Characterization of pectin chemistry in unpollinated and pollinated pistils of Petunia hybrida

1. Characterization of pectin chemistry in unpollinated and pollinated pistils of Petunia hybrida Faculty Mentors: Gerry Prody (Chemistry) Anu Singh-Cundy (Biology) Undergraduate Research Team: Iudita Repta (Biology; Cell emphasis) Peggy Leviton (Chemistry; Biochemistry) Justin Lamb (Chemistry; Biochemistry) Phil Moulton (Biology; Botany emphasis)

2. Project goals for the summer: • To compare the composition of pectic polysaccharides • in the 3 different functional zones of • the pistil: the stigma, • the transition zone (TZ), and the style • To understand how pectic polysaccharides are altered in response to the gaseous plant hormone, ethylene.

5. stigma transition zone style

9. Thursday’s data

11. After reduction

12. The Glycome Rules!

15. Pollen Tube Growth in vitro (culture medium) In the pistil

16. Background: The Physiological Context • Pistil structure: • Pistils contain a tract of cells--called the transmitting • tissue--that provides a specialized matrix for supporting • the growth of pollen tubes. • The extracellular matrix of the transmitting tissue is • unusually thick, and is presumed to be rich in pectic • polysaccharides. Pollen tubes grow (by tip extension) • in this extracellular matrix. • The pistil is composed of three structurally and • functionally distinct zones: the stigma, the transition • zone (TZ), and the style. Pollen tubes behave differently • (for example, in terms of growth rates) in each of these • 3 zones.

17. Background: The Biochemical Context Extracellular polysaccharides in plants and pistil cell wall chemistry: Plant cell walls contain three main classes of extracellular polysaccharides: cellulose, hemicellulose, and pectins.  In living plant cells, the walls are frequently re-modeled such that their physical, chemical, and biological properties are altered.  Pollen tubes secrete pectinases, and pectinases such as polygalacturonase are also constitutively present in the stigma and TZ. It is assumed that these pectinases hydrolyze the pectin matrix in the transmitting tissue; softened matrix may offer less resistance to pollen tube growth, released sugars may be used as energy source.

18. Cell wall architecture Common sugars found in plant polysaccharides

19. Pectin structures

20. Cross-bridging and esterification in pectins

22. Project Goals for This Summer • 1. Are pistils rich in pectins in all 3 zones? • Early results indicate: Nope. Stigmas don’t seem • to have much galacturoninc acid. So, we hope • to determine: • --the sugar composition of pectic polysaccharides in the stigma, • TZ, and style • --the degree, and type, of esterification on the galacturonic acids found in each of these regions • 2. Does ethylene promote pollen tube growth by altering the pectic chemistry (leading hypothesis)? We want to find if • --ethylene treatment increases hydrolysis of pectins (increase in reducing sugars, release of low MW pectic fragments) • --ethylene treatment triggers a change in the sugar composition, and degree of esterification, of pectins extracted from the stigma, TZ, and style (to detect differential action of ethylene on the matrix in the 3 different zones)

23. Research Methods To determine sugar composition of pectic polysaccharides: --dissect pistil into stigma, TZ, and style; freeze in liquid nitrogen --extract pectic fractions: analyze for  sugar content  uronic acid content  reducing sugar content  degree and type of esterification --hydrolyze pectins, derivatize the sugars: --analyze by GC-MS --compare ethylene vs. no ethylene treatments --look for changes in any of the above as well as for overall size changes in the polysaccharides by size exclusion chromatography --section tissues and stain with pectin specific antibodies