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Journal of Experimental Botany, Vol. 65, No. 7, pp. 1829–1848, 2014 doi:10.1093/jxb/ert459 Advance Access publication 22 January, 2014 Review paper Symplasmic networks in secondary vascular tissues: parenchyma distribution and activity supporting long-distance transport Rachel Spicer Department of Botany, Connecticut College, New London, CT 06320, USA To whom correspondence should be addressed. E-mail: [email protected] Received 1 October 2013; Revised 25 November 2013; Accepted 26 November 2013 Abstract Stems that develop secondary vascular tissue (i.e. xylem and phloem derived from the vascular cambium) have unique demands on transport owing to their mass and longevity. Transport of water and assimilates must occur over long distances, while the increasing physical separation of xylem and phloem requires radial transport. Developing secondary tissue is itself a strong sink positioned between xylem and phloem along the entire length of the stem, and the integrity of these transport tissues must be maintained and protected for years if not decades. Parenchyma cells form an interconnected three-dimensional lattice throughout secondary xylem and phloem and perform critical roles in all of these tasks, yet our understanding of their physiology, the nature of their symplasmic connections, and their activity at the symplast–apoplast interface is very limited. This review highlights key historical work as well as current research on the structure and function of parenchyma in secondary vascular tissue in the hopes of spurring renewed interest in this area, which has important implications for whole-plant transport processes and resource partitioning. Key words: Cambium, contact cells, parenchyma, phloem, rays, xylem. Introduction Xylem and phloem transport are often presented in isolation, or worse, in distinct contrast: xylem transport of water and mineral nutrients occurs through dead cells in which the water is under tension (negative pressure) and is an entirely passive process; transport of photoassimilates in the phloem, on the other hand, occurs through living cells under positive hydrostatic pressure and often (although not always) requires an input of energy. The focus is traditionally on structure/ function relationships of the conducting elements: the vessels and tracheids of xylem and the sieve tubes and sieve cells of phloem. In fact, the two transport processes are highly interdependent and the activity of parenchyma cells provides a critical metabolic and energetic link. Highly specialized parenchyma cells are distributed throughout xylem and phloem and take on particular importance in secondary vascular tissues where the physical separation of water and assimilate streams, long transport distances, and perennial lifestyle place new and at times extreme demands on transport. Some parenchyma cells act as storage sites for carbohydrates and thus may function as either sources or sinks, despite being heterotrophic by definition. Other parenchyma cells serve as an intermediary between transport and storage tissues and function in protecting and maintaining the integrity of the transport system. The primary goal of this review is to draw attention to the diverse and active roles of parenchyma in supporting transport in stems with secondary growth. By integrating knowledge from traditionally distinct fields, taking a comparative approach to highlight the broad themes that emerge above natural variation, and identifying key areas of both accelerated progress and woeful neglect, it is hoped that this review will stimulate new avenues of research in this important but understudied field. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: [email protected] 1830 | Spicer The activity of parenchyma cells in secondary vasculature is particularly relevant in light of the increasing recognition that phloem source–sink relationships in trees are complex (van Bel, 1996; van Bel and Ehlers, 2000; Hafke et al., 2005; Thompson, 2006; Turgeon, 2010; Fu et al., 2011). The canonical source leaves and terminal sinks (root, fruit, or seed) are separated by a great distance, while a strong axial sink, the vascular cambium and its rapidly expanding derivatives, is just several cells away from the phloem along the entire length of the stem. Parenchyma storing carbohydrates in both xylem and phloem may function as diffuse, local sources—even of water—and contribute to phloem turgor gradients (Sevanto et al., 2011, 2013). The fact that trees do not have large pressure gradients in their phloem (Gamalei, 1989; Rennie and Turgeon, 2009; Turgeon, 2010; Fu et al., 2011) further suggests that activity along the entire length of the stem—activity that relies on energy and carbohydrates from neighbouring parenchyma cells—needs to be incorporated into models of long-distance phloem transport. In plants with secondary growth, the stem must no longer be viewed as a simple transport route linking above- and belowground organs, but instead as a distribution manifold. A great deal more work needs to be done in this area. It is impossible to discuss the role of parenchyma in stems undergoing secondary growth without frequent reference to anatomical features and a clear image of the three-dimensional arrangement of cells. To anyone not well versed in plant anatomy, the terminology can be daunting. A primer on relevant anatomy is thus included here in order to make the work accessible to newcomers from different fields, as well as to highlight classical work, some of which is quite old but still important and at risk of being forgotten. Rather than a thorough review, the anatomy covered here is meant to provide an overview of variation found in nature with a focus on the aspects that are most relevant to long- and short-distance vascular transport. Although all seed plants forming secondary vascular tissues are technically considered, the information on nonconiferous gymnosperms (cycads, Gnetales, Ginkgo) is so sparse and/or the anatomy so derived that the discussion is effectively limited to conifers and angiosperms. In addition, although some extreme cases of variation due to atypical cambial activity (e.g. successive cambia) are mentioned, the emphasis is on common patterns rather than a thorough coverage of existing variation. Lastly, although the focus of this review is the activity of parenchyma in secondary vascular tissues, evidence will at times be drawn from work on primary tissues when detailed physiological roles have only been studied in the latter and where there is good reason to believe the processes are similar. Three-dimensional arrangement and specialization of parenchyma in secondary vascular tissue Parenchyma cells in secondary vascular tissue are oriented either radially or axially, and, with both orientations typically present, they form a highly interconnected network—effectively a three-dimensional lattice—of living cells throughout the xylem and phloem (Fig. 1). A subset of both axial and radial parenchyma are in physical contact with the conducting elements of the xylem and phloem, either directly via pits in the cell wall or indirectly via the apoplast, and are therefore able to modify the composition of the transpiration stream and phloem sap, respectively. The reverse is also true, such that parenchyma cells are supplied with materials and signalling molecules travelling in both transport streams. A great deal more is known about the comparative anatomy of parenchyma in xylem than in phloem, in part due to the commercial importance of wood, diagnostic use of parenchyma characteristics in species identification, and greater longevity of the tissue. However, both xylem and phloem parenchyma show complex spatial patterns of functional significance that are often taxa specific. In general, conifers have far less radial and axial parenchyma in xylem than angiosperms, with lianas and stem succulents representing some of the most parenchyma-rich stems in the plant world, both xylem and phloem included (Table 1). Although comparable values for parenchyma in phloem are not widely reported, there is often a correlation in patterns between the two tissues. Lastly, it should be noted that while cellular structure and arrangement is described here, a brief discussion of cellular contents has been incorporated into several sections on parenchyma function. Rays are complex structures with specialized cell types Radially oriented parenchyma cells are grouped together in rays that are continuous through the cambium such that a single ray is at once both a xylem ray and a phloem ray. This is a function of the fact that secondary xylem and phloem are produced by a bifacial vascular cambium, such that all cells within a single radial file are derived from the same cambial initial (i.e. a single initial will produce both xylem and phloem derivatives; Larson, 1994). This presents a unique situation whereby materials to be exchanged between xylem and phloem must pass through meristematic tissue, including the true cambial initials and differentiating cells on either side. The structure and composition of mature xylem rays has received a great deal of attention, that of phloem rays less so, and limited work examines the nature of symplasmic connections both among parenchyma and across the cambium linking the two tissues. In the central portion of both xylem- and phloem-side rays are typically radially elongate cells (radial/longitudinal length ratio 1.5–40:1) termed ‘procumbent’ cells (Fig. 2). Based on their shape, a high density of plasmodesmata linking cells radially (Sauter and Kloth, 1986; van der Schoot and van Bel, 1989a; Chaffey and Barlow, 2001), and evidence of symplasmic continuity through both fluorescent tracer studies and membrane potential measurements (van der Schoot and van Bel, 1990; Sokolowska and Zagorska-Marek, 2012), these cells are thought to be specialized for radial transport. In conifers these cells are stacked to form narrow plates just one cell wide (i.e. the ray is ‘uniseriate’) or less frequently two cells wide (‘biseriate’). Angiosperm rays are typically ‘multiseriate’ (1–15+ cells wide) and may form two or more discrete size classes. There are Symplasmic networks in secondary xylem and phloem | 1831 Fig. 1. Cross-section of a stem with secondary vascular tissue. The cambial zone is sink tissue during active growth and is never a source, such that secondary phloem is adjacent to a strong sink along the entire length of a woody stem. A subset of axial and radial parenchyma function in carbohydrate and/or nitrogen storage, and therefore may act as sinks or sources depending on phenology. Radial parenchyma provide a route of symplasmic transport between xylem and phloem that must pass through the undifferentiated cambial initials and young derivatives. Symplasmic connections exist between ray and axial parenchyma, and subsets of both cell types are in direct contact with the transpiration stream, whereas contact with the assimilate transport stream is likely indirect. Both axial and radial parenchyma remain alive for many years after secondary xylem and phloem have ceased to conduct water and photosynthate, respectively. Xylem parenchyma die upon heartwood formation, prior to which they synthesize secondary metabolites and cell wall material (i.e. tyloses) and may further lignify. Subsets of phloem parenchyma expand, develop heavily lignified cell walls, and often proliferate via renewed mitotic divisions in nonconducting phloem, but they do not die until periderm formation. Note that the distribution of axial parenchyma shown here (short tangential bands) is generic and that many specialized configurations exist (this figure is available in colour at JXB online). important functional implications of ray width in that only the cells on the outer margins will be in contact with conducting elements. Given this, every parenchyma cell in a conifer ray will be in some contact with a tracheid or sieve cell, whereas cells in the centre of wide angiosperm rays will be isolated from vessels and sieve tubes. The terms ‘contact’ and ‘isolation’ cells have long been in use to make this distinction (Braun, 1970, 1984; Sauter and Kloth, 1986), but it is important to note that there is a great deal of variation in the configuration of conducting element–parenchyma contacts (Fig. 2). Cells at the longitudinal tips of rays (i.e. the top and bottom when viewed in tangential section) are often structurally quite different from procumbent cells and suggest functional specialization, but work in this area is almost exclusively based on anatomy. When two or more distinct cell types are present in a single ray it is described as ‘heterogeneous’, but the cell types present differ between xylem and phloem and between conifers and angiosperms such that this term is nonspecific. In conifers, xylem-side rays are either composed exclusively of procumbent cells (i.e. the ray is ‘homogenous’) or have ray tracheids at their tips. Ray tracheids are nearly identical to longitudinal tracheids in structure and hence dead at maturity, and although a function in radial transport of water has long been presumed, their importance in this capacity has recently been challenged (Barnard et al., 2013). In contrast, ‘erect’ parenchyma cells, which range from square to vertically elongate (radial–longitudinal length ratio 1:1–10), are often located at the tips of phloem-side rays in conifers and of both phloem- and xylem-side rays in angiosperms (Fig. 2). In conifers, Strasburger cells (also called ‘albuminous’ cells) are interspersed among erect cells and so considered part of the ray (Grillos and Smith, 1959; Alfieri and Kemp, 1983; Alfieri and Mottola, 1983; Allen and Hiatt, 1994). Found in all gymnosperms, Strasburger cells are thought to be functionally equivalent to companion cells in angiosperms and hence function in phloem loading (Sauter et al., 1976; Sauter, 1980). Plasmodesmatal connections between Strasburger cells and sieve cells are extensive and provide a direct link between radial and axial symplasmic transport, further highlighting the fact that short-distance transfer between diffuse local sources and sinks is occurring along the length of the long-distance transport pathway in secondary phloem. Companion cells are consistent features of the transport phloem of angiosperms, but with few exceptions they are not part of the radial system. It has been suggested that erect (also called ‘upright’) cells in angiosperm xylem-side rays are specialized to function in exchange of material with vessels (Höll, 1975; Sauter and Kloth, 1986), as in some taxa they alone are connected to vessels via pits in the cell wall (e.g. Populus and Salix). However, there is tremendous variation in patterns of ray parenchyma 1832 | Spicer Table 1. Volume proportion of secondary xylem occupied by radial and axial parenchyma Conifers have narrow rays (1 or 2 cells wide) and minimal axial parenchyma. Angiosperm rays are often much wider (commonly 3–6 cells, but up to 15 or more cells wide). Axial parenchyma ranges from minimal to abundant, especially in lianas and trees described as stem succulents. aLianas (woody vine); bPachycauls (bottle-tree growth form). Sources: DeSmidt, (1922), Myer (1922), Panshin and de Zeeuw (1980), Chapotin et al. (2006b), Brandes and Barros (2008), and Hearn (2009a). Angiosperms Acer Populus Betula Ulmus Fraxinus Carya Juglans Quercus Piptadeniaa Dalbergiaa Senegaliaa Adansoniab Adeniab Conifers Pinus Picea Larix Pseudotsuga Abies Cupressus Juniperus Radial parenchyma (% of volume) Axial parenchyma (% of volume) 12–18 10–14 10–16 10–18 12–16 17–26 10–17 15–40 15 12 8–14 70–80 + (total parenchyma) 40–95 + (total parenchyma) <0.1 <0.2 2 2–8 3–10 8–12 9–14 8–24 25 33 32–43 4–8 5–7 6–10 7 5–7 6 6–8 – – – – – <0.2 <0.2 vessel pitting and in many cases both procumbent and erect cells are highly pitted in walls shared with vessels such that both cell types are capable of exchange (Fig. 2). This would serve to maximize the symplast/apoplast contact area, which is important given that internal parenchyma cells are isolated. Similarly, the size and frequency of pits and/or plasmodesmatal connections linking parenchyma longitudinally and tangentially within a single ray have clear implications for the efficiency of transport within a ray, but these aspects are not well documented. It is striking that the erect cells typically form uniseriate portions of the ray (Fig. 2). Although the functional implications of this arrangement are not known, these radial cell files could link axial elements (either vessels or axial parenchyma) on opposite sides of a ray in a way that a given radial file in the multiseriate portion of the ray cannot. Axial parenchyma distribution also suggests functional specialization Axial parenchyma forms an intermediary between radial parenchyma and the axial conducting elements in secondary vascular tissue and is often arranged in distinct taxa-specific spatial patterns. In general these patterns are well documented in xylem where they are preserved in time (see Carlquist, 2001a for an excellent review), but far less so in phloem where parenchyma is difficult to distinguish from neighbouring sieve element and companion or Strasburger cells, and cellular organization is lost due to sieve element collapse and ray dilatation (see discussion on longevity). Individual cells are usually short in the axial direction and longitudinally aligned in strands (sometimes called ‘strand’ parenchyma, derived from a fusiform cambial initial that rapidly subdivides into several shorter cells; Larson, 1994), but in plants forming only small amounts of secondary growth (i.e. the unfortunately named ‘woody herbs’) there may be an additional type that is long and tapered, apparently derived from a fusiform initial without any further transverse divisions (Eames and MacDaniels, 1947; Zahur, 1959). Both types are present surrounding a vessel in Solanum lycopersicum in Fig. 3A (van der Schoot and van Bel, 1989a, b). In conifers, axial parenchyma constitutes a far lower proportion of secondary xylem than in angiosperms (Table 1) and may be lacking entirely, present as single strands scattered throughout the xylem, form short (2–6 cells wide) tangential bands, or be concentrated at the growth ring boundary (Panshin and de Zeeuw, 1980; Braun, 1984). In contrast, it is common for axial parenchyma in angiosperms to occupy anywhere from under 1% to over 25% of total xylem volume. It often surrounds vessels in partial or complete sheaths (Figs 3 and 4A) or forms wide tangential bands in which smaller vessels are nested (Carlquist, 2001a). This configuration may underscore the role of axial parenchyma in maintaining water transport in angiosperm vessels, several aspects of which are discussed later in the review. Where less abundant, axial parenchyma forms narrow tangential bands (Fig. 4B) or a one- to several-cell-thick band at the growth ring boundary (Fig. 4). This arrangement, which is also found in many conifers, is likely important during reinitiation of cambial activity in seasonal climates. Interestingly, in temperate regions these cells often overwinter in the cambial zone as partially differentiated cells (Jane, 1934; Chowdhury, 1936; Fuchs et al., 2010b). Together with partially differentiated phloem elements (including parenchyma; Grillos and Smith, 1959), these are the first cells of the growing season to complete maturation such that the newly activated cambium is located between two layers of parenchyma. The activities of these cells in relation to supporting new growth deserves further attention. Axial parenchyma in secondary phloem is often more abundant proportionally than in secondary xylem of both conifers and angiosperms, although it is more difficult to distinguish from other living elements. Strasburger and companion cells are in essence highly specialized parenchyma, but there is a rich body of literature on the structure and physiology of these cells and they are only included here in the context of symplasmic continuity with parenchyma. As in xylem, the volume of axial parenchyma in secondary phloem is lower and more consistent in conifers (sieve cells dominate) and typically exists as scattered strands, small Symplasmic networks in secondary xylem and phloem | 1833 Fig. 2. Ray parenchyma arrangement in angiosperm secondary xylem shown in radial (left) and tangential (right) sections. Vertical dashed lines indicate regions in contact with a vessel. Rays may be uniseriate (A–C), multiseriate (D, E, H, I), or contain both uniseriate and multiseriate regions (F, G, J–L). Erect parenchyma cells are either square or vertically elongate and occur at the upper and lower margins of rays (A, B, E, F, I, J) or in vertical bands that alternate with procumbent cells (D, G, K, L). Pits in parenchyma cell wall regions shared with vessels (delineated by a dashed outline) may occur in both types of ray parenchyma (A, D–G, I, K, L) or be absent in some (C, H) or all (B, J) procumbent cells. The degree of variation in ray structure suggests that similar variation exists in functional specialization. In some species, tile cells form via sequential radial divisions (i.e. formation of tangential walls) of developing procumbent cells (G). It is interesting to speculate on the function role of this massive proliferation of cell wall for radial transport. Rhomboid crystals, typically composed of calcium oxalate, are shown in the erect parenchyma cells of a few species (D, I, J). Species shown are (A) Guilfoylia monostylis, (B) Salix caerulea, (C) Eucalypus regnans, (D) Hopea nutans, (E) Panax elegans, (F) Endospermum peltatum, (G) Durio oxleyanas, (H) Acer pseudoplatanus, (I) Canarium mehenbethene, (J) Cyclostemon sp., (K) Madhuca utilis, (L) Palaquium galatoxylum. Figures adapted from Chattaway M. 1951. Morphological and functional variations in the rays of pored timbers. Australian Journal of Biological Sciences 4, 12–27, with permission from the publisher, CSIRO Publishing (http://www.publish.csiro.au/nid/280/ paper/BI9510012.htm). 1834 | Spicer Fig. 3. Vessel contacts and symplasmic connections among living elements in the secondary vascular cylinder of Solanum lycopersicum. (A) Elongate axial parenchyma (AP), parenchyma strands (PS), and radial contact cells (C) all form a symplast–apoplast interface at pits (not shown) in the walls shared with vessels (V) and are thus capable of exchange with the transpiration stream. (B) Intracellular injections with Lucifer Yellow CH demonstrate extensive symplasmic connections among living elements, where an asterisk indicates the cell that was impaled and arrows indicate intercellular dye transfer. The cambial zone is shown on the right as three adjacent files of four cells each, all of which have distinctly narrow radial widths. Three files of phloem cells are thus shown at the far right; everything to the left of the cambial zone is xylem. Axial parenchyma in contact with a vessel were strongly coupled (a) as were living fibres (e). Dye injected into xylem ray parenchyma moved to like-cells in the same radial file (c, d, i) as well as into adjacent living fibres (f). Dye injected into phloem ray parenchyma moved through the cambial zone into the xylem (j). Cambial cells showed axial dye transfer (h). Figures adapted from (A) van der Schoot C, van Bel AJE. 1989. Architecture of the internodal xylem of tomato (Solanum lycopersicum) with reference to longitudinal and lateral transfer. American Journal of Botany 76, 487–503, permission of the publisher, Botanical Society of America, and (B) van der Schoot C, van Bel AJE. 1990. Mapping membrane-potential differences and dye-coupling in internodal tissues of tomato (Solanum lycopersicon L.). Planta 182, 9–21, with permission of the publisher, Springer. clusters of strands, or short tangential bands that alternate with sieve and Strasburger cells, and fibres if present (Grillos and Smith, 1959; Alfieri and Evert, 1968; Alfieri and Kemp, 1983; Alfieri and Mottola, 1983; Castro et al., 2005, 2006). Axial parenchyma in angiosperm secondary phloem is more variable and although attempts have been made to categorize major pattern types, it is best described as a continuum, as with most anatomical characteristics. When production by the cambium is examined closely however, specific repeating patterns of alternating cell types are observed (Evert, 1963b; Alfieri and Kemp, 1983; Barlow and Lueck, 2006). When sparse, axial parenchyma is found in thin tangential bands; when abundant it forms the majority of the phloem in which clusters of sieve elements and companion cells are nested (+/–fibres; Zahur, 1959; den Outer, 1983, 1993). This latter condition is especially common in lianas (den Outer, 1993). Axial parenchyma in phloem appears to be symplasmically isolated from the conducting elements and associated companion and Strasburger cells (van Bel and Kempers, 1991; van Bel, 1996; van Bel and Ehlers, 2000; Evert, 2006), but prominent clusters of pits are found in the walls in contact with ray parenchyma. The nature of contacts among parenchyma and axial conducting elements Connections are formed where radial and axial parenchyma are in direct contact and where both types of parenchyma abut conducting elements of the xylem and phloem. Nearly all of these connections occur at pits, which are effectively holes in the cell wall. In the absence of pits, exchange of important materials may occur via the apoplast. When one considers the diversity of plants with secondary growth and the multitude of cell types present, the number of specific types of contacts is quite large. Only a few of these types have been studied in detail, but it is clear that in order to understand intercellular symplasmic transfer as well as exchange across the apoplast–symplast interface, the physical nature of these contacts should be studied in detail and with a comparative approach wherever possible. The apoplast–symplast interface in xylem occurs at cell wall pits occupied by a unique cell wall layer The site of exchange between the transpiration stream and either radial or axial parenchyma occurs at a unique interface Symplasmic networks in secondary xylem and phloem | 1835 Fig. 4. Axial parenchyma highlighted in black with image processing software (A, B) or left unstained and appearing white (C) in cross-sections of Fraxinus americana (A), Quercus rubra (B), and Adansonia za (C). Axial parenchyma forms a band at the growth ring boundary in F. americana and often completely surrounds latewood vessels (A; a large earlywood vessel is shown in the lower left, with small latewood vessels above), whereas in Q. rubra latewood, it forms thin tangential bands connecting latewood vessels surrounded by tracheids and limited parenchyma. In A. za, the proportion of parenchyma is extremely high (C), shown here with the cell walls of lignified elements heavily stained but the background matrix of both axial and ray parenchyma unstained and appearing white. * indicates a uniseriate ray; ** indicates a multiseriate ray. Bars, 50 µm (A, B) and 1 mm (C). Figures adapted from (A, B) Spicer R, Holbrook NM. 2007. Parenchyma cell respiration and survival in secondary xylem: does metabolic activity decline with cell age? Plant, Cell and Environment 30, 934–943, with permission of the publisher, Wiley, and (C) Chapotin SM, Razanameharizaka JH, Holbrook NM. 2006. A biomechanical perspective on the role of large stem volume and high water content in baobab trees (Adansonia spp.; Bombacaceae). American Journal of Botany 93, 1251–1264, with permission of the publisher, Botanical Society of America. within the cell wall pits. In conifers, all ray parenchyma cells have pitting in the wall shared with tracheids and show taxaspecific variation in pit size and shape (Panshin and de Zeeuw, 1980). In fact, cross-field pitting, where ‘cross-field’ refers to the rectangular region of cell wall shared between ray parenchyma and tracheids, is one of the most useful characters for identifying conifer woods. Pits range in shape from single broad ovals or quadrangles that nearly fill the cross-field to up to six small, round pits with prominent arched borders in the tracheid wall (Phillips, 1941). Although parenchyma-vessel pitting in angiosperms has received less attention in terms of comparative anatomy, there is greater variation in the types of parenchyma present in xylem-side rays and pitting ranges from closely spaced pits that are small and round to larger, oblong pits (Fig. 2). As already noted, these pits may occur along the entire length of an angiosperm ray where it contacts a vessel, or in isolated regions. A unique cell wall layer—referred to here as the amorphous layer—is found in most parenchyma in secondary xylem, and its chemical composition and function deserve a great deal more attention. Most radial and axial parenchyma cells in xylem have some form of secondary cell wall, although it typically lacks the complex laminar structure of tracheary elements and may be more comparable to a thickened primary wall (Panshin and de Zeeuw, 1980). Once secondary cell wall formation is complete, an additional layer of loosely woven and irregularly aligned microfibrils is deposited with hemicelluloses in a matrix rich in pectin but with little-to-no lignin in the space between the plasma membrane and newly deposited cell wall (Schmid and Machado, 1968; Chafe, 1974; Schaffer and Wisniewski, 1989; Barnett et al., 1993). This amorphous layer is thickest near pits that face a vessel or tracheid such that it fills the parenchyma-side pit cavity (Fig. 5), but it has consistently been shown to surround the entire parenchyma protoplast in a thin layer (Schaffer and Wisniewski, 1989; Murakami et al., 1999). Various functions have been ascribed to the amorphous layer over the past 50 years but the simple truth is that we have very little idea of its role(s), largely because work is limited to ultrastructural studies. It appears to be involved in tylosis formation (Foster, 1967; Meyer and Cote, 1968; Rioux et al., 1988; Bonsen and Kucera, 1990; Rudelle et al., 2005) but it is also present in species in which tyloses do not form, including conifers. It has been hypothesized to protect the parenchyma protoplast from changes in hydrostatic pressure in the adjacent transpiration stream (van Bel and van der Schoot, 1988), but it also surrounds the protoplast on all sides. The amorphous layer has also been suggested to form an important barrier that prevents damaging ice crystal formation and allows parenchyma cells to supercool in freezing conditions (Wisniewski and Davis, 1989; Wisniewski et al., 1991). Here, pectin in the amorphous layer is thought to function as a hydrogel and protect against freeze-induced desiccation. Perhaps the most compelling argument however is that this loose layer forms an extension of the apoplast in cells where the cell wall may be quite impermeable due to a dense laminate structure and lignification (Barnett et al., 1993), thereby greatly expanding the surface area available for exchange across the plasma membrane, much like transfer cell wall invaginations (Pate and Gunning, 1972; Offler et al., 2003). This would be in keeping with the observation that the amorphous layer also fills cavities of ‘blind pits’ (van der Schoot and van Bel, 1989a), pits 1836 | Spicer Fig. 5. Axial parenchyma (AP) cell in Cercocarpus betuloides shown adjacent to a vessel (V) and two tracheids (T). Arrowheads point to the loosely fibrillar amorphous layer filling the two halfbordered pit cavities facing the vessel and the cavity of a simple pit facing a tracheid. Bar, 2.5 µm. Figure adapted from Dute R, Patel J, Jansen S. 2010. Torus-bearing pit membranes in Cercocarpus. IAWA Journal 31, 53–66, with permission of the publisher, Brill. in parenchyma that lead simply to vessel or tracheid walls. Usually interpreted as developmental mishaps and effectively dead ends, they can be quite abundant and could also serve to increase the area of the parenchyma plasma membrane exposed to the apoplast. Most likely the amorphous layer serves many of these functions and shows taxonomic variation in its specialization. Lastly, it should be noted that many names have been used to describe this and related loosely fibrillar layers, including the ‘protective layer’ and ‘isotropic layer’. Although the protective layer has been used most widely, the amorphous layer is preferred here because it describes the structure without implying any function. Symplasmic continuity among secondary vascular parenchyma is extensive and dynamic Extensive symplasmic connections exist among the various types of parenchyma cells in secondary vascular tissue, typically in the form of simple pits (i.e. pits without arched borders) crossed by multiple plasmodesmata. A crude—yet still highly labour intensive—assessment of potential symplasmic continuity can be made through quantitative ultrastructural studies, but a complete picture of symplasmic fields requires tests to determine the size exclusion limit of plasmodesmatal connections and in vivo imaging following symplasmic dye injection. Given the thick lignified walls that are characteristic of secondary xylem elements, phloem fibres, and even many parenchyma cells, this is naturally extremely challenging work. Combine these technical difficulties with the diversity of cell types and distribution patterns and it is easy to appreciate why so little progress has been made in this area. Nevertheless, a few systems are well characterized (S. lycopersicum and Populus spp.) and suggest that symplasmic continuity shifts rapidly during cell differentiation, and that once mature there are symplasmic fields of cells that are partially or wholly isolated from each other. There is also evidence for taxonomic variation in these patterns, as well as seasonal shifts in symplasmic continuity in the temperate zone. As already noted, the central ray cells are often radially elongate with high plasmodesmatal densities in their tangential walls and thus appear specialized to transport material radially (Sauter and Kloth, 1986; van der Schoot and van Bel, 1989a; Chaffey and Barlow, 2001). Although dyecoupling experiments are scarce, there is some evidence for continuity through xylem-side and phloem-side rays (van der Schoot and van Bel, 1990; Sokolowska and Zagorska-Marek, 2012), and circumstantial evidence as well as immunolabelling suggests that carbohydrates and nitrogenous compounds (Chaffey and Barlow, 2001; Fuchs et al., 2010a) are trafficked through the rays via plasmodesmata. Little is known about symplasmic continuity within rays in the longitudinal and radial directions, but pitting in radial and transverse walls is a diagnostic feature (Panshin and de Zeeuw, 1980) and the potential for continuity can be inferred from anatomy in a few cases. Pain-staking studies of symplasmic continuity in S. lycopersicum internodes in which dye coupling, metabolic activity, and membrane potential were mapped onto a threedimensional reconstruction that includes living fibres, xylemand phloem-side rays cells, and cambial derivatives illustrate both the complexity and taxon-specific nature of these symplasmic networks (Fig. 3). An improved understanding of symplasmic continuity in secondary growth is particularly important in the context of the vascular cambium, a large and critically important sink that must not only radially import all materials needed for cell division, expansion and differentiation, but also relies on (presumably) positional signals from neighbours to determine cell fate. Recent work shows that the number of plasmodesmatal connections between cells in the cambial zone changes during differentiation (Ehlers and van Bel, 2010; Fuchs et al., 2010b, 2011). During active growth in Populus, cambial initials are linked via plasmodesmata in their tangential walls to the immediate derivatives on either side, but there is then a dramatic but transient increase in plasmodesmatal frequency in the same wall between mother and daughter cells (i.e. radial continuity increases). In later stages of maturation, tangential wall plasmodesmatal numbers decrease, and they decrease more rapidly on the xylem side. Cambial initials appear to have very few plasmodesmata in their radial walls (i.e. tangential continuity is low), but these also increase in number in their immediate derivatives. During dormancy, the number of plasmodesmatal connections is greatly reduced in all directions but increases upon cambial reactivation in the spring. Dye coupling in the cambial zone in S. lycopersicum supported roughly analogous changes in plasmodesmatal number, with injections into immediate cambium derivatives showing movement through one or more cells in the Symplasmic networks in secondary xylem and phloem | 1837 directions of both xylem and phloem, whereas older phloemside derivatives appeared isolated and longitudinal transfer of dye was extremely rare (Ehlers and van Bel, 2010). Given the potential for regulation of plasmodesmatal aperture and role of phloem- and/or neighbour-derived signalling molecules, it is interesting to speculate on the effects of transient isolation during xylem and phloem differentiation. Unfortunately, the major technical barriers to working with woody tissue are likely to continue to impede progress in this area in the absence of a major advance in live cell imaging technology. Symplasmic connections among axial and radial parenchyma in both xylem and phloem are perhaps the least characterized. Axial parenchyma surrounding vessels in S. lycopersicum show ready axial transfer of dye (Fig. 3B), as do clusters of living fibres, and dye moves freely from an injected ray cell into a fibre cluster (van der Schoot and van Bel, 1990). Similarly, injections into mature xylem and phloem parenchyma result in dye movement into like cell types both radially and longitudinally (Ehlers and van Bel, 2010). Of particular interest is the degree of symplasmic isolation of sieve element/companion cell and sieve cell/Strasburger cell complexes (each pair of course is in high symplasmic continuity) from the adjacent axial and radial parenchyma in the phloem. Substantial evidence exists for complete isolation in a number of herbaceous and woody species (van Bel and Kempers, 1991; van Bel and van Rijen, 1994; Hafke et al., 2005; Evert, 2006; with particularly good discussions in van Bel, 1996; van Bel and Ehlers, 2000). If phloem conducting elements and their respective companion/Strasburger cells are symplasmically isolated from adjacent parenchyma, any exchange of materials would require release into the apoplast and subsequent transfer across a plasma membrane. Cell-specific localization studies of transport proteins in the plasma membrane of parenchyma in both xylem and phloem are welcome additions to this field, particularly those able to resolve asymmetric localization with respect to orientation of neighbouring cells. Work in this area is minimal and almost exclusively focused on vessel-associated xylem parenchyma. Relevant findings are included in the following sections on storage and embolism repair functions. Functions of parenchyma in secondary vascular tissue Parenchyma cells function in many different and overlapping capacities to facilitate and maintain transport in secondary vascular tissue. Although a traditional breakdown of the most obvious functions includes carbohydrate storage, radial transport, and xylem–phloem exchange, these categories are too broad to be useful. For example, some axial and radial parenchyma store substantial amounts of carbohydrates in the form of starch, but the timescale over which that starch is broken down and mobilized (e.g. seasonally versus diurnally), its final point of use (e.g. spring leaf expansion, cambial growth, embolism repair, freezing tolerance), and the means by which it travels (e.g. symplasmically through rays or release into the apoplast) all vary. While much of the work on parenchyma storage and implied transport is descriptive (e.g. a fair body of work documents temporal changes in the cellular contents of parenchyma), it is rarely paired with experimental work to establish the fates of those contents. Similarly, experimental work in plant physiology may implicate parenchyma but rarely includes cell-type-specific observations. An attempt is made here to discuss specific parenchyma functions in the context of whole-plant physiology. Some of the most promising research areas are those that combine physiological manipulations with cellular level gene expression, protein localization, and quantitative measures of cell composition. Secondary vascular parenchyma serve as transfer stations for carbohydrates, nitrogenous compounds, and water The function of secondary vascular parenchyma in seasonal storage and short- and long-term redistribution of organic and inorganic compounds is one of the most important and arguably most studied. Many observations are descriptive, based on macroscopic tissue samples, and specific to the species and environment studied and therefore difficult to generalize. However, it is clear that subsets of parenchyma in secondary xylem and phloem store carbohydrates as soluble sugars, starch, and/or lipids (Höll, 2000) as well as nitrogen in the form of vegetation storage proteins (Stepien et al., 1994). Excellent reviews in this area include Höll (1975, 2000), Pate (1975) van Bel (1990, 1995), Pate and Jeschke (1995), and Hoch et al. (2003) and only a brief summary is included here. There is also increasing recognition that parenchyma in secondary xylem function in water storage and recirculation between xylem and phloem (Chapotin et al., 2006b; Holtta et al., 2006; Sevanto et al., 2011; Hearn et al., 2013). The relationships are complex, but these studies are some of the most informative regarding the role of parenchyma in xylem and phloem exchange. Despite climate- and taxa-specific variation, there are several patterns of seasonal carbohydrate storage in secondary tissues with respect to growth. Following leaf flush and expansion in temperate deciduous trees, starch stores increase in xylem parenchyma throughout the period of active growth (Sauter and Kloth, 1987; Kozlowski, 1992; Sauter and Neumann, 1994; Sauter and van Cleve, 1994; Sauter and Wellenkamp, 1998; Barbaroux and Breda, 2002; Hoch et al., 2003; Landhausser and Lieffers, 2003; Wong et al., 2003; Bazot et al., 2013). Where present, stored lipids start to increase as leaves begin to senesce (Sauter and Neumann, 1994; Hoch et al., 2003). Levels of stored starch gradually decrease throughout dormancy and then decrease significantly at the onset of bud break and cambial activity, usually corresponding to a transient increase in sugar levels in both xylem and phloem sap. There are also reports of soluble sugar stores increasing during dormancy in cold climates and in response to chilling, suggesting a role in freezing tolerance (Sauter and Kloth, 1987; Sauter and Neumann, 1994; Wong et al., 2003). Similar seasonal changes in starch and soluble sugars have been reported in the phloem (Landhausser and Lieffers, 2003; Bazot et al., 2013). Although rarely documented, lipid 1838 | Spicer stores show far less seasonal variation than starch (Hoch et al., 2002, 2003). In the seasonally dry tropics, starch storage increases prior to the onset of the dry season and there is evidence of mobilization to support leaf flush (Mooney et al., 1992; Latt et al., 2001; Newell et al., 2002; Wurth et al., 2005). In contrast, in conifers the previous-year needles are thought to provide new growth with the needed assimilates and a large decrease in stored carbohydrates at budbreak is lacking (Höll, 1985; Fischer and Höll, 1991, 1992). Overlaid onto seasonal patterns of storage are differences in tissue age and there is evidence that starch stored in parenchyma closer to the cambium (e.g. in younger cells) or the foliage (e.g. in small twigs) show the largest fluctuations (Bullock, 1992; Mooney et al., 1992; Barbaroux and Breda, 2002; Landhausser and Lieffers, 2003; Wong et al., 2003; Chapotin et al., 2006b). Nitrogen is stored in both xylem and phloem parenchyma as vegetative storage proteins localized to the vacuole (Sauter et al., 1988; Sauter and van Cleve, 1989; Sauter et al., 1989; Wetzel and Greenwood, 1991; Harms and Sauter, 1992; Stepien et al., 1994). Protein bodies increase in abundance at the end of the growing season as leaves senesce and then decrease in the spring (Harms and Sauter, 1991, 1992; Langheinrich and Tischner, 1991; Lawrence et al., 1997; Sauter and Wellenkamp, 1998; Bazot et al., 2013), concomitant with a transient appearance in xylem sap (van Cleve et al., 1991). Xylem parenchyma are active in retrieving amino acids from the transpiration stream and transferring them to the phloem, as well as effectively concentrating them in xylem streams destined for young leaves (van Bel et al., 1979; van Bel and van der Schoot, 1979; Dickson et al., 1985; Vogelmann et al., 1985; Pate and Jeschke, 1995). The extent to which secondary vascular parenchyma function in water storage and/or recirculation between the xylem and phloem is gaining increasing attention. Parenchyma in secondary xylem are thought to store and release water, most notably in stems with high parenchyma volumes (Mooney et al., 1992; Chapotin et al., 2006a,b,c; Hearn, 2009b; Hearn et al., 2013). However, carbon stores are utilized for many competing processes (e.g. supporting new growth; Mooney et al., 1992; Sauter and van Cleve, 1994; Sauter, 2000; Latt et al., 2001; Barbaroux and Breda, 2002; Newell et al., 2002; Hoch et al., 2003; Wong et al., 2003; Wurth et al., 2005), freezing tolerance (Sauter et al., 1996; Hoch et al., 2002; Kuroda et al., 2003; Decourteix et al., 2006; Kasuga et al., 2007; Takata et al., 2007), recovery from episodic defoliation (Gregory and Wargo, 1986; Landhausser and Lieffers, 2012), defence (Imaji and Seiwa, 2010; Goodsman et al., 2013; Lahr and Krokene, 2013), and transpiration stream maintenance and repair (discussed below), all of which represent important tradeoffs. A renewed interest in the effects of drought stress on forest trees highlights the roles of carbon storage and radial exchange of water between secondary xylem and phloem (Holtta et al., 2006; Sala et al., 2010; Sevanto et al., 2011; Mencuccini et al., 2013; Sevanto et al., 2013). Field measurements, carbon supply manipulations, and models of stem diameter changes in trees demonstrate the importance of radial exchange of water between xylem and phloem and the fact that axial transport of water and assimilates are linked (Holtta et al., 2006; De Schepper and Steppe, 2010; De Schepper et al., 2010; Sevanto et al., 2011; Mencuccini et al., 2013). For instance, increasing tension in the xylem under drought requires an increase in phloem osmotic pressure to sustain assimilate transport. If soluble sugars released from stored carbon are used as osmoticum, internal stores of carbon—stores that will be needed in prolonged drought if stomata close—will be depleted. Although parenchyma play key roles in this process, little is known about the path of radial water movement or ultimate source of osmoticum. This key area of research illustrates the importance of xylem–phloem exchange in whole-plant resource partitioning in trees, where mechanistic studies have largely been restricted to smaller plants of agricultural importance (Pate, 1975; van Bel, 1990, 1995; Pate and Jeschke, 1995). The mechanism of transport within the parenchyma symplast is still poorly understood One of the most striking gaps in our understanding of parenchyma function in storage is the lack of a proposed mechanism for transport through the various parenchyma symplasmic connections (i.e. how materials are physically moved once they have been signalled to be mobilized). Early work focused on the high activity of acid phosphatases in ray parenchyma, which is highest during active growth and localized more heavily to the tangential cell walls facing the cambium in both xylem and phloem (Höll, 1975; Fink, 1982). However, this would imply transmembrane transport whereas most evidence suggests that cell-cell transport is symplasmic, including plasmodesmatal counts, dye coupling, and cytoskeleton immunolocalization (Chaffey and Barlow, 2001). Particularly convincing is the immunolocalization of protein particles to within plasmodesmata during budbreak. More focus has been placed on the mechanism of symplast– apoplast exchange, where both H+-ATPases (Fromard et al., 1995; Alves et al., 2001, 2004, 2007; Arend et al., 2002, 2004) and sucrose and hexose transporters (Decourteix et al., 2006, 2008) have been localized to the plasma membranes of parenchyma cells associated directly with vessels (‘vessel-associated cells’ or ‘contact cells’). Interestingly however, both types of sugar transporters were also localized to radial parenchyma. Given that radial transport can occur both centripetally and centrifugally, it is interesting to ask whether this can occur at the same time in a given tissue (e.g. in separate rays) or whether a single transport direction dominates. A great deal more work needs to be done in this area. Parenchyma function in xylem embolism repair Due to the strong cohesive forces between water molecules, liquid water is actually able to sustain a negative hydrostatic pressure, or tension (Apfel, 1972). Indeed, it is this critical property of water coupled with its tendency to adhere to the hydrophilic components of the cell wall (namely, cellulose microfibrils) that provides the foundation for the cohesion-tension theory of xylem transport. Although a positive hydrostatic pressure in the xylem can occur, during Symplasmic networks in secondary xylem and phloem | 1839 active transpiration the xylem water column is under varying degrees of tension and hence considered ‘metastable’, and in this state is at risk of cavitating due the expansion of small gas bubbles (Sperry and Tyree, 1988; Tyree and Sperry, 1989). If these gas bubbles expand to block liquid flow through a conduit, an ‘embolism’ is said to have formed, and the hydraulic capacity of the shoot in which it forms is correspondingly reduced. The field of cavitation research is large, active, and well beyond the scope of this review, but for one fact: there is a body of evidence suggesting that parenchyma in secondary xylem play a role in refilling embolized conduits with water. Embolisms may form either under increasing tension in the xylem (e.g. drought-induced embolism; Sperry and Tyree, 1988) or following freezing when gas bubbles trapped during ice formation expand upon thawing (freeze-induced embolism; Mayr and Sperry, 2010). There is variation in the time course of both embolism formation and reversal, with freezeinduced embolism repair generally thought to occur seasonally. In contrast, drought-induced embolism in angiosperms has increasingly been viewed as highly dynamic process, with some evidence for diurnal cycles of cavitation and refilling (Zwieniecki and Holbrook, 1998; Zwieniecki et al., 2000; Bucci et al., 2003), leaving puzzling questions as to how water under positive pressure might be driven into empty conduits adjacent to conduits filled with water under tension (Holbrook and Zwieniecki, 1999; Zwieniecki and Holbrook, 2009). Far less is known about refilling conifer tracheids (but see McCulloh et al., 2011). It is especially important to note that at the time of writing this review, a recent discovery that evidence for diurnal cycles of cavitation repair in several angiosperm species is the result of an experimental artefact (Wheeler et al., 2013) may call into question a great many experimental results in the area of xylem refilling (Sperry, 2013). However, there is still compelling evidence for refilling of embolized conduits in xylem over longer time scales and/or under minimal tension (e.g. in the absence of sufficient root-generated pressure), and several lines of research suggest the importance of parenchyma in this process. Most recently, in vivo imaging with high resolution X-ray computed tomography shows water droplets forming, expanding, and coalescing during vessel refilling in grapevine (Brodersen et al., 2010). These exciting images confirm those of prior cryo-SEM studies (Canny, 1997; Utsumi et al., 1998; Canny et al., 2007) and show droplets specifically forming in the proximity of contacts with ray parenchyma. Both techniques are thus capable of discerning the contributions of specific parenchyma cell types. Many models of refilling dynamics have been proposed and all rely on the activity of parenchyma cells in contact with xylem vessels to draw water into the embolized conduit (Brodersen and McElrone, 2013). In most cases, this is thought to be achieved by the deposition of solutes in the form of sugars, inorganic cations, or a combination of both. One notable exception is a model in which a high osmotic pressure in isolated cells creates a high hydrostatic pressure surrounding the parenchyma adjacent to embolized vessels such that water is physically forced into the xylem via reverse osmosis (Canny, 1997), but this model and its parent, the compensating pressure theory of sap ascent (Canny, 1995, 1998), have received little to no acceptance (Comstock, 1999; Stiller and Sperry, 1999). There is considerable evidence that partially or fully girdling the phloem (i.e. reducing phloem transport) reduces or prevents xylem refilling (Salleo et al., 1996, 2004, 2006; Zwieniecki et al., 2000; Bucci et al., 2003) and that starch levels in parenchyma adjacent to xylem vessels decline on a timescale coincident with apparent refilling (Bucci et al., 2003; Sakr et al., 2003; Salleo et al., 2004, 2006, 2009; Secchi and Zwieniecki, 2011). There is also evidence that inhibiting the activity of several H+-ATPases in xylem parenchyma prevents refilling, while stimulating the activity of H+-ATPase promotes it (Salleo et al., 2004), suggesting that proton-coupled membrane transport is involved. Particularly exciting is a report using an elegant technique to collect xylem sap from functional and nonfunctional (i.e. embolized) vessels separately in water stressed trees. Sap from nonfunctional vessels—which was substantial in volume— had a significantly lower pH and up to 5-times the osmotic potential as functional vessels, where inorganic ions made up only half of the osmoticum, the rest coming from soluble sugars (Secchi and Zwieniecki, 2012). Gene expression studies provide further evidence for a role of parenchyma in osmotically driven water movement into embolized vessels, with parenchyma in the secondary xylem of Populus showing an upregulation of genes encoding aquaporins (primarily PtPIP2s), sucrose transporters (PtSUC2.1 and PtSUT2b, albeit transient), di- and tri-valent cation tansporters, and α- and β-amylases in response to artificially induced embolism (Secchi and Zwieniecki, 2010, 2001; Secchi et al., 2011). Although not strictly in secondary vascular tissue, similar changes in gene expression were found in petioles during diurnal cycles of water stress and recovery in grape (Perrone et al., 2012). As key candidate genes and transport proteins are identified, reporter constructs and immunolabelling studies should allow for cell-specific resolution of parenchyma activity in xylem refilling. Parenchyma in secondary xylem may directly affect hydraulic conductivity through ion exchange Both radial and axial parenchyma in secondary xylem have direct access to the transpiration stream where the plasma membrane (and amorphous layer) abut a vessel or tracheid pit, and some evidence suggests that their activity could have rapid and reversible effects on hydraulic conductivity. Flow rates through vessels have been shown to increase by up to 2.5 times in response to low (i.e. physiologically relevant) concentrations of disassociating solutes (KCl, NaCl, KNO3, CaCl2) in the xylem sap solution (Zimmermann, 1978; van Ieperen et al., 2000; Zwieniecki et al., 2001; Gasco et al., 2006). This effect is independent of the presence of living parenchyma cells and thus thought to be localized to the connections between vessels (i.e. flow through a single vessel was unaffected, but see van Ieperen et al., 2000 for contrasting results), namely the intervessel pit membranes, which are composed of partially hydrolysed primary cell wall material (Choat et al., 2008). Intervessel pit ‘membranes’ consist largely of loosely woven cellulose microfibrils and bear no relationship to true 1840 | Spicer plasma membranes. One hypothesis is that residual pectins in the pit membrane function as a hydrogel and shrink and swell in response to ionic interactions, effectively changing the size of the pores through which water flows (Zwieniecki et al., 2001; Gasco et al., 2006; van Ieperen, 2007). However, there is some evidence that pectins are effectively removed from the pit membrane during enzymatic hydrolysis of the primary wall/middle lamella complex in angiosperms (O’Brien and Thimann, 1967; O’Brien, 1970; Butterfield and Meylan, 1982; Plavcova and Hacke, 2011; van Doorn et al., 2011; Kim and Daniel, 2013). Alternative mechanisms include (a) other polymers like lignin and hemicelluloses shrinking and swelling via ionic interactions (van Doorn et al., 2011) and (b) the viscoelectric effect, in which charge interactions effectively alter the viscosity of a fluid, causing an apparent change in conductivity (Santiago et al., 2013). Regardless of the mechanism, it is thought that xylem parenchyma could significantly alter flow by changing the ionic composition and/or pH of the transpiration stream. This would allow plants to spatially regulate xylem resistance and potentially compensate for lost hydraulic capacity due to embolism, as well as favour transport to or from certain xylem sectors (Zwieniecki et al., 2003). Although the in planta significance of this effect remains unclear (van Ieperen, 2007), there is evidence that phloem-derived ions added to the transpiration stream can increase hydraulic conductivity of the xylem (Zwieniecki et al., 2004). Potassium ions (K+) could be loaded into the xylem via recycling from the phloem (Zwieniecki et al., 2001, 2004), and H+-ATPases in xylem parenchyma membranes could modify pH (Fromard et al., 1995). However, there is also evidence that the inclusion of physiologically realistic Ca2+ concentrations greatly decreases the ion-mediated effect on hydraulic conductivity (van Ieperen and van Gelder, 2006), calling into question the biological significance of the effect (van Ieperen, 2007). It is not clear how widespread this phenomenon is however, as some woody species maintain a significant effect of KCl even with the inclusion of Ca2+ (Nardini et al., 2007). Lastly, it has been shown that an inverse relationship exists across all vascular plants between the degree of primary wall lignification and the magnitude of ion-mediated hydraulic response (Boyce et al., 2004), suggesting large-scale evolutionary implications of this effect. Interestingly, the authors attribute the relationship to an effective shielding of pectins by lignin deposition, but it could also easily support the alternative hypothesis proposed by van Doorn (2011). Clearly more work is needed to understand the true in planta capacity of parenchyma to regulate xylem resistance. Parenchyma exhibit extreme longevity, remain pluripotent, and serve to protect the integrity of the stem Most axial and radial parenchyma in secondary vascular tissue remain alive after the conducting elements cease to function, underscoring that their roles are complex and may change with age. In senescing secondary xylem, parenchyma help defend tissue against microorganisms by rapidly synthesizing phenolic compounds (Chattaway, 1952; Bamber and Fukazawa, 1985; Magel, 2000) and by plugging both vessels and resin canals with newly produced cell wall material (i.e. tyloses and tylosoids, respectively), gums, and resins (Chattaway, 1949; Czaninski, 1977; Bonsen and Kucera, 1990). These activities occur shortly before parenchyma cells die and characterize heartwood formation, in which a nonconducting cylinder devoid of all living cells is compartmentalized in the centre of the stem (Chattaway, 1952; Spicer, 2005). In angiosperms in which only the outermost xylem is conductive, parenchyma may live for 5–25+ years after water transport ceases (Spicer and Holbrook, 2005, 2007; Chapotin et al., 2006b), continuing to function in storage and wound response until the onset of heartwood formation or tree death. Interestingly however, there is evidence that axial parenchyma surrounding vessels die much earlier than ray parenchyma in the highly parenchymatous Adansonia (Chapotin et al., 2006b). Parenchyma in secondary phloem also remain alive well after the tissue has ceased conducting photosynthate, which is usually at the end of the first growing season (or ‘flush’ of growth in nontemperate regions) and rarely more than two (Esau, 1948, 1950, 1964; Schneider, 1955; Evert, 1961, 1963a; Alfieri and Evert, 1968; Alfieri and Kemp, 1983). Although the Strasburger and companion cells die when their partnered conducting elements form definitive callose and lose their protoplast, many ray and axial parenchyma remain alive, in some cases for 20+ years. In nonconducting phloem, axial parenchyma cells expand in diameter, contributing to the collapse of sieve elements. Some axial parenchyma develop thick lignified walls at this point, effectively differentiating into sclereids late in life (Esau, 1950), functioning in mechanical support and defence. An equally conspicuous change is in the radial parenchyma, which expand in tangential diameter and often undergo renewed divisions in a process called ‘ray dilatation’ (Schneider, 1955). This dramatically increases the tangential diameter of a subset of the rays, thereby allowing the phloem to keep pace with the expanding circumference of the stem and remain intact. The ability of both ray and axial parenchyma cells to divide is retained throughout their life, such that these cells also function in wound response through the formation of callus tissue (Wargo, 1977; Shigo, 1984). Highly parenchymatous stems like pachycauls and lianas (discussed below; Table 1) appear to have particularly active parenchyma in this regard, which may be an adaptation to having mechanically weak, easily injured stems (Dobbins and Fisher, 1986; Fisher, 1981; Fisher and Ewers, 1989, 1991). Repeated evolution of parenchymatous secondary tissue underscores the importance of parenchyma in whole-plant physiology A very different approach to understanding the physiological significance of parenchyma at the whole-plant level is to consider its role in evolutionary diversification. Although it is beyond the scope of this review to cover these patterns in any detail, there are several growth forms in angiosperms in which an enormous amount of parenchyma is produced by the Symplasmic networks in secondary xylem and phloem | 1841 vascular cambium, including lianas, pachycauls (swollen stems, or bottle-trees), and a subset of stem or root tubers (Table 1). The diverse and often overlapping functions of parenchyma are encapsulated within these unique growth forms: parenchyma in lianas allows for increased stem flexibility (Gartner, 1991; Putz and Holbrook, 1991) and recovery from injury (Fisher and Ewers, 1991), pachycaulous stems store both carbohydrates and water as part of a complex suite of adaptations to arid environments (Borchert, 1994; Chapotin et al., 2006b; Hearn, 2009b; Hearn et al., 2013), and subterranean tubers store carbohydrates for survival through cold or dry periods (Pate and Dixon, 1981; Hearn, 2009b). Within each of these growth forms are multiple developmental paths to producing large amounts of parenchyma, some of which occur together in the same organ, while some singularly characterize whole species or genera. Each of these developmental paths has evolved independently multiple times (Hearn, 2009b; Hearn et al., 2013) and most result in wide bands of parenchyma that alternate with vascular tissue, underscoring the importance of a close association of the two tissues. This evolution of development approach also highlights the inextricable link between anatomy and physiology, where selective pressures act on both to produce adaptations to particular environments. Highly parenchymatous secondary growth may be a product of a vascular cambium functioning ‘normally’ but simply producing large amounts of parenchyma, but more typically results from cambial activity that is unusual when compared to the majority of trees and shrubs. The production of successive or ‘supernumery’ cambia is common in lianas, pachycauls, and tubers (e.g. Beta spp.; Carlquist, 2001b). Here, a normal cambium forms and produces secondary xylem and phloem in the expected positions, but later a new cambium dedifferentiates from parenchyma, first in the cortex and then later in the secondary phloem (Fahn and Zimmermann, 1982; Carlquist, 2001b, 2007; Rajput et al., 2008, 2012a,b, c; Robert et al., 2011). Each new cambium produces a disproportionate amount of parenchyma (usually axial xylem parenchyma but variation exists and standard definitions are difficult to apply) as well as conducting elements, the end result being a highly parenchymatous organ with repeating layers of both xylem and phloem. A second means to build up large volumes of parenchyma is to allow parenchyma to proliferate (renew mitotic divisions) at some distance from the cambium. This occurs in secondary phloem normally during ray dilatation but is common in the xylem (less commonly the phloem) of pachycauls (Carlquist, 1998; Chapotin et al., 2006b; Hearn, 2009a,b). A related phenomenon occurs when alternating tangential segments of the cambium produce significantly different ratios of xylem/phloem such that the cambium becomes furrowed and/or disjunct (i.e. the outward expansion of alternating segments occurs at different rates). This produces a stem with wedges or strips of phloem running deep into the xylem and is usually associated with proliferation of parenchyma as well (Carlquist, 2001b; Pace et al., 2009, 2011; Angyalossy et al., 2012). Lastly, many lianas achieve high proportions of parenchyma by having exceptionally wide, tall, unlignified rays, some of which may undergo postcambial proliferation (Carlquist, 1991; Angyalossy et al., 2012). Conclusion It is difficult to overstate the importance of parenchyma in supporting transport in secondary vascular tissues. Although our understanding of the physicochemical mechanisms driving xylem and phloem transport have improved dramatically, we still lack a fundamental understanding of the mechanism behind radial transport. Studies focused on the apoplast– symplast interface and plasma membrane transporters have provided a good foundation for xylem/parenchyma exchange, but the role of the amorphous layer filling pit cavities is still poorly understood. Although studies of transporter gene expression and protein quantification are welcome additions, emphasis should be placed on cell-specific techniques (in situ hybridization, immunolocalization, and reporter constructs) and applied to secondary tissues of different ages and physical positions within the stem. 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