Molecular cloning, sequence characterization of a novel pepper gene NADP-ICDH and its effect on cytoplasmic male sterility

Research Article
Genet.Mol.Res. 11 (3) : 3020-3031

NADP-dependent isocitrate dehydrogenase (NADP-ICDH) is an important enzyme involved in energy metabolism. The complete coding sequence of the pepper (Capsicum annuum) NADP-ICDH gene was amplified using a reverse transcriptase PCR based on the conserved sequence information of the tomato and other Solanaceae plants and known highly homologous pepper ESTs. Nucleotide sequence analysis revealed that the pepper NADP-ICDH gene encodes a protein of 415 amino acids that has high homology with the proteins of seven species, Solanum tuberosum (100%), Citrus limon (98%), Daucus carota (98%), Nicotiana tabacum (98%), Vitis vinifera (99%), Arabidopsis thaliana (97%), and Oryza sativa (98%). Tissue expression analysis demonstrated that the pepper NADP-ICDH gene is over expressed in flower, pericarp and seed, moderately in placenta, weakly in stem and leaf, hardly expressed in root. At the abortion stages, activities and expression levels of NADP-ICDH in anthers of a sterile line were strongly reduced, while those in an F1 hybrid remained normal. Activities and expression levels of NADP-ICDH were too low to maintain balanced energy metabolism in the sterile line, which indicated that stable transcripts of NADP-ICDH are necessary to maintain energy metabolism at a normal level. When the restorer gene was transferred to the cytoplasmic male sterile line, activities and expression level of NADP-ICDH were regulated by the restorer gene and became stable. The restorer gene likely plays an important role in keeping the balance of the energy metabolism within normal levels during microspore development.

Balk J and Leaver CJ (2001). The PET1-CMS mitochondrial mutation in sunflower is associated with premature programmed cell death and cytochrome c release. Plant Cell 13: 1803-1818.
PMid:11487694 PMCid:139137
Bartoli CG, Pastori GM and Foyer CH (2000). Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV. Plant Physiol. 123: 335-344.
PMid:10806250 PMCid:59007
Bendtsen JD, Nielsen H, von HG and Brunak S (2004). Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340: 783-795.
Benkert P, Biasini M and Schwede T (2011). Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics 27: 343-350.
PMid:21134891 PMCid:3031035
Bergman P, Edqvist J, Farbos I and Glimelius K (2000). Male-sterile tobacco displays abnormal mitochondrial atp1 transcript accumulation and reduced floral ATP/ADP ratio. Plant Mol. Biol. 42: 531-544.
Camacho M, Rodriguez-Arnedo A and Bonete MJ (2002). NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii: cloning, sequence determination and overexpression in Escherichia coli. FEMS Microbiol. Lett. 209: 155-160.
Carlsson J, Lagercrantz U, Sundstrom J, Teixeira R, et al. (2007). Microarray analysis reveals altered expression of a large number of nuclear genes in developing cytoplasmic male sterile Brassica napus flowers. Plant J. 49: 452-462.
Connett MB and Hanson MR (1990). Differential mitochondrial electron transport through the cyanide-sensitive and cyanide-insensitive pathways in isonuclear lines of cytoplasmic male sterile, male fertile, and restored petunia. Plant Physiol. 93: 1634-1640.
PMid:16667667 PMCid:1062722
Corpas FJ, Barroso JB, Sandalio LM, Palma JM, et al. (1999). Peroxisomal NADP-Dependent Isocitrate Dehydrogenase. Characterization and Activity Regulation during Natural Senescence. Plant Physiol. 121: 921-928.
PMid:10557241 PMCid:59455
Ducos E, Touzet P and Boutry M (2001). The male sterile G cytoplasm of wild beet displays modified mitochondrial respiratory complexes. Plant J. 26: 171-180.
Dutilleul C, Garmier M, Noctor G, Mathieu C, et al. (2003). Leaf mitochondria modulate whole cell redox homeostasis, set antioxidant capacity, and determine stress resistance through altered signaling and diurnal regulation. Plant Cell 15: 1212-1226.
PMid:12724545 PMCid:153727
Fieuw S, Muller-Rober B, Galvez S and Willmitzer L (1995). Cloning and expression analysis of the cytosolic NADP(+)- dependent isocitrate dehydrogenase from potato. Implications for nitrogen metabolism. Plant Physiol. 107: 905-913.
PMid:7716247 PMCid:157207
Fujii S, Komatsu S and Toriyama K (2007). Retrograde regulation of nuclear gene expression in CW-CMS of rice. Plant Mol. Biol. 63: 405-417.
Gálvez S and Gadal P (1995). On the function of the NADP-dependent isocitrate dehydrogenase isoenzymes in living organisms. Plant Sci. 105: 1-14.
Gálvez S, Roche O, Bismuth E, Brown S, et al. (1998). Mitochondrial localization of a NADP-dependent [corrected] isocitrate dehydrogenase isoenzyme by using the green fluorescent protein as a marker. Proc. Natl. Acad. Sci. U. S. A. 95: 7813-7818.
PMid:9636233 PMCid:22766
Gibon Y, Blaesing OE, Hannemann J, Carillo P, et al. (2004). A Robot-based platform to measure multiple enzyme activities in Arabidopsis using a set of cycling assays: comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness. Plant Cell 16: 3304-3325.
PMid:15548738 PMCid:535875
Gueguen V, Macherel D, Jaquinod M, Douce R, et al. (2000). Fatty acid and lipoic acid biosynthesis in higher plant mitochondria. J. Biol. Chem. 275: 5016-5025.
Karpova OV, Kuzmin EV, Elthon TE and Newton KJ (2002). Differential expression of alternative oxidase genes in maize mitochondrial mutants. Plant Cell 14: 3271-3284.
PMid:12468742 PMCid:151217
Kirimura K, Yoda M, Kumatani M, Ishii Y, et al. (2002). Cloning and expression of Aspergillus niger icdA gene encoding mitochondrial NADP+-specific isocitrate dehydrogenase. J. Biosci. Bioeng. 93: 136-144.
Linke B and Börner T (2005). Mitochondrial effects on flower and pollen development. Mitochondrion 5: 389-402.
Maxwell DP, Nickels R and McIntosh L (2002). Evidence of mitochondrial involvement in the transduction of signals required for the induction of genes associated with pathogen attack and senescence. Plant J. 29: 269-279.
Moller S, Croning MD and Apweiler R (2001). Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 17: 646-653.
Nakai K and Horton P (1999). PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem. Sci. 24: 34-36.
Rhoads DM, Umbach AL, Subbaiah CC and Siedow JN (2006). Mitochondrial reactive oxygen species. Contribution to oxidative stress and interorganellar signaling. Plant Physiol. 141: 357-366.
PMid:16760488 PMCid:1475474
Sabar M, Gagliardi D, Balk J and Leaver CJ (2003). ORFB is a subunit of F1F(O)-ATP synthase: insight into the basis of cytoplasmic male sterility in sunflower. EMBO Rep. 4: 381-386.
PMid:12671689 PMCid:1319156
Sassi S, Gonzalez EM, Aydi S, Arrese-Igor C, et al. (2008). Tolerance of common bean to long-term osmotic stress is related to nodule carbon flux and antioxidant defenses: evidence from two cultivars with contrasting tolerance. Plant Soil 312: 39-48.
Sienkiewicz-Porzucek A, Sulpice R, Osorio S, Krahnert I, et al. (2010). Mild reductions in mitochondrial NAD-dependent isocitrate dehydrogenase activity result in altered nitrate assimilation and pigmentation but do not impact growth. Mol. Plant 3: 156-173.
PMid:20035036 PMCid:2807928
Sun Q, Hu C, Hu J, Li S, et al. (2009). Quantitative proteomic analysis of CMS-related changes in Honglian CMS rice anther. Protein J. 28: 341-348.
Sweetlove LJ, Heazlewood JL, Herald V, Holtzapffel R, et al. (2002). The impact of oxidative stress on Arabidopsis mitochondria. Plant J. 32: 891-904.
Tadege M and Kuhlemeier C (1997). Aerobic fermentation during tobacco pollen development. Plant Mol. Biol. 35: 343-354.
Teixeira RT, Knorpp C and Glimelius K (2005). Modified sucrose, starch, and ATP levels in two alloplasmic male-sterile lines of B. napus. J. Exp. Bot. 56: 1245-1253.
Touzet P and Budar F (2004). Unveiling the molecular arms race between two conflicting genomes in cytoplasmic male sterility? Trends Plant Sci. 9: 568-570.
Wang Y, Addess KJ, Chen J, Geer LY, et al. (2007). MMDB: annotating protein sequences with Entrez's 3D-structure database. Nucleic Acids Res. 35: D298-D300.
PMid:17135201 PMCid:1751549
Wise RP and Pring DR (2002). Nuclear-mediated mitochondrial gene regulation and male fertility in higher plants: Light at the end of the tunnel? Proc. Natl. Acad. Sci. U. S. A. 99: 10240-10242.
PMid:12149484 PMCid:124896
Yui R, Iketani S, Mikami T and Kubo T (2003). Antisense inhibition of mitochondrial pyruvate dehydrogenase E1α subunit in anther tapetum causes male sterility. Plant J. 34: 57-66.