Effects of light exposure on acclimation of Digitalis Purpurea
Plants must adapt to survive in their natural environments. Environmental conditions are constantly changing and it is therefore essential for plants to adjust to their surroundings in order to endure the changes and stresses. Over long periods of time (over many generations), plants become better suited to their environment through natural selection. This evolutionary process is known as adaptation, one of the basic phenomena of biology (Williams G.C. 1966).
Over short periods of time, i.e. within a plant's lifetime, plants will evolve in response to environmental changes also. Plants can adjust their morphological, behavioural and biochemical qualities to survive in the altered environment. Whereas adaptation takes many hundreds of years to occur, these changes may be temporary, i.e. not permanent and the process is known as acclimation.
An example of acclimation to an altered environment is that of plants adapting to shady habitats. On the forest floor, plants are shaded by the tree canopy. The canopy stops a significant amount of sunlight from reaching these plants at ground level. Not only will the amount of sunlight reaching these ground level plants be reduced, but the wavelengths of light too. Compared to full sunlight, shade light contains reduced numbers of wavelengths of light between 400 and 700nm. These wavelengths are most involved in photosynthesis and are known as the photosynthetically active radiation (PAR). Shady environments therefore reduce the amount of PAR. As part of the process of photosynthesis, leaves absorb blue and red light. Red light is absorbed by leaves of the tree canopy, but far-red light passes through to the ground plants (Campbell N.A, et al 2008). The shift in the ratio of red to far-red light induces the changes in the plant for survival. This specific adjustment in response to light is known as photomorphogenesis and is an example of acclimation. The exemplary morphological changes when acclimatising to shade may include the plants growing taller, the plants' leaves being better adapted for light absorption (thinner leaves that have a larger surface area to volume which are easier to support and maintain) and longer petioles.
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Chlorophylls are pigments found in chloroplasts of plant cells and are the most important pigments in photosynthesis (Horton H. R, et al 2006), as they allow plants to harvest energy from light. In addition to chlorophyll, chloroplasts contain carotenoids. These are accessory pigments that further aid light capture and absorption. Carotenoids absorb different wavelengths of light to chlorophyll and so increase the wavelength for light absorption. Carotenoids are also involved in the protection of chlorophylls from damage when light levels are too high. This damage is known as photooxidation. Shaded plants can not only change their shape and structure but their biochemical compositions with regards to the pigments discussed. The pigment concentrations in the leaves can increase. Furthermore, light-gathering parts of protein complexes known as photosystems (which contain pigments) can increase in number and re-organise into wider antennae, with more pigments, to increase their ability to catch light and harvest energy.
Digitalis purpurea (Common Foxglove) is a flowering, biennial plant. It is a facultative shade plant preferring partial sun to fully shaded areas. The plant is native to most of Europe and is found both at woodland edges and in grassland.
The aim of the experiment was to determine whether D. purpurea undergoes acclimation when grown in different light conditions. We considered the morphology and pigment concentrations of the plant when grown in light and in shade.
We were provided with two Digitalis purpurea plants that had been grown in different light conditions in a greenhouse. Plant A was grown in the open (sunny conditions) and Plant B was grown under shade covers (shaded conditions).Identifying changes in morphology
To begin, Plant A and Plant B were examined and compared. Observations made were recorded in a table. Differences in height, colour, length of petioles, size and number of leaves, arrangement of leaves, leave thickness and whether or not the plant was flowering was noted in the table.
Next, three leaves from each plant were removed. These were placed on graph paper and drawn around. The area of the leaves was established by counting the squares. The total number of leaves on the plant were counted and the total plant leaf area was calculated in cm2: Total plant leaf area = average leaf area x number of leaves on plant.
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Subsequently, from each plant, three circular leaf cuttings were made using the bottom of a universal tube. This cutting has an area of 3.7cm2. These cuttings were weighed using a fine balance and the weights recorded in another table. Using these fresh leave weights and leaf areas calculated in the previous step, the specific leaf area (SLA) was calculated using the following equation. Standard error (S.E) was also considered.
SLA (m2g-1FW) = La/Lw
Where La is leaf area and Lw is leaf fresh weight (FW).
S.E. = standard deviation ÷ vn
Where n is the number of samples per plant.Measuring pigment absorbance
The following was carried out for each leaf sample previously weighed (three samples from Plant A, three from Plant B). The first leaf sample was placed into a mortar. Into the mortar 1ml of 96% ethanol was added using a plastic Pasteur pipette. Using the pestle, the leaf was grinded to a paste with a pinch of sand. The ethanol solution from the mortar was transferred to a graduated centrifuge tube. The mortar was washed with a little more ethanol. This too was added to the centrifuge tube. This sample was made up to 10ml using more ethanol. The sample was mixed and labelled and then placed into a bench top centrifuge. Once these steps had been completed for all six leaf samples, they were centrifuged at 2000rpm for 5 minutes. Once centrifuged, samples were kept on ice.
To measure the absorbance of the samples, firstly a cuvette was filled with clean 96% ethanol to act as a blank. A second cuvette was filled with one of the leaf sample solutions that had been centrifuged. Care was taken not to dislodge the pellet at the bottom of the centrifuge tube. The cuvette being used as the blank (ethanol only), was placed into a spectrophotometer. A wavelength of 470nm was set and the spectrophotometer zeroed. The blank cuvette was removed and replaced with the cuvette containing the leaf sample solution. The 470nm reading was recorded in a table. The leaf sample cuvette was removed from the spectrophotometer and replaced with the blank cuvette. The wavelength was changed to 649nm and the absorbance was zeroed. The blank cuvette was switched with the leaf sample solution and the 649nm reading was recorded in the table. This was then repeated again for a wavelength of 665nm. This entire process was repeated for the remaining 5 leaf samples and the readings recorded in the table. The cuvettes were rinsed between every sample to avoid any contamination and false readings. If any absorbance readings were above 1.5 at any wavelength, they were considered too high and the samples would have been diluted by a known amount of 96% ethanol.Calculating pigment concentration
The concentration of chlorophyll a, b and total carotenoids were calculated from the absorbance readings of each sample using the following equations and recorded in a table:
These pigment concentration values were then used to calculate the pigment concentration per g of fresh weight (FW). The final value was done as milligram per gram of fresh leaf weight. This was calculated using the following equation:
RESULTSChanges in morphology
From general observations, it was noted that Plant A was taller than Plant B. Plant A had lighter coloured leaves. The petioles of Plant A were thicker and slightly red in colour, whereas Plant B's petioles were green and much thinner. There were 10 leaves on Plant A and 3 on Plant B. Plant A's leaves were larger, thicker and arranged so that there was overlap between levels of leaves. Plant B's leaver were more spread out. Neither plants were flowering.
The total plant leaf area for Plant A was calculated as 581 cm2 and Plant B was 60.6cm2.
For statistical tests of significance, two sample t-tests were performed. These were to see if the pigment concentrations were significantly different as a result of different light conditions.
For a two sample t-test the significance level is 0.05%. For a significant difference, the significance value calculated must be below 0.05.
For chlorophyll a: the t value is -1.599 and the significance value is 0.034. This significance value is below 0.05 and thus, there is a significant difference between Plant A and Plant B's chlorophyll a concentrations.
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For chlorophyll ß: the t value is -0.416 and the significance value is 0.699. The value is considerably larger than 0.05 and so there is no significant difference between the chlorophyll ß concentrations of the two plants.
For total carotenoids: the t value is -2.259 and the significance value is 0.087. This value is above 0.05, however it is much closer than the two previous values and so displays more significance. Yet, the value is not below 0.05 and so not significant enough.
For Chl α/ß ratio: the t value is 1.542 and the significance value is 0.198. There is no significant difference between Plant A and Plant B.
For total Chl/total carotenoid ratio: the t value is 1.775 and the significance value is 0.041. This value is just under 0.05 and shows a significant difference between Plant A and Plant B.
It was acknowledged that Plant A was taller and larger with thicker leaves so therefore stronger than Plant B. Plant A had been grown in light conditions - it received adequate amounts of light. Its rate of photosynthesis would have been much more efficient than Plant B's. As Plant B was shorter, it is fair to conclude that its growth was slower. This would be due to sugar production in photosynthesis becoming a limiting factor as the rate of photosynthesis would have been lower for Plant B. Decreased photosynthesis results in decreased sugar production.
An adaptation plants have to shade is developing much thinner leaves. The plant will want to have a reduced surface area to volume ratio. Thinner leaves need less energy and resources to maintain and so energy needed to maximise light absorption is not wasted by supporting heavier leaves. It was noted that Plant B had thinner leaves.
Calculating the specific leaf area further supported this. An increased SLA is evidence for a thinner leaf. Considering Graph 1, it is clear that Plant B had consistently higher SLAs than Plant A.
In addition to this, the number of leaves and the total leaf area were higher in Plant A than Plant B. The way the leaves were arranged in Plant B - not overlapping - shows that energy was invested into reaching more sunlight and is known as shade avoidance syndrome. (Ballaré 1999). This further supports the fact that Plant B is not wasting its energy on growing in size but concentrating on maximising its chlorophyll production by trying to capture as much light as possible and by the morphology of the leaves.
It is valid to conclude that in reduced light exposure, a smaller plant will develop with smaller leaves but a larger SLA as the two plants were grown in the same conditions except for the differing light exposures.Pigment concentrations
When referring to Table 1, the chlorophyll a concentrations and total chlorophyll means of Plant B are higher than Plant A. From this we gather that D. purpurea can acclimatise to produce more chlorophyll in shaded conditions. This is further supported by the results from the statistical tests. The sets of results that showed a significant difference between the two plants were for chlorophyll a and total Chl/total carotenoid ratio. The significance values were below 0.05% and so we can assume that these results did not occur by chance. The higher concentrations of chlorophyll a increases the chance of plants harvesting the little amount of red and blue light that filters through the canopy as discussed in the introduction.
There were more carotenoids in Plant B. This supports evidence that carotenoids act as extra light capturing components, and the fact that there are more in the shade plant shows that the wavelengths of light will be increased to aid light capture in the environment with less light available.
To summarise, Digitalis purpurea showed acclimation in the shaded environment. Plant B grew less to produce more chlorophyll to maximise photosynthesis. Both Plant A and B grew best in their environments; the plants optimised themselves for the conditions they were living under. This is evidence for successful acclimation. Even though many of the statistical tests showed that there was not a high enough level of significance to draw upon, it can be concluded that D. purpurea does grow more in increased light conditions as a larger and stronger plant grew. Carotenoid levels proved that photooxidation was prevented. In the shaded environment, D. purpurea showed many levels of acclimation to maximise its capture of light.
- Ballaré CL (1999) Keeping up with the neighbours: phytochrome sensing and other signalling mechanisms. Trends Plant Sci 4, 97-102
- Campbell N. A. Reece J. B. Urry L. A. Cain M. L. Wasserman S. A. Minorsky P. V. Jackson R. B. (2008) Biology, Eight edition. Pearson Education Inc. San Francisco.
- Horton H. R. Moran L. A. Scrimgeour K. G. Perry M. D. Rawn J. D. (2006). Principles of Biochemistry, fourth edition. Pearson Education Inc. New Jersey. Plant Finder: Foxglove. Available from: http://www.bbc.co.uk/gardening/plants/plant_finder/plant_pages/274.shtml
- Williams, G. C. 1966. Adaptation and natural selection: a critique of some current evolutionary thought. Princeton. "Evolutionary adaptation is a phenomenon of pervasive importance in biology."