Cutting-edge research or cutting-edge artefact? An overdue control experiment complicates the xylem refilling story

Arguably, the biggest current mystery in water relations research is how embolized xylem vessels refill despite negative pressures in the surrounding transpiration stream (Nardini, Lo Gullo & Salleo 2011). In this issue, Wheeler et al. (2013) set out to investigate the phenomenon in two known refillers, Acer rubrum and Fraxinus americana (Zwieniecki & Holbrook 1998; Zwieniecki et al. 2000). Instead, they made the sobering discovery that the ‘refilling’ in these species was an experimental artefact. And the artefact was seen in other species too. They report what amounts to a series of control experiments that should have been done decades ago. The results demand attention from anyone interested in xylem function and plant water relations. Xylem studies are complicated by the remarkable fact that the liquid sap is transported under negative pressure. Negative gas pressures are impossible, but negative liquid pressures can exist as long as intermolecular forces hold. The strong bonding between water molecules combined with exclusion of large gas bubbles or other destabilizing surfaces allows liquid sap to be pulled up to the leaves by pressures far below vapour pressure. By analogy with a solid that can be placed under tension, negative sap pressures can also be referred to as (positive) tensions. The xylem water column is consequently metastable, and any gas bubble over a critical size nucleates ‘cavitation’: the phase change to vapour. The resulting vapour void can grow to occupy the entire conduit, and this gas ‘embolism’ blocks transport. The phenomena of cavitation and its reversal have grown over the years from a minor footnote to a significant chapter in plant physiology. The metastability of xylem sap creates experimental difficulties. Any observation of xylem conduit functioning, whether by direct imaging or measurement of a bulk property like hydraulic conductivity, is only accurate if the observation itself does not nucleate cavitation and embolism. The need to validate observations with the appropriate control experiment is probably the single most limiting factor to progress in the field. It can be hard to know when a control is even necessary. There have been many interesting setbacks over the years when observations have subsequently been shown by belated controls to be experimental artefacts (Tyree 1997; Cochard et al. 2000). The artefact exposed by Wheeler et al. has to do with how plant organs are excised prior to standard measurements of xylem hydraulic conductivity (see Fig. 1).As any florist knows, the excision must be made under water. If a stem is cut in air, negative pressure pulls the sap away from the surface and aspirates air into the open conduits, reducing future water uptake. But what Wheeler et al. show very convincingly, is that in 3 out of 4 species, significant blockage can be created even if the cut is made under water (see Fig. 1a,b schematics). Only if the negative pressures are relaxed before making the final underwater cut is the additional embolism avoided (Fig. 1c,d). The potential for the ‘excision artefact’ was shown to increase with more negative sap pressures, but to decrease with the amount of embolism present before cutting. The mechanism of the excision artefact is unknown. The likely explanation is that gas gets drawn into the conduits during the cut by the sap negative pressure, blocking the openings to the conduits (Fig. 1a,b). The gas could either be aspirated from existing pockets in the tissue exposed by the cutting edge, or from cavitation nucleated as the blade slices open the conduit. Greater negative pressure would pull more gas and create more blockage.The blockage may be localized close to the exposed surface (e.g. Fig. 1b) because sap pressures should moderate quickly as water and gas are taken up. Interestingly, the only species showing no sign of the artefact was paper birch (Betula papyrifera), which has scalariform perforation plates. The narrow gaps in these perforation plates may be sufficient to trap gas near the cut surface, preventing measurable blockage. Of historical interest is that the excision artefact was predicted long ago by Dixon and Joly (Dixon 1914, quoted at the beginning of the Wheeler et al. Discussion). Precautionary protocols designed to avoid the artefact were detailed in the Materials and Methods of M.H. Zimmermann’s pioneering 1978 article on hydraulic architecture (Zimmermann 1978). Early practitioners of these methods continued these practices (Sperry, Donnelly & Tyree 1988), but the precautions were gradually dropped from the Materials and Methods sections. Why? Because no one . . . neither Dixon, nor Zimmermann, nor anyone else . . . had ever bothered to demonstrate whether the excision artefact actually existed. If you do not know whether it matters, it is human nature to forget over time about taking precautions. Wheeler et al. show that the excision artefact does indeed matter. In some species, like red maple (A. rubrum), it matters a lot: cutting negatively pressurized xylem under water can decrease the hydraulic conductivity by 2–3-fold below what is arguably the correct value. From now on, researchers know that they really do have to take the precaution of relaxing xylem pressure before the final excision of a segment for assaying xylem conduit functioning. The consequences of the excision artefact are significant. As emphasized by Wheeler et al., it casts doubt on numerous Correspondence: J. Sperry. e-mail: j.sperry@utah.edu Plant, Cell and Environment (2013) doi: 10.1111/pce.12148 bs_bs_banner