i.. s. . . Mwfiuw mm“? . . i . . image... .umifl 1. » w. Pawn)? ..._.r_.nun. Win... I; :I-‘J‘l. 4}."'. V. :1. . .33... +2. .5. n innunn t... . t at 1.7!, It. than.“ 11. x u. wilhhunuu.“ $.12 .x. {2!} s. . rcviv-g-g'" I31. I! ’30.). 7 6| :1 A}!!! [1‘13145 1‘36. 1.1 Ara-rut}; :. V! I.) .. . $. ‘ ’ mtvn-MIN‘R ‘f? ”you" o-uw “qu‘Cl‘CC. W ‘7 a A, an no" on V S r nlflfltnfitda ‘ a. 7‘ . . «1 .. OI. . .» . ‘ .flflfixdwcamfjnf .021 L. a. . . .. . .. Z ‘ a ‘ . his, 1 ‘ ‘h.,r.".1o.7.. $2.95.? ‘. . FRESH: {220:3 iUlUHlIHHIItHIHHHIHIHWWHilHlMlIWWI 31293 01834 LIBRAm Michigan Sta. University This is to certify that the thesis entitled EFFECTS OF FLOODING DURATION ON THE GROWTH, PHYSIOLOGY, AND STARCH RESERVES 0F BLACK ASH (FRAXINUS NIGRA MARSH.), GREEN ASH (FRAXINUS PENNSYLVANICA MARSH.) , AND WHITE ASH (FRAXINUS AMERICANA L.) SEEDLINGS. presented by Renee Rose Cloutier has been accepted towards fulfillment of the requirements for M. S. degree in Horticulture L117 ‘fj (I?) Major pro ssor Date May 12, 1999 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINE return on or before date due. MAY BE RECAU.ED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 1m chiRG/DamepGS—p.“ EFFECTS OF FLOODING DURATION ON THE GROWTH, PHYSIOLOGY, AND STARCH RESERVES 0F BLACK ASH (FRAXINUS NIGRA MARSH), GREEN ASH (FRAXINUS PENNSYLVANICA MARSH), AND WHITE ASH (FRAXINUS AMERICANA L.) SEEDLINGS. By Renee Rose Cloutier A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1999 ABSTRACT EFFECTS OF F LOODING DURATION ON THE GROWTH, PHYSIOLOGY, AND STARCH RESERVES OF BLACK ASH (F RAXINUS NIGRA MARSH), GREEN ASH (FRAXINUS PENNSYLVANICA MARSH), AND WHITE ASH (FRAXINUS AMERICANA L.) SEEDLINGS. By Renee Rose Cloutier The effect of flood duration on growth, gas exchange, and starch content of bare root black ash (F raxinus nigra Marsh), green ash (F. pennsylvanica Marsh), and white ash (F. amerz’cana L.) seedlings was studied to characterize their flood response and compare their flooding tolerance. Seedlings were flooded for O, 4, 8, 16, and 32 days with the recovery interval extended to day 60. Green ash seedlings grew throughout the study however flooding reduced stem height, leaf number, and leaf area of this species. In contrast, black and white ash seedlings did not grow during flood stress. After 32 days of flooding, root starch concentrations were lower in flooded seedlings than in the control seedlings. Flooded green ash seedlings had no reduction in root biomass when compared to control seedlings. Root biomass was reduced in both black and white ash seedlings in response to flood. Flooding also decreased stomatal conductance followed by a reduction in assimilation for black, white, and green ash seedlings, respectively. Green ash seedlings maintained a higher assimilation rate than black and white ash seedlings throughout the 32- day flood. Hence, green ash seedlings recovered earlier from flood stress and is considered more flood tolerant than both black and white ash seedlings. Pour Rose-Helene iii ACKNOWLEDGMENTS I am grateful to my major advisor, Dr. R.E. Schutzki, for his patience and support during the course of my graduate studies. I also want to thank my committee members, Drs. J .A. Flore and F. Ewers for their encouragement and helpfiil criticisms. A special thanks to Drs. A. J anoudi, and F. Dennis for their review of the manuscripts, to Drs. W. Loescher and Z. Gao for their helpful advice on a method to determine starch and to Drs. G. Hosfield, M. Nair, and J. Hancock for the use of their laboratory equipment. Many others have contributed to this work and also deserve mention such as Susan Gruber, Dr. Shirley Owens, Dave F reville, Bill Chase and the staff of the MSU Horticulture Teaching and Research Center, Jerome Kosman, Burke Jenkins, Katie Wilkinson, Mike Meyers, Jamie Mulvaney, Carrie May, Sarah Breitzkreut, and Leo Lombardini for their assistance in the field and laboratory. I appreciate your time, effort, and most of all your friendship. Finally, I want to thank my parents, Yvon and Gaetane Cloutier, and my husband, Clifford Beninger, for their love and support. iv TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................. vi LIST OF FIGURES ............................................................................................ viii LIST OF ABBREVIATIONS ............................................................................. ix INTRODUCTION .............................................................................................. 1 Literature Cited ........................................................................... 5 CHAPTER I EFFECTS OF F LOODING DURATION ON THE GROWTH AND PHYSIOLOGY OF BLACK ASH (FRAXINUS NIGRA MARSH), GREEN ASH (FRAXINUS PENNSYLVANICA MARSH), AND WHITE ASH (FRAXINUS AMERICANA L.) SEEDLINGS. Abstract ....................................................................................... 9 Introduction ................................................................................. 10 Materials and Methods ................................................................ 12 Results ......................................................................................... 18 Discussion ................................................................................... 35 Literature Cited ........................................................................... 41 CHAPTER II EFFECTS OF FLOODING DURATION ON THE BIOMASS AND STARCH CONTENT BLACK ASH (FRAXINUS NIGRA MARSH), GREEN ASH (FRAXINUS PENNSYLVANICA MARSH), AND WHITE ASH (FRAXINUS AMERICANA L.) SEEDLINGS. Abstract ....................................................................................... 47 Introduction ................................................................................. 48 Materials and Methods ................................................................ 51 Results ......................................................................................... 55 Discussion ................................................................................... 61 Literature Cited ........................................................................... 66 SUMMARY AND CONCLUSIONS ................................................................. 70 V Table l . 1 1.2 1.3 1.4 1.5 LIST OF TABLES Mean increase in caliper and leaf area for black, green, and white ash seedlings flooded for 0, 4, 8, 16, and 32 days. The changes in caliper were presented for the entire study (0 to 60 days) whereas leaf area were presented during the flood (0 to 32 days) period. Means, followed by the same letter within a column for each main effect, do not differ significantly (Duncan, P50.05). Mean current stem height growth and leaf number for black, green, and white ash seedlings flooded for 0, 4, 8, l6, and 32 days. Stem height and leaf number were presented for the entire study (0 to 60 days). Means, followed by the same letter within a column for each species x flood duration interaction, do not differ significantly (Duncan, n=8, P50.05). Negative values in the leaf number indicate net loss of leaves. Leaf, current stem, old stem, and root dry weight means for black, green, and white ash seedlings harvested after 8, 16, and 32 days of flood stress. Means, followed by the same letter within a column for each separate harvest, do not differ significantly (Duncan, n=8, P50.05). Current stems harvested on day 16 were not available (N/A). Mean assimilation, stomatal conductance, and transpiration for black, green, and white ash seedlings flooded for 0, 4, 8, l6, and 32 days. Values presented are for the entire study including the both the flood and the recovery intervals. Means, followed by the same letter within a column for each species x flood duration interaction in the experiment, do not differ significantly (Duncan, P50.05). Summary of growth, gas exchange, and plant component dry weights for black, green, and white ash seedlings exposed flood treatment after 2, 4, 8, 16, and 32 days. Differences between control and flooded seedlings at the P50.05 or P50.0l level are indicated as * and ** , respectively (Duncan, n=8). (Blanks were included where no determination was made whereas N/A was noted when the data was not available.) vi Page 19 20 22 24 34 Table 2. 1 2.2 2.3 Leaf, current stem, old stem, and root dry weight means for black, green, and white ash seedlings harvested after 8, 16, and 32 days of flood stress. Means, followed by the same letter within a column for each separate harvest, do not differ significantly (Duncan, n=8, P5005). Current stems harvested on day 16 were not available (N/A). Mean starch concentration of leaf, current and old stem, and coarse roots for control and flooded black, green, and white ash seedlings harvested after 8, 16, and 32 days of flood stress. Current stems harvested on day 16 were not available (N/A). Means, followed by the same letter within a column for each separate harvest, do not differ significantly (Duncan, P5005). Mean starch concentration of fine roots for control and flooded black, green, and white ash seedlings harvested after 8, 16, and 32 days of flood stress. Means, followed by the same letter within a column for each species x treatment interaction, do not differ significantly (Duncan, n=8, P5005). vii Page 56 59 60 Figure 1.1 1.2 1.3 1.4 LIST OF FIGURES Regression of estimated terminal leaflet area over the actual leaflet area measured fiom three ash species. Estimated leaflet area was calculated by length x width x correction coefficient. Correction coefficients for black, green, and white ash terminal leaflets are 0.6129, 0.7154, and 0.6823. Regression line for each species was based on 20-24 terminal leaflets. Mean assimilation (:SE) of black ash, F raxinus nigra L. (a), green ash, Fraxinus pennsylvanica Marsh. (b), and white ash, F raxinus americana L. (c), seedlings flooded for 0, 4, 8, 16, and 32 days. Seedlings flooded for 0 and 32 days were measured throughout the study whereas seedlings flooded for 4, 8, and 16 days were added when released to monitor recovery. (Duncan, n=8, P5005) Mean stomatal conductance (iSE) of black ash, Fraxinus nigra L. (a), green ash, Fraxinus pennsylvanica Marsh. (b), and white ash, F raxinus americana L. (c), seedlings flooded for O, 4, 8, 16, and 32 days. Seedlings flooded for 0 and 32 days were measured throughout the study whereas seedlings flooded for 4, 8, and 16 days were added when released to monitor recovery. (Duncan, n=8, P5005) Mean transpiration (:SE) of black ash, F raxinus nigra L. (a), green ash, Fraxinus pennsylvanica Marsh. (b), and white ash, F raxinus americana L. (c), seedlings flooded for 0, 4, 8, 16, and 32 days. Seedlings flooded for 0 and 32 days were measured throughout the study whereas seedlings flooded for 4, 8, and 16 days were added when released to monitor recovery. (Duncan, n=8, P5005) viii Page 17 27 29 31 LIST OF ABBREVIATIONS General and physiological terms A ...................................................... carbon assimilation ca. ................................................... circa (approximately) E ........................................................... transpiration Eh ..................................................... soil redox potential gs .................................................... stomatal conductance Oz ........................................................... oxygen gas Rubisco ........................................... ribulose-l ,5- biphosphate WUE ................................................. water use efficiency ix INTRODUCTION Woody plants have different strategies to deal with environmental stresses such as flooding, drought, soil compaction, extremes in temperature, and high salinity. These vary according to species and include both morphological and physiological adaptations. Even closely related species will differ in flood tolerance, as seen by Norby and Kozlowski (1983) for Betula papyrifera and B. nigra. In a 60 day study, flooding reduced growth of B. nigra; but, not to the extent seen in B. papyrifera where leaf area was drastically reduced. Inhibition of new leaves and senescence decreased growth and for many seedlings, flooding lead to death. In contrast, B. nigra seedlings produced hypertrophied lenticels and adventitious roots. Both responses are viewed as adaptations to flood stress that enhance gas exchange and help eliminate the buildup of toxic metabolites whereas other metabolic processes help to tolerate anoxia. Consequently, each trait can contribute to a species’ plasticity and ecological success. Unfortunately, criteria used to determine flooding tolerance differ with each investigation. Changes in growth, gas exchange, injury, and survival are common parameters whereas some studies include hormone or enzyme activity. For example in Phung and Knipling (1976), photosynthesis rates for four citrus rootstocks were 80 to 90 % lower within ten days of flooding. Clemens et a1. (1978) determined this flood tolerance order with Eucalyptus grandis > E. robusta > E. saligna was related to the formation of adventitious roots. Anella and Whitlow (1999) ranked seven red maple cultivars by characterizing photosynthesis, lenticel intumescence, and survival in order to make cultivar recommendations for use in urban landscapes. In addition, Tang and Kozlowski (1983) reported that flooding reduced the height growth for Pinus banksiana and P. resinosa. Each study contributes to our knowledge of woody species in response to flooding stress. Although characteristics of the flood water, seasonality, and duration of flooding all affect response, such responses vary with the age of plant, seedlings being more susceptible than mature trees. Initially, flooding reduces the oxygen content of the soil and this causes variable and complex changes in plant-water relations including transient wilting, reduced transpiration, and a decline in root conductivity (Kozlowski et a1. 1991; Reid et al. 1991). Roots are sensitive to oxygen deficiency and no records of sustained growth by anoxic roots has been reported (Armstrong et a1. 1994). Under hypoxic or anoxic conditions, seedling survival by anaerobic metabolism is dependant on the availability of carbohydrate reserves and temperature, which controls respiration rate (Jackson and Drew 1984). In addition, the elimination of toxic metabolites such as ethanol, acetaldehyde, or ethylene may also be critical for the root system (Chirkova and Gutrnan 1972). Not surprisingly, flooding adversely affects stem, leaf, and cambial growth as well as reproductive growth of trees (Kozlowski 1985). Many studies have indicated that above-ground processes such as photosynthesis and stomatal conductance also decrease. For example, Kozlowski and Pallardy (1979) observed that flooding induced stomatal closure in 5 month old green ash seedlings with a gradual reopening. Pezeshki and Chambers (1986) showed that flooding green ash and baldcypress did not cause a significant decrease in photosynthesis despite a 50 % decrease in stomatal conductance. In another study, Pezeshki et al. (1996) reported a reduction in stomatal conductance of Quercus lyrata and Q. falcata but not in T axodium distichum under low soil redox conditions. Reduced net photosynthesis, root and leaf dry 2 weights were also observed for these three species. In North America, 16 native species belong to the genus Fraxinus. The ashes represent a unique group of trees because many species have markedly different native habitats. Two trees commonly planted in urban landscapes, green (Fraxinus pennsylvanica Marsh.) and white ash (Fraxinus americana L.), differ in their ability to tolerate flooding stress. Green ash is one of the most adaptable trees, with a wide geographic distribution. Green ash is a highly flood-tolerant species that can grow on land subjected to periodic flooding and display vigorous growth even when flooded for much of the growing season (Tang and Kozlowski 1984). For example, Broadfoot and Williston (1973) showed that green ash had a 50 to 100 % increase in radial growth during flood years. In contrast, white ash, which usually appears on deep, well-drained upland soils, is a flood-intolerant species (Hall and Smith 1955; Kozlowski et a1. 1991). Very little information is available for white ash and its response to flood stress. Black ash (F raxinus nigra L.) is an extremely hardy species common to low woodlands, swamps, and periodically inundated river bottoms. Often referred to as swamp or hoop ash, black ash can tolerate standing water for many weeks usually when spring or early summer flooding occurs. Black ash is a determinant species meaning that the next season’s grth is determined by climatic conditions during the previous season (Lechowicz 1984; Tardiff and Bergeron 1993). Comparison of the morphological and physiological response of three Fraxinus species will add to our knowledge of flood tolerance mechanisms of woody species. In addition, it can indicate why ashes occupy such broad ecological niches. Practical benefits of this research can also be applied in plant selection process and identifying species able to 3 tolerate flooding and soil compaction common in urban landscapes. For these reasons, observation of growth, gas exchange, and starch reserves will be critical for determining the flood tolerance of each species. Little research has been done on how closely related species in the genus F raxinus cope with environmental stresses such as flooding. On a broader scale, information on how flood duration changes carbon partitioning is needed especially since late-season stresses that affect root starch reserves can severely impact future growth and survival (Loescher et al. 1990). Therefore, our objectives were to characterize and compare the growth, physiology, and starch content of black, green, and white ash seedlings exposed to increasing durations of flooding stress during the summer. LITERATURE CITED Anella LB. and TH. Whitlow 1999. F lood-tolerance ranking of red and Freeman maple cultivars. J. of Arboriculture 25: 31-36. Armstrong W., R. Brandle and MB. Jackson 1994. Mechanisms of flood tolerance in plants. Acta Bot. Neerl 43: 307-358. Broadfoot W.M. and HL. Williston 1973. Flooding effects in southern forest. J. For. 71: 584-587. Chirkova T.V. and TS. Gutrnan 1972. Physiological role of branch lenticels in willow and poplar under conditions of root anaerobiosis. Sov. Plant Phys. 19: 289-295. Clemens J ., A.M. Kirk and PD. Mills 1978. The resistance to waterlogging of three Eucalyptus species, effect of flooding and of ethylene releasing growth substances on E. robusta, E. grandis and E. saligna. Oecologia 34: 125-131. Dirr MA. 1990. Manual of woody landscape plants: their identification, omamental characteristics, culture, propagation and uses. (4th Ed.) Stipes Publishing Co., 1012 Chester St., Champaign, IL 61820. 1007 p. Hall T.F. and GE. Smith 1955. Effects of flooding on woody plants, West Sandy dewatering project, Kentucky Reservoir, J. For. 53: 281 -285. Jackson MB. and MC. Drew 1984. Effects of flooding on growth and metabolism of herbaceous plants. In Flooding and Plant Growth (T.T. Kozlowski, Ed.) Academic Press, Orlando, FL, pp. 47-128. Kozlowski T.T. and S.G. Pallardy. 1979. Stomatal responses of Fraxinus pennsylvanica 5 seedlings during and after flooding. Physiol. Plant. 46: 155-158. Kozlowski T.T. 1984. Plant responses to flooding of soil. BioScience 34: 162-167. Kozlowski T.T. 1984. Flooding and plant growth. Academic Press, Inc. Orlando, FL, 356 p. Kozlowski T.T. 1985. Soil aeration, flooding, and tree growth. J. Arboriculture 11: 85-96. Kozlowski T.T., P.J. Kramer, and S.G. Pallardy 1991. The physiological ecology of woody plants. Academic Press, Inc., San Diego, CA, USA. Lechowicz M.J. 1984. Why do temperate deciduous trees leaf out at different times? adaptation and ecology of forest communities. Am. Nat. 124: 821-842. Loescher W.H. ,T. McCamant, and J .D. Keller 1990. Carbohydrate reserves, translocation, and storage in woody plant roots. HortScience 25: 274-281. Norby R.J. and T.T. Kozlowski 1983. Flooding and S02 stress interaction in Betula papyrz'fera and B. nigra seedlings. For. Sci. 29: Pezeshki SR. and J .L. Chambers 1986. Variations in flood-induced stomatal and photosynthetic responses of three bottom-land tree species. For. Sci. 32: 914-923. Pezeshki S.R., J.H. Pardue, and RD. DeLaune. 1996. Leaf gas exchange and growth of flood-tolerant and flood-sensitive tree species under low soil redox conditions. Tree Physiology 16: 453—458. Phung HT. and EB. Knipling 1976. Photosynthesis and transpiration of citrus seedlings under flooded conditions. HortScience 11: 131-133. Reid D.M., F.D. Beall, and RP. Pharis 1991. Environmental cues in plant growth and development. In: Plant Physiology, A Treatise Vol X: Growth and Development 6 Chapter 2, Academic Press, Inc. pp.65-l 81. Tang Z.C. and T.T. Kozlowski 1984 Water relations, ethylene production, and morphological adaptation of F raxinus pennsylvanica seedlings to flooding. Plant Soil 77: 183-192. Tardiff J. and Y. Bergeron 1993. Radial grth of Fraxinus nigra in a Canadian boreal floodplain in response to climatic and hydrological fluctuations. J. Veg. Sci. 4: 751- 758. CHAPTER 1 EFFECTS OF FLOODING DURATION ON THE GROWTH AND PHYSIOLOGY OF BLACK ASH (FRAXINUS NIGRA MARSH), GREEN ASH (FRAXINUS PENNSYLVANICA MARSH), AND WHITE ASH (FRAXINU S AMERICANA L.) SEEDLINGS. ABSTRACT Bare root black ash (Fraxinus nigra Marsh), green ash (F raxinus pennsylvanica Marsh), and white ash (Fraxinus americana L.) 2-1 seedlings 30.5 to 45.7 cm in height were planted into 19 liter plastic containers filled with a sterilized 2:1 (v/v) sand:10am mixture. Flood treatments were imposed for 0, 4, 8, l6, and 32 days with the recovery interval extended to day 60. Minimal growth was observed for black and white ash seedlings. For these two species, flooding for 32 days reduced dry weight biomass 46 % in roots. Differences in biomass were not observed for green ash seedlings regardless of treatment, but stem height, leaf number, and leaf area were reduced by flooding for 8, 16, and 32 days. Green ash seedlings were slower to react to flooding stress than were black and white ash seedlings. Flooded black, white, and green ash seedlings had reductions in stomatal conductance on days 2, 4, and 8, respectively. Lower assimilation rates were observed by day 4, 8, and 11. During recovery, opposite results were observed with green, black, and finally white ash seedlings reopening their stomata and resuming photosynthesis. Physiological recovery for each species was modified by the plant response and duration of the flood. Once a reduction in stomatal conductance occurred, Fraxinus seedlings required a 12 to 16 day period to recover. As the flood duration increased to 32 days, a shorter recovery time of 8 days was observed. Thereafter, the ability of black and white ash seedlings to recover increased to 12 days compared to green ash seedlings which recovered after 8 days. INTRODUCTION Responses of woody plants to environmental stresses are known to vary appreciably. For example, Ceulemans et a1. (1984) reported that variations in gas exchange rates among different plant species (or cultivars) are determined by both environmental factors and plant characteristics. Consequently, woody plants respond to stresses, such as flooding, drought, soil compaction, wind, and extremes in temperatures, with different morphological and physiological adaptations. Many of these adaptations are related to a species’ plasticity and ecological success. Two trees commonly seen in urban landscapes are green ash (F raxinus pennsylvam’ca Marsh.) and white ash (F raxr‘nus americana L.). Green ash is one of the most adaptable trees, with a wide geographic distribution. Green ash is a highly flood-tolerant species that can grow on land subjected to periodic flooding and display vigorous growth even when flooded for much of the growing season (Tang and Kozlowski 1984). In contrast, white ash, which usually appears on deep, well-drained upland soils, is a flood-intolerant species (Hall and Smith 1955; Kozlowski et a1. 1991). Black ash (Fraxinus nigra L.) is an extremely hardy species common to low woodlands, swamps, and periodically inundated river bottoms. Often referred to as swamp or hoop ash, black ash can tolerate standing water for many weeks. Black ash is also a determinant species meaning that the next season’s growth is determined by climatic conditions during the previous season (Lechowicz 1984; Tardiff and Bergeron 1993) Conditions of flood water, seasonality, and duration are factors that determine a 10 species’ response. The response also changes with the age of the plant, with seedlings more susceptible than mature trees. Initially, flooding reduces the oxygen content of the soil (Kozlowski et al. 1991). This affects many aspects of growth and development (Kozlowski ed. 1984). For example, flooding of roots causes variable and complex changes in plant- water relations including transient wilting, reduced transpiration, and a decline in root conductivity. Some researchers find no water deficit, while others describe symptoms of long-term water deficit (Reid et a1. 1991). In 1979, Kozlowski and Pallardy observed that flooding induced stomatal closure in 5 month old green ash seedlings with a gradual reopening. Pezeshki and Chambers (1986) showed that flooding green ash and baldcypress did not cause a significant decrease in photosynthesis despite a 50 % decrease in stomatal conductance. The physiological changes caused by flooding stress is critical to the plant’s survival, growth, and future development. The objectives of this study were to characterize and compare the growth and physiology of black, green, and white ash seedlings exposed to increasing durations of flooding stress during the summer. Determination of these variables may help to explain differences in physiological and ecological responses among these three species with respect to flood tolerance. 11 MATERIALS AND METHODS Plant materials Bare root black ash, green ash, and white ash seedlings (2-1; 30.5 to 45.7 cm in height) were obtained fi'om Lawyers' Nursery in Plains, Montana, USA, and stored in a cold room at 2-3 °C until potted. Seedlings were ranked based on uniform caliper size taken at the root collar. Seedling roots were pruned to 18 to 20 cm prior to planting into 19-liter plastic containers (Classic 1200, Mollema, 34 cm width x 38 cm height) lined at the bottom with a layer of cheese cloth and filled with a sterilized 2:1 (v/v) sand:10am mixture. Lateral branches were pruned and one terminal shoot was allowed to develop for each seedling. Seedlings were placed in an uncovered frame house at the Michigan State University Horticulture Teaching and Research Center, Lansing, MI, USA (42.8°N, 84.5°W) where they were kept well watered until the beginning of the flood treatment. Seedlings were allowed to grow 4 weeks prior to the onset of flood duration treatments. The flood treatment began on the 24'11 of June 1998, and continued for 32 days. The recovery interval began upon release from flood for each treatment and lasted until August 23'“, 1998 on day 60. Thus, seedlings flooded for 4, 8, 16 and 32 days recovered for a total of 56, 52, 44, and 28 days, respectively. Throughout the recovery period, seedlings were fertilized once a week with 200 ppm of nitrogen from a 20:10:20 NPK soluble fertilizer using a HOZON applicator. Experimental Design This factorial experiment was arranged in a completely randomized design with a total of 12 264 seedlings assigned to one of two groups. In the first group, eight replicate seedlings of each species (black, green, and white ash) were flooded for durations of 0 (control), 4, 8, 16, or 32 days and monitored for growth and gas exchange. Control seedlings were watered regularly to maintain soil moisture below or near field capacity. In the second group, eight replicate seedlings of each species were flooded for durations of 0 (control), 8, 16, and 32 days. These seedlings were used to determine the effect of flooding on biomass and carbohydrate reserves (Chapter 2). To ensure uniformity in soil moisture, soil moisture data was recorded every 4 days on the control and drained seedlings using a Theta Meter type HHl (Delta-T Devices, Cambridge, England) equipped with a Theta Probe type MLl. The flood treatments were imposed by inserting the l9-liter container inside a larger container lined with a reinforced plastic bag. The water level was maintained near the rim of the container using a drip irrigation system controlled by a time clock (Model LX-12, Rainbird, USA), so as to mimic standing water. Plants released from the flood treatment were allowed to drain and were maintained similar to the control seedlings. Growth parameters Growth measurements were taken every 4 days on 120 seedlings. Current stem lengths were measured using a ruler to the nearest mm. The current stern lengths were measured fi'om the terminal bud scar to just below the apical meristem. The average of two stem diameter measurements (45 to 90° angles from each other) were taken using 3 Max Series Electronic Digital Calipers (Fred V. Fowler Company Inc., Japan) to the nearest 0.1 mm at 2 cm above the container rim. Leaf number was determined by counting. Data is reported as the increase 13 in leaves from the onset of flooding. Leaflet area was measured by taking the length and width of the most recently expanding terminal leaflet and multiplying these by a correction coefficient previously estimated by measuring the lamina length and width of individual terminal leaflets and determining their area using a Delta-T Area Measurement System (Delta-T Devices Ltd., Cambridge, England). Correction coefficients for black, green, and white ash terminal leaflets were 0.6129, 0.7154, and 0.6823. These were determined from 20 terminal leaflets for green ash and 24 terminal leaflets for black and white ash. Regressions between the estimated leaflet area and measured area were highly correlated (r2= 0.98) for each species (Figure 1.1). Separate t-tests comparing the estimated and measured areas were not different, indicating the accuracy of the correction coefficient (black ash F=1.07, P=0.8682; green ash F=1.08, P=0.8654; white ash F=1.14, P=0.7570). After 8, 16, and 32 days of flood stress, control and flooded seedlings from the second group were harvested to determine the dry weight for each plant component. Leaves, old and current stems, and roots were washed, separated, bagged, and kept in coolers until processed in the laboratory. Fresh and dry weights were determined using an analytical balance (Mettler PM460, Mettler Instruments Corp., Hightstown, NJ, USA) to an accuracy of 0.001 g. Prior to lyophilization (Virtis, Genesis 12EL, Gardiner, NY, USA), samples stored in a -30 °C fieezer were transferred to a -80 °C freezer (Fisher Scientific, Isotemp Freezer, USA). This prevented thawing of the samples during the freeze-drying process. Gas exchange Carbon assimilation (A), stomatal conductance (gs), transpiration (E), and water use 14 efficiency (WUE) were determined using a CIRAS-l photosynthesis system (PP Systems, Haverhill, MA, USA) equipped with a CIRAS-l broad Parkinson leaf cuvette (PP Systems, Haverhill, MA, USA). Gas exchange measurements were taken on a 4-day interval, or when weather permitted between 9:30 and 1:30 HR on the terminal leaflet of a mature fully expanded leaf. Control and 32-day flooded seedlings were measured throughout the study with seedlings flooded for 4, 8, and 16 days added after release fiom their respective flood treatments until the end of the experiment. Statistical analyses Growth and gas exchange parameters were analysed for each flood treatment interval. Growth in caliper, current stem height, and leaf number were calculated by subtracting initial measurements on day 0 from the data measured on day 60. Leaf area expansion (terminal leaflet) was calculated by subtracting initial measurements on day 0 fiorn data collected on day 32 because leaf expansion had ceased. Regressions of the estimated and measured leaflet areas were conducted followed by a t-test to ensure the accuracy of the correction coefficient to estimate the leaflet area for each species. Physiological data were pooled to compare species and flood duration for the entire study. Analysis of variance was conducted using PROC GLM and regression analysis using the PROC REG procedure in SAS/PC software (SAS Institute Inc. 1985). Comparisons were tested using a Duncan’s multiple range test. Probabilities less than or equal to 0.05 were regarded as significant. 15 Figure 1.1. Regression of estimated terminal leaflet area over the actual leaflet area measured from three ash species. Estimated leaflet area was calculated by length x width x correction coefficient. Correction coefficients for black, green, and white ash terminal leaflets are 0.6129, 0.7 154, and 0.6823. Regression line for each species was based on 20-24 terminal leaflets. 16 Estimated leaf area (cmz) 25'- 20'* 15" 1' O (3' " 10" ' C) 1. . Black ash r2= 0.98 5 ‘ 0 Green ash r2= 0.98 v White ash r2= 0.93 o I I I j 0 1O 15 20 25 Actual leaf area (cm’) 17 RESULTS Growth parameters Changes in caliper and leaf area had significant species and duration main effects (Table 1 . l ). Green ash seedlings accrued a greater caliper and leaf area compared to black and white ash seedlings. In addition, white ash seedlings had a greater leaf area when compared to black ash seedlings. As the flood duration increased, stem caliper growth increased proportionally whereas the leaf area decreased. In general, ash seedlings flooded for 32 days had a greater increase in caliper (1.98 mm) than seedlings flooded for the other durations. Eight and sixteen day seedling calipers (1.39 and 1.52 mm) exceeded caliper for seedlings flooded for 0 and 4 days (1.09 and 1.02 mm). Increases in the leaf area of the terminal leaflet were greater (ca. 2 fold difference) for controls with a value of 15.72 cm2 when compared to seedlings flooded for 4, 8, 16, and 32 days with values ranging between 7.13 and 7.55 cmz. Current stem height and leaf number had a significant species x flood duration interaction (Table 1.2). Green ash control and 4-day flooded seedlings had greater stem growth (7.65 and 6.60 cm) compared to seedlings flooded for 8,16, and 32 days (2.06, 1.41 , and 3.08 cm). Minor current stem growth of 0.45 to 0.70 cm were observed for black and white ash seedlings, respectively. Leaf numbers were also greater in green ash seedlings compared to black and white ash counterparts, in which there was no increase and even a decrease in leaf number. Green ash control and 4-day flooded seedlings had a similar increase in leaf number (6.63 and 5.63). These values were greater than 8-day flooded green ash with a value of 3.75, which was greater than 16 and 32-day flooded green ash seedlings 18 Table 1.1. Mean increase in caliper and leaf area for black, green, and white ash seedlings flooded for O, 4, 8, 16, and 32 days. The changes in caliper were presented for the entire study (0 to 60 days) whereas leaf area were presented during the flood (0 to 32 days) period. Means, followed by the same letter within a column for each main effect, do not differ significantly (Duncan, P5005). Growth parameters Main effects Caliper (mm) Leaf area (cmz) Species Black ash 1.18b 2.690 Green ash 1.84a 17.78a White ash 1.1% 64% Duration Day 0 1.09cd 15.72a Day 4 1.02d 7.55b Day 8 1.39bc 7.54b Day 16 1.52b 7.21b Day 32 1.98a 7.13b 19 Table 1.2. Mean current stem height growth and increase in leaf number for black, green, and white ash seedlings flooded for 0, 4, 8, l6, and 32 days. Stem height and leaf nrunber were presented for the entire study (0 to 60 days). Means, followed by the same letter within a column for each species x flood duration interaction, do not differ significantly (Duncan, n=8, P5005). Negative values in the leaf number indicate net loss of leaves. Species x Duration of flooding Stern height (cm) Leaf number ((1) Black ash Day 0 0.46c 0.00de Day 4 0.53c 0.00de Day 8 0.560 -0.25de Day 16 0.63c -038e Day 32 0.59c —0.25de Green ash Day 0 7.65a 6.63a Day 4 6.60a 5.63a Day 8 2.06bc 3.75b Day 16 1.41bc 2.130 Day 32 3.08b 1.50c White ash Day 0 0.64c 0.00de Day 4 0.59c -O.25d Day 8 0.70c -O.13de Day 16 0.54c 0.00de Day 32 0.45c -O.50e 20 with a leaf number increase of2. 13 and 1.50. Overall, green ash seedlings increased in height and produced leaves after black and white ash seedlings ceased. Depending on the harvest interval, dry weights for each plant component differed with species regardless of the flood treatments (Table 1.3). White ash seedlings harvested after 8 days of flooding had a greater leaf, current stem, old stern, and root dry weight compared to black ash seedlings. Green ash seedlings had similar dry weights to black ash seedlings with two exceptions. First, green ash seedling had a current stem dry weight that was not different from either white or black ash seedlings. Secondly, green ash seedlings Old stem dry weights were greater than those for black ash seedlings. The significant species difference for old stem dry weight for each harvest is due to initial size differences where smaller black ash seedlings are compared with larger green and white ash seedlings. After 16 days of flood stress, no differences were observed for leaf and root dry weights. Current stem samples were not available. After 32 days of flood stress, leaf and current stem dry weights were similar for control and flooded seedlings of each species (Table 1.3). Flooded black ash seedlings, however, had a lower leaf dry weight than control and flooded green and white ash seedlings. In addition, flooded black ash seedlings had a lower current stem dry weight than control and flooded green ash and control white ash seedlings. Root dry weight had a significant species x flood treatment interaction after 32 days of flood stress (Table l .3). Control black and white seedlings had greater root dry weight compared to their flooded counterparts. Dry weights were reduced by 46 % for roots in flooded black and white ash seedlings. In contrast, control and flooded green ash seedlings had no root dry weight differences indicating that flooding did not restrict root growth. 21 Table 1.3. Leaf, current stern, old stem, and root dry weights for black, green, and white ash seedlings harvested after 8, 16, and 32 days of flood stress. Means, followed by the same letter within a column for each separate harvest, do not differ significantly (Duncan, n=8, P5005). Current stems harvested on day. 16 were not available (N/A). Harvests Dry weight biomass for each plant component Day 8 Leaf (g) Current stern (g) Old stem (g) Roots (g) Black control 7.5 lb 0.86c 11.30b 23.01b flooded 8.68ab 1.57abc 19.36b 28.34ab Green control 9.52ab 2.3lab 33.333 31.82ab flooded 8.50b 1.39bc 28.6la 22.37b White control 11.83a 2.34ab 33.82a 33.50a flooded 10.50ab 2.67a 34.60a 34.36a Day 16 Black control 9.39 N/A 14.0% 34.92 flooded 8.69 N/A 21 .34b 26.54 Green control 10.78 N/A 41 .78a 31.37 flooded 9.09 N/A 38.97a 26.90 White control 1 1.15 N/A 36.24a 36.64 flooded 8.69 N/A 33.93a 28.64 Day 32 Black control 9.9lab 1.64ab 17.41c 48.20a flooded 7.64b 1.35b 17.01c 22.30b Green control 12.273 3.11a 37.90b 33.83b flooded 11.41a 3.20a 51.35a 31 .86b White control 11.47a 2.96a 41 .58ab 54.53a flooded 11.45a 2.66ab 41 .89ab 24.83b 22 Physiological parameters Carbon assimilation (A), stomatal conductance (g,), and transpiration (E) rates showed significant species x flood duration interactions (Table 1.4). Water use efficiency (WUE), however, only had a significant species main effect wherein black ash seedlings had greater values compared to both white and green ash seedlings (data not shown). In Table 4, means pooled for the entire study showed that 16-day flooded green ash seedlings had higher A rates (9.94 umol m'2 s") than control and 8-day flooded green ash seedlings with A rates of 9.80 and 9.20 umol m'2 s", respectively. Carbon assimilation of the 8-day flooded green ash was not different fi'om 4 and 32-day flooded green ash, control and 4-day flooded white ash, and control black ash seedlings. Carbon assimilation rates for these seedlings ranged between 8.91 and 8.59 umol In2 5'1 during the entire study. White ash seedlings flooded for 8 days had an intermediate A rate (8.18 umol rn‘2 s") that did not differ from the latter group; but, was also similar to rates seen in l6-day flooded white ash and 4, 8, and 16-day flooded black ash seedlings. Finally, 32-day flooded white and black ash seedlings had lower A rates (5 .99 and 4.92 umol m’2 s“), which were also different. Overall, control green ash had higher g5 and E rates followed by either 4-day flooded green ash or control white ash seedlings when evaluating either g, or E rates. Both g,, and E rates decreased consistently with an increase in flood duration except for 4-day black ash seedlings, which had a slightly lower g,, and E rates than its’ 8 and 16-day counterparts (Table 1.4). Again, reductions in A, g, and E rates were most pronounced in 32-day flooded seedlings for black and white ash seedlings. In white ash, seedlings flooded for 8, 16, and 32 days had a proportional and gradual decrease in A, gs. and E rates (Table 1.4). In contrast, black ash seedlings flooded for 4, 8, and 16 days 23 Table 1.4. Mean assimilation, stomatal conductance, and transpiration for black, green, and white ash seedlings flooded for O, 4, 8, 16, and 32 days. Values presented are for the entire study including the both the flood and the recovery intervals. Means, followed by the same letter within a column for each species x flood duration interaction in the experiment, do not differ significantly (Duncan, P5005). Species x Duration of Assimilation Stomatal Transpiration flooding (d) (umol In2 S“) conductance (mmol In2 5") (mmol m‘2 s") Black ash Day 0 8.65cd 109.75de 1.77cde Day 4 7.58e 94.35fg 1.51f Day 8 7.83e 101.34ef 1.61ef Day 16 7.60e 99.19ef 1.52f Day 32 4.92g 62.06h 1.03h Green ash Day 0 9.80b 150.1 la 2.26a Day 4 8.90cd 136.95ab 2.02b Day 8 9.20bc 132.52b 2.04b Day 16 9.94a 129.42bc 1.88bcd Day 32 8.91cd 132.13b 189de White ash Day 0 8.97c 131.45b 2.08ab Day 4 8.59cd 126.01bc 1.94bc Day 8 8.18de 116.14cd 1.79cde Day 16 7.80e 116.46cd 1.70def Day 32 5.99f 81.45g 1.25g 24 had similar A, g,, and E rates which were greater than values in their 32-day flooded counterpart. The 32-day flooded green ash seedlings maintained similar A and g8 rates to other flooded green ash seedlings. The above trends in the gas exchange parameters are present in data taken at each measurement interval. After4 days of flooding, control black ash seedlings had a greater A rate than flooded black ash seedlings (Figure 1.2a). By day 8, white control seedlings had greater A rates than their flooded counterparts. Carbon assimilation rates of control and flooded green ash were different on day 11. Thereafter, seedlings flooded for 32 days had lower A rates from controls with one exception. Control and flooded green ash seedlings had similar A rates on day 28 (Figure 1.2b). The variation in A rates follow a similar pattern found for both g8 and E rates (Figure 1.3-1.4). Two days after the onset of the flood, control black ash seedlings had higher gs and E rates compared to flooded black ash seedlings. Differences in g5 and E rates for white and green ash seedlings occurred after 4 and 1 1 days of flooding, respectively (Figure 1.3-1.4c). Thereafter, control seedlings had greater gs and E rates compared to flooded seedlings until seedlings recovered (Figure 1.3-1.4). Once released fi'om the 32-day flood, A rates returned to control levels for green, black, and white ash seedlings by day 39, 40, and 44, respectively (Figure 1.2abc). During recovery, A rates for flooded green ash seedlings exceeded those of control seedlings on days 48 and 52 (Figure 1.2b). Green ash seedlings had similar g5 and E values by day 39 compared to both black and white ash seedlings that recovered by day 44 (Figure 1.3-1.4). On day 52, g8 and E rates for green flooded seedlings exceeded those found for green control seedlings. 25 Figure 1.2. Mean assimilation (:SE) of black ash, Fraxinus nigra L. (a), green ash, Fraxinus pennsylvanica Marsh. (b), and white ash, Fraxinus americana L. (c), seedlings flooded for 0, 4, 8, l6, and 32 days. Seedlings flooded for 0 and 32 days were measured throughout the study whereas seedlings flooded for 4, 8, and 16 days were added when released to monitor recovery. (Duncan, n=8, P5005) 26 15.0 - 12.5 - 10.0 - 7.5 4 5.0 - 2.5 a 17.5 ‘ I I I ' I I I T l r l I 15.0 ‘ 12.5 - + D 0 10.0 - 35’ —I- Day4 —4- Day8 —'V'- Day 16 Assimilation (umol rn'2 3‘1) 1" 9' .‘1 on o 0: 15.0 ~ 12.5 ~ 10.0 1 7.5 - :: / 5.0- ,. {K 2.5 1 I 0.0 I I I I I I ' I I I 0 51015202530354045505560 Duration of flood and recovery (days) 27 Figure 1.3. Mean stomatal conductance (:SE) of black ash, F raxinus nigra L. (a), green ash, Fraxinus pennsylvanica Marsh. (b), and white ash, F raxinus americana L. (c), seedlings flooded for 0, 4, 8, 16, and 32 days. Seedlings flooded for O and 32 days were measured throughout the study whereas seedlings flooded for 4, 8, and 16 days were added when released to monitor recovery. (Duncan, n=8, P5005) 28 250— + DayO —I— Day4 A- Day8 + Day 16 ""0 Day32 Stomatal conductance (mmol m"2 s" 1) C 0 51015202530354045505560 Duration of flood and recovery (days) 29 Figure 1.4. Mean transpiration (18E) of black ash, Fraxinus nigra L. (a), green ash, Fraxinus pennsylvam'ca Marsh. (b), and white ash, Fraxinus americana L. (c), seedlings flooded for 0, 4, 8, 16, and 32 days. Seedlings flooded for O and 32 days were measured throughout the study whereas seedlings flooded for 4, 8, and 16 days were added when released to monitor recovery. (Duncan, n=8, P5005) 30 Transpiration (mmol rn’2 5'1) 4 - + DayO —l— Day4 -4- Day8 + Day 16 "'"O Day32 4 . 3 4 9. g7 11* / ' c o I T I f I f T I 0 51015202530354045505560 Duration of flood and recovery (days) 31 Seedling recovery for 32-day flooded seedlings was faster or similar to seedlings flooded for 4, 8, and 16 days. For example, 4-day flooded black ash seedlings, with lower A rates than control seedlings, recovered 12 days after release from the flood stress on day 16. As the flood duration increased to 8 and 16 days, carbon assimilation rates of black ash seedlings recovered within 8 and 4 days, respectively. Thereafter, A rates of black ash seedlings flooded for 32 days recovered after 12 days. Similar recovery pattern and times were observed for flooded white and green ash seedlings which had lower A rates after 8 and 11 days of flooding, respectively. Four day flooded seedlings recovered 16 days after release. Again, a shorter recovery period of 8 days was observed for flooded green ash seedlings as the flood duration increased to 16 and 32 days. In contrast, A rates for white ash seedlings flooded for 16 and 32 days recovered within 8 and 12 days, respectively. Growth, gas exchange, and dry weights were summarized in Table 1.5 to clearly indicate when differences occurred. Caliper, stem height, and leaf number were not different for white ash and black ash except for the caliper width of flooded black ash seedlings on day 32. White ash seedlings had a significant change in leaf area throughout the study whereas black ash seedlings had none. Root dry weights for these two species was also different on day 32. Earlier changes in gas exchange for black and white ash were seen and maintained compared to the green ash seedlings. For example, the gs of black and white ash seedlings was different on day 2 and 4 followed by a difference in A rates on day 4 and 8, respectively. In contrast, A and E rates of green ash seedlings were different on day 16 followed by differences in all three gas exchange parameters (A, g,, and E) on day 32. Green ash seedling growth was also influenced by flooding with differences in the leaf area throughout the 32 study. Leaf numbers were different by day 8 whereas stern height was influenced on day 16. The caliper widths of green ash was different only on days 4 and 32 (Table 1.5). 33 2.. m2 m2 m2 m2 . : m2 m2 maaoaaom m2 m2 mz .. m2 m2 m2 m2 m2 amass 8% so mz <2 m2 m2 $2 m2 m2 32 m2 ”Haas 83m .856 m2 m2 m2 m2 m2 m2 m2 m2 m2 mamas s3 2.. r. 2.. m2 m2 2.. .. m2 m2 m2 2.. t 2. m2 : confines r. r. I .. mz .. m2 m2 m2 m2 2.. t :- .. .. 832680 3.8% t t 2.. m2 m2 2.. 2.. m2 m2 m2 9.. t r. .. mz 8:353... .. .. . .. :- : t 2.. m2 m2 m2 m2 83.28.. m2 m2 m2 m2 2.. r. : m2 m2 m2 m2 m2 55:34 m2 m2 m2 m2 : .. m2 m2 m2 m2 m2 m2 Ewe: 8% m2 m2 m2 m2 : m2 mz : .. m2 m2 m2 5&6 mm 3 w v N mm m: w v N mm 3 w v N Heaven-am :8 as? as :85 :a scam SEE 996 E 86.5 coca mo couch—Q 3323.6 we: we? 8% 05 can? 38: mm? $2 320:? 038 we? eowaemgfiov 0: 80:3 vows—2: 803 Saw—e Am": .5258 b33838 . a... was a mm 333%,: 8a 3%: 8.on co 3.on 05 «a mwemeoom Becca Ba 35:8 50253 $erme .33 mm c5 .2 .w .v .m Baa coca 3893 Hem—38 a8 BE? 93 .32» £er com flames» be Becomes Ema can .owfifioxo mam 539% we magnum .mA 033. 34 DISCUSSION Flooding black ash (Fraxinus nigra), green ash (Fraxinus pennsylvanica), and white ash (F raxinus americana) seedlings during the growing season stimulated different morphological and physiological responses. Changes in current stem height, leaf number, and leaf area expansion were negligible when comparing control and flooded black or white ash seedlings (Table 2). In contrast, green ash seedlings continued growing throughout the study. Although flooding began June 24th 1998, it is likely that both black and white ash seedlings had completed the majority of their growth for the season. For example, white ash saplings grown in Ontario are known to develop their canopy in early May (Brundrett and Kendrick 1988). In addition to phenological and developmental characteristics which vary with tree size (Fredericksen et a1. 1996), transplant shock and pruning are two additional factors that need careful consideration when dealing with bare root seedlings (Schutzki pers. com.). Planting shock is known to reduce seedling performance because of the mechanical and physiological stresses that accompany the seedlings through lifting, processing, storage, and subsequent planting (Coutts 1980; Sutton 1980). Unfortunately, very little information is available for black and white ash seedlings. One study on green ash seedlings, however, determined that root pruning had no sustained effect on growth because root regenerated quickly (Amold and Struve 1989). Flooding adversely affects stern, leaf, and cambial growth as well as reproductive growth of trees (Kozlowski 1985). Stem swelling due to increased stern hydration commonly occurs during flooding. In this study, caliper widths for all three flooded species were greater 35 than their control counterparts. Changes in current stern height, leaf number, and leaflet area for black and white ash seedlings were small compared to green ash seedlings. Cessation of growth was evident with no change or a fewer number of leaves and a small expansion in leaflet area. Black and white ash leaflets increased 2.69 and 6.49 cm2 compared to green ash leaflets with a mean increase of 17.78 cm2 (Table 1.1).Consequently, flooding stress, regardless of duration, reduced leaf expansion with control seedlings gaining twice as much leaf area. Flooding experiments on different species have shown that flooding impedes leaf initiation and expansion and in severe cases causes leaf senescence (Kozlowski 1985). Caliper grth changes induced by flooding green ash seedlings were greater in magnitude and different from control seedlings. Flooded green ash had a greater caliper increase overall whereas current stem height, leaf number, and leaf expansion were greater for the control seedlings (Table 1.2). Seedling dry weights harvested after the 32 day flood also highlight species and flood induced differences. Leaf and current stem dry weights were not different except for flooded black ash seedlings had lower leaf and current stem dry weights (Table 1.3). A proportionally smaller size for black ash seedlings may account for its lower accrued growth and an increased susceptibility to flood stress. This is important since roots are sensitive to oxygen deficiency and that no records of sustained grth by anoxic roots has been reported (Armstrong et al. 1994). Consequently, seedling survival by anaerobic metabolism is dependant on the availability of substrates present in the roots for respiration (Jackson and Drew 1984). In this study, flooded black and white ash seedlings had a 46 % reduction in root dry weights compared to their control counterparts. Similar results by Smith and Boume 36 (1989) had 50 and 48 % reduction in root mass for 28 day flooded pecan seedlings when compared to controls during bud break or active growth, respectively. Flooded pecan roots were also brown to black compared to control seedlings with light yellow roots. During the harvest, flooded roots were partly yellow with brown and black discolorations of varying degrees mainly at the tips. Root discoloration was most pronounced in black ash seedlings and least in green ash seedlings. Generally, reductions in root dry weights exhibit both a decrease in root mass due to root death and inhibited root growth (Smith and Boume 1989). Studies on various species have outlined the inhibition of root growth in response to flood stress conditions (Pezeshki and DeLaune 1990; Liu and Dickrnann 1992; Topa and Cheeseman 1992). For green ash seedlings, however, control and flooded root dry weights were similar, clearly showing a greater flood tolerance. These findings support a study by Good and Patrick (1987) where root aeration and anaerobic respiration contributed to the survival of green ash seedlings flooded for a period of 9.5 months (mid-July to May). Other studies on green ash seedlings (70 to 150 days old) with shorter flood duration (28 to 30 days) reported reductions in dry weight increment of roots, stems, and leaves combined with the formation of hypertrophied lenticels and adventitious roots (Sena Gomes and Kozlowski 1980; Tang and Kozlowski 1984). Although the leaf, current and Old stern, and root dry weights for control and flooded green ash seedlings did not differ, significant growth differences were observed during the flood. In addition, hypertrophied lenticels were observed for all three Fraxinus species exposed to flooding stress. Black and green ash seedlings developed numerous hypertrophied lenticels on their submerged stems within 8 to 12 days whereas those for white ash developed later as the flood progressed. By the end of 37 the 32-day flood, raised lenticels covered most of the submerged stems but no apparent adventitious roots developed contrary to results noted in Sena Gomes and Kozlowski (1980). Among woody plants, Hook (1984) noted that the development of prominent lenticels at the stem base and on older roots contributes to improved gas exchange. It is thought to enhance Oz diffusion to roots (Kozlowski 1984) or eliminate potentially toxic metabolites such as ethanol, acetaldehyde, or ethylene (Chirkova and Gutman 1972). The development of hypertrophied lenticels, however, does not necessarily confer flood-tolerance (Larson and Schaffer 1991). Other processes, especially those that are metabolic or physiological in nature, may be critical in determining flood tolerance. For example, many flood responses help minimize transpirational loss of water by either leaf shedding, stomatal closure, leaf epinasty, or slowing of growth. Everard and Drew (1987, 1989) emphasized that minimizing these losses may originate fi'om the low conductivity of oxygen-deficient roots to water. Not surprisingly, woody plants often exhibit a decrease in A and gs within 1 to 3 days after flooding, although longer flooding durations are required for reductions in growth (Andersen et a1 1984; Crane and Davies 1989). Sena Gomes and Kozlowski (1980) reported that stomatal closure was one of the earliest responses to flooding green ash seedlings. In comparing the three F raxinus species, stomatal closure occurred after 2 days of flooding for black ash. Lower g, for white ash seedlings were observed after day 4 whereas green ash seedlings responded after 1 1 days. Similarly, reductions in A rate were observed in black, white, and green ash seedlings after 4, 8, and 11 days. The time required for stomatal closure to inhibit A rates may be attributed to a species sensitivity to anaerobic soil conditions. Hypoxic conditions within the roots may 38 adversely affect photosynthetic processes (Bradford (1983). For example, flooded soils with low soil redox potential (Eh) could cause a reduction in net photosynthesis with either decreased leaf water potential, reduced Rubisco activity (Vu and Yelenosky 1992; Pezeshki 1994), disruption in photosynthate transport, alteration in source-sink relationships, or reduced sink demand (Warnple and Thornton 1984; Drew 1990). A flood-resistant plant can avoid oxygen deficiency either by transporting 02 fiom the leaves internally to its’ root system (Armstrong 1978) or by sustaining and tolerating anaerobic metabolism (Crawford 1982; Barclay and Crawford 1982). During flooding, A rates for flooded black and white seedlings remained lower primarily because gs were no greater than 68 rnmol rn'2 s‘l (Figure 1.3). In contrast, stomatal conductances for flooded green ash seedlings were two folds higher with a mean of 126 mmol rn‘2 s". The ability of flooded green ash to maintain open stomata may indicate an osmotic adjustment to flooding stress. Daily fluctuations in gs during the flood displays a control and capacity to optimize A while adjusting for water loss to the environment. These two physiological processes may explain why green ash biomass was not as affected by flood stress as the other two species (Figure 1.3-1.4, Table 1.3). Duration of the flood stress influenced seedling recovery time for each species. The capability to return to normal levels of photosynthesis following a period of stress is an important factor in the fitness of a plant (Bacone et al. 1976; Ormsbee et a1. 1976; McGee et a1. 1981; Peterson and Bazzaz 1984). Seedling response to flooding is undoubtedly mediated by various plant hormones which attempt to optimize A and growth. Theoretically, Jackson (1993) advises that changes in hormones between roots and shoots can involve ‘positive’, ‘negative’, ‘accumulative’, and ‘debit’ messages (Armstrong et a1. 1994). This 39 topic is beyond the scope of this study. In summary, the mechanisms whereby flood tolerance is attributed to a species involves a combination of both physiological and morphological adaptations. Within the genus Fraxinus, species from markedly different native habitats demonstrated different growth and flood responses. Overall, green ash seedlings had greater A, g,, and E rates compared to black and white ash seedlings. In addition, green ash dry weights were not influenced by the flood. Both black and white ash seedlings, however, had minimal incremental growth, a proportional reduction in root dry weights, and a slow post-flood recovery from reduced g3 and A rates. These results make it difficult to discern which of these two species (black or white ash) is more flood tolerant especially since the initial seedling size differed. Nonetheless, black and white ash possess certain adaptations to flooded soil conditions but are not as highly flood-tolerant as green ash seedlings. 4O LITERATURE CITED Andersen P.C., P.B. 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Pallardy. 1979. Stomatal responses of Fraxinus pennsylvanica seedlings during and after flooding. Physiol. Plant. 46: 155-158. Kozlowski T.T. 1984. Plant responses to flooding of soil. BioScience 34: 162-167. Kozlowski T.T. 1984. Flooding and plant growth. Academic Press, Inc. Orlando, FL, 356 p. Kozlowski T.T. 1985. Soil aeration, flooding, and tree growth. J. Arboricult. 11: 85-96. Kozlowski T.T., P.J. Kramer, and S.G. Pallardy 1991. The physiological ecology of woody plants. Acadean Press, Inc., San Diego, CA, USA. 43 Larson K.D., B. Schaffer, and F .S. Davies 1991. Flooding, leaf gas exchange and growth of mango in containers. J. Am. Soc. Hort. Sci. 116: 156-160. Lechowicz M.J. 1984. Why do temperate deciduous trees leaf out at different times? adaptation and ecology of forest communities. Am. Nat. 124: 821-842. Liu Z. and DJ. Dickrnann 1992. Responses of two hybrid Populus clones to flooding, drought, and nitrogen availability. 1. Morphology and growth. Can. J. Bot. 70: 2265- 2270. McGee A.B., M.R. Schmierbach, and FA. Bazzaz 1981. Photosynthesis and growth in populations ofPopulus deltoides from contrasting habitats. Am. Midl. Nat. 105: 305- 311. Orrnsbee P., F.A. Bazzaz, and W.R. Boggess 1976. Physiological ecology of Juniperus virginiana in oldfields. Oecologia 23: 75-82. Peterson D.L. and RA. Bazzaz 1984. Photosynthetic and growth responses of silver maple (Acer saccharinum L.) seedlings to flooding. Am. Midl. Nat. 112: 261-272. Pezeshki SR. and J .L. Chambers 1986. Variations in flood-induced stomatal and photosynthetic responses of three bottom-land tree species. For. Sci. 32: 914-923. Pezeshki SR. and RD. DeLaune 1990. Influence of sediment oxidation-reduction potential on root elongation in Spartina patens. Acta. Oecol. 11: 377-383. Pezeshki SR. 1994. Responses of baldcypress seedlings to hypoxia: leaf protein content, ribulose-1,5-bisphosphate carboxylase/oxygenase activity and photosynthesis. Photosynthetica 30:59-68. Reid D.M., F.D. Beall, and RP. Pharis 1991. Environmental cues in plant growth and 44 development. In: Plant Physiology, A Treatise Vol X: Growth and Development Chapter 2, Academic Press, Inc. pp.65-18l. SAS Institute Inc. 1988. SAS/STAT user’s guide, version 6.03 Edition, Cary, NC., 1028 pp. Sena Gomes AR and T.T. Kozlowski 1980. Growth responses and adaptations of F raxinus pennsylvanica seedlings to flooding. Plant Physiol. 66: 267-271. Smith M.W. and RD. Boume 1989. Seasonal effects of flooding on greenhouse-grown seedling pecan trees. HortScience 24: 81-83. Sutton RF. 1980. Techniques for evaluating planting stock quality. For. Chron. 56: 1 16-120. Tang Z.C. and T.T. Kozlowski 1984 Water relations, ethylene production, and morphological adaptation of F raxinus pennsylvanica seedlings to flooding. Plant Soil 77: 183-192. Tardiff J. and Y. Bergeron 1993. Radial growth of Fraxinus nigra in a Canadian boreal floodplain in response to climatic and hydrological fluctuations. J. Veg. Sci. 4: 751- 758. Topa M.A. and J.M. Cheeseman 1992. Effects of root hypoxia and a low P supply on relative growth, carbon dioxide exchange rates and carbon partitioning in Pinus serotina seedlings. Physiol. Plant. 86: 136-144. Vu J .C.V. and G. Yelenosky 1992. Photosynthetic responses of rough lemon and sour orange to soil flooding, chilling and short-term temperature fluctuation during growth. Environ. Exp. Bot. 32: 471-477. Wample R.L. and R.K. Thornton 1984. Differences in the response of sunflower (Helanthus annuus) subjected to flooding and drought stress. Physiol. Plant. 61: 611-616. 45 CHAPTER 2 EFFECTS OF FLOODING DURATION ON THE BIOMASS AND STARCH CONTENT BLACK ASH (FRAXINUS NIGRA MARSH), GREEN ASH (FRAXINUS PENNSYLVANICA MARSH), AND WHITE ASH (FRAXINUS AMERICANA L.) SEEDLINGS. 46 ABSTRACT Bare root black ash (Fraxinus nigra Marsh), green ash (Fraxinus pennsylvanica Marsh), and white ash (F raxinus americana L.) 2-1 seedlings 30.5 to 45.7 cm in height were planted into 19-1iter plastic containers filled with a sterilized 2:1 (v/v) sand:10am mixture. Flood treatments were imposed for 0 (control), 8, 16, and 32 days. Minimal biomass growth was observed for black and white ash seedlings. For these two species, flooding for 32 days reduced root dry weight biomass by 46 %. Differences in biomass were not observed for green ash seedlings regardless of treatment. Starch content varied with tissue type, flood duration and species. A species x duration interaction was present only for the starch concentrations in fine roots on day 32. On days 8 and 16, flooded seedlings had higher leaf starch than control seedlings, while there were no differences in leaf starch concentration on day 32. On day 16 and 32, white ash leaves had 32 and 54 % less starch compared to the other species. On day 8, starch in current and old stems was greater for black ash than in green ash seedlings. This difference did not persist as starch concentrations increased during the study. Stem starch ranged fiom 28.76 to 45.81 mg/g DW on day 8 and fi'om 61.48 to 76.72 mg/g DW on day 32. After 32 days, fine and coarse roots of flooded seedlings had 84 to 88 % and 60 % less starch, respectively, when compared to control seedlings. Green ash seedlings, however, had higher coarse root starch throughout the study. Consequently, green ash seedlings were more tolerant to increased duration of flood stress compared to black and white ash seedlings where root biomass and starch reserves were reduced. 47 INTRODUCTION Woody plants have different strategies to deal with environmental stresses such as flooding, drought, and high salinity. Morphological and physiological adaptations can assure survival of an individual in a species if growth and partitioning of reserves become limited. In woody species, starch is the main insoluble storage carbohydrate present in most above-ground tissues (Loescher et a1. 1990). High concentrations of starch can also be stored in the roots. These reserves fluctuate seasonally to protect the tree during winter and insure renewed growth in the spring. Little research has been done on how environmental stresses impact carbon reserves in roots during the growing season. In a review of carbohydrate reserves in roots, Loescher et a1. (1990) note that late-season stresses impact root reserves and adversely affect plant yield, flowering, and subsequent fiuiting. More information, however, is available on how environmental stresses influence photosynthesis in tree species, especially fruit crops of economic value. Starch synthesis within the chloroplasts is highly coordinated with sucrose synthesis in the cytosol (Taiz and Zeiger 1991). Source-sink relationships play an important role in determining how carbon is fixed and the regulation of photosynthesis (Guinn and Mauney 1980; Daie 1985; Roper et al. 1988). For example, high leaf starch concentrations lead to the formation of additional soluble carbohydrates, which in turn decrease the net photosynthetic rate (Mauney et a1. 1979; Luxmoore 1991). In some species, sorbitol or mannitol (nonreducing sugars) can replace sucrose as the principal photosynthetic product. Some Rosaceae species accumulate sorbitol, whereas some species of Oleaceae, Apiaceae, and Scrophulariaceae accumulate 48 mannitol (Loescher et a1. 1990). The three species studied in this paper are from the genus Fraxinus in the family Oleaceae. How production of mannitol alters starch synthesis in the chloroplasts is not known. Detailed studies are needed to show how the type and ratio of soluble carbohydrates influence carbon fixation. This topic is beyond the scope of this study where the effect of flooding stress on growth and starch reserves at the whole plant level will be examined. Trees commonly planted in urban landscapes, such as green (F raxinus pennsylvan ica Marsh.) and white ash (Fraxinus americana L.), differ in their ability to tolerate flooding stress. Green ash is one of the most adaptable trees, with a wide geographic distribution. Green ash is also a highly flood-tolerant species that can grow on land subjected to periodic flooding and displays vigorous growth even when flooded for much of the growing season (Tang and Kozlowski 1984). In contrast, white ash, which usually occurs on deep, well- drained upland soils, is a flood-intolerant species (Hall and Smith 1955; Kozlowski et al. 1991). Black ash (Fraxinus nigra L.) is an extremely hardy species common to low woodlands, swamps, and periodically inundated river bottoms. Often referred to as swamp or hoop ash, black ash can tolerate standing water for many weeks. Black ash is a determinant species, meaning that the next season’s growth is determined by climatic conditions during the previous season (Lechowicz 1984; Tardiff and Bergeron 1993). Although characteristics of the flood water, seasonality, and duration of flooding all affect response, such responses vary with the age of plant, seedlings being more susceptible than mature trees. Initially, flooding reduces the oxygen content of the soil (Kozlowski et al. 1991). Under these conditions, root respiration decreases and anaerobic metabolism 49 compensates for the reduction in ATP formation. Survival in hypoxic or anoxic conditions then depends on availability of carbohydrate reserves and temperature controlling respiration rate (Jackson and Drew 1984). The elimination of toxic metabolites such as ethanol, acetaldehyde, or ethylene may also be critical for the root system (Chirkova and Gutrnan 1972). Above-ground processes are altered by reduced water and nutrient uptake by roots. Many studies report lower photosynthetic and stomatal conductances (Jackson and Drew 1984; Reid et a1. 1991) wherein decreased growth is observed after a longer period. Good et al. (1986) showed that green ash was able to survive long periods of flood stress by oxidizing the rhizosphere and maintaining anaerobic respiration. To what extent the duration of flood stress changes carbon partitioning, future growth, and if it predisposes a tree to other environmental stresses is not known especially with respect to root reserves. The objectives of this study were to characterize and compare the biomass and starch content in several tissues of black, green, and white ash seedlings exposed to increasing durations of flooding stress during the summer. These data might help to explain differences in physiological and ecological responses among these three species with respect to flood tolerance. 50 MATERIALS AND NIETHODS Plant materials Bare root black ash, green ash, white ash seedlings (2-1; 30.5 to 45.7 cm in height) were obtained from Lawyers' Nursery in Plains, Montana, USA, and stored in a cold room at 2-3 °C until potted. Seedlings were ranked based on uniform caliper size taken at the root collar. Seedling roots were pruned to 18 to 20 cm prior to planting into 19-liter plastic containers (Classic 1200, Mollema, 34 cm width x 38 cm height) lined at the bottom with a layer of cheese cloth and filled with a sterilized 2:1 (v/v) sand:10am mixture. Lateral branches were pruned and one terminal shoot was allowed to develop for each seedling. Seedlings were placed in an uncovered frame house at the Michigan State University Horticulture Teaching and Research Center, Lansing, MI, USA (42.8°N, 84.5°W) where they were kept well watered until the beginning of the flood treatment. Seedlings were allowed to grow 4 weeks prior to the onset of flood duration treatments. The flood treatment began on the 24th of June 1998, and continued for 32 days. Experimental Design This factorial experiment was arranged in a completely randomized design with a total of 264 seedlings assigned to one of two groups. In the first group, eight replicate seedlings of each species (black, green, and white ash) were flooded for durations of 8, 16 or 32 days with an equivalent number of control seedlings reserved to compare biomass and starch reserves. Control seedlings were watered regularly to maintain soil moisture below or near field 51 - 3n capacity. In the second group, eight replicate seedlings of each species were flooded for durations ofO (control), 4, 8, 16, or 32 days. Growth and gas exchange was monitored on this second group of seedlings during and after the flood treatment (Chapter 1). To ensure uniformity in soil moisture, soil moisture data was recorded every 4 days on the control seedlings using a Theta Meter type HHl (Delta-T Devices, Cambridge, England) equipped with a Theta Probe type MLl. The flood treatments were imposed by inserting the 19-liter container inside a larger container lined with a reinforced plastic bag. The water level was maintained near the rim of the container using a drip irrigation system controlled by a time- clock (Model LX-12, Rainbird, USA) so as to mimic standing water. Plants released from the flood treatment were allowed to drain and were maintained similar to the control seedlings. Biomass and carbohydrate determination After 8, l6, and 32 days of flood stress, control and flooded seedlings were harvested to determine the dry weight for each plant component. Leaves, old and current stems, and roots were separated, washed (roots only), bagged, and kept in coolers until processed in the laboratory. Fresh and dry weights were determined using an analytical balance (Mettler PM460 ,Mettler Instrument Corp., Highstown, NJ, USA) to an accuracy of 0.001 g. Prior to lyophilization (V irtis, Genesis 12EL, Gardiner, NY), samples were then stored in a -30 °C freezer and transferred a -80 °C freezer (Isotemp, Fisher Scientific, USA). This prevented thawing of samples and minimized enzymatic reactions during lyophilization. Once weighed, samples were stored in a -30 °C freezer in sealed plastic bags with drierite as a dessicant. 52 Dried root tissues were then separated into fine and coarse roots, respectively. Fine roots were defined as extremity or peripheral segments (5 1 mm thickness) whereas coarse roots were thicker (2 1-3 mm) and bore the fine roots. Dried tissues were ground with a Wiley mill to pass a 40-mesh screen (Thomas Scientific Co., Swedesboro, NJ, USA) except for old stems which were ground to pass through a 1 mm mesh screen. A total of 100 mg was weighed into labelled 16 x125 mm test tubes for carbohydrate extraction. Soluble carbohydrates were extracted with 3.5 ml of 80% ethanol for 20 minutes three times. Between each extraction, samples were centrifuged in a Sorvall rotor at 1879 g for 6 minutes and the supematants collected and dried separately. The remaining starch pellets were dried in a Savant Speedvac SC200 fitted with a refiigerated condensation trap RT4104 at -100 °C (Savant Instruments, Inc., Farmingdale, NY, USA). Dried samples were kept in a freezer with drierite dessicant. After adding 5 m1 of 0.1 M acetate buffer (pH 5.0), each test tube was covered with a plastic cap, autoclaved for 30 minutes (121 °C, 19 PSI) (Amsco, Eagle Series 2021 Gravity) and cooled prior to adding the recommended 12 units ofamyloglucosidase (Boehringer Mannheim, Indianapolis, IN, USA). Samples were digested for16 hours at 48 °C in a water bath and the resulting glucose was determined colorimetrically using a glucose assay kit (Glucose (Trinder); Procedure No. 315, Sigma Diagnostics). Absorbance of glucose standard and samples were read at 505 nm with a Hitachi U-3110 spectrophotometer (Hitachi Ltd., Tokyo, Japan). Statistical analyses Biomass and starch content was analysed for each measurement interval to determine 53 differences over time between green, white, and black ash seedlings in response to flooding stress. Analysis of variance were conducted using PROC GLM in SAS/PC software (SAS Institute Inc. 1985) to determine differences between duration and species. Comparisons between treatment means were tested with a Duncan test. Probabilities less than or equal to 0.05 were regarded as significant. 54 RESULTS Dry weights of each plant component had significant species main effects averaged across the flood treatments depending on the harvest interval (Table 2.1). White ash seedlings harvested after 8 days of flooding had a greater leaf, current stem, old stem, and root dry weight compared to black ash seedlings. Green ash seedlings had similar dry weights to black ash seedlings with two exceptions. First, green ash seedling had a current stem dry weight that was not different from both white or black ash seedlings. Second, green ash seedlings old stem dry weights were greater than those for black ash seedlings. The significant species difference for old stem dry weight for each harvest is due to initial size differences where smaller black ash seedlings are compared with larger green and white ash seedlings. After 16 days of flood stress, no differences were observed for leaf and root dry weights. Current stem samples were not available. After 32 days of flood stress, leaf and current stem dry weights were similar for control and flooded seedlings of each species (Table 2.1). Flooded black ash seedlings, however, had a lower leaf dry weight than control and flooded green and white ash seedlings. In addition, flooded black ash seedlings had a lower current stem dry weight than control and flooded green ash and control white ash seedlings. Root dry weight had a significant species x flood treatment interaction after 32 days of flood stress (Table 2.1). Control black and white seedlings had greater root dry weight compared to their flooded counterparts. Dry weights were reduced by 46 % for roots in flooded black and white ash seedlings. In contrast, green ash seedlings had no treatment differences in root dry weight indicating that flooding did not restrict root growth. 55 Table 2.1. Leaf, current stem, old stem, and root dry weight means for black, green, and white ash seedlings harvested after 8, 16, and 32 days of flood stress. Means, followed by the same letter within a column for each separate harvest, do not differ significantly (Duncan, n=8, P5005). Current stems harvested on . day 16 were not available (N/A). Harvests Dry weight biomass for each plant component Day 8 Leaf (g) Current stern (g) Old stem (g) Roots (g) Black control 7.51b 0.860 11.30b 23.01b flooded 8.68ab 1.57abc 19.36b 28.34ab Green control 9.52ab 2.31ab 33.33a 31 .82ab flooded 8.50b 1.39bc 28.61a 22.37b White control 11.83a 2.34ab 33.82a 33.50a flooded 10.50ab 2.67a 34.60a 34.36a Day 16 Black control 9.39 N/A 14.0% 34.92 flooded 8.69 N/A 21 .34b 26.54 Green control 10.78 N/A 41 .78a 31.37 flooded 9.09 N/A 38.97a 26.90 White control 11.15 N/A 36.243 36.64 flooded 8.69 N/A 33.93a 28.64 Day 32 Black control 9.91ab 1.64ab 17.410 48.20a flooded 7.64b 1.35b 17.010 22.30b Green control 12.27a 3.11a 37.90b 33.83b flooded 11.4la 3.20a 51.35a 31.86b White control 11.47a 2.96a 41 .58ab 54.53a flooded 1 1.45a 2.66ab 41 .89ab 24.83b 56 Leaf, current stem, old stem, and coarse root starch content had a significant species and duration main effects (Table 2.2). A species x duration interaction has present, however, for starch concentrations in fine roots on day 32 (Table 2.3). Changes in starch content varied with tissue type. On days 8 and 16, flooded seedlings had higher leaf starch than control seedlings, while there were no differences in leaf starch concentration on day 32. On day 16 and 32, white ash leaves had 32 and 54 % less starch compared to the other species. (Table 2.2). The amount of starch in current and old stems were similar and increased slightly during the study. For example, stem starch concentrations ranged from 28.76 to 45.81 mg/ g DW on day 8 and fiom 61.48 to 76.72 mg/g DW on day 32. Flooded stem tissues had 19 to 28 % more starch than control seedlings during each harvest interval. Black ash seedlings had more starch present in both current and old stems than green ash seedlings on day 8. Similar results were obtained on day 16 for the old stems with white ash seedlings having intermediate values, which did not differ fi'om the other two species. By day 32, starch concentrations in the stems of black and green ash seedlings were no longer different. However, white ash seedlings now had a lower current stem starch concentration than green ash seedlings. Flooding also reduced the starch concentrations of below ground tissues. On day 8, fine root starch concentrations ranged fiom 4.57 to 12.13 mg/g DW. During this harvest, flooded seedlings consistently had lower starch concentrations (Table 2.3). The same was observed on day 16 except that differences were greater between flooded and control black and white ash seedlings and starch concentrations ranged from 4.65 to 29.86 mg/ g DW. Overall, flooded black and white ash seedlings had 65 and 76 % less starch than the control seedlings. At 32 days, fine and coarse roots of flooded seedlings had 84 to 88 % and 57 60 % less starch, respectively, when compared to control seedlings. Fine roots of flooded seedlings had lower starch concentrations with 2.95, 3.05, and 5.98 mg/g DW for white, black, and green ash seedlings, respectively (Table 2.3). For this harvest, starch fine root concentrations ranged from 2.95 to 38.63 mg/ g DW. In comparison, green ash seedlings had higher coarse root starch than the other species throughout the study. 58 Table 2.2. Mean starch concentration of leaf, current and old stem, and coarse roots for control and flooded black, green, and white ash seedlings harvested after 8, l6, and 32 days of flood stress. Current stems harvested on day 16 were not available (N/A). Means, followed by the same letter within a column for each separate harvest, do not differ significantly (Duncan, P5005). Main effects Glucose content in mg per g DW for each plant component Day 8 Harvest Leaf Current Old Coarse stem stem root Species Black ash 50.85 4063a 45.81a 27.87b Green ash 54.61 28.76b 38.30b 47.75a White ash 38.88 30.44b 42.34ab 32.43b Treatment Control 40.81b 28.05b 35.25b 44.00a Flooded 55.41a 38.51a 49.053 27.88b Day 16 Harvest Species Black ash 75.79a N/A 59.70a 36.72b Green ash 88.13a N/A 49.96b 61 .86a White ash 55.41b N/A 56.59ab 38.63b Treatment Control 66. 18b N/A 49.57b 5 l .05 Flooded 8004a N/A 61 .26a 40.76 Day 32 Harvest Species Black ash 59.95b 65.83ab 74.54 33.27b Green ash 102.97a 76.72a 74.10 57.32a White ash 34.680 61.48b 69.41 38.65b Treatment Control 68.16 58.45b 64.95b 61 .57a Flooded 63.57 77 .57a 80.42a 24.60b 59 Table 2.3. Mean starch concentration of fine roots for control and flooded black, green, and white ash seedlings harvested after 8, 16, and 32 days of flood stress. Means, followed by the same letter within a column for each species x treatment interaction, do not differ significantly (Duncan, n=8, P5005). Species x Treatment Glucose content in mg per g DW for fine root tissue Harvest Time Day 8 Day 16 Day 32 Black ash control 9.81ab 13.08ab 24.75b flooded 6.89ab 4.65b 3.050 Green ash control 12.13a 24.43ab 38.63a flooded 8.32ab 2004ab 5.980 White ash control 8.84ab 29.86a 18.11b flooded 4.57b 7.22b 2.950 60 DISCUSSION Flooding black ash (Fraxinus nigra), green ash (Fraxinus pennsylvanica), and white ash (F raxinus americana) seedlings during the growing season altered the biomass and starch concentrations in specific tissues. After the 32 day flood, dry weights from seedlings highlighted species and flood induced differences. Leaf and current stern dry weights were not different except for flooded black ash seedlings, which had lower dry weights (Table 2.1). Black ash seedlings were also shorter in height compared to the green and white ash seedlings (data not shown). This is evident when observing the old stem dry weight of black ash seedlings, which are considerably lower when compared to green and white ash seedlings. The smaller size of each black ash seedling may account for lower accrued growth and an increased susceptibility to flood stress. This is important since roots are sensitive to oxygen deficiency and that no records of sustained growth by anoxic roots has been reported (Armstrong et a1. 1994). Under hypoxic or anoxic conditions, seedling survival by anaerobic metabolism is dependant on the availability of substrates present in the roots for respiration (Jackson and Drew 1984). Root starch concentrations were negatively impacted by imposing flood stress (Table 2.2 and 2.3). After 32 days, flooded ash seedlings had approximately 60 % less starch in its’ coarse roots than control seedlings. In comparison, fine roots suffered more drastic reductions, with approximately 84 to 88 % less starch compared to control seedlings. Between day 8 and 16, coarse root starch for all seedlings increased whereas no change occurred between day 16 and 32. Throughout the study, green ash coarse roots had higher 61 starch than both black and white ash seedlings. These absolute values may indicate that more starch is present or required in the root system of green ash. Changes in the fine roots indicate that green ash accumulated starch as flooding proceeded through day 16, then declined by day 32. This starch may have been used by these roots to survive the flooding. White ash also did the same to a much lesser extent and, thus, was less able to tolerate the hypoxic conditions. In contrast, black ash did not accumulate starch in the fine roots during flooding. This information may suggest that anaerobic fermentation took place and that starch was utilized for maintenance of roots when exposed to low oxygen levels for an extended period (32 days). Another hypothesis to consider is that cell membrane injury occurs after a certain amount of time in anoxic conditions resulting in the cell contents leaking out. Both of these events can occur in response to flood stress. Not surprisingly, these costly processes can drastically reduced root dry weights. During each harvest, we observed that flooded roots were partly yellow with brown and black discolorations of varying degrees mainly at the tips. Root discoloration was most pronounced in black ash seedlings and least in green ash seedlings. Hence, flooded black and white ash seedlings had a 46 % reduction in root dry weight compared to their control counterparts. Similar results by Smith and Boume (1989) had 50 and 48 % reduction in root mass for 28 day flooded pecan seedlings when compared to controls during bud break or active growth, respectively. For green ash seedlings, however, root dry weights were similar regardless of the flood treatment. Green ash seedlings were clearly more tolerant to flooding than black and white ash seedlings. Research by Good and Patrick (1987) noted that root aeration and anaerobic respiration contributed to the 62 survival of green ash seedlings flooded for a period of 9.5 months (mid-July to May). This adaptive response to flooding may explain why green ash coarse roots had more starch and why root dry weight was not different from control seedlings. In general, studies on various species have outlined the inhibition of root growth in response to flood stress conditions (Pezeshki and DeLaune 1990; Liu and Dickrnann 1992; Topa and Cheeseman 1992). Reductions in root dry weights can be attributed to the combined effects of root death and inhibited root growth (Smith and Boume 1989). Inasmuch as the root system becomes compromised during flooding, limited water and nutrient uptake subsequently impede above-ground growth and development. Both leaves and stems starch concentrations were influenced by flooding stress. Starch concentrations in current and old stems were similar and increased throughout the study. Black ash seedlings had a greater starch concentration in stem tissue on days 8 and 16. These values were not different for old stem starch observed in white ash seedlings. In comparison, green ash seedlings had slightly higher starch content than the other two species for the final harvest. These findings may suggest that starch allocation to the stem occurs at-different rates or times in these three species. In general terms, stems are viewed as a storage compartment where reserves accumulate mostly in the absence of competition by vegetative or reproductive sinks (Goldschmidt and Koch 1996; Loescher et al. 1990). Surprisingly, stems of flooded seedlings had 19 to 28 % more starch than control seedlings regardless of the species. Possibly indicating that flooding increases starch allocation to stems because growth is inhibited and translocation of carbohydrates to the roots may be too costly or wasteful. Additional research is needed to collaborate these findings and it’s significance at the whole 63 plant level. Starch allocation to the stern, however, must be linked to photosynthesis. Therefore, starch accumulation in the leaves may also reveal how flooding stress modifies above- ground processes. For example, high leaf starch concentrations were observed on day 8 and 16 in flooded seedlings; but not on day 32. White ash seedlings had much lower starch concentrations on day 16. By day 32, white ash leaves had a lower starch content than black ash leaves with an intermediate value, which was also lower when compared to starch in the leaves of green ash seedlings. This suggests that flooding may increase leaf starch synthesis initially; but, not after a longer period of time. High leaf starch concentrations, however, can lead to a decrease in net photosynthetic rate. This occurs through feedback inhibition where high leaf starch concentrations lead to the formation of additional soluble carbohydrates, which in turn decrease photosynthesis (Mauney et al. 1979; Luxmoore 1991). Ultimately, the changes caused by flooding will control the quantity of reserves that is stored prior to the end of the growing season. Our study has shown that black and white ash seedlings had a sizeable reduction in root biomass and starch. In contrast, green ash seedlings had no change in root biomass and a higher concentration of starch in its’ coarse roots. Fine root starch concentrations, however, were similar to values in the other two Fraxinus species. The ability of a seedlings to tolerate flooding and recover depends on environmental conditions, time or stage of development, and seedling health. Additional research will be needed to improve our understanding of how and to what extent flood stress influences stored carbohydrates at the whole plant level. This is especially important with respect to root reserves, which act as an important and perhaps the major source of substrates 64 for the subsequent year’s early respiration, growth, and development (Loescher et al. 1990). 65 LITERATURE CITED Armstrong W., R. Brandle and MB. Jackson 1994. Mechanisms of flood tolerance in plants. Acta Bot. Neerl 43: 307-358. Chirkova T.V. and TS. Gutrnan 1972. Physiological role of branch lenticels in willow and poplar under conditions of root anaerobiosis. Sov. Plant Phys. 19: 289-295. Daie J. 1985. 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Effects of root hypoxia and a low P supply on relative growth, carbon dioxide exchange rates and carbon partitioning in Pinus serotina seedlings. Physiol. Plant. 86: 136-144. 69 SUMMARY CONCLUSION In summary, flooding black, green, and white ash seedlings for a period of 32 days induced various changes in growth, gas exchange, and starch content. Recognition of each response and to what extent it varies during the course of flooding can indicate how seedlings from the genus Fraxinus differ in their ability to tolerate flood stress. Differences in above and below-ground processes were observed. During flooding, a reduction in stomatal conductances occurred on days 2, 4, and 8, followed by lower assimilation rates by day 4, 8, and l l for black, white, and green ash seedlings, respectively. These findings indicate that black ash seedlings are more sensitive to the low soil oxygen levels that accompany flood stress whereas green ash seedlings were not as sensitive. Further investigation is needed to ascertain the factors mediating this reduction in stomatal conductance. Growth responses also varied according to species and duration of flood. Minimal biomass growth and minor changes in leaf number, stern height, and caliper were observed for black and white ash seedlings. Caliper growth of flooded black ash seedlings was greater after 32 days of flood stress compared to no change for the white ash seedlings. Leaf area expansion of white ash seedlings was reduced during the entire flood compared no change for black ash seedlings. Prolonged flood stress (ie. 32 days) decreased root dry weights in both black and white ash seedlings by 46 %. These findings demonstrate that as flood duration increases root death and inhibition of growth result. In contrast, differences in biomass were not observed for green ash seedlings regardless of treatment, but stem height, leaf number, and leaf area were reduced by flooding for 8, 16, and 32 days. The ability of green ash seedlings to actively grow throughout the 32 day flood suggests that other factors 70 contribute to its’ flood tolerance. Besides observations that carbon assimilation and stomatal conductances were higher during the flood than for the other two species, hypertrophied lenticels, which are thought to improve gas exchange, were observed on the submerged stems for all three species. Many were observed in black and green ash seedlings within 8 to 12 days whereas those for white ash developed later as the flood progressed. At the whole plant level, variation in growth and gas exchange must also influence the allocation of starch reserves. During the study, starch content varied with tissue type, flood duration, and species. Root starch concentrations were negatively impacted by imposing flood stress. After 32 days, flooded ash seedlings had approximately 60 % less starch in its’ coarse roots than control seedlings. In comparison, fine roots suffered more drastic reductions, with approximately 84 to 88 % less starch compared to control seedlings. Changes in the fine roots indicate that green ash accumulated starch as flooding proceeded through day 16, then declined by day 32. This starch may have been used by these roots to survive the flooding. White ash also did the same to a much lesser extent and, thus, was less able to tolerate the hypoxic conditions. In contrast, black ash did not accumulate starch in the fine roots during flooding. This information is pertinent and can tentatively explain why root dry weights for control and flooded green ash seedlings were not different. Differences in starch at the leaf and stem level were more difficult to explain over time. Our interpretation for these differences (without enumeration of each change) suggests that carbon is allocated to the stems at different times or rates for each species. In addition when flooding stress reduced leaf and stem growth, it promotes starch allocation to the stem because it may be too costly for the plants to translocate carbohydrate to the roots. Moreover, 71 roots may not act as a resource sink when anoxic conditions are present in the soil. More research is needed to improve our understanding of how and to what extent flood stress influences stored carbohydrates at the whole plant level. Ultimately, the quantity of starch stored and allocation plays an important role in seedling survival. It can maintain grth during periods of environmental stresses and ensure a faster recovery. Physiological recovery for each species was modified by duration of the flood. Once a reduction in stomatal conductance occurred, all three species required a 12 to 16 day period to recover. As the flood duration increased to 32 days, black and white ash seedlings recovered in 12 days compared to green ash seedlings which recovered after 8 days. Consequently, green ash seedlings were more tolerant to increased duration of flood stress compared to black and white ash seedlings where root biomass and starch reserves were reduced. The significance of this work was to characterize the flood tolerance of three different F raxinus species during the summer. Comparison of the morphological and physiological response will add to our knowledge of flood tolerance mechanisms of woody species. In addition, it can indicate why ashes occupy such broad ecological niches. Little research has been done on how closely related species in the genus Fraxinus cope with environmental stresses such as flooding. Practical benefits of this research can also be applied in plant selection process and identifying species able to tolerate flooding and soil compaction common in urban landscapes. For example, our findings indicate that green ash is more flood tolerant than black and white ash. 72 "11111111111111111