Comparison of lethal and semilethal chlorophyll mutants ... · the following cultivar varieties of winter rye (Secale cereale L.): Pancerne, Dankowskie Zlote, Dankowskie Selekcyjne,

CARYOLOGIA Vol. 53, no. 3-4: 227-234, 2000

Comparison of lethal and semilethal chlorophyll mutantscharacterized by different expression of genes responsiblefor colour of leaves in winter rye (Seca/e cereale L.)B. GABARA1 and H. KUBICKA2'*

1 Department of Plant Cytology and Cytochemistry, University of Lodz, Banacha 12/16, 90-237 Lodz; Poland. 2 Botanical Garden, PolishAcademy of Sciences, Prawdziwka 2, 02-973 Warszawa, and Department of Production Ecological in Agriculture, Engineering Collegeof Bialystok, Tarasiuka 2, 15-601, Bialystok, Poland

Abstract — Two groups of chlorophyll mutants of winter rye i.e. lethal and semi-lethal were compared in the present studies. Plants of the first group were charac-terized by pink orange or light violet colour of leaves at the seedlings stage. Plantswith light green leaves, with yellow necrotic spots on leaves, or with white patternand necrotic spots on leaves belonged to the second semilethal group. While plas-tidsin pink and violet mutants were deprived of internal membrane organization inorange ones they had single thylakoids. In the second group of plants granal andintergranal thylakoids were present although the number of grana was reduced. Theabove changes in the plastid ultrastructure were accompanied by a lack ofchlorophylls in pink, orange and light violet plants or strong reduction of bothchlorophylls a and b in the remaining mutants. All the described features were in-herited by single recessive genes.Key words: chlorophyll, chloroplast, inheritance, mutants, rye, thylakoid.

INTRODUCTION

Biodiversity in plants, including rye is caused,among others, by spontaneous mutations. Thesemutations in rye were observed by manyinvestigators (KUBICKI and KUBICKA 1981; DE VRIESand SYBENGA 1984; SMIRNOV and SOSNICHINA 1984)in self-pollinated populations of cultivar varieties,strongly heterozygous. Therefore, as a result ofinbreeding in homozygots the mutations ofreccessive genes appeared. Such mutantionswere not visible in heterozygous state.

Diversity in plants occurs also as a result ofmutagenesis. Therefore to increase variability inrye the seeds of cultivars or inbred lines aretreated with chemical or physical mutagens. Inthis way it is possible to obtain many mutants(MUNTZING 1963; GRZESIK and NALEPA 1974;DABROZSKI 1979; KUBICKA and MALEPSZY 1996).

* Corresponding author: fax ++22 757 6645; e-mail:[email protected]

Among spontaneous as well as induced mu-tants a significant part established chlorophyllmutants. The chlorophyll mutants are charac-terized by changes in leaf colours from whitethrough light-yellow to pink or local colourchanges from white through yellow to necroticspots. These changes are accompanied by disor-ganization of internal lamellar system in plas-tids. The bigger fragment of leaf changes colourthe higher degree in plastid disorganization isseen (GABARA and KUBICKA 1991; KUBICKA et al. 1986,1998).

The aim of the present study was compari sonlethal and sublethal groups of chlorophyllmutants in winter rye. Their genetic analysis andchlorophyll content as well as chloroplaststructure were taken into consideration.

MATERIALS AND METHODS

Different chlorophyll mutants (L69, L281, 110,L208, hs1, L158b, wch, zp and L24) were obtained inthe inbred line S2 generation after self-pollination of

the following cultivar varieties of winter rye (Secalecereale L.): Pancerne, Dankowskie Zlote, DankowskieSelekcyjne, Garczynskie, Chrobre, Smolickie and oneform from SHR Jeleniec. One mutant- hs2 — wasobtained after treatment of inbred line, self-fertile ofgeneration S10 (L299) with 1.5 mM azide sodium.

Young seedlings of chlorophyll mutants: L69,L281 and 110 were characterized by pink, orange orlight violet colour, respectively. Inheritance mode ofthese mutants was determined by rate segregation ofdark green plants and those with changed colour inS3 and S4 generations. The remaining chlorophyllmutants after numerous self-pollinations appeared inS10 (L208 and hs1) and S12 generations (Ll56b, wch, zpand L24). Subsequently these homozygotic plantswere crossed with control ones (with uniformelygreen leaves). Plant segregation both in F1, F2 and S 3,S4 generations, was observed and reliability of theobtained results was checked by means of chi-squaretest.

Chlorophyll content was determined accordingto BRUINSMA (1963) method and measured at 645, 652and 663 nm in spectrophotometer Spectronic 601(Milton Roy Company USA).

For electron microscopy analysis small pieces(0.5 ^im2) from middle part of mutant and controlleaves were fixed in 2% glutraldehyde buffered with0.1 M sodium cacodylate for 2h at 0-4° C. Afterwashing in the buffer and postfixation in 1 % osmicacid material was prepared to EM as described earlier(KUBICKA et al. 1986). Ultrathin sections stained inuranyl acetate followed by lead citrate were examinedin an Jeol 1010 electron microscope at 40 kV.

RESULTS

All chlorophyll mutants except hs2 were ob-tained in inbred generation S2, after self-polli-nation of cultivar varieties of winter rye (Secale

TABLE 2 — Origin of chlorophyll mutants in winter rye.

cereale L.). The frequency of these mutationsappearance oscillated from 2 to 6 cases and de-pended on the initial variety. Most mutants wereselected from inbreded SHR-Jeleniec short form(6) and only two from varieties: DankowskieZlote, Chrobre and Smolickie each (Table 1).Therefore, four of investigated chlorophyllmutants originated from SHR Jeleniec, two fromvarieties: Smolickie and Chrobre and only onefrom Dankowskie Selekcyjne and Garczynskieeach (Table 2).

TABLE 1 — Frequency of chlorophyll changes in winter rye afterself-pollination.

Among ten analysed chlorophyll mutantsthree (lethal) were characterized by the totalchange of leaf colours in young seedlings topink (L69), orange (L281) and light violet (110).These colours were revealed at the stage of seedgermination (autumn). All such young seedlingsdied 4 weeks after germination (Table 3). Ge-netic analysis of these mutants (L69, L281,110)made on the basis of segregation rate of mu tatedand dark green plants in inbred genera tions S3and S4 (Table 4) revealed that segregation intomutated and control plants was approximate tothe theoretical ratio 3:1. On the other hand, allthe F1 generation plants were dark green.

228 GABARA and KUBICKA

CHLOROPHYLL MUTANTS IN SECALE CEREALE

Fig. 1 — Chloroplast from dark-green plants (control). Typical thylakoid organization is visible, x 16.000.Fig. 2 — Plastids with concentric arrangement of thylakoids, mutant pink in colour, x 20.000.Fig. 3 — Plastid with numerous vesicles from orange mutant. Aggregate of plastoglobules. x 21.600.Fig. 4 — Plastid from orange mutant. Single long thylakoids among short ones, and aggregate of plastoglobules. x 18.200.

The other studied chlorophyll mutants (sub-lethal) appeared in spring. They had reducedvitality and were characterized by differentovercolouring of leaves from light green (L208),yellowish-necrotic (hs1) hs2) to white yellow ir-regular pattern (L24, L158b, wch, zp). By re-

peated self-pollination of these mutants theywere fixed in homozygous state and later treatedas inbred generation lines S10 — S12. These linesconstituted the source of the above-mentionedtraits (Table 2, 3). The mode of inheritance ofthese mutants was designed on the

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TABLE 3 — Visualization of gene activity responsible for chlorophyll changes in winter rye during vegetation season.

basis of F1 and F2 offsprings obtained aftercrossing of mutants and control plants (with darkgreen leaves). In sublethal mutants rate ofsegregation 3:1 was observed in F2 generation.This result clearly indicates that the describedtraits were inherited by monogenous recessivegenes (Table 3, 4).

Genes revealed in the investigated chloro-phyll mutants were expressed in the reduction ofchlorophyll a and b contents (Table 5). While inlethal mutants no chlorophylls were observed insemilethal ones with genes ync but especially inwyv1wyv1 and wyv2wyv2 significant reduction inboth chlorophylls was seen.

Described genes were also responsible forchanges in plastids ultrastructure. Plastids fromdark green leaves (control) had internal mem-brane organization typical for chloroplasts i.e.granal and intergranal thylakoids, mostly paral-lelly arranged to long plastid axes and in theirmatrix starch grains and numerous plastoglob-ules were seen (Fig. 1). However, in lethal mu-tants all plastids had completely changed ul-

TABLE 4 — Genetic analysis of chlorophyll mutants in winter rye.

trastructure (Table 5, Figs. 2-4) that was accom-panied by a lack of chlorophylls in them. In le-thal mutants with pink leaves plastidultrastructure significantly differed from thosewith light-violet ones. Short thylakoids in plas-tids were randomly dispersed or concentricallyarranged (Fig. 2). Sporadically vesicles variousin size and shape appeared in their matrix. Granain such plastids were never distinguished (Fig. 2).

In the second lethal mutant characterized bylight-violet leaves plastids were almost en tirelydeprived of inner membrane organiza tion (Fig.5). Sometimes only single and short thylakoidsor electron-transparent minute vesicles (arrow)were visible in granular matrix of such plastids.

In the third obtained lethal chlorophyll mu-tant with orange leaves internal membrane sys-tem of plastids was composed only of intergranalthylakoids mostly arranged longitudinally to longplastid axes (Fig. 4). Sometimes in matrix of suchplastids the vesicles as well as ag-

CHLOROPHYLL MUTANTS IN SECALE CEREALE

Fig. 5 — Plastid form light-violet mutant, completely deprived of thylakoid system x 24.000.Fig. 6 — Plastid from light-green line (L 208). Numerous grana among some intergranal thylakoids are visible, x 21.600.Fig. 7 — Yellow mutant (hsj). Granal and intergranal thylakoids less numerous than in Fig. 1. x 28.800.

gregates (1-4) of numerous plastoglobules wereseen (Fig. 3). No starch was noticed in plastids ofthese three types of mutants. Second group ofmutants, semilethal was represented by threetypes of plants characterized by different colourof leaves (Table 3) and reduced number of granaland intergranal thylakoids within plastids (Table5, Figs. 6-9). That was accompanied by a loweramount of chlorophylls content.

In the first, light green plants plastids hadslightly disorganized thylakoid arrangement (Fig.6) although granal thylakoids were easydistingished. However, grana were less numerousthan in control dark green plants, with fewernumber of thylakoids per single granum(Tabled).

In the second semilethal type of mutantscharacterized by yellow necrotic spots on leaves

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TABLE 5 — Gene expression in plastids of chlorophyll mutants in winter rye.

plastids had typical inner membrane organiza-tion although number of granal and intergranalthylakoids significantly diminished, as comparedwith control plants (Fig. 7).

Most pronounced reduction in number ofgranal and intergranal thylakoids was observedin the latter semilethal type of chloropyll mu-tants, i.e. with white-yellow irregular pattern onleaves (Fig. 8) represented by four lines. The re-duction in number of grana was especially drasticin zp line where only minute thylakoids werevisible (Fig. 9).

DISCUSSION

The majority of hereditary changes occuredas a result of recessive gene mutations, notwith-standing the traits influenced. However, reversemutations in which recessive genes mutate intodominant ones are extremely rare. Therefore, sofar described numerous chlorophyll mutants,spontaneous as well as obtained through muta-genesis, both in mono- (REDDY and SUBRAH-MANYAN 1988; MOLLER et al. 1997) and dicoty-ledonous plants (MILLER et al, 1984; CHEN et al.1999), are most frequently determined by singlerecessive genes. A similar inheritance mode ofchlorophyll mutations was also observed in rye(SMIRNOV and SOSNICHINA 1984; DE VRIES and SYBENGA1984; KUBICKA et al. 1986, 1988; GABARA et al. 1991).

Among mutants described in the present paperthere were mainly (9) spontaneous ones ob tainedby inbreeding winter rye and one (hs 2) byinduced mutation. However, independently ofthe way the mutants were obtained the samegenes were involved. For instance hs1 and hs2mutants resulted from mutation of the same

gene, marked as ync (KUBICKA et al. 1998). Al-though these mutants were characterised bysimilar phenotypes, the expression of ync genein hs1 and hs2 mutants was different on the level ofchloroplast ultrastructure and chlorophyll (a andb] contents.

On the other hand, as allelism test indicatedthe irregular white yellow patern on leaves inL158b, wch and zp mutants were determined bythe same single wyv1 gene. Similarly as in the caseof ync gene the expression of wyv1 gene causedonly small differences in the morphologicalappearance of these mutants but significant onesin the plastid ultrastructure and chlorophyll (aand b) contents.

This phenomenon can be explained by dif-ferent genetic origins of mutants selected fromdifferent varieties of cultivated winter rye thatresulted in the diverse expression of ync andwyv1 genes. Moreover, it cannot be excludedthat other genes of a given genotype also wereresponsible for the above mentioned changes.

On the other hand, gene wyv2, nonallelic towyv1, controlled the appearance of the sametrait, i.e. irregular white yellow pattern onleaves. Effect of this gene was weaker and ex-pressed itself in minimal differences betweenphenotypes (GABARA et al. 1991; GABARA andKUBICKA 1989; KUBICKA et al. 1986, 1988, 1998).

More distinct differences in the gene expres-sion were noticed in the chloroplast ultrastruc-ture and chlorophyll (a and b) contents. Genewyv2 reduced both chlorophylls (a and b) con-tent, the number of chloroplasts (within singlecell) as well as the number of grana (in chloro-plast) and thylakoids (per granum) to a lesserdegree than wyv1 gene did (KUBICKA etal. 2000).

CHLOROPHYLL MUTANTS IN SECALE CEREALE

Fig. 8 — Mutant with white yellow irregular pattern (L 24). Plastid characterized by granal and intergranal thylakoids arrangement, x24.000.Fig. 9 — Mutant with white yellow irregular pattern (L 158b). A significant reduction in the number of granal and intergranal thy-lakoids. x 24.000.

Another mutant presented in the study (L208)characterised by light green leaves wasmorphologically similar to mutants describedearlier by DE VRIES and SYBENGA (1984), hence thegene responsible for this trait was marked as asuccesive lg gene (GABARA and KUBICKA 1991). InL208 mutant the reduction of grana (14.4%) inchloroplasts and thylakoids (54%) in singlegranum as compared to the control, dark leavesplants took place. The lowering in chlorophyllcontent observed in lines with light green leaves insoya (WILCOX and KOLLER 1992) and maize(LAMBERT et al 1996) seems to prove ourobservations.

In the case of lethal chl1 chl2 and chl3 genes,which controlled the changes in colour of youngseedling leaves into pink, orange and light violet,there were significant defects in proplastidultrastructure leading to their degen-

eration and in effect, to recessive homozygotesdeath (GABARA et al. 1991; KUBICKA et al. 1986,1988). Similar disorders in the plastids organi-zation were noticed by MOLLER et al. (1997) inxantha barley lethal mutants (e.g. numerousplastoglobules).

In conclusion, ten chlorophyll rye mutants forwhich seven genes are responsible: chl1 cbl2, chl3,lg, ync, wyv1 and wyv2 were described, andsimilarities as well as differences in their expres-sion at the level of plastid ultrastructure and bothchlorophyll (a and b) contents were found.Despite of the fact that each of these chloro phyllmutants were earlier described (GABARA andKUBICKA 1991; GABARA et al. 1989; KUBICKA et al.1986, 1988, 1998, 2000), the majority aim of thispaper was comparative analysis lethal andsemilethal mutants in rye.

We hope that these studies allow us to un-derstand better the mechanisms of varied ex-

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pression of the same genes in different geno-types.

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Received 5 August 2000; accepted 21 September 2000

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