X-rays overdose: quantifying the cellular damage caused by synchrotron X-ray Micro-CT in plants

Long-distance water transport in plants relies on generation of negative pressure (= tension) at leaf level, which is propagated to root tips via xylem conduits. This mechanism ensures a passive movement of water within plants, but it is vulnerable to liquid-to-vapour phase transition. Indeed, water under tension is in a physically metastable state and, as the tension in xylem water increases, air could be aspirated in water-filled conduits through small pores present in cell walls. The formation of air emboli breaks the water columns and causes the blockage of water transport, possibly leading to plant death. For this reason, quantifying the critical tension inducing a significant blockage of water transport has become a major aim in plant ecophysiology. Vulnerability to embolism is species-specific, as species living in mesic habitats are generally more vulnerable than species living in xeric ones. Moreover, some species can eventually repair embolized conduits and recover their hydraulic conductivity after re-watering (= xylem refilling). Most of the techniques used to quantify xylem embolism and refilling are generally destructive, as they require the excision of stems, roots or petioles from plants. Due to the negative pressure in functional xylem conduits, samples’ excision might cause air entry in the xylem, producing artefactual embolism. To overcome this problem, over the last years novel non-destructive techniques have been increasingly used to visualize the functional state of xylem conduits in intact plants. In particular, Micro-CT has emerged as a very promising technology, thanks to its ease of use and to the good contrast between air-filled vswater-filled conduits. Due to its supposed non-invasive nature, synchrotron-based Micro-CT is considered as the reference technique to determine xylem vulnerability to embolism formation and to visualize in vivothe possible refilling of embolized conduits. However, the occurrence of the latter mechanism is still debated among plant physiologists. Theoretically, xylem refilling mechanism relies on the activity of living parenchyma cells surrounding xylem conduits, that releases sugars into embolized vessels to generate the osmotic forces needed to counterbalance eventual residual tension in still functioning elements. A possible drawback of Micro-CT is that the ionizing radiation emitted by X-ray sources might damage living tissues. In many studies, samples are scanned multiple times at the same point to visualize xylem refilling, and thus they might be exposed to potentially lethal doses of X-rays. In this light, we designed an experiment using X-ray micro-tomography (Micro-CT) setup available at the SYRMEP beamline at Elettra to quantify the possible damage produced by multiple scans to living tissues in plant stems (see Fig.1).
 

Figure 1.     In vivo visualization by x-ray Micro-CT of the functional status of xylem conduits in a stem of Laurus nobilis. Black circles represent gas-filled conduits and light-grey circles represent water-filled conduits.

We repeatedly exposed small portions of stems of three species, namely Helianthus annuus, Coffea arabicacv Pacamaraand Populus tremulax alba,to 3 min X-rays synchrotron radiation. We measured the relative electrolyte leakage (REL, %), a proxy of cellular membrane integrity, and the quality of RNA (RNI, RNA integrity number) after 0, 1, 2, and 3 scans. Our results showed that repeated scans negatively affected cellular membrane integrity and the quality of RNA of the selected species. Samples scanned multiple times had higher values of REL and lower values of RIN. This suggests that Micro-CT should be used carefully to investigate phenomena that depend on physiological activity of living cells. This probably applies to conduit refilling, which theoretically requires the activation of genes encoding key proteins involved in carbohydrates metabolism pathways and membrane transport of ions and sugar molecules, to generate the osmotic forces necessary to refill embolized conduits.
 

This research was conducted by the following research team:

Francesco Petruzzellis¹, Chiara Pagliarani^2,5, Tadeja Savi³, Adriano Losso⁴, Silvia Cavalletto⁵, Giuliana Tromba⁶, Christian Dullin^6,7, Andreas Bär⁴, Andrea Ganthaler⁴, Andrea Miotto¹, Stefan Mayr⁴, Maciej A. Zwieniecki⁸, Andrea Nardini¹, Francesca Secchi⁵

¹ Dipartimento di Scienze della Vita, Università di Trieste, Trieste, Italia
² Institute for Sustainable Plant Protection, National Research Council, Torino, Italia
³ University of Natural Resources and Life Sciences, Division of Viticulture and Pomology, Department of Crop Sciences, Tulln, Vienna, Austria
⁴ Department of Botany, University of Innsbruck, Innsbruck, Austria
⁵ Dipartimento di Scienze Agrarie, Forestali e Alimentari, Università di Torino,Torino, Italia 
 ⁶Elettra-Sincrotrone Trieste, Trieste, Italia
⁷ Institute for Diagnostic and Interventional Radiology, University Medical Center Göttingen, Göttingen, Germany; Max-Plank-Institute for Experimental Medicine, Göttingen, Germany
⁸ Department of Plant Sciences, University of California Davis, Davis, USA

Contact persons:

Andrea Nardini, email: