MSU LIBRARIES “ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. (L "1'. STARCHLESS AND STARCH-DEFICIENT MUTANTS OF ABABIQQPSIS IHALIANA: ANALYSIS OF GROWTH, CARBOHYDRATE METABOLISM, AND GRAVITROPISM By Timothy Caspar A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1988 ABSTRACT STARCHLESS AND STARCH-DEFICIENT MUTANTS OF ARABIQQPSIS THALIANA: ANALYSIS OF GROWTH, CARBOHYDRATE METABOLISM, AND GRAVITROPISM By Timothy Caspar The role of starch in plant growth and development has been examined using three nuclear mutations of Arabiggpsis thaljang (L.) Heynh. which affect starch metabolism. Two lines, TC7 and TL25, accumulated no detectable starch and a third line, TL46, accumulated 40% as much starch as the wild-type. The enzymatic basis for the starch phenotypes was shown to be a partial or complete lack of activity for starch biosynthetic enzymes. TC7 had no detectable activity for the chloroplast isozyme of phosphoglucomutase. TL25 was completely deficient in ADPglucose pyrophosphorylase activity and lacked both subunits of the enzyme. TL46 contained only 5% as much ADPglucose pyrophosphorylase activity as the wild-type and contained the 51 kD subunit, but not the 54 kD subunit. The gene identified by the mutation in TL25 is unusual in that heterozygotes containing one wild-type and one mutant copy of the gene produced wild-type levels of ADPglucose pyrophosphorylase activity. This lack of dependence of enzyme activity on gene dosage and the absence of both subunits of the enzyme indicates that the mutation may affect a regulatory gene. The mutants were all healthy and completely viable and when grown in continuous light they had rates of growth and photosynthesis similar to the wild-type. However, when grown in a light/dark cycle, the rates of growth, photosynthesis, and dark respiration were affected in the starchless mutants. In addition to the secondary affects of the mutations on growth, photosynthesis, and respiration, the activity of an extrachloroplastic leaf‘B-amylase was increased about 40-fold in the starch mutants. The increased activity was caused by a modification of the enzyme, rather than by an increased amount of the enzyme protein. The unidentified modification which caused the increased activity was stable during electrophoresis and did not alter the mobility of the enzyme in either native polyacrylamide gel electrophoresis or isoelectric focusing. The role of starch in the reception of gravity in gravitropism was examined using the starchless TC7 mutant. Gravitropism in roots and hypocotyls of the mutant was equal to or slightly less than the wild-type, indicating that starch is not required for the detection of gravity. ACKNOWLEDGMENTS I am indebted to many people for their help, advice, and encouragement during this work. First, I acknowledge the people with whom I have collaborated on the work described here. Some of the experiments described in this dissertation were not performed by me but by my collaborators. In order to present this as a complete story and to acknowledge the contributions made by others, I have included these results and labelled them as personal communications. My greatest thanks go to my advisor Chris Somerville for the help, encouragement and excitement which he has given me. I am also grateful to the past and present members of Chris Somerville’s lab for their advice, assistance and friendship. I also thank Jack Preiss and Ken Poff for their assistance and advice during this work; Werner Bernhard for assistance with the work in Chapter 5; Greg Rorrer for his assistance with the HPLC sugar analysis in Chapter 5; Jane Schuette, Karen Klomparens, and Ljerka Kunst for assistance with the microscopy in Chapter 6; Micha Volokita for providing the purified glycolate oxidase used in Chapter 4; Peter Summers for providing purified antibodies and helpful advice for Chapter 4; and Rainer Hertel, Fred Sack, and John Kiss for useful discussions of the work in Chapter 6. Finally, I thank Ellen Johnson for her support, assistance, and perspective. iv TABLE OF CONTENTS PAGE List of Tables .......................... ix List of Figures ......................... x List of abbreviations and notations ............... xiii Chapter I: Introduction ..................... 1 References .................. 3 Chapter 2: Alterations in growth, photosynthesis and respiration in a starchless mutant of Arabidgpsis thaliana (L.) deficient in chloroplast phosphoglucomutase activity . . 4 Abstract ................... 4 Introduction ............... °. . 5 Materials and Methods ............ 6 Results ................... 9 Discussion .................. 26 References .................. 31 Chapter 3: Isolation and characterization of a starchless mutant of Arapjggpsis thaligng lacking ADPglucose pyrophosphorylase ..... Abstract ................... Introduction ................. Materials and Methods ............ Results ................... Discussion .................. References .................. Chapter 4: A starch-deficient mutant of Arabiggpsis thaljana with reduced ADPglucose pyrophosphorylase activity lacks one of the two subunits of the enzyme Abstract ................... Introduction ................. Materials and Methods ............ Results ................... Discussion .................. References .................. Chapter 5: Metabolic, environmental, and developmental regulation of B-amylase in Anahiggpsis thaliang leaves Abstract ................... vi PAGE 34 34 35 36 39 46 51 54 54 55 56 60 72 78 82 82 PAGE Introduction ................. 82 Materials and Methods ............ 84 Results ................... 87 Discussion .................. 102 References .................. 107 Chapter 6: Gravitropism by a starchless mutant of Arabidgnsis: Implications for the starch-statolith theory of gravity sensing ...... 109 Abstract ................... 109 Introduction ................. 110 Materials and Methods ............ 112 Results ................... 119 Discussion .................. 141 References .................. 151 Chapter 7: Concluding remarks .................. 157 Summary and conclusions ........... 157 Future work using the starch mutants ..... 158 Approaches to isolate other starch mutants . . 161 References .................. 164 Appendix A: Search for revertants of TC7 ............. 165 vii Appendix B: Genetic mapping of starch mutations . . . . Appendix C: Procedures for screening for starch mutants viii Table Table Table Table Table Table Table Table Table Table Table Table Table 2-1. 2-2. 2-3. 3-1. 3-2. 3-3. 4-1. 4-2. 5-1. 6-1. 8-1. 8-2. LIST OF TABLES Carbohydrate concentrations in WT and TC7. Effects of the png mutation on the activity of certain enzymes associated with starch and sucrose metabolism in leaves. Gas exchange rates for TC7 and MT in air (350 ul l C02, 21% [v/v] 02). Comparison of the activities of several enzymes associated with starch metabolism in leaves of HT and mutant TL25. ADPglucose pyrophosphorylase activity and starch content in leaves of MT, mutant, and F1 hybrid lines of mm. Cosegregation of ADPglucose pyrophosphorylase activity and starch content in F2 plants from a cross of HT and TL25. Comparison of the activities of enzymes associated with carbohydrate metabolism in leaves of NT and TL46. ADPglucose pyrophosphorylase activity in NT, TL46, and F1 hybrid lines of Arabiggpgis. Description of starch mutant collection. The effects of various mutations on activities of several enzymes associated with starch and sugar metabolism. Enzyme activities in extracts of HT and mutant TC7 ArabjdmsLs Mapping of pgmfi. Mapping of sop}. ix PAGE 11 11 21 42 42 45 62 62 85 92 120 168 168 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 2-1. 2-2. 2-3. 2-4. 2-5. 2-6. 2-7. 3-1. 3-2. 3-3. 4-1. 4-2. 4-3. 4-4. 4-5. LIST OF FIGURES Diurnal changes in starch concentration in leaves of HT [0] and TC7 [o]. PGM and GPI isozymes in TC7 and HT. Effect of photoperiod on growth of TC7 [o] and MT [0]. Diurnal changes in soluble carbohydrate concentration in leaves of MT (0) and TC7 (0). Net C02 uptake by HT (0) TC7 (o). Light-response curve for photosynthetic COZ-fixation by HT [0] and TC7 [0]. Effect of photoperiod on activity of sucrose phosphate synthase in HT [0] and TC7 [o]. Diurnal changes of starch content in the leaves of HT (0) and TL25 (x). Effect of photoperiod on growth rate of NT (0) and mutant TL25 (x). Immunological detection of ADPglucose pyrophosphorylase in crude homogenates of leaves from MT and TL25. Starch content of leaves of the NT (0) and TL46 (X) Mails. Growth rates of MT (0) and TL46 (I) in continuous light (A) and a 12 h photoperiod (B). Immunoblots of crude homogenates of leaves of WT (A), TL46 (B), and TL3 (C). Immunoblots of crude homogenates of leaves of TL46. Immunoblots of crude leaf homogenates of WT and TL46. PAGE 10 l3 l6 18 20 23 25 41 47 48 61 65 67 68 69 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 4-6. 5-1. 5-2. 5-4. 5-5. 5-6. 5-7. 5-8. 6-1. 6-2. 6-3. 6-4. 6-5. Competition ELISA assays to measure ADPglucose pyrophosphorylase (A) or glycolate oxidase (B) in NT (0) and TL46 (I) crude leaf homogenates. Total amylase activity of leaves of HT and mutants with altered starch metabolism. Native polycrylamide gel of leaf extracts of NT Arabiggpsis and mutants with altered starch metabolism stained for amylase activity. Total amylase activity during the life cycle of HT and the pgmfl;1 starch-free mutant. Total amylase activity in NT and the pgmfl mutant during a diurnal cycle. Total amylase activity in NT (A) and the png-1 mutant (B) following a shift from 24/0 to 12/12. Activity and immunoreactive staining of amylase in leaf extracts from MT and the png-l mutant following native polyacrylamide gel electrophoresis. Activity and immunoreactive staining on IEF gels of amylase from RT and the png-l mutant. HPLC analysis of soluble sugars from WT, png-l and sopl;1 lines of Arabidopsis. Light micrographs of median longitudinal sections of MT and TC7 Arabidgpsis root tips and hypocotyls stained with PAS and toluidine blue. Electron micrographs of HT and TC7 Arabidopsis root cap columella cells. Electron micrographs of plastids from WT (A) and TC7 (8) mm; hypocotyls. The effect of gravity on the position of plastids in root-cap columella cells of NT (A) and TC7 (B) Arabidopsis. Gravitropism by roots of NT and TC7 seedlings. xi PAGE 71 89 94 95 96 98 100 101 122 125 127 129 132 Figure 6-6. Figure 6-7. Figure 6-8. Figure 6-9. Figure 6-10. Influence of sugar on gravitropism by MT roots. Gravitropic curvature developed by Arabjdgpsjs roots on a clinostat following stimulations of 2.5 - 30 min in the horizontal position. Gravitropism (A) and phototropism (B) by hypocotyls of HT and TC7 seedlings. Orientation of dark and light-grown HT and TC7 seedlings of identical ages. Illustration of the predicted effects of plastid mass and of mediational capability on gravitropic curvatures. xii PAGE 134 136 138 140 145 ELISA HPLC GPI IEF M2 PAGE PAR PAS PBS PBSB PBSBT PGI PGM Rubisco SOS-PAGE SPS 12/12 24/0 LIST OF ABBREVIATIONS AND NOTATIONS Enzyme-linked immunosorbent assay High performance liquid chromatography Glucose phosphate isomerase Isoelectric focusing Second generation after mutagenesis Polyacrylamide gel electrophoresis Photosynthetically active radiation Periodic acid-Schiff reagent Phosphate-buffered saline Phosphate-buffered saline plus bovine serum albumin Phosphate-buffered saline plus bovine serum albumin and Tween-20 Phosphoglucose isomerase Phosphoglucomutase Ribulose bisphosphate carboxylase Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Sucrose phosphate synthase Mild-type 12 hour light/12 hour dark photoperiod Continuous light xiii CHAPTER 1 INTRODUCTION Starch is a polysaccharide composed of glucose residues connected by sx-l,4 linkages and with variable numbers of ok-l,6 branchpoints. In many plant tissues, starch accumulates in substantial amounts. For example, starch comprises 10% of the dry weight of Arabiggpsis leaves (see Chapter 2) and in storage tissues such as seeds, roots, and tubers typically comprises about 70% of the dry weight (2). In higher plants starch is synthesized and generally localized only inside plastids where it forms insoluble grains ranging from 1 to 100 um in diameter (2). In some instances (e.g. maturing seeds) the plastid envelope breaks down which releases the starch grains into the cytosol. In photosynthesizing tissue, the pathway and regulation of starch synthesis is well described (3). Glucose-6-phosphate is synthesized from fructose-6-phosphate, a Calvin cycle intermediate, by PGI. Glucose-6-phosphate which is an important branchpoint in chloroplast metabolism can be converted to glucose-I-phosphate by PGM which is the first enzyme unique to the starch metabolic pathway. The glucose unit of glucose-I-phosphate is then activated by conversion to ADPglucose by ADPglucose pyrophosphorylase and subsequently added to the non-reducing end of an existing starch molecule by starch synthase, forming ancx-l,4 linkage. Branching enzyme introduces branches in the starch molecule by removing part of the chain and attaching it through an cx-1,6 linkage to an internal residue of another chain. Starch synthesis is thought to be regulated primarily at the ADPglucose pyrophosphorylase step (3). The activity of this enzyme is regulated allosterically by the ratio of 3-phosphoglycerate (an activator) and inorganic phosphate (an inhibitor). In the light the concentration of 3-phosphoglycerate increases because of photosynthetic C02 fixation and the concentration of inorganic phosphate decreases primarily because of photophosphorylation. This activates the enzyme and increases the rate of starch synthesis. In the dark, the concentration of 3-phosphoglycerate declines and inorganic phosphate increases, leading to the inactivation of ADPglucose pyrophosphorylase. The primary function of starch is to act as a storage form for carbon skeletons and energy. Storage may be short-term as in green tissues where sugars produced by photosynthesis are converted to starch in the light. The amount of starch synthesized in leaves varies considerably. For example, in Arabiggpsis leaves about 40% of the photosynthetically fixed carbon is accumulated as starch (recalculated from Chapter 2), whereas in barley leaves the value is about 10% (recalculated from reference 1). During the dark period, the starch formed the previous day is degraded to sugar which is used for cellular metabolism or is translocated to other parts of the plant. Long-term storage of starch occurs in many seeds, tubers, and roots where starch accumulates over the course of days or weeks during one phase of the development of the tissue and then is degraded during a subsequent phase to support growth. Starch, because of its high density, is also thought to function in gravity sensing by plants. Starch-filled plastids which sediment in response to gravity are generally present in tissues which are ‘ sensitive to gravity. This movement, or the pressure exerted by the plastids which this movement indicates, is thought to be the initial step in gravity sensing. This thesis describes studies using mutants of the crucifer Agabidgnsis thaliana which have lesions in starch synthesis. Chapters 2,3,4 describe the isolation and characterization of three different classes of mutants which are partially or completely deficient in starch. The storage function of starch is addressed in these chapters as well as many of the secondary effects which arise when leaf carbohydrate metabolism is altered in these mutants. Chapter 5 addresses in greater depth the secondary effects produced in these starch mutants on the regulation of a specific enzyme of carbohydrate metabolism,‘B-amylase. In Chapter 6 the role of starch in gravity sensing is investigated using several of the starchless lines. The final chapter summarizes the major findings of this work, suggests further uses of these mutants, and describes new approaches to isolate additional classes of mutants. Three appendices are attached which describe the search for second-site revertants of one of the starchless mutant lines, the mapping of two of the lines described in this thesis, and new protocols for isolating starch mutants. REFERENCES 1. Gordon AJ 1986 Diurnal patterns of photosynthate allocation and partitioning among sinks. In J Cronshaw, NJ Lucas, RT Giaquinta, eds, Phloem Transport. Alan Liss, Inc., New York, pp 499-517 2. Jenner CF 1982 Storage of starch. In FA Loewus, N Tanner, eds, Encyclopedia of Plant Physiology, New Series, Vol 13A. Springer-Verlag, New York, pp 700-747 3. Preiss J 1988 Biosynthesis of starch and its regulation. In J Preiss, ed, Biochemistry of Plants, Vol 14. Academic Press, New York, pp 182-254 Chapter 2 Alterations in Growth, Photosynthesis and Respiration in a Starchless Mutant of Arabidgnsis thaliana (L.) Deficient in Chloroplast Phosphoglucomutase Activity ABSTRACT A mutant of Arabiggpsis thaliana (L.) Heynh. which lacks leaf starch was isolated by screening for plants which did not stain with iodine. The starchless phenotype, confirmed bquuantitative enzymatic analysis, is caused by a single recessive nuclear mutation which results in a deficiency of the chloroplast isozyme of phosphoglucomutase. Hhen grown in a 12-h photoperiod, leaves of the WT accumulated substantial amounts of starch but lower levels of soluble sugars. Under these conditions the mutant accumulated relatively high levels of soluble sugars. Rates of growth and net photosynthesis of the mutant and HT were indistinguishable when the plants were grown in constant illumination. However, in a short photoperiod, the growth of the mutant was severely impaired, the rate of photosynthesis was depressed relative to the NT, and the rate of dark respiration, which was high following the onset of darkness, exhibited an uncharacteristic decay throughout the dark period. The altered control of respiration by the mutant, which may be related to the relatively high levels of soluble carbohydrate that accumulate in the leaf and stem tissue, is believed to be partially responsible for the low growth rate of the mutant in short days. The depressed photosynthetic capacity of the mutant may also reflect a metabolic adaptation to the accumulation of high levels of soluble carbohydrate which mimics the effects of Z, alterations in source/sink ratio. The activities of sucrose phosphate synthase and acid invertase are significantly higher in the mutant than in the NT whereas ADPglucose pyrophosphorylase activity is lower. This suggests that the activities of these enzymes may be modulated in response to metabolite concentrations or flux through the pathways. INTRODUCTION The accumulation of nonstructural carbohydrate in leaves has been suggested to influence both the rate of photosynthesis and the rate of dark respiration. An effect of leaf carbohydrate concentration on photosynthesis has been repeatedly proposed as a possible explanation for the depression of photosynthetic rate which may result from experimental treatments which increase the source/sink ratio or decrease the rate of translocation of photosynthate from a source leaf [1,11,18]. However, interpretation of the results of such experiments has been complicated by the possibility that hormonal or other changes may result from the treatment, and from instances in which no correlation has been observed between leaf carbohydrate concentration and photosynthesis rate [17,20,22]. An inherent problem in attempting to establish explanatory correlations is that most experimental manipulations do not permit control of the molecular species of carbohydrate which accumulates. Thus, potential regulatory effects of sucrose or other sugars may be obscured by mechanisms which regulate starch/sucrose partitioning and prevent soluble carbohydrate from accumulating [12,23,28]. The effect of carbohydrate accumulation on the rate of dark respiration has received less attention but appears to have been consistently observed by a variety of approaches [2,6,29]. The essential observation is that the rate of respiration is proportional to carbohydrate content or is stimulated by provision of exogenous carbohydrate. The implication is that the amount of respiration is regulated by substrate supply rather than demand for ATP or reducing equivalents. Indeed, it has been suggested that, under conditions of high carbohydrate supply, a substantial proportion of the reductant generated during carbohydrate catabolism may be consumed by the alternative oxidase without being linked to ATP production [3,l5]. Thus, such respiration may be considered as potentially wasteful and may represent a target for genetic manipulation. To investigate the role of starch in leaf carbohydrate metabolism, we have isolated several mutants of Arabiggpsis thaliana which are unable to synthesize leaf starch. Ne were specifically interested to observe to what extent photosynthate would accumulate in the mutants as soluble sugars rather than starch, and what effects this might have on photosynthesis, respiration and growth. Ne describe here the properties of one such mutant which is unable to convert glucose-6-phosphate to glucose-l-phosphate in the chloroplast, because of a deficiency of the chloroplast isozyme of PGM, and hence is unable to synthesize starch. MATERIALS AND METHODS w h i i . The mutant lines TC7, TC9 and TCI35 were isolated from the Columbia NT of Arabidopsis thaliana (L.) Heynh. following mutagenesis with ethyl methane sulfonate by previously described procedures [26]. The mutant lines, maintained as homozygotes by self-fertilization, were advanced for six to eight generations before being used for the physiological experiments reported here. Unless otherwise indicated, plants were grown at approximately 22°C with cool-white fluorescent illumination (200 uE m'zs'l, PAR) on a perlitezvermiculitezsphagnum (1:1:1) mixture irrigated with a mineral nutrient solution [27]. S1a:§h_ge1_g1ggtrgphgzgsis. Leaf material was ground with an equal weight of buffer containing 100 mM Tris-Cl (pH 7.5), 100 mM KCl, 100 mM sucrose, 40 mM 2-mercaptoethanol, and 5 mM EDTA. The crude extract was absorbed by a small strip of Nhatman 3MM paper which was inserted into a 12% (w/v) starch gel in 50 mM Tris-borate (pH 8.0) and 1.6 mM EDTA [24]. The sample was electrophoresed at 6 volts/cm for 9 h at 4°C, then the gel was sliced horizontally into 1 mm slabs and stained for PGM and GPI activity [24]. The stained gels were fixed with ethanol:acetic acid:glycerin:water (5:2:lz4) and photographed. The chloroplast specific isozymes were identified by first purifying isolated intact chloroplasts from protoplasts [25]. The chloroplasts were ruptured by osmotic shock and the extract was subjected to electrophoresis as described above for whole leaves. fi§§_gxgh§ngg_mg§sgrgmgnt§. Methods for short-term gas exchange measurements on single intact plants have been described [26]. For long-term gas exchange measurements, a plexiglass chamber was constructed to snugly hold a 13 cm pot with a 4.5 cm head space above the pot. Gas inlets and outlets were designed to direct the gas stream across the surface of the plants so as to minimize the boundary layer resistance of intact plants growing in the pots. The C02 concentration in the entering and exiting gas stream was continuously monitored with an Analytical Development Company Series-225 infrared gas analyzer in differential mode. The irradiance was 400 uE m'2 s-l (PAR). Measurements of photosynthesis and respiration were corrected for exchange due to the non-leaf material in the pot at the completion of an experiment by removing all leaf material and repeating the gas exchange measurements on the harvested pot. Chl was determined in ethanol [30]. Carbghydratg_mga§grgmgnts. For genetic screening the presence of starch (amylase) in leaves was qualitatively determined by staining for 30 min with a solution containing: 5.7 mM iodine, 43.4 mM potassium iodide in 0.2 N HCl. Leaves were decolorized before staining by soaking in 96% ethanol for about 6 h. Quantitative measurements of leaf carbohydrate were performed by homogenizing leaf samples in 80% ethanol. Starch was estimated as glucose released by amyloglucosidase treatment of the ethanol-insoluble fraction [23]. The ethanol soluble fraction was evaporated to dryness and then the residues were resuspended in water and assayed for hexose and sucrose content [23]. Enzymg_§§§§y§. Extracts were prepared by grinding leaf samples in cold 50 mM sodium HEPES [pH 7.5], 5 mM MgCl2, 1.0 mM EDTA, 2.5 mM DTT, 2% polyethylene glycol-20 (w/v) and 1% BSA (w/v). Insoluble matter was removed by centrifugation at 38,000 x g for 10 min and an aliquot of the extract was desalted by passage through a small column of Sephadex G-25. Assay procedures for SP5 [23], cytoplasmic fructose bisphosphatase [23], starch synthase [5], UDPglucose pyrophosphorylase 9 [25], ADPglucose pyrophosphorylase [25] and invertase [13] have been described. I ow h . Plants were grown as described above except the irradiance was 500 uE m'2 s'1 PAR. At various times (noted in the text) samples of five plants of each genotype were harvested for fresh weight determinations. RESULTS Mutant_1sglatign. The mutant isolation protocol was based on the assumption that the absence of leaf starch would impose no deleterious effect on plants growing in continuous illumination. In the first attempt at mutant isolation, leaves were removed from approximately 1500 M2 plants descended from ethyl methane sulfonate mutagenized seed, and stained for the presence of starch with iodine. Two starchless plants from this population gave rise to lines (designated TC7 and TC135) which have remained uniformly starchless for the six generations tested so far. In a subsequent screen with an independently mutagenized batch of seed, one starchless mutant line (TC9) was recovered from among approximately 700 M2 plants by the same method. Except for the starchless character, the mutants are phenotypically indistinguishable from the NT when grown in continuous illumination. A quantitative measurement of the starch content of the mutant line TC7 and the NT was obtained by measuring the ethanol-insoluble carbohydrate concentration of plants grown in a 12-h light/ 12-h dark photoperiod (Fig. 2-1) or in continuous illumination (Table 2-1). The leaves of the mutant were almost completely lacking in starch under all conditions. At the level of detection of the iodine stain, the mutants completely lacked starch in leaf, stem and root tissue. 10 g t-Ofi-u—LightOn == Light Off—- '0- 124 O) \ (I) 15 1o- 2 m .2 :3 q 0‘ 8 O) 8 o 6-0 0 £2 03 4‘ 01 EE V I. I. o 2 h CD “ ‘0 o -—¢ *-—¢ . o 4 8 12 16 20 24 Time (hours) Figure 2-1. Diurnal changes in starch concentration in leaves of NT [0] and TC7 [o]. The plants were grown in a 12 h photoperiod. The NT plants were 28-35 days old and the mutant plants were 49-56 days old. Symbols represent the means of measurements made on two samples of leaves. The data points at 0 and 24 h represent the same samples which are repeated simply for clarity. 11 Table 2-1. Carbohydrate concentrations in NT and TC7. Plants were grown in continuous illumination. Values represent the mean of two measurements. Strain Atmosphere Carbohydrate Concentration Starch Soluble Sugars mg glucose equivalents/g fwt Nild-type Ambient 7.70 1.04 TC7 Ambient 0.25 1.13 Nild-type 1%i(v/v) C02 78.30 2.54 Table 2-2. Effects of the pgmfi mutation on the activity of certain enzymes associated with starch and sucrose metabolism in leaves. Plants were grown in a 12-h photoperiod and harvested at the end of the light period. Values represent the mean +/- SE (n-3). (Steve Huber, personal communication) Specific activity Enzyme Nild-type Mutant (TC7) umol g fwt ’ h" ADPglucose pyrophosphorylase 69 i 3 23 i 1 Starch synthase (soluble) 7. 7 i 1.4 10.2 i 1.0 Fructose bisphosphatase 103 i 10 108 i 15 UDPglucose pyrophosphorylase 525 i 20 663 i 25 Acid invertase 99 i 19 212 i 16 aCytoplasmic enzyme 12 BiQgnem1;11_ghazagtgrizatign. The diversion of carbon from the Calvin cycle to amylose involves only four steps which are catalyzed by PGM, GPI, ADPglucose pyrophosphorylase and starch synthase [21]. Thus, the strategy for determining the biochemical basis for the starchless phenotype was straightforward except for the fact that GPI and PGM exist as several isozymes in all plants examined [10]. The presence of multiple isozymes in leaf tissue can complicate interpretation of activity measurements made on crude extracts. Therefore, we examined the activity of PGM and GPI isozymes by histochemical localization following resolution of the isozymes by electrophoresis in starch gels. The results of these experiments (Fig. 2-2) revealed three PGM isozymes and two GPI isozymes in the NT. As a matter of convenience the three isozymes have been designated PGMf (fast), PGMi (intermediate) and PGMs (slow). The fast isozyme of both GPI and PGM is the chloroplast isozyme since this is the only activity which was recovered from isolated intact chloroplasts from the NT (Fig. 2-2). The presence of three isozymes of PGM in A. thaliana is unusual because most species have only two [10]. This may represent a case of a single locus gene duplication as has been suggested for anomalous isozyme patterns in other species [10]. The mutant line TC7 had both of the GPI isozymes found in the NT (Fig. 2-2), normal levels of starch synthase (Table 2-2), about one third as much activity of ADPglucose pyrophosphorylase as the NT (Table 2-2) but completely lacked activity of the chloroplast isozyme of PGM (Fig. 2-2). Since the mutant appears to have adequate levels of the other enzymes required for starch biosynthesis we consider it extremely likely that the loss of chloroplast PGM activity is the biochemical pgm gpl Figure 2-2. PGM and GPI isozymes in TC7 and NT. Nhole leaf extracts from the mutant (lanes 1, 4) and NT (lanes 2, 5) or from isolated intact NT chloroplasts (lanes 3, 6) were electrophoresed in a starch gel which was then sliced and stained for activity. The direction of migration was toward the anode, at the top. 14 basis for the starchless phenotype in this mutant. The reason for the reduced activity of ADPglucose pyrophosphorylase is not known but may reflect a regulated response to the absence of flux through the starch biosynthetic pathway. Subsequent analysis of the other starchless mutant lines may be useful in resolving the basis for this effect. Genetig_gha[§gtgxizgtign. The genetic basis for the starchless phenotype was established by determining the presence of starch in the leaves of the F1 and F2 progeny from a cross between the NT and the mutant line TC7. The F1 progeny from this cross had leaf starch and had all three isozymes of PGM. 0f 84 F2 progeny from this cross, 23 lacked starch. This satisfactory fit (X2-0.25; p > 0.5) to the 3:1 hypothesis, and the properties of the F1 hybrid are entirely consistent with the presence of a single nuclear recessive mutation. Ne have designated the locus defined by this mutation pgmfi (i.e., the plastid isozyme of PGM) and the allele contained in TC7 as pgm£;1. In order to substantiate the relationship between the loss of the f PGM isozyme and the starchless phenotype, 54 F2 plants from a NT x TC7 cross were tested both for starch and for the presence of the PGMf isozyme. All of the 14 plants that lacked starch also lacked activity of the PGMf PGMf isozyme. All other plants contained both starch and the isozyme. This cosegregation of the starchless phenotype and the absence of the PGMf isozyme makes it very unlikely that two separate biochemical deficiencies are responsible for the starchless phenotype. Genetic complementation studies indicate that the starchless phenotype of one of the other lines (TC9) is also due to a mutation in pgmfi since the F1 plants resulting from the TC7 x TC9 cross lacked starch. TC9 also lacked activity of PGMf. The allele contained in TC9 15 has been designated pgm£;2. In contrast, the FI hybrids obtained by crossing the starchless line TC135 with TC7 all had normal levels of starch. This complementation indicates that the mutant line TC135 carries a lesion at another locus. Megsgrgmgn1_gf_grgwtn. In order to quantify the effects of the starchless phenotype on growth rate, the rate of increase in fresh weight of mutant and NT plants was measured for plants growing in various photoperiods. The results of this experiment (Fig. 2-3) show that when plants were grown in continuous illumination, the growth rate of the mutant was indistinguishable from that of the NT. However, as the length of the diurnal dark period was increased, growth of the mutant was differentially impaired relative to that of the NT. Thus, for example, after 37 days of growth on a 7-h light/l7-h dark photoperiod the fresh weight of the mutant was only 10% that of the NT (Fig. 2-3). The similarity of the growth rate of mutant and NT in continuous illumination indicates that the pgmfl mutation is only conditionally deleterious. A requirement for a long photoperiod has also been observed in the other starchless mutants (results not presented), suggesting that the effect is specifically related to the absence of starch per se. The indistinguishable growth rate of the mutant and NT in continuous light also indicates that there are no other significant deleterious genetic defects in the background genotype of the line TC7. dra . In order to provide a basis for interpreting the gas exchange and growth characteristics of the mutant, the nonstructural carbohydrate concentrations of leaves were determined at various times during a light/dark cycle (12-h photoperiod). The 16 .co_umo,=:ssou chomcma .Lmnzz w>mumv .mmpae~m omega go o3» co mums mucmsmgzmaws mo mcmms mew mponeaw .5 «N Auv .5 NH Amy .e A Ae=u mmcoqmme-»;mwa .m-~ me=m_e Am\~E\m_3 mocficmt. coup coop one. com omw o 11$ e m e m 0 io; io.m 30.9 Wk :06 .io.m (u/iuo Btu/zoo 5w) uonexw zoo 24 observation that the photosynthetic capacity of the mutant is not affected by photoperiod raises the possibility that the depression of NT photosynthetic capacity by growth in continuous light is related to decreased net synthesis of starch. It seems likely that in both the NT grown in continuous light, and in the mutant under all conditions, the apparent increase in synthesis of sugars may trigger an adaptation which results in relatively depressed photosynthetic capacity. In this respect, these observations are consistent with the proposal that high levels of soluble carbohydrate may depress photosynthetic capacity [1]. However, because there was no apparent reduction of the photosynthetic rate of the mutant throughout the course of a single photoperiod (Fig. 2-5) in which soluble carbohydrate concentration increased about ten-fold (Fig. 2-4), the mechanisms must involve relatively long-term metabolic adaptation. i P ivi . There is substantial evidence implicating SPS as a regulatory step in sucrose biosynthesis [7,12,23]. In view of the greater amount of sucrose accumulation in the mutant, it was of interest to compare the activity of SPS and related enzymes in the mutant and NT. In attempting to resolve some initial difficulties in obtaining reproducible SPS activity measurements on plants grown in different photoperiods. it was found that there was a pronounced effect of photoperiod on SPS activity. In both mutant and NT the amount of activity increased as the duration of the photoperiod increased. However, the effect was less pronounced in the mutant which had levels of SPS activity similar to the NT when grown in continuous illumination, but significantly higher levels when grown in a 7-h photoperiod (Fig. 2-7). These observations are consistent with the A 25 g T .1: 0 CD E 2 20" C) E :3 v )5 .t.’ :> 155'“ 1:: (J < (O O. CO 10 ; 1'2 24 Photoperiod (hrs) V—b Figure 2-7. Effect of photoperiod on activity of sucrose phosphate synthase in NT [0] and TC7 [0]. Plants were grown for 21 days in the indicated photoperiods. Leaves were harvested at the end of the 7 and 12-h photoperiods. Symbols represent the mean t SE (n-3). (Steve Huber, personal communication) 26 evidence, noted earlier, that the NT and the mutant are functionally similar in continuous light. The other enzymes of sucrose biosynthesis which were assayed (cytoplasmic fructose bisphosphatase and UDP-glucose pyrophosphorylase) showed no major difference in activity but invertase activity was two-fold higher in the mutant (Table 2-2). These observations suggest that SPS and invertase activity may be regulated in response to the amount of flux through the sucrose biosynthetic pathway. DISCUSSION In spite of substantial interest in the biochemistry of starch biosynthesis and degradation [21], the effects of starch on photosynthesis [11,28], stomatal function [19], and gravitropism [14], there have been no previous studies employing a mutant lacking leaf starch. The mutants described here were relatively easy to isolate by direct screening, and mutations at two loci have been isolated. However, it should be noted that A. thaliana is an oilseed and probably does not require starch as a seed carbohydrate reserve. Species which rely on seed starch reserves could, in principle, be dependent on the same genes for both leaf and seed starch biosynthesis. Thus, the recovery of mutants lacking leaf starch might not be possible in these species because such mutants would be seedling lethals. The presence of very low but detectable levels of starch in the mutant line TC7 may be explained in several ways. First, the lesion in the pgmfl gene in TC7 could be somewhat leaky so that sufficient PGM activity is present in the chloroplast to allow the synthesis of small amounts of starch but not enough to permit visualization of the 27 activity by histochemical techniques (Fig. 2-2). Second, small amounts of glucose-l-phosphate may enter the chloroplast from the cytoplasm and be incorporated into starch. Third, the amyloglucosidase treatment we used to degrade the starch for quantification may release small amounts of sugar from polysaccharides other than starch. In this respect, the measured levels are so close to the limit of detection of the methods used (ca. 0.1 mg g fwt'l) that it may simply represent an unavoidable background reaction. Analysis of the other starchless mutants (TC9 and TC135) may be useful in distinguishing among these possibilities. The presence of three isozymes of PGM in A. thaliana is unusual since all other diploid plant species examined to date have only two [10]. Two of the PGM isozymes in A. thaliana are extrachloroplastic and are presumably involved in cytoplasmic metabolism. It seems possible that the gene encoding the cytoplasmic isozyme has undergone gratuitous gene duplication, as suggested to explain other instances of apparently redundant isozymes [10]. Gene duplication seems particularly noteworthy in A. thaliana because the unusually small genome size [16] implicitly suggests an economy of organization. One of the distinguishing characteristics of the mutant is that, because it is unable to store net photosynthate in starch, it accumulates relatively large quantities of sucrose and hexose in both leaf and stem tissue. The accumulation of abnormally high concentrations of soluble carbohydrate pg: sg has no obvious short-term effect on photosynthesis since the photosynthetic rate of mutant plants grown in a 12-h photoperiod does not change as carbohydrate accumulates throughout the period of illumination (Fig. 2-5). However, the photosynthesis rate of the mutant is reduced, relative to the NT, when 28 both are grown in short days (Table 2-3). This effect is probably due to one of two major possibilities. First, it may be due to a specific long-term adjustment of photosynthetic capacity brought about in response to the atypical accumulation of soluble sugars. This is, in effect, similar to the hypothesis proposed to explain the depression of photosynthesis which may be observed following sink removal or other experimental treatments which alter consumption of photosynthate by non-source tissue [18]. Second, the reduced photosynthesis rate of the mutant in a 12-h photoperiod may be just one manifestation of a general reduction in metabolic capacity. Such an effect could, for example, be primarily related to the apparent enhancement of respiratory loss associated with the increased accumulation of soluble carbohydrate in the mutant. Although the results presented here do not distinguish between these possibilities, we favor the concept that the accumulation of soluble carbohydrate may trigger a negative long-term regulatory influence on photosynthetic capacity. This could explain why growth in continuous light has no effect on the photosynthetic capacity of the mutant but depresses the photosynthetic capacity of the NT to a level comparable to that of the mutant. The observation that NT plants grown in continuous illumination in air accumulated only about one tenth as much starch as plants grown in air enriched with 1% (v/v) C02 (Table 2-1), and only about one half as much as plants grown in a 12-h photoperiod (Fig. 2-1) suggests that net starch synthesis ceases in NT plants in continuous illumination not because the size of the starch pool has reached an intrinsic limit, but rather, that an unidentified regulatory mechanism is involved. Furthermore, the steady-state level of total storage carbohydrate in 29 plants of either genotype grown in continuous light is substantially lower than the maximal levels observed during growth in a 12-h photoperiod. Ne are unable to explain this observation which we believe merits further study. However, it may be related to the intriguing observation that soybeans grown in a 7-h photoperiod partitioned about twice as much photosynthate into starch and sugars as did plants grown in a 14-h photoperiod [4]. Finally, it may be worth noting that in continuous light the rate of net C02 assimilation and carbohydrate utilization for growth must be equal. Thus, since the mutant has the same photosynthesis rate in both continuous light and a 12-h photoperiod, the rate of utilization of assimilate in the light must be substantially lower for plants grown in a 12-h light/dark cycle. In addition to the effects on overall photosynthetic rate, a regulatory effect associated with increased flux of carbon through the sucrose biosynthetic pathway is suggested by the observation that SPS activity was increased in response to increased duration of photoperiod in both mutant and NT (Fig. 2-7). This enzyme has recently attracted attention because of an apparent correlation between the amount of extractable SPS activity and the partitioning of photosynthate into starch or sucrose [7,12,23,28]. The essential question which has emerged from this correlative approach is whether the SPS activity is the cause or effect of altered partitioning. The results obtained with the mutant do not directly distinguish between the two possibilities. However, it seems apparent from results presented here that SPS activity may be modulated in the long term by substrate availability or the flux of carbon into hexose phosphate. This explains why the mutant '30 has greater SPS activity than the NT when grown under a light/dark regime. Presumably, as the duration of the photoperiod is increased the NT partitions an increased proportion of photosynthate into soluble carbohydrate until, as noted earlier, it becomes functionally equivalent to the mutant in continuous light. In these circumstances the mutant and NT apparently have the same amount of flux through the sucrose biosynthetic pathway and the same amount of SPS activity. One of several interesting characteristics of the 99mg mutant is the effect of time-of-day on the respiration rate of the mutant. An analogous effect, observed in NT plants of some other species [3], has been interpreted as evidence that the amount of respiration is responsive to the amount or form of storage carbohydrate [3]. In view of the fact that the starchless mutant accumulates high levels of soluble carbohydrate in the light which rapidly declines in the dark, it seems likely that, as suggested [2,3,6] the amount of respiration is proportional to the availability of substrate rather than to the demand for ATP and NADH. It remains to be seen if the abnormally high respiration of the mutant is due to alternative oxidase activity which has been invoked to explain substrate-regulation of respiration [3,15,29]. This concept is of particular interest because of the possibility that crop productivity might be amplified by control of non-productive respiratory losses [9]. In this context the pgmfi mutant may afford a means of selecting directly for loss of the wasteful component by selecting for secondary mutations which enhance the growth rate of the pgmfl mutant in a 7-h photoperiod. If as suggested, the instability of the respiratory response indicates that some proportion of respiration is regulated by the ”)1 amount of substrate rather than demand for ATP (or demand for availability of ADP [8]), then the deleterious effects of the 29mg mutation on growth may be partially explained by proposing the existence of a non-productive competition between consumption of carbohydrate to satisfy the energetic and precursor requirements for growth and wasteful consumption by uncoupled respiration. By consuming all available storage carbohydrate during the first few hours of darkness, the mutant may deprive itself of the carbon required to maintain biosyntheses throughout the night period. The demand for substrate to support maintenance respiration during the latter phase of the dark period may actually stimulate catabolic destruction of recently synthesized macromolecules with the result that growth is severely impeded. Thus, starch may be important not only as a non-osmotic form of reserve carbohydrate, but also as a metabolically inactive reserve.' By controlling the availability of respiratory substrate, the mechanisms regulating the activity of enzymes required for starch hydrolysis may, therefore, be an indirect but important determinant of respiratory efficiency. REFERENCES l. Azcon-Bieto J, 1983 Inhibition of photosynthesis by carbohydrates in wheat leaves. Plant Physiol 73:681-686 2. Azcon-Bieto J, CB Osmond 1983 Relationship between photosynthesis and respiration. The effect of carbohydrate status on the rate of CO production by respiration in darkened and illuminated wheat leaves. Plant Physiol 71:574-581 3. Azcon-Bieto J, H Lambers, DA Day 1983 Effect of photosynthesis and carbohydrate status on respiratory rates and involvement of the alternative pathway in leaf respiration. Plant Physiol 72598-603 IO. 11. 12. 13. 14. 15. l6. 17. 32 Chatterton NJ, JE Silvius 1979 Photosynthate partitioning into starch in soybean leaves. I Effects of photoperiod versus photoperiod duration. Plant Physiol 64:749-753 Ching TM 1982 A sensitive and simple assay of starch synthase activity with pyruvate kinase and luciferase. Anal Biochem 122:139-143 Cunningham GL, JP Syvertsen 1977 The effect of nonstructural carbohydrate levels on dark CO2 release in Creosotebush. Photosynthetica 11:291-295 Doehlert DC, SC Huber 1983 Regulation of spinach leaf SPS by glucose-G-phosphate, inorganic phosphate, and pH. Plant Physiol 73:989-994 Dry IB, JT Niskich 1982 Role of the external adenosine triphosphate/adenosine diphosphate ratio in the control of plant mitochondrial respiration. Arch Biochem Biophys 217:72-79 Gifford RM, JH Thorne, ND Hitz, RT Giaquinta 1984 Crop productivity and photoassimilate partitioning. Science 225:801-808 Gottlieb L0 1982 Conservation and duplication of isozymes in plants. Science 216:373-380 Herold A 1980 Regulation of photosynthesis by sink activity - the missing link. New Phytol 86:131-144 Huber SC 1983 Role of sucrose-phosphate synthase in partitioning of carbon in leaves. Plant Physiol 71:818-821 Huber SC 1984 Biochemical basis for effects of K-deficiency on assimilate export rate and accumulation of soluble sugars in soybean leaves. Plant Physiol 76:424-430 Iversen TH 1969 Elimination of geotropic responsiveness in roots of cress [Lgpidigm sativum) by removal of statolith starch. Physiol Plant 22:1251-1262 Lambers H 1982 Cyanide-resistant respiration: A non-phosphorylating electron transport pathway acting as an energy overflow. Physiol Plant 55:478-485 Leutwiler LS, BR Hough-Evans, EM Meyerowitz 1984 The DNA of A. thaliana. Molec Gen Genet 194:15-23 Mondal MH, NA Brun, ML Brenner 1978 Effects of sink removal on photosynthesis and senescence in leaves of soybean (Glycine max L.) plants. Plant Physiol 61:394-397 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 33 Neales TF, LD Incoll I968 The control of leaf photosynthesis by the level of assimilate concentration in the leaf: A review of the hypothesis. Bot Rev 34:107-125 Outlaw NH Jr. MC Tarczynski 1984 Guard cell starch biosynthesis regulated by effectors of ADP-glucose pyrophosphorylase. Plant Physiol 74:424-429 Potter JR, PJ Breen 1980 Maintenance of high photosynthetic rates during the accumulation of high leaf starch levels in sunflower and soybean. Plant Physiol 66:528-531 Preiss J 1982 Regulation of the biosynthesis and degradation of starch. Ann Rev Plant Physiol 33:431-459 Robinson JM 1984 Photosynthetic carbon metabolism in leaves and isolated chloroplasts from spinach plants grown under short and intermediate photosynthetic periods. Plant Physiol 75:397-409 Rufty TN, SC Huber 1983 Changes in starch formation and activities of sucrose-phosphate synthase and cytoplasmic fructose 1,6-bisphosphatase in response to source-sink alterations. Plant Physiol 72:474-480 Shaw CR, R Prasad I970 Starch gel electrophoresis of enzymes - A complication of recipes. Biochem Genet 4:297-320 Somerville CR, SC Somerville, NL Ogren 1981 Isolation of photosynthetically active protoplasts and chloroplasts from A. ,tnaliana. Plant Sci Lett 21:89-96 Somerville CR, NL Ogren 1982 Isolation of photorespiration mutants in A. thaliana. In M Edelman, R Hallick, NH Chua, eds, Methods in Chloroplast Molecular Biology. Elsevier, New York, pp 129-138 Sowokinos JR 1976 Pyrophosphorylase in Sglgngm tgbgrgsgm. Changes in ADP-glucose and UDP-glucose pyrophosphorylase activities associated with starch biosynthesis during tuberization, maturation and storage of potatoes. Plant Physiol 57:63-68 Stitt M, B Herzog, HN Heldt 1984 Control of photosynthetic sucrose synthesis by fructose 2,6-bisphosphate. Plant Physiol 75:548-553 Tetlet RM, KV Thimann 1974 The metabolism of oat leaves during senescence. I Respiration, carbohydrate metabolism, and the action of cytokinins. Plant Physiol 54:294-303 Nintermans JFGM, A DeMots I965 Spectrophotometric characteristics of chlorophylls a and b and their pheophytins in ethanol. Biochim Biophys Acta 109:448-453 Chapter 3 Isolation and Characterization of a Starchless Mutant of Argbidopsis thaliana Lacking ADPglucose Pyrophosphorylase Activity ABSTRACT A mutant (TL25) of Arabiggnsis thaliana lacking ADPglucose pyrophosphorylase activity (EC 2.7.7.27) was isolated from a mutagenized population of plants by screening for the absence of leaf starch. The mutant grows as vigorously as the NT in continuous light but more slowly in a 12 h light/12 h dark photoperiod. Genetic analysis showed that the deficiencies of both starch and ADPglucose pyrophosphorylase activity were attributable to a single, nuclear, recessive mutation at a locus designated gag]. The absence of starch in the mutant demonstrates that starch synthesis in the chloroplast is entirely dependent on a pathway involving ADPglucose pyrophosphorylase. Analysis of leaf extracts by 2-dimensional polyacrylamide gel electrophoresis followed by Nestern blotting experiments using antibodies specific for spinach ADPglucose pyrophosphorylase showed that two proteins, present in the NT, were absent from the mutant. The heterozygous FI progeny of a cross between the mutant and NT had a specific activity of ADPglucose pyrophosphorylase indistinguishable from the NT. These observations suggest that the mutation in the egg] gene in TL25 might affect a regulatory locus. 34 '35 INTRODUCTION The unique pathway for starch synthesis is believed to involve three steps catalyzed by ADPglucose pyrophosphorylase, starch synthase and branching enzyme. ADPglucose pyrophosphorylase (ATP: c<~glucose-l-P adenyl transferase, EC 2.7.7.27) catalyzes the reversible synthesis of ADPglucose and PPi from ATP and glucose-l-phosphate. Regulation of the activity of this enzyme is believed to play a vital role in controlling the biosynthesis of o<-1,4-glucans in plants and bacteria (18). Mutant maize lines partially deficient in endosperm ADPglucose pyrophosphorylase activity have been reported (4,26). These lines accumulated approximately 25% as much starch as the NT. Nhile the properties of these mutants support the involvement of ADPglucose pyrophosphorylase in starch synthesis, a mutant line completely lacking ADPglucose pyrophosphorylase is required to rule out the possibility that other pathways also contribute to starch synthesis. Here we describe such an Arabidgpsis mutant which lacks both ADPglucose pyrophosphorylase and starch, thus confirming that starch synthesis in the chloroplast is dependent on the participation of this enzyme. This mutant is the third to be described in which leaf starch synthesis is partially or completely impaired. An Arabidopsis mutant has previously been characterized which specifically lacks activity for the plastid isozyme of phosphoglucomutase (Chapter 2). A glgrkig mutant was partially deficient in activity for the plastid isozyme of phosphoglucose isomerase (10). 36 MATERIALS AND METHODS Reagente. Chemicals and enzymes were obtained from Sigma. BCA protein reagent was purchased from Pierce Chemical Co., [32P]PPi was obtained from New England Nuclear Corporation. Nitrocellulose membranes (BA-85; 0.45 um) were from Schleicher & Schuell. Protein A-horseradish peroxidase conjugate was from Bio-Rad Laboratories. Elan; materiale and gnawth eonditiene. The starchless mutants were isolated from the Columbia NT of Ananigeneie thaliana (L.) Heynh following mutagenesis with ethyl methane sulfonate as previously described (6). A once backcrossed derivative of the mutant line TL25 was used for all of the physiological and biochemical experiments reported here. M2 plants were grown in a greenhouse at 22°C under natural illumination with a photoperiod of about 12 h. All other plants were grown at 22°C with illumination from cool-white fluorescent tubes of 200 uE m'2 s'1 PAR on a perlite:vermiculite:sphagnum (1:1:1) mixture irrigated with a mineral nutrient solution (6). untant_1§nla11nn. Mutant screening was carried out as previously described (Chapter 2). Leaves of M2 plants were removed at the end of the photoperiod and the starch content was qualitatively examined by IZ-KI staining. Stareh measurement. Qualitative assays of starch were done by means of IZ-KI staining as previously described (Chapter 2). For segregation analysis, seedlings were grown with continuous illumination in petri plates containing 1% sucrose, 1% agar and mineral salts (6). The plates were oriented vertically so that the roots grew along the surface of the agar. After 4-8 days, the plates were flooded with 37 Iz-KI stain and seedlings were scored for the starch phenotype by observing the staining of the root cap with a dissecting microscope. It was confirmed that the presence or absence of starch in the root cap and leaves is perfectly correlated. The method of quantitative measurement of starch was described by Rufty and Huber (22) except that cit-amylase was also included in the starch digestion mixture. Leaves (100-200 mg) were weighed, then stored in 80% (v/v) ethanol until assayed. They were then cut into 3-4 mm strips and extracted three times with 80% ethanol at 70°C. The leaf strips were then suspended in 2 ml of 0.2 N KOH, ground with a mortar and pestle, and heated at 100°C for 30 min. After cooling, the mixture was centrifuged at 17,000 x g for 10 min, the supernatant was adjusted to pH 5.5 with l N acetic acid, and the volume was adjusted to about 40-80 mg leaf fresh weight per ml with water. To each 200-ul sample solution, 7.4 units of pancreaticcx-amylase in 35 ul was added and incubated at 37°C for 30 min. About 5 units of amyloglucosidase (from Aenengillne nigen) in 165 ul of 0.1 M Na acetate (pH 4.6) was then added and incubated at 55°C for l h. After digestion, the tubes were placed in boiling water for 1 min and centrifuged to pellet denatured protein. Glucose in the supernatant was analyzed enzymatically using hexokinase and glucose-6-P dehydrogenase (9). Quantitative hydrolysis of the starch to glucose under these conditions was confirmed by the complete hydrolysis of amylopectin added to the sample solution. Aeeay_nf_AQ£glnen§e_nynnnnn§nnnnylaee. Fresh leaves of Anaaigenaie were ground with a mortar and pestle in 5 ml/g fresh weight of cold 50 mM Tris-HCl (pH 7.5), 2 mM EDTA. The homogenate was centrifuged at 27,000 x g for 10 min and the supernatant was analyzed 38 for enzyme activities. The pyrophosphorolysis of ADPglucose was followed by the formation of [32P]ATP in the presence of [32P]PPi. The standard reaction mixtures contained in 0.25 ml: 20 umol of glycylglycine (pH 7.6), 1.5 umol of MgClz, 0.25 umol of ADPglucose, 0.5 umol [32P]PPi (1.0 to 4.0 x 106 cpm/umol), 100 ug of BSA, 0.2 umol of 3-phosphoglycerate and enzyme. The reaction mixture was incubated at 37°C for 10 min and terminated by the addition of 3 ml of cold 5% TCA. [32P]ATP was measured as previously described (23). Aeaay_nf_ntnen_enzyne_aet1y111ee. Amylase, starch phosphorylase and D-enzyme were assayed as previously described (16,21). Starch synthase was assayed according to Hawker et al. (7), phosphoglucose isomerase and phosphoglucomutase as described in Chapter 2, and UDPglucose perphosphorylase according to Ozbun et al. (17). Protein concentration was determined by the method of Smith et al. (24) using bicinchoninic acid (Pierce Chemical Co.) and BSA as the standard. Enete1n_aeteet19n_by_1nmnnnbletting. Since the large subunit of ribulose bisphosphate carboxylase and the two subunits of ADPglucose pyrophosphorylase have similar mobilities on SDS-PAGE, a two-dimensional PAGE procedure was developed in order to separate the interfering ribulose bisphosphate carboxylase protein from ADPglucose pyrophosphorylase. Leaf tissue (100 mg) was homogenized in 300 ul of 20 mM Tris-HCl (pH 7.5) using a mortar and pestle. Homogenates were spun in a microfuge for 10 min. Samples of the supernatant (containing 200 ug protein) were run at 150 V for 6 h at 4°C in a nondenaturing polyacrylamide gel using a discontinuous system based on the procedure of Davis (3). Acrylamide concentration in the 1.5 mm thick slab gel was 3% (w/v) for the stacking gel and 7% (w/v) for the separating gel. 39 Following electrophoresis, the lane was excised and equilibrated with 62 mM Tris-HCl (pH 6.8), 2.3% (w/v) SDS, 5% (v/v) 2-mercaptoethanol and 10% (v/v) glycerol at room temperature for 2 h to denature the proteins. A second dimension slab gel was performed based on the procedure of Laemmli (11). The acrylamide concentration in the 1.5 mm thick slab gel was 4.5% (w/v) for the stacking gel and 10% for the separating gel. The stacking gel was poured to about 3 mm from the top of the glass plate. The top of the stacking gel was overlayed with molten gel adherence buffer: 1% (w/v) agarose containing 0.1% (w/v) SDS, 62 mM Tris-HCl (pH 6.8). The excised gel strip was placed between the glass plates in the molten gel adherence buffer. After the gel strip was positioned, the remaining space was filled with gel adherence buffer. SOS-PAGE was run at 50 V overnight at room temperature. Following electrophoresis, the gel was electroblotted onto nitrocellulose membrane using a TE 42 electroblotting apparatus (Hoeffer Scientific Instruments) according to Burnette (1). The filters were blocked for 1 h at 37°C in so mM Tris-HCl (ph 7.5), 150 mM NaCl, 0.1% (v/v) Nonidet P-40, 1% BSA (w/v). The filters were then treated with affinity-purified rabbit anti-spinach leaf ADPglucose pyrophosphorylase antibody (14) and the antigen-antibody complex was visualized by treating with goat anti-rabbit IgG-horseradish peroxidase conjugate (diluted 1:1000) (8). RESULTS Mutant ieelatign. Fifteen starchless or starch deficient plants were recovered from 4,580 M2 plants. In preliminary studies of the 40 progeny of these 15 plants, several enzymes involved in the starch synthetic pathway were assayed. Two lines designated TL24 and TL25 had no detectable activity for ADPglucose pyrophosphorylase. Genetic analysis (see below) indicated that the mutation responsible for the starch deficiency is at the same locus in TL24 and TL25. Thus, TL24 was not further analyzed. Stanen_enntent. A quantitative measurement of the starch content of the leaves of the mutant line TL25 and the NT showed that the mutant contains less than 2% of the starch of the NT when grown in a 12 h light/12 h dark photoperiod (Figure 3-1). Nhen grown in continuous light in an ambient atmosphere or one with elevated C02 (2% [v/v]) leaves of the mutant were also essentially starchless (about 0.2 mg/g fresh weight). At the level of resolution of the iodine stain, the mutant also completely lacked starch in four organs (the root, petiole, flower stalk, and flower) in which the NT normally accumulates starch. The chlorophyll content and soluble protein on a fresh weight basis were comparable in the NT and TL25. B1neneniea1_enanaetenizattnna Comparison of the levels of ADPglucose pyrophosphorylase activity in the mutant line TL25 and NT indicated that the mutant contained less than 2% of the NT ADPglucose pyrophosphorylase activity (Table 3-1). Similar results were also obtained with higher concentrations of MgClz, ADPglucose and 3-phosphoglycerate (20, 3, and 3.6 mM, respectively). The several-fold higher substrate and effector concentrations were found necessary to detect ADPglucose pyrophosphorylase activity in some Eeeherichia e911 mutants which have reduced affinity for the substrate and effector (20). Nhen equal amounts of NT and TL25 extracts were mixed together, 41 7 a—Liqhton—e 4—Liqhi off ’i ‘2 s 5.. I 3 m 54.- u. 0 o > 4‘- U o: < o ’0'; 3.. o 5 2.1- |.. 4———.* F T— T gi—==¥- O 4 8 l2 16 20 24 Time, hour Figure 3-1. Diurnal changes of starch content in the leaves of NT (0) and TL25 (x). The plants were grown in a 12 h light/12 h dark photoperiod (43 days of growth). Symbols represent the means of measurement made on two samples of leaves. (Tsan-Piao Lin, personal communication) 42 Table 3-1. Comparison of the activities of several enzymes associated with starch metabolism in leaves of NT and mutant TL25. Plants were grown in a 12-h photoperiod. All assays were carried out in duplicate on two independent extracts from 42 and 52 day-old plants. Enzyme Specific activity Nild type TL25 nmol min'i (mg protein)-1 ADPglucose pyrophosphorylase 52 0a Starch synthase 4.6 3.8 Amylase 160 550 Starch phosphorylase 29 56 D-enzyme 36 42 UDPglucose pyrophosphorylase 200 240 aDetection limit of ADPglucgse pyrophosphorylase activity is about 1 nmol min (mg protein) . Table 3-2. ADPglucose pyrophosphorylase activity and starch content in leaves of NT, mutant, and F1 hybrid lines of Anatifinnete. The plants were grown for 21 days in continuous light. Line Plants ADPglucose Starch Tested pyrophosphorylasea Contenta nmol min'I (mg proteinfi mg (g fresh weight)‘1 Nild type (NT) 12 54 1 5: 7.3 i 0.4: F1 (NT x TL 25) 12 so 1 4d 6.0 i 0.5e nu 3 0:0 opium gValues are the mean + the standard error of the mean. cNot different at 90% confidence level. dDifferent at 95% confidence level. Detection limit of ADPglucose pyrophosphorylase activity is about 1 nmol min (mg protein) . Limit of detection for starch is about 50 ug/g fresh leaf weight. 43 the activity originally present in the NT extract was not inhibited, indicating that there was no inhibitory substance present in the mutant extract. The mutant had normal levels of starch synthase (Table 3-1) and of the chloroplast isozymes of phosphoglucomutase and phosphoglucose isomerase (data not shown). However, it had consistently higher specific activities of amylase and starch phosphorylase than the NT. The reason for these increased activities is not known at the present time. However, we do not believe these increased activities are directly responsible for the starchless phenotype since other mutant lines which do accumulate starch also have elevated amylase activity. Also, the levels of phosphorylase and amylase in the mutant are not elevated over the NT level when the mutant is grown in continuous light (see Chapter 5). Moreover, the high level of amylase is due to an increase in the amount of an extra-chloroplastic amylase isozyme. Thus, we believe that the ADPglucose pyrophosphorylase deficiency is the direct cause of the starch deficiency. Genet1e_ana1y§1§. The genetic basis of the starchless phenotype was determined by crossing the mutant with the NT. The F1 heterozygous plants derived from this cross all contained starch in the root caps. The F1 individuals were also analyzed for enzyme activity and leaf starch content (Table 3-2). This analysis showed that leaf extracts of F1 plants contained the same ADPglucose pyrophosphorylase specific activity as the NT. All F1 plants were confirmed to be heterozygotes by showing that, when self-fertilized, they segregated starch-containing and starch-deficient plants. 44 Of 369 F2 seedlings from the NT x TL25 cross scored, 90 had root caps which lacked starch. This excellent fit to the 3:1 hypothesis (XZ-O.O73; P>O.9) indicates that the presence of a single, nuclear, recessive mutation in TL25 is responsible for the starchless phenotype. Ne have designated the locus defined by this mutation aggl and the allele contained in TL25 as agg1;1. To confirm that the absence of ADPglucose pyrophosphorylase activity and the starchless phenotype are caused by the same mutation, the F2 plants resulting from a cross of NT x TL25 were tested for both starch content and enzyme activity. Out of 42 F2 plants tested, 6 lacked starch and ADPglucose pyrophosphorylase activity. All other plants contained both starch and enzyme activity (Table 3-3). In an additional experiment in which 50 F2 plants were quantitatively analyzed for ADPglucose pyrophosphorylase activity but only qualitatively for starch (by iodine staining of leaves), all 10 of the starchless plants lacked detectable ADPglucose pyrophosphorylase activity and all 40 of the starch-containing plants had ADPglucose pyrophosphorylase activity (mean - 87 nmol min'l mg"1 protein). The distribution of ADPglucose pyrophosphorylase specific activities in the F2 plants from both experiments was most satisfactorily represented by 2 classes with 100% and 0% of the activity of the NT. Thus, the heterozygotes contained ADPglucose pyrophosphorylase activity comparable to the NT, consistent with the results of the F1 plants (Table 3-2). The mutation in TL24 is recessive since all the F1 progeny from the cross NT x TL24 contained starch. This mutation is also in the aggl locus since all 20 of the F1 progeny of the cross TL24 x TL25 45 Table 3-3. Cosegregation of ADPglucose pyrophosphorylase activity and starch content in F2 plants from a cross of NT and TL25. Plants were grown for 48 days in a 12 h photoperiod and were sampled 10 h after the beginning of the light period. Line Plants ADPglucose Starch Tested pyrophosphorylase Content nmol min'} (mg protein)'} mg (g fresh weight)‘1 F2 (NTxTLZS) 6 oiob o..211004 F2 (HT x TL25) 36 19 1 2 3.9 i 0.6 Nild type (NT) 1 26b 4.4 TL25 2 O 0.10 bValues are the mean + the standard error of the mean. Detection limit_ gf ADPglucose pyrophosphorylase activity is about 1 nmol min protein. 46 lacked starch and ADPglucose pyrophosphorylase activity and may represent another allele (designated agg1;2). Meaagnenent_gf_gnentn. The rate of fresh weight accumulation was measured for the mutant and NT growing in 2 different photoperiods. Figure 3-2 shows that the growth rate of the mutant was indistinguishable from the NT when the plants were grown in continuous illumination. However, the growth of the mutant was greatly reduced in a 12 h light/12 h dark photoperiod. In both conditions, the mutant appeared healthy and, apart from the slower growth rate in the 12 h photoperiod, had no apparent differences from the NT. A similar effect of photoperiod on growth rate was observed previously with another starchless mutant defective in plastid phosphoglucomutase activity (Chapter 2). Neetenn_hlgtt1ng_exnen1nent. The effect of the aggl mutation on the amount of ADPglucose pyrophosphorylase protein was examined by Z-dimensional PAGE of crude leaf extracts followed by Nestern blot hybridization. The NT contained two polypeptides which cross-reacted with affinity-purified rabbit antibodies prepared against spinach leaf ADPglucose pyrophosphorylase (Figure 3-3, upper panel). These two subunits of ADPglucose pyrophosphorylase from Ananjggneie leaf have the same apparent molecular mass, 51 kD and 54 kD, as the 2 subunits from spinach leaf (14). In contrast, neither the TL25 (Figure 3-3, lower panel) nor the TL24 (results not shown) leaf homogenates contained detectable cross-reactive polypeptides. DISCUSSION Genetic evidence has previously been cited to show that ADPglucose 47 A 24IO 0.12/12 500- 500.. WT 400« 400« E o 300-- E 300.. { g 3 0' u. \ 2000 a 200.. 3 2 u. 0 2 IOO«- 100.. ‘i ‘ ‘ 2'0 so 40 20 3O 40 O 0 DAYS OF GROWTH DAYS OF GROWTH Figure 3-2. Effect of photoperiod on growth rate of NT (0) and mutant TL25 (x). The daily period of illumination was (A) 24 h; (B) 12 h. Symbols are means of measurements of 5 plants; error bars represent the standard deviations. (Tsan-Piao Lin, personal communication) 48 3 le Native PAGE A. WT 54kD— p .. 511(0- "”‘ H p . a {—SDVdSOS Figure 3-3. Immunological detection of ADPglucose pyrophosphorylase in crude homogenates of leaves from NT and TL25. Proteins were electrophoretically separated in a nondenaturing gel followed by SOS-PAGE. The cross-reactive polypeptides were detected using affinity-purified rabbit anti-spinach leaf ADPglucose pyrophosphorylase immunoglobulins. Crude homogenate containing 200 ug protein was used for NT (upper panel, lane 8), and mutant TL25 (lower panel, lane B). Purified spinach leaf ADPglucose pyrophosphorylase (500 ng, lanes A) was run only in the second dimension with the gel strip obtained from a nondenaturing gel of the first dimension. (Tsan-Piao Lin, personal communication) 49 pyrophosphorylase is a component of the major route of starch biosynthesis (18). In two maize mutants, shrunken-2 (Sh;2) and brittle-2 (nt;2), the levels of ADPglucose pyrophosphorylase activity in the endosperms was less than 10% of that found in normal endosperm (4,26). Although the 90% reduction in activity was correlated with a 75% reduction in the level of starch compared to normal endosperm, it was not possible to exclude the involvement of another route for starch biosynthesis in maize amyloplasts using these mutants. Mutant line TL25, which contains less than 2% of the ADPglucose pyrophosphorylase activity and starch of the NT, has provided additional evidence which shows that this enzyme is responsible for at least 98% of the starch synthesis in the chloroplast. The absence of starch in the mutant in the nonphotosynthetic root cap suggests that ADPglucose pyrophosphorylase is entirely responsible for starch synthesis in amyloplasts as well. This evidence effectively excludes the possible participation of both UDPglucose pyrophosphorylase and starch phosphorylase in net starch synthesis in the chloroplast. UDPglucose pyrophosphorylase has been suggested to have a role in starch synthesis in maize endosperm (26), and in developing pea seeds (27), even though it was not detected in the amyloplast of soybean (12). Starch phosphorylase was also suggested to play a role in starch biosynthesis (13,15). However, the Kn values found for glucose-I-phosphate are at least one order of magnitude higher than the estimated glucose-l-phosphate concentration in spinach chloroplasts (19). The observation that the mutant line TL25 has more than two times higher specific activity of starch phosphorylase in the leaf crude homogenate 50 than that of the NT indicates that this enzyme does not contribute directly to starch synthesis in the leaf tissue. The previously characterized starchless mutant TC7, which lacked phosphoglucomutase activity, had physiological characteristics which were attributed to the altered carbohydrate status resulting from the inability to synthesize starch (Chapter 2). Among these were a decreased growth and photosynthetic rate and an unstable dark respiration rate when grown in a 12 h light/12 h dark photoperiod. These rates were, however, similar to the NT when the plants were grown in continuous light, suggesting that in these conditions the lack of starch had no serious effect on the mutant. The response of the growth rate of TL25 to photoperiod (Fig. 3-2) is qualitatively identical to that observed for TC7. This indicates that similar physiological changes resulted from the starchless phenotype which is caused by the aggl mutation in TL25. In the immunochemical analysis of the mutant TL25, no cross-reactive material could be detected in crude extracts using an antibody against ADPglucose pyrophosphorylase (Fig. 3-3). This could be due to a nonsense mutation in the structural gene for the enzyme, a mutation in a regulatory locus which prevents the transcription or translation of the protein, or a mutation which destabilizes the structure of the protein so that the protein is rapidly degraded. Our results tend to rule out the first and third possibilities, since ADPglucose pyrophosphorylase activity comparable to the NT was observed in heterozygous F1 plants from the NT x TL25 cross (Table 3-2). This is in contrast to numerous other reports in which heterozygotes exhibited levels of activity intermediate to that found in either 51 parent. For example, intermediate enzyme activity was found in heterozygous plants derived from crosses between NT and a barley mutant deficient in glutamine synthetase activity (28), Anantggneie mutants deficient in phosphoglycolate phosphatase (25) and chloroplast phosphoglucomutase (Caspar, unpublished results), and maize mutants deficient in ADPglucose pyrophosphorylase (5). The restoration of 50% of NT activity in the heterozygote is consistent with the expectation for a mutation within the structural gene. Thus, the presence of normal levels of ADPglucose pyrophosphorylase activity in the heterozygote suggests a more complex situation and may indicate that aggl is a gene which regulates the amount of ADPglucose pyrophosphorylase in Azaniggneie leaf tissue. Nhatever the case, the aggl mutation appears to have a specific effect on ADPglucose 'pyrophosphorylase since none of the other enzymes associated with starch biosynthesis were similarly affected. REFERENCES 1. Burnette NN 1981 “Nestern blotting" electrophoretic transfer of proteins from SOS-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112:195-203 2. Caspar T, SC Huber, C Somerville 1986 Alterations in growth, photosynthesis and respiration in a starch mutant of Arabigongis thaliana (L.) Heynh deficient in chloroplast phosphoglucomutase activity. Plant Physiol 79:1-7 3. Davis BJ 1964 Disc electrophoresis 11. Methods and applications to human serum proteins. Ann NY Acad Sci 121:404-427 4. Dickinson DB, J Preiss 1969 Presence of ADPglucose pyrophosphorylase in shrunken-2 and brittle-2 mutants of maize endosperm. Plant Physiol 44:1058-1062 10. 11. 12. 13. 14. 15. 16. 17. 18. 52 Hannah LC, OE Nelson 1975 Characterization of adenosine diphosphate glucose pyrophosphorylase from developing maize seeds. Plant Physiol 55:297-302 Haughn GN, CR Somerville 1986 Sulfonylurea resistant mutants of Azaniggnate tnaliana. Molec Gen Genet 204:430-434 Hawker JS, JL Ozbun, H Ozaki, E Greenberg, J Preiss 1974 Interaction of spinach leaf adenosine diphosphate glucose ot-l,4-, «x-I,4-glucosyl transferase and x-I,4-glucan, o<-1,4-glucan-6-glucosyl transferase in synthesis of branched cx-glucan. Arch Biochem Biophys 160:530-531 Hawkes R 1982 Identification of concanavalin A-binding proteins after sodium dodecyl sulfate-gel electrophoresis and protein blotting. Anal Biochem 123:143-146 Jone; MGK, NH Outlaw, 0H Lowry 1977 Enzymic assay of 10"7 to 10 moles of sucrose in plant tissue. Plant Physiol 60:379-383 Jones TNA, LD Gottlieb, E Pichersky 1986 Reduced enzyme activity and starch level in an induced mutant of chloroplast phosphoglucose isomerase. Plant Physiol 81:367-371 Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680-685 MacDonald F, T apRees 1983 Enzymatic properties of amyloplasts from suspension cultures of soybean. Biochim Biophys Acta 755:81-89 Mengel K, GK Judel 1981 Effect of light intensity on the activity of starch synthesizing enzymes and starch synthesis in developing wheat grains. Physiol Plant 51:13-18 Morell MK, M Bloom, V Knowles, J Preiss 1987 Subunit structure of spinach leaf ADPglucose pyrophosphorylase. Plant Physiol 85:182-187 Obata-Sasamoto H, H Suzuki 1979 Activities of enzymes relating to starch synthesis and endogenous levels of growth regulators in potato stolon tips during tuberization. Physiol Plant 45:320-324 Okita TN, E Greenberg, DN Kuhn, J Preiss 1979 Subcellular localization of the starch degradative and biosynthetic enzymes of spinach leaves. Plant Physiol 64:187-192 Ozbun JL, JS Hawker, E Greenberg, C Lammel, J Preiss, EYC Lee 1973 Starch synthase, phosphorylase, ADPglucose pyrophosphorylase and UDPglucose pyrophosphorylase in developing maize kernels. Plant Physiol 51:1-5 Preiss J 1982 Regulation of the biosynthesis and degradation of 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 53 starch. Annu Rev Plant Physiol 33:431-454 Preiss J, C Levi 1980 Starch biosynthesis and degradation. In The Biochemistry of Plants, Vol. 3, J Preiss, ed, pp 371-423. Academic Press, New York Preiss J, A Sabraw, E Greenberg 1971 An ADPglucose pyrophosphorylase with lower apparent affinity for substrate and effector molecules in an E. gall B mutant deficient in glycogen synthesis. Biochem Biophys Res Commun 42:180-186 Preiss J, TN Okita, E Greenberg 1980 Characterization of the spinach leaf phosphorylase. Plant Physiol 66:864-869 Rufty TN, SC Huber 1983 Changes in starch formation and activities of sucrose-phosphate synthase and cytoplasmic fructose 1,6-bisphosphatase in response to source-sink alteration. Plant Physiol 72:474-480 Shen L, J Preiss I964 The activation and inhibition of bacterial adenosine diphosphoglucose pyrophosphorylase. Biochem Biophys Res Commun 17:424-429 Smith PK, RI Krohn, GT Hermanson, AK Mallia, FH Gartner, MD Provenzano, EK Fujimoto, NM Goeke, BJ Olson, DC Klenk 1985 Measurement of protein using bicinchoninic acid. Anal Biochem 150:76-85 Somerville CR, NL Ogren 1979 A phosphoglycolate phosphatase-deficient mutant of Ananignneie. Nature 280:833-836 Tsai C, DE Nelson 1966 Starch-deficient maize mutant lacking adenosine diphosphate pyrophosphorylase activity. Science 151:341-343 Turner JF 1969 Physiology of pea fruits. VI Changes in uridine disphosphate glucose pyrophosphorylase and adenosine diphosphate glucose pyrophosphorylase in the developing seed. Aust J Biol Sci 22:1145-1151 Nallsgrove RM, JC Turner, NP Hall, AC Kendall, SNJ Bright 1987 Barley mutants lacking chloroplast glutamine synthase-biochemical and genetic analysis. Plant Physiol 83:155-158 Chapter 4 A Starch-Deficient Mutant of Ananiggneie tnaliana with Reduced ADPglucose Pyrophosphorylase Activity Lacks One of the Two Subunits of the Enzyme ABSTRACT A starch-deficient mutant of Ananjggnaie thaliana (L.) Heynh. has been isolated in which leaf extracts contain only about 5% as much activity of ADPglucose pyrophosphorylase (EC 2.7.7.27) as the wild- type. A single, nuclear mutation at a previously undescribed locus designated aggz is responsible for the mutant phenotype. Although the mutant contained only 5% as much ADPglucose pyrophosphorylase activity as the wild-type, it accumulated 40% as much starch when grown in a 12 h photoperiod. The mutant also contained about 40% as much starch as the wild type when grown in continuous light, suggesting that the rate of synthesis regulates the amount of starch accumulated in this steady state condition. Immunological analysis of leaf extracts using antibodies against the spinach 54-kD and 51-kD ADPglucose pyrophosphorylase subunits indicated that the mutant is deficient in a cross-reactive 54-kD polypeptide and has only about 4% as much as the NT of a cross-reactive 51-kD polypeptide. This result and genetic studies suggested that an2 is a structural gene which codes for the 54-kD polypeptide, and provides the first functional evidence that the 54-kD polypeptide is a required component of the native ADPglucose pyrophosphoryiase enzyme. 54 55 INTRODUCTION ADPglucose pyrophosphorylase (ATchK-glucose-l-P adenyl transferase, EC 2.7.7.27) is required for the biosynthesis of oe-l,4-glucans in plants and bacteria (23). Allosteric regulation of this enzyme by metabolites such as inorganic phosphate and 3-phosphoglycerate in plants and 5’-adenylate and fructose-1,6-bisphosphate in bacteria plays a vital role in controlling the rate of starch and glycogen biosynthesis (23). In bacteria and plants the size of the native ADPglucose pyrophosphorylase is similar, ranging from about 200 to 240 kD (3,24) but the subunit structure is different. The flatnenienia egli enzyme is a homotetramer with a subunit size of about 50 k0 (7). By contrast, two different polypeptides of 51 and 54 kD co-purified with the activity from spinach leaves (3, 17). Leaves of other species such as Anantggnaie (Chapter 3), wheat, rice, and maize (14) also contain immunologically related peptides of similar sizes. However, purified potato tuber ADPglucose pyrophosphorylase showed only a single band of 50 k0 on SOS-PAGE (29) and maize endosperm ADPglucose pyrophosphorylase has been reported to contain only a single subunit of 55 kD (22). It has recently been found, however, that a second polypeptide of 60 k0 is also associated with the enzyme from maize endosperm (P. Summers, J. Preiss, in preparation). Functional studies which implicate both polypeptides in enzymatic function are limited to the observation that both spinach leaf polypeptides interact with pyridoxal phosphate, an analog of the allosteric activator 3-P-glycerate (18,24). However, because it was 56 not possible to separate the two polypeptides without denaturing them (17,24), the functions of the polypeptides are not known. Ne have previously described mutants of Anantggnete which lacked ADPglucose pyrophosphorylase (Chapter 3). In this paper we describe a new Azanignnete mutant which is deficient in the 54-kD subunit and contains only about 5% as much ADPglucose pyrophosphorylase activity as the NT. This provides the first strong evidence for the involvement of the 54-kD polypeptide in the functioning of this enzyme. Despite the enzymatic deficiency, this mutant still accumulates about 40% as much starch as the NT and shows no obvious morphological effects of the mutation. MATERIALS AND METHODS Beagente. Biochemicals and enzymes were obtained from Sigma. [32P]PPi was obtained from New England Nuclear Co. Nitrocellulose membranes (BA-85; 0.45 um) were from Schleicher 8 Schuell. Goat anti-rabbit horseradish peroxidase conjugate was from Bio-Rad and goat anti-rabbit alkaline phosphatase conjugate was from Sigma. P n i io . The starchless mutants were isolated from the Columbia NT of Arabiggneje thaliana (L.) Heynh. following mutagenesis with ethyl methane sulfonate as previously described (Chapter 2). M2 plants were grown in a greenhouse at 22°C under natural illumination with a photoperiod of about 12 h. All other plants were grown in growth chambers at 22°C with illumination from cool white fluorescent tubes of about 120 uE m'2 s'1 PAR on a perlite:vermiculite:sphagnum (1:1:1) mixture irrigated with a mineral nutrient solution (8). M6 plants or lines backcrossed once to the NT 57 were used for the physiological and biochemical experiments reported here. Aaeay_gf_AQEg1g;gee_nyngnng§nngnyla§e. Leaves were ground in 4 ml (9 fresh weight)"1 of cold 50 mM Tris-HCl (pH 7.5), 2 mM EDTA. The homogenate was microfuged for 10 min at 4°C and the supernatant was used for enzyme assays. The pyrophosphorolysis of ADPglucose was followed by the formation of [32P]ATP in the presence of [32PJPPi. The standard reaction mixtures contained in 0.25 ml: 20 umol glycylglycine (pH 7.6), 1.5 umol MgClz, 0.25 umol ADPglucose, 0.5 umol [32P]PPi (1.0 - 4.0 x 103 dpm nmol'l), 100 ug BSA, 0.2 umol 3-P-glycerate, and crude extract containing 1 to 10 ug protein. The reaction mixtures were incubated at 37°C for 10 min and terminated by the addition of 3 ml of cold 5% trichloroacetic acid. [32P]ATP was measured as previously described (Chapter 3). The high concentration of 3-P-glycerate and the absence of Pi in the reaction mixture ensures that the enzyme is fully activated. AW. Amylase (19). starch phosphorylase (I9), D-enzyme (l9), soluble starch synthase (9), Rubisco (21), UDPglucose pyrophosphorylase (20), phosphoglucomutase (Chapter 2), phosphoglucose isomerase (Chapter 2), and acid invertase (10) were assayed as previously described. Protein concentration was determined using BSA as a standard with a Coomassie blue binding assay (Bio-Rad) for the experiments of Figs. 4-5 and 4-6 or, for all other experiments, by the method of Smith et al. (28) using bicinchoninic acid (Pierce Chemical Co.). Starch measurement. Qualitative starch assays using Iz-KI staining of leaves removed at the end of the light period were used for 58 mutant screening as previously described (Chapter 2). The method of quantitative measurement of starch was as previously described (Chapter 3) based on the procedure of Rufty and Huber (26) except that ct-amylase was also included in the starch digestion mixture. Glucose derived from the digested starch was analyzed enzymatically using hexokinase and glucose-6-P dehydrogenase (11). Quantitative hydrolysis of the starch to glucose under these conditions was confirmed by the complete hydrolysis of amylopectin added to the sample solution. Engte1n_geteettgn_ty_1nnnngblgtt1ng. Since the large subunit of Rubisco and the two subunits of ADPglucose pyrophosphorylase have similar mobilities on SOS-PAGE, two-dimensional gel electrophoresis as previously described (Chapter 3) was primarily used for immunoblotting. The primary antibodies used were raised in rabbits against the native spinach leaf enzyme or the isolated 54- or 51-kD subunits of the spinach enzyme (17). Cross-reacting peptides were detected using either goat anti-rabbit IgG-horseradish peroxidase conjugate (Figs. 4-3 and 4-4) or alkaline phosphatase conjugate (Fig. 4-5). The amount of cross-reactive material in the bands in Figure 4-5 was quantified by excising the region of the nitrocellulose containing the band, dissolving it in DMSO, and measuring the absorbance at 569.5 nm. Engte1n_geteet1gn_by_EL1§A. Cross-reactive polypeptides were detected using a competition ELISA. In principle, biotinylated, purified ADPglucose pyrophosphorylase (or glycolate oxidase as a control) is mixed with crude extracts containing unknown amounts of ADPglucose pyrophosphorylase protein. The ADPglucose pyrophosphorylase in the crude extract competes with the biotinylated ADPglucose pyrophosphorylase for antibody binding sites. The amount of bound 59 biotinylated ADPglucose pyrophosphorylase is then measured by its reaction with streptavidin conjugated alkaline phosphatase. The assay was initiated by adding 50 ul of diluted (in 15 mM Na CO 35 mM 2 3’ NaHCO pH 9.2) “capture” antibodies (affinity purified rabbit IgG 3. against ADPglucose pyrophosphorylase, a gift from Peter Summers, or crude rabbit sera against ADPglucose pyrophosphorylase or glycolate oxidase) to each well of a 96 well polystyrene ELISA plate (Nunc-Immuno Plate 1) and incubated overnight at 4°C. The wells were washed twice in P858 (10 mM Na-phosphate, pH 7.3, 150 mM NaCl, 1%, w/v, BSA) and blocked with 100 ul PBSB. This and all subsequent incubations were conducted at room temperature on a rotating platform at 250 rpm. Purified spinach glycolate oxidase (a gift from Micha Volokita) or ADPglucose pyrophosphorylase was biotinylated with biotin-N-hydroxysuccinimide ester (Bethesda Research Laboratories) (1). Crude extracts were prepared by grinding leaves in 0.75 ml per g fresh weight of cold 50 mM Tris-Cl, pH 7.5. The homogenate was microfuged for 10 min at 4°C and the supernatant was mixed in varying dilutions with 5 ng biotinylated ADPglucose pyrophosphorylase or glycolate oxidase in 50 ul of PBSB. These mixtures were incubated in the wells for 1 h and the wells were then washed 4 times with PBSBT (PBSB plus 0.3%, v/v, Tween-20). Fifty ul streptavidin conjugated alkaline phosphatase (Bethesda Research Laboratories, diluted 1:1000 in PBSB) was then added to each well, incubated for 1 h, and then washed 6 times with PBSBT. Fifty ul of substrate solution (5 mM p-nitrophenyl phosphate, 0.3% Tween-20, 100 mM Tris-Cl, pH 9.5, 100 mM NaCl, 50 mM MgCl2) was then added to each well and after 90-120 min the absorbance at 405 nm was measured with an ELISA plate reader (Bio-tek). 60 RESULTS Mgtant_1enlatign. Fifteen starchless or starch-deficient plants were identified from among 4580 M2 plants. Preliminary studies of the 15 lines indicated that 2 lines (TL3 and TL46) had reduced activity for ADPglucose pyrophosphorylase. These lines were advanced four generations to ensure that the mutations were stable. Two other lines (TL24 and TL25) which had no activity for ADPglucose pyrophosphorylase have already been described (Chapter 3). A quantitative measurement of the starch content of the leaves of the mutant line TL46 and the NT showed that the mutant accumulated about 40% as much starch as the NT when grown either in a 12 h photoperiod or in continuous light (Fig. 4-1). B1eenen1ea1_enanaeten11atign. The specific activity of ADPglucose pyrophosphorylase in the mutant TL46 varied slightly in different preparations but averaged about 5% as much as the NT (Tables 4-1, 4-2, unpublished results). The activity relative to the NT was not increased if 1.5 mM phenylmethylsulfonyl fluoride and 10 mg l'1 chymostatin were included in the grinding buffer (results not presented), suggesting that the low activity in the mutant was not due to proteolytic susceptibility of the enzyme in the crude homogenate. The low specific enzyme activity in TL46 extracts was obtained even when using elevated concentrations of MgClZ, ADPglucose and 3-P-glycerate (20, 3 and 3.6 mM, respectively; results not presented). The several-fold higher substrate and effector concentrations were found necessary to observe ADPglucose pyrophosphorylase activity in some it egli mutants which have reduced affinity for the substrate and effector (25). Mixing experiments with NT and TL46 extracts (Table 61 24m e—lighion——-)(<-——"9m off—- mp- .. . WT period .. x'TL46 ,_ 6. 2- ' c '5 ‘0 3 E 1: 8 g, ‘I- i- g h 23 cm c1‘\\ ‘6 '5 L ‘- |*_ ‘ n- O E: 53 -1 cm EE U _ x\ i '- 1 (3i . . . ‘\\\\‘; o 4 e :2 IS 20 24 Time (hours) Figure 4-1. Starch content of leaves of the NT (0) and TL46 (X) Aranidgpeia. The plants were grown in a 12 h photoperiod for 35 days (left panel) or in continuous light for 21 days (right panel). Symbols represent the means of measurements made on two samples of leaves; error bars represent the standard error of the mean. (Tsan-Piao Lin, personal communication) 62 Table 4-1. Comparison of the activities of enzymes associated with carbohydrate metabolism in leaves of NT and TL46. glants were 28 to 35 days old and were grown in a 12 h photoperiod at 22 C. Values represent the mean 1 SE of assays carried out on two or three extracts. Enzyme Specific Activity NT TL46 nmol min'i (mg protein)'% ADPglucose pyrophosphorylase 88 a 1.4 1.8 a 0.1 Soluble starch synthase 1.5 a 0.1 1.3 a 0.1 Ribulose bisphosphate carboxylase 180 a 8.4 170 a 5.5 D-enzyme 19 a 0.6 21 a 1.0 Amylase 47 i 22 97 i 32 Starch phosphorylase 29 a 0.7 37 a 0.7 Acid invertase 100 i 5.8 110 a 1.5 UDPglucose pyrophosphorylase 41 a 2.2 44 a 1.5 Table 4-2. ADPglucose pyrophosphorylase activity in NT, TL46, and F1 hybrid lines of Anantggnaie. Plants were grown for 16 to 28 days in continuous light. Values are reported as the percentage of the specific activity of the NT of the same age and assayed on the same day. Theloverall average specific activity for the NT was 47 i 3.9 nmol min (mg protein) . Line Plants ADPglucose Pyrophosphorylase Tested Mean SE % of NT NT 9 100 - TL46 11 6.6 0.9 TL3 4 4.4 0.2 TL25 a 2 0.0 0 0 NT + TL46 - 50 - F1 (NT x TL46) 10 63 2.3 F1 (NT x TL3) 3 37 1.8 F1 (TL3 x TL46) 3 5 0 1.0 F1 (TL25 x TL46) 5 65 3.2 Fl (TL25 x 1L3) 5 60 4.4 TL46 + TL25 - 0.6 - 3Mixture of equal amounts of the two extracts. 63 4-2) showed that the activities in the NT and mutant extracts were additive, indicating that the mutant did not contain an inhibitory substance. The mutant had normal levels of the other enzymes involved in starch synthesis: soluble starch synthase (Table 4-1), and the chloroplast isozymes of phosphoglucomutase and phosphoglucose isomerase (not shown) and Rubisco (Table 4-1). Enzymes associated with starch degradation (D-enzyme, starch phosphorylase and amylase) and sucrose metabolism (UDPglucose pyrophosphorylase and invertase) were also present in levels comparable to the NT (Table 4-1). Genette_analy§i§. The genetic basis of the ADPglucose pyrophosphorylase deficient phenotype was determined by crossing the mutant with the NT and examining the specific activity of ADPglucose pyrophosphorylase in subsequent generations. F1 plants from both the NT x TL46 and NT x TL3 crosses had specific activities of ADPglucose pyrophosphorylase intermediate between NT and TL46 (Table 4-2). Following self-fertilization, 52 F2 progeny from the NT x TL46 cross were scored for ADPglucose pyrophosphorylase activity. Eleven individuals had the mutant phenotype (5 - 17 nmol min'1 (mg protein)'1) and the other 41 had the phenotype of the heterozygote or NT (36 - 160 nmol min'l (mg protein)'l). This satisfactory fit to the 3:1 hypothesis (X2 - 0.41; P>0.5) together with the results from the analysis of the F1 plants indicates that a single, nuclear, semi-dominant mutation is responsible for the deficiency in ADPglucose pyrophosphorylase activity in TL46. Ne have designated the gene defined by the mutation in TL46 as aggz and the allele contained in this line as agg2;1. In this segregating F2 population, mutant 64 individuals had about 40% as much starch as the NT whereas heterozygous and NT individuals (as determined by their ADPglucose pyrophosphorylase activities) had statistically indistinguishable starch levels: 3.25 i 0.8 and 3.57 1 1.39 mg starch (g fresh weight)'l, respectively. Thus, at the level of the starch phenotype, the mutation in TL46 is recessive. The mutation in TL3 is also at the egg: locus since F1 hybrids from the cross TL3 x TL46 have ADPglucose pyrophosphorylase activities indistinguishable from either parent (Table 4-2). Ne have designated the allele contained in TL3 as agg2;2. In Chapter 3 a starchless mutant (TL25) was described which completely lacked ADPglucose pyrophosphorylase activity due to a lesion in the aggl gene. The mutations in TL3 and TL46 are not in the aggl gene since the F1 hybrids produced between TL25 and either TL3 or TL46 have 60 to 65% of the NT ADPglucose pyrophosphorylase activity (Table 4-2). These intermediate activities are consistent with the semi-dominant nature of the aggz mutations and the recessive nature of the egg] mutation. There was no increase in the specific activity when extracts of aggl (TL25) and aggz (TL46) mutants were mixed (Table 4-2). f i row r . Nhen grown in either continuous light or a 12 h photoperiod, the mutant TL46 appeared healthy and exhibited no apparent morphological differences from the NT. Figure 4-2 shows that the growth rate of the mutant was comparable to the NT when the plants were grown in either photoperiod. Thus, neither the ADPglucose pyrophosphorylase and starch deficiencies nor any other mutations in the background of TL46 cause any significant deleterious effects to the growth of the plant. IW’ 0 WT A <{ eTL46 ‘ .. “ 24h phOIOperiod o .. S E, 100014 0- ‘. 3 T Q 1 E it .E.’ 5 1004 4: l. N 4 g . LI. IO : : IO 20 3O 4O IOOOOE . WT B 1 oTL46 ' 1i I2h photoperiod it 'a. , E g meo- E 3 . a J» E O '3 3 '00) g 1 a 1 3 . u- 4) l) 10 c ; ‘fi IO 20 30 4O 50 Age (Days) Figure 4-2. Growth rates of NT (0) and TL46 (O) in continuous light (A) and a 12 h photoperiod (B). Symbols represent the mean fresh weight (i SE) of the shoots of five plants. 66 un l i ' . In order to examine the basis for the deficiency of ADPglucose pyrophosphorylase activity, the presence of the enzyme polypeptides was studied using two-dimensional PAGE followed by Nestern blot hybridization. Crude extracts of NT leaves contained two polypeptides which cross-reacted with affinity-purified rabbit anti-spinach leaf ADPglucose pyrophosphorylase IgG and had similar mobilities in the SOS-PAGE dimension as the two subunits (51 kD and 54 kD; 17) from spinach (Fig. 4-3A). On Nestern blots of TL46 leaf extracts a polypeptide of 51 kD cross-reacted with the antibody whereas the larger (54-kD) subunit was not detectable (Fig. 4-3B). The allelic mutant TL3 also contained cross-reactive material of 51 kD but not 54 kD (Fig. 4-3C). Antisera produced against the 51-kD polypeptide of spinach ADPglucose pyrophosphorylase reacted with a single polypeptide of similar mobility in NT (not shown) and TL46 leaf extracts (Fig. 4-4A). However, the antisera produced against the 54-kD spinach polypeptide did not show any cross-reactivity with polypeptides in TL46 extracts (Fig. 4-48) whereas it did cross-react with a 54-kD polypeptide in NT extracts (not shown). As shown in Figure 4-3, TL46 contains no detectable level of the 54-kD subunit of ADPglucose pyrophosphorylase and the level of the 51-kD subunit appears to be reduced relative to the NT. This reduction was quantified in two ways. First, varying amounts of crude leaf extracts were resolved by one-dimensional SOS-PAGE followed by Nestern blot analysis. On one-dimensional SOS-PAGE, the 51-kD subunit migrates ahead of the large subunit of Rubisco and can be detected whereas the 54-kD subunit migrates to a similar position as the Rubisco subunit which prevents its detection. As shown in Figure 4-5, TL46 extracts 67 s I; Natinve PAAGE A.WT' “o. - ‘ i are" - B. TL46 <—39Vd SOS C. TL3 \a' -- Figure 4-3. Immunoblots of crude homogenates of leaves of NT (A), TL46 (B), and TL3 (C). Proteins were electrophoretically separated in a nondenaturing gel followed by SDS-PAGE. The antigenic peptides were detected using affinity-purified rabbit immunoglobulins prepared against spinach leaf ADPglucose pyrophosphorylase. Crude homogenates containing about 200 ug protein were used for each gel. Purified spinach leaf ADPglucose pyrophosphorylase (500 ng) was run in the second dimension at the positions indicated by the S. The splitting of the spots in the native dimension was occasionally observed for both the mutant and NT extracts. Filters have been cropped for presentation to show only the regions which contain cross-reactive bands. (Tsan-Piao Lin, personal communication) 68 s I; Native PAGE A m . -U U) 5‘1"L “ “ .0 > _ a) I, [TI ... 1 Figure 4-4. Immunoblots of crude homogenates of leaves of mutant TL46. Crude homogenates containing 200 ug protein were separated by 2-dimensional electrophoresis. The antigenic peptides were detected using affinity-purified rabbit immunoglobulins prepared against the 51-kD subunit (A) or the 54-kD subunit (B) of spinach leaf ADPglucose pyrophosphorylase. Purified spinach leaf ADPglucose pyrophosphorylase (500 ng) was run in the second dimension at the positions indicated by the S. Filters were cropped without removing any bands. (Tsan-Piao Lin, personal communication) 69 ‘ ‘ TL 46 —> Spinach e WT - v 600 ug 200 ug 50ug 60009 2001.19 50 ug 0.5 ug Figure 4-5. Immunoblots of crude leaf homogenates of NT and TL46. Crude homogenates and purifed spinach leaf ADPglucose pyrophosphorylase containing the indicated amounts of total protein were separated by one-dimensional SOS-PAGE. The increased number (relative to Figures 4-3 and 4-4) of cross-reactive bands, including the large subunit of Rubisco, is because crude serum rather than affinity purifed antibodies were used. The 54-kD subunit in the NT lanes is obscured by the antigenic reaction of the large subunit of Rubisco. The position of the 51-kD subunit in the Arabidopsis lanes is indicated by the arrow labelled ADG. 70 contain much less of the 51-kD subunit than the NT. The amount of alkaline phosphatase reaction product in each band was determined by Spectrophotometric readings of the excised bands which had been dissolved in DMSO. By this criterion, the amount of 51-kD subunit in lane 4 (600 ug of TL46 crude protein) was about half that in lane 3 (50 ug of NT crude protein); thus TL46 contains about (0.5)(50)/600 . 4% as much of the 5I-kD subunit as the NT. The second method for comparing the amount of ADPglucose pyrophosphorylase in NT and TL46 leaf extracts was a quantitative ELISA. This analysis (Fig. 4-6A) showed that about 10-fold more TL46 than NT crude leaf protein was required in order to achieve a given level of competition with purified, biotinylated spinach leaf ADPglucose pyrophosphorylase. Thus, the total amount of ADPglucose pyrophosphorylase protein in TL46 is only about 10% of that in the NT. As a control, Figure 4-6B shows that glycolate oxidase, an enzyme involved in photorespiration is present in equal amounts in NT and TL46 leaves. The ELISA measures both subunits of ADPglucose pyrophosphorylase, however it is not possible to determine the relative weight each receives in the final results. Assays using the subunit-specific antibodies which wOuld have resolved this uncertainty were not possible because these antibodies have a very low affinity for the non-denatured subunits. It is also possible that in TL46, the 51 kD subunits which are present may be more tightly bound by antibodies since they lack the 54-kD subunit which in the NT may mask certain epitopes. The ELISA resuits therefore, while useful in confirming the conclusions from the one-dimensional Nestern experiments, are somewhat less informative. 71 IOG A. ADPglucose pyrophosphorylase BO, 501 A E E 40" o O J .- t o O 0‘ 204 v e 1 > '.‘: U ‘ L ‘ ““‘ ‘ ‘ ‘ :i‘“ 4 ’00 W V 'Y—rvvv v v v V v17 3 9 B. Glycolote oxidase 2 i o .c 2 . g 804- o I .5 '6 x 60" 4 40+ 20-) 9 G “ 4 es“:“ ‘ seems? e Ase-H“ 01 1 IO ioo . Competitor (pg protein) Figure 4-6. Competition ELISA assays to measure ADPglucose pyrophosphorylase (A) or glycolate oxidase (B) in NT (0) and TL46 (O) crude leaf homogenates. The binding of biotinylated, purified spinach leaf ADPglucose pyrophosphorylase or glycolate oxidase to rabbit antibodies was competed with varying amounts of crude leaf homogenates of NT or TL46. Bound biotinylated ADPglucose pyrophosphorylase or glycolate oxidase was measured by its reaction with streptavidin-conjugated alkaline phosphatase and is presented as a percentage of the controls using no crude homogenate. The results in panel A are the mean 1 SE of 3 independent experiments using affinity purified antibodies or 1:1000 or 1:5000 dilutions of crude serum. The results in panel 8 represent a single experiment using a 1:1000 dilution of crude serum against glycolate oxidase. 72 DISCUSSION n h r la i Ananiggneie. The results presented here show that the starch deficient mutants TL46 and TL3 contain only about 5% as much ADPglucose pyr0phosphorylase activity as the NT because of mutations in the aggz gene. The aggz gene is clearly distinguishable from the aggl gene (Chapter 3) based on genetic complementation tests and by the different phenotypes produced by mutations in the two loci. Both of the aggl mutants have undetectable levels both of ADPglucose pyrophosphorylase activity and of cross-reactive peptides for both subunits. By contrast, both of the aggz mutants (TL46 and TL3) have low but detectable ADPglucose pyrophosphorylase activity and only lack cross-reactive material for the larger subunit of the enzyme. Also, plants heterozygous for aggl mutant alleles had NT levels of ADPglucose pyrophosphorylase activity whereas aggz heterozygotes have ADPglucose pyrophosphorylase activities almost exactly intermediate between the mutant and NT levels. These observations for aggz are most simply explained by a model in which aggz is the structural locus for the 54-kD subunit of the ADPglucose pyrophosphorylase enzyme. The absence of the 54-kD subunit in TL46 could be due to the mutation inhibiting the synthesis of the subunit or to instability of the altered polypeptide which results in it being rapidly degraded. The decreased abundance of the 51-kD subunit in the mutant could be due to the more rapid turnover of this subunit in the absence of the 54-kD subunit or to an unknown regulatory mechanism. Ph iol i ff . Despite the presence of only 5% of the ADPglucose pyrophosphorylase activity of the NT, TL46 accumulates 40% 73 as much starch as the NT when grown either in continuous light or in a 12 h photoperiod. Since we have previously shown that starch accumulation in Azaniggnaie leaves is completely dependent on ADPglucose pyrophosphorylase (Chapter 3), this starch accumulation in TL46 must be due to the remaining ADPglucose pyrophosphorylase activity rather than to another biosynthetic pathway. There are several possible explanations for the discrepancy between the large reduction in ADPglucose pyrophosphorylase activity and the smaller effect on starch content. First, the activity measured in crude leaf homogenates from TL46 may be an underestimate of the activity in yiyn if the altered enzyme is less stable 1n,yjtng than the NT. Second, one or more other enzymes may normally limit the maximal rate of starch synthesis in the NT so that it is only when ADPglucose pyrophosphorylase activity is greatly reduced, as in the mutant, that it becomes limiting. For example, the soluble starch synthase activity in the NT is 70 times lower than the ADPglucose pyrophosphorylase activity (Table 4-1). Moreover, the starch synthase activity, 1.4 nmol min"1 (mg protein)'l, is almost exactly the amount required to support the maximal rate of starch synthesis by the NT observed between 8.5 and 12 h into the photoperiod, calculated from Fig. 4-1 to be 1.6 nmol glucose residues min'1 (mg protein)'l. However, since a particulate starch synthase may also be present and the conditions for extraction and assay of starch synthase have not been optimized for Arabidopsie, additional studies would be required in order to determine if this is, indeed, a rate-limiting step. The third explanation is that ADPglucose pyrophosphorylase is normally greatly inhibited in_vivo. Thus, the activity measured in 74 yttne represents the maximal rather than the actual 1n yiyg activity. Consistent with this idea, it can be estimated that in spinach the in 1119 activity of ADPglucose pyrophosphorylase in light-adapted chloroplasts is only about 10-40% of the fully activated level measured 1n_y1tng (recalculated from ref. 3 and 13). Decreased inhibition in 111g of the mutant enzyme, which could be caused by increased concentrations of allosteric activators (e.g. 3-P-glycerate and fructose-6-P) or a reduced concentration of the inhibitor Pi, could result in sufficient activity to support the observed rates of starch accumulation. In addition, the ADPglucose pyrophosphorylase substrate glucose-l-P may also increase in the mutant due to the limitation in the ADPglucose pyrophosphorylase activity. In spinach chloroplasts glucose-l-P has been reported to be below saturating levels for ADPglucose pyrophosphorylase (4) so any increase in glucose-I-P in TL46 should produce increased ADPglucose synthesis. l n n of h de r n. During a 12 h photoperiod TL46 accumulates starch only 40% as rapidly as the NT and the rate of starch degradation during the dark period is also only 40% as fast as the NT. Since ADPglucose pyrophosphorylase has no known role in starch breakdown, this suggests that the rate of starch breakdown is regulated in such a way that it is essentially proportional to the amount of starch accumulated. These observations are consistent with the results of other studies in barley (5), tomato (16) and clover (27) which have shown that the rate of starch degradation is regulated. Nhen grown in continuous light the rate of net starch synthesis by fully expanded mutant and NT leaves must be essentially zero, since the 75 starch pools do not increase beyond about 0.5 and 1.3 , mg (g fresh weight)'l, respectively. Obviously, this equilibrium state can only be achieved by coordinate regulation of the gross rates of synthesis and degradation such that they are equal. The mechanisms which operate to allow this coupling are not understood, but several possibilities exist. One is that a system operates which measures the size of the starch pool and regulates synthesis and degradation in order to maintain a given size of the starch pool. Alternatively, these rates may be balanced by other mechanisms which measure the overall capacity for starch synthesis and then regulate synthesis and degradation accordingly. It is also possible that these rates balance each other due essentially to mass action. Thus, for instance, as the size of the starch pool increases, the rate of degradation may increase proportionally because of the increasing substrate concentration available for the degradative enzymes. The fact that the mutant accumulates only about 40% as much starch in fully expanded leaves as the NT when grown in continuous light suggests that the regulation of the balance between synthesis and degradation is not achieved by a mechanism of the first type, but that a system which either actively (the second type) or passively (the third) measures the total capacity for synthesis and degradation is responsible for balancing synthesis and degradation. 0 f r u r w . The two previously described classes of starchless Arabigoneje mutants, which completely lacked activity of either ADPglucose pyrophosphorylase (Chapter 3) or plastid phosphoglucomutase (Chapter 2), had rates of growth indistinguishable from the NT when grown in continuous light but grew much more slowly 76 than the NT in a photoperiod of 12 h or less. The decreased growth rate in a 12 h photoperiod was correlated with decreased photosynthesis and increased dark respiration by the phosphoglucomutase mutant. The similar rates of growth of NT and TL46 in both continuous light and a 12 h photoperiod suggest that normal plant growth does not depend on the absolute size of the starch pool, but rather on the presence of a minimal level of starch. Based on the properties of TL46, this starch level required for vigorous growth must be less than 40% of the NT level. A mutant of Clankia which is partially deficient in chlorOplast phosphoglucose isomerase accumulates about 60-90% as much starch but grows more slowly than the NT (12). However, phosphoglucose isomerase is required not only for starch synthesis, but also for starch degradation, and for all of the pathways which initiate from glucose-6-P (e.g. the pentose phosphate shunt and inositol biosynthesis). Thus, the decreased growth rate in this glankia mutant is probably not solely related to its slightly reduced starch content. P r h r l . Previous work has suggested that the ADPglucose pyrophosphorylase enzyme in maize endosperm is coded for by two structural genes (6) and in spinach leaf is comprised of two polypeptides of 54 and 51 kD (3,17). However, this earlier work did not permit any evaluation of the functional role of the two polypeptides. The results with TL46 bear directly on this point. The mutant lacks the larger subunit, contains only about 4% as much of the smaller subunit as the NT, and has only about 5% of the ADPglucose pyrophosphorylase activity of the NT. Assuming that the aggl gene codes for the 54-kD subunit, the simplest interpretation of these results is that the 54-kD polypeptide is a subunit of the native 77 enzyme and is required for full ADPglucose pyrophosphorylase activity. This requirement likely includes at least a regulatory, assembly or stability role and may include a catalytic role as well. These conclusions are supported by a similar analysis of the 50:2 maize endosperm mutant which contains about 12% of the normal ADPglucose pyrophosphorylase activity (6) and also lacks the larger of the two subunits of ADPglucose pyrophosphorylase (P. Summers, S. Danner, J. Preiss, in preparation). The residual activity in the aggz and 50:2 mutants could be due to the presence of a small amount of the larger subunit or to residual activity of the smaller subunit in the absence of the other subunit. At present we cannot unequivocally distinguish between these possibilities, however, further study of the enzyme from the Araniggneia aggz or maize Sh;2 lines should provide this information. m c on. r l A P lu r h hor e. Ne have previously described another gene (aggl) which affects ADPglucose pyrophosphorylase activity in Arabjggpsie (Chapter 3). This gene was unusual in that heterozygotes containing a NT and a null allele exhibited NT levels of activity. This result was attributed either to aggl being a regulatory locus or the capability of the plant to attain normal levels of activity by increasing the activity produced by the single functional gene in these heterozygotes. The observation that TL46 heterozygotes contain intermediate levels of ADPglucose pyrophosphorylase activity shows that the latter explanation is not generally true for Aranigopeie, and supports the possibility that aggt is a regulatory gene. Furthermore, the aggl mutant lacked both of the ADPglucose pyrophosphorylase subunits. This was also consistent with a 78 regulatory function for the gene or could have been due to a mutation which directly affected only one subunit, with the second being degraded due to its instability in the absence of the first. The present results with TL46 show that the 51-kD subunit accumulates to a detectable level in the absence of the 54-kD subunit. Previous work has shown that the two subunits of the spinach ADPglucose pyrophosphorylase could not be separated from each other without using denaturing conditions (17) and thus the function of the individual subunits could not be studied. Since TL46 does not contain detectable levels of the 54-kD subunit, it may provide a source of non-denatured 51-kD subunits for detailed studies of its kinetic properties and for potential in yitnn reconstitution studies with 54-kD subunits derived from Ananiggnaie or other species. TL46 accumulates no detectable 54-kD subunits and the lesion may be in the structural gene for this subunit. Thus, it may also serve as a useful genetic background into which genes coding for the subunit may be introduced. The affects of foreign or in yttnn mutated genes on the kinetic properties of the ADPglucose pyrophosphorylase enzyme could then be studied under in ytyn conditions. Changes in the kinetic properties of an enzyme produced in such a way could then be unequivocally linked to alterations in starch/sucrose partitioning in a completely native condition. REFERENCES 1. Billingsley, ML, KR Pennypacker, CG Hoover, RL Kincaid 1987 Biotinylated proteins as probes of protein structure and protein-protein interactions. Biotechniques 5:22-30 2. Caspar T, SC Huber, C Somerville 1986 Alterations in growth, 10. ll. 12. 13. 14. 79 photosynthesis and respiration in a starch mutant of Arabidopsis thaliana (L.) Heynh deficient in chloroplast phosphoglucomutase activity. Plant Physiol 79:1-7 Copeland L, J Preiss 1981 Purification of spinach leaf ADPglucose pyrophosphorylase. Plant Physiol 68:996-1001 Dietz K-J 1987 Control function of hexosemonophosphate isomerase and phosphoglucomutase in starch synthesis of leaves. in J Biggens, ed, Progress in Photosynthesis Research, vol III. Martinus Nijhoff, Dordrecht, pp 329-332 Gordon AJ, GJA Ryle, DF Mitchell, CE Powell 1982 The dynamics of carbon supply from leaves of barley plants grown in long or short days. J Exp Bot 33:241-250 Hannah LC, 0E Nelson 1975 Characterization of adenosine diphosphate glucose pyrophosphorylases from developing maize seeds. Plant Physiol 55:297-302 Haugen TN, A Ishaque, J Preiss 1976 Biosynthesis of bacterial glycogen: Characterization of the subunit structure of Eeenenienia eglj B glucose-l-phosphate adenylyltransferase (EC 2.7.7.27). J Biol Chem 251:7880-7885 Haughn GN, CR Somerville 1986 Sulfonylurea resistant mutants of Ananjgnnele Molec Gen Genet 204:430-434 Hawker JS, JL Ozbun, H Ozaki, E Greenberg, J Preiss 1974 Interaction of spinach leaf adenosine diphosphate glucose 091,4-1,4-glucosyl transferase andixe1,4-glucan, oe1,4-glucan-6-glucosyl transferase in synthesis of branched oeglucan. Arch Biochem Biophys 160:530-551 Huber SC 1984 Biochemical basis for effects of K-deficiency on assimilate export rate and accumulation of soluble sugars in soybean leaves. Plant Physiol 76:424-430 Jongg MGK, NH Outlaw, 0H Lowry 1977 Enzymic assays of 10"7 to 10 moles of sucrose in plant tissue. Plant Physiol 60:379-383 Jones TNA, LD Gottlieb, E Pichersky 1986 Reduced enzyme activity and starch level in an induced mutant of chloroplast phosphoglucose isomerase. Plant Physiol 81:367-371 Kaiser NM, JA Bassham 1979 Light-dark regulation of starch metabolism in chloroplasts II. Effect of chloroplastic metabolite levels on the formation of ADP-glucose by chloroplast extracts. Plant Physiol 63:109-113 Krishnan HB, CD Reeves, TN Okita 1986 ADPglucose pyrophosphorylase is encoded by different mRNA transcripts in leaf and endosperm of cereals. Plant Physiol 81:642-645 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 80 Lin TP, T Caspar, C Somerville, J Preiss 1988 Isolation and characterization of a starchless mutant of Arabjdoneie thaliana (L.) Heynh. lacking ADPglucose pyrophosphorylase activity. Plant Physiol: 86:1131-1135 - Madsen E 1968 Effect of COZ-concentration on the accumulation of starch and sugar in tomato leaves. Physiol Plant 21:168-175 Morell, MK, M Bloom, V Knowles, J Preiss 1987 Subunit structure of spinach leaf ADPglucose pyrophosphorylase. Plant Physiol 85:182-187 Morrel M, M Bloom, J Preiss 1988 Affinity labeling of the allosteric activator sites(s) of spinach leaf ADP-glucose pyrophosphorylase. J Biol Chem 263:633-637 Okita TN, E Greenberg, DN Kuhn, J Preiss 1979 Subcellular localization of the starch degradative and biosynthetic enzymes of spinach leaves. Plant Physiol 64:187-192 Ozbun JL, JS Hawker, E Greenberg, C Lammel, J Preiss, EYC Lee 1973 Starch synthase, phosphorylase, ADPglucose pyrophosphorylase and UDPglucose pyrophosphorylase in developing maize kernels. Plant Physiol 51:1-5 Pierce JN, SD McCurry, RM Mulligan, NE Tolbert 1982 Activation and assay of ribulose-I,5-bisphosphate carboxylase/oxygenase. Methods Enzymol 89:47-55 Plaxton NC, J Preiss 1987 Purification and properties of nonproteolytic degraded ADPglucose pyrophosphorylase from maize endosperm. Plant Physiol 83:105-112 Preiss J 1988 Biosynthesis of starch and its regulation. In Biochemistry of Plants, Vol 14. Academic Press, New York, pp 182-254 Preiss J, M Bloom, M Morell, VI Knowles, NC Plaxton, TN Okita, R Larsen, AC Harmon, C Putnam-Evans 1987 Regulation of starch synthesis: Enzymological and genetic studies. 1n G Bruening, J Harada, T Kosuge, A Hollaender, eds, Tailoring Genes for Crop Improvement. Plenum Publishing Corp., New York, pp 133-152 Preiss J, A Sabraw, E Greenberg 1971 An ADPglucose pyrophosphorylase with lower apparent affinity for substrate and effector molecules in an E. colj B mutant deficient in glycogen synthesis. Biochem Biophys Res Commun 42:180-186 Rufty TN, SC Huber 1983 Changes in starch formation and activities of sucrose-phosphate synthase and cytoplasmic fructose 1,6-bisphosphatase in response to source-sink alteration. Plant Physiol 72:474-480 27. 28. 29. 81 Scheidegger EC, J Nosberger 1984 Influence of carbon dioxide concentration on growth, carbohydrate content, translocation and photosynthesis of white clover. Ann Bot 54:735-742 Smith PK, RI Krohn, GT Hermanson, AK Malia, FH Gartner, MD Provenzano, EK Fujimoto, NM Goeke, BJ Olson, DC Klenk 1985 Measurement of protein using bicinchoninic acid. Anal Biochem 150:76-85 Sowokinos JR, J Preiss 1982 Pyrophosphorylases in Solanun tuberosgn. III. Purification, physical, and catalytic properties of ADPglucose pyrophosphorylase in potatoes. Plant Physiol 69:1459-1466 Chapter 5 Metabolic, Environmental, and Developmental Regulation of B-amylase in Anantggneie thaliana Leaves ABSTRACT The regulation of leaf amylase activity has been studied in NT Ananign§1§,tna11ana and a diverse collection of mutants with alterations in starch metabolism. All of the mutants had increased amylase activity relative to the NT when grown in a 12 h photoperiod. This effect, however, was suppressed in plants grown in continuous light and in young plants grown in the 12 h photoperiod. A cytosolic B-amylase with elevated specific activity was responsible for the increased activity in the mutants. The alteration which regulates this activity is stable during electrophoresis, indicating that the activation/inactivation process causes a stable modification of the enzyme. The forms of this enzyme with higher and lower specific activity have the same mobilites on native polyacrylamide gel electrophoresis and native isoelectric focusing, which places constraints on the types of modifications which might be responsible for the activation. INTRODUCTION Photosynthesizing leaves may respond to changes in the ratio between source (i.e. carbohydrate exporting) regions and sink (i.e. carbohydrate importing) regions of the plant by changing the rates of 82 83 photosynthetic C02 fixation and respiration (2,9). These responses have been intensively studied since they may shed light on one of the only known means of regulating the rate of photosynthetic C02 fixation. The signal for the altered rate of photosynthesis is not known, but has been suggested to be either a hormonal signal produced by the sink regions or altered carbohydrate pools which occur in the leaf cells because of an imbalance between synthesis (photosynthesis) and utilization (respiration and export) of carbohydrate. In order to study this complex regulatory system, we have identified mutants which have alterations in photosynthetic carbohydrate metabolism (Chapters 2,3,4, unpublished results). Three classes of these mutants, which either completely lack starch, or contain reduced or elevated levels of starch, have substantial alterations in leaf carbohydrate composition which can be used to search for corresponding metabolic responses. Previous analysis of one of the mutants, which is unable to synthesize starch because of a lack of chloroplast phosphoglucomutase activity (Chapter 2), indicated that it had increased leaf sugar content and altered photosynthesis and dark respiration rates which were secondary effects of its inability to synthesize starch. In our further analysis of the secondary effects of these mutations on other aspects of leaf metabolism, we have identified a large effect on the B-amylase activity in these mutants. In this paper we show that the activity of B-amylase is highly (up to 40-fold) induced in a diverse collection of mutants with alterations in starch metabolism. The increased activity is due to an increase in the specific activity of the‘B-amylase protein rather than an increase in the amount of protein itself. The increased activity of the B-amylase 84 in the mutants is maintained even after electrophoresis, indicating the activation is likely due to a stably-maintained alteration of the protein, rather than allosteric regulation. MATERIALS AND METHODS l er' . The Columbia NT of Ananiggnate thaliana (L.) Heynh. and a starch-free line TC75 which lacks activity of the chloroplast isoenzyme of phosphoglucomutase due to the ngn£;1 allele were used for most of the experiments in this study. TC75 was derived from the previously described line TC7 (Chapter 2) by a series of five backcrosses to the NT. Other starch-free, reduced starch, or elevated starch mutant lines were also used for the experiments of Figs. 5-1 and 5-2. The phenotypes and enzymatic lesions of these lines are listed in Table 5-1. Descriptions of several of the mutations are described in this thesis (i.e. ngnE;1, pgn£;2, chapter 2; agg1;1, agg1;2, chapter 3; agg2;1, chapter 4) and others represent unpublished work (i.e. ngnflg3, §t11;1, agn1;1, and eggl;2). Plants were grown as previously described (8) in growth chambers at about 110 umol'm'z's'l photosynthetically active radiation from "cool-white" fluorescent tubes. Growth conditions were either continuous light (24/0), 21°C or a 12 h light/ 12 h dark photoperiod (12/12), 19°C. (Similar results were obtained from plants grown in 12/12 at 21°C.) n a 5. Leaves were harvested and in most cases stored at -70°C until use. Preliminary experiments showed no drop in amylase activity of leaves or crude extracts stored for at least 3 months at -20°C or -70°C. Extracts were prepared at 4°C by grinding leaves in 85 Table 5-1. Description of starch mutant collection. Original Locus-l Line 2 M2 3 Enzymatic allele Designation Batch Phenotype Deficiency Reference nng-l TC7 B Starch-free Chloroplast Chapter 2 png-z TC9 D ” phosphogluco- " png-3 TL35 E ' mutase Unpublished adg1-1 TL25 E ADPglucose Chapter 3 adgl-z TL24 E pyrophosphorylase " sth-l TL14 E ' Unknown4 Unpublished ang-I TL46 E Reduced starch5 ADPglucose 5 Chapter 4 pyrophosphorylase egn1;1 TC26 0 Elevated starch Unknown Unpublished agnl;z TL52 E ' ” " 1In the current genetic nomenclature for Arabidopsis, a gene is represented by 3 lowercase letters and a number (or a capital letter for ngnfl) and different alleles are designated by a dash and another number. Thus, agg1;1 and agg1;2 are alleles of the aggl gene, whereas 2aggz-l represents an allele of the aggz gene. Following backcrossing to the NT, lines are given new designations. Mutants derived from different M2 batches represent independently derived lines and alleles. The defect in this mutant is unknown, but it does contain normal activities of ADPglucose pyrophosphorylase and chloroplast 5phosphoglucomutase. The adg2-I line accumulates 40% as much starch as the NT and has 5% as much activity of ADPglucose pyr0phosphorylase as the NT. .a-w 86 either 1 ml/g fresh weight of 100 mM Tris-Cl, pH 7.5 for the experiments of Fig. 5-6, in 1 ml/g fresh weight of 50 mM Na-succinate, pH 5.0 for the experiments of Fig. 5-7, or in 4 ml/g fresh weight of 50 mM Tris-Cl, pH 7.5 for all other work. Extracts were microfuged for 10 min at 4°C and the supernatants were used for enzyme assays. Total amylase activity, measured as the increase in reducing sugars, was assayed as previously described (15) in samples of crude extracts containing 5-30 ug protein in 40 mM sodium acetate, pH 6.0 with amylopectin (5 mg/ml) as the substrate. Acid invertase (11), Rubisco (6), and phosphorylase, D-enzyme, and UDPglucose pyrophosphorylase (14) were assayed as previously described. Protein was measured using BSA as a standard with a Coomassie blue binding assay (Bio-Rad) for the experiments of Figs. 5-6, 5-7 or, for all other experiments, using bicinchoninic acid (Pierce). Eleetngnngneeie. Native PAGE was performed as previously described (15) at 4°C using a 4% (w/v) acrylamide stacking gel and a 7% (w/v) resolving gel. Native IEF gels contained 5.4% (w/v) acrylamide, 0.16% (w/v) bis-acrylamide, 4% (v/v) Pharmalyte pH 4-6.5, 1% (v/v) Pharmalyte pH 3-10 and 5% (w/v) glycerol and were photopolymerized using 5 uM riboflavin 5-phosphate and 0.017% (v/v) N,N,N’,N’-tetramethyl-ethylenediamine. The 1.5'100'110 mm3 gels were pre-focused at 4°C at 4 N for 1 h and the samples were focused at 4°C at 8 N for 2 h. Amylase activity staining was according to Lin et al. (15) and the activity bands are referred to following the convention they established. For immunoblots, proteins were eletrotransferred to nitrocellulose (21) and immunoreactive proteins were visualized using rabbit antibodies prepared against sweet potato (ngnoea batatae) 87 B-amylase (Boehringer) followed by Protein-A alkaline phosphatase (Sigma). e a a i . NT, ngn£;1, and egn1;1 plants were grown for 35, 58 and 58 days, respectively, in 12/12. Leaves and petioles were harvested at the end of the light period, quickly weighed, and homogenized in 80% ethanol at 55°C. Homogenates were centrifuged at 4000 x g for 30 min and the supernatant was dried under vacuum at 32°C. The residue was dissolved in water and extracted with an equal volume of chloroform to remove lipids. The aqueous phase was passed through a 0.45 um filter and concentrated under nitrogen at 32°C. The samples were digested with sweet potato B-amylase (1 unit/400-800 ug sugar) for 50 min at 37°C or were mock-digested with boiled enzyme under the same conditions. The protein was then separated from the sugars by passage through a 1 cm Dowex-SOXB column and the eluant concentrated under nitrogen at 32°C. The sugars were separated using a HPX-42 column (Bio Rad) at 85°C with water (0.6 ml/min) as the solvent and detected using a refractive index detector (SP6040). This column resolves oligosaccharides as large as (glucose)8, but does not resolve monomers and dimers well. The column was calibrated usingiaw1,4 glucans from (glucose)1 to (glucose)8. RESULTS Amylase aetivity jg elevated in etanen mutants. Total amylase activity was measured in a collection of mutants which are starch-free or contain reduced or elevated levels of starch and were grown in either a 12 h photoperiod (12/12) or continuous light (24/0). The 88 results (Fig. 5-1) show that amylase activity was much higher in the mutants than the NT in 12/12, however this effect was suppressed when the plants were grown in 24/0. It should be noted that this collection of mutants represents 5 genetic complementation groups, of which 3 have been shown to have specific enzymatic deficiencies which account for their starch phenotypes (Chapters 2, 3, 4, and unpublished results). Therefore, the increase in amylase activity is not a direct consequence of the mutations in these lines, but rather is a secondary consequence of the physiological changes produced in these mutants by the enzymatic deficiencies. The total amylase activity measured in these assays includes contributions from at least four forms ofcx- and B-amylases (15), and may also include debranching enzyme. In order to determine whether a particular enzyme species was responsible for the increase in total amylolytic activity, crude extracts were resolved on native PAGE and stained for amylase activity. The results (Fig. 5-2) indicate that one band of activity (A3) was strongly increased in parallel to the total amylase activity observed in Fig. 5-1. A second band (A5) with much lower apparent activity was also observed in the mutants and not in the NT. Small changes in the other activity bands (A1, A2, A4) were also sometimes observed, but were neither reproducible nor proportional to the total amylase activity. Ne have previously observed small differences (BO-200% of the NT) in the activities of sucrose phosphate synthase, invertase, starch synthase, and ADPglucose pyrophosphorylase in the ngnfl;1 mutant. In order to determine whether other enzymes involved in starch and sucrose metabolism were regulated in a similar manner as the amylase, starch 89 V//////////////////////////% 2-1 sopi -1 sopi -2 V/////////////////////////////////////////////////% I 2470 12712 500 100‘ 0 E203 955.5885 §>=o< $352 Genotype WT png-i png-z png-3 adgi-i adgi-z stii-i adg 13wmmm045sfia~€—St—> 9 12 independent samples. St - starchless, St' . reduced starch + SE for 4- Plants were grown either in continous illumination (24/0) for 19-28 days or in a 12 h photoperiod (12/12) for 28-36 days. 9 ues are the means - NT gl elevated starch. Figure 5-1. Total amylase activity of leaves of NT and mutants with V altered starch metabolism. Phenotypes: St 90 Genoptype WT png—i adgi-i sopi-i sop1-2 adgi-2 stii-i Photoperiod 2412 24 12 24 12 24 12 24 12 24 12 24 12 R 1234567891011121314 Figure 5-2. Native polycrylamide gel of leaf extracts of NT Arabidopsis and mutants with altered starch metabolism stained for amylase activity. Plants were grown either in continous light (24/0) for 26 days or in a 12 h photoperiod (12/12) for 35 days. Crude extracts containing equal amounts of protein were separated by electrophoresis on a 7% native polyacrylamide gel and then stained for amylase activity. Amylase isozymes A1, A2, A3, described by Lin et al. (15), are indicated, as are two additional bands A4 and A5. Ribulose bisphosphate carboxylase (R), which stains nonspecifically with iodine, is marked and serves as a useful internal control, confirming that equal amounts extract were loaded in each lane. (Tsan-Piao Lin, personal communication) 91 phosphorylase, disproportionating enzyme, acid invertase, UDP-glucose pyrophosphorylase, and Rubisco were assayed in extracts from the NT and a subset of the mutants grown in both 24/0 and 12/12 (Table 5-2). Although some slight differences in these enzyme activities were noted, they were much smaller than the effect on amylase, and with one exception not uniformly observed in all of the mutants. Thus, they do not appear to be regulated in concert with B-amylase. For example, starch phosphorylase was about two-fold higher in the ngn£;1 and agg1;l lines when grown in 12/12 but was similar to the NT in the other mutants. UDP-glucose pyrophosphorylase was about two-fold lower than the NT in eg21;1 and agnl;z lines when grown in 12/12 but was about 2.5-fold higher in these lines than in NT when grown in 24/0 and was not greatly different in the other mutants regardless of the photoperiod. In agreement with our previous observations (Chapter 2), invertase was about two-fold higher than NT in the ngn£;1 line and was about 1.5-fold higher in the egn1;1 line when these mutants were grown in 12/12. However, other mutants were comparable to the NT. Finally, there was a small but consistent 20-30% decrease in Rubisco activity in the ngn£;1, aggl;1, ennl;1 and egnl;2 lines when grown in 12/12, whereas the agg2;1 line in 12/12 was reduced only slightly. Nhen grown in 24/0 all the mutants had Rubisco activities indistiguishable from the NT. This effect on Rubisco parallels the effects of these mutations on growth rates (all except agg2;1 grow more slowly than the NT in 12/12 but all grow as well as the NT in 24/0 [Chapters 2, 3, 4, and unpublished results]) and on photosynthetic C02 fixation rates (where they have been examined, in the ngnfl;l line, they are lower than the NT in 12/12 but comparable in 24/0 [Chapter 2]). 92 Table 5-2. The effects of various mutations on activities of several enzymes associated with starch and sugar metabolism. Enzyme activities were measured in crude extracts from plants grown in 24/0 (19 days) or 12/12 (35 days). Results are the means of at least 2 independent measurements. Mutation Photo- Enzme negiod NT nqu-l adgl;1_adgz;1_§_ep_l;1_§_enl;z nmol min"l (mg protein)"1 Starch 12/12 28 51 54 371 23 25 phosphorylase 24/0 21 26 25 ND 23 21 D-enzyme 12/12 19 18 22 21 20 18 24/0 19 12 18 ND 15 18 UDPglucose 12/12 41 50 68 44 14 24 pyrophosphorylase 24/0 16 22 13 ND 42 36 Acid invertase 12/12 100 200 I40 110 160 120 24/0 78 66 63 ND 78 61 Rubisco 12/12 180 150 150 170 120 150 24/0 300 310 290 ND 290 300 1Not done. 93 nv' m n v nt r io f am l tivit . Analysis of the effect of the age of the plants on amylase activity indicated that the increased amylase activity observed in the mutants was dependent on the developmental stage of the plants. As shown in Fig. 5-3A, when plants were grown in 12/12 the amylase activity in the ngn£;1 mutant line was initially comparable to the NT. However, at later times, the activity in the mutant increased markedly, whereas the activity in the NT remained roughly constant. The timing and rate of this increase in activity varied in two independent experiments for unknown reasons. Nhen grown in 24/0 (Fig. 5-3B) there were no large variations in activity during the life cycle of either the NT or mutant. There have been reports that amylase activity exhibits a two-fold diurnal variation in spinach leaves grown in 12/12 (16). Ne did not observe any regular diurnal variation in amylase activity in 29-day-old NT or ngn£;1 lines which had been grown in 12/12 (Fig. 5-4). Because the amylase activity in mutants grown in 12/12 was much higher than those grown in 24/0, it was of interest to know how quickly, if at all, plants grown in 24/0 and then shifted to 12/12 would perceive and respond to the altered photoperiod. The results of such an experiment (Fig. 5-5) indicated that in both the NT and the ngnE;1 mutant a shift in the photoperiod induced a diurnal fluctuation in amylase activity beginning within 12 h and continuing at least 3 days. Amylase activity declined during the dark and increased during the light periods. Additionally, the activity in the mutant increased and eventually stabilized at about the levels found in plants which had been grown in 12/12 (compare with Figs. 5-1, 5-3). In contrast, the NT 94 700 ‘ A. 12/12 A~~‘ 99““ E‘ 600‘ ” ‘A--"""4 '5 ‘ .1 o-o ” 9 500 r.— g - l png-i 400- I g . I ._. g I E 3°° I E ‘ ' E 200- I 5 1 ' WT 24 100‘ 4‘ r ‘0.--” e A" ~ .__,.- m 0 0" - I I I I I r I ' I f, 10 20 30 40 50 60 3 200 E? El ERUT) . pgnwhi VVT 100‘ <( 4 j.- ‘Ar— 0 r T I T I 10 20 40 Age (days) Figure 5-3. Total amylase activity during the life cycle of UT and the pgnP-l starch-free mutant. Plants were grown in a 12 h photoperiod (12/12, A) or continuous illumination (24/0, B) and total amylase activity was measured in leaf samples. In pane] A, the results of two independent experiments are presented to indicate the range of variation observed. In the first experiment (filled symbols and solid lines) n - 3; in the second (open symbols and dashed lines) n - 3 for the NT and n - 6 for the starch-free mutant. In panel B, n - 3. Error bars have been deleted for clarity, but the SE averaged about 50% of the mean, indicating the substantial variability inherent in these measurements. In 12/12 bolting began at about 45 days in the NT and had not begun in the mutant before the end of the experiment. In 24/0 both lines began to bolt at 20 days. Plants normally begin to show signs of senescence (yellowing leaves) about 15-20 days after bolting. ‘—777777fl_ugm 0117777777/I Light On 400 ' ’c? 13.; 2 °' 300 - g png-i E = ‘e’ 5 200 .3: 225 < 3 100 " 4%. WT < 0 I I I I I I I I I I I I o 4 8 12 16 20 24 Time (hours) Figure 5-4. Total amylase activity in NT and the gng mutant during a diurnal cycle. Plants were grown for 29 days in 12/12 and total amylase activity was measured in leaf samples. Results are mean t SE, n-3. Amylase Activity (nmol/min/mg protein) Figure 5-5. following a shift from 24/0 to 12/12. 96 150‘ 100' AL VVT Unshifted . Control . " Shiited ‘ . M 0 2 4 6 3 1° 12 250 200‘ 150‘ 100‘ 50' WL””””””,,A-! Shifted 1 .11 Unshiited Control 4. P in m. 0 2 4 6 8 10 12 Time (dayS) Total amylase activity in NT (A) and the nng-I mutant (B) Plants were grown for 20 days in 24/0 then shifted to 12/12 (filled symbols) or left in 24/0 as controls (open symbols). The light conditions for the shifted plants are indicated by the filled (light off) or open (light on) boxes. Results are the mean 2 SE, n - 3. 97 reached a peak of activity 5 days after the shift and then declined to levels normally observed in the NT in 12/12 (compare with Figs. 5-1, 5-3). In general, the pattern in both the NT and mutant indicates that shifting from 24/0 to 12/12 first stimulated a rapid response which is then followed by a long-term adjustment. B1genen1ea1_baa1e_fgn_1nenea§eg_anyla§e_activitv in the mutante. The increase in total amylase activity was primarily due to a single form of amylase (A3, Fig. 5-2). The magnitude of the increase in activity of this form in the mutants varied somewhat from batch to batch of plants, but in one representative experiment, it was a least 40-fold higher in the ngn£;1 mutant than in the NT (Fig. 5-6A). This amylase comigrates on both native PAGE and IEF (results not shown) with an amylase which has been partially purified and shown to be a cytosolic exoamylase (B-amylase) (15). In order to confirm that the affected activity band was a B-amylase and to determine whether the increased activity of this form was due to an increase in the amount of protein or an increase in the specific activity of the protein, immunoblots of crude extracts resolved on native PAGE were probed with antibodies prepared against sweet potato B-amylase. The results (Fig. 5-68) confirmed that the affected band was a B-amylase. More importantly, the crude extracts from the mutant contained comparable or even slightly lower amounts of immunoreactive protein than the NT, despite the fact that in this set of crude extracts, there was at least 40 times more activity in the mutant in this band. In order to confirm that a single amylase species was responsible for both the increased activity and the immunoreactive band, crude extracts were also run on native IEF and stained for activity and for 98 (— WT—><—— png-1 —> Total protein per lane (pg) 2 5 10 25 50100 200 2 5 10 25 50 100 200 Figure 5-6. Activity and immunoreactive staining of amylase in leaf extracts from NT and the png-I mutant following native polyacrylamide gel electrophoresis. Plants were grown in 12/12 for 42 days (NT) or 70 days (nng-l). Crude leaf extracts containing the indicated amount of total protein were resolved on 7% native polyacrylamide gels. The gel in panel A was stained for amylase activity; the gel in panel B was used for immunoblot analysis with rabbit antibodies against sweet potato B-amylase. Ribulose bisphosphate carboxylase, which reacts nonspecifically in both assays, is marked (R) and serves as an internal indicator of the relative amount of crude extract in each lane. 99 immunoreactive proteins. The amylase activity and one of the two immunoreactive bands had similar isoelectric points of about 4.8 (Fig 5-7). The second band observed in the immunoblot (isoelectric point of about 4.5) may represent a B-amylase form which is inactive in both the NT and the mutant. Alternatively, it may simply be unstable or inactive in the conditions used for this experiment or it may represent a cross-reaction by an unrelated protein. Egnet1gn_gf_tne_negg1ateg_fi;anyla§e. The regulated B-amylase is located outside the chloroplast, whereas its presumed substrate starch is confined, at least in leaves, to the chloroplast. One possible explanation of this apparent contradiction is that another substrate exists for the cytosolic B-amylase. The starchless ngnfl and aggl lines have been shown to lack starch (Chapters 2, 3) which is operationally defined by the design of the starch assay as material insoluble in 80% ethanol, soluble in 0.2 N KOH at 100°C, and degraded into hexose by amyloglucosidase. Therefore, the investigation of whether an alternative substrate for B-amylase might exist was directed at finding an ethanol-soluble glucan (oligosaccharide). Material soluble in 80% ethanol was extracted from leaves of the NT and the ngnfl;1 and egnl;l mutants grown in 12/12 and harvested at the end of the light period. Analysis of these preparations by HPLC (Fig. 5-8) showed large amounts of sugars in the mono- and di-saccharide regions of the chromatogram, but only trace amounts of material which eluted in the oligosaccharide region. The major peak in this region eluted at about 6 min and contained about 1-2 ug/g fresh weight which was 0.2-0.9% (w/w) of the total sugars in the extract. The material in this peak (and all other detectable peaks), however, was not degraded by sweet potato P-amylase 100 Figure 5-7. Activity and immunoreactive staining on IEF gels of amylase from NT and the png-I mutant. Plants were grown in 12/12 for 42 days (NT) or 70 days (gng-I). Crude extracts containing 100 ug protein were separated by native IEF. Half the gel was stained for amylase activity (A) and the other half used for immunoblot analysis with rabbit antibodies against sweet potato B-amylase (B). 101 Digested Detector Response (x 10‘3) Ehtion Time (min) Figure 5-8. HPLC analysis of soluble sugars from NT, png-I and agpl-l lines of Anabidgneie. Soluble sugars were extracted from leaves of plants grown in 12/12 and separated by HPLC. Each trace contains the material extracted from 475 mg fresh leaves. The full traces represent mock-digested samples treated with boiled sweet potato B-amylase; the insets show the oligosaccharide regions at the same scale from duplicate samples which were digested with the amylase. Arrows marked G indicate the elution times of alpha-1,4 glucans with n residues. A pBak with an elution time of about 6 min (slightly larger than the 68 standard) is indicated by the arrow marked P. 102 (Fig. 5-8, insets), suggesting it was neither an cx-1,4 glucan nor a substrate for the cytosolic Anatiggneie B-amylase. The similar amount of this material in the three lines further suggests that it is not the signal for the activation ofyp-amylase in the mutants. DISCUSSION '-I_ ._ 0‘ I .1 -. ‘ ._ 1 . I .thI dr. ' ‘ th .nI geyelgnmenta1_jaetgn§. Using a collection of mutants of Anaaignnaie with alterations in starch metabolism, we have discovered a system which regulates leaf B-amylase. The mutants, which show up to 40-fold increases in activity over the NT, include three different classes of starch-free mutants, a mutant with reduced starch, and a class which contains more starch than the NT. All of the starch-free mutants examined showed this response (i.e. there were no exceptions to the correlation between the starch-free and increased amylase phenotypes). The increased total amylase activity was primarily attributable to the increase in activity of a single B-amylase form. The biochemical basis for the increased activity was an altered specific activity of the protein, rather than an alteration in the amount of protein. The activation of the B-amylase in the mutants (or inhibition in the NT) was maintained during electrophoresis in non-denaturing conditions, and thus the alteration must be due to a very stable conformational change or to a very tightly bound factor. The 40-fold variation in activity of this B-amylase makes this enzyme one of the most highly regulated enzymes known in plants. Covalent modifications which regulate enzyme activity are well known and include phosphorylation, adenylylation, methylation and 103 oxidation-reduction of sulfhydryl groups. Other covalent modifications of proteins are also known, but (to our knowledge) they have not been reported to affect enzyme activity. Although we have no direct I evidence as to the nature of the modification of B-amylase which alters its activity, the observations that the inactive form in the NT has the same isoelectric point and Rf on native gels as the active form in the mutants place certain constraints on the possibilities. For example, phosphorylation and adenylylation seem unlikely since they add charged groups to the protein which should alter its electrophoretic characteristics. Further work will be required to identify the nature of the modification of B-amylase. The regulation of B-amylase in the mutants was dependent on the developmental stage of the plants and the environmental conditions of growth. The activity was elevated when the mutants were grown in 12/12 but this response was suppressed when they were grown in 24/0. Furthermore, in 12/12 the activity in the mutants increased as the plants developed, whereas the activity in the NT was essentially constant. In addition, shifts in the photoperiod produced both a rapid diurnal fluctuation and a long-term stable adjustment in the amylase activity in both the NT and a mutant. The results of all of these experiments indicated a large amount of variability from experiment to experiment and even from plant to plant within a given experimental treatment in the absolute activities observed. This is, perhaps, not surprising, given that the amylase activity appears so easily regulated by a variety of factors, and suggests that there may be other factors which also can affect the 104 activity. Taken as a whole, however, the results provide a consistent picture of the regulation of leaf B-amylase. 329W. The regulation of the B-amylase observed in this work suggests that it has an important function in the plant. However, this B-amylase is located outside the chloroplast (15) whereas starch, its only known substrate, is present inside the chloroplast. This surprising situation is similar to that found in many plants where the majority of the amylase and starch phosphorylase activity is localized outside the chloroplast (reviewed in 17). One possible explanation is that substrates for these enzymes, other than starch, exist outside the chloroplast. However, we (Fig. 5-8) and others (3) have been unsuccessful in identifying such a substrate. Thus, no satisfactory explanations presently exist for the presence of high levels of starch degradative enzymes outside the chloroplast. Similarly, it has been argued that the major B-amylase present in soybean seeds has no role in starch metabolism (1,10) M l r d v' - m . At first sight, it seems difficult to explain why two groups of mutants with diametrically opposing phenotypes (i.e. containing no starch or elevated levels of starch - the low starch aggg;1 class will be considered later) should each have elevated'B-amylase activity in 12/12 (but not in 24/0). However, the similarities between the two classes become more obvious if the net flgxes through the starch pool are considered, rather than the size of the starch pool. In the starchless mutants, there is no flux through starch since they are deficient in starch biosynthetic enzymes. Similarly, although the enzymatic 105 deficiency in the elevated starch mutants is not known, they have been shown to have no large changes in the starch pool during a diurnal photoperiod (TC, unpublished results). In contrast to these mutants, the starch pool in leaves of the NT is completely turned over each day (Chapters 2, 3, 4). Thus, there must be great differences in the carbohydrate metabolism of the mutants versus the NT in 12/12. In continuous light, however, both the NT and the mutants must reach an equilibrium state in which there is no net starch turnover and thus the NT and mutants should be functionally equivalent. At least two possibilities exist which might explain why the mutants activate the B-amylase activity. First, it is possible that B-amylase is activated by the plant in an attempt to increase the rate of starch breakdown when the cytosolic sugar levels reach sufficiently low levels. An obvious problem with this model is that, as previously noted, the‘B-amylase is located outside the chloroplast. This model is, however, attractive in that it has been shown that sugar levels drop rapidly in the starchless pgn£;1 mutant during the night since there is rapid respiration of the existing sugars and there is no starch pool to be degraded to provide additional sugars (Chapter 2). Although similar analyses have not been conducted-with the other mutants, there is no reason to believe they should react differently. Furthermore, the elevated starch mutant snnl;1 does not degrade starch during a 12 h dark period (TC, unpublished results) and so it must also be dependent on sugars accumulated in the light to provide respiratory substrate during the dark period. ‘ Secondly, since there is no net starch synthesis in the light in the mutants, the photosynthate may instead be converted into a form 106 which triggers the activation of the B-amylase. For example, in the ngn£;1 mutant, sucrose, glucose, and fructose accumulate to high levels in the light (Chapter 2). These sugars might serve directly as the signal, or might be converted into larger oligosaccarides which could serve as the signal and (and perhaps the substrate as well) for the B-amylase. As noted earlier, however, such oligosaccharides have not been identified. The results with the agg2;1 line, which accumulates 40% as much starch as the NT (unpublished results), suggest that less drastic changes in carbohydrate metabolism are also sufficient to trigger at least a partial activation of B-amylase. Wanted. P-amylase activity increases during germination of cereal seeds and, like our results, this is due to activation of the existing protein (reviewed in 17,20). In dry seeds the inactive protein is bound by a dithiol linkage to protein bodies. Upon imbibition the B-amylase is released and activated either enzymatically by proteases or non-enzymatically. A similar mechanism could be responsible for the regulation observed in this work, although since the electrophoretic mobilities were not affected, the sulfhydryl linkage must either be intramolecular or to a small, relatively uncharged molecule. Regulation of amylases has also been observed in leaves in response to environmental and hormonal signals. For example a cytosolicex-amylase is transcriptionally induced about 3-fold by drought stress in barley (12), the synthesis of B-amylase in mustard cotyledons is regulated by phytochrome (7), :x-amylase activity is increased about 70-fold by flooding of deep-water rice (18,19), total amylase activity is increased 2-3 fold by feeding 107 gibberellic acid to the gibberellic acid deficient as maize mutant (13), and amylase activity is increased about 50% in pangola grass by feeding gibberellic acid or increasing the night time temperature from 10°C to 30°C (4). Nhether these effects and those described in this study are representative of a common regulatory system or separate ones, it seems apparent that amylase activity is regulated in a wide variety of plants and tissues by various stimuli. The elucidation of the nature of the modification to B-amylase and identification of the (presumptive) enzymes involved in activation/deactivation may shed light on the nature of the signals, the specificity of the response and its physiological significance. REFERENCES 1. Adams, CA, TH Broman, RN Rinne 1981 Starch metabolism in developing and gerrminating soya bean seeds is independent of {B-amylase activity. Ann Bot 48:433-439 2. Azcon-Bieto J, H Lambers, DA Day 1983 Effect of photosynthesis and carbohydrate status on respiratory rates and involvement of the alternative pathway in leaf respiration. Plant Physiol 72:598-603 3. Camp, PJ, SC Huber, OM Pharr 1987 A note on the role of extrachloroplastic amylase in spinach leaves (Sninaeja oleraeea L.). Plant Physiol 83 (Suppl):l47 4. Carter, JL, LA Garrard, SH Nest 1973 Effect of gibberellic acid on starch degrading enzymes in leaves of Qigitania geeumbens. Phytochem 12:251-254 5. Caspar T, SC Huber, C Somerville 1986 Alterations in growth, photosynthesis and respiration in a starch mutant of Arabidopsis thaliana (L.) Heynh deficient in chloroplast phosphoglucomutase activity. Plant Physiol 79:1-7 6. Caspar, T, CR Somerville, BG Pickard 1988 Gravitropism by a starchless mutant of Arabiggnsis: Implications for the starch-statolith theory of gravity sensing. Planta In press 7. Drumm, H, I Elchinger, J Moller, K Peter, H Mohr 1971 Induction of amylase in mustard seedlings by phytochrome. Planta 99:265-274 10. ll. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 108 Haughn GN, CR Somerville 1986 Sulfonylurea resistant mutants of Arabiggpsjs thaljana. Molec Gen Genet 204:430-434 Herold A 1980 Regulation of photosynthesis by sink activity - the missing link. New Phytol 71:818-821 Hildebrand, OF, T Hymowitz 1981 Role of B-amylase in starch metabolism during soybean seed development and germination. Physiol Plant 53:429-434 Huber SC 1984 Biochemical basis for effects of K-deficiency on assimilate export rate and accumulation of soluble sugars in soybean leaves. Plant Physiol 76:424-430 Jacobsen, JV, AD Hanson, PC Chandler 1986 Nater stress enhances expression of anixramylase gene in barley leaves. Plant Physiol 80:350-359 Katsumi, M, M Fukuhara 1969 The activity of «eamylase in the shoot and its relation to gibberellin-induced elongation. Physiol Plant 22:68-75 Lin TP, T Caspar, C Somerville, J Preiss 1988 Isolation and characterization of a starchless mutant of Arabidonsjs thaliana (L.) Heynh. lacking ADPglucose pyrophosphorylase activity. Plant Physiol: 86:1131-1135 ' Lin, TP, SR Spilatro, J Preiss 1988 Subcellular localization and characterization of amylases in Anatiggnsis leaf. Plant Physiol 86:251-259 Pongratz, P, E Beck 1978 Diurnal oscillation of amylolytic activity in spinach chloroplasts. Plant Physiol 62:687-689 Preiss, J, C Levi 1980 Starch biosynthesis and degradation. In J Preiss, ed, Biochemistry of Plants, Vol 3, Carbohydrates: structure and function. Academic Press, New York, pp 371-423 Raskin I, H Kende 1984 Effect of submergence on translocation, starch content and amylolytic activity in deep-water rice. Planta 162:556-559 Smith MA, JV Jacobsen, H Kende 1987 Amylase activity and growth in internodes of deepwater rice. Planta 172:114-120 Thoma, JA, JE Spradlin, S Dygert 1971 Plant and animal amylases. 1n PD Boyer, ed, The Enzymes, Ed 3, Vol 5. Academic Press, New York, pp 115-189 Towbin, H, T Staehelin, J Gordon 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. PNAS 76:4350-4354 Chapter 6 Gravitropism by a Starchless Mutant of Anabiggnsis: Implications for the Starch-statolith Theory of Gravity Sensing ABSTRACT The starch-statolith theory of gravitropic reception has been tested with a mutant of the crucifer Anantggnsts thaliana (L.) Heynh. which, lacking plastid phosphoglucomutase (EC 2.7.5.1) activity, does not synthesize starch. The hypocotyls and seedling roots of the mutant were examined by light and electron microscopy to confirm that they did not contain starch. In upright wild-type (NT) seedlings, starch-filled plastids in the starch sheath of the hypocotyl and in three of the five columellar layers of the root cap were piled on the cell floors, and sedimented to the ceilings when the plants were inverted. However, starchless plastids of the mutant were not significantly sedimented in these cells in either upright or inverted seedlings. Gravitropism of light-grown seedling roots was vigorous: e.g., 10o curvature developed in mutants rotated on a clinostat following a 5 min induction at l’g, compared with 14° in the NT. Thus, neither starch nor sedimenting plastids are required for the detection of gravity. Curvatures induced during intervals from 2.5 to 30 min were uniformly 70% as great in the mutant as in the NT. Because the growth rate of mutant roots was only 80% as great as that of NT roots, most and quite possibly all of the 30% discrepancy between mutant and NT curvature was attributable to impaired expression of induction rather than to 109 110 impaired reception of stimulus. Thus, under these conditions the presence of starch and the sedimentation of plastids are unnecessary for reception of gravity by Azaniggnsis roots. Gravitropism by hypocotyls of light-grown seedlings was less vigorous than that by roots, but the mutant hypocotyls exhibited an average of 70-80% as much curvature as the NT. Roots and hypocotyls of etiolated seedlings and flower stalks of mature plants were also gravitropic, although in these cases the mutant was generally less closely comparable to the NT. Thus, starch is also unnecessary for gravity reception in these tissues. INTRODUCTION Since the turn of the century it has been widely accepted that the first step in gravitropism by higher plants requires statoliths. As proposed by Haberlandt and NemeE (reviewed in Audus 1962), these have been thought to be starch-filled plastids, relatively dense organelles that often settle to the lower sides of cells in tissues displaced from their upright position of equilibrium (e.g. Larsen 1971; Shen-Miller and Hinchman 1974; Audus 1975; Juniper 1976; Volkmann and Sievers 1979; Jackson and Barlow 1981; Nilkins 1984; Feldman 1985; Moore and Evans 1986). Once settled, according to many authors (e.g. Perbal and Riviere 1976; Hillman and Nilkins 1982; Sack et al. 1984; Nendt and Sievers 1986), the statoliths might either press against or interact chemically or electrically with the lateral plasmalemma itself, the ”cortical gel”, or associated layers of the endoplasmic reticulum. Alternatively, it has been suggested (e.g. Iversen and Larsen 1973; Clifford 1979) that statoliths act by their movement through the 111 cytoplasm. Others have noted that starch-laden plastids need not settle, but might act by exerting force on hypothetical cytoskeletal structures that restrain their movement (e.g. Filner et al. 1970). Three classes of evidence have been amassed in support of the starch-statolith theory. First, mobile, starch-filled amyloplasts are usually present in gravitropic organs. Second, accumulation of starch is often correlated with the development of gravitropic competence (e.g. Barlow 1974; Perbal and Riviére 1976; Hillman and Nilkins 1982; Nright 1986). Third, low starch content or low amyloplast mobility is correlated with impaired gravitropism in a number of mutants (Roberts 1984; Olsen et al. 1984; Mirza et al. 1984; Hertel et al. 1969; Filner et al. 1970; Miles 1981). Disputing the starch-statolith theory, some authors have reported substantial gravitropic responses by plant organs naturally free of starch, low in starch or depleted of starch (e.g. Pickard and Thimann 1966; Nesting 1971; Grenville and Peterson 1981; Moore 1987). It has also been argued that the kinetics of plastid displacement are inconsistent with the kinetics of gravitropic induction (Pickard 1973; Johnsson and Pickard 1979; Clifford and Barclay 1980). However, because of the mass of evidence consistent with the starch-statolith theory and because of the lack of a satisfying alternative hypothesis, the inconsistencies have generally been viewed as problems which could be reconciled by further experimentation (e.g. Volkmann and Sievers 1979; Moore and Evans 1986). - This chapter describes a study of a previously described (Chapter 2) starchless mutant of the crucifer Arabidopsis thaliana. Both light-grown and etiolated mutant seedlings as well as mutant flower 112 stalks are capable of gravitropic curvature, precluding an obligatory role for starch in their detection of gravity. Furthermore, the response by roots of light-grown mutant seedlings is closely comparable to that by the wild-type (NT) roots, and no primary sensory role for root plastids could be demonstrated. MATERIAL AND METHODS Reagents. Agar was obtained from Difco Laboratories, Detroit, MI, USA; NaHl4CO3 was from ICN Radiochemicals, Irvine, CA, USA; biochemicals were obtained from Sigma Chemical Co., St. Louis, MO, USA. Plants. The starchless mutant lines TC7, TC9, TC21, and TL25 were independently derived from the Columbia NT of Arabigogsis thaliana (L.) Heynh. (Chapters 2, 3). The mutations in TC9 and TC2l were characterized as being allelic to that in TC7 and the mutation in TL25 as nonallelic to that in TC7. The single, recessive, nuclear mutation in TC7 causes a deficiency of the activity of the plastid isoenzyme of phosphoglucomutase (PGM; EC 2.7.5.1) and that in TL25 causes a deficiency in the activity of ADPglucose pyrophosphorylase (EC 2.7.7.27). Seedlings of these plants were grown under sterile conditions in square, gridded 100 x 100 x 15 mm3 Petri plates with a medium consisting of the nutrient salts described by Haughn and Somerville (1986) plus 1% (w/v) sucrose and solidified with 1% (w/v) agar. For the experiment of Fig. 6-6, sucrose was omitted from some plates. Seeds were surface-sterilized as described in Haughn and Somerville (1986). They were distributed on the agar medium at intervals of 2 mm in parallel rows and the Petri plates were sealed with Parafilm 113 (American Can Co., Greenwich, CT, USA). Seed sterilization and sowing were carried out in room light for both light- and dark-grown seedlings. In a few of the later experiments, after seeds were sown they were maintained in the dark for 2 or 3 d at 4°C to promote uniform germination. The plates were placed on edge, with the rows of seeds horizontal, at 24°C in the light or dark as indicated. Photosynthetically active radiation (PAR) was provided by 'cool white” fluorescent tubes (F4OCN/RS/EN-II, Nestinghouse, Somerset, NJ, USA or F40/CN/RS/SS, Sylvania, Danvers, MA, USA). For the experiment of Fig. 6-8 a flux 2's'l PAR was used because it produced 2 density of 10 umol'm' fast-growing hypocotyls; otherwise 50 umol'm' ‘s'1 PAR was used because it produced vigorous root growth and compact shoots. Light was measured with a LI-IBBB or LI-185B meter with a LI-19OSB quantum sensor (Li-Cor, Lincoln, NE, USA). Roots of light-grown seedlings were used when their typical length was 6-10 mm, and hypocotyls when about 3-4 mm.. For the NT, these lengths were achieved in about 85-90 and 90-95 h, respectively. Mutant seed were sown 14-16 h earlier because germination was delayed with respect to the NT. For production of leaves and flower stalks, sets of 15-30 plants were grown in 130-mm-diameter pots (as in Haughn and Somerville 1986) at 22°C with continuous illumination from ”cool white” fluorescent tubes (FR72T12/CN/VHO/135, Sylvania) (125 umol'm'°°s'l PAR). For biochemical assays, plants were used at the rosette stage (about three weeks old). For studying flower-stalk gravitropism, plants were used when six weeks old with flower stalks 50-150 mm in length. 114 Miengsegny. Fresh seedling and flower stalk tissue was stained with iodine as described in Chapter 2. For fixation, uniform seedlings were placed upright on approximately 10 x 20 x 30 mm3 blocks of agar, and gently secured to the blocks with moistened gauze. Care was taken to maintain the vertical orientation of the seedlings throughout these manipulations. Half the assemblies were placed upright and half were placed inverted in darkened, sealed beakers. For the study of roots, the assemblies were left for 2 h; for hypocotyls, 3 h. The seedlings were then fixed without altering their positions by filling the beakers with ice-cold 4% (v/v) glutaraldehyde in 0.1 M sodium phosphate (pH 7.2). After 90 min, the root tips or hypocotyls were excised from the seedlings and fixed for an additional 60 min. Following a rinse in the phosphate buffer, the sections were post-fixed for 90 min in 1% (w/v) 0s04 in the same buffer and the tissues were then dehydrated and embedded in a 1:1 (w/w) mixture of Mollenhauer’s and Spurr’s resins (Klomparens et al. 1986). Longitudinal 2-um sections were cut serially until the midplane of each organ was reached; then, ultrathin (approx. 80 nm) sections were cut. The 2-um sections were stained with periodic acid-Schiff’s reagent (PAS) and counterstained with toluidine blue (Feder and O’Brien 1968). The ultrathin sections were stained 30 min with uranyl acetate and lead citrate (Reynolds 1963) and viewed with either a Philips (Eindhoven, The Netherlands) EM 201 electron microscope at 60 kV or a JEOL (Tokyo, Japan) IOOCX II electron microscope at 100 kV. Managemetnie assessment at plastig gistribution. Root caps of the NT were analyzed by light microscopy; analysis of a small sample of electron micrographs gave similar results. The plastids in the 115 columella of TC7, however, were not readily visible by light microscopy, so electron micrographs were used. The tntal anea occupied by the plastids in a single section of a columella cell in the root cap was measured by a point-counting method, using a square lattice (Neibel and Bolender I972). The ayenage nesttign of the plastids in each micrograph of a columella cell was determined by a modified point-counting method in which the lattice was used to calculate the distance from the ends of the cell to each unit square of the lattice occupied by plastid material. The nelatiye plastid ngsjtjgn was calculated by counting the number of unit squares occupied by plastid material within each transverse row of the lattice and multiplying by the rank of the row; the sum of the products for all rows was divided by the sum of the unit squares occupied in all rows; finally, the resultant value was divided by the number of rows in the cell (i.e. its height) and expressed as a percentage. (This method, based on the size rather than the number of plastid sections, was chosen because, without serial reconstruction of the plastids in each cell, it was not possible to determine for the irregularly-shaped plastids whether adjacent sections of plastid in a micrograph were part of the same or different plastids.) For each experimental treatment the total area occupied by plastids and the relative mean plastid position determined for all cells within a given columella layer from 3 to 6 seedlings were further averaged to produce the overall average values for that layer. Assays fen enlgnnnlast enzymes. Plants were placed in the dark for 16 h, assuring depletion of starch in the NT. Extracts were prepared at 4°C. For crude extracts, 500 mg of leaves were ground with a mortar and pestle in 5 ml of buffer (28 mM imidazole-HCl, pH 7.4; 3.3 116 mM MgCl2; 40 mM 2-mercaptoethanol; 0.1%, w/v, defatted bovine serum albumin; 2 mM glucose-6-phosphate) and filtered through Miracloth (Behring Diagnostics, La Jolla, CA, USA). For preparation of chloroplast extracts, washed leaves were homogenized in 20 volumes of buffer (20 mM N-[2-hydroxy-1,1-bis- (hydroxymethyl)ethyl]glycine (Tricine)-KOH, pH 8.4; 10 mM ethylenediaminetetraacetic acid (EDTA); 10 mM NaHCO3; 0.1%, w/v, defatted bovine serum albumin; 450 mM sorbitol) for 6 s at the maximum speed with a Tekmar homogenizer (Tekmar, Cincinnati, OH, USA). After filtration through Miracloth, the extracts were centrifuged at 475 x g for 4 min. The pellets were gently suspended in suspension buffer (300 mM sorbitol; 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes)-KOH, pH 7.4; 5 mM MgCl2; 2.5 mM EDTA; 0.1%, w/v, defatted bovine serum albumin; 10 mM NaHCO3; 2 mM glucose-6-phosphate), using 0.5 ml per 1 g of leaf starting material, and centrifuged at 275 x g for 90 s. The pellets were resuspended (0.04 ml suspension buffer per 1 9 leaf) and used for assays. Phosphoenolpyruvate carboxylase (EC 4.1.1.31) and PGM were assayed at 22°C according to Stitt et al. (1978) and ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) was assayed at 22°C according to Pierce et al. (1982) except that the reaction was initiated by the addition of ribulose 1,5-bisphosphate. Octyl phenoxy polyethoxy ethanol (triton X-100; 0.1%, v/v) was included in all the enzyme assays to ensure complete lysis of organelles. Chloroplast intactness was measured by ferricyanide-dependent oxygen evolution (Somerville et al. 1981). Starch gels were run as described in Chapter 2. Chlorophyll was assayed in 80% (v/v) acetone according to Mackinney (1941). 117 Ktnettes gt tngnism. Gravitropism of light-grown roots was assessed at 24°C in white light of 50-60 umol‘m'z’s'1 PAR (tubes were of the same types as used for growth). For the experiment of Fig. 6-7, induced plants were mounted parallel to the axis of a 3-rpm clinostat 2 and provided with an axial light source of about 40 umol'm' 's'1 PAR and a lateral source of about 15 umol‘m'z's'l PAR. It was carefully checked that the roots were not phototropic even in continuous bright unilateral white light. Gravitropism of light-grown hypocotyls and flower stalks was assessed in the dark (except for light required for photography). Gravitropic stimulation of hypocotyls and roots was achieved by rotating the square Petri plates on edge so that the seedlings were horizontal, or, when specified, 30° from the vertical. Stimulation of flower stalks was achieved by placing the pot with the plants on its side in a room of high relative humidity (> 95%). For phototropic stimulation of hypocotyls, a fan-cooled 80‘360-mm2 panel of blue filter glass (#5543, Corning Glass Norks, Corning, NY, USA) was sealed into a window of a light-tight shield placed immediately in front of two adjacent fan-cooled "cool-white” fluorescent tubes. Petri plates were set with seedlings in their normal orientation but with the surface of the agar at an angle of 24° from the axis of incident light; this orientation prevented shoots from shading each other. Photon flux density at the surface of the agar in the center of each Petri dish was I umol‘m'z’s'l. A thermistor placed in a mock assembly indicated that the air temperature inside the Petri dishes was maintained constant at 24°C. 118 Seedlings were photographed in the vertical position at the beginning of each experiment and at all designated times for plants gravitropically reacting at 30° from the horizontal or on the clinostat, or undergoing phototropism. They were generally photographed in the horizontal position if reacting from the horizontal position. The photographic light was supplied by white fluourescent tubes except for experiments carried out in the dark, for which a red light confirmed to be phototropically inactive was used for both focusing and exposure. Film was 35 mm Kodak Plus-x Pan (Eastman Kodak,- Rochester, NY, USA); prints were made on Kodak Polycontrast Rapid 11 RC FM paper. Plants were reduced by a factor of 0.5 in the negative image, and enlarged by a factor of 3.5 in the prints. Images of plants which had germinated too late to produce roots at least 4 mm long, and plants which had grown into contact with their neighbors, were marked for exclusion. Flower stalks were photographed using infrared-sensitive film (Kodak 2481) and an electronic flash covered with a far-red filter (Kodak Nratten 87C). It was confirmed that the flash did not induce a phototropic response. Curvatures were measured by extending the axis of the apical 1 mm of the enlarged image of the root or hypocotyl with a straight-edge and a sharp pen and measuring the angle formed with the originally vertical grid lines of the Petri plate. All angles are given as increments over the starting values. To calculate elongation, fine needle points mounted in a draftsman’s divider were set at unit separation and stepped along the axis of each image, starting with a convenient crossing grid line or a line drawn parallel to it. The sum of all steps at the end of the growth period was then subtracted from the sum at the beginning. All error bars in 119 figures are standard errors of the means (SE) unless otherwise specified. All figures represent pooled data from at least two consecutively performed experiments. Seedling orientation in the experiment of Fig. 6-9 was quantified by determining the angle from vertical of the line connecting the root-hypocotyl junction with either the root tip or a point just proximal to the hypocotyl hook. RESULTS 2611 1:11.011 10 isolated 2010211012512. The previous characterization of mutant line TC7 as lacking starch because of a deficiency of the chloroplast isoenzyme of PGM (Caspar et al. 1985a) was based on a starch gel assay of leaf extracts. In order to determine more rigorously whether any chloroplast PGM activity remained in the mutant, PGM activity in extracts from isolated chloroplasts from mutant and NT plants was measured. As shown in Table 6-1, the chloroplast preparation from the mutant had less than 4% of the PGM activity of the NT chloroplast preparation. Based on the activities of ribulose 1.5-bisphosphate carboxylase (a stromal enzyme) and phosphoenolpyruvate carboxylase (a cytosolic enzyme) in the chloroplast preparations, the residual PGM activity in the chloroplast extract from the mutant was entirely attributable to contamination of the chloroplasts by the cytosolic isoenzymes of PGM. This conclusion was confirmed by electrophoresis of the chloroplast extracts on starch gels followed by staining for PGM activity. On these gels only the cytosolic PGM isoenzymes were observed in chloroplast preparations from 120 Table 6-1. Enzyme activities in extracts of NT and mutant TC7 Anabtggnsts. Values are the means of 2 to 8 assays. Ferricyanide-dependent oxygen evolution assays indicated that 22% of the NT an? 27% of the mutantlchloroplasts were intact. Activities in umol‘min “(mg chlorophyll) Enzyme Ni TC7 Crude Chloroplast Crude Chloroplast Phosphoglucomutase 2.7 0.28 2.1 0.010 Ribulose-1,S-bisphosphate 2.2 0.67 2.4 0.95 carboxylase Phosphoenolpyruvate 0.14 0.001 0.14 0.001 carboxylase 121 the mutant, whereas the plastid isoenzyme was the major species in preparations from the NT (data not shown). Qetenninatign gf stanen ny histgehemjstny ang eleetron miergscopy. ' Because the previous determinations of starch (Chapter 2) and the determinations of PGM activity shown in Table 6-1 were made with leaves, it remained possible that starch could be present in specialized tissues of gravitropically receptive organs - tissues such as the columella of the root cap and the starch sheaths of the hypocotyl and flower stalk - and even here, only at special stages of development. Therefore, these tissues were examined for starch in plants of the same age and grown under the same conditions as those used for measurements of gravitropism. Initially, fresh root caps, hypocotyls, and flower stalks were stained with iodine. Starch was clearly evident in the NT organs, but none was evident in those of the mutant (n > 20; data not shown). To improve the sensitivity of the assay, fixed, sectioned roots were examined. The Columbia NT root cap (Fig. 6-1A) generally resembled that of the Landsberg race of Ananignnsis described by Olsen et al. (1984). Five (or occasionally six) distinct columellar layers were present. The outermost layer was complete rather than represented by only a single apical cell as in Landsberg. The columella layers were numbered from 1 to 5, with 5 being outermost (Olsen et al. 1984). In roots with six intact columella layers, the innermost layer was designated layer zero. A layer of detaching cells (which lacked starch) was often seen incompletely separated from the root cap; these were not included in the numbering system. 122 Figure 6-1. Light micrographs of median longitudinal sections of NT and TC7 Arabiggpsis root tips and hypocotyls stained with PAS and toluidine blue. Sections are positioned according to their orientation during growth; arrows marked g indicate the direction of the gravity vector for the 2 h (roots) or 3 h (hypocotyls) immediately before fixation. A. Upright NT root tip. B. NT root tip inverted for 2 h before fixation. C. Upright TC7 root tip. 0. Upright NT hypocotyl. E. NT hypocotyl inverted for 3 h before fixation. F. Upright TC7 hypocotyl. SS, starch sheath; 1, vascular tissue; M, meristematic zone; 00, detaching cells; E, plastid. A, B, C x320; scale bar - 50 um. D, E, F x295; scale bar - 100 um. 123 124 The NT contained large plastids in columella layers 2, 3, 4, and 5 (and occasionally in layer 1). They were filled with starch which reacted intensely in the PAS test as judged by light microscopy (Fig. 6-1A, B), and stained densely in electron micrographs (Fig. 6-2A). In contrast, the mutant showed no evidence of starch in the cap or in any other tissue of the nine roots for which serial sections were viewed by light microscopy (Fig. 6-1C) and median longitudinal sections were viewed by electron microscopy (Fig. 6-2B, C, D). The mutant plastids were small and irregularly shaped, and frequently contained internal membranes similar to those found in proplastids. Plastoglobuli, present in both the NT and mutant, were readily distinguished from small starch grains by their characteristic size and uniform round shape. Internal membranes and plastoglobuli were less apparent in NT than in mutant plastids (Fig. 6-2A, 8). Possibly they were less obvious in the NT because of the proportionately smaller volume they occupied in the starch-filled plastids, or perhaps they were more abundant in the mutant because more carbon is channelled into lipid synthesis when starch synthesis is blocked. Longitudinal sections of fixed hypocotyls were also examined. Large, starch-filled plastids were present in the NT (Fig. 6-10, E, and 6-3A). However, in each of the three mutant seedlings examined, the plastids were small and showed no starch by either light (Fig. 6-1F) or electron (Fig. 6-38) microscopy. Otherwise, they appeared similar to those in the NT. lne ngtant plastids as net settle. Preparatory to examining whether starch-free plastids would sediment in response to gravity, the distribution of plastids was checked in undisturbed and reoriented NT 125 Figure 6-2. Electron micrographs of NT and TC7 Anaplggnsls root cap columella cells. Panels C and D are positioned according to their orientation during growth and the arrow marked g indicates the direction of gravity during the 2 h before fixation. A. Plastid from layer 3 of the root cap columella of a NT seedling. B. Plastid from layer 2 of the columella of a TC7 seedling. C. Cells from layer 3 of the columella from an upright TC7 seedling. 0. Cell from layer 3 of the columella from an inverted TC7 seedling. N, nucleus; Eg, plastoglobulus; S, starch grain; arrow heads in C and 0 point to plastids. A, B x27,800; scale bar - 1 um. C, D x4,200, scale bar - 5 um. 126 Figure 6-2 127 Figure 6-3. Electron micrographs of plastids from NT (A) and TC7 (B) Arabidopsis hypocotyls. S, starch grain; Pg plastoglobulus. A, B x18,500; scale bar - 1 um. 128 seedlings. As expected, sedimented plastids were observed in NT seedlings only in the root-cap columella and the hypocotyl starch sheath. Thus, these tissues were subjected to more detailed study. In the upright NT root, the plastids in columella layers 2 through 4 (and layer 1 when plastids were evident) appeared by simple inspection to be sedimented on the floors of the cells (Fig. 6-IA). Following a 2-h inversion, the plastids in layers 1 through 3, but not in layers 4 and 5, had settled to the ceilings of the cells (Fig. 6-lB). These observations were confirmed by morphometric analysis (Fig. 6-4A). It should be noted that, because of the large fraction of the NT columella cells occupied by plastids (13-17% in layers 2 through 5) and the loose packing of the plastids which resulted from their large size, the average position of the sedimented plastids was about a third of the distance from the lower to the upper cell wall. Combining the data on average plastid position with the average height of the cells in each layer, the average distance moved by the plastids following inversion was calculated to be 2.4, 3.2 and 4.3 um for layers 1, 2 and 3, respectively. These estimates agree reasonably well with the value of 4.2 um previously reported (Olsen et al. 1984) for a 40-min inversion of the Landsberg ecotype of Anabiggpsis seedlings (the columella layers used for these measurements were not noted). Thus it is likely that sedimentation in our experiments was essentially complete within 40 min. Both in upright and inverted mutant seedlings, plastids in all columella layers appeared by simple inspection to be randomly distributed (Fig. 6-2C, D). Morphometric analysis (Fig. 6-4B) showed that in layers 1 through 3, which contained mobile plastids in the NT, 129 :- 100 . O O upright g I inverted 9 80 - . 3. O: O H 11 11} {1 A. wild type 1 1 1 1 I ts O N O lOO 1' O upright D inverted m 0 I Relative position in cell (proximal) (distal) c> 0 re e—cr—4 'eftrli b O I e—<>—4 t-{PH N O 8. mutant TC7 O 5 4 3 2 1 (outer) (inner) Columella layer (distal) Figure 6-4. The effect of gravity on the position of plastids in root-cap columella cells of NT (A) and TC7 (B) Arabigepsjs. Seedlings were either fixed in their normal upright orientation or were inverted for 2 h and then fixed. The average positions of plastids relative to the proximal (basal) and distal (apical) ends of the cell were determined in the central columella cells from 3 to 6 roots for each treatment. Error bars represent the 95% confidence limits. The NT layer 1 inverted-treatment point has no error bars since it represents only one cell (only one out of 16 cells examined from this layer contained starch). 130 mean plastid positions were similar for upright and inverted plants and were fairly close to the midpoint of the cell. Had sedimentation been possible, it would likely have resulted in a much greater displacement of the mean position for the mutant than for the NT, because of the considerably smaller total volume of the plastids (1-3% of the total cell volume). In layer 5, the mean positions were likewise indistinguishable with plant position, but both deviated toward the proximal wall to about the same extent as the mean for the NT. The larger variability of the average positions for TC7 than for the NT attests to the more random position of the starch-free plastids in the mutant. Only cell layer 4 showed any significant difference in average position of the plastids between the upright and inverted treatments, and this difference, though ostensibly reproducible (p > 99%), may well be artifactual: much of it could be attributed to a single one of the six inverted seedlings measured. It is possible that in this particular seedling layer 4 was in developmental transition to layer 5 in which the mean position in both NT and mutant for both treatments is more proximal. Moreover, the plastids in this layer of the NT did not sediment; since they are as large as or larger than those in layers 1 through 3, this is presumably a consequence of cytoskeletal constraint. There is no reason to suppose that this constraint present in the NT is absent in the starchless mutant. Examination of PAS-stained longitudinal sections of NT hypocotyls by light microscopy showed plastids of the starch sheath sedimented in both upright (Fig. 6-10) and 3-h-inverted plants (Fig. 6-1E). In the mutant no plastid sedimentation was observed in electron micrographs of longitudinal sections through the region of the starch sheath in either 131 upright or inverted hypocotyls. Morphometric analysis of plastid sedimentation in hypocotyls was not performed because of the difficulty in unambiguously identifying the starch sheath in the starchless mutant. GnaxitrooismLof light-greun_seedling_roets. Figure 6-5 shows the time-course of downward bending by horizontally displaced roots of light-grown seedlings stimulated in the light: both the NT and mutant TC7 display vigorous gravitropism. During the period of most rapid response, the mutant lagged slightly: at 2 h, for example, it had attained only 70% as much curvature as the NT. By 4 h the mutant had almost caught up, and ultimately it and the NT achieved the same curvature. In order to exclude the possibility that the ability of TC7 to respond gravitropically is atypical of starchless mutants, the independently derived allelic mutants TC9 and TC21, which are also defective in plastid PGM activity, were gravitropically stimulated in assays comparable to that shown in Fig. 6-5. Roots of TC9 and TC2l produced curvatures closely comparable to those of TC7 (e.g. at the representative response time of 3 h, mean responses were 39.7 1 l.1°, 40.4 1 1 0°, and 38.2 i 1.0°, for TC9, 1021, and TC7, respectively). Another starchless mutant (TL25) of Ananlggnsls which is completely deficient in ADPglucose pyrophosphorylase activity (Chapter 3) also had vigorously gravitropic roots (data not shown). Limitatien of rent ataxitronism bx carbohxdrate status. The _ slight sluggishness in the graviresponse of the mutant indicates that starch or starch-related metabolism does participate in gravitropism. Is this participation direct as predicted by the starch-statolith 132 90' N—I . ° 52 3 70. wild type 9., mutant TC7 a i- .{g .. 50c 93 2 .. 9 s 30' u . /} 1 D 10* // Ag 1 1 1 1 l 1 1 I N—‘L‘ 1 2 3 4 24 Time, hours Figure 6-5. Gravitropism by roots of NT and TC7 seedlings. Plants were placed horizontally at time zero. Interpretive curves were drawn based on visual impressions to intercepts of 20 min for the NT and 30 min for TC7. n - 35. (Barbara Pickard, personal communication) 133 hypothesis, or is it indirect, such as metabolic support of the response processes? Because the mutant lacks the large starch pools present in the HT root, it is possible that the different carbohydrate status of the mutant and HT might be the cause of the altered kinetics of root gravitropism. This idea is supported by an experiment which showed that the graviresponse of roots of the HT was reduced when they were grown on media without sucrose (Fig. 6-6). Furthermore, this reduced response of the NT was similar to the response of the starchless mutant when grown with supplemental sucrose (i.e. 80% versus 70% of the curvature of the sucrose-supplemented, illuminated NT controls at 2 h - compare Figs. 6-6 and 6-5). Thus, since under our experimental conditions gravitropism in the HT is limited by carbohydrate metabolism or some consequence of metabolism, the altered gravitropic response of the mutant may likewise be a consequence of its altered carbohydrate status. Limitatjgn 9f grayjtzgpjg expression by growth, Because tropic expression depends on differential growth which depends on net elongation, the rate of root elongation was measured under the experimental conditions of Fig. 6-5. Although the long-term overall gain of weight by mutant rosette-stage plants in continuous light was indistinguishable from that of the NT (Chapter 2), during the first 4 h of the experiment of Fig. 6-5 and three comparable experiments (during which mutant and HT growth rates were constant with time) the seedling roots of the mutant elongated only about 80% as fast as WT roots. (Percentages for the individual experiments were 74, 79, 83, and 85; in all, 170 HT and 124 mutant roots were measured.) The close similarity in extent of the retardation of root elongation and gravitropism caused 134 90- Mg 70 - 1% sucrose 0% sucrose Curvature, degrees (a) (It (:3 (I) my 1* .1 J. 1 1 4L, 1 A_r\q_4_y Time, hours Figure 6-6. Influence of sugar on gravitropism by MT roots. Seedlings were grgwn_pn agar plates either with or without 1% sucrose in 50 umol’m ‘s PAR. Plants were placed horizontal at 0 h. n > 20. (Barbara Pickard, personal communication) 1'35 by the mutation indicates that the difference between gravitropism in the mutant and NT is in large part the result of impaired growth in the mutant. grayitngpig induction in the root. Gravitropic reception was separated from the late phases of response by more direct means: plants were stimulated for short intervals and permitted to develop curvature on a clinostat. Previous studies on Artemisia roots (Larsen 1957) and Aygna coleoptiles (Dolk 1936; Pickard 1973) showed that the clinostating time required to achieve maximum response under a given set of conditions is independent of the duration of the stimulus. Thus, for a population with a given growth rate, induction (which is a measure of reception) can be measured as a function of stimulus time. In order to determine when to measure response on the clinostat, an experiment was performed to check the development of curvature as a function of time on the clinostat for both mutant and HT. Contrary to previous observations with the Landsberg ecotype of Arabidopsis (Mirza et al. 1984), curvature increased rapidly during the first 2 h on the clinostat, slowly reached a peak during the next 2 h, and then declined gradually (data not shown). Considerable nutation was superimposed on these curvatures; also, the mutant appeared to reach its peak somewhat more slowly than the HT, and to decline a little later. In order to minimize the effects of nutation and of subtle differences in curvature development, curvatures for the induction experiment were assessed at both 2.25 and 3.5 h, and averaged. Induction plots for TC7 and WT roots are presented in Fig. 6-7. For each pair of points plotted, the performance of the mutant was 136 30' wild type 9‘ o o 6 .3 20 ‘ mutant TC7 ° 9 . g. 3 o 3 25 a 10 6 . ‘ 15 5 0.5 is 3 bis 50 l l I I 5 10 15 20 25 30 Induction time, minutes Figure 6-7. Gravitropic curvature developed by Arabidopsjs roots on a clinostat following stimulations of 2.5 - 30 min in the horizontal position. 47 < n < 121. Zero stimulus values were not assessed in the experiments of the graph; however, supplementary experiments with the WT indicated that comparably large sets of unstimulated plant could show both positive and negative mean curvatures as large as 2 during a 3-h period on the clinostat. Inset: A replot of the data with semilogarithmic coordinates and with interpretive curves generated by linear regression of semilogarithmically-transformed data. (Barbara Pickard, personal communication) 137 about 70% that of the HT (73, 68, 73, 73, and 71% for successive values of induction time). This emphasizes that the gravitropic impairment of the mutant is slight, and may result at least as much from expression as from reception of stimulus. Besnenses nf roets tn brjef and week stimnlj, In Fig. 6-7, it is noteworthy that the mutant and HT responded 8.70 and 11.30, respectively, following a 2.5-min stimulation at 1 x 9. Moreover, induction thresholds determined by extrapolation are brief. The inset shows that regression plots for semilogarithmically-expressed induction data for the HT and mutant extrapolate to thresholds of 0.5 min. Because of the possibility that brief inductions might reflect reception more directly than extended inductions, the approximately 70% ratio of mutant to NT performance following the 2.5-min stimulus and the brief, closely similar threshold estimates are of particular importance in establishing the mutant’s relative gravitropic effectiveness. The mutant also responded slightly more than 70% as well as the NT after weak rather than brief stimuli: seedlings stimulated with a perpendicular vector of 0.5’g by displacing them 30° from the vertical (compare Pickard 1971, 1973) curved 15.9 i 1.60 versus 21.4 i 1.00 within 3 h (n - 100 and 172, respectively; curvatures should not be compared with those of other experiments because these seedlings were shorter than normal). As 1 x g is the maximum gravitrOpic stimulus plants receive in nature, it is reassuring that stimulation with more moderate effective force yielded comparable results. Gravitropism and nhetetronism by hxneeotyls. Figure 6-8A shows the gravitropic response in the dark by hypocotyls of light-grown 1'38 A. gravitropism 20 . ‘5 b wild type 10- / mutant TC7 5 - //// ' B. phototropism Curvature, degrees 3 U! C I 30 r _ /’ O wiid type 0 mutant TC7 10 r // /. l 1 l n l 4 J A 1 l 2 3 4 5 6 Time, hours Figure 6-8. Gravitropism (A) and phototropism (B) byzhypqcotyls of NT and TC7 seedlings. Seedlings were grown in 10 umol’m ’s PAR and pre-equilibrated in the dark for 1.5 h beforezbejgg placed horizontal (A) or illuminated with blue light (1 umol'm ‘s ) (B) at time zero. For phototropism, the plates bearing the upright seedlings were oriented with the agar slab at an angle of 24 from the light beam, but photographs were taken perpendicular to the slabs; thus the component of curvature measured is only 90% the value in the axis of the beam. Photographs were taken with a red worklight. n > 50. (Barbara Pickard, personal communication) 139 mutant and WT seedlings. Mutant curvature at 1.5 and 3 h was 70-80% that of the NT. By 6 h the response by both had slowed and the mutant’s curvature exceeded that of the WT slightly, though not significantly. In one large replicate experiment, performance by mutant and HT hypocotyls was essentially identical although absolute curvatures were lower than those in Fig. 6-8A, while in another large replicate absolute curvatures were considerably higher but initial curvature by the mutant averaged only 70% of the "T. Figure 6-BB shows that mutant and HT hypocotyls had vigorous, indistinguishable phototropic responses to strong, continuous blue light. This is pertinent because in the absence of data on hypocotyl growth (which are difficult to obtain because of the smooth transition between hypocotyl and cotyledon in silhouette) it indicates that the potential for tropic response of the mutant hypocotyls is not greater than that of the NT. finey1tnenism_by_flenen_stelks. The flower stalks that are formed by the plants after a period of rosette growth showed a gravitrOpic response in both mutant and HT plants. In darkness, mutant stalks responded more slowly than those of the HT. For example, in one experiment with more than 75 plants in each set, HT stalks achieved 60° curvature in 80 min whereas the mutant required 240 min to reach the same curvature. Variability was so great, however, that quantitative comparisons were of dubious value. vi ' i dli 5. Both roots and hypocotyls of mutant and HT seedlings grown in total darkness were oriented with respect to gravity (Fig. 6-9), although alignment by the dark-grown mutant was less accurate than by either the dark-grown NT or the light-grown mutant. Growth in white light (50 umol'm'z‘s'1 PAR) in an 140 Figure 6-9. Orientation of dark and light-grown WT and TC7 seedlings of identical ages. (Normally, the mutants were grown longer than the WT in order to permit comparison of seedlings of identical sizes.) Seeds were 2sterilized and sown in Petri plates in room light (less than 10 umol' m PAR). Within 1 h of initiating imbibition, seeds f the WT (A, C) and TC7 (B, D) were placed in complete darkness at 4 C for 60 h. Plates were _§hew placed vertically in darkness (A, B) or in white light (50 umol m PAR) (C, D) for 90 h at 22 C. Arrow marked 9 indicates the direction of the gravity vector during growth. Scale bar = 30 mm, magnification = 0.48x. Measurement of the orientation relative to vertical of a larger sample (n > 87) of roots and hypocotyls of the dark-grown WT and TC7 seedlings produced the following results (values are the mean orientation from vertical t SE): WT T 7 Root 7 : o.5° 21 : 2.3° Hypocotyl 8 i 0.60 37 + 4.20 141 atmosphere containing less than 15 ul‘l'l CO2 improved the gravitropic alignment in the mutant, indicating that the light was not required simply to support photosynthetic C02 fixation. DISCUSSION mummmmmmmmm. Leaves of the Anehideesis mutant TC7 completely lack starch and any detectable activity of the chloroplast isoenzyme of PGM (Table 6-1 and Chapter 2). This enzyme is required for starch biosynthesis, and a cosegregation analysis indicated that a single mutation is responsible for the lack of both starch and chloroplast PGM activity (Chapter 2). Seedlings of the mutant also contain no starch in the root cap, hypocotyl starch sheath or elsewhere as judged by both light and electron microscopy (Figs. 6-1, 6-2, 6-3), indicating that both amyloplasts and chloroplasts must utilize the ngnfl gene to produce PGM activity in the plastid. Despite the complete absence of starch in hypocotyls, flower stalks and seedling roots of the mutant, these organs are all gravitropic. Although flower stalks of the mutant were much slower to respond than those of the WT, hypocotyls and roots of the mutant were only slightly slower. Thus, starch is not required for gravitrOpism in these organs in the mutant or, by extension, in the WT. It might be suggested that a second mutation is present in the background of TC7 which in some way permits gravitropism by compensating for the lack of starch. This possibility is effectively ruled out by the independent isolation of the mutant lines TC9 and TC21 which are also starchless because of allelic mutations at the nng 142 locus and also gravitropically competent. A related question is whether mutations in the ngnfi locus are unusual in that they themselves promote a compensatory gravitropic capacity. This possibility is excluded by the observation that the mutant line T125, which is starchless because of a mutation at another locus resulting in the absence of ADPglucose pyrophosphorylase, is also gravitropically responsive. WWW A:eh1fiens1s_neets_ene_nyneeetyls. In principle, it is possible that Anenigensis has two mechanisms of gravitropic reception, one requiring and one not requiring statolith starch. It is also possible that statolith starch participates in a nonessential but contributory manner. These possibilities have been evaluated for roots of light-grown seedlings. Curvature on a clinostat following brief horizontal treatments was 72% as great in TC7 as in the WT (Fig. 6-7). But expression of gravitropic induction depends roughly on elongation (for example Pickard and Thimann 1966), which in TC7 amounted to 80% of that in the WT. Thus, normalizing to the rate of elongation, the gravitropic curvature of TC7 was 72%/80% or 90% as large as would be expected if differential growth is assumed to be linearly related to straight growth. Under this assumption there is no evidence that statolith starch contributes to any major extent (i.e. more than 10%) to gravity reception in the WT. Evolution of special, inessential statolith mechanisms of so small a consequence to the plant seems unlikely. More likely, if the 10% discrepancy in graviresponsiveness is truly the result of processes preceding curvature, it might reflect a dependence of gravitropic 143 reception or mediation on carbohydrate metabolism or related processes. This idea is supported by the decreased gravitropism of WT roots when grown without exogenous sucrose (Fig. 6-6), an effect which was similar in magnitude to the effect of the mutation on gravitropism. Alternatively, the discrepancy might merely reflect the fact that differential growth is only approximately proportional to corresponding straight growth (see Pickard 1985a,b). For hypocotyls responding in the dark, there was not a large or consistent difference between the mutant and WT which could be invoked to support an inessential, but supplementary, participation of starch statoliths in gravitropic sensing. Because the net gravitropic response was small and variable, it might be suggested that starch participates if present but does not conspicuously limit the ultimate curvature. However, gravitropic curvature was not initially unusually slow; rather, it stopped before the horizontally displaced organs attained an upright position. Thus, statolith starch appears to be irrelevant for gravitropic sensing by the hypocotyl of the Columbia race of Aneniennsis. We are currently introgressing the ngnfl mutation into the Estland race of Anenieensis, which exhibits a more vigorous hypocotyl gravitropic response than the Columbia race (Ken Poff, Michigan State University, personal communication) in order to evaluate this argument more thoroughly. n i t o - e id a ith Although it is the presence of starch that gives the plastid its unusually high relative density (e.g. Audus 1962) and hence promotes sedimentation, it must be evaluated whether plastids might serve as statoliths even in the absence of starch. The density of starch—free root-cap plastids is 1441 probably, like that of proplastids (Quail 1979) which they resemble ultrastructurally, about 1.23 mg'mm’3. In comparison, the densities of cytoplasm and of starch-laden plastids are perhaps 1.0 and 1.5 mg'mm'3 (Audus 1962). Given that the total plastid volume per cell, averaged for the entire columella region, is about 14% for the WT and 2% for the mutant, the total force exerted by the plastids in a mutant columella cell is only about 6% that for the WT. If the difference between performance by the mutant and WT were to result solely from this substantial difference in buoyant mass of the plastids, how would the time-course of induction for the mutant be shifted with respect to that for the WT? Assuming, as is customary, a “gravitropic reciprocity relation” - that is, within a reasonable range the product of force and time necessary to produce a selected curvature is constant (see Pickard 1973), and given a roughly logarithmic dependence of induction on stimulus time (Fig. 6-7), induction must also be roughly dependent on the logarithm of force. As shown in Fig. 6-10, this implies that the effect of lowering the plastid mass would be a large increase in the threshold induction time. Induction would be shifted by a constant amount, so that for large stimulus times it would approach that for standard plastid mass. The data of Fig. 6-7 are inconsistent with such a mechanism: the thresholds of the mutant and WT are closely similar, whereas induction diverges with time. In contrast, if the difference between the mutant and WT is due to effects on mediation and expression of curvature (carbohydrate status model), the induction curves of the mutant and WT will be similar at the threshold and diverge with time (Fig. 6-10). This situation, 145 A: wild type model with 3:100, BID 3 i carbohydrate-status model with E=O.8 25 Cistatolith model with B=0.06 Q) B h 2 g 15 - S- :3 U 5 C l l l l 0.5 2 5 8.3 15 30 Time Figure 6-10. Illustration of the predicted effects of plastid mass and of mediational capability on gravitropic curvatures. Lines A versus C: Separation of threshold times and down-shift of curvature C predicted by the starch-statolith theory for plastid buoyant masses B found in the WT (B - 1 unit) versus TC7 (B - 0.06 units) as modeled by C - K log1 (Bat/Bogfug, for Bat/Bogzs> 1; here 5 ( - 1 unit) is a reference 1Buoyant statolith mass,g . 9.8 m/s 9 is standard gravitational acceleration, a ( - g)( is the relevant acceleration during the experiment, ‘270 ( - 30 s) is the threshold induction time under standard experimental conditions where B - B and a -g, t is the exposure time, and K is a constant of numerical vaTue 17. 4. LinesA versus B: Divergence of curvatures because of growth (an activity which can control expression of induced curvature) as modeled by the modified equation C - KElog 0?(Bat/B gfzg, where E (dimensionless) is the relative elongatioho rate; in the plots, K - 17.4, W30 . 2;- 30 s, and E - 1 for the WT and E - 0.8 for TC. A reasonably general demonstration of the behaviors (rather than just an Illustration) can be based on the Pi Theorem of Buckingham (Langhaar, 951 . 146 manifested in Fig. 6-7, provides the basis for normalizing the response of the mutant with respect to its elongation rate. Ceyeet. As pointed out by Rainer Hertel of the University of Freiburg and John Z. Kiss and Fred D. Sack of Ohio State University (personal communication), it is possible that the WT, TC7, or both induction curves of Fig. 6-7 change form for stimulus values below 2.5 min. In such a case, it is possible that within 2.5 min the WT has adapted to stimulation far more than has the mutant, thus masking differences in sensitivity which might have been detected by using extremely brief stimulus times. Continuity of the induction functions for low stimuli is a critical assumption underlying the evaluations of the preceding two sections, and discovery of a serious discontinuity would necessitate fresh examination of the participation of both starch-filled and starch-free plastids in gravitropism. fineyjtgenisn by etieleeee seeelings. It is unclear why gravitropism of seedlings is disproportionately weaker for the mutant than the WT in the absence of light (Fig. 6-9). Perhaps in gravity reception by dark-grown seedlings there might be a reliance on plastids which is suppressed in light-grown seedlings. Alternatively, a potential reduction in the intensity of the gravitropic response of the mutant based on its altered carbohydrate reserves might be most strongly realized under the suboptimal conditions of growth in darkness. At present it is not possible to distinguish between these and other possibilities. Bessiele extnenelegien nf neselts. Gravity detection is essential for the survival of both subterranean and aerial portions of the land plant. Probably the evolution of gravity detection was early enough 1417 and elaborate enough that a mechanism found in any given species will prove to be widely distributed. Consideration of the literature reinforces the idea that plants of many families may, like Anenieensis, be able to detect gravity without starch statoliths. For example, there is evidence against an essential role for starch in gravitropism by either coleoptiles or roots of grasses. When experimentally depleted of starch, wheat coleoptiles were shown to have nonsedimenting plastids and to curve gravitropically (Pickard and Thimann 1966). Curvature was slower than controls when the rate was time-based, but when the rate was expressed as curvature per unit elongation, controls and experimental plants were indistinguishable. Moore (1987), working with a viviparous cultivar of maize, observed that amyloplasts in the columella cells contained only about 8% as much starch as in a non-viviparous cultivar. The plastids sedimented much more slowly, but gravitropic curvature was about 80% as rapid as in the non-viviparous variety. (On the other hand, mediation of gravitropism in flower stalks of grasses has features not seen in the seedlings - e.g. Kaufman et al. 1987, Wright 1986 - and thus the reception of gravity by stalks may differ as well.) Other groups of organisms provide precedent for gravitropism in the unequivocal absence of starch statoliths. Gravitropically active organs naturally lacking amyloplast statoliths are found in many fungi (e.g. finyeenyees sporangiophores and Conrings stipes; Dennison and Shropshire 1984, Banbury 1962) and lower vascular plants (e.g. the caps of §e|aginelle rhizophores [aerial roots]; Grenville and Peterson 1981). No bodies have been unequivocally identified as statoliths in 148 these species. Furthermore, §nnegnnn shoots were still gravitropic after a cold treatment which lead to the complete loss of starch (von Bismarck 1959). Lastly, the gravitropic rhizoids of gnene are believed to utilize crystaloid bodies containing barium sulfate as statoliths (Sievers and Volkmann 1979) even though they also contain starch-laden plastids. Support for the starch-statolith theory has been inferred from studies by several workers who noted correlations between rates of gravitropism and levels of starch (e.g. Filner et al. 1970, Iversen 1969). However, it is plausible that many situations in which starch is deficient arise because energy is limiting or because of some other alteration of the metabolic or regulatory systems of the cells - such as a shift in the level of second messenger - which might also retard the gravitropic response or shift the position of gravitropic equilibrium. There is no 3 enieni reason why a stressed plant need reduce the rates of gravitropism and other processes in parallel: it might sacrifice gravitropism first. Studies on etiolated coleoptiles of maize mutants affected in endosperm starch synthesis (Hertel et al. 1969; Filner et al. 1970) have been considered particularly strong support for the starch-statolith theory. In some of these mutants (amylose extender, sugary-l, brittle and dull-1 "old") altered sedimentation of amyloplasts clearly correlated with gravitropic auxin asymmetries. Phototropic controls for amylose extender indicated it had ' light-induced auxin asymmetries comparable to the WT, suggesting that the starch defect affected gravitropism specifically. However, the complexity of dose-response relations and their sensitivity to subtle 149 shifts in experimental parameters (Pickard 1985c) suggest the desirability of more exhaustive evaluation. Importantly, however, in all of the mutants studied by Hertel and co-workers in which sedimentation was strongly affected, the enzymatic deficiencies are expressed only in the endosperm (and, in at least one case, the pollen) but not so far as is known in the embryo or mature plant. Therefore, any differences in starch content in the coleoptiles are likely attributable to secondary effects of the alteration of endosperm reserves on the nutrition of the developing seedling. Thus the observed differences in gravitropically induced lateral auxin transport might have resulted directly from the altered amyloplast sedimentation rates, as these workers concluded, but on the other hand both effects might have resulted independently from the generally lowered nutritional status. The latter interpretation seems necessary for the four instances (etched, opaque-2, shrunken-2, and dull-1 ”young”) in which marked decreases in auxin asymmetry were accompanied by only very slight decreases in amyloplast sedimentation. Thus, the four instances for which altered rates of lateral auxin transport and amyloplast sedimentation are correlated can be similarly explained. In sum, there is no solid evidence that the alteration of amyloplast sedimentation was the only changed parameter in the mutant coleoptiles which could influence gravitropism. Further support for the starch-statolith theory has derived from studies of the Lazy-1 mutant of tomato (Roberts 1984). The hypocotyls of this mutant are agravitropic and the amyloplasts in their starch sheaths do not sediment. However, lacking biochemical information on this mutation, a reasonable alternate interpretation of these results 150 is that the agravitropic behavior and the absence of amyloplast sedimentation may both be caused by an unidentified alteration in the cytosolic milieu within the starch sheath. In each of these cases favoring the starch-statolith theory, the evidence is correlational and hence less compelling than the evidence that the completely starchless Aneniennsis mutant TC7 is competent in gravity sensing. In each of the cases contradicting the starch-statolith theory, it has long been argued that the evidence is insufficient; but they must surely become more persuasive in light of the present findings with Anenieensis. memmmmw greens? The nearly ubiquitous presence of large, displaceable, starch-filled plastids in gravitropically sensitive organs has both been explained by and provided the basis for the starch-statolith theory. If this theory is to be rejected, an alternative explanation for their presence in these organs is required. Growing organs have energetic needs for starch reserves. For example, as cells mature in the columellar layers in a root, they may well accumulate reserves to support mucilage production at the periphery of the cap as well as for functions during their long lifetime after being shed from the cap (e.g. Guinel and McCully 1987). The reduction in the amount of starch in the detached cells relative to the columella cells - e.g. Fig. 6-1A and Barlow (1975) - is consistent with a role as a metabolic precursor. Similarly, cells in a seedling stem or coleoptile, by sequestering starch from the storage tissues of the seed, are assured a supply of energy for later expansion. In general, the starch sheath in shoots might be the locus for starch deposition simply because it lies next to 151 the phloem. The displaceability of amyloplasts in root-cap and starch-sheath tissues (in contrast to the immobility in nearby cells) might be the incidental result of a reduction of cytoskeletal anchoring and supporting network correlated with other cellular activities. WWW; Although the results provide no evidence to support a statolith role for amyloplasts in Anebjdensis, at least in roots, other versions of the statolith theory remain possible: small bodies of very low or very high density or very large organelles of slightly low or high density (specifically, the nucleus or vacuole) could have a statolith function. Alternatively, the roots and stems might sense their own weight. Multicellular organs of heterogeneous structure which exist in a lighter (or heavier) medium are subject to stresses and strains which depend on their orientation in the gravitational field, and which might if suitably focused serve as signals. In particular, the turgid cells and restraining interconnected walls of the plant must focus stress in an elaborate manner. Such focused stress could be transmitted from the walls to the plasma membranes, which may well be provided with their own mechanism of focusing the stress onto transductive stretch-activated ion channels (Edwards and Pickard 1987). In any case, fresh ideas may be required to explain the mechanism of gravity reception in Arabidopsis seedlings. REFERENCES Audus, L.J. (1962) The mechanism of the perception of gravity by plants. Symp. Soc. Exp. Biol. 16, 197-226 Audus, L.J. (1975) Geotropism in roots. In: Development and function of roots, pp. 327-363, Torrey, J.G., Clarkson, D.T., eds., Academic 152 Press, London New York San Francisco Banbury, G.H. (1962) Geotropism of lower plants. In: Encyclopedia of Plant Physiology, vol. 17(2): Physiology of movements, pp. 344-377, Ruhland, W.,ed., Springer-Verlag, Berlin G6ttingen Heidelberg Barlow, P.W. 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Plant Growth Regul. 5, 37-47 Chapter 7 Concluding Remarks SUMMARY AND CONCLUSIONS 1) 2) 3) 4) 5) 6) 7) Mutations in two loci which produce starchless plants and a third which produces a reduced starch phenotype were readily isolated. The starchless plants were viable indicating that starch is not absolutely required for plant growth and development. However, their vigor was conditional on the photoperiod. When grown in an alternating light/dark photoperiod, the inability to synthesize starch led to decreased growth, increased sugar accumulation, decreased photosynthesis and altered respiratory rates. A gene with the characteristics of a regulatory gene was identified. It controls the accumulation of both subunits of ADPglucose pyrophosphorylase. A mutation which did not accumulate the 54 kD subunit of ADPglucose pyrophosphorylase confirmed that this polypeptide is required for full enzymatic activity. Cytosolic B-amylase was stably activated up to 40-fold in a diverse collection of starch mutants. Starch is not required for gravitropic gravity sensing by roots or hypocotyls of Arabidensis. 157 158 Future work using the starch mutants. I hope that the mutants described in this thesis will be useful for further work; several of these potential uses are described below. The preparation of intact organelles from plant tissues is frequently hindered by the presence of starch grains, which are sufficently dense that they may disrupt membranes during centrifugation at relatively low centrifugation forces. Thus, many protocols suggest "destarching" plants by placing them in the dark for at least 8 h before use. While this normally presents no problem, in certain situations (e.g. where the effects of light are being studied) it may not be appropriate. The use of a starchless mutant as the starting material would alleviate these types of problems. More generally, in any other isolation protocol in which starch interferes, the starchless mutants should prove useful. ADPglucose pyrophosphorylase is believed to be the limiting and regulated enzyme in the synthesis of leaf starch (5). However, it has been suggested (1) that PGI and PGM which precede ADPglucose pyrophosphorylase in the pathway also limit the rate of starch synthesis. The support for this has come from the observation that in spinach and beet the concentrations of substrates and products for these two enzymes (fructose-6-phosphate and glucose-6-phosphate for PGI and glucose-6-phosphate and glucose-l-phosphate for PGM) are not present in their equilibrium (as predicted from in MiLLQ measurements) concentrations. If PGI and PGM are limiting in WT Arabidensis as they may be in spinach and beet, then the fructose-6-phosphatezglucose- 6-phosphate and glucose-6-phosphate:glucose-l-phosphate ratios should 159 be displaced from equilibrium in the WT. That this disequilibrium is due to limitation by PGI and PGM and not to some other cause could be verified by using the three mutants described in this thesis. In TC7 the reaction catalyzed by PGI should be at true (in yiye) equilibrium since flux through the pathway is blocked by the lack of PGM activity. Similarly, in TL25 both the PGI and PGM steps should be at true equilibrium due to the lack of ADPglucose pyrophosphorylase activity. In TL46 which has 5% of the WT activity for ADPglucose pyrophosphorylase, the PGI and PGM reactions might or might not be at equilibrium, depending on the relative limitations imposed by the three enzymes. For this reason, TL46 might be the most informative of the mutants. Starch has been considered important in the functioning of stomata as a source of malate which is used by guard cells in osmotic regulation (4). This should be verifiable using the starchless mutants since they lack insoluble starch and accumulate soluble sugars instead. Since the conversion of soluble sugars to malate produces relatively little change in the osmotic potential, no large fluctuations in osmotic potential should be possible in guard cells of the starchless mutants without transport of solutes across the plasmalemma. Although the starchless mutants showed no obvious differences in stomatal behavior, it is possible that any effects would be more subtle and require more sensitive measurements to be observed. The eegl gene has two properties which suggest it might be a regulatory gene. First, mutations in this gene caused the absence of both subunits of ADPglucose pyrophosphorylase. Second, the WT gene shows unusual dosage effects (i.e. a line with 2 copies of the WT gene 160 had the same ADPglucose pyrophosphorylase activity as a line with 1 WT and 1 mutant copy). Further analysis of this mutant line should allow a determination of whether the eegl is, indeed, a regulatory gene. This is worthwhile since there are relatively few well-characterized regulatory genes in plants. In order to determine whether the ngl gene represents a regulatory or a structural gene, three approaches might be considered. First, the basis for the lack of both subunits could be studied by analyzing whether messages for the two subunits are produced. Second, mapping of the eegl (by classical genetic approaches) and the two structural genes (using restriction fragment length polymorphism maps) should indicate whether eegl is linked to either structural gene. If it is not, this would support the possibility that it is a regulatory gene. Third, structural genes coding for the two subunits could be used to transform a mutant line. If the enzymatic lesion is not corrected, then this would also suggest that ng1 serves a regulatory role. At present, however, the gene encoding only one of the subunits has been cloned (3). Thus, at present, these types of studies can only be partially completed. An alternate approach to the study of the eegl gene would be to clone it by complementation. Klee et al. (2) have recently demonstrated the feasibilty of this approach using petunia, in which the efficiency of transformation is presently much greater than for Arenidonsjs. Assuming that more facile transformation protocols are developed for Anenieensis, a line homozygous for the §Q91;1 gene could be transformed with a library made from WT DNA. The transformants which accumulate starch should contain a WT egg; gene which could then be easily cloned. The screening of the large numbers of transformants 161 required for such an approach should be relatively easy since root tissue, which is the primary transformed tissue produced using the current Aneniennsis transformation protocol (6), normally accumulates large quantities of starch in the root cap (see Chapter 6). Furthermore, the presence of starch can be non-destructively assayed in root caps by staining seedlings with iodine (see Appendix 3). Finally, as discussed throughout this thesis, the starch mutants have greatly altered carbohydrate metabolism because of the storage of photosynthate as soluble sugars outside the chloroplast rather than starch inside the chloroplast. We have only begun the use of these mutants in analyzing the regulation of leaf metabolism in such processes as the partioning of photosynthate between starch and sucrose and the partioning of sugars between respiration, storage, and translocation. I feel that further work is warranted in these and other related areas. Approaches to isolate other starch mutants. The conversion of carbon from glucose-6-phosphate to starch (amylose) requires three enzymes: PGM, ADPglucose pyrophosphorylase, and starch synthase, A fourth enzyme, branching enzyme converts the linear amylose form of starch to the branched amylopectin form. About 8000 M2 plants were screened for starch-deficient mutants, resulting in the three classes described in this thesis (with lesions in the ngnfl, eegl and eegz genes) plus several classes which have resisted characterization. No mutants which affected starch'synthase were isolated. Furthermore, the simplest interpretations of the two mutations which affect ADPglucose pyrophosphorylase suggest that one 162 affects a regulatory gene and the other affects the structural gene for the larger (54 kD) of the two subunits of the enzyme. Thus, at least two types of mutations were expected but were not isolated: those affecting starch synthase and those affecting the smaller subunit of ADPglucose pyrophosphorylase. It is possible that these mutations are rare and further screening will be necessary to isolate them. In this work, the simpler seedling or dry seed screens (see Appendix 3) will be useful. Alternatively, these mutations may not have been found because they are either lethal of because they have no or only a slight effect on starch accumulation. The first possibility is unlikely because the only known function of these enzymes is in starch biosynthesis and the results with TC7 and TL25 show that starch metabolism is not required for growth, development, or reproduction of Anenieensis. The second possibility seems more likely. Multiple isozymes of starch synthase exist in many species (reviewed in 5) so the elimination of one form might produce only a moderate decrease in starch content which would be easily overlooked in the screening for mutants. In the case of the smaller subunit of ADPglucose pyrophosphorylase, the results with TL46 indicate that in the absence of the larger subunit, the activity associated with the smaller subunit is capable of producing almost half as much starch as the WT. (Thus, the isolation of TL46 by Tsan-Piao Lin was somewhat fortuitous.) A mutant which is missing the smaller subunit and contains the larger might likewise contain substantial amounts of starch and be difficult to isolate. However, an improved strategy to isolate mutations in the smaller subunit exists. If the 163 TL46 line is mutagenized, new mutations which affect the smaller subunit should be completely starchless and thus easily isolated. Another useful approach for isolating starch mutants relies on the finding that mature seeds containing the sen1;1 mutation (briefly described in Chapter 5) contain large amounts of starch in the seed coat whereas the WT does not (unpublished results). Dry seeds may be stained for starch, scored, and then germinated. Thus, if a line containing the senl;1 is mutagenized, starchless mutations could be easily identified by scoring dry seeds for starch. In a similar manner, additional sen1;1 alleles, and perhaps other classes of starch overproducers as well, could be isolated by screening mutagenized WT seeds. In both cases, however, screening would have to be done using M3 seeds since the seed coat is a maternal tissue. (M2 seeds have seed coats derived from the M1 generation which is heterozygous for induced mutations and thus would not show phenotypic differences for recessive mutations. M3 seeds, on the other hand, have seed coats derived from the M2 generation.) Finally, the increased activity of the B-amylase in the starch mutants described in Chapter 5 suggests that mutants which lack this enzyme activity could be isolated in a starch mutant background. Such a screen might simply employ a crude colorimetric amylase assay using a leaf extract incubated with starch and then stained with iodine. These mutants might be useful in understanding the regulation of this enzyme and since this cytosolic B-amylase has no known function (see Chapter 5), mutants which lack it might help to identify its function. 164 References. 1. Dietz K-J 1987 Control function of hexosemonophosphate isomerase and phosphoglucomutase in starch synthesis of leaves. In J Biggens, ed, Progress in Photosynthesis Research, Vol III. Martinus Nijhoff, Dordrecht, pp 329-332 Klee HJ, MB Hayford, SG Rogers (1987) Gene rescue in plants: A model for "shotgun” cloning by retransformation. Mol Gen Genet 210:282-287 Krishnan HB, CD Reeves, TW Okita 1986 ADPglucose pyrophosphorylase is encoded by different mRNA transcripts in leaf and endosperm of cereals. Plant Physiol 81:642-645 Outlaw WH, J Manchester 1979 Guard cell starch concentration quantitatively related to stomatal aperture. Plant Physiol 64:79-82 Preiss J 1988 Biosynthesis of starch and its regulation. In J Preiss, ed, Biochemistry of Plants, Vol 14. Academic Press, New York, pp 182-254 Van Sluys MA, J Tempe, N Federoff (1987) Studies on the introduction and mobility of the maize Aeliyeeen element in museum sneliene and Qenens geneee. EMBO J 6:3881-3889 APPENDICES Appendix A Search for Revertants of TC7 The TC7 mutant line has a reduced growth rate in short photoperiods which is believed to be caused by its a reduced rate of photosynthesis and increased rate of respiration (Chapter 2). In an attempt to determine the basis of these altered growth and metabolic rates, and to identify potentially interesting genes which regulate photosynthesis and dark respiration, I sought to isolate phenotypic revertants of TC7. The rationale was that mutations which reversed the effects of the ngnfl;1 mutation on photosynthesis or dark respiration would be expected to grow more quickly in a short photoperiod than the mutant itself and thus be easily recognizable. Once isolated, the new mutations could be separated from the ngn£;1 mutation by crossing with the WT, allowing the new mutation to be studied in the absence of MILL Assuming that the increased levels of sugars which accumulate in the leaves of TC7 are directly or indirectly responsible for the altered rates of photosynthesis and respiration, there are, in principle, three classes of revertants which might be isolated in this screen. First, true revertants which regain activity of the chloroplast isozyme of PGM might be isolated. These should likely be quite rare since there are presumably relatively few ways to restore the activity of a protein relative to the number of ways to eliminate activity. Second, mutations which reduce the accumulation of sugars in the leaves might be isolated. Third, mutations which reduce the 165 166 sensitivity of photosynthesis or respiration to elevated sugars levels might be isolated. The pgnfl;1 allele derived from TC7 was combined with two morphological mutations, gl;1 and 11:5, by crossing TC7 successively with C52 and CS287. This was done to allow revertants induced by mutagenesis to be distinguished from potential contaminating WT plants. 75,000 seeds of the resulting line TC19 were mutagenized as described in Chapter 2 and grown to maturity in continuous light. M2 seed were harvested in bulk. These seed were grown in a 7 h light/17 h dark photoperiod. In this photoperiod plants containing the ngn£;1 gene normally grow much more slowly than the WT (see Chapter 2) and are easily distinguished from the WT. Approximately 50,000 M2 seed were screened in this way. No plants were observed which grew more quickly than unmutagenized TC19. The lack of mutants was not attributable to a lack of mutagenesis since other mutations have been isolated from this seed lot at about the expected frequencies (personal communications, Barbara Moffatt and Nancy Artus). The conclusion from this work is that there is no single gene, the activity of which can be eliminated to reverse the deleterious effects on growth produced by the ngn£;1 gene. Appendix 8 Genetic Mapping of Starch Mutations Two of the genes described in this dissertation, ngnfi and senl, were genetically mapped using previously mapped morphological markers (Koorneef 1987). Mapping was accomplished by scoring markers in segregating F2 populations produced by crossing the starch mutant with lines containing multiple markers provided by M. Koorneef. Data was analyzed using the Linkage 1 program (Suiter et al. 1983) which calculates contingency X2 values and recombination percentages using maximum likelihood techniques. Recombination percentages were corrected for multiple crossovers using the Kosambi mapping function to produce map distances. Mapping results for pgnfi (Table B-1) and see; (Table B-2) are presented for linked markers only. Unweighted averaging of the mapping results for the pgnfl gene results in an estimated location on chromosome 5 at about position 75.5. The mapping of senl indicates it is located on chromosome 1, 12 map units from en. Since only one marker was used, it is not possible to determine whether seel is distal or proximal to en. Mapping was attempted for the egg; gene, but no linkage was detected between it and a set of markers on each chromosome present in the primary mapping strain (en, en, ll, m2. and 1151). REFERENCES Koorneef, M. (1987) Linkage map of Anebigopsis thaliane (2n = 10). In: 167 168 Table B-1. Mapping of nng. Marker Marker Position1 Joint Segregation Data 2 Distance Chromosome-location +/+ -/+ f/png -/gcmP X from Marker (OF-1) (Map Units) nsl 5-25 7 77 35 36 8 2.7 53.8 11 5-90.4 127 70 65 4 20.9 24.0 ELQ . 5-31.S 132 65 58 11 6.7 47.3 eer3 5-85 6 253 28 113 1 9.8 22.0 91; 5-56 1 213 71 108 7 18.6 31.2 lMap positions of markers are from Koorneef (1987). Table B-2. Mapping of seal. Marker Marker Position1 Joint Segregation Data 2 Distance Chromosome-location +/+ -/+ +/sool -/sool X from Marker (OF-1) (Map Units) en 1-0 171 68 82 1 27.2 12.3 lMap position of marker is from Koorneef (1987). 169 Genetic Maps 1987, vol. 4, pp. 742-745, O’Brien, SJ, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Koorneef, M., van Eden, J., Hanhart, C.J., Stam, P., Braaksma, F.J., Feenstra, W.J. (1983) Linkage map of Anenieensis theljane. J. Hered 74:265-272 Suiter, K.A., Wendel, J.F., Case, J.S. (1983) LINKAGE-1: A PASCAL computer program for the detection and analysis of genetic linkage. J. Hered 74:203-204 Appendix C Procedures for Screening for Starch Mutants The mutants studied in this thesis were isolated as described in Chapter 2 by staining leaves for starch. In the course of this work, I developed simpler procedures for screening for starch mutants. The basis of the first procedure is the observation that the iodine stain described in Chapter 2 but without HCl (i.e. 5.7 mM iodine, 43.4 mM potassium iodide) does not harm the seedlings of the plant. Thus, the seedlings can be immersed in the stain for several minutes, sufficient time for the starch in the root cap to be stained, and then rinsed free of the iodine and grown to maturity. WW. 1. Seeds are surface-sterilized as described in Chapter 6. 2. Sterilized seeds are sown in rows in petri plates containing the salts described in Chapter 6, plus 1% sucrose and 1% agar. 3. The plates are sealed with Parafilm and placed on edge so the surface of the agar is vertical and the rows of seeds are horizontal. The plates are illuminated from above with about 50 umol/mz's PAR from fluorescent tubes. 4. When the roots are about 5-10 mm long (about 3-5 days), the plates are flooded with iodine stain (5.7 mM iodine, 43.4 mM potassium iodide). After several minutes the starch in the root caps is stained dark blue or black, 170 171 which is easily observed in a dissecting microscope at a magnification of about 150x. Root caps of seedlings which lack starch stain a light yellow. 5. Seedlings to be saved are rinsed with water for several minutes and then placed onto fresh petri plates (nutrient salts plus 0.7% agar, no sucrose). After several days, the seedlings may be transplanted to pots and grown to maturity. Neee: Seedlings which are contaminated often do not accumulate starch. To avoid ”false mutants", it is important to ensure that plates are kept sterile. The second procedure for isolating starch mutants relies on the observation that dry seeds of TC26 (sen1;1) contain starch in the seed coat whereas the WT lacks starch in the seed coat (unpublished observations). The starch present in dry seeds can be visualized by staining with the iodine stain described above. Since this treatment also does not injure dry seeds, the iodine may be washed off and the seeds grown to maturity. Thus starch over-producer mutants may be screened directly in mutagenized seed lots. Alternatively, starchless mutants may be isolated with this screen by mutagenizing a starch-overproducer line and screening for seeds which lack starch. However, since the seed coat is a maternal tissue, both of these approaches must use M3 rather than M2 seed lots.