1. Characterizing Auxin Biosynthetic Mutants in Arabidopsis thaliana
Nicole Colón Carrión
University of Puerto Rico, Cayey campus; North Carolina State University
The phytohormoneauxin regulates numerous aspects development. Although of plant growth and
auxinwas one of the first plant hormone to be discovered, ourunderstanding of how plants produce this
hormone in a spatially and temporally regulated manner is still limited. Two main routes for auxin
production have been proposed, 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 response induces this auxin biosynthetic pathway, the
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 the root. wei8 was
mutagenized to find novel mutants which enhance its auxin deficient phenotype. In particular, I have
focused on the 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 seedlings with the 2-93 mutant phenotype were selected 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 root meristem integrity were selected for mapping.
Polymorphic markers across the whole Arabidopsis genome are being used to determine the chromosomal
location of causal mutation in the 2-93 line. The characterization of this mutant will advances our
understanding of how this essential plant hormone is produced.
Introduction:
Arabidopsis thaliana is a small
flowering plant related to the mustard
family. This type of family is the most systems, such as regulation, growth and
widely distributed; it has approximately 340 development. Indole acetic acid molecules
genera and 3,350 species. Arabidopsisis can inhibit or stimulate the expression of
highly distributed around the world, it can certain genes. Auxin can control plant
be found in central Asia, Mediterranean development at all levels; that’s why it is
regions and North and South America. This important for plants to maintain a balance of
plant is highly used as a model organism for IAA, too much or too little can be fatal for
the 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 have
better understanding of plants biological indicated thatauxinbiosynthesis is controlled
systems. by the tryptophan independent pathway and
tryptophan dependent pathway, however
Indole acetic acid, also known as how this pathways work remain poorly
auxin was one of the first hormones to be understood. Several pathways has been
discovered. It is a phytohormone that postulated for Trp-dependent pathway; the
controls numerous processes in plant indole-3- acetamide, the indole-3-pyruvic

2. acid, the trypthamine, and the indole-3- About 25 mutants that enhance the
acetaldoxime pathway.The indole-3-pyruvic auxin deficient phenotype of wei8were
acid pathway is important for IAA synthesis chosen for further characterization; however
not only in plants but also for
we focus on the 2-93mutants in Arabidopsis
microorganisms. It was postulated that
Arabidopsis seedlings contain the TAA1 Thaliana.Three-day-old seedlings
gene, also known as wei8, which encodes an wereanalyzed for ethylene and auxin
aminotransferase that is used to converts Trp response. Characteristic of auxin
into IPA (Stepanova et al., 2008). Finally biosynthetic mutants, wei8 tar2 and 2-93
IPA is converted into IAA. The Trp- lack apical hooks and displaylongerroots on
independent pathways was postulated in the ethylene-supplemented media, yet a
1991 but how this pathway designs IAA is
normal auxin response, as shown in figure 1.
not well understood. It is our objective to
shed the light on the auxin biosynthetic
pathway by finding genes that enhance the
wei8 phenotype using Arabidopsis as our
plant model. By understanding how plants
synthesis this hormone, we could understand
better how plants regulate it fine balance.
Materials and Methods
About 50,000 Arabidopsis Thaliana
plants were previously EMS mutagenized
and screened. 2,100 putative mutants were
chosen, based on long root and loss of the
apical hook phenotype, for further Figure 1. Shows Three-day-old seedlings
characterization. Since ethylene induces analyzed for ethylene and auxin response.
auxin biosynthesis it is used as a tool during
the screen. F2’s seedlings from crosses were The recombination frequency was
analyzed at twotimepoints (three-day-old calculated for 2-93 mutants based on the
and ten-day-old) for auxin sensitivity and long root phenotype. 2-93 mutant cross to
ethylene responses. Seedlings with the Col and to Ler presented a 1:16 ratio, while
parental phenotype were selected for further the cross to wei8 presented 1:4 ratio
analysis and there recombination frequency meaning that it is a recessive mutation. Root
was 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 root
Results:
meristem defect. On auxin media, wei8 tar2
and 2-93 showed a healthy meristem with
normal expression of DR5:GFP. Addition of

3. auxin to these mutants resulted in the
complementation of the root meristem The 2-93 mutant in Arabidopsis
phenotype. 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 the
Figure 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 mutation
phenotype were selected for mapping. may be workingtogether with the wei8 gene
Polymorphic markers across the whole in controlling auxin biosynthesis.
Arabidopsis genome are being used to
determine the chromosomal location of the
References:
causal mutation in 2-93. . Markers
MAC9D21K and MQD22 on ● Price, Robert et al., 1994.Arabidopsis.
chromosomefivedidnotexhibitlinkage. The United States of America: Cold Spring
causal mutation of 2-93 is Harbor Laboratory Press.
notlocatednearthese markers. Marker
T13E11-1 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 the
chromosomes to find wherethere is linkage ●Stepanova et al., 2008.TAA1-Mediated
to find the gene responsible for the 2-93 AuxinBiosynthesisIsEssential for Hormone
phenotype. Crosstalk and Plant Development.
● TAIR. [internet] [2008] USA:
Arabidopsis Information Resource (TAIR).
Discussion:

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