An ancestral role for 3-KETOACYL-COA SYNTHASE3 as a negative regulator of plant cuticular wax synthesis.

The plant cuticle, a structure primarily composed of wax and cutin, forms a continuous coating over most aerial plant surfaces. The cuticle plays important roles in plant tolerance to environmental stress, including stress imposed by drought. Some members of the 3-KETOACYL-COA SYNTHASE (KCS) family are known to act as metabolic enzymes involved in cuticular wax production. Here we report that Arabidopsis (Arabidopsis thaliana) KCS3, which was previously shown to lack canonical catalytic activity, instead functions as a negative regulator of wax metabolism by reducing the enzymatic activity of KCS6, a key KCS involved in wax production. We demonstrate that the role of KCS3 in regulating KCS6 activity involves physical interactions between specific subunits of the fatty acid elongation complex and is essential for maintaining wax homeostasis. We also show that the role of the KCS3-KCS6 module in regulating wax synthesis is highly conserved across diverse plant taxa from Arabidopsis to the moss Physcomitrium patens, pointing to a critical ancient and basal function of this module in finely regulating wax synthesis.

The Arabidopsis cytochrome P450 enzyme CYP96A4 is involved in the wound-induced biosynthesis of cuticular wax and cutin monomers.

The plant cuticle is composed of cuticular wax and cutin polymers and plays an essential role in plant tolerance to diverse abiotic and biotic stresses. Several stresses, including water deficit and salinity, regulate the synthesis of cuticular wax and cutin monomers. However, the effect of wounding on wax and cutin monomer production and the associated molecular mechanisms remain unclear. In this study, we determined that the accumulation of wax and cutin monomers in Arabidopsis leaves is positively regulated by wounding primarily through the jasmonic acid (JA) signaling pathway. Moreover, we observed that a wound- and JA-responsive gene (CYP96A4) encoding an ER-localized cytochrome P450 enzyme was highly expressed in leaves. Further analyses indicated that wound-induced wax and cutin monomer production was severely inhibited in the cyp96a4 mutant. Furthermore, CYP96A4 interacted with CER1 and CER3, the core enzymes in the alkane-forming pathway associated with wax biosynthesis, and modulated CER3 activity to influence aldehyde production in wax synthesis. In addition, transcripts of MYC2 and JAZ1, key genes in JA signaling pathway, were significantly reduced in cyp96a4 mutant. Collectively, these findings demonstrate that CYP96A4 functions as a cofactor of the alkane synthesis complex or participates in JA signaling pathway that contributes to cuticular wax biosynthesis and cutin monomer formation in response to wounding.

Acetyl-CoA Carboxylase1 Influences ECERIFERUM2 Activity to Mediate the Synthesis of Very-Long-Chain Fatty Acid Past C28.

Cuticular wax is a protective layer on the aerial surfaces of land plants. In Arabidopsis (Arabidopsis thaliana), cuticular wax is mainly constituted of compounds derived from very-long-chain fatty acids (VLCFAs) with chain lengths longer than C28. CER2-LIKE (ECERIFERUM2-LIKE) proteins interact with CER6/KCS6 (ECERIFERUM6/ß-Ketoacyl-CoA Synthase6), the key enzyme of the fatty acid elongase complex, to modify its substrate specificity for VLCFA elongation past C28. However, the molecular regulatory mechanism of CER2-LIKE proteins remains unclear. Arabidopsis eceriferum19 (cer19) mutants display wax-deficient stems caused by loss of waxes longer than C28, indicating that CER19 may participate in the CER2-LIKE-mediated VLCFA elongation past C28. Using positional cloning and genetic complementation, we showed that CER19 encodes Acetyl-CoA Carboxylase1 (ACC1), which catalyzes the synthesis of malonyl-CoA, the essential substrate for the CER6/KCS6-mediated condensation reaction in VLCFA synthesis. We demonstrated that ACC1 physically interacts with CER2-LIKE proteins via split-ubiquitin yeast two-hybrid (SUY2H) and firefly luciferase complementation imaging (LCI) analysis. Additionally, heterologous expression in yeast and genetic analysis in Arabidopsis revealed that ACC1 affects CER2 activity to influence VLCFA elongation past C28. These findings imply that CER2-LIKE proteins might function as a link between ACC1 and CER6/KCS6 and subsequently enhance CER6/KCS6 binding to malonyl-CoA for further utilization in VLCFA elongation past C28. This information deepens our understanding of the complex mechanism of cuticular wax biosynthesis.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 13 (SLP13) together with SPL9 redundantly regulates wax biosynthesis under drought stress.

Wax biosynthesis is closely controlled by many regulators under different environmental conditions. We have previously shown that the module miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 (SPL9)-DEWAX is involved in the diurnal regulation of wax production; however, it was not determined whether other SPLs are also involved in wax synthesis. Here, we report that SPL13 also regulates drought-induced wax production, by directly and indirectly affecting the expression of the two wax biosynthesis genes ECERIFERUM1 (CER1) and CER4, respectively. In addition, we show that SPL13 together with SPL9 redundantly regulates wax accumulation under both normal and drought stress conditions, and that simultaneous mutation of both genes additively increases cuticle permeability and decreases drought tolerance. However, in contrast to SPL9, SPL13 does not seem to participate in the DEWAX-mediated diurnal regulation of wax production.

Fine-tuning the activities of ß-KETOACYL-COA SYNTHASE 3 (KCS3) and KCS12 in Arabidopsis is essential for maintaining cuticle integrity.

The plant cuticle, consisting of wax and cutin, is involved in adaptations to various environments. ß-Ketoacyl-CoA synthases (KCSs) usually serve as a component of the fatty acid elongation complex that participates in the production of very long-chain fatty acids and provides precursors for the synthesis of various lipids, including wax; however, we recently reported that KCS3 and KCS12 negatively regulate wax biosynthesis. In this current study, we observed that unlike KCS3-overexpressing (OE) lines, KCS12-OE lines had fused floral organs because of abnormal cuticle biosynthesis. This prompted us to compare the functions of KCS3 and KCS12 during cuticle formation. Mutation of KCS3 caused greater effects on wax production, whereas mutation of KCS12 exerted more severe effects on cutin synthesis. The double-mutant kcs3 kcs12 had significantly increased wax and cutin contents compared to either single-mutant, suggesting that KCS12 and KCS3 have additive effects on cuticle biosynthesis. Cuticle permeability was greater for the double-mutant than for the single mutants, which ultimately led to increased susceptibility to drought stress and floral-organ fusion. Taken together, our results demonstrate the regulatory roles of KCS3 and KCS12 during cuticle biosynthesis, and show that maintaining KCS3 and KCS12 expression at certain levels is essential for the formation of a functional cuticle layer.

Alterations in the gut microbiome and metabolome profiles of septic rats treated with aminophylline.

The treatment of sepsis remains a major challenge worldwide. Aminophylline has been shown to have anti-inflammatory effects; however, the role of aminophylline in sepsis, a disease characterized by immune dysregulation, is unknown. In this study, we combined microbiome sequencing and metabolomic assays to investigate the effect of aminophylline administration on the intestinal flora and metabolites in septic rats. Sixty SD rats were randomly divided into three groups: a sham-operated (SC) group, a sepsis model (CLP) group and a CLP + aminophylline treatment (Amino) group. The intestinal flora and metabolic profile of rats in the CLP group were significantly different than those of the SC group, while aminophylline administration resulted in a return to a state similar to healthy rats. Differential abundance analysis showed that aminophylline significantly back-regulated the abundance of Firmicutes, unidentified_Bacteria, Proteobacteria, Lactobacillus, Escherichia-Shigella and other dominant bacteria (P < 0.05) and altered chenodeoxycholic acid, isolithocholic acid and a total of 26 metabolites (variable importance in the projection (VIP) > 1, P < 0.05). In addition, we found that there were significant correlations between differential metabolites and bacterial genera of the Amino and CLP groups. For example, Escherichia-Shigella was associated with 12 metabolites, and Lactobacillus was associated with two metabolites (P < 0.05), suggesting that differences in the metabolic profiles caused by aminophylline were partly dependent on its influence on the gutmicrobiome. In conclusion, this study identified a novel protective mechanism whereby aminophylline could regulate disordered intestinal flora and metabolites in septic rats.

Arabidopsis ACYL-ACTIVATING ENZYME 9 (AAE9) encoding an isobutyl-CoA synthetase is a key factor connecting branched-chain amino acid catabolism with iso-branched wax biosynthesis.

Iso-branched wax compounds are well known in plants, but their biosynthetic pathways are still mostly unknown. It has been speculated that branched waxes are derived from branched-chain amino acid (BCAA) catabolism, but the evidence for this is very limited. Gas chromatography-flame ionisation detection (GC-FID) analysis revealed that mutations in two subunits of the branched-chain ketoacid dehydrogenase (BCKDH) complex, a key enzyme complex in the degradation of BCAAs, significantly decreased the amounts of branched wax compounds, indicating that BCAA degradation may be integral to the synthesis of iso-branched wax. Substrate feeding studies further revealed that the metabolic precursor of iso-branched wax compounds is isobutyric acid (iBA), which is derived from valine degradation in Arabidopsis. We also isolated a novel mutant and found that its branched wax deficient phenotype could not be rescued by iBA. Map-based cloning together with complementation analysis revealed that mutation in ACYL-ACTIVATING ENZYME 9 (AAE9) is responsible for this phenotype. Genetic and enzyme activity analysis demonstrated that AAE9 is located downstream of the BCAA degradation pathway, and that it activates iBA to isobutyryl-CoA for use on branched wax synthesis. Taken together, our study demonstrates that AAE9 is a key factor connecting BCAA catabolism with branched wax biosynthesis.

Fatty alcohol oxidase 3 (FAO3) and FAO4b connect the alcohol- and alkane-forming pathways in Arabidopsis stem wax biosynthesis.

The alcohol- and alkane-forming pathways in cuticular wax biosynthesis are well characterized in Arabidopsis. However, potential interactions between the two pathways remain unclear. Here, we reveal that mutation of CER4, the key gene in the alcohol-forming pathway, also led to a deficiency in the alkane-forming pathway in distal stems. To trace the connection between the two pathways, we characterized two homologs of fatty alcohol oxidase (FAO), FAO3 and FAO4b, which were highly expressed in distal stems and localized to the endoplasmic reticulum. The amounts of waxes from the alkane-forming pathway were significantly decreased in stems of fao4b and much lower in fao3 fao4b plants, indicative of an overlapping function for the two proteins in wax synthesis. Additionally, overexpression of FAO3 and FAO4b in Arabidopsis resulted in a dramatic reduction of primary alcohols and significant increases of aldehydes and related waxes. Moreover, expressing FAO3 or FAO4b led to significantly decreased amounts of C18-C26 alcohols in yeast co-expressing CER4 and FAR1. Collectively, these findings demonstrate that FAO3 and FAO4b are functionally redundant in suppressing accumulation of primary alcohols and contributing to aldehyde production, which provides a missing and long-sought-after link between these two pathways in wax biosynthesis.

The Plant Fatty Acyl Reductases.

Fatty acyl reductase (FAR) is a crucial enzyme that catalyzes the NADPH-dependent reduction of fatty acyl-CoA or acyl-ACP substrates to primary fatty alcohols, which in turn acts as intermediate metabolites or metabolic end products to participate in the formation of plant extracellular lipid protective barriers (e.g., cuticular wax, sporopollenin, suberin, and taproot wax). FARs are widely present across plant evolution processes and play conserved roles during lipid synthesis. In this review, we provide a comprehensive view of FAR family enzymes, including phylogenetic analysis, conserved structural domains, substrate specificity, subcellular localization, tissue-specific expression patterns, their varied functions in lipid biosynthesis, and the regulation mechanism of FAR activity. Finally, we pose several questions to be addressed, such as the roles of FARs in tryphine, the interactions between transcription factors (TFs) and FARs in various environments, and the identification of post-transcriptional, translational, and post-translational regulators.

ArabidopsisKCS5 and KCS6 Play Redundant Roles in Wax Synthesis.

3-ketoacyl-CoA synthases (KCSs), as components of a fatty acid elongase (FAE) complex, play key roles in determining the chain length of very-long-chain fatty acids (VLCFAs). KCS6, taking a predominate role during the elongation from C26 to C28, is well known to play an important role in wax synthesis. KCS5 is one paralog of KCS6 and its role in wax synthesis remains unknown. Wax phenotype analysis showed that in kcs5 mutants, the total amounts of wax components derived from carbon 32 (C32) and C34 were apparently decreased in leaves, and those of C26 to C32 derivatives were obviously decreased in flowers. Heterologous yeast expression analysis showed that KCS5 alone displayed specificity towards C24 to C28 acids, and its coordination with CER2 and CER26 catalyzed the elongation of acids exceeding C28, especially displaying higher activity towards C28 acids than KCS6. BiLC experiments identified that KCS5 physically interacts with CER2 and CER26. Wax phenotype analysis of different organs in kcs5 and kcs6 single or double mutants showed that KCS6 mutation causes greater effects on the wax synthesis than KCS5 mutation in the tested organs, and simultaneous repression of both protein activities caused additive effects, suggesting that during the wax biosynthesis process, KCS5 and KCS6 play redundant roles, among which KCS6 plays a major role. In addition, simultaneous mutations of two genes nearly block drought-induced wax production, indicating that the reactions catalyzed by KCS5 and KCS6 play a critical role in the wax biosynthesis in response to drought.

CER16 Inhibits Post-Transcriptional Gene Silencing of CER3 to Regulate Alkane Biosynthesis.

The aerial surfaces of land plants have a protective layer of cuticular wax. Alkanes are common components of these waxes, and their abundance is affected by a range of stresses. The CER16 protein has been implicated in alkane biosynthesis in the cuticular wax of Arabidopsis (Arabidopsis thaliana). Here, we identified two new mutant alleles of CER16 in Arabidopsis resulting in production of less wax with dramatically fewer alkanes than the wild type. Map-based cloning with genetic analysis revealed that the cer16 phenotype was caused by complete loss of AT5G44150, encoding a protein with no known domains or motifs. Comparative transcriptomic analysis revealed that transcripts of CER3, previously shown to play a principal role in alkane production, were markedly reduced in the cer16 mutants. To define the relationship between CER3 and CER16, we transformed the full CER3 gene into a cer16 mutant. Transgenic CER3 expression was silenced, and levels of small interfering RNAs targeting CER3 were significantly increased. Mutating two major components of the RNA-silencing machinery in a cer16 genetic background restored CER3 transcript levels to wild-type levels, with the stems restored to wild-type glaucousness. We suggest that CER16 deficiency induces post-transcriptional gene silencing of both endogenous and exogenous expression of CER3.

Dysregulation of LncRNA ANRIL mediated by miR-411-3p inhibits the malignant proliferation and tumor stem cell like property of multiple myeloma via hypoxia-inducible factor 1α.

Long non-coding RNA (lncRNA) ANRIL has been reported to be closely related to the relapse of multiple myeloma patients. However, the functional role and underlying mechanism of lncRNA ANRIL in multiple myeloma are not known. This study aims to investigate the biological function of lncRNA ANRIL in multiple myeloma. In this study, compared with normal tissues from healthy donors, lncRNA ANRIL and HIF-1α expressions were up-regulated in tumor tissues from multiple myeloma patients. miR-411-3p expression was down-regulated in tumor tissues from multiple myeloma patients. Besides, lncRNA ANRIL can interact with miR-411-3p. HIF-1α was confirmed to be a target of miR-411-3p. Correlation analysis showed that lncRNA ANRIL expression was negatively correlated with miR-411-3p expression. HIF-1α expression was negatively correlated with miR-411-3p expression. Further transfection experiments showed that knockdown of ANRIL or overexpression of miR-411-3p significantly inhibited cell proliferation, tumor formation ability and tumor stem cell like property, promoted cell apoptosis in vitro. Finally, miR-411-3p mimic reduced tumor volume, improved survival rate, suppressed malignant proliferation and tumor stem cell like property in U266 xenograft model. Our results demonstrate that lncRNA ANRIL mediated by miR-411-3p promotes the malignant proliferation and tumor stem cell like property of multiple myeloma through regulating HIF-1α.

Molecular Cloning and Functional Analysis of GmLACS2-3 Reveals Its Involvement in Cutin and Suberin Biosynthesis along with Abiotic Stress Tolerance.

Cutin and wax are the main precursors of the cuticle that covers the aerial parts of plants and provide protection against biotic and abiotic stresses. Long-chain acyl-CoA synthetases (LACSs) play diversified roles in the synthesis of cutin, wax, and triacylglycerol (TAG). Most of the information concerned with LACS functions is obtained from model plants, whereas the roles of LACS genes in Glycine max are less known. Here, we have identified 19 LACS genes in Glycine max, an important crop plant, and further focused our attention on 4 LACS2 genes (named as GmLACS2-1, 2, 3, 4, respectively). These GmLACS2 genes display different expression patterns in various organs and also show different responses to abiotic stresses, implying that these genes might play diversified functions during plant growth and against stresses. To further identify the role of GmLACS2-3, greatly induced by abiotic stresses, we transformed a construct containing its full length of coding sequence into Arabidopsis. The expression of GmLACS2-3 in an Arabidopsis atlacs2 mutant greatly suppressed its phenotype, suggesting it plays conserved roles with that of AtLACS2. The overexpression of GmLACS2-3 in wild-type plants significantly increased the amounts of cutin and suberin but had little effect on wax amounts, indicating the specific role of GmLACS2-3 in the synthesis of cutin and suberin. In addition, these GmLACS2-3 overexpressing plants showed enhanced drought tolerance. Taken together, our study deepens our understanding of the functions of LACS genes in different plants and also provides a clue for cultivating crops with strong drought resistance.

Lupeol Accumulation Correlates with Auxin in the Epidermis of Castor.

Lupeol, a natural lupane-type pentacyclic triterpene, possesses various pharmacological properties, and its production attracts attention. Significant quantities of lupeol are deposited on the castor aerial organ surface and are easily extractable as a predominant wax constituent. Thus, castor might be considered as a potential bioreactor for the production of lupeol. The lupeol biosynthesis pathway is well known, but how it is regulated remains largely unknown. Among large numbers of castor cultivars, we targeted one accession line (337) with high levels of lupeol on its stem surface and low levels thereof on its hypocotyl surface, implicating that lupeol synthesis is differentially regulated in the two organs. To explore the underlying mechanisms, we did comparative transcriptome analysis of the first internode of 337 stem and the upper hypocotyl. Our results show that large amounts of auxin-related genes are differentially expressed in both parts, implying some possible interactions between auxin and lupeol production. We also found that several auxin-responsive cis-elements are present in promoter regions of HMGR and LUS genes encoding two key enzymes involved in lupeol production. Furthermore, auxin treatments apparently induced the expression levels of RcHMGR and RcLUS. Furthermore, we observed that auxin treatment significantly increased lupeol contents, whereas inhibiting auxin transport led to an opposite phenotype. Our study reveals some relationships between hormone activity and lupeol synthesis and might provide a promising way for improving lupeol yields in castor.

Tumor suppressor miR-139-5p targets Tspan3 and regulates the progression of acute myeloid leukemia through the PI3K/Akt pathway.

Dysregulation of microRNAs is closely implicated in the initiation and progression of human cancers including acute myeloid leukemia (AML). Though miR-139-5p was reported to be a potent tumor suppressor in adult AML, its underlying molecular mechanism in AML remains to be further defined. Herein, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis were conducted to determine the expressions of miR-139-5p and tetraspanin3 (Tspan3) in AML patients and cells. Luciferase reporter assay, qRT-PCR, and Western blot analysis were carried out to detect the interaction between miR-139-5p and Tspan3. Cell proliferation, cell cycle distribution, invasion, and migration were evaluated by cell counting kit-8, flow cytometry, transwell invasion, and migration assays, respectively. Western blot analysis was conducted to determine phosphorylated-protein kinase B (Akt) and Akt levels. We found that a significant reduction in miR-139-5p expression and a prominent increase in Tspan3 expression were observed in AML patients and cells. Tspan3 was confirmed as a direct target of miR-139-5p and was negatively modulated by miR-139-5p. Rescue experiments showed that overexpression of miR-139-5p constrained cell proliferation, invasion and migration capabilities, and induced cell cycle arrest at the S phase in AML cells, which were partially reversed by Tspan3 overexpression. In addition, we found that miR-139-5p suppressed the phosphoinositide 3-kinase (PI3K)/Akt pathway in AML cells by targeting Tspan3. In conclusion, our study concluded that miR-139-5p suppressed the leukemogenesis in AML cells by targeting Tspan3 through inactivation of the PI3K/Akt pathway, providing a better understanding of AML progression.

Evolutionarily conserved function of the sacred lotus (Nelumbo nucifera Gaertn.) CER2-LIKE family in very-long-chain fatty acid elongation.

MAIN CONCLUSION: Identification of NnCER2 and NnCER2-LIKE from Nelumbo nucifera, which are required for the very-long-chain fatty acid elongation, provides new evidence that CER2 proteins are evolutionarily conserved across the eudicots. CER2-LIKE family proteins have been described as core components of the fatty acid elongase complex in Arabidopsis, maize, and rice, having specific function in synthesis of the C30 to C34 fatty acyl-CoA precursors of cuticular waxes. Little is known about the functional conservation in this gene family across species. In this study, two CER2-LIKE family proteins, NnCER2 and NnCER2-LIKE, were characterized from sacred lotus (Nelumbo nucifera), which is an ancient basal eudicot. The transcriptional expression of NnCER2 and NnCER2-LIKE was found in floating leaf blades, emergent petioles and vertical leaves, petals, and anthers. The NnCER2 and NnCER2-LIKE proteins were localized to the endoplasmic reticulum and nucleus. Overexpressing NnCER2 and NnCER2-LIKE in Arabidopsis led to alteration of cuticle wax structure in inflorescence stems, and this was associated with elevated 30, 32, and 34 carbon length wax compounds, and their derivatives. The different substrate specificities of NnCER2 and NnCER2-LIKE were explored using co-expression with AtCER6 in yeast cells. These findings provide clear evidence that the function of CER2 family proteins in producing VLCFAs is highly conserved across the eudicots.

A Nucleus-Localized Long Non-Coding RNA Enhances Drought and Salt Stress Tolerance.

Long noncoding RNAs (lncRNAs) affect gene expression through a wide range of mechanisms and are considered as important regulators in many essential biological processes. A large number of lncRNA transcripts have been predicted or identified in plants in recent years. However, the biological functions for most of them are still unknown. In this study, we identified an Arabidopsis (Arabidopsis thaliana) lncRNA, DROUGHT INDUCED lncRNA (DRIR), as a novel positive regulator of the plant response to drought and salt stress. DRIR was expressed at a low level under nonstress conditions but can be significantly activated by drought and salt stress as well as by abscisic acid (ABA) treatment. We identified a T-DNA insertion mutant, drirD , which had higher expression of the DRIR gene than the wild-type plants. The drirD mutant exhibits increased tolerance to drought and salt stress. Overexpressing DRIR in Arabidopsis also increased tolerance to drought and salt stress of the transgenic plants. The drirD mutant and the overexpressing seedlings are more sensitive to ABA than the wild type in stomata closure and seedling growth. Genome-wide transcriptome analysis demonstrated that the expression of a large number of genes was altered in drirD and the overexpressing plants. These include genes involved in ABA signaling, water transport, and other stress-relief processes. Our study reveals a mechanism whereby DRIR regulates the plant response to abiotic stress by modulating the expression of a series of genes involved in the stress response.

The Acyl Desaturase CER17 Is Involved in Producing Wax Unsaturated Primary Alcohols and Cutin Monomers.

We report n-6 monounsaturated primary alcohols (C26:1, C28:1, and C30:1 homologs) in the cuticular waxes of Arabidopsis (Arabidopsis thaliana) inflorescence stem, a class of wax not previously reported in Arabidopsis. The Arabidopsis cer17 mutant was completely deficient in these monounsaturated alcohols, and CER17 was found to encode a predicted ACYL-COENZYME A DESATURASE LIKE4 (ADS4). Studies of the Arabidopsis cer4 mutant and yeast variously expressing CER4 (a predicted fatty acyl-CoA reductase) with CER17/ADS4, demonstrated CER4's principal role in synthesis of these monounsaturated alcohols. Besides unsaturated alcohol deficiency, cer17 mutants exhibited a thickened and irregular cuticle ultrastructure and increased amounts of cutin monomers. Although unsaturated alcohols were absent throughout the cer17 stem, the mutation's effects on cutin monomers and cuticle ultrastructure were much more severe in distal than basal stems, consistent with observations that the CER17/ADS4 transcript was much more abundant in distal than basal stems. Furthermore, distal but not basal stems of a double mutant deficient for both CER17/ADS4 and LONG-CHAIN ACYL-COA SYNTHETASE1 produced even more cutin monomers and a thicker and more disorganized cuticle ultrastructure and higher cuticle permeability than observed for wild type or either mutant parent, indicating a dramatic genetic interaction on conversion of very long chain acyl-CoA precursors. These results provide evidence that CER17/ADS4 performs n-6 desaturation of very long chain acyl-CoAs in both distal and basal stems and has a major function associated with governing cutin monomer amounts primarily in the distal segments of the inflorescence stem.

A Conserved Glycine Is Identified to be Essential for Desaturase Activity of IpFAD2s by Analyzing Natural Variants from Idesia polycarpa.

High amounts of polyunsaturated fatty acids (PUFAs) in vegetable oil are not desirable for biodiesel or food oil due to their lower oxidative stability. The oil from Idesia polycarpa fruit contains 65⁻80% (mol%) linoleic acid (C18:2). Therefore, development of Idesia polycarpa cultivars with low PUFAs is highly desirable for Idesia polycarpa oil quality. Fatty acid desaturase 2 (FAD2) is the key enzyme converting oleic acid (C18:1) to C18:2. We isolated four FAD2 homologs from the fruit of Idesia polycarpa. Yeast transformed with IpFAD2-1, IpFAD2-2 and IpFAD2-3 can generate appreciable amounts of hexadecadienoic acid (C16:2) and C18:2, which are not present in wild-type yeast cells, revealing that the proteins encoded by these genes have Δ12 desaturase activity. Only trace amounts of C18:2 and little C16:2 were detected in yeast cells transformed with IpFAD2-4, suggesting IpFAD2-4 displays low activity. We also analyzed the activity of several FAD2 natural variants of Idesia polycarpa in yeast and found that a highly conserved Gly376 substitution caused the markedly reduced products catalyzed by IpFAD2-3. This glycine is also essential for the activity of IpFAD2-1 and IpFAD2-2, but its replacement in other plant FAD2 proteins displays different effects on the desaturase activity, suggesting its distinct roles across plant FAD2s proteins.

The Putative E3 Ubiquitin Ligase ECERIFERUM9 Regulates Abscisic Acid Biosynthesis and Response during Seed Germination and Postgermination Growth in Arabidopsis.

The ECERIFERUM9 (CER9) gene encodes a putative E3 ubiquitin ligase that functions in cuticle biosynthesis and the maintenance of plant water status. Here, we found that CER9 is also involved in abscisic acid (ABA) signaling in seeds and young seedlings of Arabidopsis (Arabidopsis thaliana). The germinated embryos of the mutants exhibited enhanced sensitivity to ABA during the transition from reversible dormancy to determinate seedling growth. Expression of the CER9 gene is closely related to ABA levels and displays a similar pattern to that of ABSCISIC ACID-INSENSITIVE5 (ABI5), which encodes a positive regulator of ABA responses in seeds. cer9 mutant seeds exhibited delayed germination that is independent of seed coat permeability. Quantitative proteomic analyses showed that cer9 seeds had a protein profile similar to that of the wild type treated with ABA. Transcriptomics analyses revealed that genes involved in ABA biosynthesis or signaling pathways were differentially regulated in cer9 seeds. Consistent with this, high levels of ABA were detected in dry seeds of cer9. Blocking ABA biosynthesis by fluridone treatment or by combining an ABA-deficient mutation with cer9 attenuated the phenotypes of cer9. Whereas introduction of the abi1-1, abi3-1, or abi4-103 mutation could completely eliminate the ABA hypersensitivity of cer9, introduction of abi5 resulted only in partial suppression. These results indicate that CER9 is a novel negative regulator of ABA biosynthesis and the ABA signaling pathway during seed germination.