Identification of lily pollen 14-3-3 isoforms and their subcellular and time-dependent expression profile.

14-3-3 proteins are major regulators in plant development and physiology including primary metabolism and signal transduction pathways, typically via a phosphorylation-dependent interaction with a target protein. Four full-length 14-3-3 isoforms were identified in pollen grains of Lilium longiflorum by screening of a cDNA library and RACE (rapid amplification of cDNA ends)-PCR. Mass spectrometry analysis of partially purified 14-3-3s confirmed the presence of the four isoforms but also indicated the presence of additional, less abundant 14-3-3 isoforms in lily pollen.

Osmoregulation in Lilium pollen grains occurs via modulation of the plasma membrane H+ ATPase activity by 14-3-3 proteins.

To allow successful germination and growth of a pollen tube, mature and dehydrated pollen grains (PGs) take up water and have to adjust their turgor pressure according to the water potential of the surrounding stigma surface. The turgor pressure of PGs of lily (Lilium longiflorum) was measured with a modified pressure probe for simultaneous recordings of turgor pressure and membrane potential to investigate the relation between water and electrogenic ion transport in osmoregulation. Upon hyperosmolar shock, the turgor pressure decreased, and the plasma membrane (PM) hyperpolarizes in parallel, whereas depolarization of the PM was observed with hypoosmolar treatment.

The pollen organelle membrane proteome reveals highly spatial-temporal dynamics during germination and tube growth of lily pollen.

As a first step in understanding the membrane-related dynamics during pollen grain germination and subsequent tube growth, the changes in protein abundance of membrane and membrane-associated proteins of 5 different membrane/organelle fractions were studied at physiologically important stages (0, 10, 30, 60, and 240 min) of Lilium longiflorum pollen in vitro culture. Proteins of each fraction and time point were identified by 'shot-gun' proteomics (LC-MS/MS). Analysis of more than 270 identified proteins revealed an increase in the abundance of proteins involved in cytoskeleton, carbohydrate and energy metabolism, as well as ion transport before pollen grain germination (10-30 min), whereas proteins involved in membrane/protein trafficking, signal transduction, stress response and protein biosynthesis decreased in abundance during this time.

Production of recombinant allergens in plants.

A large percentage of allergenic proteins are of plant origin. Hence, plant-based expression systems are considered ideal for the recombinant production of certain allergens. First attempts to establish production of plant-derived allergens in plants focused on transient expression in Nicotiana benthamiana infected with recombinant viral vectors.

Nondiffusional release of allergens from pollen grains of Artemisia vulgaris and Lilium longiflorum depends mainly on the type of the allergen.

Background: Upon contact with a wet surface, mature pollen grains hydrate and release proteins including allergens. Knowledge of the release mechanism of allergens that are mainly localized intracellularly may allow the design of strategies for inhibition of allergen release and the consequent sensitization process.

Methods: An improved pollen chromatography was performed with Artemisia vulgaris and Lilium longiflorum pollen.

Release of an acid phosphatase activity during lily pollen tube growth involves components of the secretory pathway.

An acid phosphatase (acPAse) activity was released during germination and tube growth of pollen of Lilium longiflorum Thunb. By inhibiting components of the secretory pathway, the export of the acPase activity was affected and tube growth stopped. Brefeldin A (1 microM) and cytochalasin D (1 microM), which block the production and transport of secretory vesicles, respectively, inhibited the acPase secretion.

First patch, then catch: measuring the activity and the mRNA transcripts of a proton pump in individual Lilium pollen protoplasts.

Combining the patch-clamp method with single-cell reverse transcription polymerase chain reaction (scRT-PCR) a fusicoccin-induced current reflecting the activity of the plasma membrane H(+) ATPase of lily pollen protoplasts was measured and subsequently, the ATPase-encoding mRNAs were collected and amplified. Southern blot signals were observed in all 'patch-catch' experiments and could be detected even in 2560-fold dilutions of the pollen contents. H(+) ATPase mRNAs were detectable only in the vegetative but not in the generative cell of pollen as confirmed by immunolocalisation.