Comparison of biological pathways in zinc decificent Arabidopsis thaliana to zinc excess Thlaspi caerulescens BioInformatics Lab Tuesday, April 13, 2010 Kristoffer Chin Salomon Garcia Michael Piña

Outline Introduction –Van der Mortal paper was used to find the differences in gene between normal zinc accumulators and hyperaccumulators –Results from paper yield to many differences in gene expression from the conditions set –Comparison of extreme conditions in order to find specific differences form the 2,272 genes observed Materials and methods –GeneMAPP and MAPPfinder used to visualize the genes that were found in the deficient A. thaliana and excess T. caerulescens Results –GenMAPP and MAPPFinder –Processes for Criterion 0 –Processes for Criterion 1 Discussion –Terpenoid metabolic processes upregulated –Chromatin processes downregulated References

Micronutrients are essential for plant species in growth, protection, and etc. Microcuntrients play a role with the growth and duration of plant life Zinc has been one of the most importaant nutrients in plant growth –Too much zinc can be toxic to plants –Too little zinc can inhibit optimal growth fo plants Zinc homeostasis is important to plant life

Van de Mortal’s paper uses A. thaliana and T. caerulescens to better understand hyperaccumulators T. caerulescens is a plant similar to A. thaliana but is a known to be a zinc hyperaccumulator These two plants were used as subjects due to their similarity Both plants were grown in 3 conditions, Deficient, sufficient, and excess zinc, and then had their genes analyzed DNA Microarray analysis showed an altered expression of many genes in both plants, specifically Zinc transport proteins and lignin biosynthesis

A. thaliana and T. caerulescens share 88.5% DNA identity in coding regions A. thalianaT. caerulescens schaechter.asmblog.org

608 genes were identified in A. thaliana and organized into clusters Cluster ICluster IICluster IIICluster IV # of genes found 98 genes128 genes347 genes35 genes Condition found in Sufficient and excess ExcessDeficientDeficient and Sufficient Functions stress response Metabolism Heat schock proteins 15 with unkown function 20 not annotated Iron homeostasis Metal transporters Stress response Metabolism Transcription factors Metal homeostasis Metal transporter Protein stability Signal transduction Transcription regulation Metabolism 164 genes unknown Secondary metabolism Biotic stress response Transcription 5 genes with unkown function

350 genes were identified in T. caerulescens and organized into 6 clusters Cluster ICluster IICluster IIICluster IV # of genes found 98 genes128 genes347 genes35 genes Condition found in Sufficient and excess ExcessDeficientDeficient and Sufficient Functions stress response Metabolism Heat schock proteins 15 with unkown function 20 not annotated Iron homeostasis Metal transporters Stress response Metabolism Transcription factors Metal homeostasis Metal transporter Protein stability Signal transduction Transcription regulation Metabolism 164 genes unknown Secondary metabolism Biotic stress response Transcription 5 genes with unkown function

An overall 2,272 genes were found to be highly expressed between the two plants A. thaliana 420 genes not expressed in root Little variation in expression among conditions Less expression in PDF genes Less lignin biosynthesis genes Less cellular process Less transport process Less stress response Less transcription T. caerulescens 420 genes expressed in root High variation variation in expression among conditions More expression in PDF genes More lignin biosynthesis genes More cellular process More transport process More stress response More transcription

Comparison of extreme conditions yields to significant gene differences Of the different combination, deficient A. thaliana and excess T. caerulescens were chosen Extreme conditions chosen because it would show the most altered expression of genes Genes that are expected to be found would deal with zinc transporters, homeostasis, and lignin biosynthesis Each gene has its own function which ultimately helps the plant in deficient and sufficient

Genes labeled in Excel and data was normalized to calculate in GenMAPP van de Mortel’s data set was labeled in a different way and had to be altered in order to understand the significance The data on excel was then normalized in order to fit the GenMAPP protocol GenMAPP is used to visualize gene expression –Helps to group genes together and find its functional expression for the subject

MAPPfinder finds the relativity of genes that were increased or decreased in extreme conditions GeneMAPP grouping was inserted in MAPP finder in order to produce a tree that helps visualize the genes –Even though genes were found in the extreme conditions, they can be related to one another through DNA similarities MAPPfinder results were then placed in MS Excel in order to filter out the amount of genes found –Numbers changed – Greater than 3, Less than 100 –Z score – Greater than 2 –PermuteP – Less than 0.05

Results from GenMAPP and MAPPFinder 1778 errors were found in the genes –“Gene not found in TAIR or any related system” The total amount of genes found in Criterion 0 were 303 distinct genes The total amount of genes found in Criterion 1 were 597 distinct genes

Top processes for criterion 0 terpenoid metabolic process cell surface receptor linked signal transduction anchored to plasma membrane response to starvation intrinsic to plasma membrane isoprenoid metabolic process serine family amino acid metabolic process response to nutrient levels response to salicylic acid stimulus response to extracellular stimulus protein binding protein import mRNA processing response to jasmonic acid stimulus methyltransferase activity

Top processes for criterion 1 extracellular region lipid catabolic process chromatin modification establishment or maintenance of chromatin architecture glutamine family amino acid metabolic process covalent chromatin modification pyridoxal phosphate binding regulation of gene expression, epigenetic regulation of gene expression hydrolase activity, acting on ester bonds chromosome organization regulation of macromolecule metabolic process jasmonic acid mediated signaling pathway two-component response regulator activity nucleosome

Terpenoid metabolic processes are upregulated Other branched processes of terpenoid metabolic processes are also upregulated –Isoprenoid metabolic process In plants, terpenoids are sometimes added to proteins to increase attachment to cell membranes Protein binding and import also show upregulation, suggesting the plant is trying to pump out as much zinc as possible using membrane proteins

Chromosome and chromatin processes are downregulated Excess zinc may be interfering with regulation of genetic material –Can lead to plant death Lipid catabolic process also downregulated –Terpenoids are lipids –Plant may be preserving lipids to convert them in to terpenoids

Lignin biosynthesis processes were not found in our results We chose to do a comparison of deficient zinc in A. thaliana and excess zinc in T. caerulescens Paper found differences in lignin biosynthesis among T. caerulescens with deficient zinc

Areas for future study Create a new database for A. thaliana with the most up to date information Create a MAPP file with GenMAPP in order to visualize grouping of genes

References van de Mortel JE, Almar Villanueva L, Schat H, Kwekkeboom J, Coughlan S, Moerland PD, Ver Loren van Themaat E, Koornneef M, and Aarts MG. Large expression differences in genes for iron and zinc homeostasis, stress response, and lignin biosynthesis distinguish roots of Arabidopsis thaliana and the related metal hyperaccumulator Thlaspi caerulescens. Plant Physiol 2006 Nov; 142(3)