E.L. WALKER LAB

1: Mechanisms of iron uptake by grasses.
We would like to understand the mechanisms that higher plants use to acquire iron from the soil and then allocate it correctly into cells and tissues. We began this work with cloning and characterization of maize YS1, which is responsible for primary iron uptake by the world's major grain crops (rice, wheat, corn). The grasses use a chelation strategy for primary iron uptake. In response to iron starvation, grasses secrete phytosiderophores (PS): non-proteinogenic amino acid derivatives of the mugineic acid (MA) acid family that form stable Fe (III) chelates. This accomplishes solubilization of the otherwise insoluble soil iron. Specific uptake systems located at the root surface then move the Fe (III)-PS complexes into root cells. The membrane bound Fe (III)-PS transporter of maize is Yellow Stripe 1 (YS1). Maize YS1 appears to have a dual function. Not only is it responsible for primary iron uptake from the soil, but also it has a second, still undefined, role in leaves. In order to elucidate the physiological role of YS1 in maize leaves, and also to refine our understanding of the role of YS1 in roots, the student taking on this project will generate and analyze reporter gene constructs that will allow precise localization of ZmYS1 and its transcripts. By observing localization of YS1, we will discover the identity of the cells that are actively taking up iron via this transporter, and will be able to distinguish whether YS1 is involved in, e.g., phloem loading, xylem unloading, or loading of iron into mesophyll cells.

2: Mechanisms of iron uptake by grasses.
We would like to understand the mechanisms that higher plants use to acquire iron from the soil and then allocate it correctly into cells and tissues. We began this work with cloning and characterization of maize YS1, which is responsible for primary iron uptake by the world's major grain crops (rice, wheat, corn). The grasses use a chelation strategy for primary iron uptake. In response to iron starvation, grasses secrete phytosiderophores (PS): non-proteinogenic amino acid derivatives of the mugineic acid (MA) acid family that form stable Fe (III) chelates. This accomplishes solubilization of the otherwise insoluble soil iron. Specific uptake systems located at the root surface then move the Fe (III)-PS complexes into root cells. The membrane bound Fe (III)-PS transporter of maize is Yellow Stripe 1 (YS1). Cloning of YS1 allowed us to identify the Yellow Stripe-Like (YSL) family of genes, which is present in all higher plants, and, in non-grasses, participates in movement of iron bound to a compound called nicotianamine. Nicotianamine is structurally related to phytosiderophores, but is used internally in plants to maintain iron homeostasis. It is now clear that Fe (III)-PS is not a suitable substrate for any of the YSLs in the non-grass species, Arabidopsis thaliana. This suggests that maize YS1 (and presumably the YS1 orthologs in other grass species) have an additional functionality that most family members do not share. True YS1 orthologs are still difficult to identify, because we remain ignorant of the sequence differences that allow phytosiderophore versus nicotianamine transport. We have identified a putative YS1 ortholog in rice. The student taking on this project will generate clones of the rice YS1 and will use functional complementation in yeast to address whether this gene is a bona fide iron-phytosiderophore transporter. Ultimately, we will use the information gained from this project to design experiments that will address the structural aspects of YS1 function.

3: Mechanisms of iron homeostasis in Arabidopsis.
We would like to understand the mechanisms that higher plants use to acquire iron from the soil and then allocate it correctly into cells and tissues. We began this work with cloning and characterization of maize YS1, which is responsible for primary iron uptake by the world's major grain crops (rice, wheat, corn). The grasses use a chelation strategy for primary iron uptake. In response to iron starvation, grasses secrete phytosiderophores (PS): non-proteinogenic amino acid derivatives of the mugineic acid (MA) acid family that form stable Fe (III) chelates. This accomplishes solubilization of the otherwise insoluble soil iron. Specific uptake systems located at the root surface then move the Fe (III)-PS complexes into root cells. The membrane bound Fe (III)-PS transporter of maize is Yellow Stripe 1 (YS1). Cloning of YS1 allowed us to identify the Yellow Stripe-Like (YSL) family of genes, which is present in all higher plants, and, in non-grasses, participates in movement of iron bound to a compound called nicotianamine. In addition to iron, nicotianamine efficiently chelates other transition metals like manganese, copper and zinc. We would like to know whether specific members of the YSL family in Arabidopsis transport these other complexes, or whether all YSLs exclusively transport iron complexes. We have identified a candidate YSL from Arabidopsis that may not be an iron-nicotianamine transporter (AtYSL6). The student taking on this project will examine whether the YSL6 protein, if it is expressed under a YSL3 promoter, will complement the iron-associated defects of ysl3 mutant plants. Complementation would indicate that YSL6 is an iron transporter, just as other members of the YSL family are. Failure to complement would indicate that YSL6 does not function as an iron transporter, and would suggest that YSL6 transports metal-nicotianamine complexes other than iron.

4: Paclitaxel (Taxol ©) production in cell cultures. One aspect of our research program is the understanding and control of cellular metabolism. These studies are conducted in collaboration with the Roberts (Chemical Engineering) and Normanly (Biochemistry and Molecular Biology) laboratories. The primary model system used is the Taxus plant cell culture system for the production of the anticancer agent paclitaxel. Variability in metabolite production is inherent to plant cell culture systems and has placed major limitations on the commercial use of this technology. Taxane accumulation in Taxus suspension cultures is highly variable. We are seeking to understand the molecular events that govern this variability. We have previously characterized the transcript accumulation profiles for paclitaxel biosynthetic genes in cultures that are actively accumulating this metabolite. The student taking on this project will examine transcript accumulation profiles for paclitaxel biosynthetic genes in cultures that accumulate only early or intermediate taxanes, but fail to accumulate the side chain taxanes paclitaxel and cephalomannin. These profiles will elucidate whether paclitaxel synthesis in these cultures fails as the result of a failure to accumulate the relevant transcripts, or instead fails due to repression at the protein level.