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.
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.
Diurnal variation in the chilling sensitivity of tomato seedlings was examined. Sensitivity to chilling in tomato seedlings is a response to light and not under the control of a circadian rhythm. Chilling sensitivity is highest in seedlings chilled at the end of the dark period, and these seedlings become more resistant to chilling injury upon exposure to the light. Diurnal variation in chilling sensitivity was associated with changes in catalase and superoxide dismutase activities. The results show an increase in catalase and superoxide dismutase activities at the end of the light period. The recovery of the net photosynthesis rate following chilling was faster in seedlings chilled at the end of the light period. It is suggested that an increase in catalase and superoxide dismutase activities at the end of light period before the chilling plays a role in the resistance to chilling stress in tomato seedlings. Forty-eight hours of 14°C acclimation or hydrogen peroxide pretreatment conferred chilling tolerance to tomato seedlings and were correlated with elevated catalase activity. Acclimated seedlings still exhibited diurnal variation in chilling sensitivity while hydrogen peroxide treated seedlings showed little evidence of a diurnal variation in chilling sensitivity. Transgenic tomato plants expressing an antisense catalase gene were generated. A several-fold decrease in total catalase has been detected in the leaf extracts of transformants. Preliminary analysis of these plants indicated that modification of reactive oxygen species scavenging in plant system can lead to change in oxidative stress tolerance.
The senescence of carnation (Dianthus caryophyllus L.) flower petals is associated with increased synthesis of the phytohormone ethylene. This ethylene serves to initiate and regulate the processes of programmed cell death. We are using molecular approaches to study the regulation of ethylene biosynthesis in various floral organs during development and senescence of flowers. We have isolated and cloned mRNAs which encode the ethylene biosynthetic pathway enzymes s-adenosylmethionine (SAM) synthetase, 1-aminocyclopropane-1-carboxylate (ACC) synthase and the ethylene forming enzyme (EFE) from carnation flower petals. These cDNAs have been used as molecular probes to determine the steady-state mRNA levels of these transcripts in senescing flowers. The increase in ethylene associated with petal senescence is accompanied by a dramatic increase in the abundance of transcripts for both ACC synthase and EFE. In striking contrast, the level of SAM synthetase mRNA decreases significantly with the onset of petal senescence. Genomic DNA Southern blots reveal both ACC synthase and EFE are encoded by multigene families. We have recently isolated several genomic clones from carnation which represent different ACC synthase genes. The structure and organization of these gene will be presented.
Diurnal variation in the chilling sensitivity of `Rutgers' tomato (Lycopersicon esculentum Mill.) seedlings was examined. Chilling sensitivity was highest in seedlings chilled at the end of the dark period, and these seedlings became more resistant to chilling injury on exposure to the light. The development of chilling tolerance in tomato seedlings was a response to light and not under the control of a circadian rhythm. The recovery of leaf gas exchange following chilling was faster in seedlings chilled at the end of the light period. Diurnal variation in chilling sensitivity was associated with changes in catalase and superoxide dismutase activities. An increase in catalase and superoxide dismutase activities was observed at the end of the light period. Catalase activity was significantly higher in all stages of chilling following the light period compared to those chilled after the end of the dark period. Forty-eight hours of 14 °C acclimation or pretreatment with hydrogen peroxide conferred increased chilling tolerance to tomato seedlings. Hydrogen peroxide-treated seedlings showed little evidence of a diurnal variation in chilling sensitivity. These results support a role for light and oxidative stress in conferring increased chilling tolerance to tomato seedlings.
Rooted cuttings of Chrysanthemum × morifolium Ramat. ‘Gt. #4 Indianapolis White’ were grown in a greenhouse in a sand culture and supplied with either 3.75 or 15.0 mm. Changes in dry matter, reduced N and of the leaves, stems (plus petioles), roots, and inflorescence and in vivo reductase activity (NRA) of leaves were determined at various stages of development. A decrease in the supply caused a decrease in the accumulation of plant dry matter, reduced N and . Plants receiving 3.75 mm remobilized a significant amount of reduced N from vegetative tissues during inflorescence development, suggesting that newly absorbed N was inadequate to supply the flower. At both fertilization levels, the content of the leaves and stems declined during inflorescence development, suggesting an increased dependence on previously accumulated for reduction. The highest NRA of the leaves (3.4 μmoles NO2 gFW−1 hr−1) was associated with early vegetative growth. NRA, however, was detectable throughout plant development. Nitrate reductase activity was greater at 15 mM than at 3.75 mM during vegetative growth and visible bud stages, but not at later stages of growth.
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.