Physiological Response of Crassulacean Acid Metabolism in Agave Americana to Water

and Nitrogen

A thesis presented to

the faculty of

the Voinovich School of Leadership and Public Affairs of Ohio University

In partial fulfillment

of the requirements for the degree

Master of Science

Andrew J. Fox

August 2019

© 2019 Andrew J. Fox. All Rights Reserved. 2

This thesis titled

Physiological Response of Crassulacean Acid Metabolism in Agave Americana to Water

and Nitrogen

by

ANDREW J. FOX

has been approved for

the Program of Environmental Studies

and the Voinovich School of Leadership and Public Affairs by

Sarah C. Davis

Associate Professor of Leadership and Public Affairs

Mark Weinberg

Dean, Voinovich School of Leadership and Public Affairs 3

ABSTRACT

FOX, ANDREW J, M.S., August 2019, Environmental Studies

Physiological Response of Crassulacean Acid Metabolism in Agave Americana to Water and Nitrogen

Director of Thesis: Sarah C. Davis

Agave americana is an obligate CAM species currently being investigated as a promising bioenergy crop. Little is known about the physiological response of these plants to nutrients and this study sought to resolve how availability of water and nitrogen impacted biomass accumulation, gas exchange rates, and accumulation of nitrogen.

Thirty A. americana plants were subjected to differing treatments of water (260 mm y-1,

650 mm y-1) and nitrogen (0 g m2, 5 g m2, 10 g m2) for 11 months. At the end of the 11- month experiment, biomass accumulation differed significantly with water treatments, but was not dependent on nitrogen availability; there was also no apparent interactive effect between water and nitrogen. Gas exchange rates were also dependent on water availability. 24-hour photosynthetic rates were not significantly impacted by the availability of water or nitrogen, but the comparison shows stark differences. Nitrogen accumulation in leaf tissue was affected both by water and nitrogen availability. The number of clonally produced pups was also dependent on water availability. While nitrogen plays a secondary role in impacting the productivity of A. americana, accumulation in the leaf tissue suggests that there may eventually be a substantial growth response. 4

DEDICATION

This work is dedicated to my family who gave me the opportunity to follow my ambitions

wherever they may lead 5

ACKNOWLEDGMENTS

I would like to primarily thank Dr. Sarah Davis whose advice, guidance, and material assistance entirely made this work possible, Dr. David Rosenthal and Dr.

Rebecca Snell whose advice and participation enhanced the scope and quality of this project. Special thanks to Toby Adjuik who was crucial to many parts of this project as well as a great friend through the process. Thank you to Dr. Ani Ruhil whose advice with data visualization prevented many headaches. I would like the thank the rest of my colleagues in the Davis Lab who continually pushed me to do better in all aspects of my professional life. This project was partially funded by the American Electric Power

Foundation and Ohio University. 6

TABLE OF CONTENTS

Page

Abstract ...... 3 Dedication ...... 4 Acknowledgments...... 5 List of Figures ...... 7 Chapter 1: Introduction ...... 8 Chapter 2: Materials and methods ...... 18 Overview ...... 18 Treatments...... 19 Accumulated Biomass ...... 20 Leaf Carbon and Nitrogen Content ...... 20 Gas Exchange...... 21 Statistical Analysis ...... 23 Chapter 3: Results ...... 24 Accumulation of Biomass ...... 24 Peak Gas Exchange ...... 29 Gas Exchange and the Impact of Water Availability...... 31 Gas Exchange and the Impact of Nitrogen Availability ...... 33 Nitrogen Content in Leaf Tissues ...... 36 Chapter 4: Discussion ...... 39 Chapter 5: Conclusion...... 47 References ...... 48 7

LIST OF FIGURES

Page

Figure 1. Change in biomass from beginning to end of study (11 months) ...... 25 Figure 2. Leaf (A) and root (B) water content...... 27 Figure 3. Average number of pups per treatement group...... 28 Figure 4. Peak carbon assimilation rates during known active period ...... 30 Figure 5. Mean transpiration rate during know active period ...... 31 Figure 6. Average 24-hour photosynthetic rate between water treatments ...... 32 Figure 7. Average 24-hour transpiration rate between water treatments ...... 33 Figure 8. Average 24-hour photosynthetic rate between nitrogen treatments ...... 34 Figure 9. Average 24-hour transpiration rate between water treatments...... 35 Figure 10. Percent leaf sample composed of nitrogen ...... 37 Figure 11. Carbon to nitrogen ratio of leaf samples ...... 38

8

CHAPTER 1: INTRODUCTION

Agave americana L. is a succulent desert plant native to the Western United

States. This species is currently being investigated as a potential biofuel feedstock crop with advantages of low water requirements and the ability to grow on marginal lands.

There are over 200 species in the genus of Agave, and their range spans from southern

South America to the western United States (Nobel, 1988). Agave americana is native to

New Mexico, Arizona, and Texas which are arid landscapes with large temperature fluctuations between night and day (Nobel, 1988). Species in the genus of Agave have adapted crassulacean acid metabolism (CAM photosynthesis) to cope with harsh conditions and thus, can live in environments not typically associated with agricultural crops. Low water availability, marginal soils, and infrequency of rain events are the challenges in these areas which have long prevented the establishment of traditional agricultural systems (Lambers et al., 2008).

Traditionally, other species of Agave have been used in agriculture, but Agave americana has never been commercially cultivated beyond its use for ornamental plants.

Agave tequilana and other species rich in soluble carbohydrates have been used in the production of alcoholic beverages; sugars are in much higher concentrations than in other species, which makes them easy to ferment (Borland et al., 2009). Agave sisalana has been used as a fiber crop to create a product known as sisal, which has been instrumental in the development of many economies (Nobel, 1988). Management practices for fiber and beverage species are well described, but to the extent these practices translate to

Agave americana remains unknown. The species for which we have established 9 management practices have native ranges mostly limited to Mexico (Garcia-Moya et al.,

2011). The tequila and sisal industries are the driving force behind much of the data regarding plants in this genus and their response to environmental conditions.

Agave americana employs the crassulacean acid metabolism (CAM) photosynthetic pathway. This photosynthetic pathway provides significant advantages in water use efficiency (WUE) (Ehrler, 1983). A. americana has the capacity to survive in otherwise inhospitable climates, where water is a major limiting factor (Nobel, 1976).

These advantages are made possible by segregating gas exchange to only periods with cooler temperatures during the night (Borland, 2009). During the hotter daytime hours, stomata remain closed and photosynthesis relies solely on stored carbon pools (Lüttge,

2004). Restricting gas exchange to only dark periods of night limits the amount of photosynthesis that can occur regardless of the availability of water and sunlight during the day (Lüttge, 2004; Nelson & Sage, 2007).

The CAM pathway separates the “dark” and “light” reactions of photosynthesis, extending the entire cycle to 24 hours (Neales, 1975; Lüttge, 2004). CAM plants uptake, fix, then store CO2 as malic acid while their stomata are open in the cooler temperatures of the night (Lüttge, 2004). CO2 is taken in and fixed into a four-carbon malate by phosphoenolpyruvate carboxylase (PEPC) (Lüttge, 2004). Malate is transported to the vacuoles and converted to malic acid for storage. When sunlight becomes available, these acids are then decarboxylated, usually by a malic enzyme, after transport to the chloroplasts (Osmond, 1978; Lüttge, 2004). CO2 then becomes available for fixation by

RuBisCO; driving photosynthesis. All carbon used in these reactions is fixed and stored 10 as an acid before use; there is not a pathway in CAM photosynthesis that can fix carbon prior to conversion (Osmond, 1978; Lüttge, 2004; Borland et al., 2009)

CAM photosynthesis is believed to have independently evolved multiple times in different plant lineages; this process is even observed in aquatic species (Lüttge, 2004).

Internal partial pressure of CO2 has been found to be closely associated with stomatal opening and closing (Lüttge, 2004). However, there are typically four phases of CAM photosynthesis which primarily regulate the stomata. In short, phase I can be classified by prominent PEPC activity and a gradual increase in the concentration of organic carbon- rich acids. Phase I occurs while the stomata are open, and light is not present in the cooler temperatures of the night. Phase I ends when PEPC becomes dephosphorylated making it far more susceptible to inhibition (Borland et al., 2009). Phase II is classified by cells reaching maximum capacity of acid storage and the ramping up of RuBisCO activity. For a short period in Phase II, CO2 may be fixed by both PEPC and RuBisCO simultaneously, usually at dawn (Borland et al., 2009). Phase III occurs throughout the majority of daylight hours and is the most characteristic phase of “photosynthesis” where

RuBisCO fixes carbon in the traditional sense, other than the stomata being closed. Phase

IV is the final phase and serves mainly as a transition back to PEPC activity and the acquisition of more organic acids while stomata prepare to open once again (Lüttge,

2004). The length of each phase is incredibly plastic and can vary in accordance with environmental factors and leaf development (Borland et al., 2009).

Cellular respiration in CAM plants is a process in which sugars are broken down by oxidation reactions to create chemical energy (Neales, 1975). The breakdown of 11 carbon rich molecules such as glucose creates CO2 as a waste product (Neales, 1975). In

CAM plants cellular respiration is essential in recycling key enzymes in photosynthesis but can also lead to a large expenditure of energy stores.

Transpiration in CAM plants occurs when the stomata are opened, exactly like in

C3 and C4 plants. In this process water vapor, which has been carried through the plants from the roots, is evaporated through small pores in the leaf surface (Bronson et al.,

2011). There is a constant negative pressure within the plant which creates a gradient guaranteeing that water will transpire from the stomata whenever they are opened (Nobel,

1988; Bronson et al., 2011). Environmental factors and plant physiology influence how much water will be lost due to transpiration. In Carnegiea gigantea, another CAM plant, there is a relationship between stomatal conductance and transpiration rates but, the most influential factor in determining transpiration rates was air vapor deficit pressure

(Bronson et al., 2011). Transpiration is also a key factor impacting the WUE of CAM plants. WUE can be impacted when photosynthetic productivity is low or when large volumes of water are lost through transpiration. These conditions occur during high and low air vapor pressure deficit periods, respectively (Bronson et al., 2011).

Reproduction in A. americana occurs in two ways; vegetative clonal propagation and seeding. An estimated 95% of reproduction in species of Agave is vegetative (Nobel,

1988). These clones propagate through rhizomes and are called “pups”, which depend on ramets from the mother plant for survival. The average age for reproduction via seeding in A. deserti, for example, is 56 years old (Nobel, 1988). Thus, it is believed in an agricultural practice the most practical way to regenerate crops would be clonally. 12

Yields comparable with other potential bioenergy crops have been shown in a field setting with far lower water inputs as compared to other crop production systems

(Davis et al., 2017). It has been shown that CAM species can be six times as water use efficient as C3 agricultural species under ideal conditions (Yang et al., 2015). Irrigation currently utilizes 32% of yearly water supplies in the United States, which is very near the logistical maximum which can be allotted to agriculture (U.S. EPA, 2018). Early estimations predicted Agave americana could have a large range of practical yields anywhere from 2 -14 Mg ha-1 (Davis et al., 2014), which was subsequently supported by a field study, that found A. americana could produce an average of 9.3 Mg ha-1 under optimal water conditions (Davis et al., 2017). Compared to bioenergy grass species that produce 10 Mg ha-1, as was recorded with switchgrass (Panicum virgatum) (Heaton et al.,

2004), it is reasonable to assume Agave could legitimately contend with other bioenergy crops.

Agave americana can be productive on lands that would otherwise not be considered for agricultural use, meaning they would pose little competition to food cropping systems (Dale et al., 2010). Individual A. americana plants grow for much longer periods of time than other crops (Nobel, 1988). This requires understanding of how physiological changes over time impact growth rates or how these plants respond to inputs (Nobel & Valenzuela, 1987). These crops will not be harvested every year and should therefore be thought of more similarly to woody crops species.

Cellulosic ethanol is a liquid biofuel that is derived from cellulose chains which can be broken into fermentable glucose molecules (Davis et al., 2014; Chen & Smith, 13

2017). After sugar is made available via hydrolysis, it can be fermented by traditional means and concentrated enough as to be combustible (Somerville et al., 2010). Cellulosic ethanol production can use all vegetative parts of plants and yield large amounts of alcohol per plant mass in comparison to traditional production methods, which use only the plant’s fruits or seeds (Somerville et al., 2010; Corbin et al., 2015). It is expected that the bioenergy portfolio will become very diverse and utilize much more land than is currently in active agricultural production (Balan, 2014; Chen & Smith, 2017). A. americana may prove to be a strong contender in a system that will rely on biomass from a range of crops.

Vegetative portions of A. americana have been found to be far richer in soluble carbohydrates than traditional agricultural crops (Li et al., 2012). Much of the soluble sugars found A. americana are in fermentable forms and wouldn’t require processing

(Jones, unpublished). Although lignin is a problem in the biofuel conversion process as it cannot be broken down into anything fermentable and is a hinderance to conversion

(Palomo-Briones et al., 2017), A. americana has been found to contain lower amount of lignin than has been observed in other bioenergy crops species (Jones, unpublished).

CAM plants are desirable for agriculture due to low input requirements. However, not much is known about how these plants respond to changes in nutrients and water availability. Little is known about the response of A. americana to inputs, especially on a physiological level. Nitrogen is a key element in enzymes such as RuBisCO, PEPC, and malic enzymes; crucial components in CAM photosynthesis. Nitrogen makes up a large portion of molecules vital for all plants to live; in general, proteins are approximately 14

~16% nitrogen (dry weight), cholorphyll is ~6% nitrogen (dry weight), and nucleic acids are composed ~15% of nitrogen (dry weight). It is generally understood that extremely low nitrogen availability can negatively impact photosynthetic rates (Longstreth &

Nobel, 1980). CAM plants rely on a more diverse set of enzymes than plants using C3 and C4 photosynthesis. Additional reactions required in one photosynthetic cycle suggests nitrogen could limit photosynthetic capacity of CAM plants if the ability to build enzymes is hindered. Understanding nitrogen could prove to be of more importance in CAM plants than those that use other modes of photosynthesis (Osmond, 1978; Lüttge,

2004).

In a hydroponic study of Agave deserti, it was shown that nitrogen availability was more deterministic than any other nutrient in predicting dry shoot mass (Nobel et al.,

1989). This study also described an optimal concentration of nitrogen before a negative impact was shown in the accumulation of biomass (Nobel et al., 1989). A similar study on Agave deserti showed an optimal concentration of nitrogen in a field setting with the highest change in dry biomass at 10 g N m-2, or 100 kg N ha-2 (Nobel & Hartsock, 1986).

No studies have been conducted that show the response of Agave americana to nitrogen inputs, thus, hypotheses and predictions made in this study are based off generalizations from various species in the genus; it is clear further investigation is needed.

Water is crucial in photosynthesis; providing hydrogen to build basic carbohydrates and create concentration gradients which drive many plant processes

(Farquhar & Sharkey, 1982; Lüttge, 2004). Water availability has been shown to be closely linked with both photosynthesis and biomass accumulation. Variations in water 15 input is typically the most determinant factor in growth potential (Nobel, 1976). The term succulence in Agave spp. describes the plants ability to store water in vacuoles in all organs of the plant (Bergsten & Stewart, 2014). These plants can experience vast changes in turgor pressure from night to day as water is lost to the atmosphere when the stomata are open (Nobel, 1988). It has been reported that water stored in tissues can remain in the plant for well over six months (Nobel, 1988).

Water has a significant impact on the amount of dry biomass accumulated in A. americana (Davis et al., 2017). In a field study, plants that were irrigated at 530 mm/year and 780 mm/year showed statistically identical responses, while plants watered at lower rates were shown to be significantly less productive (Davis et al., 2017). It has also been shown that there may be an optimal amount of water input, beyond which there is a negative impact on biomass accumulation (Davis et al., 2017). A prior pot study determined that water inputs are positively correlated with growth and transpiration rates in Agave americana (Ehrler, 1983).

There is evidence that shows CAM plants may be amongst some of the most capable species to cope with global climate change (Nobel & Israel, 1994; Borland et al.,

2009). Uptake of CO2 in CAM plants can increase with increasing atmospheric CO2

(Nobel & Israel, 1994). These species can survive extensive periods of drought which is a concerning difficulty that may accompany global climate change (Yang et al., 2015;

Hazrati et al., 2016). Agave americana has the added benefit of requiring far fewer water resources than current crop species. This is significant because rainfall is becoming less consistent, especially in more arid regions (Yang et al., 2015). Without diverting more 16 water resources away from residential and industrial needs, it will be necessary to find ways to use less water in agriculture practices (Borland et al., 2009). It is important to recognize which crops will be productive, resilient, and sustainable as we currently have no alternative than to accept environmental fluctuations that will accompany global climate change.

Sustainability is a major component of creating agricultural systems of the future.

CAM crops have a lesser impact on water systems and potentially soil carbon than other crops (Borland et al., 2009). A. americana fields are comparable to naturalized areas in their capacity to serve as habitats for wildlife (Kuzmick, unpublished).

This study was designed to examine the physiological impacts of both nitrogen and water in A. americana. Thirty A. americana plants were propagated as clones and subjected to one of six treatment groups. Treatment groups were established at two water input regimes (260 mm/y, 650 mm/y) and three nitrogen input regimes (0g m2, 5g m2,

10g m2). Metrics used to quantify the impact of these treatments are as follows: biomass accumulation over the course of the study, carbon and nitrogen content of leaf tissues, peak carbon assimilation capacity and transpiration, and net 24-hour rates of photosynthesis and transpiration. Plants were subject to controlled inputs for 11 months

(from March 2018 to February 2019).

It is expected that biomass accumulation and photosynthetic rates would be highly dependent on water availability; this is based on our current understanding about basic

CAM photosynthesis (Nobel, 1988; Lüttge, 2004). It would also be expected that leaf nitrogen content and photosynthetic rates would be dependent on nitrogen availability 17 based on our understanding of the role of enzyme in regulating CAM photosynthesis

(Lüttge, 2004). Furthermore, nutrient transport is expected to be dependent on the availability of water in these plants.

Specific study objectives are as follows:

1. Resolve the impact of water inputs on biomass accumulation, gas exchange rates,

nitrogen accumulation in leaf tissues, and average number of pups in A.

americana.

2. Resolve the impact of nitrogen inputs on biomass accumulation, gas exchange

rates, nitrogen accumulation in leaf tissues, and average number of pups in A.

americana.

3. Resolve the interactive effect of water and nitrogen inputs on biomass

accumulation, gas exchange rates, nitrogen accumulation in leaf tissues, and

average number of pups in A. americana.

18

CHAPTER 2: MATERIALS AND METHODS

Overview

The experimental design for this study was a 2x3 fully factorial design that included six treatment groups of two levels of water availability and three levels of nitrogen availability; five plants were randomly assigned to each treatment group. Thirty

Agave americana plants were propagated in the summer of 2017 from clonal offsets

(pups) of six mother plants that were 5 years old; the plants were not of uniform size. The pups ranged from 12 – 15 months “old” at the beginning of this the study. Plants were repotted into uniformly sized 8-inch plastic pots using a substrate of 70% Quikcrete™ all- purpose sand and 30% Harvest Organic™ potting soil (0.10% N, 0.08% P, 0.06% K).

Plants were housed in a heated greenhouse at Ohio University in Athens, Ohio. They were placed on raised benches with approximately 30cm between each plant for the entirety of the study. Each pot was fit with a tray to avoid loss of liquids through the bottom of the pot. Plants were randomly assigned to one of the six treatment groups and randomly arranged on the benches, being rotated to a new random position on the bench every six weeks.

Air temperatures were set with a low of 15°C and a high of 31.6°C, this is acceptable for A. americana which is adapted to desert conditions with highly variable temperatures from day to night (Nobel. 1988). Humidity in the air varied from 50% to as high as 90%. Light levels were recorded as high as 750 μmol photons m-2 s-1 (PAR) on sunny days, however, very low light levels were recorded on cloudy days during fall and winter months. A. americana is a desert species adapted to very high light levels; thus, 19 two high pressure sodium (HPS) lights and two LED grow light panels were installed providing an additional 700 μmol photons m-2 s-1 of artificial light to supplement the natural sunlight in the greenhouse. Supplemental lights were set to a 12-hour light cycle and light levels averaged 785 μmol photons m-2 s-1 after their installation.

Treatments

This study was designed to examine both the interactive and individual effects of nitrogen and water availability in A. americana. Treatment groups of low water and zero nitrogen input served as the control group since it would be unlikely plants would survive without water inputs. There were two water treatment groups, colloquially named “low

-1 -1 water” (260 mm H2O y ) and “high water” (650 mm H2O y ). These were selected to be representative of natural low water conditions and high water conditions that would require irrigation in arid environments (Davis et al., 2017). Tap water was applied to the plants every two weeks. Water treatments were adjusted for the size of the pot and applied directly to the substrate.

There were three levels of nitrogen application in this study; 0 kg ha-1 (0 g m-2),

50 kg ha-1 (5g m-2), and 100 kg ha-1 (10 g m-2). These were selected based on the peak biomass accumulation recorded in A. americana in response nitrogen availability (Nobel et al., 1988). The source of nitrogen was granular urea 46-0-0 (CH4N2O) which was dissolved into overlapping water treatments before application. It was expected that too little water would be applied to dissolve granular urea and make it accessible to the plants. It should be noted that urea can break down into both ammonium and nitrate. 20

These applications occurred three times during the study: May 15, 2018, July 13, 2018, and September 21, 2018.

Accumulated Biomass

The wet weight of the plants was recorded at the beginning and the end of the study. Plants were removed from their pots and shaken free of any substrate before recording the total mass; including root mass. At the end of the experiment, plants were subject to a destructive harvested and roots and shoots were separated and recorded independently. Whole leaf samples and portions of the roots were collected from each plant and dried at 65°C for 72 hours. Dried leaf and root samples were used to calculate moisture content of each organ. Independent weights for roots and shoots were used to create resource allocation ratios designated by organ type. Differences in initial and ending weight was used to calculate the plant change in mass as the plants did not start at uniform sizes and treatment effects could not be resolved based on final weights alone.

At the time of harvest, pups that emerged during the experiment were counted and recorded. This data was used to determine the average number of pups that were present per treatment group of five plants each.

Leaf Carbon and Nitrogen Content

From each plant, three 1.25 cm2 leaf punches were taken from the middle of the leaf located midway between the outermost leaves (oldest) and the central spike of the rosette (youngest). Three punches were required to obtain enough material if an additional run would have been required. The positioning of punches was selected to be most representative of nitrogen content gradients that occur in the leaves of A. americana 21

(Nobel, 1989). One plant from each treatment group was sampled at the beginning of the study and all plants were sampled at the conclusion. All plants were sampled on the same day which was three days after a scheduled watering event.

Carbon to nitrogen ratios and nitrogen content of leaf tissues were determined in an elemental analyzer (Costech Analytical Technologies, Inc.; Valencia, CA) which requires samples to be dried, weighed, and ground. Samples were dried at 65°C for 24 hours and ground using an automated bead beater with 5mm glass beads. Samples were stored in sealed sample vials which successfully prevented moisture from entering the samples. Samples were individually wrapped in aluminum tins and all analyzed in one run. Standardized acetanilide (C8H9NO) was used for calibration and to test accuracy throughout the analysis.

Gas Exchange

Gas exchange rates were measured and recorded with the LiCOR LI-6400XT portable photosynthesis system (LiCOR, Lincoln, Nebraska, USA). Two approaches were used in collecting photosynthesis data, 24-hour full cycle measurements and point measurements centralized around the known carbon assimilation peak.

Point measurements at peak carbon assimilation were recorded during the known carbon assimilation peak which was determined by several preliminary 24-hour measurements. Gas exchange in CAM photosynthesis occurs at different times of day depending on the conditions of the plants. These tests determined that gas exchange was most active from approximately 7:30pm to 11:30pm in these plants; this was an hour after the lights turned off. Chamber conditions for these measurements were set as 22

-1 follows: CO2 concentration was set to 400μmol s , temperature was set to ambient, and the chamber light was turned off. Plants were measured 9 times during this period in a randomly selected order with a match of the IRGA before every recorded measurement.

24-hour photosynthetic measurements were made on a subset of the plants with data being logged every 30 minutes. These 24-hour measurements were used to identify peak photosynthesis and were also used to compare the physiological condition of plants in a subset of the treatments for which significant differences in peak photosynthetic rates were observed. Differences were found to be most considerable between plant in high and low water groups with identical nitrogen availability and plants from high water treatments with different nitrogen availability. Plants from these groups were randomly selected to undergo further measurements. Chamber conditions for 24-hour

-1 measurements were as follows: CO2 concentration was set to 400μmol s , temperature was set to ambient, and the chamber light was set to 1000 PAR (saturating light) during daylight and turned off in succession with overhead supplemental lights (12 hour light /

12 hour dark cycle). There were 46 measurements recorded for each plant.

All gas exchange measurements were taken in the greenhouse under exact conditions to which the plants were acclimated. All measurements occurred within 24 –

96 hours of a watering event, according to methods by Erhler (1983).

The Li-6400XT is an open system meaning estimates for photosynthesis and transpiration are based on the differences in H2O and CO2 from a reference sample.

Measurements are collected by an infrared gas analyzer (IRGA) which is in the sensor 23 head of the machine. This system makes estimates of gas exchange rates based on known properties of these gasses and assumptions made about leaf area and stomatal ratios.

Statistical Analysis

All statistical analysis and data visualization were conducted using R version

3.4.3 (R Core Team, 2013) and the ggplot2 graphics package (Wickham, 2016).

Comparisons made between all groups were done using two-way ANOVAs as this was the most appropriate test to compare both individual effects and interactive effects of the treatments. Comparisons between 24-hour time-series gas exchange measurements were done using a two-sample t-tests. Analyses were all conducted with at a significance level of α = 0.05. Some analyses required transformed data to meet the assumptions of the test; these are described per individual test, below. Graphics were all created using untransformed data.

24

CHAPTER 3: RESULTS

Accumulation of Biomass

Harvested biomass was analyzed based on the change in whole plant biomass recorded from the beginning to the end of the study; these weights included root tissues

(figure 1). Results from a two-way ANOVA indicated that water treatments significantly impacted the change in biomass through the course of the study (df = 1, 24, F = 35.87, p

= <0.005). This analysis also determined that there was no significant response of biomass to nitrogen availability (df = 2, 24, F = 2.47, p = 0.106), nor was there an interactive effect of water and nitrogen treatments (df = 2, 24, F = 1.315, p = 0.287). It was generally observed that low water treatments lost biomass regardless of nitrogen availability, this was likely due to excessive cellular respiration caused by a lack of photosynthetic activity, characteristic of CAM idling (Sipes & Ting, 1985) (figure 1).

25

Figure 1. Change in wet biomass in A. americana over 11 months. Coloring represents water treatment groups as described in the legend. A horizontal line is drawn from 0 to display negative or positive change.

Water content of leaves (figure 2a) and roots (figure 2b) were analyzed separately by comparing the difference in mass of wet and dried samples from each organ. These were converted to a percentage representing the percent of the sample composed of water. Since the values are represented as a percentage, they needed to be transformed before analysis using an arcsine square root transformation; this ensured the data met the assumptions of a parametric analysis. A two-way ANOVA of the transformed data determined that water content in leaf samples was significantly impacted by water availability (df = 1, 24, F = 14.064, p = <0.005), while impacts of nitrogen availability (df

= 2, 24, F = 1.848, p = 179) and the interactive effect of water and nitrogen (df = 2, 24, F

= 0.073, p = 0.929) were not statistically significant. 26

Root water content was also transformed using an arcsine square root transformation and the data successfully met the assumption of a parametric ANOVA.

The two-way ANOVA determined that while water availability did not significantly impact root water content (df = 1, 24, F = 4.130, p = 0.053), it was very close, and is likely biologically significant. This claim is further supported by calculation of effect size

(Cohen’s f) on the model which determined that water availability (f = 0.415) had a moderate relationship with root water content. The ANOVA also determined that nitrogen availability (df = 2, 24, F = 2.584, p = 0.096) and the interaction of the treatments (df = 2, 24, F = 0.167, p = 0.847) both had no significant impact on the moisture content in the roots in A. americana; however, a calculation of effect size

(Cohen’s f) determined that nitrogen availability had a moderate relationship with root water content (f = 0.464). Moisture content of leaf samples (figure 2a) and root samples

(figure 2b) followed similar trends to those described in data for biomass accumulation

(figure 1).

27

A

B

Figure 2. Moisture content of leaf (A) and root (B) samples of A. americana. Coloring describes water treatment groups as described in the legend. Y-axis describes water content as the percent of the sample composed of water.

28

Clonal propagation of pups in A. americana is the primary mechanism for short term regeneration. Analysis was conducted on the observed numbers of pups which occurred in each treatment group at the time of harvest. A two-way ANOVA was conducted on the number of pups per treatment (n = 5). It was determined that water availability had a significant impact of the total number of pups found in each group (df =

1, 24, F = 4.932, p = 0.036). Nitrogen availability (df = 2, 24, F = 0.231, p = 0.795) and the interactive effect of the treatments (df = 2, 24, F = 0.538, p = 0.591) did not significantly impact the number of pups observed per treatment group. The highest observed number of pups was recorded in high water treatment groups (650 mm y-1)

(figure 3).

Figure 3. Total number of pups recorded per treatment group (n=5). Bar graph is separated into facets representing high water treatment (650 mm y-1) and low water treatment (260 mm y-1) groups. Coloring represents membership to nitrogen treatment group. 29

Peak Gas Exchange

The highest rates of carbon assimilation during known peak gas exchange periods were analyzed with a two-way ANOVA. However, issues of non-normality required that the data be transformed before analysis to avoid violating the assumptions of ANOVA. A cube root transformation was used because some of the values were negative. The two- way ANOVA was conducted on the transformed data, which now met assumptions, and it was determined that there was a significant effect of water availability and the highest rate of carbon assimilation recorded during this known active period (df = 1, 24, F=

29.43, p-value = <0.005). It was determined that nitrogen availability (df = 2, 24, F =

1.687, p = 0.206) and the interactive effect of both water and nitrogen (df = 2, 24, F =

1.017, p = 0.377) did not significantly interact with carbon assimilation rates. Overall, it was found groups of higher water availability reached maximum carbon assimilation rates 3-4 times higher than the plants in low water groups (figure 4).

30

Figure 4. Peak carbon assimilation rates achieved in Agave americana. Rates were recorded 9 times during this period between 7:30pm and 11:30pm, and the highest rate achieved was used for analysis. Plants were measured in complete darkness. Each box represents a treatment group. Data displayed in plot is untransformed.

A two-way ANOVA of transformed data indicated that water availability had a significant interaction with mean transpiration rates (df = 1, 24, F = 11.12, p = 0.002). A cube root transformation was applied to achieve normality in the distribution of transpiration rates before analysis. Analysis showed that nitrogen treatments (df = 2, 24,

F = 0.042, p = 0.95) and the interaction of water and nitrogen (df = 2, 24, F = 0.852, p =

0.43) did not have significant impact of the mean transpiration rates in these plants

(figure 5).

31

Figure 5. Mean transpiration rates recorded during period of known gas exchange peaks in A. americana. Rates were recorded 9 times between 7:30pm and 11:30pm. Boxes represent each treatment group. Coloring describe to which water treatment group the plants belong. Data displayed in plot is untransformed.

Gas Exchange and the Impact of Water Availability

Through the course of the study it became apparent that there were stark differences between certain groups, particularly high and low water treatments. To further resolve these differences, 24-hour photosythesis measurements were conducted on plants that would isolate the effects of water availability. This was accomplished by selecting plants from the highest nitrogen groups but from groups differing in water application rates. Welch’s two-sample t-test was conducted on the sum of photosynthetic rates for each plant per selected group and determined that the photosynthetic rates over

24-hours did not differ significantly between plants that received differing amount of 32 water (df = 4, t = 2.21, p = 0.091). Photosynthetic rates were higly varied between the two groups and individuals within the groups displayed similar patterns to eachother

(figure 6). It was observed that plants in the low water groups often exhibited long periods of cellular respiration with little to no photosythetic activity (figure 6).

Figure 6. Average 24-hour photosynthetic rate of three plants per group of differing water treatments in A. americana. All plants received nitrogen inputs (10g m-2). The x- axis displayed time of day and with coloring describing membership of different water treatment groups. Dotted horizontal line displays zero.

A two-sample t-test was also conducted on net transpirations rates between these selcted groups and it was determined that net transpirations rates over 24-hours did not differ significantly between these groups (df = 4, t = 1.332, p = 0.253). Values for photosynthesis and transpiration showed very similar diurnal patterns, highlighting the 33 effect of water availablity on 24-hour transpiration rates in A. americana (figure 7). An additional t-test determined that net stomatal conductance rates over 24-hours also did not differ significiantly between the selected water availability groups (df = 4, t = 0.924, p =

0.407).

Figure 7. Average transpiration rates of three plants per group recorded over 24-hours in A. americana (n =3). Plants received identical nitrogen inputs (10 g m-2). The x-axis displayed time through the course of a day with coloring describing membership of different water treatment groups.

Gas Exchange and the Impact of Nitrogen Availability

After isolating the effect of water availability on 24-hour net photosynthetic and transpiration rates, it became apparent that the same comparison needed to be made isolating the effect of nitrogen availability. To accomplish this, three plants were selected 34 from two groups (N = 6) that both received the highest water treatment but differed in the nitrogen application rates. This means that the only differences in the treatments applied to these plants was in nitrogen and water did not differ in this comparison. These groups were compared using Welch’s two-sample t-test and these isolated groups did not significantly differ in net photosynthetic rate over 24-hours (df = 4, t = 1.494, p = 0.209).

There were distinct differences in the average photosynthetic rate over 24-hours between groups of differing nitrogen applications (figure 8).

Figure 8. Average photosynthetic rates over 24-hour period in A. americana with treatments differing in nitrogen (n=3). Water treatment was identical in this comparison (650 mm y-1). Horizontal dotted line represents a 0 photosynthetic rate to display differences between photosynthesis and respiration. Coloring describes plants from differing nitrogen groups.

35

Net transpiration rates over 24-hours of plants from differing nitrogen availability was compared using Welch’s two-sample t-test and determined that nitrogen application rate did not have a significant impact on net transpiration rates over 24-hours (df = 4, t =

1.308, p = 0.261). These comparisons highlight differences in 24-hour transpiration rates between groups treated differently only with nitrogen. Distinct and consistent differences are shown in average transpiration rates based on the availability of nitrogen in these plants (figure 9). An additional t-test was conducted on net stomatal conductance rates over 24-hours and determined that nitrogen availability also did not have a significant impact on these rates over 24-hours (df = 4, t = 1.205, p = 0.294).

Figure 9. Average transpiration rates over 24-hours in A. americana with differing treatments of nitrogen (n=3). Water treatments were identical in this comparison (650 mm y-1). Coloring describes differences in nitrogen application rates.

36

Nitrogen Content in Leaf Tissues

Nitrogen content of leaf samples was determined by elemental analysis. The percentage of leaf tissue samples composed of nitrogen was analyzed using a two-way

ANOVA and to meet assumptions of a parametric analysis was transformed with an arcsine square root transformation. This test determined that nitrogen availability had the largest impact on percent nitrogen content of the samples (df = 2, 24, F = 9.142, p =

<0.005). It was also shown in this analysis that water availability (df = 1, 24, F = 8.642, p

= 0.006) and the interaction of nitrogen and water treatments (df = 2, 24, F = 5.819, p =

0.0075) both significantly impacted percent nitrogen in leaf tissue. There was a clear stratification of the percent nitrogen in the samples as the nitrogen application rate increased through different groups (figure 10) and a general correlation between the percentage of the leaf sample composed of nitrogen and the nitrogen availability (figure

10).

A Tukey HSD post-hoc analysis was used to determine differences between specific treatments. There was a significant difference between nitrogen groups of 50 kg ha-1 and 100 kg ha-1 (p = 0.0077) as well as between nitrogen groups of 0 kg ha-1 and 100 kg ha-1 (p = 0.0007), but not between 0 kg ha-1 and 50 kg ha-1 (figure 10). 37

Figure 10. Percentage of total leaf sample composed of nitrogen in A. americana. Samples were taken February 2019. Boxes represent each treatment group (n=5).

Ratios of carbon to nitrogen in leaf samples were created using masses of each element within the sample. A two-way ANOVA was used and determined that nitrogen application rate has a significant interaction with the carbon to nitrogen ratios in leaf samples (df = 2, 24, f-value = 7.55, p-value = 0.0023). Analysis for water treatments (df

= 1, 24, F = 3.77, p = 0.061) and the interaction of treatments (df = 2, 24, F = 3.268, p =

0.052) were determined to not significantly impact the C:N ratios in these samples. Effect size (Cohen’s f) was calculated from this model and determined that nitrogen availability

(f = 0.722) had a strong relationship with the C:N in leaf tissues and the interaction of the treatments (f = 0.475) had a moderate relationship with C:N. The trends suggest that C:N ratios decreased as nitrogen availability increased (figure 11). A Tukey HSD post-hoc analysis determined that significant differences were present between 10 g m-2 and 0 g m- 38

2 as well as between 5 g m-2 and 0 g m-2 groups, but 5 g m-2 and 0 g m-2 nitrogen groups were not significantly different.

Figure 11. Carbon to nitrogen ratios of leaf samples in A. americana calculated from mass of each element within the sample. Boxes represent each treatment group (n=5). 39

CHAPTER 4: DISCUSSION

This study resolved the effects of water and nitrogen availability on the physiology of young Agave americana. Prior to this study, little was known about the general impact of nitrogen availability and if there was an interactive effect of water and nitrogen on the physiology of A. americana.

Biomass accumulation in A. americana is highly dependent on water inputs

(figure 1). Water inputs of 260 mm y-1 were far too low to allow these plants to grow in pots. Plants in this group spent much of the time respiring (figure 6) and decreased in mass through the course of the study (figure 1); this was likely a product of CAM idling in response to severe water stress (Sipes & Ting, 1985). Water input rates of 650 mm y-1 allowed A. americana to grow and increase in biomass by approximately 50% in a year.

While the water input of 260 mm y-1 was selected to best represent natural rainfall conditions in the native habitat of A. americana, this water application rate with young plants in pots inhibited growth. In a prior field study, it was shown that at 300 mm y-1 plants were able to be accumulate biomass (Davis et al., 2017). While other studies have not shown examples of decreased biomass due to low water availability, it has been shown that in other species of Agave, water availability is strongly linked to biomass production over several months (Bergsten & Stewart, 2014). It is likely that young plants are also more susceptible to dry conditions than more established plants.

The plants did not appear to be root restricted by the pots, but it is possible that there was less lateral transfer of water than would otherwise be possible in a field setting where plants can gather water from a larger volume of soil (Nobel, 1976). There was a 40 negative growth response regardless of water treatments being adjusted for the size of the pots. Other issues could be due to the distributions of watering events. A. americana is adapted to capture large amounts of water from heavy but infrequent rainfall events

(Nobel, 1988). In this study water was applied at a consistent rate with only small amounts of water applied each time. Larger volumes of water at wider increments may have a different impact on these plants.

Biomass accumulation was not strongly associated with nitrogen availability when compared to water availability (figure 1), which contrasts previous research done in a range of other species (Poorter et al., 1990). In this study, biomass declined in low water conditions with higher nitrogen inputs (figure 1). This is interesting because it seems to suggest an underlying dependency on water for A. americana to appropriately handle nitrogen. It appears without adequate water this excess nitrogen had a slight adverse effect, even though the difference was not significant. A previous study on Agave lechuguilla found an increase in nitrogen availability decreased biomass allocation to root tissues (Nobel et al., 1989). Due to measurement constraints, masses of separated roots and shoots was not recorded at the beginning of this study, so a conclusion cannot be drawn about differential growth response of root masses to nitrogen.

Water content in leaf tissue was shown to be significantly impacted by the availability of water (figure 2a). While not statistically significant, the response of root water content to water availability was trending. Differences in water content in the leaves was apparent when conducting the harvest as the leaves were thicker in higher water treatments, however, no data was collected for this. For Agave deserti, water 41 storage capacity of leaves was linked to photosynthetic capacity (Graham & Nobel,

1999). Observations from this study show the same conclusion in A. americana. The same study on A. deserti shows a response in leaf thickness to watering frequency which also appeared to be the case with A. americana in this study (Graham & Nobel, 1999). It is generally understood that the thickness of the mesophyll in the leaves is partially determined by the availability of nitrogen (Longstreth & Nobel, 1980). However, further investigation would be needed to show this in A. americana. It was interesting that so much of the root mass was composed of water, evidence of the drought avoidance strategy that Agave spp. use to prevent water loss even in extremely dry soils (figure 2b).

The presence of clonally propagated pups was also strongly associated with water availability (figure 3). This is in slight contrast to previous studies that found no concrete link to these two parameters (Davis et al., 2017). The production of pups could be agriculturally significant as it is expected that if commercially produced, these crops would be propagated by pups not by seeding which usually only occurs after 50 year of growth (Nobel, 1988). This could have implications in identifying irrigation rates that may prove useful when pups are required for continuation of commercial production.

Point measurements of carbon assimilation and transpiration showed similar trends to the response of biomass accumulation to treatments (figure 4; figure 5). Water played a far larger role in determining rates of carbon assimilation and transpiration when compared to nitrogen availability (figure 4; figure 5). Similar to the accumulation of biomass, many of the carbon assimilation rates recorded were negative, meaning the plants were respiring during a period where carbon uptake would have been expected. 42

These observations further support the claim that 260 mm y-1 is not adequate water for A. americana to accumulate biomass in a pot study. Plants subjected to low water availability appeared to enter the modified metabolic state of CAM-idling where the stomata remained completely closed to cope with extreme water stress (Sipes & Ting,

1985). However, plants that were watered at far higher rates of 650 mm y-1 exhibited photosynthetic rates that were 3-4 times higher than those in the lower water treatment groups (figure 4). A previous field study also found that photosynthetic rates increased with water availability in A. americana (Davis et al., 2017). This analysis (figure 4) suggests room for further investigation to resolve the point at which A. americana stops idling and returns to normal photosynthetic functioning.

With increased water inputs, plants responded with far higher transpiration rates

(figure 5). This likely occurs alongside higher photosynthetic rates as more carbon dioxide is taken in during the night to be assimilated during daylight hours. It has been shown in a previous study with A. americana that soil moisture and transpiration rates are closely linked, which is further affirmed by data in this study (Ehrler, 1983).

Full cycle (24-hour) photosynthetic measurements indicate that carbon assimilation rates were consistently much higher with more available water (figure 6).

Plants which received 260 mm of water per year were continually respiring for the 24- hour measurement period. This has interesting implications for how these plants may respond to long periods with inadequate water. It is believed that without water A. americana become mostly inactive; this study shows that this also occurs when water is present but in inadequate quantity. While a field study showed that the photosynthetic 43 rates of A. americana decrease with lower water availability (Davis et al., 2017), this study shows long term respiration by young plants when water is available but in inadequate quantity.

Differences in 24-hour gas exchange parameters were observed between nitrogen groups (0 g m-2 and 10g m -2). There were differences in gas exchange rates when plants were subject to high water treatments but differed only in nitrogen availability (figure 8; figure 9). These observations are seemingly in contrast to other measurements which showed little difference in peak carbon uptake rates between nitrogen treatments.

There is evidence of a relationship between transpiration rates and nitrogen availability in A. americana (figure 9). Over 24-hours, plants with greater nitrogen availability exhibited ~250% higher photosynthetic rates. Transpiration rates followed the same trend with the different nitrogen groups exhibiting transpirations rates that were consistently higher by approximately ~200%.

Nitrogen content in the leaves of A. americana was significantly affected both by water availability and nitrogen availability (figure 10). There was a significant interactive effect of water and nitrogen on percentage of nitrogen in leaf samples. Nitrogen plays a key role in the construction of enzymes used in photosynthesis (Lüttge, 2004). While the

C:N ratio in A. americana was statistically significantly different in response to nitrogen availability, there was a trend towards an effect of water on C:N ratio also (figure 11).

Most interesting in this analysis was the step-wise increase in nitrogen content of the leaves as the availability of nitrogen increased. This shows that A. americana was taking up nitrogen at the rates that it was applied, given there was adequate water available. This 44 apparent dependency on water availability to uptake nitrogen is evidence that without adequate water these plants were unable to take in additional nitrogen applied to the soil.

Groups that were treated with the same amount of nitrogen but differed in water availability contained different amounts of nitrogen in the leaves (figure 10), which also seems to be related to their photosynthetic capacity (figure 4). Correlation of peak carbon assimilation and percent nitrogen in leaf tissues shows a slight, but insignificant relationship (R = 0.26, p = 0.17). While nitrogen treatments played a role in determining the nitrogen content of the leaves, water appears to have an almost compounding effect.

The effect on physiology of nitrogen accumulation in young plants appears to be secondary, but this could have a much larger impact as these trends continue as the plant grows. This trend is also not exclusive to CAM plants as was shown in previous studies

(Patterson et al., 1997).

The response to nitrogen and water is not exclusive to CAM plants and has been measured in other studies (e.g. Patterson et al., 1997). For example, two species of spruce showed greater growth responses when receiving water but no nitrogen as opposed to receiving nitrogen but no water (Patterson et al., 1997). The C:N ratios of spruce biomass was significantly lower in groups that received nitrogen without additional water inputs

(Patterson et al., 1997).

The primary limitations of this study arose from external environmental conditions that were not favorable for growing A. americana. As these plants were grown in Ohio, they had to be housed in a greenhouse which removed much of the environmental variability, but light conditions changed significantly with seasonal 45 variation. Much of the data collection in this study took place during the winter months, when ambient light availability was relatively low, even though supplemental light was supplied. Days were short and night-time temperatures were sometimes lower than optimum (Niechayev et al., 2018). Due to the need to be housed inside, these plants were grown in pots which could have allowed for rapid change in soil moisture and temperature.

There were also several periods where systems controlling the greenhouse in which the plants were housed did not function properly allowing for large temperature fluctuations. While these plants were acclimated to regular day and night temperature fluctuations, it is not certain how rapid changes to these conditions may have impacted the long-term growth of these plants.

A final limitation of this study was the age of these plants; they were very young and likely did not represent trends that would be found in A. americana at other growth stages. While it is important to understand these plants at all stages of life, it must be considered that this study is only representative of A. americana between one and two years old that were propagated as clones.

There are several elements of this study that warrant further investigation. The most significant of these would be further investigation of the impact of nitrogen on photosynthetic rates of A. americana over longer time periods. Another interesting interaction with nitrogen was the apparent dependency of these plants on water to take up and properly store nitrogen. It is possible that nutrient transport is highly dependent on water availability in A. americana, but further investigation is required conclusively 46 describe this. There was a slight negative growth response when nitrogen was applied without adequate water.

There is room for additional investigation of several parameters related to water availability. Further study should seek to resolve the impact of frequency of water events on growth and photosynthetic rates in A. americana. More infrequent applications of water of higher quantity may prove to be better aligned with conditions to which A. americana is adapted. At low water availability photosynthesis was completely stopped, further study should investigate at what level of stress the cessation of photosynthesis occurs. These results have real world applications for avoiding improper irrigation regimes in an agricultural setting. It would also be practical to identify at what point growth per unit of transpiration is most optimal. This would mean identifying how to produce the most biomass in A. americana per unit of water lost through transpiration.

47

CHAPTER 5: CONCLUSION

In the first year of growth, biomass accumulation in A. americana is heavily dependent on water availability while not being significantly impacted by the amount of available nitrogen. Carbon assimilation and transpiration rates were also highly dependent on the availability of water. Clonal propagation of pups is dependent on water availability in this species. Somewhat surprisingly, there was a slight relationship between transpiration rates over 24-hours and nitrogen availability, which had formerly not been observed in A. americana. Nitrogen accumulation in leaf tissues was dependent on nitrogen availability but also related strongly to water availability. It appears that A. americana is unable to accumulate nitrogen when inadequate water is available.

This study sheds light on the potential impact of nitrogen which should be considered as management strategies are developed for A. americana.

48

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