Pioneers in chloroplast & plant molecular biology

Ruth Sager (Harvard) Lawrence Bogorad (Harvard) Jean-David Rochaix (Geneva)

Discovered chloroplast genetics (Chlamydomonas) and DNA

Chloroplast genes in corn; demonstrated light regulation

Chloroplast gene function; nuclear control

Chloroplast Biology
I. Structure
1. 2. 3. 4. double-membrane envelope stroma: large soluble interior thylakoid membrane system intrathylakoid space or lumen

From Gunning & Steer

Isolated Spinach chloroplast

thylakoid membrane

envelope

stroma

From Hoober

High magnification of thylakoid membranes

From Hoober

Stroma of Avena (rye) plastid at high magnification to show ribosomes

S aggregates of a glucosidase used in defense against fungi, substrate is in vacuole (avenacoside), product is a toxic saponin

II. Functions
1. many important biochemical (anabolic) pathways, e.g., photosynthesis* starch synthesis fatty acid synthesis* amino acids synthesis pigment synthesis* nucleotide synthesis nucleic acids* and protein synthesis sulfur and nitrogen assimilation 2. own genetic system*
* Indicates that pathway involves a chloroplast encoded gene in at least some organisms

Many of the biosynthetic pathways are regulated, to peak during the light period of L-D cycle.

Chlorophyll a and b synthesis during a 24 hour light-dark cycle (Chlamydomonas).

A similar result was obtained for carotenoids and other chloroplast lipids. (Janero and Barnett, 1982)

III. Reproduction
all plant and eukaryotic algal cells have plastids chloroplasts form by division; semi-autonomous Involves proteins (Fts) similar to those that mediate cell division in bacteria

Cyanidioschyzon chloroplast dividing

From Miyagishima et al.

IV. Development
There are several forms of plastids: 1. Proplastids - precursor of all plastids, found in meristems 2. Etioplasts - form in shoots of dark-grown plants, distinctive internal structure 3. Chloroplasts - in all green tissues 4. Amyloplasts - prominent in roots, store starch, colorless 5. Chromoplasts - in mature fruit, lots of carotenoids, little chlorophyll

Plastid development in dark-grown barley Oldest Mature Etioplast

Youngest Proplastid

Amyloplasts from Glycine (soybean) root-cap (peripheral cell)

S - starch grains Also have some thylakoid membranes (star) close-by.

From Gunning & Steer

From U. Wisconsin Botany Dept.

Young chromoplast from developing tomato fruit

Stars mark lycopene crystals; many plastoglobuli

From Gunning and Steer

Plastid development is plastic & mostly under nuclear control.

Shoots:
light proplastids etioplasts chloroplasts chromoplasts Roots: proplastids

amyloplasts

V. Chloroplast Genetics
1. Inheritance is typically uniparental, usually maternal. Multiple mechanisms involved, not well understood: - in Chlamydomonas (next slide), the paternal (-) cpDNA is destroyed, and the maternal (+) cpDNA is preferentially replicated - in some land plants, the paternal plastids are excluded during fertilization or absent from the sperm cell 2. Essentially all plastids have DNA, usually the same DNA throughout the organism (homoplasmy). 3. The DNA sequence does not change during differentiation.

There are exceptions to the last 2 statements.

Chlamydomonas life cycle has sexual and asexual reproduction. For sex, there are 2 mating types, mt+ and mt-,

mt- cpDNA destroyed

Digestion of cpDNA of the mt- parent in a young zygote of Chlamydomonas revealed by fluorescence staining of DNA.

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Nishimura, Yoshiki et al. (1999) Proc. Natl. Acad. Sci. USA 96, 12577-12582

From Mauseth, 1998

Acetabularia green alga, fossils known, many species likely extinct nucleus

A single giant cell (5 cm), 1 nucleus, ~1 x 106 chloroplasts

~30 % of chloroplasts dont have DNA!

Chloroplast DNA (cpDNA)

General features: 1. double-stranded, circular molecule 2. no histones, but have bound proteins (e.g., Hu), organized into nucleoids 3. G-C content typically less than nuclear DNA 4. multiple copies (~30-100) per plastid (i.e., all cp genes are multi-copy) 5. can be 10-20% of the total DNA in leaves

relaxed cpDNA molecule from lettuce

From Kolodner & Tewari