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Plant Physiology (Bot 328), Test #2 NAME: KEY
Spring 1996

Please provide succinct answers in the space provided under each question. Unless otherwise noted in
the margin the value of each question is 3 points.

1. (a) What are the gravity responses in Chara, Ceratopteris, and in an oat coleoptile?
Ans.: Chara: polar ratio of cytoplasmic streaming of vertical cells is 1.1, faster downward;
Ceratopteris: nuclear migration is downward; oat coleoptile: asymmetric growth, faster on
bottom side.

(b) What fundamental aspect of the gravity response in coleoptiles (i) could be clarified and
(ii) could not be clarified by studying the gravity response in Chara?
Ans.: (i) Cellular mechanism by which gravity signal is sensed; (ii) mechanism by which growth
gradient across multiple cell layers is established.

(c) Are the emerged rhizoids of germinated Ceratopteris spores gravitropic? Explain.
Ans.: No, many hours before the rhizoid emerges the directional polarity of rhizoid growth has
alrady been fixed by gravity.

2. (a) Treatment of Chara internodal cells with proteinase K blocks the gravity response in
these cells. Does it matter where on the cells the proteinase is applied for it to be an effective
antagonist? Explain.
Ans.: Yes, the proteinase has to be applied on the ends of the cells for it to be effective. Applying
the agent along the middle of the cells has no effect on the gravity response.

4 pts. (b) Based on the model of Staves and Wayne, indicate two likely targets of proteinase
action that could explain its inhibition of the gravity response in Chara. Explain the likely
role of each target in mediating the gravity response.
Ans.: (1) Integrins, which anchor the extracellular matrix to the cytoskeleton, thus allowing
tension and compression forces to be generated when the orientation of the protoplasm is
changed; (2) Ca channels, a portion of which may be accessible on the outer face of the PM.
Opening/closing of channels appears to be important for gravity response.

4 pts. 3.(a) What is RGDS and how might it affect the gravity response in Chara, and by what
mechanism?
Ans: RGDS is a tetrapeptide that can mimic and thus compete for the binding site of integrins
to proteins in the extracellular matrix. By dissociating integrins from its wall binding site,
RGDS can prevent the formation of tension/compression forces needed for gravity sensing.

4 pts. (b) What is EGTA and how might it affect the gravity response in oat coleoptiles, and by what
mechanism?
Ans.: EGTA is a calcium chelator. By chelating calcium in walls, it may prevent gradient of wall
calcium from developing, and this appears to inhibit gravitropism.

4 pts. (c) What is nifedipine and how might it affect the gravity response in Chara, and by what
mechanism?
Ans.: Nifedipine is a calcium channel blocker. By preventing calcium channels from opening it
appears to block the gravity response in Chara.

4. (a) What is the evidence that there is more than one blue-light photoreceptor controlling
growth responses in plants?
Ans.: The gene for one blue-light response (control of hypocotyl elongation) has been identified,
and it has been shown that this gene does not code for the blue light photoreceptor that regulates
phototropism.

4 pts. (b) Describe a light response in Arabidopsis that affects the orientation of shoots with respect
to gravity and name specifically (not generically) the photoreceptor that controls this response.
Ans.: Red light induces disorientation of hypocotyl growth. PhyB controls this response.

4 pts. (c) Briefly describe the experimental results that identified the specific photoreceptor for the
light response described in your answer to 4(b).
Ans.: Mutants lacking PhyB did not show the red-light induced hypocotyl disorientation response.

5. (a) Some phy responses are not photoreversible. Why?
Ans.: FR normally reverses effects of R, but because Pr absorbs some (very little) FR, FR sets
up an equilibrium between Pr and Pfr, with ca. 3% Pfr. For some Phy responses, 3% Pfr is
sufficient to promote the response, thus FR cannot reverse the response.

(b) Photoreversibility is normally diagnostic for a phytochrome response. What is the evidence
that non-photoreversible red-light responses are regulated by phy and not by some other
photoreceptor?
Ans.: Because mutants without PhyA do not show the non-photoreversible (VLF) responses.

4 pts. (c) We said in class that phy has to be photoreversible for it to really function as a shade
detector in natural environments. How then can non-photoreversible phy responses also have
survival value for plants?
Ans.: Most non-photoreversible responses are involved in the phy-induced changes that convert a
plant from a dark-growing form to a light-growing form. These are one-time changes, for which
photoreversibility has no advantages.

6. (a) Phy-induced volume changes in cereal protoplasts has been used as a model system to study
phy-induced signal transduction. What relevance does this response have to phy function in intact
plants?
Ans.: Cell expansion is important for the unrolling response of the first leaf that is induced by phy
in etiolated seedlings when they first emerge into the light.

(b) Table 1 (p.4) describes the results of a study to determine steps that couple Pfr to cell volume.
What effects would (i) lanthanum (LaCl₃), (ii) verapamil and (iii) A23187 be expected to have on
cells?
Ans.: (i) LaCl₃ and (ii) verapamil block calcium channels; (iii) A23187 is a calcium-channel ionophore,
and would promote the passive uptake of calcium into cells.

4 pts. (c) What conclusion follows from the results shown? Explain.
Ans.: An increase in cell [Ca²⁺] can mediate phy responses, since blocking calcium uptake blocks
the response, and artificially promoting the uptake with an ionophore can substitute for Pfr in
inducing the response.

7. (a) How could a G-protein control IP₃ levels in cells?
Ans.: G-proteins can activate phospholipase C, which can generate IP₃ (& DAG) from a PIP₂
precursor.

4 pts. (b) Describe two lines of evidence from microinjection experiments that G-proteins mediate
phy responses.
Ans.: Microinjection of GTP(S can substitute for Pfr in inducing phy responses; microinjection of
GDP$S blocks effects of Pfr.

4 pts. (c) In vision, what name is given to the G-protein that is activated by rhodopsin, and what are
the steps that couple this activation to a ) [Ca²⁺] in rod cells?
Ans.: Transducin; it activates a PDE which decreases cGMP levels, thus closing Ca²⁺ channels.

9 pts. 8. For protoplasts to increase in size (>)V) after a red-light stimulus, new membranes and
membrane proteins would have to be added to the plasma membrane (PM), being delivered
as vesicles from the Golgi that would fuse to the PM. From the results in Table 1 (p. 4) and what
you know about targets of calcium action in plant cells, propose a likely signal transduction chain
starting with red light and ending with )V.
Ans.: Phy induces increase in [Ca²⁺] which activates annexins which promote the fusion of
Golgi-derived vesicles with the plasma membrane.

4 pts. 9. (a) In Fig. 1 (p. 4), taken from an article by Bowler et al. (in Cell, 1994), cGMP actually stands
for 8-Br-cGMP. Why is 8-Br-cGMP used instead of cGMP? Your answer should include a brief
comment on how the use of 8-Br-cGMP addresses criticisms leveled against previous
microinjection experiments reported by the same laboratory (Neuhaus et al., 1993).
Ans.: 8-Br-cGMP, unlike cGMP is membrane permeable, so it does not have to be microinjected to
get inside cells. Also unlike cGMP it is non-hydrolyzable, so its level remains stably high in cells once
it gets in. In previous experiments all agonists/antagonists were microinjected, which with current
technology is a very low efficiency process, so success rates were typically lower than 8%. By
diffusing an agonist in across a membrane, microinjection is bypassed altogether.

4 pts. (b) In Fig. 1, what is the function of (i) sodium nitroprusside (SNP) and (ii) Zaprinast (Zap)?
Ans.: SNP induces the production of NO, which is an activator of cGMP cyclase, so this is a way
of increasing the synthesis of cGMP. Zap is an inhibitor of PDE, which hydrolyzes cGMP, so
treatment of cells with Zap is a way of decreasing the turnover of cGMP.

4 pts. (c) What question is being addressed by the experiment? What answer do the results support?
Ans.: Question is, does Pfr increase cGMP levels primarily by promoting synthesis or primarily
by decreasing turnover. Since SNP, but not Zap, can mimic the effects of Pfr, it would seem that
Pfr induces the expression of the chs gene primarily by a signalling pathway that promotes the synthesis
of cGMP.

9 pts. 10. Discuss the complexity of signal transduction chains, and the multi-functionality of
components in the transduction pathway, using the following terms: translation, cytoskeleton,
PIP₂, PIK-A49, kinase activity.
Ans.: PI-4 kinase promotes the conversion of PI to PIP, which is a key intermediate in the
biosynthesis of PIP₂, in turn a critical precursor for IP₃ production. PIK-A49 is an activator
of PI 4-kinase. The product of PI 4-kinase action (PIP) can enhance actin polymerization; PIK-A49
binds actin and also facilitates actin polymerization. PIK-A49 also has more than 90% sequence
identity to EF-1", which is an elongation factor needed for translation. Thus PIK-A49 can participate
in signalling by promoting PIP production, by promoting a change in cytoskeleton organization
and dynamics, and by enhancing translation.