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

Abstract

Similar to seeds, pollen tubes contain lipid droplets that store triacylglycerol (TAG), but the fate of this TAG as well as the enzymes involved in its breakdown are unknown. Therefore, two potential TAG lipases from tobacco and Arabidopsis, NtOBL1 (Oil body lipase 1) and AtOBL1, were investigated, especially with respect to their importance for pollen tube growth. We expressed NtOBL1 and AtOBL1 as fluorescent fusion proteins to study their localization by confocal microscopy. Furthermore, we overexpressed AtOBL1 in Nicotiana benthamiana leaves to characterize it enzymatically. The obl1 mutant was studied in respect to its pollen tube growth in vivo and its seed germination. Both NtOBL1 and AtOBL1 localized to lipid droplets. AtOBL1 was abundant in pollen tubes and seedlings, and acted as a lipase on TAG, diacylglycerol and 1-monoacylglycerol at a pH optimum of 5.5. The obl1 mutant was hampered in pollen tube growth, whereas seedling establishment was not affected under optimal conditions, even though AtOBL1 accounted for a major lipase activity in seeds. TAG could be a direct precursor for the synthesis of membrane lipids in pollen tubes and proteins of the OBL family involved in the flux of acyl groups.

PUX10 Is a Lipid Droplet-Localized Scaffold Protein That Interacts with CELL DIVISION CYCLE48 and Is Involved in the Degradation of Lipid Droplet Proteins

“…Another enzyme we identified enriched in the LD fraction (Table 1) is a tobacco homolog of the castor bean (Ricinus communis) oil body lipase (Rc-OBL; Eastmond, 2004). Recently, we confirmed that both Nt-OBL and its Arabidopsis homolog At-OBL1 are LD-associated and that At-OBL1 is a TAG, diacylglycerol, and 1-monoacylglycerol lipase and that the activity of At-OBL1 is important for pollen tube growth (Müller and Ischebeck, 2018). We also found a previously unknown LD protein, PTLDb (Table 1, Figure 1F), and via BLAST analysis, we identified four additional tobacco homologs (Supplemental Figure 10), all of which were enriched in LD fractions isolated from tobacco pollen tubes (Supplemental Data Set 4).…”

“…These pollen grains contain conspicuously enlarged LDs that cannot enter the pollen tube, leading to reduced fertility. LD degradation appears to be important to some degree, as mutants disrupted in a LDassociated TAG lipase show reduced pollen tube growth (Müller and Ischebeck, 2018). Hence, while these data support the importance of LDs during pollen tube growth, it remains to be determined what specific function(s) these organelles serve.…”

“…In the pollen tube data set, 18 proteins had an enrichment factor higher than 100 and an iBAQ ‰ higher than 1 (Table 1). From these proteins, at least one member of the corresponding nine gene families was cloned from tobacco pollen tube cDNA, except for Nt-OBL1, which was previously characterized (Müller and Ischebeck, 2018). All constructs were transiently expressed in tobacco pollen tubes as fusion proteins with mVenus, and Nile Red was used to stain LDs.…”

“…In addition, the LDs in pollen tubes and oilseeds differ in their neutral lipid composition: Sterol esters make up a much larger proportion of neutral lipids (24% on a weight basis; Rotsch et al, 2017) in pollen tubes in comparison to seeds from Arabidopsis (∼0.3% of the level of TAG; Bouvier-Navé et al, 2010). While seeds degrade their neutral lipids following germination, tobacco pollen tubes synthesize TAG (Mellema et al, 2002) and sterol esters (Müller and Ischebeck, 2018), and pollen tubes cultivated for 14 h still contain large amounts of these neutral lipids (Müller and Ischebeck, 2018). By contrast, pollen tubes from olive (Olea europaea) degrade their LDs during pollen tube growth (Zienkiewicz et al, 2013).…”

Tri-Hydroxy-Triacylglycerol Is Efficiently Produced by Position-Specific Castor Acyltransferases

“…Since HFA-TAG persist in developing seedlings, it is likely that Arabidopsis TAG lipases only slowly hydrolyze the storage lipid to render the FA available for growth and development. Such defective hydrolysis suggests that establishment may benefit from expression of a castor homolog of known Arabidopsis TAG lipases like SUGAR-DEPENDENT1 (Eastmond, 2006) or OIL BODY LIPASE1 (Müller and Ischebeck, 2018), based on the hypothesis that these would more rapidly hydrolyze HFA-TAG.…”