Small rubber particle proteins from Taraxacum brevicorniculatum promote stress tolerance and influence the size and distribution of lipid droplets and artificial poly(cis‐1,4‐isoprene) bodies

Abstract

Natural rubber biosynthesis occurs on rubber particles, i.e. organelles resembling small lipid droplets localized in the laticifers of latex-containing plant species, such as Hevea brasiliensis and Taraxacum brevicorniculatum. The latter expresses five small rubber particle protein (SRPP) isoforms named TbSRPP1-5, the most abundant proteins in rubber particles. These proteins maintain particle stability and are therefore necessary for rubber biosynthesis. TbSRPP1-5 were transiently expressed in Nicotiana benthamiana protoplasts and the proteins were found to be localized on lipid droplets and in the endoplasmic reticulum, with TbSRPP1 and TbSRPP3 also present in the cytosol. Bimolecular fluorescence complementation confirmed pairwise interactions between all proteins except TbSRPP2. The corresponding genes showed diverse expression profiles in young T. brevicorniculatum plants exposed to abiotic stress, and all except TbSRPP4 and TbSRPP5 were upregulated. Young Arabidopsis thaliana plants that overexpressed TbSRPP2 and TbSRPP3 tolerated drought stress better than wild-type plants. Furthermore, we used rubber particle extracts and standards to investigate the affinity of the TbSRPPs for different phospholipids, revealing a preference for negatively charged head groups and 18:2/16:0 fatty acid chains. This finding may explain the effect of TbSRPP3-5 on the dispersity of artificial poly(cis-1,4-isoprene) bodies and on the lipid droplet distribution we observed in N. benthamiana leaves. Our data provide insight into the assembly of TbSRPPs on rubber particles, their role in rubber particle structure, and the link between rubber biosynthesis and lipid droplet-associated stress responses, suggesting that SRPPs form the basis of evolutionarily conserved intracellular complexes in plants.

Uncovering mechanisms of rubber biosynthesis in Taraxacum koksaghyz – role of cis‐prenyltransferase‐like 1 protein

“…As microscopy did not allow for precise determination of particle size, we carried out dynamic light scattering to determine the intensity distribution of particle size in the isolated white pellet fractions from TkCPTL1 ‐RNAi plants as well as in the RPs from T. koksaghyz wild‐type plants (Figure d). For all samples, the highest intensity was detected at a particle diameter of approximately 450 nm (d.nm), a typical diameter for rubber particles (Laibach et al ., ), indicating that the particles in the white pellet fraction have a size similar to that of regular rubber particles. Additionally, the measured size of the rubber particles from the TkCPTL2 knockdown plants as wells as from control plants also peaked at about 450 d.nm (Figure d).…”

“…PC , phosphatidylcholine 18:2/16:0; PE , phosphatidylethanolamine 18:2/16:0; PI , phosphatidylinositol; PA , phosphatidic acid (1‐palmitoyl‐2‐linleoyl‐ sn ‐glycero‐3‐phoshpate); PIP , phosphatidylinositol monophosphate mix; RP ‐E, rubber particle extract according to Laibach et al . (). (f) Western blots of proteins of white pellet ( WP ) and the IP from one Tk CPLT 1 ‐ RNA i plant as well as the IP and RP from one Tk CPTL 2 ‐ RNA i plant and from each corresponding WT .…”

“…The isolated phospholipids were separated by TLC (Figure e). The most abundant phospholipid of the particles in the white pellet fraction was phosphatidylcholine (PC), as has been reported for rubber particles from dandelion wild‐types (>60% (w/w) PC 18:2/16:0 and PC 18:2/18:2) (Laibach et al ., ). In addition, the amounts of other phospholipids such as phosphatidylethanolamine and phosphatidylinositol that appear less frequently at RP also appeared less frequently in the white pellet particles.…”

“…TkSRPPs were detected in a western blot analysis of the white pellet fraction and, furthermore, the latex proteomes from TkCPTL1 ‐RNAi lines and the T. koksaghyz wild‐type indicated no differences in SRPPs. Due to similar phospholipid composition in the white pellet fraction, TkSRPPs might still be able to bind to particle membranes via pockets of unsaturated phosphatidylcholine and finally stabilize the particle structures, as reported for rubber particles (Laibach et al ., ). For rubber particles in TbRTA ‐RNAi plants, examined by electron microscopy, these particles were considered to be empty structures (Epping et al ., ).…”

“…The SRPP isoforms of Taraxacum brevicorniculatum are relevant for rubber particle stability and bind to the particle membrane due to their affinity to phospholipids that comprise unsaturated fatty acids. Functioning as structural proteins, SRPPs are crucial for NR synthesis (Hillebrand et al ., ; Laibach et al ., ). However, the enzymatic synthesis reaction requires an active rubber transferase complex, which is also bound to the rubber particle membrane (Cornish, ).…”