Project 4
LED light quality-induced differences in thylakoid membrane
structure in etiolated Brassica rapa seedlings: A thin section electron
microscopic study
(PIs: M.
Blackwell, K. Nordell, W. Perreault; Univ. of Wisconsin, Madison - A. Ellis, Chemistry,
P. Williams, Plant Pathology)
We hypothesize that Brassica rapa (BR) chloroplast morphology (as seen by transmission electron
microscopy (TEM) of thin sections) will change in response to LED light quality
during illumination of etiolated fast plant proplastids. The objectives for
this study are:
1) Develop an LED light illumination
regimen, including quantification of exposure times and intensities, and a thin
section TEM protocol for studying chloroplast development from proplastids in
red and blue light (summer 2002)
2) Assess thylakoid membrane
morphological responses (e.g. degree of stacking) to exposure to red and blue
light of etiolated BR proplastids
using analysis of thin section TEM images (summer 2003).
Summary of Research Results
Under the direction of Karen Nordell, the three student
collaborators quickly learned and applied a device for growing BR seedlings in film canisters, and then
subjecting them to LED light, in order to stimulate development of proplastids
into chloroplasts, thus satisfying the first objective.
Under the direction of William Perreault, the three
students then turned their attention to the second objective. They had a productive summer 2002 research
experience learning to master the intricate skills involved in preparing plant
tissues for TEM. This sometimes arcane
art involves fixation, dehydration and embedding of plant material (leaves in
this case) in plastic molds. Finding the
right combination of reagents and timing to optimize tissue preservation and
staining presented the first variable to be standardized. They learned to make
and evaluate glass knives suitable for preparing "thick" sections of
1-2 um suitable for staining and examination in the light microscope. Here the students had to become familiar with
the histological anatomy of the leaves and the thick sections produced with the
goal of actually identifying individual cells and clumps of cells that were of
interest for high-resolution examination in the electron microscope. The next
step involved reshaping the plastic blocks into segments no larger than one mm
square for ultra sectioning of the desired cells and tissues. The "thin sections" obtained from
the ultramicrotome ( 60 to 100 nm, or 1/50 the diameter of a human hair) had to
be manipulated onto copper grids and stained with heavy metal stains (uranium
and/or lead) for visualization in the electron beam. Finally, the students had to master
microphotography and darkroom techniques leading to acceptable quality photographs
and prints. Altogether, the number of
new and challenging techniques the Merck scholars, Katharine and Nicole
developed provided a generous degree of progress for novice microscopists. They both did well overall, and it was not
surprising to find that one or the other became more proficient at various
tasks. They cooperated nicely to
complement each other's strengths.
Under the supervision of Professor Blackwell, the three
students noticed that in their preliminary studies they were finding an altogether
new chloroplast thylakoid membrane morphology in BR proplastids developed under red LED illumination, while the
morphology in blue and green LED
developed plants was normal. They called
this new morphology the granal star cluster morphology. The students continued to obtain TEM data in
independent research projects carried out during the academic year, are now
beginning to define ways to quantify the TEM data they are obtaining, and plan
to continue working on the project during the summer of 2003.