Molecular cloning and characterization of ARF1 and COPI coat proteins from Medicago truncatula cv. Jemalong: Dissection of their role in vesicular transport in root cells

Abstract

The integrity of the Golgi apparatus in both plant and mammalian cells is dependent upon a coordinated flow of COPII (coatomer protein) coated vesicles in anterograde (forward) and of COPI coated vesicles in retrograde (backward) direction. Although a fair amount of work on vesicular trafficking has been published in Arabidopsis thaliana not much information is available related to the secretory pathway in other higher plants. In the present study we have used Medicago truncatula, a model plant for legume species as for symbiotic and pathogenic interactions, to identify Arf1 and COPI components of the early secretory pathway. Their localisation and interaction with the Golgi apparatus in the root cells has been identified by confocal and two-photon laser microscopy. EST databases of the M. truncatula were screened and putative homologues for all seven coatomer proteins and MtArf1 were identified. Two isoforms of the ζ- COPI subunit and MtArf1 genes were isolated from M. truncatula cDNA libraries and were sequenced. GFP fusions of MtArf1, dominant active (Q71L), dominant inactive (T31N) forms of MtArf1and two isoforms of MtζCOP1 were expressed in transgenic “hairy root” and their subcellular localisation were analyzed. Our results show that MtArf111::GFP, Mtζ-COP1:: GFP and Mtζ-COP2::GFP were localized on mobile Golgi structures, streaming along the ER network and were sensitive to brefeldin A, indicating their potential association with Golgi stacks. This study demonstrates an important role of Arf1 and COP1 proteins in early secretory pathway in root cells of M. truncatula.