Brian Seitzman GA Experiment Application of Gibberellic Acid to Leaf Surface Accelerates Stem Growth in Deficient Brassa rapa Hypothesis:

Gibberellic acid can be taken up through leaf stomata in Brassica rapa plants, the result of which will be normalization of stem elongation in mutant rosette types that do not synthesize the growth regulator on their own. Previous experimentation has shown that this is noted in Arabidopsis, and the same principles should apply to B. rapa. Abstract: Gibberellic acid is one the most important plant growth regulators, playing crucial roles in the plant life cycle. From its role in the germination of seeds to stem elongation to flowering and fruit development, this plant "hormone" is vital to the growth and reproduction of plants. Nonetheless, mutations resulting in an inability to synthesize gibberellic acid are known to occur. In this experiment, we used one such mutant strain in a model organism, Brassica rapa, to investigate whether gibberellic acid applied to the leaf surface could stimulate growth and to determine the significance of concentration in such an application. By comparing the growth of treated and untreated rosette mutants, the stems of which normally do not elongate due to the absence of a complete biochemical pathway for gibberellic acid synthesis, to that of treated and untreated wild type plants, we determined that gibberellic acid is capable of entering the plant through leaf stomata. By treating wild type plants with gibberellic acid as well, we were able to show that the addition of excess GA does not cause greater than normal stem elongation. Last, we determined that external application of gibberellic acid to rosette plants at concentrations beyond those normally present in the wild type does not cause greater growth in plants which cannot synthesize the growth regulator on their own. Introduction: Gibberellic acid is one of a class of 126 compounds known as the gibberellins. It was first theorized to exist in 1926 by Elichi Kurosawa in a study of a fungal pathogen (Gibberella fujikori) of rice seedlings. It was subsequently isolated from the fungus in 1934 by Teijiro Yabuta, and he is credited with determining its chemical structure as 5-n-butylpicolinic acid. It was Yabuta who gave gibberellic acid the name by which it is now known. Subsequent studies of the plant growth regulator have shown that it is involved in nearly every developmental stage in the life cycle of plants. It is gibberellic acid, for example, that promotes the initial growth of shoots from desiccated seeds when environmental moisture is adequate. Throughout the lifetime of a given plant, gibberellin continues to promote the elongation of stems. It furthermore is of importance in causing flowering and, thereafter, the development of fruit. Because of these many areas of involvement, it can be said that gibberellin in plants is analogous to pituitary hormone in humans. Without it, normal development is impossible. The potential for agricultural applications clearly exist. Gibberellic acid could be used to promote increased yields of fruits and stem vegetables such as celery and asparagus. The growth regulator might also be applied in order to promote early flowering or, assuming that it has suppressive actions at high concentrations as is the case with auxins, it might be used to inhibit flowering out of season in crops grown in geographic areas where the plants' blooming season is

not ideally aligned to weather conditions. Additionally, since natural mutations are known to occur wherein the biochemical pathways necessary to synthesize gibberellic acid are absent from the mutant, external application of the compound could be useful in reclaiming what might otherwise be lost crops without replanting, thus helping to maximize economic returns in agriculture. The usefulness of application, however, hinges on several questions. First, it is necessary to determine whether gibberellic acid can be taken up by a plant via the stomata, the pores in the plant cuticle which serve as the sites for the exchange of water vapor and CO2 with the atmosphere. Second, it is necessary to determine whether or not this uptake, if it exists, is of benefit to mutants lacking the synthetic pathway only or if wild type plants, capable of making gibberellic acid on their own, also exhibit increased stem length following external application. Finally, optimal concentration of gibberellic acid in the applied solution needs to be determined in order to avoid wasting money on the cost of a chemical that is of no benefit, or even of potential harm, to the plants to which it is being applied. In this experiment, we have used a mutant strain of Brassica rapa, a member of the mustard family, as a model organism for the effects of superficial application of gibberellic acid on plants. The mutant type, known as rosette, lacks the ability to synthesize the growth regulator on its own and consequentially does not undergo stem elongation under normal conditions. We compared the effects of application on the mutant to that on wild type plants which normally undergo very rapid stem elongation and a fast seed-to-seed lifecycle. Our data indicate that mutant B. rapa benefit from external gibberellic acid application, taking up the compound through leaf stomata and achieving stem lengths comparable to that of the wild type plant. We demonstrate that only low concentration of GA is necessary to achieve this benefit, with higher concentration producing no additional stem elongation. Our data also indicates that only the mutant type derives benefit from treatment with GA; wild type plants do not show greater stem elongation as a result of the increased availability of gibberellic acid. Materials and Methods: Commercially-available Brassica rapa seeds known to be either the wild type or rosette mutant were planted in pre-fertilized potting soil contained in plastic film canisters. A hole with made in the bottom of each canister, through which a knotted string wick was inserted into a container filled with approximately 360 mL of water in order to provide sufficient moisture for plant growth without the need for subsequent watering. The canisters were grouped according to phenotype (wild type or mutant) and divided up by the treatment to be applied, resulting in a total of 8 sets of two canisters each. The seeds were germinated in a greenhouse in order to insure that light and humidity conditions would be identical for all of the plants. Only the first plant to germinate were allowed to grow, with subsequently germinating seedlings being removed in order to avoid competition between multiple plants in a single canister. The plants were allowed to grow on their own for ten days after germination, at which time the application of treatments began. Four different treatments were applied to two of each of the wild type and rosette plants. Two plants of each phenotype were given 20 L of water from a syringe at the leaf surface. Two others were given a solution of 10 ppm of giberellic acid dissolved in water and tween 20 in the same fashion, another two given a 100 ppm solution, and the last two were given the tween 20 solution without gibberellic acid. In this manner, the four plants receiving only water served as overall controls and those receiving tween 20 served as

controls for any effects that might be caused by tween 20 itself. Treatment was delivered beginning on the tenth day after germination and then on every second day thereafter so that the plants received treatment for a total of 14 days after ten days of independent growth. Prior to the delivery of treatment the central stems of the plants were measured from the soil surface to the base of apical bud. When the length of the stem became so great that a plant could not support its own weight it was loosely tied to a stake to insure accurate measurement. Results:
Days after treatment began 2 4 6 8 10 12 14 16 ros H2O A 0.5 0.5 0.9 1.0 1.0 1.0 1.0 1.1 ros H2O B 0.9 1.5 1.5 1.5 1.9 2.5 5.5 7.2 ros 0 A 0.3 0.9 1.1 1.1 1.1 1.2 1.5 1.7 ros 0 B 0.4 1.0 1.0 1.0 1.0 1.1 1.1 1.3 ros 10 A 0.7 1.1 1.5 1.5 2.6 4.5 6.0 14.7 ros 10 B 1.8 2.3 3.2 5.6 7.9 16.0 19.0 21.0 ros 100 A 0.3 0.7 1.5 2.1 3.8 6.1 10.2 14.9 ros 100 B 0.4 0.8 0.9 1.5 4.6 10.0 19.0 25.7 wt H2O A 1.7 2.4 2.9 5.5 10.1 14.3 18.0 20.6 wt H2O B 2.1 2.5 3.2 6.3 10.2 15.8 20.0 22.3 wt 0 A 1.4 1.9 2.3 4.1 12.0 15.0 21.0 23.9 wt 0 B 1.3 1.6 1.6 2.8 3.4 5.7 10.5 15.2 wt 10 A 0.7 1.1 1.5 2.1 3.9 5.5 8.5 15.0 wt 10 B 1.2 1.5 2.3 3.1 7.1 9.3 15.0 18.9 wt 100 A 1.1 1.4 2.5 4.2 8.0 12.7 16.2 18.7 wt 100 B 1.9 2.3 3.9 7.0 13.5 18.3 24.0 26.9

Table 1. Plant height (cm) vs. days after beginning of treatments. ros = rosette (mutant) plants, wt = wild type plants, H2O = received water only, 0 = received tween 20 and water, 10 = received 10 ppm GA in tween 20 and water, 100 = received 100 ppm in tween 20 and water. The heights for all plants on each day of record are shown separately.
ros Days after treatment began 2 4 6 8 10 12 14 16 H2O 0.70 1.00 1.20 1.25 1.45 1.75 1.06 3.25 3.18 4.15 4.31 1.50 0.28 1.30 0.28 SD, H2O 0.28 0.71 0.42 0.35 0.64 1.15 0.07 0 0.35 0.95 1.05 1.05 1.05 SD, 0 0.07 0.07 0.07 0.07 0.07 10.2 5 12.5 0 17.8 5 10 1.25 1.70 2.35 3.55 5.25 SD, 10 0.78 0.85 1.20 2.90 3.75 8.05 8.13 9.19 4.45 14.6 0 20.3 0 2.76 6.22 7.64 100 0.35 0.75 1.20 1.80 4.20 SD, 100 0.07 0.07 0.42 0.42 0.57 H2O 1.90 2.45 3.05 5.90 10.1 5 15.0 5 19.0 0 21.4 5 SD, H2O 0.28 0.07 0.21 0.57 0.07 1.06 1.41 1.20 10.3 5 15.7 5 19.5 5 0 1.35 1.75 1.95 3.45 7.70 SD, 0 0.07 0.21 0.49 0.92 6.08 7.40 6.58 7.42 6.15 11.7 5 16.9 5 2.69 4.60 2.76 wt 10 0.95 1.30 1.90 2.60 5.50 SD, 10 0.35 0.28 0.57 0.71 2.26 100 1.50 1.85 3.20 5.60 10.7 5 15.5 0 20.1 0 22.8 0 SD, 100 0.57 0.64 0.99 1.98 3.89 3.96 5.52 5.80

Table 2. Mean plant height and standard deviation vs. days after treatment began. Calculate from the raw data shown in table 1.

Mean Plant Height vs. Day of Treatm ent 35.00 30.00 25.00 Mean Height (cm) 20.00 15.00 10.00 5.00 0.00 2 4 6 8 10 12 14 16 Days after treatm ent began ros, H2O ros, tw een20 ros, 10 ppm GA ros 100 ppm GA w ild type, H2O w ild type, tw een20 w ild type, 10 ppm GA w ild type, 100 ppm GA

Figure 1. Mean height for all groups over the course of treatments. Mean height values of all groups for each recorded measurement shown with error bars.
M e a n H e i ght v s . D a y s o f T r e a t me nt , M ut a nt T y pe M e a n H e i ght v s . D a y s o f T r ea t ment , Wi l d T y pe

30. 0 0

3 5. 0 0

25. 0 0

3 0. 0 0

2 5. 0 0 20. 0 0 ros, H2O ros, t ween 2 0 15. 0 0 ros, 10 ppmGA 15. 00 ros, 100 ppmGA 10. 0 0 10. 00 5. 0 0 5. 00 2 0. 0 0 wi t ype, t ween 20 d l wi t ype, 10 ppmGA d l wi t ype, 100 ppmGA d l wi t ype, H2 O d l

0. 0 0 2 4 6 8 10 12 14 16

0. 00 2 4 6 8 10 12 14 16

D ay af t er T r eat men t B egan

D ays of T r eat men t

Figure 2. Mean height for rosette plants over the course of treatments. Mean heights for mutant plants for each recorded measurement shown with error bars.

Figure 3. Mean height for wild type plants over the course of treatments. Mean heights for wild type plants for each recorded measurement shown with error bars.

Mean Total Grow th, all groups 30.0

25.0 Mean Height (cm) at conclusion ros, H2O 20.0 ros, tw een20 ros, 10 ppm GA 15.0 ros, 100 ppm GA w ild type, H2O w ild type, tw een20 10.0 w ild type, 10 ppm GA w ild type, 100 ppm GA 5.0

0.0 Mean Total Grow th (cm)

Figure 4. Mean total growth for all groups. The difference in mean stem lengths for all groups between the first and last measurements taken is shown with error bars. Note the large difference between total stem elongation between the ros type plants that received GA and those that did not versus the much smaller differences in elongation among those ros types that did not receive GA. Wild types that received GA in either concentration did not exhibit much greater elongation than those that did not. The final height for each treatment within each of the two phenotypic set was compared against those of the H2O controls using a one-tailed paired t-test with a significance level of p <= 0.05 interpreted as a statistically significant increase in stem length in the groups receiving either tween 20 or gibberellic acid over the controls. These results are summarized in Table 3. Determination of Significance ros, tween 20 0.282 no ros, GA 10 ppm 0.002 significant ros, GA 100 ppm 0.046 significant wt, tween 20 0.388 no wt, GA 10 ppm 0.076 no wt, GA 100 ppm 0.374 no Table 3. Results of t-test of treated groups vs. H2O controls. From the t-test results shown above, we can deduct that gibberellic acid entered the mutant plants via leaf stomata and effected stem elongation. We can also see that the application of tween 20 alone did not have any appreciable effect on stem elongation in either the mutant or 5 Phenotype and Treatment p-value

wild type plants. Moreover, we note that the wild type plants that received GA did not undergo stem elongation that was significantly different from those that received only water. As there is no reason to believe that the surface of the mutant and wild types are different, and we know that gibberellic acid did affect the mutant plants, we can also conclude that the plants which produced sufficient gibberellic acid on their own were not significantly affected by an increased concentration of gibberellic acid entering the stomata. The effects of the two concentrations of gibberellic acid solutions applied were next analyzed with the same t-test as above. Determination of Significance ros, 10 ppm GA vs. 100 ppm GA 0.219 no wt, 10 ppm GA vs. 100 ppm GA 0.115 no Table 4. Results of t-test for concentration. No significant difference can be seen between the effects of treatment with 10 ppm and 100 ppm gibberellic acid solutions in either phenotype. Phenotype and Treatment p-value From our data, it does not appear that there is any significant difference between the groups that were treated with 10 ppm and 100 ppm solutions of GA. This supports the hypothesis that there is a maximum concentration of gibberellic acid that is useful for stem elongation in B. rapa. Since we have already noted that there is no significant difference in stem elongation between plants receiving tween 20 solution and those receiving only water we can also rule out any effect of tween 20 in these results. Next, we compare the final stem lengths of the wild type and rosette plants that received the same treatments over the course of our study. Determination of Significance 100 ppm GA, ros vs. wt 0.374 no 10 ppm GA, ros vs. wt 0.415 no tween 20, ros vs. wt 0.029 yes H2O, ros vs. wt 0.016 yes Table 5. Results of t-test for final stem length compared between treatments. The mutant plants treated with GA had final stem lengths that were not significantly different from wild types. There was a significant difference between the final stem lengths of the two control groups. Phenotype and Treatment p-value Discussion: From the results obtained, it is clear that the rosette-type plants benefited significantly from the external application of a dilute gibberellic acid solution in terms of stem elongation. We have also demonstrated that receiving ten times the concentration of the growth regulator did not provide any additional benefit to the plant. It is thus made apparent that an optimal concentration of gibberellic acid between 10 and 100 ppm exists for GA-deficient plants. Determination of the exact level would require further investigation, as will the question of whether the reason that greater stem elongation is not seen is due to the nature of biochemical pathways in the plant or the inability of more gibberellic acid to diffuse into the leaf stomata

beyond a given concentration. The lack of greater stem elongation in the wild type plants could also be due to biochemical, physical, or a combination of both considerations. What is clear is that GA-deficient crop plants can benefit from the availability of exogenous GA solution. In keeping with previous experiments, rosette mutant plants in our study that were treated with GA attained stem lengths practically indistinguishable from that of wild type plants. We can thus safely conclude that our rosette mutants did not lack the ability to utilize gibberellic acid, only the ability to synthesize it. In this regard, the mutant plant may be very much like the well-studied Arabidopsis ga1-3 mutant. Furthermore, despite its lack of GA for post-germination stem elongation, the seeds of our mutant plants did germinate. This suggests that the defective gene is one that is not normally activated in the plant until after some growth has occurred, and so we can rule out several possible genes involved in the biosynthetic pathway in the embryo, such as AtGA3ox1 and AtGA3ox2 [Yamaguchi 2001]. It would be worthwhile to investigate the effect of similar GA applications to B. rapa seedlings closer to germination to see whether wild type and mutants would react differently than they have here and to see whether or not they are affected in the same way in which rice seedlings are affected by Gibberella fujikori. References: Kurosawa, Elichi. 1926. Experimental studies on the nature of the substance secreted by the "bakanae" fungus. Natural History Society of Formosa. 16: 213. Raven, Peter H., Evert, R.F, and Eichhorn, S.E. Biology of Plants, 7th Edition. W.H. Freeman and Company, New York, NY, 2005, page 613. Yabuta Teiji, Sumiki Y. 1938. On the crystal of gibberellin, a substance to promote plant growth. Journal of the Agricricultural Chemistry Society of Japan. 14: 1526. Yamaguchi, Shinjiro, Kamiya, Y. and Sun, T. 2001. Distinct cell-specific expression patterns of early and late gibberellin biosynthetic genes during Arabidopsis seed germination. The Plant Journal. 28: 443.