1. The HAT2 Homeodomain-Like Transcription Factor Family: Genes AT5G47370 and AT4G17460 Bekah Charney HC70AL Spring 2006

2. What is a HAT2 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 HAT2 gene is induced by auxin, a plant hormone that regulates growth and development

3. Where is AT5G47370 located and what does it code for? AT5G47370 is located on the 5 th chromosome The gene is 1,598 basepairs in length It codes for a HAT2 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

4. What are the Anatomical Features of AT5G47370? EXON 15’URT3’ _____ EXON 2EXON 3EXON 4 ___________ 5’UTR3’ 263-413 549-859943-10221119-1598

5. Where is AT4G17460 and what does it code for? AT4G17460 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 HAT1 homeobox protein mRNA The size of this protein is 283 amino acids

6. What are the Anatomical Features of AT4G17460? EXON 15’URT3’ _____ EXON 2EXON 3EXON 4 ___________ 5’UTR3’ 183-330 418-746832-9111013-1304

7. What Genotypes Did My Plants Reveal? SALK_055288 knockout for gene AT5G47370 Homozygous mutants were found, showing that a knockout of this gene does not cause seed lethality Homozygous Mutant Expected WT Size = 731 bps Observed WT Size = 700 bps Expected Mutant Size = 354 bps Observed Mutant Size = 737 and 354 bps 15 Plants were genotyped: -8 Homozygous WT -7 Homozygous M -0 Heterozygotes Wild Type

8. Where is the T-DNA Insert in AT5G47370? EXON 15’URTEXON 2EXON 3EXON 4 ___________ 3’UTR 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 T-DNA LBb1 RV FW

9. What were the genotypes for SALK_069162 knockout for gene AT4G17460? Homozygous Mutant Since homozygous mutant plants were found, it can be concluded that a knockout of AT4G17460 does not cause seed lethality Expected WT Size = 1,037 bps Observed WT Size = 1,000 bps Expected Mutant Size = 669 bps Observed Mutant Size = 784 bps Wild Type

10. Where is the T-DNA insert in gene AT4G17460? EXON 15’URT _____ EXON 2EXON 3EXON 4 ___________ 3’UTR T-DNA Sequencing results showed that there is a single T- DNA 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 FW RV LBb1

11. Where is the gene AT5G47370 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

12. Where is the gene AT4G17460 active? My RT-PCR data differs from the gene chip data in that I found the gene AT4G17460 to be active in both the inflorescence and the silique

13. What work was done with the upstream region of the AT5G47370 gene and the AT4G17460 gene? Amplification of upstream region using iProof High Fidelity DNA Polymerase Upstream region ligated with TOPO-vector so lacZ gene is knocked out E. Coli cells are transformed with recombinant plasmids Restriction Digest with EcoRI Plasmids isolated and sequenced with Sp6 and T7 [AT5G47370 only] Amplified upstream region is 3 kB for gene AT5G47370 Restriction digest of TOPO-vector with EcoRI shows a 3.5 kB band and an approximately 2.9 kB band AT4G17460 What further experiments can be done with this information?

14. Did the knockout Arabidopsis plants show any phenotypic differences than the WT plants? Knockout for gene AT5G47370 Wild Type Early Torpedo Stage NO! Mature Stage NO!

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

16. Knockout for gene AT4G17460 Wild Type Some siliques did not develop seeds For those that did, seeds did not contain an embryo

17. Conclusion A knockout of gene AT5G47370 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 AT4G17460 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?

18. What’s the next step? AT5G47370 –Double knockout –Use GFPs and cloned upstream region to help determine where and when gene is being expressed AT4G17460 –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

19. Acknowledgements I would like to thank Anhthu, Mike, Ria, Jonathan, Tomo, Brandon, XingJun, Jessica, the entire HC70AL class and Dr. Goldberg for all their help and guidance throughout this quarter I would also specifically like to thank Jennifer, Ria, Jonathan and XingJun for allowing me to use their Nomarski photographs in this presentation, as well as Brandon for the gene chip data used in this presentation