Multiple transcription factors of Arabidopsis thaliana that are activated by LEAFY COTYLEDON 2 regulate triacylglycerol biosynthesis.

Plant triacylglycerols (TAG) are used in food and various industrial feedstocks. LEAFY COTYLEDON 2 (LEC2), a master positive regulator of TAG biosynthesis, regulates a complex network of transcription factors (TFs) during seed development. Aside from WRINKLED1 (WRI1), the TFs regulated by LEC2 related to TAG biosynthesis have not yet been identified. Previously, we identified 25 seed-expressing TFs that were upregulated in Arabidopsis leaves that overexpressed senescence-induced LEC2. In this study, each of the 25 TFs was transiently expressed in the leaves of Nicotiana benthamiana to identify unknown TFs that regulate TAG biosynthesis. The TAG content of the transformed leaves was analyzed using thin layer chromatography and gas chromatography. We observed that five TFs, ARABIDOPSIS RESPONSIVE REGULATOR 21 (ARR21), AINTEGUMENTA-LIKE 6 (AIL6), APETALA2/ETHYLENE RESPONSIVE FACTOR 55 (ERF55), WRKY DNA-BINDING PROTEIN 8 (WRKY8), and ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN 38 (ANAC038) increased TAG synthesis in the leaves. Among these, the promoters of AIL6, ERF55, WRKY8, and ANAC038 contain RY motifs, which are LEC2-binding sites activated by LEC2. AIL6 overexpression in Arabidopsis increased the total fatty acid (FA) content in seeds and altered the FA composition, with increases in 16:0, 18:1, and 18:2 and decreases in 18:0, 18:3, and 20:1 compared with those in the wild type (WT). AIL6 overexpression activates several FA and TAG biosynthesis genes. Therefore, our study successfully identified several new TFs regulated by LEC2 in TAG biosynthesis and showed that AIL6 increased the TAG content in seeds.

PDAT1 genome editing reduces hydroxy fatty acid production in transgenic Arabidopsis.

The fatty acids content of castor (Ricinus communis L.) seed oil is 80-90% ricinoleic acid, which is a hydroxy fatty acid (HFA). The structures and functional groups of HFAs are different from those of common fatty acids and are useful for various industrial applications. However, castor seeds contain the toxin ricin and an allergenic protein, which limit their cultivation. Accordingly, many researchers are conducting studies to enhance the production of HFAs in Arabidopsis thaliana, a model plant for oil crops. Oleate 12-hydroxylase from castor (RcFAH12), which synthesizes HFA (18:1-OH), was transformed into an Arabidopsis fae1 mutant, resulting in the CL37 line producing a maximum of 17% HFA content. In addition, castor phospholipid:diacylglycerol acyltransferase 1-2 (RcPDAT1-2), which catalyzes the production of triacylglycerol by transferring HFA from phosphatidylcholine to diacylglycerol, was transformed into the CL37 line to develop a P327 line that produces 25% HFA. In this study, we investigated changes in HFA content when endogenous Arabidopsis PDAT1 (AtPDAT1) of the P327 line was edited using the CRISPR/Cas9 technique. The successful mutation resulted in three independent lines with different mutation patterns, which were transmitted until the T4 generation. Fatty acid analysis of the seeds showed that HFA content decreased in all three mutant lines. These findings indicate that AtPDAT1 as well as RcPDAT1-2 in the P327 line are involved in transferring and increasing HFAs to triacylglycerol. [BMB Reports 2024; 57(2): 86-91].

Physaria fendleri FAD3-1 overexpression increases ɑ-linolenic acid content in Camelina sativa seeds.

Camelina (Camelina sativa) is an oil crop with a short growing period, resistance to drought and cold, low fertilizer requirements, and can be transformed using floral dipping. Seeds have a high content of polyunsaturated fatty acids, especially ɑ-linolenic acid (ALA), at 32-38%. ALA is an omega-3 fatty acid that is a substrate for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the human body. In this study, ALA content was further enhanced by the seed-specific expression of Physaria fendleri FAD3-1 (PfFAD3-1) in camelina. The ALA content increased up to 48% in T2 seeds and 50% in T3 seeds. Additionally, size of the seeds increased. The expression of fatty acid metabolism-related genes in PfFAD3-1 OE transgenic lines was different from that in the wild type, where the expression of CsFAD2 decreased and CsFAD3 increased. In summary, we developed a high omega-3 fatty acid-containing camelina with up to 50% ALA content by introducing PfFAD3-1. This line can be used for genetic engineering to obtain EPA and DHA from seeds.

Characterization of fab2 T-DNA insertion mutants in terms of fatty acid composition and plant phenotype.

Fatty acid biosynthesis 2 (FAB2) is an essential enzyme responsible for the synthesis of unsaturated fatty acids in chloroplast membrane lipids found in leaves and triacylglycerols (TAG) in seeds. FAB2 functions at the junction of saturated to unsaturated fatty acid conversion in chloroplasts by converting 18:0-ACP to 18:1-ACP. In the present study, plant growth and seed phenotypes were examined in three Arabidopsis T-DNA mutants (fab2-1, fab2-2, and fab2-3). The three fab2 T-DNA mutants exhibited increased 18:0 fatty acid content in both the leaves and seeds. The degree of growth inhibition of the fab2 mutant was proportional to the increase in 18:0 and decrease in 18:3 fatty acids present in the leaves. The FAB2 mutation affected seed yield but not the seed phenotype. This result indicates that FAB2 affects the fatty acid composition of the leaf chloroplast membrane more than seed TAG. In summary, the characteristics of these three fab2 mutants provide information for studying leaf membrane lipid and seed oil biosynthesis.

Applications and prospects of genome editing in plant fatty acid and triacylglycerol biosynthesis.

Triacylglycerol (TAG), which is a neutral lipid, has a structure in which three molecules of fatty acid (FA) are ester-bonded to one molecule of glycerol. TAG is important energy source for seed germination and seedling development in plants. Depending on the FA composition of the TAG, it is used as an edible oil or industrial material for cosmetics, soap, and lubricant. As the demand for plant oil is rising worldwide, either the type of FA must be changed or the total oil content of various plants must be increased. In this review, we discuss the regulation of FA metabolism by Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, a recent genome-editing technology applicable to various plants. The development of plants with higher levels of oleic acid or lower levels of very long-chain fatty acids (VLCFAs) in seeds are discussed. In addition, the current status of research on acyltransferases, phospholipases, TAG lipases, and TAG synthesis in vegetative tissues is described. Finally, strategies for the application of CRISPR/Cas9 in lipid metabolism studies are mentioned.

Enhanced production of hydroxy fatty acids in Arabidopsis seed through modification of multiple gene expression.

BACKGROUND: Castor (Ricinus communis L.) seeds contain unusual fatty acid, hydroxy fatty acid (HFA) used as a chemical feedstock for numerous industrial products. Castor cultivation is limited by the potent toxin ricin in its seeds and other poor agronomic traits, so it is advantageous to develop a suitable HFA-producing crop. Significant research efforts have been made to produce HFA in model Arabidopsis, but the level of HFA produced in transgenic Arabidopsis is much less than the level found in castor seeds which produce 90% HFA in seed oil. RESULTS: We designed a transformation construct that allowed co-expression of five essential castor genes (named pCam5) involved in HFA biosynthesis, including an oleate [Formula: see text] 12-hydroxylase (FAH12), diacylglycerol (DAG) acyltransferase 2 (DGAT2), phospholipid: DAG acyltransferase 1-2 (PDAT1-2), phosphatidylcholine (PC): DAG cholinephosphotransferase (PDCT) and Lyso-PC acyltransferase (LPCAT). Transgenic Arabidopsis pCam5 lines produced HFA counting for 25% in seed oil. By knocking out Arabidopsis Fatty acid elongase 1 (AtFAE1) in pCam5 using CRISPR/Cas9 technology, the resulted pCam5-atfae1 lines produced over 31% of HFA. Astonishingly, the pCam5-atfae1 line increased seed size, weight, and total oil per seed exceeding wild type by 40%. Seed germination, seedling growth and seed mucilage content of pCam5-atfae1 lines were not affected by the genetic modification. CONCLUSIONS: Our results provide not only insights for future research uncovering mechanisms of HFA synthesis in seed, but also metabolic engineering strategies for generating safe HFA-producing crops.

The seed-specific transcription factor DPBF2 modulates the fatty acid composition in seeds.

Triacylglycerol (TAG), an ester derived from glycerol and three fatty acids (FAs), is synthesized during seed development and controlled by transcriptional regulation. We examined the mechanism regulating the FA composition of developing Arabidopsis thaliana seeds. The seed-specific DC3 PROMOTER-BINDING FACTOR2 (DPBF2) transcription factor was upregulated by LEAFY COTYLEDON2 (LEC2). DPBF2 showed transcriptional activity in yeast and localized to the nucleus in Arabidopsis protoplast cells. The Arabidopsis dpbf2-1 homozygous T-DNA mutant and transgenic lines overexpressing of DPBF2 using a seed-specific phaseolin promoter in wild-type (WT) Arabidopsis and in dpbf2-1 showed similar FA composition profiles in their seeds. Their 18:2 and 20:1 FA contents were higher, but 18:1 and 18:3 contents were lower than that of WT. Transcript levels of FATTY ACID DESATURASE2 (FAD2), FAD3, LYSOPHOSPHATIDYLCHOLINE ACYLTRANSFERASE1 (LPCAT1), LPCAT2, PHOSPHATIDYLCHOLINE DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE (PDCT), and FATTY ACID ELONGASE 1 (FAE1) are increased in DPBF2-overexpressing seeds. Besides, PDCT and FAE1 were upregulated by DPBF2, LEC1-LIKE (L1L), and NUCLEAR FACTOR-YC2 (NF-YC2) transcriptional complex based on tobacco protoplast transcriptional activation assay. These results suggest that DPBF2 effectively modulates the expression of genes encoding FA desaturases, elongase, and acyl-editing enzymes for modifying the unsaturated FA composition in seeds.

C-to-G Base Editing Enhances Oleic Acid Production by Generating Novel Alleles of FATTY ACID DESATURASE 2 in Plants.

The demand for vegetable oil, which is mainly used for dietary purposes and cooking, is steadily increasing worldwide. It is often desirable to reduce unsaturation levels of fatty acids in order to increase storage stability and reduce trans-fat generation during cooking. Functional disruption of FATTY ACID DESATURASE 2 (FAD2) prevents the conversion of monounsaturated oleic acid to polyunsaturated linoleic acid, thereby enhancing the production of the desirable oleic acid. However, FAD2 null alleles, due to growth defects under stress conditions, are impractical for agronomical purposes. Here, we aimed to attenuate FAD2 activity in planta while avoiding adverse growth effects by introducing amino-acid substitutions using CRISPR base editors. In Arabidopsis, we applied the adenine base editor (ABE) and cytosine base editor (CBE) to induce semi-random base substitutions within several selected FAD2 coding regions. Isolation of base-edited fad2 alleles with higher oleic acid revealed that the CBE application induced C-to-T and/or C-to-G base substitutions within the targeted sequences, resulting in an alteration of the FAD2 enzyme activities; for example, fad2-144 with multiple C-to-G base substitutions showed less growth defects but with a significant increase in oleic acids by 3-fold higher than wild type. Our "proof-of-concept" approach suggests that equivalent alleles may be generated in vegetable oil crops via precision genome editing for practical cultivation. Our targeted semi-random strategy may serve as a new complementary platform for planta engineering of useful agronomic traits.

Transcriptome Analysis and Identification of Lipid Genes in Physaria lindheimeri, a Genetic Resource for Hydroxy Fatty Acids in Seed Oil.

Hydroxy fatty acids (HFAs) have numerous industrial applications but are absent in most vegetable oils. Physaria lindheimeri accumulating 85% HFA in its seed oil makes it a valuable resource for engineering oilseed crops for HFA production. To discover lipid genes involved in HFA synthesis in P. lindheimeri, transcripts from developing seeds at various stages, as well as leaf and flower buds, were sequenced. Ninety-seven percent clean reads from 552,614,582 raw reads were assembled to 129,633 contigs (or transcripts) which represented 85,948 unique genes. Gene Ontology analysis indicated that 60% of the contigs matched proteins involved in biological process, cellular component or molecular function, while the remaining matched unknown proteins. We identified 42 P. lindheimeri genes involved in fatty acid and seed oil biosynthesis, and 39 of them shared 78-100% nucleotide identity with Arabidopsis orthologs. We manually annotated 16 key genes and 14 of them contained full-length protein sequences, indicating high coverage of clean reads to the assembled contigs. A detailed profiling of the 16 genes revealed various spatial and temporal expression patterns. The further comparison of their protein sequences uncovered amino acids conserved among HFA-producing species, but these varied among non-HFA-producing species. Our findings provide essential information for basic and applied research on HFA biosynthesis.

The Arabidopsis MYB96 Transcription Factor Mediates ABA-Dependent Triacylglycerol Accumulation in Vegetative Tissues under Drought Stress Conditions.

Triacylglycerols (TAGs), a major lipid form of energy storage, are involved in a variety of plant developmental processes. While carbon reserves mainly accumulate in seeds, significant amounts of TAG have also been observed in vegetative tissues. Notably, the accumulation of leaf TAGs is influenced by environmental stresses such as drought stress, although underlying molecular networks remain to be fully elucidated. In this study, we demonstrate that the R2R3-type MYB96 transcription factor promotes TAG biosynthesis in Arabidopsis thaliana seedlings. Core TAG biosynthetic genes were up-regulated in myb96-ox seedlings, but down-regulated in myb96-deficient seedlings. In particular, ABA stimulates TAG accumulation in the vegetative tissues, and MYB96 plays a fundamental role in this process. Considering that TAG accumulation contributes to plant tolerance to drought stress, MYB96-dependent TAG biosynthesis not only triggers plant adaptive responses but also optimizes energy metabolism to ensure plant fitness under unfavorable environmental conditions.

Three-dimensional comparative study on the accuracy and reproducibility of dental casts fabricated by 3D printers.

STATEMENT OF PROBLEM: Studies investigating the precision of 3-dimensional (3D) printed casts for fixed prosthodontics are scarce. PURPOSE: The purpose of this in vitro study was to compare the accuracy and reproducibility of dental casts made by the conventional method and by 3D printing. MATERIAL AND METHODS: A master model was designed and fabricated with polyetherketoneketone. Ten specimens were fabricated with Type IV dental stone with polyvinyl siloxane. A light scanner was used to scan the master model, and the data were converted to standard tessellation language (STL) files. Three different types of 3D printers (Objet EDEN260V, ProMaker D35, and LC-3Dprint) were used to make 10 specimens each. All specimens were scanned by the light scanner, and the scanned files were superimposed on the files of the master model with specialized software to analyze the volumetric changes. The Kruskal-Wallis test, Mann-Whitney U tests, and Bonferroni method were performed with statistical analysis software (α=.05). RESULTS: The volumetric changes in casts made by the conventional method and by the 3D printers were significantly different. The conventional casts showed smaller volumetric change than the 3D-printed casts. Significant differences (P<.05) were found among the different types of 3D printers. The ultraviolet-polymerizing polymer with digital light processing exhibited the smallest volumetric change. In 3D color maps, the deformations were in similar patterns with all the 3D printers. CONCLUSIONS: The conventional method of die fabrication was more reliable than that of 3D printers.