Pioneers in chloroplast & plant molecular biology

Discovered chloroplast genetics (Chlamydomonas) and DNA

Chloroplast genes in corn; demonstrated light regulation

Chloroplast gene function; nuclear control

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

Chloroplast Biology

I. Structure

1. double-membrane envelope

2. stroma: large soluble interior

3. thylakoid membrane system

4. intrathylakoid space or lumen

From Gunning & Steer

Isolated Spinach chloroplast

envelope

stroma

thylakoid membrane

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

From Gunning & Steer

II. Functions1. many important biochemical (anabolic)

pathways, e.g., photosynthesis*

starch synthesis fatty acid synthesis*amino acids synthesispigment synthesis*nucleotide synthesisnucleic acids* and protein synthesissulfur 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 tissues4. Amyloplasts - prominent in roots, store starch,

colorless5. Chromoplasts - in mature fruit, lots of

carotenoids, little chlorophyll

Mature Etioplast

Proplastid

Plastid development in dark-grown barley

Youngest

Oldest

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

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

From Gunning & Steer

From U. Wisconsin Botany Dept.

Young chromoplast from developing tomato fruit

From Gunning and Steer Stars mark lycopene crystals; many plastoglobuli

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 cell2. 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

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

Digestion of cpDNA of the mt- parent in a young zygote of Chlamydomonas revealed by fluorescence staining of DNA. 0 Minutes

10 minutes

From Mauseth, 1998

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

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

~30 % of chloroplasts don’t have DNA!

nucleus

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