Anatomy of the fruit of the halophyte Crithmum maritimum L. with emphasis on the endosperm structure and histochemistry

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1 Africn Journl of Biotechnology Vol. 10(45), pp , 17 August, 2011 Aville online t DOI: /AJB ISSN Acdemic Journls Full Length Reserch Pper Antomy of the fruit of the hlophyte Crithmum mritimum L. with emphsis on the endosperm structure nd histochemistry Adllh Ati*, Hfsi Chokri, Rhi Mokded, Zouhier Brhoumi, Chedly Adelly nd Aderrzk Smoui Lortoire des Plntes Extrêmophiles, Centre de Biotechnologie, Technopole de Borj Cédri, BP 901, Hmmm-Lif, 2050, Tunisi. Accepted 13 June, 2011 The hlophytes re plnts tht cn survive nd reproduce under high slinity. They show high potentility s new crops plnt for iosline griculture. Crithmum mritimum L. (Apicee) is one of the promising hlophytes. In this pper, the endosperm structure of the fruit of this oilseeds hlophyte ws investigted using scnning electrons microscopy (SEM), light microscopy (LM) nd fluorescence microscopy (FM). The fruit ws composed of spongy outer cot, secretory envelope, thin endocrp reduced to unicellulr lyer delimiting the endosperm nd n emryo. The endosperm cell ppered limited y thick cell wll nd filled with numerous reserve glooids. The histochemicl test showed tht the cell wll of the endosperm ws rich of crohydrtes s reveled y PAS (periodic cid-schiffs). Within the endosperm cells, there were minly lipid odies nd protein odies. The strch grins were less undnt. The protein odies enclose crystl glooids. The x-ry micronlysis reveled tht the reserve glooids ccumulted mostly Mg, K, C, S nd P. Tken together, these results highlight the structurl fetures, the iochemicl composition nd confirm the nutritionl qulity of C. mritimum L. fruit. Keys words: C. mritimum L., crystl glooids, the endosperm cells, histochemicl test, protein odies, x-ry micronlysis. INTRODUCTION In rid regions, desertifiction, soil sliniztion nd wter shortge constitute the common prolems due to vriety of nturl nd humn cused fctors (Kinet et l., 1998; Khn nd Qiser, 2006; Koyro et l., 2008). In these res, the slt-ffected soils contin high concentrtions of solule slts tht reduce the growth nd the productivity of the most conventionl crop species (Koyro et l., 2008). Studies conducted y the United Ntions Food nd Agriculture Orgniztion (FAO, 2005) estimted tht n dditionl 200 million hectres (h) of griculturl lnd will e required over the next 30 yers. Thus, the reserch of new species to e used in slt ffected res to overcome the mentioned prolems *Corresponding uthor. E-mil: tdllh@yhoo.fr. Tel: (+216) Fx: (+216) is needed (Ati et l., 2010). It is well known tht in these res there re specific plnts tht cquire specific dpttion which permit them to pss esily the prolems relted to moisture deficit stress nd soil sliniztion. They were nmed hlophytes. They cn tolerte nd reproduce vile seeds t concentrtion not lower thn 200 mm NCl (Flowers nd Colmer, 2008). Severl species of hlophytes show high economicl potentility nd hve een selected for economicl uses. Crithmum mritimum L. (Apicee) is one of the promising hlophytes species. This is perennil species nd thrives long rocky costl ecosystems; this hve the cpcity to mintin their growth potentil up to 300 mm (Ben Amor et l., 2005). C. mritimum L. is potentilly useful for economicl nd medicinl purposes. Indeed, its leves disply high ntioxidnt nd ntimicroil ctivities (Meot-Duros et l., 2008; Meot-Duros nd Mgné, 2009). The fruits of C. mritimum L. show high ccumultion of

9194 Afr. J. Biotechnol. em end lt A B end mic cot C D seed rd 1 mm sc Figure 1.

micropylr (end mic), the cotyledon (cot), the endosperm lterl (end lt) nd the rdicle (rd). Sc, spongy cot; em, emryo. essentil oils nd other iologicl ctive compounds (Ati et l., 2009).

Consequently, this species hve potentil s sline wter-irrigted oilseed crop (Ati et l., 2009). This species ws trditionlly consumed y humn.

2 9194 Afr. J. Biotechnol. em end lt A B end mic cot C D seed rd 1 mm sc Figure 1. () Stereomicroscope views of the longitudinl section on the mericrps showing the spongy cot, the seeds nd the emryo; () SEM view of longitudinl section t the emryo region showing the endosperm micropylr (end mic), the cotyledon (cot), the endosperm lterl (end lt) nd the rdicle (rd). Sc, spongy cot; em, emryo. essentil oils nd other iologicl ctive compounds (Ati et l., 2009). They contin up to 44% DW of lipids nd show good oils composition tht close up to olive oil composition (Ati et l., 2010). Consequently, this species hve potentil s sline wter-irrigted oilseed crop (Ati et l., 2009). This species ws trditionlly consumed y humn. In vegetle tissues, the nutriments tht re used in humn nd/or niml diet is in lrge prt loclised in the seed or fruit tissues, in the form of crohydrtes, lipids, proteins, orgnic phosphtes nd vrious inorgnic compounds (Bewley nd Blck, 1983; Coimr nd Slem, 1994). The fruit of C. mritimum L. is schizocrp divided into two mericrps; ech one contins one endospermic seed. The endosperm tissue of C. mritimum L. is rich in oil glooids (Ati et l., 2010). The fruit of C. mritimum L. contins significnt mounts of oil, up to 44.4% (w/w), potentilly edile due to its ftty cid composition close to olive oil (Ati et l., 2010). Yet, more detils out the fruit structure were needed such s the loclistion of the proteins, the crohydrtes nd the inorgnic compounds. Thus, microscopic study ws conducted to more highlight the structure of C. mritimum L. fruit. MATERIALS AND METHODS Mture fruits were collected in Decemer 2008 from plnts in the nturl popultion growing in the rocky cost of Trk (N 36 57' 12" E 08 45' 18"), locted in N-W of Tunisi. Oservtions of free-hnd sections of seeds were crried out y scnning electron microscope (SEM; type FEI Qunt 200). For light microscopy, fixed mterils were used. Fruits were fixed in 1% (w/v) prformldehyde solution. Then, they were dehydrted in n ethyl lcohol series, infiltrted nd emedded in prffin lock. Emedded tissues were then sectioned with microtome. 0.5 µm thick sections were otined. The histochemicl tests of periodic cid-schiffs (PAS) test were used for polyscchrides (Miller et l., 1999). Light green dye nd toluidine leu (TBO) solutions were used for protein odies loclistion (Miller et l., 1999). The prepred slides were oserved under light microscope (Olympus DX41). For fluorescence microscopy, free-hnd sections were prepred nd stined with 0.001% (w/v) neutrl red solution for nucleic cid (DNA) (Durovsky et l., 2006), with 0.1% (w/v) KOH solution for flvonoids (Tzonoglou et l., 2001) nd fuchsin cid for proteins (Fulcher nd Wong, 1980). The x-ry spectr for the compositionl nlyses were performed for the following elements: K, Mg, C, P nd S. The scnning electron microscope (SEM) FEI Qunt 200, equipped with x-ry (EDAX) system for micronlysis, ws used t 15 kv with working distnce of 10 to 11.4 mm. RESULTS The SEM nd stereomicroscope oservtions showed tht the fruit of C. mritimum L. ws composed of spongy outer cot, secretory envelope, thin endocrp, which constituted the seed envelopes tht delimited the endosperm nd n emryo (Figure 1). Although the ltter ws very smll in size, it ws completely differentited on rdicle nd cotyledons (Figures 1 nd 2). The SEM view of trnsversl section of the fruit showed

3 Ati et l end cot seed envelopes Figure 2. () Trnsversl section t AB level showed in Figure 1 ; this section shows the emryo cotyledons; () trnsversl section t CD level showed in, showing the endosperm crcks (rrows) tht llow the immture emryo elongtion. crck in the endosperm (Figure 2). The endosperm cells ws limited y thick cell wll nd filled with numerous reserve glooids (Figure 3). We noted tht fter distilled wter imiition of the fruit, the cell wll ppered more flexile nd the reserve glooids were lierted (Figure 3). The histochemicl tests reveled tht the cell wll of the endosperm ws rich with crohydrtes s reveled y periodic cid-schiffs (PAS) colortion (Figure 4). Some strch grins were lso stined y PAS within the cells (Figure 4). The light microscope oservtions, fter green light dye stining showed tht the cell wll ws rich in proteins (Figure 4c). The endosperm tissue ws filled with the protein odies which took green colortion nd enclosed crystl glooids (Figure 4c). The toluidine leu stined sections showed tht protein mtrix of the crystl glooids ws stined light (Figure 4). The fluorescence microscope oservtions of the fuchsin cid stined section confirmed the richness of the endosperm cells y the protein odies (Figure 5, ). Ech endosperm cells contined n intct DNA s reveled y neutrl red colortion (Figure 6, ). The fluorescence microscopic oservtions reveled the richness of C. mritimum L. with oil glooids nd the richness of the endosperm tissue with flvonoid compounds tht ppered s fluorescent corpuscles fter KOH stining (Figure 6c, d). The x-ry micronlysis reveled tht glooid reserves ccumulted mostly Mg, K, C, S nd P (Figure 7). DISCUSSION Locliztion of stored reserves inside the C. mritimum L. fruit ws chieved. The fruit ws composed of spongy outer cot, secretory envelope nd thin endocrp, which constituted the seed envelopes tht delimits the endosperm nd the emryo which ws very smll in size. Therefore, the C. mritimum L. emryo ws immture nd exhiited morphologicl dormncy. Upon imietions, the emryo must elongte within the seed through the endosperm crck. This is common in Apicee fruit; the emryo is often rudimentry nd emedded in the endosperm nd exhiits morphologicl dormncy (Nikolev, 1977; Bskin nd Bskin, 2004). Thus, the endosperm is the mjor prt of the seed nd constitutes the reserve tissue (Ati et l., 2010). In C. mritimum L., the endosperm cells ppered limited y thick cell wll nd filled with numerous glooid reserves. In ngiosperm seeds, the reserve mteril my e stored in emryo, minly in the cotyledons or in extr-emryonic tissues minly in the endosperm or in the perisperm or in oth. For instnce, in the Leguminose, nd the Crucefere, the min storge tissue is the cotyledon, in the cerels, Euphoricee nd the Apicee, the storge tissues is the endosperm (Bewley nd Blck, 1983) in the Chenopodice, the min storge is the perisperm (Prego et l., 1998). In C. mritimum L., the endosperm ccumultes high level of lipids in the oil odies (Ati et l., 2010). The histochemicl tests reveled tht the cell wll of the endosperm ws rich in crohydrtes s reveled y PAS. Mny strch grins were lso found within the endosperm cells, however, they were less undnt in comprison with other reserve forms. In C. mritimum L. endosperm, the crohydrtes were loclised in the cell wll. This is common in the Apicee. In these species the crohydrtes were minly loclised in the cell wll of the endosperm in form of

9196 Afr. J. Biotechnol. rg fcw rg cw fcw Figure 3. SEM views of the endosperm tissues.

with the glooid reserves (rg) nd showing thick nd powerful cell wll (cw); () SEM view of the

cell wll (fcw). c cw 10 µm 10 µm d 4 µm 4 µm Figure 4.

() Loclistion of the cell wll (cw) polyscchrides fter colortion with PAS; () loclistion of

colour fter light green colortion (lck rrows) nd the glooid crystls were drk stined (white

4 9196 Afr. J. Biotechnol. rg fcw rg cw fcw Figure 3. SEM views of the endosperm tissues. () SEM view of dry seed section showing detil view of the endosperm cells which ppered filled with the glooid reserves (rg) nd showing thick nd powerful cell wll (cw); () SEM view of the endosperm cells of imied seed showing the liertion of the glooid reserves (rg) nd the flexile cell wll (fcw). c cw 10 µm 10 µm d 4 µm 4 µm Figure 4. Light microscope oservtions of the endosperm tissues. () Loclistion of the cell wll (cw) polyscchrides fter colortion with PAS; () loclistion of strch grins (rrows) fter colortion with PAS; (c) loclistion of proteins odies tht took green colour fter light green colortion (lck rrows) nd the glooid crystls were drk stined (white rrows). The cell wll (cw) lso ppered green; (d) toluidine leu colortion showing the protein mtrix of the crystl glooids tht re light leu stined (discontinuous rrows) nd the glooid crystls (continuous rrows).

Ati et l. 9197 cw Figure 5. Fluorescence microscopy oservtions of endosperm tissues. () Fuschine cid stined section tht revels the protein odies (rrows); () detils view of the crystl glooids (rrows).

5 Ati et l cw Figure 5. Fluorescence microscopy oservtions of endosperm tissues. () Fuschine cid stined section tht revels the protein odies (rrows); () detils view of the crystl glooids (rrows). Note tht the crystl glooids which were loclised within the protein odies did not tke the colortion. c d Figure 6. Fluorescence microscopy oservtions of the endosperm tissues. () Autofluorescence of the endosperm cells; () oservtion of the neutrl red colored section showing the DNA within endosperm cells; (c) oservtion of KOH stined section tht revel the flvonoids (Blck rrows) nd the oil glooids (white rrows); (d) detil view of KOH stined section tht revel the flvonoid corpuscles (rrows).

6 9198 Afr. J. Biotechnol. Counts kv Figure 7. X-ry micronlysis showing the x-ry spectr derived from reserve glooids of the endosperm cells of C. mritimum L. fruit for K, Mg, C, P nd S. of glctomnnnes nd glucomnnnes (Bewley nd Blck, 1994). The photonic microscopic oservtions showed tht the endosperm tissue ws filled with protein odies tht enclose crystl glooids. This confirms the richness of C. mritimum L. fruit with inorgnic compounds. The ccumultion of the oil reserves ws ccompnied y high ccumultion of protein odies in C. mritimum L. fruit. This is lso oserved in some oleginous species such s Brssic npus (Bewley nd Blck, 1983). In ngiosperms, the proteins re ccumulted minly in reserve glooids (Prego et l., 1998). Storge proteins, which serve s source of cron, nitrogen nd sulfur, rech 90% of the totl protein frction in mture seeds (Kummru et l., 2007). The richness of seeds of ny species on protein revels its economic vlue. For instnce, the seed of Chenopodium quino L. ws vlorised for its high level of proteins (Prego et l., 1998; Konishi et l., 2004). The photonic microscopic oservtion showed tht the protein odies enclosed crystl glooids. The x-ry micronlysis reveled tht the reserve glooids ccumulted mostly Mg, K, C, S nd P. These results highlight the minerl composition of C. mritimum L. fruit nd confirm their richness with essentil elements nd inorgnic compounds. The sme composition ws lso oserved for Bet vulgris L., Coffe ric L., Zoster cpricorni L. nd C. quino L., nmely P, K, Mg nd C (West et l., 1995; Prego et l., 1998). The richness of C. mritimum L. fruit with sulphur my indicte their richness with sulphured mino cids. This study showed significnt ccumultion of flvonoids in the endosperm. These compounds re known to exhiit some ntioxidnt nd ntimicroil propriety. They inhiit nd kill mny cteril strins, nd inhiit some virl enzymes, such s reverse trnscriptse nd protese (Hvsteen, 2002). Furthermore, they re none toxic to humn cells. Thus, flvonoids re mjor functionl components of mny herl preprtions for medicl use. In food, the dily intke of flvonoids especilly fruits nd vegetles re needed for good humn helth (Hvsteen, 2002). In previous study, we showed tht C. mritimum L. fruit envelope ccumulte severl forms of iologicl ctive compounds (Ati et l., 2009). The eril prts of this species were known to ccumulte the sme molecules (Meot-Duros et l., 2008; Meot-Duros nd Mgné 2009). Tken together, these results give new insight out the structurl fetures of the fruit of the oil seed hlophyte C. mritimum L.; the loclistion of the protein odies, the crystl glooids, the polyscchrides nd the minerl composition which highlights the iochemicl composition nd confirms the nutritionl qulity of the C. mritimum L. fruit. REFERENCES Ati A, Deez A, Brhoumi Z, Adelly C, Smoui A (2009). Histochemicl locliztion of essentil oils nd ioctive sustnces in the seed cot of the hlophyte Crithmum mritimum L. (Apicee). J. Plnt Biol. 52: Ati A, Deez A, Brhoumi Z, Smoui A, Adelly C (2009). ABA, GA 3, nd nitrte my control seed germintion of Crithmum mritimum (Apicee) under sline conditions. C.R. Biol. 332: Ati A, Deez A, Zouhier B, Adelly C, Smoui A (2010). Locliztion nd composition of seed oils of Crithmum mritimum L. (Apicee).

7 Ati et l Afr. J. Biotechnol. 39: Bskin JM, Bskin CC (2004). A clssifiction system for seed dormncy. Seed Sc. Res. 14: Ben Amor N, Ben Hmed K, Deez A, Grignon C, Adelly C (2005). Physiologicl nd ntioxidnt responses of the perennil hlophyte Crithmum mritimum to slinity. Plnt Sci. 168: Bewley JD, Blck M (1994). Seeds: Physiology of developpement nd germintion, 2nd edn. New York, Plenum Press. Bewley JD, Blck M (1983). Physiology nd Biochemistry of Seeds. Springer-Verlg, New York. Coimr S, Slem R (1994). Amrnthus hypochondricus: Seed structure nd loclistion of seed Reserves. Ann. Bot. 74: Durovsky JG, Guttenerger M, Srlegeui A, Npsucily-Mendivilh S, Voigt B, Blusk F, Menzel D (2006). Neutrl red s proe for confocl lser scnning microscopy studies of plnt roots. Ann. Bot. 97: FAO (2005). Glol network on integrted soil mngement for sustinle use of slt ffected soils. Fulcher RG, Wong SI (1980). Inside cerels fuorescence microchemicl view. In: Inglett GE, Munck L, eds. Cerels for food nd everges. New York: Acdemic Press, pp Flowers TJ, Colmer TD (2008). Slinity tolernce in hlophytes. New Phytol. 179: Hvsteen BH (2002). The iochemistry nd medicl significnce of the flvonoids. Phrmcol.Therpeut. 96: Kummru T, Ogw M, Stoh H, Okit TW, Krouniotis G (2007). Protein ody iogenesis in cerel endosperms. Plnt Cell Mono. 8: Khn MA, Qiser M (2006). Hlophytes of Pkistn: Chrcteristics, distriution nd potentiels economics usges. In Khn MA, Böer B, Kust GS (eds), Skh Ecosystems; Volume II: West nd Centrl Asi, pp Kinet M, Benreih FZ, Bouzid S, Lilchr S, Dupuit P (1998). Reseu Atriplex. Atelier iotechnologies et écologie pour une sécurité limentire ccrue en régions rides et semi rides. Chiers Agric., l7: Konishi Y, Hirno S, Tsuois H, Wd M (2004). Distriution of minerls in Quino (Chenopodium quino Willd.) seeds. Biosci. Biotechnol. Bioch. 68: Koyro HW, Geiler N, Hussin S, Huchzermeyer B (2008). Survivl t extreme loctions: life strtegies of hlophytes the long wy from system ecology, whole plnt physiology, cell iochemistry nd moleculr spects ck to sustinle utiliztion t field sites. In: Adelly C, Ashrf M, Oztürk M, Grignon C (Eds.), Biosline Agriculture nd Slinity Tolernce in Plnts. Verlg, Switzerlnd, Birkhüser, pp Meot-duros L, Mgné C (2009). Antioxidnt ctivity nd phenol content of Crithmum mritimum L. leves. Plnt Physiol. Biochem. 47: Meot-Duros L, Le Floch G, Mgné C (2008). Rdicl scvenging, ntioxidnt nd ntimicroil ctivities of hlophytic species. J. Ethnophrmcol. 116: Miller SS, Bowmn LA, Gijzen M, Miki BLA (1999). Erly development of the seed cot of soyen (Glycine mx). Ann. Bot. 84: Nikolev MG (1977). Fctors controlling the seed dormncy pttern. In: Khn AA, ed. The physiology nd iochemistry of seed dormncy nd germintion. Amsterdm: North-Hollnd. pp Prego I, Mldondo S, Otegui M (1998). Seed structure nd locliztion of reserves in Chenopodium quino. Ann. Bot. 82: Tzonoglou D, Nikolopoulos D, Likopoulos G (2001). Epicuticulr phenolics over gurd cells: exploittion for in situ stomtl counting y fluorescence microscopy nd comined imge nlysis. Ann. Bot. 87: West MM, Flnnign DT, Lott JNA (1995). Elementl composition of glooids in the perisperm tissue of vrious seeds. Cn. J. Bot. 73:

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