Effect of pH on the action of Gibberellin in the growth of shoot height of green chilli

To learn from what I observe has always been my passion. Being passionate about gardening, some basic knowledge about choosing the appropriate soil for my plants and using the correct fertilizers to grow them has been my forte. The journey started from a challenge that I faced while growing a tomato plant in my roof top garden. Despite adding Gibberillin, the plant did not show sufficient symptoms of growth. To inquire the reason behind it, I tried to find the factors that could have opposed the growth. As also studied during my IGCSE Biology course, soil pH is an important factor for growth. If the soil is too acidic or too basic the growth of the plant is hindered. Thus, I tested the pH of the soil using a pH paper and found that the soil pH was too acidic to promote the growth of the plant. From my knowledge of DP-Biology, I know about the various hormones which are essential to promote the growth of plant like-auxin, cytokinnins, absicissic acid, Gibberillin and so on. It must be noted that the type and composition of soil where plants are grown will differ and thus the pH will differ too. Thus, I was intrigued to know if the pH of the soil at which the soil is grown will have an impact on the role or the mechanism of the hormone action on plants or not. Will the change in pH alter the pathway hormone follows to trigger cell growth and thus the growth of plant? Thus, I arrived at the research question stated below.

How does the effect of the concentration of Gibberellin (in ppm) on the growth (in terms of vertical shoot height) of a green chilli (Capsicum annum) varies with the pH – acidic (pH = 4.00), basic (pH = 9.00) and neutral (pH = 7.00)?

Gibberellin is a class of plant hormone that regulates various developmental stages of the plant like – ‘developmental processes, including stem elongation, germination, dormancy, flowering, flower development, and leaf and fruit senescence’. They are named as GA-1, GA-2, GA-3 and so on based on the time they are identified. This hormone is prepared in the plastid along a terpenoid pathway. Following this, they undergo certain structural modifications in endoplasmic reticulum and finally in the cytosol. In the germination stage, GA acts as a precursor of the enzyme alpha-amylase which catalyses the hydrolysis of starch into sucrose and provides nutrients to the seedlings soon after they are contact to water. Cell elongation is one of the main role performed by this hormone which aids the plant in multiple stages of development. The hormone changes certain ‘rheological properties of the cell wall’. Consequently, the cell wall becomes less rigid and the osmotic pressure in it decreases due to a decrease in the water potential. This allows the cell to inflow of water into the cell wall and thus increases its volume which finally result in elongation of the cells.

Gibberellin, being a hormone, it is highly sensitive to pH with a tolerance range that lies within 3 to 10. Changes in pH alters the hydrogen ion concentration of the endoplasm and thus affect the mechanism in which they secrete enzymes for starch hydrolysis. Lowering the pH increases the response to GA.

This is because as the pH is decreased, the dissociation of GA is inhibited. Thus, more amount of undissociated GA molecules which can penetrate into the cell increases. Moreover, with lowering of pH, there is excess of hydrogen ions in the medium which interferes with various proteins in the cell wall and degrades or decomposes them; an effect known as ‘acid effect’. This makes the cell wall more permeable and facilitates the passage of more GA molecules into the cell. Both of these together will eventually increase more penetration of GA into the cells and thus promote the elongation of cell walls via the process of water uptake. GA also triggers the production of the enzyme hydrolase by endosperm which aids in the breaking down of dormancy.

Chilli is a small shrub branched fruit bearing plants. It has two major types of fruits-red and green. The major constituent of the fruit is capsaicin which plays major role as a taste enhancer and has other nutritional benefits as well. This plant was originally grown in Mexico and then became to the cultivators across South America, India, Thailand because of its high rate of vegetative reproduction. It acts as a major anti-oxidant and contains huge amount of Vitamin-A and Vitamin-C. Because of the fact that this plant can be grown easily without the need of any major fertilizer or special conditions, this has become so popular amongst both farmers and amateur gardeners. It needs roughly an exposure to 5-6 hours of sunlight everyday and adequate amount of water. It grows well in pots with the use of requisite amounts of organic fertilizers.

In a research paper on” The effect of external pH on the gibberellic acid response of barley aleurone effect” it was observed that “a reduction in medium pH from 6.0 to 3.7 enhanced the GA3-response of isolated barley (Hordeum vulgare L.) aleurone layers.” To be specific, there are quite a few researches that reports the positive impact of responses towards GA by the endosperm in germination stage of various seeds. However, the exact mechanism behind this has not been investigated and researched much. Another research on the effect of pH on the activation of molecules towards GA was done by measuring the % germination of barley seeds and the result of that is shown below.

The result clearly indicates that at lower pH, the molecules are more activated towards GA.

Null hypotheses

• The vertical shoot height of green chilli plant does not depend on the concentration of GA added to it.
• At all pH, the response of the green chilli plant towards GA molecules is same.

Alternate hypotheses

• The vertical shoot height of green chilli plant depends on the concentration of GA added to it.
• At all pH, the response of the green chilli plant towards GA molecules is not same.

Concentration of GA (Gibberellic acid) in ppm

The concentration of GA used in this investigation was varied in the range of 100.00 ppm to 600.00 ppm; 100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm and 600 ppm. A solution of 100 ppm means 100 mg (0.10 g) of GA in 1000 cc of distilled water. All these solutions were made by weighing the required amount of GA using a digital mass balance and adding them to 1000 cc of water added using a graduated measuring cylinder. As per the biological interval, the mean level of GA in a nutritious healthy chilli plant lies within the range of 200 ppm to 400 ppm. Thus, this range is chosen to have values within the biological interval as well as above it and below it.

pH at which the plant is grown

The external pH at which the plant is grown was varied using buffer solutions. Both acidic and alkaline pH values, pH = 4.00 and pH = 9.00 were used. As a control, the neutral pH (=7.00) was also used.

To measure the growth of the plant, the vertical shoot height was measured. A ruler and a thread was used for this. As one of the major functions of GA is to promote stem elongation through increases in cell volume, the vertical shoot height was chosen as the dependent variable.

• A face mask and a laboratory coat were used.
• Any edible materials were not brought inside the laboratory.Any edible materials were not brought inside the laboratory.
• All the solutions were prepared under the careful guidance of an expert.
• Ethanol was not inhaled or exposed to skin.
• The scissor was used carefully.
• Hair was always tied up.

• The buffer solutions were given to the chemistry laboratory for re-use.

• The unused solutions of GA were re-used in the garden.
• The plants were taken back by the researcher and re-planted in the balcony garden.

• Take a 1000 cc glass beaker.
• Weigh 0.10 ± 0.01 g of GA powder using a watch glass and a spatula on a digital mass balance.
• Transfer the weighed solid into the glass beaker.
• Transfer 200 cc of ethanol into the same beaker using a graduated measuring cylinder.
• Transfer 800 cc of tap water into the same beaker using a graduated measuring cylinder.
• Stir the solution using a glass rod.
• Repeat steps 1-6 with 0.20 ± 0.01 g of GA (for 200 ppm), 0.30 ± 0.01 g of GA (for 300 ppm), 0.40 ± 0.01 g of GA (for 400 ppm), 0.50 ± 0.01 g of GA (for 500 ppm), 0.60 ± 0.01 g of GA (for 600 ppm) solutions.

• Take 35 plastic cups.
• Weigh 100.00 ± 0.01 g of loamy soil and put in each cup.
• Weigh 1.00 ± 0.01 g of NPK-20:20:20 fertilizer and mix it well with the soil in each of the cup.
• Label all the cups. Label five of them as – 100 ppm, another five as 200 ppm, another five as 300 ppm, another five as 400 ppm, another five as 500 ppm, another five as 600 ppm.
• Keep all the 35 cups in a place near a window so that all of them can receive the same amount of sunlight.
• Take a thread and a ruler to measure the initial shoot height of the plant in Day-1.
• Take 100 cc of the 100 ppm GA solution.
• Take one buffer tablet of pH = 9.00, crush it using a mortar and pestle and add it to the GA solution.
• Take a 10.00 cc graduated pipette and add 2.00 ± 0.05 cc of the 100 ppm GA solution (pH = 9.00) to the first five plastic cups labelled as 100 ppm each day for a period of 10 days.
• After 10 days, take a ruler and a thread to record the shoot height.
• Repeat steps 5-8 for other GA solutions – 200 ppm, 300 ppm, 400 ppm, 500 ppm and 600 ppm. Use 100 cc tap water instead of GA solution for control (0.00 ppm).
• Repeat Steps 1-9 using the other two buffer tablets – pH = 7.00 and pH = 4.00.

For control, distilled water – 0.00 ppm of GA solution was used. The same procedure was followed.

Key

ISH = Initial shoot height in ± 0.05 cm (Shoot height measured in Day-1)

FSH = Final shoot height in ± 0.05 cm (Shoot height measured in Day-10)

DSH = Difference in shoot height (± 0.10 cm) = Final shoot height (± 0.05 cm) – Initial shoot height (±0.05 cm)

PI = Percentage increase in shoot height = \(\frac{DSH}{ISH}\) × 100

DSH = 2.40 - 2.00 = 0.40 cm

PI = \(\frac{0.40}{2.00}\) × 100 = 20.00 %

Mean = \(\frac{20.00+15.00+...+25.00}{5}\) = 19.00 %

SD = \(\sqrt\frac{(19.00-20.00)^2+(19.00-15.00)^2+...+(19.00-25.00)^2}{5}\) = 4.18 %

Mean Percentage increase in shoot height for pH equals 4.00 = \(\frac{7.00+13.00+...+31.00}{7}\) = 19.71 %

The graph above shows the changes in mean percentage increase in shoot height on addition of GA against the concentration of GA added at acidic medium (pH = 4.00), control (pH = 7.00) and basic medium (pH = 9.00). As the graph clearly indicates that there is an increase in the mean percentage increase of shoot height increases with the increase in the concentration of GA used under all pH conditions; as the concentration of GA added increases from 100 ppm to 600 ppm, the mean percentage increase of vertical shoot height rises from 13.00 to 31.00 for pH = 9.00 (in basic medium), from 23.00 to 38.00 in pH = 7.00 (neutral, control medium) and from 52.00 to 82.00 for pH = 4.00 (in acidic medium). Thus, it can be claimed that there is a positive correlation between the concentration of GA and the increase of shoot height; more the concentration of GA more the growth of the shoot at all conditions of pH.

However, the way the mean percentage increase of shoot height depends on the concentration of GA used is not same at all conditions of pH. The graph above also displays the linear trend line obtained. Comparing the gradient the relative effect of concentration on vertical shoot height can be ascertained. The values of  gradient at pH = 4.00, pH = 7.00 and pH = 9.00 are 0.0621, 0.0339 and 0.0371 respectively. Thus, it is clear that in acidic medium, the effect of concentration on the mean percentage increase is more significant than that in basic and neutral medium. Hence, it can be claimed that in acidic medium, or lowering the pH the responses of the molecules towards GA and thus the cell elongation is more. Thus, stem elongation at a developmental stage due to GA is more effective in acidic medium.

At all values of pH, the distance between the successive points is mostly equal. This indicates that at all pH, the growth of shoot due to addition of GA is uniformly increasing as the concentration of GA increases. Hence, the relative changes in the shoot height due to increase in the concentration of the GA solution is same in all values of pH.

For all values of pH, the values at the control (distilled water, 0.00 ppm of GA) is the lowest. The values are 7.00, 19.00 and 43.00 at pH = 9.00, 7.00 and 4.00 respectively. The values at other values of concentration are higher than that in the control. This again confirms that the use of GA or increasing the concentration of GA has a positive impact on the shoot height.

The bar graph above shows the category average (mean values of all the concentration taken together) of the mean percentage increase of shoot height at each pH value. It is clear that the mean percentage is maximum in pH of 4.00 (acidic medium; value = 63.43) and minimum for pH = 9.00 (basic medium; value = 19.71).  Thus, it is clear that the action of GA on shoot height is more in the acidic medium and least effective in the basic medium. The value in the control medium (pH = 7.00) is 29.14 which is way more less than that in the acidic medium and a little more than that in the basic medium. The value in acidic medium is (63.43 – 29.14) =  34.29 units higher than that in neutral medium and the value in basic medium is (29.14-19.71) = 9.43 units lesser than that in neutral medium. This again shows that positive effect of lowering the pH and increasing the acidity is way more significant than increasing the pH and making the medium more basic in context to response of molecules towards GA and promote cell elongation.

Both Figure - 9 and Figure - 10 clearly shows that the shoot height increases with the increase in the concentration of the GA in ppm and the growth of shoot is more favored in acidic medium in comparison to neutral or basic medium. Moreover, acidity favors the growth while basicity decreases the response of the molecules towards GA.  The fact that the increase in shoot height is more with the increase in concentration can be explained based on the fact that as the concentration increases, there are more molecules of GA which finally reduces the water potential of the cell wall. As a result, there is more flow of water molecules from outside of the cell to inside and this eventually increases the volume of the cell and finally elongates it. Moreover, GA also triggers the production of certain enzymes that loosens the proteins that binds the cell wall and thus make it more permeable. This effect is more favored in an acidic medium because with the increase in the concentration of hydrogen ions in the medium, the dissociation of GA is inhibited and thus there are higher number of undissociated GA molecules which can thus promote cell elongation to a higher extent.

The aim of the statistical analysis is to understand if there is any statistical difference between the three groups - pH = 4.00, pH = 7.00 and pH = 9.00.

Null hypotheses (H₀): There is no statistical difference in between the three groups- pH=4.00, pH=7.00 and pH=9.00

Alternate hypotheses (H₁):  There is a statistical difference in between the three groups-  pH = 4.00, pH = 7.00,  and pH = 9.00

Since there are three different independent groups and they are exclusive, an ANOVA analysis was done.

Degree of freedom = 7-1 = 6

Significance level (α)=0.05

Total number of variables (N) = 21

Number of values in each category (n) =7

Number of category (a) = 3

\(df_{between}\)= a-1 = 3-1 = 2

\(df_{within}\)= N-a = 21-3 = 18

\(df_{total}=df_{between}+df_{wthin}\) = 2+18 = 20

Decision rule: If F is > 3.5546, the null hypotheses will be rejected and alternate hypotheses will be accepted. Otherwise, the null hypotheses will be accepted.

SS_between = \(\frac{(7+13+17+21+23+26+31)^2+(19+...+38)^2+(43+...+73)^2}{7}\) - \(\frac{(7+...+73)^2}{21}\) = 7409.14

SS_within = (7²+……+ 82²) - \(\frac{(7+...31)^2+(19+...+38)^2+(43+...+73)^2}{21}\) = 1826

F = \(\frac{\frac{SS_{between}}{df_{between}}}{\frac{SS_{within}}{df_{within}}}\) = \(\frac{\frac{7409.14}{2}}{\frac{1826}{18}}\) = 42.60

As the calculated value of F (42.60) is greater than the ideal value (3.5546), the null hypotheses is rejected and the alternate hypotheses is accepted. It means that the mean of the three independent groups is different. In other words, the way the molecules response to GA and thus causes cell elongation to increase shoot height depends on the pH of the medium.

How does the effect of the concentration of Gibberellin (in ppm) on the growth (in terms of vertical shoot height) of a green chilli (Capsicum annum) varies with the pH – acidic (pH = 4.00), basic (pH = 9.00) and neutral (pH = 7.00)?

• There is an increase in the mean percentage increase of shoot height increases with the increase in the concentration of GA used under all pH conditions; as the concentration of GA added increases from 100 ppm to 600 ppm, the mean percentage increase of vertical shoot height rises from 13.00 to 31.00 for pH = 9.00 (in basic medium), from 23.00 to 38.00 in pH = 7.00 (neutral, control medium) and from 52.00 to 82.00 for pH = 4.00 (in acidic medium). Thus, it can be claimed that there is a positive correlation between the concentration of GA and the increase of shoot height; more the concentration of GA more the growth of the shoot at all conditions of pH.
• The values of gradient at pH = 4.00, pH = 7.00 and pH = 9.00 are 0.0621, 0.0339 and 0.0371 respectively. Thus, it is clear that in acidic medium, the effect of concentration on the mean percentage increase is more significant than that in basic and neutral medium.
• The relative changes in the shoot height due to increase in the concentration of the GA solution is same in all values of pH.
• The ANOVA test concludes that the way the molecules response to GA and thus causes cell elongation to increase shoot height depends on the pH of the medium.
• The null hypotheses have been rejected and the alternate hypotheses has been accepted.
• As the concentration increases, there are more molecules of GA which finally reduces the water potential of the cell wall. As a result, there is more flow of water molecules from outside of the cell to inside and this eventually increases the volume of the cell and finally elongates it. Moreover, GA also triggers the production of certain enzymes that loosens the proteins that binds the cell wall and thus make it more permeable.

• The values of the independent variable have been chosen over a range that is in coherence and justified with the realistic biological interval. Moreover, the values are varied at specific intervals. This gives a more detailed scattered plot which allows better analysis. For all pH values, the range and the values of concentration chosen are same. As a result, in Figure - 9, the values in the x axes are same for all the three trend lines are same. This makes the comparison more mathematically easier and the results obtained from comparing the gradient of the trend lines more reliable.
• The investigation uses % increase in shoot height instead of directly using the shoot height. This gives an idea of relative comparison which is a more generalized approach.
• The results of the investigation are in agreement with the literature review. The literature review clearly suggests that the action of GA is more in acidic medium and lowering pH or increasing the acidity results in better responses towards GA and the current results as indicated in Figure - 10 also shows that increase of shoot height is maximum in acidic medium.

As the concentration of GA has a correlation with the growth of plants, the method of exposure is also important. Plant samples can be exposed to GA in two ways- infused media and spray method. In infused media method, the GA solution is mixed with the medium in which the plant is grown while in the spray method, a solution of GA is sprayed on the plant at regular intervals. I would like to investigate how does the relationship between the concentration of GA and the shoot height depends on the method of exposure-infused media or spraying method. I would like to use the sample – Arabidopsis Thaliana for this investigation as this plant can be grown in a very small space and it genetically resembles the phenotype of plants that shows decreased growth due to deficiency of GA. I would grow them in a plastic cup and mix GA solution with the soil for the category of – infused media and in other groups I would spray the GA solution on the plants. In both cases, I would measure the shoot height at all values of concentration of GA used.

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