Characterizing auxin biosynthetic mutants in arabidopsis thaliana - First Paper

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Characterizing auxin biosynthetic mutants in arabidopsis thaliana - First Paper

  1. 1. Characterizing Auxin Biosynthetic Mutants in Arabidopsis thaliana Nicole Colón CarriónUniversity of Puerto Rico, Cayey campus; North Carolina State UniversityThe phytohormoneauxin regulates numerous aspects development. Although of plant growth andauxinwas one of the first plant hormone to be discovered, ourunderstanding of how plants produce thishormone in a spatially and temporally regulated manner is still limited. Two main routes for auxinproduction have been proposed, the tryptophan dependent and tryptophan-independent pathways. Thisstudy aims to shed light on the tryptophan-dependent auxin biosynthetic pathway using a novel mutantscreen in Arabidopsis thaliana. Because ethylene response induces this auxin biosynthetic pathway, theethylene response is utilized as a tool in the screen. Specifically, loss of the auxin biosynthetic enzymetryptophan aminotransferase, wei8, results in a partial loss of ethylene response in the root. wei8 wasmutagenized to find novel mutants which enhance its auxin deficient phenotype. In particular, I havefocused on the characterization of the mutant 2-93 that shows dramatic reduction in the ethylene responseand reduced root meristematic activity. A mapping population from a cross between 2-93 and the Leraccession was obtained. F2 seedlings with the 2-93 mutant phenotype were selected and the geneticnature of the mutation determined to be recessive based on the segregation analysis. Plants with strongauxin deficiency phenotype marked by the loss of root meristem integrity were selected for mapping.Polymorphic markers across the whole Arabidopsis genome are being used to determine the chromosomallocation of causal mutation in the 2-93 line. The characterization of this mutant will advances ourunderstanding of how this essential plant hormone is produced.Introduction: Arabidopsis thaliana is a smallflowering plant related to the mustardfamily. This type of family is the most systems, such as regulation, growth andwidely distributed; it has approximately 340 development. Indole acetic acid moleculesgenera and 3,350 species. Arabidopsisis can inhibit or stimulate the expression ofhighly distributed around the world, it can certain genes. Auxin can control plantbe found in central Asia, Mediterranean development at all levels; that’s why it isregions and North and South America. This important for plants to maintain a balance ofplant is highly used as a model organism for IAA, too much or too little can be fatal forthe study of plant biology, since is the first the plants.plant to have it entire genome sequenced.Studying Arabidobsis thaliana can help to a Several studies havebetter understanding of plants biological indicated thatauxinbiosynthesis is controlledsystems. by the tryptophan independent pathway and tryptophan dependent pathway, however Indole acetic acid, also known as how this pathways work remain poorlyauxin was one of the first hormones to be understood. Several pathways has beendiscovered. It is a phytohormone that postulated for Trp-dependent pathway; thecontrols numerous processes in plant indole-3- acetamide, the indole-3-pyruvic
  2. 2. acid, the trypthamine, and the indole-3- About 25 mutants that enhance theacetaldoxime pathway.The indole-3-pyruvic auxin deficient phenotype of wei8wereacid pathway is important for IAA synthesis chosen for further characterization; howevernot only in plants but also for we focus on the 2-93mutants in Arabidopsismicroorganisms. It was postulated thatArabidopsis seedlings contain the TAA1 Thaliana.Three-day-old seedlingsgene, also known as wei8, which encodes an wereanalyzed for ethylene and auxinaminotransferase that is used to converts Trp response. Characteristic of auxininto IPA (Stepanova et al., 2008). Finally biosynthetic mutants, wei8 tar2 and 2-93IPA is converted into IAA. The Trp- lack apical hooks and displaylongerroots onindependent pathways was postulated in the ethylene-supplemented media, yet a1991 but how this pathway designs IAA is normal auxin response, as shown in figure 1.not well understood. It is our objective toshed the light on the auxin biosyntheticpathway by finding genes that enhance thewei8 phenotype using Arabidopsis as ourplant model. By understanding how plantssynthesis this hormone, we could understandbetter how plants regulate it fine balance.Materials and Methods About 50,000 Arabidopsis Thalianaplants were previously EMS mutagenizedand screened. 2,100 putative mutants werechosen, based on long root and loss of theapical hook phenotype, for further Figure 1. Shows Three-day-old seedlingscharacterization. Since ethylene induces analyzed for ethylene and auxin response.auxin biosynthesis it is used as a tool duringthe screen. F2’s seedlings from crosses were The recombination frequency wasanalyzed at twotimepoints (three-day-old calculated for 2-93 mutants based on theand ten-day-old) for auxin sensitivity and long root phenotype. 2-93 mutant cross toethylene responses. Seedlings with the Col and to Ler presented a 1:16 ratio, whileparental phenotype were selected for further the cross to wei8 presented 1:4 ratioanalysis and there recombination frequency meaning that it is a recessive mutation. Rootwas calculated. Root degeneration and degeneration and DR5:GFPwere examined.DR5:GFPexpression were monitored. DR5:GFPreporter was used to monitor auxin response. On the standard media, 10- day-old wei8 tar2 and 2-93 seedlings showed an auxin deficiency and rootResults: meristem defect. On auxin media, wei8 tar2 and 2-93 showed a healthy meristem with normal expression of DR5:GFP. Addition of
  3. 3. auxin to these mutants resulted in thecomplementation of the root meristem The 2-93 mutant in Arabidopsisphenotype. Thaliana presented was one of the best putative mutants found for further characterization. The characterization of 2- 93, at three days old, shows that it exhibits auxin deficiency phenotype and root meristem defectthat enhance wei8, indicating that it is an auxin biosynthetic mutant. The chromosomal location of the 2- 93 mutation for the chromosomal location is stillunknown. . Markers MAC9D21K and MQD22 on chromosomefivewere tested and didnotexhibitlinkage. The causal mutation of 2-93 is notlocatednearthese markers. Also, marker T13E11-1 on chromosometwo was tested andappears to be linked to theFigure 2.Shows the rootdegeneration and gene responsible for the 2-93 mutation,DR5:GFPof 2-93 mutant. however this cannot be concludedsincenotall the markers has been tested. In conclusión, 2-93 mutant enhance the wei8 phenotype Seedlings with the mutant and the gene responsable for this mutationphenotype were selected for mapping. may be workingtogether with the wei8 genePolymorphic markers across the whole in controlling auxin biosynthesis.Arabidopsis genome are being used todetermine the chromosomal location of the References:causal mutation in 2-93. . MarkersMAC9D21K and MQD22 on ● Price, Robert et al., 1994.Arabidopsis.chromosomefivedidnotexhibitlinkage. The United States of America: Cold Springcausal mutation of 2-93 is Harbor Laboratory Press.notlocatednearthese markers. MarkerT13E11-1 on chromosometwoappears to be ● Mano, Yoshihiro.Nemoto, Keiichirou.linked to the gene responsible for the 2-93 2012. The pathway of auxin biosynthesis in plants.mutation. I will be testing markers on all thechromosomes to find wherethere is linkage ●Stepanova et al., 2008.TAA1-Mediatedto find the gene responsible for the 2-93 AuxinBiosynthesisIsEssential for Hormonephenotype. Crosstalk and Plant Development. ● TAIR. [internet] [2008] USA: Arabidopsis Information Resource (TAIR).Discussion:
  4. 4. Available:http://www.arabidopsis.org/about/index.jsp

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