The Carthamus tinctorius L. genome sequence provides insights into synthesis of unsaturated fatty acids.

Domesticated safflower (Carthamus tinctorius L.) is a widely cultivated edible oil crop. However, despite its economic importance, the genetic basis underlying key traits such as oil content, resistance to biotic and abiotic stresses, and flowering time remains poorly understood. Here, we present the genome assembly for C. tinctorius variety Jihong01, which was obtained by integrating Oxford Nanopore Technologies (ONT) and BGI-SEQ500 sequencing results. The assembled genome was 1,061.1 Mb, and consisted of 32,379 protein-coding genes, 97.71% of which were functionally annotated. Safflower had a recent whole genome duplication (WGD) event in evolution history and diverged from sunflower approximately 37.3 million years ago. Through comparative genomic analysis at five seed development stages, we unveiled the pivotal roles of fatty acid desaturase 2 (FAD2) and fatty acid desaturase 6 (FAD6) in linoleic acid (LA) biosynthesis. Similarly, the differential gene expression analysis further reinforced the significance of these genes in regulating LA accumulation. Moreover, our investigation of seed fatty acid composition at different seed developmental stages unveiled the crucial roles of FAD2 and FAD6 in LA biosynthesis. These findings offer important insights into enhancing breeding programs for the improvement of quality traits and provide reference resource for further research on the natural properties of safflower.

CePP2C19 confers tolerance to drought by regulating the ABA sensitivity in Cyperus esculentus.

BACKGROUND: Tiger nut (Cyperus esculentus) is widely known as an additional source of food, oil and feed worldwide. The agricultural production of tiger nut has been greatly hindered by drought stress, reducing both yield and quality. Protein phosphatase 2 C (PP2Cs) plays an important role in plant responses to drought stress however, the molecular mechanism of PP2Cs in tiger nuts still unclear. RESULTS: In this study, we identified a putative group A PP2C-encoding gene (CePP2C19) from tiger nut using transcriptome analysis, which is highly induced by drought stress. The transient expression assay suggested that CePP2C19 was localized to nucleus. Furthermore, the interaction between CePP2C19 and CePYR1, a coreceptor for ABA signaling, was first detected using a yeast two-hybrid assay and then verified using a bimolecular fluorescence complementation (BiFC) analysis. In addition, the transgenic Arabidopsis lines overexpressing CePP2C19 exhibited extreme tolerance to ABA and mannitol stresses during seed germination and root growth. At the mature stage, overexpression of CePP2C19 resulted in a higher tolerance to drought stress in transgenic Arabidopsis, as confirmed by a visible phenotype and several physiological parameters. Noticeably, the silencing of CePP2C19 by virus-induced gene silencing (VIGS) showed obvious reduction in drought tolerance in tiger nut plants. CONCLUSIONS: The CePP2C19 emerges as a pivotal gene involved in the ABA signaling pathway, which likely reduce ABA sensitivity and thus enhances drought tolerance in Cyperus esculentus.

Disruption of CHORISMATE SYNTHASE1 leads to yellow-green variegation in soybean leaves.

Yellow-green variegation leaf phenotype adds more value to ornamental plants, but it is regarded as an undesirable trait in crop plants, affecting their yields. Until recently, the underlying mechanism regulating the yellow-green variegation phenotype has remained largely unexplored in soybean. In the present study, we indentified four Glycine max leaf yellow/green variegation mutants, Gmvar1, Gmvar2, Gmvar3, and Gmvar4, from artificial mutagenesis populations. Map-based cloning, together with the allelic identification test and CRISPR-based gene knockout, proved that mutated GmCS1 controls yellow-green variegation phenotype of the Gmvar mutants. GmCS1 encodes a chorismate synthase in soybean. The content of Phe, Tyr, and Trp were dramatically decreased in Gmcs1 mutants. Exogenous supply of three aromatic amino acid mixtures, or only Phe to Gmvar mutants, leads to recovery of the mutant phenotype. The various biological processes and signalling pathways related to metabolism and biosynthesis were altered in Gmvar mutants. Collectively, our findings provide new insights about the molecular regulatory network of yellow-green variegation leaf phenotype in soybean.

Genome-Wide Identification of MADS-Box Family Genes in Safflower (Carthamus tinctorius L.) and Functional Analysis of CtMADS24 during Flowering.

Safflower is an important economic crop with a plethora of industrial and medicinal applications around the world. The bioactive components of safflower petals are known to have pharmacological activity that promotes blood circulation and reduces blood stasis. However, fine-tuning the genetic mechanism of flower development in safflower is still required. In this study, we report the genome-wide identification of MADS-box transcription factors in safflower and the functional characterization of a putative CtMADS24 during vegetative and reproductive growth. In total, 77 members of MADS-box-encoding genes were identified from the safflower genome. The phylogenetic analysis divided CtMADS genes into two types and 15 subfamilies. Similarly, bioinformatic analysis, such as of conserved protein motifs, gene structures, and cis-regulatory elements, also revealed structural conservation of MADS-box genes in safflower. Furthermore, the differential expression pattern of CtMADS genes by RNA-seq data indicated that type II genes might play important regulatory roles in floral development. Similarly, the qRT-PCR analysis also revealed the transcript abundance of 12 CtMADS genes exhibiting tissue-specific expression in different flower organs. The nucleus-localized CtMADS24 of the AP1 subfamily was validated by transient transformation in tobacco using GFP translational fusion. Moreover, CtMADS24-overexpressed transgenic Arabidopsis exhibited early flowering and an abnormal phenotype, suggesting that CtMADS24 mediated the expression of genes involved in floral organ development. Taken together, these findings provide valuable information on the regulatory role of CtMADS24 during flower development in safflower and for the selection of important genes for future molecular breeding programs.

A Soybean Sucrose Non-Fermenting Protein Kinase 1 Gene, GmSNF1, Positively Regulates Plant Response to Salt and Salt-Alkali Stress in Transgenic Plants.

Soybean is one of the most widely grown oilseed crops worldwide. Several unfavorable factors, including salt and salt-alkali stress caused by soil salinization, affect soybean yield and quality. Therefore, exploring the molecular basis of salt tolerance in plants and developing genetic resources for genetic breeding is important. Sucrose non-fermentable protein kinase 1 (SnRK1) belongs to a class of Ser/Thr protein kinases that are evolutionarily highly conserved direct homologs of yeast SNF1 and animal AMPKs and are involved in various abiotic stresses in plants. The GmPKS4 gene was experimentally shown to be involved with salinity tolerance. First, using the yeast two-hybrid technique and bimolecular fluorescence complementation (BiFC) technique, the GmSNF1 protein was shown to interact with the GmPKS4 protein. Second, the GmSNF1 gene responded positively to salt and salt-alkali stress according to qRT-PCR analysis, and the GmSNF1 protein was localized in the nucleus and cytoplasm using subcellular localization assay. The GmSNF1 gene was then heterologously expressed in yeast, and the GmSNF1 gene was tentatively identified as having salt and salt-alkali tolerance function. Finally, the salt-alkali tolerance function of the GmSNF1 gene was demonstrated by transgenic Arabidopsis thaliana, soybean hairy root complex plants overexpressing GmSNF1 and GmSNF1 gene-silenced soybean using VIGS. These results indicated that GmSNF1 might be useful in genetic engineering to improve plant salt and salt-alkali tolerance.

GmUFO1 Regulates Floral Organ Number and Shape in Soybean.

The UNUSUAL FLORAL ORGANS (UFO) gene is an essential regulatory factor of class B genes and plays a vital role in the process of inflorescence primordial and flower primordial development. The role of UFO genes in soybean was investigated to better understand the development of floral organs through gene cloning, expression analysis, and gene knockout. There are two copies of UFO genes in soybean and in situ hybridization, which have demonstrated similar expression patterns of the GmUFO1 and GmUFO2 genes in the flower primordium. The phenotypic observation of GmUFO1 knockout mutant lines (Gmufo1) showed an obvious alteration in the floral organ number and shape and mosaic organ formation. By contrast, GmUFO2 knockout mutant lines (Gmufo2) showed no obvious difference in the floral organs. However, the GmUFO1 and GmUFO2 double knockout lines (Gmufo1ufo2) showed more mosaic organs than the Gmufo1 lines, in addition to the alteration in the organ number and shape. Gene expression analysis also showed differences in the expression of major ABC function genes in the knockout lines. Based on the phenotypic and expression analysis, our results suggest the major role of GmUFO1 in the regulation of flower organ formation in soybeans and that GmUFO2 does not have any direct effect but might have an interaction role with GmUFO1 in the regulation of flower development. In conclusion, the present study identified UFO genes in soybean and improved our understanding of floral development, which could be useful for flower designs in hybrid soybean breeding.

Genome-Wide Identification and Comprehensive Analysis of the FtsH Gene Family in Soybean (Glycine max).

The filamentation temperature-sensitive H (FtsH) gene family is critical in regulating plant chloroplast development and photosynthesis. It plays a vital role in plant growth, development, and stress response. Although FtsH genes have been identified in a wide range of plants, there is no detailed study of the FtsH gene family in soybean (Glycine max). Here, we identified 34 GmFtsH genes, which could be categorized into eight groups, and GmFtsH genes in the same group had similar structures and conserved protein motifs. We also performed intraspecific and interspecific collinearity analysis and found that the GmFtsH family has large-scale gene duplication and is more closely related to Arabidopsis thaliana. Cis-acting elements analysis in the promoter region of the GmFtsH genes revealed that most genes contain developmental and stress response elements. Expression patterns based on transcriptome data and real-time reverse transcription quantitative PCR (qRT-PCR) showed that most of the GmFtsH genes were expressed at the highest levels in leaves. Then, GO enrichment analysis indicated that GmFtsH genes might function as a protein hydrolase. In addition, the GmFtsH13 protein was confirmed to be localized in chloroplasts by a transient expression experiment in tobacco. Taken together, the results of this study lay the foundation for the functional determination of GmFtsH genes and help researchers further understand the regulatory network in soybean leaf development.

The antimalarial activity of 1,2,4-trioxolane/trioxane hybrids and dimers: A review.

Malaria, a mosquito-borne parasitic infection caused by protozoan parasites belonging to the genus Plasmodium, is a dangerous disease that contributes to millions of hospital visits and hundreds and thousands of deaths across the world, especially in Sub-Saharan Africa. Antimalarial agents are vital for treating malaria and controlling transmission, and 1,2,4-trioxolane/trioxane-containing agents, especially artemisinin and its derivatives, own antimalarial efficacy and low toxicity with unique mechanisms of action. Moreover, artemisinin-based combination therapies were recommended by the World Health Organization as the first-line treatment for uncomplicated malaria infection and have remained as the mainstay of the treatment of malaria, demonstrating that 1,2,4-trioxolane/trioxane derivatives are useful prototypes for the control and eradication of malaria. However, malaria parasites have already developed resistance to almost all of the currently available antimalarial agents, creating an urgent need for the search of novel pharmaceutical interventions for malaria. The purpose of this review article is to provide an emphasis on the current scenario (January 2012 to January 2022) of 1,2,4-trioxolane/trioxane hybrids and dimers with potential antimalarial activity and the structure-activity relationships are also discussed to facilitate further rational design of more effective candidates.

Genome-Wide Identification and Expression Analysis of Fatty Acid Desaturase (FAD) Genes in Camelina sativa (L.) Crantz.

Camelina sativa (L.) Crantz is an indispensable oilseed crop, and its seeds contain many unsaturated fatty acids. FAD (fatty acid desaturase) regulates the synthesis of unsaturated fatty acids. In this research, we performed CsFAD gene family analysis and identified 24 CsFAD genes in Camelina, which were unevenly distributed on 14 of the 19 total chromosomes. Phylogenetic analysis showed that CsFAD includes four subfamilies, supported by the conserved structures and motifs of CsFAD genes. In addition, we investigated the expression patterns of the FAD family in the different tissues of Camelina. We found that CsFAD family genes were all expressed in the stem, and CsFAD2-2 was highly expressed in the early stage of seed development. Moreover, during low temperature (4 °C) stress, we identified that the expression level of CsFAD2-2 significantly changed. By observing the transient expression of CsFAD2-2 in Arabidopsis protoplasts, we found that CsFAD2-2 was located on the nucleus. Through the detection and analysis of fatty acids, we prove that CsFAD2-2 is involved in the synthesis of linolenic acid (C18:3). In conclusion, we identified CsFAD2-2 through the phylogenetic analysis of the CsFAD gene family and further determined the fatty acid content to find that CsFAD2-2 is involved in fatty acid synthesis in Camelina.

Integration of sRNA, degradome, transcriptome analysis and functional investigation reveals gma-miR398c negatively regulates drought tolerance via GmCSDs and GmCCS in transgenic Arabidopsis and soybean.

BACKGROUND: Drought conditions adversely affect soybean growth, resulting in severe yield losses worldwide. Increasing experimental evidence indicates miRNAs are important post-transcriptional regulators of gene expression. However, the drought-responsive molecular mechanism underlying miRNA-mRNA interactions remains largely uncharacterized in soybean. Meanwhile, the miRNA-regulated drought response pathways based on multi-omics approaches remain elusive. RESULTS: We combined sRNA, transcriptome and degradome sequencing to elucidate the complex regulatory mechanism mediating soybean drought resistance. One-thousand transcripts from 384 target genes of 365 miRNAs, which were enriched in the peroxisome, were validated by degradome-seq. An integrated analysis showed 42 miRNA-target pairs exhibited inversely related expression profiles. Among these pairs, a strong induction of gma-miR398c as a major gene negatively regulates multiple peroxisome-related genes (GmCSD1a/b, GmCSD2a/b/c and GmCCS). Meanwhile, we detected that alternative splicing of GmCSD1a/b might affect soybean drought tolerance by bypassing gma-miR398c regulation. Overexpressing gma-miR398c in Arabidopsis thaliana L. resulted in decreased percentage germination, increased leaf water loss, and reduced survival under water deficiency, which displayed sensitivity to drought during seed germination and seedling growth. Furthermore, overexpressing gma-miR398c in soybean decreased GmCSD1a/b, GmCSD2a/b/c and GmCCS expression, which weakened the ability to scavenge O2.-, resulting in increased relative electrolyte leakage and stomatal opening compared with knockout miR398c and wild-type soybean under drought conditions. CONCLUSION: The study indicates that gma-miR398c negatively regulates soybean drought tolerance, and provides novel insights useful for breeding programs to improve drought resistance by CRISPR technology.

Tinkering Cis Motifs Jigsaw Puzzle Led to Root-Specific Drought-Inducible Novel Synthetic Promoters.

Following an in-depth transcriptomics-based approach, we first screened out and analyzed (in silico) cis motifs in a group of 63 drought-inducible genes (in soybean). Six novel synthetic promoters (SynP14-SynP19) were designed by concatenating 11 cis motifs, ABF, ABRE, ABRE-Like, CBF, E2F-VARIANT, G-box, GCC-Box, MYB1, MYB4, RAV1-A, and RAV1-B (in multiple copies and various combination) with a minimal 35s core promoter and a 222 bp synthetic intron sequence. In order to validate their drought-inducibility and root-specificity, the designed synthetic assemblies were transformed in soybean hairy roots to drive GUS gene using pCAMBIA3301. Through GUS histochemical assay (after a 72 h 6% PEG6000 treatment), we noticed higher glucuronidase activity in transgenic hairy roots harboring SynP15, SynP16, and SynP18. Further screening through GUS fluorometric assay flaunted SynP16 as the most appropriate combination of efficient drought-responsive cis motifs. Afterwards, we stably transformed SynP15, SynP16, and SynP18 in Arabidopsis and carried out GUS staining as well as fluorometric assays of the transgenic plants treated with simulated drought stress. Consistently, SynP16 retained higher transcriptional activity in Arabidopsis roots in response to drought. Thus the root-specific drought-inducible synthetic promoters designed using stimulus-specific cis motifs in a definite fashion could be exploited in developing drought tolerance in soybean and other crops as well. Moreover, the rationale of design extends our knowledge of trial-and-error based cis engineering to construct synthetic promoters for transcriptional upgradation against other stresses.

Anisotropic Nano-/Microparticles from Diversified Copolymers by Solvent-Mediated Self-Assembly.

Effective synthesis of anisotropic nano-/microparticles (APs) by the copolymers is of great significance in nanomaterials and nanotechnology. However, achieving regulation of the morphology, composition, property, and particle size of anisotropic nano-/microparticles (APs) with diversified copolymers is difficult due to complex mechanism and formation conditions. In this work, a versatile one-pot solvent-mediated self-assembly (SmSa) strategy had been proposed for the facile one-pot synthesis of shape-tunable anisotropic nano-/microparticles (StAPs). In addition, the formation mechanism of StAPs was determined through numerous characterization methods related to morphology and element distribution. The results revealed that the anisotropic architectures of StAPs were closely related to the nature of poly(methylacrylic acid-methyl methacrylate-butyl acrylate) (L1) and poly(butyl acrylate-styrene) (L2) polymer chains imparted by polymer blocks of different domains. Therefore, the ordered assembly of the rigid and hydrophobic L2 polymer chains in micelles consisting of the flexible and amphiphilic L1 and solvent could be successfully carried out under the mediation of increasing solvent polarity and the strong adsorption of poly(vinylpyrrolidone) for L2. Furthermore, the developed versatile SmSa strategy and the obtained StAPs play an essential role in the development of nanoscience and nanotechnology. Particularly given its adjustable emulsifying properties at different pH values, as well as numerous sites for further modification by fluorescent or other components, it can be employed to synthesize a wide range of functional materials.

Overexpression of GmCAMTA12 Enhanced Drought Tolerance in Arabidopsis and Soybean.

Fifteen transcription factors in the CAMTA (calmodulin binding transcription activator) family of soybean were reported to differentially regulate in multiple stresses; however, their functional analyses had not yet been attempted. To characterize their role in stresses, we first comprehensively analyzed the GmCAMTA family in silico and thereafter determined their expression pattern under drought. The bioinformatics analysis revealed multiple stress-related cis-regulatory elements including ABRE, SARE, G-box and W-box, 10 unique miRNA (microRNA) targets in GmCAMTA transcripts and 48 proteins in GmCAMTAs' interaction network. We then cloned the 2769 bp CDS (coding sequence) of GmCAMTA12 in an expression vector and overexpressed in soybean and Arabidopsis through Agrobacterium-mediated transformation. The T3 (Transgenic generation 3) stably transformed homozygous lines of Arabidopsis exhibited enhanced tolerance to drought in soil as well as on MS (Murashige and Skoog) media containing mannitol. In their drought assay, the average survival rate of transgenic Arabidopsis lines OE5 and OE12 (Overexpression Line 5 and Line 12) was 83.66% and 87.87%, respectively, which was ~30% higher than that of wild type. In addition, the germination and root length assays as well as physiological indexes such as proline and malondialdehyde contents, catalase activity and leakage of electrolytes affirmed the better performance of OE lines. Similarly, GmCAMTA12 overexpression in soybean promoted drought-efficient hairy roots in OE chimeric plants as compare to that of VC (Vector control). In parallel, the improved growth performance of OE in Hoagland-PEG (polyethylene glycol) and on MS-mannitol was revealed by their phenotypic, physiological and molecular measures. Furthermore, with the overexpression of GmCAMTA12, the downstream genes including AtAnnexin5, AtCaMHSP, At2G433110 and AtWRKY14 were upregulated in Arabidopsis. Likewise, in soybean hairy roots, GmELO, GmNAB and GmPLA1-IId were significantly upregulated as a result of GmCAMTA12 overexpression and majority of these upregulated genes in both plants possess CAMTA binding CGCG/CGTG motif in their promoters. Taken together, we report that GmCAMTA12 plays substantial role in tolerance of soybean against drought stress and could prove to be a novel candidate for engineering soybean and other plants against drought stress. Some research gaps were also identified for future studies to extend our comprehension of Ca-CaM-CAMTA-mediated stress regulatory mechanisms.

Comprehensive Genomic Analysis and Expression Profiling of Diacylglycerol Kinase (DGK) Gene Family in Soybean (Glycine max) under Abiotic Stresses.

Diacylglycerol kinase (DGK) is an enzyme that plays a pivotal role in abiotic and biotic stress responses in plants by transforming the diacylglycerol into phosphatidic acid. However, there is no report on the characterization of soybean DGK genes in spite of the availability of the soybean genome sequence. In this study, we performed genome-wide analysis and expression profiling of the DGK gene family in the soybean genome. We identified 12 DGK genes (namely GmDGK1-12) which all contained conserved catalytic domains with protein lengths and molecular weights ranging from 436 to 727 amino acids (aa) and 48.62 to 80.93 kDa, respectively. Phylogenetic analyses grouped GmDGK genes into three clusters-cluster I, cluster II, and cluster III-which had three, four, and five genes, respectively. The qRT-PCR analysis revealed significant GmDGK gene expression levels in both leaves and roots coping with polyethylene glycol (PEG), salt, alkali, and salt/alkali treatments. This work provides the first characterization of the DGK gene family in soybean and suggests their importance in soybean response to abiotic stress. These results can serve as a guide for future studies on the understanding and functional characterization of this gene family.

Comparative Study of Early Cold-Regulated Proteins by Two-Dimensional Difference Gel Electrophoresis Reveals a Key Role for Phospholipase Dα1 in Mediating Cold Acclimation Signaling Pathway in Rice.

To understand the early signaling steps that regulate cold responses in rice, two-dimensional difference gel electrophoresis (2-D DIGE)(1)was used to study early cold-regulated proteins in rice seedlings. Using mass spectrometry, 32 spots, which represent 26 unique proteins that showed an altered expression level within 5 min of cold treatment were identified. Among these proteins, Western blot analyses confirmed that the cellular phospholipase D α1 (OsPLDα1) protein level was increased as early as 1 min after cold treatment. Genetic studies showed that reducing the expression ofOsPLDα1makes rice plants more sensitive to chilling stress as well as cold acclimation increased freezing tolerance. Correspondingly, cold-regulated proteomic changes and the expression of the cold-responsive C repeat/dehydration-responsive element binding 1 (OsDREB1) family of transcription factors were inhibited in thepldα1mutant. We also found that the expression ofOsPLDα1is directly regulated by OsDREB1A. This transcriptional regulation ofOsPLDα1could provide positive feedback regulation of the cold signal transduction pathway in rice. OsPLDα1 hydrolyzes phosphatidylcholine to produce the signal molecule phosphatidic acid (PA). By lipid-overlay assay, we demonstrated that the rice cold signaling proteins, MAP kinase 6 (OsMPK6) and OsSIZ1, bind directly to PA. Taken together, our results suggest that OsPLDα1 plays a key role in transducing cold signaling in rice by producing PA and regulatingOsDREB1s' expression by OsMPK6, OsSIZ1, and possibly other PA-binding proteins.

A phosphoinositide-specific phospholipase C pathway elicits stress-induced Ca2+ signals and confers salt tolerance to rice.

In animal cells, phospholipase C (PLC) isoforms predominantly hydrolyze phosphatidylinositol-4,5-biphosphates [PtdIns(4,5)P2 ] into the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3 ] to regulate diverse biological processes. By contrast, the molecular mechanisms and physiological significance of PLC signaling in plants still awaits full elucidation. Here, we identified a rice (Oryza sativa cv) PI-PLC, OsPLC1, which preferred to hydrolyze phosphatidylinositol-4-phosphate (PtdIns4P) and elicited stress-induced Ca2+ signals regulating salt tolerance. Analysis by ion chromatography revealed that the concentration of PtdIns4P was c. 28 times of that of PtdIns(4,5)P2 in shoots. OsPLC1 not only converted PtdIns(4,5)P2 but also - and even more efficiently - converted PtdIns4P into DAG and Ins(1,4,5)P3 in vitro and in vivo. Salt stress induced the recruitment of OsPLC1 from cytoplasm to plasma membrane, where it hydrolyzed PtdIns4P. The stress-induced Ca2+ signaling was dependent on OsPLC1, and the PLC-mediated Ca2+ signaling was essential for controlling Na+ accumulation in leaf blades, thus establishing whole plant salt tolerance. Our work identifies a conversion pathway and physiological function for PtdIns4P pools in rice and reveals the connection between phosphoinositides and Ca2+ signals mediated by PLC during salt stress responses.

Seed-specific expression and analysis of recombinant anti-HER2 single-chain variable fragment (scFv-Fc) in Arabidopsis thaliana.

Antibodies to human epidermal growth factor receptor 2 (HER2) are a key element of breast cancer therapy; however, they are expensive to produce and their availability is limited. A seed-specific expression system can be used to produce recombinant proteins. We report a seed-specific expression system for the manufacture of anti-HER2 ScFv-Fc in Arabidopsis thaliana, driven by the Phaseolus vulgaris ß-phaseolin promoter. Recombinant anti-HER2 ScFv-Fc was successfully and specifically expressed in seeds, and identified by protein analysis. The highest protein accumulation level, with a maximum of 1.1% of total soluble protein, was observed in mature seeds. We also demonstrated the anti-tumor potency of the plant-derived antibody against SK-BR-3 cells. These results suggest that seed-expression systems could contribute to the manufacture of commercial antibodies such as anti-HER2 ScFv-Fc.

The AaDREB1 Transcription Factor from the Cold-Tolerant Plant Adonis amurensis Enhances Abiotic Stress Tolerance in Transgenic Plant.

Dehydration-responsive element binding (DREB) transcription factors (TFs) play important roles in the regulation of plant resistance to environmental stresses and can specifically bind to dehydration-responsive element/C-repeat element (DRE/CRT) proteins (G/ACCGAC) and activate expression of many stress-inducible genes. Here, we cloned and characterized a novel gene (AaDREB1) encoding the DREB1 transcription factor from the cold-tolerant plant Adonis amurensis. Quantitative real-time (qRT)-PCR results indicated that AaDREB1 expression was induced by salt, drought, cold stress, and abscisic acid application. A yeast one-hybrid assay demonstrated that AaDREB1 encodes a transcription activator and specifically binds to DRE/CRT. Furthermore, transgenic Arabidopsis and rice harboring AaDREB1 showed enhanced tolerance to salt, drought, and low temperature. These results indicated that AaDREB1 might be useful in genetic engineering to improve plant stress tolerance.

Mining and identification of polyunsaturated fatty acid synthesis genes active during camelina seed development using 454 pyrosequencing.

BACKGROUND: Camelina (Camelina sativa L.) is well known for its high unsaturated fatty acid content and great resistance to environmental stress. However, little is known about the molecular mechanisms of unsaturated fatty acid biosynthesis in this annual oilseed crop. To gain greater insight into this mechanism, the transcriptome profiles of seeds at different developmental stages were analyzed by 454 pyrosequencing. RESULTS: Sequencing of two normalized 454 libraries produced 831,632 clean reads. A total of 32,759 unigenes with an average length of 642 bp were obtained by de novo assembly, and 12,476 up-regulated and 12,390 down-regulated unigenes were identified in the 20 DAF (days after flowering) library compared with the 10 DAF library. Functional annotations showed that 220 genes annotated as fatty acid biosynthesis genes were up-regulated in 20 DAF sample. Among them, 47 candidate unigenes were characterized as responsible for polyunsaturated fatty acid synthesis. To verify unigene expression levels calculated from the transcriptome analysis results, quantitative real-time PCR was performed on 11 randomly selected genes from the 220 up-regulated genes; 10 showed consistency between qRT-PCR and 454 pyrosequencing results. CONCLUSIONS: Investigation of gene expression levels revealed 32,759 genes involved in seed development, many of which showed significant changes in the 20 DAF sample compared with the 10 DAF sample. Our 454 pyrosequencing data for the camelina transcriptome provide an insight into the molecular mechanisms and regulatory pathways of polyunsaturated fatty acid biosynthesis in camelina. The genes characterized in our research will provide candidate genes for the genetic modification of crops.

De Novo Sequencing and Analysis of the Safflower Transcriptome to Discover Putative Genes Associated with Safflor Yellow in Carthamus tinctorius L.

Safflower (Carthamus tinctorius L.), an important traditional Chinese medicine, is cultured widely for its pharmacological effects, but little is known regarding the genes related to the metabolic regulation of the safflower's yellow pigment. To investigate genes related to safflor yellow biosynthesis, 454 pyrosequencing of flower RNA at different developmental stages was performed, generating large databases.In this study, we analyzed 454 sequencing data from different flowering stages in safflower. In total, 1,151,324 raw reads and 1,140,594 clean reads were produced, which were assembled into 51,591 unigenes with an average length of 679 bp and a maximum length of 5109 bp. Among the unigenes, 40,139 were in the early group, 39,768 were obtained from the full group and 28,316 were detected in both samples. With the threshold of "log2 ratio ≥ 1", there were 34,464 differentially expressed genes, of which 18,043 were up-regulated and 16,421 were down-regulated in the early flower library. Based on the annotations of the unigenes, 281 pathways were predicted. We selected 12 putative genes and analyzed their expression levels using quantitative real time-PCR. The results were consistent with the 454 sequencing results. In addition, the expression of chalcone synthase, chalcone isomerase and anthocyanidin synthase, which are involved in safflor yellow biosynthesis and safflower yellow pigment (SYP) content, were analyzed in different flowering periods, indicating that their expression levels were related to SYP synthesis. Moreover, to further confirm the results of the 454 pyrosequencing, full-length cDNA of chalcone isomerase (CHI) and anthocyanidin synthase (ANS) were cloned from safflower petal by RACE (Rapid-amplification of cDNA ends) method according to fragment of the transcriptome.