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).     

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.     

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.     

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).     

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.     

Investigations Concerning Cavitation and Frost Fatigue in Clonal 84K Poplar Using High-Resolution Cavitron Measurements

Both drought and freezing-thawing of stems induce a loss of hydraulic conductivity (percentage loss of conductivity [PLC]) in woody plants. Drought-induced PLC is often accompanied by physical damage to pit membranes, causing a shift in vulnerability curves (cavitation fatigue). Hence, if cavitated stems are flushed to remove embolisms, the next vulnerability curve is different (shifted to lower tensions). The 84K poplar (Populus alba × Populus glandulosa) clone has small vessels that should be immune from frost-induced PLC, but results demonstrated that freezing-thawing in combination with tension synergistically increased PLC. Frost fatigue has already been defined, which is similar to cavitation fatigue but induced by freezing. Frost fatigue caused a transition from a single to a dual Weibull curve, but drought-fatigued stems had single Weibull curves shifted to lower tensions. Studying the combined impact of tension plus freezing on fatigue provided evidence that the mechanism of frost fatigue may be the extra water tension induced by freezing or thawing while spinning stems in a centrifuge rather than direct ice damage. A hypothesis is advanced that tension is enhanced as ice crystals grow or melt during the freeze or thaw event, respectively, causing a nearly identical fatigue event to that induced by drought.Water transport in xylem conduits of trees occurs while water is under tension (negative pressure; Tyree and Zimmermann, 2002). The xylem water-transport system is vulnerable to cavitation and embolism because tensile water is metastable, so if a gas bubble appears in a conduit it will rapidly expand to fill the conduit whenever the fluid tension is 0.1 MPa or greater, where a tension of 0.1 MPa is equivalent to vacuum pressure. A cavitation event occurs whenever a tensile water column breaks, which results in a water vapor-filled void. Because of Henry’s law of gas solubility in water, this vapor void will eventually equilibrate with air at atmospheric pressure, at which point the conduit is fully embolized (Tyree and Zimmermann, 2002). Embolism has been identified as a limiting factor of primary production (Hubbard et al., 2001). As a result, tree growth and fitness are probably negatively impacted temporarily or seriously limited permanently if embolism is extensive (Christensen-Dalsgaard and Tyree, 2013).The two main factors causing cavitation and embolism are drought and frost (Mayr et al., 2003; Christensen-Dalsgaard and Tyree, 2013). Drought-induced cavitation is caused by the high xylem tension attributed to water stress. The high tension in the sap forces air bubbles into functional conduits from neighboring embolized ones through shared pit membranes (Jarbeau et al., 1995; Sperry et al., 1996; Hacke et al., 2001; Stiller and Sperry, 2002; Christman et al., 2012) according to an air-seeding mechanism (Sperry and Tyree, 1988; Cochard et al., 1992). Hence, the continuity of water flow is disrupted due to cavitation. Frost-induced cavitation, on the other hand, occurs when dissolved gases in the sap freeze out and create bubbles during ice formation because air is not soluble in ice (Mayr et al., 2003; Christensen-Dalsgaard and Tyree, 2013, 2014) but remains entrapped between ice crystals. Once the sap melts and tension is regenerated, the entrapped bubbles may expand to embolize the conduits instead of dissolving (Pittermann and Sperry, 2006). Current thinking is that freezing-induced embolism occurs when the tension exceeds a critical value determined by the surface tension of the bubbles, which mainly depends on the xylem water potential and the bubble radius (Yang and Tyree, 1992; Tyree et al., 1994; Hacke and Sperry, 2001). Larger bubbles may form in conduits with a larger diameter, so species with larger conduits are more vulnerable to frost-induced embolism (Langan et al., 1997; Davis et al., 1999; Pittermann and Sperry, 2006). Furthermore, enhanced loss of hydraulic conductivity (K_h) of trees may occur when stems are subjected to a combination of frost and drought causing low xylem water potential (Mayr et al., 2003; Willson and Jackson, 2006) and repeated freeze-thaw cycles (Sperry and Sullivan, 1992; Cox and Zhu, 2003; Mayr et al., 2003). However, even trees with small conduits are found to suffer severe embolism in winter (Sperry et al., 1988; Améglio et al., 2002) due mostly to freezing-drying of stems.Resilient species are those that suffer no significantly different cavitation resistance before and after a cavitation-refilling cycle (Hacke et al., 2001; Christensen-Dalsgaard and Tyree, 2013). In contrast, species that are weakened by cavitation or frost are said to suffer cavitation fatigue or frost fatigue. Cavitation or frost fatigue is quantified by how much the vulnerability curve is shifted before versus after a fatigue-inducing event, and it is typically reported as a shift in P₅₀, which is either the pressure (negative value) or tension (positive value) that produces 50% loss of K_h. In the rest of this article, we will use T₅₀ to indicate the tension at 50% loss of conductivity or T_x to indicate the tension that induces x% loss of conductivity. Vulnerability curves (VCs) are usually measured by a centrifuge technique (Alder et al., 1997; Cochard et al., 2005), but most researchers measure just five or six points to determine a VC. High-resolution VC curves with nine to 27 points per curve can be collected quickly using the Cochard rotor. Recent studies have successfully used high-resolution VC to characterize the detailed shape of VCs, revealing dual Weibull curves (e.g. r- and s-shaped or dual s-shaped curves; Cai et al., 2014; Wang et al., 2014a) because a complex shape to a VC cannot be identified with just a few points. Furthermore, we used a centrifuge to induce tension while simultaneously freezing in order to study the combined impact of tension and freezing-thawing on frost fatigue and freeze-thaw-induced embolism.In this article, we intend to investigate whether drought and freeze-thaw cycles could have an effect on the cavitation resistance in terminal shoots from adult trees of 84K poplar (Populus alba × Populus glandulosa), with high-resolution analysis of VCs and an artificial freeze-thaw simulation technique. Populus spp. are known to be water-demanding, drought-sensitive species with T₅₀ ranging from 1.07 to 2.5 MPa (Fichot et al., 2015) and vulnerable to winter damage (Feng et al., 2010). Among poplars, 84K poplar is known by foresters to be relatively resistant to water stress, low temperature, diseases, and insects (Zhou et al., 2007). As the main afforestation species in Shaanxi, Gansu, and Qinghai Province, 84K poplar is of great ecological importance.     

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.     

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.     

Noninvasive Measurement of Vulnerability to Drought-Induced Embolism by X-Ray Microtomography

Hydraulic failure induced by xylem embolism is one of the primary mechanisms of plant dieback during drought. However, many of the methods used to evaluate the vulnerability of different species to drought-induced embolism are indirect and invasive, increasing the possibility that measurement artifacts may occur. Here, we utilize x-ray computed microtomography (microCT) to directly visualize embolism formation in the xylem of living, intact plants with contrasting wood anatomy (Quercus robur, Populus tremula × Populus alba, and Pinus pinaster). These observations were compared with widely used centrifuge techniques that require destructive sampling. MicroCT imaging provided detailed spatial information regarding the dimensions and functional status of xylem conduits during dehydration. Vulnerability curves based on microCT observations of intact plants closely matched curves based on the centrifuge technique for species with short vessels (P. tremula × P. alba) or tracheids (P. pinaster). For ring porous Q. robur, the centrifuge technique significantly overestimated vulnerability to embolism, indicating that caution should be used when applying this technique to species with long vessels. These findings confirm that microCT can be used to assess the vulnerability to embolism on intact plants by direct visualization.Theory describing the physiological mechanism that allows plants to extract water from the soil and transport it many tens of meters in height has often been the subject of intense debate (Tyree, 2003). Plants have evolved a water-transport system that relies on water sustaining a tensile force; as a result, xylem sap is at negative absolute pressures (Dixon and Joly, 1895; Melcher et al., 1998; Wei et al., 1999). However, this transport mechanism comes with its own set of problems. Most notably, water under tension is prone to cavitation, which results in the formation of gas bubbles (emboli) that block xylem conduits. Embolism reduces the capacity of the xylem tissue to deliver water to the canopy, where it is required to maintain adequate levels of cellular hydration (Tyree and Sperry, 1989). The probability of embolism occurring in the xylem increases during drought, with increasing tension in the xylem sap. During prolonged and severe droughts, xylem embolism can reach lethal levels, causing branch dieback and, ultimately, plant death (Davis et al., 2002; Brodribb and Cochard, 2009; Hoffmann et al., 2011; Choat, 2013; Urli et al., 2013). Water stress-induced embolism is now recognized as one of the principal causes of plant mortality in response to extreme drought events (Anderegg, 2015). In the face of increasingly severe droughts expected with rising global temperatures, hydraulic failure due to embolism has the potential to cause widespread dieback of trees across all major forest biomes (Choat et al., 2012).The majority of techniques used to estimate cavitation resistance are indirect and/or invasive, increasing the possibility of artifacts occurring during measurement (Cochard et al., 2013). Artifacts relating to invasive techniques are particularly relevant in this case, since xylem sap under tension is in a metastable state and may easily vaporize as a result of disturbance. Noninvasive imaging techniques offer the potential to make direct observations of xylem function in intact plants at high resolution and in real time. Noninvasive techniques include magnetic resonance imaging (MRI; Holbrook et al., 2001; Kaufmann et al., 2009; Choat et al., 2010) and, more recently, x-ray computed microtomography (microCT; Brodersen et al., 2010; Charra-Vaskou et al., 2012; McElrone et al., 2012). MicroCT provides superior spatial resolution to MRI, with resolutions below 2 μm attainable for a plant stem of 4 to 5 mm in diameter. This allows for detailed analysis of embolism formation and repair in the xylem, including spatial patterns of embolism spread between conduits (Brodersen et al., 2013; Dalla-Salda et al., 2014).However, noninvasive imaging techniques have seldom been used to validate indirect or invasive techniques used to estimate cavitation resistance. At this stage, only a handful of studies have utilized imaging technology to measure cavitation resistance in trees (Torres-Ruiz et al., 2014; Cochard et al., 2015), and these studies employed a destructive mode of the technique in which small branches were cut off the plant before scanning took place. Thus far, noninvasive imaging on intact plants has only been used to measure cavitation resistance in two species, grapevine (Vitis vinifera; Choat et al., 2010; Brodersen et al., 2013) and Sequoia sempervirens (Choat et al., 2015). Further measurement of cavitation resistance using noninvasive imaging on intact plants across a range of species, therefore, is a high priority.These comparisons are particularly important because of the current debate surrounding the invasive techniques (Cochard et al., 2013). Specifically, evidence from a variety of experiments suggests that centrifuge and air injection techniques underestimate cavitation resistance in species with long xylem vessels (Choat et al., 2010; Cochard et al., 2010; Ennajeh et al., 2011; Martin-StPaul et al., 2014; Torres-Ruiz et al., 2014; Wang et al., 2014). This artifact occurs when samples placed into centrifuge rotors or air injection collars have a large proportion of vessels that are cut open at both ends of the segment. A number of studies have disputed this open-vessel hypothesis and suggested that some versions of the centrifuge and air injection techniques provide reliable estimates of cavitation resistance (Jacobsen and Pratt, 2012; Sperry et al., 2012; Tobin et al., 2013). Because there will always be uncertainties associated with indirect measurements, noninvasive imaging using intact plants provides the best option for resolving these methodological issues.In this study, synchrotron-based microCT was utilized to investigate the formation of drought-induced embolism in the xylem of intact, potted plants. Three species were selected to provide a range of contrasting xylem structures: Quercus robur (ring porous), Populus tremula × Populus alba (diffuse porous), and Pinus pinaster (tracheid bearing). Visualizations of xylem embolism in the stems of these species during a sequence of natural dehydration were used to construct embolism vulnerability curves. We hypothesized that (1) vulnerability curves based on microCT observations would match vulnerability curves based on the centrifuge technique for species with short vessels (P. tremula × P. alba) or tracheid-based xylem (P. pinaster) and (2) the centrifuge technique would overestimate vulnerability to embolism in the long-vesseled species (Q. robur) due to the open-vessel artifact.     

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.     

Mechanical Failure of Fine Root Cortical Cells Initiates Plant Hydraulic Decline during Drought

Root systems perform the crucial task of absorbing water from the soil to meet the demands of a transpiring canopy. Roots are thought to operate like electrical fuses, which break when carrying an excessive load under conditions of drought stress. Yet the exact site and sequence of this dysfunction in roots remain elusive. Using in vivo x-ray computed microtomography, we found that drought-induced mechanical failure (i.e. lacunae formation) in fine root cortical cells is the initial and primary driver of reduced fine root hydraulic conductivity (Lp_r) under mild to moderate drought stress. Cortical lacunae started forming under mild drought stress (−0.6 MPa Ψ_stem), coincided with a dramatic reduction in Lp_r, and preceded root shrinkage or significant xylem embolism. Only under increased drought stress was embolism formation observed in the root xylem, and it appeared first in the fine roots (50% loss of hydraulic conductivity [P₅₀] reached at −1.8 MPa) and then in older, coarse roots (P₅₀ = −3.5 MPa). These results suggest that cortical cells in fine roots function like hydraulic fuses that decouple plants from drying soil, thus preserving the hydraulic integrity of the plant’s vascular system under early stages of drought stress. Cortical lacunae formation led to permanent structural damage of the root cortex and nonrecoverable Lp_r, pointing to a role in fine root mortality and turnover under drought stress.Root systems of woody plants consist of both coarse and nonwoody fine roots. Fine roots can constitute as little as 1% of the total root surface area (Kramer and Bullock, 1966), yet are critically important for biogeochemical cycling in terrestrial ecosystems as they constitute the primary exchange surface between plants and soil (Jackson et al., 1997). They are responsible for the vast majority of water absorption in woody root systems (Gambetta et al., 2013; Kramer and Boyer, 1995; Kramer and Bullock, 1966) and mediate backward flow of water from a plant to the soil via a process called hydraulic redistribution, which can alter regional climate (Richards and Caldwell, 1987; Lee et al., 2005). Fine roots also modify the soil through carbon exudation and stimulation of microbial activity (McCormack et al., 2015), and their production and annual turnover represent 33% of global net primary productivity (Jackson et al., 1997; McCormack et al., 2015). Elucidating details of fine root function and responses to stress can thus improve our understanding of how these plant organs can influence ecosystem carbon, nutrient, and water cycles.Fine roots are traditionally defined as all roots <2-mm diameter, but recent work has emphasized the need to delineate this diameter class into distinct functional groups. By separating fine roots into a shorter-lived absorptive pool and a longer-lived transport pool, McCormack et al. (2015) showed that fine root functionality can alter estimates of global net primary productivity by 30%. This work highlights our still-limited understanding of fine root functionality, the mechanisms underlying their lifespan and turnover, and how those traits respond to abiotic stress (Lukac, 2012; Tierney and Fahey, 2002; Guo et al., 2008). Fine root mortality during drought has been linked to increased root respiration and inhibited photosynthate transport to roots (e.g. Marshall, 1986) but could also be attributed to hydraulic dysfunction (Jackson et al., 2000). Portions of root systems are thought to operate analogously to a hydraulic fuse in an electrical circuit and designed to fail hydraulically when carrying excessive current under drought stress (Zimmermann, 1983; Jackson et al., 2000). However, the exact location of these hydraulic fuses in the root system has yet to be identified. Axial water transport in the xylem is considered a weak link, as roots of numerous species have been shown to be more susceptible to drought-induced xylem embolism compared to other organs within the same plant (i.e. trunks, stems, tap roots; Alder et al., 1996; Hacke and Sauter, 1996; McElrone et al., 2004; Pratt et al., 2015; Johnson et al., 2016). Moreover, Sperry and Ikeda (1997) found that smaller roots were the most vulnerable plant organ to xylem embolism, which would localize failure to inexpensive, distal, and easily replaceable portions of a root system. Such a design is considered effective, because it is widely assumed that the hydraulic capacity of smaller distal roots is readily repaired upon rewatering via xylem embolism removal (Domec et al., 2006; Jackson et al., 2000). While much work has demonstrated that xylem embolism reduces hydraulic capacity under severe drought stress (Brodribb et al., 2016a, 2016b; Choat et al., 2012), its contribution under mild to moderate stress is less clear (Choat et al., 2016; Cochard and Delzon, 2013; Cochard et al., 2013; McElrone et al., 2012; Wheeler et al., 2013; Choat et al., 2010; Torres-Ruiz et al., 2015). Work is still needed to resolve the location and sequence of root hydraulic dysfunction under drought and what tissues are involved in each stage of this process, especially under mild stress where fine root hydraulic conductivity (Lp_r) is known to decrease dramatically (Aroca et al., 2012).Before entering the xylem for long distance transport, water absorbed by roots must traverse a series of cell layers that include the epidermis, cortex, and endodermis (Steudle and Peterson 1998). Hydraulic resistance is much greater along this radial pathway compared to the axial transport pathway in the xylem (e.g. Frensch and Hsiao, 1993; Frensch and Steudle, 1989). The resistance differential between radial and axial pathways persists or increases in magnitude as fine Lp_r decreases under mild to moderate drought stress. While decreased fine root permeability under drought has been attributed to root shrinkage (Passioura, 1988; Nobel and Cui, 1992), changes in membrane permeability via aquaporins (Maurel et al., 2015; Aroca et al., 2012; North, 2004), development of suberized apoplastic barriers over longer periods of drought (Barrios-Masias et al., 2015; North and Nobel, 1991), or mechanical damage in cortical cells (i.e. lacunae formation; North and Nobel, 1991), the integration of these responses particularly under mild stress is still lacking. Elucidating the physiological mechanism that drives this response could help to resolve long-standing questions about fine root functionality, lifespan, and turnover.Here, we originally aimed to study whether fine roots function as the primary hydraulic fuse that disconnects a plant from drying soil. We studied the sequence of events during soil drying from saturated to severe drought conditions in coarse and fine roots of grapevines, which are considered a model species and have long been characterized as highly susceptible to drought-induced embolism. While performing these experiments, we discovered that the fine root cortex was radically changing under mild drought stress that preceded any embolism formation. We then performed hydraulic measurements and fluorescence light microscopy to investigate how fine Lp_r is affected by the formation of cortical lacunae that should significantly increase radial hydraulic resistance to flow.     

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.     

Herb Hydraulics: Inter- and Intraspecific Variation in Three Ranunculus Species

The requirements of the water transport system of small herbaceous species differ considerably from those of woody species. Despite their ecological importance for many biomes, knowledge regarding herb hydraulics remains very limited. We compared key hydraulic features (vulnerability to drought-induced hydraulic decline, pressure-volume relations, onset of cellular damage, in situ variation of water potential, and stomatal conductance) of three Ranunculus species differing in their soil humidity preferences and ecological amplitude. All species were very vulnerable to water stress (50% reduction in whole-leaf hydraulic conductance [k_leaf] at −0.2 to −0.8 MPa). In species with narrow ecological amplitude, the drought-exposed Ranunculus bulbosus was less vulnerable to desiccation (analyzed via loss of k_leaf and turgor loss point) than the humid-habitat Ranunculus lanuginosus. Accordingly, water stress-exposed plants from the broad-amplitude Ranunculus acris revealed tendencies toward lower vulnerability to water stress (e.g. osmotic potential at full turgor, cell damage, and stomatal closure) than conspecific plants from the humid site. We show that small herbs can adjust to their habitat conditions on interspecific and intraspecific levels in various hydraulic parameters. The coordination of hydraulic thresholds (50% and 88% loss of k_leaf, turgor loss point, and minimum in situ water potential) enabled the study species to avoid hydraulic failure and damage to living cells. Reversible recovery of hydraulic conductance, desiccation-tolerant seeds, or rhizomes may allow them to prioritize toward a more efficient but vulnerable water transport system while avoiding the severe effects that water stress poses on woody species.The resistance of terrestrial plant species to water stress is an important determinant of their spatial distribution (Engelbrecht et al., 2007), and in a globally changing climate, resistance to water stress-induced damage to the water transport system is an essential parameter for the prediction of species survival and future changes in biodiversity and species composition. The hydraulic vulnerability to drought-induced embolism in woody species is adjusted to their environmental conditions on a global scale (Choat et al., 2012), but increased hydraulic safety entails several tradeoffs, such as transport efficiency, as plants can reduce the risk of hydraulic failure by the formation of smaller and hydraulically less efficient conduits (Tyree et al., 1994; Wagner et al., 1998, Gleason et al., 2016).While many studies have dealt with the hydraulics of woody species (Maherali et al., 2004; Choat et al., 2012), the hydraulic characteristics of small herbaceous species are largely understudied, and our knowledge regarding their water transport system, including vulnerability to drought-induced loss of conductivity, remains very limited (Kocacinar and Sage, 2003; Brodribb and Holbrook, 2004; Iovi et al., 2009; Holloway-Phillips and Brodribb, 2011a; Tixier et al., 2013). Yet, herbs play an important ecological role in many biomes, such as grasslands and the alpine zone (Billings and Mooney, 1968; Gilliam, 2007; Scholz et al., 2010), and represent a significant percentage of crop plants (Monfreda et al., 2008); therefore, they are interesting from an ecophysiological viewpoint.The requirements of the water transport system of herbs differ in several ways from those of woody species (Mencuccini, 2003). First, transport distances and supported leaf areas are much smaller in herbs, and reversible extraxylary limitations (Brodribb and Holbrook, 2005; Kim and Steudle, 2007) may have a stronger effect on hydraulic conductivity. Second, herbs often do not feature an essential, lignified main axis designed for long-term functioning, in which hydraulic failure may mean whole-plant mortality. Third, small plants may more easily restore water transport capacity in the case of embolism formation, because, due to smaller size, a water potential (Ψ) at or near zero can be reached quickly under favorable conditions due to positive root pressure (Tyree and Sperry, 1989; Tyree and Ewers, 1991; Stiller and Sperry, 2002; Tyree and Zimmermann, 2002; Ganthaler and Mayr, 2015). Thus, herbs may not be as threatened by drought-induced failure as taller woody species.Hydraulic failure occurs when vulnerability thresholds are exceeded under water stress and the xylem tension is suddenly released as water is replaced by air, effectively blocking water transport in the affected conduits (Tyree and Zimmermann, 2002). Vulnerability to drought-induced embolism can thus be analyzed hydraulically by measuring decreases in conductance during increasing water stress (Sperry et al., 1988a; Cochard, 2002; Cochard et al., 2013), by noninvasive imaging of xylem water content (Choat et al., 2010; Brodersen et al., 2013; Cochard et al., 2015), or indirectly by monitoring of ultrasonic acoustic emissions (UE) which occur during the spontaneous energy release at embolism formation (Milburn and Johnson, 1966; Mayr and Rosner, 2011; Ponomarenko et al., 2014). However, the usability of UE analysis to noninvasively study hydraulic vulnerability remains under discussion (Sandford and Grace, 1985; Kikuta, 2003; Wolkerstorfer et al., 2012; Cochard et al., 2013; Rosner, 2015).On the leaf level, desiccation may induce turgor loss, decreases in mesophyll conductance (Kim and Steudle, 2007; Scoffoni et al., 2014), or cell collapse (Blackman et al., 2010; Holloway-Phillips and Brodribb, 2011a), and limit hydraulic conductance, gas exchange, and thus photosynthesis before embolism occurs. When water stress increases further, living cells may incur damage resulting in tissue and leaf mortality.In this study, we compared key hydraulic features (vulnerability to drought-induced loss of conductance as measured via hydraulic flow and xylem staining, pressure-volume relations, and the onset of cellular damage) of three species from the genus Ranunculus, which is distributed almost worldwide and contains species adapted to dry and humid habitats as well as species with broad ecological amplitude. We hypothesized that herbaceous species would be more vulnerable to water stress than woody species but also would show interspecific and intraspecific adjustments in hydraulic vulnerability based on the water availability of their respective habitats. Namely, the hydraulics of species with narrow ecological amplitude were hypothesized to reflect their respective habitat conditions, and broad-amplitude species should show adequate intraspecific variation to enable growth in dry and humid habitats.