Targeting of the soybean leghemoglobin to tobacco chloroplasts:
effects on aerobic metabolism in transgenic plants
Reinaldo M. Barata
a, Alejandro Chaparro
a, Sabrina M. Chabregas
a,
Raquel Gonza´lez
c, Carlos A. Labate
a, Ricardo A. Azevedo
a, Gautam Sarath
b,
Peter J. Lea
c, Marcio C. Silva-Filho
a,*
aDepartamento de Gene´tica,Escola Superior de Agricultura‘Luiz de Queiroz’,Uni6ersidade de Sa˜o Paulo,A6.Pa´dua Dias,11,Caixa Postal83, 13400-970Piracicaba,SP,Brazil
bN-226,Beadle Center,Protein Core Facility-Center for Biotechnology and Department of Biochemistry,Uni6ersity of Nebraska,Lincoln, Lincoln,NE68588-0664,USA
cDi6ision of Biological Sciences,Uni6ersity of Lancaster,Bailrigg,Lancaster LA1 4YQ,UK
Received 27 December 1999; received in revised form 7 February 2000; accepted 7 February 2000
Abstract
Several attempts have been made to alter the aerobic metabolism of plants, especially those related to the oxygenation or carboxylation of Rubisco. However, designing a more efficient Rubisco protein is rather problematic since its structural manipulation leads frequently to an enhancement of oxygenase activity, which is responsible for photorespiratory losses. In order to reduce oxygen availability inside the chloroplast, a chimeric gene consisting of a soybean leghemoglobin cDNA (lba) ligated to the chloroplast targeting signal sequence of the Rubisco small subunit gene, was introduced and expressed in Nicotiana tabacum. Lb was efficiently imported and correctly processed inside the chloroplasts of transgenic tobacco plants. Furthermore, the level of Lb expression in leaf tissue ranged from 0.01 to 0.1%. Analysis of photosynthesis, starch, sucrose and enzymes involved in aerobic metabolism, revealed that despite the high affinity of Lb for oxygen, no significant difference was observed in relation to the control plants. These results suggest that higher Lb concentrations would be required inside the chloroplasts in order to interfere on aerobic metabolism. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Leghemoglobin; Oxygen; Photorespiration; Photosynthesis
www.elsevier.com/locate/plantsci
1. Introduction
In a leaf at 25°C, oxygen in the air equilibrates with dissolved O2 in aqueous solution to produce
a concentration of aproximately 250 mM. Under
these conditions, oxygen concentrations within photosynthesizing chloroplasts have been experi-mentaly estimated to be 10 – 20% higher than the external aqueous environment, which is 275 – 300
mM O2[1]. Oxygen acts as a substrate or cofactor
in many biochemical reactions in both the primary and secondary metabolism of plant cells. For
ex-ample, chlorophyll and protoporphyrin biosynthe-sis [2], an alternative Hill oxidant: Mehler reaction [1], and substrate to the oxygenase activity of
ribulose-1,5-bisphosphate carboxylase/oxygenase
(Rubisco) in C3 plants. Photorespiration in these
plants is assumed to be responsible for as much as 40% loss of net CO2 assimilated. In this manner,
the photorespiration can be considered a wasteful process since the CO2released must be fixed again
within the leaf [3].
Control of photorespiration has emerged as a primary objective in efforts to increase plant pro-ductivity in C3crops [4]. Elevated CO2
concentra-tions generally enhance photosynthesis over the
short term (days) because of higher CO2
concen-* Corresponding author. Tel.: +55-19-4294125; fax: + 55-19-4336706.
E-mail address: mdcsilva@carpa.ciagri.usp.br (M.C. Silva-Filho)
trations at the carboxylation site, results in an increased availability of carbohydrate for plant growth [5,6]. Nevertherless, due to practical limita-tions of increasing CO2 concentrations on a large
scale, many efforts have been made to modify the properties of Rubisco to reduce the oxygenation reaction, especially using molecular biology tech-niques. The majority of results reported so far, indicate that modification of Rubisco structure reduces the oxygenase activity as well as the car-boxylase activity [7,8]. An alternative to alter the active site of Rubisco is to modify the ratio of CO2/O2in the proximity of the enzyme, by
target-ing leghemoglobin (Lb) to the chloroplast. Lb has a very high affinity to oxygen, and is in – planta synthesized exclusively in root nodules due to a symbiotic association ofRhizobium spp. with legu-minous plants [9,10]. Lb functions by facilitating oxygen diffusion through the cytoplasm of bacte-ria-infected nodule cells to membrane-enclosed bacteroids, and thereby, protecting O2 damage to
nitrogenase [11,12].
In previous studies, transgenic tobacco plants expressing functional different hemoglobins have been produced [13 – 15]. It has been demonstrated that bacterial hemoglobin promoted enhanced growth and altered metabolite production in to-bacco plants [14], whereas expression of functional human hemoglobin in transgenic tobacco had no phenotypic effects reported [13]. In the present work, targeting of heterologous Lb to tobacco chloroplasts was performed to determine whether Lb can be correctly imported and processed inside the organelle and to examine the effects on photo-synthesis, carbohydrates, chlorophyll and the ac-tivities of enzymes involved in oxidative stress. We have shown that the presence of Lb in chloroplasts does not interfere with aerobic metabolism in transgenic plants, that may be due to the presence of insufficient quantities of Lb to alter the rate of diffusion of the high concentrations of oxygen found inside the chloroplast.
2. Materials and methods
2.1. Gene constructions
Standard procedures were used for DNA ma-nipulations [16]. The constructs assembling the Rubisco transit peptide and Lb were made as follows.
The prbcS plasmid carries a DNA fragment
corresponding to the 5%-non-coding region, and the
full-length cDNA of the small subunit precursor of Rubisco from pea [17] (kindly provided by Kenton Ko, Queen’s University, Canada). Using the polymerase chain reaction specific restriction sites were added to the flanking regions of the transit peptide encoding region. Synthetic primers
provided with HindIII and EcoRI sites were as
follows. The rbcS1 upstream primer was 5%-
CC-CAAGCCTTTAACAATGGCTTCC, and the
rbcS2 downstream primer was 5%
-CCCCGATTC-CTGCATGCATTGCAG.
After polymerase chain reaction amplification,
the fragment was digested with HindIII and
EcoRI and inserted into the corresponding sites of SK(+) Bluescript (Stratagene), resulting in the SK(+) rbcS plasmid.
The leghemoglobin a encoding sequence from
soybean [18] (kindly provided by K. Marcher, Aarhus University, Denmark) was modified at the flanking regions by the polymerase chain reaction.
Synthetic primers provided with EcoRI and
BamHI cloning sites were as follows. The leg1
upstream primer was 5%
-CCCGAAATTCA-GAAATATGGTTGC, and the leg 2 downstream
primer was 5%
-CCCGGATCCTACTAATTATG-CC.
After polymerase chain reaction amplification, the fragments were double digested respectively
with EcoRI and BamHI and cloned in the
corre-sponding sites of SK(+)rbcS (Stratagene),
previ-ously digested with EcoRI and BamHI, resulting
in the SK(+)rbcS-Lb plasmid. The authenticity of this construct was verified by DNA sequencing. To prepare the rbcS-Lb construct for tobacco transformation, the Bin2-35ScatE9% [19] was
di-gested with HindIII and treated with Klenow
DNA polymerase so that the end was filled in. Immediately after, the plasmid was digested with
BamHI to release the chloramphenicol acetyltrans-ferase encoding sequence. In parallel, the SK(+)
rbcS-Lb plasmid was digested with HindIII, filled in with Klenow DNA polymerase and further digested with BamHI, releasing the chimeric gene
rbcS-Lb. This fragment was inserted into the cor-responding sites of the modified Bin2-35ScatE9%
vector, producing the plant transformation vector Bin2-35SrbcS-LbE9%.
plas-mid SK(+)rbcS-Lb was digested with EcoRI and
BamHI, releasing the Lb gene. This fragment was
then inserted into the corresponding sites of Bin2-35ScatE9% vector previously digested with EcoRI
and BamHI.
2.2. Plant transformation
Transformation experiments were performed
with Nicotiana tabacum as previously described
[20,21]. Regenerated plants were selected for kanamycin resistance.
2.3. Plant material and growth conditions
Seeds of SRI Petite Havana N. tabacum lines
were sown in a commercial substrate and fed with commercial fertilizer at least twice a week. Plants were grown in a glasshouse under a photoperiod of 14/10 h day/night and a temperature of 20/ 35°C. For metabolite extraction analyses, leaf
discs (approximately 10 cm2) from control and
four independent S2 transformants (R3, R6, R9,
and R10) were harvested from fully expanded leaves (third or fourth from the apex).
2.4. Chlorophyll assay
Leaf samples from 4-week-old plants were ho-mogenized in 1.5 ml tubes and the chlorophyll was extracted with ice-cold 80% buffered acetone. The debris were removed and the supernatants col-lected. The absorbance of the chlorophyll extracts was monitored at 663 and 646 nm. The amounts
of chlorophyll-a and b were calculated as
de-scribed [22].
2.5. Metabolite measurements
Sucrose contents were extracted from leaf discs according to the method of [23] and determined as described previously [24]. For estimations of starch, the samples were assayed essentially as described by Jones et al. [25].
2.6. Catalase and glutathione reductase acti6ities
Enzyme extraction was carried out as described previously by Azevedo et al. [26]. Catalase activity was determined as described by Kraus et al. [27] with some modifications [26].
Glutathione reductase activity was determined according to Smith et al. [28] with some modifica-tions [26].
2.7. Protein quantitation
Protein concentration was determined spec-trophotometrically at 595 nm as described by Bradford [29] using the Bio-Rad Protein Assay Dye Reagent with bovine serum albumin as a standard.
2.8. Fractionation of tobacco cells and protein
analysis
Subcellular fractions were obtained from 10 g of leaves as described previously [19], except that homogenization was performed in 100 ml of ho-mogenization buffer and that 0.2% (w/v) insoluble polyvinylpyrrolidone was added to the buffer.
Purification of chloroplasts and thylakoids on a continuous Percoll gradient was performed ac-cording to Bruce et al. [30].
2.9. Western blot analysis
After SDS-polyacrylamide gel electrophoresis, proteins were transferred to a nitrocellulose mem-brane and immunodetected with antibodies raised against purified leghemoglobin (1/1000), ribulose 1,5-biphosphate carboxylase (1/8000), followed by an anti-mice IgG alkaline phosphatase conjugate (Sigma Chemical Co).
2.10. Gas exchange measurements
Net photosynthesis, stomatal conductance, tran-spiration and internal CO2concentration were
de-termined in fully expanded leaves of each line using a portable infra red gas analyzer (CIRAS P/P systems, Hitchin, UK). Photosynthetic active
radiation (PAR) was approximately 600 mmol
m−2 s−1 at leaf level. The air supply unit of the
system was connected to two cylinders, one with
21% O2 concentration and another with 1% O2
concentration. Gas exchange parameters were first measured at ambient oxygen concentration (21% O2). The air supply was then changed to 1% O2by
af-ter the switch between the two O2concentrations).
The effects of line, oxygen concentration and their interaction were analyzed by factorial analysis of variance (ANOVA) using SPSS (version 6.1.4) software.
2.11. Leghemoglobin quantitation in transgenic tobacco plants
All operations at 4°C unless stated otherwise. Leaves (1 g) of tobacco transformants (3.6, 6.6 and 9.9) were ground in 3 ml, 50 mM Tris – Cl buffer containing 2 mM PMSF and 0.2 g polyvinylpolypyrollidone. The homogenate was filtered through miracloth and centrifuged at
14 000×g for 15 min. The clarified supernatant
was assayed for protein using a dye binding proto-col (BCA, Pierce Chemical Co, IL, USA) using bovine serum albumin as a standard.
Aliquots of extracts in SDS-PAGE sample buffer (Laemmli) containing 50 mg of protein and
purified and quantitated leghemoglobin a were
separated on a 14% denaturing gel. Separated proteins were electroblotted onto nitrocellulose membranes and probed with antibodies raised to Lba. Antigen – antibody complexes were verified by chemiluminescence, using peroxidase
conju-gated secondary antibodies and luminol reagent (Pierce Chemical Company, super signal) follow-ing manufacturers directions. Chemiluminescence was captured by exposing X-ray film (Kodak,
BioMax) and developed on an automated
developer.
3. Results
3.1. The rbcS-transit peptide targets the soybean
leghemoglobin into chloroplasts
The Rubisco transit peptide (rbcS) has been used successfully to introduce foreign proteins in vivo into chloroplasts [31 – 33]. In the present work, a gene construct was prepared to express the soybean leghemoglobin (Lb) into tobacco chloroplasts (Fig. 1). The Rubisco transit peptide
was fused to the soybean leghemoglobin a [34].
The construct (rbcS-Lb) retained the whole rbcS targeting sequence, followed by four amino acid residues of the mature protein to allow cleavage of the transit peptide that might have required the surrounding residues. As a control, Lb was pre-pared without the targeting sequence assuring ex-pression in the cytosol. Both chimaeric genes were placed under the control of the 35S transcription promoter of cauliflower mosaic virus and the 3% -non-coding region of a pea Rubisco small subunit gene [35]. These genes were introduced into to-bacco, using an A. tumefaciens Ti plasmid-derived
vector. Transgenic plants were selfed and several independent S2 plants were characterized for both
constructs.
The level of Lb expression using the 35S pro-moter was 0.01 – 0.1% of total soluble proteins for rbcS-Lb plants. These values were based on the relative intensity of the immunoreactive band to authentic Lb also loaded on the gels as a standard. As can be seen in Fig. 2, there was considerable variation in the amount of Lb present in the three different tobacco transformants analyzed. Maxi-mal amounts were seen in 6.6, which appeared to be between 0.005 and 0.5 mg Lb in 50 mg of total protein, which is about 0.01% at the low end and 0.1% at the high end. The two other transformants had less than this value of Lb. Similar results were also observed with the expression of human hemoglobin in tobacco seeds driven by the 35S promoter [13].
Fig. 1. ChimericrbcS-Lbgene construct. Below the scheme of therbcS-Lbconstruct is shown the nucleotide and amino acid sequences from the Rubisco (rbcS) transit peptide, the re-tained mature Rubisco, as well as the linker region upstream of the Lb initiation codon. Linker amino acid residues are in italics. TheHindIII restriction site is underlined. The Rubisco and Lb initiation codons are in bold. The6ertical arrowhead
Fig. 2. Leghemoglobin expression levels in transgenic tobacco plants. Lb quantitation was carried out as described in Sec-tion 2. Lb expression from three independent tobacco trans-formants (3.6, 6.6 and 9.9) was compared with purified Lb at different concentrations (0.005, 0.05 and 0.5mg).
showed that leghemoglobin was targeted and cor-rectively processed inside the chloroplasts (Fig. 3a), since Rubisco enrichment in the chloroplast fraction was similar (Fig. 3b). Lb enrichment in the chloroplast fraction was low, but this was to be expected since chloroplast proteins of meso-phyll cells represent up to 50% of the total proteins of the leaves [35]. In plants carrying the 35S-Lb construct, Lb was found in the superna-tant (cytosol) only and no Lb was detected in the chloroplast fraction. As expected, the size of the mature Lb observed for rbcS-Lb in the chloroplast fraction was larger than that of the cytosolic Lb. This difference can be accounted by the presence of four amino acid residues of mature Rubisco and one residue from the linker region.
3.2. Gas exchange
Reduced O2 concentration significantly
in-creased net photosynthesis (PB0.001) with
in-creases in the range 30 – 35% in respect to 21% O2.
There were no significant differences in net photo-synthesis between lines at either O2concentration.
The Line xconcentration interaction was also not significant for net photosynthesis or any other measured parameter (Table 1). Significant differ-ences between lines and between O2concentrations
were observed for stomatal conductance and
inter-nal CO2 concentration (Table 1). Increases in
stomatal conductance were approximately 8 – 12% at 1% O2for all lines; the wild type and R10 were
also significantly different from the other two lines at both O2concentration. At 1% O2, internal CO2
concentration decreased (8 – 10%) in all lines. No significant differences were observed for
transpira-tion in any line or between O2 concentration
(Table 1).
3.3. Effect of chloroplast-leghemoglobin on
metabolite le6els
Sucrose and starch provide confirmatory evi-dence over a longer time period that the photosyn-thetic rate is not changed. To determine the effects of the presence of soybean leghemoglobin inside the chloroplasts on carbohydrate levels, leaf sam-ples from the control (WT) and four independent S2
F2 transformants were harvested and assayed for
their sucrose (Fig. 4A) and starch (Fig. 4B) con-tents. The results indicate that the concentration
Fig. 3. Immunodetection of Lb in subcellular fractions of transgenic tobacco plants. Homogenization and subcellular fractionation of rbcS-Lb and Lb transgenic plants were car-ried out as described in Section 2. Western blot analysis was carried out on 10mg proteins of the homogenate (H) and the chloroplast (C) enriched fraction. (a) size comparison of the mature Lb (lane 5), and rbcS-Lb (lanes 7 and 8) products. A soybean Lb protein extract (0.1 mg) was used as a control (lane 1). (b) Western blot analysis of Rubisco, a chloroplast marker, in a Lb (lanes 3 and 4), and rbcS-Lb (lanes 5 and 6) plant. Molecular mass (kDa) is indicated.
of sucrose and starch are not affected, suggesting that primary carbon metabolism is not altered by the presence of Lb inside the organelle. Consistent with this, was the observation that plant growth and dry weight measurements were not statistically different (data not shown). In contrast, expression of a bacterial hemoglobin in the cytosol of trans-genic tobacco promoted a significant increase in plant growth and higher chlorophyll-b levels re-lated to the untransformed plants arguing that the bacterial hemoglobin could supply oxygen for chlorophyll biosynthesis [14]. To verify whether Lb could alter chlorophyll biosynthesis, the chlorophyll-aandb concentrations in the leaves of the transgenic and control plants were analyzed. There was no modification of chlorophyll contents in any of the transformed rbcS-Lb (Fig. 4C) and 35S-Lb plants (data not shown).
3.4. Catalase and glutathione reductase acti6ities
in transgenic plants
During photorespiration, H2O2 is produced in
the peroxisome by oxidation of glycolate and then rapidly removed by catalase (CAT, EC 1.11.1.6) inside the organelle. Previously published data has shown that there is a correlation between catalase activity and the rate of photorespiration [3,24]. Therefore catalase activity was used as an indica-tor of phoindica-torespiration in the tobacco plants in this study. At high light intensities, there is often
an excess of PSI reduction and NADP+ pools
become fully reduced. Under these conditions oxy-gen is reduced by PSI, leading to the oxy-generation of superoxide radicals through the Mehler reaction. This superoxide radical is then rapidly dispropor-tionated to H2O2 by superoxide dismutase (SOD,
EC 1.15.1.1), which may be associated with the thylakoids or in the stroma. The H2O2produced is
quickly scavenged via the ascorbate/glutathione pathway [36]. Regeneration of these compounds is carried out by glutathione reductase (GR, EC 1.6.4.2). Therefore, CAT and GR activities have been investigated in transgenic and wild type plants. It was demonstrated that both enzyme activities were not affected by the presence of the leghemoglobin inside the chloroplasts (Fig. 5).
4. Discussion
Enhanced plant growth and yield by the manip-ulation of the photosynthetic process has been a major goal of plant physiologists. As approxi-mately 90% of the plant dry weight is derived from CO2 assimilated by photosynthesis, increasing the
carboxylation reaction of Rubisco may be a great benefit to plant produtivity [3]. Using molecular
biology techniques, the soybean leghemoglobin a
gene fused to a chloroplast targeting sequence has been introduced into the tobacco genome, in an attempt to alter the aerobic metabolism of the transgenic plants. Western blot analysis showed that the Lb was targeted to chloroplasts and the
Table 1
Measurements of transpiration (E), stomatal conductance (gs), net photosynthesis (A) and internal CO2concentration (Ci) at two
concentration (21 and 1%) on the 8th leaf of each plant linea
A(mmol CO2m−2s−1)
O2concentration ns
ns
Intercation ns ns ns
aData are means of 20 plants 9S.E. The results of analysis of variance are shown: ns indicates no difference;
Fig. 4. The metabolite concentrations in transgenic and con-trol tobacco leaf discs. Sucrose (A) and starch (B) accumu-lated in leaves over the early morning (06:00 h) and the late afternoon (18:00 h) periods. Ten replicate measurements were taken from each of five different plant lines and the results are shown as the mean sucrose and starch accumulation (ex-pressed inmmol hexose equivalents mg−1chl). Chlorophyll-a
and b amounts (C) in the transgenic lines and a wild-type control. Data represents mean values from ten individual plants of each line 9S.E.
and reduce the build up of toxic compounds re-leased during fermentation [38]. On the other hand, high temperatures stimulate the oxygenation reaction of Rubisco, which is responsible for the
wasteful photorespiratory process in C3 plants.
Therefore, increasing the carboxylation reaction of
Rubisco by a reduction of the O2 concentration
inside the chloroplast, by the expression of Lb has been the major aim of this work. In addition, expression of Lb in the cytosol of transgenic to-bacco was also attempted in order to promote higher growth, enhanced dry matter, chlorophyll content and faster germination, as observed previ-ously by Holmberg et al. [14]. Contrary to these results, expression of Lb in the cytosol did not affect any of the parameters analyzed. This is unexpected since soybean Lb has a much higher affinity for oxygen than the bacterial hemoglobin. It has always proved very difficult to obtain a direct measurement of photorespiration due to the reassimilation of CO2. However, a comparison of
the rates of photosynthetic CO2 assimilation at
atmosferic concentrations of O2 (21%) and at 1 –
2% O2, has been frequently used as an indirect
measurement [39]. It is clear from Table 1, that there were no significant differences between the
rates of photosynthetic CO2 assimilation at the
two O2 concentrations, between the wild type and
any of the three transgenic rbcS-Lb plants.
Inter-Fig. 5. Antioxidant enzyme activities. (A) CAT (mmol mg−1
protein min−1) and (B) GR (mmol mg−1 protein min−1)
activities in leaves of wild type and transgenic plants (R3, R6, R9, and R10) after growth for 8 weeks in a glass-house (14/10 h day/night). Data represents means of ten measurements
9S.E.
estingly, Couture et al. [40] have demonstrated that a chloroplast Hb was located in the pyrenoid
and the thylakoid region in Chlamydomonas eu
-gametos. The role of the pyrenoid has been
sug-gested as creating a microaerobic environment where the CO2to O2ratio is maintained at a high
level, through the action of carbonic anhydrase, thus at the expense of the competing O2 [41]. In
cyanobacteria, it has been proposed that Hb is
involved in delivering O2 to cytochrome oxidase
under microaerobic conditions [42]. What could account for the absence of any discernible effect in the transgenic rbcS-Lb plants? It may be argued that the soybean Lb is not in a functional state in order to interfere on oxygen binding kinetics or additional residues present at the N-terminus could influence the oxygen-binding activity of Lb. First, the N-terminus in Lb is free and does not appear to influence ligand-binding properties of the protein [43]. Addition of a few to several amino acids to the N-terminus would thus not to be expected to alter the conformation of the protein, nor impact its ligand-binding properties. The residues critical to the heme pocket are on helices distant from the N-terminus. Second, the more complex tetrameric human hemoglobin was functional in tobacco chloroplasts [13], and the observation that a chloroplast hemoglobin is natu-rally found in the unicellular green algae [40], strongly suggest that the soybean Lb is in a func-tional form within the transformed plants. Simi-larly, the observations that functional plant Lb
and Hbs have been expressed in E. coli [44,45],
would also indicate that the chloroplast located Lb could be able to operate as an O2 carrier.
Chloroplasts are organelles with a high rate of oxygen flux during the day, as oxygen is able to act as a product as well as a substrate in several metabolic reactions. To function as an oxygen carrier and to create a microaerobic environment in root nodules, the Lb exceeds the oxygen con-centration by 10 000-fold [46]. It has been shown that in the transgenic rbcS-Lb plants, Lb expres-sion varied from 0.01 to 0.1% of the total soluble protein, suggesting that to act effectively, the Lb concentration inside the chloroplast should be sev-eral times higher in order to create a gradient that removes oxygen faster than the rate of diffusion of free oxygen. Estimating a ratio of 1:30 for barley Hb when compared to myoglobin in muscle cells, Hill [47] pointed out that it would require a free
oxygen concentration of less than 1 mM, before
Hb became effective in oxygen diffusion. At 25°C, the O2 concentration inside chloroplasts has been
estimated at being between 275 and 300mM [1]. At this concentration, extremely high levels of Lb would be required to alter the carboxylation effi-ciency of Rubisco and influence other oxygen-de-pendent metabolic pathways. This would support the observations for a lack of significant biochem-ical changes in the transgenic plants, despite the high affinity of the expressed leghemoglobin for oxygen.
Acknowledgements
This work was supported by a grant from the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, FAPESP (94/03561-0) and CNPq. R.M.B. was supported by a graduate fellowship from FAPESP. A.C.G. and S.M.C. were sup-ported by grants from CAPES and CNPq re-spectly. M.C.S.F and R.A.A. are research fellows of CNPq.
References
[1] J.M. Robinson, Does O2 photoreduction occur within
chloroplasts in vivo?, Physiol. Plant 72 (1988) 666 – 680. [2] D. von Wettstein, S. Gough, C.G. Kannangara,
Chloro-phyll biosynthesis, Plant Cell 7 (1995) 1039 – 1057. [3] I. Zelitch, Control of plant produtivity by regulation of
photorespiration, Bioscience 42 (1992) 510 – 516. [4] A.J. Keys, Biochemistry of photorespiration and the
consequences for plant performance, in: J.A. Bryant, M.M. Burrell, N.J. Kruger (Eds.), Plant Carbohydrate Biochemistry, BIOS, Oxford, 1999, pp. 147 – 162. [5] M. Stitt, Metabolic regulation of photosynthesis, in:
N.R. Baker (Ed.), Photosynthesis and the Environment, Kluwer, Dordrecht, 1996, pp. 151 – 190.
[6] B.D. Moore, S.-H. Cheng, J. Rice, J.R. Leeman, Sucrose cycling, Rubisco expression and prediction of photosyn-thetic acclimation to elevated atmospheric CO2, Plant
Cell Environ. 21 (1998) 905 – 916.
[7] F.C. Hartman, M.R. Harpel, Structure, function, regula-tion, and assembly of d-ribulose-1,5-biphosphate car-boxylase/oxygenase, Annu. Rev. Biochem. 63 (1994) 197 – 234.
[8] I. Kanevski, P. Maliga, D.F. Rhoades, S. Gutheridge, Plastome engineering of ribulose-1,5-biphosphate car-boxylase/oxygenase in tobacco to form a sunflower large subunit and tobacco small subunit hybrid, Plant Physiol. 119 (1999) 133 – 141.
[10] M. Becana, R.V. Klucas, Oxidation and reduction of leghemoglobin in root nodules of leguminous plants, Plant Physiol. 98 (1992) 1217 – 1221.
[11] F.J. Bergersen, Delivery of O2 to bacteroids,
Proto-plasma 191 (1996) 9 – 20.
[12] R. Arredondo-Peter, M.S. Hargrove, J.F. Moran, G. Sarath, R.V. Klucas, Plant hemoglobins, Plant Physiol. 118 (1998) 1121 – 1125.
[13] W. Dieryck, J. Pagnier, C. Poyart, M.C. Maarden, V. Gruber, P. Bournat, S. Baudino, B. Merot, Human haemoglobin from transgenic tobacco, Nature 386 (1997) 29 – 30.
[14] N. Holmberg, G. Lilius, J.E. Bailey, L. Bulow, Trans-genic tobacco expressingVitreoscillahemoglobin exhibits enhanced growth and altered metabolic production, Nat. Biotech. 15 (1997) 244 – 247.
[15] L. Bulow, N. Holmberg, G. Lilius, J.E. Bailey, The metabolic effects of native and transgenic hemoglobins on plants, Trends Biotechnol. 17 (1999) 21 – 24.
[16] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, New York, 1989, pp. 1.25 – 1.31.
[17] K. Ko, A. Cashmore, Targeting of proteins to the thy-lakoid lumen by the bipartite transit peptide of the 33 kD oxygen-evolving protein, EMBO J. 8 (1989) 3187 – 3194.
[18] J.J. Hyldig-Nielsen, E.O. Jensen, K. Paludan, O. Wiborg, R. Garret, P. Jorgensen, K.A. Marcker, The primary structures of two leghemoglobin genes from soybean, Nucleic Acids Res. 10 (1982) 689 – 701.
[19] M.C. Silva-Filho, F. Chaumont, S. Leterme, M. Boutry, Mitochondrial and chloroplast targeting sequences in tandem modify protein import specificity in plant or-ganelles, Plant Mol. Biol. 30 (1996) 769 – 780.
[20] M. Bevan, BinaryAgrobacteriumvectors for plant trans-formation, Nucleic Acids Res. 12 (1984) 8711 – 8721. [21] F. Chaumont, M.C. Silva-Filho, D. Thomas, S. Leterme,
M. Boutry, Truncated presequences of mitochondrial F1-ATPase b-subunit from Nicotiana plumbaginifolia
transport CAT and GUS proteins into mitochondria of transgenic tobacco, Plant Mol. Biol. 24 (1994) 631 – 641. [22] A.R. Wellburn, The spectral determination of chloro-phyll-a and chlorophyll-b, as well as total carotenoids, using various solvents with spectrophotometers of differ-ent resolution, J. Plant Physiol. 144 (1994) 307 – 331. [23] R.C. Leegood, R.T. Furbank, Stimulation of
photosyn-thesis by 2% oxygen at low temperatures is restored by phosphate, Planta 168 (1986) 84 – 93.
[24] O.H. Lowry, J.V. Passoneau, A Flexible System of Enzy-matic Analysis, Academic Press, New York, 1972. [25] M.G.K. Jones, W.H. Outlaw, O.H. Lowry, Procedure
for the assay of sucrose in range 10−7– 10−14 moles,
Plant Physiol. 60 (1977) 379 – 383.
[26] R.A. Azevedo, R.M. Alas, R.J. Smith, P.J. Lea, Re-sponse of antioxidant enzymes to ozone fumigation in the leaves and roots of wild-type and a catalase-deficient mutant of barley, Physiol. Plant. 104 (1998) 280 – 292. [27] T.E. Kraus, B.D. McKersie, R.A. Fletcher,
Paclobutra-zol-induced tolerance of wheat leaves to paraquat may involve increased antioxidant enzyme activity, J. Plant Physiol. 145 (1995) 570 – 576.
[28] I.K. Smith, T.L. Vierheller, C.A. Thorne, Assay of glu-tathione reductase in crude tissue homogenates using 5,5%-dithiobis (2-nitrobenzoic acid), Anal. Biochem. 175 (1988) 408 – 413.
[29] M.M. Bradford, A rapid and sensitive method for the quantification of microgram quantities of protein utiliz-ing the principle of protein-dye bindutiliz-ing, Anal. Biochem. 72 (1976) 248 – 254.
[30] B.D. Bruce, S. Perry, J. Froehlich, K. Keegstra, In vitro import of proteins into chloroplasts, in: S.B. Gelvin, R.A. Schilperoort (Eds.), Plant Molecular Biology Man-ual, Kluwer, Dordrecht, 1994, pp. 1 – 15.
[31] G. Van de Broeck, M.P. Timko, A.P. Kausch, A.R. Cashmore, M. Van Montagu, L. Herrera-Estrela, Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose-1,5-biphosphate carboxylase, Nature (1985) 358 – 363. [32] M. Boutry, F. Nagy, C. Poulsen, K. Aoyagi, N.-H.
Chua, Targeting of bacterial chloramphenicol acetyl-transferase to mitochondria in transgenic plants, Nature 328 (1987) 340 – 342.
[33] J. Wan, S.D. Blakeley, D.T. Dennis, K. Ko, Transit peptides play a role in the preferential import to proteins into leucoplasts and chloroplasts, J. Biol. Chem. 271 (1996) 31227 – 31233.
[34] J.J. Hyldig-Nielson, E.O. Jensen, K. Paludan, O. Wilborg, R. Garret, P. Joergensen, K.A. Marcker, The primary structure of two leghemoglobin genes from soy-bean, Nucleic Acids Res. 10 (1982) 689 – 701.
[35] M.C. Silva-Filho, M.C. Wieers, U.-I. Flugge, F. Chau-mont, M. Boutry, Different in vitro and in vivo targeting properties of the transit peptide of a chloroplast envelope inner membrane protein, J. Biol. Chem. 272 (1997) 15264 – 15269.
[36] K. Asada, Mechanisms for scavenging reactive molecules generated in chloroplasts under light stress, in: N.R. Baker, J.R. Bowyer (Eds.), Photoinhibition of Photosyn-thesis: from Molecular Mechanisms to the Field, BIOS, Oxford, 1994, pp. 129 – 142.
[37] A.W. Sowa, S.M.G. Duff, P.A. Guy, R.D. Hill, Altering hemoglobin levels changes energy status in maize cells under hypoxia, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 10317 – 10321.
[38] P. Hoffman, G. Walter, E.-M. Wiedenroth, G. Peine, How the cereal cope with oxygen defficiency, Photosyn-thetica 4 (1993) 495 – 513.
[39] Z. Daı´, M.S.B. Ku, G.E. Edwards, C4 photosynthesis:
the CO2concentrating mechanism and photorespiration,
Plant Physiol. 103 (1993) 83 – 90.
[40] M. Couture, H. Chamberland, B. St-Pierre, J. Lafontaine, M. Guertin, Nuclear genes encoding chloro-plast hemoglobins in the unicellular green algaChlamy
-domonas eugametos, Mol. Gen. Genet. 243 (1994) 185 – 197.
[41] J.V. Moroney, A. Somanchi, How do algae concentrate CO2to increase the efficiency of photosynthetic carbon
fixation?, Plant Physiol. 119 (1999) 9 – 16.
[43] M.S. Hargrove, J.J. Barry, E.A. Brucker, M.B. Berry, G.N. Phillips, J.S. Olson, R. Arredondo-Peter, J.M. Dean, R.V. Klucas, G. Sarath, Characterization of re-combinant soybean leghemoglobin a and apolar distal histidine mutants, J. Mol. Biol. 267 (1997) 1032 – 1042. [44] S.M.G. Duff, J.B. Wittenberg, R.D. Hill, Expression
purification, and properties of recombinant barley (Hordeum sp.) hemoglobin, J. Biol. Chem. 272 (1997) 16746 – 16752.
[45] D.H. Jones, R. Badii, F.I. Rossel, E. Lloyd, Bacterial expression and spectroscopic characterization of soy-bean leghaemoglobin a, Biochem. J. 330 (1998) 983 – 998.
[46] J.B. Wittenberg, B.A. Wittenberg, Mechanisms of cyto-plasmic hemoglobin and myoglobin function, Annu. Rev. Biophys. Chem. 19 (1990) 217 – 241.
[47] R.D. Hill, What are hemoglobins doing in plants?, Can. J. Bot. 76 (1998) 707 – 712.
