At anthesis, petunia pollen contains large amounts of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). This ACC is thought to contribute to the rapid burst of ethylene produced by the pistil following pollination. An analysis of ACC content in developing anthers revealed that ACC began to accumulate the day before anthesis, indicating its synthesis was a late event in pollen development. We employed degenerate DNA primers to conserved amino acid sequences of ACC synthesis to amplify a cDNA from anther mRNA by RT-PCR. The resulting cDNA (pACS2) was sequenced and found to represent ACC synthase. Use of pACS2 as a hybridization probe revealed an increase in ACC synthase mRNA concomitant with the increase in ACC content. Further analysis indicated the ACC synthase mRNA was localized specifically to the haploid pollen grain. In an attempt to determine the function of ACC in pollen maturation or pollen–pistil interactions, we have generated a series of transgenic petunias designed to inhibit the accumulation of ACC in pollen. For these experiments, we have employed a pollen-specific promoter (LAT52) from tomato to drive the expression of antisense pACS2 or the coding region of ACC deaminase. The results of the experiments will be discussed.
Chih-Hsien Lei, Jon T. Lindstrom, and William R. Woodson
Jon T. Lindstrom, Chih-Hsien Lei, and William R. Woodson
Petunia hybrida pollen accumulates significant levels of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) late in development. This pollen ACC is thought to play a role in the rapid burst of ethylene produced by pollinated pistils. To investigate this further, we have expressed the ACC deaminase gene product from Pseudomonas in transgenic petunias under the control of three different promoters including CaMV-35S, LAT52, and TA29 directing construction expression, pollen-specific expression and tapetum-specific expression, respectively. Several transgenic plants expressing the LAT52-ACC deaminase gene exhibited significant reduction of ACC in pollen. Two independent transformants contained only trace amounts of ACC in pollen. In contrast, the other promoters did not lead to reduced ACC in pollen. Pollination of wild-type pistils with pollen from LAT52-ACC deaminase plants elicited increased ethylene similar to wild-type pollen. Fecundity was unaffected by the reduction in pollen ACC content. Taken together, we conclude pollen-borne ACC is not the elicitor of pollination-induced ethylene production by pistils.
Jon T. Lindstrom, Chih-Hsien Lei, Michelle L. Jones, and William R. Woodson
Mature pollen from Petunia hybrida contains significant levels of 1-aminocyclopropane-1-carboxylic acid (ACC), and this ACC is thought to play a role in pollination-induced ethylene by the pistil. We investigated the developmental accumulation of ACC in anthers and pollen. The level of ACC in anthers was very low until the day before anthesis, at which time it increased 100-fold. A 1.1-kb partial ACC synthase cDNA clone (pPHACS2) was amplified from total RNA isolated from mature anthers by reverse transcriptase, followed by polymerase chain reaction using oligonucleotide primers synthesized to conserved amino acid sequences in ACC synthases. The expression of pPHACS2 mRNA during anther development was correlated with the accumulation of ACC and was localized to the pollen grain. The pPHACS2 cDNA was used to identify the PH-ACS2 gene from a library of genomic DNA fragments from Petunia hybrida. PH-ACS2 encoded an ACC synthase transcript of four exons interrupted by three introns. The ACC synthase protein encoded by the PH-ACS2 gene shared >80% homology with ACC synthases from tomato (LE-ACS3) and potato (ST-ACS1a). A chimeric PH-ACS2 promoter-β-glucuronidase (GUS) gene was used to transform petunia and transgenic plants were analyzed for GUS activity. GUS staining was localized to mature pollen grains and was not detected in other tissues. Despite similarities to LE-ACS3, we did not detect GUS activity under conditions of anaerobic stress or in response to auxin. A series of 5-prime-flanking DNA deletions revealed that sequences within the PH-ACS2 promoter were responsible for pollen-specific expression.