This study aims to characterize auxin biosynthetic mutants in Arabidopsis thaliana to better understand auxin production. Over 100,000 seedlings were screened to identify mutants with auxin-deficient phenotypes. One mutant, 2-93, showed dramatic reduction in ethylene response and reduced root growth. 2-93 has a recessive mutation that causes auxin deficiency and loss of root meristem integrity. The researchers are using genetic mapping to determine the chromosomal location of the causal mutation in 2-93, which may help identify new genes involved in auxin biosynthesis.

1. Characterizing Auxin Biosynthetic Mutants in Arabidopsis thaliana
Nicole Colon Linda Anna Carrion 1,
and Jose Robles 2, Stepanova 2, Alonso 2
University of Puerto Rico, Cayey 1 ; Department of Genetics, North Carolina State University
Abstract Methods wei8 Enhancer 2-93 Shows Root Meristem
Identification of Putative Auxin Defects
The phytohormone auxin regulates numerous aspects of
plant growth and development. Although auxin was one of
the first plant hormones to be discovered, our Biosynthetic Mutants Col wei8 wei8 tar2 2-93
understanding of how plants produce this hormone in a
spatially and temporally regulated manner is still limited. 50,000 wei8 DR5:GFP plants were EMS
Standard
Two main routes for auxin production have been proposed, mutagenized
the tryptophan-dependent and tryptophan-independent
pathways. This study aims to shed light on the tryptophan-
dependent auxin biosynthetic pathway using a novel
mutant screen in Arabidopsis thaliana. Because ethylene ~100,000 three-day-old M2 seedlings were
response induces this auxin biosynthetic pathway, the screened in the triple response assay
Standard
ethylene response is utilized as a tool in the screen.
Specifically, loss of the auxin biosynthetic enzyme
tryptophan aminotransferase, wei8, results in a partial loss
of ethylene response in roots. wei8 was mutagenized to ~2,100 M2 “putants” were selected
find novel mutants which enhance its auxin deficient Criteria for selection:
phenotype. In particular, I have focused on the Longer roots and/or loss of apical hooks
Auxin
characterization of the mutant 2-93 that shows dramatic
reduction in the ethylene response and reduced root
meristematic activity. A mapping population from a cross
between 2-93 and the Ler accession was obtained. F2 ~200 plants retested
seedlings with the 2-93 mutant phenotype were selected
Auxin
and the genetic nature of the mutation determined to be
recessive based on the segregation analysis. Plants with
strong auxin deficiency phenotype marked by the loss of ~25 best mutants were chosen
root meristem integrity were selected for mapping.
Polymorphic markers across the whole Arabidopsis genome
for crossing and mapping
-Characterization of these ”putants” includes ethylene and auxin Figure 3. Root degeneration and DR5:GFP were examined. DR5:GFP
are being used to determine the chromosomal location of response assays and a detailed analysis of the root meristem. reporter was used to monitor auxin response. On the standard
causal mutation in the 2-93 line. The characterization of this -Of these 200 “putants”, 25 mutants that enhance the auxin media, 10-day-old wei8 tar2 and 2-93 seedlings showed an auxin
mutant will advance our understanding of how this deficient phenotype of wei8 were selected for a more detailed deficiency and root meristem defect. On auxin media, wei8 tar2 and
essential plant hormone is produced. analysis. 2-93 showed a healthy meristem with normal expression of DR5:GFP.
Addition of auxin to these mutants resulted in the complementation
of the root meristem phenotype.
Objective Results Chromosomal Location of the Causal Mutation
Auxin Biosynthesis via Trp- dependent 2-93 Exhibits Auxin Deficient
in 2-93
Pathway Phenotype
B Characterization of 2-93
TRP
CYP79B2/B3
TAA1 Col wei8 wei8 tar2 2-93
TAR1
TAR2
TAM IAOx IPyA IAM
UC Standard
HTAM IAN IAM media
NIT
SUR2
SUR1
YUC AMI
UGT74B1
IAN
Ethylene
media
IGs IAA
Auxin
Figure 1. The auxin biosynthetic pathway has been studied for many Figure 4. Seedlings with the mutant phenotype were selected for
years, however it is not completely understood. There are many
media mapping. Polymorphic markers across the whole Arabidopsis genome are
genes regulating these different routes in the auxin biosynthetic being used to determine the chromosomal location of the causal mutation
pathway that have not yet been identified. Two main routes have in 2-93. Figure 4 shows the 5 chromosomes in Arabidopsis and the markers
been proposed for auxin biosynthesis: trp- dependent and trp- Figure 2. Three-day-old seedlings were analyzed for ethylene and auxin used to determine the location of this mutation. Markers MAC9D21K and
independent pathways. This study aims to shed light on the auxin response. Characteristic of auxin biosynthetic mutants, wei8 tar2 and 2-93 lack MQD22 on chromosome five did not exhibit linkage. The causal mutation
biosynthetic pathway using a novel mutant screen in Arabidopsis apical hooks and display longer roots on the ethylene-supplemented media, of 2-93 is not located near these markers. Marker T13E11-1 on
thaliana to find mutants defective in auxin biosynthesis. Our yet a normal auxin response. chromosome two appears to be linked to the gene responsible for the 2-93
objective is to try to find genes that enhance auxin deficient wei8 that mutation. I will be testing markers on all the chromosomes to find where
may be contributing to the auxin biosynthetic pathway. there is linkage to find the gene responsible for the 2-93 phenotype.
Acknowledgments:
● Dr. Alonso ● Dr. Karen Merchante
● Dr. Stepanova ● Jeonga Yun
● Dr. Linda Robles ● Dr. Nelson
● Dr. Sue Carson ● RISE Program , R25 GM059429