Research

Comparison of phloem and xylem hydraulic architecture in Picea abies stems Tuula Jyske1 and Teemu H€ oltt€a2 1

Vantaa Research Unit, Finnish Forest Research Institute, PO Box 18, FI-01301 Vantaa, Finland; 2Department of Forest Sciences, University of Helsinki, PO Box 27, FI-00014 Helsinki,

Finland

Summary Author for correspondence: Tuula Jyske Tel: +358 40 801 5588 Email: [email protected] Received: 31 March 2014 Accepted: 6 July 2014

New Phytologist (2015) 205: 102–115 doi: 10.1111/nph.12973

Key words: allometry, conductivity, conduit frequency, conduit tapering, conifers, phloem sieve cells, Picea abies, xylem tracheids.


The hydraulic properties of xylem and phloem differ but the magnitude and functional consequences of the differences are not well understood.
Phloem and xylem functional areas, hydraulic conduit diameters and conduit frequency along the stems of Picea abies trees were measured and expressed as allometric functions of stem diameter and distance from stem apex. Conductivities of phloem and xylem were estimated from these scaling relations.
Compared with xylem, phloem conduits were smaller and occupied a slightly larger fraction of conducting tissue area. Ten times more xylem than phloem was annually produced along the stem. Scaling of the conduit diameters and cross-sectional areas with stem diameter were very similar in phloem and xylem. Phloem and xylem conduits scaled also similarly with distance from stem apex; widening downwards from the tree top, and reaching a plateau near the base of the living crown.
Phloem conductivity was estimated to scale similarly to the conductivity of the outermost xylem ring, with the ratio of phloem to xylem conductivity being c. 2%. However, xylem conductivity was estimated to increase more than phloem conductivity with increasing tree dimensions as a result of accumulation of xylem sapwood. Phloem partly compensated for its smaller conducting area and narrower conduits by having a slightly higher conduit frequency.

Introduction Xylem and phloem lie side by side along the vertical axis of a tree from roots to leaves. Carbon gain and whole-tree survival are dependent on the functioning and interplay between these two vascular subsystems (Jensen & Zwieniecki, 2013; Nikinmaa et al., 2013; Sevanto et al., 2014). Their hydraulic performance is tightly connected to the spatial arrangement and structural properties of the conduits of which they are built. Research on xylem transport physiology and its dependence on structure in trees has long been flourishing, whereas studies relating phloem structure to function are surprisingly scarce (Mencuccini et al., 2011; Ryan & Asao, 2014). Phloem research has concentrated on elucidating the cellular and ultrastructural processes of phloem function (van Bel, 2003; Knoblauch & Peters, 2013). Regarding large trees and particularly conifers, however, we have only limited information on even the simple metrics of phloem (Ryan & Asao, 2014; Woodruff, 2014). For example, few studies exist that have analysed the differences in scaling allometry between transporting phloem and xylem (Mencuccini et al., 2011; H€oltt€a et al., 2013). Within a tree, however, the transport conductances of phloem and xylem should scale in approximately similar fashion when the tree grows taller in order to sustain the ratio between water 102 New Phytologist (2015) 205: 102–115 www.newphytologist.com

and carbon exchange, and to maintain the driving forces of phloem and xylem transport (H€oltt€a et al., 2013). In conifers, water is conducted through xylem tracheids in sapwood, whereas photoassimilates are transported through phloem sieve cells. Tracheids, which also provide the tree with structural support, are biologically dead at maturity. Vascular phloem, in contrast, is metabolically more active tissue. In Pinaceae, it is mainly composed of highly specialized, living sieve cells. They are characterized by nonlignified cell walls with secondary thickenings (Abbe & Crafts, 1939), and constitute a great proportion of the axial phloem network (up to 90%; Evert, 2006). When functioning, the cytoplasm of sieve cells is greatly reduced and the cells are interconnected through sieve area pores and median cavities originating in plasmodesmata, providing a lower resistance pathway for translocates (Schulz, 1998; van Bel, 2003). Phloem transport is assumed to be driven by an osmotically generated pressure differential that is endogenously maintained by sugar loading at source tissues and unloading at sink tissues, as postulated by the M€ unch pressure flow hypothesis (M€ unch, 1927; van Bel, 2003; Thompson, 2006; De Schepper et al., 2013). Unloading (or leakage) and reloading (or retrieval) may also occur within the conducting pathway (Thorpe et al., 2005). Thus, phloem flow with increasing tree height may be possible Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist without large increases in turgor pressure difference (Turgeon, 2010), if phloem conduits widen enough (similarly to the xylem conduits) to diminish the hydraulic resistance (Jensen et al., 2011; Mencuccini et al., 2011), which is proportional to the conductive path length (Hagen–Poiseuille law; Tyree & Zimmermann, 2002). For a conduit of constant diameter, the resistance to flow linearly increases with increasing conductive path length. Conduit widening partly compensates for the increasing resistance as a tree becomes taller (M€akel€a & Valentine, 2006; Sperry et al., 2008). Xylem conduits have been repeatedly shown to widen basipetally from the stem apex towards the base (i.e. conduit tapering towards the stem apex) in both conifers and angiosperms (Sanio, 1872; Bailey & Shepard, 1915; Zimmermann, 1978; Anfodillo et al., 2006; Mencuccini et al., 2007; McCulloh et al., 2010). By contrast, very little empirical data have been assembled to assess the axial pattern of phloem conduit variability, especially on conifers (Rosner et al., 2001; Mencuccini et al., 2011). Fragmentary empirical evidence seems to support the hypothesis of phloem conduit widening down the stem and/or with increasing stem diameter (Mencuccini et al., 2011; Petit & Crivellaro, 2014). Recently, theoretical scaling has attracted much scientific interest in relation to water transport: theories predict the degrees by which the conduits should widen to ensure the independence of flow resistance from conducting path length (e.g. the West, Brown and Enquist (WBE) model; West et al., 1999). The WBE theory, assuming constant conduit widening with increasing distance from the stem apex (i.e. a power-law function with a scaling exponent of at least 1/4 (West et al., 1999) or c. 1/5 as suggested by Anfodillo et al. (2006) and Petit & Anfodillo (2009)), has been rigorously tested and has received some criticism (M€akel€a & Valentine, 2006; Mencuccini et al., 2007) but also empirical support (Mencuccini, 2002; Anfodillo et al., 2006, 2013; Weitz et al., 2006; Petit et al., 2008, 2010). In phloem, the WBE predictions have not been systematically tested for conduit widening

Research 103

along the longitudinal axis of large conifers (cf. Petit & Crivellaro, 2014). A recent vascular network scaling model by Savage et al. (2010) proposed a value of optimal scaling between the basal conduit diameter and stem diameter (i.e. a scaling exponent of 1/3), taking into account the trade-offs between hydraulic efficiency and safety. A scaling exponent higher than 1/3 would increase the risk for hydraulic failure which would soon offset any gain in transport efficiency, whereas a scaling exponent lower than 1/3 would decrease the hydraulic efficiency (and therefore the carbon gain) which would soon offset any gains from higher embolism resistance. Empirical evidence supporting the prediction of 1/3 exists in xylem (Savage et al., 2010; Olson & Rosell, 2013). In phloem, the prediction has not been tested to our knowledge. Note that the scaling predictions for conduit diameter as functions of distance from the stem apex and stem diameter yield closely comparable results, as the latter two variables scale very well with each other. As long-living organisms, trees have evolved mechanisms to adjust to changing demands for hydraulic and biomechanical functionality as they grow taller and larger. The tradeoffs between hydraulic, mechanical and developmental constraints are proposed to result in widely recognized radial (pith-to-cambium) and axial (parallel to the cambium or parallel to the pith) patterns in xylem properties, including conduit size and frequency (Domec & Gartner, 2002; Lachenbruch et al., 2011). The first few rings from the pith are produced by a physiologically young cambium and are generally termed juvenile wood or corewood (Fig. 1; Zobel & Sprague, 1998), characterized by smaller conduits of lower hydraulic efficiency but higher resistance to embolism (Lachenbruch et al., 2011). Xylem further from the pith – commonly termed mature wood or outerwood – is produced by physiologically ageing cambium and has wider conduits that permit higher conductivity (Zobel & Sprague, 1998; Lachenbruch et al., 2011). This typical asymptotic radial progression (from juvenile to mature tissue) is usually strong in conifers, and detected also axially down the stem in a given ring as counted

Fig. 1 Schematic presentation of the sampling procedure and ontogeny within a stem, and demonstration of the phloem microstructure of Picea abies at 1.3 m and 95% relative height on the stem. Conducting phloem is identified as a narrow strip next to the vascular cambium consisting of noncollapsed sieve cells and a tangential band of axial parenchyma. CB, base of the living crown. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist (2015) 205: 102–115 www.newphytologist.com

New Phytologist

104 Research

In the light of current understanding of the significance of phloem transport for whole plant functioning, and the still very fragmentary knowledge of phloem hydraulic architecture, we focus here on the following fundamental questions. How does conducting area–stem diameter allometry vary between phloem and xylem? Do phloem conduits widen downwards from the stem apex similarly to what is observed in xylem? Does conduit frequency differ between phloem and xylem? What are the determinants of conduit diameter variations in phloem and xylem? How do phloem and xylem conductivity scale along the stems?

from the cambium, for example parallel to the newest ring of xylem and phloem (Fig. 1; Duff & Nolan, 1953; Burdon et al., 2004; Weitz et al., 2006). As xylem and phloem are derived from cambial divisions – xylem produced inwards and phloem outwards – one would expect similar radial/axial progression in phloem and xylem conduit sizes. The changes in the number of conducting elements along the stem also affect the hydraulic conductivities of the conducting tissue. For instance, conduit widening is accompanied by decreasing conduit frequency: the larger the conduit diameter, the smaller the number of conduits that can occupy the given unit area/space in the conducting tissue (Sperry et al., 2008; Savage et al., 2010). The degrees of conduit widening and furcation along a stem can have a major influence on whole-tree conductance (McCulloh et al., 2010). Moreover, the differences in phloem and xylem cross-sectional areas along the stem propagate to the hydraulic efficiency of the tissues. These factors have been neglected in the WBE and in most subsequent analyses when inferring hydraulic conductivity from anatomical data (Sperry et al., 2008). This may partly be a consequence of the fact that the ratio of conducting area to leaf area changes at each branching in the tree, making a quantitative analysis tedious. For the xylem, the pipe model theory states that the cross-sectional area of the xylem of the sapwood is conserved at branching junctions (Shinozaki et al., 1964). This allows a first-order approximation of the trend in axial xylem cross-sectional area per unit leaf area, at least for species and conditions where the pipe model assumption has been empirically verified, for example for Picea abies in boreal conditions (Kantola & M€akel€a, 2004). However, the ratio of phloem to xylem crosssectional areas decreases considerably with increasing stem diameter and distance from the leaf apex (H€oltt€a et al., 2013). Thus, the scaling of cross-sectional areas affects at least the ratio of xylem to phloem conductivities in different parts of the tree.

Materials and Methods Tree material and sampling The trees used in the study were 18 Norway spruce (Picea abies (L.) Karst.) trees from six different clones of rooted cuttings growing in two forest stands in southern Finland (Loppi; 60°440 N, 24°300 E, 120 m above sea level), within a boreal zone. The stands were on fertile old agricultural land providing trees with similar environmental conditions. The selected clones represented two different age groups: four clones of older trees planted in the 1970s, and two clones of younger trees planted in the 1990s (Table 1). Both age groups included clones of higher and lower growth and quality performance, on the basis of visual assessments regarding growth, vigour and stem form. The cuttings of each clone originated from three seedlings that were the progeny of three mother trees originating from southern and eastern Finland. The use of clonal trees diminished random variation in phloem and xylem anatomy, providing a convenient data set for scaling analysis of phloem and xylem. From each clone, three sample trees of different sizes (small, medium, and large) were harvested during late October to early

Table 1 Characteristics of rooted cutting clones of Picea abies ( SD) Clone Variable

19

20

51

61

67

255

Growth and quality classa Age (yr)b D1.3 m (cm) D6 m (cm) Vigour indexc Tree height (m) H/D ratiod Crown basee (m) Crown length (m) Crown ratiof Crown width (m)g

L

H

L

14.0 (1.00) 9.0 (2.71) 5.4 (2.31) 0.10 (0.03) 8.9 (0.48) 104.85 (26.93) 2.15 (0.18) 6.77 (0.31) 0.76 (0.01) 2.21 (0.25)

H 28.7 (0.58) 22.5 (3.46) 16.6 (3.50) 0.08 (0.02) 20.1 (1.44) 89.84 (7.92) 7.99 (1.48) 12.09 (1.12) 0.60 (0.06) 3.55 (0.61)

L

12.7 (1.15) 9.1 (2.95) 3.8 (3.13) 0.11 (0.07) 8.5 (2.04) 94.13 (7.48) 1.77 (0.66) 6.69 (1.47) 0.79 (0.04) 2.62 (0.45)

26.0 (1.00) 19.6 (3.38) 16.3 (2.58) 0.09 (0.01) 17.7 (1.10) 91.62 (10.10) 5.70 (1.86) 12.03 (2.72) 0.67 (0.12) 3.57 (0.37)

26.0 (1.73) 15.7 (0.96) 13.9 (7.57) 0.05 (0.01) 17.5 (0.10) 111.70 (6.84) 7.61 (1.68) 9.85 (1.58) 0.56 (0.09) 2.82 (0.18)

H 30.3 (0.58) 23.9 (5.22) 20.7 (4.47) 0.07 (0.02) 22.2 (0.29) 95.96 (20.63) 10.41 (1.13) 11.79 (1.31) 0.53 (0.05) 4.27 (1.64)

a

L, low growth and quality; H, high growth and quality by visual assessments in the 1960s. The number of annual rings at a height of 1.3 m (breast height (BH)) on the stem. c The ratio of the basal area of the last annual ring to the basal area of sapwood at BH. d Tree height (mm) divided by tree diameter at 1.3 m (mm). e The height of the first living branches from the ground. f The ratio of live crown length to tree height. g Crown width was measured as the arithmetic mean of the crown widths at the north–south and east–west directions. n = 3 trees/clone. b

New Phytologist (2015) 205: 102–115 www.newphytologist.com

Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist November in 2011. Tree dimensions and stem taper are shown in Table 1 and Supporting Information Table S1. At the time of sampling, the formation of phloem and xylem had ceased (i.e. the cambium was dormant) and no winter-related collapsing of conducting sieve cells was expected. To account for the vertical variation in phloem and xylem along the stem, five discs were sampled at: the stem base (0 m); breast height (BH; 1.3 m); the base of the living crown (CB); a relative height of 75% (RH75) and 95% (RH95) of the total stem height as counted from the stem base (Table S1). Immediately after felling, small blocks (c. 3 9 2 9 2 cm, longitudinal 9 radial 9 tangential), including the outermost xylem rings, cambium, phloem and outer bark, were cut from the discs and fixed with 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7) and stored at +4°C until further analysis. The remaining discs were stored at 20°C. In the laboratory, the border between sapwood and heartwood (Fig. 1) in each cross-section (disc) was visually identified based on moisture difference, and the widths of sapwood and heartwood were measured along the northsouth and west-east radii. In each cross-section, the number of annual growth rings of xylem was determined, and their widths, as well as the widths of earlywood and latewood, were measured (with an accuracy of 0.001 mm) with a computer-aided system consisting of a stereomicroscope (Olympus SZ51, Tokyo, Japan) attached to a transducer (Heidenhein VRZ 480, Traunreut, Germany).

Research 105

ring and sapwood were computed for each cross-section assuming a circular cross-section. The radial diameters of functional sieve cells and xylem tracheids were measured along three representative radial files in each section with the image analysis software IMAGE-PRO PLUS v. 7.0. (Media Cybernetics Inc., Bethesda, MD, USA) or PixeLINK. In the case of sieve cells, the measurements of cell widths included thin double cell walls, except in one sample tree in which the widths of cell lumens and double cell walls (t; i.e. the thickness of the double cell wall between two adjacent lumens) were measured separately. Sieve cell wall thickness did not vary with increases in lumen diameter (R2 = 0.00; b = 0.002; Fig. S1). Thus, the measurement of phloem conduit lumen diameter (a) was obtained by subtracting the arithmetic mean double wall thickness (t) from the measurement of cell width. Regarding tracheids, the widths of cell lumens (a) and double cell walls (t) were measured separately. Additionally, tangential diameters of sieve cells were measured on three locations in each radial cell row: next to the cambium, at the border between conducting and nonconducting phloem, and in the middle of each row; and their arithmetic mean was calculated (denoted here as b). Tangential diameters of tracheids were not measured as they did not deviate from those in phloem (data not shown). The number of measured cells in each section is shown in Tables S2 and S3. Calculating indices for conduit tapering and frequency

Phloem and xylem anatomical measurements In the laboratory, the sample blocks were cut using a razor blade into smaller specimens (8 9 3 9 3 mm, radial 9 tangential 9 longitudinal) that included either cambium and phloem or cambium and the outermost xylem ring. The specimens were dehydrated in a graded series of ethanol and ethanol–resin mixture (LR White Resin, hard grade; London Resin Co Ltd, London, UK). The specimens were then embedded in resin blocks and transverse radial sections (c. 5– 8 lm thick) were cut using a rotary microtome (Leica RM2265; Leica Microsystems, Wetzlar, Germany). The sections were then stained with an aqueous solution of 1% toluidine blue, air-dried and mounted in Ultrakitt M540 mountant (TAAB, Reading, UK). Images were taken of the outermost rings of phloem and xylem with a digital camera (MicroPublisher 3.3 RTV; QImaging, Surrey, BC, Canada; 6.6 PL-B686CF-KIT; PixeLINK, Ottawa, ON, Canada) attached to a light microscope (Olympus BX60 or Olympus BX50) at 9100–200 magnification and with a resolution of 0.343–0.234 lm pixel1. One to several overlapping images were taken and merged to cover the whole width of each ring. The widths of conducting phloem and the outermost xylem ring were measured from the images. The conducting phloem was identified as a narrow strip outside the cambium consisting of uncollapsed, conducting (i.e. functional) sieve cells and one to two tangential bands of axial parenchyma cells (Fig. 1; Rosner et al., 2001; Evert, 2006). Based on the widths and mean stem diameter, the areas of conducting phloem, the outermost xylem Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

The radial cell lumen diameter a and the average tangential diameter b were used to calculate the hydraulic cell diameter dh (Mencuccini et al., 1997): dh ¼

2ab : aþb

Eqn 1

The weighted averages of hydraulic diameters Dh for both phloem and xylem in each stem section were calculated as P 5 d Dh ¼ P h4 ; dh

Eqn 2

which weights the hydraulic diameters of sieve cells and tracheids according to hydraulic conductance (Sperry et al., 1994), as commonly done in allometric analysis where hydraulic conductivity is of interest. To analyse conduit frequency (i.e. the number of conduits per unit xylem and phloem area), we determined the areas of sections in which conduit diameters were measured, and calculated the number of conduits (n). For analysing the conduit frequency as a function of conduit diameter, we used the area-weighted mean conduit diameter (DA) for each cross-section (McCulloh et al., 2010): DA ¼ ð

X

ðabÞ=nÞ1=2

Eqn 3

New Phytologist (2015) 205: 102–115 www.newphytologist.com

New Phytologist

106 Research

compared using Akaike’s information criterion (AIC), residual scatterplots, and P-values. The coefficient of determination (R2) of the full model was computed as follows:

Scaling of conduit diameter with stem diameter and distance from apex For each tree, Dh values were employed to express the withinstem variation in conduits of phloem and xylem as power-law functions of distance from the stem apex (m) or stem diameter (cm): Dh ¼ ax b

Eqn 4

(x, the independent factor of distance from the apex or stem diameter; a, the base; b, the exponent for the scaling relation.) Given the stem diameter–stem height allometry, both the distance from the apex and stem diameter may equally well describe the variation in conduit diameter within the stem. Thus, both scaling relationships were applied and the goodness of fit of the models (R2) compared. For the pooled data, Dh values were used to compare the common shapes of vertical tapering in phloem and xylem as a function of distance from the stem apex (Eqn 4), and to compare the empirical scaling exponents of conduit tapering with those of a theoretical WBE power-law. From the WBE theory, the value for the scaling exponent is expressed as a function of branching architecture (i.e. WBE segments; West et al., 1999) instead of the distance from the apex. To compare the WBE theory with the empirical scaling exponent as a function of distance from the apex, we used an exponent of 1/5, as frequently proposed (Anfodillo et al., 2006; Lintunen & Kalliokoski, 2010). The conduit tapering was studied separately for younger and older trees. To compare the scaling relationship of conduit diameter–stem diameter with the scaling exponent of 1/3 by Savage et al. (2010), a linear model was fitted to the log10-transformed Dh values of phloem and xylem. The fitting was performed separately for juvenile and mature tissues (Fig. S2a). The conduit and stem diameters were log10-transformed to meet the statistical assumptions of normality and to linearize the relationship between the variables. Ninety-five per cent confidence intervals (CIs) for the scaling exponents were calculated. If the 95% CIs excluded the theoretical scaling exponents, we concluded that they significantly deviated. Modelling conduit diameter variation To analyse the differences in conduit diameter variation between phloem and xylem, and to describe the main factors of variation, a hierarchical linear mixed model was fitted to the log10-transformed hydraulic mean conduit diameters (Dh) of phloem and xylem. The models were fitted using the MIXED procedure of IBM SPSS STATISTICS (v. 20) with a restricted maximum likelihood (REML) criterion. Phloem and xylem were included in the model as dummy variables. The search for other explanatory variables included testing the following factors and their interactions: log10 stem diameter, log10 distance from apex, log10 height in the tree (m; measured from ground level), and log10 cambial age (i.e. the number of annual rings in each stem section). Alternative models were New Phytologist (2015) 205: 102–115 www.newphytologist.com

R 2 ¼ ðVn  Vf Þ=Vn ;

Eqn 5

(Vf, the sum of variance components of the full model; Vn, that of the null model (which includes the same random variables as the full model but only the intercept as a fixed part; Xu, 2003).) The final mixed linear model describing the log10 hydraulic conduit diameter of phloem and xylem (Dh) was as follows: log10 ðDh Þ ¼ a0 þ phloem þ a1 log10 ðdiamthp Þ þ a2 log10 ðheightthp Þ þ a3 log10 ðdiamthp Þ  phloem

Eqn 6

þ a4 log10 ðheightthp Þ  phloem þ t þ ht þ ethp (a0, the intercept; phloem, a dummy variable indicating phloem (1) and xylem (0); a1–a4, the fixed regression coefficients; diamthp, stem diameter (cm); heightthp, height on the stem (m; measured from the ground upwards on the stem); t and ht, random parameters for tree, and height level within a tree; ɛthp, a residual error.) The interaction terms diamthp 9 phloem, and heightthp 9 phloem allow the effects of diamthp and heightthp on conduit diameter to be dependent on tissue type (phloem or xylem). The dummy variable was treated as a repeated factor within height level h in a tree t. A scaled identity (ID) covariance structure for residuals was applied as it resulted in the best (i.e. smallest) AIC. Calculation of xylem and phloem conductivities The phloem and xylem conductivities were estimated based on the measurements conducted in this study. For the calculations, the cross-sectional areas of conducting phloem, outermost xylem ring and xylem sapwood, and conduit radius and conduit frequency in the phloem and outermost xylem ring were expressed as allometric functions of the stem diameter (Table S4). The approach for calculating conductivities is described in detail in Methods S1. Briefly, xylem and phloem conductivities were calculated from the xylem and phloem cross-sectional areas, conduit frequencies, and conduit radii according to the Hagen–Poisseuille law, assuming that pit membranes and sieve pores always accounted for three-quarters of the hydraulic resistance within both the xylem and the phloem. Note that the actual values used for the fraction accounting for pit membranes and sieve pore resistances do not affect the scaling between xylem and phloem conductivities as long as these fractions are independent of xylem and phloem conduit diameters. The viscosity of xylem sap was assumed to be that of pure water at 20°C, and the viscosity of phloem was twice that of the xylem, thus making xylem conductivity twice as high as phloem conductivity for a given configuration of conduit size and number. No xylem embolism was assumed; that is, all conduits were assumed to be water-filled and conductive. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist

Research 107

Results

Scaling of phloem and xylem conduit diameter with stem diameter and distance from apex

Functional areas of phloem and xylem Diameters of stem cross-sections varied from 0.81 to 34.2 cm (Table S1). Tree heights ranged from 6.5 to 22.5 m. Pooling the data together yielded a high correlation (R2 = 0.96) between the distance from the apex and stem diameter (Fig. 2a). We found highly significant correlations between functional areas of xylem and phloem and stem diameter (Fig. 2). With increases in stem diameter, the functional area of xylem (i.e. sapwood) accumulated faster than that of conducting phloem (Fig. 2b). Thus, the ratio of conducting phloem to sapwood area decreased with increasing stem diameter, following a power-law scaling with an exponent of 0.76 (Fig. 2c). Annually produced phloem (approximately conducting phloem) and xylem (i.e. the outermost xylem ring) were highly correlated (R2 = 0.91) and showed a constant (1 : 10) phloem to xylem ratio with increasing stem diameter (Fig. 2d).

Phloem and xylem conduit diameter rapidly increased with increasing cambial age in the first 10 rings from the pith (Fig. S2a). Thereafter, conduit diameter remained rather constant with increasing cambial age. Thus, the first 10 rings of phloem and xylem were considered to be juvenile tissue, and the rings produced thereafter to be mature tissue. The ratio of phloem to xylem conduit diameter was constant regardless of stem diameter (Fig. S2b). On average, the phloem conduits were c. 27% (range 17–45%) smaller than the xylem conduits. The log10 conduit diameter scaled with log10 stem diameter in a similar manner in phloem and xylem (Fig. 3). The scaling slopes were steeper in juvenile than in mature phloem and xylem. The scaling slopes deviated slightly from the 1/3 prediction by Savage et al. (2010), except that of juvenile phloem. Tree-wise power-law scaling of conduit diameters with stem diameter and distance from the apex is shown in Tables S2 and

4

1.5

(b) Sapwood R 2 = 0.99, y = –0.275 + 1.980x Outermost xylem ring R 2 = 0.80, y = –0.294 + 1.131x Conducting phloem R 2 = 0.90, y = –1.376 + 1.165x

R 2 = 0.96, y = –0.38 + 1.22x 1.0

Distance from ground (m): 2

Log10 area (cm )

Log10 distance from stem apex (m)

(a)

0–4 4–8 8–12 12–16 16–20 20–24

0.5

2

0

0.0

–2 0.5

1.0

1.5

2.0

0.0

0.5

Log10 stem diameter (cm)

10

(d)

2

R = 0.92, slope = 0.59

1

0.1

0.01 0.1

1

10

100

1000

2

Sapwood (cm )

0.04

0.00

Ratio of conducting phloem to outermost xylem ring area (cm2 cm2)

2

Conducting phloem (cm )

Ratio of conducting phloem to sapwood area (cm2 cm2)

0.08

0.6 0m 1.3 m CB 75% 95% R 2 = 0.87, slope = –0.76

2

0.12

(c)

1.0

1.5

2.0

Log10 stem diameter (cm 2)

Conducting phloem (cm )

0.0

R 2 = 0.004, slope = 0.00

0.4

10

2

R = 0.91, slope = 0.93

1

0.1

0.01 0.1

1

10

100 2

0.2

Outermost xylem ring (cm )

0.0

0

10

20 Stem diameter (cm)

30

40

0

10

20

30

40

Stem diameter (cm)

Fig. 2 (a) Allometry of stem diameter–distance from the apex across the measured data for Picea abies; (b) the areas of conducting phloem, the outermost xylem ring, and sapwood as a function of stem diameter; (c) the ratio of conducting phloem area to sapwood area; (d) the ratio of conducting phloem to the outermost xylem ring as plotted against stem diameter. The insets show the log–log scaling relationships between the area of conducting phloem and sapwood area (c), and the area of conducting phloem and the area of the outermost xylem ring (d). Different symbols refer to sampling height from the ground. Brown symbols, juvenile tissues; turquoise symbols, mature tissues (see Fig. 1 and Supporting Information Fig. S2). Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist (2015) 205: 102–115 www.newphytologist.com

New Phytologist

108 Research 2.0

2.0

Log10 mean conduit diameter Dh (µm)

(a) Phloem 1.8

1.6

1.4

(b) Xylem

Juvenile tissue R 2 = 0.86, y = 1.113 + 0.298x (0.257–0.339) Mature tissue R 2 = 0.36, y = 1.065 + 0.239x (0.151–0.327) All, R 2 = 0.61, y = 1.152 + 0.180x (0.150–0.210) Slope = 1/3 (Savage et al., 2010)

1.8

R2 = 0.91, y = 1.259 + 0.264x (0.236–0.292) R2 = 0.44, y = 1.266 + 0.193x (0.133–0.253) R2 = 0.78, y = 1.288 + 0.183x (0.163–0.203)

1.6

1.4

1.2

1.2

1.0

1.0

0.8

0.8 0.0

0.5

1.0

1.5

0.0

Log10 stem diameter (cm)

0.5

1.0

1.5

Log10 stem diameter (cm)

Fig. 3 The log10 hydraulic mean diameters of phloem (a) and xylem (b) conduits in juvenile (brown symbols) and mature (turquoise symbols) tissue of Picea abies plotted against log10 stem diameter with the theoretical scaling exponent (1/3) suggested by Savage et al. (2010). Dotted and dashed lines are the 95% prediction and confidence bands for the regression, respectively. The values in parentheses are the 95% confidence interval for the slope.

S3, respectively. In the majority of cases (55%), stem diameter explained slightly better the within-stem variation in both phloem and xylem. Similar widening of xylem and phloem conduits was observed with increasing distance from the apex (Fig. 4). Both in phloem and xylem, conduits rapidly widened next to the apex and then plateaued out lower on the stem (Fig. 4). The degree of conduit widening deviated slightly from the predicted scaling exponent of 1/5 in older trees (Fig. 4b,d). Determinants of conduit diameter variations in phloem and xylem The constructed linear mixed model for log10 conduit diameter of phloem and xylem (Dh) explained 89% of the total variation in the pooled data. The model coefficients are shown in Table 2. The behaviour of the model is shown in Fig. 5. The dummy variable indicating whether a conduit belongs to phloem or xylem explained 34% of the total variation in conduit diameter. Stem diameter alone predicted 45% of the variation. The effect of height (i.e. distance from the ground) was significant (Table 2) and explained 10% of the variation. In particular, height from the ground described the phenomenon of decreasing basal conduit diameter for a given stem diameter below a living crown (Fig. 5a–c). The interaction terms heightthp 9 phloem and diamthp 9 phloem were significant but explained < 0.5% of the total variation, and indicated a slightly faster rate of increase in phloem than in xylem conduit diameter with increasing height and stem diameter (Fig. 5c,d). Other two- and three-way interaction terms (i.e. diamthp 9 heightthp, and diamthp 9 heightthp 9 phloem) were insignificant and were omitted from the final model. The results imply that phloem and xylem conduit diameters scale very similarly with stem diameter, and show similar conduit diameter–stem diameter scaling regardless of height on the stem New Phytologist (2015) 205: 102–115 www.newphytologist.com

(Fig. 5). Cambial age and stem diameter were highly correlated (r = 0.93; P < 0.01) but stem diameter better described the variation in conduit diameters (R2 = 0.45 versus R2 = 0.21; data not shown). Similarly, distance from the apex and stem diameter showed a high positive correlation (r = 0.96; P < 0.01) but stem diameter was a slightly better predictor for conduit diameter (R2 = 0.45 versus R2 = 0.40; data not shown). Conduit frequency Phloem and xylem conduit frequency and conduit lumen diameter showed a typical inverse relationship: the larger the conduits, the fewer of them were packed into a given cross-sectional area of conducting tissue (Fig. 6a). The scaling exponent for the relationship was smaller in phloem (1.8) than in xylem (2.1). This indicated that, in phloem, wider conduits occupied proportionately more of the conducting area than narrower conduits. By contrast, in xylem, narrower conduits occupied a higher fraction of the possible wood area than wider conduits. On average, conduit frequency was 1.6 times higher in phloem compared with xylem, and phloem showed a smaller decrease in conduit frequency with increasing stem diameter (Fig. 6b). Conductivity of phloem and xylem Using the allometric scaling relations (see Table S4 for the bases and scaling exponents), the conductivity of the outermost xylem layer kx,outer was 3.5 9 1014 x1.43 m4 Pa1 s1 (where x is the stem diameter), the conductivity of the xylem sapwood kx,sapwood was 3.7 9 1014 x2.33 m4 Pa1 s1 (this relation is strictly not allometric but very close it), and the conductivity of phloem kp was 5.3 9 1016 x1.56 m4 Pa1 s1 (Fig. 7a). Phloem conductivity varied from 1.5% to 2.4% of the conductivity of the outermost xylem layer and from 1.8% to 0.18% of the conductivity of the xylem sapwood (Fig. 7b). Phloem conductivity thus increased Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist

Research 109

Younger trees

Older trees 50

Mean phloem conduit diameter Dh (µm)

50

(b) Phloem

(a) Phloem

R2 = 0.382, y = 19.082x0.082 (CI: –0.089–0.267)

All, R2 = 0.50, y = 14.935x0.143 (CI: 0.081–0.205) Scaling exponent = 0.2 (Anfodillo et al., 2006) Juvenile tissue Mature tissue

40

40

30

30

20

20

10

10

0

0 0

2

4

6

8

10

0

12

50

10

15

20

25

20

25

50

(c) Xylem Mean xylem conduit diameter Dh (µm)

5

(d) Xylem

2

R = 0.69, y = 21.002x

40

0.158

(CI: 0.111–0.205)

40

30

30

20

20

10

10

0

2

R = 0.62, y = 24.695x

0.107

(CI: 0.083–0.131)

0 0

2

4

6

8

10

Distance from the stem apex (m)

12

0

5

10 15 Distance from the stem apex (m)

Fig. 4 Hydraulic conduit diameters of phloem (a, b) and xylem (c, d) in younger (a, c) and older (b, d) trees of Picea abies plotted against distance from the stem apex with the theoretical West, Brown and Enquist (WBE) model (West et al., 1999) scaling exponent (1/5; as suggested by Anfodillo et al. (2006) and Petit & Anfodillo (2009)). Variability in conduit diameters between juvenile (brown symbols) and mature (turquoise symbols) tissues is shown. The 10 first annual rings of phloem and xylem from the pith were considered as juvenile; the rings thereafter as mature tissue (for determination, see Figs 1, S2). Dotted and dashed lines are the 95% prediction and confidence bands for the power-law scaling, respectively. The values in parentheses are the 95% confidence interval for the scaling exponent.

less in comparison to the conductivity of the xylem sapwood, but increased slightly more in comparison to the conductivity of the outermost xylem ring with increasing stem diameter. This latter finding was mainly attributable to the smaller decrease in conduit frequency with increasing stem diameter in phloem, while the scalings of the conduit radius and cross-sectional area were very similar.

Discussion Because of the difficulties in measuring intact phloem, our current means of determining actual phloem conductivity are limited, particularly in large trees (Ryan & Asao, 2014). Even the widely, yet not completely, agreed assumption of M€ unch pressure flow relies more on a solid theoretical hypothesis than on real empirical evidence (Knoblauch & Peters, 2013). However, the conductive capacity of the phloem transport system, similarly to Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

that of xylem, must be strongly affected by its anatomical structure: conduit size and number, and sieve pore size and frequency along the pathway (Mullendore et al., 2010; Patrick, 2013). To gain new insights into the coordination of phloem and xylem transport capacities in conifers, we compared the axial withinstem patterns of phloem and xylem hydraulic networks, and estimated the conductivity of phloem and xylem. We observed a constant ratio of annually produced phloem to xylem area (1 : 10) along the tree stems. The required transport capacity of the xylem exceeds that of phloem, as the transport rate of fluids is higher in xylem than in phloem (H€oltt€a et al., 2009). Accordingly, in most tree species, more xylem is produced annually. The ratio of phloem to xylem production varies between 1 : 4 and 1 : 10 (Fromm, 2013). Our result supports earlier findings on P. abies and Abies alba: the widths of yearly increments of phloem and xylem were interrelated (Gricar et al., 2006) and the ratio of annually produced phloem to xylem cell number varied New Phytologist (2015) 205: 102–115 www.newphytologist.com

New Phytologist

110 Research Table 2 Linear mixed model predicting the log10 conduit diameter (Dh; lm) in phloem and xylem of Picea abies based on log10 stem diameter (Diam), log10 height on the stem (Height), Height 9 tissue, and Diam 9 tissue interaction terms (Eqn 6) Parameters

Estimate

SE

P

Fixed part

R2 89

0.000

Intercept Phloem Xylem Diam Height Height 9 Phloem Height 9 Xylem Diam 9 Phloem Diam 9 Xylem

1.0808 1.2445 0.2232 0.0259 0.0176 0 0.0249 0

0.020728 0.011439 0.011199 0.003964 0.003216 0 0.009089 0

treet heighth residual error ɛthp

0 0.000916 0.000449

0 0.000179 0.000069

0.000 0.000 0.000 0.007

Random part, variance estimates 0.000 0.000

Parameter estimates, their standard errors (SEs), and P-values of the parameters are shown. The coefficient of determination (R2) describes the proportion of the variation explained by the fixed part of the model (Eqn 5). Tissue type (phloem or xylem) is applied as a dummy variable in the model. n = 88 cross-sections from 18 trees.

between 1 : 10 and 1 : 20 in vigorous trees (Gricar et al., 2009). Declining trees, by contrast, may produce more phloem than xylem, or almost exclusively phloem (Gricar et al., 2009). The growth of phloem differs from that of xylem in terms of time and space partly because phloem sieve cells remain functional for 1–2 yr only (Huber, 1939; Evert, 2006). The cessation of conductive capacity of sieve cells is initiated by the accumulation of callose in sieve areas, followed by disappearance of cell organelles and breakdown of the cell membrane (Schulz, 1990; Evert, 2006). Finally, with the loss of turgor pressure and growth adjustments in the neighbouring tissues, the sieve cells collapse. Thus, the layer of conducting phloem is narrow: 0.14–0.27 mm has been reported for Picea (Huber, 1939). In addition to P. abies, similar trends have been reported for Pinus sylvetris, Betula bendula, Populus tremula and Eucalyptus globulus (Quilho et al., 2000; H€oltt€a et al., 2013). As xylem tracheids sustain their water-conducting capacity for much longer, sapwood accumulates over several years (Pittermann et al., 2006). Consequently, the ratio of phloem to xylem functional area decreases from tree top to base. In this study, the ratio of phloem to xylem functional areas ranged from 1 : 10 at the tree top to 1 : 100 at the base. As our study included samples along the main stems only, the smallest stem diameters were c. 8 mm. In addition to stems, H€oltt€a et al. (2013) analysed inner bark (i.e. including the conducting and nonconducting phloem) areas from branches. In the smallest branches (Ø c. 1 mm), the inner bark area exceeded the xylem area. With increasing branch/stem diameter (> 2.5 mm), xylem accumulated faster than inner bark. In other words, the volume of phloem increases relative to xylem volume from the stem base towards the tree top and with decreases in stem/branch diameter. New Phytologist (2015) 205: 102–115 www.newphytologist.com

The increase in phloem area relative to xylem area towards the tree apex may also be related to a greater requirement for refilling of embolized xylem conduits as a result of more negative water potentials. Carbohydrates and phloem functions are presumed to have a key role in refilling (Nardini et al., 2011; Secchi & Zwieniecki, 2011). The inner bark of Picea is characterized by a ray and axial parenchyma network, which potentially serves as an important carbohydrate and water reserve (Rosell et al., 2014; Spicer, 2014). In our study, phloem conduits were slightly narrower than xylem conduits, and the ratio of phloem to xylem conduit diameter decreased only marginally with increasing stem diameter. This was a result of very similar conduit widening in xylem and phloem. For both phloem and xylem, conduit diameter rapidly increased with increasing distance from the stem apex at the tree top, and then reached a plateau or even decreased lower on the stem. This is in accordance with earlier studies showing that the rate of xylem conduit widening varies along the conductive path, being first rapid next to the stem apex and then decreasing towards the stem base (Anfodillo et al., 2006; Mencuccini et al., 2007; Petit et al., 2010). However, we found the overall degree of conduit widening of older trees to be slightly less than the WBE predictions (Anfodillo et al., 2006; Savage et al., 2010). Conduit diameter has been shown to increase asymptotically with increasing cambial maturation (i.e. with increasing distance from the pith) up to a steady-state value in mature tissue in several woody species (Spicer & Gartner, 2001; Lachenbruch et al., 2011; Franceschini et al., 2012; Eugenio et al., 2014). Next to the stem apex and in younger trees, rapid cambial maturation is reflected in rapidly increasing conduit diameter. The transition from juvenile to mature tissue is a gradual process, and the exact mechanism is still not known. Several hypotheses have been proposed: in addition to the hydraulic constraints, developmental and mechanical factors interact in determining the anatomical properties throughout the tree’s life (Lachenbruch et al., 2011). The tree is assumed to produce juvenile tissues next to the apex to maintain hydraulic safety (Spicer & Gartner, 2001; Domec & Gartner, 2002; Domec et al., 2009; Rosner, 2013). The decreasing trend in phloem and xylem conduit diameters below the living crown may be related to mechanical needs and/or increasing construction costs of wider conduits with thicker walls (Mencuccini et al., 2007). Also, the well-documented increase in the proportion of latewood (with thicker walls and smaller conduits than in earlywood) from the pith to the bark in conifers may support this assumption (Spicer & Gartner, 2001). The below-crown sapwood, with several rings and a radially increasing proportion of latewood, possibly plays a role in providing the tree with maximal mechanical support. Heartwood, in contrast, contributes less to stem stiffness as it is mainly composed of juvenile wood of lower elasticity (Mencuccini et al., 1997). In P. abies, Rosner et al. (2001) have also reported decreasing phloem conduit diameter from the middle of the stem to the stem base. Accordingly, earlier studies have shown that the widening of xylem conduits may vary with phylogenetic group, tree size and compartment, and within the stem (Sanio, 1872; Mencuccini et al., 2007; Lintunen & Kalliokoski, 2010; Petit & Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist

Research 111

(a)

(b)

(c)

(d)

Fig. 5 Demonstration of measured within-stem variation of mean hydraulic conduit diameter in a large (tree no. 24) and a small (tree no. 3) sample tree of Picea abies (a), and predicted within-stem variation of mean hydraulic conduit diameters in theoretical trees of different sizes (b). For theoretical trees, we used the mean allometric relation for stem diameter–distance from the apex from the measured data (see Fig. 2a for the equation). Demonstration of model predictors on phloem and xylem conduit diameters: conduit diameters show a rapid increase with increasing height in the lowermost stem, followed by a levelling off (c); a fast initial increase in conduit diameter for the first 5–10 cm close to the pith is followed by a lower rate of increase with increases in stem diameter (d). The values in (c) were computed for a fixed stem diameter of 10 cm and those in (d) for the fixed height of 5 m on the stem. The predicted log10 values were transformed back to the original scale. Dotted lines are the 95% confidence interval for the prediction. Arrows in (a) indicate the base of the living crown (CB).

Crivellaro, 2014). Our results diverged from the assumption of universal phloem scaling proposed by Jensen et al. (2011, 2012) except in the juvenile tissue at the tree top (data not shown). From the hydraulic point of view, our results would mean that, at least in large conifers, the axial xylem conduit size variation is not optimal and that the tree top should experience water stress (Anfodillo et al., 2006) and/or decline in xylem area specific transpiration rate. In phloem, this would mean an increased sugar concentration especially near the sources and/or a decline in phloem area specific photosynthesis rate. However, the effect of conduit widening is moderated by the concurrent changes in conduit number. When conduits become smaller, their number typically increases; a fact often neglected in tapering studies (Sperry et al., 2008). We observed the typical pattern of higher conduit frequency with smaller conduits in both phloem and xylem. Compared with xylem, Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

phloem seemed to at least partly compensate for its lower functional area and smaller conduit size by achieving a slightly higher conduit frequency than xylem. Moreover, in phloem, there was a tendency for wider conduits to occupy proportionately more of the conducting area than smaller conduits. Phloem had a lower wall-to-lumen ratio than xylem. Xylem, by contrast, has different requirements for mechanical and hydraulic safety (Sperry et al., 2008) as evidenced here by larger functional areas and more rigid walls (higher wall-to-lumen ratio) as compared with phloem, thus resulting in lower conduit frequency per unit conducting area but more mechanical support (Hacke et al., 2001). As tree height is scaled from stem diameter, xylem and phloem conduit diameters scale with stem diameter (Olson & Rosell, 2013). We found that conduit diameter of both phloem and xylem scaled slightly better with stem diameter than with the New Phytologist (2015) 205: 102–115 www.newphytologist.com

New Phytologist

112 Research (a) 6000

(b) 6000 Phloem R2 = 0.95, scaling exponent = –1.773 Outermost xylem ring R2 = 0.79, scaling exponent = –2.081

–2

Conduit frequency (mm )

5000

Square-packing limit, scaling exponent = –2

4000

R 2 = 0.67, y = 3576.7x –0.381 R 2 = 0.82, y = 2675.9x –0.463

5000

4000

3000

3000

2000

2000

1000

1000

0

0 10

20

30

40

0

10

Mean area-weighted conduit lumen diameter (µm)

20

30

40

Stem diameter (cm)

Fig. 6 Conduit frequency of Picea abies phloem and xylem plotted against the mean area-weighted conduit lumen diameter (a) (n = 88 cross-sections of 18 trees) and stem diameter (b). The scaling exponent of theoretical square-packing limit (2) shows the maximum possible number of conduit lumens per unit area, when square-packing is assumed. The square-packing limit was calculated as 1/d2 (McCulloh et al., 2010).

distance from the apex, indicating that the radial progression is somewhat more stable than the axial. Both hydraulic and hormonal signals, which depend on within-tree axial and radial position and tree size, can influence cambial growth. The driving force for cell expansion is turgor pressure, which arises as a result of the interplay between local water potential and osmotic concentration (Pantin et al., 2012). The cell turgor is presumed to decline with increasing water stress high up in the tree, and thus reduce cell expansion. Woodruff (2014) tested the assumption about the effect of increasing water stress on phloem conduit size in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco), comparing cell size and water potential in the upper branches of trees of differing heights. Increasing tree height generated a decreasing water potential, which in turn was associated with decreasing phloem conduit size. However, water potential alone is not the only determinant of turgor pressure and conduit size, as osmoregulation and hormonal signals may offset the effect of lowering water potential on turgor. Hormonal signals, for example auxin, are involved in the control of cell division and tuning of the plastic properties of cell walls to irreversibly stretch under the mechanical force created by turgor pressure. Hormonal concentrations also depend on the within-tree position (Sorce et al., 2013). The polar auxin concentration gradient is hypothesized to be involved in the control of axial conduit widening from the tree top towards the stem base by providing a dose-dependent effect on the duration of the cell expansion phase; that is, longer cell expansion results in larger conduits from the tree top to the stem base (Anfodillo et al., 2012). In our study, the largest conduits were found close to the base of the living crown; the position within the living crown, and the distance from auxin sources, may play a role in the control of conduit size. Sucrose is also supposed to act as a morphogen-like substance that can strongly influence conducting tissue morphogenesis (Novitskaya & Kushnir, 2006). For instance,

New Phytologist (2015) 205: 102–115 www.newphytologist.com

(a)

(b)

Fig. 7 Conductivity of phloem, xylem sapwood and outermost xylem ring of Picea abies as a function of stem diameter (a), and the ratio of phloem to xylem sapwood and phloem to outermost xylem ring conductivity as a function of stem diameter (b).

Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist high sucrose concentrations have been found to lead to an increased phloem to xylem tissue ratio, as a result of increased phloem parenchyma production (Novitskaya & Kushnir, 2006). As phloem and the outermost xylem ring showed similar scaling of the conduit radius and cross-sectional areas with stem diameter, phloem conductivity was estimated to scale very similarly to the conductivity of the outermost xylem ring within a stem. The ratio of phloem to outermost xylem ring conductivity was estimated to be c. 2%. However, phloem conduit radii will vary according to turgor pressure, as they are elastic. When conducting the measurements the sieve cells have lost their turgor pressure. Their radii could be up to 10% larger when at their operating turgor pressure (Thompson & Holbrook, 2003). This would translate into an almost 50% increase in their conductivity, but would not affect the scaling of phloem versus xylem conductivities. Also, we did not account for embolism in the xylem which, if present, would decrease xylem conductivity. Xylem sapwood conductivity was predicted to increase faster with increasing stem diameter/distance from the apex in comparison to the phloem. This indicates that the ratio of whole tree level phloem to xylem conductance decreases with increasing tree size. A similar prediction was arrived at in the study by H€ oltt€a et al. (2013). This seems contrary to the finding that the ratio of CO2 to water exchange at the leaves (i.e. water use efficiency), and hence the transport need of sugars in phloem versus water in the xylem, are likely to increase with increasing tree size (Koch et al., 2004; Martınez-Vilalta et al., 2007), although also a decrease in water use efficiency with increase in tree size has been found (K€ostner et al., 2002). Reasons for this can only be surmised, but could be related to, for example, the effect of gravity, which aids phloem transport but slows down xylem transport, or compensation of decreased phloem conductance by active metabolism along the transport pathway or increased turgor pressure and osmotic gradients within the phloem, or by a relative shift in sugar usage towards the upper crown with increasing tree size.

Acknowledgements We thank M. V. Alonso Pablos, S. Guo, E. Simon Vicente, T. J€arvinen, T. Nevalainen, and M-L. Napola for skillful assistance. H. M€akinen, J. Hyv€onen, and J. Heinonen are thanked for valuable comments during the study. S. Rosner is thanked for her comments on phloem anatomical analysis. The study was funded by the Academy of Finland (nos. 250299 and 1268342), and carried out within the framework of the COST FP1106 network STReESS.

References Abbe LB, Crafts AS. 1939. Phloem of white pine and other coniferous species. Botanical Gazette 100: 695–722. Anfodillo T, Carraro V, Carrer M, Fior C, Rossi S. 2006. Convergent tapering of xylem conduits in different woody species. New Phytologist 169: 279–290. Anfodillo T, Deslauriers A, Menardi R, Tedoldi L, Petit G, Rossi S. 2012. Widening of xylem conduits in a conifer tree depends on the longer time of cell Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

Research 113 expansion downwards along the stem. Journal of Experimental Botany 63: 837– 845. Anfodillo T, Petit G, Crivellaro A. 2013. Axial conduit widening in woody species: a still neglected anatomical pattern. IAWA Journal 34: 352–364. Bailey W, Shepard HB. 1915. Sanio’s laws for the variation in size of coniferous tracheids. Botanical Gazette 60: 66–71. van Bel AJE. 2003. The phloem, a miracle of ingenuity. Plant, Cell & Environment 26: 125–149. Burdon RD, Kibblewhite RP, Walker JCF, Megraw RA, Evans R, Cown DJ. 2004. Juvenile versus mature wood: a new concept, orthogonal to corewood versus outerwood, with special reference to Pinus radiata and P. taeda. Forest Science 50: 399–415. De Schepper V, De Swaef T, Bauweraerts I, Steppe K. 2013. Phloem transport: a review of mechanisms and controls. Journal of Experimental Botany 64: 4839– 4850. Domec JC, Gartner BL. 2002. Age- and position-related changes in hydraulic versus mechanical dysfunction of xylem: inferring the design criteria for Douglas-fir wood structure. Tree Physiology 22: 91–104. Domec JC, Warren JM, Meinzer FC, Lachenbruch BL. 2009. Safety for xylem failure by implosion and air-seeding within roots, trunks and branches of young and old conifer trees. IAWA Journal 30: 101–120. Duff GH, Nolan NJ. 1953. Growth and morphogenesis in the Canadian forest species. I. The controls of cambial and apical activity in Pinus resinosa Ait. Canadian Journal of Botany 13: 471–513. Eugenio M, Olano JM, Fonti P. 2014. Disentangling the effects of ontogeny and environmental factors on xylem anatomy in a semiarid chamaephyte. Journal of Arid Environments 104: 1–8. Evert RF. 2006. Esau’s plant anatomy. Meristems, cells, and tissues of the plant body: their structure, function, and development. Hoboken, NJ, USA: John Wiley & Sons, Inc. Franceschini T, Lundqvist S-O, Bontemps J-D, Grahn J, Olsson L, Evans R, Leban J-M. 2012. Empirical models for radial and tangential fibre width in tree rings of Norway spruce in north-western Europe. Holzforschung 66: 219– 230. Fromm J. 2013. Xylem development in trees: from cambial divisions to mature wood cells. In: Fromm J, ed. Cellular aspects of wood formation. Berlin, Germany: Springer, 3–39.  Gricar J, Krze L, Cufar K. 2009. Number of cells in xylem, phloem and dormant cambium in Silver fir (Abies alba), in trees of different vitality. IAWA Journal 30: 121–133.  Gricar J, Zupancic M, Cufar K, Koch G, Schmitt U, Oven P. 2006. Effect of local heating and cooling on cambial activity and cell differentiation in the stem of Norway spruce (Picea abies). Annals of Botany 97: 943–951. Hacke UG, Sperry JS, Pockman WT, Davis SD, McCulloch KA. 2001. Trends in wood density and structure are linked to prevention of xylem implosion by negative pressure. Oecologia 126: 457–461. H€oltt€a T, Kurppa M, Nikinmaa E. 2013. Scaling of xylem and phloem transport capacity and resource usage with tree size. Frontiers in Plant Science 4: 496. H€oltt€a T, Mencuccini M, Nikinmaa E. 2009. Linking phloem function to structure: analysis with a coupled xylem–phloem transport model. Journal of Theoretical Biology 259: 325–337. Huber B. 1939. Das Siebr€ohrensystem unserer B€aume und seine jahreszeitlichen Ver€anderungen. Jahrb€ u cher f€ u r Wissenschaftliche Botanik 88: 175–242. Jensen KH, Lee J, Bohr T, Bruus H, Holbrook NM, Zwieniecki MA. 2011. Optimality of the M€ unch mechanism for translocation of sugars in plants. Journal of the Royal Society Interface 8: 1155–1165. Jensen KH, Liesche J, Bohr T, Schulz A. 2012. Universality of phloem transport in seed plants. Plant, Cell & Environment 35: 1065–1076. Jensen KH, Zwieniecki MA. 2013. Physical limits to leaf size in tall trees. Physical Review Letters 110: 018104. Kantola A, M€akel€a A. 2004. Crown development in Norway spruce (Picea abies [L.] Karst.). Trees – Structure and Function 18: 408–421. Knoblauch M, Peters WS. 2013. Long-distance translocation of photosynthates: a primer. Photosynthesis Research 117: 189–196. Koch GW, Sillett SC, Jennings GM, Davis SD. 2004. The limits to tree height. Nature 428: 851–854. New Phytologist (2015) 205: 102–115 www.newphytologist.com

114 Research K€ ostner B, Falge E, Tenhunen JD. 2002. Age-related effects on leaf area/ sapwood area relationships, canopy transpiration and carbon gain of Norway spruce stands (Picea abies) in the Fichtelgebirge, Germany. Tree Physiology 22: 567–574. Lachenbruch B, Moore JR, Evans R. 2011. Radial variation in wood structure and function in woody plants, and hypothesis for its occurrence. In: Meinzer FC, Lachenbruch B, Dawson TE, eds. Size- and age-related changes in tree structure and function, vol 4. Dordrecht, the Netherlands: Springer, 121–164. Lintunen A, Kalliokoski T. 2010. The effect of tree architecture on conduit diameter and frequency from small distal roots to branch tips in Betula pendula, Picea abies and Pinus sylvestris. Tree Physiology 30: 1433–1447. M€akel€a A, Valentine HT. 2006. The quarter-power scaling model does not imply size-invariant hydraulic resistance in plants. Journal of Theoretical Biology 243: 283–285. Martınez-Vilalta J, Vanderklein D, Mencuccini M. 2007. Tree height and age-related decline in growth in Scots pine (Pinus sylvestris L.). Oecologia 150: 529–544. McCulloh K, Sperry JS, Lachenbruch B, Meinzer FC, Reich PB, Voelker S. 2010. Moving water well: comparing hydraulic efficiency in twigs and trunks of coniferous, ring-porous, and diffuse- porous saplings from temperate and tropical forests. New Phytologist 186: 439–450. Mencuccini M. 2002. Hydraulic constraints in the functional scaling of trees. Tree Physiology 22: 553–565. Mencuccini M, Grace J, Fioravanti M. 1997. Biomechanical and hydraulic determinants of tree structure in Scots pine: anatomical characteristics. Tree Physiology 17: 105–113. Mencuccini M, H€oltt€a T, Martınez-Vilalta J. 2011. Comparative criteria for models of the vascular transport systems of tall trees. In: Meinzer FC, Lachenbruch B, Dawson TE, eds. Size- and age-related changes in tree structure and function, vol 4. Dordrecht, the Netherlands: Springer, 309–339. Mencuccini M, H€oltt€a T, Petit G, Magnani F. 2007. Sanio’s Laws revisited. Size-dependent changes in the xylem architecture of trees. Ecology Letters 10: 1084–1093. Mullendore DL, Windt CW, Van As H, Knoblauch M. 2010. Sieve tube geometry in relation to phloem flow. The Plant Cell 22: 579–593. M€ unch E. 1927. Versuche u €ber den Saftkreislauf. Berichte der Deutschen Botanischen Gesellschaft 45: 340–356. Nardini A, Lo Gullo MA, Salleo S. 2011. Refilling embolized xylem conduits: is it a matter of phloem unloading? Plant Science 180: 604–611. Nikinmaa E, H€oltt€a T, Hari P, Kolari P, M€a kel€a A, Sevanto S, Vesala T. 2013. Assimilate transport in phloem sets conditions for leaf gas exchange. Plant, Cell & Environment 36: 655–669. Novitskaya LL, Kushnir FV. 2006. The role of sucrose in regulation of trunk tissue development in Betula pendula Roth. Journal of Plant Growth Regulation 25: 18–29. Olson ME, Rosell JA. 2013. Vessel diameter-stem diameter scaling across woody angiosperms and the ecological causes of xylem vessel diameter variation. New Phytologist 197: 1204–1213. Pantin F, Simonneau T, Muller B. 2012. Coming of leaf age: control of growth by hydraulics and metabolics during leaf ontogeny. New Phytologist 196: 349– 366. Patrick JW. 2013. Fundamentals of phloem transport physiology. In: Thompson GA, van Bel AJE, eds. Phloem: Molecular Cell Biology, Systemic Communication, Biotic Interactions. Hoboken, NJ, USA: Wiley-Blackwell, 30–60. Petit G, Anfodillo T. 2009. Plant physiology in theory and practice: an analysis of the WBE model for vascular plants. Journal of Theoretical Biology 259: 1–4. Petit G, Anfodillo T, Mencuccini M. 2008. Tapering of xylem conduits and hydraulic limitations in sycamore (Acer pseudoplatanus) trees. New Phytologist 177: 653–664. Petit G, Crivellaro A. 2014. Comparative axial widening of phloem and xylem conduits in small woody plants. Trees – Structure and Function 28: 915–921. Petit G, Pfautsch S, Anfodillo T, Adams MA. 2010. The challenge of tree height in Eucalyptus regnans: when xylem tapering overcomes hydraulic resistance. New Phytologist 187: 1146–1153. New Phytologist (2015) 205: 102–115 www.newphytologist.com

New Phytologist Pittermann J, Sperry JS, Wheeler JK, Hacke UG, Sikkema EH. 2006. Mechanical reinforcement of tracheids compromises the hydraulic efficiency of conifer xylem. Plant, Cell & Environment 29: 1618–1628. Quilho T, Pereira H, Richter HG. 2000. Within-tree variation in phloem cell dimensions and proportions in Eucalyptus globulus. IAWA Journal 21: 31–40. Rosell JA, Gleason S, Mendez-Alonzo R, Chang Y, Westoby M. 2014. Bark functional ecology: evidence for tradeoffs, functional coordination, and environment producing bark diversity. New Phytologist 201: 486–497. Rosner S. 2013. Hydraulic and biomechanical optimization in Norway spruce trunkwood – a review. IAWA Journal 34: 365–390. Rosner S, Baier P, Kikuta S. 2001. Osmotic potential of Norway spruce [Picea abies (L.) Karst.] secondary phloem in relation to anatomy. Trees – Structure and Function 15: 472–482. Ryan MG, Asao S. 2014. Phloem transport in trees. Tree Physiology 34: 1–4. Sanio K. 1872. Ueber die gr€osse der holzzellen bei der gemeinen kiefer (Pinus silvestris). Jahrb€ u cher f€ u r Wissenschaftliche Botanik 8: 401–420. Savage VM, Bentley LP, Enquist BJ, Sperry JS, Smith DD, Reich PB, von Allmen EI. 2010. Hydraulic trade-offs and space filling enable better predictions of vascular structure and function in plants. Proceedings of the National Academy of Sciences, USA 107: 22722–22727. Schulz A. 1990. Conifers. In: Behnke H-D, Sjolund RD, eds. The sieve element– comparative structure, induction and development. Berlin, Germany: Springer-Verlag, 63–88. Schulz A. 1998. Phloem. Structure related to function. Progress in Botany 59: 429–475. Secchi F, Zwieniecki MA. 2011. Sensing embolism in xylem vessels: the role of sucrose as a trigger for refilling. Plant, Cell & Environment 34: 514– 524. Sevanto S, McDowell NG, Dickman LT, Pangle R, Pockman WT. 2014. How do trees die? A test of the hydraulic failure and carbon starvation hypotheses. Plant, Cell & Environment 37: 153–161. Shinozaki K, Yoda K, Hozumi K, Kira T. 1964. A quantitative analysis of plant form – the pipe model theory. I. Basic analyses. Japanese Journal of Ecology 14: 97–105. Sorce C, Giovannelli A, Sebastiani L, Anfodillo T. 2013. Hormonal signals involved in the regulation of cambial activity, xylogenesis and vessel patterning in trees. Plant Cell Reports 32: 885–898. Sperry JS, Meinzer FC, McCulloh KA. 2008. Safety and efficiency conflicts in hydraulic architecture: scaling from tissues to trees. Plant, Cell & Environment 31: 632–645. Sperry JS, Nicholas KL, Sullivan JEM, Eastlack SE. 1994. Xylem embolism in ring porous, diffuse porous, and coniferous trees of northern Utah and interior Alaska. Ecology 75: 1736–1752. Spicer R. 2014. Symplasmic networks in secondary vascular tissues: parenchyma distribution and activity supporting long-distance transport. Journal of Experimental Botany. 65: 1829–1848. Spicer R, Gartner BL. 2001. The effects of cambial age and position within the stem on specific conductivity in Douglas-fir (Pseudotsuga menziesii) sapwood. Trees – Structure and Function 15: 222–229. Thompson MV. 2006. Phloem: the long and the short of it. Trends in Plant Science 11: 26–32. Thompson MV, Holbrook NM. 2003. Application of a single-solute non-steady-state phloem model to the study of long-distance assimilate transport. Journal of Theoretical Biology 220: 419–455. Thorpe MR, Minchin PEH, Gould N, McQueen JC. 2005. The stem apoplast: a potential communication channel in plant growth regulation. In: Holbrook NM, Zwieniecki MA, eds. Vascular transport in plants. Burlington, MA, USA: Elsevier, 355–371. Turgeon R. 2010. The puzzle of phloem pressure. Plant Physiology 154: 578–581. Tyree MT, Zimmermann MH. 2002. Xylem structure and ascent of sap. Berlin, Germany: Springer-Verlag. Weitz JS, Ogle K, Horn HS. 2006. Ontogenetically stable hydraulic design in woody plants. Functional Ecology 20: 191–199. West GB, Brown JH, Enquist BJ. 1999. A general model for the structure and allometry of plant vascular systems. Nature 400: 664–667. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist Woodruff DR. 2014. The impacts of water stress on phloem transport in Douglas-fir trees. Tree Physiology 34: 5–14. Xu RH. 2003. Measuring explained variation in linear mixed effects models. Statistics in Medicine 22: 3527–3541. Zimmermann MH. 1978. Hydraulic architecture of some diffuse-porous trees. Canadian Journal of Botany 56: 2286–2295. Zobel BJ, Sprague JR. 1998. Juvenile wood in forest trees. Berlin, Heidelberg, Germany: Springer.

Research 115

Table S1 Characteristics of Picea abies sample trees and sampling positions Table S2 Allometry between phloem and xylem conduit diameter and stem diameter Table S3 Allometry between phloem and xylem conduit diameter and distance from the stem apex

Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 Cell wall thickness versus lumen diameter in phloem and xylem. Fig. S2 Phloem and xylem conduit diameter versus cambial age, and phloem to xylem conduit diameter ratio versus stem diameter.

Table S4 Coefficients used for calculating xylem and phloem conductivities Methods S1 Calculation of xylem and phloem hydraulic conductivity. Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

New Phytologist is an electronic (online-only) journal owned by the New Phytologist Trust, a not-for-profit organization dedicated to the promotion of plant science, facilitating projects from symposia to free access for our Tansley reviews. Regular papers, Letters, Research reviews, Rapid reports and both Modelling/Theory and Methods papers are encouraged. We are committed to rapid processing, from online submission through to publication ‘as ready’ via Early View – our average time to decision is