The number of known proteins associated with plant lipid droplets (LDs) is small compared with other organelles. Many aspects of LD biosynthesis and degradation are unknown, and identifying and characterizing candidate LD proteins could help elucidate these processes. Here, we analyzed the proteome of LD-enriched fractions isolated from tobacco (Nicotiana tabacum) pollen tubes. Proteins that were highly enriched in comparison with the total or cytosolic fraction were further tested for LD localization via transient expression in pollen tubes. One of these proteins, PLANT UBX DOMAIN-CONTAINING PROTEIN10 (PUX10), is a member of the plant UBX domain-containing (PUX) protein family. This protein localizes to LDs via a unique hydrophobic polypeptide sequence and can recruit the AAA-type ATPase CELL DIVISION CYCLE48 (CDC48) protein via its UBX domain. PUX10 is conserved in Arabidopsis thaliana and expressed in embryos, pollen tubes, and seedlings. In pux10 knockout mutants in Arabidopsis, LD size is significantly increased. Proteomic analysis of pux10 mutants revealed a delayed degradation of known LD proteins, some of which possessed ubiquitination sites. We propose that PUX10 is involved in a protein degradation pathway at LDs, mediating an interaction between polyubiquitinated proteins targeted for degradation and downstream effectors such as CDC48.

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.

Pollen tubes require a tightly regulated pectin secretion machinery to sustain the cell wall plasticity required for polar tip growth. Involved in this regulation at the apical plasma membrane are proteins and signaling molecules, including phosphoinositides and phosphatidic acid (PA). However, the contribution of diacylglycerol kinases (DGKs) is not clear.We transiently expressed tobacco DGKs in pollen tubes to identify a plasma membrane (PM)-localized isoform, and then to study its effect on pollen tube growth, pectin secretion and lipid signaling. In order to potentially downregulate DGK5 function, we overexpressed an inactive variant.Only one of eight DGKs displayed a confined localization at the apical PM. We could demonstrate its enzymatic activity and that a kinase-dead variant was inactive. Overexpression of either variant led to differential perturbations including misregulation of pectin secretion. One mode of regulation could be that DGK5-formed PA regulates phosphatidylinositol 4-phosphate 5-kinases, as overexpression of the inactive DGK5 variant not only led to a reduction of PA but also of phosphatidylinositol 4,5-bisphosphate levels and suppressed related growth phenotypes.We conclude that DGK5 is an additional player of polar tip growth that regulates pectin secretion probably in a common pathway with PI4P 5-kinases.

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