By probing the mechanism behind plant responses to drought, understanding the components of a plant’s internal clock, and investigating ways to harness plants to produce vaccines, undergraduates at UCSD are finding ways to use plants to improve people’s lives.
by Priva Patkar, Safwanul Haque, Catherine Nakao | staff writers | UTS Vol. 3 (2012-2013)
Over the past fifty years, researchers at UCSD have been putting their plants to work. The Yanofsky lab has made groundbreaking discoveries of fruit development genes involved in fruit formation and opening. The Estelle lab studies the plant hormone auxin and its impact on plant growth pathways. These are only some of the labs striving to elucidate how plants perform their core functions. Contrary to common belief, plants possess intricate internal processes and functions that are highly relevant to our own lives.
Photosynthesis is a crucial process for plant growth. Leaves have structures called stomata on their lower surface that regulate carbon dioxide and allow for water exchange with the atmosphere. In transpiration, plants undergo water exchange to maintain fitness. Most plants adaptively use photosynthesis to make sugars and store them as starch during the day. They break down this starch for energy during the nighttime, allowing the plants to grow and flourish.
When Plants Face Drought
Stress conditions, like drought or lack of nutrients, cause plants to produce the hormone abscisic acid (ABA). ABA stimulates stomatal guard cell closure to prevent further water loss and cytosolic calcium ions are involved in this process. A cell’s surface is embedded with a multitude of proteins for sensing signals and compounds, like calcium ions, with great specificity. Desiree Nguyen, an undergraduate researcher in the laboratory of Dr. Julian Shroeder, attempted to characterize the molecular role of two types of calcium sensors in the stress response: Calcium Dependent Protein Kinases (CPKs) and Calmodulin-like (Cml) proteins.
CPK sensors were found to be more effective than Cml sensors in inducing cytosolic calcium production. Nguyen explored various plant strains with different mutations in these sensors. She discovered two different strains of plants, cpk3/6 and cpk4/11. Each strain consisted of plants with mutations in the two respective sensor kinases, making them “double mutant” plants. These double mutants displayed impaired stomatal opening, a process that relies on ABA and calcium. Meanwhile, plants with “knocked out,” and thus non-functional, Cml9 protein were drought sensitive and their seedlings showed ABA-dependent growth. Nguyen noted that although the Cml sensors did not display significant involvement in ABA-dependent growth, the calcium dependent kinases hold some promise. If so, the impact is great as the kinases could be harnessed to better control plant transpiration and growth in stress conditions.
Today, the underlying mechanisms are not fully understood, but Nguyen and the Shroeder lab continue to study the proteins, hoping to decipher the molecular pathway. They plan to isolate cpk3/cpk4/cpk6/cpk11 and cpk5/cpk6/cpk11/cpk23 “quadruple mutant plant lines,” each with mutations in 4 different calcium dependent kinases. Presently, Nguyen has isolated one quadruple mutant plant line and is in the process of studying the resulting root growth phenotypes. She hopes to compare the double and quadruple mutant in future experiments.
All the confirmed mutant lines will undergo biological experiments for stomatal movement in response to various stimuli, such as ABA. These phenotypic studies can help elucidate the role of the proteins in a plant’s stress tolerance to environmental factors. Improved understanding of this complex transpiration molecular pathway can facilitate the development of innovative strategies to combat harmful environmental conditions faced by plants. Global climate change is likely to bring drastic changes in weather conditions including extreme drought. Research by Nguyen, and others like her, holds the potential to counter the agricultural impact of these environmental stresses. With more study, plants could survive drought and produce increased crop yields.
Tiny Molecules, Ticking Clocks
Other undergraduate researchers at UCSD have illustrated an alternate role for ABA in plants: in their circadian rhythms. The ABA pathway resides deep at the molecular basis of plants but circadian behaviors can be observed even externally. Ying Sun, a student in Dr. Steve Kay’s lab, investigated the details of this circadian clock in Arabidopsis thaliana.
Arabidopsis is often the model plant of choice in research. Like most plants we eat, it is a dicot and has pathways applicable to most other plant systems. Plant internal processes like photosynthesis and metabolism of starch all occur in distinct and repetitive cycles within a period of about 24 hours.This phenomenon is known as a circadian rhythm and such rhythms are present in animals, plants, and fungi. For example, photosynthetic activity occurs during the day as it needs light to proceed. Understanding circadian rhythms in plants can affect our daily food sources, and future global warming will require plants to acclimate to dynamic environments, weather and periods of daylight and night.
The main components of the circadian clock are transcription factors. Genes in a plant are transcribed into RNA which is translated into functional protein that help the plant perform certain functions. Specific transcription factors can determine the timing and quantity of this protein translation. TOC1 protein is a key player in the plant circadian clock produced during the night and it encourages CC1 and LHY production. The latter are transcription factors that interact with clock genes. CC1 and LHY form a negative feedback loop inhibiting production of TOC1 during the day. Over the course of the day, as CC1 and LHY break down, their lower levels allow TOC1 to reaccumulate overnight and continue the cycle.
Sun was able to identify transcriptional co-activators that may help in the transcription process and enable transcription factors to recruit RNA polymerase and nucleotides. She screened and selected mutant plants showing possibly altered circadian pathways. The output of such pathways can often be observed externally. For example, some outputs that follow distinct rhythms are petal opening and flowering. Circadian rhythms similarly affect the expression of transcription factors, and changing the genes for these factors can affect the whole function of a plant.
Sun will examine plants with various disruptions in these co-activators for altered phenotypes. Her observations will focus on circadian-related outputs and whether the experimental plants will maintain circadian responses to various stresses. Though the research may be academic in its pursuits, the resulting knowledge can go far in establishing a foundation to enhance agricultural yield.
Turning Chloroplasts Into Vaccine Machines
Plants can also be a good resource for achieving medical progress. For thousands of years, malaria has caused millions of deaths around the world. People previously believed there was no possible cure or prevention for this fatal disease. However, in 2012, the biological science research division at UCSD kindled new hope for malaria victims. The possibility of eradicating malaria, as with smallpox, finally seemed within our grasp. Lauren Tomosada, a student researcher in the lab of the renowned Dr. Stephen Mayfield, was a part of this research.
The female mosquito is the vector that transmits malaria. Within the gut of the mosquito, malaria agents reproduce sexually, producing zygotes. Tomosada aimed to stop this stage of reproduction by using algae plants. The algal chloroplast is used as a platform to produce subunit vaccines. These vaccines cause the production of antigens in the body. The specific antigens Plasmodium falciparum surface protein 25 (Pfs 25) and 28 (Pfs 28) were difficult to produce in other forms of vaccines due to their complex structure and lack of sugar coat.
However, these unique features were formed in the algal chloroplast. Using western blot, a process used to detect specific proteins, it was clear that the chloroplast was a successful agent in producing these antigens. Although subunit vaccines are generally more costly to produce than traditional vaccines, algae are widely used as a platform for subunit vaccine production because they are relatively easy and inexpensive to grow, have a short lifespan, and do not have viral contaminants within their systems, making them an ideal implement in vaccine production.
This algae vaccine has yet to be tested on humans but experiments in mice models have shown successful results. The surface proteins elicit antibodies against Plasmodium falciparum, which disrupts the sexual development of malaria parasites within the mid-gut of the mosquito. The disruption can prevent malaria transmission from one host to the next, as malaria is most often transmitted when mosquitoes bite human hosts.
The past year showed promising progress in plant research at UCSD. Examining the effects of calcium dependent sensors and abscisic acid on leaf stomatal guard cells is important in helping plants cope with drought conditions and preserving their essential internal water supply. Similarly, knowledge from continued study of transcription factors involved in the plant circadian clock will further come into play for producing ideal plant growth. Both hold great importance as the effects of global climate change take effect on our world. On an alternate note, as diseases like malaria continue to claim lives, new methods for vaccine production give a promising hope for a cure. The ability to produce algae efficiently and sustainably is a great solution not only for producing alternative fuels, but also for producing new vaccines in the future. The plant world holds great and extensive potential and further study is sure to bring a brighter and better future for generations to come.
Priya Patkar is a Biochemistry and Cell Biology major from Sixth College. She will be graduating in 2013. Safwanul Haque is a Microbiology major from Thurgood Marshall College. He will be graduating in 2014. Catherine Nakao is a Human Biology major from Sixth College. She will be graduating in 2016.