Arsenic Speciation in Phloem and Xylem Exudates of Castor Bean

How arsenic (As) is transported in phloem remains unknown. To help answer this question, we quantified the chemical species of As in phloem and xylem exudates of castor bean (Ricinus communis) exposed to arsenate [As(V)], arsenite [As(III)], monomethylarsonic acid [MMA(V)], or dimethylarsinic acid. In the As(V)- and As(III)-exposed plants, As(V) was the main species in xylem exudate (55%–83%) whereas As(III) predominated in phloem exudate (70%–94%). The ratio of As concentrations in phloem to xylem exudate varied from 0.7 to 3.9. Analyses of phloem exudate using high-resolution inductively coupled plasma-mass spectrometry and accurate mass electrospray mass spectrometry coupled to high-performance liquid chromatography identified high concentrations of reduced and oxidized glutathione and some oxidized phytochelatin, but no As(III)-thiol complexes. It is thought that As(III)-thiol complexes would not be stable in the alkaline conditions of phloem sap. Small concentrations of oxidized glutathione and oxidized phytochelatin were found in xylem exudate, where there was also no evidence of As(III)-thiol complexes. MMA(V) was partially reduced to MMA(III) in roots, but only MMA(V) was found in xylem and phloem exudate. Despite the smallest uptake among the four As species supplied to plants, dimethylarsinic acid was most efficiently transported in both xylem and phloem, and its phloem concentration was 3.2 times that in xylem. Our results show that free inorganic As, mainly As(III), was transported in the phloem of castor bean exposed to either As(V) or As(III), and that methylated As species were more mobile than inorganic As in the phloem.Arsenic (As) is an environmental and food chain contaminant that has attracted much attention in recent years. Soil contamination with As may lead to phytotoxicity and reduced crop yield (Panaullah et al., 2009). Food crops are also an important source of inorganic As, a class-one carcinogen, in human dietary intake, and there is a need to decrease the exposure to this toxin (European Food Safety Authority, 2009). Paddy rice (Oryza sativa) is particularly efficient in As accumulation, which poses a potential risk to the population based on a rice diet (Meharg et al., 2009; Zhao et al., 2010a). Other terrestrial food crops generally do not accumulate as much As as paddy rice; however, where soils are contaminated, relatively high concentrations of As in wheat (Triticum aestivum) grain have been reported (Williams et al., 2007; Zhao et al., 2010b). On the other hand, some fern species in the Pteridaceae family are able to tolerate and hyperaccumulate As in the aboveground part to >1,000 mg kg⁻¹ dry weight (e.g. Ma et al., 2001; Zhao et al., 2002); these plants offer the possibility for remediation of As-contaminated soil or water (Salido et al., 2003; Huang et al., 2004). A better understanding of As uptake and long-distance transport, metabolism, and detoxification is needed for developing strategies for mitigating As contamination, through either decreased As accumulation in food crops or enhanced As accumulation for phytoremediation.The pathways of As uptake by plant roots differ between different As species; arsenate [As(V)] enters plant cells via phosphate transporters, whereas arsenite [As(III)] is taken up via some aquaporins (for review, see Zhao et al., 2009). In rice, a silicic acid efflux protein also mediates As(III) efflux toward stele for xylem loading (Ma et al., 2008). Methylated As species, such as monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)], which may be present in the environment as products of microbial or algal methylation of inorganic As or from past uses of methylated As pesticides, are taken up by rice roots partly through the aquaporin NIP2;1 (for nodulin 26-like intrinsic protein; also named Lsi1; Li et al., 2009). Once inside plant cells, As(V) is reduced to As(III), possibly catalyzed by As(V) reductase(s) such as the plant homologs of the yeast (Saccharomyces cerevisiae) ACR2 (Bleeker et al., 2006; Dhankher et al., 2006; Ellis et al., 2006; Duan et al., 2007). As(III) has a high affinity to thiol (-SH) groups and is detoxified by complexation with thiol-rich phytochelatins (PCs; Pickering et al., 2000; Schmöger et al., 2000; Raab et al., 2005; Bluemlein et al., 2009; Liu et al., 2010). As(III)-PC complexation in roots was found to result in reduced mobility for efflux and for long-distance transport, possibly because the complexes are stored in the vacuoles (Liu et al., 2010). Excess As(III) causes cellular toxicity by binding to the vicinal thiol groups of enzymes, such as the plastidial lipoamide dehydrogenase, which has been shown to be a sensitive target of As toxicity (Chen et al., 2010). The As hyperaccumulating Pteris species differ from nonhyperaccumulating plants because of enhanced As(V) uptake (Wang et al., 2002; Poynton et al., 2004), little As(III)-thiol complexation (Zhao et al., 2003; Raab et al., 2004), and efficient xylem loading of As(III) (Su et al., 2008). Recently, an As(III) efflux transporter, PvACR3, has been found to play an important role in As(III) detoxification by transporting As(III) into vacuoles in Pteris vittata (Indriolo et al., 2010).With the exception of As hyperaccumulators, most plant species have a limited root-to-shoot translocation of As (Zhao et al., 2009). The chemical species of As in xylem exudate have been determined in a number of plant species. As(III) was found to be the predominant species (80%–100%) in the xylem sap of rice, tomato (Solanum lycopersicum), cucumber (Cucumis sativus), and P. vittata even when these plants were fed As(V) (Mihucz et al., 2005; Xu et al., 2007; Ma et al., 2008; Su et al., 2010), suggesting that As(V) is reduced in roots before being loaded into the xylem. In other plant species, such as Brassica juncea (Pickering et al., 2000), wheat, and barley (Hordeum vulgare; Su et al., 2010), As(V) accounted for larger proportions (40%–50%) of the total As in the xylem sap. Studies using HPLC-inductively coupled plasma (ICP)-mass spectrometry (MS) coupled with electrospray (ES)-MS showed no evidence of As(III)-thiol complexation in the xylem sap of sunflower (Helianthus annuus; Raab et al., 2005). When rice plants were exposed to MMA(V) or DMA(V), both As species were found in the xylem sap (Li et al., 2009). Generally, methylated As species are taken up by roots at slower rates than inorganic As, but they are more mobile during the xylem transport from roots to shoots (Marin et al., 1992; Raab et al., 2007; Li et al., 2009).It has been shown that phloem transport contributes substantially to As accumulation in rice grain (Carey et al., 2010). However, little is known about how As is transported in phloem (Zhao et al., 2009). There are no reports on the chemical species of As in phloem exudate. The speciation of As in phloem is important because it dictates how As is loaded in the source tissues and unloaded in the sink tissues, such as grain. Questions with regard to the oxidation state, methylation, and complexation of As in phloem sap remain to be answered. Unlike xylem sap, phloem sap is much more difficult to obtain in sufficient quantities for analysis. In this study, we investigated As speciation in phloem and xylem exudates of castor bean (Ricinus communis), which is widely used as a model plant to investigate phloem transport of solutes (e.g. Hall et al., 1971; Hall and Baker, 1972; Allen and Smith, 1986; Bromilow et al., 1987).     

Tyloses and Phenolic Deposits in Xylem Vessels Impede Water Transport in Low-Lignin Transgenic Poplars: A Study by Cryo-Fluorescence Microscopy

Of 14 transgenic poplar genotypes (Populus tremula × Populus alba) with antisense 4-coumarate:coenzyme A ligase that were grown in the field for 2 years, five that had substantial lignin reductions also had greatly reduced xylem-specific conductivity compared with that of control trees and those transgenic events with small reductions in lignin. For the two events with the lowest xylem lignin contents (greater than 40% reduction), we used light microscopy methods and acid fuchsin dye ascent studies to clarify what caused their reduced transport efficiency. A novel protocol involving dye stabilization and cryo-fluorescence microscopy enabled us to visualize the dye at the cellular level and to identify water-conducting pathways in the xylem. Cryo-fixed branch segments were planed in the frozen state on a sliding cryo-microtome and observed with an epifluorescence microscope equipped with a cryo-stage. We could then distinguish clearly between phenolic-occluded vessels, conductive (stain-filled) vessels, and nonconductive (water- or gas-filled) vessels. Low-lignin trees contained areas of nonconductive, brown xylem with patches of collapsed cells and patches of noncollapsed cells filled with phenolics. In contrast, phenolics and nonconductive vessels were rarely observed in normal colored wood of the low-lignin events. The results of cryo-fluorescence light microscopy were supported by observations with a confocal microscope after freeze drying of cryo-planed samples. Moreover, after extraction of the phenolics, confocal microscopy revealed that many of the vessels in the nonconductive xylem were blocked with tyloses. We conclude that reduced transport efficiency of the transgenic low-lignin xylem was largely caused by blockages from tyloses and phenolic deposits within vessels rather than by xylem collapse.Secondary xylem in woody plants is a complex vascular tissue that functions in mechanical support, conduction, storage, and protection (Carlquist, 2001; Tyree and Zimmermann, 2002). The xylem must provide a sufficient and safe water supply throughout the entire pathway from roots to leaves for transpiration and photosynthesis. It is well established that enhanced water conductivity of xylem can increase total plant carbon gain (Domec and Gartner, 2003; Santiago et al., 2004; Brodribb and Holbrook, 2005a). According to the Hagen-Poiseuille equation, xylem conductivity should scale with vessel lumen diameter to the fourth power (Tyree and Zimmermann, 2002). Indeed, xylem conductivity largely depends on anatomical features, including conduit diameters and frequencies (Salleo et al., 1985; McCulloh and Sperry, 2005). However, there are hydraulic limits to maximum vessel diameters, because xylem conduits have to withstand the strong negative pressures of the transpiration stream that could cause cell collapse or embolisms within vessels that are structurally inadequate to withstand these forces (Tyree and Sperry, 1989; Lo Gullo et al., 1995; Hacke et al., 2000). To some extent, stomatal regulation of transpiration limits the negative pressures that the xylem experiences (Tardieu and Davies, 1993; Cochard et al., 2002; Meinzer, 2002; Brodribb and Holbrook, 2004; Buckley, 2005; Franks et al., 2007; Woodruff et al., 2007). Nevertheless, plants rely on an array of structural reinforcements of xylem to ensure the safety of water transport. The size of xylem elements, vessel redundancy, intervessel pit and membrane geometries, and the thickness, microstructure, and chemical composition of cell walls are among the features that regulate tradeoffs between efficiency and safety of xylem water transport (Baas and Schweingruber, 1987; Hacke et al., 2001; Domec et al., 2006; Ewers et al., 2007; Choat et al., 2008).The xylem cell wall is made up of cellulose bundles that are hydrogen bonded with hemicelluloses, which are in turn embedded within a lignin matrix (Mansfield, 2009; Salmén and Burgert, 2009). Besides providing this matrix for the cell wall itself, lignin is thought to contribute to many of the mechanical and physical characteristics of wood as well as conferring passive resistance to the spread of pathogens within a plant (Niklas, 1992; Boyce et al., 2004; Davin et al., 2008). Lignin typically represents 20% to 30% of the dry mass of wood and therefore is among the most abundant stores of carbon in the biosphere (Zobel and van Buijtenen, 1989). The complex molecular structure and biosynthetic pathway of various types of lignins have been studied extensively (Boerjan et al., 2003; Ralph et al., 2004, 2007; Higuchi, 2006; Boudet, 2007; Davin et al., 2008). The monomeric composition of lignin varies between different cell types of the same species depending on the functional specialization of the cell (Yoshinaga et al., 1992; Watanabe et al., 2004; Xu et al., 2006). The composition and amount of lignin in wild plants varies in response to climatic conditions (Donaldson, 2002) or gravitational and mechanical demands (Pruyn et al., 2000; Kern et al., 2005; Rüggeberg et al., 2008). It is clear that plants are capable of regulating the lignification pattern in differentiating cells, which provides them with flexibility for responding to environmental stresses (Donaldson, 2002; Koehler and Telewski, 2006; Ralph et al., 2007; for review, see Vanholme et al., 2008).Whereas some level of lignin is a requisite for all vascular plants, it is often an unwanted product in the pulp and paper industry because it increases the costs of paper production and associated water treatments necessary for environmental protection (Chen et al., 2001; Baucher et al., 2003; Peter et al., 2007). Reducing the lignin content of the raw biomass material may allow more efficient hydrolysis of polysaccharides in biomass and thus facilitate the production of biofuel (Chen and Dixon, 2007). With the ultimate goal of development of wood for more efficient processing, much research has been aimed at the production of genetically modified trees with altered lignin biosynthesis (Boerjan et al., 2003; Boudet et al., 2003; Li et al., 2003; Halpin, 2004; Ralph et al., 2004, 2008; Chiang, 2006; Coleman et al., 2008a, 2008b; Vanholme et al., 2008; Wagner et al., 2009). It is now technically possible to achieve more than 50% reductions of lignin content in xylem of poplar (Populus spp.; Leplé et al., 2007; Coleman et al., 2008a, 2008b), but the consequences of such reduction on plant function have received relatively little attention (Koehler and Telewski, 2006). In-depth studies on the xylem structure and functional performance of transgenic plants with low lignin are limited, despite the need to assess their long-term sustainability for large-scale production (Anterola and Lewis, 2002; Hancock et al., 2007; Coleman et al., 2008b, Voelker, 2009; Horvath et al., 2010).Genetically modified plants are suitable models for studying fundamental questions of the physiological role of lignin because of the possibility of controlling lignification without the confounding effects encountered when comparing across plant tissues or stages of development (Koehler and Telewski, 2006; Leplé et al., 2007; Coleman et al., 2008b). Research on Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) has shown that down-regulation of lignin biosynthesis can have diverse effects on plant metabolism and structure, including changes in the lignin amount and composition (p-hydroxyphenyl/guaiacyl/syringyl units ratio) as well as the collapse of xylem vessel elements (Lee et al., 1997; Sewalt et al., 1997; Piquemal et al., 1998; Chabannes et al., 2001; Jones et al., 2001; Franke et al., 2002; Dauwe et al., 2007). Among temperate hardwoods, poplar has been established as a model tree for genetic manipulations because of its ecological and economic importance, fast growth, ease of vegetative propagation, and its widespread use in traditional breeding programs (Bradshaw et al., 2001; Brunner et al., 2004). The question of how manipulation of lignin can affect the anatomy and physiological function of xylem in poplar has been addressed in part by several research groups (Anterola and Lewis, 2002; Boerjan et al., 2003; Leplé et al., 2007; Coleman et al., 2008b). Some studies that involved large lignin reductions reported no significant alterations in the xylem anatomy (Hu et al., 1999; Li et al., 2003). However, in many other experiments, reduced total lignin content was associated with significant growth retardation, alterations in the lignin monomer composition, irregularities in the xylem structure (Anterola and Lewis, 2002; Leplé et al., 2007; Coleman et al., 2008b), and the patchy occurrence of collapsed xylem cells (Coleman et al., 2008b; Voelker, 2009). Furthermore, severely down-regulated lignin biosynthesis has resulted in greatly reduced xylem water-transport efficiency (Coleman et al., 2008b; Lachenbruch et al., 2009; Voelker, 2009). It is generally assumed that the reduced water transport ability of xylem with very low lignin contents is caused by collapsed conduits and/or increased embolism due to the entry of air bubbles into the water-conducting cells (Coleman et al., 2008b; Wagner et al., 2009), but detailed anatomical investigations of the causes of impaired xylem conductivity of low-lignin trees are lacking. Analysis of the anatomical basis for the properties of xylem conduits in plants with genetically manipulated amounts and composition of lignin can provide a deeper understanding of the physiological role of lignin as well as the lower limit of down-regulation of lignin biosynthesis at which trees can still survive within natural environments.One of the approaches for the suppression of lignin biosynthesis is down-regulation of 4-coumarate:coenzyme A ligase (4CL), an enzyme that functions in phenylpropanoid metabolism by producing the monolignol precursor p-coumaroyl-CoA (Kajita et al.,1997; Allina et al., 1998; Hu et al., 1998; Harding et al., 2002; Jia et al., 2004; Costa et al., 2005; Friedmann et al., 2007; Wagner et al., 2009). In a 2-year-long field trial on the physiological performance of poplar (Populus tremula × Populus alba) transgenic clones, out of 14 genotypes with altered lignin biosynthesis (down-regulated 4CL), five showed dramatically reduced wood-specific conductivity (k_s) compared with that of control trees (Voelker, 2009). Those mutants with the severely reduced k_s were also characterized by having the lowest wood lignin contents (up to an approximately 40% reduction) in the study. Trees with transgenic events characterized by the formation of abnormally brown wood exhibited regular branch dieback at the end of the growing season, despite having been regularly watered (Voelker, 2009). Our objective was to identify the structural features responsible for reduced transport efficiency in the xylem of transgenic poplars with extremely low lignin contents. We employed fluorescence and laser scanning confocal microscopy for anatomical analyses of xylem structure as well as dye-flow experiments followed by cryo-fluorescence microscopy to visualize the functioning water-conductive pathways in xylem at the cellular level. We report the frequent occurrence of tyloses and phenolic depositions in xylem vessels of strongly down-regulated trees that may be the cause of their reduced xylem conductivity.     

Phloem as Capacitor: Radial Transfer of Water into Xylem of Tree Stems Occurs via Symplastic Transport in Ray Parenchyma

The transfer of water from phloem into xylem is thought to mitigate increasing hydraulic tension in the vascular system of trees during the diel cycle of transpiration. Although a putative plant function, to date there is no direct evidence of such water transfer or the contributing pathways. Here, we trace the radial flow of water from the phloem into the xylem and investigate its diel variation. Introducing a fluorescent dye (0.1% [w/w] fluorescein) into the phloem water of the tree species Eucalyptus saligna allowed localization of the dye in phloem and xylem tissues using confocal laser scanning microscopy. Our results show that the majority of water transferred between the two tissues is facilitated via the symplast of horizontal ray parenchyma cells. The method also permitted assessment of the radial transfer of water during the diel cycle, where changes in water potential gradients between phloem and xylem determine the extent and direction of radial transfer. When injected during the morning, when xylem water potential rapidly declined, fluorescein was translocated, on average, farther into mature xylem (447 ± 188 µm) compared with nighttime, when xylem water potential was close to zero (155 ± 42 µm). These findings provide empirical evidence to support theoretical predictions of the role of phloem-xylem water transfer in the hydraulic functioning of plants. This method enables investigation of the role of phloem tissue as a dynamic capacitor for water storage and transfer and its contribution toward the maintenance of the functional integrity of xylem in trees.Physiological and hydraulic functioning of the two long-distance transport systems in trees, xylem and phloem, have intrigued plant researchers for more than a century. Since the pioneering work of Dixon and Joly (1895; cohesion-tension theory for xylem) and Münch (1930; pressure flow hypothesis for phloem), the majority of studies have investigated these systems independently of each other. Although the work of Stout and Hoagland (1939) as well as Biddulph and Markle (1944) laid the foundation for the physiological nexus between xylem and phloem, it is only recently that we have begun to understand the importance of the hydraulic nexus (Hölttä et al., 2006, 2009; Sevanto et al., 2011, 2014). Processes related to both nexus occur in parallel, and here the term physiological nexus covers all metabolite exchange, including the bidirectional flow of amino acids, minerals, and carbohydrates (Wardlaw, 1974; Ferrier et al., 1975; Pfautsch et al., 2009, 2015; De Schepper et al., 2013; for review, see van Bel, 1990, 2003). The term hydraulic nexus refers to the function of phloem as a capacitor, where water stored in phloem moves into xylem vessels to maintain the integrity of the transpiration stream (Zweifel et al., 2000, and refs. therein). Throughout this article, we use the term phloem collectively for cells that make up the transport phloem of woody plants (including sieve element/companion cell complexes, parenchyma cells, etc.), as opposed to collection and release phloem tissue, which differ in structure and function. Transport phloem is characterized by the retention of “high hydrostatic pressure by retrieval of leaked osmotica accompanied by water flux” (Patrick, 2013).According to the cohesion-tension theory, water in xylem vessels is constantly under tension and moves in a metastable state from roots to leaves along a hydrostatic pressure gradient. Depending on both the availability of soil moisture and the vapor pressure deficit of the atmosphere, this tension can exceed the cohesive forces that bind water molecules, resulting in the formation of a gas void that, after expanding, can lead to rupture of the water column inside individual vessels (termed cavitation; Zimmermann, 1983). Once cavitated, vessels become dysfunctional, and the transport of water and nutrients to leaves declines. However, water stored in woody tissues of trees can be mobilized to alleviate the risk of cavitation, and recent theory suggests that both water and carbohydrates from phloem may aid in the reversal of vessel embolism (i.e. air intrusion), although the evidence is indirect (Salleo et al., 2009; Brodersen et al., 2010; Nardini et al., 2011).All parts of plants have a water storage capacity (symplastic and apoplastic), and this capacitance increases with tree size and age (Phillips et al., 2003). The ability to mobilize stored water varies according to the force required to drag it out of storage (Holbrook, 1995). One-half century ago, Reynolds (1965) highlighted the importance of the volume of internally stored water to support the transpiration of trees. Since then, studies have quantified the fraction of stored water in total daily transpiration for a range of tree species. This fraction varies between 2% and 20% (Tyree and Yang, 1990; Čermák et al., 2007, and refs. therein; Barnard et al., 2011; Pfautsch and Adams, 2013) and is generally smaller in angiosperms compared with gymnosperms, where a maximum fraction of 50% was reported for Pinus sylvestris (Waring et al., 1979). Given that the daily water use of large adult trees can easily reach 260 to 380 L (Čermák et al., 2007; Pfautsch et al., 2011), considerable volumes of stored water must be mobilized from and restored back into capacitors on a daily basis. Remobilization of stored water also can prolong stomatal opening and thus increase carbon gain (Goldstein et al., 1998).The volume of stored water released depends on the elasticity of the storage tissue, its connectivity to xylem vessels, and the gradient of water potential (ψ) between the storage tissue and vessels. Cells with elastic walls represent ideal capacitors because they can change their volume as a consequence of small changes in ψ. Thus, phloem, cambium, and juvenile xylem cells are well suited for water storage and release (Yang and Tyree, 1992; Zweifel et al., 2014). The magnitude of release and refill of stored water in trees can be approximated by separately measuring the change in thickness of phloem and xylem during a diel cycle using high-precision dendrometers (Zweifel et al., 2014). Whitehead and Jarvis (1981) have calculated that around 90% of the diurnal change in stem radius can be attributed to changes in the water content of cambial and phloem tissues. To date, it is commonly accepted that tree bark, independent of the wood below, swells during the night and shrinks during the day (Zweifel et al., 2000), reflecting the water flow dynamics that characterize the dynamic exchange of water between phloem and xylem.Phloem and xylem tissues are separated by rows of intermediary cambial cells. However, depending on the species, phloem and xylem are connected through uniseriate or multiseriate strands of radially aligned ray parenchyma cells, commonly termed wood rays. These rays have been shown to be capable of symplastic water transport through plasmodesmata (Höll, 1975). Based on measurements of radial conductance, Sevanto et al. (2011) suggested that aquaporins also might be involved in the radial transfer of water. Both theoretical and experimental approaches have been developed to better understand the dynamic exchange of water between xylem and phloem. Hölttä et al. (2006, 2009) developed a model based on Münch’s hypothesis and included a term that represents the hydraulic connection between the two tissue types. Through incorporating this term, model outputs suggest the occurrence of a constant exchange of water between xylem and phloem along gradients of ψ. However, some authors suggested that changes in ψ of xylem alone might be insufficient to account for the observed diurnal shrinkage and swelling of bark (Sevanto et al., 2003). Along this line of argument, loading and unloading of carbohydrates in phloem tissue has been suggested to further impact the radial transfer of water and associated changes in bark thickness (Mencuccini et al., 2013).Nevertheless, to date, all approaches remain indirect, and the routes of water transfer between phloem and xylem have yet to be determined. Here, we present a technique that enables the visualization of water transfer from phloem to xylem tissues and resolves the apoplastic and symplastic pathways and cell types. The method involves the injection of an aqueous solution that contains fluorescent dye into phloem followed by analyses of woody tissues using confocal laser scanning microscopy. We assess the effectiveness of three different dyes to stain possible transfer pathways. We also introduce dye during different time intervals of the diel transpiration cycle to test the effect of predicted dynamic changes in ψ between phloem and xylem on the transfer processes. We hypothesized that radial transfer of water would be most pronounced during periods where conditions of the hydraulic nexus between phloem and xylem differ the most. These differences are expected during high rates of transpiration that cause a steep decline in xylem ψ, commonly observed during morning hours. We use leaf water potential (ψ_L) and high-precision dendrometer measurements to identify relevant time intervals. The simultaneous assessment of ψ_L and the independent diameter fluctuation of phloem and xylem may provide empirical evidence for the role of phloem as a water storage capacitor that helps mitigate increasing tension in the transpiration stream.     

Turnip mosaic virus Moves Systemically through Both Phloem and Xylem as Membrane-Associated Complexes

Plant viruses move systemically in plants through the phloem. They move as virions or as ribonucleic protein complexes, although it is not clear what these complexes are made of. The approximately 10-kb RNA genome of Turnip mosaic virus (TuMV) encodes a membrane protein, known as 6K₂, that induces endomembrane rearrangements for the formation of viral replication factories. These factories take the form of vesicles that contain viral RNA (vRNA) and viral replication proteins. In this study, we report the presence of 6K₂-tagged vesicles containing vRNA and the vRNA-dependent RNA polymerase in phloem sieve elements and in xylem vessels. Transmission electron microscopy observations showed the presence in the xylem vessels of vRNA-containing vesicles that were associated with viral particles. Stem-girdling experiments, which leave xylem vessels intact but destroy the surrounding tissues, confirmed that TuMV could establish a systemic infection of the plant by going through xylem vessels. Phloem sieve elements and xylem vessels from Potato virus X-infected plants also contained lipid-associated nonencapsidated vRNA, indicating that the presence of membrane-associated ribonucleic protein complexes in the phloem and xylem may not be limited to TuMV. Collectively, these studies indicate that viral replication factories could end up in the phloem and the xylem.Plant viruses use the host preexisting transport routes to propagate infection to the whole plant. After replication in the initially infected cells, viruses move cell to cell through plasmodesmata (PD) and start a new round of replication in the newly infected cells. This cycle is repeated until viruses reach vascular tissues, where they enter into the conducting tubes for systemic movement. Several studies have indicated that plant viruses are passively transported along the source-to-sink flow of photoassimilates and thus are believed to move systemically through the phloem (for review, see Hipper et al., 2013).The conducting tube of the phloem is the sieve element. The mature sieve element is enucleated and relies on the associated companion cells for the maintenance of its physiological function (Fisher et al., 1992). The specialized PD connecting one sieve element with one companion cell is called the pore plasmodesmal unit (PPU). Different from the other PDs, PPUs are always branched on the companion cell side but have only one channel on the sieve element side (Oparka and Turgeon, 1999). It is believed that the loading and uploading of viral material during phloem transport are through PPUs. Even though the size exclusion limit of PPUs (Kempers and Bel, 1997) is larger than that of the other PDs (Wolf et al., 1989; Derrick et al., 1990), PPUs should not allow, in their native state, virions or viral ribonucleoprotein (vRNP) complexes to pass through. It is thus believed that specific interactions between virus and host factors are required to allow the viral entity to go through. For instance, the movement protein of Cucumber mosaic virus (CMV) is targeted to PPUs (Blackman et al., 1998), suggesting that this viral protein modifies the size exclusion limit of PPUs and helps viral entry into sieve elements.Most plant viruses are assumed to move systemically through the phloem as virions. This assumption is based on the observation that Coat Protein (CP) deletions debilitating virus assembly prevent systemic infection (Brault et al., 2003; Zhang et al., 2013; Hipper et al., 2014). Some investigations showed the actual presence of virions in sieve elements. This is the case for the icosahedral Tobacco ringspot virus (Halk and McGuire, 1973), Carrot red leaf virus (Murant and Roberts, 1979), Potato leaf roll virus (Shepardson et al., 1980), and Beet western yellows virus (Hoefert, 1984). In addition, virions also were observed in phloem sap, such as the icosahedral CMV (Requena et al., 2006) and the rigid rod-shaped Cucumber green mottle mosaic virus (Simón-Buela and García-Arenal, 1999). Some viruses also are believed to move as ribonucleic protein (RNP) complexes, since systemic movement was observed in CP mutants where virion assembly was hindered. For instance, Tobacco rattle virus, Potato mop-top virus, Brome mosaic virus, and Tomato bushy stunt virus can still move systemically when the CP gene has been deleted from the viral genome (Swanson et al., 2002; Savenkov et al., 2003; Gopinath and Kao, 2007; Manabayeva et al., 2013). For potyviruses, it is still not clear if long-distance transport involves exclusively viral particles or if vRNP complexes also are implicated (Dolja et al., 1994, 1995; Cronin et al., 1995; Schaad et al., 1997; Kasschau and Carrington, 2001; Rajamaki and Valkonen, 2002). But whether virions or vRNP complexes are involved in viral movement, the full nature of the viral entity being implicated has not been defined.Xylem also is used for systemic infection of viruses, but its importance in viral transport generally has been overlooked. Vessel elements are the building blocks of xylem vessels, which constitute the major part of the water-upward-transporting system in a plant. The side walls of mature vessel elements contain pits, which are areas lacking a secondary cell wall; the end walls of the mature vessel elements are removed, and the openings are called perforation plates (Roberts and McCann, 2000). CP or virions of some plant viruses of all different shapes have been detected in the xylem vessels and/or guttation fluid, suggesting that these viruses may move systemically through xylem vessels. For example, the CP of the icosahedral Tomato bushy stunt virus (Manabayeva et al., 2013) and Rice yellow mottle virus (Opalka et al., 1998), the CP of the rigid rod-shaped Soilborne wheat mosaic virus (Verchot et al., 2001) and Cucumber green mottle mosaic virus (Moreno et al., 2004), and the flexuous rod-shaped Potato virus X (PVX; Betti et al., 2012) were detected in xylem vessels. Colocalization of anti-Rice yellow mottle virus antibodies and a cell wall marker for cellulosic β-(1-4)-d-glucans over vessel pit membranes suggests that the pit membranes might be a pathway for virus migration between vessels (Opalka et al., 1998). Moreover, flexuous rod-shaped virions of Zucchini yellow mosaic virus were found in both xylem vessels of root tissue and the guttation fluid (French and Elder, 1999). Finally, icosahedral Brome mosaic virus (Ding et al., 2001) and rigid rod-shaped Tomato mosaic virus and Pepper mild mottle virus (French et al., 1993) virions were found in guttation fluid. Guttation fluid originates from xylem exudate, indicating that these plant viruses can move through xylem within the infected plant. The above studies, however, mainly relied on electron microscopy and infection assays and may have missed the presence of other viral components that might be involved in transport.Turnip mosaic virus (TuMV) is a positive-strand RNA virus belonging to the family Potyviridae, genus Potyvirus, which contains around 30% of the currently known plant viruses and causes serious diseases in numerous crops (Shukla et al., 1994). Potyviruses are nonenveloped, flexuous rod-shaped particles of 680 to 900 nm in length and 11 to 13 nm in diameter. The genomic approximately 10-kb RNA encodes a polyprotein, which is processed into at least 11 mature proteins. TuMV remodels cellular membranes into viral factories, which are intracellular compartments involved in viral replication and movement. These compartments take the form of vesicles of approximately 100 nm in diameter originating from the endoplasmic reticulum (Grangeon et al., 2012). These vesicles contain viral RNA (vRNA) and viral and host proteins involved in vRNA replication (Beauchemin et al., 2007; Beauchemin and Laliberté, 2007; Dufresne et al., 2008; Huang et al., 2010; Grangeon et al., 2012). The viral membrane 6K₂ protein is involved in the membrane alterations and vesicle production (Beauchemin et al., 2007). The membrane-bound replication complexes can move intracellularly and cell to cell (Grangeon et al., 2013) at a rate of one cell being infected every 3 h (Agbeci et al., 2013). Intercellular trafficking of the replication complex is likely mediated by the PD-localized potyviral proteins Cytoplasmic Inclusion (CI) and P3N-PIPO (for N-terminal Half of P3 fused to the Pretty Interesting Potyviridae ORF; Carrington et al., 1998; Wei et al., 2010; Vijayapalani et al., 2012) as well as CP (Dolja et al., 1994, 1995), Viral Protein genome-linked (VPg; Nicolas et al., 1997; Rajamaki and Valkonen, 1999, 2002), and Helper Component-Proteinase (HC-Pro; Cronin et al., 1995; Kasschau et al., 1997; Rojas et al., 1997; Kasschau and Carrington, 2001), which are involved in both cell-to-cell and vascular movement.It is expected that, ultimately, TuMV reaches the vascular tissues of the plant, but how and under what form it is released into the conducting tubes are not known. To further understand viral spread and systemic movement, we investigated the distribution of 6K₂-tagged TuMV factories in all of the leaf and stem tissues other than the epidermal cells. We found TuMV factories in all tissues. Interestingly, we observed 6K₂-tagged vesicles, containing vRNA and viral replication proteins, in both phloem sieve elements and xylem vessels. We confirmed that TuMV could move systemically through xylem by a so-called stem-girdling assay, which induces cell death of the phloem without affecting xylem integrity. Hence, our study indicates that membrane-associated TuMV replication complexes are involved in the systemic movement of the virus.     

Direct X-Ray Microtomography Observation Confirms the Induction of Embolism upon Xylem Cutting under Tension

Direct visualization shows enhanced embolism of xylem samples when they are collected under tension.Embolism resistance is a critically important trait for evaluating the ability of plants to survive and recover from drought periods and predicting future drought-induced forest decline (Choat et al., 2012). However, recent publications have provided evidence that some measurement techniques used to evaluate the hydraulic function and vulnerability to cavitation of plant organs may be prone to artifacts (Sperry et al., 2012; Cochard et al., 2013; Torres-Ruiz et al., 2014; Trifilò et al., 2014). The discovery of these artifacts has raised questions regarding the reliability of some previously published plant hydraulics data, in particular data relating to the refilling of embolized xylem conduits while the xylem is under tension. In this context, Wheeler et al. (2013) reported that sampling plant organs by cutting while the xylem is under tension can induce artificial increases in the degree of embolism at the moment of sample excision, even when cuts are made under water. The methodology applied by Wheeler et al. (2013), however, did not allow the visualization of embolized or functional vessels, and native embolism levels could not be determined in intact plants before any cutting was done.Whereas Scoffoni and Sack (2014) showed that the artifact described by Wheeler et al. (2013) has no impact on leaf xylem hydraulic conductance, there is some uncertainty about its importance in stems or shoots (Trifilò et al., 2014; Venturas et al., 2014). The results of Wheeler et al. (2013) indicate that more embolism could be induced by cutting samples that are under midrange xylem tension (e.g. at midday or under conditions of water stress). Potential overestimation of embolism due to changes in the xylem tension during the day has important implications for our understanding of plant water relations, since they could erroneously suggest that daily patterns of embolism formation and repair are routine in many woody plant species. Debate continues regarding the implications of a cutting artifact for the existence of a mechanism that allows plants to repair embolism while the xylem is under tension, so-called novel refilling (Salleo et al., 1996; Cochard and Delzon, 2013; Sperry, 2013; Delzon and Cochard, 2014). To avoid the excision artifact, Wheeler et al. (2013) recommended the relaxation of the xylem tension prior to excision by rehydrating plant tissue for anywhere between 2 min and 2 h. However, recent results from Trifilò et al. (2014) indicated that the rehydration procedures used by Wheeler et al. (2013) for relaxing the samples might favor xylem refilling and embolism repair (rehydration artifact), suggesting that the artifact resides in the relaxing procedure rather than in the cutting procedure. In light of these data, the assessment of the artifact described by Wheeler et al. (2013) using noninvasive techniques on intact plants, such as direct observation using x-ray microtomography (micro-CT; McElrone et al., 2013; Cochard et al., 2014) or magnetic resonance imaging (Choat et al., 2010; Zwieniecki et al., 2013), is useful to visually assess changes in embolism after cutting stems.     

Grain Unloading of Arsenic Species in Rice

Rice (Oryza sativa) is the staple food for over half the world''s population yet may represent a significant dietary source of inorganic arsenic (As), a nonthreshold, class 1 human carcinogen. Rice grain As is dominated by the inorganic species, and the organic species dimethylarsinic acid (DMA). To investigate how As species are unloaded into grain rice, panicles were excised during grain filling and hydroponically pulsed with arsenite, arsenate, glutathione-complexed As, or DMA. Total As concentrations in flag leaf, grain, and husk, were quantified by inductively coupled plasma mass spectroscopy and As speciation in the fresh grain was determined by x-ray absorption near-edge spectroscopy. The roles of phloem and xylem transport were investigated by applying a ± stem-girdling treatment to a second set of panicles, limiting phloem transport to the grain in panicles pulsed with arsenite or DMA. The results demonstrate that DMA is translocated to the rice grain with over an order magnitude greater efficiency than inorganic species and is more mobile than arsenite in both the phloem and the xylem. Phloem transport accounted for 90% of arsenite, and 55% of DMA, transport to the grain. Synchrotron x-ray fluorescence mapping and fluorescence microtomography revealed marked differences in the pattern of As unloading into the grain between DMA and arsenite-challenged grain. Arsenite was retained in the ovular vascular trace and DMA dispersed throughout the external grain parts and into the endosperm. This study also demonstrates that DMA speciation is altered in planta, potentially through complexation with thiols.Paddy rice (Oryza sativa) is particularly effective, compared to other cereals, at accumulating arsenic (As) in shoot and grain (Williams et al., 2007b). Rice is the staple food for over half the world''s population (Fageria, 2007) and rice represents a significant dietary source of inorganic As, a class 1, nonthreshold carcinogen, particularly in Southeast Asia (Meharg et al., 2009). Inorganic As levels in rice grain are problematic even where soil As is at background levels, derived from geogenic sources (Lu et al., 2009; Meharg et al., 2009). However, widespread pollution of paddy soils with As, leading to further elevation of grain As, has occurred in some regions due to base and precious mining (Liao et al., 2005; Zhu et al., 2008), irrigation of paddies with As-elevated groundwaters (e.g. Meharg and Rahman, 2003; Williams et al., 2006), and the use of arsenical pesticides (Williams et al., 2007a). Unlike other cereal grains, paddy rice cultivation is dependent of soils being anaerobic, and it is this anoxia that gives rise to elevated As concentrations in the plant. Anaerobic soil conditions lead to the mobilization of As as arsenite, where under aerobic systems arsenate dominates (Xu et al., 2008). Arsenite is efficiently assimilated by rice roots through silicic acid transport pathway (Ma et al., 2008).Knowledge of As metabolism and partitioning within plants, particularly rice, is still developing rapidly (Zhao et al., 2009). Several studies have now shown that As in rice vegetative tissue and grain is predominantly speciated as inorganic As and the methylated species dimethylarsinic acid (DMA), with variable, though low, levels of monomethyl arsonic acid (MMA; Abedin et al., 2002a; Williams et al., 2005, 2006; Norton et al., 2009). Arsenate is an analog of phosphate and competes with phosphate for rice root uptake (Abedin et al., 2002a) while arsenite is taken up by rice roots via silicic acid transporters (Ma et al., 2008). Abedin et al. (2002b) demonstrated that the methylated species DMA and MMA are also taken up by rice plants although at a much slower rate than inorganic As, with the protonated neutral forms also transported through silicic acid pathway (Li et al., 2009). Arsenate is reduced to arsenite within the rice root (Xu et al., 2008; Zhao et al., 2009), which then enters the xylem via a silicic acid/arsenite effluxer (Ma et al., 2008; Zhao et al., 2009). Arsenite may be detoxified through complexation with thiol-rich peptides including phytochelatins (PCs) and glutathione followed by sequestration into vacuoles (Bleeker et al., 2006; Raab et al., 2007b; Zhao et al., 2009). Raab et al. (2007a) found that while methylated As species are taken up by rice roots much less efficiently than inorganic species, they appear to be translocated within the plant more efficiently. The comparative contributions of xylem and phloem transport, in translocation of As to the grain, are unknown.The main species within rice grain, along with DMA, are inorganic As, particularly arsenite, which may be complexed with thiols (Williams et al., 2005; Lombi et al., 2009). Nutrients are unloaded into the grain from the ovular vascular trace (OVT) into the nucellar tissue and from there are uploaded, via the apoplast into the filial tissue (the aleurone and the endosperm; Krishnan and Dayanandan, 2003). Lombi et al. (2009) recently suggested that this may represent a physiological barrier that As species cross with differential efficiency. However, the transport and unloading of As to/into the grain, which are key processes in terms of human exposure to this contaminant, are far from being fully understood.This study investigated the differential efficiency with which important As species are translocated and unloaded into the rice grain and the comparative contributions of phloem and xylem transport. Rice panicles were excised below the flag leaf node during grain development, 10 DPA, and treated to a hydroponically administered 48-h pulse of arsenite, arsenate, arsenite glutathione, or DMA. Total As concentrations in flag leaf, grain, and husk samples for each treatment were quantified by inductively coupled plasma mass spectroscopy (ICP-MS), and As speciation in the fresh grain was determined by x-ray absorption near-edge spectroscopy (XANES) analysis. To evaluate the contributions of phloem versus xylem transport, a stem-girdling treatment was applied, using steam to destroy phloem cells in a second set of panicles prior to a pulse of either DMA or arsenite. The spatial unloading of As species into the developing grain was examined by synchrotron x-ray fluorescence (XRF) mapping, and fluorescence microtomography for the DMA and arsenite treatments.     

Water Transport Properties of the Grape Pedicel during Fruit Development: Insights into Xylem Anatomy and Function Using Microtomography

Xylem flow of water into fruits declines during fruit development, and the literature indicates a corresponding increase in hydraulic resistance in the pedicel. However, it is unknown how pedicel hydraulics change developmentally in relation to xylem anatomy and function. In this study on grape (Vitis vinifera), we determined pedicel hydraulic conductivity (k_h) from pressure-flow relationships using hydrostatic and osmotic forces and investigated xylem anatomy and function using fluorescent light microscopy and x-ray computed microtomography. Hydrostatic k_h (xylem pathway) was consistently 4 orders of magnitude greater than osmotic k_h (intracellular pathway), but both declined before veraison by approximately 40% and substantially over fruit development. Hydrostatic k_h declined most gradually for low (less than 0.08 MPa) pressures and for water inflow and outflow conditions. Specific k_h (per xylem area) decreased in a similar fashion to k_h despite substantial increases in xylem area. X-ray computed microtomography images provided direct evidence that losses in pedicel k_h were associated with blockages in vessel elements, whereas air embolisms were negligible. However, vessel elements were interconnected and some remained continuous postveraison, suggesting that across the grape pedicel, a xylem pathway of reduced k_h remains functional late into berry ripening.In grape (Vitis vinifera), fruit growth by water accumulation follows a double sigmoid pattern and is influenced by the diurnal and developmental changes in water flow between fruit and the parent plant (Matthews and Shackel, 2005). Until the onset of fruit ripening (i.e. veraison), water enters the fruit predominantly via the xylem and thereafter mainly through the phloem (Greenspan et al., 1994, 1996). Choat et al. (2009) showed that the hydraulic conductance (i.e. 1/resistance) of the grape berry and pedicel declines substantially at later ripening stages predominantly due to a decline in pedicel conductance. Significant developmental changes in pedicel hydraulic properties were also reported for tomato (Solanum lycopersicum) and were found to be associated with xylem anatomical changes (Lee 1989; Van Ieperen et al., 2003; Rancić et al., 2008, 2010). Due to its position along the vascular transport pathway between fruit and the parent plant, the pedicel can play an important role in affecting fruit growth, as in kiwi (Actinidia deliciosa; Mazzeo et al., 2013). However, for grape, it needs to be elucidated how pedicel hydraulic properties change developmentally in relation to xylem anatomy and function.The location and nature of the loss in hydraulic conductance between the parent plant and the fruit is unclear and may differ among fruits. For tomato, Malone and Andrews (2001) showed that most of the loss of hydraulic conductance occurs in the fruit per se, but Van Ieperen et al. (2003) reported important and decreasing hydraulic conductance in the pedicel abscission zone over fruit development. For Citrus spp., Garcia-Luis et al. (2002) reported that xylem vessels in the pedicel remain largely functional late into fruit ripening. For grape, although vessel breakage in the berry was thought to lead to xylem dysfunction (Coombe and McCarthy 2000), several studies and methods have shown that xylem vessels in the fruit remain functional (Rogiers et al., 2001; Bondada et al., 2005; Chatelet et al., 2008a, 2008b). In line with these findings, data by Keller et al. (2006) suggest that the pedicel xylem also remains at least partially functional in ripening grape berries and can conduct water to and from the parent plant. Nevertheless, a reduction in the ability to transport water during ripening has been reported for grape (Tyerman et al., 2004; Choat et al., 2009) and other fleshy fruits, such as apple (Malus domestica; Lang and Ryan, 1994) and kiwi (Mazzeo et al., 2013), and it still remains unclear what causes this loss in xylem hydraulic conductance. For the grape pedicel, Choat et al. (2009) detected higher concentrations of xylem solutes postveraison and proposed that this is related to the deposition of gels into the xylem vessel lumen. However, direct evidence for the presence of xylem blockage and/or embolism formation in the grape pedicel is missing.This study of the grape ‘Cabernet Sauvignon’ pedicel was conducted with the goal to obtain a comprehensive understanding of how changes in hydraulic properties relate to changes in xylem structure and function over fruit development. Over the course of fruit development from 20 to 90 d after anthesis (DAA), water transport properties of pedicels were investigated under osmotic and hydrostatic driving forces using a modified pressure-probe system. This was combined with analyses of spatial and temporal changes in pedicel xylem anatomy and function using fluorescent light microscopy and x-ray computed microtomography (microCT; Brodersen et al., 2010, 2013; Rancić et al., 2010).     

Vascular Function in Grape Berries across Development and Its Relevance to Apparent Hydraulic Isolation

During the latter stages of development in fleshy fruit, water flow through the xylem declines markedly and the requirements of transpiration and further expansion are fulfilled primarily by the phloem. We evaluated the hypothesis that cessation of water transport through the xylem results from disruption or occlusion of pedicel and berry xylem conduits (hydraulic isolation). Xylem hydraulic resistance (R_h) was measured in developing fruit of grape (Vitis vinifera ‘Chardonnay’) 20 to 100 d after anthesis (DAA) and compared with observations of xylem anatomy by light and cryo-scanning electron microscopy and expression of six plasma membrane intrinsic protein (PIP) aquaporin genes (VvPIP1;1, VvPIP1;2, VvPIP1;3, VvPIP2;1, VvPIP2;2, VvPIP2;3). There was a significant increase in whole berry R_h and receptacle R_h in the latter stages of ripening (80–100 DAA), which was associated with deposition of gels or solutes in many receptacle xylem conduits. Peaks in the expression of some aquaporin isoforms corresponded to lower whole berry R_h 60 to 80 DAA, and the increase in R_h beginning at 80 DAA correlated with decreases in the expression of the two most predominantly expressed PIP genes. Although significant, the increase in berry R_h was not great enough, and occurred too late in development, to explain the decline in xylem flow that occurs at 60 to 75 DAA. The evidence suggests that the fruit is not hydraulically isolated from the parent plant by xylem occlusion but, rather, is “hydraulically buffered” by water delivered via the phloem.The development of grape (Vitis vinifera) berries is typical of many fleshy fruits, following a double sigmoid pattern of growth with three distinct phases: an initial phase of rapid cell division and expansion in green berries, a short transitory phase of very little growth, and a final phase in which growth is reinitiated and the fruit ripens. The transition to the ripening phase is accompanied by many physiological changes, such as the production of anthocyanins and fruit softening. In grape, these distinctive and highly visible physiological changes are collectively referred to as veraison. The rapid accumulation of sugars that is initiated in the berry mesocarp around the time of veraison is accompanied by a dramatic shift in the proportion of xylem and phloem transport (Lang and Thorpe, 1989; Greenspan et al., 1994, 1996). This same shift, albeit more gradual, occurs in many other fleshy fruits such as tomato (Solanum lycopersicum; Ho et al., 1987), apple (Malus domestica; Lang and Ryan, 1994; Drazeta et al., 2004), and kiwifruit (Actinidia deliciosa; Dichio et al., 2003) as well as in the flowers of tropical trees (Chapotin et al., 2003). The sudden reduction in xylem transport to the fruit is perceived as a mechanism to hydraulically isolate the fruit and buffer them from environmental stresses experienced by the parent plant.Using mass balance techniques, Greenspan et al. (1994, 1996) reported major changes in the role of the xylem and phloem in water transport to the grape berry at veraison. During the first growth phase, the xylem provides the majority of water transport into the berry. In the final growth stage, the phloem provides more than 80% of the berry''s water requirements and the contribution of the xylem becomes negligible. Berry water status also becomes apparently uncoupled from plant water status after veraison. Before veraison, diurnal contractions in berry diameter were strongly related to changes in plant (stem) water potential, while after veraison, diurnal contractions were greatly reduced and unrelated to changes in stem water potential (Matthews and Shackel, 2005). A similar lack of response was also observed for mesocarp cell turgor after veraison (Thomas et al., 2006). Thus, it is clear that some mechanism acts to decouple berry water relations from the water status of the parent plant.Over the past two decades, a general consensus has developed that the berry xylem becomes physically disrupted after veraison, effectively blocking the xylem pathway and isolating the fruit essentially as a whole from the parent plant (During et al., 1987; Findlay et al., 1987; Lang and Ryan, 1994). Evidence for this has been provided by observations of dye uptake into the berry through the xylem. When the cut pedicel of a preveraison berry is submerged in dye, the dye is taken up into peripheral and axial xylem conduits of the entire berry (Findlay et al., 1987; Creasy et al., 1993; Rogiers et al., 2001). After veraison, dye uptake is limited to the base of the berry vasculature (brush). From this evidence, together with micrographs that appeared to show stretched and ruptured xylem conduits in postveraison berries, it was inferred that the lignified tracheids present at veraison were physically torn apart by the expansion of the berry that occurred postveraison.Recent experimental work using a range of techniques suggests that the hypothesis of physical disruption may be oversimplified and that the berry xylem remains at least potentially functional after veraison (Bondada et al., 2005; Keller et al., 2006; Chatelet et al., 2008b). The results of Chatelet et al. (2008a, 2008b) demonstrate that the majority of xylem conduits in the berry remain intact after veraison and suggest that xylem development (growth of new conduits) continues well into the postveraison growth phase. Using both a modified pressure plate/membrane apparatus and a wicking technique, it was demonstrated that dye moved through the xylem of postveraison berries when a hydrostatic pressure or matric gradient was applied between the pedicel and the cut stylar surface (Bondada et al., 2005; Chatelet et al., 2008b). Keller et al. (2006) demonstrated this in reverse, showing that berry xylem was still capable of conducting a dye tracer back to the parent plant if the dye was introduced at the cut stylar end while the plant was transpiring. Thus, given a large enough pressure gradient, the xylem of postveraison berries retains the potential to transport water between the parent plant and the berry or vice versa. However, anatomical measurements and dye tracer studies can only be used to infer the degree to which fruit may become isolated from the parent plant. Knowledge of changes in hydraulic resistance (R_h) is required to determine whether xylem dysfunction is actually responsible for declining xylem flows reported with the progression of ripening. It is also important to differentiate between xylem flows and R_h, as these variables are sometimes confused in the literature; xylem flow rates can vary independently of R_h if water potential gradients along the pathway are altered.Previous studies examining changes in R_h associated with the development of fleshy fruit generally indicate that R_h increases during ripening but show differences in the timing and location of the increase. Some fruits develop an abscission zone in the pedicel or receptacle that is associated with vascular constriction and high R_h (Mackenzie, 1988; Lee, 1989; Van Ieperen et al., 2003). However, although some table grapes are believed to develop an abscission zone, there is no evidence of an abscission zone in wine grapes (Pratt, 1971). Tyerman et al. (2004) reported a substantial increase in R_h of grape berries after veraison, although this increase in resistance did not occur in the pedicel or receptacle but mainly in the distal section of the berries. Similarly, Malone and Andrews (2001) evaluated R_h in developing tomato fruits and stems and found that R_h increased in the fruit, but not proximal to the calyx. In apple, Lang and Ryan (1994) observed an increase in R_h at 80 d after anthesis (DAA) and also reported an increasing proportion of samples in which the xylem was completely occluded with age. Although they described these data as pedicel R_h, their measurements actually included the fruit vascular pathway; therefore, it is difficult to determine if the increase in R_h was manifested in the fruit or the pedicel.Increases in pedicel and receptacle R_h should be associated with changes in the dimensions or conductive state or xylem conduits. An increase in the R_h within the fruit may relate either to xylem dysfunction or to extravascular resistance beyond the xylem. Although they were not able to partition an increase in fruit R_h between the apoplast and symplast, Tyerman et al. (2004) suggested that the site of increased resistance may be the plasma membranes of vascular parenchyma cells separating xylem conduits and mesocarp cells rather than the xylem itself. A likely candidate driving hydraulic isolation at the cellular level is changes in plasma membrane R_h resulting from the differential expression and activity of aquaporins. Aquaporins are a family of transmembrane proteins considered to be largely responsible for the high permeability to water exhibited by plasma membranes. The regulation of R_h by aquaporins is now well documented in roots (Martre et al., 2001; McElrone et al., 2007; Vandeleur et al., 2009), and oxidative gating of aquaporins has been reported to reduce hydraulic conductivity by 90% in cells of the giant algae Chara (Henzler et al., 2004). The results of previous work suggest that aquaporins play an important role in the regulation of water movement during the development of flowers, seeds, and fruits (Maurel et al., 1995; Gao et al., 1999; Picaud et al., 2003; Shiota et al., 2006; Zhou et al., 2007). Changes in the expression of the plasma membrane intrinsic protein (PIP) PIP1 and PIP2 aquaporin gene families have been noted in ripening grapes (Picaud et al., 2003; Fei et al., 2004), although the effects of these changes on water transport (membrane conductivity) have not been documented. An increase of R_h between the mesocarp cells and the xylem within the fruit could provide a mechanism to restrict water movement between the parent plant and the berry if a large gradient in xylem tension existed between the two (Tyerman et al., 2004).While the concept of hydraulic isolation is generally accepted as part of the physiology of fleshy fruit development, we note that no studies have demonstrated an increase in R_h that is coincident with the decline in xylem flow. Additionally, measured variation in the R_h of the fruit and pedicel has not been quantitatively related to the water requirements of the fruit, taking into account water potential gradients between the fruit and the parent plant. In this study, we examined changes in the R_h of the berry, receptacle, and pedicel of Chardonnay grape over the course of fruit development. These measurements were compared against observations of xylem anatomy and aquaporin gene expression in order to investigate the hypotheses that (1) occlusion and/or disruption of xylem conduits results in the hydraulic isolation of ripening grape berries, and (2) an increase in the R_h of the berry is associated with changes in the expression of aquaporin genes in the mesocarp.     

Cavitation Resistance in Seedless Vascular Plants: The Structure and Function of Interconduit Pit Membranes

Plant water transport occurs through interconnected xylem conduits that are separated by partially digested regions in the cell wall known as pit membranes. These structures have a dual function. Their porous construction facilitates water movement between conduits while limiting the spread of air that may enter the conduits and render them dysfunctional during a drought. Pit membranes have been well studied in woody plants, but very little is known about their function in more ancient lineages such as seedless vascular plants. Here, we examine the relationships between conduit air seeding, pit hydraulic resistance, and pit anatomy in 10 species of ferns (pteridophytes) and two lycophytes. Air seeding pressures ranged from 0.8 ± 0.15 MPa (mean ± sd) in the hydric fern Athyrium filix-femina to 4.9 ± 0.94 MPa in Psilotum nudum, an epiphytic species. Notably, a positive correlation was found between conduit pit area and vulnerability to air seeding, suggesting that the rare-pit hypothesis explains air seeding in early-diverging lineages much as it does in many angiosperms. Pit area resistance was variable but averaged 54.6 MPa s m⁻¹ across all surveyed pteridophytes. End walls contributed 52% to the overall transport resistance, similar to the 56% in angiosperm vessels and 64% in conifer tracheids. Taken together, our data imply that, irrespective of phylogenetic placement, selection acted on transport efficiency in seedless vascular plants and woody plants in equal measure by compensating for shorter conduits in tracheid-bearing plants with more permeable pit membranes.Water transport in plants occurs under tension, which renders the xylem susceptible to air entry. This air seeding may lead to the rupture of water columns (cavitation) such that the air expands within conduits to create air-vapor embolisms that block further transport. (Zimmermann and Tyree, 2002). Excessive embolism such as that which occurs during a drought may jeopardize leaf hydration and lead to stomatal closure, overheating, wilting, and possibly death of the plant (Hubbard et al., 2001; Choat et al., 2012; Schymanski et al., 2013). Consequently, strong selection pressure resulted in compartmentalized and redundant plant vascular networks that are adapted to a species habitat water availability by way of life history strategy (i.e. phenology) or resistance to air seeding (Tyree et al., 1994; Mencuccini et al., 2010; Brodersen et al., 2012). The spread of drought-induced embolism is limited primarily by pit membranes, which are permeable, mesh-like regions in the primary cell wall that connect two adjacent conduits. The construction of the pit membrane is such that water easily moves across the membrane between conduits, but because of the small membrane pore size and the presence of a surface coating on the membrane (Pesacreta et al., 2005; Lee et al., 2012), the spread of air and gas bubbles is restricted up to a certain pressure threshold known as the air-seeding pressure (ASP). When xylem sap tension exceeds the air-seeding threshold, air can be aspirated from an air-filled conduit into a functional water-filled conduit through perhaps a large, preexisting pore or one that is created by tension-induced membrane stress (Rockwell et al., 2014). Air seeding leads to cavitation and embolism formation, with emboli potentially propagating throughout the xylem network (Tyree and Sperry, 1988; Brodersen et al., 2013). So, on the one hand, pit membranes are critical to controlling the spread of air throughout the vascular network, while on the other hand, they must facilitate the efficient flow of water between conduits (Choat et al., 2008; Domec et al., 2008; Pittermann et al., 2010; Schulte, 2012). Much is known about such hydraulic tradeoffs in the pit membranes of woody plants, but comparatively little data exist on seedless vascular plants such as ferns and lycophytes. Given that seedless vascular plants may bridge the evolutionary transition from bryophytes to woody plants, the lack of functional data on pit membrane structure in early-derived tracheophytes is a major gap in our understanding of the evolution of plant water transport.In woody plants, pit membranes fall into one of two categories: the torus-margo type found in most gymnosperms and the homogenous pit membrane characteristic of angiosperms (Choat et al., 2008; Choat and Pittermann, 2009). In conifers, water moves from one tracheid to another through the margo region of the membrane, with the torus sealing the pit aperture should one conduit become embolized. Air seeding occurs when water potential in the functional conduit drops low enough to dislodge the torus from its sealing position, letting air pass through the pit aperture into the water-filled tracheid (Domec et al., 2006; Delzon et al., 2010; Pittermann et al., 2010; Schulte, 2012; but see Jansen et al., 2012). Across north-temperate conifer species, larger pit apertures correlate with lower pit resistance to water flow (r_pit; MPa s m⁻¹), but it is the ratio of torus-aperture overlap that sets a species cavitation resistance (Pittermann et al., 2006, 2010; Domec et al., 2008; Hacke and Jansen, 2009). A similar though mechanistically different tradeoff exists in angiosperm pit membranes. Here, air seeding reflects a probabilistic relationship between membrane porosity and the total area of pit membranes present in the vessel walls. Specifically, the likelihood of air aspirating into a functional conduit is determined by the combination of xylem water potential and the diameter of the largest pore and/or the weakest zone in the cellulose matrix in the vessel’s array of pit membranes (Wheeler et al., 2005; Hacke et al., 2006; Christman et al., 2009; Rockwell et al., 2014). As it has come to be known, the rare-pit hypothesis suggests that the infrequent, large-diameter leaky pore giving rise to that rare pit reflects some combination of pit membrane traits such as variation in conduit membrane area (large or small), membrane properties (tight or porous), and hydrogel membrane chemistry (Hargrave et al., 1994; Choat et al., 2003; Wheeler et al., 2005; Hacke et al., 2006; Christman et al., 2009; Lee et al., 2012; Plavcová et al., 2013; Rockwell et al., 2014). The maximum pore size is critical because, per the Young-Laplace law, the larger the radius of curvature, the lower the air-water pressure difference under which the contained meniscus will fail (Jarbeau et al., 1995; Choat et al., 2003; Jansen et al., 2009). Consequently, angiosperms adapted to drier habitats may exhibit thicker, denser, smaller, and less abundant pit membranes than plants occupying regions with higher water availability (Wheeler et al., 2005; Hacke et al., 2007; Jansen et al., 2009; Lens et al., 2011; Scholz et al., 2013). However, despite these qualitative observations, there is no evidence that increased cavitation resistance arrives at the cost of higher r_pit. Indeed, the bulk of the data suggest that prevailing pit membrane porosity is decoupled from the presence of the single largest pore that allows air seeding to occur (Choat et al., 2003; Wheeler et al., 2005 Hacke et al., 2006, 2007).As water moves from one conduit to another, pit membranes offer considerable hydraulic resistance throughout the xylem network. On average, r_pit contributes 64% and 56% to transport resistance in conifers and angiosperms, respectively (Wheeler et al., 2005; Pittermann et al., 2006; Sperry et al., 2006). In conifers, the average r_pit is estimated at 6 ± 1 MPa s m⁻¹, almost 60 times lower than the 336 ± 81 MPa s m⁻¹ computed for angiosperms (Wheeler et al., 2005; Hacke et al., 2006; Sperry et al., 2006). Presumably, the high porosity of conifer pits compensates for the higher transport resistance offered by a vascular system composed of narrow, short, single-celled conduits (Pittermann et al., 2005; Sperry et al., 2006).Transport in seedless vascular plants presents an interesting conundrum because, with the exception of a handful of species, their primary xylem is composed of tracheids, the walls of which are occupied by homogenous pit membranes (Gibson et al., 1985; Carlquist and Schneider, 2001, 2007; but see Morrow and Dute, 1998, for torus-margo membranes in Botrychium spp.). At first pass, this combination of traits appears hydraulically maladaptive, but several studies have shown that ferns can exhibit transport capacities that are on par with more recently evolved plants (Wheeler et al., 2005; Watkins et al., 2010; Pittermann et al., 2011, 2013; Brodersen et al., 2012). Certainly, several taxa possess large-diameter, highly overlapping conduits, some even have vessels such as Pteridium aquilinum and many species have high conduit density, all of which could contribute to increased hydraulic efficiency (Wheeler et al., 2005; Pittermann et al., 2011, 2013). But how do the pit membranes of seedless vascular plants compare? Scanning electron micrographs of fern and lycopod xylem conduits suggest that they are thin, diaphanous, and susceptible to damage during specimen preparation (Carlquist and Schneider 2001, 2007). Consistent with such observations, two estimates of r_pit imply that r_pit in ferns may be significantly lower than in angiosperms; Wheeler et al. (2005) calculated r_pit in the fern Pteridium aquilinum at 31 MPa s m⁻¹, while Schulte et al. (1987) estimated r_pit at 1.99 MPa s m⁻¹ in the basal fern Psilotum nudum. The closest structural analogy to seedless vascular plant tracheids can be found in the secondary xylem of the early-derived vesselless angiosperms, in which tracheids possess homogenous pit membranes with r_pit values that at 16 MPa s m⁻¹ are marginally higher than those of conifers (Hacke et al., 2007). Given that xylem in seedless vascular plants is functionally similar to that in vesselless angiosperms, we expected convergent r_pit values in these two groups despite their phylogenetic distance. We tested this hypothesis, as well as the intrinsic cavitation resistance of conduits in seedless vascular plants, by scrutinizing the pit membranes of ferns and fern allies using the anatomical and experimental approaches applied previously to woody taxa. In particular, we focused on the relationship between pit membrane traits and cavitation resistance at the level of the individual conduit.     

Evidence for Hydraulic Vulnerability Segmentation and Lack of Xylem Refilling under Tension

The vascular system of grapevine (Vitis spp.) has been reported as being highly vulnerable, even though grapevine regularly experiences seasonal drought. Consequently, stomata would remain open below water potentials that would generate a high loss of stem hydraulic conductivity via xylem embolism. This situation would necessitate daily cycles of embolism repair to restore hydraulic function. However, a more parsimonious explanation is that some hydraulic techniques are prone to artifacts in species with long vessels, leading to the overestimation of vulnerability. The aim of this study was to provide an unbiased assessment of (1) the vulnerability to drought-induced embolism in perennial and annual organs and (2) the ability to refill embolized vessels in two Vitis species X-ray micro-computed tomography observations of intact plants indicated that both Vitis vinifera and Vitis riparia were relatively vulnerable, with the pressure inducing 50% loss of stem hydraulic conductivity = −1.7 and −1.3 MPa, respectively. In V. vinifera, both the stem and petiole had similar sigmoidal vulnerability curves but differed in pressure inducing 50% loss of hydraulic conductivity (−1.7 and −1 MPa for stem and petiole, respectively). Refilling was not observed as long as bulk xylem pressure remained negative (e.g. at the apical part of the plants; −0.11 ± 0.02 MPa) and change in percentage loss of conductivity was 0.02% ± 0.01%. However, positive xylem pressure was observed at the basal part of the plant (0.04 ± 0.01 MPa), leading to a recovery of conductance (change in percentage loss of conductivity = −0.24% ± 0.12%). Our findings provide evidence that grapevine is unable to repair embolized xylem vessels under negative pressure, but its hydraulic vulnerability segmentation provides significant protection of the perennial stem.The plant hydraulic system is located at the interface between soil water and the atmosphere. Evaporative demand from the atmosphere generates a tension within a continuous xylem water column, pulling water from the soil, through roots, stems, petioles, and leaves (Dixon, 1896). Under drought conditions, the overall resistance to water flow through the soil-plant continuum increases. Increased resistance to water flow results from changes in the resistance at multiple specific locations along the flow pathway: in the soil, at the soil-root interface, and in the roots, the main plant axis (i.e. stems and branches), the petioles, and the leaves. Two primary mechanisms controlling the resistance are stomatal closure (leaf-to-air water flow) and the loss of xylem hydraulic conductivity (soil-to-leaf water flow; Cochard et al., 2002). Stomatal closure is closely related to decreasing plant water status (Brodribb and Holbrook, 2003) and is often considered to be a protective mechanism against the loss of xylem hydraulic conductivity (Tyree and Sperry, 1988; Jones and Sutherland, 1991). Loss of xylem hydraulic conductivity occurs when the water potential of xylem sap reaches levels negative enough to disrupt the metastability of the water column, potentially resulting in embolism.Generally, high resistance to embolism is observed in species distributed in dry environments, whereas highly vulnerable species are distributed in wet environments (Maherali et al., 2004; Choat et al., 2012). Although grapevine (Vitis spp.) is widely cultivated, including in regions where it is frequently exposed to water deficit during the growing season (Lovisolo et al., 2010), recent studies have produced contrasting estimates of its resistance to embolism. Grapevine has been described as either vulnerable (Zufferey et al., 2011; Jacobsen and Pratt, 2012) or relatively resistant (Choat et al., 2010; Brodersen et al., 2013). In Vitis spp., and Vitis vinifera especially, stomatal closure is typically observed for midday leaf water potentials less than −1.5 MPa (Schultz, 2003). Thus, according to some studies, significant losses in xylem hydraulic conductivity should be observed before stomatal closure (Ψ₅₀ > −1 MPa; Jacobsen and Pratt, 2012; Jacobsen et al., 2015), implying that embolism would be commonplace.The risk of hydraulic dysfunction is mitigated along the hydraulic pathway by hydraulic segmentation (i.e. more distal organs such as leaves and petioles will be at greater risk to embolism than more basal organs such as the trunk; Tyree and Zimmermann 2002; Choat et al., 2005). This could promote hydraulic safety in larger, perennial organs, which represent a greater investment of resources for the plant. Hydraulic segmentation may occur in two ways. During transpiration, the xylem pressure will always be more negative in more distal parts of the pathway (leaves and petioles). All else being equal, this translates to a greater probability of embolism in distal organs. However, organs also may differ in their vulnerability to embolism, compensating or exacerbating the effects of differences in xylem pressure along the pathway. If leaves or petioles were more vulnerable to embolism than branches and the trunk, then they would be far more likely to suffer embolism during periods of water stress. This would allow petioles, leaves (Nolf et al., 2015), or even young branches (Rood et al., 2000) to become embolized without significant impacts on the trunk and larger branches. In grapevine, petioles have been described as extremely sensitive to cavitation (Ψ₅₀ of approximately −1 MPa; Zufferey et al., 2011). However, the hydraulic methods employed in those previous studies have been shown to be prone to artifacts (Wheeler et al., 2013; Torres-Ruiz et al., 2015), necessitating the use of a noninvasive assessment of drought-induced embolism.High-resolution computed tomography (HRCT) produces three-dimensional images of xylem tissue in situ, allowing for a noninvasive assessment of embolism resistance. This technique has provided robust results in various plant species with contrasting xylem anatomy (Charra-Vaskou et al., 2012, 2016; Dalla-Salda et al., 2014; Torres-Ruiz et al., 2014; Cochard et al., 2015; Knipfer et al., 2015; Bouche et al., 2016). Synchrotron-based tomography facilities allow the visualization of intact plants, offering a noninvasive, in vivo estimation of the loss of hydraulic conductivity within the xylem (Choat et al., 2016). Moreover, the quality of the x-ray beam in the synchrotron facilities provides high resolution and signal-to-noise ratio, making image analysis simple and accurate.If grapevine were as vulnerable to xylem embolism as suggested in some studies, refilling of embolized vessels would be expected to occur on a frequent (daily) basis in order to maintain hydraulic continuity (Sperry et al., 1994; Cochard et al., 2001; Hacke and Sperry, 2003; Charrier et al., 2013). Various refilling mechanisms have been proposed to date, including positive root/stem pressure and refilling while the xylem is under negative pressure via water droplet growth (Salleo et al., 1996; Brodersen et al., 2010; Knipfer et al., 2016). Positive pressure in the xylem sap can be related to mineral nutrition and soil temperature in autumn or spring (Ewers et al., 2001) and to soluble carbohydrate transport into the vessel lumen during winter (Améglio et al., 2001; Charrier et al., 2013). Refilling under negative pressure is based on the hypothesis that embolized vessels are isolated from surrounding functional vessels, permitting positive pressures to develop and the embolism to dissolve (Salleo et al., 1996; Tyree et al., 1999). This process has been related to the chemistry of conduit walls (Holbrook and Zwieniecki, 1999), the geometry of interconduit bordered pits (Zwieniecki and Holbrook, 2000), and phloem unloading (Nardini et al., 2011). While refilling via positive pressure has been described frequently (Sperry et al., 1987, 1994; Hacke and Sauter 1996; Cochard et al., 2001; Améglio et al., 2004; Cobb et al., 2007), refilling under negative pressure remains controversial (Cochard et al., 2013, 2015). In grapevine particularly, imaging techniques have provided evidence of refilling in embolized vessels (Brodersen et al., 2010), but uncertainties remain regarding the xylem water potential measurement at the position of the scan.The goal of this study was to provide a noninvasive assessment of (1) the vulnerability to drought-induced embolism in two widespread grapevine species in perennial (V. vinifera and Vitis riparia) and annual (V. vinifera) organs and (2) the ability to refill embolized vessels under positive or negative pressure (V. vinifera). This approach would indicate whether embolism formation and repair are likely to occur on a daily basis and/or if hydraulic segmentation could protect perennial organs from drought stress. Stems and petioles from intact V. vinifera ‘Cabernet Sauvignon’ and V. riparia plants were scanned using Synchrotron-based HRCT, characterizing their vulnerability to embolism and quantifying their ability to refill at different positions along the plant axis (base and apex) in relation to bulk xylem pressure. These data were integrated with other noninvasive techniques assessing leaf hydraulics and transpiration.     

In Vivo Visualizations of Drought-Induced Embolism Spread in Vitis vinifera

Long-distance water transport through plant xylem is vulnerable to hydraulic dysfunction during periods of increased tension on the xylem sap, often coinciding with drought. While the effects of local and systemic embolism on plant water transport and physiology are well documented, the spatial patterns of embolism formation and spread are not well understood. Using a recently developed nondestructive diagnostic imaging tool, high-resolution x-ray computed tomography, we documented the dynamics of drought-induced embolism in grapevine (Vitis vinifera) plants in vivo, producing the first three-dimensional, high-resolution, time-lapse observations of embolism spread. Embolisms formed first in the vessels surrounding the pith at stem water potentials of approximately –1.2 megapascals in drought experiments. As stem water potential decreased, embolisms spread radially toward the epidermis within sectored vessel groupings via intervessel connections and conductive xylem relays, and infrequently (16 of 629 total connections) through lateral connections into adjacent vessel sectors. Theoretical loss of conductivity calculated from the high-resolution x-ray computed tomography images showed good agreement with previously published nuclear magnetic resonance imaging and hydraulic conductivity experiments also using grapevine. Overall, these data support a growing body of evidence that xylem organization is critically important to the isolation of drought-induced embolism spread and confirm that air seeding through the pit membranes is the principle mechanism of embolism spread.Water is transported through the xylem under tension and in a metastable state, making it inherently vulnerable to cavitation, the rapid phase change of liquid water to vapor (Dixon and Joly, 1895; Hayward, 1971; Tyree and Sperry, 1989). The resulting gas embolisms block water transport in the affected xylem vessel. It is widely accepted that embolisms spread between adjacent conduits when the pressure differential between gas-filled and water-filled conduits reaches a critical point where water vapor is aspirated through the pit membrane from the neighboring conduit (Tyree and Sperry, 1989; Tyree and Zimmermann, 2002). The resulting spread of embolisms through the xylem effectively reduces the hydraulic conductivity of the network, impairing the capacity to replace transpired water. The consequences of embolism formation can be dramatic, and it is now considered to be one of the major physiological factors driving reductions in forest primary productivity and drought-induced mortality in woody plants (Anderegg et al., 2012; Choat et al., 2012).Embolism spread between conduits is necessarily dependent on the number and orientation of the interconduit connections, but little is known about the organization of those connections or the spatial dynamics of embolism spread in vivo (Tyree and Zimmermann, 2002; Brodersen et al., 2010). This knowledge gap is largely due to the lack of a nondestructive visualization tool with sufficient resolution to study the propagation and spread of embolism. Previous efforts to visualize embolism in vivo utilized either cryo-scanning electron microscopy (cryo-SEM) or NMR imaging. Cryo-SEM yields fine resolution of frozen plant tissue, revealing the functional status of xylem conduits (i.e. water- or air-filled) at the time of freezing (Canny, 1997; Melcher et al., 2003; Cobb et al., 2007; Mayr et al., 2007; Johnson et al., 2012). Both transverse (Hukin et al., 2005; Sun et al., 2007; Johnson et al., 2012) and longitudinal (Utsumi et al., 1999) cryo-SEM sections have been prepared, but only provide a snapshot of a single point in time and in a single, two-dimensional plane. Similarly, NMR imaging was used in several studies as a nondestructive visualization tool to study the functional status of the xylem in vivo (Holbrook et al., 2001; Clearwater and Clark, 2003). However, the resulting images are typically of insufficient resolution to determine anything other than whether xylem conduits were filled with water or air. Three-dimensional (3D) imaging with NMR is challenging and is not frequently employed (Kuroda et al., 2006). Despite the availability of NMR, studies using this technology are largely focused to the spread of embolism over long periods of time (e.g. weeks [Umebayashi et al., 2011] or months [Pérez-Donoso et al., 2007]) rather than the short-term dynamics of embolism spread over the course of a few hours.Recently, high-resolution x-ray computed tomography (HRCT), a nondestructive diagnostic imaging tool, has been successfully used to study plant tissue in vivo (Brodersen et al., 2010, 2011). Synchrotron-based HRCT is based on the same principles as medical computed tomography systems but yields data with a spatial resolution of less than 5 µm and a temporal resolution of less than 30 min. Brodersen et al. (2011) expanded on this technology to study and map the 3D organization of grapevine (Vitis vinifera) stems and found that the functional status of the xylem could be determined in vivo. Brodersen et al. (2010) visualized the dynamics of embolism repair (i.e. the metabolically active refilling of embolized xylem conduits) in live plants using HRCT, including the growth of water droplets emerging from xylem parenchyma surrounding embolized vessels that eventually led to the dissolution of trapped gas inside the vessels. While we now have a better understanding of embolism repair and the physiological consequences of embolism spread are well documented (Tyree and Zimmermann, 2002; McDowell et al., 2008; Cochard et al., 2009; Zwieniecki and Holbrook, 2009; Choat et al., 2012), the spatial dynamics and biophysics of embolism formation and spread in vivo have yet to be fully explored. Clearly, the spatial organization of xylem conduits plays a critical role in embolism repair and is likely even more influential in embolism spread, as direct connections between conduits are the most likely pathway through the network. Building on these findings and new techniques, we aimed to take advantage of HRCT imaging to provide the first high-resolution visualization of the spread of drought-induced embolism.     

In Planta Localization of Stilbenes within Picea abies Phloem

Phenolic stilbene glucosides (astringin, isorhapontin, and piceid) and their aglycons commonly accumulate in the phloem of Norway spruce (Picea abies). However, current knowledge about the localization and accumulation of stilbenes within plant tissues and cells remains limited. Here, we used an innovative combination of novel microanalytical techniques to evaluate stilbenes in a frozen-hydrated condition (i.e. in planta) and a freeze-dried condition across phloem tissues. Semiquantitative time-of-flight secondary ion-mass spectrometry imaging in planta revealed that stilbenes were localized in axial parenchyma cells. Quantitative gas chromatography analysis showed the highest stilbene content in the middle of collapsed phloem with decreases toward the outer phloem. The same trend was detected for soluble sugar and water contents. The specimen water content may affect stilbene composition; the glucoside-to-aglycon ratio decreased slightly with decreases in water content. Phloem chemistry was correlated with three-dimensional structures of phloem as analyzed by microtomography. The outer phloem was characterized by a high volume of empty parenchyma, reduced ray volume, and a large number of axial parenchyma with porous vacuolar contents. Increasing porosity from the inner to the outer phloem was related to decreasing compactness of stilbenes and possible secondary oxidation or polymerization. Our results indicate that aging-dependent changes in phloem may reduce cell functioning, which affects the capacity of the phloem to store water and sugar, and may reduce the defense potential of stilbenes in the axial parenchyma. Our results highlight the power of using a combination of techniques to evaluate tissue- and cell-level mechanisms involved in plant secondary metabolite formation and metabolism.The bark of conifers has anatomically and chemically integrated defense strategies that are either constitutive (i.e. continuously produced) or inducible (i.e. activated as a response to insect or pathogen attack; Krokene, 2015). Many defense traits exist in both forms (Franceschi et al., 2005). For example, axial phloem parenchyma cells (or polyphenolic parenchyma) are critical in conifer bark defense. These cells regularly form in Pinaceae during annual phloem formation (Franceschi et al., 1998, 2000; Krekling et al., 2000; Jyske et al., 2015) but also are produced on invasion (Franceschi et al., 2005; Krokene, 2015). In Norway spruce (Picea abies) phloem, axial parenchyma forms distinctive, continuous tangential sheets across conducting (i.e. noncollapsed) and nonconducting (i.e. collapsed) tissue.Pioneering studies using microscopy with different dye agents and autofluorescence showed that the large vacuole is a special feature of the axial phloem parenchyma that contains phenolic substances (i.e. phenolic bodies; Franceschi et al., 1998). Microscopic imaging techniques also showed that polyphenolic content is highly dynamic (Franceschi et al., 1998, 2000, 2005) and changes seasonally (Krekling et al., 2000). Within the last 5 years, progress in laser microdissection (LMD) has facilitated the sampling of individual tissues and cells, providing information about the exact chemical composition of phenolic content. Li et al. (2012) used LMD to show that the axial parenchyma is the main site of phenolic accumulation in spruce bark, including that of stilbene compounds.Stilbenes are secondary metabolites that are composed of two phenol moieties linked by a C₂ bridge. These compounds are derived from the phenylpropanoid pathway, in which the last steps of biosynthesis are catalyzed by stilbene synthase (Chong et al., 2009). There is increasing interest in these antioxidant, antibacterial, and antiinflammatory compounds for use in healthy human diets, therapeutic approaches, and as protective agents in materials sciences (Shibutani et al., 2004; Metsämuuronen and Siren, 2014; Reinisalo et al., 2015; Hedenström et al., 2016; Sirerol et al., 2016). The tetrahydroxystilbene glucosides trans-astringin (3,3ʹ,4ʹ,5-tetrahydroxystilbene 3-O-β-d-glucoside) and trans-isorhapontin (3,4ʹ,5-trihydroxy-3ʹ-methoxystilbene 3-O-β-d-glucoside) are the most abundant constitutive stilbene compounds of Norway spruce, while the trihydroxystilbene glucoside trans-piceid (resveratrol 3-O-β-glucoside) and stilbene aglycons (i.e. without the sugar moiety) are less abundant. Stilbene synthesis in spruce probably proceeds through the formation of resveratrol (i.e. aglycon of piceid) followed by further modifications (i.e. hydroxylation, O-methylation, and O-glycosylation) to yield tetrahydroxystilbene glucosides (Hammerbacher et al., 2011). Stilbenes are assumed to provide protection against a wide variety of environmental stressors (Franceschi et al., 2005; Witzell and Martin, 2008; Chong et al., 2009). Stilbenes appear to contribute to antifungal defense in spruce (Hammerbacher et al., 2011, 2013). The fungal inoculation of spruce bark with the blue-stain fungus Endoconidiophora polonica (previously named Ceratocystis polonica; de Beer et al., 2014) causes astringin levels to decrease, in parallel with increasing dimeric stilbene glucoside levels in the LMD-isolated axial phloem parenchyma (Li et al., 2012) or increasing levels of corresponding aglycons in bulk tissue (Viiri et al., 2001). During the annual formation of phloem in Norway spruce, the accumulation of stilbene glucosides inside the newest, LMD-isolated phloem ring is preceded by the formation and cellular development of a new band of axial parenchyma (Jyske et al., 2015). These observations strongly indicate that the inducible and constitutive stilbene compounds of spruce phloem are both stored and synthesized in the axial parenchyma.New mass spectrometry imaging techniques provide significant improvements in the mapping of plant metabolites (Briggs and Seah, 1993; Vickerman and Briggs, 2001; Burrell et al., 2007; Cha et al., 2008; Lee et al., 2012; Bjarnholt et al., 2014; Aoki et al., 2016). To elucidate the synthesis, distribution, and metabolism of secondary plant metabolites, it is essential to gather positional information about them in a living state, as pretreatment of specimens, such as drying, may change the distribution and concentration features of soluble chemicals (Metzner et al., 2008; Li et al., 2012; Kuroda et al., 2013). In this study, we used a unique system of time-of-flight secondary ion mass spectrometry and scanning electron microscopy connected with a cryo-shuttle (cryo-TOF-SIMS/SEM) to study the localization and accumulation patterns of stilbenes within cells and tissues of phloem. This system has been developed to study chemical distributions at high-spatial resolution (1 µm) directly from the surfaces of plant specimens in a frozen-hydrated state (i.e. in planta) representing living tissues (Kuroda et al., 2013; Aoki et al., 2016). Time-of-flight secondary ion mass spectrometry (TOF-SIMS) directly detects organic and inorganic compounds on the specimen surface over a broad mass-to-charge ratio (m/z) range by mass spectrometry with high chemical sensitivity. Specimen surface morphology is visualized by the detection of total secondary ion content. The quality of cellular integrity may be further observed by scanning electron microscopy connected with a cryo-shuttle (cryo-SEM) imaging of the frozen surface of the same specimen. The cryo-TOF-SIMS/SEM system has still rarely been applied to the analysis of plant physiology (Metzner et al., 2008, 2010; Iijima et al., 2011; Kuroda et al., 2013; Aoki et al., 2016).Mass spectrometer imaging techniques consist of an ionizer and a mass analyzer. In the TOF-SIMS system, secondary ion mass spectrometry is used as an ionizer and time-of-flight as a mass analyzer. In another mainstream imaging mass spectrometry technique, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), matrix-assisted laser desorption/ionization is used as ionizer. Compared with TOF-SIMS, MALDI-MS is more quantitative and has high-M_r acceptance, but the resolution of MALDI-MS is not high enough for cell-level detection (Aoki et al., 2016). Instead, the spatial resolution of TOF-SIMS is superior to focus on cell functions. The disadvantage of TOF-SIMS is that the ionization and fragmentation phenomenon may be affected by the matrix effect, causing some degree of uncertainty. However, when time-of-flight secondary ion mass spectrometry connected with a cryo-shuttle (cryo-TOF-SIMS) is used in combination with quantitative gas chromatography, it is very powerful to study the positional and temporal distributions of metabolites within living plants.To complement TOF-SIMS analysis, we applied quantitative chemical microanalysis methods to study the amounts of stilbene glucosides and to correlate those with the amounts of total extractives, monosaccharides and disaccharides, and water across phloem and bark. The methods include tangential cryo-sectioning of tissues and their chemical microanalysis by gas chromatography with flame-ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS).To combine the chemical information with phloem morphology, the cellular and subcellular features of the axial phloem parenchyma were analyzed by three-dimensional (3D) synchrotron radiation microtomography (µCT). µCT is a prominent tool that has gained popularity for 3D analysis of xylem structure and physiology (Brodersen, 2013; Cochard et al., 2015), but only recently has it been applied to the 3D analysis of phloem (Jyske et al., 2015). This method offers advantages over traditional light microscopic approaches, as high-throughput data at the submicrometer level can be produced from significantly larger tissue volumes. The data allow for representative volumetric analysis of cellular distributions along with 3D visualization of subcellular features.In this study, we used a novel combination of cutting-edge techniques to analyze in parallel (1) in planta cellular localization and accumulation of stilbene glucosides across phloem and bark by semiquantitative cryo-TOF-SIMS/SEM; (2) tissue-level quantitative amounts of stilbene glucosides, total extractives, and monosaccharides and disaccharides across phloem and bark by tangential cryo-sectioning and GC-FID and GC-MS; (3) 3D cell abundance distributions across phloem and bark by µCT; and (4) variation in water content across phloem and bark (Fig. 1).Open in a separate windowFigure 1.Schematic presentation of the specimen structure and preparation for different analyses. Sample blocks were taken from living tree stem (A) or stem discs (B) at 1.3 m on the stem. The blocks (C) containing outer bark (periderm), phloem, cambium, and part of the outermost xylem ring (D; transverse view of phloem and bark) were further divided into subblocks (1–3; C and E). Subblocks 1 and 2 were quick frozen, and subblock 3 was fixed chemically. Subblock 1 was used for the direct chemical mapping of stilbenes across the phloem from the cambium to the outer bark (i.e. semiquantitative analysis of stilbene localization and accumulation across transverse and radial surfaces [purple] of the tissue block by TOF-SIMS; E-1). To obtain quantitative data on the amounts of stilbenes, other extractives, and carbohydrates across phloem and bark, tangential cryo-sections (250 or 450 µm each; cut slices illustrated with purple in E-2) were cut across subblock 2 and directed for chemical microanalysis by GC-FID (E-2). Subblock 3 was divided into four to six zones, and from each zone, small cuboids (illustrated with purple in E-3) were cut and directed for morphological analysis of phloem by phase-contrast µCT (E-3). Water content across the phloem and bark was analyzed from separate fresh blocks, which were further cut tangentially into thin sections. Black arrows indicate the radial direction from the cambium toward the outer bark. Purple areas show the analyzed locations of each subblock (E). Note that schematic drawings are not to scale.