Identification, expression analysis and molecular modeling of Iron deficiency specific clone 3 (Ids3) like gene in hexaploid wheat

Graminaceous plants secrete iron (Fe) chelators called mugineic acid family phytosiderophores (MAs) from their roots for solubilisation and mobilization of unavailable ferric (Fe3+) ions from the soil. The hydroxylated forms of these phytosiderophores have been found more efficient in chelation and subsequent uptake of minerals from soil which are available in very small quantities. The genes responsible for hydroxylation of phytosiderophores have been recognized as iron deficiency-specific clone 2 (Ids2) and iron deficiency-specific clone 3 (Ids3) in barley but their presence is not reported earlier in hexaploid wheat. Hence, the present investigation was done with the aim:(i) to search for the putative Hordeum vulgare Ids3 (HvIds3) ortholog in hexaploid wheat, (ii) physical mapping of HvIds3 ortholog on wheat chromosome using cytogenetic stocks developed in the background of wheat cultivar Chinese Spring and (iii) to analyze the effect of iron starvation on the expression pattern of this ortholog at transcription level. In the present investigation, a putative ortholog of HvIds3 gene was identified in hexaploid wheat using different bioinformatics tools. Further, protein structure of TaIDS3 was modelled using homology modeling and also evaluated modelled structure behavior on nanoseconds using molecular dynamics based approach. Additionally, the ProFunc results also predict the functional similarity between the proteins of HvIds3 and its wheat ortholog (TaIds3). The physical mapping study with the use of cytogenetic stocks confines TaIds3 in the telomeric region of chromosome 7AS which supports the results obtained by bioinformatics analysis. The relative expression analysis of TaIds3 indicated that the detectable expression of TaIds3 induces after 5th day of Fe-starvation and increases gradually up to 15th day and thereafter decreases till 35th day of Fe-starvation. This reflects that Fe deficiency directly regulates the induction of TaIds3 in the roots of hexaploid wheat.

Cloning and Characterization of Deoxymugineic Acid Synthase Genes from Graminaceous Plants

Graminaceous plants have evolved a unique mechanism to acquire iron through the secretion of a family of small molecules, called mugineic acid family phytosiderophores (MAs). All MAs are synthesized from l-Met, sharing the same pathway from l-Met to 2′-deoxymugineic acid (DMA). DMA is synthesized through the reduction of a 3″-keto intermediate by deoxymugineic acid synthase (DMAS). We have isolated DMAS genes from rice (OsDMAS1), barley (HvDMAS1), wheat (TaD-MAS1), and maize (ZmDMAS1). Their nucleotide sequences indicate that OsDMAS1 encodes a predicted polypeptide of 318 amino acids, whereas the other three orthologs all encode predicted polypeptides of 314 amino acids and are highly homologous (82–97.5%) to each other. The DMAS proteins belong to the aldo-keto reductase superfamily 4 (AKR4) but do not fall within the existing subfamilies of AKR4 and appear to constitute a new subfamily within the AKR4 group. All of the proteins showed DMA synthesis activity in vitro. Their enzymatic activities were highest at pH 8–9, consistent with the hypothesis that DMA is synthesized in subcellular vesicles. Northern blot analysis revealed that the expression of each of the above DMAS genes is up-regulated under iron-deficient conditions in root tissue, and that of the genes OsDMAS1 and TaDMAS1 is up-regulated in shoot tissue. OsDMAS1 promoter-GUS analysis in iron-sufficient roots showed that its expression is restricted to cells participating in long distance transport and that it is highly up-regulated in the entire root under iron-deficient conditions. In shoot tissue, OsDMAS1 promoter drove expression in vascular bundles specifically under iron-deficient conditions.