Orchestration of Three Transporters and Distinct Vascular Structures in Node for Intervascular Transfer of Silicon in Rice

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Orchestration of three transporters and distinct vascular structures in node for intervascular transfer of silicon in rice

Naoki Yamajia,1, Gen Sakuraib,1, Namiki Mitani-Uenoa, and Jian Feng Maa,2

aInstitute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan; and bEcosystem Informatics Division, National Institute for Agro-Environmental Sciences, Tsukuba 305-8604, Japan

Edited by Maarten J. Chrispeels, University of California, San Diego, La Jolla, CA, and approved July 27, 2015 (received for review May 7, 2015) Requirement of mineral elements in different plant tissues is not After EVB phase, the VB is continued to a VB of the leaf asso- often consistent with their transpiration rate; therefore, plants ciating the node (2–4). As an exception, DVBs in the uppermost have developed systems for preferential distribution of mineral two nodes (nodes I and II), in which VBs have no corresponding elements to the developing tissues with low transpiration. Here leaves, become VBs in the panicle without EVB phase as detailed we took silicon (Si) as an example and revealed an efficient system in ref. 2. Based on these repetitive vascular constructions, inter- for preferential distribution of Si in the node of rice (Oryza sativa). vascular transfers between axial VBs are required for preferential Rice is able to accumulate more than 10% Si of the dry weight in delivery of nutrients to apical tissues such as developing leaf or the husk, which is required for protecting the grains from water panicle (2). Furthermore, the node also shows distinct structures at loss and pathogen infection. However, it has been unknown for a cellular and subcellular level, which are characterized by (i)ex- long time how this hyperaccumulation is achieved. We found that panded xylem area of EVBs; (ii) differentiation of xylem transfer three transporters (Lsi2, Lsi3, and Lsi6) located at the node are cells (XTCs), which have expanded cell surface due to cell wall involved in the intervascular transfer, which is required for the ingrowth facing to the xylem vessel; (iii) increased number of preferential distribution of Si. Lsi2 was polarly localized to the DVBs around the EVBs; and (iv) parenchyma cell bridge (PCB) bundle sheath cell layer around the enlarged vascular bundles, with dense plasmodesmata between EVB and DVB (2, 4, 5). PLANT BIOLOGY which is next to the xylem transfer cell layer where Lsi6 is local- However, the role of these structures in intervascular transfer of ized. Lsi3 was located in the parenchyma tissues between en- nutrients has not been examined. larged vascular bundles and diffuse vascular bundles. Similar to In the present study, we examined the preferential distribution Lsi6, knockout of Lsi2 and Lsi3 also resulted in decreased distribu- system of silicon (Si) in rice. Silicon, the most abundant mineral tion of Si to the panicles but increased Si to the flag leaf. Further- element, shows beneficial effects for plant growth (6). The more, we constructed a mathematical model for Si distribution and beneficial effects are characterized by protecting plants from revealed that in addition to cooperation of three transporters, an abiotic and biotic stresses (6). Silicon is especially important for apoplastic barrier localized at the bundle sheath cells and devel- high and sustainable production of rice (Oryza sativa), a typical Si opment of the enlarged vascular bundles in node are also required accumulating species. Rice is able to accumulate Si up to 10% of for the hyperaccumulation of Si in rice husk. the shoot dry weight, which is severalfold more than macronu- trients such as nitrogen, phosphorus, and potassium (7). Low node | rice transporter | apoplastic barrier | silicon distribution | accumulation of Si in rice results in significant reduction of the mathematical model yield (8); therefore, Si fertilization has been considered as a

lants have different requirement for mineral elements Significance Pdepending on organs and tissues. For examples, developing tissues such as new leaves and reproductive organs require more Requirement of mineral elements differs with different organs mineral elements for their active growth (1). However, these tissues and tissues; therefore, plants have developed systems for usually have low transpiration; therefore, transpiration-dependent preferentially delivering mineral elements to tissues with high distribution of mineral elements is not sufficient to meet their high requirement. However, the molecular mechanisms for these requirements. Plants must have developed systems for preferential systems are poorly understood. We took silicon (Si) as an ex- distribution of mineral elements depending on the requirements. ample and revealed an efficient distribution system occurring However, the molecular mechanisms for these systems are poorly in the node of rice, which is a hub for distribution. We found understood. Recently, several studies have shown that nodes func- that hyperaccumulation of Si in the husk (more than 10%) is tion as a hub for distribution of mineral elements in graminaceous achieved by cooperation of three different Si transporters lo- plants (2). calized at the different cell layers in the node. Furthermore, Node is a key component of the phytomer in graminaceous mathematical modeling showed that an apoplastic barrier and plants, which is connected with a leaf, a tiller (or a tiller bud), and development of enlarged vascular bundles are also required. crown roots (or these primordia) to the culm or panicle (2, 3). Our work revealed a set of players for efficient distribution Therefore, graminaceous plants are composed of repeated nodes. control in node. Nodes have apparently complicated but regularly organized vascular bundles (VBs). No VBs are continuously connected from the Author contributions: N.Y. and J.F.M. designed research; N.Y., G.S., N.M.-U., and J.F.M. per- bottom to the top of the culm, but a unit of VB is repeated with a formed research; N.Y., G.S., N.M.-U., and J.F.M. analyzed data; and N.Y., G.S., and J.F.M. wrote two-thirds overlapped rule; one axial VB is connected with three the paper. nodes and a leaf and specialized at different nodes as diffuse VB The authors declare no conflict of interest. (DVB), transit VB (TVB), and enlarged VB (EVB) from basal to This article is a PNAS Direct Submission. apical (2, 3). In a node, three different axial VBs with different 1N.Y. and G.S. contributed equally to this work. phases coexist and connected transversely by nodal vascular anas- 2To whom correspondence should be addressed. Email: [email protected]. tomosis (NVA). TVB is a passage phase, but its appearance and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. function within the node are similar to one of the multiple DVBs. 1073/pnas.1508987112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1508987112 PNAS | September 8, 2015 | vol. 112 | no. 36 | 11401–11406 Downloaded by guest on September 29, 2021 practice of rice production in many countries, especially under A heavy application of nitrogen fertilizers (7). SIET3 SIET4 SIET5 Silicon in soil solution is present in the form of silicic acid, an m Lsi1 Lsi6 Lsi2 Lsi3 500 uncharged molecule (7). Silicic acid is taken by the roots through 400 300 two different transporters: Lsi1 and Lsi2 in rice (9, 10). Lsi1 be- 200 longs to NIP subgroup of aquaporin and preamble to silicic acid, 100 whereas Lsi2 is an efflux transporter of silicic acid (9, 10). Both Lsi1 and Lsi2 are localized at the exodermis and endodermis cells of rice roots, but in contrast to Lsi1, which is localized at the distal B Caryopsis side, Lsi2 is localized at the proximal side (9, 10). Knockout of Rachis either Lsi1 or Lsi2 results in defect of Si uptake (9, 10). Recently, in Peduncle silico mathematical modeling revealed that in addition to co- Node I operation of Lsi1 and Lsi2, presence of the Casparian strips at the Node II exodermis and endodermis is also required for efficient Si uptake Node III and high accumulation (11). Unelongated stem After uptake, more than 95% of Si is rapidly translocated from the roots to the shoots (7). Silicon is also present in the form of 0.0 0.5 1.0 1.5 silicicacidinthexylemsap(12), which is unloaded by Lsi6, a Relative expression homolog of Lsi1 (13). Lsi6 is polarly localized in the adaxial side of the xylem parenchyma cells in the leaf sheaths and leaf blades (13). With water loss due to transpiration, silicic acid is gradually con- C Caryopsis centrated and polymerized to amorphous silica, which is deposited Rachis beneath the cuticle, forming cuticle–silica double layers, and inside Peduncle of particular cells of leaf epidermis (motor cells and short silica Node I cells) (7). Knockout of Lsi6 results in leakage of large amount of Node II silicic acid into the leaf guttation and altered deposition of Si in the Node III leaves (13). Unelongated stem At the reproductive stage, higher Si is finally deposited in the husk, which can reach more than 10% of the dry weight (7). 0.0 0.5 1.0 1.5 Silicon in the husk is also deposited between the epidermal cell Relative expression wall and the cuticle, forming a cuticle–silica double layer (7). This heavy deposition of Si in the husk is very important for rice Fig. 1. Expression analysis. (A) Semiquantitative RT-PCR of Lsi1-like genes grain fertility because it prevents water loss and pathogen (e.g., (Lsi1 and Lsi6) and Lsi2-like genes (Lsi2, Lsi3, SIET3, SIET4, and SIET5) in node – I of rice sampled at the heading stage. PCR products (25 cycle) with ladder panicle blast) infection (6 8). However, the question arises on marker (m) were electrophoresed and stained by ethidium bromide. (B and how Si is highly deposited in the husk. The rice husk does not C) Expression pattern of Lsi2 (B) and Lsi3 (C) determined by quantitative real- have stoma at the outside. Furthermore, compared with the time RT-PCR in various organs at flowering stages. Expression level relative leaves, the surface area is much smaller. Therefore, transpira- to node I was shown. Actin was used as an internal standard. Data are means ± tion-dependent distribution is unlikely responsible for the high Si SD of three biological replicates. accumulation in the husk. Previously, Lsi6 highly expressed in the nodes was reported to be involved in the intervascular transfer for preferential distribution of These samples were also used for analysis of Lsi6 previously (14). Si. Lsi6 in nodes is polarly localized at the xylem parenchyma cells Similar to Lsi6, the highest expression of both Lsi2 and Lsi3 was (mainly XTCs) of EVBs (14). Knockout of Lsi6 resulted in de- found in the uppermost node I (Fig. 1 B and C). Expression of Lsi2 creased Si accumulation in the husk but increased Si in the flag leaf and Lsi3 was also detected in lower nodes including nodes II and III (14). However, Lsi6 is a channel-type passive transporter, which (Fig. 1 B and C). In contrast to Lsi3, which was also expressed in the transports silicic acid following concentration gradient (13, 15). peduncle (internode I) and rachis, there was no expression of Lsi2 in Therefore, Lsi6 alone cannot explain hyperaccumulation of Si in these organs (Fig. 1 B and C). Expressions of both Lsi2 and Lsi3 were the husk. In the present study, we found that the other two active alsonotdetectedincaryopsis(Fig.1B and C). efflux transporters are also involvedintheintervasculartransfer required for high accumulation of Si in the husk. Furthermore, our Functional Characterization of Lsi3. Among Si transporter genes mathematical modeling revealed that the distinct nodal structures highly expressed in node I, Lsi6 and Lsi2 proteins have been including EVBs, XTCs, and apoplastic barrier are also required for functionally characterized, but Lsi3 is uncharacterized in terms the efficient intervasculartransferofSiinrice. of transport activity and subcellular localization. Lsi3 shares 80% identity with Lsi2 at amino acid sequence (Fig. S1). To examine Results whether Lsi3 also has efflux transport activity for silicic acid, we Expression Profiles of Si Transporters in Nodes. To find out trans- expressed Lsi3 in Xenopus laevis oocyte and determined its efflux porters involved in the intervascular transfer of Si in nodes, we transport activity. Similar to Lsi2, Lsi3 also showed efflux transport investigated the expression profile of both passive Si channel (Lsi1- activity for Si (Fig. S2). like) and active Si efflux transporter (SIET/Lsi2-like) homolog Furthermore, we expressed Lsi3 in the lsi2 rice mutant under genes using node I RNA at heading stage. In rice genome, there is the control of Lsi2 promoter. Analysis of two independent only one homolog of Lsi1 (Lsi6) (9) but four homologs of Lsi2/ transgenic lines showed that introduction of Lsi3 significantly SIET1 (Lsi3/SIET2 and SIET3–5) (10). Among them, Lsi6, Lsi2, increased Si uptake (Fig. S2). and Lsi3 showed high-level expression in the node I (Fig. 1A). By To investigate subcellular localization of Lsi3 protein, we pre- contrast, expression of Lsi1 and SIET3 was not detected, and only pared a fusion gene of GFP and Lsi3 (GFP-Lsi3) and transiently trace expression of SIET4 and SIET5 was found in node I introduced it into onion epidermal cells together with DsRed as a (Fig. 1A). marker for cytosol and nucleus by particle bombardment. Fluores- A detailed expression pattern was further investigated by quan- cence signal from GFP-Lsi3 was observed mainly at outer region of titative RT-PCR using various organs of rice at the flowering stage. the cell, which is located outside of fluorescence of DsRed (Fig. S3).

11402 | www.pnas.org/cgi/doi/10.1073/pnas.1508987112 Yamaji et al. Downloaded by guest on September 29, 2021 In situ subcellular localization of Lsi3 protein was also investigated back-to-back polar localization (Fig. 2 A and B), although at by immunohistochemical staining in rice node I. No signal in the different cell layers next to each other. To understand the sig- lsi3 knockout mutant as described below was detected (Fig. S4), nificance of this polar localization, we examined whether apo- indicating high specificity of this antibody against Lsi3. Double plastic barrier is also present in node. We fed periodic acid, a staining with 4′,6-diamidino-2-phenylindole (DAPI) for nuclei tracer of apoplastic flow (16), from cut end of the internode showed that the fluorescence signal from Lsi3 antibody was lo- below node II, followed by Schiff’s staining. At the cross section calized mainly at the peripheral region of the cells, which was of node I, the xylem regions of the EVBs were heavily stained, circumscribed but did not envelope the nuclei (Fig. S4). Taken but the phloem regions of EVBs and DVBs were hardly stained together, these results show that Lsi3 is a plasma membrane- (Fig. S5). No staining was observed at the bundle sheath cell localized efflux transporter of silicic acid. layer of EVBs and outer parenchyma cells between EVBs and DVBs (Fig. S5). This result shows that the apoplastic tracer was Tissue-Specific Localization of Si Transporters in Node. Lsi6 is local- blocked inside the bundle sheath cell layer, indicating the pres- ized at the xylem parenchyma cells of EVBs of node I with polarity ence of an apoplastic barrier at the bundle sheath of EVBs. facing toward the xylem vessels (14), but the tissue localizations of Lsi2 and Lsi3 in nodes are unknown. To examine the tissue spec- Stem-Fed Si Distribution to Different Organs. To investigate the ificity of localization of Lsi2 and Lsi3, we performed coimmuno- exact roles of three Si transporters highly expressed in node, we staining of Lsi2 or Lsi3 with Lsi6. Lsi2 protein (magenta) was compared the distribution pattern of Si to different organs be- localized at the bundle sheath (bs) cells around the EVBs (Fig. tween knockout lines and their wild-type rice. 2A), which is next to the cell layer where Lsi6 is localized (green). Knockout lines of Lsi2 and Lsi6 were used in previous studies Furthermore, Lsi2 showed a polar localization at the distal side of (6, 9), whereas lsi3 mutant is a T-DNA insertion line (Fig. S1). The the vasculature (Fig. 2B), which is the opposite side of Lsi6. On the expression of Lsi3 at the 3′-untranslated region was hardly detected other hand, Lsi3 protein (magenta) was detected in the paren- in the lsi3 mutant (Fig. S1). Because Lsi2 and Lsi6 are also chyma cells between the bundle sheath cell layer of EVBs and expressed in the roots (10, 13), to rule out the effect from root Si DVBs (Fig. 2 C and D). Unlike Lsi2 and Lsi6, Lsi3 did not show uptake, we fed Si as well as Rb and Sr as tracer of symplastic and polarity (Fig. 2D). apoplastic transfer from the cut end of the internode II for 24 h. Sr is a phloem-immobile and xylem-mobile element, whereas Rb is Detection of Apoplastic Barrier in Node. In rice roots, presence of transported through the phloem (17). Knockout of either Lsi6,

Casparian strips at the exodermis and endodermis prevents Lsi2,orLsi3 resulted in decreased distribution of Si to the panicle PLANT BIOLOGY apoplastic flow of water and solutes; therefore, cooperation of organs including spikelet, rachis, and peduncle but increased dis- polarly localized Lsi1 and Lsi2 at these cell layers is required for tribution to the flag leaf (Fig. 3A). Among Lsi6, Lsi2,andLsi3, efficient Si uptake (9–11). In node, Lsi2 and Lsi6 also showed knockout of Lsi6 showed the largest effect in decreasing Si distribution to the panicles. By contrast, there was almost no difference in the distribution of Rb and Sr to different organs between mutants and their corresponding WTs (Fig. 3 B and C). Based on Sr ABDVB – DVB DVB distribution, it was estimated that 70 80% of transpiration oc- B EVp curred from the flag leaf in this experiment (Fig. 3 ). EVx DVB DVB DVB Construction of Mathematical Model for Si Distribution in Node I. To DVB comprehensively understand the distribution system of Si in

EVx EVx node, we constructed a mathematical model. We simulated the EVp transfer of Si in the node I by using a diffusion equation on bs the 2D grids (11) and a 2D explicit finite difference method.

DVB DVB The simulations represent a 2D longitudinal section of node I. DVB For simplicity, the simulation region includes only one EVB and one DVB connected by NVA (Fig. S6). Areas of EVB and DVB EVx DVB (2), the location of the Si transporters (Fig. 2), apoplastic barrier CDEVp (Fig. S5), and transpiration rate (i.e., distribution of Sr) from EVx DVx panicle and flag leaf (Fig. 3C) were estimated according to the DVB experimental data. Then, we calibrated the model parameters, Si DVB DVp EVx transport activity of three transporters, and background of Si DVB DVx DVx DVx permeability of cell membrane per unit simulation area based on DVB DVB DVB the results of the stem-fed in vivo experiment shown in Fig. 3. Using these parameter sets, we established a model for Si dis-

EVx bs tribution in node I. Using this model, we were able to reproduce EVp the Si distribution pattern in lsi6, lsi2, and lsi3 mutants as ob- EVx served in the feeding experiment (Fig. S7). DVB DVB DVB DVB In Silico Simulation Experiments of Si Distribution. Based on the model constructed, we conducted in silico modeling experiments Fig. 2. Tissue specificity of localization of Lsi6, Lsi2, and Lsi3 in node I. Node to estimate the importance of the cooperative effect of three I of the wild-type rice at heading stage was sampled for immunostaining. transporters and the contribution of nodal structural features to Coimmunostaining using Lsi6 antibody (green) and Lsi2 antibody (magenta the distribution. In the normal setting of the model (corre- in A and B) or Lsi3 antibody (magenta in C and D) was performed. Auto- sponding to the wild-type rice), the Si concentration in the DVB fluorescence of the cell wall is shown in blue/cyan color. Nonspecific signals was much higher than that in EVB, indicating a preferential in nuclei by Lsi2 antibody was also observed in A and B. B and D are mag- A nified image of yellow boxes area in A and C, respectively. DVB, xylem and transfer of Si to DVB in node I (Fig. 4 ). However, if Lsi6 was phloem regions of EVBs (EVx and EVp), xylem and phloem regions of DVBs absent, the difference in Si concentration between DVB and (DVx and DVp), and bundle sheath cell layer of EVBs (bs) are indicated. (Scale EVB disappeared, indicating the significant role of Lsi6 in the bar, 100 μm.) intervascular transfer of Si (Fig. 4A). If Lsi2 or Lsi3 was absent,

Yamaji et al. PNAS | September 8, 2015 | vol. 112 | no. 36 | 11403 Downloaded by guest on September 29, 2021 Spikelet expressing more transporters, we further simulated the distri- A Rachis and Peduncle B Node I bution ratio of Si on the assumption that the transport activity in 120 Flag leaf sheath 120 Flag leaf blade EVB was simply decreased by 1/3.16, which corresponded to the 100 100 decrease of circumference of one-tens area circle, together with ** ** 80 ** 80 10× faster velocity of the xylem flow. Under these settings, Si * – – 60 * ** 60 * distribution to the panicle was decreased from 56 66% to 22 ** 40 ** 40 32%, regardless of Si concentration supplied and extension of Si distribution (%) ** Rb distribution (%) * ** node length (Fig. S9). 20 20 * 0 0 Discussion WT1 lsi6 lsi3 WT2 lsi2 WT1 lsi6 lsi3 WT2 lsi2 Essential mineral elements taken up by the roots are translocated C 120 to different organs and tissues in the aboveground part through 100 the transpiration stream. However, it has been unknown for a

80 long time how plants deliver these mineral elements to different

60 tissues according to their different demands. There is an in- consistency between the transpiration rate and mineral element 40 Sr distribution (%) requirement. For example, developing tissues such as new leaves 20 and reproductive organs have low or no transpiration but usually 0 require more mineral elements for their active growth. Therefore, lsi6 lsi3 lsi2 WT1 WT2 plants must have systems for preferentially delivering essen- Fig. 3. Distribution pattern of stem-fed Si, Rb, and Sr. Three mutants (lsi6, tial mineral elements to the developing tissues independent of lsi3, and lsi2) and their corresponding wild types (WT1, cv. Dongjin for lsi6 transpiration rate. and lsi3; WT2, cv. Taichung-65 for lsi2) were grown hydroponically until Nodes of graminaceous plants are characterized by very compli- flowering in the absence of Si and cut at the internode II. Solution con- cated but well-organized vascular systems (2–4). Recently, identifi- taining 2 mM Si and 50 μM of Rb and Sr was fed from the cut end. After 24 h, each organ was separately harvested and subjected to determination of Si, cation of mineral nutrient transporters in node and physiological Rb, and Sr by ICP-MS. Distribution ratio of Si (A), Rb (B), and Sr (C) in dif- studies shed a new light on the importance of node for preferential ferent organs above node I was calculated. Data are means ± SD of three distribution of mineral nutrients to developing and reproductive biological replicates. Asterisks indicate significant differences from corre- organs (2, 14, 18–23). The xylem flow in EVBs and DVBs, which sponding WT at *P < 0.05 and **P < 0.01 by Tukey’s test. are connected by nodal vascular anastomosis (NVA) at the base of the node, are simply divided following the transpiration rate of each upper organ. Therefore, for preferentially delivering mineral the Si concentration in the DVB was higher than that in EVB due to presence of Lsi6 and another efflux transporter (Lsi3 or Lsi2), but compared with normal setting, the Si concentration in the DVB was much lower (Fig. 4A). Lack of both Lsi2 and Lsi3 A resulted in no difference in the Si concentration between EVB and DVB (Fig. 4A). This simulation confirmed that Lsi2 and 8 DVB EVB Lsi3 also play important roles in the intervascular transfer of Si. to panicle 6 Enlarged vascular bundle in node is structurally characterized to flag leaf lsi6 lsi2 lsi3 lsi2lsi3 by more than 10× enlargement of the xylem area, xylem transfer 4 cells with folded plasma membrane, and apoplastic barrier at the bundle sheath (10–12) (Fig. S5). Simulation experiments showed 2 that the apoplastic barrier is also essential for raising Si con- normal 0 Si / input concentration ratio centration in DVB (Fig. 4A). On the other hand, 10× faster from root

velocity of the xylem in EVB (to mimic no enlargement of EVB) no barrier fast EVB no XTC no all or lower permeability parameter of Lsi6 (to mimic no development of XTCs) only resulted in a slight effect on the Si con- B to panicle to flag leaf A normal centration in the DVB (Fig. 4 ). If all these transporters and lsi6 structure features are defective, the Si enrichment in DVB lsi2 A lsi3 completely disappeared (Fig. 4 ). lsi2lsi3 We also simulated the Si distribution ratio to panicle and flag no barrier fast EVB leaf using the model. Lack of Lsi6, Lsi2, Lsi3, and apoplastic no XTC barrier resulted in significant decrease of Si distribution to the no all panicle but increase to the flag leaf (Fig. 4B). In particular, lack 0 102030405060708090100 of both Lsi2 and Lsi3 and absence of apoplastic barrier de- Distribution ratio (%) creased Si distribution to the panicles from 51% to 18% (Fig. B Fig. 4. In silico modeling experiments for Si distribution in node I. Diagram 4 ), indicating their important role in preferential distribution of of the model is shown in Fig. S6.(A) Estimated Si concentration in each grid Si to the panicles. On the other hand, increase of xylem velocity of the model at 10 min after 2 mM Si supply and (B) estimated Si distribution and lower permeability parameter of Lsi6 did not have a sig- ratio to the panicle and flag leaf, under multiple experimental settings. nificant effect on the Si distribution ratio (Fig. 4B). These trends Normal, normal setting as wild-type rice; lsi6, lack of Lsi6; lsi2, lack of Lsi2; in the distribution ratio did not change whether 2 mM or 20 mM lsi3, lack of Lsi3; lsi2lsi3, lack of both Lsi2 and Lsi3; no barrier, no apoplastic Si was used for simulation input (Fig. S8). Another additional barrier at the bundle sheath of EVB; fast EVB, 10× faster velocity of the simulation was twofold axial extension of node. With this as- xylem in EVB; no XTC, permeability parameter of Lsi6 replaced by that of Lsi1 in root; no all, combined defect of all above factors. Results of the simulation sumption, more Si was distributed to the panicle under normal with the parameter set that had the maximum likelihood among 3,000 condition, but defect of each transporter or nodal feature also simulations are shown for A.ForB, the error bars indicate SDs estimated by resulted in decreased distribution of Si to the panicles (Fig. S8). 30 simulations that were sampled from 3,000 simulations according to the Because enlargement of EVB also provides more space for likelihoods.

11404 | www.pnas.org/cgi/doi/10.1073/pnas.1508987112 Yamaji et al. Downloaded by guest on September 29, 2021 elements, an intervascular transfer from EVBs to DVBs in node is flag leaf (Fig. 3), indicating that the cooperation of these three required (2). Based on the structure of the node, this intervascular transporters is required for the preferential distribution of Si to the transfer includes at least three steps: unloading of a mineral ele- panicles and subsequently high accumulation of Si in the husk ment from xylem flow in EVB, transfer through symplast or apo- (Fig. 5). plast of PCB, and reloading to xylem or phloem of DVB (2) (Fig. Second, we constructed a mathematical model for distribution 5). Although different transporters are supposed to be involved in of Si in node I (Fig. S6), which is well fit to the experimental data these steps, only a few of them in some steps have been identified. (Fig. S7). We found that the apoplastic barrier identified at the For instance, Lsi6 is involved in Si unloading from EVB (14, 18); bundle sheath of EVBs in this study is also required for efficient OsHMA5 and OsYSL16 are involved in Cu exclusion from xylem intervascular transfer of Si in node based on the mathematic and loading to phloem of DVB, respectively (19, 20), whereas modeling (Fig. 4 and Fig. S8). This barrier is essential for effi- OsHMA2 is required for Zn/Cd reloading to phloem of DVB (21); cient cooperation of three transporters by generating a concen- and OsNramp3 has a dual role for both xylem unloading from tration gradient from xylem of EVB to XTC–PCB for promotion EVB and phloem reloading to DVB of Mn (22). In the present of Si transport via Lsi6 and further directional transport by Lsi2 study, by integrating in vivo experiments with mathematical mod- and Lsi3 at the opposite side of the barrier (Figs. 4 and 5). This eling, we were able to identify all factors involved in the inter- axisymmetric arrangement of passive channel (Lsi6) and active vascular transfer for preferential distribution of Si in node (Fig. 5). efflux transporters (Lsi2 and Lsi3) with an apoplastic barrier is First, we identified two Si transporters (Lsi2 and Lsi3) highly very similar to the roots, where Lsi1 and Lsi2 for Si uptake are expressed in the node in addition to Lsi6 reported previously (Fig. localized at the exodermis and endodermis with Casprian strips 1) (14). Both of them are efflux transporters of Si but localized at (9, 10). A previous modeling study also demonstrated that these the different cell layers between EVB and DVB of node (Figs. 2 apoplastic barriers in root are required for the efficient uptake and 5). Lsi2 was localized at the distal side of bundle sheath cells of and enrichment of Si to the stele (11). However, there are two EVB having apoplastic barrier, which is the next cell layer where differences in these cooperative systems between the root and Lsi6 was localized (14) (Fig. 2), whereas Lsi3 was localized at the node. One is that two efflux transporters (Lsi2 and Lsi3) are parenchyma cells without polarity between the bundle sheath and involved in node, but only one efflux transporter Lsi2 is involved DVB (Fig. 2). The parenchyma cells between EVBs and DVBs are in root. The second difference is that Lsi1 and Lsi2 are localized symplastically connected by dense plasmodesmata, so-called PCB at the same cells with different polarity in root, but in node, Lsi6, (5)(Fig.5).Therefore,SiinthexylemofEVBistakenupbyLsi6 Lsi2, and Lsi3 are localized at the different cell layers (Fig. 5). and symplastically moved to the next cell layer, the bundle sheath However, these two cooperative systems show similar function in PLANT BIOLOGY root and node. This is because different from root, in node, cell through the plasmodesmata (Fig. 5). Part of Si is released to the layers expressing Lsi6, Lsi2, and Lsi3 are tightly connected to apoplastic space of parenchyma cells by Lsi2, which is transferred each other by plasmodesmata (5) (Fig. 5). These cell layers to the xylem of DVB through apoplastic pathway, whereas therefore behave like a single cell in the model. This connected remaining Si is moved to the parenchyma cells through the plas- multilayer structure by cells specialized for different functions is modesmata, followed by releasing to the xylem of DVB by Lsi3 presumably more suitable for the more integrated multivascular (Fig. 5). Knockout of either Lsi2 or Lsi3 resulted in decreased structure of nodes compared with multifunctional single-cell distribution of Si to the panicles but increased distribution to the layer(s) in the root. In fact, single knockout of Lsi2 or Lsi3 had smaller effect on the Si distribution to the panicle compared with that of Lsi6 (Fig. 3A), indicating that both Lsi2 and Lsi3 are Panicle Flag leaf involved in generation of the Si concentration gradient for mo- tive force of Lsi6 channel (Figs. 4 and 5). This is supported by the

Apoplastic barrier fact that Si enrichment in the DVB was completely lost if both

: Apoplast Lsi2 and Lsi3 were knocked out in the model (Fig. 4). Lsi2Lsi3 Lsi6 : Symplast Xylem transfer cells where Lsi6 is located are characterized by : Plasmodesma folded plasma membrane due to cell wall ingrowth, resulting in : Apoplastic barrier increased uptake surface (2, 5). We simulated the importance of : Xylem/apoplastic flow of Si this distinct structure in Si distribution by using the model con- : Symplastic flow of Si structed. In our model, the estimated transport activity param- − : Influx transport of Si by Lsi6 eter of Lsi2 per unit modeling grid (7.05 ± 2.10 μm·s 1) in node I

Xylem of DVB : efflux transport of Si by Lsi2 Xylem of EVB

Node I was almost the same as that estimated in the root in a previous −1 : efflux transport of Si by Lsi3 study (4.48 ± 0.26 μm·s ) (11). These are also comparable to the − EVB : Enlarged vascular bundle parameter of Lsi3 in node I (6.19 ± 2.17 μm·s 1). By contrast, the − DVB : Diffuse vascular bundle estimated permeability of Lsi6 (136.25 ± 68.49 μm·s 1) was much BS XTC NVA : Nodal vascular anastomosis PCB ± μ · −1 DVB EVB PCB : Parenchyma cell bridge higher than that estimated for Lsi1 in the root (16.38 3.40 m s )

NVA BS : Bundle sheath of EVB (11). When the permeability parameter of Lsi6 was replaced by XTC : Xylem transfer cell that of Lsi1 in the root to mimic no XTC development (no XTC), the estimated contribution of XTCs to Si distribution was relatively small (Fig. 4). Lsi6 is an aquaporin-like passive channel, which has much faster transport capacity compared with Fig. 5. Schematic presentation of intervascular transfer pathway of Si in node I other types of active transporters (15). Therefore, transport ca- silicon in the xylem of EVB is unloaded by Lsi6 polarly localized at the XTCs, pacity of Lsi6 for Si in the node is possibly high enough even if followed by symplastical transfer through plasmodesmata to the bs cells of EVB the XTC was not developed. with an apoplastic barrier. Part of Si is then released by Lsi2 localized at the The xylem area of EVB is a mosaic of numerous vessels and distal side of bs to the apoplast of PCB and transported to the xylem of DVB, parenchyma cells, which has more than 10× larger area com- whereas the remaining Si is simplistically transferred to the PCB and exported – and reloaded to the xylem of DVB by Lsi3. The cooperation of these three pared with connecting VBs in both internode and leaf (2 4). This transporters across the apoplastic barrier is required for the intervascular enlargement has two meanings: slowdown of the xylem mass flow transfer of Si and subsequently for hyperaccumulation of Si in the rice husk. and increase of space for expressing transporters. Both of them Modeling shows that a physical barrier on the bs and development of EVB are may contribute to efficient intervascular transfer. However, our also required for efficient intervascular transfer of Si. For details, see text. simulation showed that if the velocity of xylem mass flow in EVB

Yamaji et al. PNAS | September 8, 2015 | vol. 112 | no. 36 | 11405 Downloaded by guest on September 29, 2021 was increasing 10×, the distribution ratio of Si to the panicles was described previously (10, 25). Furthermore, the transport activity of Lsi3 was only slightly decreased, probably due to the invariable concen- determined by transforming the promoter region of Lsi2 (2.0 kb) (25) and tration of Si input in this model (Fig. 4 and Fig. S8). By contrast, ORF of Lsi3 into lsi2 mutant using an Agrobacterium-mediated trans- when the transport capacity was simply decreased to 1/3 to mimic formation system. The subcellular localization of Lsi3 was investigated by reduced space for transporters, the distribution of Si to the introducing plasmids carrying GFP-Lsi3 fusion gene and DsRed-monomer panicles was significantly decreased (Fig. S9). This simulation (Clontech) into onion epidermal cells using Helios Gene Gun system (Bio- suggests that increased transport capacity due to enlargement Rad). Immunostaining was performed for observing localization of Lsi6, Lsi2, of EVB is also important for the efficient intervascular transfer of Si. and Lsi3 in node I as described previously (22). Fluorescence signals were In conclusion, we identified a complete set of players involved observed with a confocal laser-scanning microscopy (LSM700; Carl Zeiss). in the intervascular transfer of Si in the node, which is required Apoplastic permeability test in node I was performed according to Shiono for the preferential distribution of Si to the husk. Transporters et al. (16). For stem feeding experiment, WT1, WT2, and three mutants (lsi6, localized at the different cell layers are responsible for move- lsi2, and lsi3) were grown hydroponically without Si until flowering and cut at the internode II below the node I. Nutrient solution containing 2 mM Si ment of Si from EVB to DVB, and the distinct structure of the and 50 μM of Rb and Sr was fed from the cut end for 24 h. Concentrations of node helps to enhance this movement. Although we focused on Si, Rb, and Sr in each part were determined by inductively coupled plasma Si distribution in uppermost node I in this study, distribution mass spectrometry (7700X; Agilent Technologies). controls of all mineral nutrients at each node throughout the For mathematical modeling of Si transport in node I, we divided the node I developing processes are required. Furthermore, graminaceous by 32 grid points in a transverse direction and 48 grid points in a longitudinal plants are composed of repeated nodes, and the structure of direction, with each grid point representing 4 × 40 μm(Fig. S6). We simulated each node is basically similar. Therefore, results shown in this the transfer of Si in the region by using a diffusion equation on the 2D grids study provide a model for studying the systems of preferential (11, 26) and a 2D explicit finite difference method. distribution of all minerals in graminaceous plants in future. For more detail, see SI Materials and Methods. Materials and Methods ACKNOWLEDGMENTS. We thank Sanae Rikiishi and Akemi Morita for their Seeds of T-DNA insertion mutants of Lsi6 (PFG_4A-01373) (13) and Lsi3 technical assistance. We also thank Drs. Akiko Satake and Masayuki Yokozawa (PFG_3C-00346; Fig. S1) [Rice T-DNA Insertion Sequence Database (RISD), cbi. for their valuable discussion and Drs. Shunsaku Nishiuchi and Mikio Nakazono khu.ac.kr/RISD_DB.html] and their wild type (WT1, cv. Dongjin) and lsi2 single- for kindly providing the protocol of the permeability staining. This research was nucleotide substitution mutant (10) and its wild type (WT2, cv. Taichung-65) supported by a Grant-in-Aid for Scientific Research on Innovative Areas from were grown hydroponically in a half-strength Kimura B nutrient solution the Ministry of Education, Culture, Sports, Science and Technology of Japan [22119002 and 24248014 to J.F.M., 70452737 to G.S.], and Joint Research Pro- (24). For expression analysis of Si transporter genes, different organs of WT1 gram implemented at the Institute of Plant Science and Resources, Okayama including unelongated stem, node I to III, peduncle, rachis, and caryopsis University, Japan (G.S.). Computations were carried out on a cluster system at were sampled at the flowering stage and subjected to semiquantitative and the Agriculture, Forestry and Fisheries Research Information Technology Center quantitative RT-PCR with gene-specific primers shown in Table S1. Efflux for Agriculture, Forestry and Fisheries Research, Ministry of Agriculture, Forestry transport activity assay of Lsi3 for Si in Xenopus oocytes was performed as and Fisheries, Japan.

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