Progress 01/01/04 to 12/31/09
Outputs
OUTPUTS: Over the 5 year period 12 different experiments were conducted using Plectranthus species and Arabidopsis. The purpose of these experiments was to determine if leaves can be re-greened after nitrogen deficiency and if so, how the plant does this. These experiments utilized state-of -the-art techniques not typically employed for foliage plants such as chlorophyll analysis, proteomics, confocal and transmission electron microscopy, nutrient element and carbohydrate analysis. Techniques created include development of a re-greening time line and protocol for TEM. Target audiences include undergraduate (2) and graduate (1) students, commercial growers of horticulture crops as well as scientific colleagues. This was accomplished through students conducting this research, the incorporation of this research into plant nutrition lectures for our distance plant nutrition class and senescence and leaf abscission lectures for our woody plant growth and development and nursery management classes. Presentation of this research at national meetings with publication in the abstract books and the journal publications including one directly on-line are available to my colleagues. PARTICIPANTS: In addition to my role as PI, there have been two undergraduate and one graduate students plus a research technologist involved in this research. I have cooperated with with Dr. Radka Vankova, Institute of Botany, Academy of Sciences, Czech Republic for the cytokinin studies. Colleagues, Dr. Michelle Jones at Ohio State University, Dr. Rosanna Freye at University of Florida and Dr. Mark Bridgen, Cornell University were very helpful in providing plants and expert advice. TARGET AUDIENCES: Target audiences include undergraduate (2) and graduate (1) students, commercial growers of horticulture crops as well as scientific colleagues. This was accomplished through students conducting this research, the incorporation of this research into plant nutrition lectures for our distance plant nutrition class and senescence and leaf abscission lectures for our woody plant growth and development and nursery management classes. Presentation of this research at national meetings with publication in the abstract books and the journal publications including one directly on-line are available to my colleagues. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Leaves of Arabidopsis like Plectranthus can be re-greened after nitrogen deficiency. Unfortunately, the proteins expressed are different so the use of Arabidopsis as a model system for gene identification is not possible. However, genes involved in nitrogen deficiency of Arabidopsis (TTG2 and BLG1) were identified and may be involved as part of a stress response by the plant. Under nitrogen deficiency, Plectranthus stores starch which is slowly broken down over time. Proteins expressed corroborate this finding. Not all Plectranthus species can be re-greened after nitrogen deficiency. Plectranthus parviflorus can be grown without nitrogen for up to six months and then re-greened. This phenomenon has spawned a new area of research. Through this project the future of science has been positively impacted as 1 graduate student completed his MS degree then continued to a PhD; 1 undergraduate student completed his research and continued on to a MS degree/possibly a PhD; 1 undergraduate student completed an independent/grant funded research program and will graduate soon.
Publications
- Korus, K. M. E. Conley, E. E. Blankenship and E. T. Paparozzi. 2010. Storage and breakdown of starch aid P. parviflorus in leaf re-greening after nitrogen deficiency. Review of Undergraduate Research in Agriculture and the Life Sciences (RURALS) 5(1):1-16. http://digitalcommons.unl.edu/rurals/vol5/iss1/1/
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Screening studies Petunia experiments. These 3 experiments were conducted to determine if petunia would repeatedly re-green after nitrogen deficiency. We are searching for a plant which has a small genome that will eventually be sequenced since Arabidopsis did not express similar proteins to Plectranthus.Dr. Michelle Jones, who gave us the seed, was told of our findings. Species screening for leaf abscission. Through colleagues at the university of Florida and Cornell University at Riverhead, NY, cuttings of three different Plectranthus - ambiguus, Red (a red-leaved species purportedly coleoides ) and Lime y Cerveza were grown in our 0/150 nitrogen hydroponic setup to determine if there was a species in which leaves senesced and abscised due to nitrogen deficiency. The results of this study were presented at the 2009 Undergraduate Research Symposium at UNL. Cytokinin experiments. Two experiments designed to investigate the effect of endogenous cytokinins on nitrogen loss and subsequent leaf re-greening have been just been completed. Leaves from these two experiments will be analyzed for chlorophyll and for the different forms of cytokinins (stored, precursors, active, free hormones). These samples will be sent to a colleague in the Czech Republic to determine if cytokinin levels change during nitrogen deficiency and re-greening. There are only four laboratories in the world capable of performing this specific kind of analysis and we were fortunate to be accepted for collaboration. PARTICIPANTS: In addition to my role as PI, there were one undergraduate and one graduate students plus a research technologist involved in this research. I am responsible for designing all experiments, keeping track of all progress, providing funds, some training and all written communications. The undergraduate student was responsible for growing the Species Screening experiments which included growing and monitoring the plants, harvesting, collecting visual and dry weight data, analyzing leaves for chlorophyll and comparing leaf loss among 3 species of Plectranthus. The graduate student is a MS student who has conducted the 3 cytokinin experiments (preliminary and two complete experiments). Without my technologist training these students and performing this research would not be possible. My graduate student is from India and despite a stellar record had to be taught how to weight chemicals and wash glassware! She also supervises their projects and came in on weekends to change hydroponic solutions for our petunia screening studies. Additionally she coordinated all space usage, double checked all experimental designs, performed general trouble-shooting for chemical safety adherence as well as rechecked nutrient and carbohydrate calculations. We use toxic elements in our analyses and I rely on her to make sure EPA safety precautions are always followed. Cooperation with Dr. Radka Vankova, Institute of Botany, Academy of Sciences, Czech Republic is essential for the cytokinin studies. Colleagues, Dr. Michelle Jones at Ohio State University, Dr. Rosanna Freye at University of Florida and Dr. Mark Bridgen, Cornell University were very helpful in providing plants and expert advice. TARGET AUDIENCES: Target audiences include undergraduate and graduate students, commercial growers of horticulture crops as well as scientific colleagues. This was accomplished through the incorporation of this research into plant nutrition lectures for our distance plant nutrition class and senescence and leaf abscission lectures for nursery management classes. Presentation of this research at one University sponsored meeting and journal publication of the carbohydrate information served my colleagues. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Petunia experiments. This cultivar of petunia, obtained from Dr. Michelle Jones at Ohio State repeatedly showed iron deficiency as well as nitrogen when growing in our hydroponic system. Thus, petunia is not a candidate for our research. Species screening for leaf abscission. All 3 species responded differently to nitrogen deficiency. P. ambiguus lost leaves under nitrogen stress and despite the return to 150 ppm did not recover. Red did not respond visually to the no nitrogen treatments. However, chlorophyll levels did decrease with treatment in a similar pattern to P. ambiguus. Leaves did not abscise. Leaves of Lime y Cerveza initially turned yellow and started to abscise, but soon the plants adjusted to the hydroponics and no longer lost leaves. Future experiments will compare the response of P. parviflorus with P. ambiguus. Impacts: The re-greening phenomenon is not unique to P. parviflorus, thus, hypothetically it could be induced and change the way cuttings/plants are shipped. P. parviflorus also displays a possibly unique ability to sit in solutions for as long as 6 months without growing and then upon the introduction of nitrogen resume growth. Research on this phenomenon could change the way germplasm is stored and lead to a greater understanding of how plants adjust to climate change.
Publications
- Korus, K. M. E. Conley, E. E. Blankenship and E. T. Paparozzi. 2010. Storage and breakdown of starch aid P. parviflorus in leaf re-greening after nitrogen deficiency. Review of Undergraduate Research in Agriculture and the Life Sciences. Accepted.
- Fallon, M. and E. T. Paparozzi. 2009. Using nitrogen deficiency to determine timing of yellowing and leaf abscission in three different species of the genus Plectranthus. UCARE Undergraduate Research Poster Session. April 2009.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Two experiments on leaf re-greening of Plectranthus after nitrogen deficiency have been completed. Leaves from these two experiments were analyzed for soluble carbohydrates and visualized for structural carbohydrates using confocal and transmission electron microscopy. Another recently completed experiment trialed different species of Plectranthus. The purpose was to help determine if many species of Plectranthus respond similarly. The fourth experiment that is underway is a preliminary study on the role of cytokinins and re-greening after nitrogen deficiency. These samples will be sent to a colleague in the Czech Republic to determine if cytokinin levels change during nitrogen deficiency and re-greening. PARTICIPANTS: In addition to my role as PI, there were two students and a research technologist involved in this research. I am responsible for designing all experiments, keeping track of all progress, providing funds, some training and all written communications. Additionally, as a trained electron microscopist, I worked with Mr. Elowsky and Dr. Chen in the UNL Microscopy Center on the confocal and electron microscopy. The first student was an undergraduate student who was responsible for growing the plants, harvesting and conducting carbohydrate analyses on leaves. The second student is a new MS student who is running the preliminary cytokinin experiment. Without my technologist training these students and performing this research would not be possible. She supervises their projects and comes in on weekends to change hydroponic solutions. Additionally she coordinated all space usage, double checked all experimental designs, performed general trouble-shooting for chemical safety adherence as well as rechecked nutrient and carbohydrate calculations. We use toxic elements in our analyses and I rely on her to make sure EPA safety precautions are always followed. TARGET AUDIENCES: Target audiences include undergraduate and graduate students, commercial growers of horticulture crops as well as scientific colleagues. This was accomplished through the incorporation of this research into plant nutrition lectures for our distance plant nutrition class and senescence and leaf abscission lectures for our woody plant growth and development and nursery management classes. Presentation of this research at one national meeting and subsequent printing of this information in the abstract book served my colleagues. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have found that not all Plectranthus spp. re-green without leaf loss. Plectranthus fruticosa displays classic nitrogen deficiency symptoms by turning yellow, lower leafves withering and browning and then abscising. This is important as it could allow us to do some comparative genomics to try to find which genes are involved in the re-greening process. To do this we will need additional funding and the appropriate genomic sequence in microarray format. The undergraduate student that worked on the carbohydrate project has entered graduate school. If this opportunity was not available, through hatch funding, he probably would be working at a greenhouse or nursery/garden center now. We have discovered that Plectranthus appears to be a plant that under nitrogen deficiency accumulates and stores starch. This has implications for the long term plant survival and for starch production for biofuels. This would not be possible without the monies that Hatch provides to land grant universities as most granting agencies want most of the research done before they will fund a project.
Publications
- Korus, K., Conley, M. E. and E. T. Paparozzi. 2008. Qualitative and Quantitative Analysis of Soluble Sugar Content in Leaves of hydroponically grown Swedish ivy at varying periods of nitrogen deficiency and subsequent re-greening. HortScience 43:1285.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Two experiments on leaf re-greening of Arabidopsis(Columbia O) after nitrogen deficiency were conducted. Plant samples were analyzed for protein differences (confirmed by MS/MS) and semi-quantitative RT-PCR was performed in ordered to relate the proteins back to their respective genes. TGG2-1 mutant plants were also obtained and used in one of the above experiments in order to differentiate between 2 genes of interest. Results have been shared in poster format at the 2007 American Society of Plant Biologists meetings and a paper is being re-edited for resubmission to Planta. One experiment on leaf-re-greening of Plectranthus is just being completed. Samples have been viewed under the confocal microscope, will be used for carbohydrate analysis and will be preserved for light and electron microscopy.
PARTICIPANTS: In addition to my role as PI, there were two students and a research technologist involved in this research. I am responsible for designing all experiments, keeping track of all progress, providing funds, some training and all written communications. The first student was a MS student who became proficient in protein analysis and RNA methods. He successfully defended his thesis at the end of August and has entered a PhD program. The second student is currently a senior undergraduate student. He learned basic lab techniques and how to conduct carbohydrate analyses on leaves. He is currently finishing his first solo greenhouse experiment and thanks to this project is considering applying to graduate school. Without my technologist this research would not be possible. She was responsible for the leaf anatomy portion of this project. Additionally she trained both students on hydroponic techniques, coordinated all space usage, double checked all experimental designs,
performed general trouble-shooting for chemical safety adherence as well as rechecked nutrient and carbohydrate calculations. We use toxic elements in our analyses and I rely on her to make sure EPA safety precautions are always followed. The National Foliage Foundation has provided funding for some of the protein work.
TARGET AUDIENCES: Target audiences include undergraduate and graduate students, commercial growers of horticulture crops as well as scientific colleagues. This was accomplished through the incorporation of this research into plant nutrition lectures for our distance plant nutrition class and senescence and leaf abscission lectures for our woody plant growth and development and nursery management classes. Presentation of this research at one national meeting and subsequent printing of this information in the abstract book served my colleagues. A poster presentation to the Florida Foliage growers international convention was the venue for initial commercial exposure. This was followed up by a short article in a grower publication - Ornamental Outlook.
Impacts
Using 1-dimensional SDS-PAGE gels, proteins expressed upon induction of nitrogen deficiency and disappearing upon resupply of nitrogen were present at approx. 60kD and were identified by MS/MS as beta-glucosidase and myrosinase. They were traced back to two genes BGL1 and TGG2, respectively. Using RT-PCR, under nitrogen deficiency BGL1 transcript levels showed increases while TGG2 did not. This means that when Arabidopsis plants experience nitrogen deficiency BGL1 is turned on and a Beta-glucosidase is produced. Its production is then gradually turned off when nitrogen is re-supplied. It was not found in senescent leaves. Additionally, this enzyme is linked with ABA which is involved in a number of plant stress responses including plant senescence. It is possible that this enzyme affects ABA in such a way as to help the plant tolerate the stress until the stress can be relieved. This is the first evidence of proteome changes in Arabidopsis when plants are deprived of
nitrogen. When leaves of Plectranthus (Swedish ivy) are re-greened after nitrogen deficiency, different proteins (than in Arabidopsis) are expressed. In addition to proteins of the heat-shock group (HSP-70), we found that transketolases were expressed when the leaves were yellow and the plant was nitrogen stressed. These proteins were no longer expressed when the plant re-greened. These proteins are key in both the Calvin Cycle and the oxidative phosphorylation pathway.
Publications
- Widhalm, J. R. J. M Stone and E. T. Paparozzi. 2007. Nitrogen deficiency-induced senescent A. thaliana rosette leaves accumulate defense-related proteins. Plant biology and Botany Poster Abstracts. P. 59.
- Widhalm, J. R. 2007. Accumulation of defense-related proteins in nitrogen deficiency-induced senescent A. thaliana rosette leaves. MS Thesis University of Nebraska, Lincoln, NE.
- Paparozzi, E. T. 2007. Powerful Proteins.Ornamental Outlook. February p.34-36.
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Progress 10/01/05 to 09/30/06
Outputs
Two experiments were run in which Arabidopsis was tested to see if leaves would re-green twice. The plants were grown under short days in hydroponics at 120 ppm N (control) with all other nutrient elements present for approx. 8 weeks. Then all were switched to 0 nitrogen for approx. 4 weeks and then half of the plants were returned to their original 120 ppm N concentration. At six weeks, a few of the plants from each treatment were harvested and nutrient concentrations were switched again. Upon harvest, leaves were analyzed for protein content. Nitrogen deficiency in Arabidopsis is typified by reddening of the undersides of all leaves. In these experiments the reddened leaves were able to be re-greened upon the re-introduction of nitrogen. Despite receiving short days the majority of plants flowered due to nitrogen stress. In terms of protein content, Rubisco was plentiful after 6 weeks of N deprivation, but not after 12 weeks. When nitrogen stress was induced a new
band of proteins appeared at the 60 kDA line and these were identified to be associated with myrosinases and beta glucosidases (gene family BLG1). From this is it appears that BGL1 is expressed under nitrogen deprivation and down-regulated upon re-greening. During the second re- greening, old leaves turned yellow/brown and died while down-regulating BGL1. Simultaneously, the new leaves fully re-greened, but in one experiment mysteriously also expressed BGL1. Further experiments are underway to determine what is occurring. Swedish ivy re-greening experiments indicated that Rubisco appears to be serving as a storage protein to aid in repeated re-greening. Preliminary leaf anatomical and chlorophyll observations indicate that chloroplasts are present, but not functioning when N is deficient.
Impacts
Leaves are an essential as well as esthetic part of plants. Without healthy green leaves, plants will not flower and fruit. Keeping leaves on plants means producing the highest quality as well as quantity of apples, ears of corn, florets of broccoli, beautiful cut flowers and so forth. Figuring out how growers can make yellowing leaves return to green means giving them the tools to produce more and better quality corn, apples, broccoli and cut flowers. It will also allow them to apply nitrogen more efficiently and thus, reduce groundwater contamination.
Publications
- Paparozzi, E. T., Widhalm, J. R. and M. E. Conley. 2006. The role of proteins in leaf re-greening. HortScience 41:1018.
- Stroup, W., Guo, S. Paparozzi, E. T. and M. E. Conley. 2006 Design and Modeling Stategies for Nonlinear Response Surfaces. Proceedings 18th Conference on Applied Statistics in Agriculture. Manhattan, KS: Kansas State University Department of Statistics.
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Progress 10/01/04 to 09/30/05
Outputs
Two experiments inducing and reversing leaf yellowing in Swedish ivy were conducted. The first experiment focused on water source - misting cuttings with tap water versus hand watering with deionized distilled water during rooting in preparation for hydroponic experiments. As expected mineral nutrient analysis indicated misted cuttings had substantially more potassium, calcium, sulfur, zinc, manganese, copper, iron and boron in yellowed leaves than cuttings hand watered with deionized distilled water due to elements in the tap water. However, magnesium, a key nutrient in the chlorophyll molecule, remained at a similar concentration under both conditions. This situation was also true at the end of previous experiments in that magnesium was not affected by treatment or time under treatment whether N was present or not. From this we conclude that a plant's ability to conserve magnesium may be key for leaf re-greening. In the second experiment, proteins were extracted
from yellow, green and re-greened leaves of Swedish ivy. Antibodies to the large and small subunit of Rubisco, the most abundant protein in plants were obtained; 2-D gels and Westerns were run in order to quantify the proteins. Initial results indicate that yellow leaves are lacking the nuclear-encoded small subunit, but have an increased quantity (about 33% more) of the chloroplast-encoded large sub-unit. Replications to confirm this observation are underway. Arabidopsis was grown hydroponically for 5 months under six levels of nitrogen from 0 to 120 ppm N with all other elements present. After approx. 8 weeks in the nutrient solutions, all plants were switched to 0 nitrogen for approx. 8 weeks and then returned to their original N concentration. Chlorophyll and anthocyanin concentrations were measured before and after nutrient concentrations were changed. Data is currently under analysis.
Impacts
Leaves are an essential as well as esthetic part of plants. Leaves produce compounds that make fruits and grains healthy to eat. Without healthy green leaves, plants will not flower and fruit. Keeping leaves on plants means producing the highest quality as well as quantity of apples, ears of corn, florets of broccoli, beautiful cut flowers and so forth. Figuring out how growers can make yellowing leaves return to green means giving them the tools to produce more and better quality corn, apples, broccoli and cut flowers.
Publications
- Kocamaz, C, E. T. Paparozzi and S. Kachman. 2006. Evaluation of alternative methods of applying sulfur fertilizers to chrysanthemums. J. Plant Nutrition 29(2): in press.
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Progress 10/01/03 to 09/30/04
Outputs
Hydroponic experiments were run using the plant Swedish ivy. Treatments were 0 nitrogen (N), 0 N switching to150 ppm N, and 150 ppm N continuously. All treatments received the same rates of all other macro and micronutrients. Leaf nutrient analysis revealed that, as planned, all nutrients were at adequate concentrations except for nitrogen. However, nutrient concentrations did shift with treatment. For example, in plants receiving no N (or any other treatment) magnesium and sulfur leaf concentrations did not change. Whereas calcium, phosphorus and potassium leaf concentrations increased when placed into the complete solution whether nitrogen was present or not. As cuttings take 4 weeks to fully root, experiments are underway to determine if mist and water components influenced the initial leaf nutrient concentrations or if this plant has a mechanism that allows key nutrients involved in chlorophyll (magnesium) and photosynthesis (sulfur) to be conserved. A
reproducible method for extracting proteins from these leaves has been developed. Results from preliminary 2-D gels have shown that Rubisco is still very present in yellow leaves. This was unexpected. While there appears to some protein changes, these may not be as dramatic as predicted. The key for re-greening may actually be preserving certain enzymes so the leaf can recover rather when conditions are right rather than synthesis of new or different enzymes. Re-greening experiments using Arabidopsis have revealed that unlike Swedish ivy, Arabidopsis leaves do not yellow due to nitrogen deficiency. Rather, the petiole and undersides of the leaves turn reddish purple. Attempts to keep Arabidopsis vegetative for 1-3 months have been successful. This sets the stage for subsequent re-greening experiments.
Impacts
Leaves are an essential part of plants. Leaves produce compounds that make fruits and grains healthy to eat. Without healthy green leaves, plants will not flower and fruit. Keeping leaves on plants means producing the highest quality as well as quantity of apples, ears of corn, florets of broccoli, beautiful cut flowers and so forth. Figuring out how growers can make yellowing leaves return to green means giving them the tools to produce more and better quality corn, apples, broccoli and cut flowers.
Publications
- Paparozzi, E. T., W. W. Stroup and M. E. Conley. 2005. How to investigate four-way nutrient interactions in plants: A new look at response surface methods. Journal American Society for Horticultural Science in press.
- Paparozzi,E.T. and W.W. Stroup. 2004. Website for response surface methods. http://statistics.unl.edu/Tools/
- Meyer, Christine, Ellen T. Paparozzi, Scott J. Josiah and Erin M. Blankenship. 2004. Assessing color change in woody floral stems. Hort Science 39(4):836.
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