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Some Aspects of Nitrogen Nutrition of Boronia Megastigma Nees

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Some Aspects of Nitrogen Nutrition of Boronia Megastigma Nees SOME ASPECTS OF NITROGEN NUTRITION . OF BORONIA MEGASTIGMA NEES by K. S. Reddy, M. Sc. (Agri.) Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Tasmania Hobart November 1987 This thesis contains no material which has been accepted for the award of any other degree or diploma in any university and to the • best of my knowledge, contains no material previously published or . written by another person except where due reference is made in the text of the thesis. K. S. Reddy University of Tasmania Hobart CONTENTS Acknowl edqemen t s ........................................... vii Abstract ................................................... viii I. Introduction ............................................ 1 II. Review of literature •............................ ~ ...... 5 1 . N and yield .......................................... 5 2. N and yield components ............................... 6 3. Utilization of NH4+ and NOi by plants ............. ~ .. 7 3.1. Differences between plant species ............... 7 .3.1.1 NH4+ plus NOi as N source ................ 11 3.2. N form in soils of natural habitat of boronia .. 13 3.2.1. Moisture and aeration ................... 14 3. 2. 2. Temperature ............................. 14 3.2.3. Acidity ................................ 15 3.3. N form and pH interactions ..................... 16 3.4. N form and other ions in plant ................. 19 3.5. N form and amino acids in plants ............... 22 3.6. N form and flowering ........................... 24 4. Application of N .................................... 25 4.1. Source of N fertilizer ......................... 27 4.2. Time of N application .......................... 28 5. Diagnosis of N deficiency/sufficiency ............... 29 6. N assimilation ...................................... 31 6.1. Kinetic characteristics of NR .............. : ... 32 6.2. Level of NR activity ........................... 33 6.3. Sites of NOi reduction ......................... 35 6. 4 . N iR enzyme .............•....................... 3 7 7. Responses of NOi excess ..................•.......... 37 III. General materials and"methods .......................... 39 Chemical analysis ..................................... 39 ,. pH •••••••••...•••••••••.••.•......•.•.•...••........• 3 9 L Ana1Jsis of Nin nutrient solutions .................. 39 t I NH4 •••••••••••••••••••••••· ••••••••••••••••••••••••• 3 9 Noa· ••••••••••••••••• ·••••••••••.••.••.....••...•...• . 39 Elemental analysis of plant material ................. 40 Preparation of leaf samples ......................... 40 Determination of N ................................. 40 Determination of P, K, Ca and Mg ·.................... 41 Wet ashing ......................................... 41 Phosphorus ..................... • ................... 41 Calcium .............................· ............... 4 2 Magnesium ......................................... 42 Potassium ......................................... 42 Determination of N0 - in plant tissue .................. 42 i,. 3 : Determination of N02- in plant tissue ................. 42 Determination of protein ............................. 42 Statistical analysis .................................. 42 III.l. Analysis of amino acids in plant materials by reverse- phase HPLC •......•.. · •..•.•............•............... 44 Materials and methods ................................ 45 Extraction .......................................... 4 5 Dinitrophenylation .................................. 46 Extraction of DNP-amino acids ....................... 47 iii t·. t. 1· iv Chromatography 47 Results and discussion 48 IV. Effects of source, rate and application time of nitrogen on flower yield and leaf nitrogen concentration in boronia 54 Materials and methods 54 Site 54 Layout 54 Treatments 54 Maintenance 55 Measurements 55 Results 56 Leaf N concentration 56 Flower yield 61 Relationship between yield and leaf N concentration .66 Discussion 68 V. Effects of different levels of nitrogen as NH 4+ , NO3- or NH4+ plus NO3- on growth, flowering and nutrient concentration in leaf tissue in boronia 79 Materials and methods 79 Application of nutrient solutions 79 Plant culture 83 Growth conditions 83 Composition of nutrient solutions 83 Measurement of responses 85 Results 87 General growth and morphology 87 1984 experiment 88 1984-85 experiment 90 Stem diameter 90 Nodes on the main shoot 92 Lateral shoots 92 Nodes on lateral shoots 95 Flowering 98 Axils with flower buds 98 Total number of flower buds 101 Developed flowers 102 10-flower weight 105 Nutrient concentration in leaves 105 Nitrogen 105 Phosphorus 108 Potassium 108 Calcium 108 Magnesium 110 Discussion 110 Nutrient concentration in leaves 118 Practical implications 122 VI. Effect of nitrogen form and pH on uptake and assimilation of nitrogen in boronia 124 Materials and methods 124 Plant material 124 Culture techniques 125 Growth conditions 125 Composition of nutrient solutions 125 Treatments 125 Measurements 126 Results 126 pH 127 N depletion from solutions 127 Nodes 132 Lateral shoots 132 Amino acids in plant tissues 132 Discussion , 132 VII. Optimization of assay conditions for nitrate reductase and nitrite reductase enzymes from boronia 139 Materials and methods 140 Plant culture 140 NR activity in vivo in roots and leaves 141 NiR activity in vivo in leaves 141 Preparation of cell-free enzyme extract from leaves 141 NR activity in vitro assay 142 NiR activity in vitro assay 142 Results and discussion 144 NR activity in vivo in roots and leaves 144 Effect of NO3- concentration 144 Effect of slicing the tissue 144 Effect of propanol 144 Effect of anaerobiosis with air or N2 147 Time course of NR activity 148 NiR activity in vivo in leaves 148 Effect of buffer pH 148 Effect of NO2- concentration 148 Time course of NiR activity 150 Preparation of cell-free enzyme extract from leaves 150 Effect of homogenization method 150 Effect of cysteine 153 Effect of bovine serum albumin (BSA) 153 Effect of extraction buffer system 154 Effect of polyvinylpyrrolidone (PVP) 154 NR activity in vitro in leaves 154 Effect of NO3- concentration 155 Effect of enzyme concentration 155 Time course of NR activity 155 NiR activity in vitro in leaves 155 Effect of NO2- concentration 155 Time course of NiR activity 158 Conclusions 158 NR activity in vivo assay 158 NiR activity in vivo assay 158 Preparation of cell-free enzyme extracts from leaves 159 NR activity in vitro assay 159 NiR activity in vitro assay 159 VIII. Nitrate uptake, reduction and accumulation in relation to nitrate toxicity in boronia 160 Materials and methods 161 Plant culture 161 Treatments 162 vi Observations 162 Analyses 164 Results 164 NO; content in roots 166 NOi in roots 166 Actual NR activity in roots (in vivo minus NOi assay) 166 Potential NR activity in roots (in vivo plus NO; assay) • 170 NO; content in leaves 172 NOi in leaves 174 Actual NR activity in leaves (in vivo minus NOi assay) 174 Potential NR activity in leaves (in vivo plus NOi assay) 177 NR activity in vitro in leaves 177 NiR activity in leaves 182 Discussion 182 Practical implications 190 IX. General discussion 190 X. References 196 Appendixes 209 Publications 243 ACKNOWLEDGEMENTS I am grateful to my supervisor Dr R. C. Menary, Reader in Horticultural Science and Head of the Department of Agricultural Science, for his advice and encouragement throughout this project. He also made successful efforts for funding from the Tasmanian Development Authority which supported part of this project. I thank Dr J. J. Yates for his advice with statistics; Mr. G. F Richardson for his assistance in design and construction of the automatic nutrient supply system used in a part of the study, for If help in planting in the field (at Kingston) and for general technical help; Mr W. R. Peterson for his help in obtaining chemicals and in repairing instruments; Mrs A. McEldowney for teaching me HPLC; Mr G. Beckett for his help in planting at Kingston; Dr R. J. Clark for useful suggestions while writing the thesis and Mrs J. Everett for access to computer and printer for printing the text. I thank Mr G. A. Luckman and Mr N. Roberts and other postgraduate students for their helpful discussions during the project. I gratefully acknowledge the University research award. I am grateful to my parents for their support and encouragement. vii ABSTRACT Boronia is a native plant with potential as a new essential oil crop. Various growth and metabolic responses of boronia to N were investigated to improve N nutrition of boronia. When N was supplied to boronia at 50 or 100 kg hal , two fast- release N sources (ammonium sulfate and calcium nitrate) caused toxicity while a slow-release N source (IBDU) did not cause toxicity. When N was supplied in a single dose in October during the active vegetative growth phase, highest flower yield was obtained and when the same amount of N was supplied in split doses at different phases of plant growth, the yield decreased. Increasing N levels from 0 to 25 mM in the nutrient solution increased the plant's production of nodes, lateral shoots from these nodes and further nodes on these lateral shoots. With the same level of N, production of nodes and lateral shoots was in the order: NH4++NO3- > NH4+ > NOi. The increase in the number of nodes subsequently translated into increased number of axils initiating flower buds and then into fully developed flowers. However, increasing N levels decreased the percentage of total flower buds that developed
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