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About 50% of the world's arable land is strongly acidic (pH ≤ 5). The low pH solubilizes root-toxic ionic aluminium (Al3+) species from clay minerals, driving the evolution of counteractive adaptations in cultivated crops. The food crop Sorghum bicolor upregulates the membrane-embedded transporter protein SbMATE in its roots. SbMATE mediates efflux of the anionic form of the organic acid, citrate, into the soil rhizosphere, chelating Al3+ ions and thereby imparting Al-resistance based on excluding Al+3 from the growing root tip. Here, we use electrophysiological, radiolabeled, and fluorescence-based transport assays in two heterologous expression systems to establish a broad substrate recognition profile of SbMATE, showing the proton and/or sodium-driven transport of 14C-citrate anion, as well as the organic monovalent cation, ethidium, but not its divalent analog, propidium. We further complement our transport assays by measuring substrate binding to detergent-purified SbMATE protein. Finally, we use the purified membrane protein as an antigen to discover native conformation-binding and transport function-altering nanobodies using an animal-free, mRNA/cDNA display technology. Our results demonstrate the utility of using Pichia pastoris as an efficient eukaryotic host to express large quantities of functional plant transporter proteins. The nanobody discovery approach is applicable to other non-immunogenic plant proteins.

Plants have evolved a number of different mechanisms for dealing with toxic metals in the environment, and these can involve both avoidance (exclusion of the metal from a plant tissue/organ) or true tolerance, which presumably involves chelation and/or sequestration of the metal in an internal compartment. The best characterized mechanism of plant metal tolerance is associated with crop aluminum (Al) tolerance. Al toxicity is a worldwide problem that arises when soil pH values drop to 5 or below; in these acidic soils rhizotoxic forms of Al are solubilized into the soil solution, damaging roots and resulting in reduced water and nutrient uptake. This talk will focus on a major Al tolerance mechanism which involves Al exclusion from the root tip mediated by Al activation of specialized transporters that release organic acids into the rhizosphere, where they chelate and prevent Al from entering the root. Several Al tolerance genes have been cloned recently and these belong to two different families of novel membrane transporters. The function and regulation of these tolerance genes will be discussed, as well as the implications of these findings in facilitating plant improvements for growth on degraded or marginal soils.

The efficient quantification of root traits remains a critical factor in exploiting many genetic resources during the study of root function and development. This is particularly true during the phenotyping of large populations under abiotic stress conditions, including aluminum (Al) toxicity where root growth is quantified in the presence and absence of Al, and phosphorous (P) deficiency, where root architectural responses and differences are often examined. Our present study highlights two root imaging and quantification systems that are being developed and used in our laboratory. We have focused on designing flexible growth, imaging and software tools that enable a multitude of 2D and 3D root system characteristics to be investigated.

Metal hyperaccumulating plant species are plants that are endemic to metalliferous soils and are able to tolerate and accumulate metals in their above ground tissues to very high concentrations. One such hyperaccumulator, Thlaspi caerulescens, has been widely studied for its remarkable properties to tolerate toxic levels of zinc (Zn) and cadmium (Cd) in the soil, and accumulate these metals to very high levels in the shoot. Thlaspi caerulescens has the ability to accumulate as much as 3% Zn and 1% Cd in the shoots of the plant with no signs of toxicity. Early molecular investigations in our lab into how this plant functions as a metal hyperaccumulator led to the identification of TcZNT1 as a gene encoding a high affinity Zn transporter and low affinity Cd transporter which is expressed at very high levels in the roots of T. caerulescens. Our lab and other labs have subsequently found that high expression of TcZNT1 and other metal transporters is a hallmark of metal hyperaccumulators. To begin to understand the molecular basis of this “hyperexpression”, we have identified two different members of the E2F family of transcription factors that complement a yeast mutant defective in Zn uptake through disruption of the yeast transcription factor that regulates expression of the yeast high affinity Zn transporter. We have also found that only one of these E2Fs is able to bind with high affinity directly to a putative E2F-binding motif in the promoter of TcZNT1 in T. caerulescens, possibly regulating its expression. Further characterization of the interactions between ZNT1 and the E2F family members is being conducted to more fully elucidate the role these transcription factors may play both in plant Zn homeostasis, and the hyperaccumulation of heavy metals.

GEOCHEM–EZ is a multi-purpose chemical speciation program, designed to replace Geochem-PC, which can only be used in DOS consoles. This program is used in plant nutrition to perform equilibrium speciation computations to estimate the activity of ions in solution and to consider simple complexes and solid phases. It is also important in molecular biological research to modify solution composition for phenotyping and screening transformed organisms (e.g., transgenic Arabidopsis plants), controlling pH, redox conditions, ionic strength (e.g., for protoplast isolations) or for using heterologous systems, such as yeast expressing plant genes.To enhance the usability and to address several weaknesses of Geochem-PC, we upgraded the program using a Java graphical user interface, improving both the power and function, and allowing it to run on any computer that supports Windows XP, Vista, or Windows 7.This program includes improvements, such as interactive and illustrated help files, hierarchical organization of options, logical output, and real-time error checking, while maintaining complete backward compatibility to the Geochem-PC format. We added a customizable database of common salts, eliminating the need to parse and calculate the concentration of each metal or ligand. This last feature will make data input more rapid and help eliminate the most common user errors. One can instantly preview input and output files and make necessary corrections (e.g. charge balance the solution), something that formerly involved having to save these files and run the calculations a second time. GEOCHEM-EZ is available as a free download from http://www.plantmineralnutrition.net/Geochem/geochem%20home.htm

Aluminum (Al) toxicity is a major constraint for crop production on acid, Al toxic soils, that occupy over 50% of the potentially arable lands in the world. Aluminum is ubiquitous in soils and when solubilized at pH values below 5.0, becomes highly phytotoxic as Al3+, causing severe yield reduction to sensitive crops. Al tolerance in species such as wheat, barley and sorghum appears to rely largely on a small number of genes within each species. However, quantitative inheritance for Al tolerance appears to be the general pattern observed in rice and maize. In maize, citrate exudation has been suggested as an important tolerance mechanism. However, lack of correlation between citrate exudation and differential Al tolerance observed in some maize lines suggests that other Al tolerance mechanisms may take place in maize. In this study, we undertook a physiological study of Al tolerance mechanisms focusing on citrate exudation in selected maize recombinant inbred lines for which a QTL map had been previously developed. Our results are being assessed in light of the genetic constitution of these RILs for the major Al tolerance QTLs segregating in the population. Our results support the role of citrate exudation as a major Al tolerance mechanism in maize but suggest that other mechanisms probably take place in this population.

Zinc (Zn) is an essential nutrient for all living organisms. Understanding how plants respond to low Zn is important, as Zn deficiency is a major contributing factor in reducing crop yield and productivity throughout the world. Zn efficiency (ZE) is the ability of plants to maintain high yield under low-Zn conditions. The objective of this study was to conduct a large-scale screening of 30,000 T-DNA activation tagged Arabidopsis lines to identify mutants with superior ZE. Our hypothesis was that overexpression of certain genes will lead to ZE in Arabidopsis. We have established a hydroponic screening system and isolated a number of putative Arabidopsis mutants, which we are currently screening for confirmation. The hydroponic solution contained 1 mM KNO3, 1mM Ca(NO3) 2, 0.05 mM NH4H2PO4, 0.25 mM MgSO4, 0.1 mM NH4NO3, 50 uM KCl, 12.5 uM H3BO3, 0.1 uM H2MoO4, 0.1 uM NiSO4, 0.4 uM MnSO4, 1.6 uM CuSO4, 96uM Fe(NO3) 3-H3HEDTA, 0.1 pM ZnSO4-H3HEDTA and 2 mM MES at pH 6.0. Zn-efficient Arabidopsis mutants were recovered and moved to agar plates with full nutrients for three days and then to soil. After growing the mutants in soil until flowering and seed set, T3 seeds were harvested for secondary screening. Thus our hydroponic screening appears to be a reliable and manageable method to screen Arabidopsis for ZE. However, the mutants identified in this study need to be validated in secondary screening.

Under depleted external phosphate (Pi), many plant species adapt to this stress by initiating downstream signaling cascades. In plants, the vascular system delivers nutrients and signaling agents to control physiological and developmental processes. Currently, limited information is available regarding the direct role of phloem-borne long-distance signals in plant growth and development under Pi stress conditions. Here, we report on the identification and characterization of a cucumber protein, Cucumis sativus Phloem Phosphate Stress-Repressed 1 (CsPPSR1), whose level in the phloem translocation stream rapidly responds to imposed Pi-limiting conditions. CsPPSR1 degradation is mediated by the 26S proteasome; under Pi-sufficient conditions, CsPPSR1 is stabilized by its phosphorylation within the sieve tube system through the action of CsPPSR1 kinase. Further, we discovered that CsPPSR1 kinase was susceptible to Pi starvation-induced degradation in the sieve tube system. Our findings offer insight into a molecular mechanism underlying the response of phloem-borne proteins to Pi-limited stress conditions. [ABSTRACT FROM AUTHOR]