1. By Emily Keator and Phoebe Parrish : Part 04-17-14

2. What Does Iron Do? Highly reactive—involved in many redox reactions Involved in photosynthesis & cellular respiration as part of the ETC Toxic in high amounts

3. Uptake Genes AHA o Extrudes H + ions FRO2 o Reduces Fe 3+ → Fe 2+ IRT1 o Transports Fe 2+ into cell (regulated by ubiquitin) o Also involved in uptake of other heavy metal cations

4. Transport Genes YSL1&2 o Influx (phloem) o Transport Fe-NA complexes FRD3 (MATE efflux family proteins) o Efflux (xylem) o Transport Fe-citrate complexes; provide shoots with usable forms of Fe

5. Storage Genes Ferritin o Complexes Fe 2+ molecules together o Stores readily-usable form of Fe in vacuoles & amyloplasts PIC1 o Transports Fe into chloroplasts IREG1 o Moves Fe into vacuole

6. Regulatory Genes bHLH38,39,100&101 o Regulate IRT1 and FRO2 genes FIT1 o Forms heterodimers with bHLH → control transcription of Fe uptake genes Both transcribed when Fe deficiency is sensed in the roots

7. QTLs C011,923,307 C0313,636,922 C0543,302,017 C0833,379,515 C09-13,558,943 C09-245,002,150

8. BLAST Results

9. C01 QTL C01: 1,923,307 AHA1,2,9: 4,691,639* IRT1: 3,007,445* YSL2: 11,431,126* YSL1: 11,431,687 FIT1: 6,411,902* 0 +10,000,000 C03 -10,000,000 +10,000,000 AHA1,2,9: 19,835,760* QTL C03: 13,636,922 FRD3: 12,416,349* FIT1: 16,455,349* QTL * = confirmed w/ IGB Questionable presence on IGB

10. C05 -10,000,000 +10,000,000 AHA1,2,9: 43,882,387* QTL C05: 43,302,017 IRT1: 41,537,794* FRD3: 43,624,447* FPN1/IREG1:34,728,143 FER1: 42,532,200* FIT1: 39,453,920 C08 -10,000,000 +10,000,000 QTL C08: 33,379,515 AHA1,2,9: 32,394,505* FRO1,2: 41,520,823* IRT1:39,033,254* FER1:30,370,173* bHLH38&39: 30,863,851* bHLH100&101: 30,878,266, 30,890,636* QTL * = confirmed w/ IGB Questionable presence on IGB

11. C09 -10,000,000 +10,000,000 QTL C09-2: 45,002,150 AHA1,2,9:40,597,697* IRT1: 49,050,708* YSL1: 40,697,628 YSL2: 36,138,179* QTL * = confirmed w/ IGB Questionable presence on IGB QTL C09-1: 3,558,943 AHA1, AHA2: 514,992* AHA9: 8,682,603* FRO1: 137,473* YSL1,2: 1,879,450* PIC1: 7,315,088* +10,000,000 0 C09

12. IGB Results

13. C01 QTL C01: 1,923,307 2,114,850: Heavy metal transport/detoxification superfamily protein^ 3,007,445 (ZIP9)^ -1,000,000 +1,000,000 QTL Confirmed BLAST result Genes found only on IGB ^ = gene identified through BLAST/TAIR method

14. ●ZIP9 (homolog of IRT3) ○ Location: nucleus, plasmodesma ○ Expression: petal differentiation & expansion stage ○ Function: transports zinc and other cations; contains zinc/iron permease domain ● Heavy metal transport/detoxification superfamily protein ○ Location: nucleus, plasmodesma ○ Expression: petal differentiation and expansion stage ○ Function: metal ion transport and binding Confirmed BLAST result Genes found only on IGB

15. C03 QTL C03: 13,636,922 12,418,215: Multidrug resistance protein^ 12,415,632: MATE efflux family protein (FRD3)^ -1,200,000 +1,200,000 QTL Confirmed BLAST result Genes found only on IGB ^ = gene identified through BLAST/TAIR method

16. FRD3 (MATE efflux family protein) o Location: plasma membrane o Expression: petal differentiation & expansion stage o Function: iron-citrate transport MdtK (multidrug resistance protein) o Location: chloroplast & plasma membrane o Expression: petal differentiation & expansion stage o Function: ion transporter/antiporter activity Confirmed BLAST result Genes found only on IGB

17. C05 QTL C05: 43,302,017 42,532,200: ferritin (FER1)^ 42,731,745: Heavy metal transport/detoxification superfamily protein^ -1,000,000 +1,000,000 43,536,616: MATE efflux family protein (FRD3)^ 43,624,447: MATE efflux family protein (FRD3)^ 41,537,794: zinc transporter (IRT1)^ QTL Confirmed BLAST result Genes found only on IGB ^ = gene identified through BLAST/TAIR method 43,882,387: calcium transporting ATPase^

18. IRT1 o Location: membrane o Expression: petal differentiation & expansion stage o Function: cellular response to iron ion starvation, iron transport, etc. FER1 o Location: chloroplast o Expression: petal differentiation & expansion o Function: iron ion transport and homeostasis, ferric iron binding Heavy metal transport/detoxification (homolog of ATX1) o Location: plasma membrane o Expression: pollen, petal, petiole o Function: metal ion transport & binding FRD3 (MATE efflux family protein) o Location: plasma membrane o Expression: roots, flower, petiole o Function: iron ion and citrate transport & homeostasis Confirmed BLAST result Genes found only on IGB

19. C08 QTL C08: 33,379,515 32,394,505: H+-ATPase (AHA) 33,828,404: Ferrochelatase -1,000,000 +1,000,000 QTL Confirmed BLAST result Genes found only on IGB ^ = gene identified through BLAST/TAIR method

20. AHA (H + -ATPase) o Location: plasma membrane, Golgi apparatus, vacuolar membrane o Expression: petal differentiation & expansion o Function: ion transport Ferrochelatase o Location: chloroplast o Expression: petal differentiation & expansion o Function: terminal enzyme of heme biosynthesis Confirmed BLAST result Genes found only on IGB

21. QTL Confirmed BLAST result Genes found only on IGB ^ = gene identified through BLAST/TAIR method C09 QTL C09-1: 3,558,943 3,501,200: Ferrochelatase^ -1,000,000 +1,000,000

22. C09 -1,000,000 +1,000,000 QTL C09-2: 45,002,150 45,568,877: MATE efflux family protein 45,927,322: heavy metal transport/detoxification superfamily protein QTL Confirmed BLAST result Genes found only on IGB ^ = gene identified through BLAST/TAIR method

23. MATE efflux family protein o Location: (?) o Expressed in: (?) o Function: transport Heavy metal transport/detoxification superfamily protein o Location: extracellular space (?) o Expression: petal differentiation & expansion o Function: exact function unknown Confirmed BLAST result Genes found only on IGB

24. Conclusions IGB contained almost all genes found on BLAST Used IGB to confirm genes found on BLAST (+/- 1,000,000 bp) IGB yielded several new candidates (more speculative) Overall: a number of good candidate genes, close to QTL and expressed in relevant tissues

25. References Barberon M et al. Monoubiquitin-dependent endocytosis of the iron-regulated transporter 1 (IRT1) transporter controls iron uptake in plants. PNAS, 2011. 108(32):E450-E458. Briat, JF et. al. Regulation of plant ferritin synthesis: how and why. Cellular and Molecular Life Sciences, 1999. 56:155-166. Grotz, N et. al. Molecular aspects of Cu, Fe, and Zn homeostasis in plants. Biochemica et Biophysica Acta, 2006. 1763:595-608. Harper JF et al. The Arabidopsis thaliana plasma membrane H + -ATPase multigene family. The Journal of Biological Chemistry, 1990. 265(23):13601-13608. Hell, R et. al. Iron uptake, trafficking, and homeostasis in plants. Planta, 2003. 216:541-551. Kim, S et. al. Mining iron: iron uptake and transport in plants. FEBS Letters, 2007. 581:2273-2280. Kobayashi T, Nishizawa NK. Iron uptake, translocation, and regulation in higher plants. Annual Review of Plant Biology, 2012. 63:131-52. Matsuoka, K et. al. Gibberellin-induced expression of Fe uptake-related genes in Arabidopsis. Plant Cell Physiol., 2014. 55(1):87-98. Robinson, NJ et. al. A ferric-chelate reductase for iron uptake from soil. Nature, 1999. 397:694-697. Roschzttardtz, H et. al. New insights into Fe localization in plant tissues. Frontiers In Plant Science, 2013. 4:1-11. Seo, PJ et. al. A golgi-localized MATE transporter mediates iron homeostasis under osmotic stress in Arabidopsis. BIochemical Journal, 2012. 442:551-561. Sussman MR. Molecular analysis of proteins in the plant plasma membrane. Annual Review of Plant Physiology and Plant Molecular Biology, 1994. 45:211-34. Vert, G et. al. IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth. The Plant Cell, 2002. 14:1223- 1233.