Frequencies of Plasmodesmata in Allium Cepa L. Roots: Implications for Solute Transport Pathways

Journal of Experimental Botany, Vol. 52, No. 358, pp. 1051±1061, May 2001

Frequencies of plasmodesmata in Allium cepa L. roots: implications for solute transport pathways

Fengshan Ma1 and Carol A. Peterson2 Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada

Received 21 August 2000; Accepted 27 December 2000

Abstract Introduction Plasmodesmatal frequencies PFs) were analysed Intercellular transport between living plant cells is medi- in Allium cepa L. roots with a mature exodermis ated by plasmodesmata PD). These cytoplasmic channels 100 mm from the tip). For all interfaces within the provide a low-resistance pathway that is available for a root, the numbers of plasmodesmata PD) mmÀ2 wall wide range of ions and molecules typical size exclusion surface Fw) were calculated from measurements of limit -1kDa). PD are dynamic structures that can be 60 walls on ultrathin sections. For tissues ranging regulated by a number of internal and external factors from the epidermis up to the stelar parenchyma, the Overall and Blackman, 1996; Ding et al., 1999). When frequencies were also expressed as total PD num- dealing with symplastic transport across a tissue, what À1 bers mm root length Fn), which is most instructive is observed is the collective, rather than the individual, for considering the radial transport of ions and photo- functioning of PD on that particular interface. Therefore, synthates because the tissues were arranged in the frequency of functional PD is the most important concentric cylinders). The Fn values were constantly parameter that will determine the direction, extent and high at the interfaces of exodermis±central cortex, rate of symplastic transport under given conditions. Data central cortex±endodermis and endodermis±pericycle regarding plasmodesmatal frequencies PFs) must be 5 5 5 4.05 3 10 , 5.13 3 10 , and 5.64 3 10 , respectively). If obtained with transmission electron microscopy TEM); the plasmodesmata are functional, a considerable the time required for this approach has largely con- symplastic transport pathway exists between the strained efforts to perform large-scale surveys. Available exodermis and pericycle. Two interfaces had espe- frequency information has been principally produced for 4 cially low PFs: epidermis±exodermis Fn ˆ 8.96 3 10 ) leaves from decades of painstaking endeavour to elucid- 4 and pericycle±stelar parenchyma Fn ˆ 6.44 3 10 ). This ate photosynthate transport processes Gamalei, 1991; suggests that there is significant membrane transport Van Bel, 1993; Van Bel and Oparka, 1995; Turgeon, across the interface of epidermis±exodermis through 1996). Also, exciting advances have been made, yet again, short cells) and direct transfer of ions from pericycle in leaves, in the structural and functional regulation of to protoxylem vessels. In the phloem, the highest PD during macromolecule transport Ding et al., 1999; PF was detected at the metaphloem sieve element± Lucas, 1999; Oparka and Santa Cruz, 2000). In compar- companion cell interface Fw ˆ 0.42), and all other ison, PD in root systems have received limited attention. interfaces had much lower PFs around 0.10). In the In a recent study of the Arabidopsis thaliana L. root apical pericycle, the radial walls had a high PF Fw ˆ 0.75), a meristem, a tissue-speci®c pattern of PD distribution feature that could permit lateral circulation of solutes, was observed, and dye-coupling experiments established a thus facilitating ion inward) and photosynthate correlation between this pattern and the potential for outward) delivery. symplastic diffusion of small molecules Zhu et al., 1998). For root tissues proximal to the root tip, only a very Key words: Allium cepa L., phloem unloading, plasmo- few species have been examined and in these, PFs were desmata, plasmodesmatal frequency, root, symplastic measured only for selected interfaces Robards et al., transport, transmission electron microscopy. 1973; Robards and Jackson, 1976; Warmbrodt, 1985a, b,

1 Present address: Department of Plant Biology and Plant Biotechnology Centre, The Ohio State University, Columbus, Ohio 43210, USA. 2 To whom correspondence should be addressed. Fax: q1 519 746 0614. E-mail: [email protected] À1 À2 Abbreviations: Fn, number of plasmodesmata mm root length 5at individual tissue interfaces); Ft, number of plasmodesmata mm tissue interface; À2 Fw, number of plasmodesmata mm wall surface; PD, plasmodesmata; PF, plasmodesmatal frequency; TEM, transmission electron microscopy.

ß Society for Experimental Biology 2001 1052 Ma and Peterson 1986a; Kurkova, 1989; Wang et al., 1995). To date, a types.) Most authors have assumed that ions are trans- complete picture of symplastic connections in any root ported from the cortex to the stelar parenchyma from has been lacking. whence they are ®nally transferred into the vessels Special consideration is needed for some plants that Sanderson, 1975; Stelzer et al., 1975; Robards and have an exodermis in their roots. The exodermis is an Clarkson, 1976; Clarkson, 1993). This idea gained outermost cortical layer that develops Casparian bands support from experiments in which stelar parenchyma Peterson and Perumalla, 1990) and suberin lamellae cells proved to be capable of actively accumulating ions Kroemer, 1903; Von Guttenberg, 1968). The Casparian from the cortex in Z. mays LaÈuchli et al., 1971a, b, bands, since they are in the anticlinal walls, do not affect 1974a, b). However, studies on Hordeum vulgare L. roots the roots' symplastic transport in the radial direction. The suggested that the pericycle might play a major role in suberin lamellae, lying all around the cells' protoplasts, xylem loading Vakhmistrov et al., 1972; Kurkova et al., may or may not affect the symplastic connections within 1974; Vakhmistrov, 1981). In the latter species, the the root, according to the type of the exodermis. In the pericycle±stelar parenchyma interface had a much lower uniform exodermis all cells elongate), as seen in Zea PF than the pericycle±endodermis interface, and it was mays L. the only species of this category examined envisaged that the majority of ions would proceed from by TEM), suberin lamellae do not interfere with the the pericycle directly to the xylem vessels, rather than symplastic continuity of the layer Wang et al., 1995). through the stelar parenchyma cells Vakhmistrov et al., However, in the dimorphic exodermis with long and 1972; Kurkova et al., 1974; Vakhmistrov, 1981). In the short cells alternating along the axis of the root), as present study, the relative signi®cance of these two tissues in Citrus sp. Walker et al., 1984) and Allium cepa L. stelar parenchyma and pericycle) at this critical point of Ma and Peterson, 2000), suberin lamella deposition in radial ion transport will be examined in Allium cepa L. long cells severs all their PD. Accordingly, the short cells roots. without suberin lamellae) must play a paramount role in Phloem unloading in roots is another ®eld that is the symplastic ¯uxes of ions and photosynthate-derived poorly understood. In the root tip, symplastic transport nutrients). In an earlier paper, an overall view of PD phloem unloading and post-phloem transport) is intens- relationships but not details of frequencies) of A. cepa ive to sustain cell division and growth Dick and ap Rees, roots was provided Ma and Peterson, 2000). A closer 1975; Oparka et al., 1994; Zhu et al., 1998). More mature examination of PD distribution in the exodermis short zones proximal to the tip) are apparently weaker sinks cells), as well as in all other tissues, will provide further for photosynthates but here all the living cells still need insights into the symplastic connections in mature roots. a continuous supply of photosynthates for their normal There are some other major issues that remain unclari- respiratory activities. Phloem unloading and post-phloem ®ed, one of which concerns xylem loading. Are the xylem transport of photosynthates in mature zones could be vessels loaded by stelar parenchyma cells intervening accomplished by a symplastic pathway in several species between the xylem and phloem strands) or by pericycle or examined Giaquinta et al., 1983; Warmbrodt, 1985a, b, by both? See Fig. 1for locations of these and other cell 1986a, b). However, in A. thaliana, no symplastic phloem unloading was observed under normal conditions of root growth Wright and Oparka, 1997). In the present study, it was noted whether or not the PD associated with the phloem were structurally normal, and what the related PFs may imply for solute transfer. There are several ways of expressing PFs, three of which were used in the present study. 1) The number À2 of PD mm wall surface Fw). This is the basic and most commonly used value as in most of the studies cited

above). Fw is most useful for a comparison of cell inter- faces to predict their relative capacity for symplastic transfer and, thus, is applicable to the phloem region of the root where the paths concerned are very short. 2) The number of PD on a tissue interface over a unit root length

Fn). This treatment is more instructive than the previous Fig. 1. Diagram of a cross-section of Allium cepa L. root showing cell one for predicting symplastic transport capacity for and tissue interfaces studied for plasmodesmatal frequencies. For the tissues ranging from epidermis to pericycle. This is simply À2 measurement of PD mm wall surface Fw), cell walls are marked with À1 because all the tissues except for the central cortex) are short, thick lines. For PD mm root length Fn), tissue interfaces are drawn in circles of thin lines numbered 1through 5). The boxed area organized into concentric cylinders; the interface areas indicates the location of Fig. 3A. of which are determined by their radii Fig. 1). The Frequencies of plasmodesmata in onion roots 1053

À1 interfaces will be traversed by both ions in the inward PD mm root length 1 Fn ) direction moving from the soil solution to the stele) and The interfaces measured were: 1) epidermis±exodermis, 2) photosynthates in the outward direction moving from exodermis±central cortex, 3) central cortex±endodermis, 4) the stele to the cortex and epidermis). 3) The number of endodermis±pericycle, and 5) pericycle±central stele Fig. 1). It À2 should be noted that this approach is not suitable for the central PD mm tissue interface cylinder Ft). The values of cortex, because intercellular spaces had developed and the cells Ft, which may or may not be equal to Fw see Materials were not arranged in concentric cylinders. The formula is and methods), was employed in the present study for 3 Fn ˆ 10 pDFw 2) the purpose of comparing the present results to related 3 2 literature data that were expressed in this way. where 10 pD is area mm ) of the cylinder D, diameter, in mm) into which each tissue interface ®ts. Fw, corresponding values obtained from formula 1. Clearly, formula 2 was not applicable to interfaces 1and 2 Materials and methods above) since intact exodermal PD did not occur in the tangential walls of exodermal long cells but only of short cells Ma and Plant materials Peterson, 2000). Therefore,

Bulbs of Allium cepa L. cv. Ebeneezer were planted in moist Fn ˆ AscFw 2-1) vermiculite as described previously Ma and Peterson, 2000). At 7±14 d, root systems were collected. Any adhering vermiculite where Asc is the `functional area' i.e. occupied by short cells) was gently rinsed away from the roots prior to further sampling. over the exodermis at the outer or inner tangential side within a 1mm root length see the previous section; Fig. 1). Fw, corresponding value obtained from formula 1. Interface 5 is highly heterogeneous, consisting of three Transmission electron microscopy sub-interfaces, i.e. pericycle±stelar parenchyma, pericycle± Root segments were excised 100 mm from the tip for TEM. companion cells, and pericycle±protophloem sieve elements). For a full account of the technique, see Ma and Peterson, 2000.) For the ®rst sub-interface, the Fn value was calculated from

At this distance, the roots had a mature exodermis, i.e. Fn ˆ AspFw 2-2) Casparian bands had developed in both short and long cells, and the latter also had suberin lamellae. The section thickness where Asp is the outer tangential area of the stelar parenchyma À2 was set at 8 3 10 mm. over a 1mm root length. This value was obtained from TEM images. Light microscope images would not give precise results for this particular case, as the area was very small.) Fw is the corresponding value obtained from formula 1. For the second Calculation of tissue interface areas and third sub-interfaces, the corresponding interface areas and Free-hand cross-sections were made 100 mm from the root tip Fw values were substituted in formula 2-2. and photographed using colour slide ®lms). Diameters and thus areas) of individual tissue interfaces were measured from the À2 transparencies under a dissecting microscope. The surface area Number of PD mm tissue interface 1 Ft ) of exodermal short cells was obtained from tangential sections The formula for this calculation is of the exodermis. These data were used to calculate Fn below). Ft ˆ FnuA 3) where A is the area of a given tissue interface over a 1mm root 2 Calculation of plasmodesmatal frequencies length mm ). For interfaces 3 and 4, Ft ˆ Fw Fig. 1) because, for each, only one cell type occurred on either side of the PFs were calculated in three different ways. The rationale for interface. Values of Ft were not estimated for the central cortex each was presented in the Introduction. for the reasons stated above.

À2 Statistics PD mm wall 1 Fw) Calculations were performed following the formula of Gunning Paired comparisons t-tests were performed at a ˆ 0.05 Sokal in Robards, 1976): and Rohlf, 1981) for the values of Fw of the phloem region and some other interfaces see Results). Analyses were not done for F ˆ NuwLTq1:5R)x 1) w Ft and Fn, since these values are secondary in nature i.e. derived from the means of F ). where L is the length of a given cell wall; N is the number of w PD along the wall; T is the thickness of sections, and R is the radius of PD. The units of L, T, and R are mm. For each interface Fig. 1), the diameters of PD were measured from Results TEM negatives mean of 5 PD at their largest dimensions in the Plasmodesmatal frequencies in tissues from epidermis longitudinal view). To obtain N and L, 60 walls were randomly sampled from 5±30 non-serial ultrathin sections which were to stelar parenchyma cut from 5±15 roots). On each wall, N was counted directly in The PD at the interfaces examined were similar in dia- the microscope. The same wall was digitized at 3 2600 by the program Analysis 2.0 Soft-Imaging Service GmbH) and its meter ranging from 59 to 65 nm) but different in fre- length was subsequently measured with another program, quency. Considering that the radial symplastic path spans Northern Exposure Empix Imaging, Inc.). between the epidermis and the stelar parenchyma, the 1054 Ma and Peterson Table 1. Frequencies ofplasmodesmata in A. cepa roots !for interfaces external to the central stele)

bEP±EX EP±SC EX±CC SC±CC CC±CC CC±EN EN±PE PE±CP PE±PSE PE±SP a c Fw n.a. 0.21"0.16 n.a. 1.03"0.44 1.59"0.43 0.58"0.22 0.70"0.19 0.16"0.12 0.08"0.04 0.12"0.09 4 5 5 5 d 4 e 4 Fn 8.96"10 4.05"10 n.a 5.13"10 5.64"10 PE±CS 6.44"10 , 1.25"10 À2 À1 À1 À1 d À2 e À2 Ft 2.77"10 n.a. 1.68"10 n.a. n.a. 5.83"10 7.00"10 PE±CS 8.96"10 , 1.74"10

a À2 À1 À2 Fw,PDmm wall surface; Fn,PDmm root length; Ft,PDmm tissue interface. bCC, central cortex; CP, companion cell; CS, central stele stele excluding pericycle); EN, endodermis; EP, epidermis; EX, exodermis; PE, pericycle; PSE, protophloem sieve element; SP, stelar parenchyma. cn.a., not applicable. dBased on the assumption that all plasmodesmata on the PE±CS interface are functional. eBased on the assumption that only plasmodesmata on the PE±CS interface are functional. Both sets of data d and e) are for the entire PE±CS interface see text). interfaces of concern were ranked, on the basis of their Fw values, in the following order: central cortex)short cells± central cortex)endodermis±pericycle)central cortex± endodermis)epidermis±short cell)pericycle±stelar par- enchyma. Related data are displayed in Table 1. To provide a clearer visual indication of PD distribution, a plasmodesmogram Van Bel and Oparka, 1995) was constructed, based on the Fw values Fig. 2). The Fn values were rather constant in the region from the exodermis up to the pericycle, in spite of the uncertainty about the central cortex Table 1). For this latter tissue, although it is theoretically possible to obtain an Fn value, the estimate would be misleading if applied to any explanation of radial transport, due to the presence of intercellular spaces and the cells' irregular arrange- ment. Nevertheless, the abundance of PD in the cell walls

Fw) of the central cortex apparently renders it ef®cient for symplastic transport. At the outermost interface of the root, epidermis±exodermis, a very small number of PD was observed. To provide an even sharper picture of PD distribution along the radial path across the con- centric cylinders of tissue interfaces), this set of data was ranked by ratios Fig. 2). In this diagram, two different Fig. 2. Plasmodesmogram of A. cepa root: PFs from the epidermis to treatments were made for interface 5 as indicated in À2 the stele. The PFs Fw,PDmm wall surface) are approximately Fig. 1). First, it was assumed that all PD between the represented by short lines that link neighbouring cells. The more the pericycle and its adjacent inner living cells are functional; lines, the higher the frequencies but not drawn in perfect proportion). the ratios are shown in the ®rst ®le of numbers. Second, The PFs associated with the phloem are not shown in this diagram but see Fig. 4). The dotted line between adjacent epidermal cells indicates based on the assumption that the pericycle±phloem the presence of a few PD, but quantitative data were not obtained. This interface is non-functional in transferring ions, the radial diagram can also be used to show the overall pattern of PFs expressed À1 symplastic pathway is then reduced to the pericycle± as Fn PD mm root length). The numbers on the right side are the ratios of Fn values: the ®rst ®le is based on the assumption that all stelar parenchyma interface. In this case, the second ®le of plasmodesmata between pericycle and inner living cells are functional, numbers applies. and the second ®le is based on the assumption that symplastic transport of ions is only across the pericycle±stelar parenchyma interface see text). In both cases, the lowest frequency was set equal to 1.0. For the central Plasmodesmatal frequencies associated with the stele cortex, Fn was not calculated, but the number of PD available for and the endodermis symplastic transport must be very high assumed to be higher than the highest known frequency among the interfaces; see text). CC, central The root usually contained six phloem strands Fig. 1). cortex; EN, endodermis; EP, epidermis; LC, exodermal long cells; MX, metaxylem vessel member; PE, pericycle; PH, phloem; PX, protoxylem In each, the components and their spatial relationships vessel member; SC, exodermal short cell; SP, stelar parenchyma. were as depicted in Fig. 3A. Each strand contained 1or 2 protophloem sieve elements, 3±5 metaphloem sieve ele- ments and their associated companion cells. At its outer companion cells. On its ¯anks, the phloem was connected tangential side, the phloem was in contact with the to the stelar parenchyma by companion cells. Metaphloem pericycle by 1or 2 protophloem sieve elements and 3±4 sieve elements were associated neither with the pericycle Frequencies of plasmodesmata in onion roots 1055

Fig. 3. Structure of phloem and surrounding tissues. A) Detail of the boxed area in Fig. 1. An overview of phloem and its relationship with surrounding tissues. B) Pericycle and neighbouring cells. The radial walls of pericycle cells are marked by asterisks. C) PD between metaphloem sieve element and companion cell. The wall was thickened. D) Companion cells. Note thick cytoplasm with ER and mitochondria MI). E) Interface of pericycle and companion cell. The PD looks normal in its structure. C, cytoplasm; CP, companion cell; EMX, early metaxylem vessel member; EN, endodermis; IEMX, immature early metaxylem vessel member; MSE, metaphloem sieve element; PD, plasmodesmata); PE, pericycle; PSE, protophloem sieve element; PX, protoxylem vessel member; SP, stelar parenchyma. Bars ˆ 50 mm A) or 0.5 mm B±E). nor with the stelar parenchyma. In the phloem, compan- All PD in the entire phloem region were structurally ion cells had the densest cytoplasm of any cell type, normal as in other areas of the root, see Ma and characterized by numerous mitochondria and ER pro®les Peterson, 2000) and did not appear to be damaged or

Fig. 3B, C, D, E). Pericycle cells had denser cytoplasm blocked e.g. Fig. 3E). In general, the PFs Fw) in this than stelar parenchyma cells Fig. 3B). Those stelar par- region were much lower than in the tissues external to the enchyma cells deep in the stele had a thin cytoplasm and stele Table 2). Within the phloem, the values were very few organelles, comparable to immature metaxylem vessel uniform except for the one at the metaphloem sieve members. element±companion cell interface Table 2; Fig. 4), but 1056 Ma and Peterson Table 2. A comparison ofplasmodesmatal frequenciesin A. cepa roots with those in the literature À2 Plasmodesmatal frequencies are expressed as numbers of plasmodesmata mm cell or tissue interface Fw or Ft, where applicable, see text), unless speci®ed otherwise.

Interface Allium cepa L. Hordeum vulgare L. Zea mays L.

aa bcd e f ghi

EP±EXb 0.03 g0.1 0.54 EX±CC 0.17 1.14 0.21b CC 1.59 1.50d 0.45 0.43 CC±EN 0.58 0.48 0.37 0.44 0.22c EN±EN 0.06 0.34 0.32 0.32 EN±PE 0.70 0.56 0.75 43% 0.48 PE±PE 0.75 0.30 0.54 45% 0.35 PE±SP 0.12 0.52 0.25 12% 0.54 SP±MSE e 0.28 0.45 SP±SP 0.11 0.31 0.32 SP±CP 0.17 0.57 0.49 PSE±PE 0.08 0.32 0.36 CP±PE 0.16 0.37 0.35 PSE±CP 0.07 0.68 0.76 CP±CP 0.11 0.31 0.34 MSE±CP 0.42 0.72 0.88 MSE±PSE 0.13 ff MSE±MSE 0.10 gg

aa, Present results. b, Peterson et al., 1978. c, Tyree, 1970. d, Warmbrodt, 1985a. e, Robards and Jackson, 1976. f, Vakhmistrov et al., 1972, plasmodesmatal frequencies in % of the total. g, Warmbrodt, 1985b. h, Clarkson et al., 1987. i, Wang et al., 1995. bAbbreviations: MSE, metaphloem sieve element. For the other abbreviations, see Table 1. cIn nodal branch roots. dTyree, 1970, calculated from micrographs in Scott et al., 1956. eCell association absent. fCell association rare. gData not available.

The pericycle and endodermis each exhibited unique patterns of PD distribution. Pericycle cells had very high

PFs Fw) in both their radial and outer tangential walls, but signi®cantly lower PF at a ˆ 0.05) in their inner tangential walls Fig. 2; Table 2). In the endodermis, the PFs on the outer and inner tangential walls were high and comparable to each other, but the radial walls had an extremely low PF which was signi®cantly lower at a ˆ 0.05) than that of corresponding walls of pericycle Fig. 2, Table 2). Fig. 4. Plasmodesmogram of A. cepa roots: PFs associated with the phloem, endodermis and pericycle. This diagram is based on Fw values. MX, metaxylem vessel member. For a complete list of abbreviations, see Fig. 3.) Discussion The present study provided a complete assessment of PFs that were associated with the symplastic transport, both this was not signi®cantly different at a ˆ 0.05). At the inward for ions) and outward for photosynthates), in phloem±pericycle interface, the Fn value was estimated in Allium cepa L. roots. This is the ®rst such comprehensive the following way. As seen in a cross-section of the root treatment for any root system. Fig. 1), the phloem was connected to about half of the inner tangential surface area of the pericycle; this Plasmodesmatal frequencies: implications for ion interface area was shared between companion cells and transport protophloem sieve elements at a ratio of about 4 : 1

Fig. 3A). The Fn values for these two sub-interfaces were Symplastic transport is made possible in A. cepa roots 4 3 estimated at 4.61 3 10 and 5.76 3 10 , respectively, total- by the presence of PD in all tissue interfaces. Previous 4 ling 5.19 3 10 . This number was added to that of the understanding of symplastic transport in the root has pericycle±stelar parenchyma interface to express the total been largely based on studies of the cortex Anderson and Fn on the entire pericycle inner face Table 1). Reilly, 1968; Ginsburg and Ginzburg, 1970a, b; Jarvis and Frequencies of plasmodesmata in onion roots 1057 House, 1970; Baker, 1971; Clarkson and Sanderson, 1974; as recorded in Robards et al., 1973; see Table 2). But in Van Iren and Boers-Van de Sluijs, 1980). The present another work on the same species, the difference was results on A. cepa roots strongly support a symplastic trivial Warmbrodt, 1985a; Table 2). Due to this discrep- pathway across this tissue, since its PF Fw) was rather ancy in the two studies, a ®rm conclusion will require high Table 1). In addition, this major, if not the only, a thorough re-examination of the system. In both A. cepa, pathway is most likely to occur both near the root surface and Z. mays, the inner and outer tangential endodermal from short cells to central cortex) and deep in the root walls had comparable PFs Table 2). It is interesting from central cortex to endodermis to pericycle); high and to note that in these latter species, the exodermis with constant PD numbers Fn) were found on these interfaces Casparian bands) is an effective apoplastic barrier Table 1; Fig. 2). Peterson, 1998). It is tempting to postulate that Short cells apparently take up ions from the apoplast, symplastic transport across the cortex is the principal as indicated by the following observations. As shown mechanism in both species. In A. cepa, the high PF in Table 1, the exodermis through short cells) was Fw, Table 1) would facilitate such movement. In this connected with the epidermis with a much lower PF connection, since H. vulgare is a non-exodermal species than with the cortex. When Ft values were compared Perumalla et al., 1990), a relatively signi®cant apoplastic À2 À1 2.77 3 10 versus 1.68 3 10 ; Table 1), there was a pathway across the cortex would be expected. Ideally, a

6-fold difference. When Fn values which are more instruc- comparison of Fn values of exodermal and non-exo- tive for examining radial transport) were compared, dermal species would be more informative; unfortunately, the difference was also obvious 5-fold). Assuming that these values are lacking in the literature. a constant ¯ow of material is sustained from the soil The PFs at the periphery of the stele are important in solution to the cortex and the PD function close to their considering the relative signi®cance of pericycle and stelar capacity, there should be a supplementary mechanisms) parenchyma in loading the xylem. In H. vulgare roots it that makes the ¯ow possible across the epidermis±short was found that the pericycle was poorly connected by PD cells interface. It is hypothesized that ions diffuse through to the internal stele tissues Vakhmistrov et al., 1972). the epidermal walls and enter the short cells at their They suggested that ions would move directly from the outer tangential plasma membranes. Short cells normally pericycle to the xylem vessels, rather than through display features typical of metabolically active cells the internal stele tissues see also the last section of this Peterson and Enstone, 1996; Ma and Peterson, 2000). Discussion). It is inferred from a later contribution In extreme situations, short cells exhibit elevated potential Vakhmistrov, 1981) that the `internal stele tissues' are for ion uptake by developing wall ingrowths Atriplex equivalent to `stelar parenchyma'. Although Warmbrodt hastata L., Kramer et al., 1978; A. cepa, Wilson and Warmbrodt, 1985a) was unable to con®rm the results Robards, 1980). In both Citrus sp. Walker et al., 1984) of Vakhmistrov et al. Vakhmistrov et al., 1972), the latter and A. cepa Barrowclough and Peterson, 1994, Kamula authors' results were in general agreement with those et al., 1994), the epidermis tends to die; the movement of Robards and Jackson Robards and Jackson, 1976; of ions from the apoplast to the symplast of short cells see Table 2). In further support of Vakhmistrov's idea, then becomes the only pathway for ion uptake in this a recent study of H. vulgare showed that the pericycle had zone of the root. In the Z. mays exodermis uniform, more intense expression of the plasma membrane all cells symplastically connected to adjacent tissues), Hq-ATPase than the stelar parenchyma Samuels et al., the PFs on its outer and inner tangential walls are not 1992). Assuming that the PD at the pericycle±phloem as different as in A. cepa Table 2). Therefore, even if interface are non-functional in transferring ions, then the the hypothesized supplementary mechanism exists in frequency of proposed functional PD at the pericycle± Z. mays, it is unlikely that it plays a role as signi®cant stelar parenchyma interface) is even lower in A. cepa than as in A. cepa. in H. vulgare Fig. 4; Table 2). If, on the other hand, The PFs of the endodermis on its outer and inner the pericycle±phloem PD were functional, then ions could tangential walls) have important implications for the func- diffuse into the phloem. However, ions in the phloem tion of the endodermis and also of the root as a whole, would need to cross the companion cells±stelar paren- especially in ion transport. It is generally believed that chyma interface before entering the vessels Figs 1, 3A); the endodermal Casparian bands divert apoplastic ¯ows this interface is unlikely to support a signi®cant amount of solutes from the central cortex across the endodermal of symplastic transport in view of the extremely low PF outer tangential plasma membrane into the symplast Table 2). Now the major concern is how the xylem of the endodermis). Supposing that all solutes in the is loaded. For A. cepa roots, there is evidence that an endodermis are then constantly moved to the pericycle active step exists from the symplast to the xylem symplastically, this interface would be expected to have for ClÀ; Hodges and Vaadia, 1964), which is probably a higher PF Fw) than the outer side. In H. vulgare, achieved by a proton pump Clarkson and Hanson, indeed, the value Fw) is doubled 0.75 versus 0.37) 1986). The exact location of the active step, however, has 1058 Ma and Peterson not been clearly determined; it could be in the stelar surrounding cells are held closed under normal condi- parenchyma or the pericycle or both. Traditionally, an tions; they open only during lateral primordium forma- active role has been designated to the xylem parenchyma tion Oparka et al., 1995) or upon application of LaÈuchli et al., 1971a, b, 1974a, b; De Boer, 1999). metabolic inhibitors, as detected by ¯uorescent tracer However, this term has frequently been used to denote a dyes Wright and Oparka, 1997). Tracer experiments collective of stelar parenchyma and pericycle. From the also demonstrated functional phloem isolation in stems of physiological point of view, there is a need to separate several species Hayes et al., 1985; Aloni and Peterson, these two tissues. They are different from each other both 1990; Van Bel and Kempers, 1990; Van Bel and in their ontogeny and in their spatial relationships to Van Rijen, 1994). In support of these results, the adjacent tissues, which in turn could well re¯ect their expression of the A. thaliana AtSUC2 sucrose-Hq sym- different functional roles. porter has been localized exclusively in the companion cells of root and stem and some other organs, Truernit and Sauer, 1995; Stadle and Sauer, 1996). A similar On phloem unloading and post-phloem transport of expression pattern of sucrose-Hq symporter was found photosynthates in mature roots in Plantago major L. Stadle et al., 1995). The new results Both outside and inside the phloem, transport of photo- are indicative of apoplastic unloading through compan- synthates can be achieved by PD. Across the root cortex, ion cells. If this is the case in roots, photosynthates in there is physiological evidence for this kind of transport the apoplast will have to enter the symplast internal to the in several species Dick and ap Rees, 1975; Giaquinta endodermal Casparian band an apoplastic barrier) prior et al., 1983; Fisher and Oparka, 1996). Inside the phloem, to their outward transport. Several questions concerning it seems to be a common feature that companion cells are phloem unloading in roots remain to be investigated: 1) the principal receivers of the translocated photosynthates is a sucrose Hq-symporter present in all plant roots and, from the shoot through sieve tubes Warmbrodt, 1985a, b). more particularly, in their mature zones? 2) is the carrier These ideas gained support from the present analysis functional and, if so, how is it regulated? and 3) how are of PFs Fig. 4; Table 2). Yet, as to the initial steps of the PD regulated along with the carriers? the post-phloem transport or, more speci®cally, post- companion cell transport), variations might occur among A further note on the pericycle species. For instance, in H. vulgare Warmbrodt, 1985a), Z. mays Warmbrodt, 1985b) and some other species In addition to the involvement of PD on the tangential Patrick and Of¯er, 1996), the preferred symplastic paths walls in the radial transport as discussed above, the peri- would be from companion cells to stelar parenchyma cycle may also conduct ions and photosynthates across cells and to the pericycle, along a decreasing PF gradient. its radial walls. Based on results obtained from H. vulgare, A parallel solute concentration gradient was detected in Vakhmistrov suggested that this cell layer could act as H. vulgare Warmbrodt, 1986b) and Z. mays Warmbrodt, an `annular collectorudisperser' Vakhmistrov et al., 1972; 1987). Although A. cepa shares certain similarities with Vakhmistrov, 1981). In brief, substances received from H. vulgare Warmbrodt, 1985a) and Z. mays Warmbrodt, the endodermis tend to move symplastically across the 1985b) with respect to phloem construction, no such radial walls with a high PF), toward those pericycle preference is expected on an anatomical basis in the cells that lie near the xylem vessels i.e. the `annular former since its PD were distributed approximately evenly collector' role; Fig. 5A) where the concentration of ions around the phloem Fig. 4; Table 2). is kept low by the transpiration stream. In this scenario, By what pathway are photosynthates unloaded from it was assumed that, in the stele, little symplastic trans- the phloem into the pericycle in the mature zone of port of ions would occur toward the vessels see the the root? Unlike the richness of literature on phloem previous section). At the same time, the pericycle func- unloading in expanding leaves and storage organs, there tions as an `annular disperser' Fig. 5B) in the outward is a paucity of knowledge regarding the root Patrick, transport of photosynthates Kurkova et al., 1974; 1990; Fisher and Oparka, 1996). In A. cepa, the interfaces Vakhmistrov, 1981). This is based on the following of phloem±pericycle and stelar parenchyma±pericycle consideration. Physically, the pericycle cells that are 4 4 À1 had Fn ˆ 5.19 3 10 and 1.25 3 10 PD mm root, respect- connected with the phloem and, to a lesser extent, ively. The PD appeared structurally normal Fig. 3E). probably also with the stelar parenchyma, see above) Assuming the PD on both interfaces are functional in are able to receive substances from the phloem if the PD transferring photosynthates to the pericycle, the combined are functional). But, these substances are not directly 4 value 6.443 10 , close to that at the exodermis±epidermis accessible to those pericycle cells that are in contact with interface, Table 1) would support a signi®cant amount the xylem see also Figs 1and 4). Thus, some of the of symplastic transport. In the mature root zone of photosynthates would move directly to the adjacent A. thaliana, the PD at the interface of phloem and endodermal cells, and the rest would tend to equilibrate Frequencies of plasmodesmata in onion roots 1059

Fig. 5. Pericycle as `annular collector-disperser' from Vakhmistrov et al., 1972, but adapted to A. cepa root structure). A) The `annular collector' model for ion transport. The radial walls of pericycle cells are drawn with broken lines to indicate the high PF. Possible routes and intensities of symplastic ion transport are labelled with arrows of different sizes. Hollow arrows indicate apoplastic pathways. B) The `annular disperser' model for photosynthate transport. Possible routes and intensities of symplastic phloem-unloading anduor post-phloem transport are labelled with arrows of varying sizes. See text for a discussion of various ¯ows. across the pericycle via PD, reaching the xylem-associated the research. The Natural Sciences and Engineering pericycle cells, and then the adjacent endodermal cells. Research Council of Canada provided funding to CAP, and the University of Waterloo awarded Graduate Student The present results on A. cepa roots are in favour of Scholarships to FM. this model. In Z. mays, a large number of PD was also observed in pericycle radial walls Warmbrodt, 1985b; see also Table 2). Vakhmistrov's brilliant insights deserve References to be better known and research should be extended to the examination of other species to see if this model is Aloni R, Peterson CA. 1990. The functional signi®cance of generally applicable, both structurally and physiologically. phloem anastomoses in stems of Dahlia pinnata Cav. Planta 182, 583±590. Anderson WP, Reilly EJ. 1968. A study of the exudation of excised maize roots after removal of the epidermis and outer Conclusions cortex. Journal of Experimental Botany 19, 19±30. Baker DA. 1971. Barriers to radial diffusion of ions in maize The ®ndings reported here show that PD connections roots. Planta 98, 285±293. are present across all root tissues of A. cepa. At the Barrowclough DE, Peterson CA. 1994. Effects of growing condi- epidermis±short cells interface, both symplastic transport tions and development of the underlying exodermis on the and transmembrane transport are anticipated. The peri- vitality of the onion root epidermis. Physiologia Plantarum 92, cycle could play a signi®cant role in symplastic transport 343±349. Clarkson DT. 1993. Roots and the delivery of solutes to the of ions and photosynthates, not only directly along xylem. Philosophical Transactions of the Royal Society of the radial path) but also indirectly by circulation in London, Series B 341, 5±17. the tangential direction across radial walls). Symplastic Clarkson DT, Hanson JB. 1986. Proton ¯uxes and the activity of photosynthate distribution could occur in the phloem a stelar proton pump in onion roots. Journal of Experimental region and beyond. These results will be useful for a func- Botany 37, 1136±1150. tional examination of ion and photosynthate movement Clarkson DT, Robards AW, Stephens JE, Stark M. 1987. Suberin lamellae in the hypodermis of maize Zea mays) across the roots of A. cepa and other species. roots: development and factors affecting the permeability of hypodermal layers. Plant, Cell and the Environment 10, 83±93. Clarkson DT, Sanderson J. 1974. The endodermis and its devel- Acknowledgements opment in barley roots as related to radial migration of ions and water. In: Kolek J, ed. Structure and function of primary We thank Mr Dale Weber University of Waterloo, Waterloo) root tissues. Bratislava: Veda, Publishing House of the Slovak for his expert support with the TEM, Dr William Diehl-Jones Academy of Sciences, 87±100. University of Waterloo) for instructions on cell wall measure- De Boer AH. 1999. Potassium translocation into the root xylem. ment, Dr Tony Robards University of York, UK) for advice Plant Biology 1, 36±45. on plasmodesmatal frequency calculation, Dr Pat Newcombe Dick PS, ap Rees T. 1975. The pathway of sugar transport University of Waterloo) for statistical advice, and Ms Daryl in roots of Pisum sativum. Journal of Experimental Botany 26, Enstone University of Waterloo) for her assistance throughout 305±314. 1060 Ma and Peterson Ding B, Itaya A, Woo Y-M. 1999. Plasmodesmata and cell- of Arabidopsis thaliana. Journal of Experimental Botany to-cell communication in plants. International Reviewof 46, 187±197. Cytology 190, 251±316. Oparka KJ, Duckett CM, Prior DAM, Fisher DB. 1994. Real- Fisher DB, Oparka KJ. 1996. Post-phloem transport: principles time imaging of phloem unloading in the root tip of and problems. Journal of Experimental Botany 47, 1141±1154. Arabidopsis. The Plant Journal 6, 759±766. Gamalei YV. 1991. Phloem loading and its development related Oparka KJ, Santa Cruz S. 2000. The great escape: phloem to plant evolution from trees to herbs. Trees 5, 50±64. transport and unloading of macromolecules. Annual Review Giaquinta RT, Lin W, Sadler NL, Franceschi VR. 1983. Path- of Plant Physiology and Plant Molecular Biology 51, 323±347. way of phloem unloading of sucrose in corn roots. Plant Overall RL, Blackmann LM. 1996. A model of the macro- Physiology 72, 362±367. molecular structure of plasmodesmata. Trends in Plant Science Ginsburg H, Ginzburg BZ. 1970a. Radial water and solute ¯ows 1, 307±311. in roots of Zea mays. I. Water ¯ow. Journal of Experimental Patrick JW. 1990. Sieve element unloading: cellular pathways, Botany 21, 580±592. mechanism and control. Physiologia Plantarum 78, 298±308. Ginsburg H, Ginzburg BZ. 1970b. Radial water and solute ¯ows Patrick JW, Of¯er CE. 1996. Post-sieve element transport of in roots of Zea mays. II. Ion ¯uxes across root cortex. Journal photoassimilates in sink regions. Journal of Experimental of Experimental Botany 21, 593±604. Botany 47, 1165±1177. Hayes PM, Of¯er CE, Patrick JW. 1985. Cellular structures, Perumalla CJ, Peterson CA, Enstone DE. 1990. A survey of plasma membrane surface areas and plasmodesmatal fre- angiosperm species to detect hypodermal Casparian bands. II. quencies of the stem of Phaseolus vulgaris L. in relation to Roots with a uniseriate hypodermis and epidermis. Botanical radial photosynthate transfer. Annals of Botany 56, 125±138. Journal of the Linnean Society 103, 93±112. Hodges TK, Vaadia Y. 1964. Uptake and transport of radio- Peterson CA. 1998. The exodermis and its interactions with chloride and tritiated water by various zones of onion roots the environment. In: Flores HE, Lynch JP, Eissenstat D, of different chloride status. Plant Physiology 39, 104±108. eds. Radical biology:advances and perspectives on the function Jarvis P, House CR. 1970. Evidence for symplastic ion transport ofplant roots . Rockville: American Society of Plant Physiolo- in maize roots. Journal of Experimental Botany 21, 83±90. gists, 131±138. Kamula SA, Peterson CA, May®eld CI. 1994. The plasmalemma Peterson CA, Enstone DE. 1996. Function of passage cells in the surface area exposed to the soil solution is markedly reduced endodermis and exodermis of roots. Physiologia Plantarum by maturation of the exodermis and death of the epidermis 97, 592±598. in onion roots. Pant, Cell and Environment 17, 1183±1193. Peterson CA, Perumalla CJ. 1990. A survey of angiosperm Kramer D, Anderson WP, Preston J. 1978. Transfer cells in the species to detect hypodermal Casparian bands. I. Roots with root epidermis of Atriplex hastata L. as a response to salinity: a multiseriate hypodermis or epidermis. Botanical Journal of a comparative cytological and X-ray microprobe invest- the Linnean Society 103, 113±125. igation. Australian Journal ofPlant Physiology 5, 739±747. Peterson CA, Peterson RL, Robards AW. 1978. A correlated Kroemer K. 1903. Wurzelhaube, Hypodermis und Endodermis histochemical and ultrastructural study of the epidermis and der Angiospermenwurzel. Bibliotheca Botanica 12, 1±59. hypodermis of onion roots. Protoplasma 96, 1±21. Kurkova EB. 1989. Three-dimensional distribution of plasmo- Robards AW. 1976. Plasmodesmata in higher plants. In: desmata in the rhizodermis of Trianea bogotensis Karst. Gunning BES, Robards AW, eds. Intercellular communication In: Loughman BC, GasÏparõÂkova O, Kolek J, eds. Structural in plants: studies on plasmodesmata. Berlin: Springer-Verlag, and functional aspects of transport in roots. Dordrecht: Kluwer 15±57. Academic Publishers, 57±59. Robards AW, Clarkson DT. 1976. The role of plasmodesmata in Kurkova JB, Vakhmistrov DB, Solovev VA. 1974. Ultrastructure the transport of water and nutrients across roots. In: Gunning of some cells in the barley root as related to transport of sub- BES, Robards AW, eds. Intercellular communication in plants: stances. In: Kolek J, ed. Structure and function of primary root studies on plasmodesmata. Berlin: Springer-Verlag, 181±201. tissues. Bratislava: Veda, Publishing House of the Slovak Robards AW, Clarkson DT, Sanderson J. 1973. The structure of Academy of Sciences, 75±86. barley roots in relation to the transport of ions into the stele. LaÈuchli A, Kramer D, Pitman MG, LuÈttge U. 1974a. Ultra- Protoplasma 77, 291±311. structure of xylem parenchyma cells of barley roots in relation Robards AW, Jackson SM. 1976. Root structure and functionÐ to ion transport to the xylem. Planta 119, 85±99. an integrated approach. In: Sunderland N, ed. Perspectives in LaÈuchli A, Kramer D, Stelzer R. 1974b. Ultrastructure and experimental biology, Vol. 2. Botany. Oxford: Pergamon ion localization in xylem parenchyma cells of roots. In: Press, 413±422. Zimmermann U, Dainty J, eds. Membrane transport in plants. Samuels AL, Fernando M, Glass ADM. 1992. Immuno- Berlin: Springer-Verlag, 363±371. ¯uorescent localization of plasma membrane Hq-ATPase in LaÈuchli A, Spurr AR, Epstein E. 1971a. Lateral transport of ions barley roots and effects of K nutrition. Plant Physiology 99, into the xylem of corn roots. I. Kinetics and energetics. Plant 1509±1514. Physiology 48, 111±117. Sanderson WP. 1975. Ion transport through roots. In: Torrey JG, LaÈuchli A, Spurr AR, Epstein E. 1971b. Lateral transport of ions Clarkson DT, eds. The development and function of roots. into the xylem of corn roots. II. Evaluation of a stelar pump. London: Academic Press, 437±463. Plant Physiology 48, 118±124. Scott FM, Hamner KC, Baker E, Bowler E. 1956. Electron Lucas WJ. 1999. Plasmodesmata and the cell-to-cell transport microscope studies of cell wall growth in the onion root. of proteins and nucleoprotein complexes. Journal of Experi- American Journal of Botany 43, 313±324. mental Botany 50, Special issue, 979±987. Sokal RR, Rohlf FJ. 1981. Biometry, 2nd edn. San Fransisco: Ma F, Peterson CA. 2000. Plasmodesmata in onion Allium WH Freeman and Company, 321±371. cepa L.) roots: a study enabled by improved ®xation and Stadle R, Brandner J, Schulz A, Gahrtz M, Sauer N. 1995. embedding techniques. Protoplasma 211, 103±115. Phloem loading by the PmSUC2 sucrose carrier from Oparka KJ, Prior DAM, Wright KM. 1995. Symplastic Plantago major occurs into companion cells. The Plant Cell communication between primary and developing lateral roots 7, 1545±1554. Frequencies of plasmodesmata in onion roots 1061 Stadle R, Sauer N. 1996. The Arabodopsis thaliana AtSUC2 plasmalemmas lose absorption capacity during differentiation. gene is speci®cally expressed in companion cells. Botanica Planta 148, 130±137. Acta 109, 229±306. Von Guttenberg H. 1968. Der primaÈre Bau der Angio- Stelzer R, LaÈuchli A, Kramer D. 1975. Pathways of intercellular spermenwurzel. In: Linsbauer K. ed, Handbuch der chloride transport in roots of intact barley seedlings. P¯anzenanatomie, Vol. 8. Berlin: GebruÈder Borntraeger, Cytobiologie 10, 449±457. 141±159. Truernit E, Sauer N. 1995. The promoter of the Arabidopsis Walker RR, Sedgley M, Blesing MA, Douglas TJ. 1984. thaliana SUC2 sucrose-Hq symporter gene directs expressin Anatomy, ultrastructure and assimilate concentrations of of b-glucuronidase of the phloem: evidence for phloem roots of Citrus genotypes in ability for salt exclusion. Journal loading and unloading by SUC2. Planta 196, 564±570. of Experimental Botany 35, 1481±1494. Turgeon R. 1996. Phloem loading and plasmodesmata. Trends in Wang XL, McCully ME, Canny MJ. 1995. Branch roots of Plant Science 1, 418±423. Zea. V. Structural features that may in¯uence water and Tyree MT. 1970. The symplast concept: a general theory of nutrient transport. Botanica Acta 108, 209±219. symplast transport according to the thermodynamics of Warmbrodt RD. 1985a. Studies on the root of Hordeum vulgare irreversible processes. Journal of Theoretical Biology 26, L.Ðultrastructure of the seminal root with special reference 184±214. to the phloem. American Journal of Botany 72, 414±432. Vakhmistrov DB. 1981. Specialization of root tissues in Warmbrodt RD. 1985b. Studies on the root of Zea mays ion transport. In: Brouwer R, GasÏparõÂkovaÂ, O, Kolek J, L.Ðstructure of the adventitious roots with respect to phloem Loughman BC, eds. Structure and function of plant roots. unloading. Botanical Gazette 146, 169±180. The Hague: Martinus NijhoffuDr. W. Junk Publishers, Warmbrodt RD. 1986a. Structural aspects of the primary tissues 203±208. of the Cucurbita pepo L. root with special reference to the Vakhmistrov DB, Kurkova EB, Soloviev VA. 1972. Some char- phloem. NewPhytologist 102, 175±192. acteristics of plasmodesmae and lomasome-like formations Warmbrodt RD. 1986b. Solute concentrations in the phloem and in barley roots and their relation to transport of substances. associated vascular and ground issues in the root of Hordeum Fiziologiia Rastenii 19, 951±960. vulgare L. In: Cronshaw J, Lucas WJ, Giaquinta RT, eds. Van Bel AJE. 1993. Strategies of phloem loading. Annual Phloem transport. New York: Alan R. Liss, 435±444. Review ofPlant Physiology and Plant Molecular Biology Warmbrodt RD. 1987. Solute concentrations in the phloem and 44, 253±281. apex of the root of Zea mays. American Journal of Botany 74, Van Bel AJE, Kempers R. 1990. Symplastic isolation of the 394±402. sieve element±companion cell complex in the phloem Wilson AJ, Robards AW. 1980. Observation on the pattern of of Ricinus communis and Salix alba stems. Planta 183, secondary wall development in the hypodermis of onion 69±76. Allium cepa) roots. Protoplasma 104, 149±156. Van Bel AJE, Oparka KJ. 1995. On the validity of plasmo- Wright KM, Oparka KJ. 1997. Metabolic inhibitors induce desmograms. Botanica Acta 108, 174±182. symplastic movement of solutes from the transport phloem Van Bel AJE, Van Rijen HVM. 1994. Microelectrode-recorded of Arabidopsis roots. Journal of Experimental Botany 48, development of the symplasmic autonomy of the sieve 1807±1814. elementucompanion cell complex in the stem phloem of Zhu T, Lucas WJ, Rost TL. 1998. Directional cell-to-cell Lupinis luteus L. Planta 192, 165±175. communication in the Arabidopsis root apical meristem. I. Van Iren F, Boers-Van der Sluijs P. 1980. Symplasmic and An ultrastructural and functional analysis. Protoplasma 203, apoplasmic radial ion transport in plant roots: cortical 35±47.