Characterizing Auxin Biosynthetic Mutants in Arabidopsis thaliana


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  • Auxin is a key regulator of many aspect of plant growth and development, including cell division and elongation, differentiation, tropisms, apical dominance, senescence, abscission, and flowering . determining the molecular mechanisms of auxin biosynthesis may provide new tools for solving difficult plant development questions.
  • Here is the current model of tryptophan mediated auxin biosynthesis. There are three main branches named for their intermediate compounds. IAOx, IPyA, and IAM. TAA1, also known as taa1, catalyzes the conversion of TRP to IPA. From there YUC act downstream eventually leading to IAA.
  • Because ethylene induce auxin sensitivity it is used as a tool in this screen.
  • The recombination frequency can be used to measure the genetic distance between two markers: the longer distance, the more recombination events, the higher recombination frequency.
  • IAA it has the expected phenotype meaning that auxin is able to contribute to that phenotype
  • 2-93 on AT present a degenerated meristem and an abnormal expression of GFP, while on IAA it presents a normal meristem with a normal expression of GFP. Auxin at AT media has a deficiency, the plant has a low amount of auxin on the AT media. The DR5:GFP reporter was used to monitor auxin response. Both taa1 tar2 and 2-93 display reduced auxin response at 10 days post germination: their root meristems degenerate. Addition of 100 nM IAA to growth media partially reverts this defect.
  • No solo es lacalculaciongenetica de 2-93 sinotambien la de wei8 yaque 2-93 es un doblemutante.
  • Characterizing Auxin Biosynthetic Mutants in Arabidopsis thaliana

    1. 1. CHARACTERIZING AUXINBIOSYNTHETIC MUTANTS INARABIDOPSIS THALIANA Nicole Colón Carrión Direction: Linda Robles Alonso-Stepanova Lab
    2. 2. Objective This study aims to shed light on the auxin biosyntheticpathway by identifying genes involved in the pathway using a novel screen in Arabidopsis.
    3. 3. Auxin• Auxin biosynthesis can occur via tryptophan dependent or independent pathways• There are many unknowns in the auxin biosynthetic pathway and we hope to find regulating factors
    4. 4. TAA1 gene encodes B TRP an aminotransferase TAA1 that is used to CYP79B2/B3 TAR1 TAR2 convert TRP into IPA.M IAOx IPyA IAMM IAN IAM NIT SUR2 SUR1 YUC AMI UGT74B1 IGs IAA
    5. 5. Ethylene : Induce Auxin synthesis Col-O Ethylene Col wei8 wei8 tar2Air Ethylene Figure 1. Represent the effect of the lost of the aminotransferase
    6. 6. Enhancer screen for auxin biosynthetic mutants~50,000 wei8 DR5:GFP plants have been EMS mutagenized.~100,000 plants have been screened.~2100 “putants” have been picked.Retesting of putants has been completed.~ 200 have the loss of apical hook. Of these, ~25 have a longer root.  Further characterization  Goal: To find other factors contributing to auxin biosynthesis.
    7. 7. F2’s from crosses were analyzed at two timepoints for auxin sensitivity and ethylene responses. Seedlings with the parental phenotype were selected for further analysis. The recombination frequency was calculated.Recombination freq = total no of mutants/ total of offsprings X 100% Root degeneration and DR5:GFP expression were monitored. Seedlings with the mutant phenotype were propagated for mapping.
    8. 8. Mutants 18-40 Semi- dominant22-25-4 Recessive mutant21-44-5 Dominant12-20-2 Recessive mutant 12-8-3 Recessive mutant 2-93 Recessive mutant
    9. 9. Characterization of 2-93 at 3 days old Col wei8 wei8 tar2 2-93Standard mediaEthylene media Auxin media
    10. 10. Characterization of 2-93 at 10 days old Col wei8 wei8 tar2 2-93 AT10 days old AT DR5:GFP10 days old IAA10 days old IAA DR5:GFP10 days old
    11. 11. F2’s for 2-93 cross Cross Long root Total RatioCol 3 100 ~1:16Ler 4 105 ~1:16wei8 8 98 ~1:4 Results: *2-93 2-93 mutant cross to Col and to Ler presented a 1:16 ratio, 10% while the cross to wei8 presented 1:4 ratio meaning 5% *2-93 that it is a recessive mutation. 0% xCol x Ler x W8-1
    12. 12. Chromosomal location of causal mutation in the 2-93 line
    13. 13. Acknowledgements• Dr. Alonso• Dr. Stepanova• Dr. Linda Robles• Dr. Sue Carson• Dr. Karen Merchante• Jeonga Yun• Dr. Nelson
    14. 14. Questions