Reprogramming the Metabolism of Yeast for High-Level Production of Miltiradiene.

Miltiradiene serves as a crucial precursor in the synthesis of various high-value abietane-type diterpenes, exhibiting diverse pharmacological activities. Previous efforts to enhance miltiradiene production have primarily focused on the mevalonate acetate (MVA) pathway. However, limited emphasis has been placed on optimizing the supply of acetyl-CoA and NADPH. In this study, we constructed a platform yeast strain for miltiradiene production by reinforcing the biosynthetic pathway of geranylgeranyl diphosphate (GGPP) and acetyl-CoA, and addressing the imbalance between the supply and demand of the redox cofactor NADPH within the cytoplasm, resulting in an increase in miltiradiene yield to 1.31 g/L. Furthermore, we conducted modifications to the miltiradiene synthase fusion protein tSmKSL1-CfTPS1. Finally, the comprehensive engineering strategies and protein modification strategies culminated in 1.43 g/L miltiradiene in the engineered yeast under shake flask culture conditions. Overall, our work established efficient yeast cell factories for miltiradiene production, providing a foothold for heterologous biosynthesis of abietane-type diterpenes.

An Investigation of the JAZ Family and the CwMYC2-like Protein to Reveal Their Regulation Roles in the MeJA-Induced Biosynthesis of ß-Elemene in Curcuma wenyujin.

ß-Elemene (C15H24), a sesquiterpenoid compound isolated from the volatile oil of Curcuma wenyujin, has been proven to be effective for multiple cancers and is widely used in clinical treatment. Unfortunately, the ß-elemene content in C. wenyujin is very low, which cannot meet market demands. Our previous research showed that methyl jasmonate (MeJA) induced the accumulation of ß-elemene in C. wenyujin. However, the regulatory mechanism is unclear. In this study, 20 jasmonate ZIM-domain (JAZ) proteins in C. wenyujin were identified, which are the core regulatory factors of the JA signaling pathway. Then, the conservative domains, motifs composition, and evolutionary relationships of CwJAZs were analyzed comprehensively and systematically. The interaction analysis indicated that CwJAZs can form homodimers or heterodimers. Fifteen out of twenty CwJAZs were significantly induced via MeJA treatment. As the master switch of the JA signaling pathway, the CwMYC2-like protein has also been identified and demonstrated to interact with CwJAZ2/3/4/5/7/15/17/20. Further research found that the overexpression of the CwMYC2-like gene increased the accumulation of ß-elemene in C. wenyujin leaves. Simultaneously, the expressions of HMGR, HMGS, DXS, DXR, MCT, HDS, HDR, and FPPS related to ß-elemene biosynthesis were also up-regulated by the CwMYC2-like protein. These results indicate that CwJAZs and the CwMYC2-like protein respond to the JA signal to regulate the biosynthesis of ß-elemene in C. wenyujin.

Protein Engineering of a Germacrene A Synthase From Lactuca sativa and Its Application in High Productivity of Germacrene A in Escherichia coli.

Germacrene A (GA) is a key intermediate for the synthesis of medicinal active compounds, especially for ß-elemene, which is a broad-spectrum anticancer drug. The production of sufficient GA in the microbial platform is vital for the precursors supply of active compounds. In this study, Escherichia coli BL21 Star (DE3) was used as the host and cultivated in SBMSN medium, obtaining a highest yield of FPP. The GA synthase from Lactuca sativa (LTC2) exhibited the highest level of GA production. Secondly, two residues involved in product release (T410 and T392) were substituted with Ser and Ala, respectively, responsible for relatively higher activities. Next, substitution of selected residues S243 with Asn caused an increase in activity. Furthermore, I364K-T410S and T392A-T410S were created by combination with the beneficial mutation, and they demonstrated dramatically enhanced titers with 1.90-fold and per-cell productivity with 5.44-fold, respectively. Finally, the production titer of GA reached 126.4 mg/L, and the highest productivity was 7.02 mg/L.h by the I364K-T410S mutant in a shake-flask batch culture after fermentation for 18 h. To our knowledge, the productivity of the I364K-T410S mutant is the highest level ever reported. These results highlight a promising method for the industrial production of GA in E. coli, and lay a foundation for pathway reconstruction and the production of valuable natural sesquiterpenes.

Transcriptome analysis reveals regulation mechanism of methyl jasmonate-induced terpenes biosynthesis in Curcuma wenyujin.

Curcuma wenyujin is the source plant of three traditional Chinese medicines, which have been widely used in clinical treatment over 1000 years. The content of terpenes, the major medicinal active ingredients, is relatively low in this plant. Studies have shown that MeJA can promote terpenes biosynthesis in plants. However, the mechanism underlying the effect of MeJA in C. wenyujin remains unclear. In this work, the transcriptome of C. wenyujin leaves with MeJA treatment was analyzed to elucidate the regulation mechanism of MeJA-mediated terpene biosynthesis. Based on the RNA-seq data, 7,246 unigenes were differentially expressed with MeJA treatment. Expression pattern clustering of DEGs revealed that unigenes, related to JA biosynthesis and signal transduction, responded to exogenous MeJA stimulation on the early stage and maintained throughout the process. Subsequently, unigenes related to terpene biosynthesis pathway showed a significant up-regulation with 6 h treatment. The analysis results suggested that MeJA induced the expression of JA biosynthesis genes (such as LOXs, AOSs, AOCs, OPRs, and MFPs) and JA signal transduction core genes (JAZs and MYCs) to activate JA signaling pathway. Meanwhile, downstream JA-responsive genes presented up-regulated expression levels such as AACT, HMGSs, HMGRs, DXSs, DXRs, MCTs, HDSs, and HDRs, thus promoting terpenes biosynthesis. The transcriptional expressions of these genes were validated by qRT-PCR. In addition, six CwTPS genes in response to MeJA were identified. With MeJA treatment, the expression levels of CwTPSs were increased as well as those of the transcription factors MYB, NAC, bZIP, WRKY, AP2/ERF, and HLH. These TFs might potentially regulate terpenes biosynthesis. These results provide insights for regulation mechanism of terpenes biosynthesis.

Functional characterization of key polyketide synthases by integrated metabolome and transcriptome analysis on curcuminoid biosynthesis in Curcuma wenyujin.

Leaf and tuber extracts of Curcuma wenyujin contain a mixture of curcuminoids. However, the curcuminoid constituents and their molecular mechanisms are poorly understood, and the relevant curcumin synthases remain unclear. In this study, we comprehensively compared the metabolite profiles of the leaf and tuber tissues of C. wenyujin. A total of 11 curcuminoid metabolites were identified and exhibited differentially changed contents in the leaf and tuber tissues. An integrated analysis of metabolomic and transcriptomic data revealed the proposed biosynthesis pathway of curcuminoid. Two candidate type Ⅲ polyketide synthases (PKSs) were identified in the metabolically engineering yeasts, indicating that CwPKS1 and CwPKS2 maintained substrate and product specificities. Especially, CwPKS1 is the first type Ⅲ PKS identified to synthesize hydrogenated derivatives of curcuminoid, dihydrocurcumin and tetrehydrocurcumin. Interestingly, the substitution of the glycine at position 219 with aspartic acid (G219D mutant) resulted in the complete inactivation of CwPKS1. Our results provide the first comparative metabolome analysis of C. wenyujin and functionally identified type Ⅲ PKSs, giving valuable information for curcuminoids biosynthesis.

Identification and Analysis of MYB Gene Family for Discovering Potential Regulators Responding to Abiotic Stresses in Curcuma wenyujin.

MYB superfamily is one of the most abundant families in plants, and plays critical role in plant growth, development, metabolism regulation, and stress response. Curcuma wenyujin is the main source plant of three traditional Chinese medicines, which are widely used in clinical treatment due to its diverse pharmacological activities. In present study, 88 CwMYBs were identified and analyzed in C. wenyujin, including 43 MYB-related genes, 42 R2R3-MYB genes, two 3R-MYB genes, and one 4R-MYB gene. Forty-three MYB-related proteins were classified into several types based on conserved domains and specific motifs, including CCA1-like type, R-R type, Myb-CC type, GARP-like type, and TBR-like type. The analysis of motifs in MYB DBD and no-MYB regions revealed the relevance of protein structure and function. Comparative phylogeny analysis divided 42 R2R3-MYB proteins into 19 subgroups and provided a reference for understanding the functions of some CwMYBs based on orthologs of previously characterized MYBs. Expression profile analysis of CwMYB genes revealed the differentially expressed genes responding to various abiotic stresses. Four candidate MYB genes were identified by combining the results of phylogeny analysis and expression analysis. CwMYB10, CwMYB18, CwMYB39, and CwMYB41 were significantly induced by cold, NaCl, and MeJA stress treatments. CwMYB18 and CwMYB41 were proved as regulators with activity of transcriptional activation, whereas CwMYB39 and CwMYB10 were not. They may participate in the response to abiotic stresses through different mechanisms in C. wenyujin. This study was the first step toward understanding the CwMYB family and the response to abiotic stresses in C. wenyujin.

Transcriptome sequencing and functional characterization of new sesquiterpene synthases from Curcuma wenyujin.

Tubers of Curcuma wenyujin are rich in essential oils, mainly various sesquiterpenes, showing antibacterial, anti-viral and anti-tumor effects. However, the molecular mechanism of C. wenyujin is deficient and related sesquiterpene synthases are still unclear. In this study, the transcriptome data of tubers and leaves from C. wenyujin were obtained and assembled into 78 092 unigenes. Of them, 244 unigenes were predicted to be involved in terpenoid biosynthesis while 131 unigenes were categorized as the "Terpenoid backbone biosynthesis" (TBB) term. Twenty-two unigenes possessed terpene synthase domain; five were predicted to be sesquiterpene synthases. Of the 208 unigenes annotated as cytochromes P450, 8 unigenes with full-length coding sequences were part of the CYP71 clade that primarily may perform hydroxylations of specialized metabolites. Furthermore, Ten DEGs related to the C5 precursor supply and sesquiterpene synthesis were validated by Real-time PCR; that showed a close correspondence with transcriptome sequence. A novel germacrene B synthase (CwGBS) and α-santalene synthase (CwSS) were identified in metabolically engineering E. coli. This study provided the first de novo transcriptome comparative analysis of leaf and tuber tissues from C. wenyujin, aiming to understand genetic mechanisms. Key genes involved in the biosynthesis of sesquiterpene will help for revealing the underlying mechanisms of C. wenyujin.

TaASR1-D confers abiotic stress resistance by affecting ROS accumulation and ABA signalling in transgenic wheat.

Cultivating new crop cultivars with multiple abiotic stress tolerances is important for crop production. The abscisic acid-stress-ripening (ASR) protein has been shown to confer abiotic stress tolerance in plants. However, the mechanisms of ASR function under stress condition remain largely unclear. In this study, we characterized all ASR family members in common wheat and constitutively overexpressed TaASR1-D in a commercial hexaploid wheat cultivar Zhengmai 9023. The transgenic wheat plants exhibited increased tolerance to multiple abiotic stresses and increased grain yields under salt stress condition. Overexpression of TaASR1-D conferred enhanced antioxidant capacity and ABA sensitivity in transgenic wheat plants. Further, RNA in situ hybridization results showed that TaASR1-D had higher expression levels in the vascular tissues of leaves and the parenchyma cells around the vascular tissues of roots and stems. Yeast one-hybrid and electrophoretic mobility shift assays revealed that TaASR1-D could directly bind the specific cis-elements in the promoters of TaNCED1 and TaGPx1-D. In conclusion, our findings suggest that TaASR1-D can be used to breed new wheat cultivars with increased multiple abiotic stress tolerances, and TaASR1-D enhances abiotic stress tolerances by reinforcing antioxidant capacity and ABA signalling.

Stable and efficient immobilization of bi-enzymatic NADPH cofactor recycling system under consecutive microwave irradiation.

One of the challenges in biocatalysis is the development of stable and efficient bi-enzymatic cascades for bio-redox reactions coupled to the recycling of soluble cofactors. Aldo-keto reductase (LEK) and glucose dehydrogenase (GDH) can be utilized as the NADPH recycling system for economic and efficient biocatalysis of (R)-4-chloro-3-hydroxybutanoate ((R)-CHBE), an important chiral pharmaceutical intermediate. The LEK and GDH was efficiently co-immobilized in mesocellular siliceous foams (MCFs) under microwave irradiation (CoLG-MIA). while they were also co-immobilized by entrapment in calcium alginate without MIA as control (CoLG-CA). The relative activity of CoLG-MIA was increased to 140% compared with that of free LEK. The CoLG-MIA exhibited a wider range of pH and temperature stabilities compared with other preparations. The thermal, storage and batch operational stabilities of microwave-assisted immobilized LEK-GDH were also improved. The NADPH recycling system exhibited the potential as the stable and efficient catalyst for the industrial preparation of (R)-CHBE.

Genome-wide identification of R2R3-MYB family in wheat and functional characteristics of the abiotic stress responsive gene TaMYB344.

BACKGROUND: MYB superfamily is one of the most abundant families in plants, which plays important roles in plant growth, development, and productivity. However, to date, researches on MYBs in wheat (Triticum aestivum L.) are scattered mostly, not comprehensive. RESULTS: In this study, a total of 393 R2R3-MYBs and 12 R1R2R3-MYBs were identified and analyzed including gene structure, chromosomal distribution, synteny relationship, and evolutionary relationship. Then, 29 clusters tandem duplication and 8 clusters segmental duplication genes were discovered. The expression profile of the identified genes under abiotic and biotic stress was analyzed using RNA-seq data. Based on expression patterns analysis, we screened many candidate genes involved in plant response to abiotic and biotic stress. Among them, the functional characteristics of TaMYB344 were further studied. TaMYB344 was localized in the nucleus and functioned as a weak transcriptional activator. We demonstrated that TaMYB344-overexpressing transgenic tobacco plants had enhanced tolerance to drought, heat, and high salt stress. CONCLUSIONS: In this study, 393 R2R3-MYBs and 12 R1R2R3-MYBs in wheat were systemically identified and analyzed. Differential expression analysis indicated that many R2R3-MYBs were involved in abiotic and biotic stress response. We identified a potential candidate gene TaMYB344, overexpression of which in tobacco plants enhanced drought, heat, and salt stress tolerance. These results will provide abundant molecular data for breeding new varieties of wheat in the future.

[Advances in synthesis of artemisinin based on plant genetic engineering].

Artemisinin is a kind of sesquiterpene lactone containing endoperoxide bridge,which is the most effective anti-malarial drug at present. However,low content of artemisinin in Artemisia annua,ranging from 0. 1%-1. 0% of dry weight,as well as the complicated extraction process have resulted in low yield and high cost of artemisinin,making it difficult to meet market demand.Based on the development of high-throughput sequencing and molecular biology,the related enzyme genes and transcription factors involved in the artemisinin metabolic pathway were cloned and identified. Metabolic engineering and synthetic biology methods to modify the original metabolic pathway of A. annua and genetic engineering in heterologous host cells have become one of the hotspots in this field. Therefore,the molecular mechanism of artemisin biosynthesis,different strategies of genetic modifications of A. annua,and the research status and application prospect of artemisinin synthesis in heterologous host cells( Nicotiana benthamiana,Physcomitrella patens) were summarized in our review,hoping to provide molecular basis and theoretical basis for breeding new varieties of A. annua with high artemisinin output.

Expression of TaGF14b, a 14-3-3 adaptor protein gene from wheat, enhances drought and salt tolerance in transgenic tobacco.

MAIN CONCLUSION: TaGF14b enhances tolerance to multiple stresses through ABA signaling pathway by altering physiological and biochemical processes, including ROS-scavenging system, stomatal closure, compatible osmolytes, and stress-related gene expressions in tobaccos. The 14-3-3 proteins are involved in plant growth, development, and in responding to abiotic stresses. However, the precise functions of 14-3-3s in responding to drought and salt stresses remained unclear, especially in wheat. In this study, a 14-3-3 gene from wheat, designated TaGF14b, was cloned and characterized. TaGF14b was upregulated by polyethylene glycol 6000, sodium chloride, hydrogen peroxide, and abscisic acid (ABA) treatments. Ectopic expression of TaGF14b in tobacco conferred enhanced tolerance to drought and salt stresses. Transgenic tobaccos had longer root, better growth status, and higher relative water content, survival rate, photosynthetic rate, and water use efficiency than control plants under drought and salt stresses. The contribution of TaGF14b to drought and salt tolerance relies on the regulations of ABA biosynthesis and ABA signaling, as well as stomatal closure and stress-related gene expressions. Moreover, TaGF14b expression could significantly enhance the reactive oxygen species (ROS) scavenging system to ameliorate oxidative damage to cells. In addition, TaGF14b increased tolerance to osmotic stress evoked by drought and salinity through modifying water conservation and compatible osmolytes in plants. In conclusion, TaGF14b enhances tolerance to multiple abiotic stresses through the ABA signaling pathway in transgenic tobaccos by altering physiological and biochemical processes.

Ectopic expression of BdCIPK31 confers enhanced low-temperature tolerance in transgenic tobacco plants.

Calcineurin B-like protein (CBL), the Ca2+ sensor, and its interacting protein kinases (CIPKs) play essential roles in plants' response to stress. However, few studies have focused on the functions of CIPKs in low-temperature response. In the present study, BdCIPK31, a cold-responsive CIPK in Brachypodium distachyon, was found to participate in low-temperature response. Ectopic expression of BdCIPK31 conferred cold tolerance in transgenic tobaccos. Further analyses indicated that expression of BdCIPK31 improved ROS detoxication and omsoprotectant biosynthesis in transgenic plants under low-temperature treatment, suggesting that the BdCIPK31 functions positively in plant adaption to the cold-induced oxidative and osmotic stresses. Moreover, BdCIPK31 could upregulate the expressions of some representative stress-related genes under cold stress. In conclusion, these findings suggest that BdCIPK31 functions positively in plant cold response.

A wheat MYB transcriptional repressor TaMyb1D regulates phenylpropanoid metabolism and enhances tolerance to drought and oxidative stresses in transgenic tobacco plants.

MYB transcription factors are involved in the regulation of plant development and response to biotic and abiotic stress. In this study, TaMyb1D, a novel subgroup 4 gene of the R2R3-MYB subfamily, was cloned from wheat (Triticum aestivum L.). TaMyb1D was localized in the nucleus and functioned as a transcriptional repressor. The overexpression of TaMyb1D in tobacco (Nicotiana tabacum) plants repressed the expression of genes related to phenylpropanoid metabolism and down-regulated the accumulation of lignin in stems and flavonoids in leaves. These changes affected plant development under normal conditions. The expression of TaMyb1D was ubiquitous and up-regulated by PEG6000 and H2O2 treatments in wheat. TaMyb1D-overexpressing transgenic tobacco plants exhibited higher relative water content and lower water loss rate during drought stress, as well as higher chlorophyll content in leaves during oxidative stress. The transgenic plants showed a lower leakage of ions as well as reduced malondialdehyde and H2O2 levels during conditions of drought and oxidative stresses. In addition, TaMyb1D up-regulated the expression levels of ROS- and stress-related genes in response to drought stress. Therefore, the overexpression of TaMyb1D enhanced tolerance to drought and oxidative stresses in tobacco plants. Our study demonstrates that TaMyb1D functions as a negative regulator of phenylpropanoid metabolism and a positive regulator of plant tolerance to drought and oxidative stresses.

A Wheat R2R3-type MYB Transcription Factor TaODORANT1 Positively Regulates Drought and Salt Stress Responses in Transgenic Tobacco Plants.

MYB transcription factors play important roles in plant responses to biotic and abiotic stress. In this study, TaODORANT1, a R2R3-MYB gene, was cloned from wheat (Triticum aestivum L.). TaODORANT1 was localized in the nucleus and functioned as a transcriptional activator. TaODORANT1 was up-regulated in wheat under PEG6000, NaCl, ABA, and H2O2 treatments. TaODORANT1-overexpressing transgenic tobacco plants exhibited higher relative water content and lower water loss rate under drought stress, as well as lower Na+ accumulation in leaves under salt stress. The transgenic plants showed higher CAT activity but lower ion leakage, H2O2 and malondialdehyde contents under drought and salt stresses. Besides, the transgenic plants also exhibited higher SOD activity under drought stress. Our results also revealed that TaODORANT1 overexpression up-regulated the expression of several ROS- and stress-related genes in response to both drought and salt stresses, thus enhancing transgenic tobacco plants tolerance. Our studies demonstrate that TaODORANT1 positively regulates plant tolerance to drought and salt stresses.

Functional Characterization of TaFUSCA3, a B3-Superfamily Transcription Factor Gene in the Wheat.

The end-use quality of wheat, including its unique rheology and viscoelastic properties, is predominantly determined by the composition and concentration of gluten proteins. While, the mechanism regulating expression of the seed storage protein (SSP) genes and other related genes in wheat remains unclear. In this study, we report on the cloning and functional identification of TaFUSCA3, a B3-superfamily transcription factor (TF) gene in wheat. Sequence alignment indicated that wheat and barley FUSCA3 genes are highly conserved. Quantitative reverse-transcription (qRT)-PCR analysis showed that the transcript of TaFUSCA3 was accumulated mostly in the stamens and the endosperms of immature wheat seeds. Yeast-one-hybrid results proved that the full-length TaFUSCA3 and its C-terminal region had transcriptional activities. Yeast-two-hybrid and bimolecular fluorescence complementation assays indicated that TaFUSCA3 could activate the expression of the high molecular weight glutenin subunit gene Glu-1Bx7 and interact with the seed-specific bZIP protein TaSPA. DNA-protein-interaction enzyme-linked immunosorbent assay demonstrated that TaFUSCA3 specifically recognizes the RY-box of the Glu-1Bx7 promoter region. Transient expression results showed that TaFUSCA3 could trans-activate the Glu-1Bx7 promoter, which contains eight RY-box sequences. TaFUSCA3 was unable to activate the downstream transcription when the RY-box was fully mutated. TaFUSCA3 could activate the transcription of the At2S3 gene promoter in a complementation of loss-of-function experiment using the Arabidopsis thaliana line fus3-3, which is a FUSCA3 mutant, demonstrating the evolutionary conservation of the TaFUSCA3 gene. In conclusion, the wheat B3-type TF, TaFUSCA3, is functional conserved between monocot and dicot, and could regulate SSP gene expression by interacting specifically with TaSPA.

BdCIPK31, a Calcineurin B-Like Protein-Interacting Protein Kinase, Regulates Plant Response to Drought and Salt Stress.

Calcineurin B-like protein interacting protein kinases (CIPKs) are vital elements in plant abiotic stress signaling pathways. However, the functional mechanism of CIPKs has not been understood clearly, especially in Brachypodium distachyon, a new monocot model plant. In this study, BdCIPK31, a CIPK gene from B. distachyon was characterized. BdCIPK31 was downregulated by polyethylene glycol, NaCl, H2O2, and abscisic acid (ABA) treatments. Transgenic tobacco plants overexpressing BdCIPK31 presented improved drought and salt tolerance, and displayed hypersensitive response to exogenous ABA. Further investigations revealed that BdCIPK31 functioned positively in ABA-mediated stomatal closure, and transgenic tobacco exhibited reduced water loss under dehydration conditions compared with the controls. BdCIPK31 also affected Na+/K+ homeostasis and root K+ loss, which contributed to maintain intracellular ion homeostasis under salt conditions. Moreover, the reactive oxygen species scavenging system and osmolyte accumulation were enhanced by BdCIPK31 overexpression, which were conducive for alleviating oxidative and osmotic damages. Additionally, overexpression of BdCIPK31 could elevate several stress-associated gene expressions under stress conditions. In conclusion, BdCIPK31 functions positively to drought and salt stress through ABA signaling pathway. Overexpressing BdCIPK31 functions in stomatal closure, ion homeostasis, ROS scavenging, osmolyte biosynthesis, and transcriptional regulation of stress-related genes.

Brachypodium distachyon BdPP2CA6 Interacts with BdPYLs and BdSnRK2 and Positively Regulates Salt Tolerance in Transgenic Arabidopsis.

The phytohormone abscisic acid (ABA) is essential in plant responding to biotic and abiotic stresses. Although ABA signaling model is well established in Arabidopsis, ABA receptor PYL family and clade A PP2C subfamily are not yet characterized in monocot model plant Brachypodium distachyon. In this study, we identified 12 PYLs and 8 clade A PP2Cs from B. distachyon genome and successfully cloned 12 PYLs and 7 clade A PP2Cs. Bioinformatic and expression analyses showed that most of the identified genes respond to several signal molecules and abiotic stresses. Protein-protein interaction analysis revealed that many BdPYLs and BdPP2CAs participate in the classic ABA-PYL-PP2C-SnRK2 signaling pathway. A clade A PP2C, designated BdPP2CA6, interacted with BdPYL11 in the absence of ABA and localized in nucleus. Most clade A PP2C members from Arabidopsis showed negatively regulation in ABA signaling pathway, whereas BdPP2CA6-overexpression transgenic Arabidopsis showed ABA hypersensitive phenotype, resulting in enhanced stomatal closure and salinity tolerance. Our results indicate that BdPP2CA6 positively regulates ABA and stress signal pathway in transgenic Arabidopsis plant seedlings.

A Member of the 14-3-3 Gene Family in Brachypodium distachyon, BdGF14d, Confers Salt Tolerance in Transgenic Tobacco Plants.

Plant 14-3-3 proteins are involved in diverse biological processes, but for the model monocotyledonous species, Brachypodium distachyon, their roles in abiotic stress tolerance are not well understood. In this study, a total of eight Bd14-3-3 genes were identified from B. distachyon and these were designated respectively as BdGF14a-BdGF14g. The qRT-PCR analyses of 3-month-old plants of B. distachyon showed that these genes were all expressed in the stems, leaves, and spikelets. By contrast, most of the plants had relatively lower transcriptional levels in their roots, except for the BdGF14g gene. The different expression profiles of the Bd14-3-3s under various stress treatments, and the diverse interaction patterns between Bd14-3-3s and BdAREB/ABFs, suggested that these gene products probably had a range of functions in the stress responses. The NaCl-induced Bd14-3-3 gene, BdGF14d, was selected for overexpression in tobacco. BdGF14d was found to be localized throughout the cell and it conferred enhanced tolerance to salt in the transgenic plants. Lowered contents of malondialdehyde, H2O2, and Na+, and lower relative electronic conductance (Rec%), yet greater activities of catalase and peroxidase, were observed in the overexpressing plants. Higher photosynthetic rate, transpiration rate, stomatal conductance, and water use efficiency were measured in the transgenic lines. Following abscisic acid (ABA) or NaCl treatment, stomatal aperture in leaves of the BdGF14d-overexpression plants was significantly lower than in leaves of the wild type (WT) controls. The stress-related marker genes involved in the ABA signaling pathway, the reactive oxygen species (ROS)-scavenging system, and the ion transporters were all up-regulated in the BdGF14d-overexpressing plants as compared with WT. Taken together, these results demonstrate that the Bd14-3-3 genes play important roles in abiotic stress tolerance. The ABA signaling pathway, the ROS-scavenging system, and ion transporters were all involved in enhancing the tolerance to salt stress in the BdGF14d-overexpression plants.