- Email: [email protected]
Cytochemical localisation of adenosine triphosphatase activity in salt glands of Sporobolus virginicus (L.) Kunth
S. Afr. J. Bot. 1999,65(5 & 6): 370- 373
370
Cytochemical localisation of adenosine triphosphatase activity in salt glands of Sporobolus virginicus (L.) Kunth Y. Naidoo* and G. Naidoo' "Electron Microscope Un it and 1 Department of Botany, University of Durban-Westville, Private Bag X54001, Durban, 4000
Rep ublic of South Africa Received j
Ju~v
1999; re\'ised I Seplember 1999
The ultracytochernical localisation of adenosine triphosphatase activity in the bicellular salt
gland~
of the haloph ytic
grass, SporoboJus virgin/cus (L ) Kunth , was investigated using a modified lead precip itation technique . Electron dense lead deposits in the celis, following the lead phosphate precipitation, suggested the localisation of membrane proton pumps and/or membrane associated soluble phosphatase. Staining was prominent in the plasma membrane of the basal cell , especially in the suberised neck-like protrusion, and in the plasmodesmata. Staining was less prominent along the plasma membrane invaginations , at the common wall between basal and cap celis, and along the partitioning membranes lining the channels within the basal cell. The plasma membrane of th e cap cell exhibited weak staining. Control tissue, incubated in the absence of ATP , exhibited no stainin g. Evidence suggests th at ion lo ad ing into the basal cell and unloading rrom the cap cell are active processes. Keywords : adenosine triphosphatase, lead preCipitation, salt glands , Sporobolus virginicus. "To whom correspondence should be addressed . (Email: [email protected] .ac.za).
Introduction
used variat ions of the lead prec ipitation techn iq ue de ve loped by
Several plants that inhabi t saline areas possess salt glands in the leaves. The gland s are regarded as specialised epidermal cells that are ac ti vely involved in the el iminati on o f mi neral element s. Salt secreto ry ac ti vity is observed in a great variety of ha lophytes and is com l11o n in many families of an giosperms . On a structural basi s, three types of glands are recognised (Thom son 1975); the bicellular glands of the Poaceae, the bladde r cells of the Chenopod iaceae , Mesemb ryanthemaceae and Oxalidaceae and the 111111ticcl1111ar gla nds of the dicotyledons. In the Poaceae, glandular structures are usually bice llul ar, cOlllprising n basa l and cap ce ll. and are referred to as sa lt g lands . tric homes or microhai rs. Salt secretion is considered as an adaptive strategy to regu late leaf ion concentrations (Naidoo & Naidoo 1998a) and the process is thought to be active (Thom son el al. 1988). There is no agreement , however, o n th e mechanism of iOIl movement via th e glands . T he tcrm ATPase is used to describe a range of enzymes which catalyse the hyd rolysis of ATP to yield inorganic phosphate. In bot h plant (Sexton & Hall 1978) and anima l cells, this term describes a group of enzymes associated with differen t ce llular membranes and physiologica l fUllcti ons, and includes the Na--. K' -, Mg2' _ transport ATPase from ani mal plasma membra nes, the coup ling ATPase of m itochond ri a and chl o roplasts. the Ca ~ ' -ATPases assoc iated wi th the plant plasma membrane and ER. and the different H '-A TPases associated with the plant plasma membrane an d tonoplast (Sexton & Hall 1978, 199 1). There is cons iderable eviden ce that these enzymes are also involved in plant secretory processes such as salt secretion. Biochemi cal stu dies have dem onstrated that A TPase act ivity is associated with the plasma membrane, nucleus, mitochondria, chlorop lasts and Iysosomes, and involves processes such as ion transpo rt. nuc le ic acid metabolism and oxidati ve phosphory lation (Serrano 1989). The plasma membrane H ' -ATPase of pl ant cell s is a key enzyme in th e control ofa wide variety ofbiochemical reactions including the regul ation of cellular pH, solute tra nsport an d phloem loading (Serrano 1989). The importance of this enzymc has generated interest in techniques for its ultracytochemicallocalis:ltion in spec ifi c cells and tissues . Early methods
Wachstein and Meisel (1957). The va li dity of this procedure. however, has been q uestioned by several autho rs (van Steveni nck 1979; Hall el al. 1980). Criticisms of the lead-based met hod included that it may ca use inhibition of ATPase activity, cata lysis of non enzymatic hydrolysis of ATP. irregular clumpy deposits and diffusi on of the precipitate to other s ites (Chauhan el al. 1991). rn plants, however, thi s procedure is widely lI sed to localise ATPase acti vi ty in variolls tissues (Sexton & Hall 1978, 199 1), although it has been criticised on the basis that the cytochemical observations are inco nsistent w ith ill \'ill"o assays for H ' -ATPases (Katz el al. 1988). Desp ite t hese limitations, modifications of the lead prec ipitation procedure have contributed significantl y to data on A TPase activity in organelles and cells (Chauhan el al. 1991). Several studies suggest that H I-A TPase drives io n tra nsport as we ll as salt secreti o n (Atkinson e/ al. 1967; Hil l & Hill 1973) but the specific location of ion pumps in halophytes is not clear. Sporobolils l'irginiclIs (L.) Kunth, an ecologicall y important ha lophytic grass, possesses bicellu lar sa lt g lands that regu late ion leve ls in the leaves. In previous work we described the morphology and ultrastructure of the gland s of S. virginicus (Naidoo & Na idoo 1998b) and the importance of salt secretion in leaf ion balance (Nai doo & Na idoo 1998a). Our resu lts suggested that salt secretion was effective in ma intaining ion ba lance at low to moderate salinities and ineffective at high salinities (Naidoo & Naidoo 1998a). Ultrastructural studies indi cted that the basa l cell of th e salt g land possessed a comp lex system of partitioning membranes that accumu lated and directed ions through the cytoplasm via the cap cell to the exterior (Naidoo & Naidoo 1998b). The association of abundant mitoch ondria with the partitioni ng membranes suggested that ion transport via th e basa l ce ll may be an active process. In this investigation, we test this hypothesis, using a modified lead precipitation techniC]ue to locali se sites of intense H'-ATPase activi ty in the salt glands of S. virginiclfs. Th is info rmati on wo uld provide cl ues to locat ion of io n pu m ps and to possible pathways of ion transport through the salt gland cells.
S. Atr. J. Bot. 1999,65(5 & 6) Materials and Methods Plant material Plant; of S t'lrgillfcllS \"~["I': collected from the Rcac!l\vood Man~ gron:s Nature Rl.:savc. Durban. and cult ivated in a mixture of coarse sand and compos t (3: I by vo lullle). Plants wcn: irrigated daily \\ith tap miter and fatilised weekly with half-strength Hoagland's llutrient so lution (Hoagl and & Arnon 1950). Plants were wlltc-n:d \\ ilh 30% seawakr to encourage salt secretion.
Fixation and Cytochemistry ATPase activity was localised in /lag leaves (i.t:. the last fully expanded !caves) using a lead phosphate precipitation protoco l moditied li'om '-Ia ll (1971). ATPase activity in salt glands \vas demonstrated by incubating I 111m2 pieces of tissue pre-tixed on icc in 2% glutara lddl)de in a 0.0 5 tv[ sodium cacody late buffer (pH 7.2) for 2 h at 4 D C. T isslie segments \vere then \vashed for 2 h in 0.05 M sod ium ctlcodyi
371 Tris maleate bu ller 1'01' 1. 5 h after which th!.! tissue \vas post~li:xed in 2% osmium Idroxide buffered \",ith 0.05 M sodium cacody late (p i I 7.2) overnight at 4 D C. Tissue was dehydrated through a graded ethanol series and intil trated and embedded in Spurr's (1969) epoxy resin. In the contro ls. substratt: (ATP) was omitted (Hall 1971: Drennan ef al. 1992). Unstained ultrathin sections were observed and p hoto ~ graphed with a Philips 301 TEM at 60 Kv.
Energy dispersive X-ray microanalysis (EDX) Ultrath in. unstained sections cuI from leaf tissues processed for ATPase loca li sation \verc collected on copper grids. The electron dense reaction product associated with the gland cells was quaJita~ li vely analysed by EDX microanalysis using the Philips e M 120 Bio Twin at 100Kv. Spot analyses ( 100 nm) were perform ed on tht.: dectron dense deposits associated with the gland ce lls .
Results T he bicellular salt glands of S. l'irgilliclls consist of a flask~shaped basal cell embedded in the mesophyJl and a dome~shaped cap cell which protrudes beyon d the epidermis (Figure 1). The upper portion of the basal cell is constricted into
/
.-
I
E
Figure I Longitudinal section of bicellular salt gland \vith flask-shaped basal cell (8C) and dome-shaped cap cell (CC) showing intensive ATPase aeti\"ity along the plasma membrane orthe basal cell (BC, long arrows) especially in the neck-li kt.: protrusion . Note: li ttle ATPase activity associated with the plasma membrane of the cap ce ll (CC, urrO\vheads) and the plasma membrane invaginations (short arrows) at the common wall (W) between basa l and cap cells. Also note apoplastlsymp last interfact.: (ASI) at this common wall (W). Mitochondria (M) t.:;..;hibitcd no ATPase activity . Plasma membrane of t.:pidermal trichomes show some staining (E. arrow s).
S. Afr. J. Bot. 1999. 65(5 & 6)
Ph
Be ')
p
('
Ca
•
Figure 2 i nh!llsive ATPase
a neck-like protrusion. An unu sual feature of the basal cell is the partitioning of the entire cytoplasm by numerous and extensive plasma membrane invaginations that form intra-cytoplasmic
membrane-bollnd channels that are referred to as partitioning membranes (F igure I). The cap cell cytoplasm, while not parti-
tioned.
is
characterised
by
th e
prese nce
of numerous
mini-vacuoles.
w
Figure 4 X-ray ~ l1l iss ion Spcctflllll of electron (knsc reaction product along plasma mcmbranc nf the basal cell of th e gland (11' S \·irgilliclIs. J'\!r~ior peaks detected \\crc Ph and P.
In all stai ni ng proced ures. at least three m icrographs were an<1lysed from each of th ree replicate pla nt s to determine the locati on of electron dense lead deposits. The micrographs prescnted in Figures I and 2 were se lected for being the Illost representative . Electron dense le ad deposits in the cells, foll owing the lead phos phate precipitati on procedure, suggests the location of mclllbrane proton pumps andlo r me mbra ne assoc iated so luble phosphatase (Katz et at. 1988). In the salt gland cells, inte nse stain ing was prominent in the plasma membrane of the basal ce ll including the plasmodesmata (Figure 2). The stain ing, as well as plasmodesmata, were not as clear in Figure 1 as in Figure 2 because of the oblique secti on. Particul arly heavy stai ning occurred a long the plasma membrane of the basal cell. especially in the suberised neck-like protrusio n (Fi gure 1). Staining was le ss promi nent along the plasma membrane invaginations at th e COlli 111 on wall between basal and cap ce lls and along the partiti oning membranes lining the channels within the ba sal ce ll (Figures I and 3 ). Adj ace nt mi tochondria were unstained (Figure I). Whil e the plasma membrane of the basal cells was intensively stained. that of the cap cell exhibited weak staining (Figure I). Some staining was associated with the cytoplasmic matrix and the ch loroplasts o rthe mesophy ll cells (Figures 1 and ::1:). Plasma membranes of the epidermal tri c ho mes ex hibited weak stain ing (Figure 1). Cont ro l tissue, incubated in th e absence of A TP e xhi bited no staining (results not presented) . The lack of staining in the con trols suggested that the staining in the experimental material \vas associated with sites of ATPase activity (Figure 1). Spot EDX microanal yses ind icated that the electron dense sta ining deposits assoc iated wit h the plasma membranes of the sa lt gla nd cells were lead phosphate, a precipi tate which results as a con.seque nee of lead catalysed hydroly sis of A TP by aden osi ne triphosphatase (Figure 4) .
Discussion
Figure J Plasma membrane invaginations (arrows) arising from common \\illl (W) bet wee n basa l (BC) and cap cd Is. Partiti oning lllem hr
The basal cell of the salt g land in S vi/gil/iclts (Figu re 1), as well as in Sparlina fo/iosa TrilL (vnodoJl dacly/on (L.) Pers. x C transl"{I/ellsis and Disl;c'hlis .wkfa (Torr.) Rydb. (Levering & Thomson 1971 ~ Oross & Thomson 198:2) may be regarded as anal ogous to a transfer cell. characterised by numcro us plasma merilbrane invaginati o ll s that amplify surface area and facil itate ion exc hange between the apoplast and symplast (Gunning & Pate 1969. Oross & Thomson! 982). Excretory cel1s of mult ieellular glands of the dicoty ledon s such as LimOI1;lIIll I'ti/gare Hill and Aricenn;a marina (Forssk.) Vie rh also exhibit llumero us plasma membrane ingrowth s ( Hill & Hi ll 1973. Sh imony et ({/. 1973: Lultge 1975). These cOl11 mo n ultrast ruc tural fcatures suggest that the basal cell of bicellular gla nd s and the excreto ry cells
S. Atr. J. Bot. 1999,65(5 & 6) of Illu lticel lular g lands are designed to access and d irect ions from surrou nding mesop hyll and epidermal cells to the ex terior. Ultrastructural ev idence and ATPase loca lisation appear to suggest that salt loading into the basal cell from surrounding cells is ooth apoplastic and sY lll plastic. The lateral walls of the basa l cel l in the v ici nity of the 'bo ttle-neck ' are vcry th ick and suherised (Naidoo & Naidoo 1998b) so that apoplastic sa lt appears to be directed into the basal ce ll symplast pri or to secretioll . Apop lastic salt appears to enter the basal cell protop last across the apop last-symplast interface (Figure 1) formed by the partitioning membranes. Intense A TPusc activity in the plasma ll1e11l brane and the partitioning m cm branes suggest that these are sites of active io n influx into the basal cell symplas t. The nUlllcrO llS mitocho ndria, in dose proximity to the partitioni ng membranes. coul d prov ide the energy source for ion pumps (LUttge 1(71). Association of num erous mitochondria with partitioning membranes in the basal cell of salt g lands in members of the Poaccae has also been reported in .)/mrl;najo!;osa (Levering & Thomson 1971). ()-'f/odoll d(lCIY/OII and Distich!;s sp;cula (Oross & Thomson 1(82). The apparent lack of endocy tos is (vcsicles) along the parti tion ing mcmbranes in the salt g lands of ,\. l'irgil1/CItS suggests that the movement of salts into the basa l cell symplast is probably t~lCilitated by ion pumps located on these membranes. Little infor ill ation. however. is available on ion pumps and their location in sal t gland cells . The excretory ce lls ofmuticcllular glands in . J 1'icel1l1ia marillo and Lil110l1illl11 \'lllgare are similar to th e basal cell of bicellular glands in possessing an elaborate sur1:1ce area and a high mitochondrial vo lume. T hese ultrastructural adaptations suggest that ion efflux rather tha n influx is the active step in the secretory process (Hill & Hill 1973; Shi mony eI af 1973: LUnge 1(75). The localisation of ATPase suggests that ion loading into the basal cell symplast is active and leads to the maintenanc e of an electrochemical potential gradi ent from the basal cell to the exterior of tile g lan d. Ion tra nsport through the basal cell is sy mp last ic, along the channe ls formed by the plasma memb rane invag inations , poss ibly aided by energy derived from the numerous mitochondria in th is region . Subsequent ion movement to the cap cell, via the plasmodesmata, is pro bably down the electrochemical po ten tial gradient. In the cap cell, th e ions appear to be compartmentalised and transported in small vacuoles prior to el imi nation through the cut icle. Localisati on of some ATPase activi ty in the plasma membrane or the cap cell suggests that sal t unloading may be an active process. In Al'h:ellllia marina. salt unloading frolll the exc retory cells also appears to be an active process (Drennan el (If. 1(92).
In th is study, evidence is presented which suggests that ion loadin g into the symplast of the basal cells is activ e, with pumps probably located on the plasma membranes and on the i nvag i na~ lions in the neck-l ik e prot rusion of the basa l cell . T he res ults of this study support the generally ac cepted view that load ing o f ions into th e basal cell and un loadi ng fr0111 the cap cel l are active processes.
Acknowledgements Thi s research was funded by the University o f Durban- Westville
References ATKINSON. M.R.. FINDLAY, G.P .. HOPE, A.U .. PITMAN , M.G .. SADDLER. H .D.W. & WEST. K.R . 1967. Salt regu lation in mangroves RhcopllOnl /11ucyonala Lam. and Aegialitis anl1u/ala R. Br.
373 :/usl. J BIOI. SCI. 20: 589- 599. CHAUHAN. F COWAN . 0 S & H ALl .. J.t. 1991. Cytochemical
localisation of pl asmamembrane ATPase
occurrence. structure and development. l'rofOJ)/OSII/O ()X : 107- 133 . IIALL. ll.. 1971. Cytochemicallm.:alis atio!l ofATl'ase acti, it) in plant ront ceI!s. J ;\!icroscoJ~~' 93(3) : 2 J!) ·-225. HALL. J.L.. I3ROWNING. A.J . &. f IARVEY. D.M.R. 19XO. The ,alidity or the lead precipitQtioll technique fo r the localisation or ,\ I ['ase activity in p lant cells . f'rolojJllislI/t/ 1O../.: 193- 200. I IlLl.. A.E . & HILL, B.S. 1973. The clectrogenit.: ch loride plll1lp llfthe Lillloniwn salt gland. J .\lemhI' Bioi 12: 121)- I-l-J.. IIOAGLAND. D.R. & ARNON. D.I 1950. The water-culture mdhod Ilx growing plants" itllllut so il. eire ('ulit' Agne F XjJ .\1II . 3·:1/' 1-
32. KATZ. D.B.. SllSSMANN. M IL MIElUWA. IU & EV ERT. R.I . 1988. Cytochemical locahsation nf AT Pase act ivity in oat root l()cal ~ ises a plasma memhrane associated solu ble phos phatase. IwL the proton pump. Pi{/1l1 PhyslOl X6: X41-X47. LEV ERING. CA . & THOMSON. W.\V. 1971. The ultrustrllctnre nfs
:/1111
!?n'
Plant Physioi. 22: 23-4../.. LlJTTGE. U. 1975. Salt Glands . In: Inn Transport in Plants Cells and
T Issues. cds. i).A. Baker ,IIHJ .I.L. Iiall, pp. 335 - 336. Nnrth II(lHand Puh lish ing Company. Amsterdam. NAIDOO , G. & NAIDOO. Y . 1995a. SaIL tolerance in .)iJorohotll.\· ginic/ls: the importance orion relations and salt secrelion.
\'/1'-
FlOi'll 193:
337- 344. NAJDOO. Y. & NAIDOO. Ci. I 998b . !)iJOrobolus t'/rgilllcw' lear salt glands: morphology and uhrasttllClt\l'l:. ,\' ,.IF .J Bot. (,4(3 ) 198- 204. OROSS. J.W. & T HOrv1S0N, W.W. 1982. The ultrastructure or tile salt glands or (vnodoll and lJisllchlis (PoiH:eae) . :Im('/' .J HOI . ()9: 939949 . PEARSE. A.G.L 1972. Ilistochemistry. Theoretical and Applied. Yo!. 2. Churchill. London. p. 99 8. SERRANO. R. 1989. StrucLure and lilllctinn nf p lasma membrane ATPase . AliI/II. Rev. PlmJl Physw l. .\101 BlOt. 40: () 1-94. SEXTON . T. & HALL, J.1-. 1918. Enzyme c)tochem istry. In: Electron lIlicroscopy and cytochemistry o f plan t cd Is. cd. J.L. II alL All1stcr~ dam : Elsevier/Noflh Holland . pp . 63- 147. SEXTON . T. & HALL J.L. 1991. Enzyme c)lot.:hemistry. [n: Electron microscopy of plant ce lls. cds . J .1.. Iiall & C. !la\ves, Acadel ni c Press. Harcourt, London, pp. lOS- ISO. SH IMONY. c., FAliN, A. & RI IE INIIOLD. L. 1973. IJitrastructure and ion gradients in the sail gl;,mds n f ..Jv/cell/lia IJW/,///(/ (Fol'ssk.) Vierh. New Phytol 72: 27- 36. SPURR. A.R. 1969. A low-viscos!lY epoxy resin embedding medium for electron microscopy. J Ullmstruct. Res. 26: 31 --.0. THOMSON. W.W. 1975. T he structure an d function of salt glands . In: Solute transport in p lant cells and tissues. cds. R. Poljakof.. Mayher & l Gale, pp. 118- 146.
TllOMSON, W.W.. FARADAY.
e.D.
& (lROSS . .I.W. 19RR. Salt
glands. In: Solute transport in plant cells ilnd tissues. cds . D.A. Baker &. .I.L Hal l. pp. 498- 537. Longman Scie nti fie and Techn ical. Esse.\; . VAN STEVENINCK. R.F.M. 1979. The verilication of C) Lochemical tests for ATPase activity in plant cells using X-ray m icwanalysis.
Proloplas/1/a 99: 211 - 220. WA CHSTEIN . M. & MEISEL. E. 1957. Il istoei1em islly o f hepatic phosphatases al a phys iologic pll . Amer J Cfill. /'alll 27 : 13- 23 .
370
Cytochemical localisation of adenosine triphosphatase activity in salt glands of Sporobolus virginicus (L.) Kunth Y. Naidoo* and G. Naidoo' "Electron Microscope Un it and 1 Department of Botany, University of Durban-Westville, Private Bag X54001, Durban, 4000
Rep ublic of South Africa Received j
Ju~v
1999; re\'ised I Seplember 1999
The ultracytochernical localisation of adenosine triphosphatase activity in the bicellular salt
gland~
of the haloph ytic
grass, SporoboJus virgin/cus (L ) Kunth , was investigated using a modified lead precip itation technique . Electron dense lead deposits in the celis, following the lead phosphate precipitation, suggested the localisation of membrane proton pumps and/or membrane associated soluble phosphatase. Staining was prominent in the plasma membrane of the basal cell , especially in the suberised neck-like protrusion, and in the plasmodesmata. Staining was less prominent along the plasma membrane invaginations , at the common wall between basal and cap celis, and along the partitioning membranes lining the channels within the basal cell. The plasma membrane of th e cap cell exhibited weak staining. Control tissue, incubated in the absence of ATP , exhibited no stainin g. Evidence suggests th at ion lo ad ing into the basal cell and unloading rrom the cap cell are active processes. Keywords : adenosine triphosphatase, lead preCipitation, salt glands , Sporobolus virginicus. "To whom correspondence should be addressed . (Email: [email protected] .ac.za).
Introduction
used variat ions of the lead prec ipitation techn iq ue de ve loped by
Several plants that inhabi t saline areas possess salt glands in the leaves. The gland s are regarded as specialised epidermal cells that are ac ti vely involved in the el iminati on o f mi neral element s. Salt secreto ry ac ti vity is observed in a great variety of ha lophytes and is com l11o n in many families of an giosperms . On a structural basi s, three types of glands are recognised (Thom son 1975); the bicellular glands of the Poaceae, the bladde r cells of the Chenopod iaceae , Mesemb ryanthemaceae and Oxalidaceae and the 111111ticcl1111ar gla nds of the dicotyledons. In the Poaceae, glandular structures are usually bice llul ar, cOlllprising n basa l and cap ce ll. and are referred to as sa lt g lands . tric homes or microhai rs. Salt secretion is considered as an adaptive strategy to regu late leaf ion concentrations (Naidoo & Naidoo 1998a) and the process is thought to be active (Thom son el al. 1988). There is no agreement , however, o n th e mechanism of iOIl movement via th e glands . T he tcrm ATPase is used to describe a range of enzymes which catalyse the hyd rolysis of ATP to yield inorganic phosphate. In bot h plant (Sexton & Hall 1978) and anima l cells, this term describes a group of enzymes associated with differen t ce llular membranes and physiologica l fUllcti ons, and includes the Na--. K' -, Mg2' _ transport ATPase from ani mal plasma membra nes, the coup ling ATPase of m itochond ri a and chl o roplasts. the Ca ~ ' -ATPases assoc iated wi th the plant plasma membrane and ER. and the different H '-A TPases associated with the plant plasma membrane an d tonoplast (Sexton & Hall 1978, 199 1). There is cons iderable eviden ce that these enzymes are also involved in plant secretory processes such as salt secretion. Biochemi cal stu dies have dem onstrated that A TPase act ivity is associated with the plasma membrane, nucleus, mitochondria, chlorop lasts and Iysosomes, and involves processes such as ion transpo rt. nuc le ic acid metabolism and oxidati ve phosphory lation (Serrano 1989). The plasma membrane H ' -ATPase of pl ant cell s is a key enzyme in th e control ofa wide variety ofbiochemical reactions including the regul ation of cellular pH, solute tra nsport an d phloem loading (Serrano 1989). The importance of this enzymc has generated interest in techniques for its ultracytochemicallocalis:ltion in spec ifi c cells and tissues . Early methods
Wachstein and Meisel (1957). The va li dity of this procedure. however, has been q uestioned by several autho rs (van Steveni nck 1979; Hall el al. 1980). Criticisms of the lead-based met hod included that it may ca use inhibition of ATPase activity, cata lysis of non enzymatic hydrolysis of ATP. irregular clumpy deposits and diffusi on of the precipitate to other s ites (Chauhan el al. 1991). rn plants, however, thi s procedure is widely lI sed to localise ATPase acti vi ty in variolls tissues (Sexton & Hall 1978, 199 1), although it has been criticised on the basis that the cytochemical observations are inco nsistent w ith ill \'ill"o assays for H ' -ATPases (Katz el al. 1988). Desp ite t hese limitations, modifications of the lead prec ipitation procedure have contributed significantl y to data on A TPase activity in organelles and cells (Chauhan el al. 1991). Several studies suggest that H I-A TPase drives io n tra nsport as we ll as salt secreti o n (Atkinson e/ al. 1967; Hil l & Hill 1973) but the specific location of ion pumps in halophytes is not clear. Sporobolils l'irginiclIs (L.) Kunth, an ecologicall y important ha lophytic grass, possesses bicellu lar sa lt g lands that regu late ion leve ls in the leaves. In previous work we described the morphology and ultrastructure of the gland s of S. virginicus (Naidoo & Na idoo 1998b) and the importance of salt secretion in leaf ion balance (Nai doo & Na idoo 1998a). Our resu lts suggested that salt secretion was effective in ma intaining ion ba lance at low to moderate salinities and ineffective at high salinities (Naidoo & Naidoo 1998a). Ultrastructural studies indi cted that the basa l cell of th e salt g land possessed a comp lex system of partitioning membranes that accumu lated and directed ions through the cytoplasm via the cap cell to the exterior (Naidoo & Naidoo 1998b). The association of abundant mitoch ondria with the partitioni ng membranes suggested that ion transport via th e basa l ce ll may be an active process. In this investigation, we test this hypothesis, using a modified lead precipitation techniC]ue to locali se sites of intense H'-ATPase activi ty in the salt glands of S. virginiclfs. Th is info rmati on wo uld provide cl ues to locat ion of io n pu m ps and to possible pathways of ion transport through the salt gland cells.
S. Atr. J. Bot. 1999,65(5 & 6) Materials and Methods Plant material Plant; of S t'lrgillfcllS \"~["I': collected from the Rcac!l\vood Man~ gron:s Nature Rl.:savc. Durban. and cult ivated in a mixture of coarse sand and compos t (3: I by vo lullle). Plants wcn: irrigated daily \\ith tap miter and fatilised weekly with half-strength Hoagland's llutrient so lution (Hoagl and & Arnon 1950). Plants were wlltc-n:d \\ ilh 30% seawakr to encourage salt secretion.
Fixation and Cytochemistry ATPase activity was localised in /lag leaves (i.t:. the last fully expanded !caves) using a lead phosphate precipitation protoco l moditied li'om '-Ia ll (1971). ATPase activity in salt glands \vas demonstrated by incubating I 111m2 pieces of tissue pre-tixed on icc in 2% glutara lddl)de in a 0.0 5 tv[ sodium cacody late buffer (pH 7.2) for 2 h at 4 D C. T isslie segments \vere then \vashed for 2 h in 0.05 M sod ium ctlcodyi
371 Tris maleate bu ller 1'01' 1. 5 h after which th!.! tissue \vas post~li:xed in 2% osmium Idroxide buffered \",ith 0.05 M sodium cacody late (p i I 7.2) overnight at 4 D C. Tissue was dehydrated through a graded ethanol series and intil trated and embedded in Spurr's (1969) epoxy resin. In the contro ls. substratt: (ATP) was omitted (Hall 1971: Drennan ef al. 1992). Unstained ultrathin sections were observed and p hoto ~ graphed with a Philips 301 TEM at 60 Kv.
Energy dispersive X-ray microanalysis (EDX) Ultrath in. unstained sections cuI from leaf tissues processed for ATPase loca li sation \verc collected on copper grids. The electron dense reaction product associated with the gland cells was quaJita~ li vely analysed by EDX microanalysis using the Philips e M 120 Bio Twin at 100Kv. Spot analyses ( 100 nm) were perform ed on tht.: dectron dense deposits associated with the gland ce lls .
Results T he bicellular salt glands of S. l'irgilliclls consist of a flask~shaped basal cell embedded in the mesophyJl and a dome~shaped cap cell which protrudes beyon d the epidermis (Figure 1). The upper portion of the basal cell is constricted into
/
.-
I
E
Figure I Longitudinal section of bicellular salt gland \vith flask-shaped basal cell (8C) and dome-shaped cap cell (CC) showing intensive ATPase aeti\"ity along the plasma membrane orthe basal cell (BC, long arrows) especially in the neck-li kt.: protrusion . Note: li ttle ATPase activity associated with the plasma membrane of the cap ce ll (CC, urrO\vheads) and the plasma membrane invaginations (short arrows) at the common wall (W) between basa l and cap cells. Also note apoplastlsymp last interfact.: (ASI) at this common wall (W). Mitochondria (M) t.:;..;hibitcd no ATPase activity . Plasma membrane of t.:pidermal trichomes show some staining (E. arrow s).
S. Afr. J. Bot. 1999. 65(5 & 6)
Ph
Be ')
p
('
Ca
•
Figure 2 i nh!llsive ATPase
a neck-like protrusion. An unu sual feature of the basal cell is the partitioning of the entire cytoplasm by numerous and extensive plasma membrane invaginations that form intra-cytoplasmic
membrane-bollnd channels that are referred to as partitioning membranes (F igure I). The cap cell cytoplasm, while not parti-
tioned.
is
characterised
by
th e
prese nce
of numerous
mini-vacuoles.
w
Figure 4 X-ray ~ l1l iss ion Spcctflllll of electron (knsc reaction product along plasma mcmbranc nf the basal cell of th e gland (11' S \·irgilliclIs. J'\!r~ior peaks detected \\crc Ph and P.
In all stai ni ng proced ures. at least three m icrographs were an<1lysed from each of th ree replicate pla nt s to determine the locati on of electron dense lead deposits. The micrographs prescnted in Figures I and 2 were se lected for being the Illost representative . Electron dense le ad deposits in the cells, foll owing the lead phos phate precipitati on procedure, suggests the location of mclllbrane proton pumps andlo r me mbra ne assoc iated so luble phosphatase (Katz et at. 1988). In the salt gland cells, inte nse stain ing was prominent in the plasma membrane of the basal ce ll including the plasmodesmata (Figure 2). The stain ing, as well as plasmodesmata, were not as clear in Figure 1 as in Figure 2 because of the oblique secti on. Particul arly heavy stai ning occurred a long the plasma membrane of the basal cell. especially in the suberised neck-like protrusio n (Fi gure 1). Staining was le ss promi nent along the plasma membrane invaginations at th e COlli 111 on wall between basal and cap ce lls and along the partiti oning membranes lining the channels within the ba sal ce ll (Figures I and 3 ). Adj ace nt mi tochondria were unstained (Figure I). Whil e the plasma membrane of the basal cells was intensively stained. that of the cap cell exhibited weak staining (Figure I). Some staining was associated with the cytoplasmic matrix and the ch loroplasts o rthe mesophy ll cells (Figures 1 and ::1:). Plasma membranes of the epidermal tri c ho mes ex hibited weak stain ing (Figure 1). Cont ro l tissue, incubated in th e absence of A TP e xhi bited no staining (results not presented) . The lack of staining in the con trols suggested that the staining in the experimental material \vas associated with sites of ATPase activity (Figure 1). Spot EDX microanal yses ind icated that the electron dense sta ining deposits assoc iated wit h the plasma membranes of the sa lt gla nd cells were lead phosphate, a precipi tate which results as a con.seque nee of lead catalysed hydroly sis of A TP by aden osi ne triphosphatase (Figure 4) .
Discussion
Figure J Plasma membrane invaginations (arrows) arising from common \\illl (W) bet wee n basa l (BC) and cap cd Is. Partiti oning lllem hr
The basal cell of the salt g land in S vi/gil/iclts (Figu re 1), as well as in Sparlina fo/iosa TrilL (vnodoJl dacly/on (L.) Pers. x C transl"{I/ellsis and Disl;c'hlis .wkfa (Torr.) Rydb. (Levering & Thomson 1971 ~ Oross & Thomson 198:2) may be regarded as anal ogous to a transfer cell. characterised by numcro us plasma merilbrane invaginati o ll s that amplify surface area and facil itate ion exc hange between the apoplast and symplast (Gunning & Pate 1969. Oross & Thomson! 982). Excretory cel1s of mult ieellular glands of the dicoty ledon s such as LimOI1;lIIll I'ti/gare Hill and Aricenn;a marina (Forssk.) Vie rh also exhibit llumero us plasma membrane ingrowth s ( Hill & Hi ll 1973. Sh imony et ({/. 1973: Lultge 1975). These cOl11 mo n ultrast ruc tural fcatures suggest that the basal cell of bicellular gla nd s and the excreto ry cells
S. Atr. J. Bot. 1999,65(5 & 6) of Illu lticel lular g lands are designed to access and d irect ions from surrou nding mesop hyll and epidermal cells to the ex terior. Ultrastructural ev idence and ATPase loca lisation appear to suggest that salt loading into the basal cell from surrounding cells is ooth apoplastic and sY lll plastic. The lateral walls of the basa l cel l in the v ici nity of the 'bo ttle-neck ' are vcry th ick and suherised (Naidoo & Naidoo 1998b) so that apoplastic sa lt appears to be directed into the basal ce ll symplast pri or to secretioll . Apop lastic salt appears to enter the basal cell protop last across the apop last-symplast interface (Figure 1) formed by the partitioning membranes. Intense A TPusc activity in the plasma ll1e11l brane and the partitioning m cm branes suggest that these are sites of active io n influx into the basal cell symplas t. The nUlllcrO llS mitocho ndria, in dose proximity to the partitioni ng membranes. coul d prov ide the energy source for ion pumps (LUttge 1(71). Association of num erous mitochondria with partitioning membranes in the basal cell of salt g lands in members of the Poaccae has also been reported in .)/mrl;najo!;osa (Levering & Thomson 1971). ()-'f/odoll d(lCIY/OII and Distich!;s sp;cula (Oross & Thomson 1(82). The apparent lack of endocy tos is (vcsicles) along the parti tion ing mcmbranes in the salt g lands of ,\. l'irgil1/CItS suggests that the movement of salts into the basa l cell symplast is probably t~lCilitated by ion pumps located on these membranes. Little infor ill ation. however. is available on ion pumps and their location in sal t gland cells . The excretory ce lls ofmuticcllular glands in . J 1'icel1l1ia marillo and Lil110l1illl11 \'lllgare are similar to th e basal cell of bicellular glands in possessing an elaborate sur1:1ce area and a high mitochondrial vo lume. T hese ultrastructural adaptations suggest that ion efflux rather tha n influx is the active step in the secretory process (Hill & Hill 1973; Shi mony eI af 1973: LUnge 1(75). The localisation of ATPase suggests that ion loading into the basal cell symplast is active and leads to the maintenanc e of an electrochemical potential gradi ent from the basal cell to the exterior of tile g lan d. Ion tra nsport through the basal cell is sy mp last ic, along the channe ls formed by the plasma memb rane invag inations , poss ibly aided by energy derived from the numerous mitochondria in th is region . Subsequent ion movement to the cap cell, via the plasmodesmata, is pro bably down the electrochemical po ten tial gradient. In the cap cell, th e ions appear to be compartmentalised and transported in small vacuoles prior to el imi nation through the cut icle. Localisati on of some ATPase activi ty in the plasma membrane or the cap cell suggests that sal t unloading may be an active process. In Al'h:ellllia marina. salt unloading frolll the exc retory cells also appears to be an active process (Drennan el (If. 1(92).
In th is study, evidence is presented which suggests that ion loadin g into the symplast of the basal cells is activ e, with pumps probably located on the plasma membranes and on the i nvag i na~ lions in the neck-l ik e prot rusion of the basa l cell . T he res ults of this study support the generally ac cepted view that load ing o f ions into th e basal cell and un loadi ng fr0111 the cap cel l are active processes.
Acknowledgements Thi s research was funded by the University o f Durban- Westville
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