Cation diffusion facilitator (CDF) proteins belong to a family of heavy metal efflux transporters that might play an essential role in homeostasis and tolerance to metal ions. We investigated the subcellular localization of Arabidopsis thaliana AtMTP1, a member of the CDF family, and its physiological role in the tolerance to Zn using MTP1-deficient mutant plants. AtMTP1 was immunochemically detected as a 43 kDa protein in the vacuolar membrane fractioned by sucrose density gradient centrifugation. The expression level of AtMTP1 in suspension-cultured cells was not affected by the Zn concentration in the medium. When AtMTP1 fused with green fluorescent protein was transiently expressed in protoplasts prepared from Arabidopsis suspension-cultured cells, green fluorescence was clearly observed in the vacuolar membrane. A T-DNA insertion mutant line for AtMTP1 displays enhanced sensitivity to high Zn concentrations ranging from 200 to 500 microM, but not to Zn-deficient conditions. Mesophyll cells of the mtp1-1 mutant plants grown in the presence of 500 microM Zn were degraded, suggesting that Zn at high concentrations causes serious damage to leaves and that AtMTP1 plays a crucial role in preventing this damage in plants. Thus we propose that AtMTP1 is localized in the vacuolar membrane and is involved in sequestration of excess Zn in the cytoplasm into vacuoles to maintain Zn homeostasis.

Plants are constantly exposed to threats from pathogenic microbes and thus developed an innate immune system to protect themselves. On the other hand, many plants also have the ability to establish endosymbiosis with beneficial microbes such as arbuscular mycorrhizal (AM) fungi or rhizobial bacteria, which improves the growth of host plants. How plants evolved these systems managing such opposite plant-microbe interactions is unclear. We show here that knockout (KO) mutants of OsCERK1, a rice receptor kinase essential for chitin signaling, were impaired not only for chitin-triggered defense responses but also for AM symbiosis, indicating the bifunctionality of OsCERK1 in defense and symbiosis. On the other hand, a KO mutant of OsCEBiP, which forms a receptor complex with OsCERK1 and is essential for chitin-triggered immunity, established mycorrhizal symbiosis normally. Therefore, OsCERK1 but not chitin-triggered immunity is required for AM symbiosis. Furthermore, experiments with chimeric receptors showed that the kinase domains of OsCERK1 and homologs from non-leguminous, mycorrhizal plants could trigger nodulation signaling in legume-rhizobium interactions as the kinase domain of Nod factor receptor1 (NFR1), which is essential for triggering the nodulation program in leguminous plants, did. Because leguminous plants are believed to have developed the rhizobial symbiosis on the basis of AM symbiosis, our results suggest that the symbiotic function of ancestral CERK1 in AM symbiosis enabled the molecular evolution to leguminous NFR1 and resulted in the establishment of legume-rhizobia symbiosis. These results also suggest that OsCERK1 and homologs serve as a molecular switch that activates defense or symbiotic responses depending on the infecting microbes.

Plants contain a large number of ATP-binding cassette (ABC) transporters belonging to different subclasses. AtPDR8 is the only member of the pleiotropic drug resistance (PDR) ABC transporter subclass in Arabidopsis that is constitutively highly expressed. In transgenic Arabidopsis plants harboring the AtPDR8 promoter fused to beta-glucuronidase (GUS), reporter expression was shown to be strong in the stomata and hydathode. In the stomata, transcripts of AtPDR8 were particularly frequent in the cells surrounding air spaces. Subcellular fractionation and immunochemical analysis showed that AtPDR8 was localized in the plasma membrane. When a knockout mutant of AtPDR8 (atpdr8) was infected with bacterial and oomycete pathogens, the plants exhibited chlorotic lesions and a hypersensitive response (HR)-like cell death. Cell death was detected in the atpdr8 mutants within 10 h of infection with the virulent bacterial pathogen, Pseudomonas syringae. As a result, the growth of P. syringae in the leaves of the atpdr8 mutant was reduced to 1% of that in the wild type. The defense response genes, PR-1, PR-2, PR-5, VPEgamma, AtrbohD and AtrbohF were highly expressed when the mutant plants were grown under non-sterile conditions. The expression of the AtPDR8 gene was enhanced by infection of virulent and avirulent bacterial pathogens. Our results indicate that AtPDR8 is a key factor controlling the extent of cell death in the defense response and suggest that AtPDR8 transports some substance(s) which is closely related to the response of plants to pathogens.

Arabidopsis thaliana AtMTP1 belongs to the cation diffusion facilitator family and is localized on the vacuolar membrane. We investigated the enzymatic kinetics of AtMTP1 by a heterologous expression system in the yeast Saccharomyces cerevisiae, which lacked genes for vacuolar membrane zinc transporters ZRC1 and COT1. The yeast mutant expressing AtMTP1 heterologously was tolerant to 10 mM ZnCl 2 . Zinc is a trace element essential as a cofactor for many enzymes and a structural element for various regulatory proteins (1-3). These proteins and enzymes include zinc-finger proteins, RNA polymerases, superoxide dismutase, and alcohol dehydrogenase. Thus, zinc deficiency causes severe symptoms in all organisms including plants. However, when present in excess, zinc can become extremely toxic, causing symptoms such as chlorosis in plants. The essential but potentially toxic nature of zinc necessitates precise homeostatic control mechanisms to satisfy the requirements of cellular activity and to protect cells from toxic effects.Plants have many kinds of zinc transporters and zinc channels (4 -6). Typical zinc transporters include metal tolerance protein (MTP) 3 (7, 8), ZIP (ZRT (zinc-regulated transporter)/ IRT (iron-regulated transporter)-like protein) (4), and HMA (heavy metal ATPase) (9) families. The MTP family in Arabidopsis thaliana consists of 12 members, and 4 members, MTP1-MTP4, form a subfamily. Both A. thaliana AtMTP1 (10, 11) and AtMTP3 (12) are localized on the membrane of central vacuoles. A similar transporter in the zinc-hyperaccumulating plant species Arabidopsis halleri, AhMTP1-3, has also been investigated (13). Zinc tolerance of A. halleri has been suggested to be due to an increased copy number of the MTP1 gene and enhanced level of transcription (13,14). AtMTP1 has high identity with A. halleri 14). AtMTP1, AhMTP1-3, and AtMTP3 belong to a ubiquitous family of transition metal transport proteins called the cation diffusion facilitator protein family, which have been identified in bacteria, Archaea, and eukaryotes and have been demonstrated to transport zinc, cobalt, and cadmium (15).AtMTP1 has been demonstrated to transport zinc from the cytosol into the vacuole in A. thaliana (10 -12) as well as AtMTP3 (12). AtMTP1 has been predicted from the hydropathy to have six transmembrane domains, long N-and C-terminal tails, and a long histidine-rich (His-rich) hydrophilic region between the fourth and fifth transmembrane domains (10, 16). A multiple histidine domain is also present in mammalian orthologues such as mouse . The His-rich domain in these members has been estimated to serve as a zinc binding region (16,17). However, the transport mechanism and structure-function relationship of these zinc transporters have not been clarified, although zinc transport activity of MTP1 was detected by using reconstituted proteoliposomes of the protein expressed in Escherichia coli (18) and by a yeast complementation assay with a zinc-hypersensitive double mutant of ZRC1 and COT1 (zrc1 cot1) (13,19).A. thaliana v...

In arbuscular mycorrhizal (AM) symbiosis, host plants supply photosynthates to AM fungi and, in return, they receive inorganic nutrients such as phosphate from finely branched fungal arbuscules. Plant cortical cells envelope arbuscules with periarbuscular membranes that are continuous with the plant plasma membranes. We prepared transgenic rice (Oryza sativa) plants that express a fusion of green fluorescent protein with rice AM-inducible phosphate transporter, OsPT11-GFP, and grew them with AM fungi. The fluorescence of the fusion transporter was observed in the arbuscule branch domain, where active nutrient exchange seems to occur. In contrast, a signal was not detected around intracellular hyphal coils on colonization by either Glomus mosseae or Gigaspora rosea, making the difference between Arum- and Paris-type mycorrhizae ambiguous. We also invented a simple device involving glass-bottomed Petri dishes for in planta observation of fluorescent proteins in living AM roots with an inverted fluorescence microscope. The plant bodies remain completely intact, avoiding any stressful procedure such as cutting, staining, etc. Since rice roots exhibit a very low level of autofluorescence, the device enabled clear time-lapse imaging to analyze the formation, function and degeneration of arbuscules. In cortical cells, arbuscules seemed to be functional for only 2-3 d. Suddenly, the arbuscular branches became fragile and they shrank. At this stage, however, the periarbuscular membranes appeared intact. Then, the fluorescence of the transporter disappeared within only 2.5-5.5 h. The collapse of arbuscules occurred in the subsequent several days. Thus, our device has a great advantage for investigation of dynamic features of AM symbiosis.