Relationship Between Vitamin C Content And Evolution Of The genus Citrus

How do the type of species of the genus Citrus affects concentration of vitamin C in different species- citron (Citrus medica), grapefruit (Citrus paradisi), key lime (Citrus aurantiifolia), lemon (Citrus limon), mandarin (Citrus reticulata), Orange (Citrus sinensis), pomelo (Citrus maxima), and tangerine (Citrus tangerina) within the genus?

Vitamin-C is an essential component of my daily diet as my doctor has prescribed it for me to boost my immunity. Because of personal interest, I explored across a lot of websites and research paper to know more about this particular compound. This gave me an idea about what role does the molecule play and how. I came across a lot of research articles as well where they have mostly studied the effect of factors like temperature, storage time and even type of fruits on the Vitamin-C content. Comparison of mean content of Vitamin-C is a widely done research work. I was intrigued by the fact that while justifying the various factors that could lead to the variation in the content of Vitamin-C in various fruits and vegetables is mainly done based on the process in which the plant has grown or some specific biological process happening in it but are there no other factors that influences it? The theory of natural selection and survival of fittest has always evoked great interests in me pertaining to the fact that why certain species emerge and others perish. I wanted to study if there was a connection between the mean Vitamin-C content and the history of their evolution. This finally brought me to the research question stated above.

Vitamin C, also known as ascorbic acid, is a major nutrient in the human diet necessary for the growth, development, and repair of many tissues in the body.

• Vitamin C plays an important role in the synthesis of neurotransmitters - norepinephrine, serotonin, as well as bile acids from cholesterol, then some experts are trying to explain the beneficial effect of vitamin C on its metabolism.
• Vitamin C is an antioxidant, it provides direct protection of proteins, fats, DNA and RNA of cells from the damaging effects of free radicals, which are often formed in cells during life. Ascorbic acid maintains the level of reduced glutathione, which in itself is the leading antioxidant in the body, providing protection against free radicals, toxins, heavy metals at a biochemical level. In addition, vitamin C has a significant effect on the metabolism of other micronutrients and vitamins.
• Ascorbic acid enters the human body mainly with plant foods. When they are consumed in the proper amounts, obtaining vitamin C will correspond to the physiological needs of a healthy person or even surpass them (which is not terrible, the body will remove excess vitamin C in the urine). However, this usually does not happen; vitamin C deficiency is the most common vitamin deficiency. This is due to two main problems: a decrease in the consumption of fresh vegetables and fruits and a high degree of technological processing of food products in which certain parts of plants are used. The fact is that the content of vitamin C in different parts of the fruits is not the same - it accumulates in the peel, outer layer leaves more than in the pulp, petiole, and stem.
• With severe vitamin C deficiency, scurvy develops. This disease is accompanied by swelling and bleeding of the gums, loosening and loss of teeth, hair loss, varicose veins, hemorrhoids, overweight, irritability, poor concentration, depression, insomnia, early wrinkles, fatigue, impaired vision, and haemorrhage
• Some animals such as Guinea pigs, bats and primates have lost the ability to synthesize their own vitamin C in their cells, due to a mutation in GLO gene which codes for the enzyme responsible for catalyzing the last step of vitamin C biosynthesis; hence, the necessary vitamin C should be taken through feeding. Plants, on the other hand, possess the feature of producing vitamin C, and among them, citrus fruits such as orange and tangerine are accounted as major providers for the ascorbic acid. I just wondered how a particular trait like the biosynthesis of a particular molecule, may constitute an evolutionary advantage for plants and most animals: having your own source of vitamin C might confer great chances of survival. Furthermore, I started to think how this particular feature would be different among species that are closely related, and how natural selection would’ve removed such an important trait from humans: this combination of genetic characteristics and the regulation and expression in the environment and life of organisms, has always been of great interest in the topic of evolution.

In plants, the metabolic pathway for the vitamin C biosynthesis consists of the series of reactions that transform the carbohydrate mannose into galactose and finally into ascorbic acid, assisted in all steps and intermediates by the corresponding enzymes (Figure 1). The complete metabolism of vitamin C in green plants has been possible by the collaborative and international research of the Arabidopsis, which is the most studied plant in this regard (Smirnoff & Wheeler, 2000). Vitamin C has been characterized as a multifunctional component in plants because it plays a key role in many processes: serves as an enzyme cofactor for the synthesis of enzymes involved in the light-independent reactions of photosynthesis, helps in the detoxification of oxygen excess that might be produced after photosynthesis, regulates the plant responses to biotic and abiotic stress, degradation of hydrogen peroxide which is a frequent toxic subproduct in plant metabolism, regulation of the cell cycle and cell division during embryo development, as well as the regulation of the flowering time (Gallie, 2013).

The most cultivated plants as source of vitamin C in humans are the citrus fruits, which belong to the genus Citrus from the family Rutaceae, and are characterized by high content of vitamin C (up to 20mM) and low content of protein and fats; they’re also a good source of dietary fibre (Liu et al., 2012).

The most common method for the determination of the vitamin C in fruits is the titration with DCPIP (phenol- indo-2:6-dichlorophenol), which in absence of vitamin C is coloured blue and when reduced by the vitamin C turns colourless, serving as self-indicator for the chemical reaction (Figure 2). Sometimes the decoloration is not total so a stable pink must be seeked, in order to determine the final point for the chemical reaction. 1.00 g of the sample will be converted into an aqueous extract and titrated with 0.10 molar DCPIP.

As per the chemical reaction above,

1 mole of DCPIP = 1 mole of Vitamin-C

Thus, mass of Vitamin-C in 1.00 g of sample = moles of DCPIP * Mass of 1 mole of Vitamin-C

= 0.10 ∗ \(\frac{V}{1000}\) ∗ 176.12 [ as moles of DCPIP = molarity * Volume in dm₃]

V = volume of DCPIP that reacts with Vitamin-C (burette reading)

Thus, mass of Vitamin-C in 100 g of sample = 0.10 ∗ \(\frac{V}{1000}\)∗ 176.12 ∗ 100 = 1.76 V

For both groups of Citrus species (experimental and database), a phylogenetic tree was generated to establish the phylogeny among them, using the tool designed by BioByte Solutions (2019).In order to establish the phylogeny among species, it was generated the phylogenetic tree for all the eight species involved in this stage which displays the evolutionary relationship based on the molecular evidence available hitherto:

This investigation is a correlational study that seeks to establish possible relationships between genetic and epigenetic traits and the phylogeny of some species in the genus Citrus. In this sense, the independent variable is the phylogeny of the species as determined by the molecular evidence and visualise in a phylogenetic tree based on taxonomy provided by the NCBI (BioByte Solutions, 2019). The dependent variable is the vitamin C content, an indicator of the genetic trait of L-ascorbic acid biosynthesis, which is determined experimentally using titration with DCPIP in eight Citrus species: citron (Citrus medica), grapefruit (Citrus paradisi), key lime (Citrus aurantiifolia), lemon (Citrus limon), mandarin (Citrus reticulata), Orange (Citrus sinensis), pomelo (Citrus maxima), and tangerine (Citrus tangerina).

Type of species within the genus Citrus: citron (Citrus medica), grapefruit (Citrus paradisi), key lime (Citrus aurantiifolia), lemon (Citrus limon), mandarin (Citrus reticulata), Orange (Citrus sinensis), pomelo (Citrus maxima), and tangerine (Citrus tangerina).

Vitamin C content (mg per 100g of fruit tissue)

My prediction is that there is relationship between vitamin C content and the phylogeny of some species in the genus Citrus. Species that are later evolved in the phylogeny will tend to have higher vitamin C content as natural selection will leave plants that can synthesize vitamin C

There are several variables within my investigation that can’t be controlled.

Intensity of light-This aspect was not controlled in my investigation given that the fruits used for the experiment were acquired with no certain knowledge all the specimens were grown in the same light conditions.

Age of the sample-The age at which the citrus fruits taken will have an impact on the amount of Vitamin-C in them. The fruit samples were collected from the local supermarket and thus the exact age of this sample is not known.

● DCPIP (phenol-indo-2:6-dichlorophenol) solution 0.10%
● 500.00 cm3 distilled water
● Peeler
● 10.0 cm3 pipettes
● Pipette suction bulb
● 25.00 cm3 burette
● Blender

Throughout all the stages in the investigation, several measures were taken to reduce safety issues and environmental impact. During the experimental stage, after consulting the DCPIP solution MSDS, all manipulations were carried out wearing a lab coat, gloves, and safety goggles. Organic residuals from Citrus specimens were disposed for gardening compost and remaining titrated solutions were disposed in residuals management vessels as instructed by the teacher and lab technician. During the database stage, exposure to PC screen was reduced at maximum and time optimized as possible. All analyses were carried on-screen to avoid printing.

• Rinse the burette using a distilled water
• Clam the burette carefully to a stand and tighten it in a vertical position
• Fill the burette with an excessive volume of DCPIP using a funnel
• Open the stopcock until the zero points of the burette are reached
• Tap the burette to remove any air bubbles

• Peel one fruit
• Put the 1.00 g of the fruit into the blender. The electronic mass balance was used to record this. The mass of the fruit sample was controlled in this way.
• Blend it on the highest speed for 3.00 min using stopwatch. The extent of blending might affect the nature of the juice we produce. Thus, the blending time was controlled at 3.00 minutes using a stop-watch.
•  Measure 25 cc of distilled water using a graduated measuring cylinder.
•  Put the water into 100 cc conical flask and add the fruit mesh we got from the blender to it.
•  Filter the extract and collect the filtrate in a different conical flask.
•  Measure 1 cc of extract using a clean graduated pipette.
•  Pour the juice into the conical flask with the water.
•  Shake the conical flask with hand
•  Put the conical flask under the burette
•  Close the stopcock immediately after a drop that changes the colour.
•  Record the final volume of DCPIP in the burette in Table 2
•  Rinse all the glassware equipment except the titration burette

Sample calculation

Average = \(\frac{Sum\ of\ all\ trial\ values}{5}\)

Standard deviation = \(\frac{Sum\ of \ square\ of\ difference\ of\ data\ value\ and\ mean\ value}{5}\)

Formula used

Mass of Vitamin-C in 100 g of sample = 1.76 * V g

[Refer to background section]

All the species has been given a rank as per the position in the phylogeny series. This has been done to elucidate their position in the phylogeny tree. The rank-1 indicates the species that is oldest while rank-10 indicates the species that is most new.

The graph featured here is a scattered plot where basically any trend or correlation cannot be predicted. A general comparison of the values can be done though. It is seen that C.sinesis has the maximum Vitamin-C content (6.32 g per 100 g of fruit tissue) while the minimum is for C.medica (3.45 g per 100 g of fruit tissue). A very close value to the minima is obtained for C.maxima (3.54 g per 100 g). C.reticulata, C.aurantifolia and C.tangerina has almost similar values and then again C. limon and C.paradisi has almost similar values.

The next aim to study if there is a correlation between the values or not. The correlation test done was a regression correlation test. The value of correlation coefficient (R²) is found to be 0.0005. This value was found by inserting a linear trend line in MS-Excel. As the value of regression coefficient is 0.005, it is clear that there is no correlation between the mean Vitamin-C content and the phylogeny tree.Thus the result contradicts the hypothesis, I made and can be easily rejected.

How do the type of species of the genus Citrus affects concentration of vitamin C in different species- citron (Citrus medica), grapefruit (Citrus paradisi), key lime (Citrus aurantiifolia), lemon (Citrus limon), mandarin (Citrus reticulata), Orange (Citrus sinensis), pomelo (Citrus maxima), and tangerine (Citrus tangerina) within the genus?

Based on the results, it is possible to state that there is no relationship between vitamin C content and the evolution of species in the genus Citrus. Species that arose earlier in the phylogeny must have lower amounts of vitamin C. However, the association between phylogeny and vitamin C content is very weak (R₂ ≤ 0.90) so they cannot be used as statistically significant predictors for phylogeny in the species for the genus Citrus.

The previous fact can be explained as the vitamin C acts as an antioxidant in plant metabolism so it is affected by several abiotic conditions that cause oxidation such as light. For example, Arabidopsis plants are grown in the continuous dark for 2 days only contained 20% of ascorbate relative to plants grown in the light (Fenech et al., 2019)., as well as other important oxidative factors. Greater differences observed in C. medica and C. sinensis might probably be due to higher light exposure than other specimens in the assay. Actually when these two species are removed the fit greatly increases to R₂=0.762. In this sense, an improvement for further investigations is to ensure that cultivation conditions are the same for all the species involved in the experimental determination of vitamin C content.

I’d like to highlight a possible contribution of this investigation regarding the utilization of phylogeny as a ranked variable (McDonald, 2014) to represent evolutionary time. The utilization of phylogenetic trees to establish which species came first in evolution according to the molecular evidence registered in the NCBI seems an interesting way to study and analyze evolution in connection with experimental aspects. Nevertheless, in this investigation only the evolutionary sequence was considered but not the estimated time between the rise of each species, namely, the distance between species was equal which is not accurately with evolutionary evidence. Perhaps, that is another explanation of the lower fit in the results.

• There are several sources of random and experimental errors in this investigation. The beakers if not washed properly will contain some unused chemical traces from the previous investigations. So, all beakers must be washed thoroughly with distilled water before re-use.
• Vitamin-C is a molecule that can quickly oxidize to dehydroascorbic acid. This can happen at room temperature and without any oxidising agent too. Thus, the Vitamin-C extracts were freshly prepared and not kept exposed to air for long time.
• The readings were taken as the colour change has occurred. Detecting the change of colour depends a lot on human perception. Thus, to ensure that human error do not interfere with the result, all data were collected solely by me and the colour was compared against a white paper to understand it better.
• Sampling error is a major methodical limitation of the investigation. It needs to be made sure that all the fruit samples used must be of the same age and grown under the same condition. It was not possible to control this as the fruits were bought from the market. To do this, only genetically engineered samples must be used.

Vitamin-C is a major anti-oxidant. Often, we use lemon juice to inhibit browning of apples. Browning of apples happens due to oxidation of phenolic compounds into brown pigments. This is done in presence of the enzyme catechol oxidase. I would like to see how the enzyme activity of catechol oxidase is inhibited in presence of Vitamin-C. I would like to take cut pieces of apples and immerse them in solutions of Vitamin-C tablets. I would vary the concentration of the Vitamin-C solution used from 1.00 % (1.00 g of Vitamin-C in 100 cc water) to 8.00 % (8.00 g of Vitamin-C in 100 cc of water). In each case, I would measure the time taken for the apples to turn brown (in number of days). Longer the time taken by the apples to turn brown, lower the activity of the enzyme catechol oxidase. This would help me to understand if there is a correlation between the % concentration of Vitamin-C used and the activity of the enzyme catechol oxidase.

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