Characterization of four new beta-tubulin genes and their expression during male flower development in maize (Zea mays L.)

Abstract

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 clones together with the tub1 and tub2 genes reported previously encode six beta-tubulin isotypes with 90-97.5% amino acid sequence identity. Results from phylogenetic analysis of 17 beta-tubulin genes from monocot and dicot plant species indicated that multiple extant lines of beta-tubulin 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-tubulin clones were used to quantify the levels of corresponding tubulin transcripts in different maize tissues including developing anthers and pollen. The results from these dot blot hybridization experiments showed that all of the beta-tubulin genes were expressed in most tissues examined, although each gene showed a unique pattern of differential transcript accumulation. The tub1 gene showed a high level of transcript accumulation in meristematic tissues and almost no accumulation in the late stages of anther development and in pollen. In contrast, the level of tub4 transcripts was very low during early stages of male flower development but increased markedly (more than 100 times) during the development of anthers and in pollen. Results from RNAse protection assays showed that this increased hybridization signal resulted from expression of transcripts from one or two genes closely related to tub4. The tub4-related transcripts were not present in shoot tissue. Transcripts from the tub2 gene accumulated to very low levels in all tissues examined, but reached the highest levels in young anthers containing microspore mother cells. RNAse protection assays were used to measure the absolute levels of alpha- and beta-tubulin transcripts in seedling shoot and in pollen. The alpha-tubulin gene subfamily I genes (tua1, tua2, tua4) contributed the great majority of alpha-tubulin transcripts in both shoot and pollen. Transcripts from the beta-tubulin genes tub4, tub6, tub7, and tub8 were predominant in shoot, but were much less significant than the tub4-related transcripts in pollen.