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First published online February 4, 2009; 10.1104/pp.108.128884 Plant Physiology 149:1638-1647 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
In Situ Investigation of Leaf Water Status by Portable Unilateral Nuclear Magnetic Resonance1,2,[C],[W],[OA]Consiglio Nazionale delle Ricerche-Istituto di Metodologie Chimiche (D.C., L.M., N.P.) and Istituto di Biologia Agroambientale e Forestale (F.B., F.L.), 00015 Rome, Italy; and Dipartimento di Scienze e Tecnologie Agroalimentari Ambientali e Microbiologiche, Universitá degli Studi del Molise, 86100 Campobasso, Italy (L.M.)
A portable unilateral nuclear magnetic resonance (NMR) instrument was used to detect in field conditions the water status of leaves of herbaceous crops (Zea mays, Phaseolus vulgaris), mesophyllous trees (Populus nigra), and natural Mediterranean vegetation characterized by water-spending shrubs (Cistus incanus) and water-saving sclerophyllous trees (Quercus ilex). A good relationship was observed between NMR signal, leaf relative water content, and leaf transpiration in herbaceous leaves undergoing fast dehydration or slowly developing a drought stress. A relationship was also observed between NMR signal and water potential of Populus leaves during the development of a water stress and when leaves recovered from the stress. In the natural vegetation, the relationship between NMR signal and water status was found in Cistus, the species characterized by high transpiration rates, when measured during a drought stress period and after a rainfall. In the case of the sclerophyllous Quercus, the NMR signal, the relative water content, and the transpiration rate did not change at different leaf water status, possibly because a large amount of water is compartmentalized in cellular structures and macromolecules. The good association between NMR signal and relative water content was lost in leaves exposed for 24 h to dehydration or to an osmotic stress caused by polyethylene glycol feeding. At this time, the transverse relaxation time became longer than in leaves maintained under optimal water conditions, and two indicators of membrane damage, the ion leakage and the emission of products of membrane lipoxygenation [(Z)-3-hexenal, (Z)-3-hexenol, and (E)-2-hexenol], increased. These results taken all together give information on the physiological state of a leaf under a developing stress and show the usefulness of the NMR instrumentation for screening vegetation health and fitness in natural and cultivated conditions. It is concluded that the portable unilateral NMR instrument may be usefully employed in field conditions to monitor nondestructively the water status of plants and to assist agricultural practices, such as irrigation scheduling, to minimize stomatal closure and the consequent limitation to plant production.
The application of in vivo NMR to plants and plant tissues has brought significant contributions across a wide range of topics, reviewed by Ratcliffe and Shachar-Hill (2001)  and Verpoorte et al. (2007). High-field magnets have been used to perform techniques, such as magnetic resonance imaging and magnetic resonance microscopy (Bottomley et al., 1986; Scheenen et al., 2000; Van As, 2007), that provided an integrated view of the topological distribution of water in plant tissues such as seeds (Manz et al., 2005), stems (Van As, 2007), and leaves (Veres et al., 1993). These techniques may also help to detect water movement through xylem vessels (Van As, 2007) and may reveal a contribution of bound water consequent to membrane injury (Maheswari et al., 1999) or changes of membrane permeability during osmotic stresses (van der Weerd et al., 2002). Low-field proton NMR relaxometry is a particularly important tool to investigate plant water relations as well as water compartment, diffusion, and movement, since it detects protons predominantly contributed by 1H2O contained in plant tissues. This technique allows the proton spin density, the NMR signal, the longitudinal relaxation time (T1), and the transverse relaxation time (T2) to be measured.
The proton spin density (i.e. the amount of 1H2O per unit of volume) can be used by itself as a marker of bulk water content in tissues (McCain and Markley, 1985
In ivy bark tissues (Stout et al., 1978
Whereas many of the commonly used measurements of water content of plant tissues are destructive (e.g. water potential or relative water content [RWC]), the NMR technique is not invasive, an important feature allowing us to sample the same material throughout a sequence of stress or dependent on its ontological status. Portable NMR instruments, with low-field magnets, have been designed such that the investigations made possible by NMR relaxation technology can now be extended to field conditions (Raich and Blümer, 2004 In this article, we report the results obtained using a portable, noninvasive unilateral NMR instrument in field conditions to detect the water status of different plants. Leaves of potted herbaceous and agroforestry plants, and leaves of vegetation naturally growing in the field in Mediterranean conditions, composed principally of sclerophyllous evergreen plants and enduring a spring to summer drought stress, were used. We specifically aimed at investigating (1) whether leaf water content (as indicated by destructive assays such as RWC and pressure/volume relationships) and water loss through transpiration can be detected by changes of the NMR signal, and (2) whether the physiological state of a leaf under a developing stress (as also shown by indicators of membrane degradation) is associated with changes of the T2.
Dehydration and Rapidly Developing Water or Osmotic Stress In the first experiment, leaves of corn (Zea mays) were subjected to a fast-developing drought stress after stopping the irrigation of the entire plants (Fig. 1A ) or to a very rapid dehydration (3–5 h) after cutting the leaf from the main stem (Fig. 1B). In both cases, the reduction of RWC was clearly associated with a reduction of the integral of the NMR echo signal. More interestingly, a very good association was also found between the integral of the NMR echo signal and the transpiration rate, a nondestructive measurement of the water lost by the plant mainly through stomatal opening. RWC and transpiration were also associated with the NMR signal in bean (Phaseolus vulgaris) leaves rapidly dehydrated after cutting them from the plant (Fig. 1C).
Measurements of T2 and T1 were performed by applying the CPMG (for Carr-Purcell-Meiboom-Gill) sequence on leaves of black poplar (Populus nigra) and bean undergoing dehydration for 24 h after being cut from the plant. In Figure 2, A and B , the CPMG decays of these leaves are reported in a semilogarithmic scale. A shortening of the decay was observed during the first 3 h of stress, whereas a longer decay was observed in all leaves after 24 h of dehydration (Fig. 2, A and B). To better visualize this trend, data were inverted by obtaining the distribution of T2 (Fig. 2, C and D). This representation is particularly suitable in the case of heterogeneous systems showing multiexponential relaxation decays. In this representation, the maxima (peaks) of the distribution are centered at the corresponding most probable T2 values, while peak areas correspond to the populations of the T2 components. During the first 3 h after the cutting, the three leaves showed at least two significant peaks, the first one in the 0.2- to 1.2-ms range, and the second one centered at about 10 ms. In all leaves, after 24 h of dehydration, a third peak centered at about 40 ms appeared.
The results of T1 measurements performed on the same leaves of poplar and bean during the 24-h-long dehydration period are shown in Figure 2G. Although the effects of the water compartmentalization on T1 measurements are generally less pronounced than they are on T2 measurements (Snaar and Van As, 1992 ), due to a different averaging over the compartments, a clear reduction of T1 values was also found during the first 3 h after cutting, whereas, similar to T2, an increase of the T1 value was recorded after 24 h of dehydration. In both poplar and bean, the NMR signal integral decreased together with RWC from 3 h after starting the dehydration treatment. However, measurements after 24 h revealed that the NMR signal integral, and the longer CPMG decay, did not match the expected further reduction of RWC in wilting leaves (Fig. 2, E and F). CMPG experiments were performed several times on different leaves, and the same behavior of the CPMG decays was observed (Supplemental Fig. S1). A second experiment was run on poplar leaves undergoing an osmotic stress as rapid as the dehydration stress. Again, early stages of the osmotic stress induced a shortening of the CPMG decay (Fig. 3A ) and a decrease of NMR signal integral (Fig. 3C) similar to those reported for dehydrated leaves. Also similar to the findings of the dehydration experiment, both an increase of NMR signal integral and a longer CPMG decay were observed in leaves 24 h after the application of the osmolyte. The T2 distribution reported in Figure 3B shows three peaks centered about at 0.3, 4, and 10 ms during the first 3 h of osmotic stress. After 24 h from the osmolyte application, a new peak corresponding to a much longer T2 value (i.e. 100 ms) was present.
As the stress further proceeds, a reduction of the CPMG decay occurred until a very short decay, observed in wilted leaves (Fig. 3D), indicated that only tightly bound water contributes to the NMR signal. Two markers of cell damage were also investigated in poplar leaves subjected to the dehydration or to the osmotic stress experiments. Both in vivo measurements of the emission of lipoxygenase (LOX) products formed by membrane degradation (Fig. 4A ) and measurements of ion leakage from leaf discs (Fig. 4B) showed clear increases of the two parameters with the time of exposure to the stresses. The increase was particularly significant and occurred somewhat earlier in leaves exposed to the osmotic stress. During the dehydration and osmotic stress experiments, the increase of the two markers was associated with the value of the magnetization intensity calculated at a time of 40 ms on the best fit curve of the CPMG decays (i.e. to the intensity of the echo at a time of 40 ms; Fig. 5 ).
Slowly Developing and Reversible Water Stress With the following experiments, we wanted to investigate whether NMR reliably assesses water content when the water stress develops slowly and is recovered upon rewatering. Measurements of T2 were performed on poplar leaves of potted plants that were watered with 30% of the transpired water and then again irrigated to full soil capacity. A shortening of CPMG decay was observed in the first 3 d of water stress, which was followed by a lengthening of the decay after 4 and 6 d of stress (Fig. 6A ). In the corresponding T2 distribution (Fig. 6B), the long T2 component appeared and was centered at about 20 and 40 ms after 4 and 6 d of stress, respectively. After a 2-d rewatering, a shortening of the decay was again observed, which corresponded to the appearance of the long T2 component back to about 20 ms. After the 2-d-long rewatering, leaves recovered about 80% of the photosynthetic rates measured before stress occurrence (16 ± 2 µmol m–2 s–1), while photosynthesis was almost completely suppressed at the end of the water stress period (data not shown).
It is worth noting that the CPMG trend observed in the case of the slow-developing stress is very similar to the one observed in the case of fast-developing stresses, with a slight shortening of the decay during the first period of stress and a net lengthening of the decay during the last period of stress. This lengthening corresponds to the appearance of a long T2 component in the T2 distribution, centered at about 40 ms after 6 d and 24 h of stress, respectively (Figs. 2, C and D, 3B, and 6B). The lengthening of the decay should not be attributed to leaf wilting, which is characterized by an extremely short decay (Fig. 3D), but rather is typical of a reversible stress from which plants can recover. In another experiment, destructive measurements of leaf water potential were coupled to T2 measurements on leaves of black poplar under a slow-developing water stress (20 d) followed by a 6-d recovery upon rewatering. The water stress was generated by supplying 50% of the transpired water to potted poplars. The value of the leaf water potential was correlated to the value of the NMR echo integral. As the water potential decreased, the echo integral also decreased during the stress (Fig. 7A ). After the rewatering, the echo integral as well as the water potential returned to similar prestress values. When plotted together, a clear correlation between the two parameters was observed (Fig. 7B).
Finally, we performed some field measurements on the natural Mediterranean vegetation to expand the experimental range to plant species adapted to summer drought and to verify the potentiality of our instruments under field conditions. Measurements were carried out on rockrose (Cistus incanus) and holm oak (Quercus ilex) plants before and after a rainfall during May. In rockrose leaves, which are characterized by high transpiration even under drought conditions, a concurrent increase of NMR echo integral and of water status parameters (RWC and transpiration) was observed after the rainfall (Fig. 8A ). By contrast, in the water-saving, sclerophyllous leaves of holm oak, the rainfall did not change substantially either NMR echo integral or water status indicators (Fig. 8B). In rockrose leaves, the CPMG decay was faster when the plants were drought stressed than after the rainfall (Fig. 8C). By contrast, no significant differences were observed in CPMG decays measured in holm oak leaves during the drought stress and after the rainfall (Fig. 8D). The corresponding T2 distributions of rockrose and holm oak leaves are reported in Figure 8, E and F, respectively. In the drought-stressed rockrose leaves, two distributions centered at about 0.1 and 10 ms were observed, whereas after the rainfall two new distributions centered at 4 and 40 ms were also found. In holm oak leaves, however, the same distributions of T2 were observed before and after the rainfall, but two peaks, centered at about 2 and 5 ms, that were observed in drought-stressed leaves, collapsed to just one larger peak centered at about 3.5 ms after the rainfall (Fig. 8F).
1H NMR measurements carried out with a unilateral instrument operating at low magnetic field are indeed a good proxy for leaf water status, confirming results previously obtained with laboratory instrumentation at high magnetic fields (Colire et al., 1988 ; Reinders et al., 1988) and with a different portable NMR instrument (Van As et al., 1994). Our experimentation on potted plants indicates that NMR signal integral is associated with the leaf water status. In unstressed conditions, a higher NMR signal integral was found in mesophyllous plants, either herbs or trees (e.g. maize or poplar) than in oak, the only sclerophyllous plant that was tested. This may be due to the different water content (fresh weight) in unstressed samples, an issue that was not investigated in this work. In stressed leaves, the relationship between NMR signal integral and leaf water status indicators was found both when the water stress was induced rapidly and when it developed slowly and was fully recovered. Interestingly, a relationship was also found between NMR signal integral and transpiration rate. This indicates that NMR measurements may effectively be used to monitor the water lost by plants through stomata and cuticle and the degree of stomatal opening. Stomatal closure may in turn limit CO2 acquisition and photosynthesis, especially in stressful conditions (Flexas et al., 2004). If these results are confirmed by a more complete and focused screening, including uncoupling of changes of transpiration and water potential, unilateral NMR technology may become a useful tool to improve irrigation practices for maximizing crop production and water saving.
The relationship between RWC and NMR signal integral was also observed in herbaceous leaves and in the leaves of the mesophyllous tree black poplar during the early induction of dehydration or osmolysis. However, this relationship was lost after 24 h of dehydration. This effect could be caused by shrinking of the dehydrating leaves, in turn reducing the volume of air proportionally more than the volume of water contained in the leaf. However, we did not observe significant changes in leaf geometry after 24 h of dehydration. To better investigate why RWC and NMR signal integral are not associated in heavily dehydrated leaves, the T2 was measured. Measurements of T2 may reveal different water populations in the leaf. Free, extracellular water molecules show longer T2 than water molecules bound to cellular structures and macromolecules (Walter et al., 1989
Both the ion leakage, indicating higher permeability of membranes as a consequence of stress induced to their structures (Gupta, 1977
The measurements conducted in a natural environment in two species with contrasting strategies with respect to water relations gave further information and confirmed some results discussed previously. A significant relationship between NMR signal integral and RWC and transpiration was obtained in rockrose, a plant species characterized by a small leaf thickness and by a water-spending strategy, that is, with a small degree of control of stomatal conductance on water flux (Correia and Catarino, 1994 Moreover, in leaves of the water-spending Cistus, CPMG decay of drought-stressed plant was faster than the decay observed in the same plant after the rainfall. In contrast, no significant differences were observed in CPMG decays measured in Quercus leaves during the drought stress and after the rainfall, confirming that in this sclerophyllous plant the cellular compartmentalization of water is maintained even during a stressful event and that mechanisms facilitating water diffusion do not play a relevant role in these plants.
The investigation of T2 distributions in the leaves of natural vegetation also showed that only in Cistus leaves, but not in Quercus leaves, two more peaks appeared after the rainfall, possibly indicating a water uptake in other preferential leaf compartments. Alternatively, the new peaks might be attributed to increased release of water due to cell expansion (van der Weerd et al., 2001
Summarizing, these observations indicate that the unilateral NMR instrumentation is a useful tool for revealing in situ changes in the water status of leaves, for screening vegetation health and fitness in natural and cultivated conditions, and for identifying mechanistic processes as the basis of the observed response. However, to perform correct measurements, it is important to consider that the magnetic field generated by the portable instrument is inhomogeneous. Therefore, the instrument does not allow the direct measurement of the free induction decay and the signal is always observed as an echo signal. Moreover, the inhomogeneous field is a further source of relaxation, which shortens the measured T2 values. To minimize this effect, the CPMG pulse sequence must be carried out with echo time values (
Plant Material and Growth Conditions
Potted Plants
Natural Vegetation
Plants were subjected to treatments to manipulate the water content of the leaves. Only fully developed and healthy leaves were used. Corn and poplar plants were exposed to a slow-developing drought by stopping irrigation of the pots. Corn, poplar, and bean leaves were also quickly dehydrated by cutting them off the plants. Cut poplar leaves maintained with the petiole in a beaker with water to allow for optimal water conditions were osmotically stressed by adding polyethylene glycol (PEG; Aldrich) to the water to obtain a 50% PEG aqueous solution. Field measurements on natural vegetation were carried out on leaves that were naturally water stressed by a 1-month-long drought period during May and were repeated on the same leaves on the day after an 8-mm rainfall.
All measurements were performed at 18.153 MHz with a commercial unilateral NMR instrument (Eureka project Measurements were carried out nondestructively on single leaves attached to the plants or detached from the plants according to the different experimental protocols. The sampled leaf was positioned in contact with the probe head and kept immobile with a piece of glass free of any 1H NMR signal. Each measurement was carried out within few minutes under experimental conditions that perturbed the leaf functionality only minimally, as indicated by concurrent physiological measurements of water relations.
Since in a nonhomogeneous magnetic field the NMR signal decays very quickly, the NMR signal can be recovered stroboscopically (Blümich et al., 2003 The total water content in the leaf was estimated by performing single Hahn echo measurements with an echo time of 50 µs. An echo time of 50 µs was purposely chosen for suppressing the signal of the solid component of the leaves (cellulose, lignin). In fact, the average T2 value of the solid component is about 20 to 30 µs. In all single Hahn echo experiments, the number of scans was fixed to 512. A recycle delay of 2 s was used. As a result of Hahn echo experiments, the intensity, the integral, the average noise, and the signal-to-noise ratio were obtained directly from the software. It is worth noting that the intensity and the integral of the Hahn echo, in all cases, showed the same trend. However, in the figures, the integral of the NMR echo signal was reported as a percentage with respect to the free water integral value. Real data were always used in the processing of the experimental data. The values of the magnetization intensity reported in Figure 5 were calculated at a time of 40 ms on the best fit curve of the CPMG decays; in fact, at this time, the decays are well distinguishable and the corresponding magnetization values are not affected by the noise.
Transverse T2 values were measured with the CPMG sequence following the procedure published previously (Carr and Purcell, 1954
The echo decays, obtained by applying the CPMG sequence, were treated as multiexponential decays as follows:
A regularized inverse Laplace transformation (Press et al., 1994
T1 values were measured with the aperiodic saturation recovery sequence (Farrar and Becker, 1971
Transpiration was measured with a portable photosynthetic system (LI 6400; Li-Cor) immediately before NMR measurements and on the same leaf portion. The leaf was clamped in a gas-exchange cuvette and maintained under environmental conditions set to reproduce the growth conditions (leaf temperature = 25°C, incident light intensity = 800 µmol m–2 s–1, relative humidity = 40%, CO2 concentration in air = 370 µmol mol–1). The leaf was exposed to a 0.3 L min–1 air flow, and the difference of water vapor in the air before and after the leaf was used to calculate leaf transpiration and stomatal conductance (von Caemmerer and Farquhar, 1981
The RWC of a leaf is defined as follows:
To measure leaf water potential, fully expanded black poplar leaves were detached from different plants during the water stress treatment. Leaves were weighed and placed in a pressure chamber (Soil Moisture Equipment Corp.) to determine the pressure at which water is exuded from the petiole (Scholander et al., 1965
Ion Leakage Measurements
LOX Product Emission Measurements
All measurements were obtained on at least three different leaves of different plants. Results from all sampled leaves are shown for measurements of NMR signal integral, while T2 is reported for a single leaf, with more replications shown in Supplemental Figure S1. Linear regression coefficients and means ± SE were calculated by Sigma Plot 9.0 (Systat Software). Means of the indicators of cell damage, and the NMR echo integral in relation to leaf water potential, were separated in an ANOVA scheme by a Tukey's test. Differences at P < 0.01 and 0.10 are represented in the figures by different single and double letters, respectively.
The following materials are available in the online version of this article.
We thank Silvano Fares, Marco Gobbino, and Sara Cozzolino for their help with laboratory and field measurements. Received September 2, 2008; accepted February 2, 2009; published February 4, 2009.
1 This work was supported by the European Commission project ACCENT, the European Science Foundation program VOCBAS, and the Scientific Committee of the Presidential Estate of Castelporziano. 
2 This article is dedicated to Prof. Annalaura Segre, in memory. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Francesco Loreto (francesco.loreto{at}ibaf.cnr.it).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.128884 * Corresponding author; e-mail francesco.loreto{at}ibaf.cnr.it.
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) values as short as possible, but T2 values measured with unilateral NMR may still be shorter than those measured in a highly homogeneous field. As a rule, only CPMG decays collected using the same 
12214) from Bruker Biospin Italy. The probe head is made of a U-shaped magnet assembly with two permanent magnets mounted on an iron yoke and includes a radio frequency resonator properly designed for avoiding detuning effects. The maximum echo signal, corresponding to a 
/2 pulse, was obtained with a pulse width of 3 µs, and the dead time was 13 µs. 
