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

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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

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Interpretation

• 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.

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Abstract

• 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 proteinfunction
  relationships, and the characterization of
  plastid-to-nucleus signaling mutants reveals
  cellorganelle interactions. Recent advances in
  transcriptomics and proteomics of the chloroplast
  make this organelle one of the best understood of
  all plant cell compartments.

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Article Terminology

• cTPs (chloroplast transit peptides)- nucleus
  encoded proteins used by the vast majority of
  chloroplasts which require an N-terminal
  presequence
• 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

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Key Terms Cont

• YCFs- Conserved ORFs (open reading frames) or
  gene types when followed by a and written
  lowercase/noted in italics
• 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

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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.

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Article-relevant Organisms

• Arabidopsis Thaliana
• Maize
• Chlamydomonas

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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 methods.

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Chloroplasts Parts are of particular interest

• A plastid containing chlorophyll, developed only
  in cells exposed to the light.
• Central site of the photosynthetic process in
  plants
• Contained in the cytoplasm of plant cells.

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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.

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Evolutionary/Functionality 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 60200 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.

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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.

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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 launched.
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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.

Examples Meristem-Identity Mutants
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Using Diverse methods In combination

• 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.)

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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.)

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Key Methodology Cont

• Transcriptomics- The genome-wide study of mRNA
  expression levels.
• Proteomics- The study of the full set of proteins
  encoded by a genome.
• previous methods used in combination, advances in
  software and equipment sensitivity increases have
  also improved plant cell understanding

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Future Implications/Outlook

• 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 decade
• Increase understanding of photosynthesis role on
  plastid signaling
• The assignment of proteins to the chloroplasts
  subcompartments

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