:Planta (Ber!.) 85, 11--34 (1969)

Uhrastructure and Functioning of the Transport System of the Leguminous Root Nodule J . S . PATh, B . E . S . GU~M~G a n d L. G. B ~ T Y Botany Department, Queen's University, Belfast, N.Ireland Received November 14, 1968

Summary. The structure of the vascular tissues of nitrogen-fixing nodules of 27 genera of legumes and some non-legumes has been investigated by light microscopy. Pisum and Tri/olium nodules have been examined by electron microscopy. Attention is directed to the presence of a pericycle in the vascular bundles of the nodules. In 7 of the legumes the pericycle cells possess a wall labyrinth consisting of branched filiform protuberances. The ultrastructure of the pericycle cell cytoplasm is described: its most striking feature is its abundant rough endoplasmic rcticulum. These cells surround the xylem and phloem of the bundles, and are in turn surrounded by a layer of endodermal cells with Casparian strips. The perieycle cells develop their wall labyrinth in the levels of the nodule at which the bacterial tissue becomes pigmented; in nodule senescence their cytoplasm is disrupted level with the breakdown of the bacterial tissue. A pathway for symplastic lateral transfer of assimilates exists, from the sieve elements through the pericycle, endodermis and cortex to the bacterial tissue. The apoplast within the endodermis consists largely of the pericycle wall labyrinth and the xylem. The ultrastructure of the Casparian strip resembles that of roots. Intact, detached nodules can be induced to bleed a fluid from their severed vascular tissue. This fluid is exceptionally rich in organic nitrogen, particularly amides, but does not appear to contain sugars. Comparison between its amino acid composition and that of other parts of the nodule suggests that an active uptake or secretion of nitrogenous compounds precedes export from the nodule. Special functions are suggested for the nodule endodermis and the pericycle cells in this export process. Introduction There can be few organs of higher p l a n t s more active in m e t a b o l i s m a n d t r a n s p o r t t h a n the nitrogen-fixing root nodule of the leguminous plant. E x p e r i m e n t s on n o d u l a t e d legumes reveal daffy rates of fixation of 3 0 - - 1 0 0 mg N/g fresh weight of nodules, d a t a which suggest t h a t the nodule is capable of a t u r n o v e r of some 3 - - 1 0 times its own n i t r o g e n c o n t e n t i n a d a y (PATE, 1958). This fixed n i t r o g e n does n o t a c c u m u l a t e to a n y appreciable e x t e n t w i t h i n the nodule (BOND, ]936; PATE, 1962) a n d it m a y therefore be inferred t h a t the export processes are both rapid a n d efficient, geared, i n fact, to the performance of the nitrogen-fixing tissue.

12

J. S. :PATE, :B. E. S. G u ~ I ~

and L. G. BRIA~TY:

I m p o r t of carbon m u s t be even more effective. I t has been e s t i m a t e d t h a t the e q u i v a l e n t of 3 - - 1 9 mg of c a r b o h y d r a t e m a y be respired or otherwise c o n s u m e d for each milligram of n i t r o g e n fixed b y the root nodules (see GIbSOn, 1966; BOl~D, 1968). I n addition, e x p e n d i t u r e of c a r b o n is specifically associated with export, for fixed n i t r o g e n leaves the root as amides a n d a m i n o acids (WI~m~GA a n d BAxI~VIs, 1957), with two or more carbon a t o m s accompanying" each a t o m of fixed nitrogen. Despite the obvious i m p o r t a n c e of this r a p i d exchange of organic c o m p o u n d s b e t w e e n the host p l a n t a n d its bacterial p a r t n e r , there is still r e m a r k a b l y little k n o w n a b o u t the detailed f u n c t i o n i n g of the t r a n s p o r t systems operating w i t h i n the i n d i v i d u a l nodule. The ultras t r u c t u r e of the cells of its t r a n s p o r t p a t h w a y has n o t been described, n o t h i n g is k n o w n of the c o n c e n t r a t i o n s a n d d i s t r i b u t i o n of solutes w i t h i n its tissues, a n d the cellular m e c h a n i s m s i n v o l v e d i n export a n d i m p o r t have n o t been fully investigated. This paper provides i n f o r m a t i o n r e l e v a n t to these topics.

Materials and Methods Unless otherwise stated, effective, haemoglobin-pigmented nodules were used, these being obtained from a range of legumes grown outdoors during the summer in cultural conditions deficient in nitrogen. :For light microscopy thin sections (0.5--2~) were cut from the lower, fully mature, regions of nodules fixed with acrolein and embedded in glycol methacrylate. Nodules from a total of 27 genera of legumes were examined. The sections were stained with toinidine blue 0, or with periodic acid-Schiff's reagent counterstained with the toinidine blue. The relevant techniques are described by FEDE~ and O'BRIE:N (1968). Nodules of field pea (Pisum arvense L.) and clover (Tri/olium repens L.) were investigated by electron microscopy. Intact nodules or transverse slices were fixed for 16 hours at room temperature in 2.5 % glutaraldehyde in 0.025 ~ phosphate buffer (pH 6.8), rinsed in cold buffer and post-fixed for 3 hours at 0~ in 2% osmium tetroxide in the same buffer. After dehydration in ethanol solutions the tissues were embedded in Araldite-Epon (MoLL~I~UnR, 1964). Sections were stained in uranyl acetate and lead citrate (R~NOLDS, 1963) and examined using an AEI EM6B electron microscope. The large nodules of broad bean (Vicia /aba L.) were used for examining the bleeding phenomenon of detached nodules. Nodules were detached from their parent root and immediately placed mcristem downwards with their lower half in contact with d~mp filter paper. Many nodules then commenced to exude drops of liquid from their uppermost, proximal ends, and examination under a dissecting microscope revealed that this exudation took place through vascular tissue severed at the original point of attachment to the root. It was possible to collect nodule bleeding sap in fine microcapillaries (Drummond "Microcaps" of 5 ~zlcapacity) mounted above the bleeding tissue of the nodule. Sap for analysis was collected from nodules detached at noon. That exuding during the first five minutes was discarded to minimise contamination from the contents of cells damaged on the cut surface, while that exuding over the next hour was collected. Sap volume was measured

Transport System of the Leguminous Root Nodule

13

directly in the mieroeapillaries, and the samples from some 60--100 nodules were combined and immediately frozen at --20 ~C. Nodules identical to those used in collecting bleeding sap provided material for further study of the ethanol soluble nitrogenous constituents of different regions of the nodule. Aliquots of nodule bleeding sap and of ethanolie extracts of these parts of the nodule were subjected to amino acid analysis using a Technieon autoanMyzer (see PATE, 1968). Fresh and dry weight determinations were made for the nodule parts so that their amino acid values could be expressed on the basis of tissue water content.

Results

Gross Structure of the Nodule The spatial distribution of specialized tissues of the nodule would not, at first sight, appear to be conducive to an efficient exchange of essential materiMs with the parent root. The vascular network is sparse and peripheral, its area in typical transections representing only a few per cent of the surface area of the nodule section (Fig. 1). Also, it can be estimated from published micrographs and present observations that materials synthesized in the central cells of the bacterial tissue would have to traverse distances of the order of 0.3--1.3 mm before reaching the nearest vascular tissues (see Fig. 1, and, for example, BIn~RDO~F, 1938; HARris, ALL~r and ALLEN, 1949; ALLE~ and ALLn~, 1954; A L L ~ , Gg~GORY and ALLE~, 1955). Estimates of the maximum distances of assimilate movement between mesophyll cells and the minor veins of leaves of various species of higher plants fall considerably below this range (WYLIe, 1939; EsAv, 1967). Each vascular bundle consists typically of a small number of centrifugally-oriented xylem elements and a centripetal patch of phloem (Figs. 2 and 4). Surrounding these elements is an investment of highly specialized cells with protuberant walls (Fig. 3) hence referred to as the "pericycle" of the bundle. Outside this pericycle is an endodermis with Casparian thickenings (Fig. 3). The presence of this "bundle" endodermis and, in some nodules, a further endodermis in the outer co ttexof the nodule has been previously described by several authors (e.g., Bgn~cuz~Y and THOrnTOn, 1925; DA~OEAgD, 1926; T ~ o ~ o ~ and Rvoo~F, 1936; BOND, 1948). F a A z ~ (1942) has suggested that the endodermM layers restrict the diffusion of gases and solutes into or out of the nodule.

Soluble Nitrogen o/the Nodule and its Bleeding Sap Mature nodules of Vicia ]aba L. were dissected carefully into the following three parts:

1. Bacterial Tissue. This fraction consisted of bacteroid filled cells pigmented with haemoglobin, i.e., the presumed site of nitrogen fixation in the nodule.

Figs. 1--3

J. S. PATE et al. : Transport System of the Leguminous Root Nodule

15

2. Nodule Apex. This comprised the apex of the nodule, including the nodule meristem and all immature tissues down to the level where haemoglobin pigmentation was evident. 3. Nodule Cortex and Vascular Tissues. This part consisted of the sheath of mature tissue remaining after the bacterial tissue (Part 1) and the nodule apex (Part 2) had been removed from the nodule. The purity of composition of these parts was checked by taking random samples of the three classes of tissues, embedding these in glycol methacrylate and examining sections under the light microscope. There was no evidence of contamination of the samples with tissues derived from other regions of the nodule. Amino acid analyses of the three parts of the nodule are recorded in the Table, the concentrations being expressed on the basis of water content of the tissues. The levels of the various solutes are found to be of the same order of magnitude as those of other parts of the host plant, suggesting that the nodule as a whole does not accumulate abnormally large amounts of nitrogenous solutes as a result of its fixation activities. However, the data do show that certain amino acids (e.g., aspartic acid, asparagine, glntamic acid, glutamine, glycine, alanine) are somewhat more concentrated at the site of fixation in the bacterial tissue than in surrounding parts of the nodule, suggesting that a concentration gradient of these compounds might exist between sites of synthesis and sites of export. One conspicuous difference in composition between the parts of the nodule relates to the unknown compound X, which is one of the more abundant constituents of the non-infected parts of the nodule and, indeed, of other parts of the host plant, yet is present in only minute amounts in the nodule bacterial tissue. The bleeding phenomenon of the detached nodule has several features in common with the bleeding mechanism of the decapitated root system. I t requires an external source of water, it is arrested or reversed by adding an osmotically active solution to the nodule surroundings and it exhibits a diurnal periodicity in which nodules detached during daytime bleed more profusely and over a longer period than those detached at night. Of the several occasions on which it was possible to measure rates of output from single large, healthy nodules, the maximum rate of Fig. 1. Cross section of a Tri/oIium repens L. nodule with central bacterial tissue (B) and peripheral vascular strands (arrows), one of which (asterisk) is shown at higher magnification in Fig. 2. • 60 Fig. 2. Vascular bundle stained with toluidine blue. • 700. Details: B bacterial cell, E endodermis, P pericycle, PH phloem, X xylem Fig. 3. Part of a nodule vascular strand of Tri/olium repens L. lightly stained with toluidine blue after PAS reaction. • Details: E endodermis, C Casparian strip, X xylem, P pericycle, showing wall protuberances

16

J . S, PATE et al. : Transport System of the Leguminous Root Nodule

Table, Amino acid a~alysis o~ root and nodule bleeding sap and the three parts of the

root nodules o~ broad bean (Vicia ]aba L.) ~moles amino acid/ml bleeding sap or tissue water a Root bleeding sap

Nodule bleeding sap

Nodule bacterial tissue

Nodule cortex -~ vascular tissues

Nodule apex tissues

Aspartic acid Asp~ragine Glutamie acid Glutamine Threonine Serine Glycine Alanine Valine )/Iethionine Isoleucine Leucine Xb Tyrosine Phenylalanine ;~-amino butyric acid Lysine Histidine Arginine

2.12 5.91 0.24 0.60 c c 0.05 0.05 0.06 c 0.02 0.02 0,07 c 0.01 0.01 0.05 0.03 0.06

31.82 124,40 6.76 I6,17 0,41 0,88 0.42 3.72 0.92 0.39 0.45 0.59 c 0.30 0.46 1,18 1.15 0,46 1.25

1.88 13.09 3.88 1,57 0,41 e 1.48 2.60 1.36 c 0.64 0.45 c 0,74 0.61 1.69 0,65 0.25 e

0.97 6.31 3.35 0.12 1.12 1.08 0.27 2.07 0.24 c 0.09 c 17.14 0.27 0.20 2.24 0.51 0.77 e

1.52 I0.97 1.15 0.12 0,90 0,97 0.27 0.62 0.63 c 0.29 e 7,25 0,33 0.38 0.45 0.23 0.38 0.12

Total (fzmoles/ml)

9.3

191.7

31.3

36.7

26.6

a Water is 72.6 % of fresh weight of bacterial tissue, 89.7 % of nodule meristem and 86.1% of nodule cortex + vascular tissue. b Runs as a shoulder past the peak for leucine in the separation described by PAT~ (1968). Determinations based on an assumed integration constant of 3.50. c Present in trace amounts (less than 0.002 Izmoles/ml). b l e e d i n g d u r i n g t h e first h o u r a f t e r e x c i s i o n a t m i d d a y was f o u n d t o be e q u i v a l e n b t o 120 m g sap or 4.5 m g n i t r o g e n / g n o d u l e f r e s h w e i g h t / h o u r . H o w e v e r , t h e r a t e of o u t p u t q u i c k l y d e c l i n e d a n d a n o d u l e r a r e l y b l e d for m o r e t h a n 3 h o u r s a f t e r its d e t a c h m e n t f r o m t h e r o o t . T h e T a b l e i n c l u d e s i n f o r m a t i o n on t h e a m i n o a c i d c o m p o s i t i o n of b l e e d i n g sap c o l l e c t e d f r o m n o d u l a t e d r o o t s y s t e m s a n d f r o m d e t a c h e d n o d u l e s , b o t h d e r i v e d f r o m p l a n t s i d e n t i c a l w i t h t h o s e u s e d for t h e a m i n o ~cid a n a l y s e s of t h e p a r t s of t h e n o d u l e . T h e d a t a m a y be s u m m a r i s e d as f o l l o w s : 1. T h e t o t a l c o n c e n t r a t i o n of a m i n o acids in b l e e d i n g sap f r o m det a c h e d n o d u l e s is m o r e t h a n 6 t i m e s g r e a t e r t h a n in o t h e r gross c o m p a r t m e n t s of t h e n o d u l e , s u g g e s t i n g t h a t t h e r e a r e m e c h a n i s m s w h e r e b y

:Fig. 4. Section of part of a nodule vascular strand of P i s u m arvense L., cut adjacent to ultra-thin sections used for electron microscopy. • 1300. Details: 1, 2 endodermal cells, 3 - - 9 pericycle cells, 10 and 11 sieve elements, 12 xylem. These numbers identify cells in the electron micrographs of Figs. 5, 7, 17 and 18. Note the presence of wall protuberances in all cells of the pericycle 2 Planta (]~erl.), Bd. 85

18

J.S. PAT~ et al. : Transport System of the Leguminous Root Nodule

nitrogenous components become concentrated prior to export. However, bleeding sap from nodulated root systems is some 20 times less concentrated than t h a t from individual nodules, a dilution t h a t presumably reflects the much greater absorption of water by the whole root. 2. Asparagiue carries over 70% of the nitrogen leaving the nodule. I n fact, this compound m a y be dissolved in the sap almost at the limit of its solubility, and it was frequently observed t h a t erystallisation of this amide ensued if samples of sap were cooled to 2~ I t s concentration in the sap (10(?--150 ~moles/ml) is more than twice t h a t encountered in analyses of the soluble fraction of various organs of a variety of asparagine-rich species of plants (FL:?:~, OCHOG~O~IE, WALLACE and PATE, unpublished). 3. The data suggest t h a t export from the nodule is a selective process. Certain compounds (e.g., sucrose and hexoses) are present in high coneentration in the nodule tissues but do not appear to be exported from the nodule. Others (e.g., y-amino butyric acid and compound X) are present in much lower concentration in the sap than in the nodule tissues, while, by contrast, compounds such as glutamic acid, alanine, aspartie acid and the amides glutamine and asparagine leave the nodule at higher concentrations than in the donor tissues of the nodule. This latter effect is most noticeable with the amides and aspartic acid. Specialized Tissues o/the Nodule Vascular Bundle The Endodermis. A bundle endodermis was present in each of the nodules examined (Figs. 2--4). I t s Casparian thiekenings encircle the constituent cells and are visible as green areas, suggestive of a phenolic composition, when stained with toluidine blue. Ultrastructural studies showed t h a t the endodermis is often badly preserved, with evident distortion or destruction of the tonoplast. Mitoehondria, proplastids, dietyosomes and endoplasmie retieulum are present (Figs. 5 and 6). The plasma membrane, elsewhere only loosely associated with the cell wall, is very closely appressed to the Casparian strip, and in this region the triple layering of the membrane is very conspicuous (Fig. 6). The strip itself is remarkably homogeneous in texture, perhaps reflecting the deposition of matrix materials between

Fig. 5. Casparian strip (between arrowheads) in cells 1 and 2 (Fig. 4) of the bundle endodermis. • 9,500. PL plasmodesmata Fig. 6. Detail of the edge of a Casparian strip. X 76,000. The smooth profile of the plasma membrane (PM), its conspicuous triple layering, and its tight adherence to the strip (left hand side) contrasts with the situation existing in ~he other regions of the cell (e.g., right hand side)

Figs. 5 a n d 6 -2*

Fig. 7

J. S. PA~E eta]. : Transport System of the Leguminous Root Nodule

21

the microfibrils of the cell wall. I n all these features the bundle endodermis of the nodule resembles its counterpart in the root (FALx and SITT]~, 1960; LEDBETTER, 1967; BO~ETT, 1968). The Pericycle. This tissue is 1--5 cells in thickness, each cell being slightly elongated in the direction of the axis of the bundle. Light microscopy shows that the pericycle cells possess dense contents and a prominent nucleus. I n 7 of the 27 genera examined the cells exhibited a highly protuberant wall structure (Figs. 3 and 4). The periodic acidSchiff's reaction of these wall protuberances is similar to that given by the cell wall from which they extend (Fig. 3). The distribution of wall protuberances among pericycle cells may vary slightly from one bundle to another within the same nodule, but more consistent and striking differences are encountered when nodules of different species are examined. The most usual distribution pattern in the material examined was one in which the wall protuberances were distributed uniformly throughout the perieycle, and regularly over the surface of each cell [species of Vicia, Pisum (Fig. 4), Lathyrus and Medicago]. I n such cases the perieycle cells collectively present a complete and interconnected labyrinth of wall material, this extra-cytoplasmic compartment being opposed on its inner face to the walls of xylem and phloem elements, and on its outer face to the walls of the endodermal sheath. A modification of this distribution pattern was observed in nodule bundles of species of Tri]olium and Lupinus where the wall protuberances were especially abundant in that sector of the pericycle adjacent to the nodule xylem. In one case (a nodule from Ononis repens L.) protuberances were almost entirely restricted to walls bordering xylem vessels and, in the body of the pericycle, to those corners of walls apposed to intercellular spaces. A more comprehensive study of the distribution of these specialized cells in the nodule vascular tissue is in progress. One fact already evident is that wall protuberances are not universally present in the nodule pericycle of legumes. For instance, they were not detected in effective nodules of species of Phaseolus, Thermopsis, Cytisus, Amorpha, Ulex, Genista, Spartium, Dorycnium, Lotus, Lespedeza and Coronilla. Since these nodules were shown to be active in fixation it must be concluded that the presence of the wall protuberances is not essential for the successful functioning of a root nodule. The pericycle cells of ineffective nodules

Fig. 7. Pericycle cells (3 and 4) and part of an endodermal cell (1). • 9,500. In the former wall protuberances extend to all parts of the cytoplasm. Mitoehondria (M), proplastids (PP), endoplastic retieulum (EI~), and dictyosomes (D) lie within the labyrinth. Other symbols: MV multivesiculate body, PL plasmodesmata, cell numbers as in Fig. 4

Figs. 8 and 9

J. S. PATE et al. : Transport System of the Leguminous l~oot Nodule

23

o b t a i n e d from Colutea, Baptisia, Piptanthus, Thermopsis, Sutherlandia, Pueraria, Hardenbergia; Albizzia a n d Acacia also l a c k e d wall p r o t u b e r ances. I~evertheless, t h e pericycle cells of these nodules d i d possess t h e dense c o n t e n t s shown b y t h e i r c o u n t e r p a r t s with p r o t u b e r a n t walls. Specialized cells w i t h wall p r o t u b e r a n c e s were also a b s e n t from t h e n o n - l e g u m e r o o t nodules of Alnus, Myrica a n d Hippophae a n d from t h e leaf nodules of Ardisia. E l e c t r o n m i c r o s c o p y confirmed (Fig. 7) t h a t t h e m a t u r e pericycle cells h a v e a surface to v o l u m e r a t i o t h a t is e n o r m o u s l y increased t h r o u g h t h e d e v e l o p m e n t of v e r y n u m e r o u s p r o t u b e r a n c e s on their cell walls. The p r o t u b e r a n c e s are of considerable l e n g t h a n d are t w i s t e d a n d b r a n c h e d , so t h a t a given u l t r a - t h i n section includes m a n y more profiles of a p p a r e n t l y u n a t t a c h e d p r o t u b e r a n c e s t h a n it does p o i n t s of a t t a c h m e n t to t h e p a r e n t walls. As shown in Fig. 7, t h e r a m i f i c a t i o n s of t h e wall reach to v i r t u a l l y all p a r t s of t h e c y t o p l a s m : w i t h i n t h e limits of this m i c r o g r a p h t h e surface d e n s i t y ( F n ~ E ~ a n d W~IB~n, 1967) of t h e p l a s m a m e m b r a n e is in fact nine t i m e s w h a t i t would be in t h e absence of wall p r o t u b e r ances. This wall l a b y r i n t h develops in t h e y o u n g pericycle cells of t h e apical region of t h e nodule, in step with the differentiation of t h e s u r r o u n d i n g tissues. These m o r p h o g e n e t i c processes occur in a definite sequence, which has been o b s e r v e d b y light a n d electron m i c r o s c o p y of a set of serial sections passing r i g h t t h r o u g h a nodule of Tri]olium repens L. The nodule was 2.1 m m long a n d was senescent a t its p r o x i m a l end. The s p a t i a l sequence was as follows:

Distance ]rom Apical End o] Nodule 0.25 mm first evidence of infection of bacterial tissue; 0.25--0.275 mm secondary thickening of living xylem elements commences, Casparian thiekenings first observed on endodermis, first sieve tubes with empty lumina; 0.30 mm initiation of wall protuberances on perieycle cells observed, p~rticul~rly in cells adjacent to xylem (electron microscopy); 0.35--0.40 mm wall protuberances first discernible by light microscopy; 0.475 mm mature bacteroids first observed in bacterial tissue; 0.6 mm bacterial tissue fully pigmented with haemoglobin; 0.9 mm wall labyrinth of pericycte fully developed in all pericycle cells; 1.45 mm disorganization of bacterial tissue first observed; 1.55 mm disorganization of cytoplasm of pericycle cells first apparent. Figs. 8 and 9. Components of pericycle cells. Fig. 8 • 38,000, Fig. 9 • 26,000, inset • 100,000. Details: M mitochondria, ER endoplasmic reticulum, D dictyosomes, C V coated vesicles, NE nuclear envelope. The inset shows polysomes, with ribosomaI subunits visible at arrows on a cisterna of endoplasmic reticulum. The polysome on the left is at least 5,200 A in length with a spiral of at least 25 ribosomes

J. S. PATE et al. : Transport System of the Leguminous Root Nodule

25

When the wall labyrinth is fully developed, it is clear that no parts of the cytoplasm of the mature pericycle cells are far from the involuted plasma membrane. Mitochondria and proplastids often lie in pockets of cytoplasm within the wall labyrinth (Figs. 7 and 8). Dictyosomes are not abundant, nor are they hypertrophied (Figs. 7 and 9). The most conspicuous cytoplasmic component is the endoplasmic reticulum. I t is richly clothed with chains and spirals of ribosomes (Figs. 7--9). The cisternae do not appear to be specially oriented or situated with respect to the plasma membrane. There is, however, an association between rough endoplasmic reticulum and multivesiculate bodies (Figs. 7, 11 and 18), which are common and are located at the plasma membrane, either adjacent to protuberances or to the normal cell wall. Eu (1967) has commented in some detail on the spatial relationships of these two membrane systems in the secretory cells of certain nectaries. In contrast with his findings, the multivesiculate bodies of the nodule pericycle do not appear to be especially associated with plasmodesmata. Clusters of coated vesicles are also quite common (Figs. 9 and 10). Microtubules occur in the pericycle cells. Figs. 12 and 13 show groups of these structures passing between wall protuberances, and Fig. 14 illustrates how they m a y be oriented parallel to one another. In this case the presence of plasmodesmata indicates t h a t the tubules are in the cell cortex close to the normal cell wall, rather than in inner regions of the cytoplasm. I t is perhaps not unexpected to find that the microtubules do not follow the irregular profiles of the wall protuberances.

Ultrastructure o] the Transport Pathway Solutes passing between bacterial and vascular tissues must traverse the bundle endodermis, and plasmodesmata have been seen, presumably providing a symplastic pathway all along this route. Plasmodesmatal connections between the endodermis and the cells on either side of it are shown in Figs. 5, 7, 15 and 16. The pericycle cells (Figs. 7 and 17), like the bacterial cells and the endodermal cells (Fig. 5), are interconnected, and plasmodesmata are also visible between sieve elements and pericycle cells (Fig. 17). Fig. 18 shows a typical junction between pericycle cells and a xylem element. Wall protuberances are particularly well developed on the walls contiguous with the xylem and it is clear t h a t the enlarged apoplast

Figs. 10--14. Components of pericyele cells. Fig. 10, coated vesicles; Fig. 11, multivesiculate body with associated endoplasmic reticulum (arrows). • Figs. 12--14, microtubules (arrows). • 23,000, • 66,000 and • 46,000, respectively. PL plasmodesmata

Figs. 15 and 16

J. S. PAT~.et al. : Transport System of the Leguminous Root Nodule

27

created by the walls and protuberances of the pericycle is in direct communication with the lumina of the xylem. Discussion

The fine structure of the nodule vascular tissue and its symplastic link with the bacterial cells provides a background for discussing the transport phenomena associated with functioning of the root nodule. As far as the translocation of assimilates to the nodule is concerned, there is no evidence against the existence of a conventional source-sink flow system, utilizing the sieve tubes of the nodule phloem as avenue of import and a symplastic route through pericycle, endodermis and cortex to reach sites of consumption of sugars in the bacterial tissues. Difficulties arise, however, when considering the reverse movement of produets of nitrogen assimilation from bacterial tissues to the nodule bundles, and, with present information, several interpretations of the export process are possible. The demonstration of bleeding from the cut vascular strands of detached nodules immediately suggests an osmotically-operated transport system akin to that displayed by decapitated plant roots. One widely accepted explanation of the root bleeding phenomenon is t h a t the epidermis and cortex together perform the bulk of the work of collecting ions from the external medium and the root apoplast, and t h a t these ions then diffuse inwards through the symplast of the root, along concentration gradients existing between the epidermal and cortical cells and the perieycle of the stele. Cells of the perieycle are pictured as being highly permeable to ions and as continually losing ions to the surrounding apoplast (C~A~Ts and BROYE~, 1938; A~Isz, 1956, LUTTGE and LATI]~S, 1966). This interpretation ascribes a passive role to the root endodermis, its main function being one of providing an effective physical barrier to ions diffusing between the extracytoplasmic compartments of stele and cortex and thereby allowing an osmotic flow of water into the stele from the more dilute apoplast of the root cortex. The completeness of the Casparian thickenings and the tight adherence of the plasma membranes to these thiekenings are fully compatible with the endodermis functioning as a semi-permeable investment to the stele (LEDB]~TTEIr 1967; BONNETT, 1968). Conversely, the ultrastructure of the cytoplasm of the

Fig. 15. Plasmodesmatal connections (PL) between an endodermal cell (g) and two of the nodule cortex cells separating it from the bacterial tissue. • 24,000 Fig. 16. Plasmodesmata (PL) between an endodermal call (E) and a pcricycle cell (P). X 32,000

J. S. PATE et al. : Transport System of the Leguminous Root Nodule

29

endodermal cells does not suggest that they have any major role in the uptake or secretion of solutes (BoN~TT, I968). The structural resemblances between the endodermal layers of the nodule bundles and of the root suggest corresponding functional similarities, and also that, as in the root, the export mechanism of the nodule consists basically of the maintenance of a difference in solute concentration between apoplasts of the stele and the rest of the nodule. However, the analogy with the root must not be overdrawn, since the major comloounds exported from nodules are not ions absorbed from the external medium but organic compounds of nitrogen generated internally at sites diametrically opposed to the presumed site of water absorption at the nodule surface. The most reasonable interpretation is that the nitrogenous solutes are delivered to the nodule bundle in relatively concentrated form, diffusing directly from the bacterial tissues via plasmodesmata to the surrounding cortex and thence, by a similar cytoplasmic route, through the endodermis to the perieycle cells. Such movement demands the existence of a source-sink gradient in concentration of amino compounds in the opposite direction to that postulated above for translocation of sugars to the bacterial tissues. While the amino acid analyses do indeed suggest the existence of concentration gradients from bacterial cells to the nodule cortex, the question remains of how nitrogenous solutes are selected and discharged into the bundle apoplast. Two events in the life of the pericycle cells - the development of their wall labyrinth and, later, the disorganization of their cytoplasm - - are correlated respectively with the initiation and the eventual senescence of the bacterial cells. This relationship, when considered together with the location of the perieyele ceils and their specialized cell wall, implies a speeiM function in the lateral transport of solutes. Two mechanisms may be envisaged. One, similar to that proposed for the root, would assume that the pericyele cells leak solutes passively into the apoplast of the stele, and that the wall protuberances and specialized cytoplasmic apparatus of the pericyele function predomi-

Fig. 17. Sieve elements (t0 and 11) and adjacent pericycle cells (5--7). • 5,700. P L plasmodesmata. A portion of sieve element retieulum (SER) and a mitoehonch,ion (M) are shown at higher magnification in the insets (both • 28,000). (Cell numbering as in Fig. 4) Pig. 18. Wall protuberances in the perieyele cells (8 and 9) adjacent to xylem element (12). • 17,000. Details: M V multivesieulate bodies, P L plasmodesmata. On either side of the lignified thickening (L) the matrix of the xylem element wall is hydrolyzed down to the level of the middle lamella (arrows), leaving only a network of fine fibrils F. (Cell numbering as in Fig. 4)

30

J. 8. PATE, ]3. E. S. GUNNINGand L. G. BRIARTY:

nantly in the efficient retrieval from this apoplast of substances (e.g., sugars, and certain amino acids) not destined for export in the xylem. An almost identical type of cell wall is found in the haustorial foot of mosses (MAIE~, 1967; EYMs and SUIRE, 1967) and of an Angiosperm parasite (D6Rn, 1968), in stem pith of a Gymnosperm (WooDING and NOICTtICOTE, 1965), in the epidermis of a water plant (FALK and SITT]L 1963), in plant embryo sacs (PLVlJ•, 1963; JENSEN, 1965; SC~ULZ and JENSEN, 1968; I)IBOLL, 1968) and in the phloem parenchyma of minor veins of leaves and cotyledons (GUNNING, PATE and B~IA]aTu 1968). In each of these situations an absorptive function has been suggested, the wall labyrinth being assumed to increase the efficiency of absorption, b y increasing the area of plasmalemma in contact with the fluids present in the cell walls and intercellular spaces of the tissue. The model described above, postulating a passive loss of solutes from the nodule pericycle, is, at first sight, difficult to reconcile with the fact that fluids exported from the nodule are many times more concentrated in certain amino acids than are the surrounding donor tissues. However, the tissues analysed comprise mixed populations and ages of cells and might well contain a cellular or subcellular compartment relatively small in volume, particularly rich in amino acids and directly concerned with transport. From this compartment solutes would pass to the bundle apoplast. A second mechanism m a y be proposed, fully consistent with the analytical data on amino acid export. It would contend that the dominant function of the specialized cells of the nodule pericycle is an active and selective secretion of nitrogenous compounds, particularly amides, into the bundle apoplast. I t is pictured that the pericycle cells would secrete these compounds at rates maintaining a steep concentration gradient from the bacterial tissues, and generating a rapid influx of water from the bundle surroundings. The main difficulty in accepting this mechanism is to reconcile the structural specialization of the pericycle with an active role in secretion. Some plant and animal gland cells possess rather similar f e a t u r e s - - t h e i r walls are protuberant or possess microvilli, their cytoplasm is dense, and they certainly have a secretory function [e.g., plant salt glands (ZIEGLER and LOTTGE, 1966; THONSOlV and LIU, 1967 ; SHIgONu and FAHN, 1968), other leaf glands (RENAVDIN, 1966), digestive glands of insectivorous ptants (ScH~pF, 1960, 1963; VOGEL, 1960 ; Lff$~OE, 1965 ; SCAJ~A, SCI~WAEand SIMMONS, 1968), plant nectaries (W~IscHE~, 1962; SC~NEPF, 1964; FIGIER, 1968), the tapetum of anthers (MARQUARDT,BARTtt, and ]~AHDEN, 1968) and the parietal cells of gastric mucosa of the higher animal (ItELANDEE, 1965)]. However, as with the cells of the nodule pericyelc, it is open to question whether the high surface : volume ratio contributes to the efficiency of

Transport System of the Leguminous l~oot Nodule

31

a secretory process, or whether a faculty for selective resorption (following non-selective export) is denoted (see SCH~EPF, 1964, 1965; ZIEGL~R, 1965, L/~TTGE, 1965, 1966). The absence of large populations of vesicles in the pericycle cells would suggest t h a t a n y secretory or absorptive activity t h a t t h e y might possess would involve a molecular flux across the plasma

membrane

rather than

transport

mediated

by pinocytotic

phenomena. Nevertheless, the cytoplasmic features of this ceil t y p e would suggest intense metabolic activity, and the possibility, therefore, of enzymatic transformation of the solutes traversing the cells. W h a t e v e r the mechanism of export, it is clear t h a t it involves a continued access b y the nodule to an external source of water. Since some of the solutes leave the nodule at almost the limit of their solubility, the nodule exhibits a most efficient conservation of water, b u t even under such circumstances it m u s t absorb some 20 ml of water to export a mere 0.1 g of nitrogen from its tissues. Nodules are most usually found in upper, often d r y horizons of the soil, and it is not surprising to find t h a t t h e y have been recorded to be highly sensitive to drought (WILsocq, 1942). I t m a y be t h a t if water is in short supply products of fixation m a y accumulate h~ the nodule, and this might then impair the further functioning of the nodule and might even cause it to be sloughed off the root system.

The financial support of the Agricultural and Science Research Councils is gratefully acknowledged. We thank Miss L. GRs.EX,1Vlr.T. FRASERand Mr. J. DALY for expert assistance.

References ALLEN, E.K., K.F. GREgOrY, and O.N. ALLE~: l~[orphological development of nodules on Caragana arborescens Lain. Canad. J. Bot. 38, 139--147 (1955). ALLEN, O.N., and E.K. ALLEN: Morphogenesis of the leguminous root nodule. Brookhaven Syrup. Biol. No 6, 209--234 (1954). A~Isz, W. H. : Significance of the symplasm theory for transport in the root. Protoplasma 46, 5--62 (1956). BIEBEaDO~F, :F.W. : The cytology and histology of the root nodules of some Leguminosae. J. Amer. Soc. Agron. 30, 375--389 (1938). BOND, G. : Quantitative observations on the fixation and transfer of nitrogen in the soya bean, with especial reference to the mechanism of transfer of fixed nitrogen from bacillus to host. Ann. Bot. (Lond.) 50, 559--578 (1936). -- Some biological aspects of nitrogen fixation. Symp. Long Ashton, Bristol, p. 15--25. London: Academic Press 1968. BOND, L. : Origin and developmental morphology of root nodules of Pisum sativum.

Bot. Gaz. 109, 4 1 1 ~ 3 4 (1948). BO~NETT, H. T. : The root endodermis: fine structure and function. J. Cell. Biol. 37, 199--205 (1968). B~ENC~LEV, W.E., and H.G. T~O~TON: The relation between the development, structure and functioning of the nodtfles on Vicia /c~ba, as influenced by the presence or absence of boron in the nutrient solution. Proc. roy. Soc. B 98, 373--398 (1925).

32

J. S. PATE, B. E. S. Gu~I~rG and L. G. B~naR~Y:

CRAFTS, A.S., and T.C. BROXrE~: Migration of salts and water into xylem of the roots of higher plants. Amer. J. Bet. 25, 529--535 (1938). DANGEARD, P . A . : Recherches sur les tubercles radicaux des L6gumineuses. Botaniste 16, 1--260 (1926). DIBOLL, A. G. : Fine structural development of the megagametophyte of Zea mays following fertilization. Amer. J. Bet. 55, 787--806 (1968). D6R~, I. : Feinbau der Kontakte zwischen Cuscuta-Hyphen und den SiebrShren ihrer Wirtspflanzen. Vortr. Bet., N. F. 2, 24~26 (1968). EsAv, K. : Minor veins in Beta leaves: structure related to function. Prec. Amer. phil. Soc. l l l , 219--233 (1967). EYM~, J. : Nouvelles observations sur l'infrastructure de tissus nectarigbnes floraux. Botaniste 50, 169--183 (1967). - - , and C. Sus Au sujet de l'infrastructure des cellules de la r~gion placentaire de Mnium cuspidatum Hedw. (Mousse bryale acrocarpe). C. 1~. Acad. So. (Paris) 265. 1788--1791 (1967). FALX, H., u. P. SITTE: Untersuchungen am Caspary-Streifen. Prec. Eur. Reg. Conf. on Electron Microscopy, Delft 2, 1063--1066 (1960). - - - - Zellfeinbau bei Plasmolyse I. Der Feinbau der Elodea-Blattzellen. Protoplasma (Wien) 57, 290--303 (1963). FEDE~, N., and T.P. O'B~IE~: Plant mierotechnique: some principles and new methods. Amer. J. Bet. 55, 123--144 (1968). FmIE~, J. : Localisation infrastrueturale de la phosphomonoestgrase aeide dans la stipule de Vicia/aba L. au niveau du neetaire. Planta (Berl.) 811, 60--79 (1968). FaAZE~, H.L. : The occurrence of endodermis in leguminous root nodules and its effect upon nodule function. Prec. roy. See. Edinb. B 61, 328--343 (1942). FREERE, I~.H., and E.R. WEI~EL: Stereologie techniques in microscopy. J. roy. micr. See. 87, 25--34 (1967). GIBson, A.I-I.: The carbohydrate requirements for symbiotic nitrogen fixation. A "whole plant" growth analysis approach. Aust. J. biol. Sei. 19, 499--515 (1966). G y v i N G , B.E.S., J . S . PATE, and L.G. BRIA~TY: Specialized "Transfer Cells" in minor veins of leaves and their possible significance in phloem translocation. J. Cell Biol. 117, C 7--12 (1968). tIA~RIS, J.O., E.K. ALLEN, and O.N. ALLEN: Morphological development of nodules on Sesbania grandiflora Pelt., with reference to the origin of nodule rootlets. Amer. J. Bet. 36, 651--661 (1949). HELAlgDER, H . F . : Morphology of animal secretory gland cells. In: Sekretion und Exkretion, funktionelle und morphologische Organisation der Zelle. Bd. 2, S. 2--21. Berlin-Heidelberg-New York: Springer 1965. JENSEN, W.A. : The ultrastructure and histochemistry of the synergids of cotton. Amer. J. Bet. 52, 238--256 (1965). LEDBETTER, M. G. : The Casparian strip: a site of a functional tight junction in plant roots. J. Cell Biol. 115, 79A (1967). LtiTTGE, U. : Untersuehungen zur Physiologic der Carnivoren-Dr/isen II. t~ber die Resorption verschiedener Substanzen. Planta (Berl.) 66, 331--344 (1965). -Funktion und Struktur pflanzlicher Driisen. Naturwissenschaften 511, 96--103 (1966). - - , and G.G. LATIES: Dual mechanisms of ion absorption in relation to long distance transport in plants. Plant Physiol. 41, 1531--1539 (1966). MAIER, K.: Wandlabyrinthe im Sporophyten yon Polytriehum. Planta (Berl.) 77, 108--126 (1967).

Transport System of the Leguminous Root Nodule

33

]~ARQUARDT,H., O. IV[.BARTH,and U. v. RA~DE~: Zytophotometrische und elektronenmikroskopisehe Beobachtungen fiber die Tapetumzellen in den Antheren yon Paeonia tenui]olia. Protoplasma (Wien) 65, 407--421 (1968). MOLLENHAV~R, H.H. : Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111--114 (1964). PATE, J.S.: Nodulation studies in legumes I. The synchronization of host and symbiotic development in the field pea, Pisum arvense L. Aust. J. biol. Sci. 11, 366--381 (1958). - - Root-exudation studies on the exchange of C14-13belled organic substances between the roots and shoot of the nodulated legume. Plant and Soft (Den Haag) 17, 333--356 (1962). - - Physiological aspects of inorganic and intermediate nitrogen metabolism. Syrup. Long Ashton, Bristol, p. 219--240. London: Academic Press 1968. PLVlJ•, J.E.: An electron microscopic investigation of the filiform apparatus in the embryo sac of Toreuia ]ournieri. In: Pollen physiology and fertilization, p. 8--16. Amsterdam: North Holland Publ. Co. 1963. RENAVDIN, S.: So~rles glandes de Lathraea clandestina L. Bull. See. Bet. Fr. 113, 379--385 (1966). RnYNOLDS, E.S. : The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208--212 (1963). SCXLA, J., D. SCHWAn,and E. S I ~ o ~ s : The fine structure of the digestive gland of Venus's flytrap. Amer. J. Bet. 55, 649--657 (1968). SCHN~P~, E. : Zur Feinstruktur der Drfisen yon Drosophyllum lusitanicum. Planta (Berl.) 54, 641--674 (1960). - - Zur Cytologic und Physiologie pflanzlicher Drfisen I. ~ber den Fangschleim der Insektivoren. Flora (Jena) 153, 1--22 (1963). - - Zur Cytologic und Physiologic pflanzlicher Drfisen IV. Licht- und elektronenmikroskopische Untersuchungen an Septalnektarien. Protoplasma (Wien) 58, 137--171 (1964). - - Physiologic und Morphologie sekretorischer Pflanzenzellen. In: Sekretion und Exkretion, funktionelle und morphologische Organisation der Zelle, Bd. 2, S. 72--88. Berlin-Heidelberg-New York: Springer 1965. SctruLz, R., and W.A. J~NSEN: Capsella embryogenesis: the synergids before and after fertilization. Amer. J. Bet. 55, 541--552 (1968). Sm~o~r C., and A. FAHN: Light- and electron-microscopical studies on the structure of salt glands of Tamarix aphylla L. J. Linn. Soc. (Bet.) 60, 283--287 (1968). T~o~soN, W.W., and L. LIU: Ultrastructural features of the salt gland of Tamarix aphylla L. Planta (Berl.) 73, 201--220 (1967). THORNTON,H.G., and J.E. R u D o ~ : The abnormal structure induced in nodules of Lucerne (Medicago sativa L.) by the supply of sodium nitrate to the host plant. Prec. roy. See. B 120, 240--252 (1936). VOGEL, A. : Zur Feinstruktur dcr Driisen yon Pinguicula. Beih. Z. schweiz. Forstverw. 30, 113--122 (1960). WI~INGA, K.T., and J.A. BAKn~ZS: Chromato~phy as a means of selecting effective strains of Rhizobia. Plant and Soil (Dell Haag) 8, 254 260 (1957). WILSON, J.K. : The Ioss of nodules from legume roots and its significance. J. Amer. Soc. Agron. 34, 460~--464 (1942). WOODING, F.B.P., and D.H. NORTHCOTE:An anomalous wall thickening and its possible role in the uptake of stem-fed tritiated glucose by Pinus pinea. J. Ultrastruct. Res. 12, 463--472 (1965). 8

Planta (Berl.), Bd. 85

34

J . S . PArE et al. : Transport System of the Leguminous Root Nodule

WtuSCHER, 1VI.: Elektronenmikroskopisehe Beobachtungen an extrafloralen Nektarien von Vicia/aba L. Aeta bot. Croatiea 21, 75--94 (1962). W ~ I E , R.B. : Relations between tissue organization and vein distribution in dicotyledon leaves. Amer. J. Bot. 26, 219--255 (1939). ZIEQLE~, It. : Die Physiologie pflanzlicher Drfisen. Ber. dtseh, bot. Ges. 78, 466--477 (1965). - - , u. U. LiiTTQE: Die Salzdriisen von Limonium vulgare. I. Die Feinstruktur. Planta (Berl.) 79, 193---206 (1966). Dr. JOHN S. PATE The Queens University of Belfast, Department of Botany Belfast 7, Northern Ireland