A. Chaparro-Giraldo 7 R.M. Barata 7 S.M. Chabregas. R.A. Azevedo 7 M.C. Silva-Filho (Ð). Departamento de Genética, Escola Superior de Agricultura. 'Luiz de ...
Plant Cell Reports (2000) 19 : 961–965
Q Springer-Verlag 2000
CELL BIOLOGY AND MORPHOGENESIS A. Chaparro-Giraldo 7 R.M. Barata 7 S.M. Chabregas R.A. Azevedo 7 M. C. Silva-Filho
Soybean leghemoglobin targeted to potato chloroplasts influences growth and development of transgenic plants
Received: 20 December 1999 / Revision received: 28 May 2000 / Accepted: 16 June 2000
Abstract Potato tubers were transformed with a chimeric gene made by the fusion of the soybean leghemoglobin encoding gene (lba) with the chloroplastic targeting sequence from Rubisco. This construct was placed under the control of the strong constitutive 35S promoter and the 3b nontranslated region of Rubisco from pea. Leghemoglobin expression on kanamycinresistant plants was monitored by RT-PCR. Furthermore, immunodetection of subcellular fractions of transgenic plants revealed that leghemoglobin was imported and correctively processed inside the organelle. In addition, analysis of transgenic plants revealed reduced growth and decreased tuber production compared with the untransformed plants. It is suggested that leghemoglobin expression in potato chloroplasts interferes with aerobic metabolism, leading to physiological and morphological changes. Key words Leghemoglobin 7 Potato 7 Aerobic metabolism Abbreviations CAT Catalase 7 GR Glutathione reductase 7 Hbs Plant hemoglobins 7 Lb Soybean leghemoglobin 7 VHb Vitreoscilla hemoglobin 7 PCR Polymerase chain reaction 7 rbcS Small subunit
Communicated by S. Gleddie A. Chaparro-Giraldo 7 R.M. Barata 7 S.M. Chabregas R.A. Azevedo 7 M.C. Silva-Filho (Y) Departamento de Genética, Escola Superior de Agricultura ‘Luiz de Queiroz’, Universidade de São Paulo, Av. Pádua Dias,11, Caixa Postal 83, 13400–970 Piracicaba, SP, Brazil e-mail: mdcsilva6carpa.ciagri.usp.br Fax: c55-19-4336706 Permanent address: A. Chaparro-Giraldo Departamento de Biologia, Facultad de Ciencias, Universidad Nacional de Colombia, AA 1440, Santafé de Bogotá, Colombia
precursor of the ribulose 1,5-biphosphate carboxylase/oxygenase (Rubisco) from pea 7 RT-PCR Reverse transcriptase-polymerase chain reaction 7 SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Introduction Oxygen acts as a cofactor or substrate in many biochemical processes in both primary and secondary plant metabolism. One possible way to increase oxygen availability inside the cell, in order to stimulate oxygendependent reactions, is through oxygen binding proteins which increase the intracellular oxygen concentration (Bülow et al. 1999). Plant hemoglobins (Hbs) are widespread in the plant kingdom, and generally display a high affinity to oxygen. Hbs may be classified into two broad groups: symbiotic and nonsymbiotic types (Arredondo-Peter et al. 1998). Symbiotic Hbs have been extensively characterized in legume plants and are involved with the facilitation of oxygen diffusion within infected tissues (Appleby 1992). One of the most widely studied and best characterized class of symbiotic Hbs is the soybean leghemoglobin (Lb), which represents a small family of closely related genes (Brisson and Verma 1982). Soybean Lb accumulates in high levels in infected tissue and has a high avidity for oxygen and a relatively fast oxygen-dissociation rate which permits a low cellular concentration of free oxygen (Arredondo-Peter et al. 1998). On the other hand, the function of nonsymbiotic Hbs is still a matter of controversy (Appleby et al. 1988; Anderson et al. 1996; Hill 1998). Three possible physiological roles have been proposed for nonsymbiotic Hbs: as an oxygen carrier, a sensor for oxygen (Appleby et al. 1988) and acting in the glycolytic metabolism in stressed tissues (Sowa et al. 1998). It has recently been shown that a Vitreoscilla hemoglobin (VHb), when expressed in tobacco plants, promoted enhanced growth and changes in metabolite
962
production (Holmberg et al. 1997). However, contrary to these findings, targeting of the soybean Lb to tobacco chloroplasts did not produce any significant alteration to either primary or secondary metabolism in transgenic plants (Barata et al. 2000). In order to verify whether the presence of the symbiotic leghemoglobin inside the chloroplasts of another C3 plant would interfere with aerobic metabolism, we introduced the soybean Lb into potato chloroplasts. The effects of Lb on plant growth, development, tuberization, and enzyme activities involved in oxidative stress have been determined.
Materials and methods Plant transformation Transformation experiments were performed with potato tuber disc cv. Bintje as previously described (Dale and Hampson 1995). Regenerated plants were selected for kanamycin resistance. The gene construct contains the lba gene fused to the transit peptide of the small subunit of Rubisco (rbcS) from pea. The chimeric gene was placed under the control of the 35S promoter, as previously described (Barata et al. 2000).
Plant material and growth conditions Transgenic plants grown in vitro for 4 weeks were transferred to the greenhouse, placed in a commercial substrate and sprayed with a commercial fertilizer at least twice a week. Plants were grown under a photoperiod of 14/10 h day/night and a temperature of 15/28 7C. For metabolite extraction, leaves (third from the apex) from control and three independent transformant plants (B1, B2 and B3) were harvested and immediately placed in liquid nitrogen and stored at P80 7C for further analysis.
Catalase and glutathione reductase activities Enzyme extraction was carried out as described previously by Azevedo et al. (1998). Catalase and glutathione reductase activities were determined as described by Azevedo et al. (1998). Protein quantification Protein concentration was determined spectrophotometrically at 595 nm as described by Bradford (1976) with bovine serum albumin as a standard. Fractionation of potato cells and protein analysis Subcellular fractions were obtained from 10 g of leaves as described previously (Silva-Filho et al. 1997) except that homogenization was performed in 100 ml of homogenization buffer and that 0.2% (w/v) insoluble polyvinylpyrrolidone was added to the buffer. Purification of chloroplast and thylakoids on a continuous percoll gradient was performed according to Silva-Filho et al. (1996). Western blot analysis After SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), proteins were transferred to a nitrocellulose membrane and inmmunodetected with anti-goat leghemoglobin antibodies (kindly provided by Gautam Sarath, University of Nebraska, USA) raised against purified leghemoglobin (1/1000), followed by an anti-mice IgG alkaline phosphatase conjugate (Sigma). Statistical analysis Overall data of sucrose and starch contents, catalase and glutathione reductase activities, and tuber and shoot fresh weights were analyzed using a variance (ANOVA) analysis for completely randomized design. When a difference was found between treatments, this was then tested at the P~0.05 level using the Duncan-test
DNA extraction and PCR analysis DNA was extracted according to Edwards et al. (1991). PCR amplification was carried out with the following primers: upstream (Leg 1 : 5b-CCCGAATTCAGAAATATGGTTGC-3b) and dowstream (Leg 2 : 5b-CCCGGATCCTACTAATGCC-3b) primers.
RT-PCR RNA extraction was carried out with Trizol (Gibco BRL) and first-strand cDNAs were synthesized from 2.5–20 mg of total DNA-free RNA, using Superscript II RNAse H-reverse transcriptase, following the manufacturer’s instructions (Gibco BRL). cDNAs were amplified using specific primers (Leg 1 and Leg 2) in the PCR reaction.
Metabolites measurements Sucrose contents were extracted from leaf discs according to the method of Leegood and Furbank (1986) and determined as described previously (Lowry and Passoneau 1972). For estimations of starch, the samples were assayed essentially as described by Jones et al. (1977).
Results and discussion rbcS-leghemoglobin protein fusion import into potato chloroplasts rbcS-leghemoglobin fusion protein contained the entire rbcS transit peptide, the cleavage site and four amino acids from mature rbcS, followed by the soybean leghemoglobin (Lb; Barata et al. 2000). The chimeric gene expression was driven by the strong cauliflower mosaic virus (CaMV) 35S promoter. The construct was introduced into potato, and three out of the seven independent transgenic plants obtained were selected for further characterization. Lb expression of kanamycinresistant plants was monitored by RT-PCR (Fig. 1A), and western blotting analysis on a chloroplast-enriched fraction showed that Lb was targeted and correctively processed inside the organelle (Fig. 1B). The size of Lb observed in the chloroplast fraction of transgenic plants was shown to be larger than that of the soybean Lb control. This difference can be accounted for by the
963
Fig. 1 A RT-PCR in transgenic potato plants. Lane 1 Molecular weight marker; lane 2 wild type potato; lanes 3–5 transgenic potato; lane 6 positive control (lba gene). B Immunodetection of Lb in subcellular fractions of transgenic potato plants. Western blot analysis was carried out on 20 mg proteins of the chloroplastenriched fraction. A soybean leghemoglobin protein extract (0.1 mg) was used as a control (lane 1). Lanes 2–4 Transgenic potato; lane 5 wild type potato
presence of four amino acid residues of mature Rubisco and one residue from the linker region (Barata et al. 2000). The rbcS transit peptide generally possesses sufficient information for the correct targeting of proteins in vivo to the chloroplast stroma (Van Der Broeck et al. 1985; Boutry et al. 1987; Barata et al. 2000). In agreement with this is the fact that Lb was not found in a mitochondrial fraction of the transgenic potato plants (not shown). However, one exception was observed with the transport of the 5-enolpyruvyl 3phosphoshikimate synthase to chloroplasts, in which additional residues were required for efficient chloroplast delivery (Comai et al. 1988).The presence of an unprocessed form of Lb in cytoplasmic fractions was not detected mainly because organellar proteases were released during leaf homogenization, as previously reported (Silva-Filho et al. 1997). Catalase and glutathione reductase activities in transgenic plants Increasing evidence suggests a relationship between plant and animal hemoglobins and enzymes involved on oxidative stress protection inside soybean nodules (Puppo and Halliwell 1988; Becana and Klucas 1990) and in animal cells, respectively (D’Agnillo and Chang 1998). Lb autoxidation or reaction with superoxide anions makes the ferric form of this protein unable to carry oxygen (Puppo et al. 1982). Therefore, the presence of active superoxide dismutase (SOD) isoenzymes would be fundamental for the scavenging of the superoxide anion, avoiding Lb oxidation. In addition to SOD, catalase (CAT) and glutathione reductase (GR) also prevent Lb inactivation by scavenging hydrogen peroxide as soon as it is formed. Thus, enzymes
involved in oxidative stress protection appear to be related to the Lb oxido-reduction changes. Moreover, pseudo-peroxidatic activity has been proposed for Lb based on the ability of the heme group to decompose H2O2 to H2O in the presence of artificial electron donors (Sievers and Rönnberg 1978). In order to determine the effects of the presence of Lb inside the potato chloroplast on the activity of the oxidative stress enzymes, CAT and GR activities were assayed from leaf samples of control and transformed plants. Both CAT and SOD enzyme activities were not affected by the presence of the Lb (data not shown). A similar observation has been recently reported in an attempt to reduce photorespiration by the presence of Lb inside tobacco chloroplasts (Barata et al. 2000). One possible explanation could be related to the low Lb expression level, which was similar to the one observed for the tobacco plants (Barata et al. 2000), but not to Lb functionality. In agreement with this, expression of functional human hemoglobin in tobacco plastids has been reported despite its complex tetrameric structure (Dyerick et al. 1997). Expression of Lb negatively affects potato growth and development Expression of a bacterial hemoglobin (VHb) in transgenic tobacco improved overall growth and reduced seed germination and flowering period (Holmberg et al. 1997). Surprisingly, targeting of Lb into chloroplasts promoted pleiotropic effects such as: reduced growth (Fig. 2), a 3.5-fold tuber weight reduction (Fig 3A), a threefold decrease in shoot fresh weight (Fig. 3B) and increased pigmentation (data not shown), in comparison to the control plants. However, no significant difference was observed in tuber number (data not shown). These observations are similar to those seen in water stressed plants (Deblonde et al. 1999). Since water stress appears to be correlated with an altered carbohydrate content (Geingenberger et al. 1999), we decided to verify whether such physiological alterations could be related to carbon metabolism. Leaf samples from control (WT) and three independent transformants were harvested and assayed for their sucrose and starch contents. The results indicated that sucrose and starch levels were not affected when compared to the control plants (data not shown), suggesting that carbon metabolism is not affected in the transgenic potato plants. It has been suggested that hemoglobin would increase the availability of oxygen or energy in the cell by releasing oxygen rapidly (Bülow et al. 1999). In addition, hemoglobins could also scavenge reactive oxygen species, consequently reducing the oxidative stress and contributing to enhanced growth of transgenic tobacco plants (Holmberg et al. 1997). Apparently, this appears not to be the case in potato plants expressing Lb inside the chloroplasts.
964 Fig. 2 The growth of wild type (far left) and three independent transgenic plants b1, b2, b3, (second left to right), demonstrates the obvious difference in growth after 40 days
What could account for the differences between the present work and data observed by Holmberg et al (1997) and Barata et al. (2000)? In the first case, a possible explanation would be the different oxygen
binding affinity between soybean Lb and Vitreoscilla hemoglobin. While soybean Lb has a high affinity to oxygen (KDp47 nM), VHb has a relatively low affinity, with a KD of 6 mM (Bülow et al. 1999). In addition, the high dissociation constant of VHb may be responsible for a rapid release of oxygen inside the cell compared with Lb. The mechanism by which the soybean Lb interferes with potato physiology is still unknown. On the other hand, no pleiotropic effects were observed when soybean Lb was targeted to tobacco chloroplasts (Barata et al. 2000). This suggests that plants respond differently to the heterologous expression of hemoglobins, which might also result in uncharacterized interactions with the host plant. Acknowledgements We thank Dr. Gautam Sarath (University of Nebraska) for the leghemoglobin antibody. This work was supported by a grant from the Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (94/03561-0) and CNPq. A.C.G. was recipient of graduate fellowships from CNPq and Colciências. R.M.B. and S.M.C. were supported by graduate fellowships from FAPESP and CNPq respectively. M.C.S.F. and R.A.A. are research fellows of CNPq.
References
Fig. 3 Total average weight of tuber (A) and shoots (B) of wild type and three independent transgenic plants. Data represent mean values from 12 plants of each line BSE
Anderson CR, Jensen EO, Llewellyn DJ, Dennis ES, Peacock WJ. (1996) A new hemoglobin gene from soybean: a role for hemoglobin in all plants. Proc Natl Acad Sci USA 93 : 5682–5687 Appleby CA (1992) The origin and function of haemoglobin in plants. Sci Prog 76 : 365–392 Appleby CA, Boguzs V, Dennis ES, Peacock WJ. (1988) A role for hemoglobin in all plant roots? Plant Cell Environ 11 : 359–367 Arredondo-Peter R, Hargrove MS, Moran JF, Sarath G, Klucas R (1998) Plant hemoglobins. Plant Physiol 118 : 1121–1125
965 Azevedo RA, Alas RM, Smith RJ, Lea PJ (1998) Response of antioxidant enzymes to ozone fumigation in the leaves and roots of wild-type and catalase-deficient mutant barley. Physiol Plant 104 : 280–142 Barata RM, Chaparro-Giraldo A, Chabregas SM, Gonzáles R, Labate CA, Azevedo RA, Sarath G, Lea PJ, Silva-Filho MC (2000) Targeting of the soybean leghemoglobin to tobacco chloroplasts: effects on aerobic metabolism in transgenic plants. Plant Sci 155 : 193–202 Becana M, Klucas R (1990) Enzymatic and nonenzymatic mechanisms for ferric leghemoglobin reduction in legume root nodules. Proc Natl Acad Sci USA 87 : 7295–7299 Boutry M, Nagy F, Poulsen C, Aoyagi K, Chua N-H (1987) Targeting of bacterial chloramphenicol acetyltransferase to mitochondria in transgenic plants. Nature 328 : 340–342 Bradford M (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 : 248–254 Brisson N, Verma DPS (1982) Soybean leghemoglobin gene family: normal, pseudo, and truncated genes. Proc Natl Acad Sci USA 79 : 4055–4059 Bülow L, Holmberg N, Lilius G, Bailey JE (1999) The metabolic effects of native and transgenic hemoglobins on plants. Trends Biotech 17 : 21–24 Comai L, Larson-Kelly N, Kiser J, Mau CJD, Pokalsky AR, Shewmaker CK, McBride K, Jones A, Stalker DM (1988) Chloroplast transport of a ribulose biphosphate carboxylase small subunit-5-enolpyruvyl 3-phosphoshikimate synthase chimeric protein requires part of the mature small subunit in addition to the transit peptide. J Biol Chem 263 : 15104–15109 D’Agnillo F, Chang TMS (1998) Polyhemoglobin-superoxide dismutase-catalase as a blood substitute with antioxidant properties. Nat Biotechnol 16 : 667–671 Dale PJ, Hampson KK (1995) An assessment of morphogenic and transformation efficiency in a range of varieties of potato (Solanum tuberosum L.). Euphytica 85 : 101–108 Deblonde PMK, Haverkort AJ, Ledent JF (1999) Responses of early and late potato cultivars to moderate drought conditions: agronomic parameters and carbon isotope discrimination. Eur J Agron 11 : 91–105 Dyerick W, Pagnier J, Poyart C, Marden MC (1997) Human hemoglobin from transgenic tobacco. Nature 386 : 29–30 Edwars K, Johnstone C, Thompson CAA (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19 : 1349
Geigenberger P, Muller-Robert B, Stitt M (1999) Contribution of adenosine 5-bdiphosphoglucose pyrophosphorylase to the control of starch synthesis is decreased by water stress in growing potato tubers. Planta 209 : 338–345 Hill RD (1998) What are hemoglobins doing in plants? Can J Bot 76 : 707–712 Holmberg N, Lilius G, Bailey JE, Bülow L. (1997) Transgenic tobacco expressing Vitreoscilla hemoglobin exhibits enhanced growth and altered metabolite production. Nat Biotechnol 15 : 244–247 Jones MGK, Outlaw WH, Lowry OH (1977) Procedure for the assay of sucrose in range 10 –7–10 –14 moles. Plant Physiol 60 : 379–383 Leegood RC, Furbank RT (1986) Stimulation of photosynthesis by 2% oxygen at low temperatures is restored by phosphate. Planta 168 : 84–93 Lowry O, Passoneau JV (1972) A flexible system of enzymatic analysis. Academic Press, New York Puppo A, Halliwell B (1988) Generation of hydroxyl radicals by soybean nodule leghaemoglobin. Planta 173 : 405–410 Puppo A, Dimitrijevic L, Rigaud J (1982) Possible involvement of nodule superoxide dismutase and catalase in leghemoglobin protection. Planta 156 : 374–379 Sievers G, Rönnberg M (1978) Study of the pseudoperoxidatic activity of soybean leghemoglobin and sperm whale myoglobin. Biochim Biophys Acta 533 : 293–301 Silva-Filho MC, Chaumont F, Leterme S, Boutry M (1996) Mitochondrial and chloroplast targeting sequences in tandem modify protein import specificity in plant organelles. Plant Mol Biol 30 : 769–780 Silva-Filho MC, Wieers M-C, Flügge U-I, Chaumont F, Boutry M (1997) Different in vitro and in vivo targeting properties of the transit peptide of a chloroplast envelope inner membrane protein. J Biol Chem 272 : 15264–15269 Sowa AW, Duff SMG, Guy PA, Hill RD (1998) Altering hemoglobin levels changes energy status in maize cells under hypoxia. Proc Natl Acad Sci USA 95 : 10317–10321 Van Der Broeck G, Timko MP, Kaush AP, Cashmore AR, Van Montagu M, Herrera-Estrella L (1985) Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose-1,5-biphosphate carboxylase. Nature 313 : 358–363
