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1 PHYSIOLOGICAL AND BIOCHEMICAL ASPECTS OF NATURAL AREA WEEDS By KURT MATTHEW VOLLMER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER O F SCIENCE UNIVERSITY OF FLORIDA 20 10
2 20 10 Kurt Matthew Vollmer
3 To my family
4 ACKNOWLEDGMENTS I would like to express my sincere appreciation to my advisor Dr. Gregory MacDonald for his time, guidanc e, and financial support. His expertise has been instrumental throughout the course of my study. I would also like to thank Kathy Pietro and her team at South Florida Water Management District for the ir funding, assistance, and collaboration. I would li ke to thank Dr. John Erickson for his time and assistance with various aspects of project as well as my other committee members Dr. Maria Gallo and Dr. Brent Sellers. I would like to give special thanks to Bob Querns and Jing Jing Wang for assistance with laboratory analyse s. I would also like to e x tend my appreciation to Anna Gr ei s and Maninderpal Singh for their assistance in data collection. I would also like to express my gratitude to Dr. Jason Ferrell Dr. Ken Langeland, and Dr. William Haller for t heir expertise and support. I would like to extend my appreciation to all the Weed Science graduate students for their encouragement and camaraderie. Finally, I would like to thank my family, especially my parents. Their support has been unwavering an d it is a blessing to have such loving people in my life.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ .......................... 7 ABSTRACT ................................ ................................ ................................ ................................ .. 10 INTRODUCTION ................................ ................................ ................................ ......................... 12 1 BIOCHEMICAL RESPONSE OF TYPHA DOMINGENSIS TO WATER DEFICIT IN STORMWAT ER TREATMENT AREAS OF SOUTH FLORIDA .............................. 15 Introduction ................................ ................................ ................................ ........................... 15 Materials and Methods ................................ ................................ ................................ ........ 20 Results ................................ ................................ ................................ ................................ ... 26 Carbohydrate Content ................................ ................................ ................................ .. 26 Chlorophyll and Carotenoid Content ................................ ................................ .......... 27 Protein Content ................................ ................................ ................................ ............. 27 Abscisic Acid Content (ABA) ................................ ................................ ....................... 27 Peroxidase Activity ................................ ................................ ................................ ....... 28 Catalase Activity ................................ ................................ ................................ ............ 28 Glutathione Reductase Activity ................................ ................................ ................... 28 Superoxide Dismutase (SOD) Activity ................................ ................................ ....... 29 Ascorbate Content ................................ ................................ ................................ ........ 29 Discussion ................................ ................................ ................................ ............................. 29 Conclusions ................................ ................................ ................................ ........................... 33 2 RECOVERY OF TYPHA DOMINGENSIS AFTER PERIODS OF PROLONGED DROUGHT ................................ ................................ ................................ ............................ 41 Introduction ................................ ................................ ................................ ........................... 41 Materials and Methods ................................ ................................ ................................ ........ 43 Results ................................ ................................ ................................ ................................ ... 45 Decline in Chlorophyll and Photosynthetic Rates ................................ .................... 45 Recovery Leaf Analysis ................................ ................................ ............................... 45 Rhizome Status ................................ ................................ ................................ ............. 46 Discussion ................................ ................................ ................................ ............................. 46 Conclusions ................................ ................................ ................................ ........................... 49 3 PHOTOSYNTHETIC RESPONSES OF NATIVE AND NON NATIVE INVASIVE SPECIES IN NORTH CENTRAL FLORIDA ................................ ................................ .... 56 Introduction ................................ ................................ ................................ ........................... 56 Overview of Invasive Species Examined ................................ ................................ .. 56 Materials and Methods ................................ ................................ ................................ ........ 60
6 Results ................................ ................................ ................................ ................................ ... 62 Trees ................................ ................................ ................................ ............................... 62 Vines ................................ ................................ ................................ ............................... 62 S hrubs ................................ ................................ ................................ ............................ 63 Discussion ................................ ................................ ................................ ............................. 63 Conclusions ................................ ................................ ................................ ........................... 65 4 SUMMARY AND CONCLUSIO NS ................................ ................................ ..................... 71 Response of Typha domingensis to Water Deficit in Stormwater Treatment Areas ................................ ................................ ................................ ................................ .. 71 Recovery of Typha domingensis following Dr ought ................................ ....................... 71 Photosynthetic Responses of Native versus Invasive Plants ................................ ....... 72 LIST OF REFERENCES ................................ ................................ ................................ ............ 74 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ........ 80
7 LIST OF TABLES Table page 2 1 Carbohydrate concentration of shootbase tissue in Typha domingensis as a function of water deficit level at 7 months ................................ ................................ ... 37 2 2 Carbohydrate concentration of rhizome tissue of Typha domingensis as a function of water deficit level at 4 and 7 months. ................................ ....................... 37 2 3 Chlorophyll, carotenoid, total protein, and abscisic acid content in leaf tissue of Typha domingensis as a function of water deficit level at 4 and 7 months. ...... 38 2 4 Total protein content in rhizome tissue of Typha domingensis as a function of water deficit level at 4 and 7 months. ................................ ................................ ........... 38 2 5 Antioxidant concentration of leaf ti ssues as a function of water deficit level of Typha domingensis at 4 and 7 months. ................................ ................................ ....... 39 2 6 Antioxidant concentration of rhizome tissue of Typha domingensis as a function of water deficit level at 4 and 7 months. ................................ ....................... 40 3 1 The effect of drought period on the dry weight of new Typha leaves produced during a 5 week recovery period after drought was terminated. ............................. 53 3 2 The effect of drought period on the number of new Typha shoots produced during a 5 week recovery period after drought was terminated. ............................. 53 3 3 The eff ect of drought period on chlorophyll content of Typha leaves produced during a 5 week recovery period after drought was terminated. ............................. 54 3 4 The effect of drought period on the photosynthetic ra te of recovered Typha leaves produced during a 5 week recovery period after drought was terminated. ................................ ................................ ................................ ........................ 54 3 5 The effect of drought period on dry weights of Typha rhizomes produced during a 5 wee k recovery period after drought was terminated. ............................. 55 3 6 The effect of drought on total nonstructual carbohydrate (TNC) content of Typha rhizomes produced during a 5 week recovery period recover y period after drought was terminated. ................................ ................................ ....................... 55 4 1 Photosynthetic responses of invasive versus native tree species found in North central Florida. ................................ ................................ ................................ ...... 68 4 2 Photosynthetic responses of invasive versus native vine species found in North central Florida. ................................ ................................ ................................ ...... 69
8 4 3 Photosynthetic responses of invasive versus native shrub species fo und in North central Florida. ................................ ................................ ................................ ...... 70
9 LIST OF FIGURES Figure page 2 1 Phosphorous filtration in stormwat er tre atment areas ................................ ............... 35 2 2 Antioxidant pathways ................................ ................................ ................................ ...... 36 3 1 Chlorophyll content in Typha domingensis following periods of drought (weaks aft er treatment initiation (WAT)) ................................ ................................ ..... 52 3 2 Photosynthesis in Typha domingensis following periods of drought (weeks after treatment intitiat ion (WAT)) ................................ ................................ .................. 52
10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science PHYSIOLOGICAL AND BIOCHEMICAL ASPECTS OF NATURAL AREA WEEDS Kurt Matthew Vol lmer May 20 10 Chair: Gregory E. MacDonald Major: Agronomy Excessive loading of nutrients into the low nutrient adapted Everglades Protection A rea Native plants including Typha domingensis u tilize excess nutrients such as phosphorous, forming monocultures and out competing surrounding vegetation. Mana gement of nutrient flow in the E verglades is dependent on stormwater treatment areas (STA s ), which use plant material as nutrient filters. T domingensis is a vital component of these STA s Water levels in STA s fluctuate constantly, making it difficult to maintain healthy Typha populations. Therefore, managers are in need of methods to detect stress especially drought, and its relationship to plant performance. An experiment was conducted at the STA facilities in south Florida to determine the effect of prolonged water stress on Typha. Plants were placed at three water depths with the surface of the water 1 cm above the soil surface, 30 cm be low surface, or 46 cm below the soil surface. Plants were harvested after four and seven months of drought conditions and the following biochemical tests were performed to analyze stress response: carbohydrate content (TNC), total chlorophyll, caroten oi d, protein and abscisic acid (ABA) and enzyme assays for peroxidase, superoxide dismutase, glutathione reductase, and ascorbate. There was a decrease in rhizome TNC and an
11 increase in rhizome protein indicating utilization of carbohydrates for maintenan ce and repair. Drought conditions also corresponded with a decrease in chlorophyl l and carotenoid content. Anti oxidant assays showed little correlation with prolonged drought stress. A separate study was conducted to determine the length of time T. doming ensis could survive complete drought conditions. Drought conditions were imposed for 5 through 9 weeks. After drought conditions were initiated, plant health was monitored for changes in photosynthesis and chlorophyll content. Analysis was performed on recovered rhizome and leaf dry weight, shoot number, and rhizome TNC. Additional measurements were also taken to compare photosynthetic rate and chlorophyll content. Despite a drop in rhizome dry weight, all plants were able to recover from all drought p eriods, and showed no differences for all parameters tested. A study of the photosynthetic responses of invasive plants versus native plants was conducted in natural areas of Alachua County, Florida. Plants were divided into 3 categories: trees, vines, and shrubs and photosynthetic measurements were taken on mature sun and shade adapted leaves. Invasive tree species showed higher light compensation points (LCP s) rates of light saturated photosynthesis (A max ) and respiration rates than native tree species; indicating a propensity to invade high light environments. Native vine species showed higher A max LCP, and respiration rates, as well as greater plasticity when moving from sun to shade when compared to invasive vines There were no significant differ ences in shrub species for any of the parameters tested.
12 CHAPTER 1 INTRODUCTION Cattails ( Typha spp.) are a family of native plants that inhabit both fresh and brackish waters. They often form dense, monotypic stands that can be problematic in certai n habitats. Anthropogenic nutrient inputs from agricultural and urban areas of South Florida have served to alter the vegetation of the Florida Everglades. This has resulted in the expansion of southern cattails ( Typha domingensis Pers.) in the Florida E verglades. As cattail populations increase, other native species that once dominated the area such as sawgrass ( Cladium jamaicense Crantz) are unable to compete with the expanding cattail populations. Several studies have confirmed that excessive nutrient loading is the cause of this increase. Cattails act as luxury consumers of phosphorous, whereby plants can accumulate phosphorous even when it is not necessary for biological function, a feature that also makes them excellent for phosphorous storage. The solution to managing these cattail populations lies in the regulation of nutrients into the Everglades ecosystem. To effect this regulation, South Florida artificial wetlands known as stormwater treatment areas (STA s ) have been constructed at the headwat ers of the everglades. These STAs remove and store nutrients from the water through plant growth and accumulation in humus. Due to the ability of T. domingensis to take up nutrients, it is a key component in these areas. Therefore, T. domingensis can be utilized in STAs to filter nutrient rich waters before they reach the Everglades, thus limiting the ecological effects of nutrient loading in the Everglades.
13 However, STAs also serve as flood control structures and water retention bodies, and undergo con stant fluctuation in water levels. As such STAs can be under long periods of water deficit. This can severely damage Typha populations, thus inhibiting nutrient regulation by STAs. The first part of this study was to determine the physiological and bioche mical responses ca ttails have to different levels of water deficit over time Cattails were exposed to three declining water levels over a period of seven months. Analysis of carbohydrate, protein, chlorophyll and abscisic acid (ABA) content, as well as e nzyme assays of ascorbate, catalase, peroxidase, glutathione reductase, and superoxide dismutase were used in an attempt to correlate water deficit with biochemical stress response. It was expected that an increase in antioxidant activity or abscisic acid or a decrease in chlorophyll or carbohydrate content could be used to correlate intensity of water deficits in Typha The second part of this study monitored the physiological response of cattails during the recovery period following complete drought C attails were subjected to up to 9 weeks of complete drought and allowed to recover. Photosynthetic rates, chlorophyll content, carbohydrate content, new shoot number, and rhizome and leaf dry weights were measured to examine the extent of Typha recovery a nd its correlation with drought period. It was expected that plants exposed to longer periods of drought would be slow or unable to recover from the imposed drought conditions. Cattails in the first two experiments showed efficient growth and photosynthet ic activity under greenhouse conditions. Photosynthesis appeared to be a viable measure of plant health and performance in Typha therefore it was thought that photosynthetic
14 efficiency could be a tool to study invasiveness. It was believed that invasive p lants would have greater photosynthetic efficiency compared to native plants in the same area. To test this hypothesis, a third study measured the photosynthetic responses of several invasive plants of various growth habits in North central Florida. Thes e responses were compared with those of surrounding native species and used to determine the type of light environment that would be inclined to favor the dominance of one species over another.
15 CHAPTER 2 BIOCHEMICAL RESPONSE OF TYPHA DOMING ENSIS TO WATER DEFICIT IN STORMWATER TREATMENT AREAS OF SOUTH FLORIDA Introduction Cattails ( Typha spp.) are a native wetland species found throughout Florida and the United States. Typha are typically characterized by a long cylindric al flower spike, app roximately 30 cm thick, linear, flattened leaves and an extensive rhizome system ( Bryson and DeFelice 2009 ). Cattails occur in most wetland areas, including lake margins shorelines, and drainage areas, and provide a vital nesting habitat for birds and othe r animals ( Baker et al. 1995 ). The southern cattail ( Typha domingensis Pers. ) can often be found in brackish and nutrient rich waters. Cattails are able to reproduce by two different me chanisms The primary me ans of rep roduction is via an extensive rhizome system. In early spring and throughout the summer, new sh oots will arise from the expanding rhizome system often forming dense monotypic stands. Cattails will also reproduce via seed. Plants generally flower from J uly to September with flowers being tightly condensed into a characteristic flower spike. Seeds tend to remain on the flower through winter and are dispersed in the spring by wind, water, and animals (Apfelbaum 1985). Cattails are often problematic alo ng lake margins and shoreline s, or areas where other vegetation is desirable. Abundant cattail populations can impact the ecology of an area by competing with other native plants for light, nutrients, and space. The expansion of T domingensis into the F lorida evergl ades is of particular concern. Over the past 60 years altered hydrology and increased loading of nutrients from agricultural and urban runoff has caused an expan sion of T. domingensis into sawgrass co mmunities (Chiang et al. 2000). As of 200 7, over 11,000 ha of T. domingensis had
16 replaced the historic Everglades landscape of sawgrass ( Cladium jamaicense Crantz ) ( Sklar et al. 2007 ). Phosphorous availability has been shown to influence growth and biomass partitioning in T. domingensis and it has phosphorous uptake rates three to thirteen times higher than that of s awgrass (Lorenzen et al. 2001). Furthermore, c attail/sawgrass competition is directly related to nutrient availability and hydroperiod. Cattails are adapted to high resource enviro nments, have expeditious growth rates, and high population expansion compared to species such as sawgrass Sawgrass and related species are adapted to low resource environments, have slow growth rates, and low population growth (Miao and Sklar 1998). Th erefore, excess phosphorous directly correlates to the over expansion of Typha populations in the Everglades. H istorically, the Everglades have been characterized as being a low nutrient ecosystem. At one time, runoff from these areas drained through the Everglades as sheet flow This ecosystem however, has been modified by the presence of over one thousand miles of canals, over one dozen pumping stations, and hundreds of flood al. 1989). Artificial wetlands, known as stormwater treatment areas (STAs), have been constructed in South Florida to curb the effects of excessive nutrient loading into the Everglades ecosystem. STAs use plant growth and the accumulation of dead plant material in a layer of peat to remove and store excess nutrients before the nutrient rich water reaches the E verglades (Figure 1 1). STAs utilize several submersed floating, and eme rgent plants, including T. domingensis In 2008 STAs reduc ed average flo w weighted mean concentrations of phosphorous from 129 to 26 parts per billion ( Pietro et
17 al. 2009 ). STAs rely primarily on the presence of T. domingensis which is responsible for the uptake of the majority of the phosphorous entering the system STAs serve not only to filter runoff, but also as water retention bodies and flood control structures. Due to weather and other environmental conditi ons water levels in the STAs fluctuate constantly. This can lead to difficulties in maintaining adequate popu lations of Typha when water le vels become too low or too high. Hydrologic stress, particularly drought, causes a multitude of problems including disruptions in membrane shape and integrity, disruption of water potential and loss of integrity, and denatura tion of cellular proteins ( Bray 1997 ). Many species are able to withstand changes in hydrologic stress due to tolerance or other mechanism s ( Bray 1997 ). Whole plant mechanisms can contribute to this tolerance but resistance to water deficit can also oc cur at the cellular level. Changes in several factors including: carbohydrate and protein partitioning, leaf photosynthetic potential, abscisic acid co ntent, and the activity of anti oxidant enzymes could be correlated with stress, including water deficit A dequate carbohydrate and protein suppl ies are important for g rowth and maintenance in plants. Carbohydrate skeletons are an integral part of the amino acids that comprise proteins. R hizome carbohydrate supply is particularly important in Typha species Rhizomes serve as carbohydrate storage organs during unfavorable conditions and periods of inactivity. Under stress conditions, there is a greater demand for carbohydrates to support cellular metabolism. Accordingly, i t has been shown that plants with larger carbohydrate reserves are able to withstand longer perio ds of stress (Sharma et al. 2008 ). Carbohydrates are a vital component of proteins and other
18 biological molecules. A decline in carbohydrate content under stress could be an indicator of carb ohydrates being used for growth and repair of damage tissues. Stomata are openings on a leaf surface that facilitate the uptake of CO 2 for photosynthesis and the release of water during transpiration. Stomates will open and close in response to environmen tal factors such as light, temperature, CO 2 concentration, and hormones. These changes in stomatal aperture are regulated by changes in turgor of the surrounding guard cells, which is influenced by ion efflux/influx. Cellular signaling is an important fa ctor in stomatal regulation and water deficit tolerance Abscisic acid (ABA) is a hormone that is known to inhibit growth and seedling germination, but it also plays a role in plant tolerance to water deficit. During embryo development levels of ABA incr ease when plants are under abiotic stress, leading to desi ccation tolerance and seed dormancy (White et al. 2000). ABA regulates most physiological responses through changes in gene expression which affect other physiological processes (Gonzalez Garcia et al. 2003) During periods of water deficit soil dries and ABA accumulates in roots and then leaves initiating a signal transduction cascade that results in stomatal closure (Finklestein et al. 2002). ABA promotes stomatal closure and decreases stomatal ap erture via a com plex pathway that involves influencing anion and cation channels increasing cytosolic pH activating phospholipase, and regulating various protein kinases and phosphatases (Hetherington 2001). As a result of stomatal closure water c annot b e lost via transpiration but carbon cannot be assimilated for photosynthesis. T his process can result in increased oxygen uptake via photorespiration, which can result in an increase in free radicals and radical oxygen species (ROS)
19 Oxygen forms radical oxygen by accepting unpaired electrons from various sources. These reactive oxygen species (ROS) disrupt the integrity of plasma membranes, causing serious cellular damage Three common ROS are superoxide (O 2 ), hydrogen peroxide (H 2 O 2 ), and the hydroxid e anion (OH ). Superoxide is often generated when NADPH is oxidized by heavy metal cations such as cadmium (Cd +2 ) (Tang et al. 2005), and under hypoxic conditions The oxidation of hydrogen peroxide by iron and copper cations forms water and the hydroxid e anion. The formation of ROS is usually quenched by the activity of antioxidants, substances that inhibit the reaction of molecules with oxygen radicals ( McKee and McKee 2003 ). Examples of common antioxidants are glutathione reductase, catalase, peroxid ase, ascorbate, and superoxide dismutase. Superoxide dismutase catalyzes the conversion of O 2 into H 2 O 2 (Figure 1 2a) Catalase or peroxidases are responsible for catalyzing the conversion the H 2 O 2 into O 2 and H 2 O (Figure 1 2b) Glutathione reductase re generates glutathione (GSH) from is oxidized form GSSH. GSH is a primary component in guaiacol peroxidase (GPX) (Figure 1 2 c ). Ascorbate catalyzes the reaction of superoxide, hydrogen peroxide, or the tocopheroxyl radical (Figure 1 2d) to form monodehydr oascorbic acid and/or dehydroascorbic acid (Shaikh 2004). Increasing levels of water stress could lead to the increase in RO S in such a case the activity of antioxidants would also increase. Populations of T domingensis in the STAs have been shown to de cline with fluctuating water levels; however, few studies have been performed to examine the cellular response of this species to these conditions. The objective of this study was to identify various physiological and biochemical parameters that may be as sociated with
20 hydrologic stress in T domingensis imposed by water deficit and correlate s tress with Typha decline and death, particularly as it relates to plant performance in the STA system. It was hypothesized that 1) there would be an increase in the activity of antioxidant enzymes and abscisic acid and 2) there would be a decrease in chlorophyll and carbohydrate content as period and duration of water stress increased Materials and Methods Experiments were conducted in outdoor greenhouse enclosure s located in the STA 1W research site near West Palm Beach, Florida in conjunction with the South Florida Water Management District. Shoot and rhizome material were collected from STA 1W, Cell 1B. Two plants each were established in pots constructed from heavy duty trash cans (150 L.) modified with drainage holes and a central well for water delivery. Growth medi um consisted of peat soil ( Euic hyperthermic Lithic Medisaprist ) with 75% organic content and pH 6.3 excavated from the STA site of plant collect ion. This was an organic soil consisting of the highly decomposed remains of plants and other organisms commonly found in South Florida. Plants were allowed to establish for six weeks before water deficits were imposed. The experiment utilized a randomiz ed block design with two treatments, water depth and time the depth was maintained. Three different water level regimes were implemented: completely saturated soil (control), soil water depth held to 30 cm below the soil surface, and soil water depth held to 46 cm below the soil surface. There were five replications per treatment. Plants were subjected to four and seven months of continuous water deficit Plants were harvested July of 2008 (initial), November of 2008 (4 MAT), and February of 2009 (7 MAT). Translucent c anopies were constructed over the top of all plants to prevent rainfall from altering the imposed h ydrologic regime
21 At harvest plants were divided into leaf, rhizome, and shootbase tissue Samples desig nated for biochemical analysis were placed in freezer bags and immediately frozen in liquid nitrogen to halt all cellular activity. Samples were then transported on ice to the University of Florida in Gainesville for biochemical analysis. Tissue samples we re ground in liquid nitrogen prior to analysis and stored at 2 0 C All analyses were perfor med on leaf and rhizome tissue. To determine carbohydrate content, 30 cm sections of the rhizome and shootbase tissue were placed in a forced air oven at 105 C fo r one hour in order to denature respiratory enzymes, and the n dried for three days at 60 C T he tissue was then ground through a 20 mesh stainless steel screen using a Wiley Mill 1 Carbohydrate content was determined based on procedures of Smith (1981) a nd Christenson (1982). An enzyme mix consisting of 47.5 mL diH 2 O, 2.5 mL acetate buffer (0.2 M acetic acid + 0.2 M sodium acetate), 2.5 mL invertase concentrate, 1.25 g amyloglucosidase and 0.1 g thymol was used to digest starch and oligosaccharides into sugar monomers over a 24 hour period M onomers were quantified spectrophotometrically at 540 nm after reaction with an alkali reagent followed by an arsenomolybdate reagent. A standard glucose curve was used for quantification. In addition to the glucose standard curve, several additional standards were used to ensure accuracy. These include separate glucose standards of known concentration, starch standards of known concentration to ensure enzyme activity, known enzyme standar ds, and water control blanks. Total nonstructural carbohydrates (TNC) consist both of starch and free sugars therefore to separate starch and sugars samples were split. Free sugars were extracted prior to 1 Thomas Scientific, Swedesboro, NJ
22 enzymatic digestion and the remaining samples we re extracted by the means previously mentioned. Starch content was then obtained by subtracting sugars from TNC content. Chlorophyll and carotenoid content was determined using methods from MacDonald (1994). Tissue was homogenized in a 2:1 chloroform/met hanol and solids extracted using Fischer P8 filter paper 2 Samples were evaporated to dryness and resuspended in 80% acetone. Absorbance was measured spectrophotometrically at 470, 646, and 663 nm. Chlorophyll a was determined using the equation: 12 .21 X A663 2.81 X A646 and chlorophyll b was determined using the equation: 20.13 X A646 5.03 X A663. Total chlorophyll was determined using the equation: ((chlorophyll a + chlorophyll b ) X 2)/ sample weight. Total carotenoid content was determined using the equation: (1000 x A 470 3.27 x chl a 104 x chl b )/229. Abscisic acid (ABA) content of leaves was quantified in leaves using an ABA assay kit from Phytodetek 3 The assay uses an enzyme linked immunoassay (ELISA) procedure with monoclonal antibodi es that are specific to ABA. Samples were quantified using standard curves outlined in the protocol. Quantification was determined based on percent absorbance of the sample, with lower absorbanc e indicating higher ABA content. Total protein content was determined using the Bradford mircoprotein assay (Bollag 1991). A set of quantitative standards were made using bovine serum albumen at concentrations of 200, 100, 50, 25, and 0 ppm in 100 mL protein extraction buffer (100 mM potassium phosphate, pH 7.5) Samples were also ground in the protein 2 Thermo Fischer Scientific, Inc., Waltham, MA 3 Sigma Aldrich, Inc., St. Louis, MO
23 L of Bradford working solution [ 425 mL DI water + 30 mL 85% phosphoric acid + 15 mL 95% ethanol + 30 mL Bradford stock solution (700 mg Coomassie Brilliant Blue G + 400 mL 85% o phosphoric acid + 200 mL 95% ethanol)] The solution was allowed to react for 2 minutes and then transferred to a 1 mL quartz cuvette and absorbance determined spectrophotometrically at 595 nm. The peroxidase assay was ba sed on Bergmeyer 1972. Frozen plant tissue was ground in 2 mL of chilled 100 mM potassium phosphate buffer (pH 7.0), then centrifuged at 4000xG for 10 minutes. The solution was prepared using 2.8 mL of the of sample or enzyme standard (horseradish peroxidase in kphos buffer, 25units/ml). Kinetic analysis of enzyme activity was done spectrophotometricall y, at a wavelength of 436 nm for 360 seconds at 20 second intervals The unit of per cent activity was calculated based on the enzyme standard (assumed to have 100% activity). Per cent inhibition was calculated as % inhibition = (100 % sample activity). Catalase activity was based on methods established by Johansson and Borg (1988), and Wheeler et al. (1990). Plant material was homogenized in ice cold 25 mM potassium phosphate buffer, pH 7.0 and 50% (w/v) homogenates were prepared, and diluted with buff er as appropriate. Homogenates were treated in an ultrasonic ice bath (35 Hz; 95 W) for 15 seconds to liberate catalase from sub cellular particles. A standard assay reagent consisting hydrogen peroxide was used. The enzymatic
24 Standard solutions of formaldehyde at concentrations of 500, 250, 125 62.5, 37.5 and 0.0 M were prepared in 25 mM phosphate buffer, pH 7.0. The reaction mixture was incubated at 20C with continuous shaking for 20 minutes. The reaction was terminated by the addition of 50 mM purp ald in 480 mM hydrochloric acid. This was mixed briefly on a vortex mixer and incubated for another 20 minutes. The product of the reaction was then oxidized by dissolved in 470 mM potassium hydroxide to each tube and vorte xed again. Samples were then placed in microcuvettes and absorbance read spectrophotometrically at 550 nm. Glutathione reductase was activity was based on the colorimetric assay kit provided by Sigma Aldrich Inc., USA. The reaction mixture contained 500 oxidized glutathione 0 mM potassium phosphate, pH 7.5 containing 1 mM EDTA), 100 mL of enzyme sample or positive control ( lyophilized powder containing yeast glutathione reductase in phosphate buffer, pH 7.5 + EDTA and tr ehalose) This was placed in a 1 mL quar tz cuvette and the reaction was initiated by the Kinetic analysis of activity was done spectrophotometrically at a wavelength of 340 nm. Each cycle lasted 2 minutes with readings at 10 second intervals. Enzyme concentration was calculated using the following formula: Units/ml = ( A sample A blank ) x (dilution factor)/ mM x (volume of sample in ml) Where A sample = change in absorbance for the plant samp le A blank = cha nge in absorbance for the blank Dilution factor = the quantity of sample used compared to the total volume assayed.
25 mM (the extinction coefficient of NADPH) = 6.22 mM 1 cm 1 (Shaikh 2004). Superoxide dismutase (SO D) activity was based on the indirect developed by Forman and Fridovich (1973). Samples were homogenized in 0.1 M sodium phosphate buffer ( pH 7.0 ) at 4C for 2 minutes and 50% (w/v) homogenates were prepared, and diluted with buffer The homogenate was t hen centrifuged at 4C for 15 minutes at 4,000 g, and the supernatant was filtered using No. 1 Whatman filter paper 4 The buffer was exchanged and l ow molecular weight components were removed by applying 1 mL of the filtrate to a 10 mm Sephadex G 25 co arse gel filtration column equilibrated in 50 mM sodium carbonate solution (pH 10.2) The reaction mixture consisted of 2 mL of 50 mM sodium carbonate buffer, 0.3 mL 0.1 mM EDTA, 0. 3 mL 0.01 ferricytochrome c, 0.3 mL of 0.5 mM xanthine, of the samp le filtrate was added to the reaction mixture in a 3 mL cuvette at 25 C. xanthine oxidase The cuvette was placed in the spectrophotometer and a kinetic program was set up to read absorbance at 550 nm fo r 2 minutes at 10 second intervals. Blanks were run to determine activity of all reaction mixture components except the sample filtrate. Units of percent activity were calculated based on the blank ( 100 units of activity ) Units of per cent inhibition wer e calculated as 100 % activity of the sample, and expressed as Ascorbic acid content was determined using the AOAC method (43.056 43.060; 1980 ed.) P lant material (0.5 g) was pulverized by gentle grinding in 5 mL metaphos ph oric acid acetic acid solution (pH 1.2), and centrifuged for 15 minutes at 5000g. A 2 mL aliquot of the supernatant was taken and mixed with 5 mL 4 Whatman, Inc., Piscataway, NJ
26 metaphos ph oric acid acetic acid solution for a final volume of 7 mL The sample was then titrated with 2,6 dichloroindophenol (DCIP) until the solution turned pink in color, indicating a reduction in the solution by ascorbic acid. Ascorbic acid levels were calculated based on the amount of DCIP required for a visual color change to pink. A standard curve was generated based on ascorbic acid standards of 0, 200, 400 600, 800 and 1000 ppm prepared in metaphos ph oric acid acetic acid solution. Blanks were calculated using only metaphosphoric acid acetic acid solution. Levels of ascorbic acid were calculated using the following formula : mg ascorbic acid/g plant materi al = (X B) x (F/E) x (V/Y); where X = average ml of sample titrated with DCIP, B = average ml for blank titrated with DCIP, F = mg ascorbic acid equivalent to 1.0 ml indophenol standard solution (based on the standard curve), E = number of grams of plant m aterial assayed, V = volume of initial assay solution and Y = volume sample aliquot titrated. Data were subjected to analysis of variance using SAS 9 1 to test for main factor and effects and interactions. There were significant treatment by time i nteractions; therefore data is presented for each harvest time. Results Carbohydrate Content There w as an observed increase in TNC levels in sho otbase tissue at the 30 and 46 cm water levels co mpared to the control following 7 months of water deficit but these results did not differ significantly (Table 2 1). S imilar results were observed with starch levels, but there w ere no differences in soluble sugar content (Table 2 1). C omparisons could not be made over time due to a lack of material from the harvest 4 months after treatment (MAT). In contrast, there was a n observed decrease in TNC content in rhizome tissue as water levels decreased below the soil surface at both 4 and
27 7 MAT harvests; however, these values did not differ significantly (Tab le 2 2). Starch content of rhizomes decreased as water l evels below the soil increased (Table 2 2). There was a slight increase in rhizome sugar content as water level below the surface decreased 7 MAT (Table 2 2). It was also interesting to note that h igher rhizome TNC and starch levels were found 7 MAT, with concomitantly lower levels at the 30 and 46 cm water levels compared to the controls. Chlorophyll and Carotenoid Content Chlorophyll content did not differ significantly across all water levels 4 MAT Chlorophyll content decrease d at the 30 and 46 cm water levels 7 MAT (Table 2 3). Carotenoid content was also similar at all water levels 4 MAT There was a decrease in carotenoid content as water level below the soil surface decreased 7 MAT (Table 2 3). Protein Content There were no significant differences in l eaf total protein 4 MAT at all water levels. There was an overall decrease in leaf protein content 7 MAT, but no significant differences among treatment levels (Table 2 3). There were no s ignificant differences in r hizome total protein 4 MAT for all water levels but there was a marked increase in protein levels 7 MAT (Table 2 4). At this later harvest, there was greater protein content at the 46 cm water level, although this was not stati stically significant. Abscisic Acid Content (ABA) There were no significant differences in l eaf abscisic acid content at all water levels 4 MAT (Table 2 3). There was an observed increase in ABA 7 MAT compared to 4 MAT but values were similar and highl y variable. The lowest level of ABA detected 7 MAT was at the 46 cm water level; presumably the level that induced the highest level of stress.
28 Peroxidase Activity Results 4 MAT show a concomitant decrease in leaf peroxidase activity from the 0 to the 46 cm water levels, although this was not statistically significant (Table 2 5). There was also a concomitant decrease in leaf peroxidase from the 0 to 46 cm water depth 7 MAT, but these values did not differ significantly. Rhizome peroxidase activity was 10 times less than observed for leaf tissue, and also showed an observed decreased from the 0 to the 46 cm water levels 4 MAT but these results did not vary significantly (Table 2 6). There were no significant differences among treatments for rhizome peroxidase activity 7 MAT. Catalase Activity There were no significant differences in l eaf catalase levels 4 MAT; however levels were observed to be lower at the 30 cm water level compared to control and 46 cm depth plants. There were no observable differences in l eaf catalase levels 7 MAT compared to 4 MAT and there was no significant difference between water defici t treatments (Table 2 5). Rhizome catalase levels were observed to be the lowest at the 30 cm w ater level 4 MAT, bu t these differences were not significant. There were no differences in catalase levels 7 MAT compared to 4 MAT, and with no significant differences among treatment levels (Table 2 6). Glutathione Reductase Activity There were no significant differences in leaf glutathione reductase activity 4 MAT (Table 2 5). There was an observed increase in leaf glutathione reductase activity 7 MAT, but no differences among treatments. In contrast, rhizome glutathione reductase activity was highest 4 MAT, with the high est activity occurring at the 30 cm water level (Table 2 6). There was an observed reduction in r hizome glutathione reductase activity
29 7 MAT, with higher levels found at the 30 and 46 cm water levels compared to control levels but these values were not s ignificantly different. Superoxide Dismutase (SOD) Activity There were no differences in l eaf SOD activity 4 MAT or 7 MAT (Table 2 5 ). There were no significant differences in rhizome SOD activity 4 MAT or 7 MAT. However, there was an observed increase in r hizome SOD activity 7 MAT at the 30 and 46 cm water levels compared to the control (Table 2 6). Ascorbate Content There no significant differences in leaf ascorbate at all treatment levels 4 MAT (Table 2 5) with the highest levels being observed at the control depth. There were no significant differences in ascorbate 7 MAT, and a dramatic increase in leaf ascorbate content at the 46 cm water levels. There was no difference in rhizome ascorbate content for all treatments 4 MAT. There was an observed increase in ascorbate content 7 MAT compared to 4 MAT, with the highest levels of ascorbate being found at the 46 cm water depth (Table 2 6). Discussion C arbohydrate content appear s to be a good indicator of water deficit in T. domingensis Carbohydrates generated by photosynthesis are an integral part of Typha growth and persistence. As CO 2 uptake is diminished a plant must rely on stored carbohydrate supplies for growth and repair of proteins and other essential molecules. There was a decrease in tota l nonstructural carbohydrate as water levels below the soil surface decreased. Rhizomes serve as the primary storage tissue in Typha spp., and the storage of carbohydrates reflects the regenerative capacity of these plants as well as the ability of the pl ants to propagate via the spread of new shoots. The observed
30 increases in soluble sugar coupled with the observed decreases in starch suggest that starch is being converted to soluble sugars for growth and repair. However, there was an increase in TNC 7 M AT compared to 4 MAT It is possible that environment, season, and life cycle stage could be partially responsible for changes in carbohydrate levels irrespective of drought stress Typha angustifolia has been shown to translocate carbohydrates acropeta lly to above ground biomass initially during the growing season followed by a linear increase in basipedal carbohydrate movement (Sharma et al. 2008). Chl orophyll and carotenoid content also decreased as water levels decreased with significant differences 7 MAT. During the light reactions of photosynthesis, chlorophyll molecules absorb light to produce ATP and NADPH for CO 2 fixation in the dark reactions. As part of the energy capture process, an electron is liberated as water into hydrogen an d oxygen. As water becomes limiting that energy flow is interrupted but chlorophyll continue s to absor b light energy This excesses energy cannot be used due to limiting CO 2 from stomatal closure T his lead s to the production of ROS which ultimately cause s chlorophyll degradation. There was little change in leaf and rhizome p rotein content 4 MAT regardless of water stress condition. How e ver, at 7 MAT there was a trend towards increased levels of protein with increasing water stress. It is likely that these levels are artificially higher due to the decrease in carbohydrate co ntent. In addition, protein is expressed on a fresh weight basis ; therefore lower water content would show greater levels of protein. Abscisic acid (ABA) is a hormone that is produced as an imm ediate response to water stress. This hormone accumulates i n roots and is translocated to leaf tissue. Changes in gene expression result in rapid stomatal closure preventing water loss via
31 transpiration as well as carbon uptake by the plant (Bray 1997). T he immediate response of this hormone, as well as its inabi lity to be stored in plant tissue, suggests that long term water deficit could not be accurately assessed using this hormone. ABA is also involved with other environmental factors such as light and hypoxia (Bray et al. 1997). Furthermore, most leaf t issue at the 46 cm depth was severely wilted with a significant loss of fresh weight; this could have artificially elevated the levels since ABA is expressed as pmol/mg of fresh weight. The majority of the anti oxidant assays did not statistically correlate with levels of water deficit As menti oned previously, plants absorb excess energy due to decrea sed carbon assimilation and increasing oxygen uptake via photorespiration. This creates a number of radical oxygen species (ROS) (Luna et al. 2005). These ROS ar e quenched by antioxidants such as catalase, peroxidase, glutathione reductase, superoxide dismutase, and ascorbate (Reddy et al. 2004). Zhang and Kirkham ( 1994 ) showed that catalase and SOD activity in wheat was increased or maintained following initial stages of drought, but there was a decrease in activity as drought stress persisted. In contrast, they found that peroxidase activity increased in response to water deficit. This conflicts with the overall decrease over time in peroxidase activity of our studies with T. domingensis Glutathione reductase has been shown to increase in response to severe water deficits in barely (Smirnoff and Colombre 1988). Typha also showed increased in glutathione reductase activity, but levels were inconsistent with w ater level and time of water deficit Catalase is the primary hydrogen peroxide detoxifying enzyme in aerobic organisms, in plants it removes hydrogen per oxide produced by mitochondrial
32 oxidation of fatty acids, and photorespiratory oxidation (Yang and Poovaiah 2002). ROS formed as a result of water deficit should lead to an increase in catalase activity; however, catalase activity has also been shown to be influenced by photochemical reactions. Appleman (1952) found that the catalase activity of barely seedlings decreases as rapid chlorophyll synthesis occurs, and catalase activity increases when chlorophyll synthesis is blocked. Therefore changes in chlorophyll content may have a more profound impact on the catalase activity than water deficit in plants. Nayyar and Gupta (2006) showed that levels of ascorbate in wheat and corn declined with an increase in water stress. However, our studies showed an increase in ascorbate measured in Typha leaf and rhizome tissue at all depths following an extended period of water deficiency The role of ascorbate in plants is not merely to alleviate damage caused by cellular water deficits. Ascorbate also plays a role in photosynthesis, photoprotection, cell wall growth, and the synthesis hormones such as ethylene and gibberellins (Smirnoff and Wheeler 2000). These additional uses of ascorbate could help to explain the incr ease in content following the 7 MAT, a period when active growth is beginning to occur in non stressed plants. ROS species have been shown to play a role in cell signaling and programmed cell death ( Mittler 2002 ). Therefore, ROS may also be present as a r esult of normal cellular metabolism, rather than just in response to water deficit conditions. Furthermore, the activity of one antioxidant can be directly correlated with that of a diffe rent antioxidant. SOD converts superoxide into hydrogen peroxide, and catalase or peroxidase converts hydrogen peroxide into oxygen and water. At the same time
33 glutathione reductase is responsible for replenishing the g uaiacol necessary for peroxidase activity. An inhibition of any of a particular enzyme in one of thes e pathways would have a direct effect on another. Conclusions It was hypothesized that there would be an increase in antioxidant levels with an increase in water stress as a result of radical oxygen formation. While most of the anti oxidant parameters meas ured showed changes in response to water deficit many of these changes were unrelated to the levels and timing of stress. This may be because levels of most anti oxidants change rapidly with the onset of stress, and are readily recycled following use. It is possible that antioxidants may be used as a factor in evaluating water stress, but this would require a much shorter time frame. The fact that some of the control plants exhibited hig h levels of anti oxidant activity also raises quest ions about the quan tity of anti oxidants produced during normal metabolic activity in T. domingensis ROS are often generated as a result of factors such as toxic compounds, excessive light, or other environmental factors; however, cells will also generate ROS to destroy inv asive or foreign material. Whether or not water deficit is solely responsible for changes in antioxidant concentration is difficult to determine. The decrease in rhizome TNC and starch as water levels decreased is indicative of carbohydrates being used fo r growth and repair. However, accurate conclusions cannot be made due to seasonal and life cycle changes between harvests. The increase in carbohydrate content from 4 MAT to 7 MAT suggests that these factors may play a more important role in addition to water deficit in determining patterns of carbohydrate allocation and translocation.
34 Chlorophyll content was the best measurement used in determining impacts from long term water deficit There was an observable decrease in chlorophyll cont ent as water levels decreased and as periods of water deficits increased. The chlorophyll content of leaf tissue is easily detectable by a portable soil plant analysis development (SPAD) chlorophyll meter 5 This device uses light emitting diodes to determ ine the amount of light transmitted at 650 nm (chlorophyll) to the ratio of light transmitted at 940 nm (reference point) (Read et al. 2003). There are several advantages to using a SPAD meter; it is rapid, nondestructive, and allows several measurements to be taken over the course of a growing season. This would allow for an instantaneous assessment of plant health during periods of water deficit. It should be noted that these plants were never subjected to complete drought (lowest water level was 46 cm below the soil surface). While there was visible senescence of aboveground biomass following 7 months of water deficit all plants were able to recover when water levels were returned to normal and those at the 30 cm water depths had the greatest number of new shoots ( Pietro et al 2010). Therefore, T domingensis is better adapted to tolerating prolonged water deficits than previously set of environmental changes than simply prolonged water deficits 5 Spectrum Technologies, Inc, Plainfie ld, Illinois
35 Figure 2 1. Phosphorous filtration in stormwater treatment areas. (Source: https://my.sfwmd.gov/portal/page/portal/common/pdf/bts/b ts_sta.pdf Last accessed February, 2010)
36 Figure 2 2. Antioxidant pathways of A ) superoxide dismutase (SOD), B ) catalase (Cat), C ) glutathione reductase (GR) peroxidase (GPX) and D ) ascorbate.
37 Table 2 1 C arbohydr ate concentration of shootbase tissue in Typha domingensis as a function of water deficit level at 7 months. Months After Treatment Water below soil surface (cm) 0 30 46 Soluble sugar (mg/g) 7 38.0 1 a 2 11.6 36.5a 6.2 34.0a 4.6 Starch (mg/g) 7 131a 47.7 194a 23.1 195a 31.7 TNC (mg/g) 7 169a 43.9 230a 26.02 229a 32.9 1 Means of 5 replications followed by standard error 2 Means followed by the same letter within the same category and month after treatment are not significantly differ 95% confidence intervals Table 2 2 C arbohydrate concentration of rhizome tissue of Typha domingensis as a function of water deficit level at 4 and 7 months. Month s After Treatment Water below soil surface (cm) 0 30 46 Soluble su gar (mg/g) 4 47.4 1 a 2 18 46.0 a 9. 6 48.9 a 7. 3 7 29.8 a 6.65 37. 8 a 5.95 42.4 a 6.42 Starch (mg/g) 4 30 2a 117 225 a 48.1 147 a 73.9 7 404 a 11 6 318 a 59.9 288 a 64.1 TNC (mg/g) 4 349 a 130 271 a 51.7 195 a 80.2 7 433 a 11 0 349 a 60.6 331 a 63. 1 1 Means of 5 replications followed by standard error 2 Means followed by the same letter within the same category and month after treatment are not significantly di
38 Table 2 3 Chlorophyll, carotenoid, total protein, and abscisic acid content in leaf tissue of Typha domingensis as a function of water deficit level at 4 and 7 months. Month s After Treatment Water bel ow soil surface (cm) 0 30 4 6 Chlorophyl l ( g/g 1 ) 4 293 1 a 2 37.9 255 a 26. 8 258 a 39.4 7 303 a 29. 6 147 b 3 3 6 107 b 17.7 Carotenoid ( mg g 1 ) 4 0.95 a 0.30 0.90 a 0. 20 0.88 a 0.27 7 1.41a 0.16 0.50 b 0.0 9 0.39b 0.15 Total Protei n 4 135 a 16 147 a 9. 8 124 a 16 7 89.1 a 5.09 92.9 a 5.96 107 a 9.78 Abscisic Acid (pmol/mg) 4 0.36 a 0.12 0.2 2 a 0.0 2 0.198 a 0.0 2 7 0.75 a 0.35 0.96 a 0.33 0.47 a 0.08 1 Means of 5 replications followed by standard error 2 Means fol lowed by the same letter within the same category and month after treatment Table 2 4 Total protein content in rhizome tissue of Typha domingensis as a function of water deficit lev el at 4 and 7 months. Month s After Treatment Water below soil surface (cm) 0 30 46 Total Protein 4 95.2 1 a 2 30. 1 99.2 a 8.56 86.1 a 24.3 7 153 a 34.2 146 a 19. 9 188 a 13.6 1 Means of 5 replications followed by standard error 2 Means f ollowed by the same letter within the same category and month after treatment
39 Table 2 5 Antioxidan t concentration of leaf tissues as a function of water deficit level of Typha d omingensis at 4 and 7 months. Antioxidant Month s After Treatment Water below soil surface (cm) 0 30 46 P eroxidase (activity/mg) 4 0.004 4 1 a 2 0.001 0 0.00 37 a 0.00 16 0.002 2 a 0.00 05 7 0.00 18 a 0.001 1 0.001 4 a 0.00 51 0.001 a 0.0004 C atalase ( M 4 0.005 a 0.004 0.001 a 0.0004 0.003 a 0.002 7 0.004 a 0.00 08 0.003 a 0.0 004 0.003 a 0.001 Glutathione reductase (activity/mL) 4 0.004 a 0.00 3 0. 0 11a 0.004 0.019a 0.018 7 0.0 89 b 0.004 0.086 a 0.002 0.09 1 a 0.009 Superoxide di smutase (activity/g) 4 6.89a 0.77 6.89a 0.33 8.46a 1.49 7 7.29a 0.23 6.79ab 0.39 6.02b 0.11 A scorbate 4 118 a 48 102 a 24 110 a 4 5 7 182 a 54 156a 35 357 a 195 1 Means of 5 replications followed by standard error 2 Me ans followed by the same letter within the same cat egory and month after treatment are not significantly different
40 Table 2 6 Antioxidant concentration of rhizome tissue of Typha domingensis as a function of wa ter deficit level at 4 and 7 months. Antioxidant Month s After Treatment Water below soil surface (cm) 0 30 46 Peroxidase (activity/mg) 4 0.0007 1 a 2 0.0003 0.0005a 0.0002 0.0002a 0.0001 7 0.000 3 a 0.0002 0.0003 a 0.0001 0.0003 a 0.0002 Cat alase M 4 0.0029 a 0.00 08 0.00 47 a 0.00 0 1 0.005 0 a 0.0007 7 0.002 2 a 0.00 08 0.00 29 a 0.0004 0.003 5 a 0.000 8 Glutathione reductase (activity/mL) 4 0.081 ab 0.01 1 0.111 a 0.01 1 0.07 2 b 0.00 59 7 0.054 a 0.003 0.07 3 a 0.00 8 0.067 a 0.005 Superoxide dismutase (activity/g) 4 5.00a 0.22 5.44a 0.29 5.10a 0.66 7 5.90a 0.84 9.79a 1.22 10.9a 1.7 8 Ascorbate 4 45.4 a 24. 3 31.6 a 10.6 35.4 a 20. 4 7 60.4 a 23.9 81.7 a 25.8 96.2 a 24. 3 1 Means of 5 replications followed by standard error 2 Means followed by the same letter within the same cat egory and month after treatment are not significantly different
41 CHAPTER 3 RECOVERY OF TYPHA DOMINGENSIS AFTER PERIODS OF PROLONGED DROU GHT Introduction Cattails ( Typha spp.) are a littoral wetland species found throughout the world. These species are typically characterized by flat, strapped shaped leaves and Bryson and DeFelice 2009 ). Although this species does produce viable seed, the primary means of reproduction are via vegetative shoots that arise from underground rhizomes. Cattails tend to form monotypic stands, excluding other aquatic plant species. This often categorizes them a s a nuisance along lake margins and shorelines. However, cattails provide vital nesting habitats for birds and other animals, prevent erosion, and help to maintain water quality ( Baker et al. 1995 ). Over the past several years there has been concern over excessive nutrient loading into the Florida Everglades, which is a naturally nutrient poor ecosystem. An increase in available nutrient levels often promotes the growth and expansion of certain species, such as Typha domingensis Pers. which out competes o ther vegetation such as sawgrass ( Cladium jamaicens e Crantz ). The problem created by this excessive nutrient loading is being ad dressed by the construction and maintenance of stormwater treatment areas (STAs). These are artificial wetlands that remove and store nutrients through plant growth and biomass accumulation in the soil. T. domingensis i s a principal component in the STA s ; aiding nutrient uptake by acting as functional remover of nutrients. In order to maintain a STA, Typha populations must be heal thy. This is challenging because STAs also function as flood control and runoff containment areas.
42 This aspect dictates that water levels fluctuate drastically, from flood to drought conditions. Recent observations have shown that T domingensis is able to recover following a water deficit up to 7 months (Pietro et al. 2010) ; however, these plants were never subjected to a complete water deficit. A decline in photosynthetic activity and chlorophyll content is one possible way to monitor plant status duri ng drought. Li et al. ( 2004 ) reported a decline in net photosynthesis and chlorophyll content following periodic drought conditions in Typha latifolia Rhizomes are the primary carbohydrate storage tissues in Typha spp. In grass species non structural ca rbohydrates are frequently translocated throughout the plant where they function in various metabolic proc esses (Steen and Larsson 1986). A decrease in rhizome biomass and total nonstructural carbohydrate content (TNC) often lead s to a decrease in leaf an d shoot production due to the lack of an adequate carbohydrate supply, and may be an indicator of plant recovery following periods of prolonged stress. The objective of this study is to monitor the decline and subsequent recovery of T. domingensis followin g the exposure of prolonged drought conditions. Several parameters including photosynthetic rate, chlorophyll content, rhizome biomass, and rhizome carbohydrate content were tested in an attempt to correlate physiological response to physical decline and recovery. It was hypothesized that plants exposed to longer periods of water deficit would be slow or unable to recover from the imposed drought conditions.
43 Materials and Methods Whole plant material (leaf shoots and rhizomes) was collected from stormwater treatment areas in South Florida, and transplanted into 7.5 L pots containing Fafard N o. 4 1 potting soil and fertilized with Osmocote fertilizer 2 Potted p lants were then placed in concrete vaults with a water depth of 30 cm and grown under greenhouse c onditions. Greenhouse temperatures ranged from 24 27 C, while daily light levels ranged from 1200 1800 2 s 1 Plants were allowed to grow and mature for 9 months (December 2008 July 2009) with dead leaf material being removed before treatments were initiated. The experiment was of a randomized design testing the main effect of length of drought period There were a t otal of 5 vaults with 5 pots (replicates) per vault; A non water stress control was also included utilizi ng 2 pots in a separate container. The s tudy was initiated by draining water from the experimental vaults. Plant status was monitored over time by measuring p hotosynthesis with a LI COR 6400 XT portable photosynthesi s system 3 and chlorophyll content with a Minolta SPAD 502 chlorophyll meter 4 The LI COR 6400XT portable photosynthesis system 5 is a device that measures leaf photosynthetic rate by utilizing a closed system. A leaf is inserted into a sealed chamber and infrared gas analysis (IRGA) is used to measure the change in concentration over time for CO 2 and H 2 O. Photosynthetic rate is calculated by 1 Fafard et Frres Ltd, Canada 2 Sierra Chemical Co., Milpitas, CA 3 LI COR Biosciences, Lincoln, Nebraska 4 Spectrum Technologies, Inc, Plainfield, Illinois 5 LI COR Biosciences, Lincoln, Nebraska
44 measuring the drawdown in CO 2 concentration in the light, respiration is measured by the increase in CO 2 concentra tion in the dark, and the change in H 2 O concentration measures transpiration. A soil plant analysis development (SPAD) meter 6 is a device that measures leaf chlorophyll content. Light emitting diodes compare the absorbance of light transmitted at 650 nm to the ratio of light transmitted at 940 nm, where 650 nm is the absorbance of the chlorophyll molecule and 940 nm is the absorbance of a reference sample (Read et al. 2003). Both the LI COR and SPAD meters provide quick measurements of photosynthetic rate and chlorophyll content, respectively, allowing repeated measurements to be made on the same leaves thus avoiding a destructive harvest. Photosynthetic and SPAD readings were taken between the hours of 11 AM and 3 PM on sunny days to ensure maximum photos ynthetic activity and to negate a ffects of irradiance on chlorophyll content. Chlorophyll content from SPAD readings was calculated based on 2 ) = 10 (M^0.265) where M is equal to the SPAD reading (Markwell et al. 1995). All measurements were made on a weekly basis. Drought conditions were maintained until photosynthesis dropped to 0 5 .0 2 /s 1 ( which was approximately 5 weeks after imposing drought ). Water was then returned to one vault to the original 30 cm level with water subsequently returned to the remaining vaults on a weekly basis; thus establishing d rought periods of 5, 6, 7, 8, and 9 weeks respectively. After the water was returned, e ach vault was allowed a five week recovery period At this time the chlorophyll content (SPAD) and photosynthetic rate of new leaves was determined and the number of new shoots that had arisen during 6 Spectrum Technologies, Inc, Plainfield, Ill inois
45 recovery was noted. Plants were sectioned into leaf and rhizome tissue and dried in a forced air oven and weighed for biomass analysis. Total nonstructural carbohydrate analysis was performed on rhizome tissue based on procedures based on the work of Smith ( 1981) and modified by Christianse n et al. (1982). Data were subjected to analysis of variance using SAS 9.1 to detect treatment procedure at the 0.05 level. Presented values for all studies represent the mean of 5 replications followed by standard error of the mean. Results Decline in Chlorophyll and Photosynthetic Rates 2 There was a significant de cline in chlorophyll content 2 weeks after drought stress was initiated; followed by a slower rate of continued decline (Figure 3 1). Initial leaf photosynthetic rates averaged 12.8 2 /s 1 and declined significantly during the first week of drought (F igure 3 2). A lag occurred during the second and third week of drought before declining again during the third week. At 5 and 6 WAT photosynthetic 2 /s 1 for the remainder of the study (data not shown). Recovery Leaf Ana lysis The length of drought condition showed little or no influence on overall leaf/shoot recovery although t here was an increase in leaf dry weight of recovered plants following 8 and 9 weeks of drought ( Table 3 1 ). This correlated with greater shoot num ber for the 8 and 9 week drought period ( Table 3 2 ). Chlorophyll content remained stable for all plants after the recovery period but there was significantly higher content in leaves following the 9 week drought period ( Table 3 3 ). Recovered leaves had v ariable
46 photosynthetic rates with the highest following 5 and 8 weeks of drought ( Table 3 4 ). Chlorophyll content returned to slightly lower levels than before the drought period and photosynthesis was higher in new leaf material. Rhizome Status There we re no significant differences in the rhizome dry weights of recovered plants. However, there was an observed decrease in the r hizome dry weight s of recovered plants as drou ght period increased ( T able 3 5). There were no significant differences in the tota l nonstructural carbohydrate content of recovered plants during this same time period ( Table 3 6 ). Discussion Photosynthesis requires an inflow of CO 2 through stomatal opening on the leaf surface. When plants experience a water deficit, stomata will often close to prevent water loss. Chlorophyll is necessary for converting the light energy necessary to form CO 2 into carbohydrates. During photosynthesis, the energy from sunlight is used to split water into hydrogen and oxygen and the resulting electrons are used to create ATP and NADPH necessary for carbohydrate formation. Chlorophyll will continue to absorb light energy, even when stomates are closed. If CO 2 or water is absent then electrons will not be used in the conversio n process and will cause damage to the chlorophyll molecule. The decline in photosynthetic rate directly correlated with the decline in chlorophyll content observed during the drought portion of this study. All plants in all vaults were able to recover fr om all drought periods tested in this study, up to 9 weeks of drought. Therefore, factors such as l eaf age, nutrient availability, and rhizome health may have a greater role in Typha decline following drought. This study showed no differences in chlorophy ll content or photosynthetic rate
47 with the length of drought, indicating that new shoot growth is independent of drought period as long as rhizome health allows for re growth. Higher photosynthetic rates and chlorophyll levels in the new versus the old lea ves are likely due to the age of the leaves. C hlorophyll content in plants has been known to de crease with leaf age. A decline in light saturated photosynthesis has also been known to be associated with an increase in leaf age (Kitajima et al 1997). H ow ever, Hikosak a ( 1994 ) report ed that photosynthesis has been shown to be independent of leaf age and changes depending on light environment and photosynthetic components of the leaf The loss of rhizome dry weight following the 5 week recovery period sugge sts the allocation of rhizome carbohydrates for new shoot production following drought. It also suggests the loss of carbohydrates to respiration following periods of non photosynthetic activity. Furthermore, rhizome TNC content is independent of rhizome weight. Rhizomes with weights as low as 10 g were able to recover and there were no differences in TNC content of rhizomes following recovery periods suggesting t hat TNC is not a good indicator of plant recovery. T domingensi s undergo es drought stress i n the field and often decline and die (Kathy Pietro personal communication) ; however, this study maintained complete drought over a more prolonged period than observed in the STA s and y et recovered with no problems. One possible explanation for the decli ne of T domingensis in the STAs may be that water levels are returned to o quickly and at levels that completely submerse cattail shoots and leaves. This creat es anoxic conditions New shoots are the key to reproduction in Typha spp. and they must b e able to carry out cellular respiration as they grow through the water column in order to reach the air. Typha
48 latifolia has been shown to be able to carry out anaerobic respiration for a limited period of time under submerged conditions (Sculthorpe 19 67 ); however, if the plant is unable to reach t he water surface due to fluctuating high water conditions it will no longer have the oxygen requ i red for cellular respiration. Continuous flooding of soil can also cause a decrease in soil redox potential (Eh) These situations will l ead to a greater demand for oxygen by the roots. Many naturally occurring wetland plants are able to withstand periods of O 2 deficiency, but vary in their ability to withstand differing levels of intense soil redox conditions (Pezeshki 2001). Typha and other wetland plants possess aerenchyma, a tissue containing extensive gas spaces that serve as a pathway for O 2 and CO 2 movement within the plant. In Typha these gas spaces are present in all parts of the plant (leaves, shoots, and rhizomes) (Constable et al. 1992). Aerenchyma can help to facilitate the transport of O 2 to the roots during flooded conditions; however, s oil reduction is known to produce a variety of phytotoxic compounds such as lactic acid butyric acid, f ormic acid hydrogen sulfide, and some c yanogenic compounds (Pezeshki 2001). These compounds can damage to aerenchyma tissue and vascular bundles, preventing or inhibiting nutrient uptake P hytotoxic compounds may also damage rubisco and other enzymes es sential for photosynthesis. In addition, p hotosynthetic rates of T domingensis have been shown to decrease w ith decreasing redox potentials ( Pezeshki et al 1996 ). Phy t otoxic damage to rubisco and other enzymes necessary for normal photosynthesis and met abolism may help to explain this decr ease in photosynthetic activity, and may help to explain an inability to recover from flood following a n extended drought period.
49 It is unlikely that photorespiration resulting from higher temperatures play s a major r ole in Typha decline and death. In most C3 species as temperature increases the affinity of rubisco for CO 2 decreases while the affinity of rubisco for O 2 increases. This leads to the binding of O 2 instead of CO 2 initiating photorespiration and causing a net loss in carbon uptake/assimilation Unlike other C3 plants, Typha is able to utilize concentrated CO 2 in aerenchyma tissue. Constable and Longstreth ( 1994 ) found that the partial pressure of CO 2 (pCO 2 ) in aerenchyma gas spaces could be 10 times that of atmospheric pCO 2 ; however, under normal conditions CO 2 conductance from aerenchyma to photosynthetic cells was found to be lower than conductance of CO 2 from atmospheric cells. This indicates that aerenchyma can serve as storage reserves when ade quate atmospheric CO 2 is unavailable. Insect predation may also contribute to Typha decline and death. Red spider mites ( Tetranychus urticae ) were found on older Typha leaves throughout the experiment. Leaves of T domingensis collected from study s ites in South Florida also exhibited insect damage. Insects such as the cattail borer ( Bellura oblique ) lay their eggs on the leaf surface. Larvae will progressively move deeper into the plant, through the crown and sometimes into the rhizome, causing leaf death and preventing flower formation (Center et al. 2002). In Florida the cattail caterpillar ( Simyra henrici ) is known to destroy large expanses of Typha stands by damaging entire leaves, causing leaf browning and disrupting water transport (Cente r et al. 2002). Conclusions Photosynthesis and chlorophyll content of T. domingensis declined as the length of drought period increased and results were easily measured by the LI COR and SPAD meter, respectively. Both these tools could be used as predicto rs of Typha status in
50 STA s but did not correlate with long term plant health and recovery as all plants were able to regrow regardless of length of drought period. The lack of di fferences in rhizome TNC content suggests this is also not an accurate method when assessing Typha status. S easonal carbohydrate fluctuations form rhizomes to shoots and leaves play s an important role in determining tissue carbohydrate content. Additi onal studies should be conducted to determine carbohydrates fluctuate in response to stress during Leaf submergence and r oot redox potential may play an unknown factor in the decline of this species f ollowing intense flooding after prolonged drought. It is also possible that T domingensis is able to withstand longer drought periods that were conceived in this experiment. The ability of Typha to fully recover following prolonged drought is further ev idence of this p ability to survive periods of water deficit. However, this experiment did not assess the ability of Typha to survive repeated drought conditions. Asamoah and Bork ( 20 10 ) showed that carbohydrate reserves in root tissue of Typha la tifolia w ere reduced following a drying period; however years of drought followed by less than average rainfall are most likely necessary to curb population growth In such a case, rhizome carbohydrate reserves may be depleted before they can be restored by photosynthetic activity. Cattails are well suited for competition with other plants. They adapt to extended hydroperiods and increased water depths compared to sawgrass which has been found to survive complete submergence for approximately six weeks ( Newman et al. 1998). Asamoah and Bork (2010) showed that T latifolia was able to better tolerate drought when compared to spikerush ( Eleocharis palustrus L.). It is possible that an increased
51 nutrient supply aid s in the ability of cattails to tolerate dr ought conditions or high water conditions, providing more expedient growth thus aiding its competitive ability.
52 Figure 3 1. Chlorophyll content in Typha domingensis following periods of drought (weak s after treatment initiation (WAT)). Means of 10 replications followed by standard error. Figure 3 2. Photosynthesis in Typha domingensis following periods of drought (weeks after treatment intitiation (WAT)). Means of 10 replications followed by standard error.
53 Table 3 1. The effect of drought period on the dry weight of new Typha leaves produced during a 5 week recovery period after drought was terminated. Weeks of Drought Dry Weights (g) 1 5 9.3ab 2.6 6 8.1b 2.8 7 8.4b 1.2 8 13ab 1.4 9 15a 2.2 1 Means of 5 replications followed by standard error 2 protected least significant difference procedure. LSD (0.05) = 5.45 Table 3 2. The effect of drought period on the number of new Typha shoots produced during a 5 week recovery period after drought was terminated. Weeks of Drought Shoot # 1 5 1.8c 0.22 6 3.4b 1.04 7 3.6b 0.27 8 5.8a 0.4 2 9 4.4ab 0.67 1 Means of 5 replications followed by standard error 2 protected least significant difference procedure. LSD (0.05) = 1.59
54 Table 3 3 The effect of drought period on chlorophyll content of Typha leaves produced durin g a 5 week recovery period after drought was terminated. Weeks of Drought 2 ) 1 5 479 b 5 4 6 442 b 3 4 7 461 b 6 6 8 506 ab 19 9 618 a 32 1 Means of 5 replications followed by standard error 2 Means followed by the same letter a protected least significant difference. LSD (0.05) = 6.99 Table 3 4 The effect of drought period on the photosynthetic rate of recovered Typha leaves produced during a 5 week recovery period after drought was terminated. Weeks of Drought 2 s 1 ) 1 5 31 a 1. 5 6 19 c 1.3 7 27 b 2. 6 8 35 a 2. 6 9 27 b 1.0 1 Means of 5 replications followed by standard error 2 Means followed by the same letter are not significantly protected least significant difference. LSD (0.05) = 3.79
55 Table 3 5. The effect of drought period on dry weights of Typha rhizomes produced during a 5 week recovery period after drought was terminated. Weeks of Drou ght Dry Weight (g) 1 5 27.3 a 18 .9 6 19.3 a 5.56 7 12 .7 a 5.85 8 9.38a 2.41 9 11.1 a 3.59 1 Means of 5 replications followed by standard error 2 protected least significant difference procedure. LSD (0.05) = 25.81 Table 3 6. The effect of drought on total nonstructu r al carbohydrate (TNC) content of Typha rhizomes produced during a 5 week recovery period recovery period after drought was terminated. Weeks of Drought TNC (mg/g) 1 5 165 a 5 1 6 80 a 2 6 7 12 a 45 8 136 a 39 9 103 a 1 9 1 Means of 5 replications followed by standard error 2 protected least significant difference procedure. LSD (0.05) = 97.7
56 CHAPTER 4 PHOTOSYNTHETIC RESPONSES OF NATIVE AND NON NATIVE INVASIVE SPECIES IN NORTH CENTRAL FL ORIDA Introduction A non native invasive plant is a plant found in an area other than its natural rang e that can has profound ecological and /or economic impacts (Cronk and Fuller 1995). The majority of invasive species often have high growth and reproducti ve capacities, and no natural enemies to limit their spread and persistence (Callaway and Aschehoug 2000, Sakai et al. 2001). These attributes allow these species to crowd out native plant species and often animal species, limiting the biodiversity of a n e cological community (Clavero and Garca Berthou 2005). Economic damage caused by invasive plants includes, but is not limited to the disruption of grazing and timber production, the restriction of irrigation and flood control activities, and interference with outdoor activities such as hiking and boating. Damages caused by invasive species (both plant and animal) approximate $ 120 billion per year in the United States. T he state of Florida spends over $14.5 m illion for the control of hydrilla ( Hydrilla v erticillata (L.f.) Royle ) alone ( Pimente l et al. 2005). Overview of Invasive Species Examined Air potato ( Dioscorea bulbifera L. ) is a twining herbaceous vine with large cal Asia and sub Saharan Africa. It was originally and brought over from the slave trade and was introduced to Florida in 1905 (Langeland and Meisenburg 2008). It is found in a variety of habitats including pinelands, thickets, fence rows, disturbed areas, and hammocks. This species quickly engulfs native vegetation by climbing high into tree canopies forming large mats of leaves and vines that are impenetrable by other plants
57 or sunlight. Air potato has been shown to smother seedlings as well as fully gr own trees (Invasive Species Specialist Group 2010). Macfadyena unguis cati (L.) A.H. Gentry ) is a high high climbing woody vine. It is native to the West Indies and from Mexico to Argentina It was introduced into Florida in the 1940 tendril forks that are stiffly hooked and claw like. It is commonly found in disturbed hammocks where it often becomes the dominant ground cover. It can also be found in orchards, gardens, open urba n areas, roadsides, and grasslands. It tolerates low light conditions, but growth is more vigorous in an open habitat (Invasive Species Specialist Group 2010). This results in a dense mat of vines and leaves of the forest floor that can out compete unders tory plants and preventing native seedling germination. This species will also grow into the forest canopy and spread laterally, killing host trees with its weight (Invasive Species Specialist Group 2010) Chinese tallow ( Sapium sebiferum (L.) Small ) is a deciduous tree that is commonly found along roadways, disturbed areas, forest edges, and waterways. It is native to ornamental, but also for its seed oil, which can b e used to make soap. It can thrive across a wide range of ecological habitats from sun to shade and swampy or well drained sites. It often overtakes large areas, forming monotypic stands. Chinaberry ( Melia azed a rach L. ) is a deciduous tree found in fore sts, along roadsides, disturbed areas, urban areas, shorelines, and wetlands. It is native to Asia and for fuel wood. It grows in full sun or shade and it able to toler ate a range of soil
58 types and soil pH. This species possesses numerous defenses against attacks from both insects and pathogens, providing it with a competitive advantage over native species (Invasive Species Specialist Group 2010). It quickly invades un disturbed areas, decreasing native biodiversity. Camphor tree ( Cinnamomum camphora (L.) J. Presl ) is an evergreen species that is typically found in dry, disturbed areas such as roadsides and fencerows, but recently has been shown to invade natural areas such as upland pine woods and mesic hammocks. It is native to China and Japan, where it is used for timber and oil. Plantations of camphor were established in Florida in 1875, but were not profitable for growers (University of Florida, IFAS 2010). It gro ws well in full sun or partial shade, but does not perform well in wet soils. Coral ardisia ( Ardisia crenata Sims ) is an upright evergreen shrub found in mesic hardwood hammocks and other moist woodland areas throughout Florida. It is native to areas of J apan and northern India, and was introduced as an ornamental. It often grows in dense clumps, shading out native understory plants, and can reduce the dim light of forest under stories by an additional 70%. This dense shade prevents the growth of native seedlings (Dozier 1999). This plant is able to tolerate a wide range of soil pH, from pH 4 to pH 10 (Langeland and Burkes 1998). Lantana ( Lantana camera L. ) is a perennial shrub found in a variety of areas including roadsides, forests, and pastures. It is native to the West Indies, and was introduced primarily as a hedge plant, but has other medicinal and practical uses. It also grows in both sunny and shaded environments and under moist or dry conditions. Lantana creates a dense understory that inhib its native species establishment. This
59 species has also been shown to produce allelopathic compounds that inhibit the growth and establishment of surrounding natives (Gentle and Duggin 1997). The life history of many invasive plants suggests that they ar e able them to utilize their environment more efficiently than surrounding native species, thus increasing competitiveness for light water, nutrients, and space (Levine et al. 2003). A more efficient photosynthetic rate could provide a competit ive advantage over co occurring plant species These photosynthetic rates can be optimized by altering the biochemical capacity for photosynthesis (McDowell 2002). Light compensation point (LCP), the net rate of light saturated photosynthesis (A max ) and r espiration rates are import ant factors that contribute to plant carbon gain This, in turn, relates directly to overall plant status and development. T he light compensation point (LCP) is the point where carbon uptake from photosynthesis equals respirati on, so overall photosynthetic rate is 2 m 2 s 1 Above the LCP there is a net carbon gain and below there is net carbon loss Light compensation points can vary among different plant species and different growth conditions due to varying light levels. Sparling (1967) showed that s hade tolerant species had lower light compensation points and photosynthetic rates than shade intolerant species. However, these measurements can be dependent on the morphology and life history of the individual plant. Certain winter annuals have been sh own to utilize a wide range of light intensities, with lower light compensation points at low leaf temperatures in the winter and elevated light compensation points with higher leaf t emperatures in the summer (Bazz az 1979). Several invasive exotic plants such as hydrilla ( Hydrilla verticillata ) and Japanese honeysuckle ( Lonicera japonica Thunb. ) have low light compensation points. These plants utilize low light levels more efficiently
60 than surrounding native species, allowing for faster growth and an incr eased competitive advantage (Gordon 1998). Durand and Goldstein (2001) showed that the invasive Australian tree fern ( Sphaeropteris cooperi ) had higher photosynthetic rates than native fern species in the genus Cibotium regardless of light environment Th is plasticity suggests that certain plants are more adept at using differing light levels for photosynthesis, and more efficient photosynthesis may help to give certain plants competitive advantages over others. There have been limited studies on the res ponse of invasive plants to variation s in light intensity compared to native species. Such information may provide a better understanding as to how and why plants invade a certain area. Therefore, t he objective of this study was to observe differences i n photosynthetic rates between native and non native invasive plant species in natural areas of North central Florida. It was hypothesized that i nvasive plants would have more efficient photosynthetic rates when compared to their native counterparts, thus helping to partially explain the competitive advantage invasive plants often have over native plants. Materials and Methods Invasive and complem entary native plants were utilized in this study and separated into 3 categories based on growth habit (trees, vines, and shrubs) V ine s sampled consisted of the native species muscadine grape ( Vitis rotundifolia Michx ), greenbriar ( Smilax spp.), trumpet creeper ( Campsis radicans (L.) Seem. ex Bureau ) and Virginia creeper ( Parthenocissus quinquefolia (L.) Planch. ); and the invasive exotic species air potato ( Dioscorea bulbifera Macfadyena unguis cati ). Trees sampled consisted of the nati ve species oak ( Quercus spp.) and holly ( Ilex spp.) ; and the invasive exotic species Chinese tallow ( Sapium sebiferum ), Chinaberry ( Melia
61 azederach ), and camphor tree ( Cinnamomum camphora ). Shrub species consisted of the native American beautyberry ( Calli carpa Americana L. ), the invasive lantana ( Lantana camera ), and coral ardisia ( Ardisia crenata ) Healthy s pecimens were randomly selected from various sites throughout Alachua County, Florida during July October 2009. Measurements were taken on fully matu re sun and shade adapted le aves during peak sunlight hours to ensure optimal photosynthetic activity. Average light levels ranged from 1700 1800 2 s 1 for sun adapted leaves and 5 25 2 s 1 for shade adapted leaves. Photosynthetic analysis w as conducted using a Li COR 6400 XT portable photosynthesis system 7 This device measures leaf photosynthetic rate and the photosynthetic response of leaves to differing light levels. This unit utilizes a closed system to measure the change in CO 2 and H 2 O c oncentration over time via infrared gas analysis. A drawdown in [CO 2 ] in the light is used to measure photosynthesis, an increase in [CO 2 ] in the dark measures respiration, and the change in [H 2 O] can be used to measure transpiration. This closed system al so provides a light source to eliminate variability in photosynthesis associated with different light levels, and allows for photosynthesis to be measured at defined light levels. Light response curves were generated by measuring the photosynthetic respon se of leaves to declining light levels (2000, 1800 1200 m 2 s 1 ) and calculated based on the following asymptotic exponential equation: A = A max *[1 (1 (Rd/Amax)) (1 (PPFD/lcp)) ] (Hanson et al. 1987). Light compensation point (LCP), the net rate of light saturated photosynthesis (A max ), and respiration rates were 7 LI COR Biosciences, Lincoln, Nebraska
62 calculated based on the curve generated for each plant. A Minolta SPAD 502 chlorophyll meter 8 was used to correlate chlorophyll content and plant health with photosynthetic rate. Sample leaves were harvested in order t o correlate leaf area and dry weight with photosynthetic rate. Leaf area was calculated using a LI COR 3100 leaf area meter 9 Leaves were dried in a forced air oven at 60 C for 24 hours before weighing Specific leaf area (SLA) was calculated based on ob served leaf area and dry weight All d ata was analyzed using SAS (2009) and PROC GLIMMIX and Tukey Kramer analysis of least square means. Results Trees Invasive tree species had higher light compensation points (LCP s), higher light saturated photosyntheti c rates (A max ), and higher respiration rates compared to the native tree species (Table 4 1). Sun leave s were found to have higher LCP s and respiration rates compared to shade leaves. Inv asive sun leaves had higher LCP s, and higher respiration rates amon g all species sampled. Invasive plants had higher specific leaf area (SLA s) than native plants but this was not statis tically significant. Higher SLA s would help to aid the plant in absorbing more sunlight, potentially increasing photosynthetic efficiency. Vines Native vines species had higher rates of light saturated photosynthesis (A max ) compared to invasive vine species (Table 4 2). Sun leaves had a higher light compensation point A max and respiration rate comp ared to shade leaves. Native su n 8 Spectrum Technologies, Inc, Plainfield, Illinois 9 LI COR Biosciences, Lincoln, Nebraska
63 leaves had higher A max and respiration rates compared to all others. There were no significant differences in chlorophyll content among species tested. Invasive plants had higher SLA s than native plants but were not significantly different This would aid in th e invasive plants being able to receive more sunlight than their native counterparts. Shrubs There were no significant differences in any of the para meters tested for shrub species (Table 4 3) However, invasive plants did appear to have higher light comp ensation points, respiration rates, and SLA s compared to native plants. This suggests that these species thrive better in high light environments, rather than shaded areas. Discussio n The competitive ability of plants is augmented by the ability to assimi late CO 2 and utilize the photosynthate to extend foliage and increase size. Plants with high photosynthetic rates have an initial advantage that can make them high yielding crops or serious weed problems (Black et al. 1969). In most cases sun leaves, irre spective of species, were shown to have a higher light compensation point, A max and respiration rate than shade leaves. Sun leaves are adapted to higher light environments; therefore, photosynthetic efficiency decreases as these leaves are moved from sun to shade (Timm et al 2002, Giv nish 1988). Sun adapted leaves tend to be thicker and have longer palisade cells than shade adapted leaves. Sun leaves also have higher rubisco nitrogen, and soluble protein per unit mass as well as higher chlorophyll a to c hlorophyll b ratio than shade leaves (Givnish 1988). Therefore, sun adapted leaves have lower SLAs than shade adapted leaves This allows sun leaves to reach light saturation at much higher irradiance levels. Reich et al. (1998) showed that plants with a h igher SLA
64 have a higher A max per unit leaf nitrogen and a more variable A max per unit leaf nitrogen when compared to plants with low SLAs. These morphological differences between sun and shade leaf directly affect LCP. In general, species grown under h igher light intensities will have higher light compensation points that than those grown under lower light intensities (Bazzaz 1979, Meyers et al. 2005). Light compensation point is strongly affected by photosynthetic induction. Timm et al. (2002) showed that a decrease in the photosynthetic induction rate of rainforest tree saplings from full (1.0) to low (0.2), led to a 400% increase in light compensation point. While some plants may be able to assimilate CO 2 at lower light levels, other factors can al so attribute to a low light compensation point. Givnish (1998) also showed that the light compensation point of Liriodendron had little effect on net carbon gain, and was heavily influenced by factors such as leaf respiration, leaf construction, and const ruction associated with root support. Invasive tree species measured in this study showed higher rates of light saturated photosynthesis compared to co occurring native tree species suggesting these species may be more competitive in a high light enviro nment. The fact that invasive trees had higher respiration rates and light compensation points also suggests that these trees are not as efficient as native trees under low light environments. Sun leaves of t he n ative vine species evaluated in this stud y were shown to have higher rates of light saturated photosynthesis compared to invasive vine species, suggesting that these species perform better under high light conditions. However, all of the vine species tested are considered to be weeds in most are as, and factors such as growth rate climbing and mechanics also contribute to the abundance of invasive
65 plants ov er native plants or vice versa C arter and Teramura ( 1988 ) hypothesized that twining vines are not well adapted for climbing under closed canop ies due to support diameter limitations, but tendrilled vines can establish on the forest floor and eventually climb into the canopy where there is more sunlight. This could give vines with tendrils or other forms of direct attachment greater plasticity w hen moving from high to low light environments since they would be more apt to reach the upper canopy. Invasive shrub species appeared to have higher light compensation points compared to native species, although these results were not significantly differ ent. This would suggest that they thrive better at higher light levels. However, this cannot be confirmed given the limited number of shrubs tested Conclusions The fact that differences in light response were observed between native and invasive pl ants suggests photosynthesis plays an important role in pl ant invasion and establishment. Invasive trees in this study were found to have very high photosynthetic rates in high light environments but respiration rates increased when plants were moved fro m sun to shade. This suggests that high light intensities help to provide invasive trees with a competitive advantage; however, that advantage is quickly diminished in the shade where respiration rates are increased and there is a net loss of carbon in th e plant. This feature may also help to explain why certain invasive tree species appear to have a competitive advantage in disturbed areas. Conversely, native vines had higher photosynthetic rates in high light environments, and showed a greater plasti city under the range of high to low light environments. All vine species, invasive and native, showed lower respiration rates compared to tree species in both sun and shade. Therefore, there is not a great deal of
66 carbon lost as light intensity changes. This feature helps to explain why many vine species are able to begin growth in a shaded understory and gradually progress into the forest canopy. With the exception of tree species, this study found few significant differences among the light compensatio n points of invasive versus native plants. Sun adapted versus shade adapted leaves were also shown to significantly affect light compensation point. Therefore, this quality most likely has more to do with leaf adaptation to light than the inherent physiolo gy of a particular species. It is possible that having a lower light compensation point will give a plant a competitive advantage ; however, this advantage is dependent on the light environment in which that particular plant grows. Having a higher SLA an d chlorophyll content could be correlated with having higher photosynthetic rates. However, there were no significant differences in either of these features for any of the species examined. Again, light adaptation and environmental conditions are likely to play a more prominent role in photosynthetic efficiency. This study showed that photosynthetic differences can exist between invasive and native plant species. M ore species should be tested over a broader geographic area to determine if these result s accurately correlate to these situations. It would also be beneficial to examine plants at various stages of their life cycle to see how the ratio of competitiveness to photosynthetic rate changes with age. Various models, such as the Australian Risk As sessment, use factors such as growth habit and climate data for a species native range to assess invasive potential. This research could help to
67 complement those types of models, by giving further detail on how a species will respond to a certain light en vironment.
68 Table 4 1. Photosynthetic responses of invasive versus native tree species found in North central Florida. LCP 1 A max 2 Resp. 3 SPAD 4 SLA 5 Status Invasive 22.05a 37.61a 1.93a 42.90a 220.03a Native 13.27b 22.97b 3.30b 45.31a 98.27a Light Sun 24.14a 34.89a 1.78a 45.02a 100.92a Shade 11. 18 b 25.69a 3.46b 43.19a 217.37a Light x status I sun 31.75a 47.69a 4.71 a 43.50a 121 .57a N sun 16.54b 23.85ab 2.19 b 46.53a 80.28a I shade 12 .35b 27.52ab 8.89 b 42.30a 318 .49a N sha de 10.01b 22.08b 1.67 b 44.08a 116.26a Tukey means with the same letter within the same category are not significantly different. 1 2 s 1 ). 2 Net rate of light saturated photosynthesis ( 2 m 2 s 1 ) 3 R CO 2 m 2 s 1 ) 4 Chlorophyll content as a function of SPAD value 5 Specific leaf area ( cm 2 g 1 )
69 Table 4 2. Photosynthetic responses of invasive versus native vine species found in North central Florida. LCP 1 A max 2 Resp. 3 SPAD 4 SLA 5 Status Invasive 12.9 a 8.39a 0.58a 45.99a 347.57a Native 9.98a 13.8 a 1.47b 38.89 a 217.16a Light Sun 17.1 a 15.9 a 1.61b 42.86a 230.98a Shade 5.78b 6.23b 0.45a 42.02a 333.75a Light x status I sun 16.0 a 9.83 a 0.92a 39.76a 261.78a N sun 18 .2 a 22.1 b 2.29b 45.95a 200. 18 a I shade 3.94b 6.96 a 0.24a 46.03a 433.37a N shade 7.61b 5.51 a 0.65a 38.01a 234.13a Tukey means with the same letter within the same category are not significantly different. 1 2 s 1 ). 2 Net rate of light saturated photosynthesis ( 2 m 2 s 1 ) 3 CO 2 m 2 s 1 ) 4 Chlorophyll content as a function of SPAD value 5 Specific leaf area ( cm 2 g 1 )
70 Table 4 3. Photosynthetic responses of invasive versus native shrub species found in North central Florida. LCP 1 A max 2 Resp. 3 SPAD 4 SLA 5 Status Invasive 13.6 a 27.86a 2.23a 38.13a 347.57a Native 8.95a 27.76a 1.57a 31.65a 217.16a Light Sun 12 .1 a 33.86a 1.98a 36.81a 230.98a Shade 10.4 a 27.77a 1.82a 32.96a 333.75a Light x status I sun 14.2 a 30.61a 2.20a 39.23a 261.78a N sun 10.1 a 37.10a 1.76a 34.40a 200. 18 a I shade 12 .9 a 25. 12 a 2.26a 37.02a 433.37a N shade 7.84a 18 .41a 1.39a 28.90a 234.13a Tukey Kramer grouping for least means with the same letter within the same category are not significantly different. 1 2 s 1 ). 2 Net rate of light saturated photosynthesis ( 2 m 2 s 1 ) 3 CO 2 m 2 s 1 ) 4 Chlorophyll content as a function of SPAD value 5 Specific leaf area (cm 2 g 1 )
71 CHAPTER 5 SUMMARY AND CONCLUSI ONS Response of Typha domingensis to Water Deficit in Stormwater Treatment Areas Enzymatic analyses of antioxidants, absicic acid lev els, and chlorophyll, protein, and carbohydrate content were examined to determine the responses Typha domingensis had to different levels of water deficit Plants showed a decline in carbohydrate content as water levels decreased. This indicates that ca rbohydrates may be utilized for growth and repair of damaged tissue during or following a drought situation. There was also a decline in chlorophyll content as water levels decreased. This effect can be measured quickly and easily using a portable SPAD m eter. This would be a very efficient way to monitor the status of Typha populations in the stormwater treatment areas ( STA s ) Drought conditions will often cause oxidative stress in plants leading to the formation of free radicals that can cause cellu lar damage. Various antioxidants are produced to quench the activity of those free radicals. Biochemical tests were performed to determine the role antioxidants play in Typha drought. The majority of antioxidants tested for showed no correlation with wa ter level or time of drought. Antioxidant enzymes are constantly being recycled after their use and may not persist long enough to efficiently monitor drought conditions. Certain antioxidant enzymes are also subject to regulation by other antioxidant enzy mes and as a result may not appear in sufficient quantities to monitor drought stress. Recovery of Typha domingensis f ollowing D rought Fully mature plants were placed in concrete vaults and exposed to drought conditions for several weeks in order to det ermine their recovery rate. Photosynthetic
72 rate and chlorophyll content showed a consistent decline as drought period increased, reiterating the fact that these measurements can be used to assess Typha health in STA s. All plants were able to fully recove r following up to 9 weeks of drought. There were no significant differences in the number of new shoots, dry weights of new leaves, photosynthetic rate, chlorophyl l, and carbohydrate content of recovered plants. This suggests that Typha is able to withst and drought periods in excess of 9 weeks. Another explanation for the decline of Typha populations in the STA s is too much water following a drought situation. Excessive flooding will often create anoxic to fully recover. These conditions are also known to allow the formation of phytotoxic compounds that can cause further damage to plant tissues. Further research should be conducted to examine whether or not these phytotoxic compounds are contributing to the decline of Typha i n STA s If is also possible that a frequent shifting from water deficit to water excess in STAs is depleting the reserves of Typha in the STAs. Unless new leaves are generated to allow for photosynthetic activity, then carbohydrate s cannot be stored for use during periods of plant stress. Photosynthetic R espo nses of N ative versus Invasive P lants The photosynthetic responses of several non native invasive plants in North central Florida were compared with those of surrounding nati ve plant species. Growth habit appeared to play a n important role in determining whether or not an invasive plant would be more prolific in a certain area. Invasive tree species have been shown to readily colonize disturbed areas and other areas that rec eive a high amount of sunlight. The photosynthetic responses of the invasive trees tested showed higher light compensation poi nts, light saturated photosynthesis ( A max ) and respiration rates under
73 high light. This indicates that invasive tree species ha ve the propensity to colonize an open area more quickly than native tree species In contrast to trees, native vines were shown to have higher A max and respiration rates compared to invasive vines. This suggests that native vines have more efficient photo synthesis under high light. However, all vines examined are considered to be weeds in most areas. Additional factors such as growth rate and climbing mechanics may also play an important role in determining whether or not invasive or native vines would b e more abundant in a particular area There were no significant differences among shrub species of any of the parameters tested. This is most likely due to a lack of species sampled. Leaf adaptation to sunlight played an important role in photosynthetic e fficiency. Sun leaves tend to have higher photosynthetic rates than shade leaves, therefore, species that grow in full sunlight are more competitive in higher light environments. Knowledge of the photosynthetic rates of invasive pl ants may be useful in fo rmulating an assessment model to predict whether or not a particular plant will become invasive if introduced into a certain area. It may also aid in developing management strategies for existing populations of invasive plants.
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BIOGRAPHICAL SKETCH Kurt Vollmer was born in Abingdon, VA. He was raised and received his primary education in Richlands, V irginia graduating from Richlands High School in 2002. In 2006 he received his Bachelor of Science degree in b iology from Emory & Henry Colleg e, Emory, VA. While at Emory & Henry he was active in the Blue Key National Honor Society, Beta Beta Beta National Biological Honor Society, and various instrumental ensembles. Upon completing his degree, he spent 9 months as an instructor/naturalist at the Environmental Education Center at YMCA Camp Thunderbird in Lake Wylie, S outh Carolina In June of 2007, he entered a Master of Science degree program at the University of Florida, Gainesville, F lorida He has presented at annual meetings of the Weed Science Society of America, Southern Weed Science Society, Florida Weed Science Society, and Florida Exotic Pest Plant Council. He has also participated in the annual s tudent w eed c ontest of the Southern Weed Science Society and co authored several extens ion publications on invasive species biology and management.