Iron is a critical micronutrient for both plant and animal nutrition, serving as a required co-factor for a variety of cellular processes. Iron deficiency anemia is one of the leading human nutritional disorders worldwide, affecting 43% of the population of developing countries [1
]For most of the world's population, legumes are a major source of dietary iron [1
]. Though iron composes 5% of the earth's crust [3
] it is largely unavailable to plants, particularly in calcareous soils with a pH greater than 7.5. Calcareous soils are especially prevalent in the upper Midwest of the US [4
] and have been implicated in iron deficiency in soybeans. Iron deficiency chlorosis (IDC) in soybeans is characterized by interveinal chlorosis of the developing trifoliates [6
]contributing to yield loss directly proportional to the severity of the chlorosis [6
Plants have evolved two systems to uptake iron from the soil. These systems are termed strategy I and II [7
]. Soybeans and other dicots utilize strategy I, in which the rhizosphere is acidified by the release of protons to produce a favorable environment for the release of iron from chelating agents in the soil. A membrane bound reductase reduces iron to the usable Fe+2
form. The iron is then transported across the plasma membrane by a specific transporter for distribution and use within the plant. The reduction of the iron from Fe3+
has been shown to be the rate-limiting step in IDC [9
]. Graminaceous monocots utilize strategy II, whereby the roots release chelators called phytosiderophores to bind Fe+3
ions. Once bound, the entire complex is transported into the root where it is uncoupled. The Fe+3
ion is reduced to Fe+2
and the phytosiderophores are re-released into the soil.
The quantitative nature of IDC makes field studies problematic. Previous studies have identified multiple Quantitative Trait Locus (QTL) associated with IDC [4
]. Many of the same QTL have been identified in both field and greenhouse studies, where plants are grown in a hydroponics system designed specifically to induce IDC[10
]. Growing plants in a controlled greenhouse environment with regulated nutritional availability allows for reproducible induction of iron deficiency stress. In addition, the advent of microarray technology now allows for the identification of individual transcripts whose expression levels are affected by iron availability[11
]. The availability of a whole-genome sequence assembly for the soybean genome has, for the first time, allowed us to genetically position differentially expressed genes induced by iron deficiency.
Genomic studies in many organisms have shown genes in close proximity to one another in the genome are often co-expressed. These co-expressed genes create clusters of expression neighborhoods [13
] which are conserved by natural selection [14
] A study in Arabidopsis showed clusters of up to 20 different genes were coordinately regulated, with a median cluster size of 100 kb [15
]. In rice, approximately five percent of the genome has been associated with co-expressed gene clusters [16
]. Initially co-expressed genes were thought to belong to similar biological pathways [15
], but further studies have shown co-functionality to be a poor predictor of co-expression [17
]. Instead, promoter analysis has found co-regulated genes are often regulated by common transcription factors [13
] The co-expression of clustered genes may be partially regulated by the interaction of common promoter elements and transcription factors [18
]. Co-regulated genes often have common transcription factors [17
], so an increase in the number of transcription factor binding sites in promoter regions would increase the likelihood of the transcription factor binding and aiding in the expression of the gene cluster.
The objectives of our research are to identify a list of candidate genes with a potential involvement in soybean iron deficiency and to associate these genes with the genome sequence to determine any correlation with previously identified QTL. We also wanted to determine whether the changes in candidate gene expression were due to structural or sequence differences in the candidate genes. The results from these analyses confirmed the co-expressed genes were co-localized and possibly coordinately regulated.