Metabolism of carbohydrates during the development of seeds of the ...

Acta Physiol Plant (2011) 33:211–219 DOI 10.1007/s11738-010-0540-8

ORIGINAL PAPER

Metabolism of carbohydrates during the development of seeds of the brazilian rubber tree [Hevea brasiliensis (Willd. Ex Adr. de Juss) Muell.-Arg.] Lisandro Tomas da Silva Bonome • Suerlani Aparecida Ferreira Moreira Luiz Edson Mota de Oliveira • Anderson de Jesus Sotero

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Received: 16 November 2009 / Revised: 1 May 2010 / Accepted: 24 May 2010 / Published online: 23 June 2010 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2010

Abstract This work aimed at the assessment of the metabolism of carbohydrate during the development of the seeds of Brazilian rubber trees. The enzymatic activity of the acid invertase, neutral invertase and sucrose synthase (SuSy) and the levels of total soluble sugars (TSS), reducing sugars (RS) and sucrose were evaluated separately in each part of the fruit and seed—pericarp, seed coat, embryo and endosperm—on different days after the pollination (DAP). Based on the results obtained in this study, it is possible to conclude that in the beginning of the development of the rubber tree seeds, until 95 DAP, the endosperm presents high concentration of RS and low concentration of sucrose. After this period, the endosperm of the seed initiates starch accumulation and the concentration of RS decreases followed by the increase in the concentration of sucrose, presenting, after 120 DAP, an inversion of concentration of these two sugars. In the

Communicated by S. Weidner. L. T. da S. Bonome (&) S. A. F. Moreira L. E. M. de Oliveira A. de J. Sotero Departamento de Biologia, Universidade Federal de Lavras, Lavras, Minas Gerais State, Brazil e-mail: [email protected] S. A. F. Moreira e-mail: [email protected] L. E. M. de Oliveira e-mail: [email protected] A. de J. Sotero Departamento de Fitopatologia, Universidade Federal de Lavras, Lavras, Minas Gerais State, Brazil e-mail: [email protected]

embryo, the levels of TSS, RS and sucrose show significant increase with the progress of the seed development. In the endosperm, the transition of the division phase and cell expansion for the storage of reserve material seem to occur around 120 DAP and is to be controlled mainly by the enzymes acid invertase and SuSy, while in the embryo, such transition seems to occur around 135 DAP and is to be controlled mainly by the enzymes acid and neutral invertases. Keywords Invertases Sucrose synthase Reducing sugars Sucrose

Introduction Sugars such as sucrose, glucose and fructose present essential role in the plant metabolism. Those sugars— besides serving as substrate for the respiration—may provide carbon source for the production of a wide variety of plant metabolites including amino acids, lipids, proteins, more complex carbohydrates such as cellulose and starch, and a range of other compounds such as chlorophylls, carotenoids and phytohormones. Recently, it has been attributed to sugars, the role of molecular signalling which may interact with different phytohormones and cause alterations in the gene expression (Dekkers et al. 2008; Mishra et al. 2009). The role of metabolites—such as sugars—acting in the molecular signalling of the seed development besides its role as nutrient, has been calling the researcher’s attention. However, this type of research has been carried out mainly with annual species of leguminous plants, not giving much attention to woody and perennial species such as rubber tree.

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The sucrose plays an essential role in the growth and development of the seeds as a primary form of transportation of carbon and energy for most plant species and as a regulator of the gene expression (Kock 2004). Non-photosynthetic sink organs such as seeds in formation are highly dependent on sucrose importation. The hydrolysis of sucrose is the first step for the metabolism and for the synthesis of storage products; thus, the cleavage of sucrose in hexose is vital for plants, not only in the carbon allocation but also for the induction of signals in important structures (Kock 2004). The only enzyme pathways known for the hydrolysis of sucrose in plants are catalysed by invertases and sucrose synthase (SuSy). Both pathways degrade sucrose in vivo, however, one of the by-products of each reaction is different (Tymowska-Lalanne and Kreis 1998; Winter and Huber 2000). The invertases form glucose and fructose, which are phosphorylated posteriorly by several hexo and fructokinases that will require two molecules of ATP. SuSy forms UDP-Glucose and fructose, requiring only one molecule of PPi (Geigenberger 2003). Generally, evidences show that the presence of hexoses favours the division and the expansion of the seed cells while sucrose favours the differentiation and the maturation (Baud et al. 2002; Borisjuk et al. 2002; Borisjuk et al. 2003; Weschke et al. 2003). This information suggests that the invertase/SuSy control is fundamentally important for the transitions of the seed developmental stages (Borisjuk et al. 2002; Borisjuk et al. 2003; Weschke et al. 2003; Wobus et al. 2005). The high activity of acid invertase in the cell wall and the high hexose/sucrose ratio have been found in the early stages of the seed development of several cereals such as maize (Cheng and Chourey 1999), rice (Hirose et al. 2002), and barley (Weschke et al. 2000). According to De´jardin et al. (1997), the transition of the embryonic expansion stage to the storage stage is followed by the decrease of the invertase activity, which remains considerably low in the seed coat as well as in the embryo. In this stage, SuSy increases its activity, which induces the synthesis of the cell wall, starch and other reserve components (Winter and Huber 2000; Weschke et al. 2000). In seeds, results of several researches indicate that the gene expression for the invertases occur predominantly in the first stages while for the SuSy, the expressions occur in the intermediate and final stages of the development. Such change is correlated with the transition from the division and expansion stage to the storage of reserve substances and was understood initially as a response to the regulation by sugar sensor (Borisjuk et al. 1998). Thus, besides presenting the role as a source of energy and storage of carbon source, molecular signalling has been suggested to be an additional role for sugars, where monosaccharides such as glucose and fructose induce mitosis and cell expansion, and sucrose induces the gene

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Acta Physiol Plant (2011) 33:211–219

expression associated to the storage (Heim et al. 1993; Weber et al. 2005) and to the starch accumulation (Weber et al. 1998). The assessment of biochemical changes that occur during the seed development—such as the presence and the level of sugars, the enzymes involved in the cleavage of sucrose, and the accumulation of reserves such as starch— is necessary for a better understanding of the events that rule the formation of seeds of the rubber tree. This work aimed at the assessment of the changes that occur in the metabolism of carbohydrate during the rubber tree seed development.

Materials and methods The experiment was carried in a non-grafted rubber tree, approximately 20 years of age, cultivated in the experimental area of the Plant Physiology Sector of the Biology Department of the Federal University of Lavras (UFLA). The flowering of the tree was followed and the branches marked with labels when the senescence reached more than 50% of the female flowers, which occurs 96 h after their opening. At this moment, it was considered as the fecundation of the ovule by the pollen grain. In each sampling period—38, 65, 95, 120, 155, and 195 days after the pollination (DAP)—ten fruits (30 seeds) were sampled and separated manually in groups of pericarp, seed coat, embryo and endosperm. The sampling on the 38th and 65th DAP collected exceptionally 30 and 20 fruits, respectively. After the separation of the fruit and seed parts, they were frozen at -80°C until the performance of biochemical analyses. The carbohydrate levels were separately rated in each part of the fruit/seed: pericarp, endosperm, embryo and seed coat, for each sample, except for the embryo and seed coat, where the carbohydrate levels were rated from the fourth month of development of the fruit/seed, the levels were rated when its separation from the other parts of the seed was possible. For the carbohydrate quantification, 0.2 g of the material of each part of the seed—ground in mortar in the presence of liquid nitrogen—was centrifuged with 0.1 M potassium phosphate buffer solution, pH 7.0 at 11,000g for 15 min, at 18°C and afterwards, placed in water bath, at 40°C, for 30 min and again centrifuged. This procedure was repeated twice and the supernatant was collected and stored at -20°C for further quantification of reducing sugars and total soluble sugars. The resulting pellets were resuspended in 2 mL of 30% perchloric acid and placed on ice for 40 min. After the conclusion of this procedure, it was centrifuged at 11,000g for 15 min at 18°C, the supernatant being frozen for further starch quantification. Three repetitions were made for each treatment.


The quantification of reducing sugars followed the protocol described by Miller (1959). The method of Yemm and Coccking (1954) was employed for the quantification of total soluble sugars and starch. The concentrations of sucrose were determined by the difference between the level of total soluble sugars and reducing sugars, multiplied by the factor 0.95, according to Martim (2003). The extraction and assessment of the activity of the enzyme SuSy and invertase (and its isoforms) were performed in the same parts of the fruit/seed in which the carbohydrates were quantified. For SuSy, the plant material of each part of the fruit/seed was ground in mortar in the presence of liquid nitrogen, followed by the addition of 0.1 g of this sample to 200 lL of 1 M HEPES–KOH extraction buffer solution, pH 6.0; 100 lL of 0.1 M MgCl2; 100 lL of 0.1 M uridine diphosphate (UDP) and 400 lL of 1.0 M sucrose; the rest being completed with water until totalising 2,000 lL. The assay was kept in water bath at 25°C for 60 min and the reaction was stopped by the transference of the micro tubes to ice. The enzymatic activity was determined by the reducing sugar rates (Miller 1959). For the acid invertase, 0.2 g of the material of each part of the fruit/seed, properly ground, was added to 200 lL of 1 M sodium acetate buffer solution, pH 4.7; 100 lL of 0.1 M MgCl2, and 400 lL of 1 M sucrose, the rest being completed with water until totalizing 2,000 lL. Afterwards, the samples were incubated in water bath, at 37°C for 60 min and the reaction was terminated by transferring the micro tubes to ice. The same procedure was used for the extraction of neutral invertase, but the sodium acetate buffer solution was replaced by 0.1 M phosphate buffer solution at pH 7.0. The activity of the invertases was determined by the rates of the reducing sugars (Miller 1959).

Results and discussion The highest level of starch in the endosperm of the rubber tree seeds was found in the end of the fruit ripening, close to the seed physiological maturation. The starch accumulation in the endosperm occurred gradually along the fruit/ seed development, varying from 2 mg g-1 FW, on the 38th DAP to 8.93 mg g-1 FW on the 195th DAP (Fig. 1). This study corroborates with the observations of Amaral et al. (2001) in seeds of Bixa orellana, who observed small amount of starch granules in the early stages of the seed development. However, from the 5th stage on, the number of starch granules increased and remained high until the end of the development—the 7th stage. The pericarp has shown an opposite behaviour than observed for the endosperm with regard to the level of

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Fig. 1 Estimation of the level of starch in the endosperm of seeds and in the pericarp of rubber tree fruits according to the developmental stage. The bars show the standard deviation of the mean of the three repetitions

starch, where a maximum accumulation on the 65th DAP was noticed followed by its reduction until the end of the development of the fruit/seed (Fig. 1). In several species such as soybean, pea and kidney bean, the sucrose produced through photosynthesis in the leaves and pods may be stored temporarily as starch in the pods before the remobilization and transference to the seeds (Marcos Filho 2005). This process may occur in rubber trees, that is, the sucrose from the leaves and even carbohydrates produced in the fruit (pericarp)—whose colour is green in the early stages of development—are stored as starch in amyloplast present in this involucre. However, in later stages of the fruit/seed development, when the endosperm and the embryo are found to be fully formed, it is possible that the starch stored on the pericarp be cleaved to become sucrose, and posteriorly, transported to the seed under formation, supplying it with carbon source for the synthesis of protein and lipids or even serve as a substrate for the respiration. In the embryo (Fig. 2), it is observed around 120 DAP 2 mg g-1 FW of starch, which has increased to 4.5 and 5.5 mg g-1 FW on the 155th and 195th DAP, respectively. Bhattacharya et al. (2002) have also found higher increase in the starch level in embryos of seeds of Camellia sinensis, in the last seed developmental stages, the accumulation along the embryo maturation being coincidently higher. On the other hand, in the seed coat, the starch level remained low and constant until the end of the fruit/seed development. Figure 3 shows alterations in the levels of total soluble sugars (TSS), reducing sugars (RS) and sucrose (Suc) in the endosperm of the rubber tree seeds during their development. A gradual increase in the TSS is observed during the seed development, reaching up to 60 mg g-1

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Fig. 2 Estimation of the starch level in the embryo and in the seed coat of the rubber tree seed according to the developmental stage. The bars show the standard deviation of the mean of the three repetitions

Fig. 3 Estimation of the levels of total soluble sugars (TSS), reducing sugars (RS) and sucrose (Suc) in the endosperm of the rubber tree seed according to the development. The bars show the standard deviation of the mean of the three repetitions

FW when the mother plant dispersed the seeds. High levels of RS are found until 95 DAP, decreasing gradually from this period until the end of the fruit/seed development. The sucrose level remained stable in the first 3 months of the fruit/seed development, increasing its level abruptly from this period on, reaching a value near 45 mg g-1 FW in the endosperm of the seeds dispersed by the mother plant. This result suggests that the increase of RS in the seed endosperm in the initial months of development is resulting from the sucrose cleavage. Several works have evidenced high concentration of hexoses in the initial stages of the seed development, correlating it with the cell division fostering, while the increase in the sucrose level is normally observed later, and correlated to the seed differentiation and maturation fostering (Borisjuk et al. 2003; Ohto

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Fig. 4 Estimation of the levels of total soluble sugars (TSS), reducing sugars (RS) and sucrose (Suc) in the rubber tree embryo according to the development. The bars show the standard deviation of the mean of the three repetitions

et al. 2005; Rolland et al. 2006; Weschke et al. 2003). The RS and sucrose level inversions plus the development of the seeds were also observed in Vicia spp by Wobus and Weber (1999) and in canola by King et al. (1997). Several authors associate this sugar content change during the seed development with the enzymatic activity changes such as invertases (acid and neutral) and SuSy (Rolland et al. 2006; Weber et al. 1996; Weschke et al. 2000). According to Borisjuk et al. (1998), the gene expression for the invertases occurs predominantly during the first stages, while the gene expressions for SuSy occurs in the final stages of the development. An increasing tendency of the levels of RS, TSS and sucrose is found for the embryo in the progress of the fruit/ seed development (Fig. 4). Moreover, superiority in the concentration of TSS to the concentration of RS and sucrose is observed during the seed development, the sucrose being found in considerably less amount in the embryo. Figure 5 shows that in the seed coat, the levels of TSS and RS decrease along the fruit/seed development. On the other hand, the sucrose level remained low and stable in this involucre until the end of the fruit/seed ripening. The fruit/seed development depends on the photoassimilates from the source tissues. Certainly, the leaves are the main source of photoassimilates, but the green tissue can also contribute substantially in some plants (Zamski 1995). Every photoassimilate from the source tissues with final destination to the seeds arrive initially through the phloem in the seed coat that is a maternal tissue. As the maternal tissue is not connected with the embryo and/or endosperm, the photoassimilate is transported to the filial tissues through a determined apoplastic pathway through extra cellular space.


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Fig. 5 Estimation of the levels of total soluble sugars (TSS), reducing sugars (RS) and sucrose (Suc) in the seed coat of the rubber tree seed according to the development. The bars show the standard deviation of the mean of the three repetitions

Fig. 6 Estimation of the levels of total soluble sugars (TSS), reducing sugars (RS) and sucrose (Suc) in the pericarp of the rubber tree fruit according to the development. The bars show the standard deviation of the mean of the three repetitions

Thus, it is possible that a high RS concentration in the seed coat on the 120th DAP could be due to the sucrose cleavage by an acid invertase that is being taken by the phloem to this tissue. Weber et al. (1995) found high activity of acid invertase in the seed coat of seeds of Vicia faba under development. This fact justifies the low level of sucrose in the seed coat throughout the seed development. On the other hand, the reduction in the RS level in the seed coat, after 120 DAP, suggests the occurrence of sugar transportation to the embryo (Fig. 4) which will be used as a substrate for the respiration as well as carbon source for the production of proteins, lipids, and complex carbohydrates. In the pericarp, the levels of TSS and RS increased up to the 95th DAP, decreasing gradually with the progress of the fruit/seed development (Fig. 6). On the 195th DAP— period in which the seed dispersion occurred—difference among the levels of TSS and RS was not verified. The sucrose concentration increased slightly until the 95th DAP, reducing drastically until reaching a value close to zero at the time of seed dispersion (Fig. 6). Probably, the highest concentration of TSS, RS and sucrose in the fruit pericarp in the first three months of development is due to the highest photosynthetic activity of this involucre in this period. The photosynthetic machinery of the pericarp may have lost its functionality due to fruit ripening and the sugars accumulated in the involucre is translocated to the seed coat, and later directed to the endosperm and to the embryo under formation, which justifies the decline of the levels of such sugars in the seed coat after 95 DAP. The increase in the RS levels of the pericarp, in the early stages of the development, may still be related to the fruit water needs, since RS decreases the tissue-osmotic potential, guaranteeing the water supply (Cavalari 2004; Rogers et al. 1999).

A high increase in the activity of the acid invertase (AI) is verified in the endosperm in the first 95 DAP (Fig. 7). On the 120th DAP, an abrupt decrease in the activity of this enzyme is noticed, reaching, at the end of the fruit/seed development, a value inferior to the one observed on the 38th DAP. On the other hand, the SuSy activity was stable until the 95th DAP, presenting increase from this day until the end of the fruit/seed development. An inversion in the activities of the enzymes AI and SuSy is noticed from the 100th DAP until the seed maturation. Some works have evidenced the high invertase activity and high hexose/sucrose ratio in the early stages of development of the seeds of cereals such as rice (Hirose et al. 2002) and barley (Weschke et al. 2000). Such results corroborate with those observed in the present work (Figs. 3, 7). According to Hill et al. (2003) through the availability of hexoses—from the hydrolysis of sucrose, mainly—the cell division and expansion take place by the action of acid invertase of the cell wall until pressing the endosperm cells. In this stage of the seed development, the SuSy activity increases and the invertase activity decreases and as consequence, the level of hexose decrease and the sucrose becomes the main sugar in the endosperm, inducing change in the gene expression for the accumulation of storage products. This change in the concentration of hexose and sucrose along the progress of seed development was also observed in the endosperm of peas by Borisjuk et al. (1998). In this research, the inversion in the concentration of hexose occurred around the 120th DAP (Fig. 3) suggesting that, in rubber tree, the change in the cell division and cell expansion stage to the storage of reserve substances occur around this time. For the NI (Fig. 7), a little increase in its activity is observed until the 95th DAP; from this day on, a more

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Fig. 7 Activities of the acid invertase (AI), neutral invertase (NI) and sucrose synthase (SuSy) in endosperm of the rubber tree seed according to the development. The bars show the standard deviation of the mean of the three repetitions

Fig. 8 Activities of the acid invertase (AI), neutral invertase (NI) and sucrose synthase (SuSy) in the embryo of the rubber tree seed according to the development. The bars show the standard deviation of the mean of the three repetitions

expressive activity of this enzyme is observed, surpassing the activity of AI at the end of the seed development. In the embryo, the enzymatic activity was assessed from the 120th DAP when a high activity for AI was noticed, which tends to decrease along the progress of the seed/ embryo development (Fig. 8). On the other hand, the NI activity, on the 120th DAP, is relatively low in comparison to the one observed for AI; however, its activity increases during the seed maturation, becoming superior to AI activity on the 155th and the 195th DAP. The high ratio regarding the acid/neutral invertase activity has been evidenced during the maturation of several seeds of leguminous plants (Cooper and Greenshields 1961; Pridham and Walter 1964; Silva et al. 1988).

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Fig. 9 Activities of the acid invertase (AI), neutral invertase (NI) and sucrose synthase (SuSy) in the seed coat of the rubber tree seed according to the development. The bars show the standard deviation of the mean of the three repetitions

These results suggest that on the 120th DAP, the embryo is found in full mitotic activity different from that observed for the endosperm, which during this period, seems to have initiated the stage of reserve material accumulation (Figs. 3, 7). Concerning the embryo, the mitotic activity seems to be prolonged until mid 135 DAP (Fig. 8), period in which an inversion of the activity of the enzymes AI (decrease) and NI (increase) is observed, although the level of reducing sugars has not decreased yet (Fig. 4). The SuSy activity in the embryo was lower than the one observed for AI and NI invertase in the entire time of assessment (Fig. 8). A tendency of decrease in the activity of such enzyme is observed in the seed maturation. Such result differs from the ones observed by Hill et al. (2003), who verified increase in the SuSy activity in the embryo of Brassica napus during the seed development. The possible explanations for the low activity of SuSy in the embryo are its compensation by the high activity of NI, since both enzymes act on the same substrate (sucrose) under the same pH—approximately 7.0—(De´jardin et al. 1997) and its location at the same cell region—the cytoplasm (Taiz and Zeiger 2004). In the seed coat (Fig. 9), a reduction in the activity of the three enzymes assessed during the fruit/seed development was observed, except for AI, which has reduced its activity abruptly between the 120th and 155th DAP and posteriorly kept constant until the end of the fruit/seed maturation. Figure 9 shows that the activity of AI, on the 120th DAP, was nearly twice as intense as the activity of NI and SuSy and that on the 155th DAP, the assessed enzymes presented similar activities, differing again on the 195th DAP with AI surpassing the activities of NI and SuSy.


The high AI activity in the seed coat on the 120th DAP, probably, is related to the formation of the cotyledons. According to Weber et al. (1995), in the seed coat, the invertase activity, specially the activity of acid invertase, whose function is to create an environment with high concentration of monosaccharides which are directed to the cotyledons through transfer cells (Borisjuk et al. 2003) is high; therefore, inducing the mitotic activity in the tissue. Nevertheless, as the cotyledons end their development, the activity of the invertases as well as the activity of SuSy decline (Fig. 9), possibly because the seed coat looses its functions—receptor and photoassimilate transferrer—and becomes more lignified, related to the seed protection (Boesewinkel and Bouman 1995). Based on the results presented in the Fig. 10, a high activity of AI followed by NI and SuSy is observed on the 38th DAP. Probably, the high AI activity in the pericarp of the seed in the beginning of the development is associated to a higher mitotic activity in this period. Numerous works associate the high AI activity to the mitotic activity (Weber et al. 1996; Weschke et al. 2000). In the period comprehended between the 65th and 120th DAP, the decline tendency in the activities of acid and neutral invertases is verified, being both enzymatic activities being similar. On the 155th and 195th DAP, the NI activity remained under decrease; however, the AI activity remained stable. With regard to SuSy, an abrupt increase in its activity is verified on the 65th DAP, decreasing posteriorly until the 120th DAP and remaining stable from this time until the end of the fruit development. The high SuSy activity in the pericarp, in the beginning of the fruit development, may justify the high concentration of sucrose in this involucre in the same period (Fig. 6), since this enzyme can hydrolyse as well as synthesise sucrose. Cavalari (2004) also

Fig. 10 Activities of the acid invertase (AI), neutral invertase (NI) and sucrose synthase (SuSy) in the pericarp of the rubber three fruit according to the development. The bars show the standard deviation of the mean of the three repetitions

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associated the highest activity of SuSy in the seed pericarp of coffee under development, in the synthesis of sucrose. However, different from the observations in this study, the increase of SuSy and sucrose in the pericarp of the coffee seeds occurred in the end of the fruit ripening and not in the beginning. In summary, it is possible to conclude that the starch accumulation in the endosperm and in the embryo occurs gradually with the progress of seed development, reaching the maximum values close to the physiologic maturity on the 195th DAP. On the other hand, in the pericarp, the highest accumulation level occurs on the 65th DAP, the period in which such cover is green, therefore, photosynthetically active. From this period forth, the pericarp starch level decreases gradually with the fruit/seed ripening probably due to its use as energy source and carbon skeleton to form the embryo and the endosperm of the seeds. The AI activity increases in the seed endosperm on the first 95 DAP coinciding with the presence of RS, while the SuSy activity remains unchanged coinciding with the low level of sucrose. Around 100 DAP, the enzymatic activity inversion takes place, where the activity of SuSy surpasses the activity of AI. The change in the activity of such enzymes leads to change in sugar content in the endosperm, promoting an inversion in the sugar concentrations, where the level of sucrose surpasses the level of RS around the 120th DAP. In the embryo, the AI activity decreases with the progress of the seed development, whereas, the NI activity increases, surpassing the acid activity around 150 DAP. The SuSy activity remains low during the entire embryonic development. It is likely that the low SuSy activity in the embryo during the whole development of the rubber tree seed has been compensated by the high NI activity, since both enzymes act upon the same substrate, the same pH, and are located at the same cell region. Such results suggest that in the endosperm, the transition of the division phase and cell expansion for storing the reserve material occur around 120 DAP and is controlled, mainly, by the enzymes acid invertase and SuSy, while in the embryo, such transition seems to occur around 135 DAP and to be controlled, mainly, by the enzymes acid and neutral invertases. The seed coat shows reductions in the activities of all assessed enzymes as the development of the rubber tree seed progresses. Such enzymatic reductions are accompanied by decreases in the levels of TSS and RS, whereas the level of sucrose remains unchanged with the seed ripening. The AI activity in the pericarp on the 38th DAP is high when compared to the other enzymes assessed, indicating high mitotic activity during this period. The levels of TSS, RS and sucrose increased in the pericarp until the 95th DAP probably in function to the photosynthetic activity of this covering during this period. From this day forth, the

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sugar levels start to decrease until the end of the fruit/seed development. Such decrease in the sugar levels in the pericarp after 95 DAP may be attributed to the functionality loss of the photosynthetic machinery of the pericarp by the fruit/seed ripening associated to the translocation of the sugars which functions to form the embryo and the endosperm. Acknowledgments This research was funded by the Minas Gerais State Foundation for Research Development (FAPEMIG) and National Council for Scientific and Technological Development (CNPq).

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