Molecular machinery of auxin synthesis,secretion, and perception in the unicellular chlorophyte alga Chlorella sorokiniana UTEX 1230

Indole-3-acetic acid is a ubiquitous small molecule found in all domains of life. It is the predominant and most active auxin in seed plants, where it coordinates a variety of complex growth and development processes. The potential origin of auxin signaling in algae remains a matter of some controversy. In order to clarify the evolutionary context of algal auxin signaling, we undertook a genomic survey to assess whether auxin acts as a signaling molecule in the emerging model chlorophyte Chlorella sorokiniana UTEX 1230. C. sorokiniana produces the auxin indole-3-acetic acid (IAA), which was present in both the cell pellet and in the supernatant at a concentration of ~ 1 nM, and its genome encodes orthologs of genes related to auxin synthesis, transport, and signaling in higher plants. Candidate orthologs for the canonical AUX/IAA signaling pathway were not found; however, auxin-binding protein 1 (ABP1), an alternate auxin receptor, is present and highly conserved at essential auxin binding and zinc coordinating residues. Additionally, candidate orthologs for PIN proteins, responsible for intercellular, vectorial auxin transport in higher plants, were not found, but PILs (PIN-Like) proteins, a recently discovered family that mediates intracellular auxin transport, were identified. The distribution of auxin related gene in this unicellular chlorophyte demonstrates that a core suite of auxin signaling components was present early in the evolution of plants. Understanding the simplified auxin signaling pathways in chlorophytes will aid in understanding phytohormone signaling and crosstalk in seed plants, and in understanding the diversification and integration of developmental signals during the evolution of multicellular plants

A thraustochytrid diacylglycerol acyltransferase 2 with broad substrate specificity strongly increases oleic acid content in engineered \u3ci\u3eArabidopsis thaliana\u3c/i\u3e seeds

Diacylglycerol acyltransferase (DGAT) catalyses the last step in acyl-CoA-dependent triacylglycerol (TAG) biosynthesis and is an important determinant of cellular oil content and quality. In this study, a gene, designated TaDGAT2, encoding a type 2 DGAT (DGAT2)-related enzyme was identified from the oleaginous marine protist Thraustochytrium aureum. The deduced TaDGAT2 sequence contains a ~460 amino acid domain most closely related to DGAT2s from Dictyostelium sp. (45–50% identity). Recombinant TaDGAT2 restored TAG biosynthesis to the Saccharomyces cerevisiae H1246 TAG-deficient mutant, and microsomes from the complemented mutant displayed DGAT activity with C16 and C18 saturated and unsaturated fatty acyl-CoA and diacylglycerol substrates. To examine its biotechnological potential, TaDGAT2 was expressed under control of a strong seed-specific promoter in wild-type Arabidopsis thaliana and the high linoleic acid fad3fae1 mutant. In both backgrounds, little change was detected in seed oil content, but a striking increase in oleic acid content of seeds was observed. This increase was greatest in fad3fae1 seeds, where relative amounts of oleic acid increased nearly 2-fold to \u3e50% of total fatty acids. In addition, \u3e2-fold increase in oleic acid levels was detected in the triacylglycerol sn-2 position and in the major seed phospholipid phosphatidylcholine. These results suggest that increased seed oleic acid content mediated by TaDGAT2 is influenced in part by the fatty acid composition of host cells and occurs not by enhancing oleic acid content at the TAG sn-3 position directly but by increasing total oleic acid levels in seeds, presumably by limiting flux through phosphatidylcholine-based desaturation reactions. Includes supplementary information

Synthesis, secretion, and perception of abscisic acid regulates stress responses in \u3ci\u3eChlorella sorokiniana\u3c/i\u3e

Abscisic acid (ABA) is a phytohormone that has been extensively characterized in higher plants for its roles in seed and bud dormancy, leaf abscission, and stress responses. Genomic studies have identified orthologs for ABA-related genes throughout the Viridiplantae, including in unicellular algae; however, the role of ABA in algal physiology has not been characterized, and the existence of such a role has been a matter of dispute. In this study, we demonstrate that ABA is involved in regulating algal stress responses. Chlorella sorokiniana strain UTEX 1230 contains genes orthologous to those of higher plants which are essential for ABA biosynthesis, sensing, and degradation. RNAseq-based transcriptomic studies reveal that treatment with ABA induces dramatic changes in gene expression profiles, including the induction of a subset of genes involved in DNA replication and repair, a phenomenon which has been demonstrated in higher plants. Pretreatment of C. sorokiniana cultures with ABA exerts a protective effect on cell viability in response to ultraviolet radiation. Additionally, C. sorokiniana produces and secretes biologically relevant amounts of both ABA and the oxylipin 12-oxo-phytodienoic acid (OPDA) into the growth medium in response to abiotic stressors. Taken together, these phenomena suggest that ABA signaling evolved as an intercellular stress response signaling molecule in eukaryotic microalgae prior to the evolution of multicellularity and colonization of land

ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activity

Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures sufficient sphingolipids to support growth while limiting the accumulation of sphingolipid metabolites that induce programmed cell death. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is considered the primary sphingolipid homeostatic regulatory point. In this report, Arabidopsis (Arabidopsis thaliana) putative SPT regulatory proteins, orosomucoidlike proteins AtORM1 and AtORM2, were found to interact physically with Arabidopsis SPT and to suppress SPT activity when coexpressed with Arabidopsis SPT subunits long-chain base1 (LCB1) and LCB2 and the small subunit of SPT in a yeast (Saccharomyces cerevisiae) SPT-deficient mutant. Consistent with a role in SPT suppression, AtORM1 and AtORM2 overexpression lines displayed increased resistance to the programmed cell death-inducing mycotoxin fumonisin B1, with an accompanying reduced accumulation of LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Conversely, RNA interference (RNAi) suppression lines of AtORM1 and AtORM2 displayed increased sensitivity to fumonisin B1 and an accompanying strong increase in LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Overexpression lines also were found to have reduced activity of the class I ceramide synthase that uses C16 fatty acid acyl-coenzyme A and dihydroxy LCB substrates but increased activity of class II ceramide synthases that use very-long-chain fatty acyl-coenzyme A and trihydroxy LCB substrates. RNAi suppression lines, in contrast, displayed increased class I ceramide synthase activity but reduced class II ceramide synthase activity. These findings indicate that ORM mediation of SPT activity differentially regulates functionally distinct ceramide synthase activities as part of a broader sphingolipid homeostatic regulatory network

A thraustochytrid diacylglycerol acyltransferase 2 with broad substrate specificity strongly increases oleic acid content in engineered \u3ci\u3eArabidopsis thaliana\u3c/i\u3e seeds

Diacylglycerol acyltransferase (DGAT) catalyses the last step in acyl-CoA-dependent triacylglycerol (TAG) biosynthesis and is an important determinant of cellular oil content and quality. In this study, a gene, designated TaDGAT2, encoding a type 2 DGAT (DGAT2)-related enzyme was identified from the oleaginous marine protist Thraustochytrium aureum. The deduced TaDGAT2 sequence contains a ~460 amino acid domain most closely related to DGAT2s from Dictyostelium sp. (45–50% identity). Recombinant TaDGAT2 restored TAG biosynthesis to the Saccharomyces cerevisiae H1246 TAG-deficient mutant, and microsomes from the complemented mutant displayed DGAT activity with C16 and C18 saturated and unsaturated fatty acyl-CoA and diacylglycerol substrates. To examine its biotechnological potential, TaDGAT2 was expressed under control of a strong seed-specific promoter in wild-type Arabidopsis thaliana and the high linoleic acid fad3fae1 mutant. In both backgrounds, little change was detected in seed oil content, but a striking increase in oleic acid content of seeds was observed. This increase was greatest in fad3fae1 seeds, where relative amounts of oleic acid increased nearly 2-fold to \u3e50% of total fatty acids. In addition, \u3e2-fold increase in oleic acid levels was detected in the triacylglycerol sn-2 position and in the major seed phospholipid phosphatidylcholine. These results suggest that increased seed oleic acid content mediated by TaDGAT2 is influenced in part by the fatty acid composition of host cells and occurs not by enhancing oleic acid content at the TAG sn-3 position directly but by increasing total oleic acid levels in seeds, presumably by limiting flux through phosphatidylcholine-based desaturation reactions. Includes supplementary information

The Origin and Biosynthesis of the Benzenoid Moiety of Ubiquinone (Coenzyme Q) in \u3ci\u3eArabidopsis\u3c/i\u3e

It is not known how plants make the benzenoid ring of ubiquinone, a vital respiratory cofactor. Here, we demonstrate that Arabidopsis thaliana uses for that purpose two separate biosynthetic branches stemming from phenylalanine and tyrosine. Gene network modeling and characterization of T-DNA mutants indicated that acyl-activating enzyme encoded by At4g19010 contributes to the biosynthesis of ubiquinone specifically from phenylalanine. CoA ligase assays verified that At4g19010 prefers para-coumarate, ferulate, and caffeate as substrates. Feeding experiments demonstrated that the at4g19010 knockout cannot use para-coumarate for ubiquinone biosynthesis and that the supply of 4-hydroxybenzoate, the side-chain shortened version of para-coumarate, can bypass this blockage. Furthermore, a trans-cinnamate 4-hydroxylase mutant, which is impaired in the conversion of trans-cinnamate into para-coumarate, displayed similar defects in ubiquinone biosynthesis to that of the at4g19010 knockout. Green fluorescent protein fusion experiments demonstrated that At4g19010 occurs in peroxisomes, resulting in an elaborate biosynthetic architecture where phenylpropanoid intermediates have to be transported from the cytosol to peroxisomes and then to mitochondria where ubiquinone is assembled. Collectively, these results demonstrate that At4g19010 activates the propyl side chain of para-coumarate for its subsequent β-oxidative shortening. Evidence is shown that the peroxisomal ABCD transporter (PXA1) plays a critical role in this branch. Includes supplementary files

Downregulation of a CYP74 Rubber Particle Protein Increases Natural Rubber Production in \u3ci\u3eParthenium argentatum\u3c/i\u3e

We report functional genomics studies of a CYP74 rubber particle protein from Parthenium argentatum, commonly called guayule. Previously identified as an allene oxide synthase (AOS), this CYP74 constitutes the most abundant protein found in guayule rubber particles. Transgenic guayule lines with AOS gene expression down-regulated by RNAi (AOSi) exhibited strong phenotypes that included agricultural traits conducive to enhancing rubber yield. AOSi lines had higher leaf and stem biomass, thicker stembark tissues, increased stem branching and improved net photosynthetic rate. Importantly, the rubber content was significantly increased in AOSi lines compared to the wild-type (WT), vector control and AOS overexpressing (AOSoe) lines, when grown in controlled environments both in tissue-culture media and in greenhouse/growth chambers. Rubber particles from AOSi plants consistently had less AOS particle-associated protein, and lower activity (for conversion of 13-HPOT to allene oxide). Yet plants with downregulated AOS showed higher rubber transferase enzyme activity. The increase in biomass in AOSi lines was associated with not only increases in the rate of photosynthesis and non-photochemical quenching (NPQ), in the cold, but also in the content of the phytohormone SA, along with a decrease in JA, GAs, and ABA. The increase in biosynthetic activity and rubber content could further result from the negative regulation of AOS expression by high levels of salicylic acid in AOSi lines and when introduced exogenously. It is apparent that AOS in guayule plays a pivotal role in rubber production and plant growth

Identification of Homogentisate Dioxygenase as a Target for Vitamin E Biofortification in Oilseeds

Soybean (Glycine max) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. As a tool for gene function discovery and improvement of this important crop, a mutant population was generated using fast neutron irradiation. Visual screening of mutagenized seeds identified a mutant line, designated MO12, which produced brown seeds as opposed to the yellow seeds produced by the unmodified Williams 82 parental cultivar. Using forward genetic methods combined with comparative genome hybridization analysis, we were able to establish that deletion of the GmHGO1 gene is the genetic basis of the brown seeded phenotype exhibited by the MO12 mutant line. GmHGO1 encodes a homogentisate dioxygenase (HGO), which catalyzes the committed enzymatic step in homogentisate catabolism. This report describes to our knowledge the first functional characterization of a plant HGO gene, defects of which are linked to the human genetic disease alkaptonuria. We show that reduced homogentisate catabolism in a soybean HGO mutant is an effective strategy for enhancing the production of lipid-soluble antioxidants such as vitamin E, as well as tolerance to herbicides that target pathways associated with homogentisate metabolism. Furthermore, this work demonstrates the utility of fast neutron mutagenesis in identifying novel genes that contribute to soybean agronomic traits

Disruption of long-chain base hydroxylation alters growth and impacts sphingolipid synthesis in Physcomitrella patens

Sphingolipids have roles as membrane structural components and as bioactive molecules in plants. In Physcomitrella patens, 4-hydroxysphinganine (phytosphingosine, t18:0) is the predominant sphingolipid long-chain base (LCB). To assess the functional significance of t18:0, CRISPR-Cas9 mutagenesis was used to generate mutant lines lacking the sole SPHINGOID BASE HYDROXYLASE (SBH) gene encoding the hydroxylase responsible for converting sphinganine (d18:0) to t18:0. Total sphingolipid content in sbh protonemata was 2.4-fold higher than in wild-type. Modest changes in glycosyl inositolphosphorylceramide (GIPC) glycosylation patterns occurred. Sphingolipidomic analyses of mutants lacking t18:0 indicated modest alterations in acyl-chain pairing with d18:0 in GIPCs and ceramides, but dramatic alterations in acyl-chain pairing in glucosylceramides, in which 4,8-sphingadienine (d18:2) was the principal LCB. A striking accumulation of free and phosphorylated LCBs accompanied loss of the hydroxylase. The sbh lines exhibited altered morphology, including smaller chloronemal cell size, irregular cell shape, reduced gametophore size, and increased pigmentation. In the presence of the synthetic trihydroxy LCB t17:0, the endogenous sphingolipid content of sbh lines decreased to wild-type levels, and the mutants exhibited phenotypes more similar to wild-type plants. These results demonstrate the importance of sphingolipid content and composition to Physcomitrella growth. They also illuminate similarities in regulating sphingolipid content but differences in regulating sphingolipid species composition between the bryophyte P. patens and angiosperm A. thaliana

A Specialized Diacylglycerol Acyltransferase Contributes to the Extreme Medium-Chain Fatty Acid Content of \u3ci\u3eCuphea\u3c/i\u3e Seed Oil

Seed oils of many Cuphea sp. contain .90% of medium-chain fatty acids, such as decanoic acid (10:0). These seed oils, which are among the most compositionally variant in the plant kingdom, arise from specialized fatty acid biosynthetic enzymes and specialized acyltransferases. These include lysophosphatidic acid acyltransferases (LPAT) and diacylglycerol acyltransferases (DGAT) that are required for successive acylation of medium-chain fatty acids in the sn-2 and sn-3 positions of seed triacylglycerols (TAGs). Here we report the identification of a cDNA for a DGAT1-type enzyme, designated CpuDGAT1, from the transcriptome of C. avigera var pulcherrima developing seeds. Microsomes of camelina (Camelina sativa) seeds engineered for CpuDGAT1 expression displayed DGAT activity with 10:0-CoA and the diacylglycerol didecanoyl, that was approximately 4-fold higher than that in camelina seed microsomes lacking CpuDGAT1. In addition, coexpression in camelina seeds of CpuDGAT1 with a C. viscosissima FatB thioesterase (CvFatB1) that generates 10:0 resulted in TAGs with nearly 15 mol % of 10:0. More strikingly, expression of CpuDGAT1 and CvFatB1 with the previously described CvLPAT2, a 10:0-CoA-specific Cuphea LPAT, increased 10:0 amounts to 25 mol % in camelina seed TAG. These TAGs contained up to 40 mol % 10:0 in the sn-2 position, nearly double the amounts obtained from coexpression of CvFatB1 and CvLPAT2 alone. Although enriched in diacylglycerol, 10:0 was not detected in phosphatidylcholine in these seeds. These findings are consistent with channeling of 10:0 into TAG through the combined activities of specialized LPAT and DGAT activities and demonstrate the biotechnological use of these enzymes to generate 10:0-rich seed oils