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.     

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.     

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.     

The Dynamics of Embolism Repair in Xylem: In Vivo Visualizations Using High-Resolution Computed Tomography

Water moves through plants under tension and in a thermodynamically metastable state, leaving the nonliving vessels that transport this water vulnerable to blockage by gas embolisms. Failure to reestablish flow in embolized vessels can lead to systemic loss of hydraulic conductivity and ultimately death. Most plants have developed a mechanism to restore vessel functionality by refilling embolized vessels, but the details of this process in vessel networks under tension have remained unclear for decades. Here we present, to our knowledge, the first in vivo visualization and quantification of the refilling process for any species using high-resolution x-ray computed tomography. Successful vessel refilling in grapevine (Vitis vinifera) was dependent on water influx from surrounding living tissue at a rate of 6 × 10⁻⁴ μm s⁻¹, with individual droplets expanding over time, filling vessels, and forcing the dissolution of entrapped gas. Both filling and draining processes could be observed in the same vessel, indicating that successful refilling requires hydraulic isolation from tensions that would otherwise prevent embolism repair. Our study demonstrates that despite the presence of tensions in the bulk xylem, plants are able to restore hydraulic conductivity in the xylem.Vascular plants have evolved a simple but elegant system for long-distance transport of water and minerals through a network of nonliving, pipe-like cells. Whereas long-distance transport in animals is actively driven by positive pressure, most water transport in plants is passively driven by tension as explained by the Cohesion-Tension (C-T) theory (Dixon and Joly, 1894; Tyree, 2003). Water under tension is metastable however (Hayward, 1971), making the transport system inherently vulnerable to cavitation and blockage by gas embolisms (Tyree and Sperry, 1989). Direct measurements of negative pressures (tensions) in xylem (Wei et al., 1999) have confirmed the fundamental basis for the C-T theory of water transport in plants (e.g. Tyree, 2003), but many details regarding the susceptibility of the xylem network to cavitation and blockage by embolisms, and a thermodynamically plausible mechanism for the repair of these embolisms, remain unclear (Clearwater and Goldstein, 2005).Plants have apparently evolved mechanisms, including root pressure, to remove embolisms and restore water transport in vessels (Sperry et al., 1987; Tibbetts and Ewers, 2000; Isnard and Silk, 2009). Refilling of embolized vessels far from roots (Holbrook et al., 2001) and under a state of tension (Salleo and Gullo, 1986) is not well understood, but most hypotheses involve localized solute export into embolized vessels from adjacent living xylem parenchyma, osmotic movement of water into these vessels, and isolation of the refilling vessel from the tension in its local water environment (Tyree et al., 1999; Hacke and Sperry, 2003; Clearwater and Goldstein, 2005; Salleo et al., 2006). Embolism repair is complicated by the fact that xylem conduits (tracheids and vessels) form an interconnected network. While such a network will provide a low-resistance pathway for the bulk flow of water when the conduits are filled, if a cavitation event and subsequent embolism (gas bubble) either spontaneously occurs within a conduit, or spreads to it from another conduit, the presence of tension in this network should also quickly drain a conduit of its water and prevent its refilling. The spread of embolisms is limited by the small effective pore size of the connections between conduits (known as pit membranes), but under conditions of low plant water availability, embolisms do occur and spread (Tyree and Zimmermann, 2002; Choat et al., 2008), and evidence for the repair of embolized vessels, despite the presumed presence of a tension throughout the plant xylem, has been obtained in many species (Salleo et al., 1996; McCully et al., 1998; Zwieniecki and Holbrook, 1998; Kaufmann et al., 2009).A major limitation to the testing of these hypotheses and to our understanding of embolism repair has been the lack of in vivo observations at a sufficient resolution and an appropriate temporal scale to document how the refilling occurs. Here we present a new method for imaging the functional status of vessels using high-resolution x-ray computed tomography (HRCT), providing, to our knowledge, the first in vivo visualization of the refilling process for any species. Previous in vivo measurements of vessel refilling have been performed using NMR imaging, but the resolution was insufficient to determine the source of the refilling water (Holbrook et al., 2001; Scheenen et al., 2007). In vivo imaging at this scale allows for nondestructive visualization and measurement of the change in both air and water volume within the vessel lumen, giving unprecedented access to the mechanisms of embolism repair.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

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).     

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.     

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.     

Characterization of the Possible Roles for B Class MADS Box Genes in Regulation of Perianth Formation in Orchid

To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, three paleoAPETALA3 genes, O. Gower Ramsey MADS box gene5 (OMADS5; clade 1), OMADS3 (clade 2), and OMADS9 (clade 3), and one PISTILLATA gene, OMADS8, were characterized. The OMADS8 and OMADS3 mRNAs were expressed in all four floral organs as well as in vegetative leaves. The OMADS9 mRNA was only strongly detected in petals and lips. The mRNA for OMADS5 was only strongly detected in sepals and petals and was significantly down-regulated in lip-like petals and lip-like sepals of peloric mutant flowers. This result revealed a possible negative role for OMADS5 in regulating lip formation. Yeast two-hybrid analysis indicated that OMADS5 formed homodimers and heterodimers with OMADS3 and OMADS9. OMADS8 only formed heterodimers with OMADS3, whereas OMADS3 and OMADS9 formed homodimers and heterodimers with each other. We proposed that sepal/petal/lip formation needs the presence of OMADS3/8 and/or OMADS9. The determination of the final organ identity for the sepal/petal/lip likely depended on the presence or absence of OMADS5. The presence of OMADS5 caused short sepal/petal formation. When OMADS5 was absent, cells could proliferate, resulting in the possible formation of large lips and the conversion of the sepal/petal into lips in peloric mutants. Further analysis indicated that only ectopic expression of OMADS8 but not OMADS5/9 caused the conversion of the sepal into an expanded petal-like structure in transgenic Arabidopsis (Arabidopsis thaliana) plants.The ABCDE model predicts the formation of any flower organ by the interaction of five classes of homeotic genes in plants (Yanofsky et al., 1990; Jack et al., 1992; Mandel et al., 1992; Goto and Meyerowitz, 1994; Jofuku et al., 1994; Pelaz et al., 2000, 2001; Theißen and Saedler, 2001; Pinyopich et al., 2003; Ditta et al., 2004; Jack, 2004). The A class genes control sepal formation. The A, B, and E class genes work together to regulate petal formation. The B, C, and E class genes control stamen formation. The C and E class genes work to regulate carpel formation, whereas the D class gene is involved in ovule development. MADS box genes seem to have a central role in flower development, because most ABCDE genes encode MADS box proteins (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994; Purugganan et al., 1995; Rounsley et al., 1995; Theißen and Saedler, 1995; Theißen et al., 2000; Theißen, 2001).The function of B group genes, such as APETALA3 (AP3) and PISTILLATA (PI), has been thought to have a major role in specifying petal and stamen development (Jack et al., 1992; Goto and Meyerowitz, 1994; Krizek and Meyerowitz, 1996; Kramer et al., 1998; Hernandez-Hernandez et al., 2007; Kanno et al., 2007; Whipple et al., 2007; Irish, 2009). In Arabidopsis (Arabidopsis thaliana), mutation in AP3 or PI caused identical phenotypes of second whorl petal conversion into a sepal structure and third flower whorl stamen into a carpel structure (Bowman et al., 1989; Jack et al., 1992; Goto and Meyerowitz, 1994). Similar homeotic conversions for petal and stamen were observed in the mutants of the AP3 and PI orthologs from a number of core eudicots such as Antirrhinum majus, Petunia hybrida, Gerbera hybrida, Solanum lycopersicum, and Nicotiana benthamiana (Sommer et al., 1990; Tröbner et al., 1992; Angenent et al., 1993; van der Krol et al., 1993; Yu et al., 1999; Liu et al., 2004; Vandenbussche et al., 2004; de Martino et al., 2006), from basal eudicot species such as Papaver somniferum and Aquilegia vulgaris (Drea et al., 2007; Kramer et al., 2007), as well as from monocot species such as Zea mays and Oryza sativa (Ambrose et al., 2000; Nagasawa et al., 2003; Prasad and Vijayraghavan, 2003; Yadav et al., 2007; Yao et al., 2008). This indicated that the function of the B class genes AP3 and PI is highly conserved during evolution.It has been thought that B group genes may have arisen from an ancestral gene through multiple gene duplication events (Doyle, 1994; Theißen et al., 1996, 2000; Purugganan, 1997; Kramer et al., 1998; Kramer and Irish, 1999; Lamb and Irish, 2003; Kim et al., 2004; Stellari et al., 2004; Zahn et al., 2005; Hernandez-Hernandez et al., 2007). In the gymnosperms, there was a single putative B class lineage that duplicated to generate the paleoAP3 and PI lineages in angiosperms (Kramer et al., 1998; Theißen et al., 2000; Irish, 2009). The paleoAP3 lineage is composed of AP3 orthologs identified in lower eudicots, magnolid dicots, and monocots (Kramer et al., 1998). Genes in this lineage contain the conserved paleoAP3- and PI-derived motifs in the C-terminal end of the proteins, which have been thought to be characteristics of the B class ancestral gene (Kramer et al., 1998; Tzeng and Yang, 2001; Hsu and Yang, 2002). The PI lineage is composed of PI orthologs that contain a highly conserved PI motif identified in most plant species (Kramer et al., 1998). Subsequently, there was a second duplication at the base of the core eudicots that produced the euAP3 and TM6 lineages, which have been subject to substantial sequence changes in eudicots during evolution (Kramer et al., 1998; Kramer and Irish, 1999). The paleoAP3 motif in the C-terminal end of the proteins was retained in the TM6 lineage and replaced by a conserved euAP3 motif in the euAP3 lineage of most eudicot species (Kramer et al., 1998). In addition, many lineage-specific duplications for paleoAP3 lineage have occurred in plants such as orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009), Ranunculaceae, and Ranunculales (Kramer et al., 2003; Di Stilio et al., 2005; Shan et al., 2006; Kramer, 2009).Unlike the A or C class MADS box proteins, which form homodimers that regulate flower development, the ability of B class proteins to form homodimers has only been reported in gymnosperms and in the paleoAP3 and PI lineages of some monocots. For example, LMADS1 of the lily Lilium longiflorum (Tzeng and Yang, 2001), OMADS3 of the orchid Oncidium Gower Ramsey (Hsu and Yang, 2002), and PeMADS4 of the orchid Phalaenopsis equestris (Tsai et al., 2004) in the paleoAP3 lineage, LRGLOA and LRGLOB of the lily Lilium regale (Winter et al., 2002), TGGLO of the tulip Tulipa gesneriana (Kanno et al., 2003), and PeMADS6 of the orchid P. equestris (Tsai et al., 2005) in the PI lineage, and GGM2 of the gymnosperm Gnetum gnemon (Winter et al., 1999) were able to form homodimers that regulate flower development. Proteins in the euAP3 lineage and in most paleoAP3 lineages were not able to form homodimers and had to interact with PI to form heterodimers in order to regulate petal and stamen development in various plant species (Schwarz-Sommer et al., 1992; Tröbner et al., 1992; Riechmann et al., 1996; Moon et al., 1999; Winter et al., 2002; Kanno et al., 2003; Vandenbussche et al., 2004; Yao et al., 2008). In addition to forming dimers, AP3 and PI were able to interact with other MADS box proteins, such as SEPALLATA1 (SEP1), SEP2, and SEP3, to regulate petal and stamen development (Pelaz et al., 2000; Honma and Goto, 2001; Theißen and Saedler, 2001; Castillejo et al., 2005).Orchids are among the most important plants in the flower market around the world, and research on MADS box genes has been reported for several species of orchids during the past few years (Lu et al., 1993, 2007; Yu and Goh, 2000; Hsu and Yang, 2002; Yu et al., 2002; Hsu et al., 2003; Tsai et al., 2004, 2008; Xu et al., 2006; Guo et al., 2007; Kim et al., 2007; Chang et al., 2009). Unlike the flowers in eudicots, the nearly identical shape of the sepals and petals as well as the production of a unique lip in orchid flowers make them a very special plant species for the study of flower development. Four clades (1–4) of genes in the paleoAP3 lineage have been identified in several orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009). Several works have described the possible interactions among these four clades of paleoAP3 genes and one PI gene that are involved in regulating the differentiation and formation of the sepal/petal/lip of orchids (Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009). However, the exact mechanism that involves the orchid B class genes remains unclear and needs to be clarified by more experimental investigations.O. Gower Ramsey is a popular orchid with important economic value in cut flower markets. Only a few studies have been reported on the role of MADS box genes in regulating flower formation in this plant species (Hsu and Yang, 2002; Hsu et al., 2003; Chang et al., 2009). An AP3-like MADS gene that regulates both floral formation and initiation in transgenic Arabidopsis has been reported (Hsu and Yang, 2002). In addition, four AP1/AGAMOUS-LIKE9 (AGL9)-like MADS box genes have been characterized that show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis (Hsu et al., 2003; Chang et al., 2009). Compared with other orchids, the production of a large and well-expanded lip and five small identical sepals/petals makes O. Gower Ramsey a special case for the study of the diverse functions of B class MADS box genes during evolution. Therefore, the isolation of more B class MADS box genes and further study of their roles in the regulation of perianth (sepal/petal/lip) formation during O. Gower Ramsey flower development are necessary. In addition to the clade 2 paleoAP3 gene OMADS3, which was previously characterized in our laboratory (Hsu and Yang, 2002), three more B class MADS box genes, OMADS5, OMADS8, and OMADS9, were characterized from O. Gower Ramsey in this study. Based on the different expression patterns and the protein interactions among these four orchid B class genes, we propose that the presence of OMADS3/8 and/or OMADS9 is required for sepal/petal/lip formation. Further sepal and petal formation at least requires the additional presence of OMADS5, whereas large lip formation was seen when OMADS5 expression was absent. Our results provide a new finding and information pertaining to the roles for orchid B class MADS box genes in the regulation of sepal/petal/lip formation.     

Focus on Water: Down-Regulation of Plasma Intrinsic Protein1 Aquaporin in Poplar Trees Is Detrimental to Recovery from Embolism

During their lifecycles, trees encounter multiple events of water stress that often result in embolism formation and temporal decreases in xylem transport capacity. The restoration of xylem transport capacity requires changes in cell metabolic activity and gene expression. Specifically, in poplar (Populus spp.), the formation of xylem embolisms leads to a clear up-regulation of plasma membrane protein1 (PIP1) aquaporin genes. To determine their role in poplar response to water stress, transgenic Populus tremula × Populus alba plants characterized by the strong down-regulation of multiple isoforms belonging to the PIP1 subfamily were used. Transgenic lines showed that they are more vulnerable to embolism, with 50% percent loss of conductance occurring 0.3 MPa earlier than in wild-type plants, and that they also have a reduced capacity to restore xylem conductance during recovery. Transgenic plants also show symptoms of a reduced capacity to control percent loss of conductance through stomatal conductance in response to drought, because they have a much narrower vulnerability safety margin. Finally, a delay in stomatal conductance recovery during the period of stress relief was observed. The presented results suggest that PIP1 genes are involved in the maintenance of xylem transport system capacity, in the promotion of recovery from stress, and in contribution to a plant’s control of stomatal conductance under water stress.Long-distance water transport in vascular plants occurs in a conduit network of nonliving cells connecting roots to leaves (Sperry, 2003). Often under drought conditions, the water column within the lumen of xylem vessels or tracheids can be subjected to tensions that result in cavitation and the subsequent formation of embolisms (Holbrook and Zwieniecki, 2008). This hydraulic failure within the xylem network can cause tissue damage, loss of plant productivity, and ultimately, plant death (Tyree and Sperry, 1989; Sperry et al., 1998; Zwieniecki and Holbrook, 2009). Plants have evolved several strategies to prevent and/or mitigate the effects of hydraulic failure caused by embolism and restore xylem transport capacity after embolism occurs (Stiller and Sperry, 2002; Nardini et al., 2011; Secchi and Zwieniecki, 2012). These strategies include passive, often long-term responses, like the growth of new vessels/tracheids or dieback followed by the growth of new shoots (shrubs), or active, often fast responses that result in the restoration of hydraulic conductivity by (1) creating positive pressure through root or stem pressure in the complete transport system (xylem level; Cochard et al., 1994; Ewers et al., 1997; Yang et al., 2012) or (2) enabling positive pressures in specific, embolized conduits, despite negative pressure in the surrounding xylem (conduit level; Salleo et al., 2004; Nardini et al., 2011; Brodersen and McElrone, 2013).Although embolism formation is a purely physical process related to the degree of tension in the water column and a wood’s physicochemical properties (Brennen, 1995; Tyree and Zimmermann, 2002), embolism removal requires that empty vessels fill with water against existing energy gradients as the bulk of water in the xylem remains under tension caused by transpiration. Thus, recovery from embolism cannot happen spontaneously and necessitates some physiological activities that promote water flow into embolized vessels (Holbrook and Zwieniecki, 1999; Thomas Tyree et al., 1999; Salleo et al., 2004; Zwieniecki and Holbrook, 2009; Secchi et al., 2011). Visual evidence from cryo-scanning electron microscopy studies, magnetic resonance imaging observations, and computed tomography scans showed that water (xylem sap) can return to empty vessels, suggesting that plants do have the ability to restore functionality in the xylem (Holbrook et al., 2001; Clearwater and Goldstein, 2005; Scheenen et al., 2007). Brodersen et al. (2010) showed that water droplets preferentially form on the vessel walls adjacent to parenchyma cells and that these droplets grow until the lumen completely refills. In addition, scientific support for the existence of embolism/refilling cycles in intact stems of Acer rubrum are provided using magnetic resonance imaging (Zwieniecki et al., 2013). Droplet formation on the walls of empty vessels that are in contact with parenchyma cells support predictions that these living cells supply both water and energy to drive the restoration of xylem hydraulic function.Processes related to water transport across the cellular membrane involve plasma intrinsic protein (PIP; aquaporins) moderators, and thus, the role of PIPs must be considered when contemplating how plants recover from embolism formation. Plant aquaporins show a great diversity and are classified into five major homologous groups that reflect specific subcellular localizations (Prado and Maurel, 2013). Among different aquaporin gene families (26-like intrinsic proteins, tonoplast intrinsic proteins, X unrecognized intrinsic proteins, small basic intrinsic proteins, and PIPs; Danielson and Johanson, 2008), the PIPs represent the largest number of members and can be further divided into two subfamilies, PIP1 and PIP2. There is a large body of evidence that aquaporins from the PIP2 subfamily contribute to water transport. The generation of data has been multidisciplinary and involved the use of chemical blockers, the down-regulation and up-regulation of genes in plants, and the expression of these proteins in oocytes (Hukin et al., 2002; Postaire et al., 2010; Shatil-Cohen et al., 2011). Expression levels of several PIP and TIP members change after the dynamic of increasing water stress and recovery in many woody plants, including walnut (Juglans regia), poplar (Populus trichocarpa.), and grapevine Vitis vinifera; (Sakr et al., 2003; Secchi et al., 2011; Perrone et al., 2012a, 2012b; Laur and Hacke, 2013; Pou et al., 2013). Furthermore, an increase in the expression of PIP2.1 and PIP2.2 genes was observed in vessel-associated parenchyma cells in walnuts at the same time that recovery from embolism was taking place (Sakr et al., 2003). The role of genes from the PIP1 subfamily in tree responses to water stress is less well-understood. PIP1s were shown to have little to no water channel activity when expressed in oocytes on their own. However, coexpression of PIP1.1 proteins with an isoform from the PIP2 subfamily led to higher membrane permeability than that observed with the expression of a single PIP2 protein (Fetter et al., 2004; Secchi and Zwieniecki, 2010). With respect to their role in mediating water stress, it was shown that the expression level of several PIP1 genes in poplar changed significantly during the onset of stress, during recovery, during the formation of embolisms after water stress, and under no stress conditions but with induced embolism, whereas the expression of PIP2 genes remained mostly unresponsive (Secchi and Zwieniecki, 2010; Secchi et al., 2011; Secchi and Zwieniecki, 2011).Despite significant effort invested in elucidating the contribution of aquaporins to the regulation of xylem hydraulic capacity throughout the progression of drought and recovery from water stress, evidence of their active role in vivo is only partially confirmed. Genetic approaches provide a reliable and effective strategy for determining the physiological function of aquaporin genes in plant water relations. However, most studies thus far have been conducted on herbaceous plants (Kaldenhoff et al., 1998; Postaire et al., 2010). For example, Arabidopsis (Arabidopsis thaliana) plants expressing PIP antisense genes exhibit an impaired ability to recover from water stress (Martre et al., 2002), and knockout mutants exhibit reduced leaf hydraulic conductivity (Da Ines et al., 2010). The Nicotiana tabacum aquaporin1 (NtAQP1) down-regulated tobacco plants show reduced root hydraulic conductivity and lower water stress resistance (Siefritz et al., 2002). RNA technology, although not often used for woody plants, has been adapted for grapevine (Perrone et al., 2012a, 2012b) and Eucalyptus spp. trees (Tsuchihira et al., 2010); in both cases, analysis focused on overexpressing specific isoforms of aquaporin genes. The PIP2;4 root-specific aquaporin enhanced water transport in transformed Vitis spp. plants under well-watered conditions but not under water stress (Perrone et al., 2012a, 2012b), whereas Eucalyptus spp. hybrid clones overexpressing two Raphanus sativus genes (RsPIP1;1 and RsPIP2;1) did not display any increase in drought tolerance (Tsuchihira et al., 2010). To date, no research on the recovery from embolism formation in woody plants with impaired aquaporin expression has been conducted.In this study, we used poplar transgenic plants characterized by a strong down-regulation of PIP1 genes to test the role of this aquaporin subfamily in the plant response to water stress and subsequent recovery from stress. Although transformed poplars did not show morphologically different phenotypes compared with wild-type plants, they were found to be more sensitive to imposed water stress, resulting in increased vulnerability to embolism formation and the loss of stomatal conductance. We also noted a reduced capacity of transformed plants to restore xylem water transport.     

Patterns of Metabolite Changes Identified from Large-Scale Gene Perturbations in Arabidopsis Using a Genome-Scale Metabolic Network

Metabolomics enables quantitative evaluation of metabolic changes caused by genetic or environmental perturbations. However, little is known about how perturbing a single gene changes the metabolic system as a whole and which network and functional properties are involved in this response. To answer this question, we investigated the metabolite profiles from 136 mutants with single gene perturbations of functionally diverse Arabidopsis (Arabidopsis thaliana) genes. Fewer than 10 metabolites were changed significantly relative to the wild type in most of the mutants, indicating that the metabolic network was robust to perturbations of single metabolic genes. These changed metabolites were closer to each other in a genome-scale metabolic network than expected by chance, supporting the notion that the genetic perturbations changed the network more locally than globally. Surprisingly, the changed metabolites were close to the perturbed reactions in only 30% of the mutants of the well-characterized genes. To determine the factors that contributed to the distance between the observed metabolic changes and the perturbation site in the network, we examined nine network and functional properties of the perturbed genes. Only the isozyme number affected the distance between the perturbed reactions and changed metabolites. This study revealed patterns of metabolic changes from large-scale gene perturbations and relationships between characteristics of the perturbed genes and metabolic changes.Rational and quantitative assessment of metabolic changes in response to genetic modification (GM) is an open question and in need of innovative solutions. Nontargeted metabolite profiling can detect thousands of compounds, but it is not easy to understand the significance of the changed metabolites in the biochemical and biological context of the organism. To better assess the changes in metabolites from nontargeted metabolomics studies, it is important to examine the changed metabolites in the context of the genome-scale metabolic network of the organism.Metabolomics is a technique that aims to quantify all the metabolites in a biological system (Nikolau and Wurtele, 2007; Nicholson and Lindon, 2008; Roessner and Bowne, 2009). It has been used widely in studies ranging from disease diagnosis (Holmes et al., 2008; DeBerardinis and Thompson, 2012) and drug discovery (Cascante et al., 2002; Kell, 2006) to metabolic reconstruction (Feist et al., 2009; Kim et al., 2012) and metabolic engineering (Keasling, 2010; Lee et al., 2011). Metabolomic studies have demonstrated the possibility of identifying gene functions from changes in the relative concentrations of metabolites (metabotypes or metabolic signatures; Ebbels et al., 2004) in various species including yeast (Saccharomyces cerevisiae; Raamsdonk et al., 2001; Allen et al., 2003), Arabidopsis (Arabidopsis thaliana; Brotman et al., 2011), tomato (Solanum lycopersicum; Schauer et al., 2006), and maize (Zea mays; Riedelsheimer et al., 2012). Metabolomics has also been used to better understand how plants interact with their environments (Field and Lake, 2011), including their responses to biotic and abiotic stresses (Dixon et al., 2006; Arbona et al., 2013), and to predict important agronomic traits (Riedelsheimer et al., 2012). Metabolite profiling has been performed on many plant species, including angiosperms such as Arabidopsis, poplar (Populus trichocarpa), and Catharanthus roseus (Sumner et al., 2003; Rischer et al., 2006), basal land plants such as Selaginella moellendorffii and Physcomitrella patens (Erxleben et al., 2012; Yobi et al., 2012), and Chlamydomonas reinhardtii (Fernie et al., 2012; Davis et al., 2013). With the availability of whole genome sequences of various species, metabolomics has the potential to become a useful tool for elucidating the functions of genes using large-scale systematic analyses (Fiehn et al., 2000; Saito and Matsuda, 2010; Hur et al., 2013).Although metabolomics data have the potential for identifying the roles of genes that are associated with metabolic phenotypes, the biochemical mechanisms that link functions of genes with metabolic phenotypes are still poorly characterized. For example, we do not yet know the principles behind how perturbing the expression of a single gene changes the metabolic system as a whole. Large-scale metabolomics data have provided useful resources for linking phenotypes to genotypes (Fiehn et al., 2000; Roessner et al., 2001; Tikunov et al., 2005; Schauer et al., 2006; Lu et al., 2011; Fukushima et al., 2014). For example, Lu et al. (2011) compared morphological and metabolic phenotypes from more than 5,000 Arabidopsis chloroplast mutants using gas chromatography (GC)- and liquid chromatography (LC)-mass spectrometry (MS). Fukushima et al. (2014) generated metabolite profiles from various characterized and uncharacterized mutant plants and clustered the mutants with similar metabolic phenotypes by conducting multidimensional scaling with quantified metabolic phenotypes. Nonetheless, representation and analysis of such a large amount of data remains a challenge for scientific discovery (Lu et al., 2011). In addition, these studies do not examine the topological and functional characteristics of metabolic changes in the context of a genome-scale metabolic network. To understand the relationship between genotype and metabolic phenotype, we need to investigate the metabolic changes caused by perturbing the expression of a gene in a genome-scale metabolic network perspective, because metabolic pathways are not independent biochemical factories but are components of a complex network (Berg et al., 2002; Merico et al., 2009).Much progress has been made in the last 2 decades to represent metabolism at a genome scale (Terzer et al., 2009). The advances in genome sequencing and emerging fields such as biocuration and bioinformatics enabled the representation of genome-scale metabolic network reconstructions for model organisms (Bassel et al., 2012). Genome-scale metabolic models have been built and applied broadly from microbes to plants. The first step toward modeling a genome-scale metabolism in a plant species started with developing a genome-scale metabolic pathway database for Arabidopsis (AraCyc; Mueller et al., 2003) from reference pathway databases (Kanehisa and Goto, 2000; Karp et al., 2002; Zhang et al., 2010). Genome-scale metabolic pathway databases have been built for several plant species (Mueller et al., 2005; Zhang et al., 2005, 2010; Urbanczyk-Wochniak and Sumner, 2007; May et al., 2009; Dharmawardhana et al., 2013; Monaco et al., 2013, 2014; Van Moerkercke et al., 2013; Chae et al., 2014; Jung et al., 2014). Efforts have been made to develop predictive genome-scale metabolic models using enzyme kinetics and stoichiometric flux-balance approaches (Sweetlove et al., 2008). de Oliveira Dal’Molin et al. (2010) developed a genome-scale metabolic model for Arabidopsis and successfully validated the model by predicting the classical photorespiratory cycle as well as known key differences between redox metabolism in photosynthetic and nonphotosynthetic plant cells. Other genome-scale models have been developed for Arabidopsis (Poolman et al., 2009; Radrich et al., 2010; Mintz-Oron et al., 2012), C. reinhardtii (Chang et al., 2011; Dal’Molin et al., 2011), maize (Dal’Molin et al., 2010; Saha et al., 2011), sorghum (Sorghum bicolor; Dal’Molin et al., 2010), and sugarcane (Saccharum officinarum; Dal’Molin et al., 2010). These predictive models have the potential to be applied broadly in fields such as metabolic engineering, drug target discovery, identification of gene function, study of evolutionary processes, risk assessment of genetically modified crops, and interpretations of mutant phenotypes (Feist and Palsson, 2008; Ricroch et al., 2011).Here, we interrogate the metabotypes caused by 136 single gene perturbations of Arabidopsis by analyzing the relative concentration changes of 1,348 chemically identified metabolites using a reconstructed genome-scale metabolic network. We examine the characteristics of the changed metabolites (the metabolites whose relative concentrations were significantly different in mutants relative to the wild type) in the metabolic network to uncover biological and topological consequences of the perturbed genes.