The Smi-miR858a-SmMYB module regulates tanshinone and phenolic acid biosynthesis in Salvia miltiorrhiza.

Tanshinones and phenolic acids are two major classes of bioactive compounds in Salvia miltiorrhiza. Revealing the regulatory mechanism of their biosynthesis is crucial for quality improvement of S. miltiorrhiza medicinal materials. Here we demonstrated that Smi-miR858a-Smi-miR858c, a miRNA family previously known to regulate flavonoid biosynthesis, also played critical regulatory roles in tanshinone and phenolic acid biosynthesis in S. miltiorrhiza. Overexpression of Smi-miR858a in S. miltiorrhiza plants caused significant growth retardation and tanshinone and phenolic acid reduction. Computational prediction and degradome and RNA-seq analyses revealed that Smi-miR858a could directly cleave the transcripts of SmMYB6, SmMYB97, SmMYB111, and SmMYB112. Yeast one-hybrid and transient transcriptional activity assays showed that Smi-miR858a-regulated SmMYBs, such as SmMYB6 and SmMYB112, could activate the expression of SmPAL1 and SmTAT1 involved in phenolic acid biosynthesis and SmCPS1 and SmKSL1 associated with tanshinone biosynthesis. In addition to directly activating the genes involved in bioactive compound biosynthesis pathways, SmMYB6, SmMYB97, and SmMYB112 could also activate SmAOC2, SmAOS4, and SmJMT2 involved in the biosynthesis of methyl jasmonate, a significant elicitor of plant secondary metabolism. The results suggest the existence of dual signaling pathways for the regulation of Smi-miR858a in bioactive compound biosynthesis in S. miltiorrhiza.

Aquaporin AtTIP5;1 as an essential target of gibberellins promotes hypocotyl cell elongation in Arabidopsis thaliana under excess boron stress.

Aquaporins play essential roles in growth and development including stem elongation in plants. Tonoplast aquaporin AtTIP5;1 has been proposed to positively regulate hypocotyl elongation under high concentrations of boron (high-B) in Arabidopsis thaliana (L.) Heynh. However, the mechanism underlying this process remains unanswered. Here, we show that paclobatrazol, an inhibitor of GA biosynthesis, significantly suppressed the hypocotyl cell elongation of wild-type (WT) seedlings, and more strongly suppressed that of AtTIP5;1 overexpressors under high-B stress. Two AtTIP5;1 null mutants displayed arrested elongation of cells in the upper part of hypocotyls compared with the WT in the presence of high-B or GA3. Moreover, paclobatrazol treatment completely inhibited the increases in AtTIP5;1 transcripts induced by high-B, whereas GA3 application upregulated AtTIP5;1 expression in the WT. In addition, treatment with high-B remarkably elevated the expression levels of GA3ox1, GA20ox1 and GA20ox2 - key biosynthesis genes of GAs - in WT seedlings. The GA3 and GA4 content also increased in WT seedlings grown in MS medium containing high-B. Additionally, application of high-B failed to enhance AtTIP5;1 expression in the double mutant rga-24-gai-t6 of DELLA genes. Together, these results suggest that AtTIP5;1 is an essential downstream target of GAs. High-B induces the accumulation of GAs, which activates AtTIP5;1 through modulation of the DELLA proteins Repressor of ga1-3 and GA-insensitive, further promoting hypocotyl elongation in A. thaliana.

ARGONAUTE Genes in Salvia miltiorrhiza: Identification, Characterization, and Genetic Transformation.

Small RNA-mediated gene silencing is a vital regulatory mechanism in eukaryotes that requires ARGONAUTE (AGO) proteins. Salvia miltiorrhiza is a well-known traditional Chinese medicinal plant. Therefore, it is important to characterize S. miltiorrhiza AGO family genes as they may be involved in multiple metabolic pathways. This chapter introduces the detailed protocol for SmAGO gene prediction and molecular cloning. In addition, an Agrobacterium-mediated genetic transformation method for S. miltiorrhiza is presented. These methodologies can be used to functionally study SmAGO genes as well as other genes of interest in S. miltiorrhiza.

Overexpression of the tonoplast aquaporin AtTIP5;1 conferred tolerance to boron toxicity in Arabidopsis.

Boron (B) toxicity to plants is responsible for low crop productivity in many regions of the world. Here we report a novel and effective means to alleviate the B toxicity to plants under high B circumstance. Functional characterization of AtTIP5;1, an aquaporin gene, revealed that overexpression of AtTIP5;1 (OxAtTIP5;1) in Arabidopsis significantly increased its tolerance to high B toxicity. Compared to wild-type plants, OxAtTIP5;1 plants exhibited longer hypocotyls, accelerated development, increased silique production under high B treatments. GUS staining and quantitative RT-PCR (qRT-PCR) results demonstrated that the expression of AtTIP5;1 was induced by high B concentration treatment. Subcellular localization analysis revealed that the AtTIP5;1-GFP fusion protein was localized on the tonoplast membrane, which was consistent with the prediction based on bioinformatics. Taken together, our results suggest that AtTIP5;1 is involved in B transport pathway possibly via vacuolar compartmentation for B, and that overexpression of AtTIP5;1 in plants may provide an effective way to overcome the problem resulting from high B concentration toxicity.

Removal of the selectable marker gene from transgenic tobacco plants by expression of Cre recombinase from a tobacco mosaic virus vector through agroinfection.

Selection markers are often indispensable during the process of plant transformation, but dispensable once transgenic plants have been established. The Cre/lox site-specific recombination system has been employed to eliminate selectable marker genes from transgenic plants. Here we describe the use of a movement function-improved Tobacco Mosaic Virus (TMV) vector, m30B, to express Cre recombinase for elimination of the selectable marker gene nptII from transgenic tobacco plants. The transgenic tobacco plants were produced by Agrobacterium-mediated transformation with a specially designed binary vector pGNG which contained in its T-DNA region a sequence complex of 35S promoter-lox-the gfp coding sequence-rbcS terminator-Nos promoter-nptII-Nos terminator-lox-the gus coding region-Nos terminator. The expression of the recombinant viral vector m30B:Cre in plant cells was achieved by placing the viral vector under the control of the 35S promoter and through agroinoculation. After co-cultivating the pGNG-leaf discs with agro35S-m30B:Cre followed by shoot regeneration without any selection, plants devoid of the lox-flanked sequences including nptII were obtained with an efficiency of about 34% as revealed by histochemical GUS assay of the regenerants. Three of 11 GUS expressing regenerants, derived from two independent transgenic lines containing single copy of the pGNG T-DNA, proved to be free of the lox-flanked sequences by Southern blot analysis. Excision of the lox-flanked sequences in the three plants could be attributed to transient expression of Cre from the viral vector at the early stage of co-cultivation, since the cre sequence could not be detected in the viral RNA molecules accumulated in the plants, nor in their genomic DNA. The parental marker-free genotype was inherited in their selfed progeny, and all of the progeny were virus-free, apparently because TMV is not seed-transmissible. Therefore, expression of Cre from a TMV-based vector could be used to eliminate selectable marker genes from transgenic tobacco plants without sexual crossing and segregation, and this strategy could be extended to other TMV-infected plant species and applicable to other compatible virus-host plant systems.

[A simple and visible assay for cre recombinase activity in Escherichia coli by using two incompatible plasmids].

The Cre/loxP system derived from bacteriophage P1 is widely used to carry out complex manipulations of DNA molecules both in vitro and in vivo. In order to further characterize and modify the Cre/loxP system, a convenient method for assaying the recombination efficiency is needed. A simple and visible assay is described, in which two incompatible plasmids, separately carrying the cre gene and loxP-flanked gfp gene, were co-transferred into E. coli. The cre gene was inserted into a kanamycin-resistant bacterial expression vector, designated pET30a-Cre. The gfp gene, flanked by directly repeated loxP sites, was cloned into an ampicillin-resistant expression vector to generate pET23b-loxGFP. E. coli BL21 (DE3) was cotransformed with pET30a-Cre and pET23b-loxGFP, and cultured in the presence of both ampicillin and kanamycin. Under UV illumination, the Cre-mediated recombination events can be easily detected. The fidelity of recombination was verified by SDS-PAGE analysis and restriction analysis followed by DNA sequencing. Thus, this cotransformation method provides a straightforward assay that can be used to modify the Cre/loxP system.

[Using green fluorescent protein as a reporter to monitor elimination of selectable marker genes from transgenic plants].

In genetic modification of plants, once the transformants are obtained, selection markers are no longer required in mature plants. At present, the Cre/lox site-specific recombination system is most widely used to eliminate the selectable marker genes from the transgenic plants. In this study, attempt was made to favour the selection of marker-free plants in the re-transformation method. Green fluorescent protein (GFP) can be directly visualized in living cells, tissues or organisms under UV illumination. This advantage of GFP is exploited in the development of a practical approach in which GFP is used as a visual marker to monitor the removal of the selectable marker gene from transgenic plants. For that purpose, the pGNG binary vector was constructed, in which the GFP gene (gfp) was linked to the expression cassette Nos P-nptII-NosT and the two units were cloned between two directly-orientated lox sites. The CaMV 35S promoter was placed before the first lox site and used to drive GFP expression. The beta-glucuronidase gene (gus) of Escherichia coli was cloned behind the second lox site without a promoter, thus would not be expressed in this position. Tobacco plants were first transformed with pGNG and selected on kanamycin (Kan)-containing media. Regenerated transgenic shoots were readily singled out by GFP fluorescence. The GFP-expressing plants were then re-transformed with pCambia1300-Cre containing hygromycin phosphotransferase gene (hpt) as a selectable marker gene. The Cre-mediated recombination resulted in the elimination of lox-flanked genes, herein gfp and nptII, from the plant genome and brought the GUS gene next to the 35S promoter. Our data demonstrated that transgenic plants free of nptII were easily selected by monitoring the loss of green fluorescence, and at the same time, GUS (here as a target protein) was expressed in the nptII-free plants. Finally, hpt and cre were removed from the progenies of the nptII-free plants by gene segregation.