Xylem functioning and water relations of the elastic living tissue of the bark: new insights about their coordination

163 Xylem Functioning and Water Relations of the Elastic Living Tissue of the Bark: New Insights about Their Coordination A. Díaz-Espejo1, E. Nicolás3, P. Nortes3, C.M. Rodriguez-Dominguez1,2, M.V. Cuevas1, A. Perez-Martin1 and J.M. Torres-Ruiz1 1 Irrigation and Crop Ecophysiology Group, Instituto de Recursos Naturales y Agrobiología, IRNAS-CSIC, Apartado 1052, 41080, Sevilla, Spain 2 Dpto. Biología Vegetal y Ecología, Universidad de Sevilla, Avda. Reina Mercedes 6, 41012-Sevilla, Spain 3 Dpto. Riego, Centro de Edafología y Biología Aplicada del Segura (CSIC), Apartado 164, 30100 Espinardo (Murcia), Spain Keywords: stem water potential, dendrometers, vulnerability curve, osmotic pressure, turgor pressure, water use, water stress Abstract The interesting species-specific relationship between maximum daily shrinkage (MDS) and stem water potential (  stem) was investigated in order to understand the mechanisms involved. Vulnerability curves of Vitis, Citrus, Olea and Prunus were analysed and the air entry  stem in the xylem (Pe) was calculated. The water relations parameters of the living tissue of the bark were also studied by building P-V curves. A strong linear correlation was obtained between the  stem at which MDS occurs (  MDS) and Pe in the studied species. Furthermore, P-V curves showed that, the species with the highest resistance to cavitation, i.e., Prunus, had the lowest osmotic pressure and the largest bulk elastic modulus. The conclusions of this study reinforce the idea of a tight correlation among plant capacitance, transpiration and maintenance of water status of the plant. These results illustrate the importance of studying the integration of long-distance transport systems in plants. INTRODUCTION Monitoring of trunk diameter variations (TDV) on the bark of woody plants is becoming a useful tool for managing irrigation in commercial orchards. In addition to being a valuable feature for agronomists, these sensors supply important information about the physiological mechanisms that allow the plant to transpire under a wide range of environmental conditions. Currently, it is not possible to mechanistically explain the daily and seasonal dynamics of transpiration without considering the role played by water storage in trunks, branches and even leaves (Steppe et al., 2006). Trunk diameter fluctuations have been reported to reflect the addition of four components: irreversible radial growth, reversible living-cell dehydration/rehydration, thermal expansion and contraction, and expansion of dead conducting elements due to the increase and relaxation of internal tensions. Therefore, the interaction between both the living parenchyma in the cortex and the xylem is important to understand the dynamics of TDV and its consequences on the water relations observed in the plant. Maximum daily shrinkage (MDS) is one of the most used indicators of water stress in fruit trees (Fernandez and Cuevas, 2010). When plotted against stem water potential (  stem) MDS shows a parabolic response, i.e., as  stem decreases MDS increases up to a maximum value after which MDS decreases following further decreases in  stem. Furthermore, the  stem value at maximum MDS has been reported to be species-specific (Ortuño et al., 2010). The aim of this work was to investigate potential relationships between the pattern of MDS described above and the vulnerability to embolism of the xylem of several fruit tree species of high agronomical interest. MATERIALS AND METHODS Four species of high agronomical interest were selected to search for shared Proc. 9th International Workshop on Sap Flow Ed.: K. Steppe Acta Hort. 991, ISHS 2013 164 features in the response of MDS and percentage of loss of conductivity of the xylem (PLC) to  stem: grapevine (Vitis), olive (Olea), clementine (Citrus) and peach (Prunus). For most of them we were able to find information about the relationship of MDS and PLC with  stem. In the cases where this was not possible, specific measurements were carried out as described below. MDS and  stem MDS values and their relationship with  stem were obtained from Ortuño et al. (2010) for Vitis, Olea and Citrus. In the case of Prunus, four linear variable displacement transducers (LVDTs) were installed in 8-year-old almond trees (Prunus dulcis (Mill.) D.A. Webb ‘Marta’ on Mayor rootstock) planted in a 1 ha experimental orchard located in Murcia (Spain). Three irrigation treatments were assessed: (i) full irrigation (FI), irrigated to satisfy maximum crop water requirements (ETc) throughout the whole growing season; (ii) regulated deficit irrigation (RDI), irrigated at 20% ETc during kernelfilling (stage IV, from end of May to end of August) and at 100% ETc throughout the remainder of the growth cycle; (iii) not irrigation (NI). MDS was recorded daily automatically; meanwhile  stem was measured with a pressure chamber twice per week. PLC and  stem Data for Vitis was obtained from Choat et al. (2010), Poggi et al. (2007) for Citrus, Diaz-Espejo et al. (2012) for Olea, and Cochard et al. (2010) for Prunus. Information was extracted from figures in those works and vulnerability curves were then generated by plotting PLCs against their corresponding  stem values. Following Meinzer et al. (2009) the air entry pressure (Pe) can be estimated from the x-intercept of a tangent drawn through the midpoint of a sigmoid function fitted to the vulnerability curve data. This parameter Pe can be defined as the threshold at which loss of conductivity begins to increase rapidly. P-V Curves of Bark Tissue Bark samples were collected from six trees per species at 1.5 m height (0.6 m in the case of Vitis). We used a 5-mm-diameter increment borer to take cylinders of outer parenchyma (living parenchymatous cells, cambium and phloem) down to sapwood. Samples were immediately sealed in glass vials in the field and transported to the laboratory where they were allowed to rehydrate. The water potential isotherms were determined with a thermocouple psychrometer with six standard C-52 sample chambers (Wescor Inc., Logan, UT, USA) connected to a datalogger (PSYPRO, Wescor Inc.). RESULTS AND DISCUSSION Figure 1 shows the response of MDS to  stem in four species of high agronomical impact with contrasting characteristics. In all of them MDS increases with  stem up to a specific  stem value at which MDS is maximum and from which MDS starts declining. Vitis and Prunus presented the highest and lowest  stem at maximum MDS (  MDS), respectively. As an example, the calculation of Pe in Olea is plotted in Figure 2. While P50 is estimated as ca. -4.0 MPa, Pe is achieved just at -1.55 MPa. Pe for Vitis, Citrus and Prunus were -1.05, -1.20 and -2.10 MPa, respectively. These values indicate a different vulnerability to cavitation for each species, Vitis being the most vulnerable and Prunus the most resistant. When Pe was plotted against  MDS a linear correlation was found, very close to the line 1:1 (Fig. 3). This suggests a tight coordination between both aspects of water transport in plants. Pe is related to the capacity of water transport by the xylem, and any further decrease in  stem below Pe would induce major losses in the hydraulic conductivity of the xylem. On the other hand, MDS is related to the capacitance of the plant (Steppe et al., 2006), and therefore plays an important role in slowing down changes in xylem pressure following transpiration-induced changes in xylem water flux (Meinzer et al., 2009). The occurrence of the largest MDS at a given  stem means that, at that  stem, the capacity of water transfer from the elastic living tissue of the bark to the xylem 165 becomes maximum. If  stem keeps on declining, there is a concomitant decrease in both hydraulic conductivity and the capacitance of surrounding living tissue to supply water stored. From this point of view, it is not surprising that both parameters appear to be so well coordinated. Another key “actor” driving transpiration, the stomatal conductance, is also very much determined by the values of  stem found in Figure 3. Again in all these species (Vitis: Schultz, 2003; Olea: Fernandez et al., 1997; Citrus: Garcia-Tejero et al., 2010; Prunus: Egea et al., 2011) there is a close relationship between Pe and the minimum value of  stem (as defined by Meinzer et al., 2009) that is targeted by a strong and regulated stomatal closure. How is this coordination between the xylem and the surrounding living tissue of the bark achieved? In order to respond to this question the water relations of the bark tissue were studied by analysing P-V curves (Table 1, Fig. 4). Similarly to what happened with Pe and  MDS, Prunus showed the most extreme values with a  100 of -2.81 MPa,  0 of -5.20 and  of 9.20 (MPa). However, although, once more, Olea and Citrus behaved in a similar fashion, Vitis did not present the other extreme values, and joined the two previous species. This is clearly seen in Figure 5a where Prunus is easily distinguished from the other three species. However, if RWC0 was plotted against  stem at maximum MDS (Fig. 5b) no relationship emerged and all four species fixed their RWC0 in a narrow range between 0.60 and 0.64. There are very scarce data about water relation parameters of this plant tissue in the literature. To the best of our knowledge, the only information available can be found in Scholz et al. (2007) in several neotropical savannah trees. These authors concluded that osmotic adjustment and values of the  of living parenchyma in the bark are critical to determine the stem capacitance and for the plant to achieve the homeostasis in the diurnal and seasonal water balance of trees. Scholz et al. (2007) interpreted the contribution of outer parenchyma to total stem capacitance as intriguing, since sapwood was found to be a major contributor to water release during high evaporative periods. However, it has to be considered that the capacity of sapwood to release water is limited by its consequence, i.e., xylem embolism. In this sense, our results show how the different species adjust their water relation parameters to optimise the water flow between tissue compartments. Parenchyma of Prunus was able to lower its osmotic potential to lower values than the other species, favouring the water exchange at  stem down to Pe. It has to be noted as well that the radial resistance between xylem and bark can vary in a diurnal and seasonal basis, affecting the water flux between these two tissues (Steppe et al., 2012). Therefore, the correlations found in this work are expected to change along the season. Indirectly, the coordination between xylem functioning and living tissues of the bark could have implications also on the coordination and regulation of stomatal conductance to avoid catastrophic embolism formation in the xylem. The decrease in water content of the living cells of the bark, among which phloem is, would affect the transport of photoassimilates from the leaves (Thompson and Holbrook, 2003), and therefore acts as a hydraulic signal influencing the photosynthesis processes, very much coordinated with stomatal conductance (Franks, 2006). The key conclusions of this work can be tested with existing model approaches, like Steppe et al. (2006) or Zweifel et al. (2007), whose performance surely will improve, especially when applied along the season. CONCLUSIONS Our results confirm the hypothesis that in long transport processes, as those occurring in xylem and phloem, the coordination among tissues in plant water relations parameters is very important. Since living tissue in the bark plays a key role in the plant water used, buffering diurnal fluctuations in the water demand by the canopy, we found in this study a tight correlation between the vulnerability to cavitation of xylem and the water potential at which the maximum daily shrinkage of the trunk occurs. It is demonstrated that the way that living cells in the bark adjust their osmotic pressure and bulk elastic modulus is essential to achieve this synchronization. Obviously, this has implications on the transport of photoassimilates in the phloem, and on the interactions in 166 the regulation of leaf gas exchange, particularly in the stomata control of transpiration. Finally, the increase in our knowledge of the integration of the processes underneath the observed response of dendrometers or sap flow is of vital importance for understanding these responses under water stress, and to conduct a rational use of these plant-based sensors in deficit irrigation management. ACKNOWLEDGEMENTS This work was funded by the Spanish Ministry of Science and Innovation (research project AGL2009-11310/AGR), by the Junta de Andalucía (research project AGR-6456-2010) and by the FEDER programme. Antonio Montero helped us with the field and laboratory work. Thanks to the owners of Internacional Olivarera, S.A.U. (Interoliva), for allowing us to make the experiments in the Sanabria orchard. We also thank Silvia Seller, agronomist, and Juan Francisco Bernabé, foreman, for their technical assistance. Literature Cited Choat, B., Drayton, W.M., Brodersen, C., Matthews, M.A., Shackel, K.A., Wada, H. and McElrone, A.J. 2010. Measurement of vulnerability to water stress-induced cavitation in grapevine: a comparison of four techniques applied to a long-vesseled species. Plant Cell Environ. 33:1502-1512. Cochard, H., Herbette, S., Barigah, T., Badel, E., Ennajeh, M. and Vilagrosa, A. 2010. Does sample length influence the shape of xylem embolism vulnerability curves? 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Main parameters determined after analysing pressure-volume curves in the four studied species. 100 (MPa) is the water potential at full turgor, 0 (MPa) is the water potential at zero turgor, RWC0 is the tissue relative water content at zero turgor,  (MPa) is the average bulk elastic modulus of tissue.  100  0 RWC0  Vitis -1.97 -3.36 0.64 6.93 Citrus -1.72 -3.38 0.60 5.99 Olea -1.62 -3.07 0.62 4.64 Prunus -2.81 -5.20 0.62 9.20 Figures  stem (MPa) -3 -2 -1 0 MDS (mm) 0.0 0.2 0.4 0.6 0.8 Vitis Citrus Olea Prunus Fig. 1. Maximum daily shrinkage in the trunk (MDS) as a function of stem water potential (  stem) for four different species. Dotted lines indicate the  stem at which the MDS is reached. Vitis, Citrus and Olea data from Ortuño et al. (2010). Prunus data from this work. 168  stem (MPa) -10-8-6-4-20 PLC (%) 0 20 40 60 80 100 P e Olea P 50 Fig. 2. Percentage of loss of hydraulic conductivity (PLC) as a function of stem water potential (  stem) in Olea. P50 indicate the  stem at which hydraulic conductivity is 50% of maximum, and Pe the  stem at which the loss of hydraulic conductivity is enhanced. MDS (MPa) -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 Pe (MPa) -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 Line 1:1 Vitis Citrus Olea Prunus Fig. 3. Relationship between air entry stem water potential (Pe) and the  stem at which maximum daily shrinkage is maximum (  MDS) for each species. 169 Vitis 1-RWC (%) 0.00.20.40.60.81.0 1/ w (-MPa) 0.0 0.5 1.0 1.5 2.0 Citrus 1-RWC (%) 0.0 0.2 0.4 0.6 0.8 1.0 1/ w (-MPa) 0.0 0.5 1.0 1.5 2.0 Olea 1-RWC (%) 0.00.20.40.60.81.0 1/ w (-MPa) 0.0 0.5 1.0 1.5 2.0 Prunus 1-RWC (%) 0.00.20.40.60.81.0 1/ w (-MPa) 0.0 0.5 1.0 1.5 2.0 Fig. 4. Pressure-volume curves of the elastic living tissue of bark of the four studied species. Dotted line indicates the osmotic component.  MDS -3.0 -2.5 -2.0 -1.5 -1.0 -0.5  0 -6 -5 -4 -3 -2 -1 0  MDS -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 RWC 0 0.5 0.6 0.7 0.8 0.9 1.0 Prunus Vitis Olea Citrus Fig. 5. Relationship between water potential of living tissue of bark at zero turgor (  o) and relative water content at zero turgor (RWC0) with stem water potential (  s) at maximum trunk diameter shrinkage (MDS). Dashed lines represent the narrow range of RWC0 in all four species.