Plasma membrane aquaporins are involved in winter embolism recovery in walnut tree

In perennial plants, freeze-thaw cycles during the winter months can induce the formation of air bubbles in xylem vessels, leading to changes in their hydraulic conductivity. Refilling of embolized xylem vessels requires an osmotic force that is created by the accumulation of soluble sugars in the vessels. Low water potential leads to water movement from the parenchyma cells into the xylem vessels. The water flux gives rise to a positive pressure essential for the recovery of xylem hydraulic conductivity. We investigated the possible role of plasma membrane aquaporins in winter embolism recovery in walnut (Juglans regia). First, we established that xylem parenchyma starch is converted to sucrose in the winter months. Then, from a xylem-derived cDNA library, we isolated two PIP2 aquaporin genes (JrPIP2,1 and JrPIP2,2) that encode nearly identical proteins. The water channel activity of the JrPIP2,1 protein was demonstrated by its expression in Xenopus laevis oocytes. The expression of the two PIP2 isoforms was investigated throughout the autumn-winter period. In the winter period, high levels of PIP2 mRNA and corresponding protein occurred simultaneously with the rise in sucrose. Furthermore, immunolocalization studies in the winter period show that PIP2 aquaporins were mainly localized in vessel-associated cells, which play a major role in controlling solute flux between parenchyma cells and xylem vessels. Taken together, our data suggest that PIP2 aquaporins could play a role in water transport between xylem parenchyma cells and embolized vessels.     

Patterns of PIP gene expression in Populus trichocarpa during recovery from xylem embolism suggest a major role for the PIP1 aquaporin subfamily as moderators of refilling process

Embolism and the refilling of xylem vessels are intrinsic to the ability of plants to handle the transport of water under tension. Although the formation of an embolized vessel is an abiotic process, refilling against the pressure gradient requires biological activity to provide both the energy and the water needed to restore xylem transport capacity. Here, we present an analysis of the dynamics of embolism and refilling in Populus trichocarpa and follow temporal dynamics of co‐occurring changes in expression level of aquaporins. Under mesic conditions, we found that the percent loss of conductance (PLC) varied diurnally by as much as 20%, suggesting a continuous embolism/refilling cycle. An increase in water stress tilted the balance between the two processes and increased the PLC to as much as 80%. Subsequent re‐watering resulted in the reversal of water stress and recovery of PLC to pre‐stress levels. Stem parenchyma cells responded to drought stress with considerable up‐regulation of the PIP1 subfamily of water channels but not the PIP2 subfamily. Even more significant was the finding that PoptrPIP1.1 and PoptrPIP1.3 genes were up‐regulated in response to embolism, but not to water stress, and were down‐regulated after embolism removal, suggesting a local ability of plants to sense an embolism presence.     

木质部导管空穴化研究中的几个热点问题

 导管的空穴化和栓塞化现象是目前国际上水分生理生态研究的一个热点。该文对导管空穴化和栓塞化研究中出现的几个热点问题进行了概括和总结。1）在研究导管空穴化的实验手段上,超声波传感器探测法具有一定的局限性;目前至少存在4种可能的原因来解释木质部压力探针法（XPP）和压力室法所测得的导管水柱张力不一致的现象;近来出现的核磁共振成像法可以进行导管空穴化的无损伤检测。2）导管解剖学特征与形成空穴的可能性之间的关系可能与树种相关。3）导管空穴化引起气孔关闭的作用机制目前还不太清楚。4）植物防止空穴化产生的能力与适应干旱能力之间的关系还没有定论。5）单独用根压来解释空穴化导管的重新注水机制是不全面的,还存在其它重新注水机制。     

Embolized conduits of rice (Oryza sativa, Poaceae) refill despite negative xylem pressure

Embolism reversal in rice plants was studied by testing the plant's ability to refill embolized conduits while xylem pressures were substantially negative. Intact, potted plants were water-stressed to a xylem pressure of -1.88 ± 0.1 MPa and a 66.3 ± 3.8% loss of xylem conductivity (PLC) by cavitation. Stressed plants were carefully rewatered, allowing xylem pressure to rise, but not above the theoretical threshold of c. -0.15 MPa for embolism collapse. Despite xylem pressures being more negative than this threshold, the PLC fell significantly (28.5 ± 5.6%), indicating the refilling of vessels. Above c. -1.0 MPa, almost all plants regained their maximum hydraulic conductivity. Dye uptake experiments showed the same pattern of embolism refilling despite negative pressure. Refilling was prevented in plants that were light-starved for 5 d, suggesting the unknown mechanism is dependent on metabolic energy. Results are among the first showing that herbaceous plants can reverse embolism without bulk xylem pressures rising near or above atmospheric.     

Applications of the compensating pressure theory of water transport

Some predictions of the recently proposed theory of long-distance water transport in plants (the Compensating Pressure Theory) have been verified experimentally in sunflower leaves. The xylem sap cavitates early in the day under quite small water stress, and the compensating pressure P (applied as the tissue pressure of turgid cells) pushes water into embolized vessels, refilling them during active transpiration. The water potential, as measured by the pressure chamber or psychrometer, is not a measure of the pressure in the xylem, but (as predicted by the theory) a measure of the compensating pressure P. As transpiration increases, P is increased to provide more rapid embolism repair. In many leaf petioles this increase in P is achieved by the hydrolysis of starch in the starch sheath to soluble sugars. At night P falls as starch is reformed. A hypothesis is proposed to explain these observations by pressure-driven reverse osmosis of water from the ground parenchyma of the petiole. Similar processes occur in roots and are manifested as root pressure. The theory requires a pump to transfer water from the soil into the root xylem. A mechanism is proposed by which this pump may function, in which the endodermis acts as a one-way valve and a pressure-confining barrier. Rays and xylem parenchyma of wood act like the xylem parenchyma of petioles and roots to repair embolisms in trees. The postulated root pump permits a re-appraisal of the work done by evaporation during transpiration, leading to the proposal that in tall trees there is no hydrostatic gradient to be overcome in lifting water. Some published observations are re-interpreted in terms of the theory: doubt is cast on the validity of measurements of hydraulic conductance of wood; vulnerability curves are found not to measure the cavitation threshold of water in the xylem, but the osmotic pressure of the xylem parenchyma; if measures of xylem pressure and of hydraulic conductance are both suspect, the accepted view of the hydraulic architecture of trees needs drastic revision; observations that xylem feeding insects feed faster as the water potential becomes more negative are in accord with the theory; tyloses, which have been shown to form in vessels especially vulnerable to cavitation, are seen as necessary for the maintenance of P, and to conserve the supplementary refilling water. Far from being a metastable system on the edge of disaster, the water transport system of the xylem is ultrastable: robust and self-sustaining in response to many kinds of stress.     

Mechanism of water stress-induced xylem embolism

We investigated the hypothesis that water stress-induced xylem embolism is caused by air aspirated into functional vessels from neighboring embolized ones (e.g. embolized by physical damage) via pores in intervessel pit membranes. The following experiments with sugar maple (Acer saccharum Marsh.) support the hypothesis. (a) Most vessels in dehydrating stem segments embolized at xylem pressures < −3 megapascals; at this point the pressure difference across intervessel pits between air-filled vessels at the segment's ends and internal water-filled vessels was >3 megapascals. This same pressure difference was found to be sufficient to force air across intervessel pits from air injection experiments of hydrated stem segments. This suggests air entry at pits is causing embolism in dehydrating stems. (b) Treatments that increased the permeability of intervessel pits to air injection also caused xylem to embolize at less negative xylem pressures. Permeability was increased either by perfusing stems with solutions of surface tension below that of water or by perfusion with a solution of oxalic acid and calcium. The mechanism of oxalic-calcium action on permeability is unknown, but may relate to the ability of oxalate to chelate calcium from the pectate fraction of the pit membrane. (c) Diameter of pores in pit membranes measured with the scanning electron microscope were within the range predicted by hypothesis (≤0.4 micrometer).     

不同水分条件下两个树种木质部栓塞对P素添加的响应

 在两种水分供给(干旱胁迫和适宜水分,土壤含水量分别为田间持水量的30%～40%和70%～80%)下,研究了耐旱树种元宝枫(Acer truncatum)和 中生树种女贞(Ligustrum lucidum )木质部栓塞(以导水率(Percentage loss of hydraulic conductivity, PLC)损失程度衡量)对P素添加的 响应。结果发现,两个树种PLC的日变化均呈现出先上升后降低的规律,表明木质部栓塞的形成与恢复是植物体的一种平常事件;除适宜水分条 件的女贞外,P素可以显著提高元宝枫和遭受干旱胁迫时女贞的PLC;两种水分条件下,干旱胁迫时元宝枫木质部栓塞明显高于适宜水分供给时 。女贞的PLC在两种水分状况下无显著差异;树种间,干旱胁迫促进了元宝枫木质部的栓塞形成,明显高于同等水分条件下的女贞。该研究结果 证实了“木质部限流耐旱假设”。     

树木树液上升机理研究进展

水分在植物体内的运输一直是很多植物生理生态学家所关注的一个重要问题。介绍了内聚力学说的基本假设和其存在争议,总结了近年来这一研究领域的几个热点问题,主要包括：（1）木质部栓塞及其恢复机理;（2）木质部压力探针和压力室法测定的木质部张力值不一致的现象及其可能原因;（3）补偿压学说;（4）不同界面层张力以及输水管道的毛细作用力、薄壁细胞膨压和木质部渗透压、逆向蒸腾等在树木汁液上升中的贡献;（5）最近发现的存在于木质部导管伴胞和韧皮部薄壁细胞等质膜中的水孔蛋白在植物水分运输中的调控作用等。这些方面在解释树木的树液上升中都起着重要的作用。     

Possible Mechanisms Limiting Movement of Ralstonia solanacearum in Resistant Tomato Tissues

The distribution and appearance of Ralstonia solanacearum in the upper hypocotyl tissues of root‐inoculated tomato seedlings of resistant rootstock cultivar LS‐89 (a selection from Hawaii 7998) and susceptible cultivar Ponderosa were compared to clarify the mechanism that limits the movement of the bacterial pathogen in resistant tomato tissues. In stems of wilted Ponderosa plants, bacteria colonized both the primary and the secondary xylem tissues. Bacteria were abundant in vessels, of which the pit membranes were often degenerated. All parenchyma cells adjacent to vessels with bacteria were necrotic and some of them were colonized with bacteria. In stems of LS‐89 plants showing no discernible wilting symptoms, bacteria were observed in the primary xylem tissues but not in the secondary xylem tissues. Necrosis of parenchyma cells adjacent to vessels with bacteria was observed occasionally. The pit membranes were often thicker with high electron density. The inner electron‐dense layer of cell wall of parenchyma cells and vessels was thicker and more conspicuous in xylem tissues of infected LS‐89 than in xylem of infected Ponderosa or mock‐inoculated plants. Electron‐dense materials accumulated in or around pit cavities in parenchyma cells next to vessels with bacteria, and in vessels with bacteria. Many bacterial cells appeared normal in vessels, except for those in contact with the pit membranes. These results indicate that R. solanacearum moves from vessel to vessel in infected tissues through degenerated pit membranes and that restricted movement in xylem tissues was the characteristic feature in LS‐89. The limitation in bacterial movement may be related to the thickening of the pit membranes and/or the accumulations of electron‐dense materials in vessels and parenchyma cells.     

In vivo observation of cavitation and embolism repair using magnetic resonance imaging

Magnetic resonance imaging (MRI) was used to noninvasively monitor the status of individual xylem vessels in the stem of an intact, transpiring grape (Vitis vinifera) plant over a period of approximately 40 h. Proton density-weighted MRI was used to visualize the distribution of mobile water in the stem and individual xylem vessels were scored as either water or gas filled (i.e. embolized). The number of water-filled vessels decreased during the first 24 h of the experiment, indicating that approximately 10 vessels had cavitated during this time. Leaf water potentials decreased from -1.25 to -2.1 MPa during the same period. Watering increased leaf water potentials to -0.25 MPa and prevented any further cavitation. Refilling of xylem vessels occurred as soon as the lights were switched off, with the majority of vessels becoming refilled with water during the first 2 to 3 h in darkness. These measurements demonstrate that MRI can be used to monitor the functional status of individual xylem vessels, providing the first method to study the process of cavitation and embolism repair in intact plants.     

Rice sucrose transporter1 (OsSUT1) up‐regulation in xylem parenchyma is caused by aphid feeding on rice leaf blade vascular bundles

The role of the sucrose transporter OsSUT1 in assimilate retrieval via the xylem, as a result of damage to and leakage from punctured phloem was examined after rusty plum aphid (Hysteroneura setariae, Thomas) infestation on leaves from 3‐week‐old rice (Oryza sativa L. cv Nipponbare) plants. Leaves were examined over a 1‐ to 10‐day infestation time course, using a combination of gene expression and β‐glucuronidase (GUS) reporter gene analyses. qPCR and Western blot analyses revealed differential expression of OsSUT1 during aphid infestation. Wide‐field fluorescence microscopy was used to confirm the expression of OsSUT1‐promoter::GUS reporter gene in vascular parenchyma associated with xylem elements, as well as in companion cells associated with phloem sieve tubes of large, intermediate and small vascular bundles within the leaf blade, in regions where the aphids had settled and were feeding. Of great interest was up‐regulation of OsSUT1 expression associated with the xylem parenchyma cells, abutting the metaxylem vessels, which confirmed that OsSUT1 was not only involved in loading of sugars into the phloem under normal physiological conditions, but was apparently involved in the retrieval of sucrose leaked into the xylem conduits, which occurred as a direct result of aphid feeding, probing and puncturing of vascular bundles. The up‐regulation of OsSUT1 in xylem vascular parenchyma thus provides evidence in support of the location within the xylem parenchyma cells of an efficient mechanism to ensure sucrose recovery after loss to the apoplast (xylem) after aphid‐related feeding damage and its transfer back to the symplast (phloem) in O. sativa leaves.     

Hydraulic characteristics of water-refilling process in excised roots of Arabidopsis

Plants have efficient water-transporting vascular networks with a self-recovery function from embolism, which causes fatal discontinuity in sap flow. However, the embolism-refilling process in xylem vessel is still unclear. The water-refilling processes in the individual xylem vessels of excised Arabidopsis roots were visualized in this study using synchrotron X-ray micro-imaging technique with high spatial resolution up to 1 μm per pixel and temporal resolution up to 24 fps. In normal continuous water-refilling process, we could observe various flow patterns affected by the morphological structures of the xylem vessels, especially when water passed through perforation plates. A simple criterion based on the variation in dynamic pressure was suggested to evaluate the contribution of individual perforation plates to the water-refilling process. Meanwhile, the water-refilling embolized sections of xylem vessels through radial pathways were also observed. Separated water columns were formed from this discontinuous water-refilling process and the water influx rates through the radial pathways were estimated to be 478 and 928 μm³ s^?1. The dynamic behavior of the separated water columns were quantitatively analyzed from the stoppage of volume growth to the translational phase. These water-refilling processes in excised roots of Arabidopsis may shed light on understanding the water refilling in the embolism vessels of intact plants and the interconnectivity of xylem vessel networks in vascular plants.     

Tyloses and the Maintenance of Transpiration

During a study of transpiration and embolism-formation in petiolesof sunflower, tyloses were frequently observed in early metaxylemvessels. Tyloses were confined to the inner ends of the xylemarcs, remote from the phloem. Vessels in this position are especiallyvulnerable to embolism. All stages of the invasion of vessellumens by xylem parenchyma cells were observed, from the earlyprotuberance of a cell through a pit to the complete occlusionof the lumen by one to several cells. The lumen space not occupiedby tyloses was seen both filled with xylem sap, or embolizedand gas-filled. Thus, during the early stages of tylosis formationthe vessel remained active in carrying the transpiration stream.Thin-walled vessels of the protoxylem or early metaxylem werenot tylosed, but were squashed and disappeared. These observationsare interpreted as evidence that vessels vulnerable to embolismare decommissioned and replaced by parenchyma tissue, whilenew and less vulnerable vessels are added to the xylem arcsat the cambial side. It is proposed that tylosis formation istriggered by the frequent embolization of the vulnerable vesselsto give, ultimately, an incompressible tissue. Then tyloseswould be necessary to preserve the tissue pressure which expresseswater to refill embolisms in the remaining vessels, and maintaintranspiration, as explained by the compensating pressure theoryof water transport. Compensating pressure theory; embolisms; starch sheath; tissue pressure; transpiration; tyloses; vessel diameter     

Easy Come,Easy Go: Capillary Forces Enable Rapid Refilling of Embolized Primary Xylem Vessels

Protoxylem plays an important role in the hydraulic function of vascular systems of both herbaceous and woody plants, but relatively little is known about the processes underlying the maintenance of protoxylem function in long-lived tissues. In this study, embolism repair was investigated in relation to xylem structure in two cushion plant species, Azorella macquariensis and Colobanthus muscoides, in which vascular water transport depends on protoxylem. Their protoxylem vessels consisted of a primary wall with helical thickenings that effectively formed a pit channel, with the primary wall being the pit channel membrane. Stem protoxylem was organized such that the pit channel membranes connected vessels with paratracheal parenchyma or other protoxylem vessels and were not exposed directly to air spaces. Embolism was experimentally induced in excised vascular tissue and detached shoots by exposing them briefly to air. When water was resupplied, embolized vessels refilled within tens of seconds (excised tissue) to a few minutes (detached shoots) with water sourced from either adjacent parenchyma or water-filled vessels. Refilling occurred in two phases: (1) water refilled xylem pit channels, simplifying bubble shape to a rod with two menisci; and (2) the bubble contracted as the resorption front advanced, dissolving air along the way. Physical properties of the protoxylem vessels (namely pit channel membrane porosity, hydrophilic walls, vessel dimensions, and helical thickenings) promoted rapid refilling of embolized conduits independent of root pressure. These results have implications for the maintenance of vascular function in both herbaceous and woody species, because protoxylem plays a major role in the hydraulic systems of leaves, elongating stems, and roots.There is a pressing need to understand how plants manage the maintenance of water transport from roots through leaves under changing environmental conditions (Allen et al., 2010; Choat et al., 2012). The problem arises because water is transported through the xylem under tension (i.e. under negative absolute pressure). As tension increases, conduits become increasingly vulnerable to cavitation, which causes the conduits to lose their ability to transport water. Conduits can become embolized during normal diurnal function as a result of tensions induced by transpiration and in response to environmental conditions such as drought or freezing stress (Zimmermann and Tyree, 2002). Vulnerability to cavitation and embolism formation suggests that plants have mechanisms to regain lost hydraulic capacity, either through the formation of new conduits or by refilling embolized ones.The vulnerability of conduits to embolisms and the capacity for repair are related to the structural diversity of xylem tissue (Zwieniecki and Holbrook, 2009; Lens et al., 2011; Cai et al., 2014). In vascular plants, the classification of xylem tissues depends on the meristem that produced them (Evert and Eichhorn, 2006). Primary xylem is produced by apical meristems and includes both protoxylem and metaxylem conduits, which are distinguished by their wall structure and the timing of their development. Protoxylem matures during organ elongation, which results in loss of function due to stretching in some tissues and species, while in many others, functionality is maintained throughout the life of the organ. In contrast, metaxylem matures in elongated tissue. In herbaceous plants, primary xylem is the major hydraulic system of the roots, stems, and leaves. In woody plants, the primary xylem remains the main hydraulic system of the leaves, while the radial growth of stems occurs through the activity of a vascular cambium, which produces secondary xylem with only metaxylem conduits. As a woody plant grows, the secondary xylem (and hence the metaxylem) thus becomes of increasing importance to stem hydraulic function. However, protoxylem remains an integral component of the plant hydraulic system due to its function in leaves and elongating stems and roots.As discussed in a recent review (Brodersen and McElrone, 2013), refilling of embolized vessels has been shown to depend on the generation of positive pressure by roots in many monocots, herbaceous plants, and a few woody species. However, many species lack root pressure; thus, attention has focused on so-called novel refilling, which involves adjacent living cells in the repair of embolized metaxylem or secondary xylem in stems of mature plants. Novel refilling has been studied with a variety of methods to visualize temporal variation in the presence and subsequent absence of embolized vessels, including cryo-scanning electron microscopy (Cryo-SEM; Canny, 1997; McCully et al., 2014), double staining (Zwieniecki and Holbrook, 1998; Zwieniecki et al., 2000), NMR imaging (Holbrook et al., 2001; Zwieniecki et al., 2013), and high-resolution x-ray computed tomography (Lee and Kim, 2008; Brodersen et al., 2010; Kim and Lee, 2010; Lee et al., 2013; Suuronen et al., 2013). These observations, in combination with other measurements, led to a working hypothesis of an osmotically driven repair mechanism in which sugars pumped into embolized vessels by adjacent paratracheal parenchyma provide the osmotic pressure difference that refills the vessel (Nardini et al., 2011).Little is known about embolism and its repair in protoxylem, which has structural features that make it potentially more vulnerable to embolism than metaxylem in the same plant or tissue (Choat et al., 2005). These include a greater exposed area of the primary cell wall with annular or helical thickenings instead of secondary walls. This could enhance stretching of the primary wall when large pressure differences develop between functional and embolized vessels, thereby decreasing the pressure required for air seeding of bubbles (Choat et al., 2004). Choat et al. (2005) suggested that greater vulnerability of protoxylem to embolism might underpin the roles of petioles, leaves, and small stems in the hydraulic segmentation hypothesis of Zimmermann (1983), in which sacrifice of the most easily replaceable tissues protects the function of the main structure of a plant during water stress. If ease of protoxylem embolism were to contribute to the function of hydraulic fuses during mild water stress, then ease of refilling would be required to rapidly reset the system.This study focuses on embolism repair in two distantly related, vascular species, Azorella macquariensis (Apiaceae) and Colobanthus muscoides (Caryophyllaceae), that depend exclusively on protoxylem for vascular water transport. Both species form cushions, with the former being an endemic, keystone species in the alpine zone of subantarctic Macquarie Island and the latter being a regional endemic that plays a major role in rocky coastal areas often within the supralittoral zone (Selkirk et al., 1990; Orchard, 1993). Both species are of ecological interest, because the subantarctic region is under increasing threat from climate change (Adams, 2009). Specifically, the climate on Macquarie Island is progressively changing from one that is perpetually wet and misty to one with increased exposure to periodic drying (Bergstrom et al., 2015). Dieback of alpine vegetation was first observed in 2008, and by 2010, extensive and unprecedented decline of A. macquariensis led to its listing as critically endangered (Bricher et al., 2013).In this study, protoxylem structure was studied in relation to embolism repair. Refilling of gas-filled vessels was compared between excised tissue and that in intact, detached shoots. The results showed that the physical properties of the protoxylem facilitated refilling by capillary forces and that rapid refilling in detached shoots supplied with water occurred without root pressure.     

Wood allocation trade-offs between fiber wall,fiber lumen,and axial parenchyma drive drought resistance in neotropical trees

Functional relationships between wood density and measures of xylem hydraulic safety and efficiency are ambiguous, especially in wet tropical forests. In this meta-analysis, we move beyond wood density per se and identify relationships between xylem allocated to fibers, parenchyma, and vessels and measures of hydraulic safety and efficiency. We analyzed published data of xylem traits, hydraulic properties and measures of drought resistance from neotropical tree species retrieved from 346 sources. We found that xylem volume allocation to fiber walls increases embolism resistance, but at the expense of specific conductivity and sapwood capacitance. Xylem volume investment in fiber lumen increases capacitance, while investment in axial parenchyma is associated with higher specific conductivity. Dominant tree taxa from wet forests prioritize xylem allocation to axial parenchyma at the expense of fiber walls, resulting in a low embolism resistance for a given wood density and a high vulnerability to drought-induced mortality. We conclude that strong trade-offs between xylem allocation to fiber walls, fiber lumen, and axial parenchyma drive drought resistance in neotropical trees. Moreover, the benefits of xylem allocation to axial parenchyma in wet tropical trees might not outweigh the consequential low embolism resistance under more frequent and severe droughts in a changing climate.     

Xylem dysfunction caused by water stress and freezing in two species of co-occurring chaparral shrubs

Water transport from the roots to leaves in chaparral shrubs of California occurs through xylem vessels and tracheids. The formation of gas bubbles in xylem can block water transport (gas embolism), leading to shoot dieback. Two environmental factors that cause gas embolism formation in xylem conduits are drought and freezing air temperatures. We compared the differential vulnerabilities of Rhus laurina and Ceanothus megacarpus, co-dominant shrub species in the coastal regions of the Santa Monica Mountains of southern California, to both water stress-induced and freezing-induced embolism of their xylem. Rhus laurina has relatively large xylem vessel diameters, a deep root system, and a large basal burl from which it vigorously resprouts after wildfire or freezing injury. In contrast, Ceanothus megacarpus has small-diameter vessels, a shallow root system, no basal burl and is a non-sprouter after shoot removal by wildfire. We found that R. laurina became 50% embolized at a water stress of –3 MPa and 100% embolized by a freeze–thaw cycle at all hydration levels. In contrast, C. megacarpus became 50% embolized at a water stress of –9 MPa and 100% embolized by freeze–thaw events only at water potentials lower than –3 MPa. Reducing thaw rates from 0·8 °C min^?1 to 0·08 °C min^?1 (the normal thaw rate measured in situ) had no effect on embolism formation in R. laurina but significantly reduced embolism occurrence in well-hydrated C. megacarpus (embolism reduced from 74 to 35%). These results were consistent with the theory of gas bubble formation and dissolution in xylem sap. They also agree with field observations of differential shoot dieback in these two species after a natural freeze–thaw event in the Santa Monica Mountains.     

Stem anatomy of Caryophyllaceae

The xylem and phloem of 88 Caryophyllaceae from subtropical and temperate regions mainly in Western Europe and the Canary Islands are described and analysed. They are compared with their taxonomic classification, and assigned to their ecological range. The xylem of different life forms (herbaceous plants, dwarf shrubs and shrubs) consists mainly of parenchyma and small vessels that are 20–50 μm in diameter in earlywood. They have simple perforations and pits are pseudosclariform and scalariform. The axial parenchyma is mostly pervasive or paratracheal, and the ray cells are exclusively upright or square. The anatomy of the subfamily Alsinoideae is homogeneous and characterised by the absence of libriform fibres, large rays, crystal druses and sclereids in the cortex. The subfamily Caryophylloideae is less homogeneous and mainly characterised by the presence of crystal druses on the xylem and phloem, as well as the presence of intra-annual fibre bands. The subfamily Paronychioideae is heterogeneous; included phloem is most characteristic. Ecological trends are clearly expressed by the age of plants and the average annual radial growth rates. Plants tend to grow older and slower at higher altitudes. The presence of intra-annual fibre bands in the xylem is characteristic of Caryophylloideae at lower altitudes. The study suggests that taxonomic and ecological classifications and large-scale ecological trend studies must be based on large and homogeneous datasets and well-defined anatomical features.