secondary metabolites, such as phenolic and terpene, at elevated [CO2]. In the present study, this hypothesis is likely to explain the observation of higher tannin accumulation in guava since C/N ratios of leaves and stem have increased in high CO2 treatment, suggesting a lower demand for amino acids. Tannins showed significant increase in leaf tissues after 90 days in high [CO2]. It may suggest that this class of substance could be related to a mechanism of signaling and/or defense of this species to environmental alterations, being accumulated or reduced in situations of surplus atmospheric carbon and under oxidative stress (Rezende and Furlan, 2009; Sandre et al., 2014). The results observed on three months of elevated [CO2] exposure can be explained by the growthedifferentiation balance theory (GDB theory): the increase of resource availability for P. guajava induced the gross primary production of this species, observed as starch accumulation and higher C/N rations; it also induced the defense-related metabolism, observed as tannins accumulation. Instead, the growth-related metabolism showed a slow increase. It is undeniable that the elevated [CO2] affects the production of primary and secondary plant compounds, but responses can vary according to the kind, location, and period of exposure (Way and Oren, 2010). Literature shows a lack of studies using tropical tree species as models for investigation on the defense-related metabolism. It is important to conduct studies interrelating changes in environmental parameters such as temperature, availability of water, ozone and elevated CO2 with primary and secondary metabolism. Guava has a great economic importance in Brazil related to the production and quality of its fruits. Studies that analyze the effect of high atmospheric CO2 in the quantity and quality of fruits produced by this species are necessary. Being a slow-growing species, longer periods of exposure will be important to better analyze the susceptibility of this cultivar to elevated [CO2] and also to confirm whether the prediction of GDB prevails. Our results can not be used for extrapolations to the composition of guava fruits in elevated CO2, but the finding of the potential that starch and tannins can increase in the plant highlights the necessity of future studies to evaluate fruit composition in elevated CO2. 5. Conclusions The results observed on three months of CO2 experiment can be explained by the growthedifferentiation balance theory (GDB theory): an initial fast increase on the gross primary production (starch accumulation and higher C/N rations) and on the defense-

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related metabolism (tannins accumulation), but the growthrelated metabolism showed a slow increase. The increase in C/N ratio is likely to have decreased the demand for amino-acids for protein synthesis and the surplus of carbon has been allocated to starch and tannins in leaves. In this study the presence of high [CO2] during growth of P. guajava cv. Pedro Sato seem to be favorable for this cultivar, since there is an accumulation of starch and anti-herbivore substances (such as tannins), which may result on higher protection of this species against herbivores and pathogens. Acknowledgment ~o de Amparo a  Pesquisa do The authors thank FAPESP (Fundaça ~o Paulo) (Process 2011/094354 and 2012/06942-5) and Estado de Sa CAPES (Coordenaç~ ao de Aperfeiçoamento Pessoal de Nível Superior) for funding this research. Mouriza Ferreira, Aline Bertinatto Cruz, Paula Alecio e Leila Sampaio da Silva for their technical assistance during chemical analysis. References Aidar, M.P.M., Martinez, C.A., Costa, A.C., Costa, P.M.F., Dietrich, S.M.C., Buckeridge, M.S., 2002. Effect of atmospheric CO2 enrichment on the estab
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Please cite this article in press as: Mendes de Rezende, F., et al., Is guava phenolic metabolism influenced by elevated atmospheric CO2?, Environmental Pollution (2014), http://dx.doi.org/10.1016/j.envpol.2014.07.028