“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