Analysis of sequence, map position, and gene expression reveals conserved essential genes for iron uptake in Arabidopsis and tomato

Abstract

Arabidopsis (Arabidopsis thaliana) and tomato (Lycopersicon esculentum) show similar physiological responses to iron deficiency, suggesting that homologous genes are involved. Essential gene functions are generally considered to be carried out by orthologs that have remained conserved in sequence and map position in evolutionarily related species. This assumption has not yet been proven for plant genomes that underwent large genome rearrangements. We addressed this question in an attempt to deduce functional gene pairs for iron reduction, iron transport, and iron regulation between Arabidopsis and tomato. Iron uptake processes are essential for plant growth. We investigated iron uptake gene pairs from tomato and Arabidopsis, namely sequence, conserved gene content of the regions containing iron uptake homologs based on conserved orthologous set marker analysis, gene expression patterns, and, in two cases, genetic data. Compared to tomato, the Arabidopsis genome revealed more and larger gene families coding for the iron uptake functions. The number of possible homologous pairs was reduced if functional expression data were taken into account in addition to sequence and map position. We predict novel homologous as well as partially redundant functions of ferric reductase-like and iron-regulated transporter-like genes in Arabidopsis and tomato. Arabidopsis nicotianamine synthase genes encode a partially redundant family. In this study, Arabidopsis gene redundancy generally reflected the presumed genome duplication structure. In some cases, statistical analysis of conserved gene regions between tomato and Arabidopsis suggested a common evolutionary origin. Although involvement of conserved genes in iron uptake was found, these essential genes seem to be of paralogous rather than orthologous origin in tomato and Arabidopsis.

Figure 1.

Phylogenetic trees of Arabidopsis and tomato iron uptake homologs. Sequences were aligned using ClustalX. N-J Trees were generated; relative branch lengths are indicated. Arabidopsis sequences can be retrieved by their gene locus number from http://mips.gsf.de/proj/thal/db/search/search_frame.html. Tomato sequences can be retrieved using the TC numbers at http://www.tigr.org/tdb/tgi/lgi/searching/reports.html. The comparisons include homologs of IRT metal transporter (A) and NAS (B).

Figure 2.

Mapping of tomato iron uptake genes. A, LeIRT1, LeIRT2, and LeNRAMP3 were located on chromosome 2; B, LeNRAMP1 on chromosome 11; C, LeFRO-TC124302 on chromosome 3; D, LeFRO1 and LeFRO-TC129233 on chromosome 1. LeNAS (chloronerva) was previously fine mapped to chromosome 1 (Ling et al., 1996, 1999), and LeFER was fine mapped to chromosome 6 (Ling et al., 1996, 2002).

Figure 3.

COS marker analysis of tomato and Arabidopsis chromosomal regions containing iron uptake genes. Represented were two tomato chromosome linkage maps that were modified from the high density molecular marker map (on the left side: Tanksley et al., 1992) and the COS marker map (on the right side: Fulton et al., 2002). The original maps were downloaded from http://www.sgn.cornell.edu/maps/tomato_arabidopsis/synteny_map.html. Gene locus names of the corresponding Arabidopsis iron uptake genes were indicated. Arabidopsis gene locus names reflect the chromosomal position. In most cases, multiple Arabidopsis iron uptake homologs were found corresponding to a tomato homolog. Such multiple homologs were boxed. COS markers that were located within ±10-cM distance of tomato iron uptake genes and corresponded to Arabidopsis COS markers located within a range of 400 genes upstream or downstream of the iron uptake homologs (about ±2 Mb) were indicated. Multiple Arabidopsis COS markers that were homologs of a single tomato COS marker were boxed. To better illustrate the presence of conserved gene clusters in between tomato and Arabidopsis iron uptake gene regions, a color code was used to indicate clustered Arabidopsis genes (compare Table I and Supplemental Table I). Homologous iron uptake genes within ±400 genes (about ±2 Mb) are denoted by the same color (e.g. At4g19680-IRT2 and At4g19690-IRT1). Homologous Arabidopsis iron uptake genes that were located in different regions of the genome (at a distance of more than 400 genes) were highlighted by different colors. Similarly, Arabidopsis COS markers received the colors of the neighboring iron uptake genes if they were located within ±400 genes (about ±2 Mb; e.g. COS marker At4g20410 has the same color as At4g19690-IRT1). Therefore, iron uptake genes and COS markers represented by the same color on the same map form a cluster on a chromosome. The tomato regions analyzed contain LeIRT1, LeIRT2 on chromosome 2 (A); LeNRAMP1 on chromosome 11 (B); LeNRAMP3 on chromosome 2 (C); LeFRO-TC124302 on chromosome 3 (D); LeFRO1 and LeFRO-TC129233 on chromosome 1 (E); LeNAS on chromosome 1 (F); LeFER on chromosome 6 (G). With the exception of LeFER-AtFRU, tomato-Arabidopsis regions harboring homologous iron uptake genes showed levels of conserved gene content.

Figure 4.

Expression analysis of Arabidopsis iron uptake homologs in roots and leaves of plants grown upon low (−Fe) and sufficient iron supply (+Fe). A, IRT/ZIP genes; B, FRO genes; C, NAS genes; D, FRU gene. Expression analysis was performed by semiquantitative RT-PCR and Southern-blot analysis. Amplification of elongation factor cDNA EF1b-α (At5g19510) served as constitutive control.

Figure 5.

Expression analysis of tomato LeFRO-TC124302 in roots and leaves of wild-type and fer mutant tomato plants, grown upon sufficient iron supply and iron deficiency. Expression analysis was performed by semiquantitative RT-PCR and Southern-blot analysis. Amplification of elongation factor cDNA LeEF1a served as constitutive control. No expression was detected for LeFRO-TC129233 (data not shown).