Czaplicki, Andrzej Z.

Konieczny, Robert

Pilarska, Maria

Acta Biologica Cracoviensia s Botanica

English

Jagiellonian Univeristy Repository

INTRODUCTIONPectins are heterogenous acidic polysaccharidesthat occur in the matrix of the primary cell walls ofall land plants. Pectic substances account for muchof the mechanical properties of the cell wall, but alsohave signaling roles in cell-to-cell communication,development and defense (for review see: Willats etal., 2001). Several studies have shown that mor-phogenic processes such as somatic embryogenesisand organogenesis are accompanied by differentialexpression of specific pectic epitopes (e.g., Kikuchiet al., 1996; Chapman et al., 2000; Verdeil et al.,2001; Willats et al., 2004). In grasses, having signif-icantly lower cell wall pectin content than in otherspecies, the role of these polysaccharides in devel-opmental processes is poorly documented(Wiethölter et al., 2003; Šamaj et al., 2006).Recently we found that shoot formation from andro-genic callus of Polish wheat cv. Apollo was accom-panied by abundant expression of two homogalac-turonan epitopes (JIM5 and JIM7) at cell-cell adhe-sion sites and in the outer extracellular surfacematrix network (ECMSN) (Konieczny et al., 2007).The experiment described here continues that studyof the distribution of pectic epitopes and ECMSNformation in androgenic culture of the Apollo culti-var. We also studied another Polish wheat cultivar,Kaspar, by immunolabelling, and compared the pec-tic epitope expression patterns of these two culti-vars. The data from immunolabelling are supple-mented with scanning electron microscope (SEM)observations and discussed in terms of the explants'ability to form ECMSN. MATERIALS AND METHODSThe Polish wheat cultivars Triticum aestivum (L.)cv. Apollo and T. aestivum cv. Kaspar were used inthe experiment. The conditions for donor plantgrowth and the procedure for culture in vitro wereas described previously (Konieczny et al., 2003).Briefly, anthers containing microspores at the mid*e-mail: robert.konieczny@uj.edu.plPATTERNS OF PECTIN EPITOPE EXPRESSION DURING SHOOTAND ROOT REGENERATION IN ANDROGENIC CULTURESOF TWO WHEAT CULTIVARSMARIA PILARSKA1, ANDRZEJ Z. CZAPLICKI2, AND ROBERT KONIECZNY1*1Department of Plant Cytology and Embryology, Jagiellonian University, ul. Grodzka 52, 31-044 Cracow, Poland2Plant Breeding and Acclimatization Institute, 05–870 Błonie, PolandReceived August 5, 2007; revision accepted November 21, 2007Spatiotemporal patterns of expression of pectic epitopes during organogenesis were compared in androgenic cul-tures of two wheat cultivars: Apollo and Kaspar. The LM8 epitope (against xylogalacturonan) was found in thecell walls of loosely attached peripheral cells of Apollo and Kaspar callus. The LM8 binding signal was muchstronger in Kaspar than in Apollo culture, and was localized in conspicuous extracellular material coveringalmost all surface cells of Kaspar callus. The LM8 epitope was also expressed in extracellular strands and lay-ers that coated the root caps of adventitious roots in the two cultivars studied. The JIM7 epitope (against methylester-rich pectins) was not detected in Kaspar culture, but the JIM5 epitope (against methyl ester-poor pectins)was abundant in the cell walls lining the intercellular spaces of callus parenchyma. The LM5 epitope (againstgalactans) was scattered in the epidermis of shoot buds and in inner parenchymatic cells of Kaspar and Apollocallus; the LM6 epitope (against arabinans) was not found in either of the cultivars. The type and location of par-ticular pectin epitopes are discussed in relation to their possible function in wheat androgenesis.Key words: Triticum aestivum, wheat, androgenesis, cell wall, extracellular surface matrix network(ECMSN), immunolabelling, pectins. ACTA BIOLOGICA CRACOVIENSIA Series Botanica 49/2: 69–72, 2007PL ISSN 0001-5296 © Polish Academy of Sciences, Cracow 2007to late uninucleate stage were cultured in darknessfor 6 weeks on induction medium, followed bytransfer of the calluses to regeneration medium.Regenerative cultures were kept in light for 4 weeks.Calluses maintained for 6 weeks on induction medi-um and after 1, 2, 3 and 4 weeks of subculture onregeneration medium were taken for microscopicstudies. Sample preparation for conventional lightmicroscopy, scanning electron microscopy and theimmunolabelling procedure were as described earli-er (Konieczny et al., 2005, 2007). The following anti-pectin antibodies were used in this study: LM5(1→4)-α-galactan (Jones et al., 1997); LM6 (1→5)-β-arabinan (Willats et al., 1999) and LM8 binding toxylogalacturonan (Willats et al., 2004) for labellingof both Apollo and Kaspar callus, and JIM5 andJIM7 reacting with methyl ester-poor and methylester-rich homogalacturonan epitopes (Willats et al.,2000) for labelling of Kaspar callus. The results ofimmunolabelling of the Apollo cultivar with JIM5and JIM7 were described elsewhere (Konieczny etal., 2007). The JIM5 and JIM7 antibodies wereobtained from PlantProbes (UK), and the LM5, LM6and LM8 antibodies were kindly provided by Dr.Jozef Šamaj (University of Bonn, Germany).RESULTS AND DISCUSSIONBy light microscopy the six-week-old calluses ofApollo and Kaspar cultivars appeared similar instructure, consisting of loosely attached parenchy-matic cells at the periphery and more compact tis-sue inside the callus (Fig. 1a). Strong fluorescencedue to LM8 binding was observed at the surface cellsof Kaspar callus, while the peripheral cells of Apollocallus were only weakly labelled by it (Fig. 1b,c).SEM showed almost all peripheral cells of Kaspar tobe covered by coarse strands and fibrils which occa-sionally formed a continuous layer over a large areaof callus (Fig. 1d,e). Extracellular material (ECMSN)of similar appearance accompanies early stages ofsomatic embryogenesis and organogenesis in situand/or in vitro (for review see: Šamaj et al., 2006).The composition of the extracellular network mightbe different; it can contain proteins such as ara-binogalactan proteins (e.g., Šamaj et al., 2008). Thepresence of pectic substances in ECMSN has beenreported in chicory (Chapman et al., 2000), coconut(Verdeil et al., 2001) and maize (et al., 2006). Inwheat cv. Apollo also, pectic epitope JIM7 was abun-dantly expressed at sites of ECMSN formation(Konieczny et al., 2007). In all the above-mentionedreports, however, the occurrence of ECMSN wasfound to be restricted to the meristematic regions ofexplants and young embryos/shoot buds, while non-regenerative cells were devoid of any fibrils orstrands. Studies on different plant tissues showedthat LM8-binding epitopes are specifically associat-ed with plant cell detachment and appear when thecells separate from each other, as was observed incarrot suspension cell cultures or the parenchymacells of maturing seeds (Willats et al., 2004).Notably, after one week of subculture, large, LM8epitope-bearing peripheral cells dissociated fromApollo and Kaspar callus, which became meristem-atic at the periphery and parenchymatous in itsinner region (Fig. 1b,c,f). As in the Apollo culture (Konieczny et al., 2007),in Kaspar callus, shoot buds regularly formed fromperipherally located meristematic clumps or layersof callus (Fig. 1g). Interestingly, unlike the Apollocultivar in which the JIM7 epitope was abundantlyexpressed at sites of ECMSN deposition (Koniecznyet al., 2007), Kaspar callus showed no signal result-ing from JIM7 binding (Fig. 1j). Moreover, themeristematic area of Kaspar callus as well as thewhole shoot bud surface were smooth, with no visi-ble extracellular outgrowths (Fig. 1h,i). Instead, theLM5 antibody only bound weakly to the epidermis ofyoung buds and the inner parenchyma cells of callusat this stage of culture (Fig. 1k). The rhamnogalac-turonan I epitope recognized by LM6 was not detect-ed throughout culture of both Kaspar and Apollo(data not shown). The homogalcturonan epitopeJIM5 showed similar distribution patterns in thetwo cultivars and was localized at the cell walls lin-ing the intercellular spaces between inner parenchy-ma cells of callus (Konieczny et al., 2007). In both cultivars, roots originating from meris-tematic centers deep in the callus were formed after1 to 2 weeks of subculture. Immunolabellingrevealed that in Apollo and Kaspar callus the cellwalls within root initials were not labelled by any ofthe antibodies used, while the walls of surroundingparenchyma cells showed similar scattered distribu-tions of LM5 (Fig. 1l). Root cap cells of newly formedApollo and Kaspar roots were highly reactive withthe LM8 antibody and were coated by layers andstrands of extracellular material (Fig. 1m–p). Willatset al. (2004) reported abundant expression of LM8pectins in cell walls of root caps and in some regionsof mucilage coating the root cap surface in severalspecies. Based on the above findings, we suggestthat the extracellular layers and strands observedover the apices of Apollo and Kaspar roots mightrepresent mucilage secretions containing the LM8xylogalacturonan epitope.This study highlights one role of pectic sub-stances in wheat androgenesis, that is, their possibleinvolvement in regulation of cellular adhesion.Earlier the dissociation of large, loosely attachedparenchyma cells from the callus periphery wasobserved to be a prerequisite for shoot regenerationin the Apollo cultivar (Konieczny et al., 2007). Herewe found that the walls of dissociating nonregenera-Pilarska et al. 70tive cells of both Apollo and Kaspar callus bore thesame LM8 xylogalacturonan epitope, specificallyassociated with cells detached from or only barelyconnected to the callus. Thus we suggest a relationbetween expression of the LM8 epitope and the sub-sequent onset of regeneration in wheat callus cul-ture. The most striking difference observed betweenApollo and Kaspar callus was the lack of detectablefluorescence of the JIM7 epitope in Kaspar tissue. Inprevious work on the Apollo cultivar, the JIM7homogalacturonan epitope was seen to be abundantin the cell walls of almost all meristematic cellsPectin epitope expression in androgenic wheat cultures 71Fig. 1. Shoot and root regeneration from androgenic callus of two Polish wheat cultivars. (a) Toluidine blue-stained sec-tion of six-week-old callus of Kaspar cultivar, (b,c) Immunofluorescence of peripheral cells of Kaspar (b) and Apollo (c)callus due to binding of LM8 antibody, (d,e) SEM images of Kaspar callus after 6 weeks of culture on induction medi-um, (f,g) Toluidine blue-stained section through regenerative region of Kaspar callus after one (f) and two (g) weeks ofsubculture, (h,i) SEM images of whole shoot buds (h) and their surface cells after two weeks of subculture of Kasparcallus (i), (j,k) Immunofluorescence due to JIM7 (j) and LM5 (k) binding within adventitious meristems of Kaspar cal-lus, (l) Immunofluorescence of inner parenchyma cells of Kaspar callus due to LM5 binding, (m–p) Root regeneration ofApollo. Toluidine blue-stained section (m), Immunofluorescence due to LM8 binding (n) and SEM images of apex (o)and surface cells of cap (p). Arrowheads on (d), (e), (o) and (p) point to extracellular strands and layers of ECMSN; starson (l) show groups of meristematic cells within parenchyma unlabelled by LM5. Bars in (a, b, d, f-h, m-o) = 50 μm, in(j-l) = 20 μm and in (i,p) = 5 μm. (Konieczny et al., 2007). Pectins are developmental-ly regulated, and changes in their composition havebeen found to accompany the growth and differenti-ation of various plant organs (e.g., Ermel et al.,2000; Majewska-Sawka et al., 2004).We cultured Apollo and Kaspar callus under thesame in vitro conditions; they followed the samedevelopmental pathways, so the observed discrep-ancy in the expression of JIM7 epitope betweenthese two cultivars seems genetically based.Additionally, the peripheral meristematic cells ofApollo callus showed a conspicuous extracellularcoat (Konieczny et al., 2007). That structure wasnever observed over regenerative areas of Kaspar tis-sue; instead, extracellular material of similarappearance was found to cover LM8-reactiveparenchyma cells of Kaspar callus. These differingobservations indicate tissue-specific secretory activ-ity in the studied cultivars. The question of whetherthe role of the extracellular coat in wheat is relatedonly to its specific pectic composition requires fur-ther studies.ACKNOWLEDGMENTSWe are grateful to Dr. Jozef Šamaj of the Universityof Bonn for providing antibodies. REFERENCESCHAPMAN A, BLERVACQ AS, HENDRIX T, SLOMIANNY C, VASSEUR J,and HILBERT JL. 2000. Cell wall differentiation duringearly somatic embryogenesis in plants. II. Ultrastructuralstudy and pectin immunolocalization on chicoryembryos. Canadian Journal of Botany 78: 824–831.ERMEL FF, FOLLET-GUEYE ML, CIBERT C, VIAN B, MORVAN C,CATESSON AM, and GOLDBERG R. 2000. 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Patterns of pectin epitope expression during shoot and root regeneration in androgenic cultures of two wheat cultivars

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Patterns of pectin epitope expression during shoot and root regeneration in androgenic cultures of two wheat cultivars

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