Long distance transport and seed loading of nutrients and toxic - - PDF document

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David Mendoza University of Missouri

• Columbia

Des Moines, IA April 28th, 2014

Plant Sciences

College of Agriculture, Food and Natural Resources

Long‐distance transport and seed loading

• f nutrients and toxic metals.

Now what?

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Today’s plan…

• Allocation of nutrients within plant tissues.
• Cell-specific translatome analyses to map

seed loading processes. (Trends & current tools)

• Dynamic view of nutrient mobilization within

the plant.

(Bottlenecks and needs) Modeling / sensing / data integration

Transition metals are extremely reactive

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Essential (Fe, Zn, Mn, Cu) catalysis, redox centers, structure. Non essential (Cd, Hg, Ag) properties similar to essential metals

Fe2+% S2'% thiol%

• r%%imidazole%

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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

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Companion ¡cells ¡ (phloem ¡loading ¡cells) ¡

Phloem image from Zhang et al. (2010) Plant Cell; Mendoza-Cozatl et al., 2011 Curr Opin Plant Biol

Transporters expressed in companion cell are key to move molecules into seeds.

Which transporters mobilize metals (and other nutrients) into seeds?

!"#$% #&'()*+% %,-.+)/*% "0123% 456% 756% 81$9%),/+.,)%

Companion cell Ribo-seq

(Polysome-IP)

SUC2p Nost HF-RPL18

RPL18

≈ 250 ng total RNA HQ RNA-seq library Without pre-amplification

RIN 8.5

SUC2

Total RNA sample Companion cell sample RPM

Total RNA CCell RNA

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Ribo-seq of phloem-loading cells was used to identify phloem-specific transporters

Transporters (raw signal)

Phloem-expressed transporters

OPT3 SUC2 YSL1 YSL3

gene1…n

Organized in 96-well plates for high throughput screening

Mather A. Khan, Andrew Riga and Mendoza-Cozatl, unpublished Z value

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• 2

2 4 6 8 10 12 14 16

Ba Ca Cd Fe K Mg Mn Na P S Zn

No ¡Cd ¡ ¡ ¡ ¡ ¡ ¡ +Cd ¡ ¡ ¡ ¡ ¡ ¡

Phloem transporters individually expressed in yeast.

A" C" F"

%0.6" %0.4" %0.2" 0" 0.2" 0.4" 0.6" 0.8" 1" 1.2"

1" 2" 3" 4" 5" 6" 7" 8" 9" 10" 11" 12"

Normalized to empty vector

Cd content in yeast seeds ¡ ¡ ¡ ¡ ¡ ¡ leaves ¡ roots ¡

Col transp1

+ Cd

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Nga T. Nguyen

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0.00 ¡ 5.00 ¡ 10.00 ¡ 15.00 ¡ 20.00 ¡ 25.00 ¡ 30.00 ¡ 35.00 ¡ 10 ¡ 13 ¡ 16 ¡ 19 ¡ 22 ¡ 25 ¡ 28 ¡ 31 ¡ 34 ¡ Leaf ¡1 ¡ Leaf ¡2 ¡ Leaf ¡3 ¡ Leaf ¡4 ¡ Leaf ¡5 ¡ Leaf ¡6 ¡ Leaf ¡7 ¡ Leaf ¡8 ¡ Leaf ¡9 ¡ Moved ¡to ¡soil ¡ Plate ¡stage ¡

Leaf ¡length ¡(mm) ¡ ¡ Days ¡aHer ¡germinaIon ¡ ¡

Ionome dynamics through plant development

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5 10 15 20 25 Ba Ca Cd Fe K Mg Mn Na P S Zn

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Integration of transcriptomics, development and phenomics

WT (Col) mutants

Z value

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2 4 6 8 10 12 14 16

Ba Ca Cd Fe K Mg Mn Na P S Zn

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So…

top-down approach (reductionist) bottom-up approach (systems) ?

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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

≠

CJ

1 + CJ 2 …+ CJ n = 1 Flux control coefficient Enzyme with high CJ Enzyme with low CJ

Modeling ¡allows ¡predicIons ¡ ¡ (new ¡hypothesis) ¡ ¡

How ¡do ¡you ¡know ¡you ¡have ¡a ¡good ¡model? ¡

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Summary

• 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

• We have the technology to track the expression of plant genes

at tissue-specific resolution (link to metabolites missing!).

Expression during development? Stress? Drought?

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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 ¡