Plastids

Plastids

Hey everyone, hope you had a wonderful Valentine's day last week! Sorry I forgot to mention it on the day! Anyway, we've been looking at a range of topics situated around the subject DNA. So I'm now going to delve into another topic that has bugged me for very long time. Plastids. Let's get straight to it!

What are they?

They are organelles. That means that they are parts of a cell. They are a type of ''organ'' of a cell, performing a function that is necessary for the overall function of the cell.

They are found in plants. Chloroplasts are plastids. Crazy, but true. Chloroplasts are weird in the sense that they are green (caused by the pigment, chlorophyll) - the only plastids to be of that insane amazing colour. Most plastids do not have a coloured pigment. However, all plastids contain a double-stranded DNA molecule that is free and not encased in a nucleus. The DNA is circular, like that of prokaryotes.

All plastids are able to change from a single type to another one. There are factors that affect the type that it is changed to:
          1. The environment
          2. The stage at which the plastid and cell are in terms of development
          3. The tissue that the plastid's cell resides in

Features of plastids

Plastids display a huge variety of size and structure. The most remarkable thing about them, I believe, is that they display a large array different functions.

Plastids have a common feature: they produce and store substances. Another common feature is that all plastids are derived from a proplastid. These are found in meristematic parts of the organism. Meristematicism refers to the region that contains undifferentiated stem cells - e.g. the active regions or growing points of plants (like in the roots and shoots).

Much like a mitochondrion, plastids all have an envelope that surrounds a matrix. The matrix holds membranes, storage material and droplets containing pigments. The pigment is dependent on the type of plastid. For example, the pigment will be nice and green in a chloroplast.

Remember when I said the DNA wasn't in a nucleus? (don't worry, I was saying the truth!) Basically, the circular DNA is held in nucloids. These are DNA-protein complexes. Nucloids are linked directly to the inner membrane of the matrix.

The number of DNA copies in a nucloid and the number of nucloids are both dependent on the plastid type and its stage of development.

The chloro of the plastids

The chloroplast. The famous king of all the plastids. It is my personal favourite. Why? Because it's green! :D Their primary function is to photosynthesise. This is why they contain the green pigment, chlorophyll.

Chloroplasts have a matrix that is commonly referred to as the stroma. This is surrounded by an envelope (as is a common feature of all plastids). Inside the stroma, there are flattened sacs called thylakoids. These are stacked up to form... stacks. The stacks are called grana.

It is within the thylakoid membrane that the protein-chlorophyll complexes that trap the photons in the light from the sun are embedded.

If this ain't a chloroplast, I give up.

Protoplasts develop into chloroplasts in the presence of light. How much light? I have no idea. But I'll do an experiment, find an answer, and tell you in 5 years! :)

In low levels of light, protoplasts form etioplasts. There are some leaves you see around you that have a yellow colour. These are partly due to the presence of these organelles. The yellow pigment is protochlorophyll. Etioplasts can quickly be converted to chloroplasts through the exposure of light. They have a prolamellar body that is quickly changed into the stroma and thylakoids upon contact with photons.

An etioplast. See the big red thing? That's the ball of tubules that are quickly converted to flat thylakoid membranes as soon as there is an introduction of light.

The transformation of etioplasts to chlroplasts is actually a reversible one. Chloroplasts can thus transform into etioplasts at will.

Right, so I think that's enough brain food for you! Next week, I think I shall continue with this plastid business - there's a lot more in the bigger picture. I'll talk about chromoplasts, leucoplasts and their transformations too. Remember to send in your feedback at praveenprathapan28@gmail.com! So, until then, see ya!

Exocytosis

Onions and crying

Why do onions make you cry?

It's an interesting question. I've always been wondering about it myself for years. It's comforting to know that there is a simple biochemical reason for this strange phenomena. :)

What others might tell you...

People normally say that there is a chemical that is released from an onion that irritates the eyes. This is kinda right... but not quite! There are a few chemical processes that occur naturally first. The chemical released from the onion is not the irritant: it is not the chemical that irratates your eyes.

Let's get to the biochemistry!

Imagine an onion cell with two compartments. One compartment holds the enzymes which are known as alliinases (yeah, it has two ''i''s in it). Imagine that the second compartment, adjacent to the first, holding the sulfur-containing amino acids (AKA amino acid sulfoxides). The name sulfoxide implies that there exists an S-O bond. The difference in electronegativity causes an overall dipole. The oxygen is more electronegative than sulfur.

This is the structure of sulfoxide which is found in a compartment of onion cells. There are two resonance forms pictured above. Resonance is a type of electron delocalisation. This occurs because electrons repel. In a double bond, they move away from each other to make the molecule more stable. In this picture, we can see the two different arrangements of the electrons.

When you cut an onion, the chemicals in the separate compartments are able to mix and form a type of sulfenic acid. This happens because you break the cells open when chopping an onion. The specific name for the acid produced is 1-propenesulfenic acid. Here's the formula:

CH3CH=CHSOH

Right, now something a little weird happens next. Another enzyme called lachrymatory factor synthase (or LFS for short) assures the rearranging of the acid molecule to form propanethial S-oxide. The rearranging, I believe, is what probably causes a decrease in boiling point in the molecule. It probably makes the structure less regular and thus decreases the number and strength of dipole-dipole interactions between the molecules. Temperature can more easily overcome the now weaker intermolecular forces and the boiling point is consequently lowered (the substance becomes more volatile).




Propanethial S-oxide before exposure to LFS enzyme

Right, so what exactly makes us ''cry''?

The gas diffuses through the air and initiates contact with the eye. Three chemicals are formed from the interaction: hydrogen sulfide, propanol and sulfuric acid. The sulfuric acid is dilute but it is in a high enough concentration to cause irritation to the eye. This triggers a sensory neural response which creates a stinging sensation and tear-production by tear glands. Tears are released as a method of flushing out and diluting the sulfuric acid from the eye.

We need sulfuric acid in onions. It sounds quite deadly, but it's true. Without it, onions wouldn't have the nice scent they they have. They would taste nothing like they do now. The same applies to garlic too.

Old wives' tales

Alright, I did promise to do this last week! And I keep my promises. Here are some ways to cut down on crying whilst chopping onions. Decide for yourself which ones really work:

1. Cover your eyes with safety goggles/specs. This provides a barrier against the gas released from
    the onions.
2. Use a fan to blow away the gas.
3. Put a teaspoon in your mouth.
4. Put a piece of bread in your mouth and let it hang there whilst chopping.
5. Wear contact lenses
6. Use a sharp knife (careful when handling sharp objects!)
7. Chill the onions
8. Chop the onion whilst holding it in water. The water becomes acidic due to the reaction between
    water and propanethial S-oxide. However, your hands could slip whilst slicing - so be extra careful.
9. Breathe with your mouth when chopping onions. You will breathe in the gas released by the
    onions.
10. Close your eyes when chopping onions. I'm kidding! That would be suicide.

Well, I hope I have shed some light on an every-day mystery. Now go and impress your mates! I'll be back next week with some more biochemistry as always! Don't forget to send me feedback too! If you are or know anyone doing or is interested in biochemistry, don't hesitate to contact me. I'm nearly always available for any help and discussion on top of other niceties! :)



Exocytosis

How to isolate DNA

How to isolate DNA

Today, I'm going to present a method of a very exciting experiment. It strongly ties in with what I have been discussing over the past week: DNA. Doing a biochemistry degree involves a lot of experimentation and this experiment is what I hope to be doing frequently during my education :)

The method of isolating DNA that I will present is the same across many parts of the world and is one of the most essential experiments in molecular biology.



Alright, gimme the main jist of it...

Okay, first we need to get an organism. I used an onion. Yes, it was a teary experiment. :')

We break up the tissue and use detergent to break down the cell surface membranes of... the cells and the nuclear membranes of the... nucleus. This is homogenisation.

The cell fragments are separated through filtration and we are left with DNA and soluble proteins.

To remove the protein, we use enzymes. Protease is a wise choice.

Finally, the DNA is precipitated using ethanol at around 250K (I personally kept it at 273K).

Apparatus

Liquidiser (mixer)
Knife and chopping board to cut onions
Water both that is ice cold
Thermometer
Funnel
Filter paper
500cm^3 Beaker
Glass rod
Stopwatch
Boiling tube and rack
25^3 measuring cylinder
Pipette
Sodium chloride solution (I used 4%M)
Low temperature ethanol (I used 95%M)

Detailed method please!

1. Slice onion with a knife into 5mm cubes and place in beaker with 100cm^3 detergent.

2. Stir and keep at 333K in a water bath for 15 minutes. The temperature breaks down the cell membranes of the onion and the detergent forms complexes surrounding the membrane phospholipids and proteins causing them to solidify out of the colution. Also, the Na+ sodium ions shield the negatively charged phosphate groups of DNA molecules causing them to coalesce.

(By this point, you will be crying. I shall give you the biochemical reason for this next time!)

3. Cool the mixture in iced water for 5 minutes whilst stirring. This slows down any breakdown of DNA if you kept it in high temperatures any longer.

4. Liquidise the mixture for ONLY 5 seconds - yeah it's that quick. This degrades the cell walls and membranes which allows more DNA to be released. The reason why liquidisation should be quick is because this stops the DNA strands from being completely and utterly broken up, which kinda destroys the experiment...

5. Filter it into a measuring cylinder and the filtrate should contain the DNA and soluble proteins.

6. Store this in a fridge for around 1 or 2 days

7. Add 1cm^3 enzyme to 20cm^3 of the onion tissue extract in a boiling tube and mix. The enzyme will hydrolyse the proteins associated with DNA.

8. Induce a layer of ice-cold ethanol on top of the mixture by pouring it slowly down the SIDE of the boiling tube.

9. Leave the tube for 3 minutes. The DNA is insoluble in ice-cold ethanol. Bubbles may form and the DNA will precipitate out.

10. Gently rotate the glass rod in the liquid at the interface of the alchol/detergent mix. The interface is a flash term for the point at which the two layers meet. Be careful not to stir too much as the mixing of layers would break up the fragile strands of DNA ~

11. A white web of DNA (which would look a lot like mucus) can be viewed. It can be drawn from the tube with a pipette and suspended again in a solution of sodium chloride or distilled water.


I hope this experiment helps people who may be getting ready to do their EMPAs or ISAs in the UK. This will help anyone who has a more practical approach to this very practical science of Biochemistry. Next week, inspired by my crying during when I carried out this experiment, I'm going to explain why we cry when we chop onions (and maybe a few old-wives tales too!).



Exocytosis