Early

Seedling Growth

1. Assign mutants to letters with their correct names. Explain your assignment of each
mutant.

o Ahk3 ahk4 (cytokinin signalling mutants) are in tray D. These mutants displayed the
triple response, as they were perceiving the ethylene gas, but were not producing it
themselves.
o Ein2-1 (ethylene receptor mutants) are in tray E. These mutants did not display the
triple response but did display normal hypocotyl growth, as they were not perceiving
the ethylene gas.

2. What is the evidence that ethylene is involved in early seedling growth?

o Seedlings with the ein2-1 mutation were unable to perceive the ethylene gas, these
seedlings did not display altered growth. The ahk3 ahk4 mutants were not producing
ethylene but when exposed to it, produced the triple response growth. This triple
response to ethylene is hypothesised to protect the delicate apical meristems while
emerging from the soil (Guzman and Ecker 1990). If the seedling doesn’t grow with a
definitive apical hook, it risks damaging the growth tip. When the seedling emerges
from the soil and is exposed to light, ethylene then causes elongation of the
hypocotyl, to maximise competitive growth.

3. Thinking of the early seedling growth experiment you measured, what would be a good
screen for ethylene mutants and of what type (mutants affected in ethylene response or
ethylene level)?

o Growing the seedlings in light conditions for entire experiment should expose which
mutant is being dealt with, given the crosstalk between phytochromes and ethylene.
Ethylene triple response should mostly occur in dark conditions and then, in the
presence of light, ethylene should contribute to normal elongation of hypocotyl. So,
if the mutant is deficient in ethylene production rather than one with an inability to
perceive it, there will likely be a lack of triple response and exhibit cell elongation. If
the mutation is on ethylene perception, the plant will exhibit a lack of hypocotyl
elongation.

4. What other mutants might you discover and why?

o There might be mutants exhibiting auxin mutations. There is evidence that there are
strong links between auxin and ethylene mutants and hypocotyl elongation (Smalle
et al. 1997).








Root Growth

5. Assign mutants to letters with their correct names. Explain your assignment of each
mutant.

§ Ahk3, Ahk4 (double mutant) = F. No response to cytokinin exposure. Normal
root growth.
§ Ein2 = G. Cytokinin exposure caused increased hairiness of roots but not
reduced length.
§ Ipt1 ipt5 ipt7 (triple mutant) = H. Exposure to cytokinin caused reduced
growth of root and increased hairiness.

6. Using whole class data, plot your own graph and add a legend (use a similar style as seen in
one of the recommended publications – plot mean and standard error).
20.00

18.00

16.00

14.00
Mean Root Length (mm)

12.00

10.00

8.00

6.00

4.00

2.00

0.00
CON BA CON BA CON BA CON BA
WT F G H
Treatment

Figure 1: Mean effect of BA on early root growth of Arabadopsis wild type (WT) and mutants: ahk3 ahk4 (F), ipt1 ipt5 ipt7 (H)
and ein2(G) grown in the dark. Control treatment for each group is shown in green. Standard errors are shown for each mean.
Seedling root lengths were measured seven days after growing in 21°C day temperature and 19°C night temperature, in an
upright position for a 16-hour day length.

 7. What is the evidence that cytokinins are involved in root growth?

o Mutants that were unable to perceive the BA (synthetic cytokinin) had normal root
growth with no increase to root hairs. Those that were able to perceive it but were
not producing it had reduced growth and increased hairiness when exposed to BA.
8. Why does the ein2 mutant prevent cytokinin from inhibiting root growth?

o Because there is some crosstalk and feedback loops between cytokinin and ethylene
biosynthesis and transduction. The ein2 mutant is preventing the cytokinin from
inhibiting root growth because the ein2 receptor is a part of the signal transduction
pathway and ein2 is, to some degree, linked with elongation of the root.

9. What could you do to distinguish between the cytokinin reception and ethylene signal
transduction mutants?

o Expose the plants to increased levels of ethylene and cytokinin for the otherwise
same experiment. If normal root length with increased hairiness and no triple
response is observed, the plants are ethylene signal transduction mutants. If the
roots are showing increased root elongation, exhibit no extra hairiness and display
the triple response in the hypocotyl, then they are cytokinin reception mutants.

Overall

10. Where do plant hormones act and why? Give an example to illustrate your point.

o Depending on the action of the hormone, they can act in either young and growing
parts of the plant and meristems, as well as in mature parts of the plant. These are
the parts of the plant that will be entering the environment, so should be exhibiting
the best possible phenotype, or in parts of the plant that are constantly being
exposed to changed conditions and must respond to that environmental change.
§ An example of hormone action is Phototropism. Light being perceived by a
growing part of a plant from one side will cause auxin to promote cell
elongation/growth on the other side of the plant. The auxin acts by
increasing acidity within the cell, allowing cleavage of cross-linking
polysaccharides. This allows the cell to expand with increased turgor, angling
the plant towards light and increasing the photosynthetic potential.

11. Are plant hormones produced in the same place that they act or are they mostly
translocated there from sites of higher synthesis elsewhere, and why? You may choose to
give an example or two to help explain your answer.

o They are mostly produced elsewhere, and then carried by xylem, phloem or
transporters to the site of signal transduction and gene expression can happen
locally or long-distance. For example, auxin is moved around the plant via PIN
proteins during polar auxin transport. It may be produced in one cell, and then
movement through the plant is directed by the PIN proteins in cells. Auxin is
potentially responsible for organogenesis and the accumulation of auxin in different
 parts of the plant can determine where particular organs (e.g. leaves or roots) occur
and at which orientation. There always are exceptions to the rule though, and some
hormones will act locally.


References

Guzmán, P., and Ecker, J. R. (1990) Exploiting the triple response of Arabidopsis to identify
ethylene-related mutants. Plant Cell2, 513–23.

Smalle J, Haegman M, Kurepa J, Van Montagu M, Straeten DV (1997) Ethylene can
stimulate Arabidopsis hypocotyl elongation in the light. Proceedings of the National Acadey of
Sciences of the United States of America. 94: 2756–2761