Mycorrhiza DOI 10.1007/s00572-016-0684-5

ORIGINAL ARTICLE

Pathway and sink activity for photosynthate translocation in Pisolithus extraradical mycelium of ectomycorrhizal Pinus thunbergii seedlings Munemasa Teramoto 1,2 & Bingyun Wu 1 & Taizo Hogetsu 1

Received: 9 August 2015 / Accepted: 29 January 2016 # Springer-Verlag Berlin Heidelberg 2016

Abstract The purpose of this study was to identify the pathway and sink activity of photosynthate translocation in the extraradical mycelium (ERM) of a Pisolithus isolate. We labelled ectomycorrhizal (ECM) Pinus thunbergii seedlings with 14CO2 and followed 14C distribution within the ERM by autoradiography. 14C photosynthate translocation in the ERM resulted in 14C distribution in rhizomorphs throughout the ERM, with 14C accumulation at the front. When most radial mycelial connections between ECM root tips and the ERM front were cut, the whole allocation of 14C photosynthates to the ERM was reduced. However, the overall pattern of 14C distribution in the ERM was maintained even in regions immediately above and below the cut, with no local 14C depletion or accumulation. We inferred from this result that every portion in the ERM has a significant sink activity and a definite sink capacity for photosynthates and that photosynthates detour the cut and reach throughout the ERM by translocation in every direction. Next, we prepared paired ECM seedlings, ERMs of which had been connected with each other by hyphal fusion, alongside, labelled the left seedling with 14 CO2, and shaded none, one or both of them. 14C photosynthates were acropetally and basipetally translocated from the left ERM to ECM root tips of the right seedling through rhizomorphs in the left and right ERMs, respectively. With * Munemasa Teramoto [email protected]

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Department of Forest Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan

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Present address: Center for Global Environmental Research, National Institute for Environmental Studies, Onogawa 16-2, Tsukuba 305-8506, Japan

the left seedling illuminated, 14C translocation from the left to the right ERM increased by shading the right seedling. This result suggests that reduced photosynthate transfer from the host to its ERM increased sink activity of the ERM. Keywords Acropetal and basipetal . Common mycelial network . Ectomycorrhizal symbiosis . Extramatrical mycelium . Soil nutrients

Introduction Many trees live in a sym biotic association with ectomycorrhizal (ECM) fungi, which form hyphal nets and thick fungal sheaths inside and outside fine root tips of the host tree, respectively (Smith and Read 2008). Hyphae extending from the fungal sheath into soil construct mycelium called extraradical mycelium (ERM) and then form a mycelial network connecting different trees called common mycorrhizal network (CMN) by infecting root tips of ambient trees. ERM markedly enhances host absorption efficiency for soil nutrients such as nitrogen (Abuzinadah and Read 1986; Finlay et al. 1992; Turnbull et al. 1995; Plassard et al. 2000; Taylor et al. 2004) and phosphorus compounds (Finlay and Read 1986b; Bougher et al. 1990; Rousseau et al. 1994). Soil nutrients absorbed into the ERM are translocated to ECM root tips then are transferred to the host tree and consequently promote host growth. Such growth promotion facilitates tree establishment and survival (Nara and Hogetsu 2004; Nara 2006; Simard et al. 2012) and, thereby, forest development and maintenance. Thus, the structure and function of the ERM may greatly contribute to forest development and maintenance. The extension and physiological activities of the ERM may be largely supported by carbon resources (Nehls 2008; Nehls

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et al. 2010). Although the ERM can absorb organic carbon in the form of amino acids and carbohydrates from soil (Abuarghub and Read 1988a; 1988b; Chalot et al. 1996; Martin et al. 1998; Wallenda and Read 1999), carbon used for the ERM extension and activity is mainly derived from photosynthates directly transferred from the host tree to its ERM (Söderstrom and Read 1987; Högberg et al. 2001; Fransson et al. 2005; Nehls 2008; Nehls et al. 2010). A large amount of photosynthate, as much as 15% of the net primary production, is translocated to ECM fungal tissue (Vogt et al. 1982; Hobbie 2006). It has been repeatedly demonstrated by autoradiography that after transfer from the host to its ERM, photosynthates are translocated from the ECM root tips to regions of demand within the ERM (Brownlee et al. 1983; Finlay and Read 1986a; Bending and Read 1995; Leake et al. 2001; Wu et al. 2001; 2002; Rosling et al. 2004). Because such photosynthate translocation in the ERM may influence the spatial patterns of ERM extension and activity, it is expected to be a major determinant of the ECM symbiotic functions and deserves much more attention. Elucidation of the pathway of photosynthate translocation within the ERM may provide a clue to a deeper understanding of this translocation in the ERM. The ERM may develop primarily via radial growth of hyphae emanating from each ECM root tip. Thus, it is possible that resistance to photosynthate translocation is much smaller in the radial than in the lateral direction and, therefore, that most photosynthates are translocated radially within the ERM. However, fusion between contacting hyphae forms a hyphal anastomosis in the ERM. Using an experimental system composed of two ECM pine seedlings of which their ERMs tightly contacted each other, Wu et al. (2012) showed by autoradiography that hyphal fusion between the ERMs allowed relatively free translocation of 14C photosynthates between them. Thus, it may also be that a considerable proportion of photosynthates are translocated laterally as well as radially through fused hyphal anastomoses. Knowledge regarding sink activity in the ERM involved in photosynthate translocation may also promote understanding of the translocation in the ERM. As for the sink activity in the ERM, there are alternative possibilities: sink activity for photosynthates might be much stronger in a few local regions within the ERM and might be otherwise similar. Bending and Read (1995) cultivated a Pinus sylvestris seedling colonised by Suillus bovinus on the surface of peat in a rhizobox with trays of litter inserted near the front of the ERM, and after hyphae of the ERM covered the trays, they labelled the seedling with 14CO2. They found by autoradiography that on some of the litter trays, much 14C was accumulated. In a similar 14 CO 2 -labelling experiment with a P. sylvestris seedling colonised by Paxillus involutus, Leake et al. (2001) also presented results obtained using an

autoradiogram showing localised 14C accumulation on some of the litter trays. Wu et al. (2001) followed, by autoradiography, the translocation of 14C photosynthate in the ERM of an ECM fungus colonising a pine seedling and found that radioactivity accumulated much more at the marginal front of the ERM than in the inner region. These observations suggest a possibility that the sink activity of hyphal cells is much stronger in restricted regions like the nutrient-rich and the front regions of the ERM than that in other regions and that the ERM portions in the translocation pathway to the 14C-accumulating region simply function as translocation paths without their own sink activity. However, it has also been pointed out that hyphae proliferated more densely in the litter trays (Bending and Read 1995) and at the marginal front of ERM (Wu et al. 2001) where 14C photosynthates obviously accumulated. Thus, the 14C accumulation in localised regions of the ERM could also be explained as reflecting localised hyphal cell density if hyphal cells exhibit similar sink activity in 14C-accumulating regions to that in other regions. In relation to the sink activity for photosynthates in the ERM, Simard and collaborators (Simard et al. 1997; Teste et al. 2009; 2010) reported that photosynthates synthesised by a seedling (donor) were transferred to a neighbouring one (receiver) which was connected with the donor by the CMN and that the amount of transferred photosynthates was increased by shading the receiver. Because this transfer process contains transfer from the donor to the CMN, translocation through the CMN and transfer from the CMN to the receiver, it is not clear which of these three processes is influenced by the shading. However, their result suggests a possibility that shading of the receiver increases sink activity for photosynthates in the CMN portion under the receiver, and this possibility may be worth investigating. Our aim of this study is to challenge the following questions: (1) is the pathway of photosynthate translocation in the ERM fixed inflexibly in a specific direction, (2) is the sink activity for photosynthates localised in restricted areas within the ERM and (3) is the sink activity of the ERM increased by shading the host tree? In the present study, we physically cut the ERM of Pinus thunbergii seedlings colonised by a Pisolithus isolate, labelled the seedling needles with 14CO2 photosynthetically, and constructed time courses of 14C distribution within the ERM by sequential and quantitative autoradiography. Furthermore, using an experimental system in which ERMs of paired ECM pine seedlings were connected by hyphal fusion, we labelled one of the two seedlings with 14 CO2, then shaded none, one, or both of the paired seedlings. We constructed time courses of 14C distribution in the ERM under these shading treatments. Based on changes in the 14C distribution according to the cutting and shading, we inferred the pathway and the sink activity of photosynthate translocation in the ERM.

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Materials and methods Preparation of ECM inoculum From a modified Melin-Norkrans (MMN) agar plate (Marx 1969) on which mycelium of a Pisolithus isolate, PS, (Wu et al. 2012) was grown, 1-cm square agar blocks with the mycelium were cut out and placed onto cellophane filmcovered plates of MMN agar medium. After incubation at 25 °C for 2 weeks, 5-mm squares of extended mycelium were cut out together with the cellophane film and used as inocula.

Preparation of ECM seedlings on a flat flower foam plate in rhizoboxes Seeds of P. thumbergii Sieb. et Zucc. were sown and grown for 2 months on Shibanome soil (2-mm granulated volcanic sand, pH 5.8–6.0) which had been autoclaved at 121 °C for 90 min. Two-month-old seedlings were transplanted onto an autoclaved mixture (1:1, v/v) of coppice soil collected from the Koishikawa Arboretum of the University of Tokyo (black sandy loam, pH 5.3) and Shibanome soil packed into rectangular flat rhizoboxes (140 × 205 × 15 mm). After fungal inocula were placed on fine roots in the seedling rhizosphere and the transparent lid was put on the rhizobox, the inoculated seedlings in rhizoboxes were cultivated for 4 weeks as ‘mother seedlings’ in a phytotron (16 h light: 350–500 μmol m−2 s−1 of photosynthetically active radiation at 25 °C; 8 h darkness: 23 °C). After mother seedlings were colonised by PS isolate, several 2-month-old, non-ECM seedlings were placed beside each mother seedling as ‘daughter seedlings’ and cultivated for an additional 4–8 weeks in the phytotron until they were colonised by PS. During cultivation, rhizoboxes were always covered with aluminium foil to shade the rhizosphere. One or two ECM daughter seedlings were transplanted onto a flat floral foam plate (Smithers-Oasis Japan, Tokyo, Japan) fitted into a rhizobox. The foam plate was always moistened with a 1000-fold diluted nutrient solution that contained 60, 100 and 50 mg L−1 of N, P and K, respectively, with inorganic micronutirents (6-10-5; Hyponex Japan, Osaka). The seedlings were cultivated for 2–4 weeks in the phytotron until their ERMs covered most of the foam plate surface and were then used for labelling experiments. In the experiments with the cut ERM, a rectangular area (114 × 22 mm) of the foam plate with one daughter seedling was cut away together with the ERM and roots on it with a knife as the majority of hyphae connecting radially between ECM root tips and the front were interrupted, and the space was packed with the same size of unused foam plate. Three daughter seedlings with the uncut ERM and three with the cut ERM were labelled with 14CO2 immediately after packing.

Autoradiograms were made 0, 1, 2, 3, 7 and 14 days after labelling. In experiments with connected ERMs of paired ECM seedlings, a foam plate, on which two daughter seedlings had fully extended its ERMs, was cut longitudinally in half, and the two halves with a seedling each were placed in a new rhizobox with ERMs of both seedlings in tight contact side by side (Fig. 1). To prevent transfer of 14C photosynthates from the left to the right half through pathways other than the ERM hyphal connection on the surface of the foam plate, the bottom and side surfaces of the right half were wrapped with a waterproof polyester film of 0.1-mm thickness (Kokuyo, Osaka, Japan). After the rhizoboxes with paired seedlings was incubated for 2 weeks in the phytotron to allow the ERMs to connect again by hyphal fusion, the left seedling was labelled with 14CO2 as described below. Four labelled rhizoboxes were prepared as a set, and the aboveground parts of none, the right, the left, and both of the paired seedlings were shaded with aluminium foil immediately after 14CO2 labelling to prevent photosynthesis, respectively. These four shade treatments were designated L/L (both seedlings in light), L/D (left in light, right in darkness), D/L (left in darkness, right in light) and D/D (both in darkness), respectively. Each rhizobox of the set was autoradiographed 0, 1, 2, 3, 7 and 14 days after labelling. These experiments were performed for three sets of rhizoboxes. 14

CO2 labelling

Labelling of the seedling with 14CO2 was performed in an i l l u m i n a t e d f u m e h o o d ( 2 3 – 2 5 ° C ; PA R , 1 5 0 – 200 μmol m−2 s−1) with continuous ventilation, following the protocol of Teramoto et al. (2012). Briefly, the aboveground part of the seedling was covered with a reclosable transparent polyethylene bag, inside of which a silicon-plugged microtube containing 925 kBq of NaH14CO3 (36.39 μg) was attached with double-sided tape. Immediately after 200 μl of 10 % lactic acid was injected into the microtube with a syringe through the silicon plug, the bag was sealed around the stem with Plasticine to prevent air leakage. Gas containing 14CO2 produced in the microtube was released inside the bag by removal of the plug (Fig. 1). The seedling was photosynthetically labelled with 14CO2 under the light condition for 2 h in the fume hood. After the labelling, any remaining 14CO2 in the bag was trapped using 1 N NaOH and then the bag was removed. The rhizobox with a labelled seedling was kept in the fume hood and autoradiographed repeatedly. In experiments with paired seedlings, only the left seedling was labelled with 14CO2, and the right one was covered with aluminium foil during the labelling to avoid 14CO2 fixation (Fig. 1). Immediately after the labelling and the shade treatment, the aboveground part of the right seedling was covered

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Cutting connected ERMs of two daughter seedlings NaH14CO3+Lactic acid

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Fig. 1 Schematic diagram of sample preparation, 14C labelling and following incubation of paired seedlings

with a reclosable transparent polyethylene bag that had vent holes at the top. The bottom of the bag was sealed with Plasticine around the stem together with a thin tube. Through the tube, ambient non-radioactive air was continuously sent into the bag with a small air pump for fish domestication. With this system, no fixed radioactivity was found in needles of the right seedling during experiments for 14 days, indicating that the fixation of expired 14CO2 from the seedlings and ERMs by respiration was prevented. Autoradiography of labelled ECM seedlings Following the protocol of Teramoto et al. (2012), an autoradiogram was obtained by laying an imaging plate (BASSR2040, Fuji Film, Tokyo, Japan) over the aboveground part of the seedling(s) and the whole rhizobox surface, which had been covered with thin wrapping film (Asahi Kasei, Tokyo, Japan) to prevent 14 C contamination,

for 90 min in the dark. In order to press the needles and the stem flat against the imaging plate, a foam polystyrene plate of appropriate thickness was inserted under the aboveground parts of the seedling. As 14C radioactivity standards, a set of three circles (6 mm in diameter) of filter paper containing 1.24, 7.4, or 37 kBq of [14C(U)]sucrose were simultaneously exposed to the imaging plate together with the rhizobox. Radioactivity distribution recorded on the imaging plate was then visualised with an imaging analyser (FLA-2000, Fuji Film). For quantitative analysis of radioactivity in an area, the photo-stimulated luminescence (PSL) value in that area was measured from the autoradiogram using Multi Gauge version 3.1 software (Fuji Film). The PSL value was converted to the absolute radioactivity in the unit of becquerel by multiplying the PSL value by the average of three ratios of absolute radioactivity to PSL of the standard circles. The value of density of radioactivity (DR) in the area was calculated by dividing the absolute

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value of radioactivity by the area and expressed in the unit of becquerel per square millimeter. Statistical analysis All statistical calculations were performed with Excel 2010 (Microsoft, Redmond, USA) with the add-in software Statcel 3 (EMS Publishing, Saitama, Japan).

Results Photosynthate translocation in uncut and cut ERMs Serial autoradiograms of ECM seedlings with uncut and cut ERMs acquired 0, 1, 2, 3, 7 and 14 days after 14C labelling are shown in Fig. 2a, b, respectively. In uncut seedlings, 14C was distributed only in needles just after labelling and spread within 1 day to the whole seedling, ECM root tips and the ERM. In the ERM, 14C was distributed mainly in rhizomorphs that extended all over the inner part and accumulated conspicuously in separate hyphae in the front (Figs. 2a and 3a). During 14 days of experiments, the ERM extended outside the front, forming the new ERM front with no hyphal accumulation. 14C

was also distributed in the new front region (Fig. 2a; 7 days and 14 days). In the cut ERM, 14C was also distributed in rhizomorphs all over the inner part and accumulated in separate hyphae in the front, as in the uncut ERM (Figs. 2b and 3b). The overall pattern of 14C distribution in the ERM was maintained even in regions immediately above and below the cut. 14C was distributed up to the cut ends of rhizomorphs at the upper and lower edges of the cut, with no local 14C depletion or accumulation (Figs. 2b and 3c). Hyphal extension to the outside of the front formed the new front with no hyphal accumulation, and 14C was also distributed in the new front region, as in the uncut ERM (Fig. 2b; 7 days and 14 days). The amounts of 14C in the seedling and various ERM areas were quantified from serial autoradiograms, and their time courses were followed (Figs. 4 and 5). The ‘seedling’ defined as the aboveground part and roots with ECM root tips were outlined on autoradiograms, and PSL values in the outlined areas were measured and converted to absolute radioactivity values. When the ERM was uncut and cut, the amounts of radioactivity in seedlings were relatively constant during the 14 days after 14C labelling, and reached to 17.3 and 15.7 kBq at the end of experiment, respectively (Fig. 4).

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Fig. 2 Serial autoradiograms of Pinus thunbergii seedlings colonised by a Pisolithus isolate, PS, after 14CO2 labelling. Before 14CO2 labelling, the ERM of each seedling was uncut (a) or cut (b). Both seedlings were autoradiographed 0, 1, 2, 3, 7 and 14 days after 14CO2 labelling. In each of rows (a) and (b), the first photograph from the left is a scan of the ECM seedling on a foam plate that was taken just before 14CO2

labelling and aligned with its sequential autoradiograms. Red frames in (b) indicate areas A, B and C where the density of radioactivity (DR) was measured (see Fig. 5) and corresponding measured areas in uncut samples are also shown as red frames in (a). The rectangular area that was cut away and packed with the same size of unused foam plate is marked with an asterisk in (b). Scale bar = 5 cm

Mycorrhiza Fig. 3 Enlargements of scans and the corresponding autoradiograms in Fig. 2. Pairs of a scan and its corresponding autoradiogram 3 days after labelling show rhizomorph meshworks in the inner part and the front of the uncut ERM (a), in the inner part and the front of the cut ERM (b), and around the cut (c). Note that 14C is distributed mainly in rhizomorphs both in the uncut and cut ERM, and up to the cut ends of rhizomorphs at the edge of the cut without any local depletion and local accumulation of 14C. Scale bar = 1 cm

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The ‘whole ERM’ regions from which roots and ECM root tips were excluded were outlined, and radioactivity values in the outlined regions were calculated. The radioactivity in the

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Fig. 4 Time course of total radioactivity in each part of symbiotic system within the rhizobox. (a) ERM-uncut seedlings; (b) ERM-cut seedlings. Closed circle, seedlings including the aboveground part, roots and ECM root tips; grey triangle, whole ERM; open triangle, ERM newly formed after labelling. Each symbol with a bar represents mean ± standard error of the mean (SEM) (n = 3)

whole ERM was greater in the uncut than the cut ERM throughout the 14 days, reaching maxima of 15.6 kBq after 3 days in the uncut and 6.5 kBq after 7 days in the cut ERM,

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followed by a slight decline in these levels. These levels reached 13.5 kBq in the uncut and 6.1 kBq in the cut ERM 14 days after labelling (Fig. 4). In the inner part of the uncut ERM in each of triplicated samples, three areas (146–239 mm2; ex. red frames in Fig. 2a) were arbitrarily set, and their DR values were averaged for each exposure day. Three averaged DR values obtained from the triplicated samples were averaged again for each exposure day, and the time course of the double averaged DR value was followed (Fig. 5). The double averaged DR values in the inner part of the uncut ERM peaked 2 days after labelling (0.44 Bq mm−2) and, thereafter, gradually declined up to the time point of 14 days (0.32 Bq mm−2) (Fig. 5). In the inner part of the cut ERM in each of triplicated samples, three areas (126–239 mm2; red frames in Fig. 2b) were set in three types of position, i.e. between the ECM root tips and the cut (area A), at the side of the cut (area B) and between the cut and the ERM front (area C). For each exposure day, DR values were calculated for areas of each position type in the triplicated samples and averaged. The time course of the averaged DR values for each position type was followed (Fig. 5). The double averaged DR value for each exposure day was also calculated by averaging DR values of the three areas 0.8

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Fig. 5 Time course of the densities of radioactivity (DR) in the inner part of the uncut and cut ERM. Three areas were defined in the ERM of each of triplicated cut samples between the ECM root tips and the cut (area A), at the side of the cut (area B) and between the cut and the front of the ERM (area C), respectively. Corresponding three parts or ERM was also defined in triplicated uncut samples as area A’–C’. Positions of areas A’– C’ and A–C are shown on the scan in Fig. 2a and b, respectively, as red rectangular frames. Open square, area A; open diamond, area B; open triangle, area C; open circle, the average of A’–C’; closed circle, the average of A–C. Each symbol with a bar represents mean ± SEM (n = 3)

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in each sample of the day and then averaging again the three averaged DR values from the triplicated samples. Its time course was also followed (Fig. 5). In area A, the DR peaked at 0.28 Bq mm−2 2 days after labelling and gradually declined to 0.18 Bq mm−2 after 14 days. In areas B and C, the DR reached respective maxima of 0.49 and 0.22 Bq mm−2 after 7 days, and gradually declined to 0.46 and 0.19 Bq mm−2 after 14 days. The DR was 2.4–2.6 times higher in area B than in areas A and C 14 days after labelling. Although the DR was higher in area A than that in area C until 3 days, the DRs in both areas were the same at the end of experiment, 14 days after labelling (Fig. 5). The double averaged DR value in the inner part of the cut ERM peaked 3 days after labelling (0.31 Bq mm−2) and, thereafter, gradually declined up to the time point of 14 days (0.28 Bq mm−2) (Fig. 5). I n t he f r o nt of e ac h E R M , n i ne s m a l l c i r cl e s (diameter = 5 mm) were arbitrarily set, and the average of their DR values was regarded as the DR value of the front in this ERM. The average of DR values of the front in triplicated uncut ERMs peaked in 3 days (5.0 Bq mm−2) and slightly declined until 14 days (4.32 Bq mm−2), and those in triplicated cut ERMs peaked in 7 days (1.68 Bq mm−2) and slightly declined until 14d (1.51 Bq mm−2) (Fig. 6). The value of the front in the uncut ERM was about three times higher than that in the cut ERM 14 days after 14CO2 labelling. In regions of the newly-formed front in the uncut and cut ERMs, 14C amounts reached 1.3 kBq and 0.5 kBq 14 days after 14C labelling, respectively (Fig. 4).

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Fig. 6 Time course of the densities of radioactivity (DR) in the front part of the uncut and cut ERM. Open circle, uncut; closed circle, cut. Each symbol with a bar represents mean ± SEM (n = 3)

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Photosynthate translocation between connected ERMs of paired seedlings In the experiments with connected ERMs of paired seedlings, 14 C photosynthates produced in the needles of the left seedling spread to the stem, roots, ECM root tips and the ERM of the left seedling within 1 day (Fig. 7). Thereafter, appreciable amounts of 14C were also distributed in the ECM root tips and the ERM of the right seedling (Fig. 7). Most of the 14C in the left and right ERMs were localised in rhizomorphs (Fig. 8). This observation suggests that the 14C photosynthates were translocated through rhizomorphs in the left and right ERM acropetally and basipetally, respectively. Until 14 days after labelling, no 14C was found in aboveground parts of the right seedling under any shading treatment (Fig. 7). The 14C amount in the right side part containing both the seedling and ECM root tips and that in whole the rhizobox except the left seedling with ECM root tips were measured from serial autoradiograms. Because the former and the latter correspond to 14C photosynthates translocated from the left to the right half and those transferred from the left seedling to its ERM, respectively, the ratio of the former to the latter could be defined as a coefficient expressing the efficiency of translocation from the left to the right ERM. Values of translocation efficiency were 23.6, 44.5, 24.2 and 26.2 % in the L/L, L/D, D/L, and D/D treatments 14 days after labelling, respectively (Fig. 9). The coefficient in L/D treatment was 1.7–1.9 times larger than in the other treatments (L/L, D/L, D/D).

Discussion Pathway of photosynthate translocation and sink activity of each portion in the ERM In the present study, we physically cut most radial mycelial connections between ECM root tips and the ERM front of P. thunbergii seedlings colonised by a Pisolithus isolate, and after 14CO2 labelling of the needles under light, we followed the time course of 14C distribution within the ERM by sequential and quantitative autoradiography. The resulting autoradiograms provided information about the pathway and sink activity of 14C photosynthate translocation. 14 C allocation to the ERM, especially to the ERM front, was reduced by the ERM cut treatment. This reduction may be caused by decrease in 14C inflow into the ERM like the 14C photosynthate transfer from the host or increase in 14C outflow from the ERM like 14C expiration by respiration in the ERM. If the ERM cutting reduces 14C transfer from the host seedling to its ERM, it would increase 14C contents in the seedling, on the supposition that irrespectively of the ERM cutting, the respiration rate in the seedling is constant. However, the 14C amounts were almost the same in seedlings with the uncut and

cut ERM (Fig. 4). This result may suggest that the ERM cutting did not decrease the 14C transfer from the host to the ERM but increased 14C expiration by respiration in the ERM which represented 14C outflow from the ERM. Even with cutting, the uniform 14C distribution pattern in the inner part and 14C accumulation in the ERM front were maintained similar to that in the uncut ERM, and no local depletion of 14C was induced in the region immediately below the cut (Figs. 2b and 3c). Although the DR value in area C in the region below the cut was smaller than that in areas in the inner part of the uncut ERM (Fig. 5), no appearance of local 14 C depletion in the region below the cut indicated that 14C photosynthates were translocated from the ECM root tips to the region below the cut through the detour around the cut that included lateral paths along the lower edge of the cut (via area B to area C). Thus, the pathway of 14C photosynthate translocation in the ERM could include not only radial paths from the ECM root tips but also lateral ones in hyphal anastomosis, and 14 C photosynthates could be translocated freely in every direction through sites of hyphal branching and interconnection. Enlargements of autoradiograms present another piece of information about the translocation pathway. In the ERM, a considerable amount of 14C was distributed in rhizomorphs that formed a spreading meshwork (Fig. 2a). This observation suggests that 14C photosynthates are translocated mainly through the rhizomorph meshwork. Rhizomorphs of many ECM fungi are bundles of vessel-like thick hyphal tubes that lack cytoplasm and septal cross-walls and thin cytoplasmic hyphae (Cairney and Clipson 1991), and such hollow vessel-like hyphal tubes have also been observed in rhizomorphs of Pisolithus species (Kammerbauer et al. 1989; Moyersoen and Beever 2004). If rhizomorphs of the Pisolithus isolate used in this study have the same structure, 14 C photosynthates may be translocated through the hollow hyphal tubes and/or cytoplasmic hyphae of the rhizomorph meshwork in the ERM. The autoradiograms also provided information about the sink activity regarding 14C photosynthates. Local depletion of 14C in the region immediately above or below the cut by ERM cutting was not observed. Given that the ERM front accumulated 14C photosynthates more than the inner regions of the ERM after 14CO2 labelling (Figs. 2 and 3a, b), it is possible that hyphal cells in the ERM front have much stronger sink activity for photosynthates than those in the other regions and that ERM areas on the translocation pathway to the front function as only path elements almost without sink activity of their own. If this hypothesis is true, ERM cutting would lead to loss of radial 14C inflow from the ECM root tips to the regions above and below the cut, and also to one-sided 14 C outflow from the region below the cut to the front, resulting in local 14C depletion in both regions. However, as no local 14C depletion was observed in either regions by cutting (Figs. 2b and 3c), it may not be the case that stronger

Mycorrhiza Fig. 7 Serial autoradiograms of paired Pinus thunbergii seedlings colonised by a Pisolithus isolate, PS, under four shading treatments. Immediately after 14 CO2 labelling of the left seedling, none (L/L), right (L/D), left (D/L), or both (D/D) of the paired seedlings were shaded with aluminium foil. Each seedling pair was autoradiographed 0, 1, 2, 3, 7 and 14 days after 14CO2 labelling. Four rows represent different shading treatments (L/L, L/D, D/L and D/D). The first photograph from the left in each row is the scan of the seedling pair on the foam plate that was taken just before 14CO2 labelling and aligned with its serial autoradiograms. Scale bar = 5 cm

0d

1d

2d

3d

7d

14d

(a) L/L

(b) L/D

(c) D/L

(d) D/D

hyphal sink activity was localised in the ERM front and attracts 14C photosynthates directionally to the front. In the cut ERM, 14C reached up to the cut ends of rhizomorphs at the upper and lower rims of the cut (Fig. 3c). This result suggests that each hyphal cell in rhizomorphs Fig. 8 Enlargements of the scan and autoradiogram in Fig. 7b. Pairs of a scan and corresponding autoradiogram 14 days after labelling show a rhizomorph meshwork in the ERM of the right seedling. Note that 14C is distributed in rhizomorphs emanated from the right seedling and in its ECM root tips. Scale bar = 1 cm

immediately above and below the cut itself has appreciable sink activity and that according to the integral of sink activity in every hyphal cell in both regions, 14C photosynthates are translocated from the ECM root tips to the region above the cut radially and to the region below the cut through the detour

Mycorrhiza

Percentage of the 14C amount in the right half to that in the whole system except the left seedling (%)

60 D/D D/L 50

L/D L/L

40

30

20

10

0 0

5 10 Days after labeling

15

Fig. 9 Time course of the ratios of the 14C amount in the right half to that in the whole experimental system with paired ECM seedlings except the left seedling under different shade treatments. Open circle, L/L; open square, L/D; closed square, D/L; closed circle, D/D. Each symbol with a bar represents mean ± SEM (n = 3)

in the rhizomorph meshwork around the cut. Sink activity may be much stronger in the front of the ERM by area units but not by hyphal cell units. No local 14C accumulation in the region immediately above the cut was observed due to ERM cutting (Fig. 3c). If each portion in the ERM had an unlimited sink capacity for photosynthate translocation, the cut would generate local 14C accumulation in the region because of continuous 14C inflow from the ECM root tips and the loss of 14C outflow to the ERM front. Thus, no appearance of local 14C accumulation above the cut suggests that the region has a definite sink capacity for photosynthates, avoiding excessive 1 4 C accumulation. 14 C also allocated to newly formed hyphal front even 7– 14 days after labelling (Fig. 2a, b; 4, 7–14 days). The amount of non-radioactive photosynthates fixed after labelling should be much more compared with 14C-labelled photosynthate at the time of 7–14 days after labelling. This result suggests that not only recently fixed photosynthate but also previously fixed carbon storage is also used for hyphal growth. Photosynthate translocation between connected ERMs of paired seedlings In experiments with connected ERMs of paired seedlings, 14C photosynthates were translocated from the left side to the ECM root tips of the right seedling through rhizomorphs in the right ERM (Figs. 7 and 8). Because rhizomorphs essentially develop acropetally from ECM root tips, this result suggests that 14C photosynthates are translocated acropetally

from the ECM root tips through the rhizomorph meshwork in the left ERM and then basipetally from the left ERM through the rhizomorph meshwork in the right ERM to ECM root tips of the right seedling. The mechanisms of such co-occurrence of acropetal and basipetal translocation manners of carbon resources through rhizomorphs still remain to be investigated. In the same experiments, it was also found that shading treatments differentially affected translocation from the left side to the right ERM. When the left seedling was illuminated, shading of the right seedling enhanced translocation of 14C photosynthates from the left side to the connected right ERM. The enhancement of 14C photosynthate transfer from the donor to the receiver seedling by shading of the receiver has been reported in a hyphal network system of paired ECM seedlings. Simard et al. (1997) prepared a pair consisting of a Betula papyrifera and a Pseudotsuga menziesii seedling between which the CMNs connected and labelled the former with 14CO2 and the latter with 13CO2. In their experiment, net transfer of photosynthates from B. papyrifera to P. menziesii seedling was promoted when the latter was shaded. They explained the results by source-sink relationships based on transfer along a gradient of photosynthate concentration. Although photosynthate transfer between connected ECM seedlings is not the same phenomenon as photosynthate translocation between connected ERMs, similar explanation could also be applied to the present results. Assuming that ERMs of shaded and illuminated seedlings have strong and weak sink activities because of lesser and greater supplies of nonradioactive photosynthates to the ERM, respectively, the L/L treatment would lead to a sink balance in which the left and right ERM both have weak sink activity. Designating such sink balance as weak/weak, L/D, D/L and D/D would lead to sink balances of weak/strong, strong/weak and strong/ strong, respectively. Thus, if the translocation of 14C photosynthates is parallel to that of non-radioactive photosynthates, the amounts of 14C photosynthates translocated from the left side to the right ERM would be small, large, small and small under L/L, L/D, D/L and D/D treatments, respectively. This speculation agrees well with the observed results. In the present Pisolithus isolate, the amount of photosynthates transferred from the host to its ERM might influence the distribution of photosyntate concentration in the ERM, and then 14C photosynthate translocation in the ERM that might occur along the concentration gradient of photosynthates. The above shading effects also leads to implications for functions of the CMN that actually develops under the forest floor in nature (Kennedy et al. 2003; Lian et al. 2006; Beiler et al. 2010, 2012, 2015). The CMN may generally connect together host trees that produce more and less photosynthates such as canopy and understory trees, respectively. Thus, photosynthate supply to the CMN may be different from host to

Mycorrhiza

host, and photosynthates transferred to the ERM would be translocated from CMN portions under more productive hosts to those under less productive ones through the CMN connection. Because photosynthates may promote mycelial extension and physiological activity such as soil nutrient absorption of the ERM, the CMN connection among host trees with various photosynthate productivities could indirectly facilitate the growth and survival of the hosts with less productivities. As a function of the CMN, direct supply of carbon resources from more productive to less productive hosts has been already reported (Simard et al. 1997; Teste et al. 2009; Teste et al. 2010). Such direct supply of carbon resources may directly facilitate growth and survival of less productive hosts. Thus, the CMN could facilitate both indirectly and directly cooperations between its hosts. However, actual contributions of such indirect and direct facilitations to the growth and survival of forest trees have not been estimated yet, and should be investigated in future.

Conclusion The present study has shown that every region of the Pisolithus ERM may have sink activity with a definite sink capacity for photosynthates and could thereby attract photosynthates mainly through rhizomorph anastomosis from all directions until the capacity is saturated. Our results also suggested that photosynthates can be translocated acropetally and basipetally through rhizomorphs and that sink activity for photosynthate translocation in the ERM may increase when photosynthate transfer from the host decreased. Acknowledgments This work was supported in part by Grants-in-Aid for Scientific Research (No. 21248018 and No. 23380080) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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