Dynamic 1-aminocyclopropane-1-carboxylate-synthase and -oxidase transcript accumulation patterns during pollen tube growth in tobacco styles

Abstract

In flowering plants, pollination of the stigma sets off a cascade of responses in the distal flower organs. Ethylene and its biosynthetic precursor 1-aminocyclopropane-1-carboxylate (ACC) play an important role in regulating these responses. Because exogenous application of ethylene or ACC does not invoke the full postpollination syndrome, the pollination signal probably consists of a more complex set of stimuli. We set out to study how and when the pollination signal moves through the style of tobacco (Nicotiana tabacum) by analyzing the expression patterns of pistil-expressed ACC-synthase and -oxidase genes. Results from this analysis showed that pollination induces high ACC-oxidase transcript levels in all cells of the transmitting tissue. ACC-synthase mRNA accumulated only in a subset of transmitting tract cells and to lower levels as compared with ACC-oxidase. More significantly, we found that although ACC-oxidase transcripts accumulate to uniform high levels, the ACC-synthase transcripts accumulate in a wave-like pattern in which the peak coincides with the front of the ingrowing pollen tube tips. This wave of ACC-synthase expression can also be induced by incongruous pollination and (partially) by wounding. This indicates that wounding-like features of pollen tube invasion might be part of the stimuli evoking the postpollination response and that these stimuli are interpreted differently by the regulatory mechanisms of the ACC-synthase and -oxidase genes.

Figure 1

Accumulation of ACC-synthase and -oxidase transcripts in various plant tissues. Each lane contained 1 μg of poly(A⁺) RNA from seedlings (SD), stems (S), leaves (L), sepals (SE), petals (PE), flower stage 12 pistils (PI), flower stage 12 anthers (AN), mature pollen (P), 12-HAP stigmas and styles (ST), and 12-HAP ovaries (OV). Autoradiogram exposure times: 48 h for ACCS2 probe and 3 h for TEFE probe.

Figure 2

Accumulation of ACC-synthase and -oxidase transcripts during development of the stigma and style. Each lane contained 20 μg of total RNA from stigma and style of flower stages 1 through 12 (see “Materials and Methods”). Autoradiogram exposure times: 2 d.

Figure 3

ACC-synthase and -oxidase transcript accumulation induction in flower stage 12 and 6 stigmas and styles. Each lane contained 5 μg of total RNA of 0-, 3-, 6-, 12-, and 24-HAP stigmas and styles of flower stage 12 or 6. Autoradiogram exposure times: stage 12, 3 d for ACCS2 and 1 d for TEFE probe; and stage 6, 3 d for ACCS2 and 1 d for TEFE probe.

Figure 4

ACC-synthase and -oxidase mRNA localization patterns in pollinated and non-pollinated styles. The lower part of 16-HAP or non-pollinated styles were fixed, embedded in paraffin, sliced into 10-μm sections, and hybridized with ³³P-labeled sense or antisense probes as described in “Materials and Methods.” Photographs were taken by dark-field microscopy. A and B, Hybridization of ACCS2 antisense probe with non-pollinated (A) and pollinated (B) styles. C and D, Hybridization of TEFE antisense probe with non-pollinated (C) and pollinated (D) styles. E and F, Hybridization of ACCS2 sense probe with non-pollinated (E) and pollinated (F) styles. G and H, Hybridization of TEFE sense probe with non-pollinated (G) and pollinated (H) styles. Emulsion exposure times: 44 d for ACCS2 probes and 4 d for TEFE probes. c, Cortex; ep, epidermis; tt, transmitting tissue; v, vascular bundle.

Figure 5

ACC-synthase mRNA accumulation patterns in stigma and style parts at different time intervals after congruous and incongruous pollination and after wounding. Graphical representation of ACC-synthase transcript accumulation levels in four consecutive segments of non-pollinated (X), tobacco-pollinated (▪), P. hybrida-pollinated (●), and wounded (♦) pistils at 0, 3, 6, 12, 24, and 48 h after treatment. Segment one represents the stigma and segments two, three, and four represent upper, middle, and lower style (including the style-ovary transition zone), respectively. The accumulation levels are expressed in picograms per microgram total RNA and were obtained from calibrated RNA gel blots as described in “Materials and Methods.” In the pistil drawing over each graph, the position of the front of the pollen tube tips or needle is indicated by the respective symbol. Autoradiogram exposure times: 1 week for non-pollinated pistils and 2 d for other treatments.

Figure 6

ACC-oxidase mRNA accumulation patterns in stigma and style parts at different time intervals after congruous and incongruous pollination and after wounding. Graphical representation of ACC-oxidase transcript accumulation levels in four consecutive segments of non-pollinated (X), tobacco-pollinated (▪), P. hybrida-pollinated (●), and wounded (♦) pistils at 0, 3, 6, 12, 24, and 48 h after treatment. Segment one represents the stigma and segments two, three, and four represent upper, middle, and lower style (including the style-ovary transition zone), respectively. The accumulation levels are expressed in picograms per microgram total RNA and were obtained from calibrated RNA gel blots as described in “Materials and Methods.” In the pistil drawing over each graph, the position of the tip of the pollen tubes or needle is indicated by the respective symbol. Autoradiogram exposure times: 16 h.

Figure 7

Stylar ACC-synthase- and -oxidase mRNA accumulation levels related to number of ingrowing pollen tubes. A through D, Aniline blue-stained 100-μm vibratome sections of stigma and style of wild-type (A and B) and stigmaless (C and D) tobacco plants, pollinated with either tobacco (A and C) or P. hybrida (B and D) pollen. All pollinations of stigmaless tobacco plants were performed with added stigmatic exudate. Photographs were taken by epi-fluorescence microscopy. E through H, Line graphs of ACC-synthase and -oxidase mRNA accumulation at 0, 3, 6, 12, and 24 HAP in styles of wild-type (E and F) and stigmaless tobacco plants (G and H), pollinated with either tobacco (E and G) or P. hybrida (F and H) pollen. The mRNA levels were measured as described in “Materials and Methods” from autoradiograms of calibrated northern blots containing 5 μg of total RNA per lane and exposed for 2 d (ACCS2 probe) or 1 d (TEFE probe). mRNA levels are expressed as picogram per microgram total RNA. pt, Pollen tubes.