ABSTRACT

Soil water saturation requires different adaptative strategies by tree species that live under such conditions. We aimed to study the responses that ensure flooding tolerance by tree species and so provide support for recovery projects with degraded areas subject to flooding. We evaluated the ecophysiology of Cedrela fissilis under different water saturations, including anatomical traits, gas exchange parameters, antioxidant system analysis and growth. We subjected 100 day-old plants to three treatments: Control (FC) where the substrate was kept at field capacity; Flooded Roots (FR), where the substrate remained submerged but with no surface layer of water, and Flooded Stem (FS), with a water layer accumulation of around 3.0 cm over the substrate, flooding part of the stems. The plants were kept under such conditions for 90 days. Plants survived in all treatments due to a marked development of cortical intercellular spaces in flooded plants. However, photosynthesis and other gaseous exchange were limited under FR and FS treatments. In addition, the periderm thickness was reduce by flooding and there was an accumulation of starch grains in the parenchyma cells of the xylem, cortex and pith of the stem. There was also a significant lipid peroxidation on the leaves under FR and FS treatments. The superoxide dismutase activity was decreased, but the activities of the ascorbate perioxidases and catalase in the leaves were increased. We concluded that young Cedrela fissilis plants partially tolerate flooding since they developed ecophysiological changes in order to survive in this condition. Consequently, this species is a good candidate for the reforestation projects in environments where intermittent flooding occurs, such as riverside and riparian forests.

Keywords:
Cedar; Flood conditions; Hypoxia; Antioxidant system; Lipid peroxidation

INTRODUCTION

Climate change has affected the water cycle around the world impacting on the availability of water resources in ecosystems (Marengo et al., 2011MARENGO, J.A.; NOBRE, C.A.; CHOU, S.C.; TOMASELLA, J.; SAMPAIO, G.; ALVES L. M.; OBREGÓN, G.O.; SOARES, W.R.; BETTS, R.; KAY, G. Riscos das Mudanças Climáticas no Brasil: Análise conjunta Brasil-Reino Unido sobre os impactos das mudanças climáticas e do desmatamento na Amazônia. CCST/INPE&Met Office Hadley Centre, 2011. 55p.). A great oscillation of water availability is estimated to occur in tropical forests, promoting unusual periods of rainfall which may affect and the occurrence and duration of flooding events (IPCC, 2014INTERGOVERNMENTAL PANEL ON CLIMATE CHANGE. Climate Change 2014-Impacts, Adaptation and Vulnerability: Regional Aspects. Cambridge University Press, 2014, 1142p.).

In this scenario, in which flooding events are likely to increase, plant life may be impaired once soil water saturation decreases oxygen availability (Loreti et al., 2016LORETI, E.; VAN VEEN, H.; PERATA, P. Plant responses to flooding stress. Current Opinion in Plant Biology, v. 33, p. 64-71, 2016.). Moreover, since O₂ diffusion is 10,000 times lower in water than in saturated air, this reduces oxygen exchange between the atmosphere and the flooded soil (Colmer and Pedersen, 2008COLMER, T.D.; PEDERSEN, O. Underwater photosynthesis and respiration in leaves of submerged wetland plants: gas films improve CO2 and O2 exchange. New Phytologist, v. 177, n. 4, p. 918-926, 2008.; Kreuzwieser and Rennenberg, 2014KREUZWIESER, J.; RENNENBERG, H. Molecular and physiological responses of trees to waterlogging stress. Plant, cell & environment, v. 37, n. 10, p. 2245-2259, 2014.).

Low oxygen availability may trigger several disorders in plants, such as reduced growth, chlorosis, premature abscission of leaves, decreased water potential and leaf expansion, among other effects and may even, in some cases, result in plant death (Jackson and Colmer, 2005JACKSON, M.B.; COLMER, T.D. Response and adaptation by plants to flooding stress. Annals of Botany , v. 96, n. 4, p. 501-505, 2005.; Medri et al., 2012MEDRI, C.; PIMENTA, J.A.; RUAS, E.A.; SOUZA, L.A.; MEDRI, P.S.; SAYHUN, S.; BIANCHINNI, E.; MEDRI, M.E. O alagamento do solo afeta a sobrevivência, o crescimento e o metabolismo de Aegiphila sellowiana Cham.(Lamiaceae)?. Semina: Ciências Biológicas e da Saúde, v. 33, n. 1, p. 123-134, 2012.).

However, there are plants that are capabler of growing in oxygen-deficient soils (De Carvalho Gonçalves et al., 2013DE CARVALHO GONÇALVES, J.F.; GURGEL DE FREITAS MELO, E.; FERREIRA, M. J.; MOURA DA SILVA, C.E.; BARRONCAS GOMES, I. Crescimento, partição de biomassa e fotossíntese em plantas jovens de Genipa spruceana submetidas ao alagamento. Cerne, v. 19, n. 2, p. 193-200, 2013.; Voesenek and Bayley-Serres, 2015VOESENEK, L.A.C.J.; BAILEY-SERRES, J. Flood adaptive traits and processes: an overview. New Phytologist , v. 206, n. 1, p. 57-73, 2015.). The mechanisms for flooding tolerance are complex and entail a combination of anatomical and physiological changes such as: activation of an anaerobic metabolism, biochemical adjustments and the activation of the antioxidant system (Martizzaro et al., 2013MARTINAZZO, E.G.; PERBONI, A.T.; OLIVEIRA, P.V.D.; BIANCHI, V.J.; BACARIN, M.A. Photosynthetic activity in japanese plum under water deficit and flooding. Ciência Rural, v. 43, n. 1, p. 35-41, 2013.; Polacik and Maricle, 2013POLACIK, K.A.; MARICLE, B. R. Effects of flooding on photosynthesis and root respiration in saltcedar (Tamarix ramosissima), an invasive riparian shrub. Environmental and experimental botany , v. 89, p. 19-27, 2013.; Voesenek and Bailey-Serres, 2015). In addition, flooding causes changes in the biomass allocation among the plants organs and changes in carbohydrate storage (Ferreira et al., 2009FERREIRA, C.S.; PIEDADE, M.T.F.; FRANCO, A.C.; GONÇALVES, J.F.C.; JUNK, W.J. Adaptive strategies to tolerate prolonged flooding in seedlings of floodplain and upland populations of Himatanthus sucuuba, a Central Amazon tree. Aquatic Botany, v. 90, n. 3, p. 246-252, 2009.), hypertrophy of lenticels, and development of adventitious roots and aerenchyma (Arruda and Colbo, 2004ARRUDA, G.M.T.; CALBO, M.E.R. Efeitos da inundação no crescimento, trocas gasosas e porosidade radicular da carnaúba (Copernicia prunifera (Mill.) HE Moore). Acta Botanica Brasilica, v. 18, n. 2, p. 219-224, 2004.; Medri et al., 2007MEDRI, M.E.; FERREIRA, A.C.; KOLB, R.M.; BIANCHINI, E.; PIMENTA, J.A.; DAVANSO-FABRO, V.M.; MEDRI, C. Alterações morfoanatômicas em plantas de Lithraea molleoides (Vell.) Engl. submetidas ao alagamento. Acta Scientiarum. Biological Sciences, v. 29, n. 1, p. 15-22, 2007.; Voesenek and Bailey-Serres, 2015).

Although the responses of plants under low oxygen availability conditions are known in general, the tolerance mechanisms vary among species and depend on multiple factors, such as the intensity and length of stress, the plant’s age among others (Colmer and Pedersen, 2008COLMER, T.D.; PEDERSEN, O. Underwater photosynthesis and respiration in leaves of submerged wetland plants: gas films improve CO2 and O2 exchange. New Phytologist, v. 177, n. 4, p. 918-926, 2008.; Lira et al., 2013LIRA, J.M.S.; ANASTÁCIO FERREIRA, R.; DA SILVA JUNIOR, C.D.; DOS SANTOS NETO, E.M.; DA SILVA SANTANA, W. Análise de crescimento e trocas gasosas de plantas de Lonchocarpus sericeus (Poir.) DC sob alagamento para uso na recuperação de matas de ciliares. Ciência Florestal , v. 23, n. 4, p. 655-665, 2013.; Kreuzwieser and Rennenberg, 2014KREUZWIESER, J.; RENNENBERG, H. Molecular and physiological responses of trees to waterlogging stress. Plant, cell & environment, v. 37, n. 10, p. 2245-2259, 2014.). Moreover, our knowledge of the responses of tree species under flooding is limited compared to herbaceous species (Grandis et al., 2010GRANDIS, A.; GODOI, S.; BUCKERIDGE, M.S. Respostas fisiológicas de plantas amazônicas de regiões alagadas às mudanças climáticas globais. Revista Brasileira de Botânica, v. 33, n. 1, p. 1-12, 2010.; Kreuzwieser and Rennenberg, 2014).

Cedrela fissilis Vell. (Meliaceae), popularly known as pink-cedar, is a woody species native to the Atlantic Rainforest. It presents a wide geographical distribution and develops as an emergent species in primary forests (Oliveira-Filho et al., 2006OLIVEIRA - FILHO, A.T.; JARENKOW, J.A.; RODAL, M.J.N. Floristic relationships of seasonally dry forests of eastern South America based on tree species distribution patterns. Systematics Association Special Volume, v. 69, p. 159, 2006.). In addition, this species can be found in flooded areas (Lorenzi, 1992LORENZI, H. Árvores brasileiras: manual de identificação e cultivo de plantas arbóreas nativas do Brasil. Nova Odessa: Plantarum, 1992.) and occurs mainly in semi-deciduous seasonal rainforests and mixed ombriophilous forests (Antoniazzi et al., 2013ANTONIAZZI, A.P; BINOTTO, B.; NEUMANN, G.M.; BUDKE, J.C.; SAUSEN, T.L. Eficiência de diferentes recipientes no desenvolvimento de mudas de Cedrela fissilis Vell.(Meliaceae). Revista Brasileira de Biociências, v. 11, n. 3, P. 313-317, 2013.). This plant has been economically importance mainly for timber production, and as a consequence this species has been over/severely exploited, and it now listed in the redbook of threatened species (IUCN, 2017The IUCN Red List of Threatened Species. Available at: Available at: http://dx.doi.org/10.2305/IUCN.UK.1998.RLTS.T33928A9821890 .en. Accessed in: 20 November 2017.
http://dx.doi.org/10.2305/IUCN.UK.1998.R... ). Because of its wide distribution in Brazilian forests, and its high value as timber, many reforestation projects make use of C. fissilis (Binotto et al., 2016BINOTTO, B.; ANTONIAZZI, A.P.; MARTA NEUMANN, G.; SAUSEN, T.L.; BUDKE, J.C. Tolerância de plântulas de Cedrela fissilis VELL. a diferentes amplitudes e intensidades de inundação. Ciência Florestal, v. 26, n. 4, p. 1339-1348, 2016.). Thus, it is important to study this species to improve its conservation as well as to provide information on its physiological mechanisms to overcome the flooding condition.

Studies on the ecophysiological events/responses in different water levels might contribute to our knowledge on the floating responses of plant tolerance, may contribute to conservation of this critically endangered species and also to the success of projects that restore areas subject to flooding. We hypothesize that C. fissilis has the capacity to tolerate flooding and so has potential to be used in the reforestation of areas with periodic flooding and riparian forest.

In the present work, the tolerance of young C. fissilis plants under different water availability was evaluated, in order to analyze the capacity of this species to survive flooding during the initial stages of development and also to evaluate whether survival is due to morphological changes in stem anatomy that facilitate gas exchanges, and in the leaf antioxidant system. It also intended to contribute to the available information about tree species used for restoring degraded areas subject to flooding, in order to guide the success of such environmental projects.

MATERIAL AND METHODS

Seed collection and plant production

Seeds of Cedrela fissilis Vell. were obtained through direct collection in areas of secondary forestation at Alfenas, Minas Gerais (21º25’28’’S and 45º56’57’’O). The seeds were placed on polypropylene trays (45 cm x 35 cm x 4 cm) containing sand and vermiculite in the ratio of 1:2 v v^-1 irrigated to field capacity and kept in a greenhouse until germination. At 100 days after germination, seedlings, which were now, in average, around 4 cm, were transferred to 7.0 L plastic trays (30 cm diameter x 15 cm depth) containing sand and vermiculite at the ratio of 1:2 v v^-1 as the substrate which were used for the experimental treatments.

Both the seedling culture and the experiment installation were carried in a greenhouse located at the Federal University of Alfenas, Santa Clara campus, Alfenas, Minas Gerais (21º25’15’’S and 45º58’56’’O). The greenhouse maintained an average internal temperature of around 28º C (minimum of 13ºC and maximum of 36ºC), the relative air humidity from 55 to 80% and a maximum radiation intensity of 700 W m^-2.

Experimental design

The water treatments wereas follows: Control (FC) where the substrate was kept at the field capacity; Flooded roots (FR), where the substrate remained submerged, however without accumulating water line on its surface, and Flooded stem (FS), in which the trays remained with a layer of water 3.0 cm above the substrate, flooding part of the seedling stem. Field capacity was determined by weighing 1.0 L of the substrate and adding controlled volumes of water until the maximum capacity to water retention without accumulation of water in the bottom of the beaker. The volume of water used was than divided by the volume of the substrate (1.0 L) and this proportion was applied to the experiment. The FC was kept with constant irrigation, with no trace of substrate saturation or drought; in a condition similar to the field capacity. Plant nutrition was provided by the application of nutrient solution according to Hoagland and Arnon (1950HOGLAND, D.R.; ARNON, D.I. The water-culture method for growing plants without soil. California Agricultural Experiment Station, v. 347, n. 2nd edit, 1950.) to 40% of ionic strength. Plants were kept in these conditions by 90 days.

The experiment was conducted in randomized block design with three blocks to each treatment, where each block comprised one tray with nine plants so that 27 plants were used per treatment.

Watering System

The experiment used a watering system that was developed especially for this study (Figure 1). This system comprised a stair-shaped metal structure with three steps, where a distinct treatment was allocated to each step. A submerged pump in the tank with a flow capacity of 1,000 L.h^-1 (SARLO, São Paulo, Brazil) permitted continuous watering and regular control of the treatments. The pump was connected to the highest level of the system and was also connected to a tank containing nutrient solution. The highest step conserved the Flooded stem (FS) treatment. These trays had two holes drilled 3 cm above the substrate which were linked via 2 mm diameter hoses with the trays for the Flooded root (FR) treatment located on the step below. These trays had two holes drilled at substrate level to prevent the accumulation of water above the soil, and the excess water dripped to the trays containing the control treatment (FC) located on the step below. These trays had holes drilled in the bottom to enable an efficient drainage of the solution. Field capacity was checked periodically by weighing the trays. The drained solution of the whole step system was returned to the tank to maintain the cycle.

The control of the level of the nutrient solutions was performed daily while their ionic strength was checked weekly with a conductivity meter MCA-150 (MS TECNOPON, São Paulo, Brazil). Water or solution was added whenever it was required.

Biometric evaluation

The biometric evaluations of the C. fissilis plants were performed at two periods: on the day of the installation of the experiment and passed 90 days under flooding. The features evaluated were: stem length, measured from the vertical distance between above substrate level the and the top of the stem; and the stem diameter at 3 cm above substrate level and the number of new leaves was counted.

Stem growth during the course of the experiment, measured to the nearest 1.0 mm, was the difference between the initial and final values. Stem diameter, taken at 3 cm above the initial stem height, was measured to 0.03 mm using as digital pachymeter.

Leaf gas exchange analyses

The physiological analyses were performed on the 60th day after the onset of the experiment, when the plants were already acclimatized and showing fully expanded leaves. The analyses were conducted with an Infra-red gas analyzer (IRGA) model LI-6400XT (LI-COR, Lincoln, USA). All measurements were performed in the morning between 9 and 11 a.m, on fully expanded and pathogen free leaves, located at third node down from the apical region. Due to the size of the leaves, the measurements were performed on the third leaflet and the area of the cuvette adjusted to 4.8 cm². The assessed parameters were: photosynthetic rate (A), transpiration rate (E), stomatal conductance (gs) and CO₂ intercellular concentration (Ci).

The light response curve was measured and the saturation point of the photosynthetic system was determined, establishing the value for the photosynthetic photon flux density (PPFD) of 500 µmol.m^-2.s^-1 as the ideal to perform the analyses. The leaf temperature was held at 28 ºC in the cuvette.

Stem anatomy analysis

At the end of the experiment, stem fragments located around 1.0 cm above the seedling height were collected and fixed in FAA_70% (Johansen, 1940JOHANSEN, D.A. Plant microtechnique. McGraw-Hill Book Company, 1940. 530p.). These samples were dehydrated in an ethanol 70%, 80%, 90% e 100% series with for 2 hours sequence. Subsequently, the stem fragments were infiltrated with hydroxyethyl- methyl acrylate in 100% ethanol (Leica Microsystems, Wetzlar, Germany) at the proportion of 1:1 and pure activated resin. They were then transferred to histomolds and a hardener was added. After 7 days in the oven at 35º C, the specimens were removed from histomolds and fixed in wooden blocks which provided a support for the microtomy. Sections at 10 µm were cut with a rotary microtome Leica RM 2235 (Leica Microsystems, Wetzlar, Germany). Sections were stained with 1% toluidine blue for starch, and they were stained with 1% Lugol (Kraus and Arduin, 1997KRAUS, J.E.; ARDUIN, M. Manual básico de métodos em morfologia vegetal. Seropédica: Edur, 1997.). Fifteen replicate sections were made for each specimen.

Photomicrography was performed using a digital camera Moticam 2300 (MOTIC, Xiamen, China), attached to a Nikon eclipse E200 microscope (NIKON, São Paulo, Brazil). Anatomical features were measured using the program Image J image analyser.

The anatomical features assessed were: the periderm thickness, percentage of intercellular spaces in the cortex, the sum of the area occupied by sieve tube elements, number of sieve tube elements, pith diameter, average area of the pith cells, total xylem area, vessel element density, percentage of vessel elements, percentage of fibers present in the xylem, number of starch granules per cortical cell, and per pith parenchyma cells and per parenchyma ray cells in the xylem.

Biochemical analyses

Fully expanded leaves from the third and fourth nodes of the C. fissilis plants were sampled after 90 days from the experiment installation. The samples were packed in aluminum foil and stored in a deep freeze at -80ºC until the analysis.

Lipid peroxidation was determined according to Buege and Aust (1978BUEGE, J.A.; AUST, S.D. Microsomallipidperoxidation. Methods and Enzymology, v.52, n. 30, p.302-310, 1978.); for this purpose, 0.1 g of plant material was macerated in liquid nitrogen, to which 20% of PVPP (m.v^-1) was added, and homogenized at 1.5 mL of 20% trichloroacetic acid (m.v^-1). The material was centrifuged at 15.000 g, for 10 minutes. Aliquots (0.75 mL) of the supernatant were added to 0.75 mL of thiobarbituric acid (TBA). Thereafter, the tubes were boiled at 90°C, for 20 minutes and then cooled in an ice bath and the readings were determined in spectrophotometer Biochrom, Libra S22 (Biochrom, Cambridge, England), at 540 nm. The concentration of the complex malondialdehyde /thiobarbituric acid (MDA/TBA) was calculated using the extinction coefficient 1.55 mM.cm^-1.

To obtain protein extracts for the analysis antioxidant enzyme activity, 200 mg of leaf were macerated in liquid nitrogen and homogenized with 1.5 mL of extraction buffer. The homogenized materials were centrifuged at 12.000 g, for 30 minutes, at 4°C. The supernatant was collected and used in the analyses of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) activities according to Biemelt at al. (1998BIEMELT, S.; KEETMAN, U.; ALBRECHT, G. Re-aeration following hypoxia or anoxia leads to activation of the antioxidative defense system in roots of wheat seedlings. Plant Physiology, v. 116, n. 2, p. 651-658, 1998.). Protein content was determined by the method of Bradford (1976BRADFORD, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, v. 72, n. 1-2, p. 248-254, 1976.) using bovine serum albumin as the standard, and the enzymatic activities were calculated following García-Limones et al. (2002GARCÍA-LIMONES, C.; HERVÁS, A.; NAVAS-CORTÉS, J.A.; JIMÉNEZ-DÍAZ, R.M.; TENA, M. Induction of an antioxidant enzyme system and other oxidative stress markers associated with compatible and incompatible interactions between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp. ciceris. Physiological and molecular plant pathology, v. 61, n. 6, p. 325-337, 2002.).

Superoxide dismutase activity (SOD) was determined by the capability of the enzyme to inhibit the reduction of nitro blue tetrazolium (NBT) in the presence of riboflavin. The tube containing the blank (incubation method without the sample) and the tubes with the phosphate buffer at 50 mM and pH 7.8, EDTA 0.1 Mm, methionine 13 mM, 7.5 µM of NBT, riboflavin 2 mM together with 20 µL of sample, were illuminated with fluorescent lighting of 20 W for 12 minutes. Afterwards, readings were performed at 560 nm in spectrophotometer. The SOD activity was established by the amount of enzyme that inhibits 50% of the reduction rate of NBT (Giannopolitis and Ries, 1977GIANNOPOLITIS, C.N.; RIES, S.K. Superoxide dismutases: I. Occurrence in higher plants. Plant physiology, v. 59, n. 2, p. 309-314, 1977.).

Catalase activity (CAT) was determined by the decreased absorbance at 240 nm, every 15 seconds for 3 minutes, monitored by the consumption of hydrogen peroxide. The reaction medium was comprised of phosphate buffer 50 mM, pH 7.0, 20 mM of H₂O₂ and 50 µL of enzyme extract. The reaction was started by adding H₂O₂ (ε = 36 mM^-1.cm^-1). The CAT activity was defined by the consumption of H₂O₂ (Beers and Sizer, 1952BEERS, R.F.; SIZER, I.W. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. Journal of biological chemistry, v. 195, n. 1, p. 133-140, 1952.).

Ascorbate peroxidase (APX) activity was determined by the decreased absorbance at 290 nm due to the consumption of ascorbate (ε=2.8 m.M^-1.cm^-1) at every 15 seconds during 3 minutes. The reaction medium was comprised of phosphate buffer 50 mM, pH 7.0, sodium ascorbate 0.25, mM, H₂O₂ 5 mM and 50 µL of enzymatic extract. The APX activity was defined by the consumption of ascorbic acid (Nakano and Asada, 1981NAKANO, Y.; ASADA, K. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant and cell physiology, v. 22, n. 5, p. 867-880, 1981.).

Statistical analysis

Data submitted to a Shapiro-Wilk normality test (α = 0.05) showed normal distribution. They were then subjected to the variance analysis (ANOVA), followed by the Tukey’s test for the comparison of the means at 5% of significance, in SISVAR software, version 5.6.

RESULTS

Biometrics

All C. fissilis plants survived for the experimental period of 90 days, regardless the treatment. However, C. fissilis plants grown under flooding showed significant decrease in shoot growth with a 44% decrease of this variable in the FR treatment and 64% for plants of FS treatment. On the other hand, flooding did not affected the stem diameter (Table 1). In addition, the development of new leaves was diminished by flooding conditions (Table 1).

Leaf gas exchange

Cedrela fissilis under flooding conditions showed lower net photosynthesis and stomatal conductance as well as lower intercellular CO₂ concentration. The FR treatment reduced the photosynthetic rate by 26% whereas the FS reduced this variable by 46% as compared to FC treatment. However, the transpiratory rate did not show significant changes (Table 2).

Stem anatomy

The thickness of the periderm was significantly reduced in the FR and FS treatments (Table 3 and Figure 2). In addition, flooding increased the percentage of cortical intercellular spaces (Table 3 and Figure 2), such that in the plants of FS treatment the percentage of intercellular spaces was up to six times greater as compared to FC treatment (Table 3 and Figure 2).

The sum of the area of sieve tube elements in the phloem was not significantly modified by the treatments. However, a decreased number of the sieve tube elements was found in the FR and FS treatments (Table 3 and Figure 2).

Flooding did not influence on the pith diameter but did decrease the size of the pith cells by 30% in the FR treatment and by 35% for the FS plants as compared to control (Table 3 and Figure 2).

There was an increase in the proportion of vessel elements and ray parenchyma in xylem for the plants under flooding (Table 3). In addition, the proportion of fibers in the xylem decreased in the FR and FS treatments. However, the diameter of vessel elements increased for FR plants (Table 3).

The amount of starch grains in the parenchyma of the cortex decreased only for the FR plants (Table 4 and Figure 3). However, the highest number of starch grains was observed in the FS treatment, followed by the FR, with the lowest mean found for the FC treatment (Table 4 and Figure 3). Likewise, in the xylem rays, there was an increase in the number of starch grains for the plants of FS treatment (Table 4 and Figure 3).

Biochemical analyses

Plants from the FS treatment showed the highest lipid peroxidation followed by the FR plants, whilst those of the FC treatment showed the lowest values (Table 5). The activity of the antioxidant system enzymes of C. fissilis showed significant differences under flooding (Table 5). The SOD activity was reduced in the plants of FR and FS treatments as compared to the control. CAT activity increased only in the FS treatment whereas the APX activity increased in both flooding conditions (Table 5).

DISCUSSION

C. fissilis plants partially tolerate flooding and we have shown they have physiological, anatomical and biochemical modifications which enabled them to survive under flooding despite some photosynthesis and growth limitations. The main adaptation of this species to overcome flooding may be the development of a higher proportion of cortical intercellular spaces, which were to be some 5 to 6 times greater in the stems of flooded plants. Intercellular spaces are known to enhance porosity of the tissue and thus, they contribute significantly to the diffusion of gases, mainly oxygen, between submerged organs and not submerged ones (Voesenek and Bailey-Serres, 2015VOESENEK, L.A.C.J.; BAILEY-SERRES, J. Flood adaptive traits and processes: an overview. New Phytologist , v. 206, n. 1, p. 57-73, 2015.; Oliveira et al., 2015OLIVEIRA, A. S.; Ferreira, C.S.; GRACIANO-RIBEIRO, D.; FRANCO, A.C. Anatomical and morphological modifications in response to flooding by six Cerrado tree species. Acta Botanica Brasilica , v. 29, n. 4, p. 478-488, 2015.; Loreti et al., 2016LORETI, E.; VAN VEEN, H.; PERATA, P. Plant responses to flooding stress. Current Opinion in Plant Biology, v. 33, p. 64-71, 2016.). It is likely, therefore, that intercellular spaces minimized the most acute harm regarding oxygen deficit and enabled the plants to survive.

However, flooded plants of C. fissilis showed some limitation to leaf gas exchange that affected photosynthesis and growth. A decrease up to 50% in stomatal conductance in plants from FS treatment, indicated that under flooding conditions, C. fissilis plants closed their stomata, promoting a stomatal limitation to photosynthesis. The lower means for the intercellular carbon concentrations in flooded plants supports this view. Moreover, many studies have shown the relation between stomatal conductance and carbon sequestration in flooded plants (Liao and Lin, 2001LIAO, C.; LIN, C. Physiological adaptation of crop plants to flooding stress. Proceedings of the National Science Council, Republic of China. Part B, Life Sciences, v. 25, n. 3, p. 148-157, 2001.; Mielke et al., 2003MIELKE, M.S.; DE ALMEIDA, A.A.F.; GOMES, F.P.; AGUILAR, M.A.G.; MANGABEIRA, P.A.O. Leaf gas exchange, chlorophyll fluorescence and growth responses of Genipa americana seedlings to soil flooding. Environmental and experimental botany, v. 50, n. 3, p. 221-231, 2003.; Striker, 2012STRIKER, G.G. Flooding stress on plants: anatomical, morphological and physiological responses. In: Botany. v. 1, p.3-28, 2012.) and so support the view that the stomatal closing may limit photosynthesis. However, the mechanism that reduces the stomatal conductance under flooding is poorly understood (Kreuzwieser and Rennernberg, 2014KREUZWIESER, J.; RENNENBERG, H. Molecular and physiological responses of trees to waterlogging stress. Plant, cell & environment, v. 37, n. 10, p. 2245-2259, 2014.). According to Striker (2012) the stomatal closing promoted by flooding prevents the leaf dehydration. However, limitation of the stomatal conductance may occur even without reduction in the leaf water potential by the abscisic acid (ABA) signalling (Jackson et al., 2002JACKSON, M.B. Long-distance signalling from roots to shoots assessed the flooding story. Journal of Experimental Botany, v. 53, n. 367, p. 175-181, 2002.). It is possible that the stomatal closing in C. fissilis happened through ABA effect, when transpiration remained unaffected by flooding.

The reduction in photosynthesis in flooded C. fissilis may explain the significant reduction in new leaf formation and the limited elongation of the stem. It is well known that plants submitted to oxygen deficiency (hypoxia), and also those which show damage photosynthesis, reduce the allocation of energy destined to growth and move it to basic metabolism in order to survive (Kolb et al., 1998KOLB, R.M.; MEDRI, M.E.; BIANCHINI, E.; PIMENTA, J.A.; GILONI, P.C.; CORREA, G.T. Anatomia ecológica de Sebastiania commersoniana (Baillon) Smith & Downs (Euphorbiaceae) submetida ao alagamento. Brazilian Journal of Botany, v. 21, n. 3, p. 305-312, 1998.; Grandis et al., 2010GRANDIS, A.; GODOI, S.; BUCKERIDGE, M.S. Respostas fisiológicas de plantas amazônicas de regiões alagadas às mudanças climáticas globais. Revista Brasileira de Botânica, v. 33, n. 1, p. 1-12, 2010.; Medri et al., 2012MEDRI, C.; PIMENTA, J.A.; RUAS, E.A.; SOUZA, L.A.; MEDRI, P.S.; SAYHUN, S.; BIANCHINNI, E.; MEDRI, M.E. O alagamento do solo afeta a sobrevivência, o crescimento e o metabolismo de Aegiphila sellowiana Cham.(Lamiaceae)?. Semina: Ciências Biológicas e da Saúde, v. 33, n. 1, p. 123-134, 2012.; Coelho et al., 2014COELHO, C.C.R.; DA SILVA, J.N.; NEVES, M. G.; DA CONCEIÇÃO, A.G.C.; DA SILVA, R.T.L; DE OLIVEIRA NETO, C.F. Aspectos ecofisiológicos e crescimento em plantas de milho ao alagamento. Revista Agroecossistemas, v. 5, n. 2, p. 41-46, 2014.).

The anatomical changes in C. fissilis under flooding treatments support the conclusion that water saturation harmed the development of the species. It is notable that the density of sieve tube elements in the phloem decreased by around 50% in plants of the FS treatment. In addition, the maintenance of the size of the sieve tube elements associated with this reduction in number, suggest that the flow of photo-assimilates through phloem was impaired. Furthermore, the limitation of the phloem transport may have harmed the translocation of carbohydrates from leaves to roots as well as the other sinks, negatively affecting the development of such organs. Similar results were shown by Souza et al. (2010SOUZA, T.C.D.; MAGALHÃES, P.C.; PEREIRA, F.J.; CASTRO, E.M.D.; SILVA JUNIOR, J.M.D.; PARENTONI, S.N. Leaf plasticity in successive selection cycles of ‘Saracura’ maize in response to periodic soil flooding. Pesquisa Agropecuária Brasileira, v. 45, n. 1, p. 16-24, 2010.). This effect is supported by the reduced number of new leaves produced by the plants under flooding (which depends on shoot apical meristem activity and photoassimilates).

The decrease in periderm thickness in C. fissilis under flooding may also be related to a lower radial transport of photoassimilates from the phloem through the cortex, which was impaired by the decreased phloem development and the excess of intercellular spaces in this region. However, the thinner periderm may have contributed to a greater diffusion of oxygen from the atmosphere to plant’s inner tissues providing benefit to plants during long periods of flooding (Voesenek & Bailey-Serres, 2015VOESENEK, L.A.C.J.; BAILEY-SERRES, J. Flood adaptive traits and processes: an overview. New Phytologist , v. 206, n. 1, p. 57-73, 2015.).

The limitation in phloem transport is corroborated by the data for starch accumulation presented. This carbohydrate accumulation was found to have a higher intensity in the innermost tissues of the stem, such as in the xylem parenchyma rays and in the pith. This supports the view that the accumulation of starch in the organs may be linked to the impairment of phloem flow (Ferner et al. 2012FERNER, E.; RENNENBERG, H.; KREUZWIESER, J. Effect of flooding on C metabolism of flood-tolerant (Quercus robur) and non-tolerant (Fagus sylvatica) tree species. Tree physiology, v. 32, n. 2, p. 135-145, 2012.).

Modifications found in the xylem also help us to understand the tolerance mechanisms of C. fissilis to flooding since we observed a greater density of vessel elements in both flooded treatments. Vessel elements can show special features which enables the transport huge amounts of water (Severo et al., 2017SEVERO, T.C.; EITELVEN, T.; LUVISON, F. Xilema: Fatores externos que influenciam no seu funcionamento, conectando o cotidiano cientifico. Revista Interdisciplinar de Ciência Aplicada, v. 2, n. 3, p. 4-9, 2017.). In addition, larger vessel diameter and density may increase the xylem water conductivity in trees (Garcia-Cervigon et al., 2018GARCÍA-CERVIGÓN, A.I.; OLANO, J.M.; VON ARX, G.; FAJARDO, A. Xylem adjusts to maintain efficiency across a steep precipitation gradient in two coexisting generalist species. Annals of Botany , v. 22, p. 461-472, 2018.). Our results indicate that despite suffering from a water saturation, the efficacy of water transport was regulated in C. fissilis, maintaining the hydraulic conductivity in flooded plants. Effective hydraulic conductivity, conferred by changes in xylem, might also explain the unchanged transpiration rate (Sperry and Pockman, 1993SPERRY, J.S.; POCKMAN, W.T. Limitation of transpiration by hydraulic conductance and xylem cavitation in Betula occidentalis. Plant, cell & environment , v. 16, n. 3, p. 279-287, 1993.). The maintenance of such hydraulic conductivity is important since flooding can decrease water absorption by plants (Coutts, 1981; Kreuzwieser and Rennernberg, 2014KREUZWIESER, J.; RENNENBERG, H. Molecular and physiological responses of trees to waterlogging stress. Plant, cell & environment, v. 37, n. 10, p. 2245-2259, 2014.).

The increase of parenchymatous rays in flooded treatments enabled the higher allocation of starch granules in flooded plants. Although a lower input for fibers on the tissue was observed, this decrease in fibers did not affect the plant’s stability. The overall anatomical response observed in C. fissilis favoured gaseous diffusion through intercellular spaces, which is an important feature under flooding conditions (Jackson et al., 2009JACKSON, M.B.; ISHIZAWA, K.; ITO, O. Evolution and mechanisms of plant tolerance to flooding stress. Annals of Botany , v. 103, p. 137-142, 2009; Loreti et al., 2016LORETI, E.; VAN VEEN, H.; PERATA, P. Plant responses to flooding stress. Current Opinion in Plant Biology, v. 33, p. 64-71, 2016.).

We observed that the action of the antioxidant system in the leaves of C. fissilis was not sufficient to avoid flooding stress since lipid peroxidation increased around 300% in flooded plants. Various studies have shown that under flooding conditions plants trigger the production and accumulation of reactive O₂ species (ROS), which may harm cell membranes through lipid peroxidation and thus impairs cell metabolism (Mittler, 2002MITTLER, R. Oxidative stress, antioxidants and stress tolerance. Trends in plant science, v. 7, n. 9, p. 405-410, 2002.; Boamfa et al., 2005BOAMFA, E.I.; VERES, A.H.; RAM, P.C.; JACKSON, M.B.; REUSS, J.; HARREN, F.J.M. Kinetics of ethanol and acetaldehyde release suggest a role for acetaldehyde production in tolerance of rice seedlings to micro-aerobic conditions. Annals of Botany, v. 96, n. 4, p. 727-736, 2005.; Larré et al., 2013LARRÉ, C.F.; FERNANDO, J.A.; MARINI, P.; BACARIN, M.A.; PETERS, J.A. Growth and chlorophyll a fluorescence in Erythrina crista-galli L. plants under flooding conditions. Acta physiologiae plantarum, v. 35, n. 5, p. 1463-1471, 2013.; Messchmidt et al., 2015MESSCHMIDT, A.A.; BIANCHI, V.J.; ZANANDREA, I.; MARTINAZZO, E.G.; RADMANN, E.B.; BACARIN, M.A. Trocas gasosas e atividade antioxidante de portaenxertos de Prunus spp. submetidos ao estresse seca e alagamento. Revista de la Facultad de Agronomía, La Plata, v. 114, n. 1, p. 71-81, 2015.).

In order to avoid ROS accumulation, plants have enzymatic and non-enzymatic defense systems that enables the scavenging of these compounds and so protecting against the oxidative stress (Larré et al., 2016LARRÉ, C.F.; MORAES, C.L.; BORRELLA, J.; AMARANTE, L.; DEUNER, S.; PETERS, J.A. Antioxidant activity and fermentative metabolism in the plant Erythrina crista-galli L. under flood conditions. Semina: Ciências Agrárias, v. 37, n. 2, p. 567-580, 2016.). Flooding triggers the activation such an antioxidant system in C. fissilis leaves, although the enzymatic activities were not sufficient to eliminate the oxidative stress, as evinced by lower SOD activity shown by flooded plants. Similar results were found by Larré et al. (2013) in Erythrina crista-galli plants under water saturation, and the authors proposed that low SOD activity is related to lower production of the superoxide (O^.- _2.). The increase of more than 100% in the CAT activity in C. fissilis plants from the FS treatment, as well as that of APX activity in plants under both levels of flooding (FR and FS), indicate that unlike the lower superoxide production, these plants showed high hydrogen peroxide (H₂O₂) levels.

High APX and CAT activities demonstrate the formation of ERO in C. fissilis when flooded, although the increase of antioxidant enzymatic activity was not enough to eliminate an excess of H₂O₂ in leaf tissue, which triggered an increase in lipidic peroxidation. Most likely, the remarkable lipid peroxidation in leaves was related to damaged thylakoid membranes within chloroplasts and the reduced photosynthesis found in C. fissilis.

The data provided by this study corroborate with those by Binotto et al., (2016BINOTTO, B.; ANTONIAZZI, A.P.; MARTA NEUMANN, G.; SAUSEN, T.L.; BUDKE, J.C. Tolerância de plântulas de Cedrela fissilis VELL. a diferentes amplitudes e intensidades de inundação. Ciência Florestal, v. 26, n. 4, p. 1339-1348, 2016.) who also found partial tolerance of C. fissilis to flooding. However, these authors did not encounter a decrease in growth in seedlings of this species when they remained partially flooded, probably due to the shorter period of time in which plants were subjected to partial flooding (5, 15 and 20 days),

CONCLUSION

We have found that young Cedrela fissilis plants partially tolerate flooding since they developed ecophysiological changes in order to survive this condition. This species would be a suitable candidate for the re-afforestation in areas where intermittent flooding occurs, such as riverside and riparian forests.

ACKNOWLEDGMENTS

The authors would like to thank Peter E. Gibbs for the English revision

REFERENCES

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