Introduction

The life cycle of plants is subjected to adverse environmental conditions that limit the development of plants and their chances of survival. One of these adverse conditions is water deficit. As a consequence of this specific environmental condition, plants can respond with the reduction of the water potential and leaf turgescence with closure of the stomata and, consequently, a lower photosynthetic rate that reflects in the cell elongation (Nogueira et al. 2005) and cell growth (Jaleel et al. 2009). Therefore, the characteristics of the internal structure of the leaves of plants may be important to determine the level of tolerance to water stress (Batista et al. 2010), as well as to detect the anatomical modifications of plants subjected to treatments that involve water conditions in a phase prior to leaf wilting (Oliveira et al. 2014).

Plants present several modifications to adapt to the stressful conditions, such as increases in the thickness of the epidermis, number of trichomes, deposition of the cuticle, and stomatal density in cavities and stomatal crypts (Castro et al. 2009), as well as a reduction in stomatal size.

In addition to leaf growth and modifications in leaf anatomy, water deficit may generate oxidative damage, caused by energy from excitation of the chlorophyll molecules not being used in photosynthesis or eliminated in the form of heat. This causes the production of reactive oxygen species (ROS), which can react with any cell molecule, causing damage to the cells and organs (Barbosa et al. 2014; Morales and Munné-Bosch 2016). Several studies have shown the induction of oxidative stress by water deficit and antioxidative enzyme activity, such as superoxide dismutase (SOD) and peroxidase (POD) (Wang et al. 2016; Rosa et al. 2017; Reis et al. 2018; Gupta et al. 2018).

However, it is important to emphasize that the effects of water deficit on plants are quite variable, because the effects depend on the intensity and speed of stress imposition and the developmental stage at which it happens (Pimentel 2004). In addition, the genetic capacity of plants and their recovery potential are determinant factors for the development and survival of each species.

Campomanesia xanthocarpa O. Berg. (Myrtaceae) is a deciduous tree, popularly known as “Guabirobeira” or “Guabiroba” and according to review of Luz and Krupek (2014) it is a heliophyte and selective hygrophyte plant, with occurrence in the “capoeiras and capoeirões” on moist soils and well drained capons and gallery forests, both in flat areas and on hillsides and near water ways. It has good adaptability and may occur in dry, compact, and low-fertility soils.

The economic importance of C. xanthocarpa comes mainly from its use in beekeeping, human food, and medicinal products. The plants’ fruits, when ripe, are rich in vitamin C and appreciated in natural consumption and industrialization in the manufacture of liqueurs and ice creams (Carvalho 2006).

The seedlings of C. xanthocarpa cultivated under water deficit shows reduction of the leaf water potential and all the characteristics of photosynthetic metabolism, which reaches close to zero only 20 days after the suspension of irrigation. However, after resumption of irrigation, the seedlings show to recover (Bento et al. 2016). No information is available on the protective mechanisms or morphoanatomical modifications that could allow this recovery after the water deficit.

Considering the occurrence of C. xanthocarpa in both moist soils and environments with low water availability, we hypothesize that this species shows plasticity in its morphoanatomical and biochemical responses that enables its survival in environments subject to temporary water deficits and allow for its recovery after irrigation restoration.

This study aims to evaluate the growth, antioxidant enzyme activity, leaf anatomy, and recovery potential of C. xanthocarpa seedlings subjected to water deficit.

Materials and Methods

Plant and cultivation material

Fruits of C. xanthocarpa O. Berg. were collected in remaining Cerrado areas, located near the municipality of Dourados/MS, Brazil. After collection, the fruits were processed manually, and the seeds were washed in running water to eliminate the remnants of pulp and dried with Germitest^® paper.

Fig. 1: Experimental scheme of the distribution of C. xanthocarpa plants in the two water regimes (Control-C and water deficit-WD) and in the six evaluation periods: T0 beginning of the experiment; 1^st and 2^nd P0 - first and second photosynthesis zero, 1^st and 2^nd REC - first and second recovery, and the END evaluation

Table 1: Water potential (Ψw) due to the evaluation periods between irrigated seedlings of C. xanthocarpa and subjected to water stress conditions

Treatments	T0^1	1^st P0	1^st REC	2^ndP0	2^nd REC	END
Control	-0.1 Aa^*	-0.1Aa	-1.3 Ba	-0.1 Aa	-1.9 Ca	-1.9 Ca
Water deficit	-0.1 Aa	-2.6 Db	-2.7 Da	-2.3 Cb	-2.3 Ca	-1.9 Ba
Uppercase letters buy the different evaluation time in the same water condition and lowercase letters bought the different hydric conditions at the same evaluation time. ^(1)T0 beginning of the experiment; 1^st and 2^nd P0 - first and second photosynthesis zero, 1^st and 2^nd REC - first and second recovery, and the END evaluation

The seedlings were produced from sowing in tubes 50 x 190 mm to one centimeter of depth containing Dystroferric Red Latosol, sand, and commercial substrate Bioplant^® in the proportion of 1:1:1, placing one seed per tube. The seedlings were transplanted to pots with the same soil and capacity of 5 kg when reached approximately 15 cm, having been acclimated for 30 days, with water retention capacity in the substrate soil of 70% and kept in a greenhouse under 40% shade. During the experiment, the plants were protected from rainfall by a plastic cover.

At the beginning of the experiment (T0), the pots were distributed in two groups: Group 1 (control (C), where plants were irrigated periodically in order to maintain 70% of the water retention capacity of the substrate, and group 2 characterized by water deficit (WD), where irrigation was suspended until the photosynthetic rate reached close to zero, at which time the plants were re-hydrated with daily irrigation for one week at a rate and maintained a water retention capacity of the substrate to 70%.

The plants were evaluated in six periods: T0 (beginning of the experiment); 1^st P0 (first photosynthesis zero), when the seedlings under suspension of irrigation showed photosynthetic rate close to zero (28 days); 1^st REC (first recovery),when the seedlings were re-irrigated until the photosynthesis values approached the control (seven days); 2^nd P0 (sec photosynthesis zero) when the seedlings were subjected to the sec cycle of suspension of irrigation and showed photosynthetic rate close to zero (nine days); 2^nd REC (sec recovery), when the seedlings were re-irrigated until the photosynthesis values reached the control in the sec cycle of irrigation suspension (seven days); and the end evaluation occurred at 172 days of experiment (Fig. 1).

Plant measurements

Growth characteristics were evaluated by collecting three seedlings of each treatment and evaluated for the number of leaves (NL), shoot length (SL), and root length (RL). For the analysis of antioxidant enzyme activity, an extract was obtained from the homogenization of 1 g of the leaves and roots, removed from each treatment, fragmented in mortar in the presence of liquid nitrogen. Next, 2 ml of extraction solution was added, consisting of EDTA 0.1 mM in potassium phosphate buffer 0.1 M, pH 6.8, containing 20 mg of polyvinylpyrrolidone. Homogenized solution was centrifuged for 20 min at 4000 rpm and the supernatant collected, which was then used in the evaluations of peroxidase - POD (Macedo et al. 2005) and superoxide dismutase - SOD (Beauchamp and Fridovich 1971; Giannopolitis and Ries 1977; Longo et al. 1993).

Free-hand para dermal and transversal cuts were made in the median region of completely expanded leaves of each treatment (n=8). Cross-sectional cuts were performed free-hand with a steel blade in fresh material being stained with astra blue and safranin, according to Bukatsch’s (1972) proposal modified and assembled between lamina and laminula in glycerinated gelatin. From this cross-sectional sections, the thickness of the cuticle adaxial (CAd; µm) and abaxial (CAb; µm), epidermis adaxial (EAd; µm) and abaxial (EAb; µm), palisade (PP; µm), and lacunous (PL; µm) were determined.

Fig. 2: Number of leaves - NL (A) root length – RL (B) and shoot length - SL (C) between irrigated seedlings of C. xanthocarpa and water deficit conditions. Lowercase letters compare the different water regimens in the same period of evaluation by the T test (P < 0.05). Uppercase letters compare the control treatment in the different evaluation periods. Uppercase letters in italics compare the stress treatment in the different evaluation periods, both by the Scott–Knott test (P < 0.05)

The paradic impressions were prepared in the morning, between 8:00 and 11:00 h, with Super Bonder^® glue of samples from the median region of the limbus on the adaxial and abaxial surfaces, being determined: polar diameter (PD µm) and equatorial diameter of the stomata (ED µm), ostiolar aperture (OA µm), stomatal functionality (considered the polar diameter/equatorial diameter ratio of stomata), and stomatal index (SI%) calculated using the formula proposed by Salisbury (1928). The laminary obtained was photographed with the aid of a Moticam 2000 digital camera coupled to the optical microscope by means of the MoticImage 2000 program and adjusted scales in the appropriate optical conditions.

Experimental design and statistical analysis

The experimental design was completely randomized in a factorial scheme with 6 evaluation periods (T0, 1^stP0, 1^st REC, 2^nd P0, 2^nd REC, and END) and 2 water regimens (control and water deficit) with four replications, where each repetition corresponded to a seedling.

The data were subjected to analysis of variance. Significant effects on the means of the water regimens were compared by the T test with a 5% probability and on the averages of the evaluation periods were compared by the Scott–Knott test at 5% probability.

Results

Effects on Water potential (Ψw)

The interaction was observed for water potential between evaluation time and water regimens (Table 1). In the first and sec cycle of photosynthesis close to zero (1^st and 2^nd P0), significant reductions were observed for Ψw. At the end of the experiment, the stressed seedlings did not differ from the control.

Effects on Growth characteristics

Fig. 3: Antioxidant activity of superoxide dismutase (SOD - U of SOD) enzymes in leaves (A) and roots (B); and Peroxidase (POD - μmol g FM^-1) in leaves (C) and roots (D) between irrigated seedlings of C. xanthocarpa and water deficit conditions

The seedlings of C. xanthocarpa under water deficit conditions showed reduction in the number of leaves (NL) in the first cycle of suspension/resumption of irrigation when compared to control (Fig. 2A). However, at the end of the experiment, the NL of seedlings under water deficit did not differ from control.

Under water deficit, root length (RL) showed an increment during the 1^st P0 period (Fig. 2B); however, at the end of the experiment, this increment was lower than the control. The shoot length (SL) was reduced during 1^st REC, 2^nd P0, 2^ndREC, and at the end of the 90 days of irrigation resumption (Fig. 2C).

Effects on antioxidant enzyme activity

The enzymatic activity of superoxide dismutase (SOD) and peroxidase (POD) was influenced by the interaction between the control and stress treatments (Fig. 3). Water deficit showed an increase in the enzymatic activity of SOD in the leaves during periods of 1^st P0 and 2^nd P0, respectively. A significant increase in the activity of this enzyme was also observed in control seedlings in the first cycle of suspension/resumption of irrigation and at the end of the experiment (Fig. 3A). However, the SOD activity in the root was higher in the 1^st REC periods and at the end of the experiment (Fig. 3B).

In relation to POD activity in the leaves, an increase was observed during the 1^st P0 and 2^nd P0 periods and at the end of the experiment in the seedlings under water deficit (1.18 mM, 0.99 mM, and 1.29 mM, respectively) (Fig. 3C). In the roots, this increase was seen in the1^st P0, 1^stREC, and 2^nd P0 periods (Fig. 3D).

Leaf anatomy

The leaf anatomy of C. xanthocarpa seedlings was influenced by irrigation conditions (Fig. 4). The seedlings under water deficit showed significant reductions in the thickness of the cuticle of the adaxial face from the 1^st P0 period and remained same until the 2^nd REC period. At the end of the experiment, the previously stressed seedlings showed an increase in the thickness of the cuticle of the adaxial face (Fig. 4A). For the cuticle of the abaxial face, the seedlings under water deficit showed an increase in thickness in 2^nd REC period (Fig. 4B).

In relation to the epidermal faces, the adaxial and abaxial faces showed an increase in the thicknesses due to the water deficit. For the epidermis of the adaxial face, this increase was observed in the 2^nd P0 and2^nd REC periods and at the end of the experiment (Fig. 4C) and for the epidermis of the abaxial face, in the periods of 1^st P0 and at the end of the experiment (Fig. 4D).

Fig. 4: Cuticle adaxial – Cad (A) and abaxial - CAb (B), epidermis adaxial – Ead (C) and abaxial – Eab (D), palisade parenchyma – PP (E) and lacunous – PL (F) of leaf structures/tissues between irrigated seedlings of C. xanthocarpa and water deficit conditions

As for the brackets, the seedlings under water deficit showed reductions in the thickness of the palisade parenchyma in the 1^st P0 period and at the end of the experiment (Fig. 4E). However, in the 2^nd P0 period, an increase in thickness with a value of 19.3 µm was observed. For the lacunous parenchyma, reductions in the thickness were observed during the 1^st REC and 2^ndREC periods and at the end of the experiment in seedlings subjected to water deficit when compared with the control seedlings (Fig. 4F).

Effects on stomatal characteristics

The ostioles aperture and the polar diameter of the stomata were influenced by irrigation conditions (Fig. 5). For the ostioles aperture, reductions observed were found in the periods of higher water stress:1^st P0 and 2^nd P0 (Fig. 5A). In relation to the polar diameter, a reduction was seen in the 1st F0; however, in the 2^nd P0 period, an inversion in the behavior was observed, with an increase in the polar diameter of the stomata in the stressed seedlings seen compared to control (Fig. 5B).

The equatorial diameter was influenced by the factors alone, and they were smaller in seedlings subjected to water deficit, reduced from the first cycle of suspension/resumption of irrigation and remaining with an average value of 2.96 µm until the end of the evaluation period (Fig. 5C, D).

The stomatal index in the seedlings under water deficit fluctuated throughout the experimental period, as seen through reductions in the index during the1^st P0, 2^nd P0, and 2^nd REC periods, and through increases in the 1^st REC period and at the end of the experiment (Fig. 5E).

It was only possible to observe stomatal functionality (PD/ED) differences promoted by the water deficit from the 2^nd P0 period, (Fig. 5F) in which the seedlings under deficit showed a value for this parameter superior to that observed for the control seedlings, which remained until the end of the experiment.

Discussion

In this study, the seedlings of C. xanthocarpa under water deficit showed a reduction in water potential (Ψw 2.4 MPa) when the photosynthetic rate was reduced to values close to zero. After the resumption of irrigation, there was recovery of the Ψw of the seedlings previously maintained under water restriction, reaching values close to of the control plants, as observed for other tree species (Bento et al. 2016; Rosa et al. 2017; Reis et al. 2018).

Fig. 5: Ostiolar aperture - OA (A), polar diameter - PD (B) and equatorial diameter of the stomata - ED (C, D), and stomatal index – SI (E) stomatal functionality – PD/ED (F) (polar diameter/equatorial diameter ratio of stomata) of leaf structures/tissues between irrigated seedlings of C. xanthocarpa and water deficit conditions

The water deficit negatively affected the growth of seedlings of C. xanthocarpa, as evidenced by the reduction in the new leaves even after the first cycle of suspension/resumption of irrigation, likely reflecting the reduction of cell division and expansion caused by the low availability of water (Martins et al. 2010).

However, in the 2^nd REC cycle, the seedlings maintained under water deficit produced a higher new leaves. This phenomenon is known as “hardening”, which allows the plant a greater osmotic adjustment when it has already undergone a first cycle of stress due to lack of water. The solutes accumulated in the first water deficit are not readily assimilated and allow a higher osmotic accumulation and adjustment in the sec cycle of water deficit (Kramer and Boyer 1995), allowing the continuity of the development processes.

As a response to the water deficit (1^st P0), the seedlings showed higher root production as a strategy to adapt to stress, since the aerial part of the plant showed reduced growth. This may be related to the recognition and signaling of stress, through the production of abscisic acid and reactive oxygen species (ROS) (Gupta et al. 2018), both of which induce root growth and stimulate the emergence of lateral roots while suppressing growth of the aerial parts.

However, after the 1^st P0 period, the stressed seedlings ceased the growth of roots and reduced the aerial portion of the plants, a state that remained until the end of the experiment. This behavior can justify the reduction of leaf water potential in this period and signals the sensitivity of C. xanthocarpa to water deficit. We emphasize that the effects of water deficit on seedling growth are due to alterations in metabolism, with observed reductions of stomatal conductance, transpiration rate, and photosynthesis (Bento et al. 2016).

The decrease in water availability affects the growth of plants by controlling the opening of the stomata (Ashraf 2010), which blocks the influx of CO₂ into the leaves, thus, affecting photosynthetic activity and biomass production.

In the face of environmental stresses, plants which exhibit tolerance tend to minimize damage to their metabolism through the regulation of enzymatic activity, showing an increase in the activity of these enzymes that inactivate and/or transform ROS immediately after stress. Oxidative stress can act as an important regulator of vegetative growth, reproduction, defense, and survival of the plant (Morales and Munné-Bosch 2016).

In present study, the highest SOD activity was recorded in the periods of higher water deficit (1^st P0 and 2^nd P0). This behavior proves that an increase in enzymatic activity is a protective response to oxidative stress that C. xanthocarpa presents in the face of increased levels of ROS. This response is produced because of water deficit, since SOD efficiently performs the removal of hydrogen peroxide (H₂O₂), which generates the toxic superoxide radical (O₂^-) (Silva et al. 2012; Hura et al. 2015).

In addition to SOD, high POD activity was observed in the leaves of seedlings under water deficit for the same periods of high water deficit. This high POD activity is associated with an increase in the intracellular level of H₂O₂ owing to removal of the superoxide radical (Pereira et al. 2012; Taiz et al. 2017). The POD has high affinity for H₂O₂ when at low concentrations (Gechev et al. 2006; Jaleel et al. 2009; Locato et al. 2010).

It should be emphasized that the increase in the activity of these antioxidant enzymes was also found in the young plants of Jatropha curcas (Arcoverde et al. 2011; Silva et al. 2012), Copaifera langsdorffii Desf. (Rosa et al. 2017) e Calophyllum brasiliense Cambess. (Reis et al. 2018), in which such behavior was related to the occurrence of a favorable adjustment of the activity of antioxidant enzymes or new synthesis of proteins (Jin et al. 2009) as a form of defense and tolerance of plants against stress.

Hydrogen peroxide is a species of ROS that may play two important roles in plants. In low concentrations, H₂O₂ acts as a molecular sign involving signaling acclimatization, triggering tolerance to various biotic and abiotic stresses. At high concentrations, it causes the programmed death of the cell (Scandalios 2005; Bhattacharjee 2012; Taiz et al. 2017). Thus, the log of the SOD and POD activity in the seedlings of C. xanthocarpa, outside the periods of water restriction, is likely related to the presence of O₂^- and H₂O₂ formed according to the stressful environmental conditions recorded in the previous periods, at levels harmful to the plants.

The activity of SOD and POD enzymes was inferior in the roots of the plants to the leaves. This is because the enzymatic activity may vary in the different organs of the plants dependent on the diversified sites of activity of each enzyme (Gechev et al. 2006; Jaleel et al. 2009; Maia et al. 2012). Furthermore, the production of H₂O₂ in peroxisomes and leaf chloroplasts during environmental stresses may be 30–100 times faster than in mitochondria (Foyer and Noctor 2003; Bhattacharjee 2012). The greater activity of enzymes in the leaves than in the roots before the water deficit indicates that the signaling for defense against this stress in C. xanthocarpa is exercised first in the leaves and then translated into other parts of the plant.

The observation of the antioxidant activity of SOD and POD in the roots of the stressed seedlings in the 1^st REC period is related to the reduction of the relative humidity of the air recorded during this period (data not presented). It is known that some enzymes are constitutively expressed while others are induced by environmental stresses, as shown in studies of low stress activities which result in less severe symptoms and high stress activities that result in more severe symptoms (Barbosa et al. 2014). Thus, the environmental conditions recorded for the 1^st REC period induced a continuity of protection from oxidative damage provided by the enzymes on the seedlings, in the face of the production of new toxic molecules.

The water deficit promoted morpho-anatomical alterations, reflected in the reduction of ostiolar aperture in the polar and equatorial diameters. Such modifications (density, index, aperture, size, and diameters) are related to the regulation of gas exchanges under stressful situations (Castro et al. 2005, 2009; Souza et al. 2010). During such situations, leaves that have smaller stomata and are under water deficit conditions have greater capacity for efficiency in water use due to smaller stomatal pore size, thus, conditioning a lower loss of water due to transpiration (Boeger and Wisniewski 2003). In case of C. xanthocarpa, the reduction in the size of the stomata was another strategy adopted by the plant to avoid water loss in the face of stress. This may indicate that the decrease observed for ostiolum opening had a greater effect on the diffusion of water than of CO₂, thereby maintaining the influx of CO₂ and lower loss of water by transpiration (Taiz et al. 2017).

The alterations evidenced in stomata size, index, and functionality, along with other modifications in the leaf structures, indicate that C. xanthocarpa can be subjected to severe water restriction situations. However, some of these alterations occur on a structural and irreversible level and therefore require time (Casson and Gray 2008), as evidenced from the 1^st REC by the increase of the stomatal index.

In present study, the increased functionality of the stomata observed for the stressed plants demonstrate that the stomata acquired a more elliptical morphology in relation to the plants maintained under irrigation, which favored the low water loss (Melo et al. 2014).

This emphasize that these alterations may serve as an indicator of plant tolerance response to their hydric state and can prove the adaptive value of the plant in investing in the protection of chlorophyllous tissues and reduce transpiration. It is interesting to note that the assessed values of some anatomical characteristics of the stressed plants were not restored to the values of the control until the end of the experiment. These results indicate that the initial priority of the species is the maintenance of turgescence, even if reduced, as a way of maintaining metabolism, and that, perhaps with a longer period of evaluation, the plants can increase metabolism or completely recover the evaluated parameters. Similar behavior was observed in other species (Arcoverde et al. 2011; Melo et al. 2014; Bento et al. 2016; Reis et al. 2018).

C. xanthocarpa showed morpho-anatomical and metabolic changes which favored minimizing water loss. This behavior can justify the occurrence of this species in environments with low water availability, proving our hypothesis that the species has plasticity to adjust to temporary water deficit.

Conclusion

The C. xanthocarpa is a native species sensitive to water deficit but presents strategies to adapt to a temporary water restrictive environment. The seedlings of C. xanthocarpa showed leaf morphoanatomical alterations as survival strategies to temporary water deficit. The species showed an active antioxidant system with increased activity of the enzymes superoxide dismutase and peroxidase in both the shoots and roots, allowing the recovery of seedlings after resumption of irrigation. This information is important because it can assist viverists or farmers, minimizing the mortality rates of seedlings and consequently the costs of production. It will also favor the success of management practices in projects of the recovery of degraded areas.

Acknowledgements

We acknowledge the CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the financial support and scholar ship.

Author Contributions

Larissa FB Araújo, Ferenanda S Junglos and Mário S Junglos collected data, Larissa FB Araújo and Daiane M Dresch conducted statistical analysis, Fernanda S Junglos e Rosilda Mara Mussury conducted leaf anatomy, Silvana de Paula Quintão Scalon supervised the experimental work and all authors wrote the article.

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