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SpotlightI Can See ClearlyNow – Embolism inLeavesChristine Scoffoni1 andSteven Jansen2,*

Deciphering how air enters theplant hydraulic transport tissuesrepresents a major challenge tounderstanding plant droughtresponses. Using a non-invasiveand cheap visualization techniqueapplied to leaves, the spread ofembolism is found to initiate inthemidrib, increasewith vein order,and is seemingly influenced by veintopology.

Vascular plants include a highly complexwater transport system that is driven by thetranspiring surfaces of leaves. The mecha-nism behind this transport system results ina negative (i.e., sub-atmospheric) pressureof xylem sap. Because of the highly cohe-sive property of the H2O molecule, watercan be transported under negative pres-sure from the soil through the entire plantsystem until it reaches the sites of evapo-ration in leaves. Although there is generalagreement about the cohesion[3_TD$DIFF]-tensiontheory to explain long-distance vasculartransport of water, the occurrence of gasbubbles, which can expand to block con-duits and disable the hydraulic transportsystem via embolism formation, representsa major challenge to understanding howplants will respond to more severe andlong-term periods of drought in many loca-tions worldwide [1]. The leaf vein systemcan be highly reticulate, and understandinghow embolism spreads across vein ordershas remained challenging. Thus, visualizingembolism events and how these varyacross species and during drought is ofcrucial importance.

Recent findings from Brodribb et al. [2],based on an indirect optical technique to

quantify the amount of drought-inducedembolism in leaves, highlight the potentialof this non-invasive and low-cost tech-nique to visualize xylem failure at the entireleaf level in vivo. The method developedby Brodribb et al. allows direct live visuali-zation of putative embolism spread acrossdistinct venation types, which is demon-strated in their paper for three fern speciesand two angiosperm trees. The techniqueis a modern, digital approach of earliervisualization techniques (see for instancereferences cited in [3]) and is based onstereomicroscopy, which allows non-inva-sive observations of embolized conduitsaccording to differences in light transmis-sion of veins between functional (i.e.,water-filled) and air-filled conduits.

Three important findings have emergedfrom this new technique. First, large dehy-dration times are necessary (up to 70 h ofshoots drying on the bench) to induceembolism in leaf veins, suggesting that leafveins are more resistant to drought-induced embolism than was previouslythought (e.g., [4]). Second, large veinsare most vulnerable to embolism, whilehigh vein orders (i.e., 3rd, 4th, and 5th veinorders) are the most resistant. Althoughthese results contradict earlier papersusing dye techniques (e.g., [5]), they areconsistent with a neutron imaging studyshowing that embolism starts in majorveins and then spreads to the minor veinsunder higher dehydration [6]. This secondfinding implies that leaves would only needto refill their major veins to become func-tional again. Moreover, the hydraulic vul-nerability segmentation hypothesis wouldnot apply within leaf veins because themost-distal high-order veins were notfound to be most vulnerable to embolism.Hydraulic vulnerability segmentation, how-ever, would be achieved through high vul-nerability of the outside-xylem pathways inleaves [7] and/or petiole vulnerability [8].The third important finding of the study isthe role of vein topology in embolism resis-tance. Their results, although limited to fivespecies, suggest that hierarchical/reticu-late systems could provide an efficient

way to avoid rapid and catastrophic failureof the entire vein network. This wasobserved in the non-hierarchical andnon-reticulate venation pattern of the fernAdiantum capillus-veneris, and could allowthe dense and embolism-resistant minorveins to remain functional during pro-longed drought, bypassing embolizedconduits in major veins.

While the method presented by Brodribbet al. [3] provides promising possibilities toanswer fundamental questions aboutembolism in leaves, there remain someshortcomings where caution is warranted.First, this method should be tested on awider range of leaf structures and anato-mies: it is unknown, for example, if thismethod would work on thick or leatheryleaves. Most importantly, the methodassumes that the captured change inrefractive index relates to embolism. How-ever, this method has not been validatedyet against an independent and moredirect visualization approach such as X-ray microtomography (microCT), whichallows direct visualization of embolizedconduits and their anatomical character-istics in different vein orders (Figure 1A,B).Although X-rays are known to potentiallydamage DNA in living cell types, they donot affect embolism formation in stemconduits after repeated scans [9]. Valida-tion of the optical technique with othertechniques such as microCT is especiallycrucial if the technique is to be used toinfer the hydraulic impact of embolisms onleaf hydraulic conductance [10]. Forinstance, the percentage of ‘embolizedpixels’ that are measured would not nec-essarily show a 1:1 correlation with theactual percentage of embolized conduits.Hence, high conduit redundancy, espe-cially in midribs (Figure 1C), could makeit hard to accurately relate the percent ofembolized pixels to actual embolized con-duits. In addition, exact quantitative dataof the embolized conduits will be neces-sary to determine their true hydraulicimpact. Thus, the percentage of ‘embol-ized pixels’ reported by this method couldoverestimate the true hydraulic impact on

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(A)

(B) (C)

Figure 1. Leaf Cross-Sections from X-Ray Computed Microtomography (microCT) and LightMicroscopy. MicroCT cross-sections provide direct visualization of embolized conduits (red arrows) in the leafmidrib of Eucalyptus sp. (A) and a needle of Pinus pinaster (B) in vivo.Note, no embolized conduits are present inthe minor veins (blue arrows) in (A). Light microscopy of a cross-section of Viburnum rhytidophyllum (C) revealsthe high number of xylem conduits in the leaf midrib, and the wide range of their conduit dimensions. A highpercentage of embolized midrib conduits would probably be necessary to induce significant hydraulic declinegiven the redundancy of conduits. Scale bars, 500 mm in A and B, 100 mm in C.

leaf hydraulic conductance. By validatingthis technique against microCT, modeling,and/or detailed anatomical observationsto support the occurrence of embolism,this technique has the potential to be auseful and cheap tool to investigate theimpact of drought on leaves.

One of the most promising outcomes ofthis technique concerns the production ofspatiotemporal maps of embolism forma-tion in entire leaves, which opens up pos-sibilities to investigate hydraulic aspects ofa large diversity of vein topologies. It hasbeen hypothesized, for instance, that abrochidodromous vein pattern (i.e., withthe secondary veins not terminating at theleaf margins, but joining together in aseries of loops or arches) would enablehigher redundancy and safety againstdamage: if a loop is damaged, water couldstill flow to the entire leaf area, whereasa leaf with craspedodromous venation

2 Trends in Plant Science, Month Year, Vol. xx, No. yy

(i.e., with secondary veins terminating atthe leaf margin) would become discon-nected from the main water source [11].

The findings from Brodribb et al. [3] alsoprovide evidence for air-seeding as themain mechanism for the spread of embo-lism. Although not discussed, the imagesand movies presented appear to showthat there is also evidence for ‘novel’ for-mation of embolism in conduits that arenot connected to the embolized ones,which supports similar observations onprogressive embolism spread in woodbased on microCT [9]. These observa-tions indicate that these conduits becomeembolized by a mechanism other than air-seeding.

Finally, because major veins were found tobe more vulnerable to embolism, Brodribbet al. [3] hypothesized this could be due tolarger conduit sizes present in these

lower-order veins. Whether or not embo-lism resistance is related to conduit sizeand the nature of the tracheary element(tracheid vs vessel element) remainsunclear and deserves more detailed ana-tomical observations. Detailed anatomywill also be necessary to understand con-duit connectivity in vein nodes, which mayfunction as a hydraulic bottleneck. Tounderstand why major veins are more vul-nerable to embolism, special attentionshould be paid to the ultrastructure andchemistry of air-seeding pores betweencellulose microfibrils in interconduit pitmembranes, which are known to play amajor role in drought-induced embolism[1,12]. In fact, almost nothing is knownabout interconduit pit membranes inleaves with respect to their physical andchemical properties, although it isassumed that these pit membranes aresimilar to those in secondary xylem ofthe stem.

In conclusion, if used in combination withother techniques, this method provides apromising and cheap means to betterunderstand water transport mechanismin plants and hydraulic failure. This couldlead to potential applications in the field ofbiomimetics such as solar-driven, nano-and microfluidic devices.

AcknowledgmentsS.J. thanks the Swiss Light Source (SLS) at the Paul

Scherrer Institute (Villigen, Switzerland) for synchro-

tron radiation beamtime at the TOMCAT beamline and

the European Synchrotron Radiation Facility (ESRF,

proposal EC-1026, Grenoble, France) for access to

the ID19 beamline. Drs Sarah Irvine (SLS) and Alexan-

der Rack (ESRF) are acknowledged for providing tech-

nical assistance.

1University of California, Los Angeles, Department of

Ecology and Evolutionary Biology, 621 Charles E. Young

Drive South, Box 951606, Los Angeles, CA 90095, USA2Ulm University, Institute of Systematic Botany and

Ecology, Albert-Einstein-Allee 11, 89081 Ulm, Germany

*Correspondence: steven.jansen@uni-ulm.de (S. Jansen).

http://dx.doi.org/10.1016/j.tplants.2016.07.001

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for air-seeding in xylem. Trends Plant Sci. 20, 199–205

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2. Brodribb, T.J. et al. (2016) Revealing catastrophic failure ofleaf networks under stress. Proc. Natl. Acad. Sci. U. S. A.113, 4865–4869

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