WO2015059236A1

WO2015059236A1 - Method and system for the determination of the water transport properties of the pathways of plants

Info

Publication number: WO2015059236A1
Application number: PCT/EP2014/072776
Authority: WO
Inventors: Prof. Dr. Ulrich Zimmermann; Wilhelm EHRENBERGER
Original assignee: Zim Plant Technology GmbH
Current assignee: Zim Plant Technology GmbH
Priority date: 2013-10-23
Filing date: 2014-10-23
Publication date: 2015-04-30
Anticipated expiration: 2016-04-23
Also published as: AR098164A1

Abstract

Method and device for determining water transport properties in plant pathways. The method for determining water-transport properties in plant pathways comprises the steps of inducing a pressure change in the plant pathways by an external change of at least one environmental parameter at a first region of the plant, which is located between a first clamping element and determining the pressure change in at least a second region of the plant, which is located between a second clamping element.

Description

METHOD AND SYSTEM FOR THE DETERMINATION OF THE WATER TRANSPORT PROPERTIES OF THE PATHWAYS OF PLANTS

Technical Field

The present invention relates in general to the field of water transport properties of the pathways (xylem) of plants. In particular, the present invention relates to a method and a system for determining the water transport properties in plant pathways.

Background of the Invention

It is known that at high transpiration rates (e.g., due to high ambient temperatures and low humidity) and the associated water loss, an interruption of the water pathways (xylem) in plants may occur, due to cavitation and embolism of the water columns in plants and trees that are under negative pressure. Especially in the summer months, the xylem vessels are only partially continuously filled with water when water is not sufficiently administered (Zimmermann U, Schneider H, Wegner LH, Haase A: "Water ascent in tall trees: does evolution of land plants rely on a highly metastable state ? ", New Phytologist 162: 575-615 (2004); West- hoff, M., Zimmermann, D., Schneider, H., Wegner, LH, Gessner, P., James, P., Bamberg, E., Shirley St Bentrup, F-W, Zimmermann, U. : "Evidence for discontinuous water columns in the xylem conduit of tall birch trees", Plant Biology 11 , 307-327 (2009). Heavy drought stress could cause the death of branches or cultures.

Up to now, the so-called Scholander pressure bomb method was used for the investigations of the water supply of plants. However, the method is very inaccurate. It is destructive and therefore cannot be automated. It also provides only "spot measurements" and is not suitable for detecting interruptions of water pathways in the xylem. Moreover, the method is very time-consuming and is associated with high operating costs. In said method, a leaf is cut off and exposed in a steel chamber to a continuously increasing gas pressure. The gas pressure at which water at the cut interface of the stem emerges (which is in equilibrium with the atmosphere), is taken as a measure for the water content (i.e., the turgor pressure or leaf water potential) in the leaf.

Porometers or gas exchange devices may determine exactly the stomatal transpiration and thus provide direct information on the water supply to the leaves. The methods are non-invasive, however expensive, labour intensive and unsuitable for automation. Detection of water pathways in the xylem is not possible.

Infrared thermometers can also be used to directly measure the water stress, to which plants are subjected. What is measured, is the temperature change on the leaf surface, which is produced by transpiration or evaporation. From the temperature change as a result of transpiration, the water flow rates in the pathways of the plants can be indirectly deduced. It is, however, often very problematic that the data are corrupted by evaporation of water from the soil. The method also requires expensive, supporting additional devices, such as microprocessors for data col- lection or thermal cameras and automated image analysis methods. Detection of interruptions in water pathways in the xylem is not possible.

Methods for determining the water transport rate in the plant, so called sap flow methods are in principle able to indirectly detect interruptions of the water pathways in the xylem, but are unsuitable for practical use be- cause they are very expensive. These methods are also invasive and extremely susceptible to interferences. The measuring accuracy is very low under water stress conditions. In this method a heat pulse is injected via an electrode, introduced into the pathways of the plant (temperature changes in the order of only 0.1 °C !) and via a thermistor, which is introduced above the injection site in the pathway, the vertical speed with which the heat pulse propagates, is measured.

Dendrometric methods have the same disadvantages as the sap flow methods. Dendrometers measure the change in thickness of the shoot or the stem due to the transpiration-induced water losses. This thickness change is mainly caused by water loss of the bark. This is one of the reasons why in water stress conditions, this method provides only very inaccurate values. In addition, these methods are very susceptible to interfer- ence.

However, in general, all listed methods provide no continuous information on local temporary interruptions of water pathways. Summary of the Invention

As is deduced from the preceding description, methods and/or devices, by means of which temporary spatial or local, irreversible interruptions of the xylem in the intact plant system can be detected, are of great interest for agriculture and also for forestry and science.

Accordingly, it is an object of the invention to provide a method and a system by means of which continuous information about local temporary interruptions of the water pathways can be determined. It is a further ob- ject of the present invention to provide a reversible and non-invasive method or system for determining the water transport properties of the pathways of plants. Further, it is an object of the present invention to provide an inexpensive and automatable method and system for determining the water transport properties of the pathways of plants.

This object is solved with the features of the independent claims. The dependent claims are related to further aspects of the invention.

According to one embodiment of the present invention, a method for the determination of water-transport properties in plant pathways of a plant is provided. The process comprises the steps of inducing a pressure change in the plant pathways by an external local change of at least one environmental parameter at a first region of the plant and of determining the pressure change that has been induced in the first region, at at least one second region of the plant that is located between a clamping element with pressure sensor.

Preferably, the first region and/or the region between the first and the second region and/or the second region does not show any transpiration.

The pressure change can be induced by a local temperature increase or temperature decrease and/or by local illumination and/or by applying an external pressure in the first region. Furthermore, the pressure change can be induced by biochemical manipulation or by gassing. Furthermore, the pressure change can be induced by an airflow.

The pressure change can be induced and/or determined at a plurality of locations of the plant, where, for example, the propagation time of the induced pressure change from the first to the second region is determined.

Preferably, the method is non-invasive.

The first region is preferably located between a clamping element for inducing the pressure change.

Further, according to one embodiment of the present invention, a system is provided which is suitable for the determination of water transport properties in plant pathways of a plant. The system according to the invention comprises at least one device which is suitable for inducing a pressure change in a first region of the plant by an external change of at least one environmental parameter in the first region of the plant. The system further comprises at least one clamping element with two contact elements and a pressure sensor, wherein the clamping element is suitable for receiving a second region of the plant between the two contact elements, wherein the pressure sensor is suitable for determining a pressure change due to the pressure change that has been induced in the first region, at the second region of the plant.

The device preferably comprises a clamping element that is suitable for receiving the first region of the plant between two contact elements. According to a preferred embodiment, at least one of the two contact elements of the first and/or the second clamping element are formed as a flat surface. However, differently designed contact elements are also possible and in each case adapted to the shape of the application site on the plant. Preferably, at least one of the two contact elements of the first clamping element is suitable for causing a local temperature change in the first region.

Further, the first clamping element may have a light source.

According to a preferred embodiment, the first clamping element can further be suitable for exerting a defined external pressure to the first region.

In a further preferred embodiment, at least one of the two contact elements of the first clamping element can have an opening for a biochemical manipulation or gassing.

According to a further embodiment, the system may comprise a device which is suitable to provide an airflow in the first region.

The system according to the invention can further comprise a plurality of first devices and/or clamping elements, wherein the plurality of devices and/or clamping elements are suitable for inducing or for determining the change in pressure in a different region of the plant.

Preferably, the system can be suitable for determining the propagation time of the induced pressure change between the clamping elements.

The system according to the invention is preferably designed so that the external change of at least one environmental parameter is non-invasive.

Brief description of drawings

Fig. 1a shows a schematic representation of an embodiment of the device of the present invention; and

Fig. 1 b shows a schematic representation of a further embodiment of the device of the present invention.

Exemplary embodiments of the invention

Hereinafter, the present invention is explained in detail by means of exemplary embodiments and the figures.

The present invention is based on the controlled manipulation of one or more environmental parameters) of a plant organ (so-called " action sites", e.g. an "action leaf") and the measurement of the pressure response, produced by the manipulation(s) in an intact organ of the same plant (so-called "measurement sites", e.g. a "measurement leaf). The pressure response is measured with probes in which a pressure sensor is integrated and which is mounted or clamped onto one or more intact leaves or onto other organs of the plants. All plant organs (including the "action sites") can be used as "measurement sites".

Figure 1a shows schematically an exemplary embodiment 1 of the present invention. Figure 1a shows a first leaf 10 with a first clamping element having two contact elements 11 , 12, a second leaf 20 with a second clamping element also having two contact elements 21 , 22, and a stem 30.

In the embodiment of according to Fig. 1a, due to the first clamping element, a pressure change in the first leaf 10 ("action leaf) is achieved by an external change in at least one environmental parameter, such as for example, the temperature, pressure, etc. The first and/or second clamp- ing element can, for example, be configured as described in PCT/EP2014/057427. PCT/EP2014/057427 is hereby incorporated by reference. In particular, the contact elements 11 , 12, 21 , 22 can be formed from at least one annular magnet and a metallic (or also mag- netic) counterpart. Alternatively, at least one of the contact elements 11 , 12, 21 , 22 can be formed from a clip or spring clip (e.g. microfix S (B3630Fz60), Wolfcraft GmbH Kempenich, Germany).

The distance of one of the two contact elements 11 , 12, 21 , 22 is pref- erably adjustable relative to the other contact element 11 , 12, 21 , 22 to change the acting force. This is, for example, achieved by means of a rotary thread. For example, the position of a magnet of one contact element 11 , 12, 21 , 22 can be adjusted relative to the counter-contact element 11 , 12, 21 , 22 by means of the rotary thread. Depending on the axial position of the magnet, the force applied by one contact element onto the other contact element can be stronger or weaker. To this end, the magnet has, for example, a central opening with an internal thread. The distance of one magnet from the other magnet can thus be adjusted. Since the magnetic interaction between the magnets depends on their mutual distance, a specific adjustment of the force or the pressure on the sample is thus obtained by the turning of the magnet.

Alternatively, the contact elements 11 , 12, 21 , 22 can also comprise electromagnets. By applying an electrical current, a magnetic field is formed in the coils of the electromagnet. By regulating the electrical current, the strength of the magnetic field can be varied in this embodiment, and thus the force or pressure on the sample can be adjusted. As described above, it is sufficient in each case to form one of the contact elements 11 , 12, 21 , 22 as a magnet or solenoid, where the counterpart consists of a metal. If the clamping element should be made of a spring clip, the adjustment of the force can be made, for example, by means of a rear rubber band. The contact surface of one contact element 11 , 12, 21 , 22 preferably has the same shape and size as the contact surface of the counter-contact element 11 , 12, 21 , 22. Whatever the size relationships are, it is important to ensure that a desired force or pressure is ensured on the sample. Preferably, the contact elements 11 , 12, 21 , 22 are formed in a flat way for attachment to flat leaves. However, the shape of the contact elements 11 , 12, 21 , 22 depends on the attachment site. Thus, for example, for the stem, for the petiole, etc. the contact elements 11 , 12, 21 , 22 can have a concave (semi-circular) shape according to the geometry of the object. The clamping elements preferably have a width in the cross-section parallel to the sample surface (leaf surface) between 1 mm and 40 mm. This ensures that the change in the environmental parameter is applied only locally, i.e. the change of an environmental parameter according to the present invention has no influence on the entire plant system.

The thus induced local pressure change in at least one pathway of the first leaf 10 spreads from there, along the pathways, as indicated in Fig. 1a by the white arrows. The pressure change (pressure response) is measured with a second clamping element having two contact elements 21 , 22 and a pressure measuring probe, which is clamped to a second leaf 20 ("measurement leaf). Preferably, the leaf 20 does not show any transpiration as far as possible. This can for example be achieved by increasing the local relative humidity (for example, by covering with hygroscopic gels), by covering the measurement leaf 20 with aluminium foil, nail polish or other films and substances, by keeping the part of the plant with the measurement leaf 20 in the dark or by spraying the measurement leaf 20 with the hormone ABA.

Furthermore, for example, in order to prevent pressure losses along the section between the action site (leaf 10) and the measurement site (leaf 20), it may be appropriate to cover all the leaves or organs of the plant, also the non-measurement leaves and non-action leaves or to stop the transpiration or reduce it to a minimum. The time between the pressure induction on the leaf 10 and pressure response at the measurement leaf 20 depends on the continuity of the water columns in the pathways between the location of the pressure induction and the pressure measurement. Both the time between the triggering of the pressure change in the action leaf 10 and the measured pressure change in the measurement leaf, and also the speed of propagation can be measured or calculated through additional knowledge of the distance covered. Thus, the resistance counteracting the pressure propagation is measured or calculated. The measured time can, in this case, for example be compared with a reference value to obtain an exact indication of the continuity of the water column in the pathways. The reference value could be a measurement of the pressure change in a well-watered plant of the same kind. Also, a previous reference measurement on the same plant would be possible, for example, at a time at which the plant is not affected by a high water loss. This could take place in a cooler season (spring) or also at a cooler time of day (morning) before transpiration begins. A very accurate measurement would thus be possible and since the clamping elements are (or remain) attached to the plant could be automated without major ex- penditure. According to Fig. 1a, the first clamping element is located on a first leaf 10 above the second clamping element on the second leaf 20. Alternatively however, it is also possible, as shown in Fig. 1 b, that the first clamping element is mounted on a leaf 10 below the second clamping element to determine the propagation of the pressure signal in the opposite direction. According to Fig. 1 b, the action site is thus interchanged with the measurement site from Fig. 1a. The pressure change is thus induced on the leaf 10 (action site) below leaf 20 (measurement site). The pressure change is accordingly measured above the leaf 10 on leaf 20. The elements from Fig. 1a were in this case designated with the same reference numbers in Fig. 1 b in order to illustrate the analogy. In general, a pressure signal will propagate in all spatial directions, so that a temporal propagation can be measured at any arbitrary location on the plant.

As described above, the pressure change can be achieved by the first clamping element in various ways. On the one hand, the temperature can be increased locally and preferably non-invasively in or on an intact leaf 10 by increasing the temperature. This can for example be achieved elec- trically, by infrared, by heating a counter-punch (contact element 11 , 12) of the ZIM-probe or "leaf patch clamp pressure probe" as described in WO 2012/107555 and incorporated herein by reference, installation of a heating element, etc. In the event of an increase in temperature, an (exponential) increase in pressure and thus a corresponding pressure change should be measured in the second clamping element on the measurement leaf 20. The pressure change in this case depends on the system to be examined. If the temperature is increased in a rather rigid system, this results in a (turgor) pressure increase, the increase in pressure is then measured at the measurement site (measurement leaf 20). If the temperature is increased in a more elastic system, this results in a (turgor) pressure decrease, the decrease in pressure is then measured at the measurement site (measurement leaf 20). Both directions are thus possible according to how the action site is selected (rigid or elastic tissue). The change of the pressure signal (increase or decrease) is in each case the same at the action site (leaf 10) and at the measurement site (leaf 20). The magnitude of the pressure response is dependent upon the duration and intensity of the increase in temperature. This can be up to +30°C. The minimum increase in temperature is preferably +5°C.

Similarly to the increase in temperature, a reduction in temperature can also lead to a pressure change at the location of the first clamping element. This can, for example, be used when the ambient temperatures are already high in any case. A Peltier-element, which for example is integrated in one of the contact elements of the second clamping element, can be used for cooling, for example, or ice or cold sprays or icing spray is simply used. In the event of a decrease in temperature at the action site (leaf 10), as with the increase in temperature, depending on the properties of the system under investigation (rigid or elastic tissue) a pressure decrease or pressure increase are induced at the action site (leaf 10) and thus a decrease or increase are measured in the region of the second clamping element. The time between the temperature decrease and pressure response in the measurement leaf 20 also depends on the continuity of the water columns in the pathways between the location of the temperature increase and the location of the pressure measurement. The magnitude of the pressure response depends on the duration and intensity of the tempera- ture decrease. This can be up to -30°C. Another alternative to bring about a pressure change is the local illumination of the plant surface, for example by the incorporation of a light guide in the first clamping element or by using planar organic LEDs (OLEDs). The OLEDs can, for example, form at least one of the two contact elements 11 , 12 of the first clamping element. The magnitude of the pressure response depends in this case on the duration and intensity of the local illumination. Further, a pressure increase in the pathways of the plant can also be achieved by external pressure on the leaf 10, for example by using the magnetic clamping elements described above. The pressure is increased in a leaf spot of an intact leaf 10, for example by an electromagnet in the first clamping element, clamping the leaf 10 into a pressure chamber, manual application of pressure, etc. The pressure response is measured with the pressure sensor of the second clamping element which is clamped to the leaf 20.

A pressure change could also be achieved by biochemical manipulation or by gassing (e.g. with ozone, CO₂) on the action leaf 10. Furthermore, the pressure change could be achieved by a deliberately induced airflow on action leaf 0. The airflow does not need to be constant, but may be a pulsating airflow. The airflow can also be applied in the form of either warm air or cold air for a few seconds or for several minutes (for example 10 to 15 minutes). The airflow thereby eliminates the hydration layer (water film) at the action site (action leaf 10) and thus induces a pressure change. The airflow may be generated by a fan, a blower or any other device for generating an airflow. The fan (or any similar device) can be provided externally to the action leaf. However, it is also conceivable that the fan (or any similar device) is integrated in the clamping element in the action leaf 10. The effect can be increased when it is measured in the direction of the water flow, that is, the airflow is provided at a location of the plant, which is located below the pressure sensor. A measurement during the filling process, i.e., when the plant absorbs water, can also be beneficial. A combination of the methods described above is also feasible, e.g. a simultaneous increase in temperature and a local illumination. The plant can thus be protected from excessive heat and nevertheless, a large pressure change can be achieved. Preferably, the temperature or one of the other methods for pressure change should be selected so that there is no damage to the plant at the site of the induced change in pressure.

Although in Fig. 1 only a first clamping element to increase the pressure on a leaf 10 is shown, it is quite possible to attach and operate a plurality of these clamping elements at various locations of the plant to increase the pressure. Further, it is possible to attach a plurality of second clamping elements for measuring pressure at a plurality of locations of the plant, in order to measure several pathways of the plant. It is further not absolutely necessary to attach the clamping elements to leaves. It is quite possible to attach the clamping elements to other plant organs such as to roots, to a stem, to a stalk etc., to measure the corresponding pathways. Furthermore, it is possible to attach a first clamping element suitable for increasing the pressure to a different action site from a second clamping element suitable for measuring the pressure (for example, a first clamping element on a leaf and a second clamping element on a stem). An arrangement on one and the same leaf, stem, etc. is naturally possible but then the clamping elements should be offset from one another. However, an arrangement of the first and the second clamping element at an opposite position is on the one hand not practical and on the other hand not suitable for measuring the water column in a pathway, and thus without significance.

Although the invention is shown and described in detail by means of the figures and the associated description, this representation and this detailed description are to be understood as illustrative and exemplary and not as limiting the invention. It is understood that the skilled person may make changes and modifications without departing from the scope of the following claims. In particular, the invention also includes embodiments with any combination of features, which are mentioned above or shown for various aspects and/or embodiments.

The invention also includes individual features in the figures even if they are shown here in conjunction with other features and/or are not men- tioned hereinbefore.

Claims

Method for the determination of water transport properties in plant pathways of a plant, said method comprising the steps of:

inducing a pressure change in the plant pathways by an external local change of at least one environmental parameter at a first region of the plant ; and

determining the pressure change, due to the pressure change that has been induced in the first region, at at least one second region of the plant that is located between a clamping element with pressure sensor.

Method according to claim 1 , wherein the first region and/or the gion between the first and the second region and/or the second gion do not show any transpiration.

Method according to claim 1 or 2, wherein the pressure change is induced by a local temperature increase or decrease in the first region.

Method according to any one of claims 1 to 3, wherein the pressure change is induced by a local illumination in said first region.

Method according to any one of claims 1 to 4, wherein the pressure change is induced by applying an external pressure.

Method according to any one of claims 1 to 5, wherein the pressure change is induced by biochemical manipulation or by gassing. 7. Method according to any one of claims 1 to 6, wherein the pressure change is induced by an airflow.

Method according to any one of the preceding claims, wherein the pressure change is induced and/or determined at a plurality of locations of the plant.

9. Method according to any one of the preceding claims, wherein the propagation time of the induced pressure change from the first to the second region is determined.

10. Method according to any one of the preceding claims, wherein the method is non-invasive.

Method according to one of the preceding claims, wherein the first region is located between a clamping element for inducing the pressure change.

System, suitable for the determination of water transport properties in plant pathways of a plant, the system comprising:

at least one device that is suitable for inducing a pressure change in a first region of the plant by an external change of at least one environmental parameter at the first region of the plant;

at least one clamping element with two contact elements and a pressure sensor, wherein the clamping element is suitable for receiving a second region of the plant between the two contact elements, wherein the pressure sensor is suitable for determining a pressure change, due to the pressure change that has been induced in the first region, at the second region of the plant. 13. System according to claim 12, wherein the device comprises a clamping element that is suitable for receiving the first region of the plant between two contact elements.

14. System according to claim 12 or 13, wherein at least one of the two contact elements of the first and/or the second clamping element are formed as a flat surface.

15. System according to any one of claims 12 to 14, wherein at least one of the two contact elements of the first clamping element is suitable for causing a local temperature change in the first region.

16. System according to any one of claims 12 to 15, wherein the first clamping element has a light source. 17. System according to claim any one of claims 12 to 16, wherein the first clamping element is suitable for exerting a defined external pressure to the first region.

18. System according to any one of claims 12 to 17, wherein at least one of the two contact elements of the first clamping element has an opening for a biochemical manipulation or gassing.

19. System according to any one of claims 12 to 18, wherein the device is suitable to provide an airflow in the first region.

20. System according to any one of claims 12 to 19, wherein the system comprises a plurality of first devices and/or clamp elements, wherein the plurality of devices and/or clamping elements are suitable for inducing or determining the change in pressure in a different region of the plant.

21. System according to any one of claims 12 to 20, wherein the system is suitable for determining the propagation time of the induced pressure change between the clamping elements.

22. System according to any one of claims 12 to 21 , wherein the external change of at least one environmental parameter is non-invasive.

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