EXPRESSION OF A METHIONINE-RICH STORAGE ALBUMIN FROM THE BRAZIL NUT ( Bertholletia excelsa H . B . K . , LECYTHIDACEAE ) IN TRANSGENIC BEAN PLANTS ( Phaseolus vulgaris L . , FABACEAE )

Bean (Phaseolus vulgaris), an important component in the diet of people in developing countries, has low levels of the essential amino acid, methionine. We have attempted to correct this deficiency by introducing a transgene coding for a methioninerich storage albumin from the Brazil nut via biolistic methods. The transgene’s coding sequence was driven by a doubled 35S CaMV promoter and AMV enhancer sequences. The transgene was stable and correctly expressed in homozygous R2 to R5 seeds. In two of the five transgenic lines the methionine content was significantly increased (14 and 23%) over the values found in untransformed plants. Embrapa/Cenargen, S.A.I.N. Parque Rural, Caixa Postal 02372, 70849970 Brasília, DF, Brasil. Send correspondence to F.J.L.A. Fax: +55-61340-3624. E-mail: aragao@cenargen.embrapa.br Centro Brasileiro de Seqüenciamento de Proteínas, Laboratório de Bioquímica e Química de Proteínas, Departamento de Biologia Celular, Universidade de Brasília, 70910-900 Brasília, DF, Brasil.


INTRODUCTION
The advent of recombinant DNA technology combined with plant tissue culture methods has made it possible to introduce foreign genes into host plants.This has created hopes that nutritionally deficient crops could be corrected through the introduction and expression of suitable transgenes.
In Latin America, India and parts of Africa, the bean (Phaseolus vulgaris L., Fabaceae) and broad bean (Vicia faba L., Fabaceae) are among the most important grain legumes used for human consumption.However, although beans are rich in some essential amino acids, e.g., lysine, threonine, valine, isoleucine and leucine, their nutritional value is limited because of the small amounts of the essential amino acid methionine and cysteine (Ma and Bliss, 1978).One strategy to correct this deficiency is to introduce transgenes encoding a methionine-rich storage albumin from the Brazil nut (Bertholletia excelsa H.B.K., Lecythidaceae) into bean hosts.
We have isolated and characterized one of the Brazil nut's 2S-albumin genes (be2s1 gene) (Gander et al., 1991).This gene codes for an 18-kDa precursor protein, which is processed by a complex series of steps to yield two subunits of 3 and 9 kDa, joined by disulfide bridges (Sun et al., 1987).This protein (2S-BN protein) contains 18.8% methionine and is targeted to the seed protein vacuoles (Altenbach et al., 1987).
A methionine-rich sunflower 2S-albumin gene has been used to increase the methionine content of grain and pasture legumes (Tabe et al., 1993).
We have reported the transient expression of the Brazil nut 2S-albumin gene in cells of the bean embryonic axis after transformation by particle bombardment (Aragão et al., 1992).However, a reproducible system for stable transformation and subsequent regeneration of bean plants was not established; thus, no transgenic seeds were obtained.Recently, we have overcome this handicap and transgenic bean plants (P.vulgaris) containing and expressing the 2S-albumin gene were obtained through biolistic processes (Aragão et al., 1996).Here we report the expression of the 2S-albumin gene from the Brazil nut in several lines of transgenic beans.

Plant material
Transgenic bean plants (P.vulgaris) cv Olathe were obtained via particle bombardment of the apical meristematic region of embryos (Aragão et al., 1996).Plants were co-transformed with plasmids pEA23 and pBI426 (circular form).Plasmid pEA23 contains the β-glucuronidase (GUS) coding region (uidA gene) under control of the 35S CaMV promoter and the 2S-albumin gene from the Brazil nut (be2s2) (Gander et al., 1991), driven by the doubled 35S CaMV promoter plus the AMV leader sequence (Datla et al., 1993).Plasmid pBI426 containing the uidA-neo gene fusion (Datla et al., 1991), driven by the doubled 35S CaMV promoter plus an enhancer sequence from AMV, was provided by Dr. William Crosby (Plant Biotechnology Institute, Saskatoon, Canada).The neo gene confers resistance to the antibiotic kanamycin.Four lines (B34-5, E36-6, F40-4 and G41-14) containing the be2s1 and uidA and one line (D35-11) containing only the gus gene were analyzed.The plants were cultivated in a greenhouse at 28 o C and a relative humidity of 70%.The R 1 seeds of 30 plants of each line were planted to obtain R 2 plants.The lines that had uidA and be2s2 genes in all plants were considered homozygous.These plants were self-pollinated until the R 5 generation.

ELISA and Western blot
Seed proteins were extracted from a pooled sample of four mature or immature (15 days after flowering) bean seeds from each line.The seeds were powdered in liquid nitrogen and homogenized in 3 ml of extraction buffer I (10 mM sodium phosphate, pH 7.2, 0.5 M NaCl, 1 mM PMSF and 10 mM 2-mercaptoethanol).The extracts were vigorously shaken at 4ºC for 30 min and centrifuged at 10,000 g for 15 min.The supernatant was recovered and used for ELISA and Western blots.Indirect ELISA was carried out according to Hornbeck (1991).Each well was loaded with 200 µg of total protein.Five wells were analyzed for each plant line.Each line was analyzed three times.The Western blot was conducted as described by Grossi de Sá et al. (1988).Seventy micrograms of total protein for each sample was fractionated using SDS-PAGE on a 12% acrylamide gel and electroblotted onto a nitrocellulose membrane.The 2S-BN protein was identified using an anti-2S-BN polyclonal antibody raised in mice, and rabbit anti-mouse IgG conjugated to alkaline phosphatase and visualized using 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitroblue tetrazolium.Purified 2S-BN protein (500 ng) was used as a positive control.The mouse antibodies utilized were produced in BALB/c mice according to Grossi de Sá et al. (1988), against HPLCand SDS-PAGE-purified 2S-BN protein.

Extraction of seed proteins and amino acid analysis
Three pooled seeds of each bean line were ground in liquid nitrogen to a fine powder.The powder was defatted three times with 25 ml of hexane for 10 min at room temperature with periodic agitation, followed by centrifugation at 6,000 g, for 6 min, at 4ºC.The supernatant was discarded.The defatted material was dried in a Savant Speed Vac Concentrator, and stored at -20ºC.The powder (110 mg) was dissolved in 880 µl of extraction buffer II (0.5 M NaCl in 50 mM phosphate buffer, pH 7.2).The extraction was carried out in a 1.5-ml Eppendorf tube with constant rotation, at 4ºC, for 4 h.The protein extract was clarified through repetitive centrifugation at 9,000 rpm in an Eppendorf centrifuge at room temperature and dialyzed in a microdialyzer system 500 (Pierce, USA) fitted with a 3.5-kDa cutoff dialysis membrane, for 15 h, at 4ºC, against Milli-RO water, at a flow rate of 10 ml/min.The dialyzed sample was dried in the Speed Vac and stored in a freezer.Each sample (0.2 mg) was oxidized with 700 µl of performic acid for 4 h at 0ºC (Moore, 1963).The reaction was stopped with 4 ml of MilliQ water, followed by Speed Vac drying.Each sample was solubilized with 100 µl of 0.1 N HCl, and transferred to a narrow glass tube, which was placed inside a hydrolysis tube in a Pico-Tag workstation (Waters, USA).The sample was dried under vacuum.Gas-phase hydrolysis was performed by adding 200 µl of 6 N HCl at the bottom of the hydrolysis tube.After three cycles of nitrogen and vacuum purge, hydrolysis took place at 150ºC, for 1 h, under vacuum.The hydrolyzed sample was dried, dissolved in 60 µl of 0.1 N HCl, and transferred to a microcentrifuge tube, which was centrifuged at 10,000 rpm for 5 min, at room temperature.Three 10-µl replicas of hydrolyzed supernatant were assayed in a Hitachi L8500 amino acid analyzer.Each line was analyzed three times.The amino acid methionine was determined as methionine sulfone, and cysteine and cystine were determined as cysteic acid.

Statistical testing
The data were analyzed using the program SigmaStat™ (Kuo et al. 1992).The Tukey test was used to make comparisons among the mean amino acid composition (percentage) and optical density in ELISA analysis of salt-soluble protein from transgenic and non-transgenic bean seeds.

RESULTS AND DISCUSSION
The 2S-BN methionine-rich albumin comprises 30% of the total seed protein of the Brazil nut (B.excelsa).The 18-kDa precursor is synthesized on membrane-bound polysomes, transferred through the secretory pathway into the endomembrane system and, presumably, from the Golgi apparatus, into the protein bodies (Shewry et al., 1995).During biosynthesis, at least three stepwise cleavages are involved at the termini and internally.The final protein consists of two polypeptide chains of 9 and 3 kDa, linked by a disulfide bridge (Sun et al., 1987).

2S-albumin expression
Expression of the 2S-BN protein in the cotyledon tissues of transgenic bean plants was determined by Western blot (Figure 1) and ELISA (Figure 2).The Western Expression of a methionine-rich albumin in transgenic beans blot analysis revealed that the 2S-BN protein was correctly expressed in some of the transgenic bean lines (B34-5, E36-6, F40-4 and G41-14), as judged from the presence of the mature 12-kDa protein (Figure 1).No signals were observed in the control D35-11, containing only the uidAneo fusion gene, and in the untransformed control.It was not possible to detect differences in the amount of 2S protein in the transgenic mature seeds.ELISAs were performed on immature and mature seeds (Figure 2).Lines B34-5, D35-11 and F40-4 revealed no significant signal increase when compared to the control plants.Both lines, E36-6 and G41-14, showed 2S-BN protein accumulation in immature and mature seeds, with greater accumulation in immature seeds.During maturation, the 2S-BN protein content in these lines decreased (Figure 2).This suggests that the 2S proteins are either not stored correctly and de-graded prematurely or that the 2S-specific mRNA is less stable in the storage cells of beans than in those of the Brazil nut.The Western blot analyses performed in this study were over-incubated and stained, so it is possible that saturation had an influence, making it impracticable to read these results quantitatively.

Amino acid composition of seeds
The amino acid composition of seeds of five transgenic lines and one control was analyzed (Table I).The methionine content significantly increased in all 2Stransgenic lines (B34-5, E36-6, F40-4 and G41-14) from 10 to 23% over the values of the control.As expected, line D35-11, which contains only the uidA-neo fusion genes, showed no significant increase.
Transgenic canola and tobacco seeds expressing the be2s1 sequence driven by the β-phaseolin promoter had an increase of 11 to 33% in methionine content (Altenbach et al., 1989(Altenbach et al., , 1992)).In V. nabornensis transformed with 2S-albumin-coding sequences under the control of the legumin B4 promoter, a 3% increase in total seed proteins was reported (Saalbach et al., 1995).Brazil nut 2S sequences driven by the analogous 2S promoter from A. thaliana caused a 20% increase in methionine content in transgenic A. thaliana (Conceição et al., 1994).However, whenever 2S-BN sequences were expressed under the control of the 35S CaMV promoter, the expression levels observed were insufficient to alter the methionine content in the target plants, e.g., S. tuberosum (Tu et al., 1994), V. narbonensis (Saalbach et al., 1995) and N. tabacum (Marcellino et al., 1996).
Since the pioneering work of Murai et al. (1983),  a expressing the bean phaseolin gene in sunflower (Helianthus annuus L., Compositae), numerous attempts to express chimeric genes encoding storage proteins such as the 2S-BN protein have been made.With the exceptions cited above, very low levels of trans-protein accumulation have been achieved (De Clercq et al., 1990;Guerche et al., 1990;Saalbach et al., 1994) and in almost no case could observable changes in seed amino acid composition be observed.
In our study, we transformed bean plants with a chimeric construct containing the doubled 35S CaMV promoter plus the AMV enhancer sequence, assuming that its performance would be superior to the native 35S promoter.Indeed, in two of the transgenic lines a 14 and 23% increase of methionine was achieved.
In view of these results, we are now assessing the possibility of achieving higher levels of methionine in transgenic beans by using homologous and seed-specific promoters such as the β-phaseolin promoter.These results will form the foundation for the production of genetically engineered commercial varieties of beans containing high levels of proteins rich in essential amino acids.

ACKNOWLEDGMENTS
We are grateful to Drs.Juan Arrieta and David John Bertioli for their critical reading of the manuscript and to Nuno Manuel Domingues for technical assistance in protein extraction and amino acid analysis.This work was supported by a grant of PADCT/CNPq.

Figure 2 -
Figure2-ELISA for detection of the 2S albumin from the Brazil nut in protein extracts from mature and immature (15 days after flowering) cotyledons from transgenic bean plants.Each well was loaded with 200 µg of total protein and the test was carried out as described in Material and Methods.B34-5, D35-11, E36-6, F40-4 and G41-14: transgenic plants; Ct: non-transgenic; BN: total protein extract from the Brazil nut.Means with different letters are significantly different (P < 0.05) according to Tukey's test.
Mean values in the same columns with different superscripts are significantly different (P < 0.05) according to Tukey's test.