Seasonal Variation of Chlorophyll and Carotenoids in Leaves of Hancornia speciosa in Three Central Areas of the Cerrado of the State of Tocantins , Brazil

This study aimed to evaluate the seasonal variation in concentrations of chlorophyll a, b, total chlorophyll and carotenoids in leaves of Hancornia speciosa, Gomes, during the periods of the year, relating them to the main phenological events, periods (rainy, dry and transitions) and populations evaluated. The survey was performed in three sites and the spatio-temporal analysis divided into four periods (rainy, rainy-drytransition, dry and dry-rainy transition), with 10 replicates (matrix plants). The data were collected in average intervals of 33 days from October 2014 to April 2017. The extraction and calculation of the chlorophyll and carotenoid contents of the leaves were expressed in mg/g DM, according to the equations of Arnon (1949) and Lichtenthaler (1987). There was a significant difference between the periods and sites analyzed for all pigments and their relationships. There was a greater amount of chlorophyll a than chlorophyll b; this difference was greater in the dry period. In the rainy period, we found a greater amount of total chlorophyll, carotenoids and total chlorophyll/carotenoid ratio. The behavior for the species follows that already observed for deciduous plants, closely related to water availability.


Introduction
The creation of a large database on functional characteristics is gaining priority in plant ecology research, with the purpose of contributing to the understanding and prediction of species distribution in present and future environments (Garnier et al., 2001).Foliar characters are often used as indicators of the relationship between the use of resources by plants, their biomass and the ecosystem functioning (Craine, Froehle, Tilman, Wedin, & Chapin, 2001).Studies have sought to establish relationships between environmental conditions and foliar properties, although leaf characteristics are usually measured at the peak of the growing season when environmental conditions favor carbon assimilation and there is an abundance of expanded and healthy leaves (Carvalho, Bustamante, Kozovits, & Asner, 2007), with scarce work conducted in savanna environments.In these ecosystems, low water availability, soil nutrient deficiencies and fire are the main causes of instability in final productivity.
In the Cerrado, soil water content decreases rapidly at the beginning of the dry season, followed by a prolonged drought period (Franco, 2002).The water deficit hinders the absorption of nitrogen, an essential component of chlorophylls, proteins, enzymes and nucleic acids, being indispensable for plant growth.Nitrogen deficiency reduces leaf area, accelerates leaf senescence, reduces the efficiency of photosynthesis, and consequently the photosynthetic activity of the plant (Uhart & Andrade, 1995).Plants native to this biome may present adaptations in response to water stress, such as decreased transpiration, leaf area and increased leaf senescence and abscission (Meir & Grace, 2005).photosynthetic activity in plants.Thus, the nutritional status of plants is directly related to the quality and quantity of chlorophyll, which comprises a family of chemically similar substances called chlorophylls a, b, c and d; chlorophyll a, b and carotenoids are directly involved in the absorption of visible light for the transfer of electrons from water to the electron transport chain in the photosynthetic mechanism (Taiz & Zaiger, 2013).Thus, the knowledge of the foliar characteristics represents an interesting measure to compose the database on a species.
Mangabeira (Hancornia speciosa, Gomes) is a fruit tree belonging to the family Apocynaceae, which is more abundant in the areas of cerrado strict sensu, mainly in soils poor in organic matter, acidic, with low nutrient content, and is tolerant to water deficit, with better vegetative development under higher temperature and lower relative humidity (Vieira Neto, Cintra, A. L. da Silva, Silva Júnior, Costa, A. A. G. da Silva, & Cuenca, 2002).It presents a diversity of phenological events, and depending on the region, it has two flowering during the year, in the summer (rain) and winter (dry), in turn, fruiting differs between the harvests and fruit aspects, with higher and better production in the summer (Silva Júnior & Lêdo, 2006).In tropical savannas, temporal patterns of plant growth and reproduction are linked to climatic seasonality (Williams, Myers, Muller, Duff, & Eamus, 1997).Pilon, Udulutsch, and Durigan (2015) studied the phenological patterns of 111 cerrado species, indicating that the mangabeira is a semideciduous or deciduous species that loses leaves during the dry season of the year, agreeing with Lorenzi (2000), and A. V. Pereira, E. B. C. Pereira, and Silva Júnior (2010).According to Carvalho et al. (2007), who analyzed the concentration of pigments and nutrients in leaves of cerrado species with different phenological strategies, detected no differences in the behavior of deciduous and evergreen plants.
Despite the relevance of photosynthetic pigments to the leaf functioning, there are still no studies that relate the seasonal variations in their concentrations in cerrado species (Carvalho et al., 2007).Based on this premise, the goal of this study was to evaluate the contents of chlorophyll a and b, chlorophyll a/chlorophyll b ratio, total chlorophyll, carotenoid and total chlorophyll/carotenoid ratio, during the periods of the year, relating them to the main phenological events, periods (rainy, dry and transitions) and populations evaluated.

Study Area
The study was conducted in the municipality of Porto Nacional (10°41′15.94″S latitude, 48°22′55.60″W longitude, 239.2 m altitude), central portion of the State of Tocantins, Brazil.The climate of the region is defined as tropical, its classification is Aw according to Köppen and Geiger.It has an average temperature of 26.1 °C (varying 3.0 °C during the year), with September being the warmest month (27.9 °C average) and July, the coldest month (24.9 °C ).The average rainfall of 1,622 mm, with a difference of 258 mm rainfall comparing the driest month (June) with an average of 4 mm and the rainiest, March with 262 mm (Climate-data.org,2017).It is characterized by two well-defined seasons: rainy (November to April) and dry (May to October).The mean annual rainfall of the ten-year period prior to the study (2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013) was 1643.75±276.85mm and mean temperature was 27.5±0.4°C.The study started in October 2014 to April 2017 (30 months).Rainfall data were recorded by the conventional meteorological station, located in the municipality of Porto Nacional, distanced approximately 10 km from each collection site, and data provided by the National Institute of Meteorology (Inmet & Bdmep, 2017).
For the purposes of analysis, the observations were divided into four collection periods (rainy, rainy-dry transition, dry and dry-rainy transition) defined according to the phytoclimatic model proposed by Walter and Lieth (1967), constructed for the region of National Harbor.The mean values of rainfall (mm) and temperature (°C) of the ten years prior to the survey of leaf characteristics (2004 to 2013) were used.For the Porto Nacional meteorological station, a Cartesian chart was drawn where the abscissa axis (X) represents the months of the analyzed years and the ordinate axis, the rainfall precipitation in mm (Y 1 ) and the average monthly air temperature, in °C (Y 2 ), in which the scale of the rainfall representation is twice that of the temperature.According to the Walter and Lieth phytoclimatic model, applied for forest situations, the year is divided into a super humid period (with monthly rainfall over 100 mm-adapted for this work-November to April), wet period (adapted in this work-transition from dry to rainy and vice versa-October and May months) and dry period (adapted in this work-June to September) (Figure 1).

Statistical Analysis
Each variable was tested analysis of variance, and the differences in pigment contents and their ratios, compared by Tukey's post-hoc test, at a 5% probability level.The errors obtained for each analyzed variable were subjected to the Shapiro-Wilk normality test and the variances compared using the variance ratio test (Zar, 1999).Data were transformed into Box-Cox, in all cases, to correct normality and homogeneity.We used the softwares Past (Hammer, Harper, & Ryan, 2001) and Microsoft Excel® for data organization and analysis.

Results and Discussion
The mangabeiras present in areas of the Cerrado of Tocantins, Brazil, is a deciduous species lose their leaves during the dry season.In Figure 2 shows the concentrations of the pigments and their relationships over time (DAFR-days after the first reading).In September 2014, the 30 matrices were marked and the first leaves were collected for standardization of the methodology.Data collection started at 32 days after the first reading (DAFR).The month of October was considered a transition period between dry and rainy seasons (represented by the readings: 32, 404, 770 DAFR); the months from November to April-rainy season (readings 69,95,131,161,222,432,455,493,526,559,587,803,823,860,895,925 and 956 DAFR); the month of May-transition period between rainy and dry seasons (readings: 250 and 620 DAFR); the months from June to September-dry period (readings: 277,315,371,647,680,711 and 744 DAFR).
Mangaba tree has arboreal habit and seed dispersal by zoochory, flowering (February, May, September to November), fruiting (May, June to August, October to December), new shoots (January to March, May, September, October and December), fall of leaves (August, September) (Pilon et al., 2015).
The highest values for chlorophyll a were observed in September (371 and 744 DAFR), period of young leaves, and October (32, 404 and 770 DAFR), end of the dry period and transition to rainy, coinciding with flowering, contributing to the observation of Franco, Bustamante, Caldas, Goldstein, Meinzer, Kozovits, Rundel, and Coradin (2005) in which the deciduous species present greater efficiency in the use of water at the end of the dry season with the emergence of new leaves and with a lower "construction cost"-a smaller specific leaf area.
It can be observed an increase in the chlorophyll content and a permanence in the levels of carotenoids for the months of September (371 and 744 DAFR) and October (32, 404 and 770 DAFR) (Figures 2A,2B,2C and 2E).Carvalho et al. (2007) also observed that, in the rainy season, pigment concentrations tend to increase, being significantly higher than at the end of the dry season for chlorophyll a and b and chlorophyll/carotenoid ratio.This increase in the concentration of chlorophylls in the rainy season is related to the presence of leaves of a new cohort, as also observed in the present work, period with a greater number of young leaves and beginning of flowering.One way to predict the nutritional level of nitrogen (N) in plants is to measure the chlorophyll content in the leaf, because there is a positive correlation between the content of this pigment and the nitrogen content in the plant (Piekielek & Fox, 1992;Smeal & Zhang, 1994;Booij, Valenzuella, & Aguilera, 2000).The variation in pigment

F
concentration and its ratios were significantly influenced by the period (rainy, dry and transitions) and by the site (Canaã, Providência and São Judas Tadeu), except for the carotenoid content that did not differ between sites (Table 2).
In general, chlorophyll a and b occur in the ratio of approximately 3, and growth conditions and environmental factors may affect this ratio (Carvalho et al., 2007).Mangaba trees evaluated in this work, in the three sites and throughout the collection period, presented annual mean values of 2.09±0.60 (mean±standard deviation), for chlorophyll a/b ratio (Figure 2D), which according to Lichtenthaler (1987)  No significant difference was detected between the rainy/dry transition and dry periods for chlorophyll a (Figure 3A), total chlorophyll (Figure 3C) and total chlorophyll/carotenoids ratio (Figure 3F).For these comparisons, the dry period always had a significantly lower effect.It should be noted that the averages presented in the Figure 3 are for the data transformed by Box-Cox, and cannot be used directly to express the levels of chlorophyll and carotenoids in each period.

Conclusion
(1) The seasonal variation of the chlorophyll a, chlorophyll b and total chlorophyll in mangaba tree (Hancornia speciosa) follows the same observed for the deciduous species, that is, is closely associated with the availability (2) The mean chlorophyll a content was 2.0 times higher than the chlorophyll b, and was 1.8 times in the rainy period (November to April) and 2.5 times in the dry period (June to September).The maximum value found reached the 3/1 ratio recommended in the literature, indicating that this ratio is associated with the moment of data collection.
(3) There was a significant difference between the analyzed periods and collection sites for all pigments and their ratios; (4) The total chlorophyllcontent was 21.8% higher in the rainy period, compared to the dry period, and its highest value occurred in the dry/rainy transition due to the higher number of young leaves in this period, whereas for carotenoids.it was 5.4% higher in the rainy period, although with little difference between the months of the year.
(5) The total chlorophyll/carotenoids ratio was 16.3% higher in the rainy period when compared to the dry period, and the highest value was observed in the dry/rainy transition period.

Figure 3 .
Figure 3. Contents of pigments: (A) Chlorophyll a; (B) Chlorophyll b; (C) Total chlorophyll; (D) Chlorophyll a/chlorophyll b ratio; (E) Carotenoids; (F) Total chlorophyll/carotenoids ratio, per period of the year (rainy/dry transition, dry, dry/rainy transition and rainy) Note.The ordinates of the graphs represent the means and the 95% confidence interval for the data transformed by Box-Cox; different letters on the comparisons indicate that the means are significantly different by the Tukey's test at the 5% probability level.
the soil, and the highest values were found shortly after the fall of leaves and the beginning of a new cohort.

Table 1 .
Geographic coordinates of the matrices sampled in three natural populations of mangaba trees, located in the municipality of Porto Nacional, State ofTocantins, Brazil, 2014 jas.ccsenet.org 1. Climatic dia g to the phytoc emperature; th ember to April-October); an

Table 2 .
Lichtenthaler (1987)de plants, although the analyzed environments are considered open environments.The a/b ratio differed statistically from the collection site (F 2, 838 = 12.837, p < 0.0001), and the highest ratio was found in the São Judas Tadeu Farm (2.20±0.60),statisticallydifferent of Canaã (2.08±0.51)andProvidência(2.01±0.66),whichdidnotdifferfrom each other.The highest values of the chlorophyll a/b ratio were verified in periods of lower water availability, reaching 3.0 in September 2015 (371 DAFR).These results corroborate withLichtenthaler (1987), who states that sun plants or exposed to conditions of high luminosity present ahigher chlorophyll a/b ratio, situation found in São Judas Tadeu.At this site, the chlorophyll b content was significantly lower (1.00±0.36)than the other two sites (Canaã: 1.08±0.38 and Providência: 1.06±0.36)thatdidnot differ from each other.Significant difference was detected for the effect of periods (rainy, rainy/dry transition, dry and dry/rainy transition) for all pigments analyzed and their ratios (Table2).Values of mean square (F) and probability (p) for the Box-Cox transformed data for the contents of pigments: Chlorophyll a; Chlorophyll b; Total chlorophyll; Carotenoids; Chlorophyll a/b; Total Chlorophyll/Carotenoids ratio between total chlorophyll and carotenoids.Values p < 0.05 are considered significant