BORON AFFECTS THE GROWTH AND ULTRASTRUCTURE OF CASTOR BEAN PLANTS

The cultivation of oleaginous plants like the castor bean guarantees employment for agricultural families and can contribute in energy and chemical sectors, especially in the northeastern semi-arid regions of Brazil. Boron (B) deficiency is a widespread nutritional disorder despite the fact that various anthropogenic sources with high B content may increase soil B to toxic levels for plants. The present study was designed to investigate the ultrastructural effects of boron deficiency and toxicity on castor bean plants which were grown under greenhouse condition using plastic containers with 10 L of nutrient solution. Boron treatments comprised: control (no B); 0.27 mg L, 5.40 mg L B pots (one plant per pot), tested in a completely randomized design with three replicates. The dry matter of all plant parts and B concentration were determined. Cellular ultrastructure was evaluated by transmission and scanning electron microscopy on samples of leaves and petioles. Dry matter yield was affected by the B absence treatment but there was no difference for the 5.4 mg L B (toxic conditions) treatment. A marginal leaf burn at edge and tips of oldest leaves and absence of starch granules in chloroplasts were noted for the B toxicity treatment. The deformation of the youngest leaves, the death of the apical meristem as well as the swelling of the middle lamella, absence of starch granules in chloroplasts and petiole vessels untidily were observed in the B absent treatment. It is concluded that the production and development of castor bean plants is affected by boron deficiency, but not for boron toxicity conditions.


INTRODUCTION
The castor bean plant (Ricinus communis L.) is an African euphorbiaceae (Joly, 2002), whose oil is the main product of the crop, supplying the medicinal, cosmetic and automotive industries.Nowadays, with increasing prices of the crude oil, the castor bean cultivation emerges as a promising activity for biodiesel production, providing income in resource poor areas of Brazil (Savy Filho, 2005).
Boron (B) deficiency occurs in a large frequency in agricultural areas,,inducing to an excessive application of this nutrient (Gupta, 1979;Blevins & Lukaszewsky, 1998;Shorrocks, 1997).Davies & Albrigo (1994), Mattos Júnior et al. (2001), and Havlin et al. (2005) have underlined the short interval that exists between the B deficiency and toxicity.The deficiency of micronutrients in Ricinus communis was described by Lange et al. (2005).However, the ultrastructure changes under B deficiency and the boron toxicity in castor bean plants are not yet known.
The determination of the B primary function is one of the current challenges related to the mineral nutrition of plants (Blevins & Lukaszewsky, 1998).The primary function of B in the cell wall structure could explain all effects related to boron deficiency (Brown & Hu, 1997).
The swelling of the cell walls under boron deficiency and its relationship with the borate-ester crosslinked rhamnogalacturonan II dimer (RG-II) was described by Ishii et al. (2001).The presence of RG-II in cell walls in families of Brassicaceae, Cucurbitaceae, Leguminosae, Apiaceae, Chenopodiaceae, Solanaceae, Asteraceae, Liliaceae, Araceae, Amaryllidaceae and Gramineae was described by Matoh et al. (1996).Thus, there is a strong evidence that suggests the existence of a RGII complex also in Ricinus communis, however, not yet been found in this species or another one from the euphorbiaceae family.
The objective of this study was to investigate ultrastructural changes under B deficiency and toxicity conditions in Ricinus communis L. as well as the effects on castor bean production.
The experiment was set up with three treatments (0; 0.27; 5.40 mg L -1 of boron) and three replicates,in a randomized experimental design.Castor beans plants (one plant per pot) were transplanted ten days after emergence to plastic pots with 10 L of nutrient solution with 1/5 of salt concentration.After a week, the solutions were replaced by 1/2, and in the following week they were replaced by complete solutions (conductivity of 1.6 mS).The solutions were replaced every three weeks.
Plants were collected 60 days after germination, and samples were visualized using a transmission and scanning electron microscope.

Transmission Electron Microscopy
Samples from the blades of new leaves that showed deficiency of boron (0 mg L -1 of B) and new leaves of the control treatment (0.27 mg L -1 of B) were collected to evaluate ultrastructural symptoms of the boron deficiency.The effects of toxicity were investigated on samples of old leaf blades presenting marginal chlorosis (5.4 mg L -1 of B) and of old leaf blades from the control treatment (0.27 mg L -1 of B).
The ultra thin cuts (60-90 nm) were obtained through the use of a diamond blade in a Porter-Blum MT Ultracut, placing on 300 mesh copper nets, and submitted to double coloration (Reynolds, 1963), using uranyl acetate and lead citrate solutions.These ultra thin cuts were examined using the Transmission Electron Microscope Zeiss EM-109 operating at 50 kV.

Scanning Electron Microscopy
The petiole tissues of new leaves from boron absent (0 mg L -1 of B) and control treatments (0.27 mg L -1 of B) were used to evaluate the symptoms of boron deficiency.There was not difference between the petiole morphology of plants treated with the boron toxicity and the control, and therefore, these samples were not analyzed.These samples were pro-Sci.Agric.(Piracicaba, Braz.), v.65, n.6, p.659-664, November/December 2008 cessed using 2% glutaraldehyde, in a 0.2M caccodilate buffer.After 2 h at 4 o C, these samples were washed in 0.1M caccodilate buffer and dehydrated with sequences of acetone series (25%, 50%, 75%, 90% and 100%).They were then dried to critical point (Balzers CPD030), and covered with gold (MED010-Balzers).The coated specimens were examined in a scanning electron microscope operating at 20 kV.

Determination of total B content of specimens
Samples weighed exactly 0.2 g after being dried for 24 h at 105°C, and were put into ceramic crucibles and ashed for 2 h at 550°C.The total boron content was determined by the azomethine-H method (Malavolta et al., 1997).

Statistical Analysis
Statistical analyses were made using Sigmaplot 2000 v.6 and SAS version 8.02 (SAS Institute, Cary, NC).The Tukey test was used to compare the means of the treatments (p < 0.05).

Boron contents in plant pots and dry matter production
The mean B content (in dry mass) in new leaves was 12 mg kg -1 for the B absent treatment and 28 mg kg -1 for the treatment of 0.27 mg L -1 B. The B content in old leaves was 40 mg kg -1 for the treatment of 0.27 mg L -1 B and 450 mg kg -1 for the treatment of 5.4 mg L -1 B. The B content (dry mass) in petioles of new leaves was 11 mg kg -1 for the absent treatment and 20 mg kg -1 for the treatment of 5.4 mg L -1 B.
The mean of dry matter weight per plant of the B deficiency treatment plants was smaller than for the treatments of 0.27 mg L -1 B and 5.4 mg L -1 B (Figure 1).The seed production of plants was strongly affected by B absence.However, there were no differences in seed production, as well as dry matter weight, between the 0.27 mg L -1 B and 5.4 mg L -1 B.

Boron deficiency and toxicity symptoms
The first symptoms of toxicity appeared for the treatment of 5.4 mg L -1 of B, on the 15 th day after transplanting.The observed symptom included chlorosis on the edges of the leaves and spots.These toxicity symptoms were evident (5.4 mg L -1 of B), although there was no significant effect on growth, fruit and seed production.This fact indicates that B does not move readily from the old leaves to growing tissues (phloem mobility) Boron generally moves through the xylem, governed by the transpiration flow, with a tendency to bind on cell wall pectin in leaves.For old leaves, the amount of transpiration along time is large, explaining the high B contents (Furlani, 2004).However, the high supply of B is not correlated with an increase of total B content in the cell walls (Matoh & Kobayashi, 2002).Besides, this micronutrient forms various biological compounds also in the cytoplasm, such as complexes of B (boric acid) with sugars, phenols, organic and polymeric acids (Dembitsky et al., 2002).
Boron deficiency symptoms appeared first in new leaves at the 40 th day after transplanting, such as deformity and necrosis of leaf edges (Figure 2).Furthermore, minus-boron petioles showed hyperplasia and necrotic spots.This can be explained by an interference in the lignin synthesis under low B supply (Marschner, 1995).These deficiency symptoms indicate that the B phloem mobility in this species is probably low or restricted.In contrast, the mobility of B in several sorbitol, mannitol, and dulcitol rich species has been verified (Brown & Hu, 1996;Hu et al., 1997).
The reproductive growth, especially flowering and seed set and seed yield, were more sensitive to B deficiency than the vegetative growth.Stem and leaf biomass were also negatively affected by the absence of B (Figure 1).Necrosis of the apical meristem of shoot tips was followed by a loss of apical dominance and highly branched shoot architecture (Figure 2).
The boron deficiency interferes in IAA levels in apical tissues, phenols and quinones levels, followed by a loss of apical dominance (Coke & Whittington, 1968).This effect was detected in an experiment with micronutrient starvation in nutrient solution in castor bean by Lange et al. (2005).However, it is possible that most effects of boron deficiency on the physiologic processes are secondary effects (Marschner, 1995).

Ultrastructural evidences of boron deficiency and toxicity
The major symptom of boron deficiency was the thickening of the middle lamellae (Figure 3).This fairly and rigid layer is a structural component, located between two adjacent primary cell walls, composed of pectin.The thickening of the middle lamella could be explained by the structural role of boron in relation to the polysaccharide present in the pectin, especially the formation of dimeric B-Rhamnogalacturonan-2 (B-RG-II) in a borate-ester crosslinking (Kobayashi et al., 1996;Ishii & Matsunaga, 1996;O'Neill et al., 1996).This crosslink forms a macromolecular complex that controls the cellular growth (Fleischer et al., 1999) and mechanical properties of primary cell walls (Ishii et al., 2001).Probably, on the B-deficient medium, the B-RG-II formation was affected, with the increase of monomers, thickening the middle lamella and affecting the cellular growth (Figure 3).
Furthermore, besides playing a role in the function and stabilization of cell walls in plants (O'Neill et al., 1996), there is also considerable information that connects B with membrane structure and function (Pollard et al., 1977), as well as cellular homeostasis, suggesting that the B exerts some functions in the cytoplasm (Gassert et al., 2002).
A low level of starch granules was observed in chloroplast of plants under B absence (Figure 3).
In B deficient plants, the carbohydrates synthesis is affected on account of the inhibition of the fosforilases action or the reduction in the uracyl synthesis, pre-  The petioles of new leaves grown under B deficient medium were thicker, irregular with necrotic spots.In a cross-sectional view, the petioles showed hyperplasia, and when observed by scanning electronic microscopy, the xylem vessels edges were more irregular (Figure 5).There was not difference between the petioles of plants grown in high B supply (5.40 mg L -1 B treatment) and control (0.27 mg L -1 of B treatment).
The irregular lignifications of the cell walls and a sensitive reduction in total lignin content as pointed out by Marschner (1995) and the effects of boron on growth and lignification in sunflower plants is due to peroxidase enzyme contents (Dutta & McIlrath, 1964).In addition, the role of boron in the lignin synthesis can be related to the formation of borate complexes with phenols, regulating the rate of free phenols that are precursors of the lignin synthesis (Lewis, 1980;Pilbeam & Kirkby, 1983;Shkolnik, 1984).Although the lignification process is associated with the secondary cell wall, it generally begins in the middle lamella and primary cell wall, sites where the boron deficiency seems to occur initially.

CONCLUSIONS
Dry matter and seed yield are negatively affected under boron deficient conditions.However, the high boron supply, twenty times more than a usual nutrient solution, was not able to decrease the development of Ricinus communis.The boron deficiency causes the swelling of middle lamellae and irregular growth of petiole vessels.Both deficiency and toxicity affects the starch synthesis in chloroplasts.
The B toxicity symptoms appeared before those of B deficiency, but were less harmful to the whole plant growth.The absence of starch granules in chloroplasts in plants grown in the 5.4 mg L -1 B treatment occurred also in all specimens (Figure 4).However, the exact cause of this effect is not known, and the same alteration in the carbohydrate metabolism due to B toxicity was verified by Scott (1960) for sunflower plants.
The starch biosynthesis occurs inside the chloroplasts and amyloplasts where the enzymes that catalyze the polymerics synthesis are located, using as basic material the sucrose produced from photosynthesis (Galliard & Bowler, 1987).Thus, a reduction in the available sucrose for metabolic processes could lead a decrease on the starch production (Zrenner et al., 1995).Most of the B is present in the apoplast (Matoh, 1997), but it is possible that B can also be present in the cytoplasm under high B supply.
It is supposed that B excess could affect indirectly the formation of starch because of the properties of the boric acid to form complexes with a large number of sugars.Although B does not form complexes with sucrose (Marschner, 1995), this micronutrient can form complexes with other sugars, phenols, organic and polymeric acids (Dembitsky et al., 2002).

Figure 1 -
Figure 1 -The effect of B treatments on dry matter yield of castor bean plant.Bar graphs of specific parts with the same letter are not different (Tukey test, p < 0.05).

Figure 3 -
Figure 3 -Spongy mesophyll of new leaves of Ricinus communis L. A: Boron absent treatment B: Boron treatment with 0.27 mg L -1 .The thickening of the middle lamellae (ML), and starch granules absence (ST) in chloroplasts (CL) in boron absent treatment.Scale bar = 1.7 µm.

Figure 4 -
Figure 4 -Spongy mesophyll of old leaves of Ricinus communis L. C: Boron toxicity treatment with 5.4 mg L -1 D: Boron treatment with 0.27 mg L -1 .The absence of starch granules in B toxicity.Scale bar = 1.7 µm.

Figure 5 -
Figure 5 -Section of petiole (new leaf).E, G: Boron treatment of 0.27 mg L -1 ; F, H: Boron absent treatment.Primary symptoms of boron deficiency were thickened petioles with necrosis.Scale bar (E, F) = 0.6 mm.Details of irregular edges part of vessels in H. Scale bar (G, H) = 250 µm.