Effects of Shading on Anatomical Aspects and Chlorophyll and Carotenoid Contents in Paricá

Paricá ( Schizolobium parahyba variety amazonicum) is an important species for reforestation programs in the Amazon; nevertheless, the anatomical and physiological parameters of the seedlings of this species under shading are not yet fully understood. This study evaluated the chlorophyll and carotenoid contents, the stomatal structure, and the stomatal position in the leaf epidermis under shading of paricá seedlings. The experiment comprised a randomized block design in a subdivided plot scheme, with two sources (Belterra and Urupá) and different shading levels (full sun, 30%, 50%, and 70%). At 100 days after sowing, we performed the analyses of photosynthetic pigment concentration and leaf anatomy. We found that the leaf is hypoestomatic with paracitic stomata and unicellular filiform trichomes on both sides. Higher levels of shading increased the contents of chlorophyll a and b and reduced the stomata density and carotenoid contents in the leaves of young paricá, showing that paricá has phenotypic plasticity to shading.


Introduction
Paricá (Schizolobium parahyba variety amazonicum (Huber × Ducke) Barneby) belongs to the Fabaceae family and is native to the Amazon region (Rosa et al., 2009;Epifanio et al., 2022). In Brazil, paricá is found in the states of Amazonas, Pará, Mato Grosso, and Rondônia. Paricá grows rapidly and can reach a height of about 30 m (Epifanio et al., 2022) with great potential use in reforestation and recovery of degraded areas (Binotti et al., 2019;Sousa et al., 2022). structure adapts to external environmental conditions (Schluter et al., 2003;Aragão et al., 2014). The morphological characteristics of the leaf surface established the amount of light absorbed or reflected. Studies have shown the positive effect of shading on the morphophysiological aspects in some forest species, such as eucalyptus and paricá (Sousa et al., 2022).
Studies have investigated the anatomical and ecophysiological aspects of forest species; however, these studies focused mostly on planted forest of eucalyptus, not on native Amazonian species. Therefore, this study investigated the chlorophyll and carotenoid contents, the stomatal structure, and the stomatal position in the leaf epidermis under shading in paricá seedlings.

Study Site
The experiment was conducted in the greenhouse of Embrapa Amazônia Oriental, Belém, Pará State, Brazil (01°24′31.6637″ S, 48°27′45.1786″ W). The nursery phase was carried out from February 2011 to August 2011. The regional climate is type Af (Köppen classification) (Alvares et al., 2013), with an average annual temperature of 26 ºC. Annual average of regional precipitation is around 3000 mm (Bastos et al., 2002).
The seeds used in the research were obtained from two natural tree populations in the Brazilian Amazon. One from the municipality of Belterra in the Mesoregion Baixo Amazonas and the Microregion Santarém, both in Pará State, with geographical coordinates 02°38′11″ South and 54°56′14″ West. The other from the Municipality of Urupá located in the micro-region and Ji-Paraná, central region of Rondônia State on parallel 13°07′, with geographical coordinates 11°06′47.2″ South and 62°17′38.3″ West.
The seeds were stored under refrigeration at 15 °C at the Laboratory of Forest Seed Analysis (Embrapa Amazônia Oriental, Belém, Pará State, Brazil) until their use in the experiment.
Seeds with tegumentary dormancy were scarified with sandpaper No. 100 and immersed in water at room temperature for 24 hours (Brasil, 2009). They were then sown directly into black polyethylene bags measuring 15 × 20 cm with lateral perforations using a substrate of black earth, tanned manure, and tanned sawdust at the ratio 3:2:1, respectively.
The experiment followed a randomized block design in subdivided plots 2 × 4 with factor A = origin: Belterra (PA) and Urupá (RO), factor B = shading levels with black "sombrite" cloth: 0%, 70%, 50%, and 30% shading with three blocks and 25 plants per plot. The reduction of solar radiation was confirmed by using a pyranometer.
At 100 days after planting, two plants were selected from each shading level. The leaflets were collected from the median region of the rachis, fixed in FAA 70 for 24 h, and stored in 70% alcohol for the anatomical analysis (Johansen, 1940).

Dissociation of the Epidermis
Sections of the median region of the leaflet were dissociated using commercial sodium hypochlorite at intervals of 4 to 5 h until complete separation of the epidermis. After complete separation, the adaxial and abaxial epidermises were washed in distilled water and then stained with 1% safranin stain.

Obtaining Histological Sections
To obtain cross sections, the median region of the leaflets underwent the usual microtomy processes: dehydration in ethyl series (butyl alcohol), infiltration in Leica synthetic resin (hydroxyethyl methacrylate), blocking, sectioning in a rotating microtome (7-5 µm thick), and staining with 0.05% toluidine blue (O'brien et al., 1965).

Stomatal Density
Three leaflets were collected from each shading level of both plant varieties and eight random fields were counted from the apical, median, and basal regions of both sides, totaling 48 measurements per leaflet. The counting was performed with the aid of a millimeter eyepiece in a Zeiss Axiolab DRB-KP optical microscope. Each visual field had an area of 0.2 mm 2 . The data obtained were expressed in number of stomas per square millimeter.

Obtaining Photomicrographs
The slides were observed under an optical microscope coupled to a digital camera and photomicrographs were captured at various magnifications with the aid of a Zeiss Axiolab HBO 50 microscope with a motican 2500 3.0 Mega Pixel digital camera (Plant Anatomy Laboratory-Embrapa Amazônia Oriental).

Determination of Chlorophyll and Carotenoid Contents
The contents of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids were determined in one plant of each treatment and repetition. Two fully expanded leaves were removed, immediately packed in a Styrofoam box with ice and taken to the laboratory where four leaflets were removed from each leaf. The leaflets were macerated and eight sub-samples (0.30 mg/leaf) of fresh material were weighed and transferred to test tubes with 5 mL of DMSO (dimethylsulfoxide, 99% purity by volume).
The test tubes were closed with rubber caps and placed in water bath with preheated water at 70 °C and centrifuged (3,600 rpm) for 2 h for chlorophyll solubilization. The extraction process was considered complete when the sample leaves became transparent in the visual examination (Arnon, 1949). After, readings were taken in a spectrophotometer (Beckman, model 640 B) at wavelengths of 645 and 663 nm.
The extraction and quantification of total carotenoids were performed according to the methodology described by Duke and Kenyon (1986), using the molar absorptivity coefficients of Sandmann and Böger (1983).

Statistical Analysis
Averages of variables were evaluated using Shapiro-Wilk and Bartlet tests (p > 0.05) to verify normality and homoscedasticity. Data that did not achieve the assumptions of parametric tests were transformed using the Box-Cox method (Box & Cox, 1964). Next, the variables were evaluated in the variance analysis followed by Tukey test at 5% probability and the means were compared using the BioEstat 3.0 program (Ayres et al., 2003). Regression equations of the polynomial type were adjusted for the different variables analyzed as a function of the shading level.

Results and Discussion
We found significant effects (p < 0.05) only for the shading factor on the variables stomatal density (DE), chlorophyll a (ca), chlorophyll b (cb) and total chlorphyll (ct). On the other hand, there was origin × shading interaction for the carotenoid contents (carot). Note. df = degree of freedom; * Significant at the 0.05 level of error probability, ** Significant at the 0.01 probability level of the error and ns: not significant at the 0.05 level of probability by the F test.
The paradermal sections in leaf blades showed that the highest values of stomatal density were observed in the full sun treatment (69.35 mm 2 ). In the shades of 30, 50, and 70%, the number of stomas was lower than in full sun, with a reduction of 68%, 72.9%, and 61% respectively (Figure 1). jas.ccsenet.

Figure 1
The larger classified a solar irrad of Curatel