PCB6528 Spring 2015 - Exam 2 – Chase
Name Key - 100 pt total Av = 80
The exam was written based on 100 points. Dr. Cline will weight its contribution to your final grade
based on the fact that the exam covers material presented in 6 classes.
Important – please keep your answers short; confine your answers to the space provided; do not
write on the back of any pages !
1 [35 pt] In our discussion paper, Stegmann et al. (Proc. Natl. Acad. Sci. USA 109:2434) used four
different transgenes in their work to look for chloroplast capture in the context of tissue grafting:
• Plastid rrn promoter driving gfp
• Plastid psbA promoter driving aadA (spectinomycin/streptomycin resistance)
• Nuclear nos promoter driving nptII (kanamycin resistance)
• Viral 35S promoter driving yfp
1-a) (5 pt) Describe what is meant by chloroplast capture
The replacement of one plastid genotype by another while the nuclear genetic background is
unchanged.
1-b) (5 pt) What was the specific function of the antibiotic resistance genes in this study?
They allowed for the selection of a new genetic combination the “recipient” nuclear genotype in
combination with “replacement” plastid genotype.
1-c) What was the specific function of the gfp and yfp genes in this study?
(5 pt) They allowed for visual confirmation that the selected transgenes were found and expressed
in the same cell.
1-d) What behaviors of plant mitochondria and plant mitochondrial genomes might be expected to
create mitochondrial genetic exchange in the chloroplast capture plants, even in the absence of a
mitochondrial selection agent? 10 pt any 2:
(5 pt) Mitochondria undergo frequent fusion
(5 pt) Mitochondrial genomes undergo frequent recombination
(5 pt) Mitochondrial coding sequences are highly consered
Therefore if mitochondria from the plastid donor also entered the recipient cell, the possibility of a
recombinant mitochondrial genome exists.
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PCB6528 Spring 2014 - Exam 2 – Chase
Name ____________________________
1-e) Successful chloroplast capture through tissue grafts was mentioned as one way to overcome
barriers to chloroplast transformation in some plant species. In theory one could transform the
desired constructs into tobacco plastids and move them into another species. Briefly discuss two
reasons why this strategy might be limited to species that are closely related.
(10 pt for any two):
• Divergent species may not be graft compatible
• Plastid genome coding content is not necessarily identical between species due to on-going
functional transfer of genes to the nucleus. If neither the donor plastid nor the recipient nucleus
contains an essential gene, capture could not occur
• Myriad nuclear genes are required for correct expression of plastid genes. If RNA binding sites
for any of these nuclear factors have altered, the recipient nuclear genome might not be able to
support correct plastid gene expression or function. Examples could include :
RNA splicing factors
PPR proteins that recognize RNA editing sites
PPR proteins that protect RNA termini
PPR proteins or other positive regulators of translation
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PCB6528 Spring 2014 - Exam 2 – Chase
Name ____________________________
2 (33 pt)
2-a) (12 pt)Plant organelles have a unique RNA editing process that is essential to organelle gene
expression. In tobacco plastids, for example, the initiation codon of the psbL gene must be created
by RNA editing. The genomic sequence at the 5' end of the tobacco gene begins with ACG. In the
table below, fill in the nucleotide sequences that correspond to this codon for each different
molecule.
molecule
psbL initiation codon
(all in the 5’ to 3’ orientation on
the coding strand)
tobacco psbL genomic DNA
5' ACG 3'
tobacco psbL un-edited RNA
ACG
tobacco psbL cDNA corresponding to the un-edited RNA
ACG
tobacco psbL edited RNA
AUG
tobacco psbL cDNA corresponding to the edited RNA
ATG
2 -b) (6 pt) Briefly describe one line of evidence that plastid RNA editing sites are defined by specific
nucleotide sequences “in cis” (nearby) the edited nucleotide.
Plastid transformation constructs carrying an RNA editing site and varying amounts of native
flanking sequence were deployed. The frequency of editing at the transgenic site was found to
depend upon the amount of 5’ and 3’ upstream sequence
Or
Specific PPR proteins are required to accomplish specific RNA edits, and PPR proteins are known to
function as site-specific RNA binding factors
2-c) List two protein families that have been implicated as trans-acting factors in plant organelle RNA
editing.
(6 pt) PPR and MORF
2-d) Discuss why plastid and plant mitochondrial RNA editing likely occur by the same mechanism.
(3 pt) They both involve C-to-U editing
(3 pt) They involve different members of the SAME protein families
(3 pt) The plastid-targeted MORFs and the mitochondria-targeted MORFs can interact with each
other in vivo and the plastid-targeted MORFs can interact with mitochondrial editing factor PPRs
(MEFs) in vivo
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PCB6528 Spring 2014 - Exam 2 – Chase
3 (32 pt) The scheme for
photosynthetic electron
transfer is shown in the
diagram to the right. If
light conditions are
altered, plastid
transcription will be
adjusted so that the two
photosystems can
maintain the balance of
electron flow through the
pathway.
Name ____________________________
PSI
PSII
PET
FD
3-a) If light is changed from a wavelength favoring photosystem II (PSII) activity (light II) to a
wavelength favoring photosystem I (PSI) activity (light I) would the ratio of transcripts for the
PSII/PSI core subunits (psbA / psaA) be expected to increase or decrease? Explain your reasoning.
(12 pt) The ratio would increase so additional PSII could be synthesized to feed sufficient electrons
to PSI.
3-b) Briefly describe the experimental observations supporting the hypothesis that the redox status
of plastoquinone (PQ) is key for the signaling that adjusts plastid transcription to achieve redox
balance between the two photosystems.
(6 pt) The redox status of PQ can be altered by treatment with electron transfer inhibitors DCMU
and DBMIB to give effects similar to those observed upon changing the light regime.
(4 pt for any one specific example such as):
DCMU will result in oxidized PQ and lead to effects similar to light I
DBMIB will result in reduced PQ and lead to effects similar to light II
Sig1 phosphorylation associated with DCMU less efficient for psaA promoter
Sig1 non-phosphorylated with DBMIB highly efficient for psaA promoter
3-c) Define retrograde regulation and explain why it this process would also be necessary to
maintain optimal redox balance in the plastid.
(5 pt) The regulation of nuclear genes by organelle signals
(5 pt) Some subunits of the photosystems are encoded by nuclear genes and the amounts of these
must also be adjusted to change abundance of the entire complex
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