ABSTRACT
A cDNA coding for a putative organic cation transporter (OCT) was isolated from Phaseolus vulgaris roots by differential display RT-PCR and the corresponding full-length cDNA (named PvOCT1) was subsequently obtained by RACE-PCR. Hydropathy profiles of the deduced amino acid sequence (547 residues) predicted the existence of twelve membrane-spanning domains, which are highly conserved in the major facilitator superfamily (MFS). Three specific domains, which characterize organic ion transporters in animals, can also be observed in the predicted protein. In the non-stressed plants, northern analysis showed that PvOCT1 is strongly expressed in roots and stems, while in situ hybridization revealed the presence of PvOCT1 transcripts in phloem cells. In roots PvOCT1 transcript levels transitorily increased after one hour of dehydration and then dramatically decreased. This decrease was associated with enhanced abundance of PvNeED1 mRNA encoding the enzyme thought to catalyze the limiting step of abscisic acid biosynthesis.
Subject(s)
Organic Cation Transporter 1/genetics , Phaseolus/genetics , Plant Proteins/genetics , Plant Structures/metabolism , Water/pharmacology , Amino Acid Sequence , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Gene Expression Regulation, Plant/drug effects , Molecular Sequence Data , Organic Cation Transporter 1/metabolism , Plant Proteins/metabolism , Plant Structures/genetics , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
Plant development requires stringent controls between cell proliferation and cell differentiation. Proliferation is positively regulated by cyclin dependent kinases (CDKs). Acting in opposition to CDKs are CDK inhibitors (CKIs). The first tobacco CKI (NtKIS1a) identified was shown to inhibit in vitro the kinase activity of CDK/cyclin complexes and to interact with CDK and D-cyclins. However, these features, which are common to other plant and animal CKIs already characterised, did not provide information about the function of NtKIS1a in plants. Thus, to gain insight into the role of NtKIS1a and especially its involvement in cell proliferation during plant development, we generated transgenic Arabidopsis thaliana plants that overexpress NtKIS1a. These plants showed reduced growth with smaller organs that contained larger cells. Moreover, these plants displayed modifications in plant morphology. These results demonstrated that plant organ size and shape, as well as organ cell number and cell size, might be controlled by modulation of the single NtKIS1a gene activity. Since in mammals, D-cyclins control cell cycle progression in a CDK-dependent manner but also play a CDK independent role by sequestering the CKIs p27(Kip1) and p21(Cip1), we tested the significance of cyclin D-CKI interaction within a living plant. With this aim, NtKIS1a and AtCycD3;1 were overexpressed simultaneously in plants by two different methods. Our results demonstrated that overexpression of the CKI NtKIS1a restores essentially normal development in plants overexpressing AtCycD3;1, providing the first evidence of cyclin D-CKI co-operation within the context of a living plant.
