Chemical and functional analyses of the plant cuticle as leaf transpiration barrier Chemie-Funktionsanalysen der pflanzlichen Kutikula als Transpirationsbarriere Doctoral thesis for a doctoral degree at the Graduate School of Life Sciences, Julius-Maximilians-Universität Würzburg, Section Integrative Biology submitted by Ann-Christin Schuster from Ingolstadt Würzburg 2016 Submitted on: ………………………………………………………… Members of the Promotionskomitee: Chairperson: Prof. Dr. Thomas Müller Primary Supervisor: Prof. Dr. Markus Riederer Supervisor (Second): Prof. Dr. Dirk Becker Supervisor (Third): Dr. Adrian Friedmann Supervisor (Fourth): Dr. Markus Burghardt Date of Public Defence: …………………………………………….. Date of Receipt of Certificates: …………………………………….. Table of contents Table of contents Introduction ........................................................................................... 1 The plant cuticle ............................................................................................... 2 1.1 Cutin polymer ............................................................................................... 3 1.2 Cuticular waxes ............................................................................................ 4 1.3 Biosynthetic origin of cutin and cuticular waxes ........................................... 7 The plant cuticle as transpiration barrier .......................................................... 8 2.1 Definition of transport parameters ................................................................ 8 2.2 The plant cuticle as transpiration barrier .................................................... 10 Aim of the present study ................................................................................ 13 Materials and methods ....................................................................... 16 Leaf characteristics ........................................................................................ 16 1.1 Plant material and leaf harvest ................................................................... 16 1.2 Saturated fresh weight, dry weight and relative water deficit ..................... 17 1.3 Leaf area .................................................................................................... 18 1.4 Degree of succulence, specific leaf area and leaf water content ............... 18 1.5 Stomata density ......................................................................................... 19 1.6 Leaf surface properties by scanning electron microscopy.......................... 19 Leaf thermal tolerance and leaf dehydration tolerance .................................. 19 Leaf area shrinkage and leaf water potential to correct transpiration rates and conductances ................................................................................................. 20 Transpiration rate and minimum conductance ............................................... 21 Transpiration rate and cuticular permeance ................................................... 23 Temperature effect on minimum conductance and cuticular permeance ....... 24 Gravimetric and chemical analysis of the cuticular components .................... 25 7.1 Isolation of cuticular membranes ............................................................... 25 7.2 Gravimetric analysis of the cuticular components ...................................... 26 7.3 Extraction of the cuticular waxes for chemical analysis.............................. 26 7.4 Extraction of the cutin monomers for chemical analysis ............................. 27 7.5 Chemical analysis of the cuticular waxes and the cutin monomers ........... 27 7.6 Average carbon chain length ..................................................................... 28 Statistical analyses ......................................................................................... 29 I Table of contents Chapter I. The leaf minimum conductance and the temperature effect on the cuticular transpiration barrier of the desert plant Rhazya stricta ...................................................................................... 30 Introduction .................................................................................................... 30 Results ........................................................................................................... 33 2.1 Leaf characteristics of Rhazya stricta ........................................................ 33 2.2 Leaf thermal tolerance and leaf dehydration tolerance .............................. 34 2.3 Leaf area shrinkage and leaf water potential to correct transpiration rates and conductances ...................................................................................... 35 2.4 Leaf drying curve, minimum conductance and temperature effect on minimum conductance ............................................................................... 37 2.5 Gravimetric and chemical analysis of the cuticular components ................ 40 2.5.1 Chemical analysis of the cuticular leaf wax ........................................... 40 2.5.2 Chemical analysis of the cutin monomers ............................................. 43 Discussion ...................................................................................................... 45 3.1 Measurement of the minimum conductance: evaluation and correction .... 45 3.2 Classification of the minimum conductance ............................................... 46 3.3 Cuticular wax chemistry and minimum water permeability ......................... 49 3.4 Conclusion ................................................................................................. 52 Chapter II. Comparison of the temperature effect on the minimum conductance and the cuticular permeance of the Mediterranean sclerophyll Nerium oleander .............................................................. 53 Introduction .................................................................................................... 53 Results ........................................................................................................... 55 2.1 Leaf characteristics of Nerium oleander ..................................................... 55 2.2 Leaf thermal tolerance ............................................................................... 56 2.3 Cuticular permeance and temperature effect on cuticular permeance ....... 56 2.4 Leaf drying curve, minimum conductance and temperature effect on minimum conductance ............................................................................... 58 2.5 Gravimetric and chemical analysis of the cuticular components ................ 61 2.5.1 Chemical analysis of the cuticular leaf wax ........................................... 61 2.5.2 Chemical analysis of the cutin monomers ............................................. 64 Discussion ...................................................................................................... 66 II Table of contents 3.1 Classification and comparison of the minimum conductance and the cuticular permeance ................................................................................... 66 3.2 Temperature effect on the minimum conductance and the cuticular permeance ................................................................................................. 68 3.3 Shift of relative water deficit at stomatal closure at elevated temperatures 70 3.4 Cuticular wax chemistry and minimum or cuticular water permeability ...... 71 3.5 Conclusion ................................................................................................. 72 Chapter III. Temperature effect on the cuticular permeance of Prunus laurocerasus .......................................................................... 74 Introduction .................................................................................................... 74 Results ........................................................................................................... 76 2.1 Leaf thermal tolerance ............................................................................... 76 2.2 Cuticular permeance and temperature effect on cuticular permeance ....... 76 Discussion ...................................................................................................... 80 3.1 Classification and comparison of the cuticular permeance ........................ 80 3.2 Temperature effect on the cuticular permeance ......................................... 82 3.3 Conclusion ................................................................................................. 85 Chapter IV. Cuticular barrier properties and thermal tolerances of plants in hot and dry environments ................................................... 86 Introduction .................................................................................................... 86 Results ........................................................................................................... 89 2.1 Leaf characteristics and minimum conductance ........................................ 89 2.2 Leaf thermal tolerance ............................................................................... 93 2.3 Leaf thermal tolerance and minimum conductance .................................... 94 2.4 Chemical analysis of the cuticular leaf wax ................................................ 95 2.4.1 Cuticular leaf wax from Hippocrepis comosa ........................................ 95 2.4.2 Cuticular leaf wax from