Ketocarotenoid production in tomato triggers metabolic reprogramming and cellular adaptation: The quest for homeostasis.

Plants are sessile and therefore have developed an extraordinary capacity to adapt to external signals. Here, the focus is on the plasticity of the plant cell to respond to new intracellular cues. Ketocarotenoids are high-value natural red pigments with potent antioxidant activity. In the present study, system-level analyses have revealed that the heterologous biosynthesis of ketocarotenoids in tomato initiated a series of cellular and metabolic mechanisms to cope with the formation of metabolites that are non-endogenous to the plant. The broad multilevel changes were linked to, among others, (i) the remodelling of the plastidial membrane, where the synthesis and storage of ketocarotenoids occurs; (ii) the recruiting of core metabolic pathways for the generation of metabolite precursors and energy; and (iii) redox control. The involvement of the metabolites as regulators of cellular processes shown here reinforces their pivotal role suggested in the remodelled 'central dogma' concept. Furthermore, the role of metabolic reprogramming to ensure cellular homeostasis is proposed.

Diverting phenylpropanoid pathway flux from sinapine to produce industrially useful 4-vinyl derivatives of hydroxycinnamic acids in Brassicaceous oilseeds.

Sinapine (sinapoylcholine) is an antinutritive phenolic compound that can account for up to 2% of seed weight in brassicaceous oilseed crops and reduces the suitability of their protein-rich seed meal for use as animal feed. Sinapine biosynthesis draws on hydroxycinnamic acid precursors produced by the phenylpropanoid pathway. The 4-vinyl derivatives of several hydroxycinnamic acids have industrial applications. For example, 4-vinyl phenol (4-hydroxystyrene) is a building block for a range of synthetic polymers applied in resins, inks, elastomers, and coatings. Here we have expressed a modified bacterial phenolic acid decarboxylase (PAD) in developing seed of Camelina sativa to redirect phenylpropanoid pathway flux from sinapine biosynthesis to the production of 4-vinyl phenols. PAD expression led to a ∼95% reduction in sinapine content in seeds of both glasshouse and field grown C. sativa and to an accumulation of 4-vinyl derivatives of hydroxycinnamic acids, primarily as glycosides. The most prevalent aglycone was 4-vinyl phenol, but 4-vinyl guaiacol, 6-hydroxy-4-vinyl guaiacol and 4-vinylsyringol (Canolol) were also detected. The molar quantity of 4-vinyl phenol glycosides was more than twice that of sinapine in wild type seeds. PAD expression was not associated with an adverse effect on seed yield, harvest index, seed morphology, storage oil content or germination in either glasshouse or field experiments. Our data show that expression of PAD in brassicaceous oilseeds can supress sinapine accumulation, diverting phenylpropanoid pathway flux into 4-vinyl phenol derivatives, thereby also providing a non-petrochemical source of this class of industrial chemicals.

Monogalactosyl diacylglycerol synthase 3 affects phosphate utilization and acquisition in rice.

Galactolipids are essential to compensate for the loss of phospholipids by 'membrane lipid remodelling' in plants under phosphorus (P) deficiency conditions. Monogalactosyl diacylglycerol (MGDG) synthases catalyse the synthesis of MGDG which is further converted into digalactosyl diacylglycerol (DGDG), later replacing phospholipids in the extraplastidial membranes. However, the roles of these enzymes are not well explored in rice. In this study, the rice MGDG synthase 3 gene (OsMGD3) was identified and functionally characterized. We showed that the plant phosphate (Pi) status and the transcription factor PHOSPHATE STARVATION RESPONSE 2 (OsPHR2) are involved in the transcriptional regulation of OsMGD3. CRISPR/Cas9 knockout and overexpression lines of OsMGD3 were generated to explore its potential role in rice adaptation to Pi deficiency. Compared with the wild type, OsMGD3 knockout lines displayed a reduced Pi acquisition and utilization while overexpression lines showed an enhancement of the same. Further, OsMGD3 showed a predominant role in roots, altering lateral root growth. Our comprehensive lipidomic analysis revealed a role of OsMGD3 in membrane lipid remodelling, in addition to a role in regulating diacylglycerol and phosphatidic acid contents that affected the expression of Pi transporters. Our study highlights the role of OsMGD3 in affecting both internal P utilization and P acquisition in rice.

Basic LEUCINE ZIPPER TRANSCRIPTION FACTOR67 Transactivates DELAY OF GERMINATION1 to Establish Primary Seed Dormancy in Arabidopsis.

Seed dormancy governs the timing of germination, one of the most important developmental transitions in a plant's life cycle. The DELAY OF GERMINATION1 (DOG1) gene is a key regulator of seed dormancy and a major quantitative trait locus in Arabidopsis (Arabidopsis thaliana). DOG1 expression is under tight developmental and environmental regulation, but the transcription factors involved are not known. Here we show that basic LEUCINE ZIPPER TRANSCRIPTION FACTOR67 (bZIP67) acts downstream of the central regulator of seed development, LEAFY COTYLEDON1, to transactivate DOG1 during maturation and help to establish primary dormancy. We show that bZIP67 overexpression enhances dormancy and that bZIP67 protein (but not transcript) abundance is increased in seeds matured in cool conditions, providing a mechanism to explain how temperature regulates DOG1 expression. We also show that natural allelic variation in the DOG1 promoter affects bZIP67-dependent transactivation, providing a mechanism to explain ecotypic differences in seed dormancy that are controlled by the DOG1 locus.

Engineering the stereoisomeric structure of seed oil to mimic human milk fat.

Human milk fat substitute (HMFS) is a class of structured lipid that is widely used as an ingredient in infant formulas. Like human milk fat, HMFS is characterized by enrichment of palmitoyl (C16:0) groups specifically at the middle (sn-2 or ß) position on the glycerol backbone, and there is evidence that triacylglycerol (TAG) with this unusual stereoisomeric structure provides nutritional benefits. HMFS is currently made by in vitro enzyme-based catalysis because there is no appropriate biological alternative to human milk fat. Most of the fat currently used in infant formulas is obtained from plants, which exclude C16:0 from the middle position. In this study, we have modified the metabolic pathway for TAG biosynthesis in the model oilseed Arabidopsis thaliana to increase the percentage of C16:0 at the middle (vs. outer) positions by more than 20-fold (i.e., from ∼3% in wild type to >70% in our final iteration). This level of C16:0 enrichment is comparable to human milk fat. We achieved this by relocating the C16:0-specific chloroplast isoform of the enzyme lysophosphatidic acid acyltransferase (LPAT) to the endoplasmic reticulum so that it functions within the cytosolic glycerolipid biosynthetic pathway to esterify C16:0 to the middle position. We then suppressed endogenous LPAT activity to relieve competition and knocked out phosphatidylcholine:diacylglycerol cholinephosphotransferase activity to promote the flux of newly made diacylglycerol directly into TAG. Applying this technology to oilseed crops might provide a source of HMFS for infant formula.

Laboratory phenomics predicts field performance and identifies superior indica haplotypes for early seedling vigour in dry direct-seeded rice.

Seedling vigour is an important agronomic trait and is gaining attention in Asian rice (Oryza sativa) as cultivation practices shift from transplanting to forms of direct seeding. To understand the genetic control of rice seedling vigour in dry direct seeded (aerobic) conditions we measured multiple seedling traits in 684 accessions from the 3000 Rice Genomes (3K-RG) population in both the laboratory and field at three planting depths. Our data show that phenotyping of mesocotyl length in laboratory conditions is a good predictor of field performance. By performing a genome wide association study, we found that the main QTL for mesocotyl length, percentage seedling emergence and shoot biomass are co-located on the short arm of chromosome 7. We show that haplotypes in the indica subgroup from this region can be used to predict the seedling vigour of 3K-RG accessions. The selected accessions may serve as potential donors in genomics-assisted breeding programs.

Production of the infant formula ingredient 1,3-olein-2-palmitin in Arabidopsis thaliana seeds.

In human milk fat, palmitic acid (16:0) is esterified to the middle (sn-2 or ß) position on the glycerol backbone and oleic acid (18:1) predominantly to the outer positions, giving the triacylglycerol (TG) a distinctive stereoisomeric structure that is believed to assist nutrient absorption in the infant gut. However, the fat used in most infant formulas is derived from plants, which preferentially esterify 16:0 to the outer positions. We have previously showed that the metabolism of the model oilseed Arabidopsis thaliana can be engineered to incorporate 16:0 into the middle position of TG. However, the fatty acyl composition of Arabidopsis seed TG does not mimic human milk, which is rich in both 16:0 and 18:1 and is defined by the high abundance of the TG molecular species 1,3-olein-2-palmitin (OPO). Here we have constructed an Arabidopsis fatty acid biosynthesis 1-1 fatty acid desaturase 2 fatty acid elongase 1 mutant with around 20% 16:0 and 70% 18:1 in its seeds and we have engineered it to esterify more than 80% of the 16:0 to the middle position of TG, using heterologous expression of the human lysophosphatidic acid acyltransferase isoform AGPAT1, combined with suppression of LYSOPHOSPHATIDIC ACID ACYLTRANSFERASE 2 and PHOSPHATIDYLCHOLINE:DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE. Our data show that oilseeds can be engineered to produce TG that is rich in OPO, which is a structured fat ingredient used in infant formulas.

Allele mining in diverse accessions of tropical grasses to improve forage quality and reduce environmental impact.

BACKGROUND AND AIMS: The C4Urochloa species (syn. Brachiaria) and Megathyrsus maximus (syn. Panicum maximum) are used as pasture for cattle across vast areas in tropical agriculture systems in Africa and South America. A key target for variety improvement is forage quality: enhanced digestibility could decrease the amount of land required per unit production, and enhanced lipid content could decrease methane emissions from cattle. For these traits, loss-of-function (LOF) alleles in known gene targets are predicted to improve them, making a reverse genetics approach of allele mining feasible. We therefore set out to look for such alleles in diverse accessions of Urochloa species and Megathyrsus maximus from the genebank collection held at the CIAT. METHODS: We studied allelic diversity of 20 target genes (11 for digestibility, nine for lipid content) in 104 accessions selected to represent genetic diversity and ploidy levels of U. brizantha, U. decumbens, U. humidicola, U. ruziziensis and M. maximum. We used RNA sequencing and then bait capture DNA sequencing to improve gene models in a U. ruziziensis reference genome to assign polymorphisms with high confidence. KEY RESULTS: We found 953 non-synonymous polymorphisms across all genes and accessions; within these, we identified seven putative LOF alleles with high confidence, including those in the non-redundant SDP1 and BAHD01 genes present in diploid and tetraploid accessions. These LOF alleles could respectively confer increased lipid content and digestibility if incorporated into a breeding programme. CONCLUSIONS: We demonstrated a novel, effective approach to allele discovery in diverse accessions using a draft reference genome from a single species. We used this to find gene variants in a collection of tropical grasses that could help reduce the environmental impact of cattle production.

Engineering of tomato for the sustainable production of ketocarotenoids and its evaluation in aquaculture feed.

Ketocarotenoids are high-value pigments used commercially across multiple industrial sectors as colorants and supplements. Chemical synthesis using petrochemical-derived precursors remains the production method of choice. Aquaculture is an example where ketocarotenoid supplementation of feed is necessary to achieve product viability. The biosynthesis of ketocarotenoids, such as canthaxanthin, phoenicoxanthin, or astaxanthin in plants is rare. In the present study, complex engineering of the carotenoid pathway has been performed to produce high-value ketocarotenoids in tomato fruit (3.0 mg/g dry weight). The strategy adopted involved pathway extension beyond ß-carotene through the expression of the ß-carotene hydroxylase (CrtZ) and oxyxgenase (CrtW) from Brevundimonas sp. in tomato fruit, followed by ß-carotene enhancement through the introgression of a lycopene ß-cyclase (ß-Cyc) allele from a Solanum galapagense background. Detailed biochemical analysis, carried out using chromatographic, UV/VIS, and MS approaches, identified the predominant carotenoid as fatty acid (C14:0 and C16:0) esters of phoenicoxanthin, present in the S stereoisomer configuration. Under a field-like environment with low resource input, scalability was shown with the potential to deliver 23 kg of ketocarotenoid/hectare. To illustrate the potential of this "generally recognized as safe" material with minimal, low-energy bioprocessing, two independent aquaculture trials were performed. The plant-based feeds developed were more efficient than the synthetic feed to color trout flesh (up to twofold increase in the retention of the main ketocarotenoids in the fish fillets). This achievement has the potential to create a new paradigm in the renewable production of economically competitive feed additives for the aquaculture industry and beyond.

Cyclin-dependent kinase activity enhances phosphatidylcholine biosynthesis in Arabidopsis by repressing phosphatidic acid phosphohydrolase activity.

Coordination of endomembrane biogenesis with cell cycle progression is considered to be important in maintaining cell function during growth and development. We previously showed that the disruption of PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAH) activity in Arabidopsis thaliana stimulates biosynthesis of the major phospholipid phosphatidylcholine (PC) and causes expansion of the endoplasmic reticulum. Here we show that PC biosynthesis is repressed by disruption of the core cell cycle regulator CYCLIN-DEPENDENT KINASE A;1 (CDKA;1) and that this repression is reliant on PAH. Furthermore, we show that cyclin-dependent kinases (CDKs) phosphorylate PAH1 at serine 162, which reduces both its activity and membrane association. Expression of a CDK-insensitive version of PAH1 with a serine 162 to alanine substitution represses PC biosynthesis and also reduces the rate of cell division in early leaf development. Together our findings reveal a physiologically important mechanism that couples the rate of phospholipid biosynthesis and endomembrane biogenesis to cell cycle progression in Arabidopsis.

Awake1, an ABC-Type Transporter, Reveals an Essential Role for Suberin in the Control of Seed Dormancy.

The mother plant plays an important dynamic role in the control of dormancy of her progeny seed in response to environmental signals. In order to further understand the mechanisms by which this dormancy control takes place in Arabidopsis (Arabidopsis thaliana), we conducted a forward genetic screen to isolate mutants that fail to enter dormancy in response to variation in temperature during seed set. We show that, for the first of these mutants, designated awake1, the maternal allele is required for entry into strongly dormant states and that awake1 mutants show seed phenotypes shown previously to be associated with the loss of suberin in the seed. We identify awake1 as an allele of ABCG20, an ATP-binding cassette transporter-encoding gene required for the transport of fatty acids during suberin deposition, and show that further suberin-deficient mutants have seed dormancy defects. Seed coat suberin composition is affected by temperature during seed maturation, but this response appears to be independent of ABCG20. We conclude that seed coat suberin is essential for seed dormancy imposition by low temperature and that the exclusion of oxygen and water from the seed by the suberin and tannin layers is important for dormancy imposition.

Genome Wide Analysis of Fatty Acid Desaturation and Its Response to Temperature.

Plants modify the polyunsaturated fatty acid content of their membrane and storage lipids in order to adapt to changes in temperature. In developing seeds, this response is largely controlled by the activities of the microsomal ω-6 and ω-3 fatty acid desaturases, FAD2 and FAD3. Although temperature regulation of desaturation has been studied at the molecular and biochemical levels, the genetic control of this trait is poorly understood. Here, we have characterized the response of Arabidopsis (Arabidopsis thaliana) seed lipids to variation in ambient temperature and found that heat inhibits both ω-6 and ω-3 desaturation in phosphatidylcholine, leading to a proportional change in triacylglycerol composition. Analysis of the 19 parental accessions of the multiparent advanced generation intercross (MAGIC) population showed that significant natural variation exists in the temperature responsiveness of ω-6 desaturation. A combination of quantitative trait locus (QTL) analysis and genome-wide association studies (GWAS) using the MAGIC population suggests that ω-6 desaturation is largely controlled by cis-acting sequence variants in the FAD2 5' untranslated region intron that determine the expression level of the gene. However, the temperature responsiveness of ω-6 desaturation is controlled by a separate QTL on chromosome 2. The identity of this locus is unknown, but genome-wide association studies identified potentially causal sequence variants within ∼40 genes in an ∼450-kb region of the QTL.

PHOSPHATIDIC ACID PHOSPHOHYDROLASE Regulates Phosphatidylcholine Biosynthesis in Arabidopsis by Phosphatidic Acid-Mediated Activation of CTP:PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE Activity.

Regulation of membrane lipid biosynthesis is critical for cell function. We previously reported that disruption of PHOSPHATIDIC ACID PHOSPHOHYDROLASE1 (PAH1) and PAH2 stimulates net phosphatidylcholine (PC) biosynthesis and proliferation of the endoplasmic reticulum (ER) in Arabidopsis thaliana. Here, we show that this response is caused specifically by a reduction in the catalytic activity of the protein and positively correlates with an accumulation of its substrate, phosphatidic acid (PA). The accumulation of PC in pah1 pah2 is suppressed by disruption of CTP:PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE1 (CCT1), which encodes a key enzyme in the nucleotide pathway for PC biosynthesis. The activity of recombinant CCT1 is stimulated by lipid vesicles containing PA. Truncation of CCT1, to remove the predicted C-terminal amphipathic lipid binding domain, produced a constitutively active enzyme. Overexpression of native CCT1 in Arabidopsis has no significant effect on PC biosynthesis or ER morphology, but overexpression of the truncated constitutively active version largely replicates the pah1 pah2 phenotype. Our data establish that membrane homeostasis is regulated by lipid composition in Arabidopsis and reveal a mechanism through which the abundance of PA, mediated by PAH activity, modulates CCT activity to govern PC content.

Step changes in leaf oil accumulation via iterative metabolic engineering.

Synthesis and accumulation of plant oils in the entire vegetative biomass offers the potential to deliver yields surpassing those of oilseed crops. However, current levels still fall well short of those typically found in oilseeds. Here we show how transcriptome and biochemical analyses pointed to a futile cycle in a previously established Nicotiana tabacum line, accumulating up to 15% (dry weight) of the storage lipid triacylglycerol in leaf tissue. To overcome this metabolic bottleneck, we either silenced the SDP1 lipase or overexpressed the Arabidopsis thaliana LEC2 transcription factor in this transgenic background. Both strategies independently resulted in the accumulation of 30-33% triacylglycerol in leaf tissues. Our results demonstrate that the combined optimization of de novo fatty acid biosynthesis, storage lipid assembly and lipid turnover in leaf tissue results in a major overhaul of the plant central carbon allocation and lipid metabolism. The resulting further step changes in oil accumulation in the entire plant biomass offers the possibility of delivering yields that outperform current oilseed crops.

ACYL-ACYL CARRIER PROTEIN DESATURASE2 and 3 Are Responsible for Making Omega-7 Fatty Acids in the Arabidopsis Aleurone.

Omega-7 monounsaturated fatty acids (ω-7s) are specifically enriched in the aleurone of Arabidopsis (Arabidopsis thaliana) seeds. We found significant natural variation in seed ω-7 content and used a Multiparent Advanced Generation Inter-Cross population to fine-map a major quantitative trait loci to a region containing ACYL-ACYL CARRIER PROTEIN DESATURASE1 (AAD1) and AAD3 We found that AAD3 expression is localized to the aleurone where mutants show an approximately 50% reduction in ω-7 content. By contrast, AAD1 is localized to the embryo where mutants show a small reduction in ω-9 content. Enzymatic analysis has previously shown that AAD family members possess both stearoyl- and palmitoyl-ACP Δ(9) desaturase activity, including the predominant isoform SUPPRESSOR OF SALICYLIC ACID INSENSITIVE2. However, aad3 ssi2 aleurone contained the same amount of ω-7s as aad3 Within the AAD family, AAD3 shares the highest degree of sequence similarity with AAD2 and AAD4. Mutant analysis showed that AAD2 also contributes to ω-7 production in the aleurone, and aad3 aad2 exhibits an approximately 85% reduction in ω-7s Mutant analysis also showed that FATTY ACID ELONGASE1 is required for the production of very long chain ω-7s in the aleurone. Together, these data provide genetic evidence that the ω-7 pathway proceeds via Δ(9) desaturation of palmitoyl-ACP followed by elongation of the product. Interestingly, significant variation was also identified in the ω-7 content of Brassica napus aleurone, with the highest level detected being approximately 47% of total fatty acids.

bZIP67 regulates the omega-3 fatty acid content of Arabidopsis seed oil by activating fatty acid desaturase3.

Arabidopsis thaliana seed maturation is accompanied by the deposition of storage oil, rich in the essential ω-3 polyunsaturated fatty acid α-linolenic acid (ALA). The synthesis of ALA is highly responsive to the level of fatty acid desaturase3 (FAD3) expression, which is strongly upregulated during embryogenesis. By screening mutants in leafy cotyledon1 (LEC1)-inducible transcription factors using fatty acid profiling, we identified two mutants (lec1-like and bzip67) with a seed lipid phenotype. Both mutants share a substantial reduction in seed ALA content. Using a combination of in vivo and in vitro assays, we show that bZIP67 binds G-boxes in the FAD3 promoter and enhances FAD3 expression but that activation is conditional on bZIP67 association with LEC1-like (L1L) and nuclear factor-YC2 (NF-YC2). Although FUSCA3 and abscisic acid insensitive3 are required for L1L and bZIP67 expression, neither protein is necessary for [bZIP67:L1L:NF-YC2] to activate FAD3. We conclude that a transcriptional complex containing L1L, NF-YC2, and bZIP67 is induced by LEC1 during embryogenesis and specifies high levels of ALA production for storage oil by activating FAD3 expression.

Expression of Castor LPAT2 Enhances Ricinoleic Acid Content at the sn-2 Position of Triacylglycerols in Lesquerella Seed.

Lesquerella is a potential industrial oilseed crop that makes hydroxy fatty acid (HFA). Unlike castor its seeds are not poisonous but accumulate lesquerolic acid mostly at the sn-1 and sn-3 positions of triacylglycerol (TAG), whereas castor contains ricinoleic acid (18:1OH) at all three positions. To investigate whether lesquerella can be engineered to accumulate HFAs in the sn-2 position, multiple transgenic lines were made that express castor lysophosphatidic acid acyltransferase 2 (RcLPAT2) in the seed. RcLPAT2 increased 18:1OH at the sn-2 position of TAGs from 2% to 14%-17%, which resulted in an increase of tri-HFA-TAGs from 5% to 13%-14%. Our result is the first example of using a LPAT to increase ricinoleic acid at the sn-2 position of seed TAG. This work provides insights to the mechanism of HFA-containing TAG assembly in lesquerella and directs future research to optimize this plant for HFA production.

Multigene engineering of triacylglycerol metabolism boosts seed oil content in Arabidopsis.

Increasing the yield of oilseed crops is an important objective for biotechnologists. A number of individual genes involved in triacylglycerol metabolism have previously been reported to enhance the oil content of seeds when their expression is altered. However, it has yet to be established whether specific combinations of these genes can be used to achieve an additive effect and whether this leads to enhanced yield. Using Arabidopsis (Arabidopsis thaliana) as an experimental system, we show that seed-specific overexpression of WRINKLED1 (a transcriptional regulator of glycolysis and fatty acid synthesis) and DIACYLGLYCEROL ACYLTRANSFERASE1 (a triacylglycerol biosynthetic enzyme) combined with suppression of the triacylglycerol lipase SUGAR-DEPENDENT1 results in a higher percentage seed oil content and greater seed mass than manipulation of each gene individually. Analysis of total seed yield per plant suggests that, despite a reduction in seed number, the total yield of oil is also increased.