Identification of Low-Abundance Lipid Droplet Proteins in Seeds and Seedlings

Abstract

Several previously unknown low-abundant lipid droplet proteins were identified in Arabidopsis thaliana seeds and seedlings by quantitative proteomics combined with two cell biological approaches.

Discovering Lipid Droplet Proteins: From Seeds to Seedlings

“…The authors noted that the Secretory protein (SEC) 61g subunit of the ER protein translocation machinery (SEC translocase) was also found to be enriched in LD fractions but did not specifically localize to LDs in the transient expression systems used. The authors note that the ER-localized SEC61g was in close proximity to LDs, which may be indicative of ER-LD junctions (Kretzschmar et al, 2020).…”

“…In this issue of Plant Physiology, Kretzschmar et al (2020) carried out a quantitative proteomic analysis of LD-associated proteins in Arabidopsis at several early developmental stages: from silique development until seedling establishment. To identify potential LD proteins, the overall protein abundance profiles were examined over development and in response to desiccation and rehydration.…”

“…To identify potential LD proteins, the overall protein abundance profiles were examined over development and in response to desiccation and rehydration. Additionally, a relatively mild technique was developed for enriching LD protein fractions, whereby analysis of an enrichment factor in LD fractions compared with total protein extracts can be used to predict novel, perhaps loosely associated or low-abundance LD proteins (Kretzschmar et al, 2020). Using this method, several novel LD proteins were identified, and their localization to LDs was shown using transient expression of fluorescent protein-tagged versions in Nicotiana benthamiana via Agrobacterium tumefaciens infiltration and in Nicotiana tabacum pollen tubes transformed by biolistic bombardment (Kretzschmar et al, 2020).…”

“…confirmed. These proteins were named based on their functional annotation at TAIR and included a lipase, methyltransferase, hydrolase, dehydrogenase, and an additional LD protein with no functional annotation (Kretzschmar et al, 2020). The functions of these newly discovered LD proteins have yet to be uncovered.…”

SEIPIN Isoforms Interact with the Membrane-Tethering Protein VAP27-1 for Lipid Droplet Formation

“…Arabidopsis EF1a and N. benthamiana ACTIN were used as the reference genes for RT-PCR. Sequence information for all primers used for RT-PCRs, as well as those used for genotyping of T-DNA insertional transgenic lines (Supplemental Figure 6), are available in the Supplemental Table. Microscopy Agrobacterium-infiltrated N. benthamiana leaves, biolistically bombarded tobacco (N. tabacum) pollen tubes, and Arabidopsis (transgenic) seeds were processed for confocal laser-scanning microscopy (CLSM), including staining of LDs either with BODIPY 493/503 (Invitrogen) or MDH (Abgent), as described previously (Cai et al, 2015;Gidda et al, 2016;Müller et al, 2017;Kretzschmar et al, 2020). Micrographs of N. benthamiana leaves were acquired using either a DM RBE microscope equipped with a 633 oilimmersion objective (numerical aperture [NA] 5 1.32) and TCS SP2 scanning head, or An SP5 microscope equipped with a 633 glycerolimmersion objective (NA 5 1.3) and a Radius 405-nm laser (Leica Microsystems).…”