A role for aquaporins during pollen tube growth?

In spermatophytes, during fertilization pollen tubes are growing through the stigma tissue and deliver the sperm cells to the egg cell. Pollen tubes are the fastest growing plant cells and transport of water across the plasma membrane of the vegetative cell is important at least at three stages of germination and growth of pollen tubes: 1. dehydrated pollen grains landing on a compatible stigma have to re-hydrate, 2. cell turgor pressure has to be adjusted, and 3. water flow into the cytoplasm increases the cell volume during tube growth. Despite its importance, water transport across the plasma membrane of pollen grains and tubes was neither investigated nor quantified before, and thus any model describing pollen tube elongation is more or less speculative with respect to water transport. Water uptake may occur through the lipid bilayer of the plasma membrane itself or by facilitated diffusion through aquaporins, i.e. members of the PIP (Plasma membrane Intrinsic Proteins) family. We will measure the water transport across the plasma membrane in a model organism in pollen physiology, i.e. intact pollen grains of Lilium longiflorum, using the turgor pressure probe, and in protoplasts obtained from pollen grains and tubes using a swelling assay. The water permeability coefficient of the plasma membrane (Pw) will be calculated from the relaxation kinetics of the turgor pressure and from volume change rates of the protoplasts, respectively. Inhibition of water transport by mercurials and the amplitude of Pw may indicate the involvement of aquaporins in the water transport during pollen tube growth. A Lilium pollen grainube cDNA-library will be screened with an aquaporin-specific probe to identify, clone and sequence putative aquaporins in pollen. Using a swelling assay the transport properties of the identified aquaporins will be measured by injection of the respective mRNA into Xenopus oocytes as well as in transiently transformed pollen. Additionally, the water permeability as well as the germination frequency and tube growth rates will be determined in transformed pollen over expressing or suppressing the expression of aquaporins. Correlation of transcription and activity of a given aquaporin in individual pollen grains can be determined by the swelling assay and by subsequent single-cell RT-PCR to measure the amount of aquaporin mRNA. These experiments should allow to identify and study the involvement of particular aquaporins during germination and tube growth and water uptake, respectively.

In spermatophytes, during fertilization pollen tubes are growing through the stigma tissue and deliver the sperm cells to the egg cell. Pollen tubes are the fastest growing plant cells and transport of water across the plasma membrane of the vegetative cell is important at least at three stages of germination and growth of pollen tubes: 1. dehydrated pollen grains landing on a compatible stigma have to re-hydrate, 2. cell turgor pressure has to be adjusted, and 3. water flow into the cytoplasm increases the cell volume during tube growth. Despite its importance, water transport across the plasma membrane of pollen grains and tubes was neither investigated nor quantified before, and thus any model describing pollen tube elongation is more or less speculative with respect to water transport. Water uptake may occur through the lipid bilayer of the plasma membrane itself or by facilitated diffusion through aquaporins, i.e. members of the PIP (Plasma membrane Intrinsic Proteins) family. We will measure the water transport across the plasma membrane in a model organism in pollen physiology, i.e. intact pollen grains of Lilium longiflorum, using the turgor pressure probe, and in protoplasts obtained from pollen grains and tubes using a swelling assay. The water permeability coefficient of the plasma membrane (Pw) will be calculated from the relaxation kinetics of the turgor pressure and from volume change rates of the protoplasts, respectively. Inhibition of water transport by mercurials and the amplitude of Pw may indicate the involvement of aquaporins in the water transport during pollen tube growth. A Lilium pollen grainube cDNA-library will be screened with an aquaporin-specific probe to identify, clone and sequence putative aquaporins in pollen. Using a swelling assay the transport properties of the identified aquaporins will be measured by injection of the respective mRNA into Xenopus oocytes as well as in transiently transformed pollen. Additionally, the water permeability as well as the germination frequency and tube growth rates will be determined in transformed pollen over expressing or suppressing the expression of aquaporins. Correlation of transcription and activity of a given aquaporin in individual pollen grains can be determined by the swelling assay and by subsequent single-cell RT-PCR to measure the amount of aquaporin mRNA. These experiments should allow to identify and study the involvement of particular aquaporins during germination and tube growth and water uptake, respectively.