A novel superoxide dismutase with a high isoelectric point in higher plants. expression, regulation, and protein localization

Abstract

Several isoforms of superoxide dismutase (SOD) with a high isoelectric point (pI) have been identified by isoelectric focusing chromatography in protein extracts from Scots pine (Pinus sylvestris) needles. One of these isoforms, a CuZn-SOD with a pI of about 10 and thus denoted hipI-SOD, has been isolated and purified to apparent homogeneity. A cDNA encoding the hipI-SOD protein was cloned and sequenced. Northern hybridization of mRNA isolated from different organs and tissues showed that hipI-SOD has a markedly different pattern of expression compared with chloroplastic and cytosolic SOD. Furthermore, the transcript levels of hipI-SOD and cytosolic SOD were found to respond differently to mechanical wounding, treatment with oxidized glutathione, paraquat, and ozone. Immunogold electron microscopy localized the hipI-SOD in the plasma membrane of sieve cells and the Golgi apparatus of albuminous cells. Moreover, high protein density was also detected in extracellular spaces such as secondary cell wall thickenings of the xylem and sclerenchyma and in intercellular spaces of parenchyma cells.

Figure 1

SDS-PAGE of purified hipI-SOD from Scots pine needles. Gel showing mobility of 25-ng hipI-SOD with and without addition of mercaptoethanol. Molecular mass calibration proteins in kilodaltons are indicated to the left: phosphorylase b (94 kD), albumin (67 kD), ovalbumin (43 kD), carbonic anhydrase (30 kD), trypsin inhibitor (20.1 kD), and α-lactalbumin (14.4 kD).

Figure 2

The deduced amino acid sequence hipI-SOD from Scots pine. The comparison of the deduced amino acid sequence of hipI-SOD with cytosolic CuZnSOD from Scots pine, tomato (Lycopersicon esculentum), and maize (Zea mays; Cannon and Scandalios, 1987; Perl-Treves et al., 1988; Karpinski et al., 1992) and chloroplastic CuZnSOD from pea, tomato, and Scots pine (Scioli and Zilinskas, 1988; Perl-Treves et al., 1988; Karpinski et al., 1992). Strictly homologous amino acid residues are shaded. Underlined amino acids indicate regions sequenced by the Edman degradation method. Asterisks show the residues binding Cu and Zn (Bordo et al., 1994). The GenBank accession no. for the hipI-SOD sequence is AJ307586.

Figure 3

Determination of pI of hipI-SOD from Scots pine by IEF, pH 6.5 to 10.5. Purified hipI-SOD of Scots pine (50 ng) was visualized by silver staining (middle lane) and by activity staining (right lane). Calibration standards (pI) are indicated to the left: γ-lactoglobulin (5.2), bovine carbonic anhydrase (5.85), human carbonic anhydrase (6.55), horse myoglobin (6.85), horse myoglobin (7.35), lentil lectin (8.15), lentil lectin (8.45), lentil lectin (8.65), trypsinogen (9.3), and cytochrome c (10.25).

Figure 4

Detection of SOD transcripts by RNA gel blot analysis and relative quantitative reverse transcriptase (RT)-PCR. A, Northern-blot hybridization of hipI-SOD, cyt-, and cp-SOD in different organs and stem tissues of Scots pine. Poly(A⁺) RNA (3.5 μg) was separated by gel electrophoresis, transferred to a filter, and hybridized to homologous, gene-specific hipI-, cyt-, and cp-SOD cDNA probes. P. needles, Primary needles; S. needles, secondary needles. B, Northern hybridization; poly(A⁺) RNA (10 μg per lane) isolated from needles of Scots pine shoots treated for 1, 3, and 6 h with water, 5 mm GSSG, or 5 mm GSH as described previously (Wingsle and Karpinski, 1996). The RNA was separated by gel electrophoresis, transferred to a filter, and hybridized to homologous hipI-SOD and cyt-SOD cDNA probes. C, Quantitative RT-PCR; separation of quantitative RT-PCR products in Scots pine cotyledons after 2, 4, and 8 h of treatment with 8 μm paraquat (PQ) and after 24 h of recovery (R). Seedlings were also fumigated with 0.5 μL L⁻¹ ozone (OZ) and samples collected after 1.5, 11, and 14 h of treatment and after recovering for 24 h (R). Wounded seedlings (W) were collected after 1.5, 4, 7, and 16 h after crushing. To control the changes of mRNA levels during the experiment, non-treated needles were collected parallel with the experimental samples and used for RT-PCR (data not shown).

Figure 5

Western-blot analysis of hipI-SOD from Scots pine after SDS-PAGE. Western-blot analysis, using crude and purified hipI-SOD antiserum, of 25 ng of purified hipI-SOD, cyt-SOD, and cp-SOD.

Figure 6

Immunogold localization of hipI-SOD in cross-sections of Scots pine needles and cotyledons. A, Primary needle and dictyosome in albuminous cell; B, cotyledon and connection between two sieve cells; C, cotyledon, intercellular, and parenchyma cells; D, secondary needle, intercellular, and parenchyma cells; E, secondary needle, control with pre-immune sera, and xylem tracheid; F, secondary needle and xylem tracheid; G, cotyledon and developing sclerenchyma; H, secondary needle and bordered pit of xylem; I, secondary needle and mature sclerenchyma; J, secondary needle and pit of transfusion tracheid. CW, Cell wall; D, dictyosome; IC, intercellular space; PM, plasma membrane; P, plastid.