Calcium dynamics in tomato pollen tubes using the Yellow Cameleon 3.6 sensor.

KEY MESSAGE: In vitro tomato pollen tubes show a cytoplasmic calcium gradient that oscillates with the same period as growth. Pollen tube growth requires coordination between the tip-focused cytoplasmic calcium concentration ([Ca2+]cyt) gradient and the actin cytoskeleton. This [Ca2+]cyt gradient is necessary for exocytosis of small vesicles, which contributes to the delivery of new membrane and cell wall at the pollen tube tip. The mechanisms that generate and maintain this [Ca2+]cyt gradient are not completely understood. Here, we studied calcium dynamics in tomato (Solanum lycopersicum) pollen tubes using transgenic tomato plants expressing the Yellow Cameleon 3.6 gene under the pollen-specific promoter LAT52. We use tomato as an experimental model because tomato is a Solanaceous plant that is easy to transform, and has an excellent genomic database and genetic stock center, and unlike Arabidopsis, tomato pollen is a good system to do biochemistry. We found that tomato pollen tubes showed an oscillating tip-focused [Ca2+]cyt gradient with the same period as growth. Then, we used a pharmacological approach to disturb the intracellular Ca2+ homeostasis, evaluating how the [Ca2+]cyt gradient, pollen germination and in vitro pollen tube growth were affected. We found that cyclopiazonic acid (CPA), a drug that inhibits plant PIIA-type Ca2+-ATPases, increased [Ca2+]cyt in the subapical zone, leading to the disappearance of the Ca2+ oscillations and inhibition of pollen tube growth. In contrast, 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of Ca2+ released from the endoplasmic reticulum to the cytoplasm in animals cells, completely reduced [Ca2+]cyt at the tip of the tube, blocked the gradient and arrested pollen tube growth. Although both drugs have antagonistic effects on [Ca2+]cyt, both inhibited pollen tube growth triggering the disappearance of the [Ca2+]cyt gradient. When CPA and 2-APB were combined, their individual inhibitory effects on pollen tube growth were partially compensated. Finally, we found that GsMTx-4, a peptide from spider venom that blocks stretch-activated Ca2+ channels, inhibited tomato pollen germination and had a heterogeneous effect on pollen tube growth, suggesting that these channels are also involved in the maintenance of the [Ca2+]cyt gradient. All these results indicate that tomato pollen tube is an excellent model to study calcium dynamics.

Transformation of the CmACS-7 gene into melon (Cucumis melo L.) using the pollen-tube pathway.

The aim of the present study was to develop a transformation system that may be useful for introducing agronomically and biotechnologically relevant traits into melon. The production of transplanted melon with maternal inheritance of the transgene could solve problems related to outcrossing between genetically modified crops and conventional crops or their wild relatives. By analyzing the main influencing factors systematically, the pollination time was ascertained and the pollen-tube pathway genetic transformation system was optimized. A screening system for resistant seeds from the T1 generation was established. The transformed seedlings were grown under standard field conditions and selected using a polymerase chain reaction-based analysis. The resistant plants were detected at a rate of 5%. These results indicate that enhanced production hastens the initiation of bisexual flowers, development of mature bisexual flowers, and fruit set in melon. We have established a melon transformation system based on the pollen-tube method.

Effects of low-energy argon ion implantation on the dynamic organization of the actin cytoskeleton during maize pollen germination.

The relationship between pollen germination and the dynamic organization of the actin cytoskeleton during pollen germination is a central theme in plant reproductive biology research. Maize (Zea mays) pollen grains were implanted with 30 keV argon ion (Ar(+)) beams at doses ranging from 0.78 x 10(15) to 13 x 10(15) ions/cm(2). The effects of low-energy ion implantation on pollen germination viability and the dynamic organization of the actin cytoskeleton during pollen germination were studied using confocal laser scanning microscopy. Maize pollen germination rate increased remarkably with Ar(+) dose, in the range from 3.9 x 10(15) to 6.5 x 10(15) ions/cm(2); the germination rate peaked at an Ar(+) dose of 5.2 x 10(15) ions/cm(2). When the implantation dose exceeded 7.8 x 10(15) ions/cm(2), the rate of pollen germination decreased sharply. The actin filaments assembled in pollen grains implanted with 5.2 x 10(15) ions/cm(2) Ar(+) much earlier than in controls. The actin filaments organized as longer parallel bundles and extended into the emerging pollen tube in treated pollen grains, while they formed random and loose fine bundles and were gathered at the pollen aperture in the control. The reorganization of actin cytoskeleton in the pollen implanted with 9.1 x 10(15) ions/cm(2) Ar(+) was slower than in controls. There was a positive correlation between pollen germination and the dynamic organization of the actin cytoskeleton during pollen germination. Ion implantation into pollen did not cause changes in the polarization of actin filaments and organelle dynamics in the pollen tubes. The effects of Ar(+) implantation on pollen germination could be mediated by changes in the polymerization and rearrangement of actin polymers.