Sean Kundinger Horticulture 550 12/16/2013 AT5G12050 and its role in Lipid composition in cellular membranes of Arabidopsis thaliana.

Overview of AT5G12050: I first chose AT5G12050 based on its relative closeness to the gene, AT5G03840, or TFL1, which controls inflorescence meristem identity, and is involved with the induction of flowering in Arabidopsis thaliana. Intending to discover a gene that could perhaps be involved in the pathways of TFL1 and its main related genes, LFY, and AP, simply by being in proximity to the aforementioned three, I scrolled through genes on the fifth chromosome of A. thaliana, and discovered AT5G12050, which was a gene of unknown function, yet the Tair page1 indicated it was involved in circadian rhythm of plants. This sparked my interest in the gene. By visiting the Comprehensive Systems Biology Projects (CSB) website2, I discovered AT5G12050 was expressed at significant levels in A. thaliana. Performing a BLAST on AT5G12050, several common plant species such as the peach, soybean, and tomato, showed highly similar amino acid sequences in the expressed protein of AT5G12050, meaning AT5G12050 could play a role in several species as it does in A. thaliana. The BLAST search also returned the lack of evidence for putative conserved domains, which could have given a greater indication in regards to the overall function of the gene. Similar proteins were seen in members of the monocot family, in mosses, and even in eukaryotes, suggesting that AT5G12050 may have homologs in species of eukaryotic organisms performing a role in the circadian rhythm, and not just in the plantae kingdom. I entered AT5G12050 through the GBrowse feature1, and discovered that it has one exon, and no introns. Also, it is not a member of a tandem gene family, as searching its protein against Tair 10 proteins returned no genes adjacent to it. Thus, a knock-out mutant phenotype is easy to achieve, which will be extremely useful to examine function of AT5G12050. Also, there existed only one splice variant, AT5G12050.1, with 6 cDNAs that appeared to span the whole gene. Combining the evidence, there appears to be very little splicing of my gene, and AT5G12050 seemingly is transcribed as a whole exon. In order to obtain a knock-out of phenotype, one can use the T-DNA offered, as an available line is WISCDSLOX377-380B4, offered by Dr. Patrick Krysans lab. T-DNA can not only be inserted to disrupt expression of AT5G12050, but it can serve as a marker to prove the existence of its insertion in the mutant plant. Using Phytozyme, synteny, or the ordering of homologous genes adjacent to a gene of interest, is shown in the five related species in the Brassicaceae family, A. thaliana, Arabidopsis lyrata, Capsella rubella, Brassica rapa, and Thellungiella halophila. Examining homologous gene structure throughout these species, it appears A. lyrata and C. rubella developed introns during evolution, perhaps offering a form of regulation on the gene. Studying the varying environments of these plants, as well as their unique genetic codes may offer reasoning as to why this occurred. Studying AT5G12050s translated protein in the Gator tool3, showed a lack of tryptic peptides (only two found), as well as no protein phosphorylation, which serves as a way to shut the protein on or off. This, coupled with the earlier finding of one splice variant, as well as there being only one exon and no intron indicates there is relatively little traditional regulation of AT5G12050 (Figure 1). Studying AT5G12050 in Epigenetics indicates that it has little methylation, which could be used as a tactic to also turn AT5G12050 on or off. Also, there

Figure 1: The single splice variant of my gene, AT5G12050.1, seen here with one exon and no intron seemed to be relatively very little methylation in neighboring genes as well. Studying AT5G12050s profile in the Electronic Fluorescent Protein Browser4 which gives a pictorial diagram of a genes expression when treated to certain environments and stresses, yielded the most important results of any of the tests run. As pictured below in Fig. 2, expression of AT5G12050 steadily climbed as the wild type Col-0 line was treated to continuous 4C, showing an 8.81 fold change from the level at 0 hours at 12 hours (extremely strong up-regulation) in Relative mode, and reaching the maximum expression of 713.19 in Absolute mode at 24 hours. These spikes in expression clearly shows AT5G12050 activity is strongly up-regulated during periods of prolonged cold stress. Another up-regulating factor appeared to be

Figure 2: Abiotic Stress search of AT5G12050 in Absolute mode (top region), and in Relative mode (bottom region) the treatment of synthetic auxin (IAA), the main plant hormone involved with plant growth, and more topically, a central hormone involved with plants response to cold stress (Rahman, 2013)5. Searching AT5G12050 through the tool Expression Angler further echoes these findings, as the highest correlation in expression levels of different genes occurs when treated to cold stress. Again, expression levels of AT5G12050 rise during the course of 24 hours of cold stress. Two important genes show a high correlation in expression levels as well as an indication of AT5G12050s apparent function: AT4G32280 (an IAA-inducible protein that responds to auxin stimulus) and AT3G03840 (member of auxin-responsive protein family). AT3G03840 has been shown to be linked to auxin transport across membranes6 (Spartz et al., 2012). Studying the Gene Ontology network of AT5G12050, further evidence of auxin response is indicated as AT4G32280 again shows relatedness, as well as the gene AT5G02760, which functions in

protein serine and threonine phosphatase activity1, and has been shown to play a role in auxin regulation and response7 (Zhao et al., 2003). Also of note, is the Map Man gene function characteristics listed on the bottom of the results page, indicating that AT5G12050 is involved in the cell wall, lipid metabolism, hormone metabolism (specifically auxin). Further, I investigated AT5G12050 using Genevestigator8, a tool similar to the eFP Browser, yet with more Gene Chip data to compare target genes to. Tests again showed an up-regulation of AT5G12050 under cold stress and auxin stimuli (NAA), showing fold changes of 10.44 and 8.18, respectively. However, maybe more interestingly, AT5G12050 was heavily down-regulated by heavy fold changes ranging -19.23, -24.7, and -38.1 when treated to Pseudomonas syringae, a rod-shaped, gramnegative bacterium. Some strains have been shown to produce Ina proteins which cause plant damage by causing intracellular water to freeze at relatively high temperatures. The combination of the above results led me to develop the hypothesis below. Hypothesis: I hypothesize AT5G12050 responds to prolonged cold stress (12-24 hours) by up-regulating genes involved in the overall auxin response and regulation signal transduction pathway to regulate auxin transport in order to change cellular membrane lipid composition to fight possible resulting plant damage. The extreme fold change of AT5G12050s expression level was unprecedented among the all the other stresses and environments AT5G12050 was exposed to, and thus the main focus of my hypothesis of function. In general, plants respond to cold stress by increasing the percentage of fatty acids among the total cellular membrane lipid structure in all cells. More specifically, Auxin responds to Pi (phosphate) starvation9 (Kobayashi et al., 2006), which is a response to acclimate a plant to cold stress, as, cold stress resulted in reduced leaf Pi levels in Arabidopsis transgenic relative to wild-type plants10 (Zhao et al., 2009). Auxin regulates membrane lipid composition during times of Pi starvation9, the response to cold stress, producing more fluid cellular membranes, and staving off the freezing of the plant, and in general plant injury (Kobayashi et al., 2006). I came to this conclusion based on several key results gained in laboratory. AT5G12050 showed strong expression levels when induced by increased auxin levels, and showed both relatedness as well as expression level patterns to genes which responded to auxin stimuli (AT4G32280, AT3G03840, AT5G02760). AT3G03840 has been shown to play a role in auxin transport to influence plant growth and development6 (Spartz et al., 2012). AT5G02760 is involved in protein serine and threonine phosphatase activity1, which has been shown to function in auxin regulation and response7, as does AT4G32280, an IAA-inducible protein1 (Zhao et al., 2003). The interaction of these three genes in the overall response to auxin activity indicates that AT5G12050 likely up-regulates the aforementioned genes to influence auxin transport, to produce membrane lipid composition changes. The Map Man terms further indicated AT5G12050s role, indicating its expression in the cell wall, lipid metabolism, hormone metabolism (specifically auxin). AT5G12050 showed a striking negative response when treated to P. syringae, which indicates that this bacterium targets AT5G12050 as a key gene that combats cold stress, more specifically manipulating cellular membrane compositions to maintain membrane fluidity in an attempt to avoid freezing, which Ina (ice nucleation) proteins (produced by P. syringae) target. Experimental Plan: To test my hypothesis, Id like to track four separate conditions. Total RNA from rosettes of A. thaliana will be grown for 10 days on soil in growth chambers. I believe that there needs to be a pattern of cold treatment in order to study how prolonged cold stress without AT5G12050 affects the overall growth of a plant. Conditions one and two will be the controls. Condition one will be

a Col-0 line of wild-type A. thaliana, reaching 18 days old at the start of the experiment. The seeds will be grown on MS-Agar-media, and transferred into MS-liquid-media at day 11. The plants will be subjected to a 16 hour light/ 8 hour dark cycle, and grown at room temperature, 24C, at 50% humidity and 150 Einstein/cm2 light intensity11. Total RNA from the rosettes of A. thaliana will be isolated by treating the rosettes to liquid nitrogen, coupled with mortar and pestle, will be used to prevent ribonuclease activity which could contaminate the samples. The isolated RNA of conditions 1 and 3 (the only lines with AT5G12050 functioning in the genome) will then be hybridized to the ATH1 Affymetrix GeneChip at a (0/6/12/15/18/21/24 hour) schedule per day in order to more completely examine expression from 12 to 24 hours, which was absent from the eFP Browser tests. Condition 2 will follow the same procedure of gene expression analysis, as well as light and temperature cycle. However, condition 2 will be a mutant line of A. thaliana. Seeds will contain the T-DNA insertion at the 3 end of AT5G12050, offered as the WISCDSLOX377-380B4 (Figure 3) Germplasm from the donors Patrick Krysan, Michael Sussman, and Rick Amasino. These seeds will exhibit the mutant phenotype in

Figure 3: WISCDSLOX377-380B4 T-DNA insertion point in AT5G12050 regards to AT5G12050, as the gene should be knocked-out as a result of the T-DNA insertion, and the resulting malfunctioning protein. Gene expression levels for AT5G12050 will not be seen. These seeds will be grown to the same treatments mentioned above as indicated by Kilian et al.11. The growth of the wild-type A. thaliana line as well as the mutant line for AT5G12050 should show relatively little variance in overall plant health, as cold stress will not be reached. In condition 3, wild type Col-0 lines will be grown in the same conditions aforementioned, except there will be a cold shock of 24 hours of 4C at days 2, 4, 6, 8, and 10 of the trial. This plant should show similar AT5G12050 expression spikes as seen in the Abiotic tests in the eFP Browser seen above in Figure 1. Condition 4 will be again be mutant lines developed by WISCDSLOX377-380B4, and GeneChip analysis need not be done. These plants will follow the same 24 hour 4C treatment at days 2, 4, 6, 8, and 10 of the trial as in condition 3. However, the major difference in plant health should be noticed here between condition 4 and condition 3. If my hypothesis is correct, plants with functioning AT5G12050 should be able to combat the cold stress by changing lipid composition to maintain fluidity in cellular membranes, influenced by increasing auxin transportation across membranes. At the end of the 10 day trial, I expect condition 4 plants to be severely injured by the intermittent cold stress (These plants will not be frozen as 4C is above freezing, 0C). Condition 3 may even show damage compared to conditions 1 and 2 due to the cumulative effect of cold stress, but I would expect similar overall plant health as a result of having AT5G12050 present to combat the cold stress. Furthermore, it

would be advantageous to study the expression levels of AT4G32280, AT3G03840, and AT5G02760 using the same Affymetrix ATH1 GeneChip. Condition 4 should show a lack of expression in these genes as a result of the lack of AT5G12050 up-regulating them in response to the cold. Also, I would expect AT5G12050 expression to correlate strongly with the three genes activity during the cold stress response. This would further support my hypothesis that AT5G12050 up-regulates genes that are involved in auxin regulation, response, and transport. Photos will be taken at the same (0/6/12/15/18/21/24 hour) cycle to further examine the overall plant health over the course of the 10 day experiment. Variance in response to light may be observed between conditions (1,3) and (2,4), as tests showed AT5G12050 responded to the 16 hr. light/ 8 hr. dark cycle in patterns of activation and silencing. The variance based on nutrients in the agar and the light treatment should be similarly seen between the presence/lack thereof of AT5G12050, as these environmental factors are conserved in all four conditions. Overall, the lack of AT5G12050 should show relatively little variation in the control condition 2, and variation should be due to how AT5G12050 reacts to light (difficult pattern to interpret). However, there should be large contrasts in responses to cold stress between plants in condition 3 (AT5G12050 +) and condition 4 (AT5G12050 -) Bibliography: 1 The Arabidopsis Information Resource (TAIR), www.arabidopsis.org/aboutarabidopsis.html, on www.arabidopsis.org, Dec 12, 2013 2 A Comprehensive Systems-Biology Database; [2013 Dec 12, cited 2013 Dec 12] . Available from: http://csbdb.mpimp-golm.mpg.de/index.html 3 Joshi, H.J., Hirsch-Hoffmann, M., Baerenfaller, K., Gruissem, W., Baginsky, S., Schmidt, R., Schulze, W.X., Sun, Q., van Wijk, K., Egelhofer, V., Wienkoop, S., Weckwerth, W., Bruley, C., Rolland, R., Toyoda, T., Nakagam, H., Jones, A., Briggs, S.P., Castleden, I., Tanz, S., Millar, A.H., and Heazlewood, J.L. (2011). MASCP Gator: An aggregation portal for the visualization of Arabidopsis proteomics data. Plant Physiol. [2013 Jan, cited 2013 Dec 12] 155, 259-270. Available from: http://gator.masc-proteomics.org/ 4 Winter et al. 2007. Arabidopsis eFP Browser . [Internet]. [Last Updated 2013 Dec 12, cited 2013 Dec 12]. Available from: http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi 5 Rahman A. 2013. Auxin: a regulator of cold stress response.. Plant Physiology (147) [Internet]. [2013 Jan, cited 2013 Dec 12] 147(1):28-35. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22435366 6 Spartz AK, Lee SH, Wenger JP, Gonzalez N, Itoh H, Inz D, Peer WA, Murphy AS, Overvoorde PJ, Gray WM. 2012. The SAUR19 subfamily of SMALL AUXIN UP RNA genes promote cell expansion.. Plant Journal. [Internet]. [2013 Dec 12, cited 2013 Dec 12] 70(6):97890. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22348445?dopt=Abstract 7 Zhao Y, Dai X, Blackwell HE, Schreiber SL, Chory J. 2003. SIR1, an upstream component in auxin signaling identified by chemical genetics.. Science (**Edition**) [Internet]. [2013 Dec 12, cited 2013 Dec 12] 301(5636):1107-10. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12893885?dopt=Abstract 8 Genevestigator ETH Zurich [Internet]:Nebion ; [2013 Dec 12, cited 2013 Dec 12] . Available from: https://www.genevestigator.com/gv/index.jsp

Kobayashi K, Masuda T, Takamiya K, Ohta H. 2006. Membrane lipid alteration during phosphate starvation is regulated by phosphate signaling and auxin/cytokinin cross-talk.. Plant Journal [Internet]. [Last Updated 2013 Dec 12, cited 2013 Dec 12] 47(2):238-48. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16762032 10 Zhao L, Liu F, Xu W, Di C, Zhou S, Xue Y, Yu J, Su Z. 2009. Increased expression of OsSPX1 enhances cold/subfreezing tolerance in tobacco and Arabidopsis thaliana.. Plant Biotechnology Journal [Internet]. [2013 Dec 12, cited 2013 Dec 12] 7(6):550-61. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19508276 11 Kilian J, Whitehead D, Horak J, Wanke D, Weinl S, Batistic O, D'Angelo C, Bornberg-Bauer E, Kudla J, Harter K. 2007. The AtGenExpress global stress expression data set: protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses.. Plant Journal (**Edition**) [Internet]. [2013 Dec 12, cited 2013 Dec 12] 50(2):347-63. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17376166