Student Sheet 20 - Can leaf discs make starch in the dark?

It is well known that photosynthesis is challenging to students and few teachers find it an easy topic to teach! Many teachers and student use leaf discs for photosynthesis investigations. Have you tried the method of sinking and then re-floating leaf discs in a syringe? With that investigation, observations are made of the buoyancy given to the submerged discs by the oxygen produced during photosynthesis. The time taken for the sunken discs of cotyledon tissue to float gives a measure of the rate of photosynthesis. The rate is directly related to different physical conditions, such as concentration of carbon dioxide in the water, the light intensity or temperature.

The method described with leaf discs in this protocol differs in that it looks at production of starch rather than production of oxygen. Yet if you compare the two methods, you can see some of the reasons why it is preferable to use leaf discs rather than adopting the classic ‘whole leaf’ approach for investigations with plants. Some of the advantages of working with discs include:

• When discs are cut from leaves, students can use several in their sample, thus encouraging replication
• Direct comparison of different experimental treatments of the discs is easier to do on a small scale
• Graded levels of experimental intervention may be designed to provide a rigorous structure to the thinking skills behind the investigation (e.g. compare glucose / water : light / dark : green disc / white disc etc.)

Let’s review the results, some possible outcomes and questions that would lead to further investigations.

• Discs in dish A (green, on water, in light) give a blue-black colour when tested for starch and so behave as expected for a whole leaf. Floating them upside down (stomatal side up) should allow maximum carbon dioxide entry.
• Discs in dish B (green, on water, in dark) give a brownish colour in the starch test. This shows they also behave as expected for a whole leaf and do not make starch in the dark.
• Discs in dish C (green, with glucose, in dark) show a dark blue-black at the edges of the discs and less dark (or even brownish) towards the centre. This result may surprise students. It shows that the discs absorb glucose, translocate it from cell to cell and convert glucose to starch. Starch formed fastest at the disc edges, confirming that glucose is absorbed through the cut edge, rather than through the stomata or leaf surface.

If you follow up the suggestion to try this protocol with white discs, taken from a variegated pelargonium, floated on water and left in the light, you find that they do not make starch. Similarly, white discs floated on glucose solution (in the dark) also do NOT make starch. We accept that the white pelargonium cells cannot produce starch as they have no chlorophyll, but it is surprising (to some teachers and to students) that those white discs cannot make starch even when the glucose has been added. Clearly these white cells lack starch grains as well as functioning chloroplasts. This result is perhaps a lesson to students (and teachers!) to be SCEPTICAL . . . for the experiment shows up as false the classic proof, given in probably every GCSE text book, of starch not being formed when chlorophyll is absent. These particular cells would not be able to make starch even if chlorophyll was there. It also helps emphasise the different stages in the process of starch synthesis.

Brush up your plastids and pelargoniums

If you look at a typical text book diagram of a 'generalised plant cell', you will find starch grains as well as chloroplasts. Neither of these is essential to all plant cells, and other plastids may be present. Plastids are membrane-bound plant cell organelles, with probable prokaryotic origins. They have their own DNA and multiply as smaller pro-plastids at an early stage in the plant meristem, then differentiate as the cell matures. Etioplasts are colourless plastids with the capacity to become chloroplasts in the light (in most higher plants, chlorophyll synthesis is light-mediated). Chloroplasts all contain several starch grains in their stroma, as well as thylakoids in the grana. Amyloplasts are characteristic of roots, stem storage tissues (such as stem tubers) and cotyledons but are absent from leaves. Generally amyloplasts are not capable of becoming green in the light, though those of the potato tuber (a stem) are an exception. Chromoplasts are specialised plastids that store coloured substances, particularly oils (e.g. ß carotene in carrots and yellow flower petals).

Pelargonium zonale has several variegated mutant forms with characteristic cell lineage variegations. This is the 'geranium' of the classroom and is easily propagated as cuttings. White areas have exclusively white plastids - mutant for their capacity to make both chlorophyll and (apparently) starch. The origin of these areas that lack functional green chloroplasts is in primordial tissue. Because of the organisation of the meristem, the white patches on the leaf may be central or peripheral. Such variegated pelargoniums commonly produce mutant branches that 'revert' - these are either all white or all green. Both types would continue as cuttings 'true' to their new form but only the green will survive, as the completely white cuttings have no means of 'feeding' themselves.

Useful reference: Kirk J.T.O. & Tilmey-Bassett R.A.E. (1967) The plastids: their chemistry, structure, growth and inheritance Freeman & Co, London and San Francisco

Stephen Tomkins, Homerton College, Cambridge