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“High Throughput Phenotyping at the Arkansas Plant Powered Production Center� Dr. Argelia Lorence Associate Professor Arkansas Biosciences Institute Arkansas State University Plant Phenomics Workshop, PAG XXI 2013 January 15, 2013


What is the P3 Center? • NSF EPSCoR-supported research consortium targeting cross-disciplinary approaches to plant biology and plant products • 5 Universities – – – – –

Arkansas State University University of AR, Fayetteville University of AR, Little Rock University of AR, Pine Bluff University Central Arkansas

• > 45 faculty (chemists, molecular biologists, biologists, bioinformaticists)


Research Focal Areas 1. Comparative metabolomics: – – –

Gene, pathway enzyme discovery Metabolite engineering HTP metabolite data management

2. Plant interactions with other organisms – –

Insects and pathogens Human and animal health and nutrition

3. Biologic “set-points” – addressing mechanisms that limit protein and biomass accumulation –

Genotype, nutrient transport, oxidative status, protein folding, trafficking, and stability

4. Next-Gen Seq and bioinformatics


We invite you to visit us at www.plantpoweredproduction.org!


A strategic investment at ABI/ASU


Available imaging units • Visible camera Size, architecture, color classification (e.g. chlorosis, necrosis) • Fluorescence camera In planta chlorophyll fluorescence, GFP, phenolic compounds • Near infrared camera In planta water content • Laser Height


Impossible Experiments Are Now Possible • Non-destructive analysis • Subtle phenotypes can be detected • Could be linked with other “omics” data • Large amounts of biological replicates can be analyzed in a short period of time 84 x 96 well plates = 8064 samples/run 15 x 3 or 32 x 3= 45 - 96 plants/run


Proof of principle: Phenotype plant lines with known altered growth Arabidopsis thaliana var. Columbia Wild type (WT, CS60000) MIOX4, GLOase AVP1-1, AVP1-2

Visible camera Leaf area (cm2) Convex hull area (cm2) Calipher length (cm) Compactness Color classification (green and yellow)

Fluorescence camera In planta chlorophyll fluorescence (low, medium, high)

Near infrared camera In planta water content (low, medium, high)


Elevated ascorbate leads to enhanced growth MIOX MIOX4 L3

D-Glucose

WT

D-Glucose-6-P

D-Fructose-6-P

D-Mannose-6-P

D-Glucose-1-P

PMM

UDP-D-Glucose

D-Mannose-1-P VTC1

UDP-D-Glucuronic acid

GDP-D-Mannose GME

GME

Phytate

GDP-L-Galactose

D-Glucuronic acid-1-P GDP-L-Gulose

Pectin

VTC2

myo-Inositol

MIOX

D-Glucuronic acid

L-Gulose-1-P

GlcUR L-Gulonic acid

L-Gulose

L-Galactose-1-P

GNL

L-Gulono-1,4-lactone

GLOase

VTC4

D-Galacturonic acid GalUR

L-Galactose GalDH

L-Galactonic acid

L-Galactono-1,4-lactone GLDH

L-Ascorbic acid Dehydroascorbate

Radzio et al., (2003) Plant Molecular Biology 53: 837-844 Lorence et al., (2004) Plant Physiology 134: 1200-1205 Nessler CL, Lorence A, Mendes P, Chevone BI (2007). US Patent Application No. 11/908,551

Lisko et al., In Vitro Plant. Submitted


Benefits of enhanced AsA content • Increased biomass • Delayed senescence

C

• Enhanced abiotic stress tolerance: – NaCl – Heat – Cold – Paraquat – H2O2 – Environmental pollutants (e.g. TCE, pyrene)

WT

0 NaCl

WT

GLOase L3

MIOX4 L3

150 mM NaCl

Lisko et al., In Vitro Plant. Submitted

Fig. 3 grow GLOa seedli conta growt Image of WT (23oC signifi


H+-Pyrophosphatase (AVP) over-expressers grow bigger and are more tolerant to stresses than controls Salt stress

Drought Stress

Dr. Roberto Gaxiola Arizona State University Gaxiola et al., 2001 PNAS 98: 11444-449 Li et al, 2005 Science 310: 121-125


Representative results (plants grown under long days (16:8 h photoperiod) WT

AVP1-1

MIOX4

GLOase


Area (cm2)

Measurements with Scanalyzer confirm results obtained using traditional methods *

20 18 16 14 12 10 8 6 4 2 0

*

*

GLOase

MIOX4

35

AVP1-1

Convex Hull Area (cm2)

WT

Values are means Âą SE, n=32

*

30

*

*

MIOX4

AVP1-1

25 20 15

10 5 0 WT

GLOase


Results for caliper length and compactness *

Caliper Length (cm)

7

*

*

6 5 4 3

Total leaf area Compactness = --------------------Convex hull area

2 1 0 WT

GLOase

MIOX4

AVP1-1

0,7

Compactness

0,6

*

0,5 0,4 0,3 0,2 0,1

Values are means Âą SE, n=32

0 WT

GLOase

MIOX4

AVP1-1


100% 80% 60% 40% 20% 0% WT

AVP1-1

MIOX4

GLOase 100%

Water content (% total leaf area)

Color classification (% total leaf area)

Color classification and in planta water content as readouts of plant health

80% Low

60%

Medium

40%

High

20%

0% WT

GLOase

MIOX4

AVP1-1


Growth curve of plants growing under short days (10:14 h photoperiod) WT GLOase MIOX4 AVP1-1 AVP1-2

Area (cm2)

50,00

40,00

30,00

20,00

10,00

0,00 11

14

15

21

25

29

Days After Germination Values are means Âą SE (n=15)

31

35

39


Color classification results indicate overall health of the foliar tissue Color classification (% total area)

100% 80% 60% 40%

20% 0% 11

Color classification (% total area)

AVP1-1

100%

14

15 21 25 29 31 35 Days After Germination

80% 60% 40% 20% 0% 11

39

14

15 21 25 29 31 Days After Germination

35

39

35

39

GLOase

MIOX4

Color classification (% total area)

Color Classification (% total area)

WT

100%

100% 80% 60% 40% 20% 0% 11

14

15 21 25 29 31 Days After Germination

35

39

80% 60% 40% 20% 0% 11

14

15 21 25 29 31 Days After Germination


Chlorophyll fluorescence (% total area)

WT

100%

60% 40%

20% 0% 15 Low

AVP1-1

100%

80%

No

Chlorophyll fluorescence (% total area)

In planta chlorophyll fluorescence as an indicator of plant health

Medium

21 High

25 29 31 35 Days After Germination

39

No

80% 60% 40% 20% 0% 15 Low

Medium

21 High

100%

No

100%

80% 60% 40% 20% 0% 15 Low

39

GLOase Chlorophyll fluorescence (% total area)

Chlorophyll fluorescence (% total area)

MIOX4

25 29 31 35 Days After Germination

Medium

21 High

25 29 31 35 Days After Germination

39

No

80% 60%

40% 20% 0% 15 Low

Medium

21 High

25 29 31 35 Days After Germination

39


Water content of unstressed plants during their life cycle WT

AVP1-1 100%

80% 60% 40% 20% 0% 15

Low

Medium

21 High

25

29

31

35

39

Water content (% total area)

Water content (% total area)

100%

80% 60% 40% 20% 0% 15 Low

Days After Germination

Medium

21

25 29 31 35 High Days After Germination

MIOX4

GLOase 100%

Water content (% total area)

Water content (% total area)

100% 80% 60%

40% 20% 0% 15 Low

39

Medium

21 High

25 29 31 35 Days After Germination

39

80% 60% 40% 20% 0% Low

15

Medium

21

High

25 29 31 35 Days After Germination

39


In summary We have confirmed the enhanced growth phenotype of high ascorbate (MIOX4, GLOase) and AVP lines using the Scanalyzer HTS Larger area Larger convex hull area Longer calipher length

We have found new readouts that better describe the architecture of the plants Compactness We have established the baseline levels for non-stress plants Color classification In planta chlorophyll fluorescence In planta water content


Next steps in this study

AVP1-1 homozygous

MIOX homozygous

Characterization of parents and crosses under stress conditions NaCl Drought


Other applications P3 faculty are interested in • Stress tolerance screening • Insect feeding • Production of phenolics by organs and tissues • Corn seed phenotyping for high-value protein accumulation • Somatic embriogenesis • Mutant screens 120

N o r m a l i z e d

100

g 80 r o 60 w t 40 h 20 0 Control

Low PEG

High PEG


Acknowledgements Arkansas State University

LemnaTec

Raquel Torres Jazmin Martin Jessica Yactayo-Chang

Dr. Dirk Vandenhirtz Dr. Jeorg Vandenhirtz Dr. Ralph Schunk Dr. Ben Niehaus Stefan Schwartz

Dr. Carole Cramer Emily Devereux Chris Groves Arizona State University Dr. Roberto Gaxiola

Funding


Man/Gal pathway D-Glucose-6-P 1

Fructose-6-P 2

PMI D-

Cell wall precursors

GDP-D-Mannose

Phytate L-myo-Inositol-1-P

5

13

Pi

Pi VTC4 myo-Inositol 14

GME

VTC2/ VTC5 ?

GalU pathway

GNL

GDP

NAD+ 8

NADH L-Galactono-1,4-lactone

L-Gulono-1,4-lactone O2 17 H2O2

6

L-Galactose

GalDH

GLOase

GLDH

L-Ascorbate

Pectin

Pi

L-Galactose-1-P H2O VTC4 7 Pi

L-Gulose

L-Gulonate

H2O

GDP-D-Glucose VTC2

GDP-L-Galactose + D-Glucose-1-P

GDP-L-Gulose

GlcUR

16

12

5

MIOX

15

Photosynthesis

Gul pathway

D-Glucuronate NADPH

3

PMM

D-Mannose-1-P GTP VTC1 4 PPi

MI pathway

H2O

Mannose-6-P

Polysaccharides and glycoproteins

D-Galacturonate

GalUR

10

L-Galactonate 11

Cyt C ox 9

Cyt C red

18

DHAR Dehydroascorbate

Oxalate, tartrate and threonate


Comparison of readouts of plants growing under long versus short days 80

Convex Hull Area (cm2)

50

Area (cm2)

40 30 20 10

60 40 20

0

0 WT

GLOase

MIOX4

AVP1-1

WT

0,7

GLOase

MIOX4

AVP1-1

MIOX4

AVP1-1

12

Calipher length (cm)

0,6

Compactness

0,5 0,4 0,3 0,2 0,1

10 8 6 4 2 0

0 WT

GLOase

MIOX4

AVP1-1

WT

GLOase


Better trays for phenotyping

QuickPot QP 35R D = 6 cm = 2.36 in

QuickPot QP 15RW D = 8 cm = 3.15 in


First publication submitted containing data acquired with the Scanalyzer HTS at ABI/ASU

Characterization of DOX1 mutant

Dr. Fiona Goggin University of Arkansas Fayetteville

Avila CA, Arévalo-Solíz ML, Lorence A, Goggin FL. Expression of α-DOX1 in tomato and Arabidopsis contributes to plant defenses against aphids. Submitted to Molecular Plant Journal


High Throughput Phenotyping at the Arkansas Plant Powered Production Center