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Effect of Nitrogen-Starvation of Anthocyanin Production in Arabidopsis thalia
Anthocyanins are a major secondary compound found in Arabidopsis thaliana plants. Under stress such as nutrient-starvation, accumulation of the compound is enhanced. Two genes play major roles in the synthesis of anthocyanins: PAP1D and MYBL2. Here, I report on the results of an experiment analysing the relative effect of nitrogen-starvation on anthocyanin production and the difference among the various genotypes (WT, PAP1D, and MYBL2). Nitrogen-starvation caused a 2-3 times increase in relative concentration of anthocyanins in all plant types tested. The enhancement of PAP1D showed 2-3 times higher concentration of anthocyanins over the wild type and MYBL2 plants, in both media treatments. The findings confirm the known response of increased anthocyanin production in Arabidopsis.
Flavonoids are an important secondary compound found in plants. Some of their roles include defense against outside harm and attraction of pollinators and seed-spreaders. This special characteristic makes it an interesting research topic for use in expression of various natural plant products. Flavonoid content rises when a plant is exposed to stressful conditions such as nutrient depletion1, and this can be useful if controlled in a lab setting. Anthocyanins are a sub-type of flavonoids and are comprised of anthocyanidins that are glycosylated with different sugars. One of these is cyanidin, which is found in the model plant Arabidopsis thaliana2.
The ability to regulate the production of these compounds is researched in depth1,3,4. This has given us knowledge of the key genes affecting the biosynthesis of anthocyanins. Two of those genes are PAP1D and MYBL2. PAP1D is essential in the activation and accumulation of anthocyanins3 while MYBL2 is responsible for inhibition of anthocyanin synthesis4.
The purpose of this experiment was to apply this theory and practice sterile working technique, while evaluating the effect of nitrogen-starvation and gene mutations on anthocyanin production in the model plant Arabidopsis thaliana.
Materials and Methods
Experiment A lasted a duration of about one week with lab work being performed on three separate days:
Day 1: Preparation of solutions, sterilization of seeds, sow seeds
Day 2: Harvest of plant material, sample preparation: addition of methanol/HCl
Day 3: Sample preparation continued, measurement of absorbance
The methods used were as described in the BIO520 lab manual2. Any deviations from or additions to the lab manual protocols are detailed below in their respective sections.
Preparations of Solutions
Day 1 of our laboratory time was used to prepare the necessary solutions for our experiment. Table 1 shows the solutions and contents made by myself and my lab partner. The pH meter was calibrated before use with the pH 7 and pH 4 standard solutions (ASY=7,178; slope=103,3%).
Table 1. Description of solutions made by myself and my lab partner. Solution: name of solution, as written on solution container; pH values are actual measured values. Volume: target volume and volume used for calculations. Contents: components used to prepare the given solutions; calculated by our lab pair; actual values used and may not match the calculations exactly. A note on the LB Broth is in the text following this table.
These solutions were labeled in their containers and left at our lab bench for autoclaving. This autoclaving step was not detailed in the lab manual, or shared with us, so we were not aware of the proper containers. Our LB Broth was mistakenly prepared and left in a 50ml plastic tube (not suitable for autoclaving, due to temperature range of the plastic and overfilling of the container). For this reason, our LB Broth did not get autoclaved. The LB Broth was not necessary for this experiment, however, and another group made extra to make up for our missing volume.
The TAE buffer is another solution (also for future experimental use) that my partner and I did not prepare. This solution was prepared by one lab pair in a volume large enough to accommodate the entire lab group.
Sowing of Seeds
Although this step is listed in the protocol following the sterilization of seeds, my partner and I performed these steps in the opposite order. In order to have a approximate growth time of a week for our plants, we needed to sow seeds on Day 1 and used seeds that had already been sterilized. The Petri dishes we sowed were left in the hood to be placed in the fridge by our lab assistant, Dugasa. He was also responsible for transferring them to a light environment the following day.
Sterilization of Seeds
This step was done for learning technique alone, since we were not going to use these seeds, as mentioned above. The 1% (w/v) Ca-hypochlorite + 1 drop of Tween solution was prepared by one lab pair for use by the entire lab group. We were supplied with the seeds to sterilize and followed the protocol as stated in the lab manual. We carried out the entire protocol at our lab bench. This was a mistake, as we should have performed this treatment in a sterile hood, starting with the ethanol washes.
Harvest of Plant Material and Sample Preparation
One week after sowing the seeds, the seedlings were harvested from the Petri dishes. The seedlings were pulled from the growth medium in the dish, and as well as possible, the leafy portion of the plants were collected, leaving the roots and media behind. The plant material was placed in eppendorf tubes and weighed, with a goal of 50mg of material per sample. As per the protocol, the samples were prepared by adding 1% (v/v) HCl in Methanol and left overnight at 4°C. The HCl/Methanol solution was prepared by one pair, in a large volume for the entire lab group.
The next day, water and chloroform were added to the plant material solution. It was noted, that when using larger plants, homogenization would be necessary at this step. Since the plant material in this experiment was so small, the amount of debris was minimal and thus homogenization was not necessary.
Measurement of Absorbance
Absorbance for the plant extracts was measured as per the protocol. The blank used in the spectrophotometer consisted of 400ul dH20 and 600ul 1% HCl/Methanol Solution, to replicate the makeup of our sample solutions.
Observation of Plant Growth
After one week in a light environment, there was already a marked difference among the plants (See Fig. 1). The first thing to notice was the color of the plants grown on nitrogen-rich media versus those grown without nitrogen. The nitrogen-rich media produced green seedlings in all three genotypes, while the nitrogen-starved plants were all red. The red color of plants is due to the higher level of anthocyanins. Secondly, the plants growing on nitrogen-rich media look much healthier, with larger leafy portions and roots. The nitrogen-starved plants were much smaller and very difficult to harvest. Next, there is a difference among each genotype. PAP1D plants were thriving in comparison to the other two plant types. MYBL2 and the wild type both yielded very tiny plants and if unlabeled, it would be hard to differientiate between them.
Figure 1. Growth after one week of wild-type (WT) and mutant (MYBL2 and PAP1D) plants on media with or without nitrogen (+N / -N). Representative sample of each plant type and treatment. N=3 for each, but the photos shown are examples of what was observed.
Harvest of Plants and Absorbance Measurement
Due to the small number and size of the plants grown in the petri dishes, all seedlings on each dish were harvested and deposited into their respective eppendorf tubes. 50 mg was desired of each sample, but only eight (about half) yielded a mass of at least that, four of those being PAP1D samples (Table 1).
The absorbance readings were taken at 530nm and 657nm for each sample, after the proper treatment to release and isolate the pigments. Readings were taken at both wavelengths to remove the effect of chlorophyll on the calculations (anthocyanins absorb at 530nm and chlorophyll at 657nm). This value was then divided by the mass of each plant to give relative concentration of anthocyanins (g-1) present in the sample (Table 2). The average and standard deviation was calculated for each plant and treatment (n=3). In both treatment situations (+N or -N) PAP1D had the most expression at 34,811 ±4,607 g-1 in nitrogen-starvation and 11,406 ±2,120 g-1 in nitrogen-rich medium. These values are well above the other plant genotypes, when comparing within the same medium. Also notable is that when comparing just the effect of the medium, the relative concentration of anthocyanins is 2-3 times higher in each plant type starved of nitrogen versus the nitrogen-rich plants.
Table 2. Relative concentration of anthocyanins in week old wild type (WT) and mutant (MYBL2 and PAP1D) plants grown in media with or without nitrogen supplementation (+N / -N) and all original data collected on plant samples during experiment. Plant mass was calculated from the difference of the mass of the tube with plant material and the mass of the empty tube. Relative conentration was calculated for each sample and then used for the average and standard deviation of each genotype plus condition. Original values are given in parentheses on the PAP1D (-N) samples 1 and 2. After analysis it seemed that the mass values for samples 1 and 2 were most likely mis-recorded, causing the wide range in results. We thus calculated average and SD assuming this error was made.
In nitrogen-starved treatments, PAP1D's relative concentration of anthocyanins is 3x higher than the wild type and twice as high at MYBL2. In nitrogen-rich medium, it is approximately 2x higher than both the wild type and MYBL2 plants. These ratios can easily be seen in Figure 2. What is also noticeable, is while PAP1D is much higher than the rest, there is no obvious difference between the wild type and MYBL2 strains. MYBL2 even seems to have higher expression of anthocyanins than the wild type under nitrogen-starved conditions. If the standard deviation is accounted for, though, these values are not statistically different.
Figure 2. Relative concentration of anthocyanins in week old wild type (WT) and mutant (MYBL2 and PAP1D) plants grown in media with or without nitrogen supplementation (+N / -N). Standard deviation shown for each sample.
The results of this experiment give a clear answer on the effect of nitrogen-starvation on anthocyanin production in Arabidopsis. As expected, under the stressful condition of nutrient limitation, all plant types produced more anthocyanin when grown in media without added nitrogen. However, our experiment was designed to explore with more detail, the effect of different mutations as well. The PAP1D plants produced the most anthocyanins, with and without nitrogen. This was expected since PAP1D expression was enhanced with a 35S promotor. MYBL2 is a negative regulator of anthocyanin biosynthesis, so it would be expected that the relative concentration in the MYBL2 samples would be lower than the wild type. However, it has been shown that under stress, MYBL2 plants accumulate high levels of anthocyanins in leaves due to increased expression of TT82. This could be the reason why the expression is essential equal to, if not greater than, the expression of the wild type plants.
Another contributing factor to this unexpected result in relative anthocyanin concentration, could be basic errors in the harvesting. These two groups of plants were very small and very few. It was much more difficult to only harvest the leafy portions of the plants, without contamination of roots or the medium, both of which would effect the mass. Which in turn, would effect the relative concentration. This effect could have been lessened if we were given more time for harvesting. I was spending quite a lot of time being careful, but given the time constraints of the laboratory, ended up being rushed to finish. Another option is to do this same experiment, but waiting longer until harvest so the plants are larger and easier to work with.
From analysis of results in this experiment, it can be concluded that nitrogen-starvation does in fact have a positive effect of anthocyanin production in Arabidopsis (by a factor of 2-3). This effect is largely amplified by the presence of the 35S-tagged PAP1D gene, and this amplification is also present in normal growth medium containing nitrogen. In addition to successful experimental results, the secondary purpose of this laboratory practice was also met. It was very good training in sterile techniques, especially applied to plant work, which I have no experience in. Getting basic information on how the lab runs in general was also an important addition, which will continue into the next experiment.
- Lillo, Cathrine, Lea, Unni S., Ruoff, Peter. (2008), Nutrient depletion as a key factor for manipulating gene expression and product formation in different branches of the flavonoid pathway. Plant, Cell, and Environment, 31: 587-601. doi: 10.1111/j.1365-3040.2007.01748.x
- BIO520 Methods in Biotechnology Laboratory Protocols. (2014), Introduction, Experiment A. pp 1-5. University of Stavanger, Stavanger, Norway.
- Borevitz, Justin O., et al. (2000), Activation Tagging Identifies a Conserved MYB Regulator of Phenylpropanoid Biosynthesis. The Plant Cell, 12: 2383-2393
- Matsui, K., Umemura, Y. and Ohme-Takagi, M. (2008), AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis. The Plant Journal, 55:954–967. doi:10.1111/j.1365-313X.2008.03565.x