Tobacco pollen tubes – a fast and easy tool for studying lipid droplet association of plant proteins

Abstract

In recent years, lipid droplets have emerged as dynamic organelles rather than inactive storage sites for triacylglycerol. The number of proteins known to be associated with lipid droplets has increased, but remains small in comparison with those found with other organelles. Also the mechanisms of how lipid droplets are recognized and bound by proteins need deeper investigation. Here, we present a fast, simple and inexpensive approach to assay proteins for their association with lipid droplets in vivo that can help to screen protein candidates or mutated variants of proteins for their association in an efficient manner. For this, a system to transiently transform Nicotiana tabacum pollen grains was used because these naturally contain lipid droplets. We designed vectors for fast cloning of genes as fusions with either mVenus or mCherry. This allowed us to assay colocalization with lipid droplets stained with Nile Red and Bodipy 505/515, respectively. We successfully tested our system not only for proteins from Arabidopsis thaliana, but also for proteins from the moss Physcomitrella patens and the alga Chlamydomonas reinhardtii. The small size of the vector used allows easy exchange of codons by site-directed mutagenesis. We used this to show that two proline residues in the proline knot of a caleosin are not essential for the binding of lipid droplets. We also demonstrated that peroxisomes are not associated with the lipid droplets in tobacco pollen tubes, which reduces the risk of false interpretation of microscopic data in our system.

Characterization of the enzymatic activity and physiological function of the lipid droplet‐associated triacylglycerol lipase AtOBL1

“…LDs in tobacco pollen tubes are not associated with peroxisomes (Müller et al ., ) and might therefore contain TAG that serves as a direct precursor for membrane lipids rather than for β‐oxidation. An indication for this could be that the FA composition of TAG and membrane lipids is similar, even though this similarity also could be based on the fact that most of the FAs used for TAG synthesis first go through the PC pool.…”

“…Pollen transformation by particle bombardment, pollen tube growth, Nile red staining, and both confocal and epifluorescence microscopy were performed as described recently (Müller et al ., ). Arabidopsis pollen tubes were grown in vitro on solid medium (see Ischebeck et al ., ) and epifluorescence recorded with an ORCA‐Flash 4.0 V2 camera (Hamamatsu Photonocs, Hamamatsu, Japan) mounted on a epifluorescence microscope (BX51; Olympus, Hamburg, Germany) using a U‐MF2 filter cube for mVenus fluorescence.…”

“…Cloning of genes into the vectors pUC‐Lat52‐mVenusC (Mähs et al ., ), pLatMVC‐GW, pLatMVN‐GW (both Müller et al ., ), pCAMBIA33‐35S (Hornung et al ., ), as well as site‐directed mutagenesis was performed as described (Müller et al ., ). For a complete list of primers and cloning strategies, see Table S1.…”

“…LDs are found – at least to a small extent – in most cells, and are most prominent in seeds and tapetosomes (Pyc et al ., ) as well as in pollen (Ischebeck, ). Here, the LDs enter the pollen tube after germination (Müller et al ., ) and, at least in olive pollen tubes, they are also degraded (Zienkiewicz et al ., ) and an inhibition of TAG degradation impaired olive pollen tube growth. Interestingly, tobacco pollen tubes form TAG de novo from radioactively labelled ethanol and glucose (Mellema et al ., ), indicating that LDs might not only be a source, but also a sink for acyl chains.…”

Plant Lipid Droplets and Their Associated Proteins: Potential for Rapid Advances

“…However, they are substantially shorter (longest being 21 residues for caleosin 299 and 24 residues for steroleosin, both one-third the length of the oleosin hairpin), and the hairpin 300 residues are only mildly conserved among isoforms within the same and among diverse species 301 (Song et al, 2014;Pasaribu et al, 2016). Two of the proline resides in the caleosin hairpin loop 302 are not essential for LD binding (Muller et al, 2017 monoacylglycerol hydrolase (to be described), possess no long hydrophobic or predicted 358 membrane-insertion segment (Gidda et al, 2016;Lopez-Ribera et al, 2014;Shimada et al, 2014 …”