Seed Germination

Seed Germination

Seed germination is when growth occurs after a period of dormancy, which is the state in which the seed is shed from the parent plant. In this dormancy period the seed is metabolically inactive and contains very little water. This process allows the seed to survive in adverse conditions, only when the temperature rises in the spring. An embryo is found within the seed, which will grow to form the new plant in germination. This is triggered by gibberellin growth regulators, which are a group of diterpenoid acids that function in a range of developmental processes in higher plants including stem elongation, germination, dormancy, flowering, sex expression, enzyme induction and leaf and fruit senescence. The embryo of the seed is surrounded by endosperm tissue which is a food store containing the polysaccharide starch, with the outer edge of the endosperm containing a protein rich aleurone layer. Active giberellin stimulates the synthesis of amylase in this layer which converts starch to maltose, which can be converted to soluble glucose for the growth of the embryo. Lead in high concentrations damages the aleurone layer, which reduces the amount of carbohydrate supplied to the embryo.      

After germination has occurred, the radicle that emerges from the seed can begin to take macronutrients and micronutrients through the process of active transport. Active transport is the energy consuming transport of molecules or ions across a membrane which is against the concentration gradient. Energy is required because the substance must be moved against its natural tendency to diffuse in the opposite direction. In plants specialised carrier proteins known as chelate proteins bind to specific metal ions and transport these ions across the membrane. There are specific chelate proteins for the macronutrients and micronutrients needed for growth, as in magnesium, copper and zinc which are needed as explained above. The chelate protein collects the micronutrient or macronutrient with the matching complementary shape and transports this across the membrane.  

Protein Structure

The chelate transport proteins contain different structural stages, known as, the primary structure, secondary structure, tertiary structure and quaternary structure.

Primary structure -this is the number and sequence of amino acids in a polypeptide chain, with peptide linkages only found in this structure. This will be different for different chelate proteins.  

Secondary structure -this is when hydrogen bonds cause a protein molecule to coil or fold into a secondary structure, examples of which are the alpha helix and the beta pleated sheet. All chelate proteins will have a secondary structure to make the protein more compact.

Tertiary Structure - this is the three dimensional shape of a protein, which is the coiling of the polypeptide chains. The structure is stabilised by hydrogen bonds, ionic bonds, disulphide bonds and hydrophobic bonds.

Disulphide bonds- A disulphide bond is a covalent sulphur-sulphur bond formed by the oxidation of two cysteine residues, which can be seen particularly in peptides and proteins. This is a strong covalent bond and most chelate proteins have a high number of these bonds to strengthen the protein.

 

Hydrogen bonds- A hydrogen bond is the attractive force between the hydrogen attached to an electronegative atom of one molecule and an electronegative atom of a different molecule. Usually the electronegative atom is oxygen, nitrogen, or fluorine, which has a partial negative charge. The hydrogen then has the partial positive charge.  

Ionic bonds- An ionic bond is an electrical attraction between two oppositely charged atoms or groups of atoms. Atoms are normally neutral, but to gain stability they either lose or gain an electron to become a positive ion (cation) or they will become a negative ion (anion). This means charged molecules will attract each other, forming a bond.  

Hydrophobic bonds-Hydrophobic bonds in proteins arise as a consequence of the interaction of their hydrophobic (water-disliking) amino acids with the polar solvent, water.  

All chelate proteins will have a specific tertiary shape to collect the desired complementary nutrient.

Quaternary Structure – this is when several different polypeptide chains are held together by bonds to form a more complex protein structure such as globular proteins. Chains are held together by hydrophobic interactions, hydrogen and ionic bonds. 90% of enzymes are globular proteins which are compact structures. Some chelate proteins have a quaternary structure.

 

Apparatus for the preliminary experiment  

• 300 cress seeds    -        All seeds were germinated so the coleoptiles and

radicles were 1mm in length before the growth                            investigation with lead nitrate and copper sulphate solutions.

• 300 radish seeds

• Distilled water

• 60 Petri dishes

• 60 circles of filter paper

• 3 x 5cm3 syringes

• Dividers

• 150mm ruler    

• Lead nitrate and copper sulphate at these concentrations:

In order to carry out the preliminary experiment, 60 petri dishes must be collected. This is so there are 3 repeats for each concentration and 12 control dishes for radish and cress seeds. Each dish needs to be labelled with a permanent marker with the correct concentration value. With the filter paper added to each dish, add 5cm3 of lead nitrate or copper sulphate to each dish and add ten seeds of cress or radish variety.  Control plates must be set up with distilled water as the solution, with all the dishes placed in the same position in the laboratory.  

Results from preliminary experiment  

Aim of experiment  

The experiment was used to investigate which out of lead nitrate and copper sulphate solutions would have the most measurable negative impact on growth of radish and cress seeds. The investigation also allows us to decide which out of the cress or radish have the easiest coleoptiles and radicles to measure.

Findings from the preliminary experiment  

1. Type of seed

      Radish- these seeds were larger and easier to see and handle.  

Cress-   these seeds were hard to see and were difficult to get on a plate.

Both the varieties of seed germinated well, but radish seeds had curly coleoptiles which broke when measured. In the main experiment, I will choose cress seeds as these will be a more reliable way of measuring coleoptiles, as they produced straighter coleoptiles and radicles.

  

2. Volume of Solution

Preliminary- In the preliminary experiment I used 5cm3, which was to large. This volume surrounded the seedlings which would have made aerobic respiration for the seed difficult. In the main experiment, I will reduce the volume to 3 cm3.  

3. Concentration and type of solution

In the preliminary experiment, I used lead nitrate and copper sulphate solutions to investigate the affect on coleoptile and radicle length. The investigation showed that copper sulphate had no effect on the growth of coleoptiles and radicles, as the highest concentration of copper sulphate with the radish seeds showed coleoptile length 25 mm and radicle length 21mm, which can be compared with the control sample at 25mm coleoptile length and 21mm radicle length. This showed that copper sulphate would not be used in the main experiment.

In contrast, lead nitrate solution did produce a difference in coleoptile and radicle length, but there were some concentrations which were too strong and killed all the seedlings. Therefore for the main experiment the concentrations will be reduced from the 0.200M solution. The mean length of cress coleoptile and radicle length at lead nitrate concentration 0.025M, showed lengths of 20mm and 15mm respectively. This can be contrasted with the control test which showed coleoptile length of 28mm with 20mm radicle length. This illustrates that lead nitrate is a suitable solution to use in the main experiment to investigate the effect on seed growth.

Prediction   

I predict that at higher concentrations of lead nitrate, which will be 0.100M and 0.0075M, the seed will have limited growth and will successively show shorter length coleoptiles and radicles than seeds which have been placed in lower lead nitrate concentrations of 0.025 and 0.05M. The coleoptiles and radicles should also be shorter than the control tests. Chelate proteins used in the active transport (see background for active transport) of nutrients into the seed are denatured by lead nitrate, as lead has an affinity for sulphur. The lead binds to the covalent sulphur-sulphur interactions which are broken which denatures the chelate protein. If the chelate protein can not bind with essential nutrients, then active transport will stop.  

If the chelate proteins are denatured, then the seed will not be able to take up magnesium which is essential for the production of chlorophyll. Magnesium is the central metal ion bonded to the porthyrin ring. If magnesium is not obtained by the seed, then chlorophyll is not produced which is used in the light dependent stage of photosynthesis. The light-dependent stage of photosynthesis uses light absorbed by chlorophyll pigments to provide ATP and NADPH which is needed for the reduction of carbon dioxide to carbohydrate in the light independent stage. Chlorophyll a has two forms of peak absorption p700 and p680, with chlorophyll b and carotenoids used in accessory pigments. If magnesium is not present than the plant cannot capture light energy from across the spectrum, which means ATP + NADPH synthesis stops.

Photophosphorylation of ADP to ATP is either carried out by cyclic or non-cyclic photophosphorylation. Non-cyclic photophosphorylation involves the use of both photosystem I and II, where light energy takes electrons to a higher state of energy. Electrons are absorbed by electron carriers which are where ATPsynthase converts ADP and inorganic phosphate to ATP. Electrons end up at photosystem I which are used to reduce NADP to NADPH for the light independent reaction. Electrons are replaced at photosystem II by photolysis. Cyclic photophosphorylation does not need NADP, so NADP is not reduced in this process, but ATP is still synthesised.    

‘Z-Scheme’/Non-Cyclic Photophosphorylation    

If photosynthesis does not take place because chlorophyll production is inhibited by the lack of magnesium, then ATP and NADPH are not available for use in the light dependent reactions in the Calvin-Benson cycle. The Calvin cycle uses carbon dioxide combined with ribulose bisphosphate (RuBP), to produce two molecules of glycerate 3-phosphate. Using ATP and reduced NADP this compound is converted to two triose phosphate molecules which is used in conversion as glucose, amino acids and lipids. The majority of triose phosphate molecules are used in the regeneration of RuBP, with the cycle having to happen 6 times for 1 molecule of glucose to be produced.  

Calvin-Benson Cycle    

If the Calvin cycle is blocked, then triose phosphate can not be used to synthesise glucose, glycerol and fatty acids. The triose phosphate would normally be converted to acetyl coenzyme A which can then be converted to amino acids and lipids. Plants use lipids for the production of plant growth hormones, with amino acids linked together to make proteins. Proteins are essential for growth and repair, as triose phosphate is not supplied from the Calvin cycle then proteins can not be synthesised. Also as nutrients are not taken up by the seed, nitrates are not absorbed which means triose phosphate that is present cannot be converted into amino acids.    

As glucose cannot be synthesised, then aerobic respiration is stopped, so the seed cannot synthesise ATP for the use in processes, such as metabolic reactions. Aerobic respiration is split into 4 processes, glycolysis, link reaction, Krebs cycle and oxidative phosphorylation. Glycolysis is the first stage of aerobic respiration, which takes place in the cytoplasm. Glucose is converted to hexose phosphate, hexose bisphosphate to produce 2 molecules of triose phosphate. Two molecules of ATP are used in the phosphorylation of glucose. ATP is used to make glucose react more easily, to gain energy from glucose for ATP production. Four ATP molecules are released from glycolysis with a net gain of 2 ATP. Hydrogen is removed from triose phosphate which is transferred to the hydrogen carrier NAD. NAD is needed for Oxidative Phosphorylation for the further gain of 28 ATP in this process.

The final product of glycolysis is two molecules of pyruvate which is used in the Krebs cycle, as pyruvate is energy rich.    

Glycolysis

The next stage of aerobic respiration is the link reaction, which is the transport of pyruvate from the cytoplasm into the mitochondrial matrix. The pyruvate is decarboxlated, dehydrogenated and combined with coenzyme A to give Acetyl coenzyme A. Coenzyme A transports acetyl groups to the Krebs cycle, with hydrogen removed by NAD used in Oxidative Phosphorylation.

Link Reaction:  Pyruvate + CoA+NAD  acetyl-CoA + Co2 + reduced NAD    

The Krebs cycle which is also known as the citric acid cycle is a closed pathway of enzyme controlled reactions. Acetyl Coenzyme A combines with a four-carbon compound (oxaloacetate) which forms a six carbon compound (citrate). The citrate is decarboxlated and dehydrogenated to yield Co2 and hydrogen which is accepted by NAD. Oxaloaceteate is regenerated to combine with another acetyl coenzyme A. The Krebs cycle’s most important function is the release of hydrogen which can be used in the electron transport chain for ATP production.  

Krebs cycle

The last stage of aerobic respiration is the electron transport chain which is used in oxidative phosphorylation, for the maximum yield of ATP from the process. The electron transport chain occurs in the mitochondrial membranes, with hydrogen from reduced NAD and FAD split into hydrogen ions and electrons which are used to synthesise ATP from ADP and inorganic phosphate. The transfer of electrons through electron carriers provides the energy for ATP synthesis. This is carried out through chemiosmosis, in which hydrogen ions are pumped through the mitochondrial matrix into the intermembrane space which creates a concentration gradient. As this gradient moves from a high concentration to a low concentration, the ions move through protein channels associated with ATPsynthase. Electrical potential energy is used to synthesise ATP.

Many electron carriers in the electron transport chain are cytochromes. Cytochromes are proteins which have a prosthetic group with an embedded metal atom for the acceptance of electrons. Copper and Zinc are used as the metal ions in the chain, with iron also being used. As lead nitrate denatures chelate proteins which transport Copper and Zinc into the seed, then the electron transport chain is stopped. As the electron transport chain yields 28 ATP from reduced NAD and FAD, the electron transport chain is essential for metabolic reactions such as growth and repair which the seed will not do if these metals are not present.    

As high concentrations of lead nitrate prevent the seed from carrying out photosynthesis and respiration the seed can not synthesize ATP and glucose. This means that a seed placed in high lead nitrate concentrations will use the limited food supply in the seed and once this supply has been used the seedling will die. At lower concentrations of lead nitrate solution I should expect coleoptiles and radicles to be shorter in length than seeds placed in a control solution, but longer than the coleoptiles and radicles in higher concentrations as chelate proteins may still be able to carry out active transport of nutrients but at a slower rate than the seeds in the control solution. As the seed will be able to do some active transport, reduced amounts of magnesium will be up taken by the seed, and so the seed can carry out light dependent reactions. This means small amounts of ATP and NADPH are available for the light independent stages of photosynthesis. As photosynthesis is still functioning then triose phosphate can be used to be converted to glucose, which means some aerobic respiration can take place. Reduced amounts of amino acids are produced from reduced nitrate uptake so this means reduced growth of coleoptiles and radicles will happen in lower concentrations of lead nitrate solution, with high concentrations preventing the growth of the seedling.  

Apparatus  

Making 1.000 M Solution of lead nitrate  

• To ensure accuracy is maintained through the investigation, apparatus must be washed three times with distilled water. Distilled water is free from impurities so no contamination will occur. All apparatus was dried so concentrations of lead nitrate solutions were not affected.

• Using the relative atomic masses, the mass of lead nitrate was calculated.

• Using an electronic balance, weigh out 331.20g of lead nitrate and place into the 1 litre volumetric flask.

• At eye level, accurately measure 1000 cm3 of distilled water in the measuring cylinder. To ensure this is achieved accurately, measure at the bottom of the meniscus up to the 1000 cm3 mark using a pipette to alter the volume if needed.

• I used this method every time I used a measuring cylinder to measure distilled water or lead nitrate solutions.  

• Pour the distilled water into the 1 litre volumetric flask, until the meniscus is below the graduation mark. Use a pipette at eye level until the meniscus is level with the graduation mark.

• Add a bung to the flask which allows mixing of the lead nitrate solution, but it also prevents water from evaporating.  

Making Serial Dilutions of lead nitrate

The serial dilutions were obtained by using the equipment and values from the table.

• Label the beakers with concentrations of solutions.

• Collect a 1000 cm3 measuring cylinder of distilled water. Use a 100 cm3 measuring cylinder for measuring a 100cm3 and 75 cm3 of lead nitrate, use a 50 cm3 syringe to measure 50cm3 and 25 cm3 of lead nitrate, use 10 cm3 syringe and subsequently 1 cm3 syringe to measure the required amounts of lead nitrate.

• Use six glass stirring rods to mix each solution.    

• Draw solution with syringe past the measurement line and flick the syringe to release air bubbles. Push the plunger so the first part of the blackline is on the measurement line.

• Cover each beaker with cling film to prevent evaporation.    

Adding the serial dilution to the seeds.

• 300 cress seeds are needed for this investigation, which need to be germinated. Extra seeds are used as not all germinate and 210 seeds are needed for the main experiment.  

• The seeds must be left until the coleoptile and radicle growth from successful germination is 1 mm in length.

• Collect 21 Petri dishes.

• The Petri dishes must be labelled with the concentration of lead nitrate to be used in the test, with the labelling round the edge of the lid. Labelling on the top will prevent seeds being moved when measurements are taken, and the labelling on the side of the lid will allow enough light to reach the seedlings for growth to occur.

• Add a small piece of warning tape to each of the lead nitrate dishes.

• Add filter paper to each Petri dish.

• The correct solution was added to each dish using a 5cm3 syringe.

• From the preliminary experiment, I reduced the measurement from 5cm3 to 3cm3.

• 10 cress seedlings need to be placed into each Petri dish using tweezers.

• Place lids on the dishes and put the Petri dishes in the same place in a laboratory.

• The seedlings must be left for a time period of a week before measurements are taken.

• Measure the coleoptiles and radicles of each seedling using dividers to accurately measure with a 300mm ruler.

• Subtract 1mm from each result to account for the initial germination and record the results in a table.

Variables

Independent

This is a variable which was changed in the main experiment, which was:

• The range of lead nitrate concentrations which were used to identify the affect of lead nitrate on growth.      

Dependent  

This is the variable which is measured, which was:

• The measurements of length for the coleoptiles and radicles in a range of lead nitrate solutions.

Control Variables

This is something in the investigation which remains the same so you can compare results.    

Health and Safety  

Results

 

Bibliography  

Dictionary of Biology-Second Edition by Unwin Hyman  

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 2621.2004.tb09947.x        

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