The purpose of my research began as finding the impact of heat stress on stigma receptivity in Arabidopsis thaliana, a model organism. At the beginning, I did not realize how broad this purpose was, it eventually developed into finding the increase or decrease in various levels of specific enzymes associated with stigma receptivity, the altered time window associated with the change in peak enzymes, thus altering the entire timeline for plant reproduction, and eventually opening up a ton of new questions about the impact of these seemingly small changes in plant growth and reproduction.
The majority of the work I have done this semester was familiarizing myself with the anatomy and physiology of Arabidopsis thaliana in order to properly analyze the importance of the changes in enzyme levels and morphological changes in the stigma due to heat stress. Stigma receptivity to pollen has been associated with the presence of specific enzymes including peroxidase, esterase, and alcohol dehydrogenase. We have been growing all plants at 23 degrees Celsius (optimal growing conditions), then in the 6th week, for two weeks, the plants were split up into the optimally grown group and the heat stressed group, the heat stressed being grown under 29 degrees Celsius. This protocol is more in line with actual field conditions (rather than the previously mentioned “cook and look” approach) and thus more applicable to what is likely to happen as plant community’s change along with climate change. Both heat stressed and optimally grown plants were grown under optimal conditions for the initial 6 weeks of growth, until they were split into their test in groups for the remaining 2 weeks.
As my plants are growing now in their appropriate groups, we are approaching the period of vigorous testing, occurring during the final 8th week around a 5 day period. The testing of a high n number of plants will allow for acceptance in scientific journals and the 5 day period occurs around the time previously determined at a peak of stigma receptivity to pollen. This period is used in order to incorporate any changes to the enzyme peak window, thus the period of receptivity. The quantitative analysis of these enzymes during the 8th week of growth is pertinent to understanding the ecological and morphological changes we assume will occur as a result of the heat stress. I have also been looking into the biochemical properties of these enzymes and processes during periods of importance for stigma receptivity because the analysis of the impact is based on the lack of function in these plants due to halting of their progression toward reproduction.
This past year has presented an incredible amount of opportunity for growth in the biology field, vastly expanding my knowledge of the research process and application of previous knowledge. Though, I have a difficult time dealing with the failures of the research experience, it only make the successes that much greater! Prof. Kipp and I have spent the past several months working on extensive literature reviews on our topic in order to interpret the bigger picture results, ultimately what this will mean for plant life as we dive further and further into a world damaged by global warming. I am lucky to be continuing research with Erica Kipp into the summer and fall semester, working towards publication in an academic journal and extending the results of my research into the biochemistry field of application. Overall, I am extremely grateful to have had this experience and am excited to continue working toward greatness!
Since the last blog, Prof. Kipp and I have had a range of achievements and setbacks. Since we are testing the plant past flowering, growing the Arabidopsis takes a long period of time and with that we have needed a large window to work with. Since we had the long Christmas break and thought we were ready to begin growing the plants for round two (round one being before Sandy), I then had a recent personal conflict. Simply said, there has been much difficultly in arranging the time for simply growing the plants through flowering. However, we have all of our test kits on deck and are currently getting back to the schedule and are ready to work! This surely has taught me about time management and how unpredictable research can be.
I am very excited to have begun working with another Pace student, Ceylan, who is joining Erica Kipp’s research assistants and will be helping us as well as bringing in another project on global warming research. Though, I have read countless academic papers on my own topic, stigma receptivity, it is refreshing to hear another part of the global warming puzzle and being involved in their process as well. This will expand my knowledge of Arabidopsis as well as expose me to other protocols and research techniques that I would not have been exposed to without this experience, so I am very grateful.
I am in the process of growing large volumes of Arabidopsis, and until this step is complete, Prof. Kipp and I are anxiously awaiting the enzyme testing. Even without our results on the table, I have learned a great deal about the research process, collaboration, and the importance of documentation. I find that this experience has helped me in other instances that require any type of research, time management, and seeking information at great lengths (past Google searches). Research documentation exceeds that of any documentation from other lab sections I have taken. The journals scientists keep are not meticulous but precise and must contain every small step or deviation from protocol.
Thus far, In our research we have done a substantial amount of preliminary work to understand the various elements of stigma receptivity, including the enzymes that are present and that dictate the window of receptivity in Arabidopsis thaliana. We have found that there are anticipated levels of esterase, peroxidase, and alcohol dehydrogenase present during this period of time. We will be testing how this window is altered due to heat stress by testing these three enzymatic markers associated with receptivity. The expected results will be an altered window and alterations in the presence and concentrations of these enzymes.
We have been working to target specific protocols for targeting esterase, peroxidase, and alcohol dehydrogenase both qualitative and quantitatively. For esterase, we will be using an alpha-naphthyl acetate test. Four separate tests will be performed for determining peroxidase activity; benzidine solution, Peroxtermo Ko Test, concanavalin A-peroxidase method, and H2O2. Alcohol dehydrogenase presence will be determined using the Baker’s test. Each of these tests will be done over certain periods of time, based on the non-heat stressed “normal” receptivity window, to determine the changes due to heat stress.
Since Hurricane Sandy shut down the Pace Lab for almost 2 weeks, much of the research has been at a hault, slowly getting back on track. However, I am confident that utilizing the winter break to grow large volumes of Arabidopsis will give us plenty of data for accurate analysis of each enzyme. We have the enzymes that need to be tested, the next step is determing the alterations of their presence and what that means for the future of these plants with an increasing threat of global warming.
The purpose of Dr. Erica Kipp and my research project is to study the effects of heat stress on plants, specifically Arabidopsis thaliana. Environmental and ecological changes due to temperature increases, such as those associated with global climate change, have been well documented, sparking research in quantifying plant responses to heat stress. Current research suggests that temperature changes can be associated with altered morphology, decreased crop production and lower fecundity for many species. This can have far-reaching effects on economically valuable ornamental and agricultural plants. Literature shows that the stigma of optimally grown (23°C) Arabidopsis thaliana, a commonly used “lab rat,” is receptive to pollen during a very narrow window of time. Stigma receptivity (to pollen) is associated with the presence of stigmatic enzymes: peroxidase, esterase, and alcohol dehydrogenase, in particular.
In order to test how heat stress can affect stigmatic receptivity, and thus reproductive success of the plant, we would like to test for the presence of these enzymes during flowering and compare heat stressed plants with optimally grown plants. How does heat affect these enzymes in terms of presence and concentrations? Is the window of receptivity altered? Are there any morphological changes to the stigma as a result of heat stress? And if so, can the morphological changes be associated with enzyme changes?
Many labs currently approach quantifying heat stress with what has become known as “cook and look,” a research approach where plants are heated to 45 degrees Celsius or more during a specific developmental phase. In this research project, plants will be heat stressed at 29 degrees Celsius, not much higher than optimum temperature, and for the life of the plant, not just during one stage. Also we will be coordinating 12/12 hr days, meaning 12 hours of day and 12 hours of night. This is more in line with actual field conditions and thus more applicable to what is likely to happen in the face of global warming. Thus far, Dr. Kipp and I are still in the premature stages of our research, and have been working on developing protocols for harvesting seeds from optimally grown Arabidopsis plants encorporating seed collection, sterilization, and storage.