Recombinant cDNA libraries to poly(A)RNA isolated from mature pollen of Zea mays and Tradescantia paludosa have been constructed. Northern blot analyses indicate that several of the clones are unique to pollen and are not expressed in vegetative tissues. The majority, however, are expressed both in pollen and vegetative tissues. Southern hybridizations show that the pollen specific sequences in corn are present in one or a very few copies in the genome. By using several of the clones as probes, it was found that there are at least two different groups of mRNAs with respect to their synthesis. The mRNAs of the first group represented by the pollen specific clones are synthesized after microspore mitosis and increase in concentration up to maturity. The second group, exemplified by actin mRNA, begins to accumulate soon after meiosis, reaches its maximum by late pollen interphase, and decreases thereafter. Although the actin mRNA and the pollen specific mRNAs studied show very different patterns of initiation of synthesis and accumulation during pollen development, the rates of decline of these mRNAs during the first 60 minutes of germination and pollen tube growth in Tradescantia are similar and reflect the previously observed declines in rates of protein synthesis during this period.The pollen grains of Tradescantia paludosa (16), corn (17), and tobacco (30) contain a store of presynthesized messenger RNAs (mRNAs) at the time of their release from the anther. These mRNAs have been shown to code in cell-free translation systems for proteins that are similar to the proteins synthesized during pollen germination and tube growth (9,17 shown that a large fraction (>64%) of the genes expressed in pollen are also expressed in vegetative tissues, whereas no more than 60% of the genes expressed in shoots are similar to those expressed in pollen (31). Similar hybridizations have been carried out with RNA from corn pollen and the results are similar to those obtained with Tradescantia (RP Willing, JP Mascarenhas, unpublished data). To further our knowledge of pollen development it is essential that the genes that are expressed in pollen, especially those unique to pollen, be isolated and characterized in some detail in order to understand their developmentally specific regulation and functions. We report here the construction of two cDNA libraries made to pollen mRNAs from Tradescantia and corn, and the utilization of some of the clones to answer questions about the nature of the pollen expressed genes and their transcription during pollen development and tube growth. MATERIALS AND METHODSPlant Material. Tradescantia paludosa L. plants were grown in the greenhouse and pollen was collected and stored as previously described (15). Corn (Zea mays L.) pollen was collected from field grown plants of the cultivar 'Gold Cup' (Harris Seeds, Rochester, NY). Pollen was quick frozen in liquid N2 and stored at -70°C. For later experiments the inbred line of corn W-22 (Illinois Foundation Seeds) was used. Various vegetative and ...

Numerous plant hormones interact during plant growth and development. Elucidating the role of these various hormones on particular tissue types or developmental stages has been difficult with exogenous applications or constitutive expression studies. Therefore, we used tissue-specific promoters expressing CKX1 and gai, genes involved in oxidative cytokinin degradation and gibberellin (GA) signal transduction, respectively, to study the roles of cytokinin and GA in male organ development. Accumulation of CKX1 in reproductive tissues of transgenic maize (Zea mays) resulted in male-sterile plants. The male development of these plants was restored by applications of kinetin and thidiazuron. Similarly, expression of gai specifically in anthers and pollen of tobacco (Nicotiana tabacum) and Arabidopsis resulted in the abortion of these respective tissues. The gai-induced male-sterile phenotype exhibited by the transgenic plants was reversible by exogenous applications of kinetin. Our results provide molecular evidence of the involvement of cytokinin and GA in male development and support the hypothesis that the male development is controlled in concert by multiple hormones. These studies also suggest a potential method for generating maintainable male sterility in plants by using existing agrochemicals that would reduce the expense of seed production for existing hybrid crops and provide a method to produce hybrid varieties of traditionally non-hybrid crops.Evidence of the involvement of cytokinins and GAs in male reproductive development of flowering plants has resulted from the studies of exogenous applications and endogenous measurements of cytokinins and GAs in various wild-type and male-sterile plants (for review, see Sawhney and Shukla, 1994). Typically, deficiencies in endogenous cytokinins and GAs result in the delay or elimination of anthesis, whereas exogenous applications shorten the time to anthesis. Anthers of several male-sterile mutants including the sl-2 (stamenless-2) mutant of tomato (Lycopersicon esculentum; Sawhney and Shukla, 1994) and a genic male-sterile line of rapeseed (Brassica napus; Shukla and Sawhney, 1993) have lower endogenous cytokinin levels. Cytokinins have also been shown to reverse cytoplasmic male sterility in barley (Hordeum vulgare;Ahokas, 1982). In the female plants of dioecious species such as hemp (Cannabis sativa) and spinach (Spinacia oleracea), and the gynoecious line of cucumber (Cucumis sativus), the formation of male flowers is promoted by exogenous applications of GAs (Mitchell and Wittwer, 1962;Pike and Peterson, 1969;Mohan Ram and Jaiswal, 1972; Khrianin, 1978a, 1978b). GAs also rescue fertility of male-sterile mutants of barley, cosmos (Cosmos bipinnatus), and tomato (Phatak et al., 1966;Kasembe, 1967;Rana and Jain, 1968;Sawhney and Greyson, 1973;Schmidt and Schmidt, 1981). Conversely, 2-chloroethyl-trimethyl ammonium chloride, a GA biosynthesis inhibitor, reduces the number of male flowers in cucumber (Mitchell and Wittwer, 1962). Moreover, when the endogenous GA le...

A pollen-specific cDNA clone, Zinc13, has been isolated from a cDNA library constructed to poly(A) RNA from mature maize pollen. The cDNA as shown by primer extension analysis is a full-length copy of the mRNA. The cDNA has been sequenced and is 929 nucleotides in length plus a 47-nucleotide poly(A) tail. Putative polyadenylation signals are identifiable in the 3'-nontranslated region. The mRNA codes for a predicted polypeptide containing 170 amino acid residues and with a molecular mass of 18.3 kilodaltons. The hydropathy profile suggests a possible signal sequence on the amino terminus. A comparison of the nucleotide and deduced amino acid sequence with sequences in data banks has not shown homology to known molecules. In situ hybridizations using RNA probes show that the mRNA is located in the cytoplasm of the vegetative cell of the pollen grain and after germination is distributed throughout the pollen tube cytoplasm.

A pollen-specific cDNA clone, Zinc13, has been isolated from a cDNA library constructed to poly(A) RNA from mature maize pollen. The cDNA as shown by primer extension analysis is a full-length copy of the mRNA. The cDNA has been sequenced and is 929 nucleotides in length plus a 47-nucleotide poly(A) tail. Putative polyadenylation signals are identifiable in the 3'-nontranslated region. The mRNA codes for a predicted polypeptide containing 170 amino acid residues and with a molecular mass of 18.3 kilodaltons. The hydropathy profile suggests a possible signal sequence on the amino terminus. A comparison of the nucleotide and deduced amino acid sequence with sequences in data banks has not shown homology to known molecules. In situ hybridizations using RNA probes show that the mRNA is located in the cytoplasm of the vegetative cell of the pollen grain and after germination is distributed throughout the pollen tube cytoplasm.