5/1/14 Plant Sciences College of Agriculture, Food and Natural Resources Long ‐ distance transport and seed loading of nutrients and toxic metals. David Mendoza University of Missouri - Columbia Des Moines, IA April 28th, 2014 Now what? 1
5/1/14 o Allocation of nutrients within plant tissues. o Cell-specific translatome analyses to map seed loading processes. (Trends & current tools) o Dynamic view of nutrient mobilization within the plant. (Bottlenecks and needs) Modeling / sensing / data integration Today’s plan … 3 Transition metals are extremely reactive Essential Non essential (Fe, Zn, Mn, Cu) (Cd, Hg, Ag) catalysis, redox centers, structure. properties similar to essential metals Fe 2+% S 2'% thiol% or%%imidazole% 4 2
5/1/14 Our goal is to understand how plants mobilize micronutrients and toxic metals Fe and Zn deficiencies are among the most prevalent nutrient deficiencies in the world. 100+ million people worldwide are exposed to As and Cd due to irrigation with contaminated water. Biofortification 3
5/1/14 Which transporters mobilize metals (and other nutrients) into seeds? Companion ¡cells ¡ (phloem ¡loading ¡cells) ¡ Transporters expressed in companion cell are key to move molecules into seeds. 7 ¡ Phloem image from Zhang et al. (2010) Plant Cell; Mendoza-Cozatl et al., 2011 Curr Opin Plant Biol Companion cell Ribo-seq (Polysome-IP) SUC2p HF-RPL18 Nost "0123% !"#$% 81$9%),/+.,)% 456% 756% #&'()*+% %,-.+)/*% RPL18 SUC2 Total RNA Companion cell sample RPM ≈ 250 ng total RNA Total RNA CCell RNA sample HQ RNA-seq library Without pre-amplification RIN 8.5 4
5/1/14 Ribo-seq of phloem-loading cells was used to identify phloem-specific transporters Phloem-expressed SUC2 gene1 … n OPT3 YSL3 transporters YSL1 Organized in 96-well plates Transporters (raw signal) for high throughput screening Mather A. Khan, Andrew Riga and Mendoza-Cozatl, unpublished Phloem transporters individually Cd content in yeast expressed in yeast. 1.2" Normalized to 1" empty vector 0.8" 0.6" 0.4" 0.2" 0" F" 1" 2" C" %0.2" 3" 4" 5" 6" 7" 8" A" 9" %0.4" 10" 11" 12" %0.6" No ¡Cd ¡ ¡ ¡ ¡ ¡ ¡ +Cd ¡ ¡ ¡ ¡ ¡ ¡ 16 seeds ¡ ¡ ¡ ¡ ¡ ¡ 14 12 10 Col transp1 Z value leaves ¡ 8 6 + Cd 4 2 roots ¡ 0 -2 Ba Ca Cd Fe K Mg Mn Na P S Zn -4 5
5/1/14 11 ¡ Nga T. Nguyen 6
5/1/14 Ionome dynamics through plant development 25 20 15 35.00 ¡ 10 5 Leaf ¡1 ¡ 30.00 ¡ 0 Leaf ¡2 ¡ Ba Ca Cd Fe K Mg Mn Na P S Zn -5 Leaf ¡length ¡(mm) ¡ Leaf ¡3 ¡ 25.00 ¡ Leaf ¡4 ¡ 20.00 ¡ Moved ¡to ¡soil ¡ Leaf ¡5 ¡ Leaf ¡6 ¡ 15.00 ¡ Leaf ¡7 ¡ ¡ Leaf ¡8 ¡ 10.00 ¡ Leaf ¡9 ¡ 5.00 ¡ 0.00 ¡ 10 ¡ 13 ¡ 16 ¡ 19 ¡ 22 ¡ 25 ¡ 28 ¡ 31 ¡ 34 ¡ Plate ¡stage ¡ Days ¡aHer ¡germinaIon ¡ ¡ Integration of transcriptomics, development and phenomics 16 14 12 10 Z value 8 6 4 2 0 -2 Ba Ca Cd Fe K Mg Mn Na P S Zn WT (Col) mutants -4 14 ¡ 7
5/1/14 So … top-down approach (reductionist) bottom-up approach (systems) ? 15 8
5/1/14 Metabolic ¡control ¡analysis ¡ (a ¡quanItaIve ¡approach ¡to ¡understand ¡metabolism) ¡ Steady state (an unvarying condition in a physical process) Constant rate of synthesis (continuous flow) without changes in intermediary metabolites. ≠ Flux Concentration C J 1 + C J 2 … + C J n = 1 Enzyme with low C J Flux control coefficient Enzyme with high C J Modeling ¡allows ¡predicIons ¡ ¡ (new ¡hypothesis) ¡ ¡ How ¡do ¡you ¡know ¡you ¡have ¡a ¡good ¡model? ¡ 9
5/1/14 Summary • We have the technology to track the expression of plant genes at tissue-specific resolution ( link to metabolites missing! ). Expression during development? Stress? Drought? • Metabolic engineering requires a good understanding of both, the individual components and system properties as a whole. Combination of top-down and bottom-up approaches • Dynamic view of nutrient mobilization throughout the plant Sensors, modeling, data integration (cross-disciplinary training) funding opportunities 19 Andrew C. Riga Sahar Dowlatshahi Norma Castro Guerrero Nga T. Nguyen Sam A. McInturf Mather A. Khan Trupti Joshi Dong Xu Special thanks to IPG faculty: Walter Gassmann David Braun Gary Stacey Scott Peck The DMC lab CAREER ¡ 20 10
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