Ljerka Kunst


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Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - April 2022)
Investigating the regulatory mechanisms of cuticular wax biosynthesis in Arabidopsis thaliana (2018)

As an integral part of plant cuticle, cuticular wax covers the primary aerial organs of plants and provides protection against desiccation and environmental stresses. It has been shown that cuticular wax biosynthesis is synchronized with the surface area expansion during plant development and is associated with plant responses to biotic and abiotic stresses. As the plant-environment interface, cuticular wax deposition is tightly regulated. The major goal of my Ph.D. study was to investigate the regulatory mechanisms that control cuticular wax accumulation during development and/or in response to environmental cues by identifying and characterizing factors that are involved in this process. In this thesis, I investigated two types of regulation of wax accumulation. Firstly, studies of the RING-type E3 ligase U37 demonstrated that U37-mediated ubiquitination is involved in the regulation of wax biosynthesis (Chapter 3). U37 ubiquitinates wax biosynthetic enzyme ECERIFERUM1 (CER1), and possibly also ECERIFERUM3 (CER3), and targets them for degradation via the 26S proteasome. Based on the U37 expression profile, I proposed that U37 might have a major function in turning off wax biosynthesis in fully developed leaves. Secondly, the identification and partial characterization of novel transcription factors that bind to the CER3 promoter revealed that CER3 is an important regulatory determinant in transcriptional regulation of cuticular wax biosynthesis (Chapter 4). Four transcription factors, MYB30, WIP5, AHL29 and AHL23, have been identified that likely control cuticular wax deposition by regulating CER3 gene expression.

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Arabidopsis ECERIFERUM2-LIKE proteins are components of the fatty acid elongation machinery required for the production of cuticular waxes (2017)

Very-long-chain fatty acids (VLCFAs) are essential molecules produced by all plant cells,and are precursors of diverse primary and specialized metabolites. VLCFAs are elongated by afatty acid elongation (FAE) complex of four core enzymes located on the endoplasmic reticulum,which sequentially adds two carbon units to a growing acyl-CoA chain. Identification andcharacterization of FAE enzymes in Arabidopsis thaliana has revealed that three of the fourenzymes act as generalists, contributing to all metabolic pathways that require VLCFAs. Afourth component, the condensing enzyme, provides substrate specificity and determines thechain length of product synthesized by the entire complex. Chain length is important because itdefines what downstream pathway a VLCFA can be used for.In Arabidopsis, characterized condensing enzymes can only elongate VLCFAs up to 28carbons in length, despite the predominance of 29- and 31-carbon components in plant cuticularwax. This suggests that elongation beyond 28 carbons is unique and requires different proteincomponents. The wax-deficient mutant eceriferum2 (cer2) of Arabidopsis lacks waxes longerthan 28 carbons, suggesting that CER2 is specifically required for VLCFA extension past thisthreshold length. Molecular characterization of the CER2 gene, both in planta and byheterologous expression in Saccharomyces cerevisiae, demonstrates that CER2 is acomponent of the elongation machinery required to synthesize 30-carbon cuticular waxprecursors (Chapter 3). Five homologous CER2-LIKE genes were identified in Arabidopsis;these CER2-LIKEs have similar metabolic functions to CER2, but different expression patternsand substrate specificities (Chapter 4). CER2-LIKEs form a distinct clade of the BAHDacyltransferase superfamily. However, structural predictions and site-directed mutagenesisreveal fundamental differences in the mechanism of activity of CER2-LIKEs relative tocharacterized BAHDs (Chapter 5). My results suggest that CER2-LIKEs enable condensingenzymes to accept longer VLCFA substrates. I suggest several mechanisms to explain theactivity of CER2-LIKEs. This peculiarity of elongation is unique to land plants, and to theproduction of cuticular wax precursors. Because the acquisition of cuticles as barriers totranspirational water loss was key to plant colonization of land, CER2-LIKE activity is animportant specialization of plant lipid metabolism.

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ECERIFERUM7 subunit of the exosome, SUPERKILLER complex and small RNA species regulate cuticular wax biosynthesis in Arabidopsis thaliana stems (2015)

The primary aerial surfaces of higher plants are covered by a continuous hydrophobic lipid layer called the cuticle, which is synthesized by the epidermal cells and provides protection against desiccation and environmental stresses. The cuticle is mainly composed of the cutin polyester matrix and cuticular waxes. Although the biosynthetic pathways of cuticular waxes are relatively well documented, how wax biosynthesis is regulated is not completely understood. The major goal of my thesis was to investigate the ECERIFERUM7 (CER7)-mediated mechanism of regulation of cuticular wax biosynthesis in stems of Arabidopsis thaliana. In particular, I was interested in investigating that how the Arabidopsis CER7 protein, a core component of the exosome complex that determines cellular RNA levels, was involved in this process. CER7 was proposed to degrade an mRNA encoding a repressor of wax biosynthetic gene CER3 to activate CER3 transcription required for stem wax biosynthesis.To identify the CER3 repressor and additional components of CER7 regulatory pathway, I carried out a cer7 suppressor screen and isolated mutants capable of restoring wild-type stem wax loads in the absence of CER7 activity. Characterization of these suppressor mutants and cloning of the affected genes resulted in a series of discoveries. First, cloning of RNA DEPENDENT RNA POLYMERASE 1 and SUPPRESSOR OF GENE SILENCING 3 from the suppressors demonstrated that small interfering RNAs (siRNAs) participate in CER7-mediated regulation of wax formation (Chapter 2). Second, forward genetics and reverse genetics, combined with small RNA sequencing confirmed that trans-acting siRNAs (tasiRNAs) are direct regulators of CER3 gene in CER7-controlled wax biosynthetic pathway (Chapter 3). Third, CER7 and tasiRNA-mediated regulation of CER3 during stem wax deposition requires the SUPERKILLER complex, which is known to be involved in cytoplasmic activities of the exosome in yeast and metazoan (Chapter 4).

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Seed Oil, Seed Coat Mucilage and Cuticular Wax Deposition Are Interconnected in Arabidopsis thaliana (2014)

Seed oil, seed coat mucilage and cuticular wax are plant-specific metabolites important for plant development and growth. Therefore, understanding biosynthesis, deposition, transport and regulation of these metabolites will benefit our daily life and the environment. The original objectives of my thesis research were to investigate the regulation of seed oil accumulation by a transcription factor GLABRA2 (GL2; chapter 2) and to explore the secretory process involved in the transport of cuticular waxes from the endoplasmic reticulum to the plasma membrane (chapter 3) in Arabidopsis thaliana. However, my research revealed two unexpected connections between seed oil and seed coat mucilage deposition and between cuticular wax export and cell wall formation.At the beginning of chapter 2, I hypothesized that GL2 may regulate seed oil biosynthesis by controlling PHOSPHOLIPASE D ZETA (PLDZ) genes in the embryo. However, my data demonstrated that GL2, and all of the transcription factors known to be required for GL2 expression, influence seed oil accumulation in the embryo by regulating transcription of a seed coat mucilage biosynthetic gene, MUCILAGE MODIFIED 4 (MUM4) in the seed coat. Based on this evidence, I propose that mucilage biosynthesis in the seed coat competes with oil biosynthesis in the embryo for available photosynthate during seed development. This information suggests a promising way to engineer high oil yields in seeds by blocking seed coat mucilage production.In chapter 3, I characterized deposition of stem cuticular wax, seed coat mucilage and secondary cell wall columella, and secretion in the cer11-1 mutant. The pleiotropic cer11-1 phenotype suggests that CER11 plays a role in secretory trafficking involved in the deposition of apoplastic matrix components, including cuticular wax, seed coat mucilage and cell wall constituents. Cloning of the CER11 gene revealed that it encodes C-TERMINAL DOMAIN PHOSPHATASE LIKE 2 (CPL2) that interacts with a vacuolar type H⁺-ATPase (V-ATPase) subunit C (VHA-C) in yeast and plants. I hypothesize that the role of the CER11/CPL2 in secretory trafficking is to determine phosphorylation levels of VHA-C involved in regulation of V-ATPase activity.

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Uncovering the mechanisms involved in regulating cuticular wax biosynthesis in arabidopsis thaliana (2013)

The cuticle is a protective layer that coats the primary aerial surfaces of land plants,and mediates plant interactions with the environment. Itis synthesizedby epidermal cells andis composed of a cutin polyester matrix that is embedded and covered with cuticular waxes.My overall interest in this thesis is to uncover the mechanisms in how cuticular waxbiosynthesis is regulated in developing stems ofArabidopsisthaliana.Previous workproposed that theCER7 exoribonuclease degrades anmRNA specifying a repressor ofCER3transcription thereby activating cuticular wax biosynthesis via the alkane pathway.In this thesis, I investigated the mechanisms ofCER7-mediated silencing ofCER3,and how this contributes to regulating cuticular wax biosynthesis. Specifically, I wanted touncover the putative repressor ofCER3andto unravel the mechanism of CER7mediatedregulation of wax production. To do this, I performed a genetic screen to isolate suppressorsofcer7-1which restorecer7-related stem wax deficiency to wild-type wax levels. Thescreen resulted in the isolation of components of theRNA silencing machinery,implicatingRNA silencing in the control of cuticular wax deposition during inflorescence stemdevelopment in Arabidopsis.Using a reverse genetics approach, I have also identifiedAGO1in this pathway.Overall, I demonstratethat in thewildtype, theCER7exoribonuclease degrades aprecursor of a small RNA that acts as a repressor ofCER3,allowing for expression ofCER3,and thus production of alkanes. However, in thecer7mutant, this small RNA is notdegraded and is used for the production of a small RNA silencing via a pathway.Thegenerated small RNA silencesCER3, leadingto the wax deficient phenotype.

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Master's Student Supervision (2010 - 2021)
The mechanism for fatty acids trafficking from plastids to the endoplasmic reticulum (2021)

Fatty acids (FAs) are building blocks for different glycerolipids, including phospholipids and galactolipids that serve as major structural components of cell membranes, as well as triacylglycerols (TAGs), which are the main form of energy and carbon storage in plants. They are also precursors of the plant surface lipids: cutin, cuticular waxes, and suberin, which play roles during the plant’s resistance to drought, pathogens, or insects. In-plant cells, de novo fatty acid synthesis happens exclusively within plastids with the growing fatty acyl chain attached to the acyl carrier protein (ACP). Once completed, the majority of these fatty acids are removed from ACP by thioesterases and re-attached to coenzyme A (CoA) under the activities of long-chain acyl-CoA synthetase (LACS) to make membrane lipids, storage or surface lipids in the ER, the major site for lipid assembly. Since the production of fatty acids and the production of complex lipids happen in different organelles, a transport system between plastids and other subcellular compartments is required in order to deliver FAs from the plastid to the ER. In Arabidopsis, neither the lacs9 single knock-out mutants which removed all plastidial LACS activities, nor the lacs4 single mutants which removed the activity of an ER LACS showed any visible phenotype while the lacs4lacs9 double mutants are dwarf, and with decreased seed oil and stem wax content. These not only suggest the functional overlap between the plastidial-LACS9 and ER-LACS4 but also suggest proposed access for the ER-LACS to the fatty acids from the plastid envelopes. In my research, I have proved such access directly by re-targeting the plastidial-LACS9 to the ER and complemented the dwarf, seed oil, and stem wax content phenotypes of the lacs4lacs9 double mutants. Besides, I also showed that the outer-envelope LACS9 and inner-envelope FAX1 could form a complex, which play roles during fatty acid export from the plastid, while the ER-LACS4 is not associate with FAX1 even it is functionally equivalent with LACS9 in such pathway.

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