This document summarizes a study using a chemical genomics approach to investigate endocytic pathways in Arabidopsis thaliana. Researchers screened several chemical libraries to identify compounds that inhibited tobacco pollen germination or caused aberrant pollen morphology. They identified 38 primary hits that were further analyzed, finding that 12.6% caused mislocalization of an endosomal marker or aberrant pollen morphology. Most of the secondary hit compounds were found to have antimicrobial, antifungal, anticarcinogenic, or antiparasitical effects. Future work will involve chemical screening of Arabidopsis seedlings to further characterize compound effects.

1. Chemical Genomics Approach to Endocytic
Pathways in Arabidopsis thaliana using
Nicotiana tabacum pollen
R.J. Washington, M. Brown, N.V. Raikhel
NSF-REU Program 2008
Source: News in Science, 8 January 2002
www.abc.net.au/science/news/stories/s453641.htm

2. ER
Golgi
vacuole
PVC
TGN
Endosomes
Plasma
membrane
PIN2
RIP1
AUX1
BRI1
A closer look at the endomembrane system

3. EXPERIMENTAL APPROACH
Classical genetics vs Chemical Genomics
Functional protein
Genetic mutation Chemical interaction
Non-functional protein
Classical genetics Chemical Approach
Cl
Cl

4. Pollen tip polarized activity
Germination: Three tier activity
– Zone 1- Apical: New materials deposited
at apical end via secretory vesicles
– Zone 2- Sub-apical: actin filaments,
mitochondria and EM organelles
– Zone 3- Shank: composed of actin
filaments
All zones constantly participate in
concert to propel tube growth via the
dynamic endocytic/exocytotic processes
Model from Cole and Fowler (2006)
Current Opinion Plant Biol. 9:1-10

5. PrimaryChemical Screen
WT Tobacco Pollen
BD Pathway microscope
Chemical Library
Inhibition of pollen germination
384 well plate
Aberrant pollen morphology
+

6. SecondaryChemical Screen
RIP1:GFP marker Tobacco Pollen
Mislocalization of marker
96 well plate
Aberrant pollen morphology
Examine with BD Pathway microscope
Primary Screen Inhibitors serially diluted
A B
+

7. Library Number of Compounds Primary Hits
Primary
Percentage Secondary Hits
Secondary
Percentage
Diverset 20,000 218 1.09% 22/176 12.5%
Novacore 10,000 18 0.18% 0/18 0%
TimTec 10,000 33 0.33% 0/30 0%
LATCA 3650 89 [76] 2.4% 16/73 21.9%
CLICK 2768 15 0.54% 0/7 0%
TOTALS 46418 360 .78% 38/302 12.6%
Summary of library progress of
pollen screens

8. ANTIMICROBIAL
3
ANTIFUNGAL
1
ANTICARCINOGEN
3
ANTIPARASITICAL
4
ANTIBACTERIAL
3
CYTOSKELETAL
EFFECTS
2
TRANSPORT
PROTEINS
4
OTHER EFFECTS
5
Bioactivity of secondary hit compounds
from database screen inquiry
Based on structure database search
Screening database used in inquiry: Science Finder Scholar Program

9. Future Work
Chemical Screening of Arabidopsis seedlings
− Root and vegetative tissues phenotype effects categorized for
each test compound
− Marker lines imaged via confocal microscopy and analyzed to
determine localization effects
Plasma membrane TGN Bri-overexpression PIN2

10. Acknowledgements
• Principal Investigator: Dr. Natasha V. Raikhel
• Mentor: Michelle Brown
• Lab members:
Glenn Hicks, Marci Surpin, Mien van de Ven, Stephanie Robert
Julie Sohn, Georgia Drakakaki, April Agee, Abel Rosado Rey
• Support Staff and Facilities:
Dr. David Carter (CEPCEB Academic Coordinator, Microscopy/Imaging)
Dr. Thomas Girke (CEPCEB Academic Coordinator, Bioinformatics)
Institute for Integrative Genome Biology (IIGB)-Chemical Libraries
Plant Transformation Research Center
• Support Funding:
National Science Foundation