EAGER: Iron sensing and signaling in Arabidopsis thaliana

University Of Massachusetts Amherst, Amherst MA

Investigators

Abstract

Living organisms keep the level of the highly reactive metal, iron, carefully controlled to avoid cellular damage. In plants, part of this control is a mechanism to allow the shoot, where iron is most heavily needed, to signal iron status to roots where primary iron uptake occurs. The molecular details of this shoot to root signaling mechanism are unknown, but are likely to be essential for successful engineering of plants to accumulate additional bioavailable iron in edible parts. This project explores the hypothesis that Yellow Stripe1-Like proteins are responsible for signaling the iron status between the shoot and the root of plants. Our current state of ignorance about many of the mechanisms involved in plant iron homeostasis is a major obstacle to devising approaches for biofortification of staple foods with iron. Biofortification refers to the genetic engineering of staple crops to accumulate additional bioavailable iron in edible parts; it is widely regarded as a sustainable means of improving the iron nutrition of the 2-3 billion people worldwide whose inadequate diet causes iron deficiency anemia. Improving our understanding of plant iron homeostatic mechanisms is also critical if we wish to improve growth of crops in marginal soils, where iron deficiency frequently limits crop growth. A double mutant with null mutations in the Arabidopsis Yellow Stripe-Like1 and Yellow Stripe-Like3 (ysl1ysl3) has severe pleiotropic growth defects related to iron. YSLs are transporters of iron and other transition metals (Zn, Cu, Mn, Ni) complexed with the plant-specific metal chelating compound nicotianamine (NA). It is important to note that both tissue localization (mainly in the leaf veins) and regulation (down-regulated by iron deficiency) argue against the idea that these proteins participate in primary metal uptake from the soil. The phenotype of ysl1ysl3 plants, which includes iron deficiency chlorosis, low Fe levels in tissues, failure to re-translocate Cu and Zn from leaves during senescence, pollen failure, and incomplete embryo development, were at first all thought to be consequences of failed metal-NA transport by AtYSL1 and AtYSL3. But ysl1ysl3 plants also exhibit defects in Fe signaling, which are difficult to explain as a simple consequence of metal transport defects in leaf veins. New evidence has indicated that ysl1ysl3 plants have a pervasive inability to respond vigorously to iron deficiency, and a new hypothesis is put forward that AtYSL1 and AtYSL3 function as "transceptors": transporters that also function as receptors for the iron status of the plant. In this project, we will investigate the hypothesis that, in addition to their roles as metal-NA transporters, AtYSL1 and AtYSL3 function in signaling the iron status of shoots. A key test of this idea is to make mutations in YSL1 and YSL3 that affect receptor function without affecting transporter function, and vice versa. In this project, transport defective versions of YSL1 and YSL3 will be made using site directed mutagenesis and will be tested for functionality in yeast. Three properly localized transport defective versions will then be introduced into the ysl1ysl3 double mutant under their own promoters, and the resulting plants will be tested for ysl1ysl3 phenotypes. In a second, more limited experiment, the acidic N-terminal domain(s) of YSL1 and YSL3 will be tested. These domains may be iron binding, and thus could be involved in iron sensing. We will test whether removing these domains affects transport function in yeast, and if it does not, the proteins will be tested in planta for complementation of ysl1ysl3 phenotypes. These experiments will elucidate whether YSL1 and YSL3 are directly involved in iron signaling. This project will be integrated into a laboratory course for undergraduates and provide training in bioinformatics and genomics methodologies, as well as the process of hypothesis-driven research.