CALEOSIN 1 interaction with AUTOPHAGY-RELATED PROTEIN 8 facilitates lipid droplet microautophagy in seedlings.

Lipid droplets (LDs) of seed tissues are storage organelles for triacylglycerols (TAGs) that provide the energy and carbon for seedling establishment. In the major route of LD degradation (lipolysis), TAGs are mobilized by lipases. However, LDs may also be degraded via lipophagy, a type of selective autophagy, which mediates LD delivery to vacuoles or lysosomes. The exact mechanisms of LD degradation and the mobilization of their content in plants remain unresolved. Here, we provide evidence that LDs are degraded via a process morphologically resembling microlipophagy in Arabidopsis (Arabidopsis thaliana) seedlings. We observed the entry and presence of LDs in the central vacuole as well as their breakdown. Moreover, we show co-localization of AUTOPHAGY-RELATED PROTEIN 8b (ATG8b) and LDs during seed germination and localization of lipidated ATG8 (ATG8-PE) to the LD fraction. We further demonstrate that structural LD proteins from the caleosin family, CALEOSIN 1 (CLO1), CALEOSIN 2 (CLO2), and CALEOSIN 3 (CLO3), interact with ATG8 proteins and possess putative ATG8-interacting motifs (AIMs). Deletion of the AIM localized directly before the proline knot disrupts the interaction of CLO1 with ATG8b, suggesting a possible role of this region in the interaction between these proteins. Collectively, we provide insights into LD degradation by microlipophagy in germinating seeds with a particular focus on the role of structural LD proteins in this process.

Recent advances and challenges in potato improvement using CRISPR/Cas genome editing.

MAIN CONCLUSION: Genome editing using CRISPR/Cas technology improves the quality of potato as a food crop and enables its use as both a model plant in fundamental research and as a potential biofactory for producing valuable compounds for industrial applications. Potato (Solanum tuberosum L.) plays a significant role in ensuring global food and nutritional security. Tuber yield is negatively affected by biotic and abiotic stresses, and enzymatic browning and cold-induced sweetening significantly contribute to post-harvest quality losses. With the dual challenges of a growing population and a changing climate, potato enhancement is essential for its sustainable production. However, due to several characteristics of potato, including high levels of heterozygosity, tetrasomic inheritance, inbreeding depression, and self-incompatibility of diploid potato, conventional breeding practices are insufficient to achieve substantial trait improvement in tetraploid potato cultivars within a relatively short time. CRISPR/Cas-mediated genome editing has opened new possibilities to develop novel potato varieties with high commercialization potential. In this review, we summarize recent developments in optimizing CRISPR/Cas-based methods for potato genome editing, focusing on approaches addressing the challenging biology of this species. We also discuss the feasibility of obtaining transgene-free genome-edited potato varieties and explore different strategies to improve potato stress resistance, nutritional value, starch composition, and storage and processing characteristics. Altogether, this review provides insight into recent advances, possible bottlenecks, and future research directions in potato genome editing using CRISPR/Cas technology.

The production of wax esters in transgenic plants: towards a sustainable source of bio-lubricants.

Wax esters are high-value compounds used as feedstocks for the production of lubricants, pharmaceuticals, and cosmetics. Currently, they are produced mostly from fossil reserves using chemical synthesis, but this cannot meet increasing demand and has a negative environmental impact. Natural wax esters are also obtained from Simmondsia chinensis (jojoba) but comparably in very low amounts and expensively. Therefore, metabolic engineering of plants, especially of the seed storage lipid metabolism of oil crops, represents an attractive strategy for renewable, sustainable, and environmentally friendly production of wax esters tailored to industrial applications. Utilization of wax ester-synthesizing enzymes with defined specificities and modulation of the acyl-CoA pools by various genetic engineering approaches can lead to obtaining wax esters with desired compositions and properties. However, obtaining high amounts of wax esters is still challenging due to their negative impact on seed germination and yield. In this review, we describe recent progress in establishing non-food-plant platforms for wax ester production and discuss their advantages and limitations as well as future prospects.

Lipases of germinating jojoba seeds efficiently hydrolyze triacylglycerols and wax esters and display wax ester-synthesizing activity.

BACKGROUND: Simmondsia chinensis (jojoba) is the only plant known to store wax esters instead of triacylglycerols in its seeds. Wax esters are composed of very-long-chain monounsaturated fatty acids and fatty alcohols and constitute up to 60% of the jojoba seed weight. During jojoba germination, the first step of wax ester mobilization is catalyzed by lipases. To date, none of the jojoba lipase-encoding genes have been cloned and characterized. In this study, we monitored mobilization of storage reserves during germination of jojoba seeds and performed detailed characterization of the jojoba lipases using microsomal fractions isolated from germinating seeds. RESULTS: During 26 days of germination, we observed a 60-70% decrease in wax ester content in the seeds, which was accompanied by the reduction of oleosin amounts and increase in glucose content. The activity of jojoba lipases in the seed microsomal fractions increased in the first 50 days of germination. The enzymes showed higher activity towards triacylglycerols than towards wax esters. The maximum lipase activity was observed at 60 °C and pH around 7 for triacylglycerols and 6.5-8 for wax esters. The enzyme efficiently hydrolyzed various wax esters containing saturated and unsaturated acyl and alcohol moieties. We also demonstrated that jojoba lipases possess wax ester-synthesizing activity when free fatty alcohols and different acyl donors, including triacylglycerols and free fatty acids, are used as substrates. For esterification reactions, the enzyme utilized both saturated and unsaturated fatty alcohols, with the preference towards long chain and very long chain compounds. CONCLUSIONS: In in vitro assays, jojoba lipases catalyzed hydrolysis of triacylglycerols and different wax esters in a broad range of temperatures. In addition, the enzymes had the ability to synthesize wax esters in the backward reaction. Our data suggest that jojoba lipases may be more similar to other plant lipases than previously assumed.

Wax synthase MhWS2 from Marinobacter hydrocarbonoclasticus: substrate specificity and biotechnological potential for wax ester production.

Wax synthases are involved in the biosynthesis of wax esters, lipids with great industrial potential. Here, we heterologously expressed the native wax synthase MhWS2 from Marinobacter hydrocarbonoclasticus in Saccharomyces cerevisiae and performed comprehensive analysis of its substrate specificity. The enzyme displayed high wax synthase (but no diacylglycerol acyltransferase) activity both in vivo and in vitro. In the presence of exogenous fatty alcohol, wax esters accounted for more than 57% of total yeast lipids. In vitro, MhWS2 produced wax esters with most of the tested substrates, showing the highest activity with 14:0-, 18:1-, 18:0-, 12:0-, and 16:0-CoA together with saturated C10-C16 fatty alcohols. Co-expression with genes encoding fatty acyl reductases resulted in the accumulation of C26-C36 wax esters. Altogether, our results provide a detailed characterization of MhWS2 which should be useful in the development of strategies for producing wax esters in various expression systems.

Metabolic engineering of fatty alcohol production in transgenic hairy roots of Crambe abyssinica.

Biotechnological production of fatty alcohols, important raw materials in the chemical industry, has been receiving considerable attention in recent years. Fatty alcohols are formed by the reduction of fatty acyl-CoAs or fatty acyl-ACPs catalyzed by a fatty acyl reductase (FAR). In this study, we introduced genes encoding FARs from Arabidopsis thaliana (AtFAR5) and Simmondsia chinensis (ScFAR) into Crambe abyssinica hairy roots via Agrobacterium rhizogenes-mediated transformation. The efficiency of the transformation ranged between 30 and 45%. The fatty alcohols were only detected in the transgenic hairy root lines expressing ScFAR gene. In all tested lines stearyl alcohol (18:0-OH), arachidyl alcohol (20:0-OH), and behenyl alcohol (22:0-OH) were produced. The content of 18:0-OH varied from 1 to 3% of total fatty acids and fatty alcohols, while the amount of either 20:0-OH and 22:0-OH did not exceed 2%. The transgenic hairy root lines produced from 0.98 to 2.59 nmol of fatty alcohols per mg of dry weight. Very low activity of ScFAR was detected in the microsomal fractions isolated from the selected hairy root lines. To our knowledge, this is the first report on the fatty alcohol production in the hairy root cultures. Biotechnol. Bioeng. 2017;114: 1275-1282. © 2016 Wiley Periodicals, Inc.

Biochemical characterization and substrate specificity of jojoba fatty acyl-CoA reductase and jojoba wax synthase.

Wax esters are used in industry for production of lubricants, pharmaceuticals and cosmetics. The only natural source of wax esters is jojoba oil. A much wider variety of industrial wax esters-containing oils can be generated through genetic engineering. Biotechnological production of tailor-made wax esters requires, however, a detailed substrate specificity of fatty acyl-CoA reductases (FAR) and wax synthases (WS), the two enzymes involved in wax esters synthesis. In this study we have successfully characterized the substrate specificity of jojoba FAR and jojoba WS. The genes encoding both enzymes were expressed heterologously in Saccharomyces cerevisiae and the activity of tested enzymes was confirmed by in vivo studies and in vitro assays using microsomal preparations from transgenic yeast. Jojoba FAR exhibited the highest in vitro activity toward 18:0-CoA followed by 20:1-CoA and 22:1-CoA. The activity toward other 11 tested acyl-CoAs was low or undetectable as with 18:2-CoA and 18:3-CoA. In assays characterizing jojoba WS combinations of 17 fatty alcohols with 14 acyl-CoAs were tested. The enzyme displayed the highest activity toward 14:0-CoA and 16:0-CoA in combination with C16-C20 alcohols as well as toward C18 acyl-CoAs in combination with C12-C16 alcohols. 20:1-CoA was efficiently utilized in combination with most of the tested alcohols.

Detailed characterization of the substrate specificity of mouse wax synthase.

Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes -- the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity.