The Involvement of Aquaporins in

Ammonia/Ammonium

Transport Across Root Cell Membranes

of Barley (Hordeum vulgare L.)

by

Alexander Becker

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Ecology and Evolutionary Ecology

University of Toronto

© Copyright by Alexander Becker 2011

ii abstract

The Involvement of Aquaporins in Ammonia/Ammonium

Transport Across Root Cell Membranes of Barley

(Hordeum vulgare L.)

Alexander Becker

Master of Science

Ecology and Evolutionary Biology

University of Toronto

2011

Abstract

Using the short-lived radiotracer 13

N, we examined the hypothesis that toxic, futile

ammonia/ammonium (NH3/NH4+) fluxes at high external concentrations are mediated by

ammonia-transporting aquaporins in roots of intact barley (Hordeum vulgare L.) plants.

Effects of the aquaporin inhibitors zinc, copper, mercury, gold, silver, hydrogen peroxide,

propionic acid, and nitrogen gas supported this hypothesis. Further tests with these

inhibitors showed that changes in plant water potential and water content could be linked to

NH3/NH4+ fluxes. An increase in external pH, causing an increase of NH3 in the nutrient

solution, resulted in large increases of 13

N influx, which can only be explained in energetic

terms if the transported solute is neutrally charged. Taken together, the evidence here

strongly supports the proposed hypothesis.

iii acknowledgments

Acknowledgments

I would like to sincerely thank everybody who was there for me on the way to the Master‘s

thesis. In particular, my thanks go to my supervisor Dr. Herbert J. Kronzucker, research

associate Dr. Dev T. Britto, and my secret lab assistant Lasse Schulze, who were always

helpful to solve any problems that I had. Research in the laboratory was a great new

experience in my life, which resulted in a lot of new and good impressions.

Of course, many thanks also go to Devrim Coskun, Mena Abdel-Nour, Imtiaz Kabir, Iman

Hussain, and Manni Singh, and to my other lab members for their assistance in

experiments.

In particular my thanks go to Geoffrey Ryan Lui for editing my thesis, baking cookies, and

listening to my problems. I am very sorry for any troubles that I made, but it is always

really enjoyable with you and I am happy having you around me.

Finally, I don‘t want to forget my friends and family. Every time they have been there for

me and have believed in me.

A thousand thank to my friends and family members for the spiritual, moral, and financial

support. ―Großen Dank an meine Freunde und Familienangehörigen für die seelische,

moralische und finanzielle Unterstützung.―

iv table of contents

Table of Contents

Abstract ii

Acknowledgements iii

Table of contents iv-v

List of figures vi-vii

1. Introduction 1

1.1. Worldwide cereals situation 1

1.2. The role of nitrogen 1

1.3. Possible ways in which nitrogen may pass 3

through the root cell membrane

1.4. The importance of aquaporins 6

1.5. Involvement of aquaporins in nitrogen transport 7

2. Materials and methods 9

2.1. Germination and growth conditions 9

2.2. Tissue Potassium analyses 10

2.3. Direct influx 10

2.3.1. Metals which bind to the extracellular side of the membrane 11

2.3.2. Inhibitors which bind to the cytosolic side of the membrane 11

2.3.3. pH changes and concentration changes of NH4+ and NH3 12

2.3.4. Harvesting of the plants 12

2.4. Measuring of root and leaf water potential with a pressure bomb 12

(Scholander bomb)

2.5. Water content of barley plants 13

2.6. Statistical analyses 13

v table of contents

3. Results and short discussion 14

3.1. Tissue potassium analyses 14

3.2. Metals which bind to the extracellular side of the membrane 17

3.3. Inhibitors which bind to the cytosolic side of the membrane 23

3.4. pH changes and concentration changes in NH4+ and NH3 concentration 25

3.5. Measuring of root and leaf water potential with a pressure bomb 28

(Scholander bomb)

3.6. Water content of barley plants 30

4. Discussion 32

4.1 How K+ increases the biomass and its relation to aquaporins 32

4.2. Metals which bind to the extracellular side of aquaporins 33

4.2.1. The metals mercury, copper, zinc, calcium, and lanthanum 33

4.2.2. Gold, silver, and mercury as aquaporin inhibitors 34

4.3. Inhibitors which bind to the cytosolic side of aquaporins 35

4.4. Who is the winner: NH4+ or NH3? 37

4.5. The linkage of NH3/NH4+ transport with water transport 43

4.6. Influence of K+ on NH3/NH4

+ fluxes 45

4.7. Conclusion 46

4.8. Future role and issues 47

5. References 48

vi list of figures

List of Figures

Figure 1 Potassium content in roots and shoots of barley plants (Hordeum vulgare L.)

grown in nutrient solution with 10 mM of NH3/NH4+. (page 15)

Figure 2 Growth of barley plants under 0.0225, 0.1125, and 5 mM K+. (page 16)

Figure 3 Influence of 1 mM cupper (Cu2+

), 10 mM calcium (Ca2+

), 10 mM mercury

(Hg2+

), 10 mM zinc (Zn2+

) or 10 mM lanthanum (La 3+

) on the influx of 13

N-

labeled NH3/NH4+ in barley roots. (page 18)

Figure 4 Influence of additional pharmacological treatments on the influx of 13

N-labeled

NH3/NH4+ into intact barley roots. (page 20)

Figure 5 Influence of additional pharmacological treatments on the influx of 13

N-labeled

NH3/NH4+ into intact barley roots when transferred from 5 mM K

+ growth

solution to 0.0225 mM K+ nutrient test solution (page 22)

Figure 6 Influence of pharmacological treatments (nitrogen gas, 20 mM propionic acid,

and 2 mM hydrogen peroxide as applied to the hydroponic rooting medium) on

the influx of 13

N-labeled NH3/NH4+ into intact barley roots. (page 24)

Figure 7 Influence of pH gradients on the influx of 13

N-labeled NH3/NH4+ into intact

barley roots. (page 26)

Figure 8 Influence of pH gradients on the influx of 13

N-labeled NH3/NH4+ into intact

barley roots (page 27)

Figure 9 Influence of pharmacological treatments (500 µM gold, 500 µM mercury,

500 µM silver) on the water potential (in MPa) in root xylem of intact barley

roots. (page 29)

Figure 10 Change of water content in leaves and roots of barley plants grown in a nutrient

solution contained 10 mM of NH3/NH4+. (page 31)

Figure 11 Illustration of an aquaporin in the plasmalemma. (page 38)

vii list of figures

Figure 12 Illustration of the molecular structures of NH3, NH4+, water, and hydronium.

(page 41)

Figure 13 Illustration of a plant cell in a barley root. (page 42)

Figure 14 Illustration of barley root tips. (page 44)

1 introduction

1. Introduction

1.1. Worldwide cereals situation

Cereals are the most important staple foods for the world population. Each year, farmers

produce over two million tons of cereals worldwide (GRABOWSKI and ROLFE, 2006).

Canada is the world leader in barley production with an annual production of about

27 000 hectogram per hectare (FAO, 2009). However, critical problems such as over-

fertilization of agricultural land and the worldwide shortage of water are decreasing crop

yields all over the world (CARVAJAL et al., 1999). It is evident that more efficient uses of

fertilizer and water in agriculture will be required in order to feed the world.

1.2. The role of nitrogen

Nitrogen is an important constituent in many molecules. For instance it can be found in

amino acids, amides, proteins, nucleic acids, nucleotides, coenzymes, alkaloids, and

chlorophyll (TAIZ and ZEIGER (2007). Thus, an optimal supply and use of nitrogen is

essential for optimal plant growth. Over the last two centuries, the importance of nitrogen

has been recognized with the discovery of nitrogen fixation and the use of nitrogen as a

fertilizer. Growing crops and cereals without any nitrogen fertilization would substantially

reduce worldwide agricultural production. Standards of modern life were reached by a

higher fertilization of crops with nitrogen and other nutrients. A present problem with the

higher fertilization is the increasing anthropogenic influence on global material cycles like

the nitrogen cycle (GRUBER and GALLOWAY, 2008). Today, anthropogenic fixation is about

20 to 30 % higher than natural biological nitrogen fixation (GALLOWAY and COWLING,

2002). Consequences of the interference with global material cycles include the salinization

of fields (SQUIRES, 1994), the eluviations of NO3- (nitrate) in soils, and the eutrophication

of lakes and rivers. These factors have to lead to a need for optimization of nitrogen

fertilization in agriculture.

2 introduction

Of the many forms of nitrogen that are applied to fields as fertilizer, the most important

forms are NO3- and NH4

+ (MARSCHNER, 1995). As mentioned, NO3

- is readily washed out

of soil. This is not, however, the only problem with nitrogen fertilization strategies that are

based solely on nitrate. The uptake and assimilation of NO3- into the plant requires more

energy and NO3- is generally taken up at lower rates than NH3/NH4

+ (MARSCHNER, 1995).

Another advantage of NH3/NH4+ fertilization is the better constancy in time and space in

soil than for NO3- (LOQUE and VON WIREN, 2004).

Numerous publications (ASLAM et al.,

1996; KRONZUCKER et al. 1999; REISENAUER, 1966) have found that NH3/NH4+ inhibits

NO3- uptake. This represents a problem for plants growing on fields that are over salted

with NH3/NH4+. A combination of both forms results in higher vegetative growth.

It is known that a lack of nitrogen can stunt plant growth. On the other hand, an excess of

nitrogen can cause nitrogen toxicity and decreased water-use efficiency. In the cultivation

of cereals, either extreme can severely limit crop yield (BRITTO and KRONZUCKER, 2002).

Nitrogen toxicity can occur in many plant species when the nitrogen source is NH3/NH4+

(BRITTO and KRONZUCKER 2002). In previous studies, it was demonstrated that toxic fluxes

of NH4+ into barley and rice plants can be greatly reduced by an increase of the potassium

(K+) availability to the plant (BALKOS et al. 2010; SZCZERBA et al. 2008). However, a better

understanding of why this is, and of NH4+ toxicity itself, is contingent upon the

identification of the primary transport mechanism(s) by which NH3/NH4+ enters the cell.

Water, which is also crucial to plant growth, is also limited. As mentioned before, an excess

of nitrogen can cause nitrogen toxicity and decreased water-use efficiency. With an

investigation of the mechanism(s) that are responsible for the water and nitrogen fluxes in

plants and an investigation of the conditions under which these fluxes are optimal for plant

growth, the biomass of many crop and cereal plants might be increased. Investigating the

mechanisms would not be as involved and expensive or as potentially hazardous as genetic

modifications or the hit-and-miss method of breeding new cultivars. With a better

understanding of the mechanisms behind nutrient transport through the plant it is easier to

optimize nutrient fertilization. This could result in an increase of biomass and a reduction

of water and nitrogen application to the environment. In this study I want to show that a

regular agricultural procedure can be optimized and can have the potential to reduce the

anthropogenic influence on the global environment.

3 introduction

1.3. Possible ways in which nitrogen may pass through the root cell membrane

The route by which nitrogen enters plant cells is of interest because nitrogen supply is the

soil factor that, after water, most strongly limits plant productivity. There are several

mechanisms by which nitrogen may be transported into the plant cell.

The five important means by which NH3/NH4+ has been proposed to be transported are

(a) simple osmotic diffusion, (b) a high-affinity ammonium transporter (AMT),

(c) potassium channels, (d) nonselective cation channels (NSCCs), and (e) aquaporins.

Before explaining in more detail the different means of NH3/NH4+

transport, it is important

to understand that NH3/NH4+ influx shows different patterns while the concentration of the

nutrient increases.

At concentrations lower than 0.5 to 1 mM NH3/NH4+, NH3/NH4

+ will be transported under

a high-affinity transport system (HATS or mechanism 1) (BLOOM and CHAPIN, 1981;

WANG et al., 1993). While increasing the concentration of nitrogen, the fluxes increase

according to Michaelis–Menten kinetics. This means at higher nitrogen concentrations the

influx does not change as strongly with increases in concentration (EPSTEIN et al., 1963).

At concentrations over 0.5 to 1 mM NH3/NH4+, another system, named the low-affinity

transport system (LATS or mechanism 2), occurs. In a study by KRONZUCKER et al. (1996),

it was shown that one of the characteristics of LATS conditions is a linear increase of the

fluxes with the increasing concentration of ions.

4 introduction

Transport via simple osmotic diffusion (a) is the semi-selective diffusion of water and ions

through gaps or spaces in the cell membrane or passage of hydrophobic materials through

the lipid bilayer. This is thought to be possible because of the movement of the cell

membrane or because of the lipid solubility of the substrate. For a complex system such as

a plant, simple osmotic diffusion cannot account for the transport of all of the required

water and ions into plant cells. It is too slow and not very selective. Amounts that could

penetrate the cell membrane by this pathway are too small to explain measured fluxes of

NH3/NH4+ through the cell membrane (NOBEL, 2009.

In the case of AMT (b), active NH4+ transport occurs under high-affinity transport

conditions (HATS) (CAMAÑES et al., 2007; KAISER et al., 2002). AMT genes have been

identified that contain the information required to express AMTs in the cell membrane

(NINNEMANN et al., 1994) and show an involvement in NH4+ transport at low NH4

+

concentrations.

Another important type of transporter acting in the HATS and LATS range

(BRITTO and KRONZUCKER, 2008; ICHIDA et al., 1994; SANTA-MARÍA et al., 2000;

SZCZERBA et al., 2008) generally transports potassium, but has some NH4+-transport

activity (c). Potassium transporters can be separated into two major groups, KUP/HAK/KT

(K+ uptake permease/high-affinity K

+ transporter/K

+ transporter) and AKT (the Arabidopsis

K+ channel). HIRSCH et al. (1998) describe a gene (AKT1) that expresses AKT potassium

channels in Arabidopsis thaliana. Furthermore, SANTA-MARÍA et al. (1997) found a gene

(HAK1) that expresses potassium channels from the KUP/HAK/KT family in barley roots.

With the identification of transporter genes, mutant plants were created or the genes were

transferred (for instance to yeast cells) to test the transport of several ions (KIM et al., 1998;

RUBIO et al. 2000; SCHACHTMANN and SCHROEDER, 1994). NH4+ and K

+ are both

monovalent cations which have similar hydrated atomic radii (KIELLAND, 1937), and it may

be that NH4+ and K

+ have similar affinities for permeation through potassium channels like

AKT1 (NIELSEN and SCHJOERRING, 1998; SZCZERBA et al., 2008; WANG et al., 1996).

5 introduction

The third important group of transporters is made up of NSCCs (nonselective cation

channels) (d). NSCCs can be separated into three main classes related to their response to

the electrical potentials of cell membranes (KRONZUCKER and BRITTO, 2011):

(1) hyperpolarization-activated NSCCs (HA-NSCCs), (2) depolarization-activated NSCCs

(DA-NSCCs), and (3) voltage-insensitive NSCCs (VI-NSCCs). There are other classes of

NSCCs (DEMIDCHIK and MAATHUIS, 2007) that are not relevant to NH4+ transport. From

the three previously mentioned NSCCs groups, it is argued that VI-NSCCs are able to

transport monovalent cations such as Na+ and NH4

+ (DEMIDCHIK and MAATHUIS, 2007;

DEMIDCHIK et al., 2002). The transport of NH4+ through these channels is described

to function under high NH3/NH4+ concentrations (DEMIDCHICK et al., 2002;

WHITE et al., 1996) and thus under LATS conditions. It is also described that the

―nonselective‖ cation channels have a permeability order. In wheat root cells, for instance,

NH4+ should be more easily transported through NSCCs than rubidium or potassium

(DAVENPORT and TESTER, 2000). The question is whether or not it makes sense that NH4+

is allowed to freely permeate the cell membrane. Under this condition NH4+ could penetrate

the cell membrane, nonselectively, as the preferred ion. Unfortunately, unlike AMTs and

potassium channels, the evidence for the existence of NSCCs on the genetic level is still

absent (ZHANG et al., 2010). Data collected from investigations based on

electrophysiological studies have many scientists believing in and arguing for the existence

of NSCCs. Most of the data is based on patch-clamp analysis, where results obtained using

isolated protoplasts or artificial lipid bilayers are used to make assumptions regarding

whole tissue or even the whole plant behaviour. Other analyses such as tissue content

measurements, radiotracer experiments, or knock-out mutants should be helpful in further

investigating NSCCs.

In addition to these previously mentioned means of transport, there is a new possible

explanation for the transport of NH3/NH4+ under low-affinity conditions: aquaporins (e).

Studies show that NH3/NH4+, urea, and NO3

- can penetrate the cell membrane through

aquaporins. (GASPAR et al., 2003; LOQUÉ et al., 2005; MAUREL et al., 2008). Because of

possible relationship between water and NH3/NH4+ transport via aquaporins

(CARVAJAL et al., 1999), this big group of channels was investigated in this study.

6 introduction

Here, unidirectional nitrogen fluxes (influx and efflux) were always investigated under

conditions of high NH3/NH4+ concentration (10 mM), i.e. under low affinity transport

(LATS or Mechanism 2) conditions (EPSTEIN et al,. 1963). NH3/NH4+ transport systems

that function under HATS condition, such as AMTs, should be down regulated at high

external NH3/NH4+

1.4. The importance of aquaporins

At the beginning of the last decade, the discovery of aquaporins (AGRE et al., 1993) was an

important milestone in the field of cell physiology. It was later discovered that plants have

more genes that are responsible for the expression of aquaporins in their cell membranes

than any other group of organisms (KALDENHOFF et al., 2007).

MAUREL et al. (2008) define aquaporins as channel proteins present in the plasma and

intracellular membranes of plant cells that facilitate the transport of water and/or small

neutral solutes (water, urea, boric acid, silicic acid) or gases (ammonia, carbon dioxide). In

the literature, plant aquaporins are identified as being a part of a group of transmembrane

protein channels named MIPs: major intrinsic proteins. MIPs are classified under four

different groupings, depending on their locations in different membranes. The groups are

PIPs (plasma membrane intrinsic proteins), TIPs (tonoplast intrinsic proteins), NIPs

(nodulin-26-like intrinsic proteins), and SIPs (small basic intrinsic proteins). The idea that

different aquaporins are only located where they were originally found, however, is now

debated. For instance, in a study done by BARKLA et al. (1999), PIPs were also found in the

tonoplast membrane.

7 introduction

Different techniques have been used to investigate water and nitrogen flow in plants in

previous studies. Most of the investigations have focused on water potential measurements

of the xylem, hydraulic water conductivity of cells, or transpiration. Water potentials have

mostly been measured with a pressure chamber (Scholander bomb) or a cell pressure probe

sensor (BRAMLEY et al., 2009; STEUDLE, 2002). Water conductivity measurements

have also been measured with a cell pressure probe (EHLERT et al., 2009;

KNIPFER and FRICKE, 2010; STEUDLE and PETERSON, 1998). Transpiration measurements

have been conducted with a potometer or gravimetrically (BARROWCLOUGH et al., 2000;

BRAMLEY et al., 2009). All the mentioned studies have used inhibitors of aquaporins to

demonstrate an involvement of aquaporins in water transport (BRAMLEY et al., 2009;

CARVAJAL et al., 1999; EHLERT et al., 2009; JAVOT and MAUREL, 2002; VOLKOV et al.,

2007; Zhu et al., 2000). Other studies have been able to demonstrate transport of NH3/NH4+

via aquaporins. These investigations were made in model systems using frog (Xenopus)

oocytes. TIP genes were transferred into the DNA of the frog eggs, and the aquaporins

expressed in the egg-cell membranes were used for experimentation (HOLM et al., 2005;

JAHN et al., 2004; MAUREL et al., 1993; LOGUÉ et al., 2005).

1.5. Involvement of aquaporins in nitrogen transport

Aquaporins as water transport channels are very important for plant growth and, therefore,

for agriculture. In a previous study done by SZCZERBA et al. (2008), it was shown that an

increase of K+ available to a plant can prevent a toxic futile cycle of NH3/NH4

+ into and out

of the plant root, induced by high NH3/NH4+ concentration. If NH3/NH4

+ is transported via

aquaporins, then it follows that K+ may have an influence on aquaporin activity. With this

knowledge, adjustments to nutrient uptake by plants can be made, eventually leading to

nutritional optimization and increased water-use-efficiency in plants. This process could

also serve to save water that is needed elsewhere.

8 introduction

All experiments were carried out under conditions known to modify water transport

through aquaporins. Aquaporin-modifying agents such as mercury(II) chloride (HgCl2) and

channel blockers as well as different pH regimes were used to investigate their effects on

aquaporin activity. Experiments were conducted to find a relationship between water fluxes

and the transportation of NH3/NH4+ across the cell membrane via aquaporins. The

assumption is that if aquaporin blockers can reduce water flow, then they should also

reduce NH3/NH4+ fluxes if NH3/NH4

+ are being transported through aquaporins.

The transport of NH3/NH4+

over potassium channels and NSCCs is described as occurring

mostly in the form of NH4+. As NH4

+ is a positively-charged ion, its transport is always

associated with a loss of energy (BRITTO and KRONZUCKER, 2006). In this study, it is

hypothesized that observed fluxes of NH4+ can be not be adequately explained solely by

transport in form of NH4+ due to the high energy requirement. On the other hand,

aquaporins are mentioned as transporting nitrogen in form of NH3, a non-charged molecule.

For the purposes of this study, we propose that influxes of nitrogen are in the form of NH3,

and that this transport does not require any energy.

In this study, the short-lived radiotracer 13

N (t½ = 9.98 min) was used to investigate the flux

of NH3/NH4+ into and out of plant cells. Experiments measuring water content in plant

tissue and plant water potentials, using a Scholander bomb, were also conducted to

investigate the relationship between a plant‘s water potential and the activity of its

aquaporins. Furthermore, the concentration of K+ in the plant tissue was measured with a

flame-photometer to demonstrate the relationship between plant K+ content, and uptake of

NH3/NH4+ and water.

9 materials and methods

2. Materials and methods

2.1. Germination and growth conditions

Seeds of barley (Hordeum vulgare L., Cultivar ‗Metcalfe‘) were used. To prevent

contamination during growth, seeds were surface sterilized for 15 min in a

1% sodium hypochlorite solution and germinated for 3 days in acid-washed, autoclaved

sand. Germination and growth took place under an irradiation of circa

200 μmol photons m-2

s-1

(lights: Philips Econ-o-watt, F96T12) at plant height for

16 h per day. The temperature was 20 °C during the daytime and 15 °C during the

nighttime. The relative humidity was set to 60%. After germination, plants were grown

hydroponically in a 1/4 strength Johnson‘s nutrient solution (as described in JOHNSON,

1980, with modifications as described below) at pH 6.3 to 6.5 for an additional

4 days.

Plants were grown in nutrient solutions with the following K+ concentrations: 0.0225 mM,

0.1 mM, 1 mM, 5 mM, 10 mM, or 40 mM. Potassium (K+) was applied as K2SO4. Also, a

high NH3/NH4+ concentration of 10 mM (total) was applied to all nutrient solutions as

(NH4)2SO4. To make sure that the plants remained at a nutritional steady state, the growth

solutions were exchanged frequently.

Plants were grown in a walk-in growth chamber. One day before the experiments were to

take place, plants were arranged into bundles held together by plastic rings. After bundling,

the plants were returned to the growth solution for the final day of growth. For experiments

with the radioisotope 13

N, plants were transferred to an experimental radiotracer facility,

with similar irradiation and temperature conditions as the growth chamber, one day before

the experiment.

10 materials and methods

2.2. Tissue Potassium analyses

Plants were grown in 0.0225 mM K+ and 5 mM K

+ growth solutions. Before harvesting, the

plants were transferred to a 10 mM CaSO4 solution to remove potassium from the

apoplastic spaces. The fresh weights (FW) of the plant roots and shoots were measured.

Plant components were then dried in an oven at 100 °C for 3 days. The dry weights (DW)

of the plant roots and shoots were measured at this point. Next, the plant components were

homogenized and placed in vials topped off with 10 ml of 30 % nitric acid. The vials were

placed on a shaker for 3 days. After shaking, 1 ml of each mixed solution was transferred to

a 1.5 ml polyethylene vial and centrifuged for 3 min at a maximum speed of 13,000 rpm

(VWR® Micro 1814, Microcentrifuges). After centrifuging, 0.5 ml of the supernatant from

the root and 0.1 ml of the supernatant from the shoot were each diluted with distilled water

in a 10 ml polyethylene plastic vial to lower the concentration for measurement. The

potassium concentrations of all samples were measured with a flame photometer

(Cole-Parmer® Single-Channel Flame Photometer).

2.3. Direct influx

Direct influx measurements can be a helpful tool in the investigation transport into plant

cells. Over a specific time a radiotracer is applied (in the form of spiked nutrient solution)

to a plant part (usually the root). After this step, the amount of radiotracer which was

transported into the plant cell can be measured with a gamma counter to determine the

influx (normalized for time and tissue weight). Here experiments were conducted with a

short-term labeling method involving the radiotracer 13

N (t½ = 9.98 min). The radiotracer

came in the form of liquid NH3/NH4+ which was applied at the same concentration as in the

growth solution.

Bundles of plants were transferred into a series of vessels containing nutrient solution, with

one vessel also containing a small amount of radiotracer. Between experiments, different

modifications (―conditions‖) were applied to the nutrient solution.

11 materials and methods

2.3.1. Metals which bind to the extracellular side of the membrane

For each condition, all vessels contained nutrient solution with the same composition as

was used for growth (0.0225 mM K+ or 5 mM K

+), and contained one metal (500 µM Hg

2+

as HgCl2, 10 mM La3+

as LaCl3, 1 mM Cu2+

as C4H4CuO6 (copper(II)-tartrate),

500 µM Ag3+

as AgNO3,10 mM Ca2+

as CaSO4 or 10 mM Zn2+

as ZnSO4). As a first step,

bundles of 3 plants each were first placed into a vessel containing the ―pre-load solution‖

(growth condition plus changed condition and no radiotracer) for 10 min, during which the

plants adjusted to the changed condition. In a second step bundles were then placed into the

―load solution‖ (growth condition plus changed condition plus radiotracer) for 5 min to

label them with radiotracer. After labeling, plants were rinsed as the third step in the ―dip

solution‖ (growth condition plus changed condition and no radiotracer) for 5 s to remove as

much radiotracer as possible from the surface of the roots. Finally, the plants were placed

as the fifth step into the ―desorption solution‖ (growth condition plus changed condition

and no radiotracer) for 5 min to desorb radiotracer from the other extracellular spaces.

In one experiment, plants that were grown in a 5 mM K+ growth solution were tested using

vessels containing 0.0225 mM K+ growth solution. To remove most of the potassium ions

from the roots, plants were first dipped for 5 s in 0.0225 mM K+ growth solution before

they were brought into the pre-load solution. This was to prevent transferring excess K+ to

the pre-load solution.

2.3.2. Inhibitors which bind to the cytosolic side of the membrane

For each condition, all vessels contained nutrient solution of the same composition as was

used for growth (0.0225 mM K+ or 5 mM K

+), and contained one inhibitor (N2 as gas

bubbling, 2 mM H2O2, or 20 mM C3H6O2 (propionic acid) at pH 5.25 and pH 6.25).

Bundles of 3 plants were first placed for 2 h into a pre-load solution (growth condition plus

the changed condition). The bundles were then transferred to a load (labeling) solution

(same conditions as pre-load solution plus tracer) for 5 minutes. After labeling, plants were

transferred to a non-radioactive nutrient solution for 5 seconds. As a last step the plants

were dipped into a desorbing solution for 5 minutes.

12 materials and methods

2.3.3. pH changes and concentration changes of NH3 and NH4+

All vessels contained the same nutrient composition as for growth (0.0225 mM K+ or 5 mM

K+), contained the same or different concentration of NH3/NH4

+ (0.1 mM, 1 mM, 10 mM or

100 mM), and were adjusted to different pH (4.25, 6.25, 7.25, 8.25, or 9.25). The pre-

loading, loading, dip and desorption steps were conducted as described in section 2.3.1.

2.3.4. Harvesting of the plants

After the labeling procedure, the plants were harvested. The roots were separated from the

rest of the plant and spun in a centrifuge for 30 seconds at low speed. The fresh weights

were determined and the radioactivity remaining in the roots was measured using a gamma

counter (Wallac 1480 WIZARD 3‖ – PerkinElmer).

2.4. Measuring of root and leaf water potential with a pressure bomb

(Scholander bomb)

Plants were grown in 0.0225 mM K+ and 5 mM K

+ growth solution and were transferred

into nutrient solutions with different metals (500 µM Hg2+ as HgCl2, 500 µM Ag

+ as

AgNO3, Au3+ as HAuCl4 (chlorauric acid)). Additionally, plants grown with 0.0225 mM K

+

were transferred into 5 mM K+ nutrient solution and plants grown with 5 mM K

+ into with

0.0225 mM K+ nutrient solution . Plants were cut with a sharp razorblade at the hypocotyl

region and brought into the pressure bomb (PMS - Model 1000 Pressure Chamber

Instrument) to measure the water potential as per the method described in SCHOLANDER et

al. (1965). Measurements were conducted in between 11 a.m. and 1 p.m. before plant

transpiration had reached its maximum. The temperature, light, and humidity during the

measurement were the same as applied under the growth conditions.

13 materials and methods

2.5. Water content of barley plants

Plants were grown in 0.0225 mM K+, 0.1 mM K

+, 1 mM K

+, 5 mM K, 10 mM K

+ and

40 mM K+ growth solutions. Before harvesting, the plants were transferred to a 10 mM

CaSO4 solution to remove potassium from the apoplastic spaces. The fresh weights (FW) of

the plant roots and shoots were measured. Plant components were then dried in an oven at

100 °C for 3 days. The dry weights (DW) of the plant roots and shoots were measured at

this point. Plant water content was calculated by subtracting DW from FW.

2.6. Statistical analyses

The estimation of the data was first conducted with a one-factorial analysis of variance

(ANOVA, F-test) in a randomized system. The test helped to identify an existence of

significant differences between means. In case of a significant difference a second F-test

with residuals was conducted. The second test led to the assumption of variance

homogeneity. A third test of the residuals for normal distribution (skewness and kurtosis)

was the base to test significance with the multiple-comparisons Tukey-test.

The data from the experiments and their significances are shown in diagrams. To calculate

significances, means, and standard errors, the computer software STATGRAPHICS

Centurion XV Version 15.2.06, Graphpad Prism Version 5.00, and Microsoft Office Excel

2007 were used.

14 results

3. Results

3.1. Tissue potassium analyses

Potassium concentrations in plant tissues were measured for plants grown under two

potassium conditions (0.0225 mM K+ and 5 mM K

+) (Figure 1). Plants that were grown

under 5 mM K+ solution were found to have 70 to 80 % more potassium in their root and

shoot tissues than the plants that were grown in solution with 0.0225 mM K+. The effect of

potassium also manifested itself in plant growth, as shown in Figure 2. Plants grown in the

solution with 5 mM K+ appeared much healthier and greener than those grown

0.0225 mM K+ in the nutrient solution.

15 results

Figure 1 Potassium content in roots and shoots of barley plants (Hordeum vulgare L.) grown in nutrient

solution with 10 mM of NH3/NH4+

. Blue columns present plants which were grown with 0.0225 mM K+.

Green columns present plants which were grown with 5 mM K+. Means of 10 replicates ± SE are shown.

70 to 80 % more potassium was found in tissue of plants grown in the higher potassium treatment.

Measurements were conducted with a flame-photometer.

16 results

Figure 2 Growth of barley plants under 0.0225, 0.1125, and 5 mM K+ (left to right). Growth solution

contained 10 mM of NH3/NH4+. After 7 days there is an obvious difference in biomass production. Barley

plants which were grown with 5 mM K+ appeared much healthier and greener.

17 results

3.2. Metals which bind to the extracellular side of the membrane

The data shown in Figure 3 corresponds to radiotracer experiments investigating the

inhibition of aquaporin activity by metals and other inhibitors that bind extracellularly, and

were applied to plants grown in a nutrient solution contained 10 mM NH3/NH4+ at

pH 6.25. Furthermore the nutrient solutions included 0.0225 mM K+ or 5 mM K

+. All

treatments resulted in significantly decreased influxes of NH3/NH4+ in the LATS range.

Lanthanum and zinc showed over 50 % inhibition—the strongest effect for both nutrient

solution compositions (no data available for zinc in the 5 mM K+ nutrient solution).

Copper, calcium, and mercury also showed significant levels of inhibition but were not as

effective as zinc or lanthanum. At a higher potassium concentration (5 mM), NH3/NH4+

influx was reduced by over 35 % compared to the influxes of plants tested at a low

potassium concentration (0.0225 mM). So far it have not been demonstrated that the

putative transport inhibitors Ca2+

and La3+

can block aquaporin. In our experiments the

application Ca2+

and La3+

resulted in significantly decreased influxes in both potassium

concentrations (0.0225 and 5 mM).

18 results

Figure 3 Influence of 1 mM cupper (Cu2+

), 10 mM calcium (Ca2+

), 10 mM mercury (Hg2+

), 10 mM zinc

(Zn2+

) or 10 mM lanthanum (La 3+

) on the influx of 13

N-labeled NH3/NH4+ in barley roots. Growth and

experimental solution contained 10 mM of NH3/NH4+. Furthermore plants were grown with either

0.0225 mM K+ (light blue columns) or 5 mM K

+ (dark blue columns). The pH in the nutrient solution during

experiment was 6.25. Means of three replicates ± SE are shown. The results show a decrease in influx with all

treatments under both levels of K+ supply. In particular, lanthanum and zinc reduced the influx by over 50 %.

Letters indicate significantly different means (p < 0.05 – Tukey test). Means of four replicates ± SE are

shown.

19 results

Figure 4 presents data from another radiotracer experiment with the established aquaporin

inhibitors silver (Niemitz and Tyermann, 2002) and mercury (BRAMLEY et al., 2009;

CARVAJAL et al., 1998; JAVOT and MAUREL, 2002; VOLKOV et al., 2007). During the

experiment the plants were placed into nutrient solutions with a pH of 5.25 to prevent

precipitation of silver or mercury. The data obtained show a large decrease in NH3/NH4+

influx in the presence of silver nitrate (AgNO3) in plants grown in a nutrient solution with

0.0225 mM K+. NH3/NH4

+ influx decreased to approximately one-third of controls.

Mercury showed a smaller but still significant inhibitory effect.

20 results

Figure 4 Influence of additional pharmacological treatments on the influx of 13

N-labeled NH3/NH4+ into

intact barley roots. Plants were grown with 10 mM NH3/NH4+ and 0.0225 mM K

+. During the experiment the

nutrient solutions (different conditions) had a pH of 5.25. Means of three replicates ± SE are shown. The

results show a significant decrease in influx with 500 µM silver or 500 µM mercury: compared to the control,

mercury lowered the influx by 5 µmol g-1

(root FW) h-1

(20 %) and silver lowered the influx by

18 µmol g-1

(root FW) h-1

(>60 %). Letters indicate significantly different means (p < 0.05 Tukey test). Means

of three replicates ± SE are shown.

21 results

Data in Figure 5 were obtained in direct-influx radiotracer experiments in which plants

grown in 5 mM K+ nutrient solution were transferred to 0.0225 mM K

+ solution adjusted to

pH 5.25. In two of the three trials, silver or mercury were applied to see if they had the

same effect on aquaporin activity as before, or if aquaporin activity was inhibited (at least

in part) by a different phenomenon. The influxes of the transferred plants were inhibited by

silver or mercury and thus it appeared that these inhibitors could prevent toxic futile

transport of NH3/NH4+ into the root cells. At the same time, the plants that were transferred

from a nutrient solution with 5 mM K+ to a solution with 0.0225 mM K

+ showed higher

influxes than the control with 5 mM K+ in the nutrient solution. Influxes for plants that

were grown with 5 mM K+ and then transferred to a nutrient solution with 0.0225 mM K

+

recovered by 50 % as compared to influxes for plants grown in 0.0225 mM K+.

22 results

Figure 5 Influence of additional pharmacological treatments on the influx of 13

N-labeled NH3/NH4+ into

intact barley roots when transferred from 5 mM K+ growth solution to 0.0225 mM K

+ nutrient test solution

(conditions 1 to 4, from left). Growth and experimental solution contained 10 mM of NH3/NH4+. The pH in

the nutrient solution during experiment was 5.25 for all conditions. Means of four replicates ± SE are shown

for conditions 1 to 4. Condition 5 was grown with 10 mM NH3/NH4+ and 0.0225 mM K

+ growth solution and

means of four replicates ± SE are shown. The results show a significant inhibition in influx with silver but not

for mercury. Compared to the control, the treatment with mercury was less effective (not significantly

different from plants transferred from 5 mM K+ to 0.0225 mM K

+). Different letters indicate significantly

different means (p < 0.05 Tukey test).

23 results

3.3. Inhibitors which bind to the cytosolic side of the membrane

Results of radiotracer experiments with inhibitors that bind to the cytosolic side of the

membrane are shown in Figure 6. Nitrogen gas only showed significant differences in

plants that were grown with 0.0225 mM K+, but these differences were not as strong as

with other inhibitors. Hydrogen peroxide decreased the influx of NH3/NH4+ in plants grown

at 0.0225 mM K+ by about 20 µmol [g(root FW)]

-1 h

-1. Under high potassium

(5 mM), the NH3/NH4+ fluxes were reduced by 8 µmol [g(root FW)]

-1 h

-1 compared to their

control. The strongest effect on NH3/NH4+ influxes was observed when propionic acid was

used as the inhibitor, at both low and high potassium concentrations. At pH 6.25 and high

potassium (5 mM) the NH3/NH4+ influx was reduced by roughly one third as compared to

the control. With low potassium (0.0225 mM), the influx was decreased by over 50 %.

More significant results were obtained at pH 5.25 with propionic acid. At low [K+], the

NH3/NH4+ influx was eight times lower than the control, while at high [K

+], propionic acid

brought influx down to the same inhibited influx as it was for the low potassium

background. Compared to the control, however, the NH3/NH4+ influx was only two thirds

smaller.

24 results

Figure 6 Influence of pharmacological treatments (nitrogen gas, 20 mM propionic acid, and 2 mM hydrogen

peroxide as applied to the hydroponic rooting medium) on the influx of 13

N-labeled NH3/NH4+ into intact

barley roots. Growth and experimental solution contained 10 mM of NH3/NH4+. Furthermore plants were

grown in 0.0225 mM K+ (light blue columns) and 5 mM K

+ (dark blue columns). Unless mentioned, the pH of

the nutrient solution during the experiment was 6.25. Means of three replicates ±SE are shown. Propionic acid

shows a significant lowering of the influx at 0.0225 mM K+ and 5 mM K

+. Minor but also significant is the

influence of hydrogen peroxide (at 0.0225 mM K+ and 5 mM K

+) and nitrogen gas (at 0.0225 mM K

+).

Different lower case letters indicate significantly different means between all treatments. Uppercase letters

indicate significantly different means between plants grown and tested with a background of 5 mM K+

(p < 0.05 – Tukey test). Means of four replicates ± SE are shown.

25 results

3.4. pH changes and concentration changes in NH4+ and NH3 concentration

Another consequence of lowering or raising the pH is the change in NH4+ and NH3

concentrations in the nutrient solution. Raising the pH results in more NH3 being available

in solution for the plant. Figures 7 and 8 show a marked increase of NH3/NH4+ flux into the

plant cell with increasing pH, up to pH 9.25. At pH 4.25, the influx was a third of the

control (pH 6.25) and at pH 8.25 it was 3 times larger. This effect was observed at both low

(0.0225 mM) and high (5 mM) potassium concentrations. When going up to

pH 9.25 at 0.0225 mM K+, NH3/NH4

+ influx increased to a very high value of

223.90 µmol [g(root FW)]-1

h-1

. Figure 6 shows the concentration change of NH4+ and NH3

and the influx of NH3/NH4+ as pH was changed. At pH 9.25 the concentration of NH3 is

equal to that of NH4+ due the fact that the pKA of NH4

+ is circa 9.25. The concentration of

NH3 and the influx of NH3/NH4+ follow an exponential pattern. In contrast the

concentration of NH4+ decreases as the pH increases.

26 results

Figure 7 Influence of pH gradients on the influx of 13

N-labeled NH3/NH4+ into intact barley roots. Growth

and experimental solution contained 10 mM of NH3/NH4+. Furthermore plants were grown with either

0.0225 mM K+

(light blue columns) or 5 mM K+ (dark blue columns). Means of three replicates ± SE are

shown. Changing external pH resulted in dramatic alterations of nitrogen influx. A pH of 8.25 increased

NH3/NH4+ influx to more than three times that of controls (pH 6.25), at both K

+ concentrations, while

lowering pH to 4.25 reduced NH3/NH4+ influx by more than 50 %. Letters indicate significantly different

means (p < 0.05 – Tukey test).

27 results

Figure 8 Influence of pH gradients on the influx of 13

N-labeled NH3/NH4+ into intact barley roots. Plants

were grown with in growth solution with 10 mM of NH3/NH4+ and 0.0225 mM K

+. The influx shows an

exponential trend, as does the concentration of NH3, when pH is increased. Means of three replicates ± SE are

shown. Changing external pH resulted in dramatic alterations of NH3/NH4+ influx. Also shown is the

increasing/decreasing of NH4+ and NH3 concentration in the nutrient solutions while pH changes. Means of

three replicates ± SE are shown.

28 results

3.5. Measuring of root and leaf water potential with a pressure bomb (Scholander

bomb)

As previously shown, silver and other inhibitors had significant influences on nitrogen

fluxes. In an attempt to demonstrate stronger evidence for a relationship between aquaporin

activity and nitrogen fluxes, root-xylem water potentials were investigated. Many studies

have used pressure measurement methods to demonstrate that aquaporins are involved in

water uptake and transport (BRAMLEY et al., 2009; EHLERT et al. 2008; STEUDLE and

PETERSON; 1998). Figure 9 shows the results from experiments in which the effects on

water potential of different potential aquaporin inhibitors were measured. When plants were

grown with 0.0225 mM K+, silver showed the highest significant change of water potential.

Plants which were grown with 5 mM K+ showed the highest significant change of water

potential with silver and with gold (highest chance). Mercury only had a strong influence

on plants grown with 0.0225 mM K+. A switch from 0.0225 mM to 5 mM K

+ or vice versa

showed a change in water potential. These results support the idea that potassium affects

nitrogen flux through aquaporins.

29 results

Figure 9 Influence of pharmacological treatments (500 µM gold, 500 µM mercury, 500 µM silver) on the

water potential (in MPa) in root xylem of intact barley roots. Growth and experimental solution contained

10 mM of NH3/NH4+. Furthermore plants were also switched from either a low potassium concentration

(0.0225 mM) to a high concentration (5 mM) or from a high potassium concentration (5 mM) to a low

concentration (0.0225 mM). Plants were grown in nutrient solution with either 0.0225 mM K+ (light blue

columns) or 5 mM K+ (dark blue columns) Means of a minimum of four replicates ±SE are shown. Lower

case letters indicate significantly different means of plants grown with 0.0225 mM K+. Upper case letters

indicate significantly different means of plants grown with 5 mM K+ (p < 0.05 – Tukey test).

30 results

3.6. Water content of barley plants

Figure 10 shows water contents of plants grown under different potassium concentrations.

Starting with the low concentration of 0.0225 mM K+, water content in root and shoot was

significantly higher compared to plants grown under much higher potassium

concentrations. At a concentration of 40 mM K+, the water content was lower by

circa 3.7 % in shoots and circa 4.4 % in roots. In general, a higher potassium concentration

resulted in higher biomass but lower water content.

31 results

Figure 10 Change of water content in leaves and roots of barley plants grown in a nutrient solution contained

10 mM of NH3/NH4+. Furthermore plants were grown under different potassium concentrations

(0.0225, 0.1, 1, 5, 10 and 40 mM). Water content is shown as a %age of the fresh weight (100 %). Means of

six replicates ± SE are shown. Lower case letters indicate significantly different means between water content

of the roots. Upper case letters indicate significantly different means between water content of the leaves

(p < 0.05 – Tukey test).

32 discussion

4. Discussion

4.1 How K+ increases the biomass and its relation to aquaporins

In previous studies done by BALKOS et al. (2010) and SZCZERBA et al. (2008), it was shown

that an increase of K+ in the nutrient medium prevents the toxic futile cycling of NH3/NH4

+

in rice and barley plants. Furthermore, BALKOS et al. (2010) showed that the activities of

important enzymes such as glutamate synthetase (GS) and PEP carboxylase were the

highest with a concentration of 5 mM K+

in the nutrient medium. GS is an important

enzyme for incorporation of inorganic NH4+ into the organic-N metabolism of the plant. It

was shown that at 5 mM K+ in the nutrient medium the biomass production was also the

highest (also shown in figure 2 of the present work). The increase of biomass is explained

by the up-regulation of GS and other enzymes that are essential to NH4+ assimilation

(BALKOS et al., 2010), in combination with the control of NH3/NH4+ concentrations inside

the cell by the K+ present in the nutrient solution. In the following chapter, the relationship

between K+

concentration in the growth medium and NH3/NH4+ influxes via aquaporins

will be explained.

33 discussion

4.2. Metals which bind to the extracellular side of aquaporins

4.2.1. The metals mercury, copper, zinc, calcium, and lanthanum

Many different methods for investigating aquaporin involvement in water transport have

used metals as inhibitors (JAIN et al., 2008; NIEMITZ and TYERMANN, 2002;

VANDER WILLIGEN et al., 2006; ZELENINA et al. 2004; ZHANG et al., 1999). Mercury is the

most commonly applied inhibitor for aquaporins (BRAMLEY et al., 2009; CARVAJAL et al.,

1998; JAVOT and MAUREL, 2002; VOLKOV et al., 2007). It has been found that aquaporins,

as intracellular proteins, are extremely sensitive to metals (MAUREL et al., 2008). The

amino acids that make up the aquaporin protein structure have many free charged positions

that readily bind metals. The binding of the metal to the surfaces of an aquaporin changes

the spatial structure of the whole aquaporin and can cause the aquaporin to become more

open or more closed (TÖRNROTH-HORSEFIELD et al., 2006). Mercury, as a sulphydryl

reagent, can bind to the cysteine groups of the aquaporin and cause the aquaporin to close

(PRESTON et al., 1992). This effect is reversible with dithiothreitol (DDT), in which the

thiol group can bind to the mercury and remove it from the aquaporin. As described in the

Introduction, the functions of aquaporins have mostly been investigated in their capacity as

water channels in cell membranes. If NH3/NH4+ are also transported via aquaporins, then

blocking of aquaporins by mercury should also lower the influx of NH3/NH4+ into the plant.

In the current study, mercury showed a significant lowering of the NH3/NH4+ influxes

(Figure 3 and Figure 4), which may be evidence for the involvement of aquaporins in

NH3/NH4+ transport. It should be noted that mercury can also have many side effects, such

as allosteric inhibition of enzymes. Additionally, it has been shown that certain aquaporins

are not sensitive to mercury (BERTL and KALDENHOFF, 2007; LU and NEUMANN, 1999).

The spatial position of the amino acids in the aquaporin seems to have an influence on the

sensitivity of the aquaporin to mercury or other metals (NÉMETH-CAHALAN et al., 2004,

TÖRNROTH-HORSEFIELD et al., 2006).

34 discussion

In the present study, calcium and lanthanum also showed a significant lowering of the

NH3/NH4+ influxes (Figure 4). While calcium and lanthanum are thought to block NSCCs

(DEMIDCHICK et al., 2002), it is nevertheless possible that they inhibit aquaporins as well.

On the other hand, mercury, zinc, and copper have been shown to be aquaporin inhibitors

(CARVAJAL et al., 1998; JAIN et al., 2008; JAVOT and MAUREL, 2002;

ZELININA et al., 2004), and these elements significantly decreased NH3/NH4+ influx in this

study (Figure 4 and Figure 5). This provides further evidence for the involvement of

aquaporins in NH3/NH4+ transport through cell membrane. In general, it seems that cations

carrying a positive charge of up to 3+ may particularly influence aquaporin activity.

4.2.2. Gold, silver, and mercury as aquaporin inhibitors

In a study conducted by NIEMITZ and TYERMANN (2002), silver and gold were used to

successfully block water transport through aquaporins. In the current study, gold, silver,

and mercury also showed effects that could be related to the function of aquaporins

(Figure 3, Figure 4, Figure 5 and Figure 9). In Figure 4, a treatment of 500 µM silver at

pH 5.25 reduced the flux of NH3/NH4+ to a third of the original flux. Mercury was not as

effective as silver, but it still influenced the fluxes significantly. In a reverse experiment

described in section 2.3.1, plants grown at 5 mM K+ were transferred for the duration of the

radiotracer experiment into a nutrient solution with a lower potassium concentration

(0.0225 mM K+) to investigate whether fluxes of NH3/NH4

+ would rebound to toxic levels.

The results show an increase in NH3/NH4+ flux of about 50 % (Figure 5). These results led

to the question of whether or not silver and mercury could also inhibit this increase. Indeed,

the silver and mercury stemmed the influx of NH3/NH4+ as compared to results obtained

without using any inhibitor, and showed more evidence for the involvement of aquaporins

in NH3/NH4+ transport.

35 discussion

4.3. Inhibitors which bind to the cytosolic side of aquaporins

Previous studies have shown that inhibitors that permeate to the inside of the cell

membrane—such as gases, hydrogen peroxide, and propionic acid—can also inhibit water

transport via aquaporins (EHLERT et al, 2009, MAUREL et al, 2008,

TOURNAIRE-ROUX et al., 2003). In the current study, it was found that the inhibitors

nitrogen gas, hydrogen peroxide, and propionic acid decreased NH3/NH4+ fluxes in barley

plants (Figure 6).

In the case of the low potassium concentration (0.0225 mM K+), all treatments were

significantly different. Plants grown with 5 mM K+ showed reduced (but still significant)

difference between treatments. For example, between the control and nitrogen gas

treatment, no significant difference was observed. This could be explained by the already

large influence of K+ on aquaporins and the fluxes of NH3/NH4

+ through them.

Two studies (KATSUHARA and HANBA, 2008; WANG et al., 2007) have shown that gases

can penetrate the plant cell membrane via aquaporins. Therefore, NH3/NH4+ may be in

competition with nitrogen gas to enter the cell via aquaporins. The favored transport of

NH3/NH4+ instead of nitrogen gas through aquaporins could mean a smaller influence of

nitrogen gas on aquaporin activity. The question is whether the aquaporin favors the

transport of NH3/NH4+ over N2 or vice versa. Nitrogen gas is also thought to inhibit

aquaporin activity by passing through the aquaporin and creating a low-oxygen condition in

the cytosol. This may lead to anoxia-induced cytosolic acidification, which can cause a

protonation of the histidine residue on the cytosolic side of the aquaporin, thereby

inhibiting it (TOURNAIRE-ROUX et al., 2003; TÖRNROTH-HORSEFIELD et al., 2006).

36 discussion

In a study conducted by BIENERT et al. (2007), it was posited that hydrogen peroxide is able

to penetrate aquaporins. If this is the case, then hydrogen peroxide would be in competition

with NH3/NH4+ to enter the cell via aquaporins, and the observed flux of NH3/NH4

+ could

decrease.

Finally, the effects of propionic acid and hydrogen peroxide on aquaporin activity could

also be explained by the known pH-driven closure of aquaporins via the protonation of a

histidine residue (FISCHER and KALDENHOFF, 2008; TÖRNROTH-HORSEFIELD et al., 2006).

With the investigation of the previously mentioned aquaporin inhibitors, there is additional

evidence for the involvement of aquaporins in NH3/NH4+ transport. The strong effect of

propionic acid on the NH3/NH4+ fluxes could be explained by the closing of aquaporins that

can be caused by the protonation of the histidine residue. An increasing of the pH can open

the aquaporins (TÖRNROTH-HORSEFIELD et al., 2006), but it also increases the concentration

of NH3/NH4+ in the nutrient solution. This prompted the testing of NH3/NH4

+ influxes

under different pH conditions.

37 discussion

4.4. Who is the winner: NH4+ or NH3?

An aquaporin is thought to be constructed like an hourglass (Figure 11) (JUNG et al., 1994).

Because of the dipolar characteristic of water molecules, they are able to move through the

narrowest part (0.2 nm) of the protein structure of an aquaporin. It is thought that the

negatively-charged oxygen binds to the positively-charged ends of the protein and then

―flips‖ its positively-charged hydrogen ions to the other side of the hourglass, after which

the oxygen is released and the water molecule has penetrated the cell membrane

(AGRE, 2006).

38 discussion

Figure 11 Illustration of an aquaporin (yellow sections) in the plasmalemma (blue sections). Water molecules

(made up of an oxygen atom—shown in red—bonded with two hydrogen atoms—shown in white) move

through the aquaporin by the binding of the negatively-charged oxygen to the positively-charged residues on

the protein. A flipping over of the hydrogen ions lets the water molecule move into the plant cell.

39 discussion

When compared, NH3 and water do have similar characteristics: they are both uncharged

molecules, they both have dipoles, and their sizes are similar. In addition, both are capable

of forming hydrogen bonds with other atoms or molecules. On the other hand, the NH4+

molecule is more similar to the K+ ion. Both carry the same charge and have similar atomic

or molecular radii (Figure 12). All of these facts lead to the assumption that NH3 is the

chemical form of nitrogen that is the most likely to penetrate via aquaporins. In fact, studies

done by of JAHN et al. (2004) and HOLM et al. (2005) have already shown that NH3 is the

form of nitrogen which penetrates aquaporins in frog embryos. The results of the current

study support the possibility that the same is true for plant cells. In Figure 7, increasing pH

levels increased the total amount of NH3 in the nutrient solutions, which resulted in an

immensely larger influx of NH3/NH4+ into the plant cells. At pH 9.25, an average influx of

223.90 µmol NH3/NH4+ [g(root FW)]

-1 h

-1 was measured. This extremely high influx

cannot be energetically explained by a transport mechanism that is required for a positive

charged cation like NH4+(BRITTO and KRONZUCKER, 2009). NH3, on the other hand, is a

neutral molecule that does not disturb thermodynamic equilibrium as much as NH4+ does.

BRITTO et al. (2001) were able to determine that the net flux of NH3/NH4+ in barley plants

at a concentration of 10 mM NH3/NH4+ is smaller than that observed under lower

concentrations of NH3/NH4+. After measuring the membrane potential, they were able to

use the Nernst equation to predict the concentrations of NH3/NH4+ in the cytosol. But

measured concentrations were lower than those predicted by the Nernst equation. This led

BRITTO et al. (2001) to make the assumption that a transport of NH4+ must be supported by

an energy consuming transport, and this was supported by a higher respiration in the barley

plants.

A main problem with NH4+ flux is the relatively high amount of energy required to drive

the transport of a large amount of NH4+ across the cell membrane (BRITTO and

KRONZUCKER, 2006). Under the assumption that NH4+ is transported via uniport, one

hydrogen proton is pumped out of the cell while consuming one ATP (BRISKIN and

RENOYLDS-NIEMAN, 1991). Furthermore, one molecule of O2 is consumed in respiration to

produce five molecule of ATP (POORTER et al., 1991). POORTER et al. (1991) also

determined that the maximum root respiration was not higher than approximately

30 µmol O2 [g (root FW)]

-1 h

-1 in a series of different plants. When considering the high

40 discussion

average influx of 223.90 µmol NH3/NH4+ [g(root FW)]

-1 h

-1 into plant roots observed in the

current study, the 45 µmol O2 molecules [g (root FW)]

-1 h

-1 required in order to facilitate

that influx exceeds the maximum root respiration rates stated by POORTER et al. (1991) by

15 µmol O2 [g (root FW)]

-1 h

-1. This does not yet take into consideration the O2 that is

needed for other processes in the cell (e.g. synthesis of organic molecules), but does

effectively illustrate that NH4+ is most likely not the main form of nitrogen that is being

transported into the plant cell. Additionally, the uniport transport of NH4+ would result in

acidification of the apoplastic space and could close aquaporins, as previously described.

An influx of 223.90 µmol NH3/NH4+ [g(root FW)]

-1 h

-1 into plant cells, therefore, suggests

that NH3 might be the preferred molecule, and aquaporin the preferred pathway.

Another argument for a transport of NH3 instead of NH4+

is that under LATS conditions the

majority of transmembrane transport has been demonstrated to occur as thermodynamically

passive transport (GLASS et al., 1997). Due to the fact that not enough energy is present to

conduct transport of NH4+ at such high levels, an electrogenic uniport is less reasonable. It

is more likely that NH3 gets transported via aquaporins passively without a loss of energy.

In Figure 13, a possible model of NH3 transport is shown. The nutrient solution in the

extracellular space is adjusted to a pH of 6.25. When circa 10 mM NH4+ (exact: 9.99) is

applied to the extracellular space it can be calculated with the Henderson–Hasselbalch

equation that concentration of NH3 must be 10 µM. In the relatively small cytosol we have

a pH of circa 7.00 (ROBERTS and PANG, 1992), which means that in case of an

equilibrium concentration of 10 µM NH3, we should have a concentration of circa

1 mM NH4+ in the cytosol. In the vacuole, the pH can fluctuate in between 5 and 6

(ROBERTS and PANG, 1992). In the case of an influx/efflux of 10 µM NH3, the concentration

of NH4+ in the vacuole should therefore be between 10-100 mM. Miller et al. (2001)

investigated that in non-stressed plants the concentration of NH4+

in the vacuole can be

between 2 to 45 mM, in agreement with the presented model (Figure 13). It is known that

the vacuole typically makes up over 90 % of the whole plant cell on a volume basis. Thus,

and because of the low concentration of NH3/NH4+ in the cytosol (here 1 mM), the cytosol

is negligible as compared to the rest of the plant cell.

41 discussion

Figure 12 Illustration of the molecular structures of NH3 (top left), NH4+ (top right), water (bottom left) and

hydronium (bottom right). NH3 and water show similar charge and structure.

42 discussion

Figure 13 Illustration of a plant cell in a barley root. The blue line represents the cell membrane, the yellow

line is the vacuole membrane, and the red arrows represent NH3 flux via aquaporins. Through different pHs

which exist between the vacuole, the cytosol and extracellular space, the concentrations of NH4+ are changing

while the concentrations of NH3 stay constant.

43 discussion

4.5. The linkage of NH3/NH4+ transport with water transport

The effects on NH3/NH4+

fluxes of metals and other putative aquaporin inhibitors provide

strong evidence that aquaporins are involved in NH3/NH4+ fluxes through plant cells. To

show a stronger correlation of the data with aquaporins, metals that were used for the

radiotracer experiments were tested with a Scholander bomb. The results supported the

hypothesis that aquaporins are involved in water uptake as well as NH3/NH4+ fluxes. The

application of silver and gold resulted in significantly large changes in the water potentials

in root xylem. From these results it can be inferred that silver and gold have an influence

on aquaporins. In an unpublished electrophysiological experiment, it was shown that silver

has a strong effect on cell membrane potential. In the same experiment, it was found that

the application of silver caused shrinking of the root cells or even destroyed the cell

membrane altogether. Figure 14 is an illustration of how this effect may occur. The active

procedure of loading ions and water into the xylem causes a constant negative pressure that

moves water molecules and ions in the direction of the xylem. If a blocking of aquaporins

or even destroying of cells happens at the tip of the roots, this movement will be

interrupted. At this point, loading of ions and water still occurs into the xylem, resulting in

higher pressure in the whole system. This is what was measured in Figure 9 and is further

evidence for the involvement of aquaporins in NH3/NH4+ and

water transport. The fact that radial water transport and therewith NH3/NH4+

transport are taking place mostly as an symplastic transport in barley roots

(KNIPFER and FRICKE, 2010) would support the lower NH3/NH4+ influx and the higher

negative pressure of the water potentials if the transport occurs via aquaporins.

44 discussion

Figure 14 Illustration of barley root tips. Grey circles represent applied silver ions, blue thinner lines

represent the cell membranes, the blue thicker lines the rhizodermis and the light yellow areas the interior of

the root cells. In the representation the root tip is cut at the right side. At this region the blue arrow represents

the negative pressure that is responsible for the water and ions which move through the root. A The relatively

small arrow indicates a normal water potential (lower negative pressure) under steady state condition. B The

relatively big arrow indicates a more negative water potential (lower negative pressure) after applying silver

to the nutrient solution. When silver inhibits the aquaporins, the cells shrink.

45 discussion

4.6. Influence of K+ on NH3/NH4

+ fluxes

Plants which have higher water concentration in their tissue show higher (less negative)

water potentials (STEUDLE, 2002; STRASBURGER, 2008). Increasing the K+ in the growth

solution resulted in lower water content and this results in a decrease of the water potential

(Figure 10). It follows that a change in K+ gives a plant a powerful osmotic tool with which

to regulate osmotic fluxes. Considering the fact that the cytoplasm can reach concentrations

of 100 to 120 mM K+ (STRASBURGER, 2008), it seems reasonable that a plant with more

potassium could build up lower water potentials. The effect of potassium is the same as

effect that was observed with all tested metals and inhibitors. An inhibition of the water and

nutrient flux into and out of the cell and through whole root cell tissue can cause a lowering

of the water potential as well.

46 discussion

4.7. Conclusion

The goal of this study was it to show an involvement of aquaporins in NH3/NH4+ transport.

A better understanding of the mechanism for NH3/NH4+ transport will provide information

to adjust and optimize the fertilization of cereals. It is essential to increase the biomass and

to lower the water use of cereal plants with the lowest effort. In Figures 3, 4, and 6, toxic

futile transmembrane NH3/NH4+ transport could be inhibited. Increasing K

+, which may

influence the activity of aquaporins by inhibiting them, resulted in an increase of biomass

(BALKOS et al., 2010). Also, plants that are grown under higher potassium concentrations

showed more biomass production and used less water (Figure 10). The plants also appeared

healthier and greener (Figure 2). The fact that NH3 and not NH4+ gets transported through

the aquaporins and that the influx of it can be controlled by the pH gives agriculture another

useful tool. Farmers could optimize the supply of NH3/NH4+ by adjusting the pH of the soil.

This is not as expensive or as complicated as the genetic manipulation of plants. Expanding

on aquaporin research that has been done in vitro and in vivo, this investigation shows that

aquaporins are very likely to be responsible for nitrogen uptake in whole plants.

47 discussion

4.8. Future role and issues

The importance of aquaporins to human medicine is of unequalled and immense

importance. For instance, there are many aquaporins to be found in kidney tissue

membranes which are thought to maintain body salt and water balance (LEE et al., 1997). In

agriculture, the consideration of aquaporins as water gating proteins will change modern

farming as water becomes more scarce in arid climates (such as in Israel). What this means

is that the mechanisms behind aquaporin activity must be better understood.

In this study, data are presented that show strong evidence for an involvement of

aquaporins in NH3/NH4+ transport. This presents the opportunity to improve the biomass

and water-use efficiency of plants. Nevertheless, more investigations have to be conducted

to better understand the mechanisms behind aquaporin function. Some examples of possible

future experiments which could contribute greatly to the scientific community‘s

understanding of aquaporins are:

A) Test of other pharmacological treatments such as original antiepileptic medicaments

(e.g. acetazolamide, bumetanide) (YOOL et al., 2009).

B) Radiotracer experiments with mutants where the genes responsible for aquaporin

expression are knocked out. (DA INES et al., 2010, KALDENHOFF and FISCHER, 2006;

SCHÜSSLER et al., 2008)

C) Testing the investigated levels of nutrients (5 mM K+ and 10 mM NH4

+) in the field.

Does the increase of biomass also result in a good seed production? Are the plants resistant

against abiotic and biotic factors?

D) How are other channels (potassium channels and NSCCs) involved in nitrogen

transport? An identification of the genes that expresses NSCCs in the cell membrane,

radiotracer experiments, tissue analyses, or respiratory experiments could be helpful.

E) Identification of genes that express aquaporins and a correlation of the roles of

aquaporins. Then, a localization of the aquaporins in the plant cells in order to set up an

identification system for plant aquaporins. Exploring different aquaporins which have

special tasks and only appear in specific plant species.

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