Maize
Genetics Cooperation Newsletter 80. 2006.
Mitochondria and chloroplasts are major sources of reactive oxygen species (ROS) in plant cells. To minimize the damaging effects of ROS, plants have evolved both enzymatic and nonenzymatic antioxidant defense systems. Superoxide dismutases (SODs, EC 1.15.1.1) are pivotal enzymes of the enzymatic defense system that are specifically compartmentalized in various cellular organelles, including mitochondria and chloroplasts. In maize mitochondria, four different MnSOD isozymes encoded by distinct nuclear genes have been identified (Zhu and Scandalios, 1993). Each member of this small maize MnSOD multigene family is differentially regulated during development in response to plant growth regulator abcisic acid and high osmoticum (Zhu and Scandalios, 1993). In contrast to mitochondria, only one nuclear-encoded Cu/ZnSOD has been found in chloroplasts (Kernodle and Scandalios, 2001). The method of accurate regulation of superoxide and other ROS levels regulation in this organelle still needs further investigation. Previously, we reported the identification of novel cDNA for the chloroplast FeSOD gene (Katyshev et al., 2005). In this report, we present data on characterization of partial cDNA corresponding to a novel maize chloroplast Cu/ZnSOD gene which differs from that reported by Kernodle and Scandalios (Kernodle and Scandalios, 2001).
Total RNA isolation from 3-day-old etiolated hybrid maize VIR46MV seedlings was performed by QIAGEN RNeasy Mini Kit according to the manufacturer�s instructions. cDNA synthesis was carried out using the Promega Universal RiboClone cDNA Synthesis System. In order to amplify 3�-ends of probable cDNA corresponding to the chloroplast Cu/ZnSOD gene, 3�-RACE (rapid amplification of cDNA 3�-ends) experiments using the previously described primers chcsc1 and 3UTR (Katyshev et al., 2006) were performed. As a result, approximately 400 bp-long RT-PCR product formation was observed. This cDNA fragment was eluted from agarose gel, blunted by Klenov fragment of E. coli DNA polymerase and ligated in pBlueScript KS(+) plasmid (Fermentas, Lithuania) by EcoRV site. After transformation, several positive XL1-Blue E. coli clones were chosen for plasmid DNA isolation and insertion sequencing using universal M13/pUC primers (Fermentas, Lithuania). The analysis of cloned sequences resulted in identification of two cDNA clones (CZ6 and CZ8) containing 3�-ends of novel maize Cu/ZnSOD cDNA (the submission of the sequences to EMBL/GenBank databases is in progress).
A search for similar nucleotide sequences by BLAST services at the PlantGDB server (http://www.plantgdb.org/PlantGDB-cgi/blast/PlantGDBblast) found several highly similar maize EST sequences (e.g., EMBL/GenBank acc. numbers CF042260, CF008113, CO443266, DN222176, DR795998, DR795999). Multiple alignment of the EST sequences found and reconstruction of the chimerical consensus sequence for the corresponding full-length cDNA, allowed us to get a probable translated sequence of novel maize Cu/ZnSOD cDNA. To predict subcellular localization of translated protein sequence, the Internet resources available at the http://www.expasy.org/ molecular biology tools server were used: a) the Predotar program at the http://www.inra.fr/predotar/; b) the TargetP V1.1 program (Emanuelsson et al., 2000) at the http://www.cbs.dtu.dk/services/TargetP/; c) the WoLFPSORT program (Horton et al., unpublished)) at the http://wolfpsort.seq.cbrc. jp/; and d) the Сhlorop v1.1 program (Emanuelsson et al., 1999) at the http://www.cbs.dtu.dk/services/ChloroP/. For comparison, subcellular localization of SOD1 protein (Kernodle and Scandalios, 2001) also was checked. The results of subcellular localization prediction are presented in Table 1 (the amino acid sequence corresponding to the novel Zea mays Cu/ZnSOD is referred to as SOD1.2).
Table 1. Probabilities of chloroplast and nonchloroplast localization
of two Cu/ZnSOD (SOD1 and SOD1.2) proteins predicted by different
programs.
|
Name of program |
Probability of chloroplast localization |
Probability of localization in other cellular compartments |
||
|
SOD1 |
SOD1.2 |
SOD1 |
SOD1.2 |
|
|
Predotar v. 1.03 |
0.01 |
0.93 |
0.18 – mitochondrion 0.01 – ER 0.81 – other |
0.16 – mitochondrion 0.03 – ER 0.06 – other |
|
Targetp v1.1 |
0.098 |
0.349 |
0.116 – mitochondrion 0.307 - extracellular 0.403 – other |
0.372 - mitochondrion 0.196 - extracellular 0.009 – other |
|
WoLFPSORT |
- |
0.839 |
0.595 – cytosol 0.118 - nucleus |
0.18 – mitochondrion |
|
Сhlorop v1.1 |
0.463 |
0.551 |
- |
- |
The results of in silico subcellular localization prediction demonstrate that the SOD1.2 protein could be referred to as chloroplast protein even to a larger extent than the SOD1 enzyme. In fact, results of multiple alignment of plant Cu/ZnSOD cDNA sequences not presented in this article provide further support for such a conclusion.
Briefly,
in this report we provide data demonstrating the existence of the novel
Cu/ZnSOD gene cDNA in maize seedlings that encode, as was predicted in silico,
chloroplast enzymes. These data
led us to suggest that in the maize chloroplast several superoxide dismutase
enzymes (two Cu/ZnSOD and one FeSOD) function together and are encoded by
distinct genes similar to MnSODs in mitochondria. Such results are not surprising since both of these
organelles are the major sources of cytotoxic superoxide radicals in plant
cells.
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