Sam Pulstudent
Jan. 14, 2014
Genetics 201: Dr. Hanson
Phenotype Prediction: Mutations in Maize
Introduction
Brassinosteroids (BR) are plant hormones that have been shown to control many
developmental process within a plant including promoting cell elongation and cell differentiation
and apical dominance. In an attempt to increase the yield in crops, the biosynthesis and signaling
of BR could play an important role because it controls cell size.
The BR biosynthesis and signaling pathways have already been proven in an experiment that has
been done using Arabidopsis. The experiment our group did was based off of this previous
experiment, with some changes.
It is first important to understand how the brassinosteroids biosynthesis occurs.
Cholesterol is the precursor of the biosynthesis pathway, which actually begins with
Campesterol. Campesterol then undergoes many changes and in the end the pathway creates
brassinolide, the most important brassiosteroid. Normally this then continues onto the signaling
pathway that consists of a receptor for the BR first. There is then a phosphoralation of BZR1,
Brassinole Resistant 1, which makes the molecule unstable, which knocks it out. This then
allows the genes for the plant to grow tall to transcribe and be played out.
However, in our experiments we blocked this pathway so the opposite would happen.
We used an inhibitor, Propiconazole (PCZ), to block the dwarf4 gene during the biosynthesis
process. This stops the production of BRs. When it comes to the signaling pathway, without the
production of the BR, there is no molecule to start the signaling pathway. BZR1 is not
phosphorylated, which keeps the molecule in the signaling pathway. Without losing this
molecule from the pathway the genes for the seed to grow tall are inhibited and thus the plant
stays short.
This was the experiment done on the Arabidopsis plant, but there are several reasons that
we could not use the exact same experiment for the maize. To begin the Arabidopsis experiment
was performed on agar plates, but because of the size of maize, we were required to do our
experiment in pots. Cost was another factor to take into account. For our experiment we used
Banner Max® instead of Brassinazole. Brassinazole is very expensive and for the amount we
used, it would have cost over a million dollars. Banner Max® is a fungicide with its active
ingredient being Propiconazole (PCZ) which works the same was as Brassinazole.
During our research, we were trying to prove three different things. First we wanted to
prove that propiconazole acts as an inhibitor of sterol/BR biosynthesis. PCZ has never been used
as a BR biosynthesis inhibitor in maize before and we wanted to prove it would work. Secondly
we were trying to find a mutant in which the BZR1 protein has been destroyed. To accomplish
this goal of finding the mutant, we will be looking for a plant, which is tall even with the use of
the inhibitor because it will show that the BR inhibitor does not affect the seeds. We also
thought that it wouldn’t be 100% the size of the wild type we were growing because there may
be more than one BZR1-like protein. Finally we knew from previous work that biosynthesis
mutants in maize have phenotypes of dwarfism, seed in tassel, curled leaves, and most
Original Author: Aaron Quigley 2013 1
importantly, red root. This phenotype was easy to screene, therefore we can screen for it
simultaneously.
Materials
Maize EMS M2 generation seeds were planted from Professor C. Weil’s Tilling
collection. In total we needed 3600 different lines, 20 seeds per line, for a total of 72,000 seeds,
separated into two rounds. The seeds came from two different backgrounds, B73 and W22.
Also needed for our experiment is extra-course and course vermiculite, about 10 bags each. Also
needed would be about 450 flats that each has 48 separate cells within them and under each flat a
water tray is required. Banner Max® is the inhibitor used in our experiments along with
fertilizer water. The plants must be grown in a dark growth camber, ours held about 220 trays,
that has a temperature control.
Methods
After obtaining all of our materials for thiss particular experiment, it was time to being.
The flats are filled about a quarter of the way with the extra-course vermiculite and the seeds are
placed in each cell, 3 or 4 per cell. There are eight separate lines per flat, each obtaining six
cells. Each flat needs to be numbered and every seed pack needs to have the flat number as well
as the cell series of the line, for future reference and track the growth. After the seeds are placed,
the flats are filled to the top with course vermiculite. Banner Max® is then mixed into two liters
of fertilizer water, 15M first round, 110M second round. Each cell is soaked, with a water
tray underneath and is put into the growth chamber. In the growth chamber the water trays are
filled with the fungicide/fertilizer water as well. The growth chamber is set to 75F and the flats
are left to germinate and grow in the dark for between eight and ten days.
After the allotted time, the chamber was opened and the screen process begins. The first
thing to screen for is a difference in height, within each line. If a height difference is found it
will only be about 25% of the seeds in that line. If a height difference is found, flat number and
cell number are recorded and the seedling mutants are removed from the media and
photographed. The mutant plants were repotted in pots filled one quarter with soil and topped
with Turface ™ and Greens Grade and labeled for future identification. If no height differences
are found, each seedling is checked for red roots. The procedure is the same as the tall mutants
for the red root seedlings. Beware that seedlings with red roots will be very short and may not
even break the soil surface, so we had to be sure to dig into the media. If no red roots are found,
the media and seedlings can be discarded. Measurements of the mesocotyl length were then
taken of the mutant plants using ImageJ.
We did two rounds due to the fact that we did not have enough space to store all 450 flats
at once, but they could be done at the same time. Also, the higher amount of M of Banner
Max® added to the fertilizer water, the easier it was to determine the tall mutants.
Results
After both rounds of growth and visual screening results, we found both things that we
were looking for, mutated tall plants, and plants with red roots. Three different candidates were
Original Author: Aaron Quigley 2013 2
found that displayed the Atbzr1-like phenotype, tall. We also found one candidate that displayed
the brassinosteriod biosynthesis mutant phenotype.
Out of our three Atbzr1-like phonotypical candidates, they came from two different
backgrounds. The first candidate and second candidate both came from the W22 background of
seed. Our final candidate that displayed the Atbzr1-like phenotype came from the B73
background. With each of these phenotypes that we found, we expected them to be about 25%
of each line that would show this growth habit because the seed that we used in our experiment
was an M2 generation.
The candidate that we discovered that had the brassinosteriod biosynthesis mutant
phenotype came from the B73 background as well. These results show the red roots but it also
shows that the plants are smaller than the other seedlings within the same line. This is because
these plants were treated with the inhibitor, PCZ, which made them small, like the short ones
within that treatment, but because they have a mutation in the brassinosteroid biosynthesis
process as well, they are extra short. The red roots are present because the plant is stressed
because of the inhibitor.
The results that we found turned out as close to perfect as possible. When we compare
the results of our experiment, you can see that the hypocotyl length, as a percent, compared to
each other and the mesocotyl length, as a percent, are almost exactly the same. All of the wildtype hypocotyl or mesocotyl lengths are at 100%. The red bars are also almost the exact same.
The first two red bars of each graph are the tall mutants we were searching for while the third red
bar in each graph is the mutant with the red roots. Each of those is at about 10%.
Candidates
Maize plant 1
Maize plant 2
Maize plant 3
Mutation?
Yes
No
No
Plant Height
185 cm
146 cm
132 cm
Discussion
Upon finishing our experiment, our conclusions, while conclusive, are not necessarily
correct. The reason that out conclusion may not be correct is simply because we did not have the
chance to see how everything grows out. While we did find four different mutant candidates, we
planted them to continue to see how they develop and we did not have the time to see the results.
My thoughts are that some of our candidates will not grow, reproduce, or produce seed correctly,
thus making that candidate not what we are looking for in the end. Therefore as to what we were
looking for in our experiment, Atbzr1-like mutants and brassinosteroid biosynthesis mutants, we
did find exactly what we were searching for. In the whole picture of the reason for the
experiment, I am not sure if we have found what they were looking for exactly but hopefully we
have and it will make a great impact on crop development.
In the near future, there are still 2400 lines to plant and grow out in the dark. The results
will then be examined and the same visual screenings will be conducted. Hopefully more of the
same results will be found in these last lines. Also in the future, more seeds of the lines which
we found the Atbzr1-like mutants and the brassinosteroid biosynthesis mutants will be planted
and grown in the same conditions as previously done to see if the same results occur. Also in the
Original Author: Aaron Quigley 2013 3
future, the inhibitor concentration could be increased even more. This would be beneficial
because the B73 wild-line creates plants that are shorter than the W22 and thus it makes it more
difficult to tell the difference in height between the expected result, of short, and the Atbzr1-like
mutant, tall.
In the end, after all is said and done, it is hopeful that our research that will help to find a
natural mutant that will produce plants which have a shorter stature, but produce maize the same
size as on a large stalk. If this is able to be complete, the plants will be much more stable and
thus will not get blown over in the wind as much as currently they do. Also, shorter plants will
require less water, which is a big deal at the moment, and less nutrients, costing the farmer less
money. Being a shorter plant will also allow the farmer to plant the plants closer together thus
having a greater bushel per acre amount.
There are many more steps within crop improvement involving this sort of experiment.
This same type of screen could be used on many different crops to see if mutations can also be
found. With some luck, in the future this experiment could help to find many different types of
important crops that would improve the input verses output ratio.
Resources Used
Article 1
Title:
By:
Publication:
Date:
Page:
Book 1
Title:
Authors:
Published Date:
Pages:
Brassinosteroid Biosynthesis and Dwarf Mutants
M. Kwon, S. Choe.
Journal of Plant Biology.
March 2005.
89-105
“Nuclear-Localized BZR1 Mediates Brassinosteroid-Induced Growth and
Feedback Suppression of Brassinosteroid Biosynthesis .“
Z. Y. Wang, et al. Developmental Cell. 2002.
April 2002
505-513
Original Author: Aaron Quigley 2013 4