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Relative expression levels of MsFAD3.1 in different tissues. The relative expression levels of the FAD3.1 gene in different tissues of M. sativa were determined by quantitative polymerase chain reaction (qPCR). The data represent the means of three independent replicates ± SE. Samples not sharing a letter are significantly different according to ANOVA
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As an important forage legume in the world, alfalfa (Medicago sativa L.) has high adaptability to various unfavorable climatic conditions and high biomass, and have been playing critical roles in animal husbandry and industrial applications. As α‐linolenic acid cannot be synthesized by animals, and most must be obtained from plants, the increasing...
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... The members of the fatty acid desaturase (FAD) gene family have been mainly studied in many plants, such as in Gossypium raimondii [28], Cucumis sativus [5], Linum usitatissimum [29], Musa spp. [30], Cicer arietinum [31], Camelina sativa [32], etc., but they have not been systematically isolated and studied in Tausonia pullulans. In Gossypium raimondii, nineteen FAD genes were cloned at low temperatures, and their expression was investigated [28]. ...
Tausonia pullulans 6A7 is a low-temperature yeast strain that can produce lipases. Yeast, which is made up of chassis cells, is an important part of synthetic biology, and the use of the lipase-producing properties of T. pullulans 6A7 for the production of fatty acids provides a new pathway for targeted synthesis in yeast cell factories. In this study, we performed RNA-seq on lipase-producing T. pullulans 6A7 at different temperatures (15 °C, 20 °C, 20 °C without corn oil, and 25 °C). Therefore, a total of 8455 differentially expressed genes were screened, and 16 of them were FAD candidate genes. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of group A (15 °C) vs. group D (25 °C) showed that the pathways of fatty acid biosynthesis (map00061) and the biosynthesis of unsaturated fatty acids (map01040) were significantly enriched. In the proposed temporal analysis of differentially expressed genes among the four temperature modulations, we found differentially expressed genes in nine clusters that had the same expression trends; these genes may be jointly involved in multiple biological processes in T. pullulans 6A7. In addition, we found 16 FAD candidate genes involved in fatty acid biosynthesis, and the expression of these genes had similar expression in the transcriptome trends with the different temperature treatments. These findings will help in future in-depth studies of the function and molecular mechanisms of these important FAD genes involved in fatty acid metabolism in yeast, and they could also be conducive to the establishment of a cellular factory for targeted fatty acid production by using yeast.
... In this study, 26 members of the tomato FAD gene family were identified and analyzed. The number of members is not significantly different from that of Arabidopsis and eggplant FAD, but the number is much lower than that of wheat (68) [27], alfalfa (62) [28], and rapeseed (84) [11], indicating that the expansion of the FAD gene family has species specificity. This expansion may be related to gene duplication events [29]. ...
... Phylogenetic analysis shows that the FAD family is significantly divided into two subfamilies, including soluble and membrane-bound FADs, which is consistent with previous research [10]. Membrane-bound FADs can be further divided into five branches: FAD2, FAD3/7/8, ADS, FAD6, and SLD, similar to wheat [27]. Tomatoes do not contain the ADS branch, which is the same as rice and bananas, indicating that ADS may have formed after the differentiation of monocots and dicots [13]. ...
The fatty acid desaturase (FAD) gene family plays a crucial regulatory role in the resistance process of plant biomembranes. To understand the role of FADs in tomato growth and development, this study identified and analyzed the tomato FAD gene family based on bioinformatics analysis methods. In this study, 26 SlFADs were unevenly distributed on 10 chromosomes. Phylogenetic analysis showed that the SlFAD gene family was divided into six branches, and the exon–intron composition and conserved motifs of SlFADs clustered in the same branch were quite conservative. Several hormone and stress response elements in the SlFAD promoter suggest that the expression of SlFAD members is subject to complex regulation; the construction of a tomato FAD protein interaction network found that SlFAD proteins have apparent synergistic effects with SPA and GPAT proteins. qRT-PCR verification results show that SlFAD participates in the expression of tomato root, stem, and leaf tissues; SlFAD8 is mainly highly expressed in leaves; SlFAD9 plays a vital role in response to salt stress; and SlFAB5 regulates all stages of fruit development under the action of exogenous hormones. In summary, this study provides a basis for a systematic understanding of the SlFAD gene family. It provides a theoretical basis for in-depth research on the functional characteristics of tomato SlFAD genes.
... At the same time, equal amounts of yeast cells were resuspended in 200 µL of sterile water at 30 • C for 36 h, to serve as a control. The cells were subsequently incubated in serial dilutions (10 0 , 10 −1 , 10 −2 , 10 −3 , 10 −4 , 10 −5 , 10 −6 ) and finally spotted on SC-Ura agar plates [62]. Then, the yeast was allowed to grow for 2−3 days at 30 • C on this plate. ...
Melilotus albus is a high-quality forage, due to its high protein content, and aboveground biomass and salt tolerance. Rab (Ras-related protein in the brain) proteins are the largest GTPase family which play a key role in intracellular membrane transport, and many Rab genes have been identified in eukaryotes. The growth and distribution of M. albus are severely hampered by soil salinization. However, little is known about candidate genes for salt tolerance in M. albus. In this study, 27 Rab family genes were identified for the first time from M. albus, and divided into eight groups (Groups A-H). The number of introns in MaRabs ranged from one to seven, with most genes containing one intron. In addition, most MaRab proteins showed similarities in motif composition. Phylogenetic analysis and structural-domain comparison indicated that Rab family genes were highly conserved in M. albus. Members of the MaRab gene family were distributed across all eight chromosomes, with the largest distribution on chromosome 1. Prediction of the protein interaction network showed that 24 Rab proteins exhibited protein–protein interactions. Analysis of the promoter cis-acting elements showed that MaRab-gene family members are extensively involved in abiotic stress responses. RNA-seq data analysis of the MaRab-gene-expression patterns suggested that the Rab gene family possesses differentially expressed members in five organs and under salt stress, drought stress, and ABA (Abscisic Acid) treatment. Differentially expressed genes under drought stress, salt stress and ABA stress were validated by quantitative real-time PCR. Furthermore, heterologous expression in yeast was used to characterize the functions of MaRab1 and MaRab17, which were upregulated in reaction to salt stress. In summary, this study provided valuable information for further research into the molecular mechanism of the response of M. albus to saline stress, as well as the possibility of developing cultivars with high salt-resistance characteristics.
... nih. gov/Structure/cdd/wrpsb.Cgi, accessed on 12 November 2021) were used to determine that these sequences contained only one AP2 domain [46]. The CD-Hit tool (http://weizhongli-lab.org/cd-hit/, accessed on 12 November 2021) was used to remove redundant sequences in the remaining ERF protein sequences [40]. ...
As an important forage legume with high values in feed and medicine, Melilotus albus has been widely cultivated. The AP2/ERF transcription factor has been shown to play an important regulatory role in plant drought resistance, but it has not been reported in the legume forage crop M. albus. To digger the genes of M. albus in response to drought stress, we identified and analyzed the ERF gene family of M. albus at the genome-wide level. A total of 100 MaERF genes containing a single AP2 domain sequence were identified in this study, named MaERF001 to MaERF100, and bioinformatics analysis was performed. Collinearity analysis indicated that segmental duplication may play a key role in the expansion of the M. albus ERF gene family. Cis-acting element predictions suggest that MaERF genes are involved in various hormonal responses and abiotic stresses. The expression patterns indicated that MaERFs responded to drought stress to varying degrees. Furthermore, four up-regulated ERFs (MaERF008, MaERF037, MaERF054 and MaERF058) under drought stress were overexpressed in yeast and indicated their biological functions to confer the tolerance to drought. This work will advance the understanding of the molecular mechanisms underlying the drought response in M. albus. Further study of the promising potential candidate genes identified in this study will provide a valuable resource as the next step in functional genomics studies and improve the possibility of improving drought tolerance in M. albus by transgenic approaches.
... The empty pYES2 plasmid and recombinant pYES2-MaGRAS12, MaGRAS33 and MaGRAS34 plasmids were transferred into Saccharomyces cerevisiae strain INVSc1. The method is consistent with previous studies [62,71]. The 4 yeast cultures were independently grown in synthetic complete (SC)-Ura liquid medium containing 2% (m/v) galactose at 30 • C for 36 h up to A600 = 0.4. ...
The GRAS gene family is a plant−specific family of transcription factors, which play an important role in many metabolic pathways, such as plant growth and development and stress response. However, there is no report on the comprehensive study of the GRAS gene family of Melilotus albus. Here, we identified 55 MaGRAS genes, which were classified into 8 subfamilies by phylogenetic analysis, and unevenly distributed on 8 chromosomes. The structural analysis indicated that 87% of MaGRAS genes have no intron, which is highly conservative in different species. MaGRAS proteins of the same subfamily have similar protein motifs, which are the source of functional differences of different genomes. Transcriptome and qRT−PCR data were combined to determine the expression of 12 MaGRAS genes in 6 tissues, including flower, seed, leaf, stem, root and nodule, which indicated the possible roles in plant growth and development. Five and seven MaGRAS genes were upregulated under ABA, drought, and salt stress treatments in the roots and shoots, respectively, indicating that they play vital roles in the response to ABA and abiotic stresses in M. albus. Furthermore, in yeast heterologous expression, MaGRAS12, MaGRAS34 and MaGRAS33 can enhance the drought or salt tolerance of yeast cells. Taken together, these results provide basic information for understanding the underlying molecular mechanisms of GRAS proteins and valuable information for further studies on the growth, development and stress responses of GRAS proteins in M. albus.
... FAD genes are essential factors which desaturate saturated fatty acids into unsaturated fatty acids for oil plants [6]. Until now, they have been identified and characterized in non-oil plants Arabidopsis (25) and rice (19) and oil plants soybean (41), rapeseed (84) and walnut (30) among others [12][13][14][15][16][17]. Olive is the only plant which extracts oil from its fresh fruits and is favored by consumers for redundant UFAs [38,39]. ...
... Sylvestris, respectively. All the FAD genes could be distinguished as the soluble FAB2/SAD genes and membrane-bound FADs, including ADS, DES, FAD4, SLD, ω-3 and ω-6 [12][13][14][15][16][17] (Figure 1, Tables 1 and 2). Protein analysis revealed that the former shared the domain PF03405 and the latter shared PF00487, except for FAD4, which shared PF10520. ...
... ω-3 and ω-6 genes have been verified and well-studied in different plants; they can catalyze C18:1 to C18:2 and C18:2 to C18:3, respectively, and included FAD3/FAD7/FAD8 and FAD2/FAD6 genes [12][13][14][15][16][17]. The mutants had a large increase in the corresponding UFAs [22,24,25], and the expression levels were induced by cold stress, salt, drought, osmotic and wound stresses [26,27,44]. ...
Olive (Olea europaea L.) is a world-famous woody oil tree and popular for redundant unsaturated fatty acids. Fatty acid desaturase (FAD) genes are responsible for fatty acid desaturation and stress regulation but have not yet been identified in olive at the whole genome level. This study identified 40 and 27 FAD genes in the cultivated olive O. europaea cv. Farga and the wild olive O. europaea var. Sylvestris, respectively. Phylogenetic analysis showed that all the FAD genes could be classified into the soluble FAB2/SAD clade and membrane-bound clade, including ADS/FAD5, DES, FAD4, SLD, ω-6 and ω-3, with the high consistency of subcellular localization, motif composition and exon-intron organization in each group. FAD genes in olive showed the diverse functional differentiation in morphology of different tissues, fruit development and stress responses. Among them, OeFAB2.8 and OeFAD2.3 were up-regulated and OeADS.1, OeFAD4.1 and OeFAD8.2 were down-regulated under the wound, Verticillium dahliae and cold stresses. This study presents a comprehensive analysis of the FAD genes at the whole-genome level in olives and will provide guidance for the improvement of oil quality or stress tolerance of olive trees.
... Particularly, ω-3 fatty acid desaturases have been proven to use C18:2 as substrates to produce 18:3 in the plastid, where FAD7 and FAD8 are located, and in the ER where FAD3 are found [5]. In plants, the accumulation of 18:3 under the activity of ω-3 fatty acid desaturases, particularly FAD3 has been demonstrated in several studies, such as in soybean [108,109], in rice [110], in olive [111][112][113], in peanut [114], in flax [115], in walnuts [116], in cowpea [117], in alfalfa [118], in peach aphid [119] and now in C. sativa, according to the current study. ...
Fatty acid desaturases add a second bond into a single bond of carbon atoms in fatty acid chains, resulting in an unsaturated bond between the two carbons. They are classified into soluble and membrane-bound desaturases, according to their structure, subcellular location, and function. The orthologous genes in Camelina sativa were identified and analyzed, and a total of 62 desaturase genes were identified. It was revealed that they had the common fatty acid desaturase domain, which has evolved separately, and the proteins of the same family also originated from the same ancestry. A mix of conserved, gained, or lost intron structure was obvious. Besides, conserved histidine motifs were found in each family, and transmembrane domains were exclusively revealed in the membrane-bound desaturases. The expression profile analysis of C. sativa desaturases revealed an increase in young leaves, seeds, and flowers. C. sativa ω3-fatty acid desaturases CsaFAD7 and CsaDAF8 were cloned and the subcellular localization analysis showed their location in the chloroplast. They were transferred into Arabidopsis thaliana to obtain transgenic lines. It was revealed that the ω3-fatty acid desaturase could increase the C18:3 level at the expense of C18:2, but decreases in oil content and seed weight, and wrinkled phenotypes were observed in transgenic CsaFAD7 lines, while no significant change was observed in transgenic CsaFAD8 lines in comparison to the wild-type. These findings gave insights into the characteristics of desaturase genes, which could provide an excellent basis for further investigation for C. sativa improvement, and overexpression of ω3-fatty acid desaturases in seeds could be useful in genetic engineering strategies, which are aimed at modifying the fatty acid composition of seed oil.
... Then, the yeast was collected and adjusted with SC-Ura including 2% galactose and cultivated up to A 600 = 1 for stress analysis. The same number of cells was resuspended in 30% PEG-6000 and 5 M NaCl [57]. The treated yeast liquid was diluted to 1:10 and cultured on SC-U/2% (w/v) glucose agar plates for 2-3 days to observe colony growth, and photos were taken to record the expression of the binding protein. ...
Coumarins, natural products abundant in Melilotus albus, confer features in response to abiotic stresses, and are mainly present as glycoconjugates. UGTs (UDP-glycosyltransferases) are responsible for glycosylation modification of coumarins. However, information regarding the relationship between coumarin biosynthesis and stress-responsive UGTs remains limited. Here, a total of 189 MaUGT genes were identified from the M. albus genome, which were distributed differentially among its eight chromosomes. According to the phylogenetic relationship, MaUGTs can be classified into 13 major groups. Sixteen MaUGT genes were differentially expressed between genotypes of Ma46 (low coumarin content) and Ma49 (high coumarin content), suggesting that these genes are likely involved in coumarin biosynthesis. About 73.55% and 66.67% of the MaUGT genes were differentially expressed under ABA or abiotic stress in the shoots and roots, respectively. Furthermore, the functions of MaUGT68 and MaUGT186, which were upregulated under stress and potentially involved in coumarin glycosylation, were characterized by heterologous expression in yeast and Escherichia coli. These results extend our knowledge of the UGT gene family along with MaUGT gene functions, and provide valuable findings for future studies on developmental regulation and comprehensive data on UGT genes in M. albus.
Fatty acid desaturase (FAD) is the key enzyme that leads to the formation of unsaturated fatty acids by introducing double bonds into hydrocarbon chains, and it plays a critical role in plant lipid metabolism. However, no data are available on enzyme-associated genes in argan trees. In addition, a candidate gene approach was adopted to identify and characterize the gene sequences of interest that are potentially involved in oil quality and abiotic stress. Based on phylogenetic analyses, 18 putative FAD genes of Argania spinosa L. (AsFAD) were identified and assigned to three subfamilies: stearoyl-ACP desaturase (SAD), Δ-12 desaturase (FAD2/FAD6), and Δ-15 desaturase (FAD3/FAD7). Furthermore, gene structure and motif analyses revealed a conserved exon-intron organization among FAD members belonging to the various oil crops studied, and they exhibited conserved motifs within each subfamily. In addition, the gene structure shows a wide variation in intron numbers, ranging from 0 to 8, with two highly conserved intron phases (0 and 1). The AsFAD and AsSAD subfamilies consist of three (H(X)2-4H, H(X)2-3HH, and H/Q (X)2-3HH) and two (EEN(K)RHG and DEKRHE) conserved histidine boxes, respectively. A set of primer pairs were designed for each FAD gene, and tested on DNA extracted from argan leaves, in which all amplicons of the expected size were produced. These findings of candidate genes in A spinosa L. will provide valuable knowledge that further enhances our understanding of the potential roles of FAD genes in the quality of oil and abiotic stress in the argan tree.
