NITRATE REDUCTA3E IN MAIZE ROOTS: LOCALIZATION AND MOLECULAR CHARACT~RIZATION By DEBORAH MARIE LONG, B.Sc. A Thesis Submitted to the School of Graduate Studies in Partial Fulfilment of the Requirements for the Degree Doctor of Philosophy McMaster University (c) Copyright by Deborah Marie Long, April 1991
NITRATE REDUCTASE IN MAIZE ROOTS
DOCTOR OF PHILOSOPHY (Department of Biology) McMaster University Hamilton, Ontario TITLE: Nitrate Reductase in Maize Roots: Localization and Molecular Characterization AUTHOR: Deborah Marie Long, B.Se.(Hons.) (University of Ottawa) SUPERVISOR: Dr. Ann Oaks NUMBER OF PAGES: xviii, 257 ii
ABSTRACT Nitrate reductase (NR) is the first enzyme involved in the pathway of nitrate assimilation in plants. It converts nitrate to nitrite. By including the serine protease inhibitor, chymostatin, in the extraction buffer, NR from maize (Zea mays L.) roots was stabilized ~n vitro. Contrary to early results, it was found in substantial amounts in the mature regions of the root. Two isozymes of NR were identified, an NADH monospecific form found predominantly in the root tip, and an NAD(P)H bispecific form which was predominant in the mature portion of the roots. Both isozymes were found to reach substantial levels of activity, approximately one-third to one-half the levels found in shoots. The levels of NR activity in both shoots and roots varied with the age of the plants and the conditions of growth. Subsequent purification and biochemical characterization of the two isozymes suggested similarities in the characteristics of the isozymes. However, the NADH form had an exceptionally high ~ for NADH which suggests that the NADH:NR may not be active in the iii
assimilatior. of nitrate as it may not be able to compete with other dehydrogenases for reductant. A partial cdna clone of root NR was isolated, sequenced and identified as a gene distinct from the gene which codes for NR in maize leaves. The NR in maize roots was affected in a positive manner by nitrate at the levels of activity and transcription. However, it did not appear to be affected by either a diurnal rhythm or directly by light, as was found for leaf NR. Through the use of the tissue print hybridization technique, NR mrna was found to be expressed throughout the maize root with the exception of the root tip. In conclusion, NR in maize roots is present in high enough amounts to account for a substantial level of nitrate reduction in the roots, suggesting that maize roots have an important role in the overall metabolism of nitrate in maize. iv
ACKNOWLE~NTS There are many people whom I would like to thank for their support and guidance throughou~ my studies. In particular, I would like to thank Dr. Ann Oaks for her supervision, guidance and constant support throughout my graduate work. I would also like to thank Dr. steven Rothstein for accepting me into his lab for the duration of my stay at the University of Guelph and for assisting in the supervision of my project during that time. I would like to thank Dr. John Greenwood and Dr. John Lott for serving on my Supervisory committee and I thank them both for their support, advice and assistance in My training as a graduate student. I would like to acknowledge the friendship and assistance of many individuals at McMaster University and the University of Guelph with special thanks for the technical assistance and guidance of Valerie Goodfellow, Lisa Thomson and Dr. Caroline Bowsher. Finally, I would like to acknowledge the support and constant encouragment of my husband, Emad, and my family. It is through their support, and that of my friends, that I have been able to reach my goals. v
TABLE OF CONTENTS Page INTRODUCTION 1 Importance of Nitrate to Crop Pla~ts 1 Uptake and Distribution of Nitrate in Higher 2 Plants The Fate of Nitrogen Within nigher Plants 7 Nitrate Reductase 12 a) Biochemistry 12 i) Isolation and purification 12 ii) Types of nitrate reductase 14 b) Regulation 16 i) Regulation by nitrate 16 ii) Regulation by light 18 iii) Regulation by other factors 19 c) Identification of structural and genetic 22 components involved in the regulation and synthesis of nitrate reductase i) Mutant analysis 22 ii) Elucidation of the structure of NR 23 iii) Identification of genes which code 27 for NR iv) Molecular regulation of NR 28 d) Localization 31 i) Localization of NR 31 vi
ii) Localization of the reductant 35 supply for NR Chapter 1: Stabilization of Root Nitrate Reductase 38 Introduction 39 Materials and Methods 41 Growth Conditions of Plants 41 a) Agar grown plants 41 b) Hydroponically grown plants 41 Nitrate Reductase Activity Assay 42 Results 44 storage Effects on Nitrate Reductase Activity 44 in Maize Roots Development of an Extraction Buffer to 44 stabilize Root NR in vitro stabilization of Root Nitrate Reductase 46 Activity with Chymostatin and Altered Growth Conditions Inhibition of Maize Root Proteinase (MRP) by 56 Chymostatin Test of Removal of NADH and NADPH from the NR 58 Assay Medium with Oxaloacetate or Alcohol Dehydrogenase and Acetaldehyde Discussion 62 Chapter 2: Purification and Biochemical Characterization 64 of Root Nitrate Reductase Introduction 65 Materials and Methods 67 Growth Conditions of Plants 67 vii
Extraction and Nitrate Reductase Assays of 67 Plant Organs Preparation of Antiserum 68 Protocol for Partial Purification of Nitrate 68 Reductase from Maize Roots MRP Activity Assay 72 Calibration of Sephadex G-150 Column 73 Results 74 Inhibition of NR Activity by Antiserum 74 Induction of NRA in Roots and Shoots of Maize 74 with 1OmM KN0 3 Induction of NRA in Maize Roots with Ammonium 76 and Nitrate Effect of Ammonium and Nitrate on Maize 78 Seedlings In vivo stability of NAD(P)H:NRA in Maize 80 upon the Removal of Nitrate Partial Purification of NADH:NR and 82 NAD(P)H:NR from Maize Roots Biochemical Characterization of NADH:NR and 90 NAD(P)H:NR from Maize Roots Discussion 102 Chapter 3: Influence of Light/Dark Cycles on the 106 Regulation of Nitrate Reductase and Nitrite Reductase Introduction 107 Materials and Methods 110 Plant Material and Growth Conditions 110 RNA Isolation and Hybridization 111 Enzyme Assays 112 viii
Results 114 Effect of Plant Age on NR and NiR Activites 114 Effecc of Altered Light Regimes on NR 116 Activity and mrna in Shoots Effect of Different Light Regimes on the 119 Expression of Root!~ Effect of Light Regimes on NiR Activity and 121 mrna in Sheots Discussion 124 Chapter 4: Molecular Characterization of Root Nitrate 129 Reductase Introduction 130 Materials and Methods 133 Screening of Root cdna Library 133 a) A Plaque Lifts 133 b) Screening of Plaque Lifts 133 c) Rescreening of Plaques 135 Purification of A DNA 135 A DNA Filter Hybridization 136 Subcloning of A cdna Inserts into puc 18 136 a) Ligation of the Insert into puc 18 136 b) Transformation of Competent Cells 137 with the Ligation Mixture Sequencing of edna Clone 137 Growth Conditions of Plants 138 RNA Extraction and RNA Blot Hybridization 138 ix
Extraction of Genomic DNA and DNA Blot 139 Hybridization Results 142 Isolation of a cdna clone for Maize Root NR 142 Sequencing of p1501 143 Comparison of p1501 to Other Known Sequences 143 of NR Induction of Maize Root mrna by Nitrate 148 Hybridization of Root and Leaf cdna clones to 150 mrna from Different Maize Organs Hybridization of Root and Leaf NRs to 153 Separate Genes Discussion 155 Chapter 5: Localization of Nitrate Reductase in Maize 160 Roots Introduction 161 Materials and Methods 165 Growth Conditions of Plants 165 Nitrate Reductase Activity Assay 166 Tissue Preparation for Elect~on Microscopy 166 Immunocytochemical Labeling 167 a) ~~tibodies and Labels 167 b) post-embedding Treatment 168 Preparation of Tissue Prints 169 Staining of Tissue Prints with India Ink 170 Hybridization Procedure 171 x
Results 172 Immunocytochemical Localization of Nitrate 177 Reductase Tissue Printing of Maize Roots 177 Expressicn of NR mrna in Maize Roots 178 Discussion 181 GENERAL DISCUSSION 185 LIST OF APPENDICES 192 BIBLIOGRAPHY 238 xi
LIST OF TABLES Table # Page 1 2 3 4 5 6 7 8 9 10 11 12 Effect of storage on the NADH:NRA of maize roots. Effect of the addition of exogenous protein and PMSF to extraction buffer on NADH:~~ in maize roots. Effect of changing the ratio of fro=en root powder to extraction buffer on NRA. Stabilization of root NR ~ith caymostatin. Effect of protease inhibitors on NRA in root extracts of agar grown plants. Effect of protease inhibitors on NADH:NRA in extracts of hydroponically grown roots. Concentration of chymostatin required to stabilize NADH:NRA in maize roots. Effect of protease inhibitors and the antibody to MRP on the recovery of nitrate reductase activity from leaves and roots in optimized assays. Effect of chymostatin on MRP activity. The disappearance of NADH or NADPH with oxaloacetate or alcohol dehydrogenase and acetaldehyde at 340nm. Effect of nitrogen source on NRA in segments of maize roots. Effect on nitrogen source on the induction of NRA in the roots and shoots of m~ize. 45 47 48 49 51 52 55 57 59 60 79 81 xii
13 14 Test of purification conditions on the stability of nitrate reductase acitivity. Test for the presence of MRP activity in ammonium sulfate pellets and supernatants. 85 86 15 Recovery of NRA protocol. from root NR purification 89 16 Summary of biochemical characteristics of the NADH ~nd NAD(P)H enzymes of maize roots and leaves. 99 17 Analy~is of nitrate treated maize tissues for 174 their NRA and for the amount of label observed on plastids in sections of the tissues. 18 Survey of ~s, molecular weights and ph optima 186 of higher plant NRs. xiii
LIST OF FIGURES Figure fi. Page 1 Nitrogen m~tabolism in maize. 8 2 Structural model of higher plant nitrate 25 reductase. 3 Disappearance of maize root NRA in vitro over 54 time. 4 Inhibition of maize leaf and root NRA by 75 antiserum made against purified leaf NR. 5 Time course of induction of NRA in maize 77 roots and shoots with 10rnM KN0 3 6 In vivo stability of NR in maize root tip and 83 mature root segments. 7 Elution profile of NADH and NADPH enzyme 88 activities from the blue Sepharose column. 8 ph optima of partially purified NAD(P)H:NR 92 and NADH: NR. 9 Calculation of Km for NADH of the NADH eluted 93 enzyme. 10 Calculation of Km for N0 3 - of the NADH eluted 94 enzyme. 11 Calculation of Km for NO; of the NADPH eluted 95 enzyme. 12 Calculation of Km for either NADH or NADPH of 96 the NADPH eluted enzyme. 13 Calculation of Km for nitrate of the maize 97 leaf enzyme. 14 Calculation of Km for NADH of the maize leaf 98 enzyme. xiv