Determination of parameters for selection of Eucalyptus clones tolerant to drought

1 Departamento de Engenharia Florestal, Universidade Federal dos Vales do Jequitinhonha e Mucuri-UFVJM. Prédio Engenharia Florestal, Campus JK Rodovia MGT 367 km 583, no 5000, Alto da Jacuba, Diamantina, MG, Brasil. 2 Departamento de Agronomia, Universidade Federal dos Vales do Jequitinhonha e Mucuri-UFVJM, Prédio Engenharia Florestal, Campus JK Rodovia MGT 367 km 583, no 5000, Alto da Jacuba, Diamantina, MG, Brasil.


INTRODUCTION
Despite the fact that planted forests represent merely 7% of the world's forest cover, they make a vital contribution to forest services (Villar et al., 2011), particularly the plants of the Eucalyptus genus.Although endemic to Australia and the nearby isles, Eucalyptus is predominantly one of the most popularly cultivated species in the tropics and Mediterranean countries (Cromer et al., 1993;Stape et al., 2001).Brazil, which boasts one of the world's most progressive technologies with respect to planted forests, has extensive Eucalyptus cultivations.
Climatic changes over the recent decades have caused the proliferation of arid conditions across the world (Allen et al., 2010;Kirono et al., 2011), resulting in a growing demand for Eucalyptus genotypes adapted to such situations.Scientists believe that the escalating emissions of greenhouse gases is one of the main reasons causing the recent spurt in the global average temperature and alterations in the global hydrological cycle, including predictable sharp surges of aridity (Sterl et al., 2008).The need of the hour, therefore, is to identify parameters which can rapidly and efficiently distinguish between genotypes that are tolerant and susceptible to drought for forest development programs.
Eucalyptus plantations vary widely in productivity due to several factors, primarily water availability (Shvaleva et al., 2006;Villar et al., 2011).Therefore, even a slight drop in soil water availability can produce negative effects on plant growth, development and productivity (Santos and Carlesso, 1998;Chaves et al., 2009).
There are many ways to express plant responses to drought.The ability of a plant to tolerate drought stress is usually determined by a combination of attributes expressed during a season of drought.
Eucalyptus exhibits very complex responses to drought which depend upon the strength and length of the dry conditions, the species, genotype and developmental stage of the plant (Santos and Carlesso, 1998;Taiz and Zeiger, 2009;Rodrigo, 2007).Morphological changes (in the size and biomass of the various plant organs) and the physiological (in the efficiency of water use and light capture) are some of the consequences observed (Li and Wang, 2003;Chaves et al., 2004;Merchant et al., 2007;Coopman et al., 2008;Pereira et al., 2010).
Distinguishing between the genotypes tolerant and susceptible to drought is possible using the values of the variables of gas exchange and also the evaluation of the photochemical efficiency of photosynthesis, obtained by measuring the chlorophyll a fluorescence.According to Krause and Weis (1991), the fluorescence emitted is equal to the amount of light energy not used by the photosynthetic apparatus and variations in the emission patterns indicate the presence of lesions (damages) in the plant's photosynthetic apparatus.Therefore, the main parameter used to evaluate these lesions is the ratio of the variable and the maximum chlorophyll fluorescence (Fv/Fm) which is the measure of the intrinsic or maximum efficiency of photosystem II (PSII).Values ranging from 0.75 to 0.85 reveal that the photosynthetic apparatus of the plant is in good condition; therefore, any decline in this ratio proves to be an excellent indicator of photoinhibitory damage when the plants are subjected to environmental stresses, including water limitation (Björkman and Powles, 1984;Bolhàr-Nordenkampf et al., 1989).
Another physiological parameter sensitive to stress due to water conditions is the chlorophyll content (Dutra et al., 2012;Ebrahimiyan et al., 2013;Huang et al., 2013), which has proven to be as effective as the other techniques used to measure gas exchange in distinguishing between the genotypes tolerant susceptible to drought.Further, it presents equally well as the variables of chlorophyll fluorescence with the extra advantage of being accurate, economical, fast and nondestructive.Therefore, this has been proven to be an important tool in ecophysiological studies (Krause and Weiss, 1991).
In this paper, we did a comparative study of the physiological responses of the chlorophyll content and chlorophyll a fluorescence in two contrasting Eucalyptus genotypes with respect to their ability to tolerate drought under two conditions of water availability.This study focused on the commercial clone sensitive of the Eucalyptus hybrid urograndis (Eucalyptus grandis vs. Eucalyptus urophylla), a genotype inefficient under water stress and clone tolerant of the hybrid Eucalyptus camaldulensis vs. E. grandis -a genotype model with high tolerance to water shortage.The objective of this study was to identify and understand some of the underlying tolerance of the photosynthetic apparatus deficit mechanisms to water level.There was need also to assess the ability of the variables in the study to distinguish the Eucalyptus genotypes of known sensitivity and tolerance.Our hypothesis states that genotype tolerant to aridity possesses a more efficient photosynthetic apparatus against the harmful effects of water shortage.

Season and experimental environment
The experiment was conducted between December 2012 and January 2013 at the Integrated Center for Propagation of Forest Species, in the greenhouse on the campus of the Federal University of JK (Jequitinhonha and Mucuri), in Diamantina,Minas Gerais,Brazil (18° 12'9.76''S,43° 34'46.13''E).The average temperature and average relative humidity inside the greenhouse were 21.9°C and 76.9%, respectively.

Plant, containers and substrates
In this study, we used Eucalyptus seedlings derived from clonal propagation, namely, commercial Eucalyptus hybrid clone sensitive urograndis (E.grandis vs. E. urophylla) and clone tolerant of the hybrid E. camaldulensis vs. E. grandis, from Aperam Bioenergy Ltd.These genotypes are selected in a breeding program.Growth performance was evaluated based on observations in field plantations subjected to drought on a dry season.First, the 56-dayold seedlings were transplanted to 2.0 and 1.5 L plastic bags, respectively, containing a substrate composed of vermiculite (40%), carbonized rice hull (30%) and coconut fiber (30%) and fertilized in line with the recommendations of Barros and Novais (1999).Initially, the seedlings were subjected to an adjustment period (30 days -period of acclimatization) in the bags, in the shade house, to ensure the establishment and survival of those seedlings receiving daily irrigation, sufficient to keep the substrate to as close to 60% of the field capacity at the time the treatments were started.

Application of treatments and statistical design
Different water regimes were started after 30 days of acclimatization in the shade house, for a 15-day period.Using the completely randomized 2 × 2 factorial design, the experiment was conducted with 25 seedlings per treatment: the water regime factor had two levels, irrigated (control) and not irrigated, while the genotype factor had two types, tolerant and sensitive.The irrigated treatments were maintained to as close to 60% of the field capacity.Every day, each bag to be irrigated with seedlings was weighed individually, and the mass plus water loss or used was replenished for each experimental unit.

Characteristics evaluated and statistical analysis
The chlorophyll and the variables of chlorophyll fluorescence were recorded from 16 plants in each treatment, just after their installation in the greenhouse and just before the treatment was started (time zero) and after that on days 6, 11 and 15.
The first fully expanded leaf (towards the apex from the base of the plant) was used to take the measurements and it was correctly identified with white wool.In the non-destructive method the total chlorophyll content was quantified indirectly using the chlorophyll ClorofiLOG brand, model CFL in 1030, according to the manufacturer's instructions.It was expressed as a dimensionless unit called Falker Chlorophyll Index (FCI) (Falker Agricultural Automation, 2008).Three measurements were recorded, avoiding the rib region and any portions damaged by pests or pathogens.These values were used to analyze the mean value.
The variables of chlorophyll fluorescence were measured using a portable PAM (JUNIOR-PAM, Heinz Walz GmbH) fluorometer.Taking the values of Fo (minimum fluorescence intensity), when the PSII reaction centers were open and at Fm (maximum fluorescence intensity), when the PSII reaction centers were closed, we calculated the variable fluorescence (Fv), obtained by Fm-Fo.Then we determined the potential quantum efficiency of PSII (Fv/Fm) (Maxwell and Johnson, 2000).Readings were taken using magnetic leaf clips attached to the fluorometer, placed in the middle region on the adaxial side of the leaf blade, avoiding the midrib.The measurements were recorded between 20 and 22 h, with the provision of a saturating light pulse of 0.3 s, at a frequency below 0.6 Khz.
The evaluations were done with the same experimental units and the same treatments four successive times.Statistical analysis was done using the nlme package in R software (R Core Team, 2013), allowing the adjustment of mixed linear models for data repeated measures (Pinheiro et al., 2013).

Index of total chlorophyll
The sensitive Eucalyptus clone sensitive showed no significant response in the total chlorophyll rate until the day 14.However, on day 15, without water supply, this rate dropped by 33.9% (Figure 1).The tolerant Eucalyptus clone tolerant, on the other hand, responded to the water restriction stress by increasing its chlorophyll rate from day 6.Emphasis was also placed on day 11 of the stress, clear differences were noted in the chlorophyll content between the genotypes under the irrigated water regime, highlighting the clone tolerant.On analysis of the behavior of the genotypes under the non-irrigated water regime, on all the evaluation days except for the first day of stress application, a statistical difference in the chlorophyll content of clone tolerant compared with that of clone sensitive was observed, with higher total chlorophyll concentrations recorded for clone tolerant.

Variables of chlorophyll a fluorescence
Minimal fluorescence (Fo), maximum (Fm) and the quantum efficiency of PSII Fv/Fm revealed significant interactions between the treatments tested (Table 1).The analysis of the opening up of the genotype vs. water regime vs. time shows that water stress caused only significant effects on the values of minimal fluorescence in both clones on day 15 of stress application (Figure 2) purposes only.On day 15, the Fo value of clone sensitive decreased by 37.76%, due to lack of irrigation, whereas in clone tolerant, the Fo increased to 39.52%.
On analyzing the maximum fluorescence upon unfolding of the genotype vs. water regime vs. time, it appears that, contrary to what was reported for the variable discussed earlier, since day 6 of stress, the water restriction induced a 36.90%reduction in the Fm in plants of clone sensitive, becoming clearer day 15 (85.36%) of the stress (Figure 3).However, in the case of clone tolerant, the effects of the non-irrigated water regime were only observed on the Fm on day 15 of the drought, which decreased by 64.20%.
According to the analysis of the interaction between water regime vs. time, from day 6 onwards effect statistically significant due to drought was seen on the Fv/Fm ratio.During the last days of the experiment (11 and 15 days), limiting the water caused the Fv/Fm ratio to plummet to near zero values.A decline in the potential PSII (Figure 4) quantum efficiency was observed, beginning from day 6 of the stress, which can be related to the damage of the photosynthetic apparatus in the plants.Plants with their photosynthetic apparatus in perfect condition express typical optimum conditions for development, showing the quantum efficiency of PSII, expressed as the Fv/Fm ratio, with values between 0.75 and 0.85 (Bolhàr-Nordenkampf et al., 1989).
The expression of genotype vs. water regime reveals that the altered quantum of efficiency of PSII occurred due to water limitation in both clones, to less than 0.75 (Figure 5).Clone sensitive revealed the most substantial drop in the Fv/Fm ratio in which the quantum efficiency decreased to 56.27% for values below 0.40.On analysis of the genotypes under each water regime, no differences in the quantum efficiency were observed between the two clones subjected to the irrigated water regime, with    recorded values greater than 0.75.However, under the water restriction regime, clone sensitive showed the Fv/Fm ratio at 31.13%, a lesser value when compared with clone tolerant, implying the greater effect of stress and its greater sensitivity to water deficit; however, clone tolerant has also expressed measurements below ideal values.
Although, the genotype vs. water regime vs. time showed no significance at the 5% level of significance; it is noted, as shown in Figure 6 that on day 6 of the water stress regime clone sensitive alone was negatively affected, probably due to the decrease in the maximum fluorescence (Figure 3).It was on this day, that the plants of clone tolerant recorded values above 0.75 for the Fv/Fm ratio, as well manifested visible symptoms in response to the water deficit.

Index of total chlorophyll
The responses of plants to drought can be expressed in different ways and the ability of a plant to tolerate a drought stress is generally determined by the combination of attributes expressed during the development of a drought.In this study, the two contrasting genotypes showed different behaviors as the studied variables.
As seen in Figure 1, the subjection of the clones to up to 11 days of water stress did not induce any changes in the chlorophyll content, although in the sensitive genotype reductions began to be observed only from day 15 of the stress.Nautiyal et al. (1996) in their study had a drop in the chlorophyll content in the Pongamia pinnata plants only under the more severe water conditions.These authors therefore suggest that the species in question is capable of survival and can remain photosynthetically active under moderate drought conditions.However, the more severe conditions exert an adverse effect on the chlorophyll content.Huang et al. (2013) stated that the substantial decrease or change in the chlorophyll content during water deficit periods varies with the intensity, duration and severity of the deficit.
On day 15 of the evaluation the effect of the severity of the stress was observed, confirmed by the visible symptoms and dryness of the substrate used.This state probably favored the appearance and accumulation of the reactive forms of oxygen, which damage the plant tissues by oxidizing the photosynthetic pigments, membrane lipids, proteins and nucleic acids (Raoudha et al., 2007;Xue et al., 2011).It is these reactive forms of oxygen that actually degrade the thylakoid membranes of the chloroplasts, by peroxidation of their lipids (Marenco and Lopes, 2005).The same phenomenon was observed in wheat plants subjected to water limitation during which the peroxidation rates were seen to rise significantly depending upon the severity of the stress (Tatar and Gevrek, 2008) they were experiencing.The drop in the chlorophyll content under the stress due to drought has therefore been suggested as a characteristic symptom of oxidative stress and could be caused by pigment photooxidation and chlorophyll degradation (Xue et al., 2011).
Clone tolerant, however, expressed an increase in the chlorophyll content when subjected to drought conditions.A similar result was observed by Silva et al. (2004) in E. grandis and by Yanqiong et al. (2007) in four shrub species under conditions of water limitation.A few other authors recorded an increase in the chlorophyll content under moderate stress conditions and explained it as likely being due to a slowing down of cell growth in relation to the chlorophyll synthesis.Ebrahimiyan et al. (2013) for instance, observed a relationship between the chlorophyll content and dry matter production during moderate stress conditions.This implied that the loss of leaf weight after moderate stress could produce a relative rise in the chlorophyll content.On the other hand, some authors did not encounter any significant difference in the total chlorophyll content of certain plant species under varying water regimes, as did Egert and Tevini (2002) in the plants of Allium schoenoprasum and Shvaleva et al. (2006) in Eucalyptus globulus.Although plants exposed to severe water stress usually undergo degradation of their photosynthetic pigments due to oxidative damage, plants can, according to Egert and Tevini (2002), protect themselves by synthesizing antioxidant molecules (carotenoids, ascorbate, a-tocopherol, flavonoids and glutathione) or by increasing the antioxidant enzymes synthesis (peroxidase, catalase and superoxide dismutase).
From these reports, the rise in the chlorophyll content of clone tolerant observed from the first day of water limitation until the last day of the stress may be attributed to a slowing down of plant growth in relation to chlorophyll synthesis in the early stages of the stress and associated with the activity of an efficient antioxidant mechanism under more severe water restriction.

Variables of chlorophyll a fluorescence
Drought affects the Fo causing light emission from the excited chlorophyll molecules, prior to the energy being dissipated to the PSII reaction center (Krause and Weiss, 1991;Baker and Rosenqvist, 2004).This has been the focus of controversy in the literature.Some authors are of the opinion that the decrease observed for this variable can be associated with the impairments in the reaction center of PSII or due to the faulty transfer of excitation energy from the antenna complex to the reaction centers, indicating an even greater sensitivity to the waterrestriction condition (Silva et al., 2006;Tatagiba and Pezzopone, 2007;Michelozzi et al., 2011).Tatagiba and Pezzopone ( 2007) reported an increase in the minimal fluorescence of the Eucalyptus plants during the dry season, the phenomenon being credited to the ability of these clones to tolerate water restriction conditions in the soil.
Authors Zlatev and Yordanov (2004) view the increase in the Fo as a negative effect of drought from their study in bean plants, as did Calatayud et al. (2006) on rosebushes.These authors hold that the highest observed Fo level can be attributed to an increase in the fraction of the PSII reaction centers in the photoinactivated state, resulting in a drop in the photochemical capacity of the PSII.Efeoğlu et al. (2009) too, in their work on corn plants, recorded an increase in the Fo under water restriction conditions.They cited the dissociation of the complex light collection of the photosystem II reaction centers as the possible cause.Photoinhibition of the reaction centers or even the reduced pool of plastoquinone (PQ) in leaves that have endured dark adaptation could be another reason.Matos et al. (2010) credited the increase in the minimal fluorescence in Cicer arietinum under water restriction to the reaction center, showing that the greatest losses occur during the energy transfer from the antenna after excitation.Silva et al. (2006) showed that the reduction in the Fm was observed in the non-irrigated water regime in both genotypes.They indicated in general the faulty photoreduction of quinone A (QA), the primary electron acceptor of photosystem II, which may be associated with PSII inactivation in the thylakoid membranes, directly affecting the electron flow between the photosystems.The same authors reported similar behavior in different forage species when subjected to water restriction.The later and less intense effect of water deficiency observed on clone tolerant implies a lower sensitivity to the higher intensity and duration of the water deficits applied (Silva et al., 2006) and indicates a greater drought tolerance compared with clone sensitive.The results concur also with the report of Zlatev and Yordanov (2004) in bean plants and that of Bączek-Kwinta et al. (2011) in Chamomilla recutita.Tatagiba and Pezzopone (2007) however, found this variable to increase in the Eucalyptus plants during the dry season, which according to these authors implies that the restricted water supply caused no decrease in the photoreduction of quinone A (QA) and none in the electron flow between the photosystems in the clones studied.
In both genotypes, the decrease in the maximal fluorescence noted could be correlated with the increase in the degradation rates of the D1 protein with increasing water limitation, as a result of the action of the reactive oxygen (Zlatev and Yordanov, 2004;Rivero et al., 2010).The D1 proteins are significant in the functioning of photosystem II as it is through these that the electron flow from the reaction center to the quinone occurs, which to achieve complete reduction emits fluorescence to the maximum level (Araújo and Deminicius, 2009;Rivero et al., 2010).Thus, with the increase in the degradation rates of these proteins due to water deficit stress, less energy is transferred from the photosystem II reaction center to the quinone, producing low maximal fluorescence levels.
A decrease in the quantum yield of PSII, as seen in Figures 4, 5 and 6, implying a reversible photoprotective regulation or an irreversible inactivation of PSII, have been recorded in many plant species due to soil water deficit (Ditmarová et al., 2010;Araújo et al., 2010;Michelozzi et al., 2011;Wang et al., 2012).
The drop in the Fv/Fm ratio in the Eucalyptus clones  due to water deficit is similar to observations of Rolando and Little (2003) in E. grandis and by Lima et al. (2003) in five species of Eucalyptus (E.grandis, E. urophylla, E. camaldulensis, Eucalyptus torelliana and Eucalyptus phaeotrica).Although, this ratio normally decreases in plants experiencing some type of stress, Rolando and Little (2008) observed no changes in the chlorophyll fluorescence when the E. grandis seedlings were subjected to water restriction conditions.These authors propose that this unexpected absence of perceptible changes in the fluorescence parameters could be the result of using plant species possessing a higher tolerance to drought or due to the short experimental time.In contrast, Susiluoto and Berninger (2007) studied the response of Eucalyptus microtheca to drought at the home of a greenhouse reported an increase in the Fv/Fm ratio under stressful water conditions.However, in a study by Susiluoto and Berninger (2007), the response of E. microtheca to drought conditions in a greenhouse revealed an increase in the Fv/Fm ratio.
The negative effects of drought on the functional integrity of PSII, which were observed in this experiment too, were certainly the result of the degradation of the PSII components other than the chlorophylls.From day 6 of the experiment, the chlorophyll content of clone sensitive showed no change due to water limitation, although, this day revealed a reduced Fv/Fm (Figure 6) relationship.This same clone revealed a drop in the chlorophyll content, which coincides with the decline of the quantum efficiency of PSII on day 15 of the stress.Clone tolerant, however, experienced an increase in the chlorophyll content throughout the experiment, under the water restriction conditions, as observed.
As interpretation in Figure 6, the later and less intense damage to PSII seen from the appearance of clone tolerant may be indicative of a photosynthetic apparatus more efficient than that of clone sensitive with respect to tolerance of the photo-inhibitory conditions resulting from the stress of water restriction.

Conclusions
The gradual rise in the chlorophyll rate as well as the reported behavior of the variables of fluorescence confirm the hypothesis that the features of genotype tolerant reveal a more efficient photosynthetic apparatus for tolerance to the photo-inhibitory conditions arising from low water availability.The potential use of such variables in the forest breeding programs whose objective is to selecting drought tolerant genetic material, emphasizing the quantum efficiency of PSII, expressed as the Fv/Fm ratio was also highlighted.The latter was found to be a reliable tool facilitating the selection for plants with drought tolerance, having the additional advantage of enabling a precise, economic, rapid and non-destructive evaluation.This is very important for forest farmers and breeders, since they need to select donor trees and Eucalyptus clones tolerant to water stress in the young stage when they are still in the seedling nursery.

Figure 1 .
Figure 1.Total chlorophyll content of the Eucalyptus clones under two water regimes at different evaluation times.

Figure 2 .
Figure 2. Minimal fluorescence (Fo) of Eucalyptus plants under two water regimes at different evaluation times.

Figure 3 .
Figure 3. Maximal fluorescence (Fm) of the Eucalyptus plants under two water regimes at different evaluation times.

Figure 4 .
Figure 4.Quantum efficiency of PSII, expressed as Fv/Fm ratio, in Eucalyptus plants under two water regimes at different evaluation times.

Figure 5 .
Figure 5.Quantum efficiency of PSII, expressed as the ratio Fv/Fm of plants of two Eucalyptus genotypes subjected to two water regimes.Different lowercase letters indicate significant differences between the genotypes in each water regime while the different capital letters indicate significant differences between the water regimes within each genotype (** P≤0.01).

Figure 6 .
Figure 6.Quantum efficiency of PSII, expressed as the Fv/Fm ratio in Eucalyptus plants subjected to two water regimes at different evaluation times.

Table 1 .
F statistic and P-value for minimal fluorescence (Fo), maximal fluorescence (Fm) and quantum efficiency of PSII (Fv/Fm) of the seedlings of two Eucalyptus genotypes under two water regimes.
For interactions of interest, significance levels (P-values) are presented in bold and with * when significant (<0.01).D.F.: Degrees of freedom.