Ultra Violet Radiation on Sex Determination in Ceratopteris Richardii

Ultraviolet (UV) radiation and its detrimental effects on plant life have been widely researched. This research studies the effects of UV radiation on sex-determination in Ceratopteris richardii. Sex determination in Ceratopteris richardii is determined by antheridiogen, a pheromone that promotes maleness. In this experiment, UV radiation was used on the wild type and mutants Her1, which are insensitive to the male-inducing pheromone (does not form male gametophytes). Ceratopteris richardii spores were exposed to UV radiation at different stages during their life cycle. Wild type gametophytes treated with UV radiation until germination and sexual maturity showed an increase of hermaphrodites as compared to wild type gametophytes only exposed to white light. Her1 mutants treated with UV radiation until germination showed no change in expected sex ratios. However, her1 mutants exposed to UV radiation until sexual maturity showed a large discrepancy in normal sex ratios and produced a huge increase of males. These results show that UV radiation does have an effect on sex determination in Ceratopteris richardii, and perhaps other plants as well.

Introduction

Ceratopteris richardii, also known as C-Fern, start out as haploid spores. After four to six days, germination takes place and rhizoids, a root-like filament which absorbs nutrition, appear. It begins to develop into a sexually mature gametophyte, an ameristic male or hermaphrodite with a well-defined meristic region. Figure 1: C-Fern Life Cycle

Source: D. Maxwell. “C-Fern Genetics”
The sexual fate of the gametophyte depends if the pheromone antheridiogen (ACE) is present in the early stages of development, approximately three to four days after spore inoculation. ACE, which is thought to be a form of gibberellin, promotes maleness in Ceratopteris richardii gametophytes and must be present during the entire duration of cell division to allow the production of antheridia. ACE is responsible for both the initiation and maintenance of the male program of expression in Ceratopteris richardii and therefore acts as a repressor for meristem and archaegonia formation. This mechanism of sexual determination allows for the ratio of males and hermaphrodites to be dependent upon the amount of ACE within the culture.

The HER (hermaphroditic), FEM1 (feminization) and TRA1 (transformer) genes are directly affected by ACE. HER genes are necessary for ameristic male development and are likely to encode factors that make up the ACE receptor and/or signal transduction pathway [Eberle and Banks, 1996]. The expression of TRA1 allows meristem and archegonia development in Ceratopteris richardii while FEM1 allows for antheridia development. ACE signals are commonly blocked by the hormone, abscisic acid (ABA). ABA is known to block antheridiogen response in undifferentiated cells of the gametophyte, indicating that ABA may repress antheridia development [Banks, Hickok, and Webb, 1993]. Figure 2: ACE Pathway

Source: Eberle and Banks, 1996.

If ACE is absent during the stage of cell division, the gametophyte will develop as a hermaphrodite, an action that is irreversible even if exposed to ACE in later stages. The HER gene is not active while TRA and FEM are expressed and the gametophyte develops an archegonia, antheridia and a meristem. In this case, TRA promotes meristem and archegonia development and FEM promotes antheridia development (Figure 2).

However, if ACE is present during the beginning parts of the stage but is withdrawn later, the undifferentiated cells of the male begin to divide and form a hermaphrodite. The HER and TRA1 genes would remain unexpressed while FEM1 will be expressed (Figure 2). In the continuous presence of ACE, ameristic males containing antheridia, male sexual organs each containing 16 sperm [Warne and Hickok, 2008], are formed. In this case, HER and FEM1 genes are expressed and repress TRA1 (Figure 2). After sexual maturity occurs, fertilization and meiosis takes place and a second generation of spores is produced. The life cycle repeats, a process that takes 60 to 80 days. The mutant, her1, which is also used in this experiment, is a spore that is insensitive to ACE, thus does not normally form male gametophytes.

Methods and materials:
The Basic C-Fern Media from Carolina Biological comes in premeasured packets for 1000 mL. Ten grams of Phytagar (Life Technologies) were added to 900 milliliters of distilled water in a 2000mL Erlenmeyer’s flask. The Basic C-Fern media packet was poured into the flask, the remaining 100 mL of distilled water, which was not placed into the flask, was placed into the packet to remove remaining powder and then poured into the flask. The media was pHed to 6.3 using sodium hydroxide (NaOH) and hydrochloric acid (HCL). A computer program from Venier called LoggerPro recorded the pH. The media was autoclaved, with a Sterilmatic autoclave, at 15 psi, at 250 ⁰ F for 20 min. The warm media was poured into 100×15 mm Petri dishes and solidified plates were inoculated with 5 drops of suspended spores. The plates were moved in a figure “eight” formation so the spores would be equally distributed on the agar. They were parafilmed to prevent desiccation. Two enclosed plastic containers were used (38.5 cm by 51 cm/15.2 in by 20 in). One for White light and the other for UV treated Ceratopteris richardii. In the containers, tin foil was placed so that it could preserve heat and reflect light waves in the container.

The boxes had a 13-Watt light bulb. To simulate a natural environment, white light was added to the UV treated groups. The distances from the white light and UV lamp to the petri dishes are equal. Control group1 was the untreated wild-type group. Control group 2 was the untreated Her1 group. The UV treated groups were Wild type with UV exposure until germination (1a) followed by regular white light, Her1 with UV exposure until germination (2a) followed by regular white light, Wild type with UV exposure until sexually mature (1b), and Her1 with UV exposure until sexually mature (2b). The UVA intensity is 209 w/m^2 and for UVB is 14.2 w/m^2. A computer program from Venier called UVA/UVB Sensor (UVA-BTA/UVB-BTA) measured the intensity in the box. All groups contained 6 Petri dishes. The sixth Petri dish was used for protein electrophoresis. The UV lamp was turned on daily from 8:00 am to 11:00 am excluding weekends. They were placed there for three hours to ensure the UV light penetration through the spores’ hard shell.

The spores in all the groups were counted. To determining the number of hermaphrodites and antheridia in each group. Sex ratio’s were then determined. (Pictures were taken of each group showing the stages of germination and sexual maturity) To make the comparison more visible a bar graph was created. Protein electrophoresis was preformed. The Ceratopteris richardii removed from the plate was weighed with a Sartorius M-prove electronic precision scale to get .125 μg. This was placed into a vile with Laemmli Sample Buffer (250 μL). Laemmli Sample Buffer helps break down the folded structure of the protein in the Ceratopteris richardii. Electrophoresis is the separation of biological macromolecules, which is done in order to observe protein bands for the gametophyte. A difference in the protein banding patterns between the control and treated groups would conclude response to UV stress. The specimens were first homogenized. A hot water bath at 95˚C was made and the specimens and the Precision Plus Protein Kaleidoscope/Standard were placed in the hot water bath.

The hot water bath, linearlizes the proteins so that the only factor during electrophoresis would be the molecular weight of the proteins moving down the gel. The specimens were placed in the hot water bath for 5 minutes. The Mini-Protean TGX precast polyacriminide gel 12% was prepared in the gel electrophoresis box and SDS buffer was added. Ten μL of the standard was used and 16 μL of the specimen was added to the wells. The unit was turned on, which causes the bands to flow down. The gel was removed from the electrophoresis unit and washed 3 times in water. The gel was placed in (Bio Rad) Coomassce Blue Stain for 6 hours. The stain was removed from the gel and the gel was washed 3 times. A best-fit line was generated using kaleidoscopic standard molecular weights. The equation generated on a TI 83 Graphing Calculator was used to determine the molecular weights of the proteins in the sample wells. The banding patterns were calculated. A statistical analysis of the sex-determining ratios was made using the ANOVA Test for significance.

The default pathway for maleness in Ceratopteris richardii is exposure to a gibberellin-like pheromone called Ace. A receptor (coded for by the HER gene) binds with the Ace and starts the signaling pathway to maleness. This pathway, in the wild type, involves inhibition of the TRA gene product (which causes archegonia and meristem formation) and activation of the Fem gene product (which produces the male). (Banks, _____)

This research shows that exposing the spores to UV radiation treatment for three hours daily, until germination (group 1a and 2a does not alter expected sex ratios). This was observed in both the wild type and Her-1 mutants. When hermaphrodites and antheridia were counted, the expected ratios were observed. The sporopollenin in spore cases apparently cannot be penetrated with UV radiation.

Those cultures exposed to UV radiation until sexual maturity (groups 1b and 2b), however, showed a marked discrepancy in sex ratios from the expected. The wild type gametophytes (group 1b) exhibited an increase in gametophytes over antheridia. Past research (_____, _____) indicates that gibberellins are broken down in UV radiation. Several of the intermediate products in the biosynthetic pathway leading to the production of gibberellins are broken down. Since it is believed that Ace is a gibberellin-like molecule, it is predicted that the increase in hermaphrodites to antheridia was a consequence of Ace breakdown.

The results in the UV treated Her-1, until sexual maturity group (group 2b), showed a marked increase in antheridia. Considering that the Her-1 mutant does not respond to Ace at all because the Ace receptor is mutated, these results are difficult to explain. The default signaling pathway for Ace is disrupted in the Her-1 mutants but the UV radiation produced males. Two hypotheses are offered as explanation. To determine, with any kind of certainty, that either of these hypotheses are correct would require biochemical studies for the presence of proteins and hormones in an alternative pathway to produce maleness. One predicted hypothesis may be that the UV wavelengths were picked up by photoreceptors (perhaps cryptochromes) which initiated an alternative signal transduction pathway, bypassing the Her-1/Ace pathway, and ultimately activating the Fem gene for maleness.

Both hormones and light receptors play essential roles in plant development, plant growth as well as plant response to stress. The second predicted hypothesis involves an alternative hormonal pathway leading to maleness. Past research (____,____) indicates that both gibberellins and auxins can stimulate maleness in plants. It may be that the UV stress induced an alternative pathway for producing maleness involving an increase in the auxin hormone; perhaps as a survival mechanism. The wild type would not be subjected to this pathway since the Her-1 gene was functional and therefore the default pathway for maleness was still in operation.

An obvious conclusion in this research is that increased UV radiation does affect the sex ratios in Ceratopteris richardii. Future research would take a look at higher plants, like angiosperms to determine if formation of gametes is effected and to what extent. The angiosperm gametes are more protected than the fragile fern gametophytes; but would fertilization and number of zygotes be reduced if UV radiation is increased? These are important ecological concerns. Plants are the primary producers of every food chain and also a major source of nutrients, clothes and medicines.

Bibliography

All the information was provided by various sources such as: 1. “The Ozone Layer, Depletion, and UV. Radiation.” Eco-action Org – Ecological Direct Action. <http://www.eco-action.org/dt/ozone.html>.

2. Stapleton, Ann E. “Ultraviolet Radiation and Plants: Burning Questions.” Plantcell.org. Department of Biological Sciences, Stanford University, Nov. 1992. Web. < http://www.plantcell.org/content/4/11/1353.full.pdf > 3. Kamachi, Hiroyuki, Orie Iwasawa, Leslie G. Hickok, Masaaki Nakayama, Munenori Noguchi, and Hiroshi Inoue. “The Effects of Light on Sex Determination in Gametophytes of the Fern Ceratopteris Richardii.” Genetics.org. The Botanical Society of Japan and Springer, 3 Aug. 2007. Web. <http://www.ncbi.nlm.nih.gov/pubmed/17674124>. 4. Eberle, James R., and Jo Ann Banks. “Genetic Interactions Among Sex-Determining Genes in the Fern Ceratopteris Richardii.” Genetics.org. Department on Botany and Plant Pathology, 1996. Web. <http://www.genetics.org/content/142/3/973.full.pdf>. 5. Hickok, Leslie G., and Thomas R. Warne. “C-Fern Web Manual.” Marginscience.com. University of Tennessee Research Foundation, 2008. Web. < http://www.magrinscience.com/images/biology/ferns/C-Fern_Manual.pdf 6. Maxwell, D. “C-Fern Genetics.” Wiki.pingry.org. N.p., n.d. Web.