The HAT 2 HomeodomainLike Transcription Factor Family Genes

The HAT 2 Homeodomain-Like Transcription Factor Family: Genes AT 5 G 47370 and AT 4 G 17460 Bekah Charney HC 70 AL Spring 2006

What is a HAT 2 Homeodomain Transcription Factor? • Type of transcription factor that is only found in plants • Has been studied in sunflowers, where it is expressed primarily in the leaves • When Hahb-4 (sunflower homeobox-leucine zipper protein) was introduced into Arabidopsis, plants were more tolerant to water stress conditions • HAT 2 gene is induced by auxin, a plant hormone that regulates growth and development

Where is AT 5 G 47370 located and what does it code for? • AT 5 G 47370 is located on the 5 th chromosome • The gene is 1, 598 basepairs in length • It codes for a HAT 2 homeobox leucine zipper protein • The size of this protein is 284 amino acids • Within the chromosome, the gene is oriented in the 3’ 5’ direction

What are the Anatomical Features of AT 5 G 47370? 5’URT 3’ EXON 1 263 -413 _____ EXON 2 549 -859 _____ EXON 3 943 -1022 ______ EXON 4 1119 -1598 5’UTR 3’

Where is AT 4 G 17460 and what does it code for? • AT 4 G 17460 is on the 4 th chromosome • The gene is 1, 467 basepairs in length • Within the chromosome, the gene is oriented in the 5’ 3’ direction • It codes for a HAT 1 homeobox protein m. RNA • The size of this protein is 283 amino acids

What are the Anatomical Features of AT 4 G 17460? 5’URT 3’ EXON 1 183 -330 _____ EXON 2 418 -746 _____ EXON 3 832 -911 ______ EXON 4 1013 -1304 5’UTR 3’

What Genotypes Did My Plants Reveal? SALK_055288 knockout for gene AT 5 G 47370 15 Plants were genotyped: -8 Homozygous WT -7 Homozygous M -0 Heterozygotes Wild Type Homozygous Mutant Expected WT Size = 731 bps Observed WT Size = 700 bps Expected Mutant Size = 354 bps Observed Mutant Size = 737 and 354 bps Homozygous mutants were found, showing that a knockout of this gene does not cause seed lethality

Where is the T-DNA Insert in AT 5 G 47370? LBb 1 T-DNA RV FW 5’URT EXON 1 EXON 2 _____ EXON 3 ______ EXON 4 • Sequencing results showed that there were two T-DNA inserts [concatomer] at nucleotide 1, 361, but oriented in opposite directions • The location of the T-DNA inserts matches SALK’s predictions 3’UTR

What were the genotypes for SALK_069162 knockout for gene AT 4 G 17460? Expected WT Size = 1, 037 bps Observed WT Size = 1, 000 bps Expected Mutant Size = 669 bps Observed Mutant Size = 784 bps Wild Type Homozygous Mutant Since homozygous mutant plants were found, it can be concluded that a knockout of AT 4 G 17460 does not cause seed lethality

Where is the T-DNA insert in gene AT 4 G 17460? LBb 1 T-DNA RV FW 5’URT EXON 1 _____ EXON 2 _____ EXON 3 ______ EXON 4 • Sequencing results showed that there is a single TDNA insert at nucleotide 336 in the first Intron • This is a 115 basepair difference than what SALK predicted, which was at nucleotide 451 in the second Exon 3’UTR

Where is the gene AT 5 G 47370 active? My RT-PCR data supports the gene chip data that this gene is active in both the stem and the silique, although the levels of activity cannot be determined

Where is the gene AT 4 G 17460 active? My RT-PCR data differs from the gene chip data in that I found the gene AT 4 G 17460 to be active in both the inflorescence and the silique

What work was done with the upstream region of the AT 5 G 47370 gene and the AT 4 G 17460 gene? • Amplification of upstream region using i. Proof High Fidelity DNA Polymerase • Upstream region ligated with TOPO-vector so lac. Z gene is knocked out • E. Coli cells are transformed with recombinant plasmids • Restriction Digest with Eco. RI • Plasmids isolated and sequenced with Sp 6 and T 7 [AT 5 G 47370 only] Amplified upstream region is 3 k. B for gene AT 5 G 47370 Restriction digest of TOPO-vector with Eco. RI shows a 3. 5 k. B band an approximately 2. 9 k. B band AT 4 G 17460 What further experiments can be done with this information?

Did the knockout Arabidopsis plants show any phenotypic differences than the WT plants? Wild Type Knockout for gene AT 5 G 47370 Early Torpedo Stage NO! Mature Stage NO!

Sterile plants were found in knockout line SALK_069162 Genotypes of Sterile Plants Number of Sterile Plants Siliques do not fully develop in the sterile plants Homozygous WT 0 Heterozygous 2 Homozygous Mutant 6

Knockout for gene AT 4 G 17460 Wild Type • Some siliques did not develop seeds • For those that did, seeds did not contain an embryo

Conclusion • A knockout of gene AT 5 G 47370 did not result in seed lethality or show any phenotypic differences – Gene not important in seed development? – Another gene is fulfilling its function? • A knockout of gene AT 4 G 17460 did not result in seed lethality, but all known mutants and two heterozygotes showed sterility – Sterility is simply due to an environmental factor? – Knockout of gene is causing sterility, either alone or with other factors?

What’s the next step? • AT 5 G 47370 – Double knockout – Use GFPs and cloned upstream region to help determine where and when gene is being expressed • AT 4 G 17460 – Check another knockout line (SAIL) to see if sterile plants result and genotype any sterile plants – Breed known heterozygotes and check to see if sterile plants result – Double knockout – GFPs

Acknowledgements I would like to thank Anhthu, Mike, Ria, Jonathan, Tomo, Brandon, Xing. Jun, Jessica, the entire HC 70 AL class and Dr. Goldberg for all their help and guidance throughout this quarter I would also specifically like to thank Jennifer, Ria, Jonathan and Xing. Jun for allowing me to use their Nomarski photographs in this presentation, as well as Brandon for the gene chip data used in this presentation