Research article
Members of a small family of nodulin-like genes are regulated under iron deficiency in roots of Arabidopsis thaliana

https://doi.org/10.1016/j.plaphy.2011.02.011Get rights and content

Abstract

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.

Highlights

► Five nodulin-like Arabidopsis genes belong to the CCC1-like gene family. ► Expression of 4 of these nodulin-like genes was positively correlated with Fe supply. ► Promoter-driven GUS expression was positively correlated with Fe supply. ► Nodulin-like T-DNA mutants showed altered Fe distribution in planta. ► A role for these nodulin-like genes in Fe storage was proposed.

Introduction

The importance of Fe as a nutritional element is explained by its crucial role in primary metabolism and stands in juxtaposition to its restricted availability in the environment [1]. The mechanisms of response to Fe deficiency and the homeostatic regulation of the Fe concentration in the cell have been frequently reviewed in the recent literature e.g.[2], [3]. In spite of the intense efforts to elucidate the molecular mechanisms that are responsible for sensing and signaling Fe deficiency, it is currently not possible to answer basic questions of how Fe deficiency is sensed by and communicated throughout plants. The response of plants to Fe deficiency involves both an increased capacity for Fe uptake [1] and a more efficient use of available Fe [4], [5]. In recent publications, we have investigated rapid changes in the Arabidopsis transcriptome in response to Fe deficiency [6], [7]. Prominent in the list of regulated transcripts are transport proteins that presumably regulate the metal concentration in cellular compartments by increasing the Fe uptake capacity into the cell or by decreasing Fe storage. As is expected, the transcripts encoding the Fe2+ transporters, AtIRT1 [8] and AtIRT2 [9], and the corresponding ferric-chelate reductases, AtFRO2 [10] and AtFRO3 [11], are increased within 6 h of Fe-deficient growth. Additionally, transcripts for an oligopeptide transporter (AtOPT3) [12] and an amino acid transporter (At5g38820) are increased in parallel with AtIRT1 and AtIRT2. Although the function of these later proteins with regard to Fe homeostasis is not known, they may catalyze transport of Fe–peptide complexes across the membrane or transport of the amino acids needed for synthesis of these peptides. Finally, transcripts for the vacuolar Fe efflux carrier, AtNRAMP4, are also increased under Fe deficiency [13], [14]. With the assumption that transcriptional changes are reflected in altered Fe fluxes, the effect of this adaptation would be to increase cytoplasmic Fe in root cells.

In contrast to transcriptional changes that indicated increased Fe uptake as an adaptation to Fe deficiency, a number of changes were observed for transporters that were not involved in Fe transport. A rapid induction was observed for MTPc3 and MTPa2 [15], [16], which catalyzed cationic metal export from the cell and Zn2+ uptake into the vacuole, respectively. The Zn2+ plasma membrane influx carrier, AtZIP3, was decreased under Fe deficiency [6], while transcript abundance increased for AtCOPT2, a plasma membrane Cu+ transporter [17]; for AtZIF1 [16], a presumptive vacuolar Zn2+-chelate transporter; and for AtIREG1 and AtIREG2 [18]. These responses to Fe deficiency could be understood in terms of regulation of an imbalance in Zn2+, Ni2+, Cu+, and/or Co2+ homeostasis in the Fe-deficient cell [18], [19]. Under Fe deficiency, an increased capacity of Zn2+ uptake, for example, might have resulted in potentially toxic concentrations of Zn in the cytoplasm. In support of this hypothesis, there was a correlation between Fe deficiency and increased shoot concentrations of Zn, Mn and Co [20].

Although much attention has been directed to transcript induction in response to Fe deficiency, we have also identified three nodulin-like genes whose transcript abundance was repressed under Fe deficiency [6], [7]. The encoded proteins were of potential importance to Fe homeostasis, because they contained a CCC1-like domain. CCC1 was initially discovered in yeast as a vacuolar Fe transporter [21], [22]. In Arabidopsis, AtVIT1 encoded a CCC1 homolog that catalyzed Fe uptake into the vacuole [23]. If the functions of these three nodulin-like proteins were similar to VIT1, then the decreased abundance of these transcripts would have been associated with a decreased capacity of the cell to store Fe in a cellular compartment.

In this report we have investigated the function of a small family of five nodulin-like proteins that all contained a CCC1-like domain and showed distant homology to AtVIT1. We presented evidence that some members of this family may participate in regulation of Fe homeostasis in plants.

Section snippets

Standardized response of nodulin-like genes to Fe deficiency

In previous reports [6], [7] we have identified a group of three so-called nodulin-like genes, whose transcript abundance was repressed under conditions of Fe-deficient growth. In an effort to identify further, related genes, the Arabidopsis genome was searched for homologies to the deduced amino acid sequences of these three nodulin-like genes. In total five genes were identified. For the purpose of this report, they were referred to as: nodulin-like1 (At1g21140), nodulin-like2 (At1g76800),

Expression of the nodulin-like gene family by Fe supply

Based on a similar response to Fe deficiency and sequence homology, we have identified a small family of nodulin-like genes whose expression was positively correlated to the Fe concentration in the growth media. Combined results from DNA microarray analyses, semi-quantitative, reverse transcription PCR and from promoter-GUS fusion constructs showed a consistent pattern of transcriptional regulation for nodulin-like1, nodulin-like2 and nodulin-like21 (Fig. 1, Fig. 2, Fig. 3, Fig. 4). In

Plant material

Arabidopsis seeds were surface-sterilized by immersion in 1.5% (v/v) NaOCl for 15 min and rinsed four times in sterile water. Seeds were vernalized for 3 d at 4 °C in the dark. Arabidopsis thaliana L. Col-0 was grown hydroponically in a growth chamber at a constant relative humidity of 75%, under a 10/14 h day/night cycle (Osram E40/ES Plantastar, 300 μmol cm−2s−1) at 21 °C (day) or 18 °C (night). Two to four seeds were sown in 1.5 ml Eppendorf® tubes that had been previously filled with 1.5 ml

Acknowledgments

We thank members of the Buckhout and Schmidt laboratories, in particular Susanne Olstowski-Jacoby and Marion Dewender, whose dedication and support were an essential part of this research effort.

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