Genome-wide Analysis Of The Nucleocytoplasmic Transport Receptors In Arabidopsis And Functional Characterization Of AtIMPα1 And AtIMPα2

The eukaryotic cell is characterized by the existence of a bilayered nuclear envelope separating the cell into two compartments: the nucleus and the cytoplasm. This segregation of nuclear transcriptional regulation from cytoplasmic metabolic and translational events necessitates the mutual communication between these two compartments, which is realized exclusively through the nuclear pore complexes that span the NE. Ions and metabolites as well as small neutral proteins that do not bind to nucleoporins run through the NPCs by passive diffusion. By contrast, most macromolecules must be actively transported through the NPCs in a receptor-mediated and energy-dependent fashion. Import and export of most proteins and RNAs is accomplished with the participation of two large evolutionarily conserved families of transport factors, importin (IMP)αandβ. IMPαs function as adaptors between NLSs and IMPβin transporting cargo from the cytoplasm to the nucleoplasm. As well,IMPβs are able to bind cargos directly.During the past few years, substantial progress has been made towards characterizing the factors participating in nucleocytoplasmic trafficking and towards elucidating the signaling pathways employing nucleocytoplasmic transport machinery in Arabidopsis. However, these findings are limited within very few receptor members and their functional mechanisms are somewhat elusive. In this study, we comprehensively analyzed the expression levels of these receptors in different tissues, and examined their responsiveness to different hormones and environmental stresses. The main results were as follows:1. The Arabidopsis genome encodes at least 8 IMPα-like proteins and 18 IMPβ-like proteins. AtIMPαs show similar gene structures and protein structures, which indicate that they could be traced back to a common ancestor. However, AtIMPβs may derive from different ancestors. They display different protein structures, implying their different functions.2. AtIMPαs/βs distribute on chromosome 1-5. AtIMPα1-AtIMPα2, AtIMPα3-AtIMPα6, AtIMPβ6-AtIMPβ12, AtIMPβ8 -AtIMPβ15 are duplicated gene pairs.3. We employed a semi-quantitative RT-PCR approach to detect the mRNA accumulations of each AtIMPα/βin root, rosette leaf, cauline leaf, stem, flower and silique tissues. The results showed that the majority of genes were constitutively expressed in different tissues with a similar level and only a few genes displayed tissue-specific expression patterns.4. We examined AtIMPαs/βs expression levels under different hormonal or environmental conditions by semi-quantitative RT-PCR analysis. The mRNA levels of most IMPαs/βs were not regulated by hormonal or environmental treatments, whereas the transcriptional enhancement or reduction was observed in some members.5. In order to characterize AtIMPα1 and AtIMPα2, we constructed impα1impα2 double mutant. impα1impα2 double mutant exhibited smaller and curled leaves, shorter petiole, fewer leaf number, delayed flowering. Compared with wild type plants, impα1impα2 double mutant plants were hypersensitive to ABA, which may result from the enhanced expression of ABA metabolic genes and responsive genes.6. The microarray data displayed that many biological pathways have been influenced in impα1impα2 double mutant plants, such as response to stress, response to biotic or abiotic stimuli, development process, protein metabolism, transcription process and so on.