1. pH Regulation in Blueberries Locating Nhx1

2. Which proteins regulate pH? The Nhe or Nhx (Na/H exchanger) family of genes – Six known members of this family in plants and animals – While these proteins regulate pH, they themselves are regulated by intercellular pH levels – Nhe/Nhx1 most studied of these proteins

3. Nhx1 - Function Controls vacuolar pH in plants 2001 study found “an increase in vacuolar pH causes blueing of flower color” Nhx1 responsible for this increase in pH – Flower with mutant Nhx1 never turned blue, stayed purple, and maintained a lower vacuolar pH Increases expression of Nhx1 causes rise in pH and blue color

4. Nhx1 - Structure 12 transmembrane protein ~820 amino acids Most conserved regions are TM 6 and 7 Regulatory binding sites located at c-terminus 14-15 exons in gene

5. Finding Nhx1 Complete cdna for grape Nhx1 obtained from NCBI No hits when blasted against illumina data 1 hit against 454 data – 82 bp, 90% ID – Using the grape genome browser, I determined this match was from exon 9 in the grape gene

6. Nhx1 in the EST db Nhx1 from grape blasted against EST db provided one large hit, 1239 bp, 79% ID Determining exons in this hit: – I blasted four exon-specific (exons 1, 2, 7 and 14) primers against these 1239 bp and got no hits – I blasted the full transcript of Arabidopsis Nhx1 (downloaded from the A. genome browser) against these 1239 bp and got no hits

7. Determining exons continued I searched for Nhx1 in the grape genome browser and found six candidate genes I blasted the full sequences of all six candidates against the 1239 bp One hit found – Analysis of the matching sequence using the grape genome browser demonstrated that this hit came from exon 10 in the grape gene

8. Further evidence that we have exon 10 I blasted the full transcript of Arabidopsis Nhx1 against the 454 data One hit: – Analysis of this hit with the Arabidopsis genome browser demonstrated it corresponds to exon 10 in Arabidopsis Nhx1

9. Attempt to expand my hits I blasted my EST hit (the 1239 bp) against the 454 db Got a total of 12 hits in two scaffolds – Turned these two scaffolds into two databases – Blasted exon-specific primers against these scaffolds and got no hits – Blasted full Arabidopsis transcript against both scaffolds and got no hits

10. Conclusions we can draw We have the equivalents of exon 9 and 10 in our sequence data The Arabidopsis transcript did not match with the largest hit obtained with grape. This indicates significant differences in the structure of the gene between Arabidopsis and blueberry

11. Further steps What information lies in the two scaffolds that contained the 12 hits? – Do these 12 hits represent 12 exons (or parts of exons)? What other information lies in the 1239 bp hit from the EST db? – Exon 10 isn’t very large, and the section of 1239 that corresponds to exon 10 is located at the end of the sequence. What other exons are located upstream? How can we identify them? If we find the full sequence: – How does the c-terminus vary from the structure of Nhx1 in other plants?

12. Beyond Nhx1 I blasted plant (grape or corn) cdnas for Nhx2, 3, 4, 5 and 6 against the 454 and found no hits As shown in the diagram earler, PIP2, CHP, CaM, p90RSK, NIK, ROCK, ERM and NIK play regulatory roles in Nhx1 – We can search for these genes in blueberry We can search for an H+ antiporter in the vacuole

13. Bibliography Putney, J.K., Denker, S.P., Barber, D. L. (2002). The changing face of the Na+/H+ exchanger, NHE1: Structure, regulation and cellular actions. Annual Reviews Pharmacology and Toxicology, 42, 527--552. Retrieved from http://www.annualreviews.org/doi/pdf/10.1146/annurev.pharmtox.42.092001.143801Yama guchi, T. e. a. (2001). Genes encoding the vacuolar Na+/H+ exchanger and flower coloration. Plant Cell Physiology, 42(5), 451--461. http://www.annualreviews.org/doi/pdf/10.1146/annurev.pharmtox.42.092001.143801Yama guchi, T. e. a. (2001). Genes encoding the vacuolar Na+/H+ exchanger and flower coloration. Plant Cell Physiology, 42(5), 451--461. Yamaguchi, T. e. a. (2001). Genes encoding the vacuolar Na+/H+ exchanger and flower coloration. Plant Cell Physiology, 42(5), 451--461. Grape genome browser: http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/ Arabidopsis genome browser: http://atensembl.arabidopsis.info/index.html