AN ABSTRACT OF THE THESIS OF Ning Li for the degree of Doctor of Philosophy in Horticulture presented on February 14. 1997. Title: Spectroscopy of Biophysical. Physiological and Pathological Responses of Plant Germplasm. Abstract approved: ^ Larry-S-BSley Imaging spectra of plant tissues yield considerable potential and useful information for basic studies and agriculture practice. We constructed an imaging spectrophotometer with spectral range from 400 nm to 1100 nm, which was used to study various physiological events of plant germplasm: location of bundle sheath and mesophyll cells in sugar cane (Saccharum officinarum L.); chlorophyll b deficiency of ornamental perennial grass (Millium effusum L. var. aureum); respiration rate of soybean roots (Glycine max Merr. var. Yuusuzumi); concentration of azosulfamide dye of apple (Malus domestica Borkh. var. Red Rome) flower buds; sucrose concentration and invertase activity in stems of hazel (Corylus avellana L. var. Barcelona); in vivo effects of a fungal pathogen {Pestalotiopsis microspord) and its phytotoxin in the foliage of the rare nutmeg (stinking) cedar or yew of Florida (Torreya taxifolia Am.) foliage; time course of water displacement by heavy water (deuterium oxide) in rossele (Jamaica sorrel, Hibiscus sabdariffa L.) leaves; water path estimation using mixtures of deuterium oxide. A novel algorithm was found to measure the estimated quantum yield image (Y'). An imaging fluorometer based on this algorithm was constructed and tested for agriculture application: photoinhibition of photosynthesis, very early determination of freeze damage, herbicide effects and invasion by fungal pathogens in Scrofularia nodosa ' Variegata'. Excellent results were also obtained in testing four toxins: pestalopyrone, hydroxypestalopyrone, pestaloside, and triticone on three varieties (non soong, red sorrel, and altissima) on Hibiscus sabdariffa L. Triticone damage was still very obvious after 24 hours. Pestalopyrone effects disappear several hours after injection showing that photosynthetic function recovers rapidly from the pestalopyrone damage. Possibility of using chlorophyll protein complexes (CPX) in vivo for digital imaging storage, and the potential of the algorithm in remote sciences are also illustrated and discussed. Using optical correlation interferometry, a novel method for plant sciences, we imaged In vivo the z-direction, perpendicular to the leaf surface, through Tradesccmtia zebrina leaves. Non-invasively we: determined the number of major cell layers, followed the time sequence of decrease in depth of the cells z-axes after exposure of tissues to high salt, and observed disruption of cells caused by freezing and thawing. © Copyright by Ning Li February 14, 1997 All Rights Reserved Spectroscopy of Biophysical, Physiological and Pathological Responses of Plant Germplasm by Ning Li A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented February 14, 1997 Commencement June 1997 Doctor of Philosophy thesis of Ning Li presented on February 14, 1997 APPROVED: Major Professor, representing Horticulture^/ Chair of Department 6rHorticulture Dean of Graduateu^e SchoolSchoi I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. -fr- L. ■- U L£. 1. Ning Li, Author ACKNOWLEDGEMENT At this point, I would like to thank everyone who has been involved in this work. I would like to thank my wife, Wenqi Bao, for all her understanding and support. I would like to thank my advisor, Larry S. Daley, for his moral and financial support during the course of my studies. I would like to thank Prof. James B. Callis, for his dedicated training on the topic of imaging spectroscopy methods. I would like to thank Prof. Gerald E. Edwards for his teaching and discussion on the subject of plant leaf fluorescence, which lead to the constructing the imaging fluorometer in the summer of 1994. I would like to thank Prof. Gary A. Strobel for his plant material and phytotoxin extractions. I wish to express my appreciation to all who cooperated with me on my research over the years for arrangement of useful advice and discussions on a broad range of subjects, particularly: Prof. Bent Petersen for his idea on the digitizing and storing data (digits of 71) on plant leaf; Walter J. Bowyer for his particular interest in the imaging fluorometer and exploration of phytotoxin bioassay; Annie M. Chozinski, Tamas Buban, Anita Azarenko for their support and providing microphotographs of dye stained apple flower buds; E. H. Piepmeier for references on the subject of Rayleigh scattering, Zhao Lu for her help in using HP 8504A interferometer and MATLAB; Mr. Ozanich Rich for his initial work on imaging spectrophotometer on plant leaf; Prof. Sonia Anderson for her training in the course of biophysics methods; Prof. Chaur-fong Chen for his help and suggestions in applying imaging fluorometer to remote sensing science; Mrs. Natalie Daley for her patient advice on improving my written English. I would like to thank committee members: Prof. Tim Righetti, and Prof. Tom K. Plant, for their encouragement, interest in my research, advice, and careful planning of my study program. Thanks go also to Prof. Kelly Falkner for serving as Graduate Representative. Funding for this research from Herman Frasch Foundation (Grant Number FRASCH 63-0738), OSU Agricultural Research Foundation, and Oregon Filbert Commission are gratefully acknowledged. CONTRIBUTION OF AUTHORS Ning Li was involved in the design, experimentation, analysis and writing of each manuscript. Dr. Larry S. Daley was involved in overseeing this work from its onset and in the design and analysis of experiments; and proofreading and editing of each manuscript. Although some of the other author's specific contributions are recognized in the acknowledgment section, the synergism, interaction and support of each member of this highly diverse and multidisciplinary group, although difficult to quantify and specifically assign credit, was essential to the production of this body of work. The diversity of the disciplines and the broad sweep of the experimental topics successfully addressed demonstrates the advantages and contributions of such a multidisciplinary group. TABLE OF CONTENTS Page CHAPTER 1: Introduction 1 CHAPTER 2: Construction Of An Imaging Visible Spectrophotometer And Its Application To Plant Sciences 15 CHAPTER 3: Five Novel Applications Of Imaging Visible And Short Near Infrared Spectrophotometry And Fluorometry In The Plant Sciences 48 CHAPTER 4: Spectroscopic Imaging Of Water In Living Plant Leaves, "Rayleigh" Corrections, And Significance For Beer's Law Based In Situ Quantification 113 CHAPTER 5: Imaging Fluorometer To Detect Pathological And Physiological Change In Plants 174 CHAPTER 6: In Vivo Plant Leaf Photosynthetic Apparatus Can Store Digital Information As Fluorescence Signal 203 CHAPTER 7: In Vivo Imaging Of The Interior Of Tradescantia Zebrina Leaves By Optical Cross Correlation Interferometry 234 CHAPTER 8: Conclusion 252 BIBLIOGRAPHY 260 LIST OF FIGURES Figure Page 1.1 Cover from the journal Applied Spectroscopy (Volume49 (10), October, 1995) illustrating the Photo-induced image on Ginko tree leaf 6 1.2 Cover from the journal Spectroscopy (Volume 11 (3), March/April, 1996) illustrating the in vivo measurement of water in slices of living Aloe miliotii leaves 10 2.1 Schematic of CCD based imaging spectrophotometer 29 2.2 Electron micrograph of bundle sheath cells and mesophyll cells and their chloroplasts 30 2.3 Spectra and chlorophyll protein complex structure 33 2.4 Spectra images of sugar cane leaf 35 2.5 Image spectra of sugar cane leaf and chlorophyll b-deficient oil yellow-yellow green (OY-YG) maize leaf 39 2.6 Respiration activity of soybean root 42 3.1 Application one: uptake of azosulfamide dye in apple flower buds 63 3.2 Model for biochemical histological changes 70 3.3 Application two: biochemical histology of invertase 73 3.4 Analysis of and calibration of spectra for fiingal pathogen detection 79 3.5 Absorbance bands of water and deuterium oxide mixtures in the range of 700-850 nm 83 3.6 Application three: imaging of pathogen advance in a leaf In vivo 86 3.7 Application four: determination of water in a living leaf 89 3.8 Application five: demonstration of fluorescence configuration used to determine differences in photosynthetic efficiency 92 LIST OF FIGURES (CONTINUED) Figure Page 3.9 Data on differences in orientation of leaves of canopy and its effect on apparent quantum efficiency of photosynthesis 96 4.1 Experimental plant material 131 4.2 "Raleigh" scattering correction 134 4.3 Spectra of deuterium oxide (D-O-D), water (H-O-H) mixtures in visible and very short NIR 139 4.4a Reconstruction of spectra of deuterium oxide diluted water compared to observed data 141 4.4b Reconstruction of a series of water/deuterium oxide mixtures using the simplified method 143 4.5 Demonstration of water peak in solid substrate 147 4.6 Utility of normalization of water in wet and living substrate 149 4.7 In vivo tests of water content and general distribution of chlorophyll-protein complexes in leaf sections of Aloe milotii and a section of living leaves of Scrofularia nodosa ' Variegata' 152 4.8 Mapping light harvestmg (LH) chlorophyll-protein complexes (CPX) in the living leaf 159 4.9 Mapping photosystem I (PSI) chlorophyll-protein complexes