Understanding the Abscisic Acid Pathway Using Guard Cell Specific Genes and the Anti-Aging Drug Spermidine

Plants must respond to environmental stress including drought and harsh winters. Overcoming these stresses depend heavily on timing of stomatal closure and seed germination. My research focused on both chemical and genetic aspects involved in the abscisic acid pathway that controls both stomatal closures in leaves and seed germination in Arabidopsis thaliana. My first study focused on recognizing specific proteins involved in the abscisic acid pathway for stomatal guard cell closure. Given specific promoters for their respective proteins, it is possible to determine whether or not proteins are guard cell specific. GatewayTM technology utilizes a series of reactions to create a clone containing a promoter of interest called an expression vector. By injecting this vector directly into the leaf of Arabidopsis, the plant will use the promoter to create the guard cell specific protein. The second study examined the effect of the anti-aging drug Spermidine on seed sensitivity to abscisic acid concentration during seed germination. By varying the concentration of Spermidine and abscisic acid exposure to seeds then observing the number of surviving seeds, the effects of Spermidine on seed germination can be measured. Spermidine is expected to reduce seed sensitivity to abscisic acid leading to increased seed germination. Introduction Plants must respond to environmental stresses such as droughts and harsh winters. Each year billions of dollars are lost due to drought and winter. The National Weather Service reports that drought alone cost the U.S. over $61.6 billion. Making plants that are better suited for combating these disasters has become a major area of concern. There are ways to do just that. Overcoming these stresses depend heavily on timing of stomatal closure and seed germination. Stomata are microscopic pores on the bottom surface of leaves. Two guard cells make up the pores which control gas exchange for the plant during photosynthesis (Pei et al., 1998; Hugouvieux et al., 2001). As gases are being exchanged water escapes. This becomes a great problem for plants during drought. Opening stomatal pores contribute to 95 percent of total water lost from the plant. By genetically controlling when stomata open and close, a plant can conserve the precious water it needs to survive when water becomes scarce. The anti-aging drug Spermidine controls many cellular processes to slow the aging process down in organisms. In plants seed germination is slowed keeping the seeds alive during the winter. This is critical for long, harsh winters during which, seeds die before the rains and rising temperatures permit optimal growth. By controlling when a seed germinates, a seed can endure the winter long enough for those favorable conditions to arrive in the spring. Greater understanding of these processes will give plants necessary tools for coping with intense weather conditions. Common to these seemingly unrelated events are their signaling mechanisms, the abscisic acid (ABA) pathway. My research focused on both chemical and genetic aspects involved in the ABA pathway. Despite ABA’s role in stomatal movements, genetic components of the humidity signaling cascade remain largely unknown. Thus, my first study focused on the genetic aspect by identifying regulatory regions in guard cell preferential genes. These genes are responsible for stomatal guard cell closure involved in the ABA pathway. Controlling when the gene is expressed means finding the promoter. So developing a faster method to identify the region in the promoter specifically that encodes functionality is of the greatest importance. Guard cells use a complex signaling network to create a “graded binary” output that can readily be observed under the microscope: stomatal ‘opening’ or ‘closing’. The study of guard cell signaling provides insights into how the many cellular processes assemble together to create a quantifiable single Celebrating 20 Years of Student Research and Scholarship 175 cell output (Kwak et al., 2008), allowing quantitative dissection of the functions of individual genes and proteins within signaling cascades. Despite its role in stomatal movements, molecular components of the humidity signaling cascade also remain largely unknown. Humidity is an environmental stimulus that regulates stomatal movements. Stomatal closure occurs very rapidly in response to a reduction in relative humidity in the atmosphere. So when the air has very little moisture, stomata close. Genomic techniques have been developed and adapted to Arabidopsis thaliana guard cell signal transduction studies in humidity. This allows for molecular genetic, cell biological, biophysical, physiological, and functional genomic analyses of single cell signaling responses (Pei et al., 1997; Pei et al., 1998; Allen et al.,1999; Wang et al., 2001; Hosy et al., 2003; Leonhardt et al., 2004) in Arabidopsis. Arabidopsis provides a model for understanding stomatal closure and seed germination. It is a small plant, allowing it to be used in vitro, meaning in controlled conditions. It has a short life cycle for quick growth. Thousands of seeds can be collected from a single plant. Its small genome was sequenced and annotated. Exchanging DNA between it and bacteria is relatively simple. This all contributes to the adundance of genetic and genomics tools available for plant experimentation. Recently, most biochemical, genetic and molecular studies on plant hormone action and biosynthesis have been carried out in the model plant Arabidopsis thaliana for its simplicity of genome, short reproductive cycle, small plant size and great amount of information available on its metabolic pathways and signaling (Tassoni, et al., 2000). Being targets of early signaling branches, ion channels provide effective functional signaling and quantitative analysis points to identify and characterize upstream regulators and identify the intermediate targets and signaling branches that are affected either directly or indirectly by these regulators. Regulatory factors that control ABA response have been identified by genetic, biochemical, and pharmacological/cell biological approaches (reviewed by Rock, 2000; Finkelstein and Rock, 2002). As mentioned previously, identifying the region in the promoter specifically that encodes functionality is necessary. Biochemical approaches have uncovered a variety of gene promoter elements such as kinases, kinase inhibitors, phosphatases, phospholipases, and transcription factors that correlate with the ABA response (Finkelstein et al., 2002). Having identified a series of transcriptional activators and their target genes, it should be possible to dissect the signaling pathway involved in the ABA activation of these [promoter elements] (Finkelstein et al., 2002). ABA mutant phenotypes may be tissue specific and subtle (Finkelstein et al., 2001). Given tissue specific promoters, it is possible to determine whether or not the genes activated are guard cell specific using Gateway technology. Gateway technology utilizes a series of reactions to create a clone containing a promoter of interest called an expression vector. By injecting this vector directly into the leaf of Arabidopsis, the plant will use the promoter to activate the promoter elements in guard cells specifically. Recall that plants must respond to both drought and harsh winters. Surviving harsh winters depends on timing of seed germination. The second study examined the effect of Spermidine on seed sensitivity to ABA concentration during seed germination. ABA is required for plant adaptation to environmental stress by affecting different plant tissues, developmental stages, and physiological processes. These include changes in seed dormancy and germination (Leung and Giraudat, 1998). Spd allows for inhibition of the phase transitions from embryonic to germinative growth and from vegetative to reproductive growth (Leung and Giraudat, 1998; Rock, 2000; Rohde et al., 2000b). Thus, seed germination is slowed keeping the seeds alive during the winter. The study examined the effect of Spermidine on seed sensitivity to ABA concentration during seed germination. Although endogenous ABA [produced by the plant] is essential for the induction of dormancy and the germination of mature Arabidopsis, the seed can be suppressed by as little as 3 uM exogenous ABA (Finkelstein et al., 2001). Genetic studies in Arabidopsis demonstrated that the first major ABA accumulation phase is maternally derived and immediately precedes the maturation phase of seeds (Karssen et al., 1983). The second major ABA accumulation in wild-type Arabidopsis seed depends on synthesis in the embryo itself (Karssen et al., 1983). Although the embryonic ABA accumulates to only one-third the level accumulated at 10 days after pollination, it is essential for the induction of dormancy, which is maintained despite a substantial decrease in ABA by seed maturity (Finkelstein et al., 2001). The ABA content of a wild-type mature dry seed is only 1.4-fold that of the peak ABA level in a nondormant ABAdeficient mutant, suggesting that endogenous ABA is not the only signal for dormancy maintenance in mature seed. Despite the strong evidence for a fundamental role of ABA in regulating dormancy, this is a complex trait controlled by many factors (Finkelstein et al., 2001). Seed maturation begins when developing embryos cease cell division and University of Maryland 176 start growing and is correlated with an increase in seed ABA content, consistent with the fact that ABA can induce the expression of a cyclindependent kinase inhibitor (ICK1) (Wang et al., 1998) that would lead to cell cycle arrest at the G1/S transition (Finkelstein et al., 2000) stopping seed germination. By varying the concentration of Spermidine and abscisic acid exposure to seeds then observing the number of surviving seeds, the effects of Spermidine on seed germination can be measured. Spermidine interacts with macromolecules like DNA, RNA, acid phospholipids and proteins (Tassoni et al., 2000). In plants, they have been implicated in a large range of growth and developmental processes including germination of seeds and response to environmental stresses (Buuren et al., 2000). Based on the results of a similar study, the authors suggest that Spd may prevent chilling injury during harsh winters in squash by a mechanism involving protection of membrane lipids. Chilling injury is thought to involve alteration of membrane structure. Raison and Lyons proposed that the primary event causing chilling injury is a phase transition in the molecular ordering of membrane lipids. The membrane phase transition would have many deleterious effects on the tissues, including increases in membrane permeability and alteration of the activity of membrane proteins. All the results support the view that Spd and may inhibit chilling injury by slowing [lipid disfigurement] in Arabisopsis (Bouchereau et al., 1999). Spermidine is expected to reduce seed sensitivity to abscisic acid leading to increased seed germination. At [0.5 mM] concentration, 100 % of seeds germinate (data not shown) to clearly demonstrate that several plants responded to low temperature acclimation with a uniform and substantial increases in Spd (Tassoni et al., 2000). An inhibition of the seed germination percentage was also observed [when too much Spd is added] (Bouchereau). Methods for Determining Guard Cell Specific Genes Stock and working solution preparation The primers were obtained and prepared by adding necessary amounts of water. Afterwards, each solution was mixed using Vortex and spun using a bench top centrifuge. The working solutions were prepared by adding 5 uL of the primer solution to 45 uL of water into a 1.5 mL Eppendorf centrifuge tube. These solutions were also mixed and spun. They were then placed in ice. Genomic DNA preparation To prepare genomic DNA, 2 Arabidopsis leaves were cut and placed into 1.5 mL microcentrifuge tubes. This was repeated 20 times. Added to each tube was 100 uL of Edward’s buffer. Using a pestle, the leaves were ground. The mixtures were centrifuged for 15 minutes at 14,000 rpms. The supernatant was transferred to new tubes. Then added to the solution was 450 uL of isopropanol to precipitate the mixture. It was inverted 5 to 10 times, then left to incubate at room temperature for 15 minutes. Afterwards, the mixtures were centrifuged for 15 minutes at 14,000 rpms. The precipitate was removed, resuspended, then centrifuged at 14,000 rpms for only 5 minutes. The DNA now could be resolubilized and separated by adding 100 uL of autoclaved water. Then the precipitate was resuspended using the pestle. The DNA was then incubated in the shaker for 15 minutes at 60C. The DNA was resuspended without the pestle and centrifuged at 14,000 rpms for 15 minutes. The supernatant, now containing the DNA, was extracted and stored at -20C. Amplified promoters using PCR for GoTaq enzyme First, the substances were thawed, mixed, and spun except for the enzymes obtained. The GoTaq solution was prepared as follows. It included 5 uL of buffer solution, 14 uL of autoclaved water, 1 uL of reverse and forward primers, 2 uL of dNTP, 1 uL of MgCl 2 , 0.5 uL of gDNA and 0.5 uL of polymerase. PCR tubes were obtained and solutions were transferred to the tubes. The PCR was run using the following settings. First, 95C for 2 minutes, 95C for 20 seconds, 55C for 20 seconds, 72C where time depends on length of primer (1 minute per kb), 72C for 5 minutes, and 4C infinitely. Agarose gel electrophoresis preparation The gel was prepared by adding 30 mL of TAE per 0.3g of agarose into a 500 mL Erlenmeyer flask. The solution can only be dissolved by heat. Thus, the solution was placed into the microwave for 1 minute. The solution was then cooled by transferring it several times between a 250 mL Erlenmeyer flask. Then, 0.3 uL of Ethidium Bromide was Celebrating 20 Years of Student Research and Scholarship 177 added to the smaller flask per 30mL of solution. The solution was swirled and then poured into the electrophoresis tray. It took approximately 15 minutes to polymerize. Agarose gel electrophoresis The GoTaq solutions were each placed into a well. Then, 6 uL of Ladders was added to the last well. It was used as a reference for size of DNA fragments. Then, the gel was run at 135 Volts until the fragments migrated to approximately two-thirds of the gel. Then using Quantity One: Gel Doc XRTM program, pictures were taken for length confirmation. If a positive match, then the promoters of interest were amplified using KOD. If not, the process was repeated to confirm the negative result from the GoTaq enzyme. Amplified promoters using PCR for KOD enzyme First, the substances were thawed, mixed, and spun except for the enzymes obtained. The KOD solution was prepared as follows. It included 5 uL of buffer solution, 32.5 uL of autoclaved water, 1.5 uL of reverse and forward primers, 5 uL of dNTP, 3 uL of MgSO 4 , 0.5 uL of gDNA and 1 uL of polymerase. PCR tubes were obtained and solutions were transferred to the tubes. The PCR was run using the following settings. First, 95C for 2 minutes, 95C for 20 seconds, 55C for 20 seconds, 70C where time depends on length of primer (25 seconds per kb), 70C for 5 minutes, and 4C infinitely. Agarose gel electrophoresis The gel was prepared in the same way as previously described. Before being added to the wells, one-fifth of KOD’s volume in 6X Sample Buffer dye was added to each PCR tube. There was excess amount of KOD solutions so each was divided into two electrophoresis wells. After adding the KOD solutions, 6 uL of Ladders was added to the final well. Then, the gel was run at 135 Volts until the fragments migrated to approximately two-thirds of the gel. Then using Quantity One: Gel Doc XRTM program, pictures were taken for length confirmation. If the lengths were correct, then the DNA would be extracted. If there was a negative result, the amount of GoTaq enzyme cycles was reduced to 10, purified, and then amplified using the KOD enzyme. PCR purification The PCR product was obtained and transferred to 1.5 mL microcentrifuge tubes. Added to the product was 62.5 uL of Binding solution I. This was then transferred to a separation column and centrifuged for 1 minute at 14,000 rpms. The flowthrough was discarded, then 500 uL of wash was added. The mixture was then centrifuged for 30 seconds at 14,000 rpms. The flowthrough was discarded and then the DNA was washed again. The residual wash was then removed via centrifugation for 1 minute at 14,000 rpms. The DNA was then eluted by adding 35 uL of water to the membrane, letting it incubate for 2 minutes, then centrifuging again for 1 minute at 14,000 rpms.