This study aims to characterize genes involved in the auxin biosynthetic pathway in Arabidopsis thaliana. The study performed an EMS mutagenesis screen of over 100,000 plants expressing the DR5:GFP auxin response reporter to identify mutants with altered auxin responses. Several hundred putative mutants were identified and further characterized. One mutant, called 2-93, showed recessive, ethylene-insensitive phenotypes. Genetic mapping placed the 2-93 mutation on a chromosome. Further experiments aim to identify the specific gene disrupted in 2-93 to gain new insights into auxin biosynthesis.

1. CHARACTERIZING AUXIN
BIOSYNTHETIC MUTANTS IN
ARABIDOPSIS THALIANA
Nicole Colón Carrión
Direction: Linda Robles
Alonso-Stepanova Lab

2. Objective
This study aims to shed light on the auxin biosynthetic
pathway by identifying genes involved in the pathway using a
novel screen in Arabidopsis.

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. TAA1 gene encodes
B TRP
an aminotransferase
TAA1 that is used to
CYP79B2/B3 TAR1
TAR2
convert TRP into IPA.
M IAOx IPyA IAM
M IAN IAM
NIT
SUR2
SUR1
YUC AMI
UGT74B1
IGs IAA

5. Ethylene : Induce Auxin synthesis
Col-O
Ethylene
Col wei8 wei8 tar2
Air Ethylene
Figure 1. Represent the effect of
the lost of the aminotransferase

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. 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. Mutants
18-40 Semi- dominant
22-25-4 Recessive mutant
21-44-5 Dominant
12-20-2 Recessive mutant
12-8-3 Recessive mutant
2-93 Recessive mutant

9. Characterization of 2-93 at 3 days old
Col wei8 wei8 tar2 2-93
Standard
media
Ethylene
media
Auxin
media

10. Characterization of 2-93 at 10 days old
Col wei8 wei8 tar2 2-93
AT
10 days old
AT
DR5:GFP
10 days old
IAA
10 days old
IAA
DR5:GFP
10 days old

11. F2’s for 2-93 cross
Cross Long root Total Ratio
Col 3 100 ~1:16
Ler 4 105 ~1:16
wei8 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. Chromosomal location of causal mutation in the
2-93 line

13. Acknowledgements
• Dr. Alonso
• Dr. Stepanova
• Dr. Linda Robles
• Dr. Sue Carson
• Dr. Karen Merchante
• Jeonga Yun
• Dr. Nelson

14. Questions

15. THANKS FOR YOUR ATTENTION