CHLOROPLAST- Structure Isolation and Function

CHLOROPLAST 

STRUCTURAL ORGANIZATION

When viewed with the light microscope, chloroplasts typically appear as oval or spherical structures exhibiting a prominent green pigmentation. They occur in the leaves and other green tissues of higher plants, as well as in algae. 

The chloroplasts of leaf cells typically measure about 2-4 11min diameter and up to 10 11min length, which is roughly twice the size of a typical mitochondrial profile. The chloroplasts of algae may even be larger, and often assume unusual shapes such as spirals, cups, and circular bands that wind around the cell. In. algae as few as one or two large chloroplasts are present per cell, but plant cells typically contain several dozen or more. 

These multiple chloroplasts appear to be largely independent of each other rather than forming an interconnected network, as often occurs in mitochondria. Relatively little internal structure can be seen when chloroplasts are viewed with the light microscope, although early light microscope did detect tiny green granules within the chloroplast, which they named grana.  Electron micrographs revealed that the organelle is surrounded by a membrane envelope. 

This chloroplast envelope consists of two closely apposed membranes, termed the outer and inner membranes, which are separated from each other by an intermembrane space.  The chloroplast envelope encloses the interior space, or stroma, of the chloroplast, which is analogous to the mitochondrial matrix. Within the stroma is another set of membranes, called thylakoid membranes, which are organized as a system of flattened sacs. 

The internal space bounded by the thylakoid membranes is called the thylakoid lumen. Thylakoid membranes are organized in two different ways, referred to as the stacked and unstacked configurations. In stacked or grana thylakoids, thylakoid sacs are stacked upon each other like a pile of coins, generating large membrane masses that correspond to the grana observed by light microscopy. Grana stacks are connected to one another by membrane-bound channels referred to as unstacked thylakoids. Each unstacked thylakoid is a large flattened sheet that makes connections to many or all of the individual thylakoids of a given granum stack. In this way, the individual thylakoid lumens of 0each granum stack become interconnected 'both with one another and with the thylakoid lumcns of other granum stacks. 

Hence the entire thylakoid membrane system of the chloroplast defines a single, enormously complex, membrane enclosed compartment.

Negative staining has revealed that the outer surfaces of thylakoid membranes are studded with spherical CFt particles, which resemble the Fl particles observed on mitochondria envelope, while mitochondrial cristae are continuous with the inner membrane surrounding  mitochondrion.

 As a result, chloroplasts consist of three separate compartments (intermembrane space, stroma, and thylakoid lumen), whereas mitochondria have only two compartments (intermembrane space and matrix)

ISOLATION OF CHLOROPLAST COMPONENTS

The development of techniques for isolating chloroplasts and their various components has played an important role in advancing our understanding of the functional organization of this organelle. 

Chloroplasts can be isolated from plant cells in one of several different ways. Some procedures utilize harsh homogenization techniques, such as grinding cells with sand using a mortar and pestle, to break the cell wall and release the chloroplasts into' solution. Following removal of nuclei by centrifugation at low speed, chloroplasts are collected by centrifuging at low speed, chloroplasts are collected by centrifuging at higher speeds. 

A more recent approach to chloroplast isolation employs the enzymes cellulase and pectinase to break down the cell wall. The advantage of this approach is that the resulting wall-less cells, or protoplasts, can be disrupted by gentler techniques prior to isolating chloroplasts by centrifugation. Chloroplasts isolated under harsh conditions are usually capable of carrying out the light-induced production of oxygen, ATP, and NADPH, but not C02 fixation. 

Electron micrographs of such defective class II chloroplasts reveal the presence of little or no stroma, and broken or missing outer envelopes. In contrast gentle disruption techniques yield completely intact class I chloroplasts that are capable of carrying out the complete pathway of photosynthesis, including C02 fixation. Class I and II chloroplast preparations have both been useful as starting material for isolating the various membranes and compartments that make up the chloroplast. In one commonly used procedure, class I chloroplasts are suspended in a hypotonic solution to rupture the chloroplast envelope, followed by isodensity centrifugation to separate the stroma, outer envelope, and. thylakoids from one another. 

Alternatively, class II chloroplasts lacking the outer envelope and stroma can be used as starting material for separating the thylakoid membranes into stacked and unstacked thylakoids. In this approach chloroplasts are disrupted in a special apparatus, called a French pressure cell,that shears the thylakoid membranes by forcing them through a small orifice following rapid decompression. 

This shearing process breaks the connections between the stacked and unstacked thylakoids, thereby allowing the two membrane fractions to be separated from one another by differential centrifugation. Biochemical studies involving components isolated in these ways have provided a great deal of information concerning the functional organization of the chloroplast

FUNCTIONS

The most important function of chloroplasts is to carry out the process of photosynthesis. The process includes the light reaction and dark reactions.

• The light reaction  takes place in the grana of the thylakoid membrane. In light reaction NADPH  and ATP are produced.
• Dark reaction occurs in stroma of the chloroplast.
• In this reaction reducing power of NADPH is used to fix co2 into carbohydrates.