The Mechanism of Somatic Association in Common Wheat, Trztzcum Aestzvum L. Iv. Further Evidence for Modification of Spindle Tubulin through the Somatic-association Genes as Measured by Vinblastine Binding

Treatment with the antitubulin vinblastine was found to disrupt the spindle system in dividing root-tip cells of common wheat, Triticum aestiuum L. Genotypes lacking the somatic association suppressor gene on 5BL, or containing the somatic-association promoter on 5BS, were found to be more sensitive to the treatment. In genetic lines carrying the somatic association suppressor, sensitivity to vinblastine was lower and there was a direct correlation between dosage of the suppressor gene (0,2, and 4) and the decrease in spindle disruption on exposure to various concentrations of vinblastine. I t is concluded that the somatic association genes affect binding ability of spindle tubulin to vinblastine. Since the same genes affect binding of colchicine to tubulin and since the two alkaloids attach to different sites it is assumed that the somatic association suppressor gene has a broad effect on the tubulin molecules which is not confined to a single site. The relevance of genetic control of antitubulin binding to somatic association is discussed. a previous study of somatic association in common wheat (AVIVI, FELDMAN 'Ynd BUSHUK 1970a; 1970b) it was found that the genes responsible for this association, which are located on the chromosomes of homoeologous group 5 , affect some of the functional characteristics of the spindle system. In dividing roottip cells, spindle sensitivity to colchine decreased with increasing dosage from zero to four of the somatic-association suppressor gene which is located on the long arm of chromosome 5B (5BL). On the other hand, the spindle became more sensitive to chochicine when the dosage of one of the somatic-association promoters, located on the short arm of chromosome 5B ( 5BS), was increased from zero to two. Since colchicine binds specifically to the subunits of microtubules, i.e., tubulin (ADELMAN et al. 1968; WEISENBERG, BORISY and TAYLOR 1968), it was postulated that the somatic-association genes regulate the association of homologous chromosomes in somatic cells by affecting some of the characteristics of spindle tubulin. The somatic association suppressor presumably diminishes the affinity of tubulin for colchicine. It was suggested by us that this lowered Genetics 73: 379-385 March, 1973. 380 L. AVIVI A N D M. FELDMAN affinity is due to genetic modification of the primary structure or allosteric conformation of spindle microtubule subunits. The alkaloid vinblastine derived from the periwinkle plant (Vinca rosea L.), resembles colchicine in producing c-mitosis and reducing the birefringence of the mitotic spindle in dividing living cells (CUTTS 1961 ; CARDINALI, CARDINALI and MEHROTA 1963; MALAWISTA, BENSCH and SATO 1968). Vinblastine, like colchicine, is an antitubulin (MARGULIS 1972) and exerts its antimitotic effect through action on the spindle microtubules (KRISHAN 1968; WISNIEWSKI. SHELANSKI and TERRY 1968; MALAWISHTA et al. 1969; BENSCH and MALAWISTA 1969). However, there appear to be differences between the mechanisms of action of the two alkaloids. There is suggestive evidence that vinblastine and colchicine do not compete for the same binding site on tubulin (CREASEY and CHAU 1968; OLMSTED et aZ. 1970) but that separate sites exist for each. The presence of different binding sites for these two antitubulins can be used for gauging the extent of tubulin modification caused by the somatic-association suppressor gene. The pattern of differential sensitivity of spindles to colchicine in genetic lines carrying different doses of the somatic-association genes is known to us. A study of the pattern of sensitivity to vinblastine in the same lines can indicate whether the somatic-association suppressor gene exerts its effect only on the colchicine binding site or whether its action is more profound and modifies also other sites on the tubulin molecule. MATERIALS A N D METHODS The material used for studying spindle microtubule sensiti\ity to vinblastine in u'uo was the same as that used in a parallel study of the disruptive action of colchicine (AVIVI, FELDMAN and BUSHUK 1970a). Lines of common wheat variety Chinese Spring having different doses of chromosomal arm 5BL, carrier of the somatic association suppressor gene (FELDMAN 1966; 1968), or 5BS, carrier of one of the somatic association promoters (FELDMAN and MELLO-SAMPAYO 1967; FELDMAN 1968), were used as set out in Table 1 of the previous paper in this series (AVIVI, FELDMAN and BUSHUK 1970a). Chromosomal constitution of each seedling used was verified by cytological examination of root-tip cells. Seeds were germinated in distilled and deionized water at room temperature (about 22°C). After 48 hours, when the seminal roots were 1.0-1.5 cm long, the seedlings were transferred for a period of four hours to 5.0 cm Petri dishes containing 2.5 ml of vinblastine sulfate (Velban) solution. Control seedlings were maintained for four hours under similar conditions in distilled and deionized water. Velban was obtained from Eli Lilly and Company, Indianapolis, Indiana, U.S.A. Solutions of the following molar concentrations were freshly prepared using distilled and deionized water: 5 x 10-6, 1 x 10-5, 2 x le5, 3 x 10-5, 5 x 10-5, and 1 x 10-4 M. Higher concentrations were not used since these affect cell viability. After being treated the root-tips were severed and squashes were made by the acetocarmine staining technique. A total of several hundred cells derived from six to eight seedlings were examined under each treatment. These cells represent three replications. The metaphase cell population was classified into two categories: ( 1 ) cells exhibiting typical c-mitosis (Figure 1) designated as totally affected cells; (2) cells exhibiting only partial suppression of the spindle designated as partially affected cells and, in the same category, unaffected cells (Figure 1). The degree of spindle sensitivity to vinblastine was estimated for each dose of the somatic association genes under different Velban treatments. This statistic was obtained by calculating the percentage of totally affected cells in the entire metaphase cell population. SOMATIC ASSOCIATION IN WHEAT. IV.