Characterizing Auxin Biosynthetic Mutants in Arabidopsis thalianaNicole Colon Carrion1, Linda Robles2, Anna Stepanova2, and Jose Alonso2

University of Puerto Rico, Cayey1 ; Department of Genetics, North Carolina State University

Abstract wei8 Enhancer 2-93 Shows Root Meristem Defects

Col wei8 wei8 tar2 2-93

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Methods

Identification of Putative Auxin Biosynthetic Mutants

Objective

The phytohormone auxin regulates numerous aspects ofplant growth and development. Although auxin was one ofthe first plant hormones to be discovered, ourunderstanding of how plants produce this hormone in aspatially and temporally regulated manner is still limited.Two main routes for auxin production have been proposed,the tryptophan-dependent and tryptophan-independentpathways. This study aims to shed light on the tryptophan-dependent auxin biosynthetic pathway using a novelmutant screen in Arabidopsis thaliana. Because ethyleneresponse 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 lossof ethylene response in roots. wei8 was mutagenized tofind novel mutants which enhance its auxin deficientphenotype. In particular, I have focused on thecharacterization of the mutant 2-93 that shows dramaticreduction in the ethylene response and reduced rootmeristematic activity. A mapping population from a crossbetween 2-93 and the Ler accession was obtained. F2seedlings with the 2-93 mutant phenotype were selectedand the genetic nature of the mutation determined to berecessive based on the segregation analysis. Plants withstrong auxin deficiency phenotype marked by the loss ofroot meristem integrity were selected for mapping.Polymorphic markers across the whole Arabidopsis genomeare being used to determine the chromosomal location ofcausal mutation in the 2-93 line. The characterization of thismutant will advance our understanding of how thisessential plant hormone is produced.

Acknowledgments:

● Dr. Alonso● Dr. Stepanova● Dr. Linda Robles● Dr. Sue Carson

● Dr. Karen Merchante● Jeonga Yun● Dr. Nelson● RISE Program , R25 GM059429

50,000 wei8 DR5:GFP plants were EMS mutagenized

~100,000 three-day-old M2 seedlings were screened in the triple response assay

~2,100 M2 “putants” were selectedCriteria for selection:

Longer roots and/or loss of apical hooks

~25 best mutants were chosenfor crossing and mapping

-Characterization of these ”putants” includes ethylene and auxin response assays and a detailed analysis of the root meristem.-Of these 200 “putants”, 25 mutants that enhance the auxin

deficient phenotype of wei8 were selected for a more detailed analysis.

Figure 1. The auxin biosynthetic pathway has been studied for manyyears, however it is not completely understood. There are manygenes regulating these different routes in the auxin biosyntheticpathway that have not yet been identified. Two main routes havebeen proposed for auxin biosynthesis: trp- dependent and trp-independent pathways. This study aims to shed light on the auxinbiosynthetic pathway using a novel mutant screen in Arabidopsisthaliana to find mutants defective in auxin biosynthesis. Ourobjective is to try to find genes that enhance auxin deficient wei8 thatmay be contributing to the auxin biosynthetic pathway.

Results2-93 Exhibits Auxin Deficient

Phenotype

Characterization of 2-93

Standard media

Ethylene media

Col wei8 wei8 tar2 2-93

Figure 4. Seedlings with the mutant phenotype were selected for mapping. Polymorphic markers across the whole Arabidopsis genome are being used to determine the chromosomal location of the causal mutation in 2-93. Figure 4 shows the 5 chromosomes in Arabidopsis and the markers used to determine the location of this mutation. Markers MAC9D21K and MQD22 on chromosome five did not exhibit linkage. The causal mutation of 2-93 is not located near these markers. Marker T13E11-1 on chromosome two appears to be linked to the gene responsible for the 2-93 mutation. I will be testing markers on all the chromosomes to find where there is linkage to find the gene responsible for the 2-93 phenotype.

Figure 2. Three-day-old seedlings were analyzed for ethylene and auxinresponse. Characteristic of auxin biosynthetic mutants, wei8 tar2 and 2-93 lackapical hooks and display longer roots on the ethylene-supplemented media,yet a normal auxin response.

Figure 3. Root degeneration and DR5:GFP were examined. DR5:GFPreporter was used to monitor auxin response. On the standardmedia, 10-day-old wei8 tar2 and 2-93 seedlings showed an auxindeficiency and root meristem defect. On auxin media, wei8 tar2 and2-93 showed a healthy meristem with normal expression of DR5:GFP.Addition of auxin to these mutants resulted in the complementationof the root meristem phenotype.

Chromosomal Location of the Causal Mutation in 2-93

Auxin media

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TRP

IAOx IPyA IAM TAM

HTAM

IAA

IAAld

IGs

IANIAM

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TAA1TAR1TAR2

CYP79B2/B3

SUR2SUR1UGT74B1

IANAMI

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PDC

AAO

TRP

IAOx IPyA IAM

IAAIGs

IANIAM

TAA1TAR1TAR2

CYP79B2/B3

SUR2SUR1UGT74B1

AMI

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YUC

A B

Auxin Biosynthesis via Trp- dependent Pathway

~200 plants retested