The analysis of rapid responses in the transcriptome of Arabidopsis roots to a decreased iron (Fe) supply was studied using DNA microarrays and revealed candidate genes with putative roles in Fe homeostasis. In addition to the frequently reported induction of gene activity in response to Fe deficiency, the expression of a number of putative cationic metal transporters was found to rapidly decrease in response to Fe deficiency. In this report we have investigated a small family of five nodulin-like genes that show protein sequence similarity to AtVIT1 and likely have a function in regulation of Fe homeostasis. DNA microarray analysis showed a rapid decrease in transcript abundance for nodulin-like1 (At1g21140), nodulin-like2 (At1g76800), and nodulin-like21 (At3g25190). This decrease was significant after 6 h of Fe deficiency and persisted at least to 72 h. Nodulin-like3 (At3g43630) and Nodulin-like4 (At3g43660) did not respond to the Fe concentration in the microarray analysis. The nodulin-like family encoded presumptive membrane proteins with five calculated transmembrane domains, and all members had significant protein sequence homology to the vacuolar Fe transporters AtVIT1 and ScCCC1p. Homologs of all five nodulin-like genes were found in both di- and monocotyledon plants, as well as in Physcomitrella and Chlamydomonas. Promoter-β-glucuronidase (GUS) assays showed expression of the nodulin-like1 gene in roots, hypocotyls, and expanded cotyledons of two-week-old Arabidopsis seedlings with the greatest activity associated with the vascular bundle and the root stele. In the absence of Fe, GUS activity was greatly reduced and was only weakly visible in the stele and vascular bundle. In an attempt to identify the function of these nodulin-like proteins, we isolated knockout mutants for nodulin-like3 and nodulin-like21 from available T-DNA insertion lines. Although these mutants did not show dramatic changes in growth or in their ability to grow on Fe-deficient media or media containing from 5 to 120 μM Fe, the nodulin-like3 mutant had a significantly higher Fe concentration in the shoots and both nodulin-like3 and nodulin-like21 mutants had significantly decreased Fe in the roots. These results were taken as an indication, that some members of this nodulin-like family were directly involved in Fe homeostasis in plants.
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