The presence of CA enzyme activity, in soybean roots and etiolated hypocotyls, was investigated spectrophotometrically. The data indicated that CA activity was observed in most tissues examined, with significantly higher levels detected in roots of etiolated soybean plants as compared to that observed in hypocotyls (p<0.05, Table 1).

The spatial distribution of CA activity was visualized by an in situ staining for activity in fresh sections of G. max roots and hypocotyls. Activity of the enzyme is coupled to the formation of a black precipitate. High levels of CA activity were detected at the meristematic zone of the primary and lateral root (Figures 1M and N). Less staining was detected in metaxylem vessels but also in phloem and in the middle enlarging cortical cells for both root and hypocotyls (Figures 1N and O). As a negative control, the histochemical assay was performed in the absence of NaHCO₃ (Figure 1P).

Extracts from roots and hypocotyls of etiolated seedlings were analyzed by Western blot using the polyclonal antibodies raised against GmCA1 coding for a beta-type cytoplasmic CA and GmPEPC7 coding for a cytoplasmic PEPC [16]. Both antibodies detected a polypteptide of about 29 and 100kDa respectively, in both roots and hypocotyls (Figure 2). Using the polyclonal antibody raised against GmCA1, CA was previously localized in the cytoplasm of the inner cortical cells and the cell walls of the endodermal cells of mature soybean nodules [32].

The spatial distribution of CA and PEPC protein in the different cell types of roots and hypocotyls of etiolated seedlings was examined by immunohistochemical localization. Transverse and longitudinal root sections as well as transverse hypocotyls sections were incubated with polyclonal antibodies raised against GmCA1 and GmPEPC7. It was observed that the PEPC protein was detected in all tissues. However, the signal was higher in cells of the region of cell division, but in the region of elongation it was restricted at the vascular cylinder and in a few neighboring cell layers (Figure 1E). The localization of CA protein essentially followed the patterns of localization of PEPC protein, with higher expression in developing metaxylem vessels, sieve elements and enlarging cortical cells, for both root and hypocotyls (Figure 1J and K). Furthermore, high signal was detected at the root apical meristem (Figure 2I). When sections were incubated with pre-immune serum, as a negative control, no significant signal was detected (Figure 1L).

To confirm the presence of GmPEPC7 and GmPK3 gene transcripts in roots (the root region of cell division, elongation, and the root region of laterals emergence) and hypocotyls of 8-days old etiolated soybean seedlings, we used a semi quantitative reverse transcription-PCR approach. A housekeeping gene coding for ubiquitin was used as an internal control. As shown in Figure 3, GmPEPC7 gene transcripts were detected in all parts of the root examined and hypocotyls, with the highest levels of accumulation to be detected in the root tip region of 8-day old etiolated seedlings. GmPK3 transcript accumulation was observed in all tissues tested with the highest levels in the root tip region (RT) of 8-day old etiolated seedlings while very low levels compared to the other tissues, were detected in the root region of laterals emergence (MRII).

Having established the presence of GmPK3 and GmPEPC7 gene transcripts, their spatial localization during root development was investigated using an in situ hybridization method. Longitudinal sections of primary and lateral roots of 8-day old etiolated soybean seedlings were hybridized with 11-digoxigenin-rUTP-labeled RNA probes transcribed from GmPK3 and GmPEPC7 cDNA clones. GmPK3 (Figure 1C and D) and GmPEPC7 (Figures 1A and B) expression was detected in the root tip region and mostly in the developing cortex parenchyma of root tip and in the central root cap region of the primary and lateral roots. Levels of both transcripts, in the vascular tissue and in a few cell layers neighbouring the vascular tissue, gradually increased from the region of cell division to the region of elongation and then decreased in the region of laterals emergence. GmPK3 and GmPEPC7 transcripts were detected in most cells in the region of cell division of lateral roots, but as the cells differentiated; GmPK3 and GmPEPC7 expression was confined to the vascular cylinder (Figures 1B and D). Moreover, the signal for both gene transcripts was very high in developing xylem vessels. As a negative control, sections were hybridized to sense RNA probes transcribed from the corresponding cDNAs. In that case, no significant signal was detected (Figure 1H).

Subsequently, the GmCA1 gene expression pattern was studied using the in situ hybridization method. Essentially, GmCA1 transcripts localization followed the patterns of localization of CA protein and activity, with higher expression in developing metaxylem vessels, sieve elements and enlarging cortical cells, for both root and hypocotyls (Figures 1R and S). Furthermore, high signal was detected at the root apical meristem (Figure 1Q) the root epidermis and some underlying cell layers (Figures 1R and S). When sections were incubated with sense probe for the GmCA1 gene, as a negative control, no significant signal was detected (Figure 1T).

Soybean (Glycine max cv. Williams) seeds were pre-germinated on two sheets of moist paper, in Petri dishes, in darkness, at 26 °C, for 8 days. RNA was isolated from hypocotyls (HYP), and different parts of roots, representing the region of cell division (RT), the root region of elongation (MRI) and the root region of laterals emergence (MRII). The different parts of the root were determined using a stereoscope. For in situ hybridization, histochemical detection of enzyme activity and immune-histochemical detection of proteins, the roots from the 8-day old seedlings were used.

By performing BLAST searches [48] among the soybean EST databases, many cDNA clones coding for a cytoplasmic isoform of PK were identified, obtained from ResGen, Invitrogen Corp., and their nucleotide sequences were determined. Alignment of these sequences allowed the identification of a distinct cDNA clone encoding a cytoplasmic isoenzyme of PK. As a part of its 5′ end was missing, total RNA was extracted from the roots of 8-day old seedlings [49] and a 5′ RACE-PCR was performed with the SMART 5′ RACE kit from Clontech (Westburg, NL). The amplified fragment was cloned into the pBlueScriptKS+ plasmid vector (Stratagene) and fully sequenced. The full-length cDNA clone was designated as GmPK3 (NCBI accession number: CAI53675). The Clustal procedure was used for both analysis of the sequence data and determination of the relatedness of the deduced polypeptide to related sequences from other species.

Semi-quantitative RT-PCR analysis was performed using total RNA extracted from the tissues of interest as described above and quantified by spectrophotometry and agarose gel electrophoresis. All RNA samples were treated with DNase I (Promega) according to the manufacturer’s instructions, to eliminate DNA contamination. In each case, the RT-PCR reactions were performed, according to the manufacturer’s directions (Qiagen) using gene-specific oligonucleotides as primers (GmPK3F: 5′-GGAGCTACTGATGTTGCC-3′, GmPK3R: 5′-CATGAGAAGCTCTAGCAG-3′, GmPEPC7F: 5′-ACCAGTTTGGATGCTGGG-3′, GmPEPC7R: 5′-ATCTGTACGGAAGGCAGC-3′). GmPEPC7 cDNA has been isolated and characterized previously [16]. RNase inhibitor was obtained from Promega. For normalization of the different RNA preparations, a fragment of GmUBQ was amplified, using two gene-specific oligonucleotides as primers (GmUBQF: 5′-GGGTTTTAAGCTCGTTGT-3′, GmUBQR: 5′-GGACACATTGAGTTCAAC-3′). The reverse transcriptase reactions were performed in a thermal cycler at 50 °C for 30 min, followed by PCR amplification of 25-30 cycles at 94 °C for 1 min, 54 °C for 1 min and 72 °C for 1 min. A complete final extension for the PCR products was performed at 72 °C for 10 min. Amplified products were separated on 1.5% agarose gels, blotted on to nylon membranes and hybridized with the respective digoxigenin-11-rUTP labeled cDNA probes.

In situ hybridization of root segments was carried out essentially as described [50]. Root segments (5 mm) were fixed in 4% (w/v) paraformaldehyde supplemented with 0.25% (v/v) glutaraldehyde in 10mM sodium phosphate buffer (pH 7.4), for 1h, in a vacuum aspirator, dehydrated with ethanol, and then exchanged with xylene before embedding in paraffin. Sections (8 μm) were mounted on poly-l-lysine slides, digested with proteinase K for 30min at 37°C, treated with acetic anhydride, dried in ethanol, and then hybridized with appropriate gene specific digoxigenin-labeled probes overnight, at 42 °C. After washing with 4xSSC containing 5mM dithiothreitol, the slides were treated with RNase A for 30 min, at 37 °C, washed again at 37 °C with RNase A buffer (500 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.5) containing 5mM dithiothreitol, and then processed for developing the digoxigenin antigen. This involved blocking with blocking reagent (0.5% blocking reagent in 100mM Tris-HCl, 150 mM NaCl, pH 7.5) for 30 min and BSA (1%BSA in 100 mM Tris-HCl, 150 mM NaCl, pH 7.5 containing 0.5% blocking reagent), followed by incubation with an anti-digoxigenin antibody conjugated to alkaline phosphatase (dilution 1:500 in 100 mM Tris-HCl, 150 mM NaCl, pH 7.5) for 2 h, and washing with blocking reagent (1%BSA in 100 mM Tris-HCl, 150 mM NaCl, pH 7.5 containing 0.5% blocking reagent) for 1h. The color was revealed by incubation in NBT and BCIP as long as necessary and the reactions were stopped with water, slides dehydrated, air-dried, and then mounted in DPX before viewing. Antisense and sense probes labeled with digoxigenin-11-rUTP (Roche) were used in parallel hybridizations. The sections were examined using a ZEISS Axiolab (Carl Zeiss, Jena, Germany) microscope and pictures were taken with SONY DSC-S75 38P/45 (SONY Corporation, Japan) digital camera system.

CA activity was histochemically localized in G. max tissues as previously described [16]. Approximately 1–5 mm free-hand sections of G. max fresh roots and hypocotyls were cutted and fixed in 2% (w/v) paraformaldehyde, 2% (w/v) polyvinilopyrolidone (PVP) and 1 mM dithiothreitol (DTT) for 30 min at room temperature. The sections were washed in distilled H₂O overnight at 4 °C and then incubated in the reaction mixture for 30 min. The reaction mixture was prepared fresh immediately before use by quickly mixing 17 mL of solution A containing 12 mM NiSO₄, 180 mM H₂SO₄ and 39 mM KH₂PO₄ with 40 mL of solution B containing 220 mM NaHCO₃. After incubation the sections were washed for 10 min in 0.67 mM phosphate buffer (pH 5.9). The developing of the histochemical color was performed by transferring the sections in 0.5% (v/v) (NH₄)₂SO₄.

Freshly harvested tissues were homogenized in liquid nitrogen. Total CA activity was determined spectrophotometrically using a modification of the pH-indicator method [51]. Tissue extracts were mixed with 200 μL of reaction buffer [190 μL of 0.1M phosphate buffer pH: 6.5 and 10 μL of 0.1% (v/v) bromothymol blue] and 700 μL of 0.1M NaHCO₃ and the increase in absorbance at 615 nm was monitored at 4 °C using a HITACHI U-2800 spectrophotometer. The observed rate was converted to equivalent μmol H⁺ consumed by comparison with a calibration established by titrating the reaction buffer with NaOH. The protein concentration was determined by the procedure of Bradford [52]. Data were analysed by one-way ANOVA.

Approximately 0.5 g of soybean roots or hypocotyls were grounded in a mortar containing liquid nitrogen and the powder was mixed with equal volume of extraction buffer (125 mM Tris-HCl pH 6.8, 20% glycerol, 6% SDS) and centrifuged. Approximately 20 μg of total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 15% polyacrylamide) and then electrotransfered to nitrocellulose (Hybond-C extra; Amersham). Nonspecific binding sites on the membranes were blocked by 30 min incubation in TBST (20 mm Tris-HCl, pH 8.0, containing 150 mm NaCl, 0.05% Tween 20 and 1% BSA). The blots were probed with the antiserum raised against GmPEPC7 and GmCA1 [16] at a 1/2.500 dilution, in the blocking buffer, at room temperature, for 30 min. Blots were washed three times in TBST and then incubated with secondary antibody (alkaline phosphatase conjugated anti-rabbit IgG at dilution 1:1000 in TBST). Blots were again washed three times in TBST and the color was revealed by incubation in NBT and BCIP as long as necessary and the reactions were stopped with water.

Approximately 5 mm root segments were embedded in paraffin and sectioned as described for the in situ hybridization method. 8 μm sections were blocked as previously reported and incubated overnight with antiserum raised against GmPEPC7 and GmCA1 [16]. Briefly, sections were incubated in 3% BSA in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween20, pH 7.5) for 1h and then in the antibody solutions (pre-immune serum and immune serum were used at dilution of 1:1500 in TBST) for 14 h. Sections were washed with TBST for 15 min and then incubated with secondary antibody (alkaline phosphatase conjugated anti-rabbit IgG at dilution 1:500 in 1% BSA in TBST). Sections incubated with pre-immune serum were used as control. The color was revealed by incubation in NBT and BCIP as long as necessary and the reactions were stopped with water. The sections were examined using a ZEISS Axiolab (Carl Zeiss, Jena, Germany) microscope and pictures were taken with SONY DSC-S75 38P/45 (SONY Corporation, Japan) digital camera system.