does acetobacter diazotrophicus live and move - annals of botany

Annals of Botany 80 : 147–158, 1997

Does Acetobacter diazotrophicus Live and Move in the Xylem of Sugarcane

Stems? Anatomical and Physiological Data

Z. DONG*, M. E. McCULLY† and M. J. CANNY

Department of Biology, Carleton Uni�ersity, Ottawa, Canada K1S 5B6

Received: 21 August 1996 Accepted: 5 March 1997

We have previously shown that the nitrogen-fixing endophyte of sugarcane, Acetobacter diazotrophicus lives in thesugar solution in the intercellular-space apoplast of the stem cortex. Various authors have claimed that it inhabits thexylem apoplast. This possibility has been investigated in the clone Ja 60-5 and shown to be most unlikely for thefollowing reasons : (1) an adequate carbon source is lacking in the xylem sap, and cannot be supplied from theintercellular-space apoplast of the cortex. Diffusion of solutes into and out of the vascular bundles is prevented bycomplete lignification and suberization of the bundle sheath cell walls except at the nodes. (2) Longitudinal movementof particles as large as bacteria is severely limited at the nodes. Vessel end walls were found in 90% of vessels at eachnode, and only 1% of open vessels extended through two nodes. None extended as far as three nodes. In additionto vessel end walls, vessel continuity at nodes was interrupted by living cells. Dye solution in the transpiration streamin metaxylem vessels did not pass through these living cells, but accumulated in crystals (sump formation) in thevessels below the node. Only in some protoxylem vessels and cavities did dye solution move through many nodes. Itis likely that selection of sugarcane clones such as Ja 60-5 for resistance to bacterial wilt diseases have selected forclones that have limited vessel continuity. (3) When cultured A. diazotrophicus was introduced into the transpirationstream, the xylem parenchyma reacted by secreting a bright red polymer which killed the bacteria and blocked themovement of water. We conclude that the xylem flow-apoplast of this clone of sugarcane is an unsuitable habitat forA. diazotrophicus and that additional habitats to those of the intercellular-space apoplast should be sought elsewhere.

# 1997 Annals of Botany Company

Key words : Acetobacter diazotrophicus, endophytic bacteria, nitrogen fixation, sugarcane, vessel end walls, xylemapoplast, xylem bacteria, xylem segmentation.

INTRODUCTION

A symbiotic association of sugarcane with an endophyticN

#-fixing bacterium has long been suspected, and attention

in recent years has focused on the newly-discoveredAcetobacter diazotrophicus (Gillis et al., 1989; Reis, Olivaresand Do$ bereiner, 1994), which has been shown to live in asugar solution in the intercellular-space apoplast of the stem(Dong et al., 1994; Canny, 1995). This bacterium alsopersists from one generation to the next of the vegetativelypropagated plant. Measurements of the acetylene reducingactivity of propagating stem cuttings suggested that N

#-

fixing bacteria (species then unknown) moved from sugar-cane stem cuttings to roots and the new stems (Patriquin,Gracioli and Ruschel, 1980). Also we found that theAcetobacter moves into new shoots from cuttings, becausethe bacteria can be isolated from second and third generationgreenhouse-grown plants growing in pasteurized soil (Donget al., 1994). Sugarcane is normally propagated asexuallyfrom stem cuttings (sette, or ‘seed’-pieces), each having twoor more nodes. At each node, above the leaf scar, there are30–120 root primordia and a bud. A few days after planting,the root primordia and the bud sprout (Van Dillewijin,1952). It has been suggested by some authors (James et al.,

* Present address : Department of Biology, Queens University,Kingston, Canada K7L 3N6.

† For correspondence.

1994; Reis et al., 1994) that the Acetobacter may movethrough the plant, and from one generation to the next, inthe flow-apoplast of the xylem. Sprent and James (1995)have speculated that the xylem is a likely habitat for this andother N

#-fixing bacteria.

There are several criteria that must be met if Acetobacter,or any other symbiotic bacteria, are to be able to live insugarcane in an apoplastic space and move freely into newlydeveloping stems: (1) A fixed carbon supply for the bacteria(for Acetobacter, about 10% sucrose is optimum) must bepresent in the space. (2) The space must be continuousthrough the plant and from the parent sette to the youngshoots, and permit free passage of the bacteria. If the spaceis not only the intercellular spaces of the stem parenchymawhere the Acetobacter has been found, then it must becontinuous with these spaces. (3) The bacteria must notblock the space and hinder their own movement. (4) Theplant must not react over-sensitively to the bacteria in thespace, limiting their growth, or hindering their movement.We investigated the extent to which the xylem vessels of thestems satisfied these criteria, as key tests of the hypothesis ofxylem-living A. diazotrophicus.

Fixed carbon supply

Because Acetobacter diazotrophicus shows optimal growthwith 10% sucrose, it would be expected that its natural

0305-7364}97}07014712 $25.00}0 bo970426 # 1997 Annals of Botany Company

148 Dong et al.—Does Acetobacter diazotrophicus Li�e and Mo�e in the Xylem of Sugarcane Stems?

environment should contain a solution of similar com-position. The amount of sugar present in the xylem sap ofsugarcane has been reported to range from 0 to C 9%.Hawker (1965) records finding 9% sucrose in sap blown outof the xylem vessels. Bull, Gayler and Glasziou (1972),sampling individual xylem vessels, showed that their sugarcontents varied widely from values close to the 9% foundby Hawker, down to 0±1% in functioning xylem. In contrast,the measurements of Welbaum et al. (1992) showed thatsugar was absent from the stem sap by the tenth node fromthe top. Although the vacuoles of sugarcane stem paren-chyma may contain 15% sucrose, and the intercellular-space apoplast about 11% (Dong et al., 1994), this sugar isunlikely to be available to bacteria in the xylem if the xylemis isolated from the parenchyma cells by apoplastic barriers,as has been reported by Jacobsen et al. (1992), Welbaum etal. (1992) and Moore (1995).

Thus the data available in the literature do not supportthe general presence of a sugar-rich sap in the trachearyelements to supply bacterial growth and migration through-out the stem. We estimated sugar concentrations in thexylem-vessel flow-apoplast from measurements of carbonconcentrations in situ by X-ray microanalysis of frozentissue. We also sought evidence of solute barriers around thexylem in our sugarcane clone. (For an account of theterminology used in describing the apoplastic spaces seeCanny, 1995.)

Barriers in the �essels which would hinder bacterialmo�ement

The xylem is the route for upward movement of water inplants and its conducting components in sugarcane (vessels)are suited for mass flow of liquid. Vessel lengths (thedistance between end plates) may range from less than amillimetre to many metres (Zimmermann and Jeje, 1981).At both ends of the vessel, there are end walls with pits. Thethin walls which form the floor of pits—the pit membranes—are porous to solution flow, but with pores small enough(a few nanometres in diameter) to stop the movement ofcolloidal suspensions, air bubbles and micro-organisms.Some sugarcane clones show morphological resistance tobacterial pathogen spread because comparatively few vesselsextend continuously from one internode to another (Teakle,Appleton and Steindl, 1978). These studies suggested that atleast some clones of sugarcane to not meet criterion (2)above for free movement of Acetobacter in the xylem flow-apoplast. Such barriers at nodes might be of two kinds,vessel end walls or living cells.

Water in the xylem flows from one vessel to another bycrossing pit membranes, either on side walls or on the endwalls of the vessels. The location of the end walls of vesselscan be revealed by finding where colloidal particles arestopped by them (Zimmermann and Jeje, 1981). We studiedthe location of the end walls in vessels of the stem by feedingcolloidal particles of latex paint via the transpiration stream.

In an analogous way, the places where water leaves theflow-apoplast and enters living cells of the symplast can berevealed by the ultrafiltration and accumulation of solutemolecules (Canny, 1995). Until recently it was thought that

all movement of water in the transpiration stream wasconfined to the apoplast, continuing beyond the flow-apoplast of the xylem in the porous cell-wall apoplast of leafmesophyll or other parenchyma (Strugger, 1943). However,study of the transpiration stream in leaves uncovered anassociated symplastic water path (Canny, 1990a, b, 1993,1995). A fluorescent dye (sulphorhodamine G) was used totrace the water pathway. This dye is negatively charged,travels almost as fast as the water, and is confined to theapoplast, since it does not normally cross cell plasmamembranes. The dye accumulates at places where waterenters the symplast through these cell membranes. Suchlocal high concentrations of dye were termed ‘sumps’(Canny, 1990a). The site and flux of water into the symplastis called a ‘flume’ (Canny, 1990a). The site of a flume canbe revealed by the presence of a sump, i.e. an accumulationof the tracer dye. If part of the path of the transpirationwater in sugarcane stems is through the symplasm thiswould be another place at which the bacteria would befiltered out. To detect such flumes and symplastic barrierswe used a simple method of locating sumps (O’Dowd andCanny, 1993).

Reaction of sugarcane to A. diazotrophicus in the xylem

There is a substantial literature which describes theinduction of xylem blocking responses by plants invaded bypathogens (e.g. VanderMolen, Beckman and Rodehorst,1977; Bishop and Cooper, 1984; Shi, Mueller and Beckman,1991; Purcell and Hopkins, 1996). Successful colonizationof the xylem by the A. diazotrophicus bacteria would beimpossible in the presence of such a response in thesugarcane, and we have documented the reaction ofsugarcane xylem to suspensions of A. diazotrophicusintroduced via the transpiration stream.

MATERIALS AND METHODS

Plant material

Sugarcane plants (Saccharum sp. Cuban clone Ja 60-5), sub-cultured from stem cuttings, were grown in pots in theglasshouses of Carleton University, Ottawa, under highintensity lamps which supplemented daylight. The plantsstudied were 1 year old and 2±5 to 3 m tall, with approx. 15internodes. Acetobacter diazotrophicus could be isolatedfrom the intercellular spaces of the stem parenchyma ofthese plants (Dong et al., 1995).

Anatomy and histochemistry of apoplastic barriers

Light microscopy. For general anatomical observationfresh transverse sections, cut either free-hand or with asliding microtome, of mature stems were made at themidpoint of mature internodes and at the base of the nodes.The sections were stained with 0±05% toluidine blue O(colour index 52040, Lot no. 73736, Polysciences, Inc.,Warrington, PA, U.S.A.) in benzoate buffer at pH 4±4 for30 s and then rinsed three times and mounted in tap water.

Dong et al.—Does Acetobacter diazotrophicus Li�e and Mo�e in the Xylem of Sugarcane Stems? 149

Lignified cell walls stain green to bright blue, unlignifiedwalls rich in acidic polysaccharides stain pink to red(O’Brien and McCully, 1981).

To reveal suberin deposits in cell walls two methods wereused on hand-cut sections: (a) Sections were immersed in asaturated solution of Sudan III (colour index 248,Anachemia Ltd., Montreal, Quebec, Canada) in 70%ethanol for 3–5 h. The sections were then quickly rinsedwith 70% ethanol, mounted in a drop of melted glycerinejelly and observed with bright-field optics. The expectedpositive reaction with Sudan III is an orange-pink colour(O’Brien and McCully, 1981). (b) Sections were examined intap water for autofluorescence with UV excitation. Toquench all but the suberin fluorescence, some sections werefirst stained with the periodic-acid Schiff’s reaction (PAS)(O’Brien and McCully, 1981). Sections were also stainedwith rhodamine B (colour index 45170, Lot no. I6249,Allied Chemical Co., New York, NY, USA) in 1:10000aqueous solution for 8 min, then mounted in tap water. Therhodamine B fluoresces a bright yellowish-white with UVexcitation when bound to hydrophobic compounds(Boerner, 1952).

To reveal lignin deposits in cell walls, hand-cut sectionswere mounted in a large drop of saturated aqueousphloroglucinol (1,3,5-trihydroxybenzene, Lot no. 792781,Fisher Scientific, Fair Lawn, NJ, USA) in 20% HCl. Othersections were cut from stem pieces fixed overnight in 30%ethanol, then water-rinsed. These sections were stained inSchiff’s reagent (Fisher Scientific) for 5 min. (Lignin stainsred-violet under bright-field optics, Jensen, 1962.)

To detect diffusive barriers in parenchyma cell walls, thepermeability was tested on fresh hand sections by stainingfor 5–10 min in a fresh solution of neutral red (Lot no.1B469, Chroma-Gesellschaft) 0±1% (w}v) in 0±025 potas-sium phosphate buffer, pH 7±8. Neutral red is widely usedas a vital dye, with cell vitality indicated by accumulation ofthe red dye in vacuole saps (Wenzel and McCully, 1991).However, the premise of this reaction is that the cell wallsmust be permeable to the neutral red, and living cells withsuberized walls will exclude the dye. Sections were mountedin buffer and observedwith bright-field optics. Only vacuolesof those living cells without a diffusive barrier on their wallswill stain red (Wenzel and McCully, 1991). Lignified wallsalso stain red.

Preparations were observed with bright-field, polarizing,Nomarski, or epifluorescence optics with an Olympus Vanoxsystem or a Zeiss Axiophot.

Electron microscopy

Scanning microscopy. The sizes and shapes of liquidspaces may be examined on the flat planed surfaces offrozen material as outlined by Huang et al. (1994). Stemportions of intact plants were frozen by laying the plants intheir containers horizontally and freezing a portion of thestem in a bath of liquid nitrogen. If the stems were cutbefore freezing the vessels became injected with sugarsolution released from cut parenchyma cells. The frozenstem piece was cut out under liquid nitrogen and kept underliquid N

#until small pieces (approx. 5¬2¬2 mm) were cut

from the stems and mounted on stubs with Tissue Tek andimmediately frozen in N

#-slush. The stub was transferred

under liquid N#to the chuck of a cryo-microtome (CR2000,

Research and Manufacturing, Inc., Tucson, AZ, USA) andthe sample was planed with a glass knife at ®80 °C,transferred under liquid N

#and then under vacuum to the

cold block in the cryo-preparation chamber (CT1500,Oxford Instruments, Eynsham, Oxford, UK), held at®180 °C. From there it was moved to the sample stage(®170 °C) in the column of the cryo-scanning electronmicroscope (CSEM) (JSM 6400, JEOL Ltd., Tokyo, Japan),and observed uncoated at 1 kV while the stage was warmedand the specimen was very lightly etched to reveal traces ofthe cell shapes. The temperature of the stage was set at®90 °C for the etching, and etching was stopped when thecell walls and intercellular spaces became visible. Thespecimen was transferred to the preparation chamber, givena standard coating (50 nm) of A1 (Hopkins, Jackson andOates, 1991), and returned to the sample stage at ®170 °Cfor observation. Specimens were observed in standardsecondary-electron mode at 5 to 7 kV.

Analytical scanning electron microscopy. The X-ray micro-analyser on the SEM (Link eXL, LZ-4) was used for twopurposes : (1) to distinguish living vessel elements frommature open vessels by their higher content of potassium[see e.g. McCully (1994)] ; and (2) to estimate the sugarconcentration in the xylem sap by measuring the con-centration of carbon. Details of the operation of the SEM inthe analytical mode, and of its use to estimate carboncontent may be found in Huang et al. (1994) and McCullyand Sealey (1996). Briefly, X-ray spectra were accumulatedto give a total of 80000 counts in the A1 peak of theevaporated coating. Net counts in the other elemental peakswere expressed as percentages of the A1 peak, and correctedfor the thickness of the A1 film using the live time.Calibrations were made with frozen standard solutions incarbon slurry, planed, etched and coated in the samemanner as the samples.

Measurements were made on the thirteenth to fifteenthinternode (C three above the soil) of five plants. The nodesand internodes were frozen intact, dissected into smallpieces under liquid N

#, mounted, planed and prepared for

analysis as described above.Transmission microscopy. Small longitudinal slices of

stem parenchyma were fixed and embedded by standardprocedures (3% glutaraldehyde in phosphate buffer at 4 °C,post-fixed in 1% osmium tetroxide, dehydrated in acetoneand embedded in Spurr’s resin: (see O’Brien and McCully,1981 for details).

Tracer studies to locate barriers in the flow-apoplast

Healthy plants were excised near soil level under water.The cut ends of the shoots were placed in clean water, recutone internode above, and used for the introduction of latexsuspension, dye, and bacteria (see below).

Latex paint. The continuity of unobstructed xylem lumenwas examined using a water suspension of a commercialgreen latex paint with minimal particle agglomeration,prepared by diluting the paint 200 times with water


(Zimmermann and Jeje, 1981). The suspension was left tostand for a week while large particles settled. This test wasused to find barriers to bacterial progress through the flow-apoplast.

The sugarcane plants were cut under water and transferredquickly to the supernatant from the paint suspension, thenallowed to transpire in the glasshouse. After 2–3 d, thestems were cut into segments and transported to thelaboratory. Transverse sections were made from eachinternode and observed under bright-field optics. The greenlatex particles stayed in the vessels during the sectioning andwere easily detected with bright-field optics.

Sulphorhodamine. The extent of water movement in theflow-apoplast was studied by locating sumps (red crystals)of the tracer sulphorhodamine G (SR, molecular weight553) (Lot no. 6301 Aldrich Chemical Co. Inc.) 0±05% inwater with the bright-field microscope. The smaller con-centrations of dye diffusing from the sump in the cell-wallapoplast are revealed by fluorescence optics. The transpiringshoots of sugarcane were transferred from water to the dyesolution and left standing for 20–120 min. Hand sections ofthe stem were cut in paraffin oil. This procedure prevents thesolution and dispersal of the water-soluble dye (O’Dowdand Canny, 1993). Sections were mounted under coverslipsin paraffin oil and viewed with bright-field optics for highconcentrations of SR, and with fluorescence optics underUV excitation for low concentrations.

Reaction to bacteria in the xylem

Inoculation. Acetobacter diazotrophicus (Type strain PAL-5, ATCC 49037) was inoculated on slopes of LGI medium(Dong et al., 1994). After incubation at 30 °C for 3 d, thecolonies were washed from slopes with 5% sucrose solutioninto liquid LGI medium (without agar). This bacterialsuspension was used to inoculate the xylem of sugarcane bytransferring cut shoots from water into the suspension, andletting them transpire for 1–5 d before observation. Controlplants were supplied with sterile water or sterile medium inthe same way.

Obser�ation. Hand sections were cut transversely fromfresh stems and stained with the PAS reaction (1% periodicacid for 10 min, a 5 min water rinse and 1 min in Schiff’sreagent). The rinsed sections were mounted in 1:10000aqueous DAPI (4«,6-diamidino 2-phenylindole, Lot no.80921, Polysciences, Inc., Warrington, PA, USA). Theautofluorescence of non-suberized tissue is quenched by thisPAS reaction, while the DNA of both bacteria and plantcells, and the cell walls of bacteria, fluoresce bright blue oryellow when excited by UV light (Coleman and Goff, 1985).Thin longitudinal slices of internode tissues were also fixedin 3% glutaraldehyde, post-fixed in osmium tetroxide, andembedded in Spurr’s resin (see O’Brien and McCully, 1981for details). Ultrathin sections were cut, stained in bothuranyl acetate and lead citrate and examined in a Phillips420 transmission electron microscope (TEM).

Micrographs. Black and white images in the opticalmicroscope and the SEM were recorded on T-Max 100,35 mm and 120 size films, respectively. Photomicrographs incolour were on Agfachrome CT-100, 35 mm film.

RESULTS

Apoplastic barriers around the xylem

The sugarcane stem is solid, unbranched, and roughlycircular or oval in cross-section. It is clearly differentiatedinto joints, each comprising a node and an internode. Eachstem has a hard, wax-covered rind (epidermis) surroundinga mass of softer tissue (parenchyma) which is interspersedwith vascular bundles.

Anatomy of �ascular bundles. Around 100 vascular bundlesare scattered throughout the internode, being more abun-dant towards the periphery than in the centre. Within aninternode the vascular bundles are parallel, and their numberremained constant. In contrast, in the node region there wasconsiderable branching and merging of bundles. Two to3 mm below the point of leaf sheath insertion, some bundlesbegan to branch and merge. In the node many large bundlesand bundle branches turned off the axis to supply the leafsheath as leaf traces. Immediately above this zone there wasa region of finer lateral bundles arising as branches from thevertical bundles. These fine bundles ramified through thenode and led to the root primordia.

A vascular bundle is made up of a sclerenchymatoussheath (mostly two or more layers of cells with thick,lignified walls) which encloses the xylem and phloem tissues(Figs 1A and B, 2A). All the sclerenchyma cells werelignified and suberized in the mature stem. The sclerenchymasheaths of peripheral bundles were conspicuously welldeveloped.

Electron micrographs revealed a suberized lamella de-veloped in the walls of the sclerenchymatous sheath cells(Fig. 1C, D and E). This lamella encloses each cell,producing barriers between the sheath cells as well asbetween the sheath and storage parenchyma cells (Fig. 1D).The suberized lamella of the sheath appeared to form acomplete barrier in the wall apoplast between the vascularbundle and the storage parenchyma, but numerous plasmo-desmata traversed the lamellae (Fig. 1D) providingsymplastic continuity.

The xylem comprised protoxylem, two (rarely one) verylarge diameter metaxylem vessels, and usually about fournarrow metaxylem vessels (Fig. 1A and B, 2A). Theprotoxylem, which consisted of one or more short rows ofannular and spirally thickened elements, was more extensivein the node than in the internodes. Protoxylem may beabsent in peripheral vascular bundles. In more centralbundles in mature internodes the protoxylem was anexpanded lacuna bounded by the walls of the surroundingvascular parenchyma cells (Fig. 1A and B). Close to andwithin nodes, the lacunae did not form, and the narrowprotoxylem vessels were intact with thick secondary walls(Figs 2A, 4A, B, C and G). Occasionally both intact vesselsand lacunae were present (Fig. 1A). The images of frozenfresh stem tissues showed that the protoxylem lacunae andvessels were filled with liquid (Fig. 1B). The appearance ofthis liquid, with almost no lines of sequestered solute,indicating a low concentration of solute (Canny and Huang,1993), was the same as that in the frozen metaxylemelements (Fig. 1B). All other cells in the vascular bundle


F. 1. Details of internal vascular bundles in mature internodes of sugarcane. All sections are transverse. A, The cell walls of the sclerenchymatoussheath which surrounds each bundle are strongly stained. Each bundle has two large metaxylem vessels (MX), prominent protoxylem (PX) andphloem (above and between the vessels). This bundle was sectioned close to the base of the internode and at that point had one protoxylem elementwhich retained an intact wall but the other protoxylem elements had expanded to form a cavity enclosed by parenchyma cells. Remains of asecondary wall cross the cavity. Hand-cut section of fresh material. Toluidine blue stain. ¬300. B, In this cryo-planed preparation from the middleof an internode, the protoxylem cavity is filled with frozen liquid as are the large and small metaxylem vessels. The absence of solute ridgesproduced by the freezing in the vessels and the protoxylem cavity indicates their very low solute content. Walls of the bundle sheath cells are alsostrongly contrasted by their low secondary electron emissivity. CSEM. ¬500. C, Bundle sheath cells (B) showing the thick, secondary walls, pitsand electron-dense suberized lamellae and primary walls. TEM. ¬4300. D, Pit between two bundle sheath cells showing the suberized lamellaof each cell traversed by plasmodesmata. TEM. ¬7900. E, Floor of a pit between a bundle sheath cell (right) and a lignified parenchyma celloutside the bundle sheath. In this case only the bundle sheath cell has a suberized lamella which is multilamellate here. This lamella is traversed

by the plasmodesmata but the points of traverse are partially or entirely out of the plane of the section. TEM. ¬67000.

(Fig. 1B), the storage parenchyma and the fluid in theintercellular spaces within the parenchyma (Fig. 3) showednumerous lines of sequestered solute.

In the middle of a mature internode all the metaxylemvessels were mature and functioning, since the tracer dyeand latex paint were transported through them until near


F. 2. All sections were cut from mature regions of sugarcane stems. All except B were from fresh tissue. Figs A, B, E and F are of transversesections, other figures are of longitudinal sections. A, Section of vascular bundle as in Fig. 1A but from just below a mature node (number 15from top of cane). Both large metaxylem elements are still immature with dark pink-staining cross walls. The wall in the right-hand vessel element


F. 3. Cryo-planed transverse face from an internode showing frozensolution in an intercellular space, and portions of three adjoining cells.Those portions of the cell walls that face the space were thinner and hadhigher secondary electron emissivity (indicated by their lightness) than

the rest of the walls. CSEM. ¬900.

the node (see details below). Below the node, from about5 mm beneath the leaf insertion, some metaxylem vesselelementswere still alive. Thick sections stainedwith toluidineblue showed that these vessel elements had intact cross walls(Fig. 2A) and strands of cytoplasm could be distinguishedby Nomarski optics (Fig. 2B).

Anatomy of stem parenchyma tissues. The ground tissue ormatrix of an internode was composed of parenchyma cellsin which the vascular bundles were embedded. Thisparenchyma consisted of longitudinally arranged files ofthin-walled cells, which were separated by long narrowintercellular spaces at their corners (Figs 2C, D, E and F, 3).Most of the intercellular spaces extended past many cells inlongitudinal section, and were triangular in cross section(Figs 2E and F, 3). Because the stem parenchyma cells lie inlongitudinal files, the spaces are continuous along the stem,but not across it. Each file was composed of parenchymacells of the same size. The size of the cells in a file increasedgradually toward the centre of the stem. There were somefiles of small cells among the files of large cells.

has been ruptured during cutting. The protoxylem elements have intact walls in this region. The heavily lignified walls of the bundle sheath havestained bluish-green. Toluidine blue stain. Bright-field optics. ¬260. B, Section as in Fig. 2A at the base of a similar mature node also passingthrough an immature large metaxylem element. A cytoplasmic strand (arrows) crosses the cell lumen. The lateral wall of the element is stainedpink indicating little lignification. Material fixed in formalin, then hand-sectioned and stained with toluidine blue. Nomarski optics. ¬230. C,Continuous intercellular spaces in the parenchyma of a mature internode. Walls bordering those longitudinal spaces that have been sectionedappear white. The section passes through a portion of a vascular bundle (left of micrograph) where such spaces are absent. Basic fuchsin staining.Nomarski optics. ¬100. D, Preparation and optics as in Fig. 2C but showing details of portions of two intercellular spaces here runningdiagonally across the micrograph. The surface of the walls lining the spaces is in focus all along the lower space. Small, irregularly spheroidstructures are attached to these walls. ¬800. E and F, Parenchyma cells in stem internodes. Some cells totally lack lignified walls or have thoseportions of the walls which border intercellular spaces unlignified. In E, strongly-stained walls are lignified, pale walls are not. In F, wallsfluorescing yellowish are lignified, those fluorescing blue are not. E, Basic fuchsin stain, bright-field optics. F, Rhodamine B stain, UV excitation,fluorescence optics. E, ¬225, F, ¬300. G, Parenchyma cells in older internode vitally stained with neutral red. Although all of these cells are alive,

only some of them have accumulated dye in their vacuoles, in others only the walls are stained. Bright-field optics. ¬120.

The widespread positive phloroglucinol-HCl reaction ofthe stem tissue, red staining with Schiff’s reagent (Fig. 2E),and greenish staining with toluidine blue showed that mosttissues were lignified. Most parenchyma cell walls werelignified, but some isolated parenchyma cells were totallyunlignified, or lignified only where they contacted othercells, leaving the portions facing intercellular spaces unlig-nified (Figs 2E and F, 3).

Tests for suberin with Sudan III, autofluorescence andrhodamine B showed that all storage parenchyma cells wallswere suberized except for the unlignified or partially lignifiedcells (Fig. 2F). These were either unsuberized or suberizedonly where they contacted other suberized cells, but alwaysunsuberized where they faced intercellular spaces (Fig. 2F).

Tests of cell wall permeability with neutral red revealed asimilar pattern of some permeable cells (in which neutral redwas accumulated in the vacuoles) in a matrix of impermeablecells where there was no dye accumulation. Permeable cellshad unstained walls, but cells that had no dye in thevacuoles had stained walls, indicating lignification (Fig.2G).

Confinement of apoplastic solutes. Sulphorhodamine dye,drawn into cut stems by transpiration, was confined in theinternodes to the vascular bundles, even after 2 h, with nooutward diffusion through the cell walls of the sclerenchymasheath (Fig. 4D). However in the node region, while somebundles were isolated and retained the dye, other bundleswere not isolated from parenchyma tissues, and the dyediffused out of them and along all the parenchyma walls,turning them pink in bright-field and fluorescent yellowwith UV excitation (Fig. 4E).

Solute concentrations in the apoplast and symplast. Thecarbon content of the xylem sap was estimated by X-raymicroanalysis of individual open vessels in frozenpreparations like that shown in Fig. 1B. Values of carboncontent converted to equivalent percent sucrose rangedfrom zero to 2%. In contrast, similar estimates of thevacuolar parenchyma and intercellular spaces gave values inthe ranges 17 to 35, and 12 to 30%, respectively. Somevessel elements near the node which were still alive (asdiscussed above), had higher carbon contents, the equivalentof C 15% sucrose, and also contained moderate con-centrations of potassium (C 50 m).

Vessel continuity

End walls. Two days after the sugarcane plants had beentranspiring in the green paint suspension, the plants began


F. 4. A–D and G are transverse sections, E, F and H show longitudinal sections. A–C and G are from the base of the first node above the entrypoint of the solution transpired up the xylem. E and F are from within the first node. D is from the middle of the third internode above the entrypoint. A, Acetobacter diazotrophicus bacteria within a xylem vessel 2 h after their introduction at the base of internode number 15. PAS reaction


to show water stress. Old leaves became wilted in 3 d,suggesting blockage of the water transport. The trans-piration stream carries the paint particles to the vessel ends.The water passes through, leaving the particles at increasingconcentration until the vessels are virtually packed withparticles. This filtration process finally leads to the blockageof the vessels.

In the internode containing the cut surface, all themetaxylem vessels and the protoxylem contained greenpaint particles, especially immediately below the first node;in all bundles both large metaxylem vessels which hadtraversed the cut internode were packed with the particles(Fig. 4C). In the second internode counted from the bottomcut surface (after one node), about 10% of the bundlescontained one metaxylem vessel with green paint and onemetaxylem clear. The other 90% of bundles contained twoclear vessels. No paint particles appeared in the protoxylem.After two nodes (in the third internode) about 1% of thebundles had one vessel filled with green paint.

In the node region large metaxylem vessels quite oftensplit into two or more smaller diameter vessels. Thesevessels were usually connected through perforations andgreen paint moved into all the vessel branches. However,sometimes two vessels were not connected directly but hadparenchyma cells in between, which let water pass throughfrom one vessel to another, but left green paint behind (Fig.4C).

Hand sections of plant stems fed with the suspension of A.diazotrophicus (see above) showed accumulation of thebacteria in the same places as the green paint particles. Allthe metaxylem vessels in the first internode contained manybacteria (Fig. 4A). In the second internode only 10% of thevascular bundles had one metaxylem vessel with bacteria.All the other vessels were free from bacteria.

Symplastic segmentation. Sulphorhodamine G drawnthrough the cut end of the canes was also used to determinethe extent of the flow apoplast. In both internodes andnodes, nearly all the protoxylem elements and lacunae haddye inside, even to the very top of the plant. This protoxylemwas light pink in bright field, and fluoresced faintly yellowishwith UV excitation. No sumps of accumulated dye were

followed by DAPI stain. UV-induced fluorescence. ¬300. B, Preparation similar to that in A but after 4 d. Unstained. Bright-field. ¬260. C,Vascular bundle in stem into which a solution of green latex particles had been introduced by transpiration. The section is through the base ofthe first node above the point of entry. There are four large metaxylem conduits, those with the smaller diameters are continuations of the twolarge conduits of the underlying internode. The ascent of the latex particles in these conduits has been stopped by a barrier at the node and theyhave not entered the two larger diameter conduits which originate in this region. Hand-cut section of fresh material, unstained. ¬100. D, Vascularbundle of a stem into which sulphorhodamine had been introduced by transpiration for 2 h. The dye, here fluorescing pale green, is confined tothe vascular bundle. The blue autofluorescence of the parenchyma walls is clearly distinguished from the dye fluorescence. The two large xylemelements appear to contain some dye so in this case may not have been segmented at the nodes. It is more likely, however, that the dye cameup the protoxylem cavity and spread from it into the xylem vessels by diffusion. With shorter times of exposure, the dye is usually confined tothe protoxylem cavity, or occasionally totally absent from the bundle. UV-induced fluorescence. ¬220. E, Sulphorhodamine in low concentration(yellow fluorescence) has leaked out of vascular bundles in this node and diffused within the walls of nearly all nodal parenchyma cells. UV-induced fluorescence. ¬100. F, A small vessel in the node is filled with concentrated sulphorhodamine (red). Such a concentration indicates thatthis is a sump in which dye has accumulated as water has passed out of the vessel through adjacent living cells, from which the dye was excluded.Note dye deposits in the pits through which water passed out of the vessel. UV-induced background fluorescence is mainly induced by smallconcentrations of dye which diffused through the wall apoplasts of adjoining cells. ¬150. G, Sulphorodamine transpired up the stem has reachedvery high concentration (red) in both metaxylem vessels, indicating both are dye sumps as explained in F. Low concentrations of the dye, asindicated by the UV-induced yellowish fluorescence, are present in the rest of the bundle. ¬150. H, Diagram showing how dye sumps (as seen inF and G) are formed. The dilute dye travelling upward in the transpiration stream is concentrated at the discontinuities in the xylem vessels asthe water moves across the living cells which partition the xylem conduits at the nodes. This water, now free of dye, continues as the transpiration

stream up the adjacent internode.

found in the protoxylem, but dye was absent from theprotoxylem in a few bundles. The pattern of continuity inthe metaxylem was quite different. In the first internodeabove the cut surface, all the large metaxylem vessels werepink, full of sulphorhodamine G and conducting the dyesolution. In the second internode, only about 10% of thebundles had one large vessel containing the dye. All theother metaxylem vessels were clear. After two nodes, only afew bundles contained the dye in one of their vessels. Afterthree nodes, dye solution was not found in any of themetaxylem vessels, but was present in many of theprotoxylem vessels.

Examination of the tissue just below the first node showedmost metaxylem vessels were full of red dye crystals (sumps)(Fig. 4F and G). There were about 10% of bundles havingonly one metaxylem vessel with a sump inside, and theother vessel carrying dye solution. Longitudinal sectionsshowed that at the node the metaxylem vessel elements wereshort and quite often split into several small vessels, andthere were sumps in most of these small vessels. The sumpswere first formed in the pit cavities and gradually built up inthe vessel lumina (Fig. 4F). These sumps of dye indicatethat water had passed at these sites from the flow-apoplastto the symplast in the adjacent living cells.

Reaction of the xylem to Acetobacter

After the first day following introduction of A. diazo-trophicus into the xylem, parenchyma cells around themetaxylem vessels showed signs of the synthesis andsecretion of unidentified mucilaginous material. This ma-terial was first deposited in the parenchyma cells betweenthe cell membrane and the cell wall at the xylem side (Fig.5). It gradually filled the pits and appeared to pass to thexylem lumen through the floor of the pits (Fig. 5). Thismucilage was colourless initially, but stained stronglymetachromatic pink with toluidine blue. After 3 d ofexposure to bacteria, the mucilage polymers accumulated inthe lumen and finally occluded it. The formerly colourlesspolymers in the xylem became bright red 4 d after the


F. 5. A section through a portion of a xylem parenchyma cell where itadjoins a large metaxylem vessel (X) at a pit. The vessel was filled witha suspension of A. diazotrophicus bacteria by transpiration. A depositof finely fibrillar material (M) has developed between the plasmamembrane and the thin wall which forms the floor of the pit. Thedeposit also extends under the portion of the secondary wall of thevessel shown at the left of the pit. Some strongly osmiophilic materialalso forms against the pit floor. Coarsely fibrillar, osmiophilic materialfills the pit cavity on the vessel side and surrounds the bacteria in thevessel lumen. Almost all of the bacteria have strongly-stained, distortedcontents and were probably dead. N, Nucleus of the xylem parenchyma

cell. TEM. ¬14500.

introduction of the bacteria (Fig. 4B), suggesting thepresence of condensed tannins. The red lumen contents werealso strongly osmiophilic. These characteristics suggest thatcondensed tannins were secreted subsequently into themucilage-filled cavities. The bacteria in the xylem of theinoculated plants appeared dead after 3 d, and weresurrounded by osmiophilic fibrillar material similar to thatwhich accumulated at the floor of the pits (Fig. 5). Nomucilage deposit was found in the xylem of control plantsfed with sterile water or sterile medium in the same periodof time. After a week or more the control plants showedfungal infections in their xylem and the coincident pro-duction of the red-coloured xylem occlusions.

DISCUSSION

Isolation of bundles

Since A. diazotrophicus needs a high concentration ofsucrose (Cavalcante and Do$ bereiner, 1988), we would

expect their natural habitat in a plant to contain sucrose. Insugarcane stems, diffusion of sucrose into the xylem mightbe thought likely, because the apoplastic fluid in the storageparenchyma tissue contains up to 13% (w}v) sucrose (Donget al., 1994), and is separated from the xylem by only a fewcells. Hawker (1965) reported concentrations of up to 9%sucrose in the xylem of sugarcane. Other reports haveshown that xylem sap is essentially free of sucrose (Welbaumet al., 1992), so a sucrose supply in the xylem may be, atbest, unreliable. This conclusion is reinforced by our studies.Direct measurement of carbon in the vessel sap showed thecontent was often below the limit of detection, andoccasionally up to 2% sucrose equivalent. As shownpreviously by Jacobson et al. (1992) for another dye,diffusion out of the vascular bundles is greatly restricted.The apoplastic tracer dye fed into the stem of sugarcanefrom the cut end was confined to the vascular bundles ininternodes, with no dye diffusion out to the parenchymatissues. This confinement of dye to bundles confirms for ourclone the earlier findings for other clones of sugarcane, thata diffusion barrier exists between the xylem and the storagetissue, restricting movement of solutes (including sucrose).The lignification and suberization found here in the cellwalls, and especially the suberized lamellae, would probablyprevent diffusion of sucrose from the intercellular-spaceapoplast into the bundles, just as diffusion of SR out of thebundles was prevented in the internodes. Jacobson et al.(1992) found that their dye did not diffuse from intercellularspaces of internodes into the vascular bundles. (At nodes,however, diffusive exchange of solutes with the bundles isclearly possible at least in clone Ja 60-5.) Thus bothstructural and experimental evidence suggests that ininternodes the solute traffic between the intercellular-spaceapoplast of the stem parenchyma and the flow-apoplast ofthe vascular bundles is much restricted, and most probablyconfined to the symplast.

Although in mature internodes most of the storageparenchyma cells are lignified and suberized to a significantdegree, some unsuberized regions remain. Jacobson et al.1992) also found some isolated cells were not lignified inyoung tissue. In advanced stages of development, their wallsbecome lignified where they contact other lignified cells, butremain unlignified where they face intercellular spaces.These isolated cells also remain unsuberized. It is possiblethat these cells may exchange solutes with the fluid in theintercellular-space apoplast where we have found A.diazotrophicus. They could be the source of the sucrosefound in the spaces, and might act to absorb nitrogenouscompounds produced by the Acetobacter. Thus all thestudies of the permeability of the cell-wall apoplast in theneighbourhood of the vascular bundles emphasize that thevessel lumens are less likely habitats for A. diazotrophicusthan the intercellular spaces outside the bundles.

Reaction to Acetobacter in �essels

Responses to the infection of the xylem by Acetobacterwere vigorous. Xylem parenchyma cells adjoining vesselswere stimulated to active synthesis and secretion of polymers


to such an extent that the vessels became filled with brightred gum. Such secretion of polymers from parenchyma intothe xylem vessels is a common plant response to pathogeninfection (Bishop and Cooper, 1984; Shi et al., 1991). Thesesecretions and the microbes occlude the xylem vessels andcause wilting of the plant. On the grounds of this reactionalone, symbiosis of A. diazotrophicus with sugarcane in itsxylem vessels seems impossible. Those bacterial pathogensthat do inhabit xylem [e.g. Cla�iceps xyli (Harrison andDavis, 1988) ; Herbaspirillum rubrisubalbicans (Do$ bereiner,Baldani and Reis, 1995)] have perhaps evolved some meansof suppressing the wound reaction stimulated by A.diazotrophicus. The basic and important question of howxylem-limited bacteria spread within the xylem system isunanswered (Purcell and Hopkins, 1996), but they can onlymove from one vessel to the next by destructive action, i.e.enzymatic dissolution of the vessel wall (Zimmermann,1983).

Transport of bacteria in the flow-apoplast

Our studies of the flow-apoplast have shown that it isunexpectedly complicated in Ja 60-5. The particle-transportexperiments are those which indicate how far bacteria couldspread in the xylem before being stopped by pit membranesin end or lateral walls, and revealed that this distance islimited to three internodes at most. Even movement to anadjacent internode is possible in only about one twentieth ofthe vessels. Not only can the Acetobacter not travel far inthe vessels, but if it were there in any quantity, it wouldaccumulate on end walls and restrict the flow of water.

Previous studies on anatomical resistance to movement ofsugarcane pathogens showed that resistant clones mainlyhad vessels that were discontinuous at nodes, whereassusceptible clones had many vessels that passed through thenode without interruption (Teakle, Appleton and Steindl,1978). With few continuous vessels in Ja 60-5, any bacteriathat got into the vessels of one internode have little chanceof spreading into vessels of another internode. It seemsprobable that, in selecting successful commercial clones ofsugarcane for pathogen resistance, selection may also havebeen for short lengths of continuous vessels.

Our anatomical study showed clearly that there wereintact cross walls (Fig. 2A) and protoplasts (Fig. 2B) insome of the large vessels close to the node. Theseobservations contrast with the conclusions of Teakle et al.(1978) that cross walls within the vessels were probablyabsent, although vessels were often discontinuous at nodesof cane clones resistant to stunting disease. However,Teakle et al. (1978) examined harshly macerated tissues, inwhich delicate cross walls and protoplasts would probablyhave been destroyed. In 1938 Atkinson (quoted in Teakle etal., 1978) concluded that the xylem vessels of resistantclones examined in that study did have cross walls at thenodes. Not all limitations to vessel lengths in our materialwere by end walls. In the vascular bundles, the two largevessels coming from the internode below lead to the twolarge vessels in the internode above. Many of these pairs ofvessels do not have direct contact with each other in the

node. There are parenchyma cells between the two vessels(Fig. 4C). So, the filtration process as water passes from thevessels below to the vessels above occurs, not by the pits, butby living cells. This finding was confirmed by the solute-transport experiments with SR.

Solutes moving through vessels in the transpiration streampass through end walls and pits, and the distribution of SRin the stream revealed two important new facts : the specialrole of the protoxylem, and the segmentation of themetaxylem by symplastic connections. The protoxylem wasthe only continuous path of the flow-apoplast from the baseof the stem to the leaves. In nodes it was restricted indiameter to the small protoxylem vessels, but withininternodes it widened to occupy the whole lacuna, whichwas a fully operational flow pathway, functioning as a largevessel. The protoxylem is still not a possible pathway for thespread of bacteria. There are barriers in the nodes whichstopped particle movement through it. But it is the onlylong-distance path for substances in solution that do notenter the symplast.

The large metaxylem vessels were not merely interruptedby end walls, but were divided into segments at the nodes byliving cells, through which the water had to pass beforeentering the vessels of a node above. These flumes wererevealed by the accumulation of sumps of SR (Fig. 4F, G,diagrammed in Fig. 4H). This finding of the segmentationof the xylem flow-apoplast by living cells is the first recordof the phenomenon.

The conclusion of earlier workers that A. diazotrophicuslives and moves through the cane by way of the xylemvessels has not been substantiated by convincing anatomicalevidence. The only published micrographs that we couldlocate were those of James et al. (1994) which show tissuefrom the base of the stem in a micropropagated sugarcaneplantlet that had been grown in the presence of the bacteria.Bacteria are shown (their Fig. 3A) in what is identified as astem xylem vessel. They are actually within a cell which hassculptured wall thickenings developed on only one side, aswell as an intact end wall, so it is not a vessel but is mostlikely a lignified xylem parenchyma cell which was deadprior to fixation or cut during preparation. There is, ofcourse, great difficulty in establishing definitively whetherbacteria seen in micrographs in empty spaces and cut cellswere really there in the living tissue or were moved thereduring preparation. Our present study suggests that con-vincing anatomical evidence for a viable population of A.diazotrophicus in functioning xylem vessels in sugarcane isunlikely to be found, particularly in clones which, like Ja 60-5 have xylem discontinuities.

CONCLUSIONS

All three lines of evidence point to the same conclusion, thatthe lumens of the xylem of sugarcane stems are not asuitable habitat for A. diazotrophicus as a symbiont : itwould have no ready access to the sugar in the stem apoplastas a carbon source, it would stimulate an intense hostilereaction from the plant, and its movement along the canewould be restricted to one or two internodes.


ACKNOWLEDGEMENTS

This work was supported by operating grants from theNatural Sciences and Engineering Research Council ofCanada to MEM and MJC, and a Graduate Fellowshipfrom the Province of Ontario to ZD. We thank the late AlanGibson, CSIRO, Canberra for helpful advice and dis-cussions, Ed Bruggink and Art Goodenough for growingthe sugarcane, and Wayne England for help with makingthe plates.

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