Heredity 70 11993) 628—633 Received 30 October 1992 Genetical Society of Great Britain

Cytology of Vicia species II. Banding patterns and chromatin organization in Vicia atropurpurea Desf

R. CREMONINI, S. FUNARI, I. GALASSO* & D. PIGNONE* Dipartimonto di Scierize Botaniche, Via L. Ghini 5, Università di Pisa, 56726 Pisa, arid *Istjtuto del Germoplasma, C.N.R., Via Amendola 125, 70700 Ban, Italia

Bandingtechniques on metaphase chromosomes of Vicia arropurpurea were carried out and nuclear DNA content in root meristematic cells was determined by cytophotometric analysis. Feulgen absorptions at different thresholds of optical density provided evidence of the organization of the chromatin in interphase nuclei and permitted an evaluation of the cytophotometrically determined heterochromatin amount. These results corroborated the analysis of the banding patterns.

Keywords:banding,chromosomes, fluorochromes, heterochromatin, interphase nucleus structure, Vicia species.

chromatin, expressed as the number and/or area of Introduction chromocentres, has been used as a parameter for Theheterochromatin of higher plants has often been heterochromatin determination (Havelange & Jeanny, studied for taxonomical and phylogenetical relation- 1984; Patankar & Ranjekar, 1984a; Hilliker & Appels, ships. Indeed, changes in genome size accompanying 1989; Samod etal.,1992). evolution and/or speciation are mainly due to an The genus Vicia comprises about 120 species and increase or decrease of the highly repeated DNA speciation in this genus is accompanied by variation component that can be partly represented by cyto- both in chromosome size and number and nuclear logical heterochromatin. C-banding is generally sup- DNA content. posed to distinguish euchromatin and constitutive In previous papers Cremonini and co-workers have heterochromatin by cytological techniques (Sumner, reported biochemical and cytophotometric data on 1990). Interphase chromocentres formed from consti- relationships between Vicia species belonging to the tutive heterochromatin also show a positive staining Faba section (Cremonini cx' al., 1 992a; Frediani cx' a!., reaction, but facultative heterochromatinized chromo- 1992). In this connection it is of interest to analyse the somes do not give positive C-banding (Verma, 1988). nuclear DNA content, the banding patterns and the Other banding techniques are useful to localize and chromatin organization evaluated by cytophotometry differentiate different types of heterochromatin such as at different thresholds of optical density of Vicia centromeric heterochromatin, NOR-associated hetero- arropurpurea and compare these results with the data chromatin and noncentromeric heterochromatin. Some from Viciafaba. fluorescent dyes or combinations of fluorescent dyes and appropriately chosen counter-stains can be used to Materials and methods characterize heterochromatin cytologically and to dis- criminate C-bands enriched in AT or GC base pairs Chromosome banding (Schweitzer 1979, 1981). The structural organization of the interphase Seeds of V. atropurpurea were soaked in running tap nucleus has long been studied (Nag!, 1979; Nag! & water overnight and germinated in Petri dishes at 22°C Fusenig, 1979; Anamthawat-Jonsson & Heslop- until secondary roots were about 1 cm long. Root Harrison, 1990) and the amount of condensed meristems were pre-treated with a paradichloroben- zene saturated solution for 2 h at 12°C in a water bath. Correspondence: Professor R. Cremonini, Dipartimento di Scienze After washing, they were fixed in ethanol—acetic acid Botaniche, Università di Pisa, Via L. Ohini, 5,1-56126Pisa, Italy. (3:1, v:v) for 24 h. The root tips were squashed in a The integralcalculationwascarriedoutonthebest-fit photometer equippedwithamirrorscannerandan trary unitsintopicogramsofDNA.FeulgenDNA (1971). C-banding showedalowheterochromatin chromosomes itresembled thatreportedbyChooi lited, threesubmetacentric andthreesubtelocentric The Banding Results each sample. curves obtainedbythreeofopticaldensityfor photometrically determinedcondensedchromatin. absorption attheinflexionpointrepresentscyto- calculation usingSimpson'srule.TheresidualFeulgen possible todiscriminatebetweentwoareasofintegral position oftheinflexionpointincurvesanditwas mathematically elaboratedinordertoobtaintheexact parison withtheinitialvalueof100. reported asapercentageofFeulgenabsorptionincom- also determined.Theresultsofthisanalysisare lower thanthepreselectedlimit.Thesurfaceareawas those partsofthenucleuswhereopticaldensityis Cremonini eta!. according tothemethodofFredianieta!.(1992)and of onenucleusatdifferentthresholdsopticaldensity interphase nuclei,weredeterminedbymeasurements fractions withdifferentcondensationlevels,in4C length, theFeulgenDNAabsorptionsofchromatin HP8 5computer. length of550nmusingaLeitzMPV3microscope absorption inearlyprophasewasmeasuredatawave- each groupofslidesandusedasinternalstandards; root tipsofViciafabawereconcurrentlystainedfor Cremonini eta!.(1992a)wasused.Squashesofthe For Pignone(1991)was followed. mycin A3andDAPIthetechniqueofGalasso& buffer theslidesweredriedandmounted.Forchromo- Mcllvaine bufferfor10mm;afterawashinthesame staining theslidesina21ugml1stainsolutionatpH7 ment to2.5 a!. (1992)wasusedwiththereductionofHC1treat- removed bythedryicemethodanddriedovernight. drop of45percentaceticacidandthecoverslips Vicia fabawasusedtoconvertrelativeFeulgenarbi- Cytophotometry The valuesofthethresholdsopticaldensitywere With thesameinstrumentandatwave- For C-bandingthetechniqueoutlinedbyGalassoet karyotype ofViciaatropurpurea: withonesate!- cytophotometry thetechniqueoutlinedby mm. (1 H33258 bandingwascarriedoutby 992a). Theinstrumentdoesnotread NUCLEAR ORGANIZATIONOFV/CIAATROPURPUREA629 reaction tobanding techniques. represent inconstantbands,the hatchingsreflectdifferent C-banding; (b)ideotypeofthe samespecies:interruptedlines Fig. 1(a)KaryotypeofVicia atropurpurea afterBSG arm involvedinthesecondaryconstrictionshowedthis the Sat-chromosomeregionofsatellited showed quenchedfluorescenceafterDAPIstaining:on and centromericregionsofthesatellitedarm.Type2 the Sat-chromosomeitwaspresentattelomeric did notshowanyreactiontoeitherfluorochrome.On heterochromatin reactedpositivelytoC-bandingbut showed allthethreetypes(Fig.2a—d,b—e).Type1of possessed type1whilethesatellitedchromosome tribution issummarizedinFig.1(b).Sixchromosomes three differentclassesofheterochromatinwhosedis- both telomeresandatinterstitialsites. highly heterochromatic,showingdifferentialstainingat on theshortarms.Thesatellitedchromosomewas extent. Threechromosomesshowedtelomericbands interphase chromocentreswerelimitedinnumberand distribution wasmainlycentromeric(Fig.la).Alsothe content. Thenumberofbandswaslimitedandtheir content wasrecorded. optimal timewas7mmwherethehighestDNA Table 1.Fromtheanalysisofhydrolysiscurves surface areaofinterphasenucleiaresummarizedin The (Fig. 2c). showed adifferentialreactionafterH33258staining regions ofthenon-satellitedarm.Nonetheseclasses was presentatthecentromericandtelomeric staining: ontheSat-chromosomethisbandingpattern ing, butshowedreducedfluorescenceafterCMA3 pattern. Type3wasshownbybothDAPIandC-band- reacted positivelytoC-bandingandCMA3,but Cytophotometry

- Fluorochrome stainingallowedustodistinguish Iiiiiiliil nuclear DNAcontentofearlyprophasesandthe I___E1 — r: liii III HID '7 aiinri

DUl I'It 630 ft CREMOMNIETAL.

Fig.2Vicia atropurpurea chromosomes stained with Chromomycin A3 (a—d), DAPI (b—e) and Hoechst 33258 (c). The symbol (v) indicates the chromatin associated to the NOR bright fluorescent after CMA and pale after DAPI; the symbol (Y) indicates chromatin segments pale fluorescent after CMA and bright fluorescent after DAPL

The results of measurements made on 4C interphase content and having the same surface area. At threshold nuclei at different thresholds of optical density are 6 of optical density Vicia atropurpurea showed 73.71 reported in Table 2 and in Fig. 3. In order to compare per cent of Feulgen absorption and Vicia faba 94.11 the values of the curves of optical density we have per cent. By increasing the thresholds of optical chosen, in each sample, interphase nuclei having density, when the instrument reads only the optically Feulgen absorption values corresponding to 4C dense, more condensed chromatin, the Feulgen NUCLEAR ORGANIZATION OF V/CIA A TROPURPUREA 631

Table 1 Nuclear DNA content of early prophases in the 00 a root meristems and surface area of interphase nuclei (4C) in Vicia samples. Each nuclear DNA content and surface area I are the mean of fifty determinations carried out in five root 80- meristems I

Vicia Nuclear DNA content Surface area 060 . (pg, mean S.E.) (urn2 S.E.) 0- species 0)0 0 U atropurpurea 14.71 1525.21 24.20 40 - A (2n= 14) 0' I A a faba 53.31* 3300.09±51.03 (2n= 12) 20-

*From Bennett and Smith (1976). I . I 3 9 5 21 27 33 Table 2 Percentages of Feulgen absorption (mean S.E.) at Thresholdsof optical density different thresholds of optical density of interphase nuclei in Fig.3 Percentage of Feulgen absorption at different thres- the root meristems of five seedlings for each sample. Twenty holds of optical density of 4C interphase nuclei in Vicia nuclei for each sample were measured atropurpurea (.) and in Viciafaba (.).Thearrows indicate the inflexion point positions. Thresholds % Feulgen absorptionin interphase nuclei of optical density V. atropurpurea V. faba banding techniques in V. melanops (D'Amato et a!., 1980). In the chromosomes V. faha,Greilhuber(1975) 3 100 100 showed different patterns when stained with C- or Hy- 6 73.71±1,48 94.11±0.22 9 34.53±1.27 86.69±0.13 banding, and Rowland (1981) showed variable expres- 12 12.83±0.63 75.59±0.33 sion of particular segments with different banding 15 3.46±0.51 62.72±1.85 techniques indicating the existence of several classes of 18 — 43.92± 1.92 heterochromatin, Perrino & Pignone (1981) demon- 21 — 28.83±2.34 strated that the C-banding and the H33258 patterns 24 — 15,01±1.68 coincided in several species of the section Faba, such 27 — 7.75±1.07 as V. faba, V. bithynica, and all the species of the V. 30 — 2.06±0.81 narbonensis group. In the metaphase chromosomes of — — 33 V faba the banding pattern prodUced by H33258 was similar to that produced by Quinacrine (Perrino & Pignone, 1981); moreover a comparison between the absorption of V. atropurpurea was reduced to zero at Q-bands and the C-bands has shown that many bands 18 of the thresholds of optical density while the are visible with both techniques on the M and on the S Feulgen absorption of V. faba was reduced to zero at a chromosomes (Vosa & Marchi, 1972; Pignone & higher threshold of optical density: 33. Attolico, 1980). In addition, nucleolar organizers and For V. atropürpurea the inflexion point is at nine of associated heterochromatin displayed very brilliant the thresholds of optical density and the residual fluorescence with CMA3 and showed negative fluore- Feulgen absorption is 34.53 per cent, for V. faba the scence after DAPI staining (Schweizer, 1976) confirm- inflexion point is at 18 of the thresholds of optical ing the heterogeneity of heterochromatin in plants. density and the residual Feulgen absorption is 43.92 After sequential fluorescent staining of metaphase per cent. chromosomes of V. atropurpurea by fluorochromes with opposite base specificity, the heterochromatin has Discussion been shown to have a specific reaction that clearly dif- ferentiates it from that of the species of section Faba Heterogeneityof heterochromatin has been shown in (Perrino & Pignone, 1981). Indeed, in Vicia atropur- plant material by various banding techniques (Filion, purea the C-banded heterochromatin has no differen- 1980; D'Amato, 1986; Pignone eta!., 1989; Galasso & tial reaction with H33258. This behaviour might be Pignone, 1991). In the genus Vicia, similar hetero- due to a different organization of the chromatin which geneity of heterochromatin has been shown by various does not favour the binding of H33258 to DNA. 632 R. CREMONINI ETAL.

Even though the cytophotometric determination of nuclear DNA by the Feulgen method is subject to Acknowledgement certain problems (Greilhuber, 1986, 1988) we have Thiswork was partially supported by a grant from tested both a different fixation (4 per cent formalin) M.U.R.S.T., Roma. and hydrolysis in SN HCI at room temperature for 30 mm and shown that the different methods gave comparable measurements of nuclear DNA content. References Our determination of nuclear DNA content (4C= 14.71 pg) differs from the value reported by ANAMTHAWAT-JONSSON,K. AND HESLOP-HARRISON, j.s.1990. Centromere, telomeres and chromatin in the interphase Chooi (1971) but this divergence may be due to the nucleus of cereals. ('aryologia, 43, 205—213. different methods of determination: biochemical vs. BASSI, i'. 1990. Quantitative variations of nuclear DNA during cytophotometric by an integrating microdensitometer. plant development. A critical analysis. Biol. 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