Student Sheet 20 – Can plants make starch in the dark?

This protocol offers an alternative technique for measuring starch production in plants, based on the popular ‘leaf disc’ technique. Rather than using whole leaves, students punch small discs from leaves, and use these as the basis for the starch test.

Students begin by measuring starch production by green leaf discs in the dark and the light. In addition, they will measure the starch production in green leaves kept in the dark, while floating in glucose solution. Students can extend this basic experiment, by repeating the procedure with discs taken from the white areas of leaves. Students then carry out the starch test using iodine. The results will encourage students to think in more detail about the role of chlorophyll, starch grains, and the plant cell.

There are a number of advantages using small leaf discs rather than whole leaves for investigations with plants. These include:

• students can use several discs 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 : discs from green leaves / disc from white leaves etc.)

Students begin by comparing the starch production in leaf discs from green leaves, under three different conditions:

• Dish A – green leaf discs placed on water in bright light for 24 hours
• Dish B – green leaf discs placed on water in the dark for 24 hours
• Dish C – green leaf discs placed on a glucose solution in the dark for 24 hours

Floating the leaves upside down (stomatal side up) should allow maximum carbon dioxide entry.

Students should then make predictions about the starch production they expect under the different conditions.

The following results would be expected from the starch test:

• Dish A (green leaf discs, on water, in light) – discs give a blue-black colour when tested for starch. They behave as expected for a whole leaf, by producing starch in the light.
• Dish B (green leaf discs, on water, in dark) – discs give a brownish colour when tested for starch. This shows they also behave as expected for a whole leaf and do not make starch in the dark.
• Dish C (green leaf discs, with glucose, in dark) – discs 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 is formed fastest at the disc edges, confirming that glucose is absorbed through the cut edge, rather than through the stomata or leaf surface.

Extension activity: Is chlorophyll necessary for starch production? What is the role of starch grains?

Students can then repeat this protocol, using discs taken from the white parts of a variegated Pelargonium. Again, they should predict their results.

Students will find that white leaf discs, floated on water and left in the light,will not make starch. Similarly, white leaf discs floated on glucose solution in the dark will also not make starch.

We accept that the white pelargonium cells cannot produce starch as they have no chlorophyll, but it is surprising to some that 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, as 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.

Starch grains, chloroplasts and other cell organelles

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