No abstract

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.

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$$\Delta$$ Δ 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 ArabidopsispCam5 lines produced HFA counting for 25% in seed oil. By knocking out ArabidopsisFatty 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 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.

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.