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Biochemical Changes - General Botany -Lab, Study notes of Botany and Agronomy

These are the lab notes of Botany. Key important points are: Biochemical Changes, Investigation, Fruit, More Particularly, Results Implicating, Hemicellulosic, Cellulosic Cell Wall, Fruit Softening, Polygalacturonase, Tomato Cultivars

Typology: Study notes

2012/2013

Uploaded on 01/24/2013

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Laboratory Four
An investigation of biochemical changes to tomato
fruit when it ripens
Introduction
According to an old country song by John Denver, the only two things money
can't buy are true love and homegrown tomatoes (I am being honest here, feel free
to check out the following link on “youtube” if you don’t believe me:
). Oh, and
PLEASE, do NOT come
to me and ask who John Denver is!!!!! Shame on YOU if you don’t know!!!!!!!!
Anyway
, the tomato has become a model system for the study of fruit
ripening and, more particularly, the changes that bring about fruit softening. In
this fruit, despite some results implicating the hemicellulosic and cellulosic cell wall
fractions in fruit softening, the most recent work in the area has focused on the
pectic fractions. In particular, there has been much interest in the polyuronide-
solubilizing enzyme, polygalacturonase. Its activity rises dramatically during
ripening, and a close correlation between polygalacturonase and pericarp softening
has been demonstrated in a range of tomato cultivars.
Naturally, tomatoes unevenly ripen, showing darker green patches when
unripe and variable redness when ripe -- traits that still show up in garden-variety
and heirloom breeds. However, in the late 1920s, commercial breeders stumbled
across a natural mutation that caused tomatoes to ripen uniformly -- from an even
shade of light green to an even shade of red. This "uniform ripening" mutation has
become indispensable to the $2 billion a year U.S. commercial tomato market,
showing up in almost all tomatoes produced for grocery stores. The uniform redness
makes it ideal for groceries, where customers expect evenly colored, red fruit.
An important fact to remember:
Whilst leaves are the primary
photosynthesis factories in a plant, developing tomato fruit can contribute up
to 20 percent of their own photosynthesis, yielding high sugar and nutrient
levels in fully ripe
fruit.
The uniform ripening mutation, which commercial
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Laboratory Four

An investigation of biochemical changes to tomato

fruit when it ripens

Introduction

According to an old country song by John Denver, the only two things money can't buy are true love and homegrown tomatoes (I am being honest here, feel free to check out the following link on “youtube” if you don’t believe me:

). Oh, and PLEASE, do NOT come

to me and ask who John Denver is!!!!! Shame on YOU if you don’t know!!!!!!!!

Anyway , the tomato has become a model system for the study of fruit

ripening and, more particularly, the changes that bring about fruit softening. In this fruit, despite some results implicating the hemicellulosic and cellulosic cell wall fractions in fruit softening, the most recent work in the area has focused on the pectic fractions. In particular, there has been much interest in the polyuronide- solubilizing enzyme, polygalacturonase. Its activity rises dramatically during ripening, and a close correlation between polygalacturonase and pericarp softening has been demonstrated in a range of tomato cultivars. Naturally, tomatoes unevenly ripen, showing darker green patches when unripe and variable redness when ripe -- traits that still show up in garden-variety and heirloom breeds. However, in the late 1920s, commercial breeders stumbled across a natural mutation that caused tomatoes to ripen uniformly -- from an even shade of light green to an even shade of red. This "uniform ripening" mutation has become indispensable to the $2 billion a year U.S. commercial tomato market, showing up in almost all tomatoes produced for grocery stores. The uniform redness makes it ideal for groceries, where customers expect evenly colored, red fruit.

An important fact to remember: Whilst leaves are the primary

photosynthesis factories in a plant, developing tomato fruit can contribute up

to 20 percent of their own photosynthesis, yielding high sugar and nutrient

levels in fully ripe fruit. The uniform ripening mutation, which commercial

breeders select for, eliminates this protein in the fruit, therefore reducing sugar

levels and nutrients in the fruit.

So, how can you look at biochemical alterations to tomato fruit as they ripen

during a three hour lab?

The act of ripening requires the expression and action of an enzyme protein to break down structural cell wall polysaccharides to smaller units and monosaccharides. So there should be a change in both sugar content and protein

content between ripe and unripe tomatoes, right?

So, write your hypothesis here:

Biochemical assays to be used in this Laboratory

Benedict's reagent assay procedure for reducing

sugars:

This reagent contains 100 g sodium carbonate and 173 g sodium citrate dihydrate in a final volume of 850 mL water. Slowly, with stirring, add a solution of 17.3 g copper sulfate pentahydrate in 100 mL of water. Bring the final volume to one liter.

The blue copper (II) ions from copper (II) sulfate are reduced to red copper (I)

ions by the aldehyde groups in the reducing sugars. This accounts for the color

changes observed. The red copper (I) oxide formed is insoluble in water and is precipitated out of solution. This accounts for the precipitate formed. As the concentration of reducing sugar increases, the nearer the final color is to brick-red and the greater the precipitate formed.

The Lasker and Enkelwitz test also utilizes Benedict's solution, although the

reaction is carried out at a much lower temperature. The color changes that are

seen during this test are the same as with Benedict's solution. In this assay, samples are heated in a 55 oC water bath for 10-20 minutes. Ketopentoses demonstrate a positive reaction within 10 minutes, while ketohexoses take about 20 minutes to react.

  • Use a 1% fructose solution and distilled water to prepare a series of

standard fructose solutions of different concentrations: 0.05%,0.075%

0.1%, 0.2%, 0.4%, 0.6%, 0.8%, and 1%

  • Add 5ml of each standard and 1 ml of Benedict’s solution together.
  • Mix the contents of each tube by gently shaking the test tubes back and forth.
  • Place the tubes in a test tube rack and set the rack in the 55oC water bath. CAUTION! The water is very hot.
  • Incubate the tubes for 10 minutes. Remove a sample for centrifugation. Replace the rest of the sample back in the 55oC water bath.
  • Remove your test tubes and allow them to cool.
  • Transfer the contents to 1.5 mL centrifuge tubes and spin for two minutes. If there are still suspended debris in any of the tubes, centrifuge for two

more minutes. **It is important that the solution be clear for the

absorbance measurements. If there is solid matter suspended in the

solution, the light being sent through the sample will be scattered and

will cause error in the measurements.

  • Decant the supernatants into clean, labeled test tubes. Be careful that any sediment remains in the pellet at the bottom of the centifuge.
  • Determine the O.D. of each of the samples using a spectrophotometer set to read at 735 nm. Use distilled water to set the 100%T.
  • Plot the average values in Excel.

Bradford assay Procedure:

The Bradford assay is a very popular protein assay method because it is simple, rapid, inexpensive and sensitive. The Bradford assay works by the action of Coomassie brilliant blue G-250 dye, which specifically binds to proteins at arginine, tryptophan, tyrosine, histidine and phenylalanine residues. Advantages

  • Fast and inexpensive
  • Highly specific for protein
  • Very sensitive
  • Compatible with a wide range of substances
  • Extinction co-efficient for the dye-protein complex is stable over 10 orders of magnitude (assessed in albumin)
  • Dye reagent is complex is stable for approximately one hour.

Disadvantages

  • Non-linear standard curve over wide ranges
  • Response to different proteins can vary widely, choice of standard is very important

Procedure:

  • Add 1.4 ml of 1X Bradford reagent in each microfuge tube.
  • Into the standard tubes place the following amounts of BSA protein standard (1 mg/ml).

Tube μl BSA μl H 2 O μg protein added 1 0.0 30.0 0. 2 0.0 30.0 0. 3 5.0 25.0 5. 4 5.0 25.0 5. 5 10.0 20.0 10. 6 10.0 20.0 10. 7 15.0 15.0 15. 8 15.0 15.0 15. 9 20.0 10.0 20. 10 20.0 10.0 20. 11 25.0 5.0 25. 12 25.0 5.0 25. 13 30.0 0.0 30.