Plant Physiology (Bot 328), Test # 3                    NAME: 
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) In Fig. 1, the cells used were transformed. What new gene was introduced into these plants 
and why?
Ans.: The introduced gene was the gene for apoaequorin. When converted to aequorin this protein serves 
as a calcium sensor, giving off more luminescence in proportion to increases in Ca.

(b) In order for the experiment to work the cells had to first take up a pigment or chromophore. 
Why?
Ans.: The pigment combines with the apoaequorin to make aequorin, which is a calcium sensor. 
Apoaequorin by itself is not a calcium sensor.

(c) Fig. 1 shows an early step in a signal transduction chain which in young growing plants would 
lead to what two specific form changes in these plants?
Ans.: Touch-stimulated plants show lower rates of stem elongation growth and higher rates of stem 
radial growth.

2. (a) In 1990 Braam and Davis reported that misting plants with water could induce gene 
expression changes. How do their data specifically relate to the results shown in Fig. 1?
Ans.: The genes turned on by misting encode calmodulin or calmodulin-like proteins, which can 
convert calcium signals induced by touch into enzyme activations and other down-stream events.

(b) The mechanostimulation given to Arabidopsis plants by Braam and Davis led these plants to 
undergo thigmomorphogenesis.  What hormone treatment would have led to the same 
morphogenetic changes without mechanostimulation?
Ans.: Ethylene.

(c) Joe and Jane Wannanobel were studying nutrient effects on gene expression in pea plants 
and, for convenience grew their test plants in a greenhouse. They found certain nutrients 
increased expression of calmodulin genes. Would you expect them to get the same results in 
plants grown outside under the same nutrient, light and temperature conditions? Explain.
Ans.: Typically field plants are more wind stimulated than greenhouse plants. Since field plants 
would already have calmodulin genes turned on (see answer to 2a), they may not show any additional 
"turn on" of these genes from nutritional changes.

3.  Regarding cell-level responses induced by auxins:
(a) When Barbier-Brygoo et al. reported that antibodies to a 22 kD ABP could block the induction 
of membrane hyperpolarization by NAA, some scientists felt that these results were incompatible 
with what they knew about the structure of the 22 kD ABP.  Explain the logic of these scientists' 
skepticism.
Ans.: The 22 kD ABP was known to have a KDEL sequence that should keep it in the ER, yet the 
antibodies to the 22 kD ABP would not be expected to penetrate the cell so their effects would be 
restricted to the plasma membrane.

(b) Antibodies raised to PS-IAA4 (a protein encoded by a gene rapidly induced by auxin) were 
used to localize where this protein occurred in cells, the results were negative; i.e., no 
immunostaining was detected. Did this mean that the cells examined had no PS-IAA4 protein? 
Explain. 
Ans.: The negative results could mean that the level of PS-IAA4 protein was too low to detect by 
antibody methods, consistent with the finding that it has a very short half-life in cells.

(c) How did the authors of the immunostaining experiment demonstrate that their antibodies 
were OK and did bind to PS-IAA4?
Ans.: The antibody successfully immunoprecipitated a protein of the predicted size of PS-IAA4.

4. (a) In their studies of the tomato mutant diageotropica (dgt), Hicks et al. (1989) showed that 
the mutant was greatly deficient in a 40/42 kD ABP. Distinguish between an ABP and an auxin 
receptor, and propose an argument based on the results of Hicks et al.  to support the notion that 
the 40/42 kD ABP could actually be an auxin receptor. 
Ans.: An auxin-binding protein simply binds auxin with a high affinity; an auxin receptor is an ABP 
that initiates physiologically significant changes in the cell as a result of binding auxin. The fact that 
diageotropica was deficient in both the 40/42 kD ABP and =in an auxin-regulated event (normal 
gravitropism) suggests that the 40/42 kD ABP was needed for auxin to mediate normal gravitropism, 
therefore it could be a receptor. 

(b) In Fig. 2 (p. 5), what is the question being asked, what is the answer to this question, and how do 
these results relate to the question of multiple auxin receptors?
Ans.: The question being asked was whether diageotropica was also deficient in its capacity to transduce 
auxin stimulus into changes in the expression of any known auxin-regulated gene. The answer is that 
diageotropica could support auxin-induced changes in the Lepar gene, but not in the LeSAUR or 
LeAUX genes. This implies there must be a different receptor that allows auxin to induce the Lepar 
gene in the dgt mutant.

5. (a) Auxin-induced growth changes typically have a lag time of about 10 min. In assaying cell-level 
changes induced by auxin prior to its induction of growth, one that is often noted is wall acidification 
(= auxin-stimulated proton pump). How might this change help mediate IAA-induced growth?
Ans.: Wall acidification could increase wall extensibility by activating expansin which is most effective 
in promoting wall extensibility at acid pHs.

(b) In the signal transduction chain leading to IAA-induced INHIBITION of growth, what gene 
expression change is critical for this inhibition? Explain your answer.
Ans.: Auxin needs to induce the gene for ACC synthase to inhibit growth. This enzyme converts 
SAM to ACC, which is a rate-limiting step for ethylene synthesis. It is auxin-induced ethylene 
synthesis that leads to growth inhbition. 

(c) Fig. 3 (p. 5) implicates auxin asymmetry in gravitropic growth. This experiment could be 
criticised because the key results require longitudinally bisecting the test plant and assaying 
the blotted exudate for protein and mRNA. Briefly describe an experiment using gene 
transformation technology that allowed the results of Fig. 3 to be independently tested without 
bisecting and blotting the plant.
Ans.: If one transforms a plant with a gene construct that has the SAUR promoter attached to a 
reporter gene, then a gravity-induced increase in the promoter activity for SAUR would yield an 
asymmetric expression of the reporter gene, which could be visualized in intact plants.   

6. (a) When Agrobacterium tumefaciens infects a wound in a plant it induces the growth of a tumor. 
By what mechanism does this bacterium induce tumor growth?
Ans.: Upon infection A. tumefaciens transforms the genome of the infected cell with a tumor-inducing 
(Ti) plasmid DNA that encodes genes for IAA and cytokinin synthesis. High expression of these two 
sets of genes leads to high levels of the hormones, which leads to tumorous growth. 

b) Scientists have used modified A. tumefaciens to examine how IAA:cytokinin ratios can affect 
morphogenesis. Describe what modifications were tested and what results were obtained.
Ans.: By selectively removing either the auxin- or the cytokinin-synthesis genes from the Ti plasmid, 
the infection results in an imbalance in the internal auxin:cytokinin ratios. If there is a relative 
overabundance of IAA, a rooty tumor results; if cytokinin is relatively overabundant, a shooty tumor 
results.

7. (a) GA20 induces enhanced stem growth in a dwarf pea variety. Is it likely that GA20 is the 
GA that binds to the GA receptor that initiates the signal transduction chain leading to enhanced 
growth?  Give experimental evidence to support your answer.
Ans.: No, because other dwarf mutants unable to convert GA20 to GA1 do not show growth 
stimulation by GA20.

(b) GA induces increased mRNA levels for an alpha-amylase gene. One could argue that GA is 
acting as a generic stabilizer of RNAs and that this effect is therefore not a specific one. What data 
would argue against this hypothesis?
Ans.: GA treatment actually induces a decrease in the expression of rRNA.

(c) Some seeds germinate only when their endogenous ABA levels are lowered, e.g., by washing out 
the ABA with rain water.  Could this phenomenon be related to the initiation of endosperm hydrolysis 
that normally accompanies seed germination? Explain.
Ans.: ABA can block the upregulation of hydrolytic enzymes by GA. Thus the induction of seed 
germination by washing out ABA can be interpreted as the removal of an inhibitor blocking GA 
induction of enzymes that hydrolyze food reserves (such as starch) in the endosperm. 

8. (a) For many fruits the onset of fruit ripening is signaled by a burst of gas release from the fruit. 
What are the two major components of this gas release, and how is each gas related to the ripening 
process?
Ans.: The two gases are ethylene and CO2. Ethylene induces genes required for ripening. CO2 increase 
is simply symptomatic of a general increase in catabolism that characterizes ripening.

(b) Is the release of either of the two gases noted above important for the release of the other gas? 
Describe experimental results to support your answer.  
Ans.: The release of ethylene is important for the CO2 increase, for fruits transformed tomatoes that 
cannot make ethylene (deficient in ACC synthesis) do not show the CO2 burst, nor do they show 
normal ripening, and treatment with exogenous ethylene corrects both of these deficiencies.

9.  (a) Describe in order of their appearance 7 documented steps of a signal transduction chain 
leading from insect feeding on a leaf to the production of protease inhibitors in intact (uninjured) 
leaves distant from the leaf being fed on. 
Ans.: 1) Systemin hormone induced at site of injury; 2) systemin leaves site of injury and travels through 
vascular system to distant leaves; 3) systemin binds to and activates receptors in distant intact leaves; 
4) activated receptor leads to increases lipoxygenase activity; 5) build up of octodecanoid precursors 
of jasmonic acid; 6) increase in jasmonic acid (methyl jasmonate); 7) activation of jasmonic acid receptors.

(b) What is the experimental evidence that the injury response of plants to insect feeding may 
confer some level of defense against the insects.
Ans.: Plants transformed with antisense to prosystemin are deficient in their systemin levels and in 
protease inhibitors, and Manduca larvae feeding on transformed plants grow larger than those feeding 
on wild-type plants.

10. (a) In studies of the phenomenon of systemic acquired resistance (SAR) a series of transormed 
plants, NahG-1, NahG-2, Nah-G3, etc. were generated that were useful in testing the role of salicylic 
acid in SAR. What "foreign" (not present in wild-type) product did these transformed plants make, 
and how did this product modify SA levels in plants. 
Ans.: The transformed plants made salicylate hydroxylase, which rapidly breaks down SA and thus 
keeps SA levels very low in plants.

(b) What correlations are shown in Table 1 that relate to the question of SA involvement in SAR?
Ans.: The more SA the smaller the lesions induced by pathogens inoculated into leaves that had received 
a prior SAR signal (i.e., the greater the resistance); the less SA, the larger the lesions (the weaker the 
resistance).

(c) Is SA the agent that moves in SAR and convers resistance to uninfected leaves against pathogen 
attack? Give experimental evidence for your answer.
Ans.: No. In grafting experiments in which wild-type scions are grafted onto a NahG stock deficient in 
SA levels, if Nah-G leaf is injected with pathogen, apparently a hormone leaves the site of inoculation 
and moves to the scion where it induces SAR. Since Nah-G leaf would have little or no SA to export, 
it is unlikely that SA is the hormone that left the Nah-G leaf, translocated to the scion and induced SAR 
there.