Promoting Photosynthetic Production of Dammarenediol-II in Chlamydomonas reinhardtii via Gene Loading and Culture Optimization.

Ginsenosides are major bioactive compounds found in Panax ginseng that exhibit various pharmaceutical properties. Dammarenediol-II, the nucleus of dammarane-type ginsenosides, is a promising candidate for pharmacologically active triterpenes. Dammarenediol-II synthase (DDS) cyclizes 2,3-oxidosqualene to produce dammarenediol-II. Based on the native terpenoids synthetic pathway, a dammarane-type ginsenosides synthetic pathway was established in Chlamydomonas reinhardtii by introducing P. ginseng PgDDS, CYP450 enzyme (PgCYP716A47), or/and Arabidopsis thaliana NADPH-cytochrome P450 reductase gene (AtCPR), which is responsible for producing dammarane-type ginsenosides. To enhance productivity, strategies such as "gene loading" and "culture optimizing" were employed. Multiple copies of transgene expression cassettes were introduced into the genome to increase the expression of the key rate-limiting enzyme gene, PgDDS, significantly improving the titer of dammarenediol-II to approximately 0.2 mg/L. Following the culture optimization in an opt2 medium supplemented with 1.5 mM methyl jasmonate under a light:dark regimen, the titer of dammarenediol-II increased more than 13-fold to approximately 2.6 mg/L. The C. reinhardtii strains engineered in this study constitute a good platform for the further production of ginsenosides in microalgae.

Metabolic Engineering of the Isopentenol Utilization Pathway Enhanced the Production of Terpenoids in Chlamydomonas reinhardtii.

Eukaryotic green microalgae show considerable promise for the sustainable light-driven biosynthesis of high-value fine chemicals, especially terpenoids because of their fast and inexpensive phototrophic growth. Here, the novel isopentenol utilization pathway (IUP) was introduced into Chlamydomonas reinhardtii to enhance the hemiterpene (isopentenyl pyrophosphate, IPP) titers. Then, diphosphate isomerase (IDI) and limonene synthase (MsLS) were further inserted for limonene production. Transgenic algae showed 8.6-fold increase in IPP compared with the wild type, and 23-fold increase in limonene production compared with a single MsLS expressing strain. Following the culture optimization, the highest limonene production reached 117 µg/L, when the strain was cultured in a opt2 medium supplemented with 10 mM isoprenol under a light: dark regimen. This demonstrates that transgenic algae expressing the IUP represent an ideal chassis for the high-value terpenoid production. The IUP will facilitate further the metabolic and enzyme engineering to enhance the terpenoid titers by significantly reducing the number of enzyme steps required for an optimal biosynthesis.

An ortholog of the MADS-box gene SEPALLATA3 regulates stamen development in the woody plant Jatropha curcas.

MAIN CONCLUSION: Overexpression of JcSEP3 causes defective stamen development in Jatropha curcas, in which brassinosteroid and gibberellin signaling pathways may be involved. SEPALLATAs (SEPs), the class E genes of the ABCE model, are required for floral organ determination. In this study, we investigated the role of the JcSEP3 gene in floral organ development in the woody plant Jatropha curcas. Transgenic Jatropha plants overexpressing JcSEP3 displayed abnormal phenotypes such as deficient anthers and pollen, as well as free stamen filaments, whereas JcSEP3-RNA interference (RNAi) transgenic plants had no obvious phenotypic changes, suggesting that JcSEP3 is redundant with other JcSEP genes in Jatropha. Moreover, we compared the transcriptomes of wild-type plants, JcSEP3-overexpressing, and JcSEP3-RNAi transgenic plants. In the JcSEP3-overexpressing transgenic plants, we discovered 25 upregulated genes involved in anther and pollen development, as well as 12 induced genes in brassinosteroid (BR) and gibberellin (GA) signaling pathways. These results suggest that JcSEP3 directly or indirectly regulates stamen development, concomitant with the regulation of BR and GA signaling pathways. Our findings help to understand the roles of SEP genes in stamen development in perennial woody plants.

Silencing of the Ortholog of DEFECTIVE IN ANTHER DEHISCENCE 1 Gene in the Woody Perennial Jatropha curcas Alters Flower and Fruit Development.

DEFECTIVE IN ANTHER DEHISCENCE 1 (DAD1), a phospholipase A1, utilizes galactolipids (18:3) to generate α-linolenic acid (ALA) in the initial step of jasmonic acid (JA) biosynthesis in Arabidopsis thaliana. In this study, we isolated the JcDAD1 gene, an ortholog of Arabidopsis DAD1 in Jatropha curcas, and found that it is mainly expressed in the stems, roots, and male flowers of Jatropha. JcDAD1-RNAi transgenic plants with low endogenous jasmonate levels in inflorescences exhibited more and larger flowers, as well as a few abortive female flowers, although anther and pollen development were normal. In addition, fruit number was increased and the seed size, weight, and oil contents were reduced in the transgenic Jatropha plants. These results indicate that JcDAD1 regulates the development of flowers and fruits through the JA biosynthesis pathway, but does not alter androecium development in Jatropha. These findings strengthen our understanding of the roles of JA and DAD1 in the regulation of floral development in woody perennial plants.

Comparative transcriptome analysis of gynoecious and monoecious inflorescences reveals regulators involved in male flower development in the woody perennial plant Jatropha curcas.

KEY MESSAGE: ABCE model genes along with genes related to GA biosynthesis and auxin signalling may play significant roles in male flower development in Jatropha curcas. Flowering plants exhibit extreme reproductive diversity. Jatropha curcas, a woody plant that is promising for biofuel production, is monoecious. Here, two gynoecious Jatropha mutants (bearing only female flowers) were used to identify key genes involved in male flower development. Using comparative transcriptome analysis, we identified 17 differentially expressed genes (DEGs) involved in floral organ development between monoecious plants and the two gynoecious mutants. Among these DEGs, five floral organ identity genes, Jatropha AGAMOUS, PISTILLATA, SEPALLATA 2-1 (JcSEP2-1), JcSEP2-2, and JcSEP3, were downregulated in ch mutant inflorescences; two gibberellin (GA) biosynthesis genes, Jatropha GA REQUIRING 1 and GIBBERELLIN 3-OXIDASE 1, were downregulated in both the ch and g mutants; and two genes involved in the auxin signalling pathway, Jatropha NGATHA1 and STYLISH1, were downregulated in the ch mutant. Furthermore, four hub genes involved in male flower development, namely Jatropha SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1, CRYPTOCHROME 2, SUPPRESSOR OF OVEREXPRESSION OF CO 1 and JAGGED, were identified using weighted gene correlation network analysis. These results suggest that floral organ identity genes and genes involved in GA biosynthesis and auxin signalling may participate in male flower development in Jatropha. This study will contribute to understanding sex differentiation in woody perennial plants.

De novo genome assembly and Hi-C analysis reveal an association between chromatin architecture alterations and sex differentiation in the woody plant Jatropha curcas.

BACKGROUND: Chromatin architecture is an essential factor regulating gene transcription in different cell types and developmental phases. However, studies on chromatin architecture in perennial woody plants and on the function of chromatin organization in sex determination have not been reported. RESULTS: Here, we produced a chromosome-scale de novo genome assembly of the woody plant Jatropha curcas with a total length of 379.5 Mb and a scaffold N50 of 30.7 Mb using Pacific Biosciences long reads combined with genome-wide chromosome conformation capture (Hi-C) technology. Based on this high-quality reference genome, we detected chromatin architecture differences between monoecious and gynoecious inflorescence buds of Jatropha. Differentially expressed genes were significantly enriched in the changed A/B compartments and topologically associated domain regions and occurred preferentially in differential contact regions between monoecious and gynoecious inflorescence buds. Twelve differentially expressed genes related to flower development or hormone synthesis displayed significantly different genomic interaction patterns in monoecious and gynoecious inflorescence buds. These results demonstrate that chromatin organization participates in the regulation of gene transcription during the process of sex differentiation in Jatropha. CONCLUSIONS: We have revealed the features of chromatin architecture in perennial woody plants and investigated the possible function of chromatin organization in Jatropha sex differentiation. These findings will facilitate understanding of the regulatory mechanisms of sex determination in higher plants.

Transcriptome analysis of two inflorescence branching mutants reveals cytokinin is an important regulator in controlling inflorescence architecture in the woody plant Jatropha curcas.

BACKGROUND: In higher plants, inflorescence architecture is an important agronomic trait directly determining seed yield. However, little information is available on the regulatory mechanism of inflorescence development in perennial woody plants. Based on two inflorescence branching mutants, we investigated the transcriptome differences in inflorescence buds between two mutants and wild-type (WT) plants by RNA-Seq to identify the genes and regulatory networks controlling inflorescence architecture in Jatropha curcas L., a perennial woody plant belonging to Euphorbiaceae. RESULTS: Two inflorescence branching mutants were identified in germplasm collection of Jatropha. The duo xiao hua (dxh) mutant has a seven-order branch inflorescence, and the gynoecy (g) mutant has a three-order branch inflorescence, while WT Jatropha has predominantly four-order branch inflorescence, occasionally the three- or five-order branch inflorescences in fields. Using weighted gene correlation network analysis (WGCNA), we identified several hub genes involved in the cytokinin metabolic pathway from modules highly associated with inflorescence phenotypes. Among them, Jatropha ADENOSINE KINASE 2 (JcADK2), ADENINE PHOSPHORIBOSYL TRANSFERASE 1 (JcAPT1), CYTOKININ OXIDASE 3 (JcCKX3), ISOPENTENYLTRANSFERASE 5 (JcIPT5), LONELY GUY 3 (JcLOG3) and JcLOG5 may participate in cytokinin metabolic pathway in Jatropha. Consistently, exogenous application of cytokinin (6-benzyladenine, 6-BA) on inflorescence buds induced high-branch inflorescence phenotype in both low-branch inflorescence mutant (g) and WT plants. These results suggested that cytokinin is an important regulator in controlling inflorescence branching in Jatropha. In addition, comparative transcriptome analysis showed that Arabidopsis homologous genes Jatropha AGAMOUS-LIKE 6 (JcAGL6), JcAGL24, FRUITFUL (JcFUL), LEAFY (JcLFY), SEPALLATAs (JcSEPs), TERMINAL FLOWER 1 (JcTFL1), and WUSCHEL-RELATED HOMEOBOX 3 (JcWOX3), were differentially expressed in inflorescence buds between dxh and g mutants and WT plants, indicating that they may participate in inflorescence development in Jatropha. The expression of JcTFL1 was downregulated, while the expression of JcLFY and JcAP1 were upregulated in inflorescences in low-branch g mutant. CONCLUSIONS: Cytokinin is an important regulator in controlling inflorescence branching in Jatropha. The regulation of inflorescence architecture by the genes involved in floral development, including TFL1, LFY and AP1, may be conservative in Jatropha and Arabidopsis. Our results provide helpful information for elucidating the regulatory mechanism of inflorescence architecture in Jatropha.

Ectopic Expression of Jatropha curcas TREHALOSE-6-PHOSPHATE PHOSPHATASE J Causes Late-Flowering and Heterostylous Phenotypes in Arabidopsis but not in Jatropha.

Trehalose-6-phosphate (T6P) phosphatase (TPP), a dephosphorylating enzyme, catalyzes the dephosphorylation of T6P, generating trehalose. In Jatropha, we found six members of the TPP family. Five of them JcTPPA, JcTPPC, JcTPPD, JcTPPG, and JcTPPJ are highly expressed in female flowers or male flowers, or both, suggesting that members of the JcTPP family may participate in flower development in Jatropha. The wide expression of JcTPPJ gene in various organs implied its versatile roles and thus was chosen for unraveling its biological functions during developmental process. We constructed an overexpression vector of JcTPPJ cDNA driven by the cauliflower mosaic virus (CaMV) 35S promoter for genetic transformation. Compared with control Arabidopsis plants, 35S:JcTPPJ transgenic Arabidopsis plants presented greater sucrose contents in their inflorescences and displayed late-flowering and heterostylous phenotypes. Exogenous application of sucrose to the inflorescence buds of wild-type Arabidopsis repressed the development of the perianth and filaments, with a phenocopy of the 35S:JcTPPJ transgenic Arabidopsis. These results suggested that the significantly increased sucrose level in the inflorescence caused (or induced) by JcTTPJ overexpression, was responsible for the formation of heterostylous flower phenotype. However, 35S:JcTPPJ transgenic Jatropha displayed no obvious phenotypic changes, implying that JcTPPJ alone may not be sufficient for regulating flower development in Jatropha. Our results are helpful for understanding the function of TPPs, which may regulate flower organ development by manipulating the sucrose status in plants.

Comparative chloroplast genomics and phylogenetics of nine Lindera species (Lauraceae).

Lindera, a core genus of the Lauraceae family, has important economic uses in eastern Asia and North America. However, its historical diversification has not been clarified. In this study, we report nine newly sequenced Lindera plastomes. The plastomes of these nine Lindera species range from 152,211 (L. nacusua) to 152,968 bp (L. metcalfiana) in length, similar to that of another Lauraceae species, Litsea glutinosa (152,618 bp). The length variation of these plastomes derived from the length variation in the loci ycf1, ycf2, ψycf1, and ndhF-ψycf1. Comparing our sequences with other available plastomes in the Lauraceae indicated that eight hypervariable loci, ihbA-trnG, ndhA, ndhF-rpl32, petA-psbJ, psbK-psbI, rps16, trnS-trnG, and ycf1, could serve as DNA barcodes for species delineation, and that the inverted repeats (IRs) showed contraction/expansion. Further phylogenetic analyses were performed using 32 complete plastomes of Lauraceae and seven barcodes from 14 additional species of Lindera and related species in the core Lauraceae. The results showed that these Lindera species grouped into two or four sub-clades, and that two Litsea species and Laurus nobilis were located in the same sub-clade as five Lindera species. These data support a close relationship between the genera Laurus, Lindera, and Litsea, and suggest that Lindera is polyphyletic.

Comparative transcriptome analysis of axillary buds in response to the shoot branching regulators gibberellin A3 and 6-benzyladenine in Jatropha curcas.

Cytokinin (CK) is the primary hormone that positively regulates axillary bud outgrowth. However, in many woody plants, such as Jatropha curcas, gibberellin (GA) also promotes shoot branching. The molecular mechanisms underlying GA and CK interaction in the regulation of bud outgrowth in Jatropha remain unclear. To determine how young axillary buds respond to GA3 and 6-benzyladenine (BA), we performed a comparative transcriptome analysis of the young axillary buds of Jatropha seedlings treated with GA3 or BA. Two hundred and fifty genes were identified to be co-regulated in response to GA3 or BA. Seven NAC family members were down-regulated after treatment with both GA3 and BA, whereas these genes were up-regulated after treatment with the shoot branching inhibitor strigolactone. The expressions of the cell cycle genes CDC6, CDC45 and GRF5 were up-regulated after treatment with both GA3 and BA, suggesting they may promote bud outgrowth via regulation of the cell cycle machinery. In the axillary buds, BA significantly increased the expression of GA biosynthesis genes JcGA20oxs and JcGA3ox1, and down-regulated the expression of GA degradation genes JcGA2oxs. Overall, the comprehensive transcriptome data set provides novel insight into the responses of young axillary buds to GA and CK.

Comparative Transcriptome Analysis between Gynoecious and Monoecious Plants Identifies Regulatory Networks Controlling Sex Determination in Jatropha curcas.

Most germplasms of the biofuel plant Jatropha curcas are monoecious. A gynoecious genotype of J. curcas was found, whose male flowers are aborted at early stage of inflorescence development. To investigate the regulatory mechanism of transition from monoecious to gynoecious plants, a comparative transcriptome analysis between gynoecious and monoecious inflorescences were performed. A total of 3,749 genes differentially expressed in two developmental stages of inflorescences were identified. Among them, 32 genes were involved in floral development, and 70 in phytohormone biosynthesis and signaling pathways. Six genes homologous to KNOTTED1-LIKE HOMEOBOX GENE 6 (KNAT6), MYC2, SHI-RELATED SEQUENCE 5 (SRS5), SHORT VEGETATIVE PHASE (SVP), TERMINAL FLOWER 1 (TFL1), and TASSELSEED2 (TS2), which control floral development, were considered as candidate regulators that may be involved in sex differentiation in J. curcas. Abscisic acid, auxin, gibberellin, and jasmonate biosynthesis were lower, whereas cytokinin biosynthesis was higher in gynoecious than that in monoecious inflorescences. Moreover, the exogenous application of gibberellic acid (GA3) promoted perianth development in male flowers and partly prevented pistil development in female flowers to generate neutral flowers in gynoecious inflorescences. The arrest of stamen primordium at early development stage probably causes the abortion of male flowers to generate gynoecious individuals. These results suggest that some floral development genes and phytohormone signaling pathways orchestrate the process of sex determination in J. curcas. Our study provides a basic framework for the regulation networks of sex determination in J. curcas and will be helpful for elucidating the evolution of the plant reproductive system.

[Application of synchronous fluorescence in identification of spilled oil at sea].

In order to screen and identify the source of spilled oils at sea, synchronous fluorescence scans combined with clustering analysis are proposed and applied to different crude oil and weathering crude oil. SFS data of deltal = 25 nm were recorded and dealt with clustering analysis. The cluster results of SFS data in the range of 300 - 500 nm show that the crude oil and the weathering oil could separate completely. And the crude oils from different sea areas, also collected at different time, clustered into different groups, respectively. The results indicate that this method could preliminarily selected, and maybe serves as an assistant method in oil spill identification.

[Studies on treatment based on differentiation of syndromes of SHA syndrome in Sha Zhang Yu Heng].

Sha Zhang Yu Heng is the earliest extant TCM classical works on SHA syndrome systematically. For the first time, it systematically summarized the basic theory and practical experiences on the treatment based on differentiation of syndromes of SHA syndrome before the Qing dynasty, and categorized it from different aspects. Based on the eight principles for the theory of treatment based on differentiation of syndromes, and the three therapeutic methods, including scraping, releasing, and medication, and treatment for all SHA syndrome, its contraindications, and nursing for its convalescence, the author of this book, Guo, laid down a good foundation for the systematic research of Sha syndrome in later generations.