Functional Analysis Of Voltage-dependent Anion Channel Genes (AtVDACs) In Arabidopsis Thaliana

Voltage-dependent anion channels (VDACs), known as the most abundant proteins in the outer mitochondrial membrane, are widely present in eukaryotic cells. VDACs mediate exchange of metabolites between mitochondria and cytoplasm, which is significant for mitochondrial functions and cellular energy transactions. In mammalian cells, VDACs are involved in mitochondria-mediated apoptosis. VDACs appear to be a convergence point for a variety of cell survival and death signals. Although functions of VDACs are predominantly investigated in yeasts and animals, research in plants was mainly focused on isoform characterization and analysis of localization and expression pattern. Relatively little is known about functions of plant VDACs. In this work, T-DNA insertion lines were used to analyze functions of AtVDAC1-AtVDAC4in Arabidopsis thaliana, which provided new evidence on functions of plant VDACs and molecular mechanism of mitochondrial regulation.Nineteen T-DNA insertion lines of AtVDAC1-AtVDAC4were characterized. Five homozygous mutants (vdacl-3, vdac2-4, vdac3-1, vdac3-2and vdac4-2) were identified, in which respective AtVDAC transcript levels were apparently reduced. vdac2-4and vdac4-2plants grew slower and could not yield seeds compared with wild-type plants. The vdacl-3plants exhibited reduced seed set.Phenotype observation indicated that mutation in AtVDACl caused abnormal petal development, shorter siliques and reduced seed set. Complemented vdacl-3plants could rescue the defective phenotype, indicating that the defective phenotype of vdacl-3plants resulted from the mutation in AtVDAC1. Genetic analysis indicated that female reproductive development was affected in vdacl-3plants. Obervation of ovule development in wild-type and vdacl-3plants using confocal laser scanning microscopy indicated that mutation in AtVDACl affected female gametophyte development. Analysis on formation and activity of pollen grains, as well as pollen germination and growth of pollen tubes showed that male gametophytic development in vdacl-3plants was not affected. RT-PCR and histochemical GUS-staining sssays showed that AtVDACl was ubiquitously expressed and its expression level in floral organs was relatively high. GFP-AtVDACl fusion proteins mainly localized to mitochondria. In addition, mitochondrial transmembrane potential (△Ψ) and ATP synthesis rate were detected, both of which are vital points in mitochondrial energetic metabolism. Results indicated that both ATP synthesis rate and△Ψ were obviously reduced in mitochondria from vdacl-3plants. As cytoskeletons and their associated proteins regulate mitochondrial respiration and a microtubule-associated kinesin AtKP1, identified by our lab previously, localized to mitochondria, analysis of interactions between AtVDAC1-AtVDAC4and AtKPl were performed in this study. Results demonstrated that only AtVDAC3specially interacted with AtKP1by yeast two-hybrid and luciferase complementation imaging assays.In summary, five mutants in AtVDAC1-AtVDAC4were isolated. vdacl-3, vdac2-4and vdac4-2plants exhibited defective phenotype in plant growth and development in various degrees, which suggested that AtVDAC1, AtVDAC2and AtVDAC4play important roles on plant growth and development. Phenotype observations indicated that AtVDAC1had crucial effect on petal growth and female reproductive development. Physiological and chemical analysis indicated that AtVDAC1is involved in maintenance of ATP level in cells via regulation of mitochondrial transmembrane potential and ATP synthesis.