Stearoyl-acyl Carrier Protein Research Articles

Comparative transcriptomic analysis provides insights into the genetic networks regulating oil differential production in oil crops

BackgroundPlants differ more than threefold in seed oil contents (SOCs). Soybean (Glycine max), cotton (Gossypium hirsutum), rapeseed (Brassica napus), and sesame (Sesamum indicum) are four important oil crops with markedly different SOCs and fatty acid compositions.ResultsCompared to grain crops like maize and rice, expanded acyl-lipid metabolism genes and relatively higher expression levels of genes involved in seed oil synthesis (SOS) in the oil crops contributed to the oil accumulation in seeds. Here, we conducted comparative transcriptomics on oil crops with two different SOC materials. In common, DIHYDROLIPOAMIDE DEHYDROGENASE, STEAROYL-ACYL CARRIER PROTEIN DESATURASE, PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE, and oil-body protein genes were both differentially expressed between the high- and low-oil materials of each crop. By comparing functional components of SOS networks, we found that the strong correlations between genes in “glycolysis/gluconeogenesis” and “fatty acid synthesis” were conserved in both grain and oil crops, with PYRUVATE KINASE being the common factor affecting starch and lipid accumulation. Network alignment also found a conserved clique among oil crops affecting seed oil accumulation, which has been validated in Arabidopsis. Differently, secondary and protein metabolism affected oil synthesis to different degrees in different crops, and high SOC was due to less competition of the same precursors. The comparison of Arabidopsis mutants and wild type showed that CINNAMYL ALCOHOL DEHYDROGENASE 9, the conserved regulator we identified, was a factor resulting in different relative contents of lignins to oil in seeds. The interconnection of lipids and proteins was common but in different ways among crops, which partly led to differential oil production.ConclusionsThis study goes beyond the observations made in studies of individual species to provide new insights into which genes and networks may be fundamental to seed oil accumulation from a multispecies perspective.

A telomere-to-telomere gap-free reference genome assembly of avocado provides useful resources for identifying genes related to fatty acid biosynthesis and disease resistance.

Avocado (Persea americana Mill.) is an economically valuable plant because of the high fatty acid content and unique flavor of its fruits. Its fatty acid content, especially the relatively high unsaturated fatty acid content, provides significant health benefits. We herein present a telomere-to-telomere gapless genome assembly (841.6Mb) of West Indian avocado. The genome contains 40 629 predicted protein-coding genes. Repeat sequences account for 57.9% of the genome. Notably, all telomeres, centromeres, and a nucleolar organizing region are included in this genome. Fragments from these three regions were observed via fluorescence in situ hybridization. We identified 376 potential disease resistance-related nucleotide-binding leucine-rich repeat genes. These genes, which are typically clustered on chromosomes, may be derived from gene duplication events. Five NLR genes (Pa11g0262, Pa02g4855, Pa07g3139, Pa07g0383, and Pa02g3196) were highly expressed in leaves, stems, and fruits, indicating they may be involved in avocado disease responses in multiple tissues. We also identified 128 genes associated with fatty acid biosynthesis and analyzed their expression patterns in leaves, stems, and fruits. Pa02g0113, which encodes one of 11 stearoyl-acyl carrier protein desaturases mediating C18 unsaturated fatty acid synthesis, was more highly expressed in the leaves than in the stems and fruits. These findings provide valuable insights that enhance our understanding of fatty acid biosynthesis in avocado.

Comparative genomics points to tandem duplications of SAD gene clusters as drivers of increased α-linolenic (ω-3) content in S. hispanica seeds.

Salvia hispanica L. (chia) is a source of abundant ω-3 polyunsaturated fatty acids (ω-3-PUFAs) that are highly beneficial to human health. The genomic basis for this accrued ω-3-PUFA content in this emerging crop was investigated through the assembly and comparative analysis of a chromosome-level reference genome for S. hispanica. The highly contiguous 321.5-Mbp genome assembly covering all six chromosomes enabled the identification of 32,922 protein-coding genes. Two whole-genome duplications (WGD) events were identified in the S. hispanica lineage. However, these WGD events could not be linked to the high α-linolenic acid (ALA, ω-3) accumulation in S. hispanica seeds based on phylogenomics. Instead, our analysis supports the hypothesis that evolutionary expansion through tandem duplications of specific lipid gene families, particularly the stearoyl-acyl carrier protein desaturase (ShSAD) gene family, is the main driver of the abundance of ω-3-PUFAs in S. hispanica seeds. The insights gained from the genomic analysis of S. hispanica will help establish a molecular breeding target that can be leveraged through genome editing techniques to increase ω-3 content in oil crops.

Genome-Wide Identification and Expression Analysis of the Stearoyl-Acyl Carrier Protein Δ9 Desaturase Gene Family under Abiotic Stress in Barley.

Stearoyl-acyl carrier protein (ACP) Δ9 desaturase (SAD) is a critical fatty acid dehydrogenase in plants, playing a prominent role in regulating the synthesis of unsaturated fatty acids (UFAs) and having a significant impact on plant growth and development. In this study, we conducted a comprehensive genomic analysis of the SAD family in barley (Hordeum vulgare L.), identifying 14 HvSADs with the FA_desaturase_2 domain, which were divided into four subgroups based on sequence composition and phylogenetic analysis, with members of the same subgroup possessing similar genes and motif structures. Gene replication analysis suggested that tandem and segmental duplication may be the major reasons for the expansion of the SAD family in barley. The promoters of HvSADs contained various cis-regulatory elements (CREs) related to light, abscisic acid (ABA), and methyl jasmonate (MeJA). In addition, expression analysis indicated that HvSADs exhibit multiple tissue expression patterns in barley as well as different response characteristics under three abiotic stresses: salt, drought, and cold. Briefly, this evolutionary and expression analysis of HvSADs provides insight into the biological functions of barley, supporting a comprehensive analysis of the regulatory mechanisms of oil biosynthesis and metabolism in plants under abiotic stress.

Genome-wide identification and a comparative transcriptomics approach reveal FaSAD3 as a strawberry fruit ripening regulator

Fatty acid desaturases (FADs) play a crucial role in plant growth as well as in the regulation of biotic and abiotic stresses. However, their function has not been well understood in the ripening of fruit. In this study, we identified 73 FADs in the octoploid strawberry genome and analyzed their physicochemical characteristics, chromosomal localization, phylogeny, gene structures, covariance, cis-acting elements, and transcript levels during fruit ripening stages. According to the predicted subcellular localization, FADs are mostly located in the plasma membrane, with a lower abundance in the cell wall, cytoplasm, and nucleus. Numerous cis-elements in the FADs promoter are responsive to light, hormones, and stress. Here, combining comparative transcriptomics, we identified a Δ9 stearoyl-acyl carrier protein (ACP) desaturase gene (FxaC_8 g24100, namely FaSAD3) that is negatively regulated by both key glycolysis genes, cytoplasmic glyceraldehyde-3-phosphate dehydrogenase (FaGAPC2) and pyruvate kinase (FaPKc2.2). The spatiotemporal expression analysis revealed that FaSAD3 is expressed in both vegetative and reproductive organs. Subcellular localization analysis confirmed that FaSAD3 is expressed in both the cytoplasm and nucleus. Meanwhile, the transient overexpression of FaSAD3 reduced anthocyanin, sugar, organic acid, total phenol, and flavonoid contents, while increased fruit firmness. Furthermore, the yeast one hybridization and dual-luciferase assay revealed that the ripening regulator FaGAMYB negatively regulates the transcriptional activity of FaSAD3 promoter, promoting strawberry fruit ripening. These findings indicated that FaSAD3 acts as a negative regulator in strawberry fruits ripening, providing a novel model for comprehending the mechanisms involved in strawberry fruit ripening.

Heterogeneous expression of stearoyl-acyl carrier protein desaturase genes SAD1 and SAD2 from Linum usitatissimum enhances seed oleic acid accumulation and seedling cold and drought tolerance in Brassica napus

Flax (Linum usitatissimum L.) is a versatile crop and its seeds are a major source of unsaturated fatty acids. Stearoyl-acyl carrier protein desaturase (SAD) is a dehydrogenase enzyme that plays a key role in oleic acid biosynthesis as well as responses to biotic and abiotic stresses. However, the function of SAD orthologs from L. usitatissimum has not been assessed. Here, we found that two LuSAD genes, LuSAD1 and LuSAD2, are present in the genome of L. usitatissimum cultivar ‘Longya 10’. Heterogeneous expression of either LuSAD1 or LuSAD2 in Arabidopsis thaliana resulted in higher contents of total fatty acids and oleic acid in the seeds. Interestingly, ectopic expression of LuSAD2 in A. thaliana caused altered plant architecture. Similarly, the overexpression of either LuSAD1 or LuSAD2 in Brassica napus also resulted in increased contents of total fatty acids and oleic acid in the seeds. Furthermore, we demonstrated that either LuSAD1 or LuSAD2 enhances seedling resistance to cold and drought stresses by improving antioxidant enzyme activity and nonenzymatic antioxidant levels, as well as reducing membrane damage. These findings not only broaden our knowledge of the LuSAD functions in plants, but also offer promising targets for improving the quantity and quality of oil, and the abiotic stress tolerance of oil-producing crops, through molecular manipulation.

Tung tree stearoyl-acyl carrier protein Δ9 desaturase improves oil content and cold resistance of Arabidopsis and Saccharomyces cerevisiae.

The seed oil of tung tree is rich in a-eleostearic acid (ESA), which endows tung oil with the characteristic of an excellently dry oil. The stearoyl-acyl carrier protein δ9 desaturase (SAD) is a rate-limiting enzyme that converts the stearic acid to the oleic acid, the substrate for the production of the α-ESA. However, the function of the two predicted VfSAD1 and VfSAD2 genes in the tung tree has not been determined. In this study, quantitative real-time PCR (qRT-PCR) analysis showed that VfSAD1 and VfSAD2 were expressed in multiple organs of tung tree but were highly expressed in the seed during the oil rapid accumulation period. Heterologous expression of VfSAD1 and VfSAD2 could promote the production of oleic acid and its derivatives in Arabidopsis thaliana and yeast BY4741, indicating that VfSAD1 and VfSAD2 possess the stearoyl-ACP desaturases function. Furthermore, both VfSAD1 and VfSAD2 could significantly improve seed oil accumulation in Arabidopsis. VfSAD1 could also significantly promote the oil accumulation in the yeast BY4741 strain. In addition, overexpression of VfSAD1 and VfSAD2 enhanced the tolerance of yeast and Arabidopsis seedlings to low temperature stress. This study indicates that the two VfSAD genes play a vital role in the process of oil accumulation and fatty acid biosynthesis in the tung tree seed, and both of them could be used for molecular breeding in tung tree and other oil crops.

Longer Duration of Active Oil Biosynthesis during Seed Development Is Crucial for High Oil Yield-Lessons from Genome-Wide In Silico Mining and RNA-Seq Validation in Sesame.

Sesame, one of the ancient oil crops, is an important oilseed due to its nutritionally rich seeds with high protein content. Genomic scale information for sesame has become available in the public databases in recent years. The genes and their families involved in oil biosynthesis in sesame are less studied than in other oilseed crops. Therefore, we retrieved a total of 69 genes and their translated amino acid sequences, associated with gene families linked to the oil biosynthetic pathway. Genome-wide in silico mining helped identify key regulatory genes for oil biosynthesis, though the findings require functional validation. Comparing sequences of the SiSAD (stearoyl-acyl carrier protein (ACP)-desaturase) coding genes with known SADs helped identify two SiSAD family members that may be palmitoyl-ACP-specific. Based on homology with lysophosphatidic acid acyltransferase (LPAAT) sequences, an uncharacterized gene has been identified as SiLPAAT1. Identified key regulatory genes associated with high oil content were also validated using publicly available transcriptome datasets of genotypes contrasting for oil content at different developmental stages. Our study provides evidence that a longer duration of active oil biosynthesis is crucial for high oil accumulation during seed development. This underscores the importance of early onset of oil biosynthesis in developing seeds. Up-regulating, identified key regulatory genes of oil biosynthesis during early onset of seed development, should help increase oil yields.

Sequence characterization and temporal expression analysis of different SADs and FAD2-2 genes in two Iranian olive cultivars

Stearoyl-acyl carrier protein (ACP) desaturases (SADs) and fatty acid desaturases (FADs) are the main genes catalyzing the first and the second desaturation steps throughout the biosynthesis of oleic and linoleic acids, respectively, hence, the major determinants of oil quality and composition. To uncover the molecular mechanisms behind differential oil content and composition in olive fruit, the nucleotide and amino acid sequences of FAD2-2 and three SAD genes were characterized in 'Mari' and 'Shengeh' as two Iranian olive cultivars displaying high contrasting oil composition. In addition, the expression levels of these genes were screened at different time points during fruit development and ripening. Despite detection of a number of nucleotide sequence variations in four characterized genes, the results of amino acid analyses have shown most of them were synonymous and caused no differences at protein level. However, some of the single nucleotide variations caused non cognate amino acid substitution indicating possible conformational changes in the resultant peptide. In particular, regarding OeFAD2-2, a nonsynonymous SNP was detected in 'Shengeh' compared with other olive's FAD2-2 causing an amino acid lysine residue substitution for a glutamic acid, as well as a nonsynonymous substitution in 'Mari' (threonine to serine). The in silico prediction of three-dimensional structure of FAD2-2 revealed these substitutions may lead to structural changes in the final protein structure and function, hence, may contribute to the higher activity of FAD2-2 protein in mesocarp of 'Shengeh' than 'Mari' cultivars. According to results obtained from sequence analysis of oil biosynthesis genes, two Iranian olive cultivars were the most similar and had high sequence differences with international varieties. Results of temporal transcript abundance revealed that OeSAD2 had higher expression in 'Mari' (the high quality oil cultivar) than 'Shengeh' during the critical period of oil biosynthesis. Results of our data corroborates the previous records suggesting that OeSAD2 as the most pronounced isoform of stearoyl-acyl carrier protein desaturases responsible for oleic acid biosynthesis in olive mesocarp. In addition our results also suggest that OeSAD3 may have contributed to the higher oil quality of 'Mari' than 'Shengeh'. Based on our results, OeSAD2 and OeFAD2-2 are suggested as the possible targets for engineering fatty acid composition to help pyramid genes to improve quality and quantity of olive oil and creating high quality olive cultivars.

The genome of oil-Camellia and population genomics analysis provide insights into seed oil domestication

BackgroundAs a perennial crop, oil-Camellia possesses a long domestication history and produces high-quality seed oil that is beneficial to human health. Camellia oleifera Abel. is a sister species to the tea plant, which is extensively cultivated for edible oil production. However, the molecular mechanism of the domestication of oil-Camellia is still limited due to the lack of sufficient genomic information.ResultsTo elucidate the genetic and genomic basis of evolution and domestication, here we report a chromosome-scale reference genome of wild oil-Camellia (2.95 Gb), together with transcriptome sequencing data of 221 cultivars. The oil-Camellia genome, assembled by an integrative approach of multiple sequencing technologies, consists of a large proportion of repetitive elements (76.1%) and high heterozygosity (2.52%). We construct a genetic map of high-density corrected markers by sequencing the controlled-pollination hybrids. Genome-wide association studies reveal a subset of artificially selected genes that are involved in the oil biosynthesis and phytohormone pathways. Particularly, we identify the elite alleles of genes encoding sugar-dependent triacylglycerol lipase 1, β-ketoacyl-acyl carrier protein synthase III, and stearoyl-acyl carrier protein desaturases; these alleles play important roles in enhancing the yield and quality of seed oil during oil-Camellia domestication.ConclusionsWe generate a chromosome-scale reference genome for oil-Camellia plants and demonstrate that the artificial selection of elite alleles of genes involved in oil biosynthesis contributes to oil-Camellia domestication.

Critical metabolic pathways and SAD/FADs, WRI1s, and DGATs cooperate for high-oleic acid oil production in developing oil tea (Camellia oleifera) seeds.

Oil tea trees produce high-quality edible oils with desirably high oleic acid (18:1) and low linoleic (18:2) and linolenic (18:3) fatty acid (FA) levels, but limited understanding of tea oil biosynthesis and regulation has become a significant obstacle for the breeding of high-yield and -quality oil tea varieties. By integrating metabolite and transcriptome analyses of developing oil tea seeds, we dissected the critical metabolic pathways, including glycolysis, fatty acid, and triacylglycerol (TAG) biosynthesis, as well as genes essential for tea seed oil production. Two plastidic stearoyl-acyl carrier protein desaturases (CoSAD1 and 2) and two endoplasmic reticulum-localized FA desaturases (CoFAD2 and 3) were functionally characterized as responsible for high 18:1 and low 18:2 and 18:3 proportions in tea oils. Two diacylglycerol O-acyltransferases (CoDGAT1 and 2) that may prefer to synthesize 18:1-TAG were functionally characterized and might be also important for high 18:1-TAG production. The highly expressed CoWRI1a and b were identified and characterized as activators of glycolysis and regulators of directing source carbon flux into FA biosynthesis in developing oil tea seeds. The upregulated CoSADs with downregulated CoFAD2 and CoFAD3 at the late seed developmental stages mainly accounted for high 18:1 levels. Two CoDGATs might be responsible for assembling TAGs with oleoyl acyl chains, whilst two CoWRI1s regulated carbons from parental sources, partitioning into oil production in oil tea embryo sinks. This study provides a deep understanding of the biosynthesis of tea seed oils and information on genes that may be used as molecular markers to breed oil tea varieties with higher oil yield and quality.

Cotton GhSSI2 isoforms from the stearoyl acyl carrier protein fatty acid desaturase family regulate Verticillium wilt resistance.

Lipids are major and essential constituents of plant cells and provide energy for various metabolic processes. However, the function of the lipid signal in defence against Verticillium dahliae, a hemibiotrophic pathogen, remains unknown. Here, we characterized 19 conserved stearoyl‐ACP desaturase family proteins from upland cotton (Gossypium hirsutum). We further confirmed that GhSSI2 isoforms, including GhSSI2‐A, GhSSI2‐B, and GhSSI2‐C located on chromosomes A10, D10, and A12, respectively, played a dominant role to the cotton 18:1 (oleic acid) pool. Suppressing the expression of GhSSI2s reduced the 18:1 level, which autoactivated the hypersensitive response (HR) and enhanced cotton Verticillium wilt and Fusarium wilt resistance. We found that low 18:1 levels induced phenylalanine ammonia‐lyase‐mediated salicylic acid (SA) accumulation and activated a SA‐independent defence response in GhSSI2s‐silenced cotton, whereas suppressing expression of GhSSI2s affected PDF1.2‐dependent jasmonic acid (JA) perception but not the biosynthesis and signalling cascade of JA. Further investigation showed that structurally divergent resistance‐related genes and nitric oxide (NO) signal were activated in GhSSI2s‐silenced cotton. Taken together, these results indicate that SA‐independent defence response, multiple resistance‐related proteins, and elevated NO level play an important role in GhSSI2s‐regulated Verticillium wilt resistance. These findings broaden our knowledge regarding the lipid signal in disease resistance and provide novel insights into the molecular mechanism of cotton fungal disease resistance.

Soybean TILLING-by-Sequencing+ reveals the role of novel GmSACPD members in unsaturated fatty acid biosynthesis while maintaining healthy nodules.

Developing soybean lines with high levels of stearic acid is a primary goal of the soybean industry. Most high-stearic-acid soybeans carry different GmSACPD-C mutated alleles. However, due to the dual role of GmSACPD-C in seeds and nodule development, all derived deleterious GmSACPD-C mutant alleles are of extremely poor agronomic value because of defective nodulation. The soybean stearoyl-acyl carrier protein desaturase (GmSACPD) gene family is composed of five members. Comparative genomics analysis indicated that SACPD genes were duplicated and derived from a common ancestor that is still present in chlorophytic algae. Synteny analysis showed the presence of segment duplications between GmSACPD-A/GmSACPD-B, and GmSACPD-C/GmSACPD-D. GmSACPD-E was not contained in any duplicated segment and may be the result of tandem duplication. We developed a TILLING by Target Capture Sequencing (Tilling-by-Sequencing+) technology, a versatile extension of the conventional TILLING by sequencing, and successfully identified 12, 14, and 18 ethyl methanesulfonate mutants at the GmSACPD-A, GmSACPD-B, and GmSACPD-D genes, respectively. Functional analysis of all identified mutants revealed an unprecedented role of GmSACPD-A, GmSACPD-B, and GmSACPD-D in unsaturated fatty acid biosynthesis without affecting nodule development and structure. This discovery will positively impact the development of high-stearic-acid lines to enhance soybean nutritional value without potential developmental tradeoffs.

Metabolomic profiling of wild-type and mutant soybean root nodules using laser-ablation electrospray ionization mass spectrometry reveals altered metabolism.

The establishment of the nitrogen-fixing symbiosis between soybean and Bradyrhizobium japonicum is a complex process. To document the changes in plant metabolism as a result of symbiosis, we utilized laser ablation electrospray ionization-mass spectrometry (LAESI-MS) for in situ metabolic profiling of wild-type nodules, nodules infected with a B. japonicum nifH mutant unable to fix nitrogen, nodules doubly infected by both strains, and nodules formed on plants mutated in the stearoyl-acyl carrier protein desaturase (sacpd-c) gene, which were previously shown to have an altered nodule ultrastructure. The results showed that the relative abundance of fatty acids, purines, and lipids was significantly changed in response to the symbiosis. The nifH mutant nodules had elevated levels of jasmonic acid, correlating with signs of nitrogen deprivation. Nodules resulting from the mixed inoculant displayed similar, overlapping metabolic distributions within the sectors of effective (fix+ ) and ineffective (nifH mutant, fix- ) endosymbionts. These data are inconsistent with the notion that plant sanctioning is cell autonomous. Nodules lacking sacpd-c displayed an elevation of soyasaponins and organic acids in the central necrotic regions. The present study demonstrates the utility of LAESI-MS for high-throughput screening of plant phenotypes. Overall, nodules disrupted in the symbiosis were elevated in metabolites related to plant defense.

Identification and functional analysis of soybean stearoyl-ACP Δ⁹ desaturase (GmSAD) gene family

Stearoyl-ACP Δ⁹ desaturase (SAD) catalyzes the synthesis of monounsaturated oleic acid or palmitoleic acid in plastids. SAD is the key enzyme to control the ratio of saturated fatty acids to unsaturated fatty acids in plant cells. In order to analyze the regulation mechanism of soybean oleic acid synthesis, soybean (Glycine max) GmSAD family members were genome-wide identified, and their conserved functional domains and physicochemical properties were also analyzed by bioinformatics tools. The spatiotemporal expression profile of each member of GmSADs was detected by qRT-PCR. The expression vectors of GmSAD5 were constructed. The enzyme activity and biological function of GmSAD5 were examined by Agrobacterium-mediated transient expression in Nicotiana tabacum leaves and genetic transformation of oleic acid-deficient yeast (Saccharomyces cerevisiae) mutant BY4389. Results show that the soybean genome contains five GmSAD family members, all encoding an enzyme protein with diiron center and two conservative histidine enrichment motifs (EENRHG and DEKRHE) specific to SAD enzymes. The active enzyme protein was predicted as a homodimer. Phylogenetic analysis indicated that five GmSADs were divided into two subgroups, which were closely related to AtSSI2 and AtSAD6, respectively. The expression profiles of GmSAD members were significantly different in soybean roots, stems, leaves, flowers, and seeds at different developmental stages. Among them, GmSAD5 expressed highly in the middle and late stages of developmental seeds, which coincided with the oil accumulation period. Transient expression of GmSAD5 in tobacco leaves increased the oleic acid and total oil content in leaf tissue by 5.56% and 2.73%, respectively, while stearic acid content was reduced by 2.46%. Functional complementation assay in defective yeast strain BY4389 demonstrated that overexpression of GmSAD5 was able to restore the synthesis of monounsaturated oleic acid, resulting in high oil accumulation. Taken together, soybean GmSAD5 has strong selectivity to stearic acid substrates and can efficiently catalyze the biosynthesis of monounsaturated oleic acid. It lays the foundation for the study of soybean seed oleic acid and total oil accumulation mechanism, providing an excellent target for genetic improvement of oil quality in soybean.

Rice OsAAA-ATPase1 is Induced during Blast Infection in a Salicylic Acid-Dependent Manner, and Promotes Blast Fungus Resistance.

Fatty acids (FAs) have been implicated in signaling roles in plant defense responses. We previously reported that mutation or RNAi-knockdown (OsSSI2-kd) of the rice OsSSI2 gene, encoding a stearoyl acyl carrier protein FA desaturase (SACPD), remarkably enhanced resistance to blast fungus Magnaporthe oryzae and the leaf-blight bacterium Xanthomonas oryzae pv. oryzae (Xoo). Transcriptomic analysis identified six AAA-ATPase family genes (hereafter OsAAA-ATPase1–6) upregulated in the OsSSI2-kd plants, in addition to other well-known defense-related genes. Here, we report the functional analysis of OsAAA-ATPase1 in rice’s defense response to M. oryzae. Recombinant OsAAA-ATPase1 synthesized in Escherichia coli showed ATPase activity. OsAAA-ATPase1 transcription was induced by exogenous treatment with a functional analogue of salicylic acid (SA), benzothiadiazole (BTH), but not by other plant hormones tested. The transcription of OsAAA-ATPase1 was also highly induced in response to M. oryzae infection in an SA-dependent manner, as gene induction was significantly attenuated in a transgenic rice line expressing a bacterial gene (nahG) encoding salicylate hydroxylase. Overexpression of OsAAA-ATPase1 significantly enhanced pathogenesis-related gene expression and the resistance to M. oryzae; conversely, RNAi-mediated suppression of this gene compromised this resistance. These results suggest that OsAAA-APTase1 plays an important role in SA-mediated defense responses against blast fungus M. oryzae.

Monitoring Adventitious Presence of Transgenes in Cotton Collections from Genebank and Experimental Plots: Ensuring GM-Free Conservation and Cultivation of Genetic Resources

Bt cotton is the commercialized genetically modified (GM) crop in India with four approved GM events, namely MON531 (Bollgard® I), MON15985 (Bollgard® II), GFM and Event 1. Several GM events of cotton with insect-resistant or herbicide-tolerant GM traits were under field trials or imported for research purposes. With the increase in adoption of Bt cotton, unintentional introgression of transgenes in the conserved diversity-rich germplasm and in the field samples needs to be monitored in a systematic manner employing efficient GM diagnostics. Adventitious presence of transgenes was checked in 100 ex situ accessions of cotton being conserved in the National Genebank. These accessions belonging to Gossypium hirsutum, G. barbadense, G. arboreum, G. hirsutum × G. barbadense, represented seven Bt cotton growing states. Pentaplex PCR assay covering screening for over 90% of globally approved GM cotton events was employed, simultaneously targeting Cauliflower Mosaic Virus 35S promoter (P-35S), Agrobacterium tumefaciens nos terminator (T-nos), neomycin phosphotransferase (nptII) and phosphinothricin-N-acetyltransferase (pat) marker genes, and endogenous stearoyl acyl carrier protein desaturase (Sad1) gene as an internal control. Real-time PCR assays targeting P-35S and T-nos, with the limit of detection up to 0.01%, were employed for further confirmation. A study was also conducted on fifty cotton samples collected from the experimental plots, which were grown at distance of 50 and 10 metres from Bt cotton event MON15985. Adventitious presence of transgenes was checked using multiplex PCR assay targeting cry1Ac, cry2Ab2 and endogenous Sad1 gene to define a strategy for screening for specific GM events. Adventitious presence of transgenes was not detected in any of the tested samples based on the screening tests conducted. This strategy targeting specific GM traits would assist in GM-free cultivation of varieties in the experimental plots. The reported approach with wide GM coverage is reliable for checking adventitious presence of transgenes in genebanks, which could be applied to test conserved germplasm on a regular basis, for ensuring GM-free conservation of genetic resources.

Theoretical study on the catalytic mechanism of human deoxyhypusine hydroxylase.

Deoxyhypusine hydroxylase is a critical enzyme for hypusination of eukaryotic translation initiation factor 5A (eIF5A). Human deoxyhypusine hydroxylase (hDOHH) has a nonheme diiron active site that resembles both in structure and function of those found in methane and toluene monooxygenases, bacterial and mammalian ribonucleotide reductases, and stearoyl acyl carrier protein Δ9-desaturase from plants. However, the detailed catalytic mechanism of hDOHH is still unclear. In this work, extensive DFT calculations reveal that the catalytic mechanism of hDOHH consists of four consecutive steps: (1) peroxo isomerization triggered by substrate binding; (2) rate-determining O-O bond cleavage and formation of the [FeIV2(μ-O)2]4+ compound; (3) H atom abstraction from the substrate; and (4) OH rebound to the substrate. This work not only rationalizes the exceptional stability of the diiron(iii)-peroxo complex in hDOHH, but also confirms that hDOHH uses a diamond shape [FeIV2(μ-O)2]4+ core to complete crucial H atom abstraction from the substrate. Our DFT calculations exclude the reaction pathway of hDOHH to use diiron(iii)-peroxo species to directly react with the substrate.

Castor Stearoyl-ACP Desaturase Can Synthesize a Vicinal Diol by Dioxygenase Chemistry.

In previous work, we identified a triple mutant of the castor (Ricinus communis) stearoyl-Acyl Carrier Protein desaturase (T117R/G188L/D280K) that, in addition to introducing a double bond into stearate to produce oleate, performed an additional round of oxidation to convert oleate to a trans allylic alcohol acid. To determine the contributions of each mutation, in this work we generated individual castor desaturase mutants carrying residue changes corresponding to those in the triple mutant and investigated their catalytic activities. We observed that T117R, and to a lesser extent D280K, accumulated a novel product, namely erythro-9,10-dihydroxystearate, that we identified via its methyl ester through gas chromatography-mass spectrometry and comparison with authentic standards. The use of 18O2 labeling showed that the oxygens of both hydroxyl moieties originate from molecular oxygen rather than water. Incubation with an equimolar mixture of 18O2 and 16O2 demonstrated that both hydroxyl oxygens originate from a single molecule of O2, proving the product is the result of dioxygenase catalysis. Using prolonged incubation, we discovered that wild-type castor desaturase is also capable of forming erythro-9,10-dihydroxystearate, which presents a likely explanation for its accumulation to ∼0.7% in castor oil, the biosynthetic origin of which had remained enigmatic for decades. In summary, the findings presented here expand the documented constellation of di-iron enzyme catalysis to include a dioxygenase reactivity in which an unactivated alkene is converted to a vicinal diol.

Identification of Climate and Genetic Factors That Control Fat Content and Fatty Acid Composition of Theobroma cacao L. Beans.

The main ingredients of chocolate are usually cocoa powder, cocoa butter, and sugar. Both the powder and the butter are extracted from the beans of the cacao tree (Theobroma cacao L.). The cocoa butter represents the fat in the beans and possesses a unique fatty acid profile that results in chocolate’s characteristic texture and mouthfeel. Here, we used a linkage mapping population and phenotypic data of 3,292 samples from 420 progeny which led to the identification of 27 quantitative trait loci (QTLs) for fatty acid composition and six QTLs for fat content. Progeny showed extensive variation in fat levels and composition, with the level of palmitic acid negatively correlated to the sum of stearic acid, oleic acid, and linoleic acid. A major QTL explaining 24% of the relative level of palmitic acid was mapped to the distal end of chromosome 4, and those higher levels of palmitic acid were associated with the presence of a haplotype from the “TSH 1188” parent in the progeny. Within this region of chromosome 4 is the Thecc1EG017405 gene, an orthologue and isoform of the stearoyl-acyl carrier protein (ACP) desaturase (SAD) gene in plants, which is involved in fatty acid biosynthesis. Besides allelic differences, we also show that climate factors can change the fatty acid composition in the beans, including a significant positive correlation between higher temperatures and the higher level of palmitic acid. Moreover, we found a significant pollen donor effect from the variety “SIAL 70” which was associated with decreased palmitic acid levels.