Source: EASTERN MENNONITE UNIVERSITY submitted to
A SABBATICAL TO INVESTIGATE THE RELATIONSHIPS OF PROTECTIVE MEASURES AGAINST EXCESS LIGHT, ANTIOXIDANTS, DEFENSE SIGNALS, AND DROUGHT STRESS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0210716
Grant No.
2007-35100-18267
Project No.
VAR-2007-01987
Proposal No.
2007-01987
Multistate No.
(N/A)
Program Code
56.0B
Project Start Date
Aug 1, 2007
Project End Date
Jul 31, 2009
Grant Year
2007
Project Director
Cessna, S. G.
Recipient Organization
EASTERN MENNONITE UNIVERSITY
1200 PARK ROAD
HARRISONBURG,VA 22802-2462
Performing Department
(N/A)
Non Technical Summary
Drought severely limits forest vitality. In addition to the obvious problems caused by drought on forest health (i.e. reduced water availability leads reduction in wood, paper and pulp production, increased susceptibility to fires, and reduction in forest biodiversity), forest trees that have been subjected to continuous or repeated drought are also more susceptible to damage from several insects, fungi, bacteria and viruses, and this susceptibility furthers the above problems. The purpose of this study is to examine connections between a plant's natural ability to tolerate drought and high light conditions, and increases in susceptibility to insects and diseases.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2030699100020%
2030699102020%
2032420100030%
2032420102030%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
0699 - Trees, forests, and forest products, general; 2420 - Noncrop plant research;

Field Of Science
1020 - Physiology; 1000 - Biochemistry and biophysics;
Goals / Objectives
The two overarching goals of this sabbatical project are enhancing my professional development and training (e.g. gaining new research skills in laboratory and field plant physiology, establishing an on-going collaboration, and improving my undergraduate teaching and research project mentoring abilities) and research (answering an important research question with applications to agriculture and forestry, publishing that research and/or presenting it at international scientific meetings, and then building on scientific findings in future grant cycles). I will work with host scientists at the University of Colorado at Boulder on a sabbatical project exploring connections between the photoprotective xanthophyll cycle and oxylipin messengers during drought stress. Oxylipins (e.g. jasmonic acid) are synthesized in the thylakoid membrane in a manner dependent on reactive oxygen species (ROS) formation and lipid-peroxidation; zeaxanthin (Z) formation reduces thylakoid ROS and lipid-peroxidation. Thus, a regulatory connection between Z and oxylipin production seems logically apparent. The research goals for this project are fourfold; to demonstrate whether: 1) Z formation reduces oxylipin formation in Arabidopsis, 2) the photoprotective capacity of Z is more readily overcome when Arabidopsis plants are under water stress, resulting in higher levels of oxylipin formation during drought, 3) oxylipins cause increases in chloroplast ascorbate, tocopherol and xanthophyll pool sizes, and thereby function in a feed-forward loop in chloroplast photoprotection and anti-oxidation, and 4) Z-modulated oxylipin formation functions differently in protecting drought-stressed, field-grown species pairs with known differences in Z accumulation (deciduous Vaccinium versus evergreen Arctostaphylos and Ponderosa pine versus Douglas fir trees). These activities will provide training for the PI in five new analytical techniques related to whole-plant-focused and field-based plant stress physiology that can easily be transferred back to his home institution in Virginia for use in locally-relevant undergraduate research projects.
Project Methods
The project will consist of an extensive greenhouse and laboratory component using Arabidopsis as a model organism, and a field component, in which four different forest species will be compared. Several different Arabidopsis mutants that are altered either in their capacity to generate zeaxanthin (Z) and/or use it in heat dissipation of excess excitatory light, or in their ability to synthesize or respond to oxylipin signals, will be tested relative to wild-type plants for the degree to which varying light and drought regiments induce the generation of oxylipins, chloroplast anti-oxidants, and xanthophyll cycle intermediates. Plants will be grown in three different light conditions (low light, high light, or low light and then transferred to high light), and in five different osmotic conditions (control, moderate or, high saline treatment, or moderate or high polyethylene glycol treatment). Oxylipins will be quantified by HPLC and GC/MS, xanthophyll pigments by HPLC, and antioxidants by enzyme-based UV-Vis spectrophotometric assays. The genotypes of the plants that will be tested will include: Wild-type: Columbia ecotype. Npq1: Columbia plants disrupted in the gene encoding the enzyme violaxanthin deepoxidase; very low levels of Z are formed in npq1 plants, even in high light. Consequently, npq1 plants have a reduced capacity for engagement of heat dissipation of excitatory light as well as for any separate antioxidant effects of Z. We expect these plants to form higher levels of oxylipins than wild-type, and likely higher levels of antioxidants. Npq2: disrupted in the gene encoding the enzyme Z epoxidase; npq2 plants have constitutively high levels of Z present, even in low light. We expect these to form lower levels of oxylipins. Npq4: disrupted in the gene encoding PsbS; npq4 plants have nearly normal xanthophylls, but cannot engage thermal dissipation. We expect these plants to show some increase in oxylipins in HL, since singlet formation in the chloroplast may be increased by the absence of thermal dissipation, however less so than in the npq1 mutants lacking the additional antioxidant effect of Z. Lut2: disrupted in the gene encoding lycopene cyclase, an enzyme involved in lutein biosynthesis; some evidence suggests that lutein enhances PsbS-dependent thermal dissipation by a mechanism similar to that of Z. Fad7 fad8 double mutants: deficient in fatty acid desaturases and thus oxylipin biosynthesis. Jar1 mutants: insensitive to oxylipin signaling. Two interspecies field comparisons will also be made: deciduous Vaccinium versus evergreen Arctostaphylos shrubs and Ponderosa pine versus Douglas fir trees. Previous work has shown that evergreens retain Z through the night when under drought stress, while deciduous plants do not. A difference in the degree of Z retention between pines and firs has also been previously shown, with pines showing less Z retention. Several plants of each of these four species will be monitored in the field for oxylipin production, NPQ, leaf water potential, midday H2O and CO2 gas exchange rates, and xanthophyll cycle pigment concentrations.

Progress 08/01/07 to 07/31/09

Outputs
OUTPUTS: The three primary categories of outputs from this completed project are 1) a new scientific collaboration, 2) new methods/skills in plant eco-physiology, for the PD (Dr. Cessna), and 3) undergraduate student learning in plant biology and agricultural science. 1) Because of the funding, Dr. Cessna was able to establish a scientific relationship with collaborators in Colorado. Drs. Barbara Demmig-Adams and Williams Adams of the University of Colorado at Boulder are some of the leading experts in the area of photosynthetic responses to stress. Dr. Cessna is now in almost monthly contact with the Adams lab, and one co-authored manuscript has so far been submitted to a peer-reviewed journal, with a few more co-authored manuscripts in their earliest stages. Dr. Cessna hopes that this collaboration will lead to greater opportunity to pursue interesting and fruitful agriculture and ecology-related research projects in the Shenandoah Valley and surrounding mountains. 2) Through the sabbatical appointment in Boulder, Dr. Cessna was able to significantly re-tool and develop several new skills in plant eco-physiology, and to develop teaching skills appropriate to research-based science classrooms. Dr. Cessna audited a course at the U. of Colorado in chromotography and mass spectroscopy, and also worked side-by-side with an analytical chemist, to develop methods for the analysis of plant compounds. Gas chromotography/Mass spectrometry methods for the analysis of free fatty acids and oxylipins from leaf tissues were compared and tested for their suitability in pine needle and arabidopsis research. HPLC-based methods for assessing pigments and antioxidants from leaf tissues were also skills that Dr. Cessna learned. Also, Dr. Cessna learned careful methods of field sampling, chlorophyll fluorescence analysis, and photosynthesis measurements. Finally, during the sabbatical year, Dr. Cessna was able to audit a graduate-level education course, and through that course and associated readings, develop his teaching skills in the botany, plant physiology, and biochemistry classroom and laboratory. 3) The sabbatical training has clearly translated into learning for EMU undergraduate students, who now get hands on training in plant compound analysis, chlorophyll fluorescence, and photosynthesis measurements. It is my hope that my students are now better prepared for careers in the plant/ag. sciences. PARTICIPANTS: Stephen Cessna (PD) was the sole individual on this project, which funded a year-long sabbatical at the University of Colorado at Boulder. Partner organizations, collaborators: The Department of Ecology and Evolutionary Biology at the University of Colorado graciously provided facilities and access to some analytical instrumentation for this research. Training and professional development: the project provided significant training for the PD, both in research methods and in teaching and learning. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Changes in knowledge: Because of this sabbatical funding, Dr. Cessna has learned several new scientific skills and methods, and new skills in teaching (see 'outcomes'). Also, new fundamental knowledge has been gained in basic plant biology: 1) There is significantly more polyunsaturated free fatty acid in leaf tissues subjected to high-light stress than leaves grown under low-light. This increase is greater in mutant Arabidopsis plants that are deficient in photoprotective measures. These findings are interesting in that they link fatty acid signaling/metabolism to photo-protection, a connection that has not previously been made in the research literature. Yet, further repetitions of these experiments are required to verify the findings, generate a working model to explain the data, and to prepare a well-reasoned manuscript. 2) Tobacco plants transformed with anti-apoptotic genes (e.g. CED-9) produce less hydrogen peroxide in their stomata in response to abscisic acid treatments than to control plants. Because hydrogen peroxide is a signaling intermediate in abscisic acid signaling, we think that this difference in peroxide synthesis may explain those plants sensitivity to drought. These findings are in a manuscript that is in preparation. These findings are important because it provides a mechanism by which plant apoptotic regulatory proteins might also affect stomatal signaling, and thereby coordinate water loss/hydration with senescence. 3) Chlorophyll fluorescence measurements of photosynthetic light use efficiency vary depending of whether red or blue light is used for excitation, in a manner depending on the amount of phenolic compounds present. The PD compared the use of a blue exciting light source to a red source in a Walz PAM fluorometer for the measurement of Fv/Fm in several leaves of several species in several environmental conditions. Overall, measurements taken with the two sources largely agree, except in the sun-exposed leaves of plants that accumulate high-levels of phenolic compounds that absorb blue light. A clear linear relationship was seen between a degree to which red vs. blue Fv/Fm measurements agree and the presence of blue-light absorbing phenolics. These data will contribute to a manuscript for a photosynthesis research journal. 4) Chlorophyll fluorescence measurements can be reliably made with relatively inexpensive fluorometers (e.g. Fluorpen and Optisci instruments). A manuscript has been submitted, which is directed towards the agriculture educator audience, advocating the use of inexpensive fluorometer measurement with undergraduates.

Publications

  • No publications reported this period