1This study investigated the effects on water transport patterns of the transverse water movement in xylem, on a whole-shoot scale, using current-year shoots of kudzu vine (Pueraria lobata (Willd.) Ohwi).
2The connections between xylem vessels were detected by dye injection and were found to be distributed throughout the internode. This means that short internodes limit the connections between vessels. The hydraulic conductance of the internode-to-petiole path decreased with successive cutting of the internodes.
3To estimate hydraulic patterns at the whole-shoot scale, the hydraulic conductance of the shoot base-to-petiole path (KBP) were measured. These were compared with two mathematical models representing extreme examples of connections between vessels – the interconnected model and the independent model. The former model assumes a high capacity for the transverse movement of water in xylem tissues. The KBP values measured were explained more accurately by the interconnected model than by the independent model.
4These results suggest that kudzu vine has a large capacity for the transverse movement of water in xylem, which contributes to the effective transport of water from internodes to leaves that offsets sectored transport at the whole-shoot scale.
Water transport in plants has been studied using models analogous to electrical circuits (Tyree & Ewers 1991). Based on the analogy between Ohm's law, describing the electric current through the conducting media, and Fick's law, describing the diffusion of substances, voltage, electric current and electrical resistance are viewed as the motive force of water transport, the rate of sap flow and hydraulic resistance, respectively. The use of such circuits helps to examine the hydraulic patterns in plants consisting of different organs and having complex branching systems (Tyree 1988; Cannell & Dewar 1994; Mencuccini & Grace 1996).
In plants, the water pathway in the xylem comprises many narrow, interconnected conduits. However, the hydraulic resistance (Calkin et al. 1986; Zwieniecki et al. 2001; Sperry & Hacke 2004) and frequency (Esau 1977; Tyree & Zimmermann 2002) of the conduit-to-conduit pathway vary considerably among plants. As a result of these features of xylem, a leaf can obtain water through the specific bundles of conduits in the stem, which results in a ‘sectored’ flow. Nevertheless, the electrical circuit models assume that the water pathway in the xylem is a tube-like structure with a central lumen typical of animal blood vessels, thus failing to express the sectored flow in plants. For woody species, a small tangential spread of sap flow has been reported (Ewers et al. 1991; Tyree & Zimmermann 2002). Therefore the electrical circuit model also fails to predict correctly the hydraulic patterns of woody species.
Many studies attempted to estimate the sectoriality of water flow at the level of whole plants and branches. At the whole-plant scale, the interactions between branches were examined mainly using conifer species. When branches were partially defoliated or shaded, stomatal conductance by leaf area increased in some cases (Troeng & Langstrom 1991; Pataki et al. 1998) but not in others (Whitehead et al. 1996; Hubbard et al. 1999; Brooks et al. 2003). In other words, neither quantitative relations nor clear general tendencies have been obtained to date. At the branch scale, the magnitude of lateral flow has been examined by measuring hydraulic conductance. Hydraulic conductance has been measured with the main axis-to-lateral branch in the Y-shaped branch segments of some woody species (Tyree & Alexander 1993; Brooks et al. 2003; Schulte & Brooks 2003; Schulte 2006). Hydraulic conductance was measured for the leaf-to-leaf path (Orians et al. 2005), or for branch segments with cut ends sealed using acrylic-based glue (Zanne et al. 2006) in several ring-porous and diffuse-porous deciduous trees. These studies confirmed the sectoriality and substantial hydraulic resistance in the lateral direction within the xylem. However, the magnitude of the lateral flow observed at the branch scale failed to explain the capacity for transverse water movement or the hydraulic patterns at the whole-plant level. As the number of transverse connections between conduits tends to increase with increasing water-transport distance, long-distance water transport may compensate for the incomplete connections between conduits and sectored flow.
To investigate mechanisms by which the transverse movement of water in the xylem influences the hydraulic pattern in the whole shoot, we evaluated the magnitude of the transverse movement of water by measuring hydraulic conductance at both branch and whole-shoot levels. We used kudzu vine (Pueraria lobata (Willd.) Ohwi), which has shoots over 10 m long, and focused on the water-transport path from internodes to petioles. This pathway is suitable for estimating the capacity of transverse water movement in the xylem, as the leaf trace connects only with the vessels in the primary xylem, in the internode (Esau 1977). Therefore the water supply from the vessels in the secondary xylem in the internode to the leaf depends largely on the lateral water flow in the xylem via vessel-to-vessel pathways. We first evaluated the magnitude of the transverse movement of water in the xylem at the internode scale using dye experiments, and by determining the hydraulic conductance of the internode-to-petiole path (KIP). To confirm the effects of the lateral movement of water in the xylem on hydraulic patterns in the whole shoot, we compared the distribution of the hydraulic conductance of the two-stem base-to-petiole path (KBP) with those predicted by two mathematical models, representing the two extreme cases of transverse water movement. Based on our results at two different scales, we discuss how the lateral movement of water in the xylem influences the hydraulic patterns in the whole shoot, and consider its ecological importance.
Materials and methods
The plant material used in this study was Pueraria lobata, the kudzu vine. The newly grown, current shoot of kudzu typically reaches over 20 m long and is thus particularly suitable for investigating long-distance water transport in vascular systems. Water can pass through all vessels in the internode. The current-year shoot consists of a series of nodes separated by 20- to 40-cm-long internodes. Each node bears a leaf. Therefore this simple form of kudzu is highly suitable for modelling hydraulic architecture and for direct measurements of shoot hydraulic patterns. Current-year, ground-creeping shoots of kudzu were collected from an open riverside field (Imaichi City, 36°44′ N, 139°45′ E; 280 m a.s.l.) either on rainy days or predawn. Shoots >10 m long and with few branches were used for measurements at the internode scale. A single, 15·2-m-long shoot bearing no lateral branches was used for measurements at the whole-shoot scale. The leaves attached to the shoot were exposed to similar light conditions. In both experiments, adventitious roots on the nodes, if present, were removed a few days before the measurements. In sampling, each shoot was coiled to about 80 cm diameter and wrapped in plastic bags. Great care was taken to avoid bending the stems and petioles. The shoot was cut at its base under water and brought into the laboratory. The cut end was kept under water until the measurements were taken. For experiments at the internode scale, the internodes between 22 and 33 cm long and ≈6 mm in diameter were collected from the 10th to the 21st node, counted from the shoot apex. Eighteen segments were obtained from 10 current-year shoots. The shoot used for the measurements at the whole-shoot scale had 36 leaves (total leaf area, 1·09 m2) and its stem diameter was, on average, 5·8 mm. The dye experiments and the measurements of hydraulic conductance of the internode-to-petiole path (KIP) were carried out between September and November 2003, and in September 2004, respectively. Measurements at the whole-shoot scale were performed in August 2003.
To identify lateral pathways in the xylem, a solution of safranin was injected from the cut end of the internode and its passage to the petiole was followed. Each segment contained two nodes (Fig. 1a,b) and was cut 5 cm above and below the respective nodes, and the petioles were cut to 5 cm long. All excisions were performed under water. The cut end of the petiole on the distal node was connected to a vacuum pump through a tube filled with water. For dye injection in the acropetal direction, the proximal end of the internode was connected with a silicone tube filled with 0·5% safranin solution (Fig. 1a). For dye injection in the basipetal direction, silicone tubes filled with the safranin solution were attached to the distal end (Fig. 1b). Water-filled silicone tubes were attached to other ends of the internode and petiole, and the tubes were closed with stopcocks. Water and the safranin solution were filtered through a 0·22-µm filter by applying negative pressure. To remove air from embolized vessels, water was forced to flow through the segment by applying a positive pressure of 0·15 MPa at the cut end of the proximal internode using a pressure bomb (Soilmoisture equipment, Goleta, CA, USA) for at least 5 min. After refilling, the safranin solution was stimulated to flow by sucking the cut end of the distal petiole at 0·03 MPa. After ≈30 min, when the dye was clearly observed on the cut end of the distal petiole, the pressure was released. In the dye-injection experiment (in an acropetal direction), transverse sections were made at 2-cm intervals starting from the distal node, using the hand-section method, to detect the connections between vessels within an internode. In the dye-injection experiment, transverse sections were made (in a basipetal direction) to observe how the dye flows from the distal end of the segment to the distal petiole.
measurement of hydraulic conductance of internode-to-petiole path (Kip)
To quantify the impact of the connections between vessels on the magnitude of lateral flow of water, the hydraulic conductance of the internode-to-petiole path, KIP, was measured when the internode length was shortened. Each segment with a single node was obtained by cutting 25 cm below and 5 cm above the node (Fig. 1c). The petiole was again cut to 5 cm. Water-filled tubes were connected to all cut ends. The tubing of the proximal end of the internode was connected to a water reservoir, and the tubing of the distal end of the internode was closed with a stopcock. The water for all measurements of hydraulic conductance was distilled, adjusted to approximately pH 2 with HCl and filtered through a 0·22-µm filter under a partial vacuum (Sperry et al. 1988). To remove air from embolized vessels, the stem segments were refilled with water from the proximal end of the internode by applying a positive pressure of 0·15 MPa for 3 min. Hydraulic conductance was determined as the mass-flow rate of water divided by the pressure applied (Tyree & Ewers 1991). After allowing for the system to reach a steady state, the outflow was collected using a cotton wool-filled micropipette tip for 1 min at a pressure of 8 kPa that was generated by normal gravitational force. The capacity for the transverse flow probably decreases with a decrease in segment length. This approach would enable us to quantify how the transverse flow changes with distance. Therefore we shortened the segment by successive cuttings at distances of 2·5, 5, 10, 20 and 25 cm from the distal node and obtained respective KIP values.
measurement of hydraulic conductance of the stem base-to-petiole path (kbp)
The distribution of hydraulic conductance of the stem base-to-petiole path (KBP) was measured to evaluate hydraulic patterns at the whole-shoot scale. After the current-year shoot was brought into the laboratory, its stem base was cut again under water with a razor, and the cut end and the water reservoir (in the pressure bomb) were connected with a water-filled tubing (Fig. 1d). To remove air from embolized vessels, the shoot was refilled with water by applying a positive pressure of 0·2 MPa for 20 min. After the bases of three leaflets were cut, water at a positive pressure of 0·2 MPa was applied for 15 min until a steady flow was reached. The rate of outflow from the proximal cut end to each of two petioles (node numbers 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35) was measured at 0·2 MPa for 3 min using the same method as described for KIP measurement. Guttation from the cut end of a petiole was barely detectable at any of the nodes, except in the case of the first four immature leaves; these were not used for measurements.
measurement of hydraulic architecture
The hydraulic conductances of an internode (KI) and a petiole (KP) were measured after the measurement of KBP. Segments, each of which had one node, were obtained by cutting 5 cm above each of the nodes. The petiole was cut at the base of three leaflets. Water-filled tubes were connected to all the cut ends. The tubing attached to the proximal end of the internode was put in a water reservoir placed on an electronic balance (±0·1 mg, Shimadzu Corp., Kyoto, Japan). To remove the embolism, water was forced to flow through the stem segments from the proximal end by applying a positive pressure of 0·15 MPa for at least 3 min using the pressure bomb. After waiting a few minutes for the system to reach a steady state, the mass flow rate of water was measured by weighing the water reservoir. KI was measured with the cut end of the petiole closed by a stopcock (Fig. 1e). Water flow at a positive pressure of 2 kPa generated by normal gravitational force was measured at 5 s intervals for 1 min. KP measurement was performed in the internode-to-petiole path, wherein the distal cut end of the internode was closed with the stopcock to include the constriction at the petiole junction (Fig. 1c; Tyree & Alexander 1993; Schulte & Brooks 2003). Water flow rate caused by a positive pressure of 8 kPa was measured at 5-s intervals for 1 min. The KP value was obtained by subtracting the value of hydraulic resistance of the internode (= 1/KI) from that of the internode-to-petiole path. This results in underestimation of KP, because the vessels used for measurement of KI include the conduits through which water did not flow in an internode-to-petiole direction. However, the resistance imposed by such vessels was small enough to be neglected. Both KI and KP measurements were obtained at two-node intervals.
To consider the effects of the transverse movement of water in the xylem, two circuit models were developed. One model assumes complete connections between vessels as water moves through a tube with a central lumen (Fig. 2a). This is similar to widely used electrical circuit models. This model was designated the interconnected model. The second model assumes that water is transported through a limited bundle of vessels (Fig. 2b). This model, designated the independent model, has the same structure as the pipe model (Shinozaki et al. 1964; West et al. 1999).
A shoot was assumed to consist of n segments, each segments being composed of an internode and a petiole. For segment i (numbered in descending order from i = 1 at the shoot tip), the mass flow rate of water and the hydraulic resistance of the internode were expressed as II(i) and RI(i), respectively, and those for the petiole as IP(i) and RP(i), respectively. For calculating hydraulic resistance, the inverse of the conductance was used. The values of RI(i) and RP(i) were obtained from the regression equations shown in Fig. 7. The total hydraulic resistance from the shoot base to the petiole of the shoot comprising segment 1 to segment i is designated as RTOT(i). Calculations were carried out using f-basic (Fujitsu, Kawasaki, Japan).
KBP of the interconnected model was obtained by mathematical induction. As the shoot in segment 1 is expressed as an electrical circuit of two resistors of an internode and a petiole in series, RTOT(1) is expressed as:
RTOT(1) = RI(1) + RP(1)((eqn 1))
The shoot comprising segments 1 and 2 was viewed as an electrical circuit having a two-way junction consisting of RI(2), RP(2) and RTOT(1). Thus the total resistance of the electrical circuit RTOT(2) is described by:
Likewise, the shoot comprising segment 1 to segment i is viewed as an electrical circuit having a two-way junction consisting of RI(i), RP(i) and RTOT(i − 1). Thus the total resistance of the electrical circuit RTOT(i) is expressed as:
The hydraulic resistance of the whole shoot RTOT(n) was derived as follows using equations 1–4.
The flow rate of water through an internode at segment n, II(n), can be described with RTOT(n) and the motive force, P, analogous to the voltage:
When the shoot is viewed as a two-way junction consisting of three resistors, RI(n), RP(n) and RTOT(n − 1), the flow rate of water through a petiole at segment n, IP(n), can be expressed using II(n), RI(n), RP(n) and RTOT(n − 1) as:
The flow rate of water through RTOT(n − 1), II(n − 1), is described as:
Likewise, IP(i) and II(i − 1) can be described with II(i) and hydraulic resistances:
IP and II values for all the segments were obtained according to equations 1–9. When P is assumed = 1, IP(i) is identical to KBP at segment i (mg H2O MPa−1 s−1).
The independent model assumes that a leaf is accessible via specific bundles of vessels isolated from others (Fig. 2b), and we followed the assumptions of the pipe model theory (Shinozaki et al. 1964; West et al. 1999). An internode includes the same number of vessel bundles as that in its distal nodes, for example the internode of segment i has i vessel bundles. RBP(i) for the independent model is expressed by a summation of the hydraulic resistance of a bundle of vessels (RB) from n to i and that of a petiole:
Each vessel bundle in a segment is assumed to have a similar hydraulic resistance value. Thus the hydraulic resistance of a vessel bundle within the internode of segment i, RB(i), can be expressed by RI(i) and the number of bundles, i:
RB(i) =RI(i) ·i((eqn 11))
Then IP(i) is predicted by the independent model as:
When P is assumed to be 1, IP(i) is identical to KBP at segment i (mg H2O MPa−1 s−1).
experiments at the internode scale
Dye experiments were performed to detect the vascular patterns within the internode. Five leaf traces were distributed evenly around the xylem (Fig. 3a). When the dye was injected into the proximal end of the internode, and its passage to the petiole (in the acropetal direction) was followed, stained vessels were observed only in the primary xylem connecting to the leaf trace at 2–6 cm below the distal node of the segment (Fig. 3b). The stained vessels were then detected in the secondary xylem adjacent to the leaf traces at 8 cm (Fig. 3c) and in the peripheral region of the secondary xylem at a distance of >12 cm below the distal node of the segment (Fig. 3d,e). When the dye was injected from the distal end of an internode and its passage to a petiole (the basipetal direction) followed, 78·9 ± 11·5% (mean ± SD, n = 5) of vessels in the secondary xylem of the nodal region were stained. The percentage of stained vessels normalized as the number of vessels at the nodal region decreased towards the proximal parts of the internode and had declined to ≈20% on reaching the next, older nodal region (Fig. 4).
To estimate the importance of the transverse movement of water in xylem, KIP values were determined in the segments with varying internode lengths. It is notable that KIP decreased gradually with decreasing internode length, although the distance of water transport was shortened. KIP of segments with the 2·5-cm internode was lower than that of the 25-cm segments by ≈40%, although the transport distance including the internode and petiole was reduced by up to ≈25% (Fig. 5).
experiments at the whole-shoot scale
The distribution of KBP was measured from the 5th to 35th node at two-node intervals in one current shoot. It was found that KBP at the 35th petiole was ≈16 times greater than that at the 5th petiole (filled circles in Fig. 6a). We tested this hydraulic pattern against the two mathematical models. The basic values of hydraulic conductance of petioles, bundles and internodes (KP, KB and KI, respectively) were obtained from the regression curves of these values vs the node number after log transformation (Fig. 7, KI: log y = 0·65 log x + 2·20, R2 = 0·64; KB: log y = −0·35 log x + 2·20, R2 = 0·34; KP: log y = 0·12 log x + 0·91, R2 = 0·028). It was found that KI was larger than KP by a factor of 20–100. KB, which was obtained by dividing KI by the number of distal nodes (equation 11) decreased slightly with node number. Both interconnected and independent models predicted that the hydraulic pattern favours basal petioles (solid and dashed curves in Fig. 6a). The KBP predicted by the independent model decreases more markedly at the basal nodes, and the values at the apical nodes were higher than those predicted by the interconnected model. The measured KBP were linearly related to those predicted by both models (Fig. 6b, interconnected model: y = 0·88x + 0·017, R2 = 0·93; independent model: y = 0·57x + 0·052, R2 = 0·86). However, it is notable that the slope and y-intercept of the interconnected model were not significantly different from 1·0 and the origin, respectively (ancova, slope, P = 0·071; y-intercept, P = 0·38), whereas those for the independent model were significantly different from 1·0 and the origin (P < 0·01).
The measured distribution of KBP was explained more accurately by the interconnected model than by the independent model (Fig. 6), suggesting a large capacity for the transverse movement of water in the xylem of the kudzu vine. The results of the internode-scale experiments could explain the mechanism by which the lateral flow is achieved in the kudzu vine.
The two dye experiments indicated that petioles have access to the secondary xylem vessels via vessel-to-vessel paths within an internode. Dye injection in the acropetal direction indicated that the connections between vessels in the leaf traces and the secondary xylem vessels occurred within 8 cm from the node (Fig. 3). According to the dye experiments in the basipetal direction, ≈80% of the secondary xylem vessels are connected to a petiole via the vessel-to-vessel paths in an internode. This is because the dye solution stained only those secondary xylem vessels where tension from the petiole was transmitted. In addition, the dye experiments showed that the vessel-to-vessel paths were distributed throughout the internode. In the dye experiment in an acropetal direction, a gradual increase was observed in stained vessels of the secondary xylem towards the proximal end of the segment (Fig. 3b–e). The decreased extent of the stained region shown in Fig. 4 indicates the existence of vessel-to-vessel paths. In this dye-injection experiment, the safranin solution in the xylem would move through the path with the minimum length along the water potential gradient. Thus the dye in a vessel moved longitudinally, so long as the vessel was in contact with an adjacent vessel with a more negative water potential, although the stained patterns may not show precisely the positions of connections between vessels, because safranin is less diffusive than water (Sano et al. 2005). However, these experiments qualitatively detect the existence of vessel-to-vessel pathways. Thus the linear reduction in the stained region suggests that the probability of transverse water movement in the xylem is nearly proportional to the internode length in the internode-to-petiole pathway. The distribution of the lateral path of water flow in the xylem can explain the decrease in KIP despite a decrease in the internode length (Fig. 5). The resistance of the transverse water movement in the xylem is due to the parallel resistance of the vessel-to-vessel paths. A shorter internode provides less chance for the transverse movement of water via vessel-to-vessel pathways, thus lowering the magnitude of the transverse water movement and KIP (Schulte 2006). Therefore the present results at the internode scale suggest that lateral flow within the internode contributes significantly to the effective water supply to the leaf. According to previous studies, the hydraulic resistance at the junction of the lateral path may limit the water flow through the internode-to-petiole path (Zimmermann 1978; Schulte & Brooks 2003). If the resistance at the petiole junction is considerably greater than that in other areas, KIP would remain unchanged in response to the shortening of internode length. However, a decrease in KIP was obvious in the present study.
The effects of internode length on the transverse movement of water (Fig. 5) are also important for hydraulic patterns at the whole-shoot scale. Long-distance transport can increase the connections between conduits and offset sectored transport over a short distance. The petiole of the kudzu vine has access to 80% of the secondary xylem vessels in the internode via the vessel-to-vessel paths within the internode. This high percentage should ensure that a greater number of vessels come into contact with the petiole, and also that a greater number of interconnections are made with vessels at internodes located near the base of the stem. These processes reduce the hydraulic resistance of the lateral paths and improve the sectored transport of water within the whole shoot. Reflecting these features, the interconnected model predicts the hydraulic features at the whole-shoot scale with greater accurately than the independent model. High KBP values on the basal part of the shoot are caused by competition for water due to the large magnitude of lateral movement of water (Fig. 6). The transpiration rate of the remaining leaves may increase in the kudzu vine, if the foliage area in the shoot's basal region is reduced.
A water-transport system with a large magnitude of transverse water movement in the xylem is beneficial to the kudzu vine, which grows to >20 m in length. Limited lateral flow in the xylem can cause restricted water transport within a shoot. The sectored transport of water supply prevents the lateral branches from extending towards light sources (Orians et al. 2004). This would be detrimental to plants that extend particularly long shoots. Moreover, the large potential for lateral water movement functions as a safety mechanism that prevents xylem dysfunction caused by embolism, because many lateral paths in the xylem can bypass the embolized vessels (Tyree et al. 1994). This is of greater significance to the kudzu vine, the xylem of which includes fewer vessels (90–130 vessels in secondary xylem) than other tree species (Ewers et al. 1991). In addition, a leaf is supported hydraulically by a great number of vessels, which would prevent an excessive increase in resistance with an increase in the transport distance of water. In the situation where a leaf connects only with specific bundles of vessels, as described in the independent model, water is supplied to a leaf with large hydraulic resistance, as KB is lower than KI (Fig. 7). Therefore sectored water transport results in a large hydraulic resistance in long-distance transport in the stem.
The lateral movement of dye in the kudzu stem is greater than in the other woody species (Tyree & Zimmermann 2002). One reason for this is the smaller distance between vessels, due to the narrow stem of kudzu compared with the trunks of tall trees (Zanne et al. 2006). Other reasons include differences in the morphological and physiological features of xylem, such as the size and frequency of pit pores (Sperry & Hacke 2004) and the sensitivity of pits to changes in ion concentrations (Zwieniecki et al. 2001). However, given the enormous variety in the anatomical features of xylem, further studies are required to confirm the general applicability of the interconnected model.
Authors thank Dr H. Nagashima, Dr A. Kume, Dr T. Kubo, Dr I. Terashima and the members of his laboratory for meaningful discussions, and also thank Mr H. Takahashi for technical support.