Effect of different light wavelengths on the overall growth of Arabidopsis

thaliana seedlings.

Mecca Clipsham, Danny Kang, Alena Safina, Dani Valgardson

Abstract

This project focused on the effect of different wavelengths of light on the overall growth
rate of the Arabidopsis thaliana plant species during their germinating stage. As for any plant
species, light is an important factor in the survival and growth of A. thaliana, it must therefore be
able to adapt to the varied sources and wavelengths of light available to them in nature. To
investigate this, seeds were grown in a controlled laboratory environment under red, green, and
blue light, as well as unfiltered lighting (which, in this experiment, was fluorescent lighting with
a clear filter), and in darkness, over a span of 11 days. Our alternate hypothesis was that
increasing wavelength of light will have a significant positive impact on hypocotyl growth, and
the null hypothesis was that increasing wavelengths of light will have no impact or will impede
hypocotyl growth. We found that seeds grown in darkness (the positive control treatment) had
grown most rapidly. For the coloured treatments, we found that red light had the most growth,
followed by the green light, and blue light having the slowest growth rate. The clear plastic
treatment was found to have growth similar to the red and green treatments. The results of an
ANOVA test done on red, green, and blue light treatments produced a p-value of less than
0.00001, which led us to infer that there is a significant difference in the growth rates of the 3
treatments. This allowed us to reject our null hypothesis and provide support for our alternate
hypothesis.

Introduction

Availability of light is one of the most important abiotic factors for growth and

development of plants during germination and throughout their life cycle (Fankhauser and

Chory, 1997). Arabidopsis thaliana, a small flowering plant from the Brassicaceae family, is no

exception. The life cycle of A. thaliana is approximately six weeks, and is affected by the

presence of light at every stage (Folta and Maruhnich, 2007). This plant can grow in a wide

range of conditions, however its optimal environmental temperature is 16 to 25oC and its optimal

light intensity is 6480 to 8100 Lux (Weigel and Glazebrook, 2002).

photoreceptors, the main ones in A. thaliana being cryptochromes, which mainly process blue

light, and phytochromes, which mainly process red light (Fankhauser and Chory, 1997). It has

been determined that A. thaliana has five different types of phytochrome receptors and two

different types of cryptochrome receptors (Fankhauser and Chory, 1997). As land plants are

stationary, they must be able to process different wavelengths of light depending on what light is

available to them. For example, plants grown in underbrush may receive more green light that is

being reflected off of the plants overhead, and less red and blue light, which are being absorbed

by those plants (Folta and Maruhnich, 2007).

Cryptochromes and phytochromes both have effects on the growth of plants during initial

seedling growth, following germination. Cryptochromes are known to inhibit hypocotyl (also

known as the early plant stem) growth and phytochromes are known to be important for the

major developmental transitions, including germination and the onset of flowering (Fankhauser

and Chory, 1997; Lin et al., 1995). Furthermore, cryptochromes and phytochromes also are able

to process green light, however, less effectively (Folta and Maruhnich, 2007).

It has been shown by Gendreau et al. (1997) that following germination, A. thaliana

seeds grown in the dark will have a faster hypocotyl growth rate, and closed cotyledons, which

are the first leaves. This pattern of growth is conducive to the seedling when grown in soil, as it

allows it to reach the surface and find a light source as early as possible (Fankhauser and Chory,

1997). Plants grown in the light will have a slower hypocotyl growth rate, and their cotyledons

will begin to open much sooner, as the presence of sufficient light will signal the onset of

vegetative growth (Fankhauser and Chory, 1997).

Arabidopsis thaliana was investigated. The null hypothesis was that an increase in wavelength

will have no effect or will decrease the growth rate of the hypocotyl. The alternate hypothesis

was that an increase in wavelength will increase the growth rate of the hypocotyl.

With human influences altering ecosystems around the world, plants need methods of

adapting to different conditions. It is important to know how different light conditions, such as

changes in wavelength, affect the germination and initial growth of plants. Previous studies have

focused on the differences in growth in seedling grown in the dark and in the light (Gendreau et

al, 1997). Our study expands on these results, by investigating hypocotyl growth under red light,

blue light and green light, in addition to unfiltered light and darkness.

Methods

Our experiment looked at the germination and initial growth of seeds under five different

treatments of light: red light (λ=680nm), green light (λ=520nm), blue light (λ=520nm), unfiltered

light, and darkness. The controls chosen for this experiment were the clear, or unfiltered

treatment, and the black or dark treatment, because we were able to predict how the seedlings

grow in these conditions based on the research of Fankhauser and Chory (1997). For each

treatment, we prepared 5 replicates containing 4 seeds each, which totalled to 100 seeds. The

mean growth of the 4 seeds was calculated for each replicate. Petri dishes were labeled from one

to twenty five to identify which replicate they were and each dish had every corner labeled from

one to four to keep track of each seed, as seen in Figure 1. Each dish was then prepared by

lining the bottom with a single layer of filter paper for a growth medium and watering. A single
seed was then carefully placed on each labeled corner. Using a micropipette, we then evenly

dampened the filter paper with 500µl of water and closed the dish.

Figure 1 - (left) Seed placement and numbering of dish and seed. The center number represented
replicate number while the corners represented seed number within replicate. (right) Petri dish
placement on tray for storage/light exposure.

Upon preparing the Petri dishes, we obtained 5 trays and placed 5 dishes, or replicates,

onto each one. Each tray was then covered; one with blue (420nm) acetate paper, one with green

(520nm) acetate paper, one with red (680nm) acetate paper, one with clear acetate paper, and one

with a black garbage bag. The trays were then taken to a 17oC incubation chamber and placed

under lighting, as seen in Figure 2. Light intensities were measured using a Lux meter placed

under the coverings. To control for light intensity, the trays were lifted or lowered closer to the

light source until the light intensities measured in at a reading of 110 +/- 10 Lux (excluding the

darkness treatment which read 3 Lux).

Each day we measured the light intensities of each tray and took them out of the chamber

to measure the samples. They were measured daily from the base of the stem to the base of the

leaf. Measuring was done using a dissecting microscope, the Dinoscope attachment, and the

ImageJ program. The Dinoscope was used to take a picture of each seedling along with a picture

of a ruler in millimeters. The ruler was then calibrated in ImageJ which allowed us to measure

each seedling by using the program’s trace feature.

When finished with our measurements, we re-watered the Petri dishes with 200µl of

water whenever we felt the filter papers were dry. The trays were then taken back downstairs

into the 17oC incubation chamber. We once again placed them accordingly on the shelves until a

reading of 110 +/- 10 was shown on the Lux meter (excluding the darkness treatment which read

3). This process was repeated every day over a period of 11 days, Mon. to Fri.

Statistical analysis was needed to determine if there were significant differences among

the treatments. The mean growth of each replicate was calculated by taking the average of the

four seeds in each replicate. The mean growth for each treatment was then determined by taking
an average of the 5 replicates. The growth rate was calculated in mm/day for each time interval,

and an overall mean growth rate was obtained. A one-way Analysis of Variance (ANOVA) test

was performed on the growth rates all five treatments, and a second one-way ANOVA test was

performed on the growth rates of the blue light, green light and red light treatments. 95%

confidence intervals were calculated for each mean growth rate for each treatment.

Results

The mean of each replicate was determined by averaging the four seeds within each Petri

dish. The averages of the replicates were then used to analyse the data and from the calculated

means, determine the overall average for each treatment, standard deviations, and 95%

confidence intervals.

The seeds did not show signs of growth until the second day of observation on day 4 (97

hours), when most had shown signs of sprouting and already lengthening of the stem. The red

treatment (680nm) showed the most growth in the first four days, but the darkness treatment

began to show faster growth on day 5 (116 hours) and continued to do so for the remaining days

as seen in Figure 3. The other treatments continued to grow less rapidly and to smaller lengths

than the samples in darkness (black; λ=0nm).

9.000

8.000

7.000

6.000
Length (mm)

Blue (λ=420nm)
5.000 Green (λ=520nm)
Red (λ=680nm)
4.000
Clear (λ=unknown)

3.000 Black (no light; λ=0nm)

2.000

1.000

0.000
0 50 100 150 200 250 300
Time (hours)

Figure 3 - The average length of stem (mm) during seed germination at 23, 97, 116, 142, 167,
190, and 265 hours under different light wavelengths.
Due to such rapid growth and longer lengths, the darkness control had the highest growth

rate (mm/hr) out of all the treatments, including controls, as shown on Figure 4. Out of the

tested, known wavelengths, excluding controls, red (680nm) had the highest growth rate. The

green (520nm) had the second highest with the slowest growth rate belonging to the blue

treatment (420nm). The unfiltered (clear) light treatment had growth rates closer to that of the

red or green treatment.

35.000

30.000
Growth Rate (μm/hr)

25.000

20.000

15.000

10.000

5.000

0.000
Blue (λ=420nm) Green (λ=520nm) Red (λ=680nm) Clear Black (no light;
(λ=unknown) λ=0nm)
Treatment

Figure 4 - The average growth rate of each treatment over the course of 11 days. The black
bars on each treatment represent the 95% confidence intervals.
The 95% confidence intervals of the means of each treatment are shown in Figure 4. The

confidence interval for the blue replicates was 4.19 to 6.41μm/hour, for green replicates, it was

7.58 to 13.2μm/hour, and the interval for red replicates was 9.92 to 15.8μm/hour. For the two

controls, the clear treatment had a confidence interval of 8.62 to 14.4μm/hour and for the black

treatment; it was 31.3 to 37.0μm/hour. The only treatments with significantly different means of

were blue and the black control treatments as neither of their confidence intervals overlap.

Statistical analysis of the data through a one way ANOVA for all treatments revealed that

the mean for the growth rate of the blue treatment (420nm) was 5.03μm/hour, 10.4μm/hour for

the green treatment (520nm), 12.9μm/hour for the red (680nm), 11.5μm/hour for the clear
control, and 34.2μm/hour for the darkness control. The standard deviation for the blue treatment

was 1.27μm/hour, for the green treatment it was 1.73μm/hour, 0.902μm/hour for the red

treatment, for the clear treatment it was 0.882μm/hour, and finally for the black treatment, the

standard deviation was 4.68μm/hour. The ANOVA reported that the calculated F value was

110.9967 and the F critical value was 2.87 giving a p-value of less than 0.00001. A second one-

way ANOVA was performed on only three of the treatments: red, green, and blue. The results of

the ANOVA excluding the unfiltered and dark controls were very similar to the ANOVA of all

five treatments. The second ANOVA test had a critical F value of 6.94 and a calculated F value

of 41.3557 resulting in a p-value for the second test to be less than 0.00001.

Discussion

A one-way ANOVA test done on all five treatments resulted in a p-value of less than

0.0001, which showed that there is a significant difference in the growth rates of at least one of

the treatments. We can see from the 95% confidence interval bars in Figure 4 that the dark

treatment is significantly different from the other four treatments, as the confidence intervals do

not overlap with any of the others and was the mean of the dark treatment was much higher than

the other growth rate averages. This was expected, as results from a study done by Gendreau et

al (1997) determined that plants that are allowed to germinate and start their growth in the dark

will have faster growth rates than plants grown in the light.

A one-way ANOVA test done on the treatments at three different wavelengths (red light

at 680nm, green light at 520nm, and blue light at 420nm) resulted in a p-value of less than

0.00001, which showed that there is a significant difference in the growth rates of at least one of

the treatments. The 95% confidence interval bars in Figure 4 showed that the blue light treatment
is significantly different from the other two treatments, as the confidence intervals do not overlap

with any of the others.

As our p-values for both ANOVA tests were less than our α of 0.05, we were able to

reject our null hypothesis, which stated that an increase in wavelength will have no effect or will

decrease the growth rate of the hypocotyl in Arabidopsis thaliana. Thus, we were able to provide

support for our alternate hypothesis, which stated that an increase in wavelength will increase the

growth rate of the hypocotyl in Arabidopsis thaliana. The mean growth rates for red light, green

light and blue light were 12.9μm/hour, 10.4μm/hour, and 5.03μm/hour, respectively. This

provided further support for our alternate hypothesis as the seedlings grown under red light,

which at 680nm was the longest wavelength that was used, had the fastest mean growth rate, and

seedlings grown under blue light, which at 420nm was the shortest wavelength that was used,

had the slowest mean growth rate. The seedlings grown under green light, which at 520nm was

the intermediate wavelength, had a mean growth rate in between the other two treatments.

Therefore, we determined that plants responded differently to light of different wavelengths, and

grew faster under longer wavelengths.

These results are consistent with the literature, which suggests that seedlings grown under

darkness will have the fastest hypocotyl growth rate and plants grown in the light will exhibit

stunted hypocotyl growth rates. Further, Fankhauser and Chory (1997) suggests that

cryptochromes, the blue light receptors, inhibit hypocotyl growth. This was observed in our

study by the slowest hypocotyl growth rate of the plants grown under blue light. In addition,

Fankhauser and Chory (1997) determined that phytochromes, the red light receptors, are needed

for the induction of growth and for development, which was observed in our study by the fastest

hypocotyl growth rate of the plants grown under red light. One of the developmental stages that
took place in the early stages of growth is de-etoliation, which includes an increase in hypocotyl

growth rate, corresponding to the fast growth rate observed in the seedlings grown under red

light. Green light is processed by phytochromes and cryptochromes (Folta and Maruhnich,

2007), and therefore it is intuitive that plants grown under these wavelengths would have a

growth rate intermediate to the other 2 treatments. Folta et al (2007) also suggest that green light

reverses the effects that blue light have on the growth, such as hypocotyl growth inhibition.

Therefore, the growth rate of seedlings grown under green light should be faster than that of

seedlings under blue light, as was observed in our experiment.

Initial growth rates were the highest for unfiltered light and red light treatments, and

lowest for the blue light, green light and dark treatments, as seen in Figure 3. This initial surge in

growth for red and unfiltered lights could be due to the fact that red light is processed by

phytochromes which are responsible for initial growth of the hypocotyl. Since unfiltered light

includes all wavelengths of visible light, this response would be present in the unfiltered light

and red light grown plants.

For the seeds grown in the dark, the initial growth rate is slow. However, at 100 hours,

we see an extreme spike in growth rate that continues throughout the 11 days of observation. The

germination of these plants grown in the dark has no light energy to power the process and

therefore it is slower initially. However, as soon as the seed has germinated, the plant needs to

find light in order to begin photosynthesis, which is why the hypocotyl growth rates spikes. In a

plant grown in the dark, it would normally be under the soil, so this spike in growth rate allows it

to reach the top of the soil, where it will encounter light, in the most efficient way (Gendreau et

al, 2007).
 A significant difference in the way the plants grown under dark conditions compared to

the other four treatments was observed. Plants grown in the dark not only had a faster growth

rate, but also had their cotyledons closed throughout the 11 days of growth that we observed.

However, the plants growth under unfiltered light, red light, green light and blue light all had

open cotyledons from the moment they sprouted. This observation is consistent with Fankhauser

and Chory (1997), who described how the plants that germinate and begin growing in the dark

will have more hypocotyl elongation, but closed cotyledons, whereas plants that germinate and

begin growing in the light will have less hypocotyl elongation, and open cotyledons. We also

observed that the plants grown in the dark were transparent, whereas plants grown under other

conditions were green. This is due to the fact that as soon as plants get access to light, their light

receptors signal physiological changes, such as the formation of chloroplasts and the opening of

the cotyledons, to provide more favourable conditions to begin photosynthesis. The seedlings

grown in the dark therefore had not yet begun to form chloroplasts.

While we minimized sources of error as much as possible, there were several factors,

apart from the treatments, that possibly influenced the results. The most prominent source of

error was likely measuring the hypocotyl length, as the seedlings were very small and even slight

deviations in measurement had the capacity to affect the results. In addition, the positioning of

the seedlings as they grew was not uniform, as some grew vertically which made it difficult to

get accurate measurements under the microscope. A related problem was the fact that measuring

the plants using ImageJ required a steady hand, and often one measurement would be quite

different from the next. Another source of error was variation in time of germination. In our

green light treatment, we had some seeds that did not germinate, or were very slow to do so. In

total, there were three seeds where germination was not observed until the 11th day and two seeds
that did not germinate at all. In addition, one seed in the blue light treatment did not germinate at

all. The non-germination and late germination of seeds was likely due to it not being viable, or

due to natural variation. This also hinted at the overall biological variation of the seedlings,

which we tried to control for by using four seeds per replicate, and five true replicates. Lastly, it

is possible that some Petri dishes were opened during measurement and exposed to mould and

fungi, which could have impeded its growth.

Conclusion

In this experiment, we rejected our null hypothesis and were able to provide support for

our alternate hypothesis was supported by the data. The growth rate of seed germination of

Arabidopsis thaliana was found to be dependent on the wavelengths of light they are exposed to,

with longer wavelengths promoting a faster growth rate. Blue light, with the shortest wavelength,

was shown to slow hypocotyl growth. Conversely, the red light was shown to increase growth

rate of the hypocotyl, due to its involvement in the promotion of developmental stages. The

control plants in the dark treatment seedlings’ growth rate was the fastest, as expected.

Acknowledgements

We would like to thank the University of British Columbia for funding, providing

equipment and laboratory space, and for giving us the opportunity to conduct this experiment

and to take the course, Biology 342. We would also like to express our gratitude to our professor,

Dr. Carol Pollock, our lab technician, Mindy Chow, our teaching assistant, Katelyn Tovey, and

our peer tutor, Kathleen Cruz for providing us with continued guidance, feedback and

encouragement.
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Gendreau, E., Traas, J., Desnos, T., Grandjean, O., Caboche, M., and Hofte, H. 1997. Cellular
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