Progress 01/15/10 to 01/14/14
Outputs
Target Audience:We have reached other scientists working in the area of plant mineral nutrition mainly by presentations at meetings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One undergraduate and one graduate student How have the results been disseminated to communities of interest?By presentations at meetings, and lectures at Monsanto and Yale University (2014-15). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have now finished characterizing the cell specific pattern of expression of six key YSL genes using the transgenic plants. To our surprise, the genes' expression is very low in vegetative tissues, and so our efforts have been focused on expression in developing seeds, where 5/6 of the genes are very strongly expressed in the Basal Transfer Layer of the developing kernel. This location is indicative that the YSLs have roles in loading of iron into developing seeds. We are just beginning to write up our results from this analysis, which will be published together with the RT-PCR data generated earlier in the project. Because we were not able to develop successful assays for YSL trasnport activity during the project period, we have turned some of our attention to understanding a previously uncharacterized aspect of iron uptake, by determining the identity of the maize ys3 gene. We identified 4 new alleles of ys3 during the final year of the project, and are finishing up sequencing of the candidate ys3 gene in these novel alleles. If our predicted candidate is correct, we will soon publish these results. Since the broader 'iron' community all believes (albeit based on inadequate evidence) that the candidate (a gene homologous to the phytosiderophore transporter TOM1) is ys3, even a finding that the wrong candidate has been developed will be an important piece of information. Having additional alleles will set the stage for future identification of the correct gene, should ZmTOM1 turn out not to be responsible for the ys3 mutant phenotype.
Publications
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Progress 01/15/12 to 01/14/13
Outputs
Target Audience:
Nothing Reported
Changes/Problems: We, and others, had previously mapped the ys3 gene to an interval on chromosome 3 (3.04). The maize genome sequence, although nearly complete, still contains some sequence gaps, including a gap in the 3.04 interval. Recently, more sequence information in the gap became available, and a partial sequence with strong similarity to TOM1 was revealed. We have used PCR to further fill in the gap, and to reveal the existance of a complete TOM1-like gene. We hypothesize that this gene is likely to be ys3. We have sequenced wild type and mutant alleles of the TOM1 gene, and have identified a frame shift mutation in the single ys3 mutant allele available from the Maize Genetics Stock Center. In future, we will characterize additional maize mutants that are available in the Maize Genetics Stock Center, and that are described as either 'yellow striped" or "green striped" to determine whether any are allelic to ys3. By identifying additional alleles, we will be able to confirm whether all of them contain mutations in the TOM1 gene, and thus gain final proof that the gene underlying the ys3 mutation is the PS effluxer, TOM1. We have embarked on this effort since other aspects (complementation in Brachypodium and yeast activity assays) have proved to be so difficult, and have not yeilded results. What opportunities for training and professional development has the project provided? One graduate student and two undergraduate students worked on the project. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? During the next one year extension period of the project, we will finish characterizing the cell specific pattern of expression of six key YSL genes using the transgenic plants now under analysis. The outcome will be a greatly improved understanding of the mechanisms that maize (and other grasses) use to move iron within their tissues and organs. We will also prepare a publication including the results of these analyses and the RT-PCR analysis of the whole gene family.
Impacts
What was accomplished under these goals?
2.) We have continued to struggle to make the yeast assays work reliably (see previous annual report). We did not measure transport activity for maize YSL proteins using this system. 3.) Completed a comprehensive expression analysis of all functional members of the maize YSL family of transporters in both vegetative and reproductive tissues, including seed maturation and germination. This has allowed us to identify the six maize YSL genes to be used for detailed functional analysis. 3.) Prepared genetic constructs to pinpoint the location of expression of 6 maize YSL genes. Plant transformation and regeneration is complete, and analysis of the plants is underway. 4.) Worked toward creating a facile in planta system for testing maize YSL activity using the model grass species Brachypodium distachyon. Use of this system would have given unequivocal evidence regarding the ability of each YSL to transport Fe(III)-PS complexes. Unfortunately, we have not been able to silence the Brachypodium YS1 gene using estradiol-inducible RNAi directed against YS1. SInce the original, consitutively expressed YS1-RNAi was lethal, this part of the project was not successful.
Publications
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Progress 01/15/11 to 01/14/12
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One postdoc was supported during this project period, and a new graduate student joined the project. Two undergraduate students assisted the postdoc and graduate student. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? Transgenic lines will be characterized in detail, once sufficient seed stocks have been accumulated.
Impacts
What was accomplished under these goals?
1. Completed during the previous project period. 2. We have put a substantial amount of effort into developing an uptake assay that can measure YSL uptake of Fe(II)-NA directly. The use of iron, which is only sparingly soluble in aerobic environments, presents special technical problems that we tried to overcome. In the uptake assay, YSLs or control genes are expressed in a yeast strain that lacks its high affinity ironuptake system (fet3fet4). Fe(III)-PS or Fe(II)-NA complexes are formed in the presence of tracer amounts of 55Fe, and then the yeast are exposed to the radioactive complexes in a simple uptake buffer containing glucose and MES. Cells are separated from the medium by vacuum filtration, and filters are counted to determine cell associated counts. These assays for uptake are severely compromised by dissolution of the iron complexes, followed by oxidation and precipitation of insoluble Fe(III)-oxides. The evidence for this problem is revealed by cell free controls, in which stochastic and often very large (up to ~5-10% of the total counts in the reaction) numbers of counts are retained on the filter. This causes a large and variable background level of radioactivity that precludes successful analysis of uptake. Even worse, the formation of precipitates highlights the fact that the complexes are in equilibrium rather than permanently bound, particularly at the acidic pHs used for the uptake assays, as predicted from direct stability measurements and theoretical calculations. This implies that the true concentration of the complexes cannot be known during the experiment, complicating the establishment of accurate biochemical parameters. The pH effect is easily seen in the no cell controls for the uptake experiments, in which the amount of precipitate present varies directly with the pH: at pH 5.5, a large amount of precipitation is observed, while at higher pH (6.2 or 7.4) the amount of precipitation is substantially less, albeit still quite variable. Unfortunately, the transporters also function poorly at high pH, and thus activity measurements cannot be made under conditions in which precipitation is minimized. This 'tug of war' between conditions that favor complex stability and those that favor transporter activity in the yeast system highlights the difficulty inherent in devising assays for transport of bimolecular complexes that have their own inherent formation and dissolution kinetics. Since we have not been able to overcome this, we are looking into using Ni as a proxy for Fe in these experiments. Ni is far more soluble than Fe. 3. All 6 YSLpromoter:GUS constructs are now complete and have been transformed into maize. The resulting lines are being propagated to generate homozygous lines for each of YSL under investigation. Preliminary expression of the transgenes was found to be very low in vegetative tissues, and efforts to identify stronger expression in reproductive tissues and in germinating seeds will be initiated. RT-PCR is complete. Conditions tested were anatomical (leaves, roots, seeds, pollen, germinating seeds) and also metal-limiting (Fe, Zn, Cu, Mn). 4. In the previous period, we learned that RNAi-directed silencing of Brachypodium YS1 is lethal. Therefore we we have placed the RNAi construct under control of an estradiol-inducible promoter, and propagating plants carrying this construct.
Publications
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Progress 01/15/10 to 01/14/11
Outputs
OUTPUTS: We have made a complete bioinformatic survey of the ZmYSL family of genes by identifying all genes and pseudogenes in the now sequenced maize genome. We then obtained and sequenced all full length cDNAs that were publicly available, and sequenced these. This surprisingly led us to conclude that several of the YSL genes in B73 contain nonsense mutations, and are apparently expressed pseudogenes. These genes were culled from further analysis. Work describing this initial characterization has been submitted for publication in Annals of Botany. We then have conducted a Q-RT-PCR study in which expression of each ZmYSL gene was characterized throughout development (including reproductive development and germination) and in response to transition metal (Fe, Mn, Zn, or Cu) starvation . Thus we have now formed a very complete picture of the pattern and level of expression of each ZmYSL gene and have settled on a set of 6 ZmYSLs (ZmYSL1, 3, 10, 12, 13, and 14a) which are abundantly expressed, have interesting (i.e., metal regulated, or developmentally specific) patterns of expression, and thoroughly represent previously uncharacterized grass-specific members of the YSL family. Our further analyses will concentrate on these 6 genes. cDNAs for most of the ZmYSLs that we are continuing to analyze were available through public resources, and we have obtained full length cDNAs for the remaining genes by RT-PCR. Gateway vectors for making promoter-GUS expression constructs suitable for use in maize have been made, and the first promoter (ZmYSL13) construct is completed, and has been sent to the Iowa Maize Transformation Facility. We are in the process of cloning promoters from the remaining 5 key ZmYSL genes, and anticipate that these will be finished shortly, and sent for transformation. We have apparently been all too successful in knocking out Brachypodium distachyon YS1 function using RNAi, as all transgenic brachypodium plants carrying this construct proceed to regeneration as green plantlets, but then bleach and die, apparently from iron deficiency. While this strong phenotype is reassuring in the sense that we appear to be knocking out YS1 successfully, it has prevented us from obtaining regenerated plants. We have tried a series of methods to supply the regenerating plantlets with iron (and other metals) but have not succeeded in rescuing the plants. As an alternative, we have placed the RNAi construct under control of an estradiol-inducible promoter, and are transforming plants with this construct. In the meantime, we are preparing Gateway vectors with the BdYS1 promoter, so that ZmYSL cDNAs can be rapidly cloned and characterized when the RNAi plants become available. PARTICIPANTS: Sarah S. Conte, postdoctoral associate Burcu K. Yordem, Ph.D. student Kenneth Vasques, M.S., student TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
This first year was a year of 'setting up'. We have achieved almost all goals for this period, which included full bioinformatic characterization of the maize YSL family, selection of 6 key ZmYSL genes for further analysis, and cloning of both full length cDNAs and large (>=2 kb in most cases) promoter fragments for all the relevant genes (and a number of others, in the case of cDNAs), and construction of high throughput vectors for maize transformation, yeast functional complementation, and cDNA expression in Brachypodium distachyon. The exception is that we have not succeeded in obtaining the Brachypodium mutant needed for in planta analysis of ZmYSL function, owing to an unexpectedly severe phenotype associcated with the mutation. We continue to work on development of the mutant (see above.)
Publications
- Conte, S. S. and E. L. Walker (Accepted with minor revisions) Transporters contributing to iron trafficking in plants. Molecular Plant Special Issue on Membrane Transport, May, 2011.
- Yordem, B. K., Conte, S. S., Ma, J. F., Yokosho, K., Vasques, K. A., E. L. Walker (submitted) Brachypodium distachyon as a new model system for understanding iron homeostasis in grasses: phylogenetic and expression analysis of Yellow Stripe Like (YSL) transporters. Annals of Botany.Publication expected 2011
- Walker E. L. and B. M. Waters (submitted) The role of transition metal homeostasis in plant reproductive development. Current Opinion in Plant Biology. Publication expected 2011
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