Current Research Projects:
Characterization of the AltSB Protein:
We have developed a peptide antibody for AltSB and verified it specifically cross-reacts with AltSB. This antibody is now being used to see if over a 6 day exposure of sorghum roots to Al there is a correlation between increases in AltSB gene expression and AltSB protein abundance. We will soon, in collaboration with Dr. Mayandi Sivaguru, Microscopy Facility Manager for the Institute for Genomic Biology at the University of Illinois, will be using this antibody to conduct immunolocalization studies of AltSB protein in root tip cells of tolerant and sensitive sorghum, to determine the membrane location of the protein. At the same time, we will also determine cell-specific localization of AltSB expression
We will be using the split ubiquitin yeast 2-hybrid system developed specifically for membrane proteins to identify cellular membrane and soluble proteins that interact with AltSB. To develop this resource for our use, a full-length AltSB ORF has been cloned into the bait vector of the split ubiquitin system and we have verified that the bait construct correctly expresses the AltSB protein in yeast cells. We have also verified that the AltSB-bait construct does not display any self-activation when transformed together with an empty library vector, a prerequisite for carrying on the following steps of the split ubiquitin system. To construct customized cDNA libraries for the split ubiquitin system, we have isolated high-quality total RNA from Al-treated root tip samples from an Al-tolerant sorghum line. From these RNA samples, we have synthesized high-quality double-stranded cDNAs. We are beginning the construction of several DNA libraries with these double-stranded cDNA samples. The cDNA libraries will then be used for screens for cellular membrane and/or soluble proteins that physically interact with AltSB, and possibly play a role in regulating AltSB function.
Characterization of the AltSB homolog in Arabidopsis:
Aluminum (Al) activated root malate and citrate exudation play an important role in plant Al tolerance. In this study, we report on AtMATE, a homolog of the recently discovered sorghum and barley Al tolerance genes, shown here to encode an Al-activated citrate transporter in Arabidopsis. Together with the previously characterized Al-activated malate transporter, AtALMT1, this discovery allowed us to examine the relationship in the same species between members of the two gene families for which Al tolerance genes have been identified. AtMATE is expressed primarily in roots and is induced by Al. An AtMATE T-DNA knock-down line exhibits very low AtMATE expression and Al-activated root citrate exudation is abolished. An AtALMT1 AtMATE double mutant lacks both Al-activated root malate and citrate exudation and exhibits greater Al sensitivity than the AtALMT1 mutant. Therefore, although AtALMT1 is a major contributor to Arabidopsis Al tolerance, AtMATE also makes a significant although smaller contribution. The expression patterns of AtALMT1 and AtMATE and the profiles of Al-activated root citrate and malate exudation are not affected by the presence or absence of the function of the other gene. These results suggest that AtALMT1-mediated malate exudation and AtMATE-mediated citrate exudation evolved independently in conferring Arabidopsis Al tolerance. However, a link between the regulation of expression of the two transporters in response to Al was found work on STOP1, a transcription factor previously shown to be necessary for AtALMT1 expression. We show that STOP1 is also required for AtMATE expression and Al-activated citrate exudation
Phytoremediation Thlaspi project
Metal hyperaccumulating plant species are plants that are endemic to metalliferous soils with the capability to tolerate and accumulate metals within their above ground tissues (shoot) to very high concentrations. In the Kochian Lab we study one such hyperaccumulator, Thlaspi caerulescens, which is widely studied for its remarkable properties to tolerate toxic levels of zinc (Zn), cadmium (Cd), and nickel (Ni) in the soil, and accumulate these metals to very high levels in the shoot. The increased awareness of metal hyperaccumulating plants by the plant biology community has spurred interest in the possible use of these plants to remove heavy metals from contaminated soils, a process known as phytoremediation. Hence there has been a focus on understanding the mechanisms that metal hyperaccumulating plant species such as Thlaspi caerulescens employ to absorb, detoxify and store metals in order to, develop plants better suited for the phytoremediation of metal contaminated soils. Continued research in the mechanisms of Zn hyperaccumulation will also help to understand the regulation and movement of Zn from soil to seed for use in other more agronomical important plant species.