V.S.Bogdanova, Y.A.Trusov, and V.A.Berdnikov

Institute of Cytology and Genetics, Novosibirsk, 630090 Russia

Instability of expression of genetic loci is usually represented by variegated phenotypes as sectors of wild-type tissue on the otherwise mutant background or vice versa. The phenomenon is rather frequent in plants and can be observed in the sectored and spotted patterns of flower or chlorophyll pigmentation. An unstable behaviour of mutable alleles with high frequency of reversion is usually attributed to the insertion and excision of transposable elements (16). Many unstable alleles of different loci in maize and some alleles in snapdragon were molecularly and genetically characterized and shown to harbor controlling elements (rev. in 8).

Transposon-like sequences were described in other plant species such as Glycine max (22), Triticum aestivum (11), Petroselinum crispum (13), Pisum sativum (4, 20). However, these sequences were not found to transpose or to be connected with genetic instability. On the other hand, there is a large number of mutable loci described in various plant species which are, as implied by genetic analysis, associated with transposable elements (see for example, 5 and references therein). Lack of appropriate molecular probes hinders establishing of unambiguous connection between genetic instability and transposition of a certain element.

Histone H1 of Pisum sativum is encoded by at least three genetic loci. The gene of the most abundant subtype 1 (His1) is localized in chromosome 5 (19), gene of subtype 7 (His7) - in chromosome 1 (15) and genes of subtypes 2 - 6 (His2-His6) are tightly linked (2) and are located, also in chromosome 1, as a gene cluster named His(2-6) (1). In the present paper we describe a phenomenon of unstable expression of a gene encoding one subtype of histone H1 in Pisum sativum L., the mutable allele being isolated from a collection of germplasm of All-Russian Plant Breeding Institute (VIR).

Materials and methods.

Plant material was originally obtained from the All-Russian Plant Breeding Institute, St.Petersburg, Russia and from Nordic Gene Bank, Weibullsholm, Landskrona, Sweden. On the base of this material in our laboratory there was constructed a series of early-ripening lines, which were included in our analysis.

Designation of allelic combinations, or haplotypes, of H1 subtypes 3-6 was as accepted in (2) (the allelic state of subtype 2, belonging to the same cluster, is not included since its "normal" allele predominates overwhelmingly). Namely, haplotype was designated by a formula consisting of a combination of four digits. The first position in the formula referred to subtype 3, the second - to subtype 4 and so on. The digit itself reflected the allelic state of the relevant subtype. Alleles of each gene were numbered according to the increase of electrophoretic mobility of the encoded protein, 1 referring to the slowest variant, 2 to the second slow and so on. 0 referred to the absence of a corresponding electrophoretic band.

Histone H1 was isolated by Johns' (14) method with further modifications. About 200-500 mg of pea leaves were homogenized in 10 ml of 0.15 M NaCl, the homogenate was filtered through a grid (1x1 mm2) of stainless steel and centrifuged at 1500 g for 5 min. Histone H1 was extracted by resuspending the pellet in 1 ml of 5% HClO4. After centrifugation the protein was recovered from the supernatant by adding 6 volumes of cold acetone and sulphuric acid to a concentration of 0.5 M. The precipitated protein was centrifuged and then dissolved in 0.02 ml of a medium containing 0.9 M acetic acid, 8 M urea, and 15% sucrose. The preparations were subjected to electrophoresis in long (up to 40 cm) slabs of 15% polyacrylamide/0.5% N,N'- methylenbisacrilamide gel containing 8 M urea and 0.9 M acetic acid following a modification (3) of Panyim and Chalkley's (18) method. After electrophoresis, the gels were stained in 0.01% Coomassi R-250 in 0.9 M acetic acid.

Results

In our previous work (2) we have performed an extensive analysis of histone H1 of peas originating from different regions of the Old World. Among 883 analyzed accessions we found one - VIR-4362 originating from the Vologda region - possessing an unusual haplotype with an absence of subtypes 3 and 5 - 0101. One plant with the haplotype 0101 was crossed (as a female parent) to a tester line from our collection possessing haplotype 1211 (in our designation "Sprint-1211"). Unexpectedly, in F1 generation we did not observe the uniformity of histone H1 phenotype. Instead of plants heterozygous for subtype 4 (second position in the formula) and hemizygous for subtypes 3 and 5 (looking like homozygote for allele 1 of subtype 3 and homozygote for allele 1 of subtype 5) we found F1 hybrids displaying two electrophoretic bands corresponding to subtype 5, namely to its allelic variants 1 and 2 (figure 1), the latter being absent from both parents. The second unusual feature of these hybrids is variable level of expression of the novel allele 2. It varies from zero (fig.1, lane D) through weak (fig.1, lanes E and C) up to the level of expression of a tester allele 1 (fig.1, lane B) forming a common heterozygous phenotype. Further in the text the allele of subtype 5 coming from the VIR-4362 stock is termed as the "Vologda" allele.

Figure 1.

We isolated histone H1 from different parts of the same F1 plant to analyze distribution of activity of the Vologda allele. As shown in the figure 2 we observed intraplant variability of its expression.

Figure 2.

Segregation of alleles of subtype 5 in F2 generation was as follows. F1 plants with equal expression of the Vologda allele and tester allele 1 (fig.1, lane B) gave rise to a progeny where all chromosomes carrying His2-6 locus from the VIR-4362 parent were of 0121 haplotype both in homozygotes and in heterozygotes (figure 3), thus indicating that an event of activation of the Vologda allele took place in the germ line tissue and generated a stable active derivative. All the other F1 plants resulted in progeny with variable expression of the Vologda allele resembling that of F1 generation.

Figure 3.

We analyzed expression of allele 2 of subtype 5 in the VIR-4362 stock. Among 328 plants only one was found to display high level of expression while others either lacked corresponding band, or it was hardly detectable and distinguishable only in heavily loaded samples. Thus, occurrence of the high level of expression of the Vologda allele was met much more rarely than after cross-pollination - one case among 328 plants analyzed (compared to 2 plants of 10 tested in F1).

It is interesting to note that the only plant with high expression of the Vologda allele appeared to be a heterozygote since in its progeny were observed 0101 haplotypes, intermediates and 0121 haplotypes with a proportion of 0101 12 plants of 39 tested, which corresponds well to 1/4 (chi-sq. equal to 0.69, p > 0.7).

Discussion

The simplest implication for observed activation of the "latent" allele after crossing is to suggest that expression of the Vologda allele in the haplotype 0101 is "turned off" by an insertion of a controlling element. After cross-pollination transposon activates and begins to excise with resulting restoration of expression of the Vologda allele. In the light of this suggestion it is reasonable to expect that heterozygous plants displaying weak expression of allele 2 are the mixtures of cell clones with active and inactive Vologda allele. As shown in the fig.2 different parts of the plant vary in the intensity of electrophoretic band corresponding to the Vologda allele. Thus, we can speak of variegated phenotype in respect of expression of the subtype 5 gene His5 of histone H1.

A mode of expression of the Vologda allele in VIR-4362 stock is in a good accordance with the assumption of a transposable element residing in the His5 gene. Weak expression appears to be a consequence of a somatic reversion while absence of detectable expression may be due to the fluctuation of activating factor(s)' concentration and its decrease below some threshold level.

We observed both somatic and germinal reversion to a high level of expression of His5 gene. Germinal reversion generated stably expressed derivative corresponding to the 2 allele of subtype 5 detectable just after cross-pollination in F1. Unfortunately, due to insufficient number of F1 plants available we can not estimate the frequency of germinal reversion. However, we can anticipate it to be rather high (2 plants of 10 tested). Preliminary data suggest that stable null derivatives also occur.

Interestingly, in 17 F1 hybrids from the crosses of the VIR-4362 plant as a pollen parent with tester lines of 1133 and 1211 haplotypes we did not observe detectable expression of the Vologda allele. However in F2 progeny from these crosses activation of the Vologda allele was apparent. Dependence of the maize Spm element transposition frequency on the direction of the cross was observed in some cases (7, 17).

The nature of activating factors responsible for the transpositions of the element remains unclear. It may be another transposon - an active member of a two-component system (rev. in 10) or some chromosomal gene coding for transposase. We know a number of genes in pea which condition variegated patterns of a seed coat coloration such as F and Fs (violet spots), M (marble spotting), Ust (purple stripes) (6). There are unstable alleles affecting other parts of the plant - for example, And produces purple spots on leaves (21), Pur (purple pod) is known to mutate frequently to weaker alleles (6). The products encoded by these genes are not known, however, one can speculate that these genes code for a kind of transposase which could be utilized by the transposon residing in the His5 gene. At least, they could serve as markers responding to the presence of transposase, of course, if these genes and His5 gene contain related transposons. It should be noted that VIR-4362 stock lacks detectable coloration of the seed coat while the majority of peas from VIR collection displays either F/Fs or M spotting, or both.

Inversely, VIR-4362 may contain a gene responsible for a repressor of transposition like a st gene of Antirrhinum majus (12), this is the subject of the further genetic analysis.

In conclusion it should be noted that the presence of a putative transposon in the His5 gene provides a good opportunity for isolation and molecular characterization of the element since the DNA sequence of a histone H1 gene from Pisum sativum is determined (9). In turn, this opens the way for transposon tagging and further investigation of the pea genome.

This work was partly supported by the Russion National Program "Basic Research Foundation".

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