The water deficit ensures the... WATER DEFICIT ENSURES THE PHOTOCHEMICAL EFFICIENCY OF Copaifera langsdorffii Desf

1 Received on 01.06.2016 accepted for publication on 29.03.2017. 2 Universidade Estadual Paulista Júlio de Mesquita Filho, Programa de Pós-Graduação em Ciências Biológicas, Botucatu, São Paulo, Brasil. E-mail: <angelicarodrigues.bio@gmail.com> and <luizfernando@ibb.unesp.br>. 3 Instituto Nacional de Pesquisas da Amazônia, Mestrado em Ciências Biológicas (Botânica), Amazônia, Brasil. E-mail: <lianelimaa@gmail.com> . 4 Universidade Estadual Paulista Júlio de Mesquita Filho, Mestrado em Botânica, Botucatu, São Paulo, Brasil. E-mail: <schley.thayssa@gmail.com>. *Corresponding author.


INTRODUCTION
The accumulation of greenhouse gases in the atmosphere has changing the climate, resulting in increasing of average temperature and redistribution of global precipitation (IPCC, 2007).According to the different climate change scenarios, regions subjected to water deficit may increase the intensity and duration of drought.Hence, the overcoming of water stress periods and the recovering with the return of the rainy season are adjustment characteristics of plants often found in these dry regions (Giorgi and Lionello, 2008;Vaz et al., 2010).Once an increased frequency of drought events is predicted for the coming decades, further investigations are needed to elucidate the mechanisms of recovery from drought in the field.This could also improve predictions of ecosystem productivity and better irrigation systems.
The effects of drought are highly variable depending on length, speed of stress imposition, and stage of plant development (Ferreira et al., 2015).Many species show morphological, physiological and biochemical mechanisms to overcome an inadequate supply of water, such as changes in the pattern of growth and development of vegetative organs, changes in stomatal conductance, adjusting osmotic potential of tissues and production of secondary metabolic substances (Smirnoff, 1993;Chaves et al., 2002;Gaspar et al., 2002;Carvalho, 2008).
Water deficit in plants leads to changes in photosynthetic rate.It can be caused by stomatal closure, or by damages in photosynthetic apparatus because of impaired photochemical reactions (Wu and Bao, 2011).Drought stress may decrease the electron transport requirement for photosynthesis and reduce PSII photochemical activity, which results in an overexcitation and photoinhibition damage to PSII reaction centers (Meng et al., 2016).The damage of the photosynthetic apparatus makes plants more susceptible to other environmental stresses.It affects plant growth and establishment (Anjos et al., 2015).The species that do not show damage to photosynthetic apparatus and exposes higher phenotypic plasticity can explain the establishment in a wide range of different environmental conditions (Barros et al., 2012).
Copaifera langsdorffii Desf., Fabaceae, subfamily Detarioideae (LPWG, 2017) is a neo-tropical tree that grows in a wide range of seasonal environments.The seedlings present great capacity to survive under contrasting light and water availability (Carvalho, 2003), being largely tolerant to environmental conditions found in fragmented environments as in Brazilian savanna (Martins et al., 2015).The wide distribution reflects the potential plasticity displayed by the species in response to different climatic regimes, presenting morphological and anatomical differences intraspecific in their populations (Melo Júnior et al., 2012).The study of Ronquim et al. (2009) showed that C. langsdorffii should present leaf physiological mechanisms and morphological adaption and it would allow this species to overcome spatial irradiance variation and seasonal water stress in vegetation areas.The authors suggest that young C. langsdorffii plants survived water stress under natural conditions due to the rapid and intense biomass accumulation in their roots in early development stages.In the dry season, the plants of Brazilian savannah lose leaves relieving water stress, which results in less intense gas exchange and water saving (Dalmolin et al., 2015).During the wet season, C. langsdorffii plants maximize carbon gain with the fully developed canopy, which influences the abundant flowering and high fruit yield promoting the establishment of the species in seasonal environments (Pedroni et al., 2002).In addition, this species can be used as a model in physiological studies due to its adjustment developed in response to different environmental regimes.
This study evaluated the photochemical performance of Copaifera langsdorffii Desf.submitted to water stress and subsequent rehydration, as well as, identified the variables contributing to this photochemical efficiency.Our hypothesis is C. langsdorffii reveals efficient photochemical performance in response to water deficit and rehydration, due to leaf physiological mechanisms that would allow this species to overcome seasonal water stress in different vegetation areas.

MATERIALS AND METHODS
The research was conducted in the Botany Department of State University of São Paulo, Botucatu Campus, Brazil.It was performed in a greenhouse under semi-controlled conditions and relative humidity between 50 and 70%, 13 hours photoperiod, maximum night and day temperatures were close to 25 and 29 °C, respectively, and maximum PPFD (photosynthetic photon flux density) 800 µmol m -2 s -1 .
The water deficit ensures the...The experiment consisted of exposing C. langsdorffii plants to severe water stress and rehydration conditions.Plants 35 cm tall were planted in 5-L pots filled with soil from a Cerrado area.The soil was characterized as red Latosol (Oxisol), whose granulometric analysis showed mean values of 788 g.kg -1 sand, 110 g.kg -1 clay, and 102 g.kg -1 silt (sandy textural class) and field capacity of -0.006 MPa.Plants were daily irrigated and kept at maximum storage capacity (MSC) of water until the beginning of the experiment.The MSC of water was calculated through pot weighing; plants were irrigated and the water was drained from the soil, according to the methodology described by Varone et al. (2012).
The study was divided in two treatments, with 4 replicates each.The pots of Control treatment, plants were kept at the MSC of water throughout the experiment, and those for the Water stress plants were subject to water deficit followed by rehydration.All plants were acclimated under favourable hydric conditions, for approximately 60 days.Only the water stress group was subjected to total irrigation suspension for more than 35 days during the experiment.The withholding of water for 35 days adopted in the present study was based on previous testing with C. langsdorffii.Stomatal conductance (g s ) values decreased to 10% of the maximum gas exchange exhibited by plants grown under favourable conditions on the 35th day of drought.Stressed pots were rehydrated until the MSC equal to that of the Control group from the initial experiment.The physiological aspects were analysed at four times: at the end of stress period (Drought); one day of rehydration (24 h); and two and four days of rehydration (48 h and 96 h).
Fully expanded younger leaves, from the second or third node, were selected for measurements per replicate.Stomatal conductance (g s ) measurements were done by using Leaf Porometer Model SC-1 (Decagon Devices Inc., USA).The relative water content (RWC) were analysed in fully expanded leaf removed from the second or third node per replicate.A leaf from each plant was cut in rectangles (3 cm x 4 cm) and immediately weighed to obtain the fresh weight (FW).Next, the samples were placed in Petri dishes covered with filter paper, immersed in deionised water and conditioned for 24 hours, at 5 °C, for rehydration, according to Elsheery and Cao (2008).After this period, the samples were weighed to obtain the turgid weight (TW); next, they were oven-dried (temperature H" 60 °C to constant weight) to obtain the dry weight (DW).An analytical scale with 0.0001 g accuracy (Bel Engineering, Italy.) was used to determine the fresh, turgid and dry weights.The RWC values were obtained according to Smart and Bingham (1974), through the formula: RWC (%) = (FW -DW)/(TW -DW) * 100.The physiological analyses on data about water relations were measured at midday (MD).
Chlorophyll fluorescence parameters were obtained through PAM Fluorometer -Junior (WALZ, Effeltrich, Germany).Different photochemical variables were calculated according to chlorophyll fluorescence parameters, namely: the F M and F 0 are the maximum and minimum fluorescence of dark-adapted leaves, respectively; the maximum quantum efficiency of PSII (F V /F M ), the effective quantum yield of  PSII ( PSII = F/F M ') measured according to Genty et al. (1989), the photochemical quenching [qP = (F M ' -F S )/(F M ' -F 0 ')], the non-photochemical quenching [NPQ = (F M -F M ')/F M '] and the electron transport rate (ETR = F/ F M '× PPFD × 0.5 × ).We used the mean value of 0.84 for leaf absorption () for green leaves (Maxwell and Johnson, 2000;Baker, 2008).The attached leaves were covered with aluminium foil and kept in the dark for approximately 30 minutes.The light curve generated in the fluorometer with eight actinic light pulses (125,190,285,420,625,820,1150 and 1500 µmol m -2 s -1 PPFD), with 10s of intervals between the pulses, were applied.The chlorophyll fluorescence parameters and water relation variables were analysed at same time (11:00h).
Soil samples were collected and analysed on a water potential analyser under controlled temperature, WP4-T (Decagon Devices Inc., USA) to evaluate the water potential of the soil ( s ).The moisture in the chamber was balanced with the water potential in the soil, which was calculated according to the soil temperature and the dew point of the air.The analysis took place immediately after the biological material collection.
The experiment was a completely random design, with 4 plants per treatment.All evaluations were performed four times.The means and calculated standard errors of mean were reported.Statistical comparisons were performed with use Student' T-test complemented by Least-significance difference (LSD) test between the means used to compare the control and the stressed treatments.Statistical significance was set at p<0.05.

Rodrigues AL et al.
All the data were analysed in SigmaPlot® version 12 (Chicago Inc., USA, 2011).The plasticity indices (PIs) of different variables were calculated through the mean of treatments as PI = (max.-min.)/max(Valladares et al., 2000) in the current study.

RESULTS
The RWC decreased sharply while drought progressed.The data demonstrated significant difference between treatments in the last irrigation suspension day (drought) when compared with control plants, thus showing minimal hydration (p<0.05).No significant difference was observed between the water stress treatment and the control even 96 h of rehydration (Figure 1a).The g s showed daily significant difference (p<0.05),except for the first day (Figure 1b).The g s and RWC values decreased in the drought day of total irrigation suspension measured in the water stress treatment.Only RWC was increased 24 h after the rehydration when C. langsdorffii plants were irrigated, thus indicating the delayed response of g s in comparison to RWC.The RWC values in the rehydrated treatments were similar to those in the control, but the g s levels remained unchanged in the water stress treatment, even 96 h of rehydration (Figure 1).
The  s in the water deficit treatment was evaluated to estimate water availability in the soil during the experiment.The value of  s was -0.005 MPa before irrigation suspension.The  s decreased to -1.050 MPa on the last day of the water stress period, after the irrigation was interrupted.The  s increased to -0.100 MPa, in the 96 hours after rehydration.
The recovery induced different responses in chlorophyll fluorescence parameters.The ETR and qP values showed significant differences between treatments (p<0.05)48 h of rehydration, when ETR was 70% higher than the control (Figure 2c).No significant difference between the treatments was observed in NPQ and F V /F M data (Figure 2a and d).
The effective quantum yield ( PSII ) showed a significant difference (p<0.05)48 h of rehydration, concurrently with the values of ETR and qP.Whilst, the minimum fluorescence (F 0 ) data showed a significant difference between treatments (p<0.05)24 hours of rehydration.Maximum fluorescence values (F M ) had no significant difference among treatments (Table 1).The water deficit ensures the...
Our studies showed that C. langsdorffii has high plasticity index (PI) of g s, according to the drought (0.775 ± 0.13 SD) and rehydration (0.533 ± 0.23 SD) analyses.However, this variable was not showing significant differences between drought and rehydration (p<0.05)(Table 2).

DISCUSSION
The decrease in RWC values during water stress is consistent with the pattern of responses frequently observed in other studies that evaluated the physiological responses to water stress (Duan et al., 2005).Our results showed minimal RWC values under water stress.C. langsdorffii plants showed 15% decrease compared to control treatment.The decreases in RWC results in stomatal closure, which protects from future irreversible cell dehydration under progressive drought conditions (Luo et al., 2014).
The g s remained lower even 96 h of rehydration.Gimenez et al. (2005) showed that g s values may require several hours, even days, to recover and get close to similar non-stressed levels.The delays in g s are possible chemical signals stimulated by irrigation suspension, as well as indicated that equilibrium through rehydration was not achieved.
The slow response of g s is contrasted by the fast RWC recovery.The quick increase in RWC data indicated Rodrigues AL et al.
tissue rehydrated due to mechanism of stomatal control represented by low g s values, even after rehydration.Thus, lower g s values indicate the presence of a mechanism used to control the water lost by C. langsdorffii to overcome water deficit.The plant can modulate g s rates through stomatal aperture regulation depending on the need for water balance (Carvalho, 2008;Zhang et al., 2010;Kudoyarova et al., 2013;Hanachi et al., 2014).
The g s variable presented the higher PI according to analyses of the drought and 96 h rehydrated (Table 2).The high plastic responses to heterogeneous environments (i.e., phenotypic plasticity) allow the species to increase their ecological breadth as well as increase in available resources (Liu et al., 2015).
The observed F V /F M showed no photoinhibition according to Baker (2008) and did not show significantly different between treatments.Cornic (2000) indicated that the instantaneous response of plants to quantum efficiency when they were rehydrated suggests that the photochemical and biochemical mechanisms were not damaged by water scarcity, even with the decreased g s .The F V /F M is a common parameter and it is inversely proportional to the damages in the PSII reaction centres (Farquhar et al., 1989).Therefore, our results indicate that there was no damage to the PSII reaction centres, even when irrigation was suspended.
With different protection strategies to alleviate the damage caused by drought, the plants exhibit different responses in their photochemical apparatus (Cooper and Farrant, 2002).Water stress period imposed for C. langsdorffii was not enough to damage the photochemical responses even with rehydration.This study showed that the F 0 of rehydrated treatment increased, while the F M value did not show significantly difference between treatments.The data indicate that the photosynthetic apparatus was not damaged and can recover within the relatively short time.This suggests that the proteins present were adequate to maintain normal photosynthetic metabolism for a considerable period subsequent to rehydration (Cooper and Farrant, 2002;Guo et al., 2016).
The photochemical performance increased 48 h of rehydration.The  PSII provides the proportion of absorbed light actually used in PSII photochemistry.It can, therefore, be used to estimate the electron transport rate (ETR) through PSII, when the light absorbed by the leaf and the photosystems is known (Murchie and Lawson, 2013).Such result corroborates the data in the current study, which showed higher  PSII values  The water deficit ensures the... in the second day after rehydration in water stress treatment.The  PSII is an indicator of real photochemical activity (Genty et al., 1989;Duan et al., 2005).So, the increased  PSII may have resulted from the increased ETR, as well as from the increase in open reactions centres (qP), as shown in our results.This increase in photochemical activity during water stress can be a characteristic of drought tolerant plants, which could alleviate the effects of stress on photosynthetic machinery and improve their resilience (Rivas et al., 2016).
The ETR and qP showed significant differences between treatments 48 hours of rehydration, when the water deficit treatment showed higher values than control treatment.The increased ETR and qP indicate that the electron transport ability was optimized i.e. enhanced the capacity of the photosynthetic apparatus to maintain QA in the oxidized state (Zhao et al., 2007;Stefanov et al., 2016).This ability of electron transport optimization improves the light absorbed efficiency by PSII, which is used for QA reduction ( PSII ) and accelerates the overall electron transport through PSII (Dobrikova et al., 2014;Stefanov et al., 2016).The ETR is a dynamic parameter and it may be a good indicator to estimate the light induction of the species' photosynthetic rate due to its great acclimation capability (Wong et al., 2012).It could be suggested that the increase of electron transport rate and the photochemical quenching in C. langsdorffii submitted to water stress is one of the reasons for the dry tolerance of the species to drought.Thus, these observations are associated with an efficient mechanism to cope with water stress.NPQ increased in both control and stressed plants 48 h of rehydration with no difference between them, as well as ETR and qP.These patterns may be effective in preventing PSII over-excitation and the consequent chronic photosynthesis photoinhibition in C. langsdorffii.The higher  PSII in water stress group reinforces the idea that it was not necessary to increase the non-photochemical dissipation of absorbed light energy in the water stress group, since the absorbed light energy was sent to photochemical reactions (Einali et al., 2013).
These adjustment strategies of C. langsdorffii found in Cerrado physiognomies corroborate data by Veiga and Habermann (2013), who suggest that species in this ecosystem exhibit photoprotection performance under water stress.Thus, the capacity of making diurnal adjustments in photochemical and non-photochemical processes in order to overcome photoinhibitory threats would play a central role in the understanding of photosynthesis acclimation in seasonal environments like in Cerrado, regardless of the prevailing irradiance levels and degree of stomata closure (Franco and Lüttge, 2002).The recovery of the photochemical efficiency observed through chlorophyll fluorescence parameters is evidenced by the high resilience of the photosynthetic apparatus in the species under drought conditions (Maxwell and Johnson, 2000;Dongsansuk et al., 2013;Zong et al., 2014).

CONCLUSION
In summary we have presented that the species demonstrated photoprotection when it was exposed to water stress. PSII , ETR and qP were higher in rehydrated plants submitted to water stress treatment than in the control treatment.Plants did not show heat dissipation similar to the NPQ values between the control and the water stress groups.The lower g s values indicated the mechanism of the plant to control water loss and our study revealed higher plasticity of g s subjected to water restriction and rehydration.Therefore, the rapid rehydration after the drought and the photochemical performance presented, allows the survival of C. langsdorffii in environments that present periods of water restriction as in Cerrado.

Table 2 -
Plasticity indices (PIs) of different variables according to the water availability in the Water stress treatment.
Values are the mean ± SD (n = 4) of the variables evaluated.