Joohyun Lee

Relatively little is known about how metals such as iron are effluxed from cells, a necessary step for transport from the root to the shoot. Ferroportin (FPN) is the sole iron efflux transporter identified to date in animals, and there are two closely related orthologs in Arabidopsis thaliana, IRON REGULATED1 (IREG1/FPN1) and IREG2/FPN2. FPN1 localizes to the plasma membrane and is expressed in the stele, suggesting a role in vascular loading; FPN2 localizes to the vacuole and is expressed in the two outermost layers of the root in response to iron deficiency, suggesting a role in buffering metal influx. Consistent with these roles, fpn2 has a diminished iron deficiency response, whereas fpn1 fpn2 has an elevated iron deficiency response. Ferroportins also play a role in cobalt homeostasis; a survey of Arabidopsis accessions for ionomic phenotypes showed that truncation of FPN2 results in elevated shoot cobalt levels and leads to increased sensitivity to the metal. Conversely, loss of FPN1 abolishes shoot cobalt accumulation, even in the cobalt accumulating mutant frd3. Consequently, in the fpn1 fpn2 double mutant, cobalt cannot move to the shoot via FPN1 and is not sequestered in the root vacuoles via FPN2; instead, cobalt likely accumulates in the root cytoplasm causing fpn1 fpn2 to be even more sensitive to cobalt than fpn2 mutants.

Arabidopsis (Arabidopsis thaliana) contains about 130 ATP-binding cassette (ABC) proteins, which are likely to contribute to the transport of diverse materials, including toxic substances. However, the substrates of ABC transporters remain unknown in most cases. We tested which ABC transporter is involved in detoxification of lead [Pb(II)]. Among the many tested, we found that the message level of only AtPDR12 increased in both shoots and roots of Pb(II)-treated Arabidopsis, suggesting that it may be involved in the detoxification of Pb(II). AtPDR12-knockout plants (atpdr12) were used to further test this possibility. In Pb(II)-containing medium, atpdr12 plants grew less well and had higher Pb contents than those of wild-type plants. In contrast, AtPDR12-overexpressing Arabidopsis plants were more resistant to Pb(II) and had lower Pb contents than wild-type plants. The mutant phenotypes and their Pb contents, as well as the localization of the GFP:AtPDR12 fusion protein at the plasma membrane, suggest that AtPDR12 functions as a pump to exclude Pb(II) and/or Pb(II)-containing toxic compounds from the cytoplasm. Inhibition of glutathione synthesis by addition of buthionine sulfoximine to the growth medium exacerbated the Pb(II)-sensitive phenotype of atpdr12 plants, consistent with a glutathione-dependent detoxification mechanism operating in parallel with an AtPDR12-dependent mechanism. Thus, we propose that AtPDR12 is an ABC transporter that contributes to Pb(II) resistance in Arabidopsis.

Cadmium (Cd) is a widespread soil pollutant; thus, the underlying molecular controls of plant Cd tolerance are of substantial interest. A screen for wheat (Triticum aestivum) genes that confer Cd tolerance to a Cd hypersensitive yeast strain identified Heat shock transcription factor A4a (HsfA4a). Ta HsfA4a is most similar to the class A4 Hsfs from monocots. The most closely related rice (Oryza sativa) homolog, Os HsfA4a, conferred Cd tolerance in yeast, as did Ta HsfA4a, but the second most closely related rice homolog, Os HsfA4d, did not. Cd tolerance was enhanced in rice plants expressing Ta HsfA4a and decreased in rice plants with knocked-down expression of Os HsfA4a. An analysis of the functional domain using chimeric proteins constructed from Ta HsfA4a and Os HsfA4d revealed that the DNA binding domain (DBD) of HsfA4a is critical for Cd tolerance, and within the DBD, Ala-31 and Leu-42 are important for Cd tolerance. Moreover, Ta HsfA4a-mediated Cd resistance in yeast requires metallothionein (MT). In the roots of wheat and rice, Cd stress caused increases in HsfA4a expression, together the MT genes. Our findings thus suggest that HsfA4a of wheat and rice confers Cd tolerance by upregulating MT gene expression in planta.

Molybdenum (Mo) is an essential micronutrient for plants, serving as a cofactor for enzymes involved in nitrate assimilation, sulfite detoxification, abscisic acid biosynthesis, and purine degradation. Here we show that natural variation in shoot Mo content across 92 Arabidopsis thaliana accessions is controlled by variation in a mitochondrially localized transporter (Molybdenum Transporter 1 - MOT1) that belongs to the sulfate transporter superfamily. A deletion in the MOT1 promoter is strongly associated with low shoot Mo, occurring in seven of the accessions with the lowest shoot content of Mo. Consistent with the low Mo phenotype, MOT1 expression in low Mo accessions is reduced. Reciprocal grafting experiments demonstrate that the roots of Ler-0 are responsible for the low Mo accumulation in shoot, and GUS localization demonstrates that MOT1 is expressed strongly in the roots. MOT1 contains an N-terminal mitochondrial targeting sequence and expression of MOT1 tagged with GFP in protoplasts and transgenic plants, establishing the mitochondrial localization of this protein. Furthermore, expression of MOT1 specifically enhances Mo accumulation in yeast by 5-fold, consistent with MOT1 functioning as a molybdate transporter. This work provides the first molecular insight into the processes that regulate Mo accumulation in plants and shows that novel loci can be detected by association mapping.