The expression of polyubiquitin and ubiquitin-extension genes is independent and stage-specific during microsporogenesis and gametophyte development
--R. A. Bouchard, J. R. H. Frappier, Ling Liu, B. G. Atkinson and D. B. Walden

Recent work employing cloned probes which represent members of the small heat shock protein genes of maize has shown that transcripts from some members of this stress-inducible gene family also accumulate in maize microsporogenic tissues during prophase and later stages of male gametophyte development (Bouchard and Walden, MNL 1990; Dietrich et al., Plant Physiol. 96:1268, 1991; Atkinson et al., this Newsletter). In light of the results with this stress-induced gene family, we have examined the developmental induction of other heat shock gene families during this important developmental sequence.

One such family is comprised of various genes that produce transcripts encoding the ubiquitin polypeptide. Maize cDNA or genomic clones representing a number of these genes have recently been isolated and characterized (Liu, PhD. Thesis, U. Western Ontario, 1991.; Liu et al., this Newsletter). We have used probes derived from these Mub (Maize-ubiquitin) clones to follow the overall accumulation of ubiquitin-encoding RNAs across maize microsporogenesis and gametophyte development by RNA-dot hybridizations. The RNAs used in these experiments were prepared from premeiotic tassels, staged and sorted maize anthers, and pollen, along with control and heat-shocked plumules of five-day seedlings. Our results indicate that there is in fact strong accumulation during development of RNA transcripts representing members of the ubiquitin gene family in maize. In addition, we find that this accumulation of transcripts is modulated independently for gene family members encoding two distinct types of ubiquitin polypeptides: polyubiquitins and "ubiquitin-fusion" proteins.

Because of the high evolutionary conservation of the ubiquitin ORF, a DNA fragment representing this region from the clone cMubC1 was used to determine the aggregate abundance of all transcripts encoding this polypeptide. The results indicate that the collective abundance of transcripts encoding the ubiquitin polypeptide itself is elevated throughout the entire developmental sequence relative to what is seen in either control or heat-shocked somatic tissue, with its apparent maximum late in development, after the haploid mitosis producing the binucleate gametophyte.

In order to compare this aggregate accumulation with that seen for individual members of the Mub gene family, subfragments of clones representing gene-specific regions were employed. Results obtained with probe MubC1-3, which represents the 3' untranslated region of a polyubiquitin gene, show a two-fold heat induced accumulation on polyribosomes in somatic tissue (Liu et al., this Newsletter). Across microsporogenesis and gametophyte development, the transcript of this gene shows an increase which parallels that seen for ubiquitin-encoding transcripts in general, but which appears to be even more pronounced at the end of the sequence, in anthers containing mature unshed pollen and in freshly shed pollen.

A contrasting pattern was found using a probe scMubG10-E, representing the 5' untranslated region of a particular type of ubiquitin gene, one encoding what is called a "fusion" or "extension" protein. This class of ubiquitin polypeptides consists of a single ubiquitin polypeptide segment fused to a distinctly different polypeptide containing a zinc-finger region; in yeast this portion of the protein has been shown to be a ribosomal component while the ubiquitin unit to which it is attached is required for efficient biogenesis of the ribosomes (Finley et al., Nature 338:394, 1989). Transcripts from this gene show their highest abundance at the beginning of the male developmental sequence, in premeiotic tassel and in anthers containing early microsporocytes. Interestingly, in addition to this independence in developmental regulation relative to MubC1, there is also a difference in accumulation of these transcripts on polyribosomes in somatic tissue during heat shock, where MubG10 transcripts show a decline in response to heat shock (Liu et al., this Newsletter). The high level of fusion-protein transcripts found in early maize PMC development (prior to and during prophase) may be significant in relation to the major turn-over of ribosomes that has been observed during meiotic prophase in other plant systems (Porter et al., J. Cell Sci. 62:177, 1983).

The maize genome also contains at least one polyubiquitin encoding gene, g/cMubC9, whose transcripts exhibit neither an increase nor a decrease in accumulation on polyribosomes of somatic tissue during heat shock (Liu et al., this Newsletter). The developmental modulation of transcripts detected by a probe specific for the 3' untranslated region of this gene is distinct from either of the others. Indeed, the accumulation of this transcript appears to parallel the pattern of aggregate abundance seen with the ubiquitin ORF probe (cMubC1).

The results of these studies indicate that, in addition to their heat shock regulation, transcripts from specific members of the ubiquitin gene family do exhibit developmental modulation during male meiosis and gametophyte development in maize. Moreover, specific members of this gene family encoding different types of ubiquitin-containing polypeptides show independent regulation. These observations are very analogous to the patterns emerging for members of the sHSP (small heat shock protein) family (Atkinson et al., this Newsletter). Programmed expression of specific gene family members may well prove to be a general feature of stress-gene families during this key sequence of development. 

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