Chloroplast Research in the Genomic Age by 3r8jZs1

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									Chloroplast Research in
    the Genomic Age
            Presented by: Andrew Brian Raupp

           Research conducted by: Dario Leister
  Abteilung für Pflanzenzüchtung und Ertragsphysiologie,
   Max-Planck-Institut für Züchtungsforschung, Carl-von-
             Linné-Weg 10, 50829, Köln, Germany

                     Trends in Genetics
       Volume 19, Issue 1 , January 2003, Pages 47-56
 This is an example of a literature
review article, therefore “success”
is based upon the clarity and
comprehensiveness of the
information compiled. The goal of
the authors is to show the trends in
chloroplast genomic research by
tying together the various methods
used into one paper. Papers such as
this, make it easier to reference
information when inquiring about a
specific topic.
 Chloroplast research takes significant
advantage of genomics and genome
sequencing, and a new picture is emerging of
how the chloroplast functions and
communicates with other cellular
compartments. In terms of evolution, it is
now known that only a fraction of the many
proteins of cyanobacterial origin were
rerouted to higher plant plastids. Reverse
genetics, Forward Genetics and novel
mutant screens are providing a growing
catalogue of chloroplast protein–function
relationships, and the characterization of
plastid-to-nucleus signaling mutants
reveals cell–organelle interactions.
Recent advances in transcriptomics and
proteomics of the chloroplast make this
organelle one of the best understood of all
plant cell compartments.
       Article Terminology
• cTPs (chloroplast transit peptides)- nucleus
  encoded proteins used by the vast majority of
  chloroplasts which require an N-terminal
• Rubisco (ribulose biphosphate carboxylase)-
  most abundant enzyme in the world, involved
  in many plant pathways
• TAT (twin-arginine translocation)- a pathway
  which requires a TAT motif
        Key Terms Cont…
• YCFs- Conserved ORFs (open reading
  frames) or gene types when followed
  by a # and written lowercase/noted in
• NPQ (Non-photochemical quenching)- a
  class of mutants which reflects the
  energy dissipated as heat following
  energization of the thylakoid
  membrane due to lumen acidifcation.
• Plastome- Chloroplast genome
• Nucleome- Nuclear genome
 Arabidopsis Thaliana Description
• Arabidopsis thaliana (L.) Heynh. (thale cress) belongs to
  the Brassicaceae (Cruciferae) family . It is a small weed
  that is distributed widely around the world. Its small size
  and rapid life cycle have made it a very popular and useful
  model organism used in the modern plant biology.

• Arabidopsis Thaliana*

• Maize

• Chlamydomonas
 Arabidopsis Thaliana Genome
The Arabidopsis genome
was completely sequenced
at the end of year 2000. It
contains about 26000
genes, of which still less
than half can be identified
or given an assigned
function. Several more or
less coordinated efforts
in the scientific community
aim at learning the role
and function of these
unknown genes using
various sophisticated
  Chloroplasts: Parts are of
     particular interest
• A plastid
  developed only in
  cells exposed to
  the light.
• Central site of
  process in plants
• Contained in the
  cytoplasm of
  plant cells.
 Chloroplast Anatomy
• The smooth outer membrane is freely
  permeable which means molecules
  come in and out freely.
• The smooth inner membrane uses
  transporters, to regulate the
  passage in an out of the chloroplast
• The thykloid lumen is the cavity
  bounded by a plant cell wall.
• A thykloid is the structural unit of
  the grana in the chloroplasts of
  plant cells. It is a saclike membrane.
 Understanding by reading into
         the Plastome
“As endosymbiotic remnants of a free-living
 cyanobacterial progenitor, plastids have,
 over evolutionary time, lost the vast
 majority of their genes. Indeed, depending on
 the organism, contemporary plastomes
 contain only 60–200 open reading frames
 (ORFs). The plastomes of green algae and
 flowering plants are remarkably similar in
 the sequences of their genes, whereas the
 organization of genes on the plastid
 chromosome differs drastically.”
  Evolutionary/ Functionality
 Understanding by reading into
    the chloroplast Cont…
“The vast majority of chloroplast
 proteins are nucleus-encoded and, with
 the exception of the outer envelope
 proteins, require N-terminal
 presequences, termed `chloroplast
 transit peptides' (cTPs), to target them to
 the chloroplast. Between 2100 and 3600
 distinct proteins are estimated to be
 located in the Arabidopsis chloroplast”
 Other proteins such as TAT are also
 similarly examined.
  Evolutionary/ Functionality
 Understanding by reading into
        the nucleome…
“In Arabidopsis, maize and rice, the targeted
mutagenesis of nuclear genes coding for plastid
proteins has been facilitated by the availability of
large collections of insertion mutants – based on
gene disruptions by T-DNA (A. thaliana), transposons
(maize and A. thaliana), or mobilized
retrotransposons (rice) – which can be
systematically searched for mutations in genes of
interest. The targeted inactivation of nuclear genes
by antisense, co-suppression and RNA interference
(RNAi) strategies has also made a significant
contribution. In principle, the mutational saturation
of all nucleus-encoded plastid proteins is feasible,
but such a large-scale effort has not yet been
         Why Study Mutations?
One of the most important parts of Arabidopsis is the
generation of mutants (defects in genes). By identifying the
mutation that affects the growth and development, or
environmental adaptation of the plant, genes that are
responsible for these traits can be identified.

         lfy-26          lfy-26        lfy-26 ap1-1    lfy-26 ap1-1
      inflorescence   inflorescence   inflorescence   inflorescence
                        (side view)                      (side view)

         lfy-26       lfy-5 flower                    ufo-2 flower
                                      ap1-1 flower
         flower          (weak)                         (strong)
            Examples Meristem-Identity Mutants
  Using Diverse methods In
• Reverse genetics- the traditional approach
  was to find a gene product and then try to
  identify the gene itself. In molecular
  genetics, the reverse has been done by
  identifying genes purely on the basis of
  their position in the genome with no
  knowledge of the gene product. This
  revolutionary approach is reverse
  genetics. Also called positional cloning. In
  this case, it is used to find nuclear genes
  encoding plasmid proteins, novel proteins
  involved in photosynthesis, small subunits
  of PSI and PSII, essential proteins and to
  learn more about Rubisco. (See distributed
  protocol for more details.)
  Key Methodology Cont…
Novel mutant screens- A screen is the
analysis of different isolates for a
given phenotype or property (like
unusual growth, the level of a given
enzyme, the presence of an interesting
metabolite, the level of a particular
antigen, or the presence of a region of
DNA capable of hybridizing to a given
probe). It should not be confused with a
selection ,which is a demand for a given
phenotype and is therefore orders of
magnitude more "powerful". (See
distributed protocol for more details.)
      Key Methodology Cont…
• Transcriptomics- The genome-wide study of mRNA expression
• Proteomics- The study of the full set of proteins encoded by a
• previous methods used in combination, advances in software and
  equipment sensitivity increases have also improved plant cell
• Since Mutant screens
  have been quite
  successful, the goal is
  to be able to find the
  functions of as many as
  1000 genes in the next
• Increase
  understanding of
  photosynthesis’ role
  on plastid signaling
• The assignment of
  proteins to the

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