Consequence of Nitrogen-Starvation of Anthocyanin Production in Arabidopsis Thalia
Anthocyanins are a major secondary compound found in Arabidopsis thaliana workss. Under emphasis such as nutrient-starvation, accretion of the compound is enhanced. Two cistrons play major functions in the synthesis of anthocyanins: PAP1D and MYBL2. Here, I report on the consequences of an experiment analyzing the comparative consequence of nitrogen-starvation on anthocyanin production and the difference among the assorted genotypes ( WT, PAP1D, and MYBL2 ) . Nitrogen-starvation caused a 2-3 times increase in comparative concentration of anthocyanins in all works types tested. The sweetening of PAP1D showed 2-3 times higher concentration of anthocyanins over the wild type and MYBL2 workss, in both media interventions. The findings confirm the known response of increased anthocyanin production in Arabidopsis.
Flavonoids are an of import secondary compound found in workss. Some of their functions include defence against outside injury and attractive force of pollinators and seed-spreaders. This particular characteristic makes it an interesting research subject for usage in look of assorted natural works merchandises. Flavonoid content rises when a works is exposed to nerve-racking conditions such as alimentary depletion1, and this can be utile if controlled in a lab scene. 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 theoretical account works Arabidopsis thaliana2.
The ability to modulate the production of these compounds is researched in deepness1,3,4. This has given us cognition of the cardinal cistrons impacting the biogenesis of anthocyanins. Two of those cistrons are PAP1D and MYBL2. PAP1D is indispensable in the activation and accretion of anthocyanins3while MYBL2 is responsible for suppression of anthocyanin synthesis4.
The intent of this experiment was to use this theory and pattern unfertile working technique, while measuring the consequence of nitrogen-starvation and cistron mutants on anthocyanin production in the theoretical account worksArabidopsis thaliana.
Materials and Methods
Experiment A lasted a continuance of about one hebdomad with lab work being performed on three separate yearss:
Day 1: Preparation of solutions, sterilisation of seeds, sow seeds
Day 2: Crop of works stuff, sample readying: add-on of methanol/HCl
Day 3: Sample readying continued, measuring of optical density
The methods used were every bit described in the BIO520 lab manual2. Any divergences from or add-ons to the lab manual protocols are detailed below in their several subdivisions.
Preparations of Solutions
Day 1 of our research lab clip was used to fix the necessary solutions for our experiment. Table 1 shows the solutions and contents made by myself and my lab spouse. The pH metre was calibrated before usage 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 spouse. Solution: name of solution, as written on solution container ; pH values are existent measured values. Volume: mark volume and volume used for computations. Contentss: constituents used to fix the given solutions ; calculated by our lab brace ; existent values used and may non fit the computations precisely. A note on the LB Broth is in the text following this tabular array.
These solutions were labeled in their containers and left at our lab bench for autoclaving. This autoclaving measure was non detailed in the lab manual, or shared with us, so we were non cognizant of the proper containers. Our LB Broth was erroneously prepared and left in a 50ml plastic tubing ( non suitable for autoclaving, due to temperature scope of the plastic and overfilling of the container ) . For this ground, our LB Broth did non acquire autoclaved. The LB Broth was non necessary for this experiment, nevertheless, and another group made excess to do up for our missing volume.
The TAE buffer is another solution ( besides for future experimental usage ) that my spouse and I did non fix. This solution was prepared by one lab brace in a volume big plenty to suit the full lab group.
Sowing of Seeds
Although this measure is listed in the protocol following the sterilisation of seeds, my spouse and I performed these stairss in the opposite order. In order to hold a approximative growing clip of a hebdomad for our workss, we needed to seed seeds on Day 1 and used seeds that had already been sterilized. The Petri dishes we sowed were left in the goon to be placed in the electric refrigerator by our lab helper, Dugasa. He was besides responsible for reassigning them to a light environment the undermentioned twenty-four hours.
Sterilization of Seeds
This measure was done for larning technique entirely, since we were non traveling to utilize these seeds, as mentioned above. The 1 % ( w/v ) Ca-hypochlorite + 1 bead of Tween solution was prepared by one lab brace for usage by the full lab group. We were supplied with the seeds to sterilise and followed the protocol as stated in the lab manual. We carried out the full protocol at our lab bench. This was a error, as we should hold performed this intervention in a unfertile goon, get downing with the ethyl alcohol washes.
Harvest of Plant Material and Sample Preparation
One hebdomad after seeding the seeds, the seedlings were harvested from the Petri dishes. The seedlings were pulled from the growing medium in the dish, and every bit good as possible, the leafy part of the workss were collected, go forthing the roots and media behind. The works stuff was placed in eppendorf tubings and weighed, with a end of 50mg of stuff 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 brace, in a big volume for the full lab group.
The following twenty-four hours, H2O and trichloromethane were added to the works material solution. It was noted, that when utilizing larger workss, homogenisation would be necessary at this measure. Since the works stuff in this experiment was so little, the sum of dust was minimum and therefore homogenisation was non necessary.
Measurement of Optical density
Optical density for the works extracts was measured as per the protocol. The space used in the spectrophotometer consisted of 400ul dH20 and 600ul 1 % HCl/Methanol Solution, to retroflex the make-up of our sample solutions.
Observation of Plant Growth
After one hebdomad in a light environment, there was already a pronounced difference among the workss ( See Fig. 1 ) . The first thing to notice was the colour of the workss grown on nitrogen-rich media versus those grown without N. The nitrogen-rich media produced green seedlings in all three genotypes, while the nitrogen-starved workss were all ruddy. The ruddy colour of workss is due to the higher degree of anthocyanins. Second, the workss turning on nitrogen-rich media look much healthier, with larger leafy parts and roots. The nitrogen-starved workss were much smaller and really hard to reap. Following, there is a difference among each genotype. PAP1D workss were booming in comparing to the other two works types. MYBL2 and the wild type both yielded really bantam workss and if unlabeled, it would be difficult to differientiate between them.
Figure 1. Growth after one hebdomad of wild-type ( WT ) and mutant ( MYBL2 and PAP1D ) workss on media with or without N ( +N / -N ) . Representative sample of each works type and intervention. N=3 for each, but the exposures shown are illustrations of what was observed.
Harvest of Plants and Absorbance Measurement
Due to the little figure and size of the workss grown in the petri dishes, all seedlings on each dish were harvested and deposited into their several eppendorf tubings. 50 milligram was desired of each sample, but merely eight ( about half ) yielded a mass of at least that, four of those being PAP1D samples ( Table 1 ) .
The optical density readings were taken at 530nm and 657nm for each sample, after the proper intervention to let go of and insulate the pigments. Readings were taken at both wavelengths to take the consequence of chlorophyll on the computations ( anthocyanins absorb at 530nm and chlorophyll at 657nm ) . This value was so divided by the mass of each works to give comparative concentration of anthocyanins ( g-1) nowadays in the sample ( Table 2 ) . The norm and standard divergence was calculated for each works and intervention ( n=3 ) . In both intervention state of affairss ( +N or -N ) PAP1D had the most look at34,811 ±4,607 g-1in nitrogen-starvation and11,406 ±2,120 g-1in nitrogen-rich medium. These values are good above the other works genotypes, when comparing within the same medium. Besides noteworthy is that when comparing merely the consequence of the medium, the comparative concentration of anthocyanins is 2-3 times higher in each works type starved of N versus the nitrogen-rich workss.
Table 2.Relative concentration of anthocyanins in hebdomad old wild type ( WT ) and mutant ( MYBL2 and PAP1D ) workss grown in media with or without nitrogen supplementation ( +N / -N ) and all original informations collected on works samples during experiment. Plant mass was calculated from the difference of the mass of the tubing with works stuff and the mass of the empty tubing. Relative conentration was calculated for each sample and so used for the norm and standard divergence of each genotype plus status. 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, doing the broad scope in consequences. We therefore calculated mean and SD presuming this mistake was made.
In nitrogen-starved interventions, PAP1D ‘s comparative concentration of anthocyanins is 3x higher than the wild type and twice every bit high at MYBL2. In nitrogen-rich medium, it is about 2x higher than both the wild type and MYBL2 workss. These ratios can easy be seen in Figure 2. What is besides noticeable, is while PAP1D is much higher than the remainder, there is no obvious difference between the wild type and MYBL2 strains. MYBL2 even seems to hold higher look of anthocyanins than the wild type under nitrogen-starved conditions. If the standard divergence is accounted for, though, these values are non statistically different.
Figure 2. Relative concentration of anthocyanins in hebdomad old wild type ( WT ) and mutant ( MYBL2 and PAP1D ) workss grown in media with or without nitrogen supplementation ( +N / -N ) . Standard divergence shown for each sample.
The consequences of this experiment give a clear reply on the consequence of nitrogen-starvation on anthocyanin production in Arabidopsis. As expected, under the nerve-racking status of alimentary restriction, all works types produced more anthocyanin when grown in media without added N. However, our experiment was designed to research with more item, the consequence of different mutants as good. The PAP1D workss produced the most anthocyanins, with and without N. This was expected since PAP1D look was enhanced with a 35S promotor. MYBL2 is a negative regulator of anthocyanin biogenesis, so it would be expected that the comparative concentration in the MYBL2 samples would be lower than the wild type. However, it has been shown that under emphasis, MYBL2 workss accumulate high degrees of anthocyanins in foliages due to increased look of TT82. This could be the ground why the look is indispensable equal to, if non greater than, the look of the wild type workss.
Another lending factor to this unexpected consequence in comparative anthocyanin concentration, could be basic mistakes in the harvest home. These two groups of workss were really little and really few. It was much more hard to merely reap the leafy parts of the workss, without taint of roots or the medium, both of which would consequence the mass. Which in bend, would consequence the comparative concentration. This consequence could hold been lessened if we were given more clip for reaping. I was passing rather a batch of clip being careful, but given the clip restraints of the research lab, ended up being rushed to complete. Another option is to make this same experiment, but waiting longer until harvest so the workss are larger and easier to work with.
From analysis of consequences in this experiment, it can be concluded that nitrogen-starvation does in fact have a positive consequence of anthocyanin production in Arabidopsis ( by a factor of 2-3 ) . This consequence is mostly amplified by the presence of the 35S-tagged PAP1D cistron, and this elaboration is besides present in normal growing medium incorporating N. In add-on to successful experimental consequences, the secondary intent of this research lab pattern was besides met. It was really good preparation in unfertile techniques, particularly applied to works work, which I have no experience in. Geting basic information on how the lab runs in general was besides an of import add-on, which will go on into the following experiment.
- Lillo, Cathrine, Lea, Unni S. , Ruoff, Peter. ( 2008 ) , Nutrient depletion as a cardinal factor for pull stringsing cistron look and merchandise formation in different subdivisions of the flavonoid tract. Plant, Cell, and Environment, 31: 587-601. Department of the Interior: 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 individual MYB sphere, acts as a negative regulator of anthocyanin biogenesis in Arabidopsis. The Plant Journal, 55:954–967. doi:10.1111/j.1365-313X.2008.03565.x