ORIGINAL ARTICLE

Development of secretory cells and crystal cellsin Eichhornia crassipes ramet shoot apex

Guo Xin Xu & Chao Tan & Xiao Jing Wei &Xiao Yan Gao & Hui Qiong Zheng

Received: 4 February 2010 /Accepted: 23 April 2010# Springer-Verlag 2010

Abstract The distribution and development of secretorycells and crystal cells in young shoot apexes of waterhyacinth were investigated through morphological andcytological analysis. The density of secretory cells andcrystal cells were high in parenchyma tissues around thevascular bundles of shoot apexes. Three developmentalstages of the secretory cells can be distinguished undertransmission electron microscopy. Firstly, a large number ofelectron-dense vesicles formed in the cytoplasm, then fusedwith the tonoplast and released into the vacuole in the formof electron-dense droplets. As these droplets fused together,a large mass of dark material completely filled the vacuole.To this end, a secretion storage vacuole (SSV) formed.Secondly, an active secretion stage accompanied withdegradation of the large electron-dense masses through anill-defined autophagic process at periphery and in thelimited internal regions of the SSV. Finally, after moststorage substances were withdrawn, the materials remainingin the spent SSV consisted of an electron-dense networkstructure. The distribution and development of crystal cellsin shoot apical tissue of water hyacinth were also studied bylight and electron microscopy. Crystals initially formed atone site in the vacuole, where tube-like membranestructures formed crystal chambers. The chamber enlargedas the crystal grew in bidirectional manner and formed

needle-shaped raphides. Most of these crystals finallyoccurred as raphide bundles, and the others appeared asblock-like rhombohedral crystals in the vacuole. Theseresults suggest that the formation of both secretory cells andcrystal cells are involved in the metamorphosis of vacuolesand a role for vacuoles in water hyacinth rapid growth andtolerance.

Keywords Water hyacinth . Shoot apex . Secretory cells .

Crystal cells . Vacuole

Introduction

Water hyacinth [Eichhornia crassipes (Martus) Solms] isone of the most productive plants in the world. The growthof this plant is indeterminate, and the major way of itsreproduction is vegetative (Spencer and Bowes 1986). Itproduces long stolons with rooted rosettes (ramets) at thenodes. Each ramet can immediately produce other stolonsfrom any axillary meristems at the basal bract, even beforethe root formation. The capacity of water hyacinth toproduce new clonal plants through stolon buds has beenreported in previous studies (Lugo et al. 1998; Kathiresan2000; Simpson and Sanderson 2002), but there are few dataavailable about the developmental anatomy of the rametswith emphasis on secretory cells and crystal cells, whichmight be important to water hyacinth for its rapid growthand adaptation to a wide range of environmental conditions.

Secretory cells, which have been found in almost allplant organs, are specialized structures filled with secretionproducts, such as tannic acid, resin, polysaccharides, pectin,salts, and calcium oxalate crystal (Ciccarelli et al. 2001;Mastroberti and Mariath 2003; Plachno and Swiatek 2008;Paiva et al. 2008, 2009). The contents of secretion

Handling Editor: Friedrich W. Bentrup

Electronic supplementary material The online version of this article(doi:10.1007/s00709-010-0157-1) contains supplementary material,which is available to authorized users.

G. X. Xu : C. Tan :X. J. Wei :X. Y. Gao :H. Q. Zheng (*)Institute of Plant Physiology and Ecology, Shanghai Institutes forBiological Sciences, Chinese Academy of Sciences,300 Fenglin Road,Shanghai 200032, Chinae-mail: hqzheng@sippe.ac.cn

ProtoplasmaDOI 10.1007/s00709-010-0157-1

substances varied with the species. The functions ofsecretory cells are related to the secretion products. Forexamples, the secretion products of floral nectars containsugar, which can attract insects to visit and pollinate theflowers. Mastroberti and Mariath (2008) suggested that arole of the secretory cells (also called mucilage cells) inyoung leaves of Araucaria angustifolia could be intranslocation and water storage of solute. The depositionof secretion was observed at interface between theplasmalemma and the cell wall (Klein et al. 2004; Paivaet al. 2008), in the reduced portion of cytoplasm(Mastroberti and Mariath 2008), or the central vacuole(Fahn and Shimony 1998). Endoplasmic reticulum (ER)has been considered to play an important role in thedevelopment of secretory cells. According to Benayoun andFahn (1979), ER produces secretion substances and trans-ports them out of cells. Langenheim (2003) also reportedthe central role the ER played in transporting terpenoidresin components from intracellular synthesis sites into thelumen of an endogenous secretory structure. They assumedthat ER could fuse with membranes of other organelles andform vesicles that move through the cell to vacuoles. Forexample, plastids are considered as the site whereterpenoid resin are synthesized (Dell and McComb 1978;Lichtenthaler et al. 1997) and fuse with ER to export theproduction to vacuoles (Dell and McComb 1978; Fahn1988; Carmello et al. 1995). Vacuoles are importantintracellular endpoints of secretion pathways in plants, butmechanism of the transformation of vacuoles of secretorycells is unknown.

Ca oxalate (CaOx) crystal is one of the most commonstorage materials that can be found in most tissues andorgans of photosynthetic plants, such as Pistia stratiotes,Morus alba, and grapevine, as an intra- or extracellulardeposit (Franceschi et al. 1993; Katayama et al. 2007;Storey et al. 2003; Li et al. 2003). Intracellular crystalsoften occurred within vacuoles of cells specialized forcrystal formation, called crystal idioblasts (Franceschi andNakata 2005). Studies on the CaOx crystal idioblastformation in developing tissues suggested that a primaryfunction of crystal cells may be to serve as a stronglocalized Ca sink to reduce the apoplastic Ca concentrationaround adjacent cells, allowing them to develop normally(Franceschi and Nakata 2005). The crystal cells were alsosuggested to work as sinks for deposition and compartmentof toxic metals to reduce physiological damage (Franceschiand Nakata 2005). Water hyacinth can absorb and accumu-late large amounts of toxic substances such as heavy metalions and pollutants from water without damage (Mahamadiand Nharingo 2010; Agunbiade et al. 2009; Rajan et al.2008; EI-Khaiary 2007). CaOx crystal, which binds heavyions such as Cd and Pb, was also observed in waterhyacinth leaf cells (Mazen and Maghraby 1997), but little

information is available for the formation of CaOxidioblasts in water hyacinth. In the present study, theprocess of vacuole metamorphosis during development ofboth secretory cells and crystal cells in water hyacinth shootapexes was examined, and its possible relevance to waterhyacinth tolerance is discussed.

Materials and methods

Plant material collection and culture

Water hyacinth (E. crassipes Solms) plants were collectedfrom a river at Jiading in Shanghai. The plant sampleswere inserted in an upright position in plastic boxes of40×40×40 cm3 containing 50 L Hoagland nutrient culturesolution (Hoagland and Arnon 1938) and grew in agreenhouse (25–30°C) as described by Zheng et al.(2006). Light intensity was about 120 µmol m−2s−1, andthe period was 16 h light/8 h dark.

Transmission electron microscopy

Water hyacinth rosette shoot apexes were cut into smallblocks (about 2×2×2 mm3) and fixed for 12 h at 4°C in 2%glutaradehyde and 2.5% paraformaldehyde in 50 mMPIPES buffer (pH 7.2), washed by 50 mM PIPES buffer(pH 7.2), then incubated in 2% osmium tetroxide in 50 mMPIPES buffer (pH 7.2) for 2 h at room temperature. Afterbeing washed by 50 mM PIPES buffer (pH 7.2) anddehydrated with an ethanol series, the samples wereembedded in Epon 812 resin. The specimens weresectioned with a diamond knife, and the thick sections(5 µm)were stained with 0.1% toluidine blue and examinedunder light microscopy (Leica DMLB).The ultra-thinsections (about 100 nm) were stained with 1% (w/v) uranylacetate (aqueous) and 1.5% (w/v) alkaline lead citrate(aqueous) and examined with a Hitachi 7650 TEM.

Light microscopy

Freehand cross sections were cut from fresh rosette shootapexes and put in a glass plate with distilled water. Thethinner sections (about 70 µm thick) were selected andexamined under a light microscopy (Leica DMLB).

Isolation of crystal protoplasts

Isolation of crystal protoplasts was according to the methoddescribed by Franceschi et al. (1993).Water hyacinth rosetteshoot apexes were cut into small blocks (6∼8 mm3) andhomogenized in four volumes of water. The homogenatewas filtered with 200- and then 70-µm nylon nets and

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rinsed with distilled water twice, and then the isolatedcrystal protoplasts were collected and examined under lightmicrocopy (Leica DMLB).

Results

Development of secretory cells in water hyacinthshoot apexes

Water hyacinth produces long stolons with rooted rosettes(ramets) at their nodes. Each rosette shoot has leaves at theapex and adventitious roots at the node end (Fig. 1a, b).Serial sections through a rosette shoot apex showed threedistinguished tissues including epidermis, cortex, andvascular cylinder. A number of vascular bundles weredistributed in the parenchyma tissue of the vascularcylinder (Fig. 1b, c). The tissues in the rosette shoot apicalarea displayed a glorious pink color (Fig. 1b), which wasdue to a lot of pink secretory cells dotted among theparenchymal tissue in vascular cylinder (Fig. 1d–f). Thedistribution density of secretory cells apparently decreasedat rooted end of the shoot (Fig. 1b). The secretory cells inshoot apical tissues displayed a heavy staining black colorin glutaraldehyde-osmium fixed sections and were distrib-uted around vascular bundles (Fig. 2a, b), corresponding tothe observation of secretory cells in the fresh sections(Fig. 1d–f). In addition, most of secretory cells wereadjacent to xylem cells of vascular bundles (Fig. 2c) orcrystal cells (Fig. 2d), which we will discuss later.

The results from the electron microscopy showed that alarge number of electron-dense vesicles formed in thecytoplasm of developing secretory cells (Fig. 3a, b). Thesedense vesicles then fused to tonoplast (Fig. 3c) anddeposited the storage materials into the vacuole, where alarge number of electron-dense droplets were presented(Fig. 3d). As development of the cells, these dark dropletsgradually filled the vacuole (Fig. 3e) and merged togetherto form a huge secretion storage vacuole (SSV), whichoccupied almost the whole cell (Fig. 3f). At this stage, a lotof mitochondria in the cytoplasm (Fig. 4a) and many smallgloboids containing heavy staining electron-dense substan-ces in the vacuole matrix were observed (Fig. 4b). Severalelectron-dense areas appeared at the periphery of the SSVand the degradation began at those areas (Fig. 4b) with anill-defined autophagic process (Fig. 4c, d), where theelectron-dense vesicles were released to cytoplasm(Figs. 4d and 5a). In the same time, the breakdown contentsin a limited region of globoids led to form a large numberof small electron-transparent holes in the SSV matrix(Fig. 5b, c).The volume of these holes increased withsecretory cell senescence, and the materials remaining inthe spent matrix of the SSV consisted of an electron-dense

network structure (Fig. 5d). Finally, after the SSVs wereexhausted, the cells lost the ability of secretion.

Development of crystal cells in water hyacinth apexes

Figure 6a showed that there were a large number of crystalcells in the water hyacinth apical tissues. The distributionpattern of crystal cells is similar to that of the secretorycells, which we have described above (Fig. 1d). Bothcrystal and secretory cells occur in high frequency in theparenchyma tissue around vascular bundles in the vascularcylinder of the rosette shoot (Fig. 2a, b). The crystal cellsare apparently larger than its neighboring parenchyma cellsand secretory cells (Fig. 6b). The morphology of crystalsdetected in isolated protoplasts of water hyacinth apexes israphide bundles (Fig. 6c) or block-like rhombohedra(Fig. 6d). Most of crystals in the cells of this tissue wereraphide bundles, while few of them were block-likerhombohedra (Fig. 6d).

Crystals appeared first in one site of the central vacuole(Fig. 7a, d) and then increased by adding new growingcrystals at the peripheral region of the crystal forming area(Fig. 7b, e). As the crystal cell developed into the maturestage, the number of crystals in the vacuole extremelyincreased and finally filled the whole vacuole (Fig. 7c, f).When the developing crystal cells were examined undertransmission electron microscopy, a large amount of ERsurrounded by vesicles throughout cytoplasm (Fig. 8a) andmany tube-like structures, measuring 8–10 nm in diameter,in the matrix of the vacuole were observed (Figs. 8b, c and9a, b). These tube-like structures might function aschambers of crystals (Fig. 8c). The volume of the smalltube-like structure obviously increased when the crystalwere initiated in its lumen (Fig. 8d, e). The chamber grewas the crystal grew in bidirectional manner and the shape ofchamber changed from oval at the beginning (Fig. 8e, f) torhombic at the mature stage (Fig. 8g, h). The size of amature crystal cell is usually three to four times larger inlength than that of parenchyma cells or secretory cellsaround it (Fig. 7c).

Discussion

The growth rate of water hyacinth seems to exceed that ofany other aquatic or cultivated plant because its shoot has astrong ability to produce new clonal plants (Gopal 1987). Inaddition, it can tolerate relatively large amounts of toxicsubstances, such as heavy metals in aqueous environment.A detailed investigation of the water hyacinth shoot apexdevelopment is needed to fully elucidate its abilities ofrapid reproduction and toxic substance tolerance. Theexperiments in this study have focused on the development

Development of secretory cells and crystal cells in Eichhornia crassipes ramet shoot apex

Fig. 1 Developmental anatomy of water hyacinth shoot and thedistribution patterns of secretory cells in a shoot apex. a A stolon bud.b A longitudinal section through the shoot apex of a stolon bud. Notethe apical area in pink color (arrow points). c A cross section throughthe shoot apex. d–f Optical micrographs of freehand sections through

the shoot apex. e, f Enlarged view of the shoot apical tissues. SHshoot, AR adventitious root, SS stolon shoot, SC secretory cell, Crcrystal cells, Ep epidermis, Co cortex, Vc vascular cylinder, Vbvascular bundle. Bars 5 mm in a; 2 mm in b, c; 0.1 mm in d; 0.05 mmin e, f

Fig. 2 Optical micrographs ofsections through glutaraldehyde-osmium fixed water hyacinthshoot apexes. a Longitudinalsection through the shoot apex.b Cross section through theshoot apex. c The secretory celladjacent to a crystal cell. d Thesecretory cell adjacent to a xy-lem cell. SC secretory cell, Crcrystal cell, X xylem cell. Bars50 µm in a, b; 20 µm in c, d

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of secretory cells and crystal cells, which may play animportant role in promoting the growth rate and stresstolerance of the water hyacinth.

Secretory cells widely exist in plant tissues and organs,such as root, shoot, leaf, flower, and fruit. The functions ofsecretory cells are various in different organs and species(Dell and McComb 1978; Paiva et al. 2008; Paiva 2009).The gland or epidermal hairs that consisted of secretorycells were often observed in the apical meristem tissue ofyoung shoot and the epidermal tissues of leaves (Klein etal. 2004; Mastroberti and Mariath 2008). According to theobservation in this study on the abundance of secretorycells in water hyacinth shoot apex and the previous reportson the rapid vegetative reproduction of this tissue, weassume that secretory cells in the shoot apex might functionas a storage compartment to store and transfer nutrientelements to meristem cells in vigorous growth stage.

The autophagic procedure has been interpreted ascatabolic activity, which was regarded as a step in the

destruction of specific metabolic machinery leading to theestablishment of a new one (Kupila-Ahvenniemi et al.1978). Krasowski and Owens (1990) considered that theautophagy resulted from nutrient deficiency caused by lackof photosynthesis in apical meristematic cells of coastalDouglas fir during embryonic shoot development. Vacuolarautophagy in plant cells have been widely found indifferent tissues, such as developing seeds, old, and diseasestressed leaves (Bassham et al. 2006), but detailed researchon autophagic procedure in the formation of secretory cellshas not been reported. In the present study, the autophagicprocedure appeared in the vacuoles, where reserve sub-stance began to be degraded. Thus, autophagic effect mightplay an important role in the transformation of secretorycells from the storage stage into the active secretion stage.

A heavy deposit of crystalline material, which wasidentified as CaOx, has been found in the water hyacinthleaves in a previous study (Mazen and Maghraby 1997).Results of our research show a dense distribution of crystal

Fig. 3 Electron and optical micrographs of developing secretory cellsin water hyacinth shoot apex. a Developing secretory cell with severaldark droplets in the vacuole. b An analogous autophagic processappeared in the cytoplasm during secretory vesicle formation.Substances with high electron density surrounded tonoplast andsecretory vesicle. c A secretory vesicle is fusing with the vacuole. d

Early stage of the developing secretory cell with a number of densedroplets in the vacuole. e Dark droplets with dense secretorysubstances fill the vacuole. f Dark droplets fused together and formeda secretion storage vacuole. V vacuole, SV secretory vesicle, DPdroplet, AV autophagic vesicle, SER smooth endoplasmic reticulum, Nnucleus. Bars 5 µm in a, 500 nm in b, 200 nm in c, 10 µm in d–f

Development of secretory cells and crystal cells in Eichhornia crassipes ramet shoot apex

Fig. 4 An ill-defined autopha-gic process appeared in secreto-ry cells during the degradationof storage substances in thevacuole. a A secretory cell withdense matrix in the vacuole anda lot of mitochondria in cyto-plasm. b Degradation in thevacuole begins with erosion atthe periphery and globoids inthe matrix. c An intermediatesecretory cell. d Enlarged viewof the collapsed matrix in theperipheral area of vacuole. Vvacuole, GL globoid, M mito-chondria, W cell wall, SV secre-tory vesicle, AV autophagicvesicle. Bars 500 nm in a, b,and d; 2 µm in c

Fig. 5 Degradation processes ofstorage substances in the vacu-ole of a secretory cell. a Highmagnification electron micro-graph of secretory vesicles in thecytoplasm and globoids in thevacuole matrix. b Globoidsappear in the heavy stainingvacuole matrix. Note that thedegradation is limited in acertain region of the globoids.c A secretory cell at the activedegradation stage. d A secretorycell at the late degradation stage.V vacuole, SV secretory vesicle,M mitochondria. Bars 1 µm ina, b; 5 µm in c, d

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cells in the rosette shoot apexes. The objective of this studywas to investigate the metamorphosis of vacuole during theprocess of crystal formation. Tube-like structures, whichmight serve as crystal chambers and determine crystal

morphology in the cells, have been found in the complexcrystal vacuole systems in developing crystal cells of waterhyacinth shoot apexes. Similarly, formation of crystals inchambers has been observed in plastids (Li and Franceschi

Fig. 6 Crystal cells in waterhyacinth shoot apex. a, b Opti-cal micrographs of cross sec-tions through a fresh shoot apexshow distribution of crystal cellsin the tissues. c Isolated crystalprotoplasts with raphide-bundlecrystals. d Isolated crystalprotoplasts with block-likerhombohedral crystals. Crcrystal, X xylem. Bars 100 µmin a, b; 50 µm in c, d

Fig. 7 Optical and electron micrographs of developing crystal cells inwater hyacinth shoot apex. a–c Phase contrast image of longitudinalsections through developing raphide crystal cells. d–f Electronmicrographs of cross sections through developing raphide crystal

cells. Note that short needle-shaped crystals appear in crystal cellvacuole (a, d), then elongate (b), and finally occupy the whole cell (c,f). Cr crystal, V vacuole, N nucleus. Bars 10 µm in a; 20 µm in b, c;5 µm in d, f; 1 µm in e

Development of secretory cells and crystal cells in Eichhornia crassipes ramet shoot apex

1990) and vacuoles (Horner and Whitmoyer 1972;Kostman and Franceschi 2000) in other plants, but theactual role of these tubules in crystal formation requiresfurther research. It has been suggested that the tubules were

involved in material transportation from the cytoplasm tothe crystal formation site in the vacuole (Frank and Jensen1970). The tubules had also been considered directing boththe orientation of the developing crystal chambers and the

Fig. 8 Ultrastructural images ofcrystal formation in the vacuole.a The interface between vacuoleand cytoplasm in a developingcrystal cell. b A group of grow-ing crystals in vacuole. c Tube-like structure in vacuole. d Acrystal chamber develops fromtube-like structure. e–g Crystalsare growing in the chambers.h Mature crystals. ER endoplas-mic reticulum, M mitochondria,VC secretory vesicle, DV densevesicle, Cr crystal, V vacuole,TU tube-like structure, iCrinitial crystal chamber. Bars500 nm in a, b, and h; 100 nmin e–g; 20 nm in c, d

Fig. 9 High magnification of ultrastructure of the developing crystalcells. a The interface between vacuole and cytoplasm of a primarycrystal cell. Note abundance of ER in the cytoplasm and tube-likestructures in the matrix of vacuole. b A number of tube-like structures

in various orientations are visible in the crystal formation region of thevacuole. ER endoplasmic reticulum, VC secretory vesicle, V vacuole,TU tube structure, iCr initial crystal chamber, Cr crystal. Bars 100 nm

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movement of the various vacuolar structures (Horner andWhitmoyer 1972). Results of ultrastructural study in thepresent research showed that crystals formed in the lumenof the tubules, which might act as the chambers of crystals.

CaOx crystals may be involved in the detoxification oftoxic heavy metals such as lead, cadmium (Cd), and copper(Mazen and Maghraby 1997; Franceschi and Nakata 2005).Different tissues of water hyacinth plant have differentabilities in accumulating heavy metals when it was grownin the medium with different concentrations of metals(Mazen and Maghraby 1997). To test if the abundance ofcrystal cells in water hyacinth shoot apexes is related tothe heavy metal tolerance, the amount of crystal cells inshoot apexes of plants grown on the medium containingdifferent concentrations of Cd was investigated. Figure S1shows that there is almost no Cd to be transported to theshoots and leaves, and most of Cd retains in roots underlow concentration Cd (less than 10 mg/L) conditions, butcontent of Cd apparently increased in shoot apexes andleaves under higher concentration Cd (more than 10 mg/L)conditions. In contrast, the amount of crystal cells did notsignificantly change in shoot apexes under both low andhigh concentration Cd conditions (Fig. 2S A and B). Inaddition, Cd, even at high concentration, could not inducethe formation of crystal cells under conditions of Cadeficiency (Fig. 2S C). Thus, the mechanism of heavymetal tolerance in water hyacinth might not depend onincreasing the amount of crystals in shoot apical tissues,but involve the sequestration of hazardous metals in theexisting CaOx crystal cells as described by previousstudies (Mazen and Maghraby 1997; Franceschi andNakata 2005).

In summary, the formation of both crystal cells andsecretory cells in water hyacinth shoot apexes are involvedin the metamorphosis of vacuoles. However, the relation-ships between these two kinds of cells in the rapid growthand the pollutant tolerance of water hyacinth and whetherion channels or transporters in tonoplast are involved inregulating the formation of these special cells are stillobscure. Further work is being carried out to clarify thesepoints.

Acknowledgment This work was supported by the ShanghaiScience and Technology Committee (072312031).

Conflict of interest The authors declare that they have no conflict ofinterest.

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