Chloroplasts

Chloroplasts
• Chloroplasts are organelles involved
in photosynthesis, the process of
formation of carbohydrates from
carbon dioxide and water in the
presence of sunlight by the plants.

• Synthesis of amino acids, fatty acids and lipid components of

their membranes.
• The important step of conversion of nitrite (NO-2) to ammonia
(NH3) occurs in the chloroplast providing the plant with the
nitrogen for the synthesis of amino acids and nucleotides.
Discovery

• The first account of photosynthesis appeared in the writings of Stephen

Hales, an English naturalist and the ‘Father of plant physiology’.
• Joseph Priestley (1772) demonstrated that plants have the capacity to
purify air.
• His experiments excited Jan Ingenhousz who in1773 noticed that
photosynthesis will occur in the presence of sunlight and only the green
parts of the plant purified air. But neither of them was aware of the
chemical nature of pure or impure air.
• It was Antoine Lavoisier, a French chemist, who in 1785, identified the
‘pure’ component of air as ‘oxygen’ and the ‘impure’ as ‘carbon dioxide’.
• Chloroplasts were first described as ‘chloroplyllkornern’ (chlorophyll granules) by German
botanist Hugo von Mohl in 1837, though because of their large size they were seen long back by
Nehemiah Grew who described them in 1682 as green precipitates!
• It was A.F.W. Schimper who in 1883 introduced the term ‘plastids’ as a substitute for
chlorophyll granule.
• In the same year A. Mayer described ‘grana’ as dense dot like structures embedded in the
‘stroma’ of these plastids. The terms grana and stroma are still in use but chloroplast has
replaced the term plastids for the green organelles of leaves.
• Julius von Sachs (1887) was the first to discover that chloroplasts are the organelles in which
carbon dioxide is fixed and oxygen is released.
A typical plant cell may have 20-40
chloroplasts. There are many alga which
have single chloroplast per cell that
occupies most of the cell (remember
cup shaped chloroplast of
Chlamydomonas).
Chloroplasts are organelles that belong
to the plastid family. This group of
organelles which are present in various
plant cells differ in their color and
function.
In young meristems small pro-plastids
develop which mature according to the
requirement of the cell and also as
dictated by the nuclear genome.
For example:
• Etioplasts- these develop in leaves
grown in dark. These have a yellow
chlorophyll precursor and semi
crystalline membranes. When exposed
to light these develop to form
chloroplasts.
• Leucoplasts- these are white colored,
non –photosynthetic and develop in
epidermal and internal tissues.
• A common type of leucoplast is the
amyloplast which accumulates
polysaccharides and has a principal
function of storage for e.g. as in potatoes.
 Structure and function
• Chloroplasts are large organelles, generally lens shaped
in higher plants, of around 2-4 µm wide 5-10 µm in long,
bound by a double membrane called chloroplast
envelope.
• In addition a third internal membrane called as the
thylakoid membrane is also present.
• The thylakoid membrane forms a network of flattened
sac like structures called thylakoids often stacked to form
grana (singular granum).
• The three membranes divide the chloroplast into three
compartments:
• The intermembrane space between the two outer
membranes,
• the stroma
• and the thylakoid lumen
Chloroplast structure can be understood under three sub-heads:
• 1) Envelope
• 2) Thylakoids
• 3) Stroma
Envelope:
• Just like nucleus and mitochondria, chloroplasts are bound by a double membrane
envelope called outer membrane and inner membrane. Both the membranes are
made up of phospholipid bilayer. The space in between the membranes is inter-
membrane space.
• The outer membrane of the chloroplast envelope contains ‘porins’ and so is freely
permeable to small molecules.
• Whereas the inner membrane is impermeable to ions and metabolites and they
need specific membrane transporters (integral membrane proteins) to enter the
chloroplast.
• The outer membrane has very less protein content about 30% and have unusually
have very little phospholipid. These have a high percentage of galactose containing
glycolipids, which have several double bonds in their fatty acids making the
membranes highly fluid. This facilitates the lateral diffusion of the protein
complexes in the membrane.
Unlike the mitochondria, the inner membrane of the chloroplast is not
involved in either electron transport or photosynthesis.
Instead the thylakoid membrane contains the electron transport system.
The protons are pumped across into the lumen of the thylakoids
creating a proton gradient that drives the ATP synthesis as protons cross
back into the stroma
Thylakoids
• The internal membrane system
(separate from the double
membrane envelope) is organized
into disc shaped/flattened
membranous structures with
empty lumen called thylakoids
that are frequently arranged in
stacks.
• Ea c h s t a c k i s c a l l e d g ra n u m
(plural: grana).
• Adjacent grana are connected by
stromal thylakoid (thylakoids in
direct contact with the stroma).
• The thylakoid membrane contains the light absorbing pigments, the
chain of electron carriers (ETC) and the ATP synthesizing machinery
(in this sense it is analogous to the inner membrane of mitochondria).
• The space inside the thyakoid is the lumen.
• The protons are pumped across this membrane from the stroma to
the thylakoid lumen. This results in formation of an electrochemical
gradient which generates ATP when protons are transported back to
the stroma.
Stroma
• The space enclosed by the inner membrane of
the chloroplast is called stroma.
• Stromal matrix is rich in metabolic
enzymes,alkaline,aqueous fluid and has small
double stranded circular DNA molecules,
ribosomes (70-S) and plastoglobuli (lipid
granules).
• Dark reaction or the C-3 cycle (Calvin cycle)
takes place in stroma.
• Enzymes for amino acid synthesis and fatty Plastoglobuli
acid synthesis are also present in stroma.
 Photosynthesis
• Photosynthesis is the main source of metabolic energy.
Chloroplasts capture energy from sunlight and use it to fix
carbon and convert it into carbohydrates.
• The reactions that occur during photosynthesis can be
divided into two stages:
• The light dependent reactions- During light reactions
energy from the sunlight drives the synthesis of ATP and
NADPH coupled with the splitting of the water molecule
and generation of O2.
• The light independent reactions (commonly called dark
reactions) – the synthesis of carbohydrates using CO2 and
the energy stored in the ATP and NADPH.
• These reactions occur in different compartments of the
chloroplast, the thylakoid membrane and stroma
respectively.
• Chlorophyll molecule consists of two parts,a porphyrin head
and a long hydrocarbon phytol tail.
• A porphyrin is a cyclic tetrapyrrole, made up of ‘four nitrogen-
containing’ pyrrole rings to which Mg2+ is chelated. Loss of
magnesium ion from chlorophyll results in the formation of a
‘non-green’ product, ‘pheophytin’

• Carotenoids also called tetraterpenoids, are yellow, orange,

and red organic pigments. They absorb light energy for use in
photosynthesis, and they provide photoprotection via non-
photochemical quenching (mechanism to protect themselves
from the adverse effects of high light intensity)

• Xanthophylls are yellow pigments. As both are carotenoids,

xanthophylls and carotenes are similar in structure, but
xanthophylls contain oxygen atoms while carotenes are purely
hydrocarbons, which do not contain oxygen. Their content of
oxygen causes xanthophylls to be more polar (in molecular
structure) than carotenes
Light reaction
• Sunlight is captured by photosynthetic pigments the
most abundant of which are the – chlorophylls.

• The photosynthetic pigments are arranged into

photocenters with each photocenter containing
hundreds of antenna pigment molecules which
absorb the photons and transfer energy to the
reaction center chlorophyll.
The energy derived from the sunlight
excites an electron (derived from the
splitting of water molecule and the
generation of O2) of the chlorophyll
thus converting the energy of the
sunlight to potential chemical energy.
This electron then moves along an
electron transport chain (a series of
electron carrier proteins located in
the thylakoid membrane).
Four protein complexes located in the
thylakoid membrane function as
electron carriers and are also involved
in the synthesis of ATP and NADPH.
Energy initially harvested by PSII
(photosystem II) is used to split a
water molecule within the thylakoid
lumen.
Electrons are then carried by
plastoquinone to the cytochrome bf
complex where they are transferred to
low energy state and protons are
pumped into the thylakoid lumen.
Electrons are transferred to
photosystem I by plastocyanin(PC).
At PSI the sunlight absorbed is again
used to generate high-energy
electrons. The photosystem I uses the
high energy electrons to reduce
NADP+ to NADPH.

Finally, the high energy electrons that move through the electron transport chain of light reaction and the H+ convert the
NADP+ to NADPH. The movement of electron through the photosystem I and II generates both ATP and NADPH which are
then used in the Calvin cycle to convert CO2 to carbohydrates.
• Another electron transport pathway that
operates to produce ATP is called the cyclic
electron flow.
• However unlike the non-cyclic pathway no
NADPH is generated. The light energy is harvested
at the photosystem I and the high energy
electrons generated are transferred to
cytochrome bf complex coupled to which protons
are transferred across the thylakoid membrane
establishing a proton gradient and thus
synthesizing ATP .
• Plastocyanin returns these electrons to
photosystem I thus completing the cycle.
• Thus PS I participates in both cyclic and non cyclic
pathways forming ATP and NADPH depending on
the requirement of the cell.
Synthesis of ATP
The electron transfer process is accompanied with the movement of H+ across the thylakoid
membrane. The resulting electrochemical proton gradient drives the ATP synthesis in the stroma
and is known as photophosphorylation.
ATP formation both in chloroplast and mitochondria is guided by chemiosmosis. Chemiosmosis is
an energy-coupling mechanism that uses energy stored in the form of a hydrogen ion gradient
across a membrane to drive cellular work (generating ATP).
During chemiosmosis and electron transport chain assembled in a membrane pumps protons
across the membrane as electrons are passed through a series of carriers in the electron transport
chain thus establishing a proton gradient.
An ATP synthase complex is built into the same membrane. The movement of the protons down
the gradient as they pass through the ATP synthase complex provides the energy by which ATP is
regenerated from ADP and phosphate. This chemiosmotic theory was proposed by Peter Mitchell
in 1961 for which he received the Nobel prize in 1978.
Dark reactions or the carbon fixation reactions
The ATP (energy source) and NADPH (reducing power), produced during light reaction, drive the conversion
of CO2 to carbohydrate (sucrose) and other organic molecules like amino acids and fatty acids. These
reactions occur partly in the chloroplast stroma and partly in the cytosol.

Dark reaction or the Calvin cycle converts CO2 to carbohydrates. This reaction occurs in three phases. Phase
one is carboxylation where one molecule of CO2 enters the Calvin cycle by joining with a 5-carbon molecule
ribulose 1,5-bisphosphate (RuBP). This reaction is catalyzed by ribulose 1,5-bisphosphate carboxylase,
perhaps the most abundant protein on earth!

The product a 6-carbon compound immediately breaks down to two molecules of 3-carbon compound, 3-
phosphoglycerate. Next phase is reduction where 3-phosphoglycerate is reduced to glyceraldehyde 3-
phosphate using ATP and NADPH.

Lastly, regeneration of RuBP occurs by converting some of the 3-phosphoglycerate to RuBP. Later, 3-
phosphoglyceraldehyde is exported to cytoplasm where it is converted to sucrose
Chloroplast genome
We know that apart from nucleus, DNA is also found in
mitochondria and chloroplast. The chloroplasts, like
mitochondria, are semi- autonomous organelles that have their
own genetic systems.
Chloroplast genomes of a number of organisms have been
sequenced and have provided a detailed information regarding
the number of genes and the products encoded by them.
The important features of chloroplast genome include:
• Circular DNA molecules present in several copies per organelle
• Size varies from 120-160 kb
• Approximately 150 genes (depending on the organism) that
can be grouped in two categories: those involved in gene
expression and those involved in photosynthesis.
• Two inverted repeats (IRs) 10-28 Kb in length divide the
genome into a large single copy region (LSC) and a small single
copy region (SSC)
The chloroplast genes code both for RNAs and proteins. Along with mRNAs, both t-RNAs and rRNAs
are encoded by the chloroplast genome. However a high percentage of the 2000-3500 polypeptides
of the chloroplasts are encoded by the nuclear genes.

For the smooth functioning of the organelle, the nuclear and the chloroplast genomes thus work
synergistically.

For example, some of the subunits of the RNA polymerase and the enzyme Rubisco found in the
chloroplast are encoded by chloroplast genome itself while rest of the subunits are encoded by the
nucleus and then imported inside the chloroplast.

Protein translation in chloroplast is strikingly similar to protein translation in prokaryotes. For

example, both in prokaryotes and chloroplast, the protein synthesis starts with N-formyl Met,
ribosomes are sensitive to antibiotics chloramphenicol and tetracycline; bacterial RNA polymerase
can transcribe chloroplast DNA, chloroplast mRNA can be translated in bacteria.
Marker Enzymes
Some enzymes are located exclusively in one particular organelle. These enzymes can be used
to establish the presence of the organelle in the isolated fraction of the cell. Such enzymes are
called marker enzymes.
For example, if in a fraction of cell activity of the enzyme succinate dehydrogenase is ascertained,
the presence of mitochondria is confirmed.
The marker enzymes also confirm the biochemical purity of the isolated organelle. Marker
enzymes can also be used to judge the biochemical purity of the fractionated organelle. If the
activity of other marker enzymes is also found in the fractionated organelle it is due to
contamination with other organelles.
Marker enzymes for chloroplast are RUBISCO and NADP-glyceraldehyde-3-phosphate
dehydrogenase. Both these enzymes participate in the Calvin cycle and are located in the stroma
of the chloroplast.
Semiautonomous nature
Some organelles enjoy some amount of autonomy or independence like in growth, division,
production of some enzymes, they are known as semiautonomous organelles.
Mitochondria and chloroplast fall in this category. They are able to do this because they have
their own DNA/ genome. Both the organelles are also known to have evolved from free living
prokaryotes, according to the endosymbiotic theory.
Endosymbiotic theory was proposed by Lynn Margulis in1960s.
She proposed that primitive anaerobic bacteria engulfed another aerobic bacteria and instead of
endocytosis endosymbiosis occurred, that is the association became mutually beneficial for both
the bacteria and resulted in early aerobic heterotrophic eukaryote. Further, when this eukaryote
engulfed photosynthetic bacteria another endosymbiotic relationship evolved and resulted in an
autotrophic eukaryote.
This theory also explains double membrane of these organelles, one derived from each of
the organism involved.Since the autonomy is not complete and the chloroplasts depend
on nuclear genome for majority of their proteins and enzymes, hence, these organelles
are known as semiautonomous in nature.
For example, in chloroplast the enzyme RuBisCo is made up of multiple units of
polypeptides assembled into two types of subunits called the large chain and small chain.
The large chain gene is a part of chloroplast DNA but the small chain genes are located on
the nuclear genome. The small chain subunit is thus synthesized in the cytosol and is
imported into the stroma.
Endosymbiontic theory
Evidences suggest that chloroplasts originated from oxygen evolving photosynthetic bacteria via
the process of endosymbiosis about 1 billion years ago. The primitive eukaryotic cells ( which
were probably anaerobic) engulfed the photosynthetic bacteria and these then later established a
symbiotic relationship with their partners. Similarly the mitochondria evolved from bacteria being
endocytosed. This probably is the reason why the basic process of ATP synthesis is similar in both
the organelles.

Most of the chloroplast proteins are encoded by the nucleus therefore it is hypothesized that an
extensive transfer of genes from organelle to the nucleus must have occurred during evolution.