Chapter 3. Measuring changes in soil nutrient availability at the root surface using ion-selective electrodes

31

Chapter 3. Measuring changes in soil nutrient availability at the root surface

using ion-selective electrodes

Wells . M . D , Smith . J . S , Borras . J , Miller . J . A

Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ , UK

uk . ac . bbsrc @ miller . tony : email ABSTRACT

For research on crop nutrition, in order to control nutrient supply to roots, plants have been usually

grown in hydroponic culture. There are some disadvantages in growing plants under these

conditions because this is an artificial situation due to the lack of a normal rhizosphere at the

root/soil interfaces. The rhizosphere and associated root morphology may have important

consequences for nutrient acquisition by roots. The aims of this research were to investigate more

fully nutrient availability in the soil/root interface by developing a method for growing roots in

rhizoboxes containing soil. This method allows direct access to the root surface and we have been

able to directly measure potassium and nitrate availability at the root surface using microelectrodes.

These measurements showed that gradients can develop along the length of the root. The

microelectrode measurements led to the idea that this methodology can be used for direct

evaluation of nutrient availability in the soil. In agriculture the efficient use of fertiliser by crops is

achieved by matching supply to demand but in practice this is very difficult to achieve, particularly

as the nutrient requirements for each type of crop are different and change throughout the season.

Regular and accurate measurement of soil nutrient status is very important in all agricultural

systems. The ability to manage soil nutrient supply to match the changing crop demand depends on

these measurements. However, soil nutrient status is dependent on a range of different factors

such as soil type, weather conditions (particularly temperature and rainfall), and previous cropping

32

method gives a value at one point in the field. Soil heterogeneity can be both spatial and temporal,

but usually soil measurements are taken shortly before a fertiliser treatment so that application

rates can be adjusted to account for what is available. The development of nutrient soil sensing

probes makes it possible to measure changes in soil nutrient availability throughout the year that

can be used to advise on future fertiliser applications.

Introduction

Soil nutrient availability is a key parameter for agriculture and the efficient use of fertiliser therefore

the methods for measuring it are very important. The methods for measuring soil nutrient status can

be divided into two types: 1) extraction methods and 2) direct soil water sampling methods.

Nutrients available for uptake by plant roots are dissolved as ions in the soil water solution and

therefore it is this pool which gives a direct measure of the nutrient supply at any one point in time.

Decisions on when and where to apply fertiliser can be made using measurements of soil available

nutrients. Such measurements can be more cost effective for farmers as the requirements of the

crop can be identified early, before yield is decreased. If action is delayed until deficiency symptoms

actually appear in crops, a sub-optimal yield will usually result.

Soil extraction methods provide a measure of the longer term potential to supply nutrients but the

release of these nutrients to make them available for uptake by plant roots depends on many

factors. These can be listed as: soil type, water content, the size of microbial populations and their

activity (in turn dependent on the available carbon supply), and pH. The extraction of plant nutrients

from soil is also complicated by the chemical strength of the extraction procedure, for example

many soil phosphate pools can be defined depending on the type of extraction procedure which is

used. These extraction procedures can be carefully defined but they have resulted in the use of

differing international standards for the measurement of soil available phosphate. For example,

‘Olsen P’ is the method used in the UK (Olsen et al., 1954) but ‘Mehlich 3’ is used in the USA

33

taken in the tests used for phosphate availability as the results can be influenced by other factors

(e.g. interfering chemicals, Axelrod and Greidinger, 1979). The Mehlich-3 assay is recommended

for any soil pH by Iowa State University, USA. Sodium bicarbonate extraction is used for Olsen P

but this extraction can be compromised by the centrifugation speed and organic carbon content of

the soil (Poile et al., 1990). Soil available potassium is usually also measured by exchanging the

cation from soil samples using high concentrations of inorganic salts (e.g. 1 M NH4NO3 or

ammonium acetate). While for measurements of soil available nitrate (NO3-) extraction washes with

a solution of 1 M KCl are used as one anion replaces the other to release the NO3- from the sample

(e.g. Djurhuus and Jacobsen, 1995). Soil water sampling methods involve the extraction of a soil

water sample for later laboratory analysis. The water samples are obtained by burying a water

permeable sampling device in the soil, commonly a ceramic suction cup, and then withdrawing and

small volume (e.g. Djurhuus and Jacobsen, 1995). This method has been modified to enable small

volume sampling to be used (Shen and Hoffland, 2007).

For crop nutrition, there has been a communication barrier between soil scientists and plant

biologists and this arises from the differing units that are used to measure soil available nutrients.

Plant biochemists and physiologists frequently use hydroponic systems for growing plants because

the nutrient supply can be easily controlled and measured by this method. On the other hand, soil

scientists commonly calculate nutrient availability using a chemical extraction procedure and then

express this figure in terms of a soil mass. Soil available nutrients are not usually expressed in

terms of the soil water content. Microelectrodes give a direct measure of the concentration of

nutrients, although more strictly it is differences in ion activity to which the electrodes actually

respond (Miller, 1995). Ion activity is the more biologically relevant parameter but it is closely

related and similar to concentration under most conditions. Ion-selective electrodes have been used

for measurements of pH in bulk soil (Quigley and Reid, 1995) and the rhizosphere (Nichol and Silk,

2001), their promise for soil K+ (Brouder et al., 2003) and NO3- (Clark et al., 2005) has been

34

producing rugged electrodes which provide an opportunity for long term in field monitoring of soil

nutrient availability and water status (in preparation). In this paper, we describe the use of

ion-selective microelectrode measurements to measure nutrient availability in the rhizosphere and

discuss how the technique may have wider applications for nutrient monitoring in the field.

Materials and methods

Soil and compost analysis. The soil was sampled from a field in Woburn, Bedfordshire, UK and it

belongs to the Stackyard series, described as a brown earth on sandy colluvium (Road Piece Field,

Catt et al., 1975). The soil pH was 7.33 and contained 1.15 % carbon with available NO3--N and

NH4+-N measured as 3.43 and 1.49 mg.kg-1 respectively. Sodium bicarbonate (pH 8.5) extractable

phosphate was measured as 55.2 mg.kg-1. Inductively coupled plasma atomic emission

spectrometry was used to measure cations following a hot acid digestion for total K+ and ammonium

acetate extraction for ‘available’ K+. Soil total and available K+ was 2148 and 132.2 mg.kg-1

respectively. The water holding capacity of an oven dried soil sample was 43 ml 100 g-1.

The compost used was obtained from Petersfield Products (Leicester, UK) and contains 75%

medium grade peat (supplemented with a wetting agent Ultrawet, Vitax Coalville, UK at 200 ml.m-3),

12% heat sterilised loam (<8 mm particle size), 3% medium grade vermiculite and 10% grit (<5 mm

particle size, lime free). The compost was supplemented with two types of fertiliser: a slow release

(3-4 months) 3.5 kg.m-3 Osmocote extract (Scotts Professional, Ipswich, UK), and 0.5 kg.m-3 PG

mix (Yara UK Ltd. Immingham, UK) quick release. The compost mix was finally amended with lime

to pH 5.5 to 6. The nutrient content of the compost for NO3--N and NH4+-N was 72.4 and 112 mg.kg -1

respectively; but the calculated total additional N available from the slow release fertiliser as NO3-

-N and -NH4+-N was 700 and 784 mg.kg-1 respectively. Sodium bicarbonate (pH 8.5) extractable

phosphate was in the range 20 to 723 mg.kg-1 with an average of 361 mg.kg-1. For compost the

amount of K+ added was 196.6 mg.kg-1, but an additional further total of 842 mg.kg-1 was available

in the slow release fertiliser. The water holding capacity of an oven dried sample of the compost

35

Plant materials. Barley seeds (Hordeum vulgare cv. Klaxon) were germinated for 2-3 days in a 10

cm Petri dish on paper towel soaked in 0.2 mM CaSO4 solution. These pre-germinated seedlings

were then transferred to a soil plastic bag system. These plastic growth pouches (Mega

International, Minneapolis, USA) were 145 x 155 mm in size and were made from gas permeable

transparent plastic containing a brown paper lining that is folded at the top to support the

germinated seed (Fig. 1). The barley seedlings were then grown for a further 4 to 7 days in these

bags that were filled with 200 g of soil or 100 g of compost also at 20˚C, 16 h daylength as

described previously (Walker et al., 1996). The soil or compost used was previously air dried and

50 mL of distilled water was added to the pouch after filling before seedling transfer. These

soil-filled pouches were covered with black plastic sheeting and placed in an upright position on a rack

that enabled the leaf to emerge into the light. These racks containing the vertical pouches were

placed in controlled environment cabinet until the seedlings were used for microelectrode

measurements. Plant tissue analysis for K+ was preformed as described previously (Walker et al.,

1996).

A.

B

Fig. 1. Barley seedlings growing in soil pouches. A. View of 145 x 155 mm seedling growth pouch.

36

Microelectrode measurements. Double-barrelled glass microcapillary ion-selective microelectrodes

were made in the manner described previously for intracellular measurements (Miller and Zhen,

1991). These glass microelectrodes had fine tips of about 1 µm diameter and they were filled as

described previously using sensor cocktails for either potassium (Walker et al., 1996) or NO3- (Miller

and Zhen, 1991). A salt bridge for ground contact was constructed as described previously using

PVC tubing and agar as described previously (Miller and Zhen, 1991) but for K+ measurements

NaCl and not KCl was used for the agar backfilling solution. Applying the same criteria used for

intracellular recordings, the microelectrode calibration curves were compared before and after the

measurements and they were rejected when these values were not identical in the range reported.

For microelectrode measurements the soil pouches containing the seedling were placed

horizontally on the stage of a microscope and light was provided by a fibre optic source as

described previously (Cookson et al., 2005). A 5 mm thick Plexiglas sheet of the same size had

been previously placed under the soil-filled pouch on the microscope stage to support the whole

area during the measurements. Roots were visible in the soil and for microelectrode access at

specific locations a small hole (approximately 3 x 2 mm) was cut in the transparent plastic using a

scalpel. After a measurement the cut section was covered with a small piece of plastic tape before

a new location was chosen. For measurements along the surface of a root the ion-selective

microelectrode tip was repositioned for up to three measurements before recalibration was

performed. The microelectrode tip was placed in various locations with the salt bridge located

always less than 1 cm away. Preliminary measurements showed that repositioning of the salt bridge

to various different distances could alter the voltage reported by the reference barrel of the

ion-selective microelectrode. Therefore the reference barrel of the double-barrelled electrode, that

would be used to report the cell membrane potential during intracellular measurements, was used

ion-37

selective barrel in the soil (Miller and Wells, 2006) before using the calibration curve to calculate the

ion activity.

B = E

A - B = a

B

A

1

2

5

7

3

6

7

8

4

9

V

7

Fig. 2. Diagrammatic representation of the arrangement for a double-barreled ion-selective

microelectrode to make measurements in the rhizosphere and soil around seedling roots. 1-

Silver/silver chloride, 2- Half-cell (electrode holder), 3- Tubing electrode, 4- 200 mM KCl or NaCl

(for K+ electrodes), 5- Agar salt bridge, 6- Silver wire, 7- 200 mM KCl, 8- Ion-selective membrane, 9- barley root A- Reference B- Ion-selective barrel C. electrometer.

Results

Figure 2 shows the experimental arrangement for measuring nutrient availability in the rhizosphere.

38

activity at various different locations were made using potassium and NO3

--selective

microelectrodes.

Fig 3. View under microscope of NO3

--selective microelectrode tip and agar salt bridge reference

alongside a barley root tip growing in soil (magnification x20).

Measurements were made on surface of the root and these were compared at various distances

along the root moving from the tip. Figure 4 shows K+ -selective microelectrode data for barley roots

growing in compost (Fig. 4 upper) and soil (Fig. 4 lower). All the measurements in compost were

higher than those obtained in soil. The average of all the measurements gave a mean K+ activity for

roots of 11.3 ± 5.4 mM (n= 30) in compost and 0.5 ± 0.5 mM (n= 21) in soil. However, in soil the

measurements provided some evidence for a general depletion of K+ in the rhizosphere moving

away from the root tip (Fig. 4 lower). An exponential relationship could be fitted through this data

Reference

electrode

Barley

root

39

supporting this idea and a fitted line is shown (Fig 4 lower). Plant tissue analysis is frequently used

as a measure of crop nutrient status so we measured and compared the K+ concentration of leaf

tissue taken from the barley seedlings at the end of the measurements. We measured a 3-fold

higher tissue K+ concentration in the leaves of seedlings growing on compost when compared with

those in soil. The mean values in mmol.g-1 dry weight with the standard deviation for seedlings in

compost was 1.3 and in soil was 0.6 ± 0.2 mmol/g dry weight (n=4).

Fig. 4. Distribution of K+-selective microelectrode measurements behind the root tip for barley

seedlings growing in two different substrates. Upper. compost (top) Lower. Woburn soil (bottom).

The decaying exponential line fitted through the Woburn soil data is y = 2.38e-0.22X (Kolmogorov-Smirnov statistic = 0.164, significance level = 0.59).

Compost

Distance from root tip (mm)

0 20 40 60 80 100

K

+ (

m

M

)

0 5 10 15 20 25 30 35

Soil

Distance from root tip (mm)

0 10 20 30 40 50 60

K

+ (

m

M

)

40

We also investigated the range of NO3

activities that could be measured at the root surface.

For these measurements the compost was given two different additional treatments. Firstly,

nutrients were leached from the compost by four washes with distilled water over 2 days. Secondly,

the compost was watered with a full standard nutrient solution containing 10 mM NO3- (Zhen et al.,

1991) and not with distilled water. Nitrate microelectrode measurements were made at the root

surface 20 mm from the tip of the primary root and the mean value obtained in each substrate in

shown in Table 1.

Table 1 Nitrate-microelectrode measurements at the root surface of barley seedlings growing in soil

and compost. Mean ± SD for n=20

Substrate Mean NO3

activity reported 20

mm from root tip (mM)

1. Distilled water leached compost 5.1 ± 0.9

2. Untreated compost 36.2 ± 3.1

3. Compost plus nutrient solution 45.4 ± 6.0

4. Woburn soil 6.3 ± 4.2

All the NO3

measurements were in the mM activity range, but the values for roots growing in soil

and leached compost were significantly lower than those obtained for the other two treatments. For

roots growing in the leached compost additional measurements were made at the root tip and these

measurements showed significantly lower NO3

activities in this region (Table 2). The mean NO3

-activity 1 mm from the root tip was 0.5 mM and this was an order of magnitude lower than the value

obtained alongside the more mature region of the root. These measurements show that the

steady-state NO3

concentration in the rhizosphere can be quite different over distances of a few

41

Table 2 Nitrate-selective microelectrode measurements at different distances behind the root tip of

a barley seedling growing in water-leached compost. Mean ± SD for n=4

Distance from root tip (mm) Mean NO3- activity (mM)

1 0.5 ± 0.4

2 1.6 ± 0.5

5 4.0 ± 1.1

Discussion

Both K+ and NO3

-selective microelectrodes were successfully used to measure the activities of

these ions at the surface of barley roots growing in soil and compost. These two different types of

substrate were chosen to provide large differences in nutrient availability. A nutrient rich compost

was compared with and a sandy poor soil with no added fertiliser. Using the soil analysis

information and water content (see Materials and Methods) we calculate the extractable K+ and

NO3

concentrations as 13 mM amd 1mM, respectively. These K+ values are 10 fold greater than

the concentrations reported by the microelectrodes near the root tip (see Fig 4) but they represent

the concentrations in the soil at the start of the experiment. For NO3

a higher concentration of 6 mM

was actually measured 20 mm behind the root tip (Table 1). These values may reflect the relative

mobility of nitrate in the soil as transpiration by the barley seedling is likely to draw water to the root

surface. Furthermore, nitrification of ammonium will tend to increase NO3- availability at the root

surface. For the compost the concentrations of both K+ and NO3- were higher. However, half the

volume was used (see Materials and Methods) and the slow release fertiliser component makes it

very difficult to predict nutrient availability accurately. Using the figures for the quick release fertiliser

we calculate the K+ and NO3

concentrations to be 10.1 mM amd 10.3 mM, respectively. These

compost values for K+ are similar to those measured with microelectrodes (Fig. 4) but as found for

soil, the NO3

concentrations at the root surface are higher (Table 1) than those calculated using the

42

seedlings growing in leached compost showed lower NO3

availability than the calculated figure

(see Tables 1 and 2). Furthermore, adding more nutrients increased the NO3

activity reported at

the root surface by the microelectrodes (Table 1).

The microelectrode measurements provided some evidence for gradients in nutrient

availability along the surface of roots growing in these solid substrates. Interestingly, the apparent

gradients for K+ and NO3- behind the root tip are in opposite directions but note that the scales are

different. We cannot explain why these gradients are different except to comment that this may

reflect root developmental differences and this requires further investigation. The results suggest

that experiments involving roots grown in hydroponics or on agar Petri dishes may not completely

reflect what happens in the rhizosphere environment .

For K+, there was some evidence for a relationship between the ion activity and the distance

of measurement from the root tip but this was only seen for seedlings growing in soil (Fig 4B).

These results suggest that under sub-optimal K+ supply a depletion zone may develop along the

root with the higher concentrations available at the root tip. The compost supplies very high

concentrations of K+ and this is reflected by the ‘luxury’ leaf tissue accumulations of the nutrient.

The mean tissue K+ concentrations were 1.3 mM/g dry weight in compost and 0.6 mM/g dry weight

in Woburn soil and the ratio is 2.3 i.e. the K concentration is 2.3 times higher than in Woburn soil.

Ion-selective microelectrode measurements of the soil K+ activity gave a 22 fold greater availability

in the compost when compared with the soil and this ratio is much larger than the leaf tissue

differences that were obtained. However, the barley seedlings were only grown for 7 days in the

pouches in these experiments. During the early growth stages the seed reserves of potassium are

important and these may lessen the effects of the large differences in soil available K+ between the

two substrates. Furthermore, the results are complicated by the fact that the mean leaf biomass

was almost two-fold greater in seedlings growing in compost when compared with those in soil

43

and error to yield optimal growth. The microelectrode measurements suggest that roots in compost

will have an optimised supply in the range that is given in hydroponic experiments. By contrast, in

soil the K+ supply is sub-optimal resulting in decreased growth by the barley seedlings and lower

leaf tissue concentrations. Only under this sub-optimal supply does a pattern of depletion appear

along the root length (Fig 4B). Such an uneven distribution of K+ supply is unlikely to be found in

hydroponically grown roots and this may have important consequences for attempts to use solution

culture to simulate the soil environment. For example, the pattern of gene expression of K+

transporters along the root may be very different and this could a significant factor in the tolerance

of drought, K+ deficiency (Walker et al., 1998) and NaCl toxicity (Carden et al., 2003).

In hydroponics it has been shown that the resting membrane potential of cells is very

dependent on the external concentration of K+ (Pitman et al., 1971; Hirsch et al., 1998). We know

from functional analysis of transporter proteins that the membrane potential is an important

parameter for regulating nutrient uptake. For example, the membrane potential not only provides

the electrical energy for nutrient uptake, but can also modify the affinity (Km) of a transporter protein

for an ion (Tong et al., 2005). Even though the resting membrane potential is negative, this is true

for both cations like K+ and anions, such as NO3

as the ion is co-transported with two protons

(Miller et al., 2007). Therefore the localised depletion of K+ along the root length may also alter

membrane potential and thereby influence the uptake of other nutrients such as NO3

from the soil.

The physiological characterisation of K+ uptake by roots has identified two mechanisms of

uptake each operating in different external concentrations (Maathuis and Sanders, 1996). Low

affinity K+ uptake activates at relatively high external K+ levels in the millimolar range and is

'passive' i.e. down the electrochemical gradient for potassium. High affinity K+ uptake from

micromolar external K+ levels is coupled to H+ transport. Measurements of root NO3- uptake have

led to the conclusion that plants have developed three types of transport system to cope with the

variations in NO3- concentrations in soils (Crawford and Glass, 1998). Two types of saturable high

affinity transport system (HATS) are able to take up NO3

44

1 mM). The constitutive system (cHATS) is available even when plants have not been previously

supplied with NO3

-. The inducible system (iHATS) is stimulated by NO3

in the external medium.

Both types of HATS are, like high affinity K+ uptake, coupled to H+ transport. The low affinity

transport system (LATS) displays linear kinetics and its contribution to NO3- uptake becomes

significant at external NO3- concentrations above 1 mM (Crawford and Glass, 1998). The

microelectrode measurements reported here suggest that in soil both mechanisms for transport of

K+ are necessary but in compost only passive uptake is required. For NO3- in both soil and compost

although the concentrations available fall in the LATS range both systems may contribute to uptake

by roots. The lowest concentration of NO3- was measured close to the root tips in leached compost

(Table 2), and at 0.5 mM the HATS uptake system is important. The microelectrode measurements

of nutrient pools on the root surface suggest that the relative importance of the different uptake

systems for K+ and NO3

may vary along the length of the root. The measurements in Table 2 show

that the steady-state NO3

activities in the rhizosphere can be quite different over distances of a few

centimetres. This local heterogeneity may be created by the root itself and to address this question

in more detail measurements in root-free bulk soil are needed to provide a comparison.

Ion-selective microelectrodes have been used to report pH and nutrient availability in the

bulk soil (Brouder et al., 2003; Clark et al., 2005). Recently design improvements have improved

the lifetime and stability of ion-selective microelectrodes. For example, the ion-selective sensor

molecules can be covalently bound into the polymer matrix of the sensor membrane to prevent

diffusion of the components into the soil (e.g. Qin et al., 2003; Puntener et al., 2004). A more

detailed comparison of this method with existing methods is required to evaluate the usefulness of

the electrode measurements. For example, more measurements of soil heterogeneity are needed.

Although soil electrode measurements provide an important biologically relevant direct measure of

nutrient ion activity in soil they are complicated by changes in soil water content. Measurements of

nutrient concentration depend on the soil moisture content and it may be necessary to monitor both

45 Conclusions

Ion-selective electrodes offer a new approach to measure soil nutrient availability. The method

provides a measure of the nutrient concentrations dissolved in the soil water and available to plant

roots and is therefore dependent on the soil water content. The electrode output provides a

measurement that is equivalent to the nutrient concentrations supplied in hydroponic culture

solutions and therefore provides a direct comparison between the conditions in these two very

different root environments.

Acknowledgements

We wish to thank Paul Dennis for the Woburn soil sample, Wendy Gregory for the soil analysis and

the University of Poitiers, France for funding JB. Rothamsted Research is grant-aided by the

Biotechnology and Biological Sciences Research Council (BBSRC) of the UK.

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