INTRODUCTION

1.0 Plants have little option but to function in the environment that they are found in. For photosynthetic organisms, it has been necessary for them to develop mechanisms to sense their light environment and to adjust their form and metabolism to optimise their performance (Willis and McElwain, 2002). Since light environments alter, these organisms have also developed the ability to continuously adjust their function to current conditions; this process is known as photomorphogensis (Beerling, 2008). Plants and their ancestors have adapted themselves over millions, if not billions of years, to use light as a source of their primary energy (Lane, 2003). This project has investigated the details of their energy source by splitting the luminescence they utilise and to grow a model species under three of the fundamental wavelengths. There has been little research carried out on the effects of excluding different wavelengths on photoautotrophs however, this project has increased knowledge of frequencies by secondary research and by applying this research to the test.

1.1 From the research, conclusions were drawn to reveal why these wavelengths are acquired by photoautotrophs and what their impacts are. Afterwards a methodology was developed to carry out the experiments to get the results. The experiments occurred over a 15 week period at Newcastle University. There were 3 test groups with 3 trials that were designed to test the hypotheses. The 3 test groups included: blue, green and red light with white light as the independent variable.

1.2 The results were recorded onto tables and processed through the discussion to either prove or disprove the three hypotheses stated. In the discussion, the results were cross referenced with the research to obtain more precise conclusions and to provide an explanation on why the outcome might have occurred. Conclusions have been drawn, invalid hypotheses rejected and explanations summarised with recommendations for improvements for future research and investigations based on this subject.

HYPOTHESES

2.0 This project aimed to prove the hypotheses and to find out what processes are involved in photosynthesis, photorespiration and how light frequencies influence these processes. The fundamental aim of the experiment was to investigate the effects of light wavelengths on photosynthesis and what would occur if certain wavebands were removed. There are 4 principle hypotheses that will be investigated in this study:

1. H₁ height will alter when grown under photoselective films.

2. H₀ height will not alter when grown under photoselective films.

3. H₁ when grown under photoselective films leaf span will modify.

4. H₀ when grown under photoselective films leaf span will not modify.

5. H₁ root length will alter under photoselective films.

6. H₀ root length will not alter under photoselective films.

7. H₁ dry weight will alter under photoselective films.

8. H₀ dry weight will not alter under photoselective films.

LITERATURE REVIEW

3.0 The aim of this investigation was to review the processes involved in photosynthesis and how different light wavelengths influenced the means of these processes. This study aimed to determine what would occur if the wavebands were split, by utilising secondary research to form a valid experiment.

Photosynthesis

3.1 Kamen (1963) describes photosynthesis as: ‘A process in which electromagnetic energy is converted to chemical energy that can be utilised for various biosynthetic processes.’ Plants require photons, CO₂, and H₂O to produce sugar and carbohydrates (Dawkins, 2010; Capon, 2010). Below is the equation to demonstrate photosynthesis:

6CO₂ + 6H₂O → (Photons & Chlorophyll) C₆H₁₂O₆ + 6O₂

Light-dependent reactions occur in the thylakoid membranes (reaction centres) of the chloroplasts and utilise photons to synthesize ATP and NADPH (Smil, 2006; Leegood, 1998).

3.2 The process commences with photosystem II (250-400 pigment molecules) when a chlorophyll molecule gains sufficient energy from the adjacent pigment, which enables photolysis of H₂O to occur (breaking of hydrogen and oxygen) (Mauseth, 2008). This process produces O₂ which is expelled and electrons and protons (H+) which are utilised. This occurs in the granum of the chloroplast or the ‘stacks of thylakoids’ (Emerson et al, 1957; Dawkins, 2006). Chitnis (1996) states that the H⁺ are then translocated through the cytochrome which energises and excites H⁺ to the second stage of light-dependent reactions which is shown in the following stages:

Photosystem II (P680) → Plastoquinone → Cytochrome b6 → Cytochrome f → Plastocyanin → Photosystem I (P700)

3.3 Photosystem I activates electrons for transfer to the Fd and finally to NADP+, where the protons from water splitting are exhausted to create NADPH⁺ H⁺ (Allaby, 2006; Harrison, 2008). To demonstrate the ability to liberate oxygen from water, Chitnis (1996) formulated the following:

The positively charged P800 exerts a strong pull on the H₂0 molecule, splitting it into H⁺ and OH^- ions. It requires CI^- and Mn²⁺ ions to act as a catalysis:

4H₂0 → (Mn²⁺, CI^-) → 4 (OH^-) + 4H⁺

OH donates its electron to oxidise P680:

4(OH^-) → 4e^- → 4OH

OH radical obtained forms H₂0 and liberates oxygen:

4(OH) → H₂0 → 4H⁺ → 4e^- → 0₂

Donation of H⁺ ion from the formation of NADPH is utilised for the Calvin-Benson Cycle (Beerling, 2008). Below is a summary of the differences between the photosystems.

3.4 The end products are transferred to the light-independent reactions, these reactions occur in the stroma found within the chloroplast between the grana and thylakoid. C0₂ is delivered to the stroma where they are reduced to ATP and combined with H⁺ to power the Calvin-Benson Cycle (B.B.C., 2011; Kratz, 2011; Devlin, 2007). This process converts ATP into the carbohydrates needed to power the photoautotrophs. The key enzyme of the cycle is RuBisCO. This enzyme fixes C0₂ to ATP by complex redox reactions to gain the carbohydrates, lipids and proteins which are required for development (Hall and Rao, 1999; Singleton, 2004). Kratz (2011) expressed the following equation for this process:

3CO₂ + 6NADPH + 5H₂O + 9ATP → glyceraldehyde-3-phosphates (G3P) + 2H⁺ + 6NADP+ + 9ADP + 8Pi

3.5 Plants convert photons into chemical energy with a photosynthetic efficiency between 3-6%, however photosynthesis varies with the frequency of light and intensity temperature and proportions of C0₂, which can vary between 0.1 – 0.8% in the atmosphere (Blankenship, 2001; Akutagawa and Ozaki, 2011; Franklin et al, 2005). Photosynthetic systems store 469 kilojoules of energy and in some cases up to 502 kilojoules. This process is more productive when both photosystems are balanced in their uptake of light. By comparison, solar panels convert photons into electrical energy at an efficiency of 6 - 20% (Blankenship, 2001).

See appendix 1 for wavelength interactivities with chloroplasts.

Research review

3.61 Research conducted by Shumin and Rajapakse (2000) explored the effects of different concentrations of far-red light utilising 3 photoselective films. 3 concentrations of far-red light were set up using a sample of 3 Dendranthemax grandifora cultivars, 20 ‘Bright Golden Anne’, 20 ‘Iriden’ and 20 ‘Yellow Snowdon’. In addition, 5 of the cultivars utilised were propagated beneath non-photoselective films for the control. The photoselective films were held in place by utilising P.V.C. piping with 2 fans to ensure continuous airflow. Plants were asexually reproduced and left to grow in nominal light conditions for a week. The cultivars were placed under the enviro-boxes for a month in a controlled glasshouse.

3.62 The study discovered the greater the concentration of far-red, the greater the reduction in height of the plants. Internodal growth was reduced by up to 35% in specimens under the higher concentration, this also decreased leaf area and dry weight (Shumin and Rajapakse, 2000). However there was a 25% reduction in light received by the specimens as the films absorbed parts of the spectrum (Graham, 2008).

3.63 A possible problem with their methodology was that a glasshouse is difficult to maintain to a constant environment due to external influences such as sunlight. Sunlight levels can easily fluctuate within a glasshouse due to the ‘greenhouse effect’ (Ingram et al, 2008). As light enters the glasshouse 50% is captured, thus increasing temperature. Glasshouses heavily rely on the external environment which can be unpredictable (Preece and Wilson, 1993). Unpredictability is a major hazard in an experiment, as variables need to be controlled to arrive at a precise conclusion.

3.64 Shumin and Rajapakse (2000) used asexual reproduction in their experiments, which has the major advantage of producing uniform specimens; subsequently leading to copies of successful individuals. Conversely if one of the copies contracted a disease or pest the others would equally be susceptible, which could result in specimens getting damaged or killed (Crops, 2009). If plants were to be reproduced sexually this would not occur as they contain different genes resulting in different susceptibilities and resistances (Beck, 2010). On the other hand, it could affect the outcome of the experiment as plants would not be uniform.

3.65 Peat is widely used for the production of plants albeit containing varying amounts of nutrition. This could have affected the outcome on their tests as one group may have received more nutrition than another. Furthermore, the above research had only 1 trial. This could have affected the outcome as it would have decreased the viability of the experiment as 1 trial’s result could have consequently produced an outliner result (Ashfield, 2008).

3.71 Wilson and Rajapakse (2001) compiled a study using photoselective films to investigate alternatives to using chemical growth retardants. They utilised 3 plants each of 3 species of Salvia (S. x ‘Indigo’, S. longispicata and S. splendens) which were purchased as plug plants. There were 3 groups which included a control that were set up using P.V.C. piping, far-red and red photoselective films and a non-photoselective film. The substrate used was a 20/80 mixture of osmocote and soiless-media. There was a fan at the bottom of the enviro-boxes with a small incision at the top to release the resulting air. The specimens were grown in these boxes for 6 weeks. After this period an additional 2 trials occurred. The minimum temperature recorded was 17°c followed by the maximum temperature of 35°c.

3.72 This research revealed that when grown under far-red the compactness, yield and greenness of the plants had increased. When grown under red film, plants showed no significant difference. Flowering and leaf area under both films was unaffected. Nevertheless the dry weight of the plants had increased under the far-red film.

3.73 A disadvantage of using enviro-boxes is that it reduces airflow. When airflow is restricted humidity increases which leads to an increased risk of diseases such as Botrytis (Harrison, 2008). The author of this study has tried to rectify this issue by inserting a fan at the bottom of the boxes and making an incision at the top. However this could have resulted in unwanted light entering the enviro-boxes as escaping air pushes the incision apart. One issue which was not addressed in this study was the sample size. Can 9 plants with 1 trial represent the population? This may have affected the validity of the study as there were not enough trials to gain an average as suggested by Greenhaun (1996). A possible solution may have been to increase the number of plants used within each group and to complete 2 further trials, this would have increased test validity.

3.74 There was a large differential between the minimum and maximum temperatures in this experiment. Plants respond to different temperatures which may have influenced their growth rates; inevitably decreasing the reliability of the tests (Sambamurty, 2005). A more controlled environment such as a growth cabinet could have ensured more variables were controlled.

3.81 A comprehensive study comparing 6 flowering species was led by Cerny et al (2003), to explore the effects of far-red and red light on plant development. The experiment occurred in an open polythene glasshouse using 3 groups. To construct the enviro-boxes P.V.C. piping was utilised with the films attached. This was followed by the control which had a non-photoselective film. 40 plants were grown under the enviro-boxes for 3 months from January to March. Light level readings were recorded every 5 minutes once a week using a spectro-radiometer. The following genera were used: Zinnia, Dendranthema, Cosmos, Petunia, Rosa and Antihrrum.

3.82 Of the genera utilised there was no significant difference in height when Antihrrum and Rosa were grown under far-red. The Zinnia, Dendranthema, Cosmos and Rosa had a considerable reduction in flowering. On the contrary, Antihrrum and Petunia had an insignificant delay in flowering (Cerny et al, 2003). All genera which when grown under far-red, had a noticeable increase in compaction. Red had no significant impact upon the growth or flowering of the specimens.

3.83 To ensure that all the groups were receiving the same conditions a non-photoselective film was utilised for the control. Although the film does not filter out specific wavebands, it ensures that variables such as humidity, temperature and light are kept constant (Horticultural Development Council, 2010). 240 plants were employed for this study which increases the outcome’s precision. Conversely there was only 1 trial completed but with increased numbers of specimens 1 trial may have been enough.

3.84 The study occurred from January to March in the southern hemisphere. During this period heavy rainfall and turbulent weather systems are widespread (Lane, 2006). This could have affected plant growth due to the polythene glasshouse being open to weather conditions. In addition specimens were measured by all the contributors, which could have resulted in inconsistencies as people measured in different ways (Rouncefield and Holmes, 1989). This could have been resolved by one person being responsible for measuring, which would have reduced the likelihood of systematic errors.

3.91 In 2008 a study was launched to overcome the problem of elongation when commercial crops were shaded during the summer. Cummings et al (2008) employed 3 groups to explore possible solutions to this problem: 20 plants were to be grown under neutral shading, 20 under blue shading, 20 under red shading and 20 un-shaded as a control. Pisum sativum were grown from seed and placed directly into each of the trial conditions. 2 seeds were planted in each pot to act as a support in the event of one not developing. Incandescent lights were used to supplement the light levels as the experiment had occurred during Spring and Autumn in a controlled-glasshouse. Light level readings were recorded using a spectro-radiometer once a week at mid-day.

3.92 Cummings (2008) revealed that when grown under blue shading, height would be significantly reduced and yield increased; whereas when grown under red shading plants would increase their height and reduce their yield. Additionally his study discovered that when plants had a reduction in height a delay in flowering occurred.

3.93 The experiment carried out considered a key confounding variable. What would occur if one of the seeds did not develop? It was decided to plant 2 seeds in each pot. This reduced the likelihood of seeds not developing which may have resulted in the collapse of the experiment (Grimaldi, 2005). Incandescent lights were used in the experiment to supplement lighting due to the season the test had occurred. Although this may appear to benefit the experiment it may have created a bias. Incandescent bulbs are well known to emit long-wave radiation (Reddy et al, 2004). This could have enhanced the red shading as red light is located on the longer frequencies on the electromagnetic spectrum. However fluorescent bulbs emit most of the spectrum and thus would have been better to use for this type of experiment (Ingram et al, 2008).

3.94 Placing the spectro-radiometer in one place once a week may not represent the light levels of the location as they can frequently fluctuate at different times of the day (Leegood and Lea, 1998). To achieve a good representation the spectro-radiometer could have been placed in several areas to gain a mean result. This would have increased the validity of the readings as the entire area would have been taken into consideration.

3.10 It is apparent from all the research which has been reviewed, that adequate controls need to be in place to ensure this investigation can draw precise conclusions. To ensure environmental specifications are met a growth cabinet with overhead fluorescent lights has been used, as growing specimens in a glasshouse would increase the amount of confounding variables (Blankenship, 2001). Furthermore this would protect against variation in the wavebands being received by the plants. To increase the validity of this experiment, 3 separate trials have been completed to gain a more precise outcome. Plants have been grown sexually which would increase disease resistance within the majority of the plants. 2 seeds had been planted per pot so it could protect against seed development failure as suggested by Cummings et al (2008).

METHODOLOGY

4.0 This study aims to determine the effects of segmented wavelengths on plant growth by measuring leaf span, height, dry weight and root length. Specimens were grown in a Snijder growth chamber. Prior to the experiment the growth cabinet at the biology department at Newcastle University was programmed and left to run for 2 weeks to ensure that environmental specifications were being followed. Illumination was provided from above to spread the light equally and to stimulate upright growth, see appendix 2 for specifications.

4.1 36 x 7.6cm sized pots were filled with 50/50 mixture of sand and terragreen and 2 seeds of Lycopersicum esculentum ‘Money Maker’ were sown in and then watered. In each group, 9 Lycopersicum were grown for 5 weeks, 36 for each trial and 108 overall. As reinforcement, 2 seeds were planted in each container reducing further confounding variables. When 2 seeds had developed 1 was withdrawn. 9 plant pots were placed in 4 drip proof trays. Each tray was placed in one of the 4 enviro-boxes. 1 with red, 1 with blue, 1 with green and 1 non-photoselective film, see appendix 3 for trial design. Subjects were then placed into the Snijder growth cabinet 37cms away from the source of light to ensure maximum illumination output which was effectively reduced by the filters.

4.2 Lycopersicum had been germinated under white light for 1 week until the first true hypocotyls developed and then they were placed into the enviro-boxes. During the time the specimens were grown, temperature and light readings were collected to monitor environmental specifications (Valiela, 2010).

4.3 There were 3 trials to gain an accurate outcome (Field and Hole, 2003). Each trial consisted of 3 groups, to test specific wavelengths: red, blue, green and white which act as a control variable (Brown, 2012. Pers. Comm. Ms Ros Brown chief technician of the microbiology at Newcastle University). Refer below for the trial layout.

4.4 Specimens were irrigated when required and supplemented with Phostrogen multi-purpose fertiliser to enrich the inert substrate to retain the health of the plants. The application has been uniform throughout the trials at 4.5 grams per 4 litres.

4.5 Once a week, specimens had their height and leaf-span measured inside the growth cabinet to ensure environmental constancy within the experiment. After each trial specimens were measured from stem base to apical meristem and from stem base to root-meristem, photographed, root architecture documented and biomass readings recorded (Montgomery et al, 2010).

4.6 To gain dry weight, specimens were removed from the substrate and briefly washed. Plants were then blotted to remove any free surface moisture. Specimens were placed into a drying oven at 100°C for 48 hours. Plants were left to cool for 3 hours and weighed on a scale which measures in milligrams (Reed et al, 2007). This trial process was repeated twice during subsequent weeks.

RESULTS

5.0 From the descriptive statistics that were analysed it was found that when grown under blue light Lycopersicum grew on 1.77cm smaller compared to the control. When grown under red light, specimens had increased height by 46mm. Specimens grew 1.41cm smaller when propagated under green light as demonstrated in the table.

Below is a chart illustrating the differences in height.

Figure 1 Plant heights (cm) (n⁴=27)

Two inferential analyses were then completed. The Anderson-Darling test provided a P-value of <0.0001 signifying normal distribution. The Levene’s test showed a P-value of <0.0001 which demonstrated unequal variance. A One-way ANOVA test was then completed to investigate if there was a significant difference between the groups. The test result was:

One-way ANOVA: F= 25.54, P<0.001 (N⁴=27)

This demonstrates a significant difference within the heights.

5.1 A post-hoc test was then required to identify differences between the groups. It revealed that there was a significant difference in height between the control and green, blue, red groups (P=<0.05). However, there was no significant difference between the red, green and blue groups (P=>0.05). This demonstrates that green, red and blue photoselective films have little impact upon the growth of Lycopersicum height, this is reported as below:

Individual 95% CIs For Mean

Based on Pooled StDev

Level N Mean StDev --------+---------+---------+--------

Blue 27 5.432 1.922 (---*---)

Control 27 7.202 3.071 (---*---)

Green 27 5.791 1.743 (---*---)

Red 27 6.743 2.814 (---*---)

--------+---------+---------+--------

Pooled StDev = 2.385 5.0 6.0 7.0

The post-hoc test can be reported as:

T Statistic = 5.32 x √ (4.32/27) = 2.12

5.2 Specimens which developed under blue light had an increase of 1.41cm in leaf span. However there was no significant difference when grown under red and green light. When cultivated under green light specimens grew 0.38cm taller and under red light they grew 0.53cm taller as illustrated in the table below.

Below is a chart demonstrating the differences in leaf span.

Figure 2 Differences in leaf span (cm) (n⁴=27)

The Anderson-Darling test indicated a P-value of <0.001 demonstrating normal distribution. The Levene’s test showed a P-value of 0.010 which demonstrated equal variance. A One-way ANOVA test was then completed to investigate if there was a significant difference between the groups. The test result was:

One-way ANOVA: F= 25.54, P <0.001(N⁴=27)

This signifies a significant difference in the leaf area.

5.3 A post-hoc test was then required to identify differences between the groups. It revealed that there was a significant difference in leaf span between the control and other groups (P=<0.05), between red and other groups (P=<0.05) and blue and the other groups (P=<0.05). However, there was no significant difference between blue and green (P=>0.05). This demonstrates that red photoselective films can have an impact upon leaf span, this is shown as below:

Individual 95% CIs For Mean

Based on Pooled StDev

Level N Mean StDev --------+---------+---------+--------

Blue 27 5.431 1.741 (---*---)

Control 27 7.234 3.082 (---*---)

Green 27 5.792 1.921 (---*---)

Red 27 6.743 2.811 (---*---)

--------+---------+---------+--------

Pooled StDev = 2.105 5.0 6.0 7.0

The post-hoc test can be reported as:

T Statistic = 4.27 x √ (4.28/27) = 1.70

5.4 This study has revealed that when cultivated under blue light the roots grew 1.8cm shorter when compared to the control. In contrast when grown under red light the root length decreased by 0.55cm. There was however a difference when cultivated under green light which decreased by 1.5cm as demonstrated below.

Figure 3 Differences in root lengths (cm) (n⁴= 27)

The Anderson-Darling test provided a P-value of <0.001 indicating normal distribution. The Levene’s test showed a P-value of 0.533 which demonstrated equal variance. A One-way ANOVA test was then completed to investigate if there was a significant difference between the groups. The test result was:

One-way ANOVA: F= 0.25, P 0.700 (N⁴=27)

This determines no significant difference in root elongation.

5.5 From the dry weight that was recorded plants that were grown under blue light saw a 0.95g decrease in dry weight, under green light specimens saw a 1.13g decrease in dry weight and finally under red light plants saw a 10g decrease in dry weight.

The chart below illustrates the differences in dry weight.

Figure 4 Dry weight of specimens (g) (n⁴= 27)

The Anderson-Darling test presented a P-value of <0.001 indicating normal distribution. The Levene’s test showed a P-value of < 0.0001 which demonstrated unequal variance.

A One-way ANOVA test was then completed to investigate if there was a significant difference between the groups. The test result was:

One-way ANOVA: F= 2.77, P 0.732 (N⁴=27)

This signifies no significant difference in the dry weights of the plants.

Refer to appendix 4 for raw data.

Refer to appendix 5 for observation log, photographic evidence and seed development chart.

5.6 The following charts are comparing the overall results for each of the readings to clearly demonstrate differences in growth patterns for each of the groups.

Figure 5 Comparison of readings recorded (cm/g) (n⁴= 108)

The chart below represents the percentage differential in growth for all the readings that were taken. Note that all specimens grown under coloured light had more variable growth when compared to the control.