Experiments in Plant Growth and Development
BIOL 305 Spring 2014
Marta Laskowski
mlaskows@oberlin.edu
Office: K114
Office hours: Monday 3-4 PM, Wednesday 11-12 and by appointment.
Course goals and structure
The goal of this course is to give students a chance to try their hands at research, a
process that includes production of a professional quality written work describing the
results. Students will read articles in the primary literature and participate in
brainstorming sessions that will result in design of semester-long projects. These projects
will be carried out jointly, most likely in pairs. The process of writing the results will take
place in stages throughout the term. Formal written and oral reports at the end of the term
will allow individuals to demonstrate what they have learned.
Grading
Grades will be based on class participation (25 %), an oral presentation (15%) and
written work (60%). Written work consists of the daily lab notebook, the project proposal
and drafts of sections that are due as the course progresses, and the final lab report (50%).
Topics of Study, Spring 2014:
Mechanisms that regulate lateral root initiation
Your first assignment is to come up with
a project that you want to do this
semester. There is some flexibility in this,
but the experiment you design should be
related to the molecular mechanisms that regulate root architecture, with emphasis on
lateral root initiation and emergence. You can begin by reading over the review articles
that are provided and the description of questions on the next page. Then, consider the
questions and think about what specific experiments appeal to you. We will talk about
your ideas during the second week of class and a project proposal will be due at the end
of next week (Feb 14, 5PM). A project plan includes details on the experiment you want
to run. You should state what hypothesis you want to test and how you will go about it.
That includes the species you want to work with, the specific experiment(s) you plan to
do, the concentration (range) of any chemicals that you want to apply, the length of time
for which you will apply them, etc.
(1) To what extent is the promotion of lateral root initiation on gravitropicallyinduced curves conserved across species?
In Arabidopsis, lateral roots arise in the pericycle, a ring of cells that lies deep
inside the root. As these cells divide and expand, they form a primordium that pushes its
way out of the root through the overlying layers of cells. Only those pericycle cells that
lie adjacent to xylem cells can form lateral roots, but any of the cells in those two files
can form a lateral root. Why do some pericycle cells become activated and form a new
organ whereas others do not?
Classical physiological experiments have shown that lateral root formation is
induced by the plant hormone auxin. Accumulation of auxin in pericycle cells appears to
suffice to induce the process. Auxin flux through the root is controlled by membranelocalized auxin transport proteins. But what causes the network of auxin transport
proteins to generate local accumulations of auxin at some times and not at others?
Recent experiments have shown that mechanically bending a root suffices to
induce lateral root formation along the outer (convex) side of the curve. One hypothesis
is that geometrical changes in the relative positions of auxin transporters (which are
located at specific points along the cell membranes) may suffice to cause changes in the
auxin flux. An alternative hypothesis is that bending plants stimulates the plants’
mechano-sensory system and that auxin accumulation lies downstream of that signaling
response.
A previous project done in our lab demonstrated that a wide range of species
across the seed plants form roots on the outside of curves. The work so far completed
really sets some boundaries on the response: it takes place in a wide range of eudicots,
and does not occur in ferns. But what about species in intermediate locations in the plant
phylogeny? It would be exciting to investigate some monocots and gymnosperms. A
previous BIOL 305 class identified useful species to work with, but work that remains
includes: (a) repeating many of the experiments so that higher quality photographs could
be obtained, (b) repeating and extending our data on the effect of curvature in monocots
(wheat) and gymnosperms (pine and gingko), (c) possibly doing new experiments on
some of the examined species in which the auxin transport inhibitor NPA is added to the
root-shoot junction.
(2) Methyl jasmonate affects lateral root formation. Can we work together to try
and piece together part of the pathway by which this mechanism operates?
We know the names of several genes that might be involved in the pathway leading from
methyl jasmonate to lateral root formation. These include:
• Methyl jasmonate (the hormone itself)
• COI1 (the methyl jasmonate receptor)
• PLETHORA 1 and PLETHORA 2 (transcription factors that affect root
elongation and meristem maintenance)
• LAX3 (an auxin importer)
What experiments can we design that will help us figure out if (and if, then how) these
players interact to generate the response?
•
We can determine the effect of methyl jasmonate on wildtype plants and
compare that to its effect on plants that carry loss-of-function mutations in
genes of interest. Mutants that we have on hand include coi1-16; a plant
carrying mutations in both plethora1 and plethora2; the lax3 auxin
importer; and a plant that carries mutations in two auxin response
genes(arf7,arf19). If we add methyl jasmonate to one of these and see a
response that is as strong as the wildtype response, what does that tell us
about the involvement of the factor that was mutated? If we entirely lose
the response, what does that mean?
•
We can grow plants with and without methyl jasmonate and see what
happens to expression of the PLETHORA or LAX3 proteins. Because we
have transgenic plants in which these proteins are fused to fluorescent
markers, we can directly the cells in which they occur. This project
requires use of the confocal (fluorescence) microscope.
•
We can use real time PCR to compare the levels of PLETHORA 1 mRNA
accumulation in untreated plants and plants treated with methyl jasmonate.
•
Lateral roots always form on the outside of curves. If we could manipulate
the system such that lateral roots formed on the inside of curves, we would
have our hands on the mechanism. One possibility is that lateral roots
form on the outside of curves because that is where the auxin accumulates.
We could try adding beads or blobs of auxin to the inside of curves on a
species that is easy to work with (cucumber?) and see what happens. If
nothing happens when the endogenous auxin, IAA, is added, we could try
adding NAA—a synthetic that has the same activity as auxin, but which
can enter cells without having to go through auxin importers. If NAA were
to induce lateral roots on the inside of curves where IAA did not, what
might that tell us about the importance of auxin importers? Could we use
this system to test the extent to which the mechanism remains conserved
across species?
Tentative Schedule
Feb 4:
Course organization; start plants, read articles, and think about what sort
of project interests you.
Feb11:
Brainstorming session in class; select projects and partners.
Project descriptions due by Friday of this week.
Feb 18:
Start projects
Feb 25:
Projects
March 4:
Projects
March 11:
Projects
March 18:
Projects
………………………………………..spring break………………………………………..
April 1:
Projects Bibliography due this week! This is a list of the articles in your
notebooks plus the original references that you have found that you can
use for your introduction. (3 of these must be found on your own.)
Friday, April 4 John Watson (IUPUI) will give a seminar on factors that regulate
auxin transport in Arabidopsis roots. This will be over the noon hour
and anyone who is able will be able to meet with him after the seminar.
April 8:
Projects
Bring a draft of your introduction to class
April 15:
Projects
April 22:
Last Project day!
April 29:
Bring a draft of the whole paper to class; you will edit each other's drafts
May 6:
Oral presentations on your projects.
Bring a draft of your methods section to class
Your research paper is the final project for this course; there will not be a final exam. The
paper is due no later than 11 AM on Saturday, May 17, the end of the final exam period
that has been scheduled for this course. You are welcome to submit your paper earlier
during finals week if you prefer, though NOT during reading period.
FORMAT FOR LAB NOTEBOOK
Lab Notebooks
Lab notebooks serve as a record of your day to day activities and observations.
You should purchase a notebook that you bring to class every time we meet. Use its first
or last page for a table of contents, which you will fill in as you perform experiments.
The contents of the notebook may be as brief as is consistent with thoroughness and
clarity, but should include all of the sections listed below:
Title:
Date:
Purpose: Why did you do this experiment? What question are you trying to answer?
What hypothesis are you testing? This can be only a sentence or two long. But it’s
amazing how helpful these cues are later when you are trying to decipher your notes.
Methods: Exactly what did you do? The more details you include here, the better. Any
calculations that you do should be included here, and don't forget to note items such as
the concentration of reagents used, and incubation times and temperatures. Invariably, if
you leave some seemingly inconsequential bit of information out of your methods, you
later discover that it is critical to the interpretation of some puzzling piece of data.
Results: What did you find? Include raw data, drawings, graphs, tables and a brief
written description of what you observed.
Conclusions: What can you conclude? What did your experiment show? Did it answer
the question you set out to answer? If not, why not? Include your speculations about
possible sources of error. What future experimental directions are suggested by today’s
results?
Every time you do lab work, there should be an entry in your lab notebook
to reflect what was done. If you did not do a section of the work, but others
did it for you, make a brief note of that as well.
FORMAT FOR LAB REPORTS
Lab Reports
Lab reports are longer and more formal than lab notebooks. They should provide
a synthesis of the work that you have done, the reason(s) that you did it, and your
conclusions. Lab reports should include the following sections:
Title: The title should be as brief as possible while still telling the reader what the report
is about. Do NOT use "Biology 305 lab report" as a title. You do not need a separate page
for the title.
Abstract: This is a one paragraph summary of the paper. The abstract should briefly state
the paper's objectives and methods, and summarize the main results and conclusions. The
abstract must be complete without reference to the text. Prepare the abstract LAST- after
all the analyses have been completed and the final conclusions stated.
Introduction: This section should provide a brief introduction to the general area of
investigation and the specific problem at hand. It should contain a statement of the
question and why the question is interesting and worth pursuing. It should include
references to journal articles that you used as a basis for your work.
Methods: Methods can be less detailed than in the notebook. For example, in your
notebook you might want to indicate that you melted agar by microwaving it for several
minutes and then added some hormone after it had cooled sufficiently that you could
handle the flask. In the report, it would be sufficient to note that the agar contained
hormone of a given concentration. In the report, it is sufficient to describe a published
method (or a method described in the lab handout) by citing the source of the method,
noting only significant departures from the standard procedure. The idea is that someone
who reads your “methods” section should be able to exactly replicate your procedure.
However, you should assume that the reader has a basic knowledge of laboratory
technique.
Results: The results section should summarize the major results of the study. One of the
key points to remember is that you are not writing a mystery. The purpose of your writing
is to convey your findings as clearly as possible. Put your most significant findings first.
Do, however, include all of your observations. The observations that you think are
extraneous may be the ones that actually hold the key to understanding. Use subheadings
to summarize the results – not to list what the experiments were. Data should be
presented in either tables or figures and these should be referred to in the text. Use words
to articulate the results shown as in the following example: "There were pronounced
differences in the two reactions; the beaker on the hot plate reached a temperature of 100
C in 5 min while the beaker in the refrigerator failed to boil, even after 12 h of incubation
(Figure 1)." In this case, Figure 1 would be a graph showing the temperatures of the two
beakers over time. Do not include calculations in this section (unless they were nonstandard). Where appropriate, report numerical data as averages of a number of
replicates.
Avoid the use of lab jargon such as “run 1” or “group A” in describing the results.
Instead, use words that detail what was done in each experiment Saying that control or
hormone-treated plants were subjected to….and responded by….. is much easier to
follow than saying that Group1 responded by….. Readers (including this one) have a
hard time remembering what “group 1” was.
Discussion: The results of the study should be related to the original question. Do your
data support or contradict your hypothesis? If they contradict it, is there a second
hypothesis that they do fit? Although it is tempting to attribute unclear results to
experimental error, avoid this unless there were obvious sources of bias or error in the
experimental design. A lack of results does not mean that the study failed. Close the
discussion with the conclusions that can be drawn from your study.
References: You must cite any sources of information that contributed to your study. It
is inappropriate to omit any sources of specific information and also inappropriate to cite
references that you haven’t read.
Specific information should first be referenced within the text by including the
author’s name(s) and the date of publication in parentheses. For example, if you were
trying to report on the findings of Jacobsen and Olszewski, you could write a sentence
that looked like this:
The spy-3 mutation in arabidopsis confers resistance to inhibitors of gibberellin
biosynthesis (Jacobsen and Olszewski, 1993).
Alternatively, if you want to draw attention to the people who did the work, you could
write:
Jacobsen and Olszewski (1993) found that the spy-3 mutation in arabidopsis
confers resistance to inhibitors of gibberellin biosynthesis.
At the end of the paper you should include a section titled "References" that is an
alphabetical list of those items mentioned in the text of the paper. For journal articles,
using the following format: Author(s), year, article title, journal, volume, pages. For
example:
Jacobsen, Steven E. and Olszewski, Neil E. (1993) Mutations at the SPINDLY
locus of arabidopsis alter gibberellin signal transduction. Plant Cell 5, 887-896.
For books, list: Author(s), year, title, publisher, place of publications.
General guidelines for writing and style:
Use topic sentences. I should be able to read only the first sentence of each paragraph and
come away with a very good idea of what your paper is about.
Good scientific writing is precise. Instead of using adjectives such as bigger and faster to
describe what you see, give measurements that document your statements.
Scientific articles are typically written as though the investigator did not exist, because
people felt that it made the results appear to be less subjective. Hence, the traditional
style never uses the first person “I”. This tradition is slowly changing, but the use of first
person is still kept to a minimum.