MACRONUTRIENT DEFICIENCY IN CARINIANA ESTRELLENSIS (Raddi) K : FROM CHANGES IN CELL ULTRASTRUCTURE TO VISUAL SYMPTOMS

The nutritional requirements of native forest species can infl uence productivity. Thus, the understanding of these requirements enables us to optimize the use of inputs and reduce the environmental impacts inforest restoration projects. The present study aimed to evaluate changes in cellular ultrastructure and the anatomy of leaf laminae as well as observe visual signs of nutrient defi ciency in young Cariniana estrellensis (Raddi) Kuntze plants, a forest species widely used in ecological restoration projects. The experiment was conducted in a greenhouse in random blocks with three replications of seven treatments using nutrient subtraction (I.e., control [plants grown with all nutrients], -N, -P, -K, -Ca, -Mg, and -S). The plants were harvested 135 days after the beginning of the treatments when the defi ciency of the macronutrients resulted in visible abnormalities. Changes in the cell ultrastructure and structure of the chloroplasts, cytoplasm, and stromal lamellae were observed, as well as starch and lipid concentrations in the cytoplasm, intercellular spaces, and parenchymal cells. Changes in the cell ultrastructures, leaf laminae, and visual signs of nutrient defi ciency hindered the development of young C. estrellensis plants; therefore, forest restoration projects that use this species in soils that require nutritional supplementation may have limited success in the absence of nutritional support.


INTRODUCTION
Forest restoration in degraded areas has expanded through the integration of strategies aimed at mitigating climate change, soil improvement, and biodiversity conservation. Seedling production and the planting of native tree species are key actions for the implementation of such programs. In many cases, soils in areas intended for forest restoration do not have adequate fertility conditions for native species to develop satisfactorily. Such nutritional defi ciency aff ects the growth, morphology, anatomy, and composition and, consequently, production of the plants (Marschner, 2012). However, few and limited studies have been conducted on native Brazilian forest species.
Knowing the nutritional requirements of native species can facilitate the development of technologies to obtain healthy seedlings intended for revegetation programs, thus enabling them to develop in previously degraded areas. For forest development to succeed, it is important to understand the nutritional requirements of each species (Lima et al., 2000). Determining the physiological stages of development in which various elements are in their highest demand can help in fertilization planning to provide them artifi cially, thereby correcting any nutritional defi ciencies.
Although such information is essential for the success of plant growth (Kramer and Kozlowski, 1960), information regarding the nutritional requirements of forest species is scarce in the literature (Schumacher et al., 2004), even though mineral defi ciencies with consequent growth disorders in forest species have been commonly observed (Dreschel and Zech, 1991). To determine the extent to which chemical elements found in plants are essential and their roles in metabolism, experiments have been undertaken using soil and nutrient solutions (Malavolta, 2006;Kerbauy, 2012;Marschner, 2012;Taiz and Zeiger, 2017). For nutritional diagnosis, the omission of nutrients in experimental nutrient solutions has been widely utilized. Although this is an indirect approach, it can provide information on the requirement for fertilizers and can improve the quality of plants (Benedetti et al., 2009).

C. estrellensis
(Raddi) (Kuntze), family Lecythidaceae, is an important tree species for heterogeneous ecological reforestation programs, although its nutritional requirements and defi ciency symptoms are unknown. It is a semi-deciduous tree in winter and prefers sunny areas; however, it can also develop in shaded areas in wet and deep soils, which are characteristics of climax forests. It is rare in the Cerrado and dry terrains. Although it has ornamental qualities, owing to its large size, it is usually used only in landscaping parks and large gardens. Its wood has great potential for use in the wood industry. It reaches 35-45 m in height and its trunk reaches 90-120 cm in diameter, it blooms from October to December, and the fruits ripen from July to September. Its seeds are consumed and distributed by monkeys (Lorenzi, 2006).
The mineral nutrition of native species has been widely studied in recent years (Sorreano, 2006;Andrade, 2010;Viégas et al., 2012;Valeri et al., 2014;Carlos et al., 2015;Sousa et al., 2018). However, there are no studies related to the mineral nutrition of this native species or how nutritional defi ciency can aff ect the cell ultrastructure and visual symptoms.
The present study aimed to evaluate the changes in the cell ultrastructure and leaf lamina anatomy as well as the visual symptoms of young C. estrellensis plants grown under defi cient nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), and sulfur (S) conditions.

MATERIAL AND METHODS
The experiment was conducted in the greenhouse of the Laboratory of Mineral Nutrition of Plants of the Center for Nuclear Energy in Agriculture, University of São Paulo, in the city of Piracicaba/SP, located at 22°42′30″ S latitude, 47°38′00″ W longitude (SIRGAS 2000). C. estrellensis seedlings were purchased from a nursery growing native species and were transported in rigid PVC plastic tubes containing 120 mL commercial substrate (Silva and Stein, 2008), 60 days after seeding. Prior to the nutrient omission treatments, a complete solution with equal mineral concentrations was used as proposed by Johnson et al. (1957). However, the C. estrellensis plants did not develop well, showing symptoms of nutrient toxicity, including burning of the lamina and leaf lamina edges. Thus, the C. estrellensis seedlings used in the experiment remained for 21 days in the greenhouse in a nutrient solution described by Johnson et al. (1957), with 50% of the concentration of the original solution of macronutrients. Micronutrients were also added as described by Johnson et al. (1957). After this procedure, the plants were subjected to diagnosis by subtraction for 135 days. Weekly nutrient solutions were replaced and descriptions of the visual symptoms were taken.
The experiment was performed based on a randomized block design with three replicates per of seven treatments using nutrient subtraction (I.e., control [plants grown with all nutrients], -N, -P, -K, -Ca, -Mg, and -S). Each replicate included a single plant in a vessel with 2 L of nutrient solution. A total of 21 plants were included in the study. The volume of the nutrient solution was maintained daily with aerated deionized water. The nutrient solutions were refreshed when the electrical conductivity of the nutrient solution decreased to 70% of the initial electrical conductivity or every 21 days.
For anatomical evaluation by light microscopy and ultrastructural evaluation by transmission electron microscopy, samples were collected from the middle region of the leaf lamina (1 × 2 mm). Semi-thin (120 nm) and ultrathin (60 to 90 nm thick) sections were prepared according to Reynolds (1963) with minor modifi cations. Drops of 2.5% uranyl acetate were placed on a glass slide and samples were added face-down. A copper screen was placed on top and the sample was allowed to air-dry for 12 minutes in a dark space. The copper screen containing the affi xed sample was then washed three times in distilled water and dried. Drops of lead citrate were added and the sample was placed on a new glass slide with NaOH tablets to remove any moisture and dried for a further 12 minutes in the dark. The samples were washed three times in distilled water and dried again. Then, the samples were examined under a Zeiss EM-900 transmission electron microscope operating at 50 kV.
To determine the nutrient content, each of the young C. estrellensis plants was separated into stems, roots, and leaves at the end of the experiment. Each tissue was washed with deionized water and dried in a greenhouse with forced air circulation at a temperature of 60 °C to a constant mass. All parts were then ground in a mill and the macromicronutrients were extracted and analyzed according to the methodology described by Sarruge and Haag (1974). Nutrient content was compared using analysis of variance (Pimentel-Gomes, 1990).

RESULTS
Macronutrient defi ciency in young C. estrellensis plants caused decreases in nutrient levels in diff erent parts of the plant as well as alterations in the ultrastructure, organization, and overall structure of the leaf lamina cells. This caused overt anatomical changes in the plant tissues and visible signs of nutrient distress. Damage to organelles and other cell structures can compromise several physiological functions (photosynthesis, transpiration, and respiration) of C. estrellensis and aff ect biomass production.

3.1.Nutrient content of leaves, stems, and roots
To direct nutritional management for optimal production, plant tissue analysis was performed to provide information on plant nutritional status (Smith and Loneragan, 1997).
The defi ciency of one nutrient leads to a lower level of the same nutrient in the leaves, stems, and roots. However, there may also be lower levels of other nutrients. In the control group, all macronutrient levels were in the normal range, demonstrating that the measured eff ects were caused by the defi ciency of the respective nutrient minerals (Table 1).
During the analysis of the macronutrient levels in the leaves, stems, and roots, we found all had lower

3.2.Changes in cell ultrastructure and leaf lamina tissues
Comparative analysis of cell ultrastructures and leaf lamina tissues among plants with macronutritional defi ciency showed alterations in chloroplasts, with disorganization of the thylakoid structure (granum) and absence of stromal lamellae in treatments defi cient in N ( Figure 1B), P ( Figure 1C), and S ( Figure 1G). A decrease in chloroplast size was also observed in the P-defi cient treatment ( Figure 1C). However, an increase in the size and number of chloroplasts in the K-defi cient treatment was observed ( Figure 1D). Mg defi ciency disrupted chloroplast membranes ( Figure 1F).
Defi ciencies in N ( Figure 1B), Ca ( Figure 1E), and Mg ( Figure 1F) caused an accumulation of starch granules in the chloroplasts. When K was defi cient ( Figure 1D), no starch granules were observed in the vacuoles. The thin cross sections revealed an increase in the number of starch granules in N-defi cient leaf lamina cells ( Figure 2B) compared to the control treatment ( Figure 2A). Meanwhile, an increase in lipid granules was observed in chloroplasts that were defi cient in N ( Figure 1A), P ( Figure 1C), Ca ( Figure 1E), and S ( Figure 1G).

3.3.Visual symptoms of nutrient defi ciency
Leaf chlorosis occurred in treatments with N defi ciency (initially in new leaves and then in old leaves, Figures 3B and 3I), P defi ciency (new leaves, Figure 3D), Ca defi ciency (old leaves, Figure 3K), Mg defi ciency (interveinal chlorosis in old leaves, Figures  3L, 3N, and 3O), and S defi ciency (new leaves, Figure 3G). P defi ciency also limited root development ( Figure 3U). K defi ciency caused the deformation of new leaves ( Figure 3F), wrinkling and chlorosis of new and old leaves ( Figures 3F and 3J), and petiole collapse ( Figure  3T); thus, causing the abscission of old and intermediate leaves from 90 days after the start of treatment.
The collapse of the petiole and abscission of old leaves also occurred in plants with Ca defi ciency ( Figures  3R and 3S). White and necrotic spots on older leaves were observed under Mg defi ciency. Newly deformed, fi liform, and wrinkled leaves, which deformed the shoot apex, were produced in plants with S defi ciency ( Figure  3C, 3G, and 3H).

4.1.Changes in cell ultrastructure and leaf lamina tissues
N defi ciency in C. estrellensis causes disorganization of the thylakoid structure and increases starch and lipid granules inside the chloroplasts, which are similar symptoms to those found in cotton (Malavolta et al., 2004), Ceiba speciosa (A. St.-Hil.) Ravenna (Sorreano, 2006), Zea mays L. (Hall et al., 1972), and Hevea sp. (Hamzah and Gómez, 1979). A lack of N forming nitrogenous compounds that combine with carbohydrates to produce amino acids and proteins can prompt the accumulation of starch and lipids (Malavolta et al., 2004).  P defi ciency has also been shown to cause the disorganization of thylakoid structures in the leaf chloroplasts of Z. mays (Hall et al., 1972) and Gossypium hirsutum L. (Zhao et al., 2001), and a decrease in the size of chloroplasts in Ceiba speciosa (Sorreano, 2006). In C. estrellensis leaf laminae, we found that P defi ciency caused a decrease in the number of starch granules inside the cells as well as in the intercellular spaces. P is a structural component of nucleotides, phospholipids, co-enzymes, phosphoproteins, and nucleic acids, and its defi ciency results in a shortage of adenosine triphosphate energy storage molecules that maintain photosynthetic processes (Mengel and Kirkby, 2001;Malavolta, 2006;Taiz and Zeiger, 2017). This may explain the disorganizations observed in the chloroplasts as well as the small number of starch granules and the decrease in intercellular spaces.
Mg defi ciency caused changes in the chloroplasts, with both starch granulation and membrane rupture. Similar observations have been made in Phaseolus vulgaris L. (Thomson and Weier, 1962), Zea mays (Hall et al., 1972), Hevea sp. (Hamzah and Gómez, 1979), Ceiba speciosa (Sorreano, 2006), and Ricinus communis L. cultivar Iris (Lavres Junior et al., 2009). Mg is a key component of the chlorophyll structure, together with N and other elements (Taiz and Zeiger, 2017). Meanwhile, leaf laminae in C. estrellensis with Mg defi ciency showed no changes compared to the control. Other signs of nutrient defi ciency included low cytoplasmic volume under K defi ciency, which has also been observed by Hamzah and Gómez (1979) in Hevea sp. Leaves, and increased intercellular spaces and deep changes in parenchymal cells associated with Ca and S defi ciency. Inadequate K has previously been observed to aff ect cell expansion, promoting tissue compaction, and resulting in a reduction in leaf thickness, chloroplast degradation, and deformation in cell ultrastructures (Dickison, 2000); however, this was not observed in C. estrellensis.
The presence of intercellular spaces and the destructuring of the middle lamella under Ca defi ciency have also been observed in Ceiba speciosa leaves (Sorreano, 2006). This occurs because Ca is a component of pectates that form the cell wall and middle lamella (Taiz and Zeiger, 2017) as well as the plasma membrane (Kirkby and Pilbeam, 1984). Ca is essential for membrane stability (Marinos, 1962) and cell wall rigidity (Christiansen and Foy, 1979). The degeneration of the cytoplasm in the tissue and disintegration of the plasma membrane have been observed in potato sprouts with Ca defi ciency, resulting in an overall reduction in growth (Hecht-Buchholz, 1979).
The formation of intercellular space, caused by the disorganization of cell ultrastructures, has been explained by Marinos (1962), who studied the submicroscopic aspects of Ca defi ciencies in the shoot apex of barley and observed that the fi rst indisputable signs of structural abnormalities appear when the nuclear membrane and plasma and vacuolar membranes are ruptured. The disorganization of other structures such as the Golgi complex and mitochondria also occurs, whereas plastids are more persistent, although eventually they also disintegrate. As Ca defi ciency progresses, darkened spots on the cell walls and gaps may appear, indicating a weakening of their structure and formation of intercellular spaces (Marinos, 1962).
In sections of C. estrellensis leaves with S defi ciency, chloroplasts with thylakoid disorganization and an absence of stromal lamellae were observed. Similar modifi cations have also been observed in S-defi cient Ceiba speciosa leaves (Sorreano, 2006). The disorganization of chloroplasts owing to S defi ciency begins with a decrease in protein synthesis based on S-containing amino acids, which leads to the accumulation of starch granules (Haneklaus et al., 2006;Malavolta and Moraes, 2007). In C. estrellensis leaves, S defi ciency also led to a decrease in intercellular spaces owing to an increase in parenchymal cell size.

4.2Visual symptoms of nutrient defi ciency
N defi ciency is the most likely cause and characteristic sign of uniform chlorosis in dicotyledon leaves (Raij, 1991;Malavolta, 2006;Taiz and Zeiger, 2017). Leaf chlorosis caused by a lack of N has been observed in other forest species, such as Amburana acreana (Ducke) A. C. Smith (Vieira et al., 2011), Croton urucurana Baill (Sorreano et al., 2011), Swietenia macrophylla King (Wallau et al., 2008), Eucalyptus citriodora Hook (Maff eis et al., 2000), Bombacopsis glabra (Pasq.) A. Robyns (Camacho et al., 2014), and Schizolobium amazonicum Herb. (Leite et al., 2017). Uniform chlorosis in N-defi cient plant leaves is associated with decreased chlorophyll synthesis or the decomposition of proteins into simpler compounds (Kramer and Kozlowski, 1960;Fasabi, 1996;Malavolta et al., 1997). When the supply is inadequate, the N of old leaves is mobilized and redistributed to the younger organs and leaves, causing chlorosis in the old leaves. However, in the present study, leaf chlorosis occurred in treatments with N defi ciency, initially in new leaves and then in old leaves.
The collapse of the petiole and chlorosis in old leaves due to Ca defi ciency in C. estrellensis has been observed previously in other species such as Croton urucurana (Sorreano et al., 2011), and Spondias tuberosa Arr. Câm (Gonçalves et al., 2006), and hybrid clones of Eucalyptus grandis W. Hill ex Maiden with Eucalyptus urophylla S.T. Blake (Silveira et al., 2002). Ca defi ciency aff ects the activity of hormones and enzymes, including those that regulate the senescence and abscission of leaves (Marschner, 2012).
Defi ciencies of N, P, K, Ca, Mg, and S are detrimental to the development of C. Estrellensis; therefore, ecological forest restoration projects using these plants in soils that require nutritional supplementation may have their success compromised if there is no nutritional complementation.

5.CONCLUSION
N, P, K, Ca, Mg, and S defi ciencies caused a decrease in their respective content in the leaves, stems, and roots of C. estrellensis. Decreased levels of these nutrients cause malformations in cell ultrastructures, such as changes in the amount and shape of chloroplasts, starch granules, and lipids, which hindered the formation of leaf lamina cells and tissues and, consequently, led to the appearance of characteristic visual symptoms.