* -n(t/rô Y,.? ,;i;b ¿øÕ ií1. .:;ti# 1t NOv 1996 Iel å 6Þì .f\:rl'|.¿ b\J PHYSIOLOGTCAL,BIOCHEI\flCALANDCHEn[rcAL Ctr DESICCATION TOLERANCE PRIMARILY IN DEVELOPING WIIEAT SEEDS by Chutima Koshawatana B.Sc.Ag. (KU), M.Sc.Ag. (KU)' Thailand Department of Plant Science, Waite Agricultural Research Institute, The University of Adelaide, South Australia Thesis submitted for the degree of Doctor of Philosophy ln The University of Adelaide (Faculty of Agricultural and Natural Resource Sciences) February,1996 I 6 0 Í.!ü';' TABLE OF CONTENTS Page v S UMMARY vu STATEMENT. ACKNOWLEDGEMENTS ' LIST OF TABLES ... LIST OF FIGURES . CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW I 1.1 lnroduction......... 3 1.2 Desiccation tolerance during seed development 3 1.2.1 Classification of seed developmental stages |.2.2 Acquisition of desiccation tolerance and desiccation sensitivity during seed develoPment. 5 5 r.2.3 Effect of drying on germination of developing seeds ' r.2.4 Transition from developmental stage to germination stage and .8 desiccation tolerance 1.2.5 Biological changes during acquisition of desiccation tolerance" " " ' .9 ,9 1.3 Desiccation tolerance of seeds during germinaúon' l1 1.4 Subcellular changes induced by desiccation ' " " ' ' ' 1.5 Possible factors conferring desiccation tolerance""" 13 1.5.1 Sugars ....... 13 tolerance. ....... 15 1.5. 1 . I Mechanisms of sugars in conferring desiccation 1.5.I.2 Negative evidence for sugars in desiccation tolerance ....... .......t7 L.5.2 Proteins ....... 18 1.5.3 Lipids .......20 I .5.3.1 Origin and structure of lipid droplets .......20 1.5.3.2 The degradation of lipid droplets 22 ll 1.5.3.3 The role of lipids in desiccation tolerance " "' 23 scavengers 24 1 .5.4 Free Radicals and free radical 25 1.5.4.1 Free radicals and their roles in damaging membranes ...... 26 1.5.4.2 The roles of the radical scavengers 28 1.5.5 Abscisic acid (ABA) 28 1 .5.5. I The role of ABA during seed development ' " 1.5.5.2 The role of ABA in conferring desiccation tolerance...... 29 1.5.5.3 Desiccation tolerance of somatic embryos' 30 CHAPTER 2 : MATERIALS AND METHODS 2.I Plantmaterials.... 34 2.I.1 Sweet com (7za maYs) 34 2.I.2 Wheat (Triticum aestivum) .... 34 2.2 Germination test 34 2.2.t Filter paper germination test ...... 34 2.2.2 Embryo germination on artificial media 35 2.3 Moisture content determination......'.., 35 2.4 Desiccation tolerance test 35 2.5 Sugar analysis 36 2.5.1 Sugar extraction .... ..36 2.5.2 Preparation of samPles for high performance liquid chromatogtaphY (HPLC) .... 36 2.6 Diffuse Reflectance Using Infrared Dispersion Spectra (DRUIDS) 37 2.7 Microscopic methods for lipid droplets 39 2.7.1 Sample preParation... 39 2.7.2 Preparation of dye... 39 39 2.7 .3 Fluorescent microscoPY . ' 2.8 Solid-state 13C CP/¡vIAS NMR analysis 40 lu CHAPTER 3 : DESICCATION TOLERANCE IN DEVELOPING SEEDS AND TIIE ROLE OF SUGARS during Experiment 1 : Sugar concentrations and desiccation tolerance seed development I Germination' sugar concentrations and desiccation tolerance of sweet corn seeds 42 3.1 Introduction 43 3.2 Materials and methods M 3.3 Results.. .. .. 48 3.4 Discussion .. Experime nt 2 z Sugar concentrations and desiccation tolerance duringseeddevelopmentllEffectofdryingonsugar concentrations in sweet corn embryos .52 3.5 Materials and methods...... .55 3.6 Results .60 3.7 Discussion Experiment 3 : Wheat embryos, desiccation tolerance' sugar concentrationsandtheeffectofdryingonsugars 3.8 Materials and methods 64 3.9 Results 65 Experiment 4 : Sugar concentrations in wheat embryos at 20 and 25 DAA after the seeds have been stored under different relative humiditY conditions 3.10 Materials and methods. 72 3.11 Results.... 73 lv Experiment5:Desiccationtoleranceandsugarconcentrationsin wheat embrYos at 15 DAA 75 3.12 Materials and methods .76 3.13 Results.... .82 3.l4 Discussion @xperiments 3-5) " .87 3.15 Conclusions (Experiments 1-5) AND CHAPTER 4 : SPECTROSCOPIC (IR AND NMR) CHROMATOGRAPHIC (TLC) STUDIES OF DESICCATION TOLERANCE IN \ryHEAT EMBRYOS 89 4.1 Introduction..... 9l 4.2 Materials and methods. .. 93 4.3 Results. t20 4.4 Discussion ... 127 4.5 Conclusions ..... CHAPTER 5 : A CONFOCAL MICROSCOPIC STUDY OF LIPID DROPLETS CHANGES IN \ryHEAT EMBRYOS DURING DEVELOPMENT AND AFTER DESICCATION r28 5.1 Introduction.. 130 5.2 Materials and methods 5.3 Results t32 5.4 Discussion t4 t47 5.5 Conclusions........ ' CIIAPTER 6 : GENERAL DISCUSSION t49 BIBLIOGRAPTIY .... 155 v SUMMARY phenomenon of desiccation This thesis is the result of my investigations into the Methods employed include the tolerance in developing seeds of sweet corn and wheat' chromatography HPLC of sugars, predominantly sucrose and raffinose, thin layer (DRUIDS), confocal together with diffuse teflectance using infrared dispersion spectra l3C and embryo microscopy of lipids, solid state NMR CP/MAS spectroscopy culture. The major results obtained were : were found to l) Neither sugar concentration nor the moleToratio of raffinose/sucrose embryos take place due be good predicters of desiccation tolerance. Sugar increases in to an alteration of atmospheric conditions affecting of detached seeds' thin layer 2) Diffuse R.eflectance Using Infrared Dispersion Spectra (DRUIDS) and carbonyl of chromatography (TLC) have established a substantial rise of the ester (15 to a triacylglycerols from a low amount at the desiccation-sensitive stage DAA) leads high amount at the desiccation-tolerant stage (20 DAA). Subsequent desiccation of free to corresponding diminution in the ester carbonyl signal and the appearance fatty acids as indicated by a carboxyl carbonyl peak at 1710 cm-l and an associated loss of the TAG ester carbonyl at t743 cm-r. 3) rlç NMR spectra are predominantly the signals corresponding to carbohydrates fresh and has enabled the comparison of the spectra from desiccated embryos and the embryos of the same coresponding age. After subtraction of one spectrum from other, there were clearly large differences in the subtracted spectra for the desiccation- tolerant stage when compared to the subtracted spectra for the desiccation-sensitive stage. vl with Nile red established 4) A cont'ocal microscope study of the neutral lipids stained measured by infrared that although there was virtually a complete loss of TAG's as in Nile red ester carbonyl diminution, there was no coffesponding decrease complete loss of Nile fluorescence suggesting that ester bond cleavage did not lead to fatty acid' red staining. This was interpreted as mostly un-ionised residual free have been The literature on desiccation tolerance has been reviewed and my results and discussed in the context of this literature. I conclude that both the carbohydrate lipid content of embryos exhibit major changes during desiccation. The independent should methods and novel observations from this thesis suggest that future workers the nature and carefully examine the role of triacylglycerols in desiccation tolerance and l3C spectra role of the carbohydrate species cha¡acterised in the NMR of the difference of embryos at the desiccation-tolerant stage' vn STATEMENT award of any other This work contains no material which has been accepted for the and' to the best of degree or diploma in any university or other tertiary institution published or written by my knowledge and belief, contains no material previously the text. another person, except where due reference has been made in Library' I give consent to this copy of my thesis, when deposited in the University being available for loan and photocopying' D s r c N E D, a r E, ....t.. l..Z.l..l.q. vlll ACKNO\ryLEDGEMENTS and I wish to acknowledge my supervisors Dr.M.E.Tate, Dr.'W'Wallace sincere appreciation must Dr.S.J.Barker for their kind assistance and comments' My and support go to Dr.M.E.Tate for his guidance, enthusiasm, encouragement throughout mY work. his comments on the sugar I am grateful to Dr.C.F.Jenner for HPLC analysis and for with the experiments. I would like to thank Dr.Peter Kolesik for his kind assistance preparation' confocal microscope and to Dr.D.E.Evans for his suggestions for sample l3C NMR analysis and his Special thanks for Dr.Philip Clarke for performing the by a kind friendship. My germination techniques have been greatly improved Giles and Ms'Rita suggestions from Dr.A.Aryan. I gratefully appreciate Ms.Lynn comments, Middelberg for the statistical analyses. For the invaluable discussions and I wish to thank Prof.J.D.BewleY. Ms'S'Jamjod, for their I also thank my Thai friends, especially Dr.C.Maneechote and English, I want to friendship and support. For his friendship and qorrecting my Thai Research thank Mr.R.Asenstorfer. I also wish to thank the staff at Waite Agricultural Institute for their help during my stay. Merit My study would not be possible here without the support from the Equity and Also Scholarship Scheme and Australian Agency for International Development' Center for thanks for Thai Government and Nakhon Sawan Field Crop Research my Mum and allowing me to have the opportunity to do this research' Finally,I thank Australia' Dad for their support and for encouraging me to continue my research in D( LIST OF TABLES of Table 3.1 Germination of fresh developing sweet com seeds and effect drying on seed moisture content and germination""""""""""'48 Table 3.2 Effect of drying on moisture content, germination and sugar concentrationsofsweetcornembryos'Datashownforseeds was dried for at different stages of development and after each 56 6 days .. Table 3.3 Effect of drying on seed moisture content,