Forum
Meetings
Current controversies and
challenges in applying plant
hydraulic techniques
International Workshop on Plant Hydraulic
Techniques, Ulm University, Germany, September
2014
The field of plant hydraulics has seen a renewed interest in recent
years from various scientific disciplines, including not only plant
scientists, but also climate modellers, foresters, and conservation
biologists. It is clear that understanding and accurately measuring
plant–water relations is highly relevant, especially when consider-
ing changes in plant distribution patterns due to climate change,
feedbacks between vegetation and climate systems, crop produc-
tivity, and food security (Choat et al., 2012; Sheil, 2014). For
instance, integration of plant hydraulic traits into dynamic global
vegetation models has the potential to improve predictions of forest
response to climate change. An international workshop on plant
hydraulic techniques at Ulm University brought together 57
experts, including leading researchers from 16 countries world-
wide.
‘. . . commonly used techniques as well as the application of
novel methods in studies on xylem transport may suffer from
the “observer effect”, indicating that measurements of the
hydraulic system cannot be made without affecting this
system.’
A major challenge with integrating plant hydraulic data into
other disciplines concerns discrepancies and ambiguity of hydraulic
data collected and published by researchers studying plant–water
relationships. Comparability of results from research groups is
increasingly important to address some of the most challenging
questions related to water transport, and is also crucial for any
interdisciplinary efforts and the compilation of large datasets.
Because there is often variation in the fine details of how hydraulic
measurements of the xylem tissue are carried out, methodological
issues have been a concern for many years. These technical
problems are frequently associated with the inherent difficulty in
observing and experimentally manipulating the xylem transport
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New Phytologist Ó 2015 New Phytologist Trust
system containing liquid under tension. Therefore, both com-
monly used techniques as well as the application of novel methods
in studies on xylem transport may suffer from the ‘observer effect’,
indicating that measurements of the hydraulic system cannot be
made without affecting this system. The field of plant hydraulics is
particularly concerned about measurement artefacts, which have
the potential to hinder progress and confuse those outside the field.
The major goals of the workshop were to: (1) recognize different
opinions and viewpoints on technical issues; (2) stimulate collab-
oration and inspire new approaches; and (3) make plant hydraulics
more accessible to students and nonexperts outside the field.
During the first day of the workshop, oral presentations were held
by Mel Tyree (Northwest A&F University, China), John Sperry
(University of Utah, Salt Lake City, UT, USA), and Herve Cochard
(INRA, Paris, France), as well as several videoconference presen-
tations and 20 poster presentations. Breakout sessions during two
additional days were held to discuss methodological issues in
smaller focus groups, including protocols related to vulnerability
curves, leaf hydraulics, sapflow, hydraulic conductivity, soil water,
and wood anatomy. Later, we report only on xylem vulnerability
curves.
Xylem vulnerability curves: can we reach consensus?
One of the major issues debated concerned the accuracy of
measuring vulnerability of the plant hydraulic pathway to the entry
of air bubbles that block conduits and impede water delivery to
transpiring plant tissues. As emphasized during the presentation by
Herve Cochard (INRA), the debate about whether xylem conduits
are filled with water or air goes back to the pioneers of microscopy in
the seventeenth century. Over the last decades, vulnerability to
embolism has been defined by plotting the percentage loss of
hydraulic conductivity (PLC, in %) against decreasing xylem water
potential (wx, in MPa). The shape of the resulting vulnerability
curve (VC) is found to vary from sigmoidal (s-shaped) to
logarithmic (r-shaped) (Fig. 1). The main methodological concern
arises from the fact that in some species the same plant material
exhibits a fundamentally different curve shape depending on the
technique used for measurement. The physiological implications of
these differences are enormous and must be viewed in the context of
the total water relations of a particular species. The r-shaped curves
imply a high amount of embolism formation or high native PLC
levels, with embolism refilling occurring on a daily basis. The
s-shaped curves, however, predict that embolism formation does
not occur below a certain water potential threshold, probably only
during periods of severe drought, and embolism refilling is either
nonexisting or assumed to occur on a seasonal rather than daily
basis. Therefore, the shape of VCs strongly influences the xylem
water potential corresponding to 50% loss of conductivity (w50, in
MPa; Fig. 1c,f). Although consensus was not achieved on this issue,
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(a) (b) (c)

(d) (e) (f)

Fig. 1 Transverse, light microscopy images of Acer pseudoplatanus from central Germany and corresponding vulnerability curves. (a, b) The root structure with
wide xylem vessels, while more narrow vessels and a more intense safranin staining is shown in branch material (d, e). Despite similarity in the size and length of
the root and branch material used, vulnerability curves obtained using a cavitron device were r- (c) and s-shaped (f), with mean w50 values of 0.82 MPa
( 0.42 standard deviation) and 3.70 MPa ( 0.03 standard deviation), respectively. The different colours in the vulnerability curves represent three
replicates measured for root and branch material.

several general guidelines were highlighted during the workshop,
and various possible explanations were discussed to better under-
stand why the plant hydraulic community is facing these current
discrepancies.
First, variation in the shape of VCs can be explained by the
method applied. While the bench dehydration method is generally
considered as the oldest, ‘gold standard’ method, this technique is
labour intensive and could be prone to some artefacts (see later).
Faster methods include the air injection and centrifugation
technique, but these can also be prone to errors (Cochard et al.,
2005; Choat et al., 2010; Ennajeh et al., 2011). New progress in
understanding the ‘open vessel artefact’ was recently reported by
Wang et al. (2014) for Robinia pseudoacacia: after spinning samples
for 4 h in a centrifuge, PLC levels were significantly higher in stem
segments that have a high mean vessel length than stem segments
with a shorter mean vessel length. This finding may suggest that the
open vessel artefact is due to bubbles moving from the stem base
towards the centre, where the tension is highest, causing them to
expand and cavitate. However, there are several versions of the
centrifuge technique and some researchers report that the original
‘static centrifuge’ method is unaffected by the open vessel artefact
(Hacke et al., 2015). The reliability and comparability of the
various centrifuge techniques thus remains a contentious issue
(Cochard et al., 2013).
Additional variation in VCs can be caused by the age of the plant
material and anatomical variation of the xylem tissue, especially the
vessel length distribution and connectivity between vessels both
within and across growth rings. Seasonality may exert influence on
VC shape via cavitation fatigue causing a lower resistance to
embolism formation after earlier drought- or frost-induced
cavitation events (Christensen-Dalsgaard & Tyree, 2014). Ideally,
different techniques for constructing VCs should be tested using
plant material from the same age, similar size, same plant
population, and within the shortest possible time frame, and the
exact age of the plant and the collecting date should be reported in
publications. Shifts in VCs could also be due to changes in the
xylem sap composition and the perfusion solution that is used
when conducting hydraulic measurements (Sperry & Tyree, 1990;
Herbette & Cochard, 2010), but may also depend on which
‘reference point’ is used to standardize PLC values. Different

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methods are applied to measure hydraulic conductivity either at
low or high pressure, which might result in PLC errors and hamper
comparability. Therefore, VCs are suggested to be expressed in
terms of xylem specific conductivity (Ks). This parameter is
generally normalized to the conductive xylem area in order to
reflect the actual conducting capacity. Variation in Ks, however,
can be influenced by several factors other than embolism,
depending on the device and protocol used. Although no ‘gold
standard’ protocol is available for Ks measurements, several tests
and recommendations to assure comparability of results were
discussed at the meeting.
An important question in the s- and r-shaped VC debate
concerns the functional starting point, that is whether or not VCs
should start with all vessels in a stem segment filled or not. In
practical terms, this means whether or not samples should be
flushed before measuring Ks. While all vessels (except for those that
have not fully developed yet) should be included for juvenile stem
segments that are < 1 yr old, there was no general agreement about
how we should deal with the ‘throw away strategy’ of ring-porous
species. Except for tracheids, functional vessels in species with ring-
porosity are mainly limited to earlywood vessels of the current year,
because embolized earlywood vessels do not refill after frost.
Interestingly, a VC that excludes these previously embolized
earlywood vessels from the measurement is always sigmoidal, which
might be considered artefactual.
The bench dehydration method is generally considered as a
reference technique for validation of other VC methods. However,
this method is also indirect and destructive, and recent work has
shown that it could be prone to artefacts caused by cutting xylem
under tension (Wheeler et al., 2013; Torres-Ruiz et al., 2014). For
many years, there has been general agreement to be careful with
cutting plant material while the xylem sap is under tension.
However, it is not clear how we should relieve the tension of the
xylem sap while preventing any refilling due to capillarity (Trifilo
et al., 2014), and whether or not multiple cuts within a short time
frame are sufficient to avoid the excision artefact.
Finally, the field of plant hydraulics should take advantage of
novel techniques to measure VCs, in addition to improving current
methods. Alternative VC protocols discussed at the workshop
included the application of acoustic emission methods, noninvasive
and direct measurement by X-ray microtomography (microCT),
magnetic resonance imaging (MRI), and sap flow methods to
estimate whole-tree VCs. An easy and novel tool was also presented
by students from Rafael Oliveira’s laboratory (State University of
Campinas, Brazil), who demonstrated how VCs could be
constructed by a vacuum meter method that is applied to the
proximal site of an intact branch.
Conclusions and future outlook
The field of plant hydraulics will benefit from standardized
protocols, definitions of parameters and units, and more intense
collaboration to develop, share, and modify techniques. One
possibility to achieve this goal will be the publication of a
plant hydraulics handbook, which is inspired by similar efforts
from plant ecologists (Perez-Harguindeguy et al., 2013). In
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Meetings Forum 963
addition, individual protocols will be uploaded on the
PrometheusWiki (Protocols in ecological and environmental
plant physiology; http://prometheuswiki.publish.csiro.au/tiki-
custom_home.php). Therefore, we are currently inviting col-
leagues working in the field of plant hydraulics to share
detailed instructions and protocols of robust methods that have
been tested repeatedly.
Acknowledgements
The authors thank all participants for valuable presentations,
discussions, and comments during the workshop, and the German
Research Foundation (DFG) for financial support. B.C. acknowl-
edges the Humboldt Foundation for his Humboldt Fellowship.
Steven Jansen1*, Bernhard Schuldt2 and Brendan Choat3
1
Institute for Systematic Botany and Ecology, Ulm University,
Ulm D-89081, Germany;
2
Plant Ecology, Albrecht von Haller Institute for Plant Sciences,
University of G€ottingen, G€ottingen, Germany;
3
Hawkesbury Institute for the Environment, University of Western
Sydney, Richmond, NSW, Australia
(*Author for correspondence: tel + 49 7315 023 302;
email steven.jansen@uni-ulm.de)
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Key words: cavitation resistance, embolism, hydraulic conductivity, plant
hydraulics, plant–water relations, vessel, vulnerability curve, xylem.
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