CHARACTERIZATION OF 4 NEW BETA-TUBULIN GENES AND THEIR EXPRESSION DURING MALE FLOWER DEVELOPMENT IN MAIZE (ZEA-MAYS L)

Four different beta-tubulin coding sequences were isolated from a cDNA library prepared from RNA from maize seedling shoots. The four genes (designated tub4, tub6, tub7 and tub8) represented by these cDNA clone s together with the tub1 and tub2 genes reported previously encode six beta-tubulin isotypes with 90-97.5% amino acid sequence identity. Res ults from phylogenetic analysis of 17 beta-tubulin genes from monocot and dicot plant species indicated that multiple extant lines of beta-t ubulin genes diverged from a single precursor after the appearance of the two major subfamilies of alpha-tubulin genes described previously. Hybridization probes from the 3' non-coding regions of six beta-tubul in clones were used to quantify the levels of corresponding tubulin tr anscripts in different maize tissues including developing anthers and pollen. The results from these dot blot hybridization experiments show ed that all of the beta-tubulin genes were expressed in most tissues e xamined, although each gene showed a unique pattern of differential tr anscript accumulation. The tub1 gene showed a high level of transcript accumulation in meristematic tissues and almost no accumulation in th e late stages of anther development and in pollen. In contrast, the le vel of tub4 transcripts was very low during early stages of male flowe r development but increased markedly (more than 100 times) during the development of anthers and in pollen. Results from RNAse protection as says showed that this increased hybridization signal resulted from exp ression of transcripts from one or two genes closely related to tub4. The tub4-related transcripts were not present in shoot tissue. Transcr ipts from the tub2 gene accumulated to very low levels in all tissues examined, but reached the highest levels in young anthers containing m icrospore mother cells. RNAse protection assays were used to measure t he absolute levels of alpha- and beta-tubulin transcripts in seedling shoot and in pollen. The alpha-tubulin gene subfamily I genes (tua1, t ua2, tua4) contributed the great majority of alpha-tubulin transcripts in both shoot and pollen. Transcripts from the beta-tubulin genes tub 4, tub6, rub7, and tub8 were predominant in shoot, but were much less significant than the tub4-related transcripts in pollen.