Integrated transcriptome and miRNA analysis uncovers molecular regulators of aerial stem-to-rhizome transition in the medical herb Gynostemma pentaphyllum.

BACKGROUND: Gynostemma pentaphyllum is an important perennial medicinal herb belonging to the family Cucurbitaceae. Aerial stem-to-rhizome transition before entering the winter is an adaptive regenerative strategy in G. pentaphyllum that enables it to survive during winter. However, the molecular regulation of aerial stem-to-rhizome transition is unknown in plants. Here, integrated transcriptome and miRNA analysis was conducted to investigate the regulatory network of stem-to-rhizome transition. RESULTS: Nine transcriptome libraries prepared from stem/rhizome samples collected at three stages of developmental stem-to-rhizome transition were sequenced and a total of 5428 differentially expressed genes (DEGs) were identified. DEGs associated with gravitropism, cell wall biosynthesis, photoperiod, hormone signaling, and carbohydrate metabolism were found to regulate stem-to-rhizome transition. Nine small RNA libraries were parallelly sequenced, and seven significantly differentially expressed miRNAs (DEMs) were identified, including four known and three novel miRNAs. The seven DEMs targeted 123 mRNAs, and six pairs of miRNA-target showed significantly opposite expression trends. The GpmiR166b-GpECH2 module involved in stem-to-rhizome transition probably promotes cell expansion by IBA-to-IAA conversion, and the GpmiR166e-GpSGT-like module probably protects IAA from degradation, thereby promoting rhizome formation. GpmiR156a was found to be involved in stem-to-rhizome transition by inhibiting the expression of GpSPL13A/GpSPL6, which are believed to negatively regulate vegetative phase transition. GpmiR156a and a novel miRNA Co.47071 co-repressed the expression of growth inhibitor GpRAV-like during stem-to-rhizome transition. These miRNAs and their targets were first reported to be involved in the formation of rhizomes. In this study, the expression patterns of DEGs, DEMs and their targets were further validated by quantitative real-time PCR, supporting the reliability of sequencing data. CONCLUSIONS: Our study revealed a comprehensive molecular network regulating the transition of aerial stem to rhizome in G. pentaphyllum. These results broaden our understanding of developmental phase transitions in plants.

Molecular characterization, expression, and regulation of Gynostemma pentaphyllum squalene epoxidase gene 1.

Gynostemma pentaphyllum (Thunb.) Makino is a perennial medicinal herb widely distributed in China. This herb contains important medicinal components called gypenosides, which belong to dammarane-type triterpenoid saponins. Squalene epoxidase (SE, EC 1.14.99.7) catalyzes the epoxidation of squalene to form oxidosqualene and is a key regulatory enzyme in triterpenoid saponin biosynthesis. In this study, a SE gene designated as GpSE1 was isolated from G. pentaphyllum leaves. The deduced protein sequence of GpSE1 contained two conserved domains involved in the catalytic function of SE. GpSE1 was expressed as inclusion bodies in Escherichia coli cells, and the HIS-tagged recombinant protein was successfully purified and renatured in vitro. Immunofluorescence indicated that the polygonal reticular fluorescence signal of GpSE1 was significantly stronger in young leaves than in mature leaves and rhizomes. This finding is consistent with the tissue-specific expression pattern of GpSE1 and suggests that the young leaves of G. pentaphyllum mainly serve as the active site of gypenoside synthesis. Methyl jasmonate (MeJA) treatment upregulated GpSE1 expression in both the young and mature leaves of G. pentaphyllum, with greater upregulation in young leaves than in mature leaves. However, the expression of GpSE1 was not enhanced continually with the increase in MeJA concentration. Moreover, the GpSE1 expression was maximally regulated in response to 50 µM MeJA but not to 100 µM MeJA. This result indicates that MeJA exerts a concentration-dependent effect on GpSE1 expression.

Molecular cloning and transgenic characterization of the genes encoding chalcone synthase and chalcone isomerase from the Tibetan herbal plant Mirabilis himalaica.

Mirabilis himalaica is an endangered medicinal plant species in the Tibetan Plateau. The two genes respectively encoding chalcone synthase (MhCHS) and chalcone isomerase (MhCHI) were isolated and characterized from M. himalaica. The sequence analysis revealed that the two genes were similar with their corresponding homologous genes in other plants. The tissue profiles showed that both MhCHS and MhCHI had higher expression levels in roots than in stems and leaves. Transgenic hairy root cultures respectively with overexpressing MhCHS and MhCHI were established. The genomic PCR detection confirmed the authority of transgenic hairy root lines, in which either MhCHS or MhCHI expression levels were much higher than that in non-transgenic hairy root line. Finally, the HPLC detection results demonstrated that the rotenoid contents in MhCHS/MhCHI-transformed hairy root lines were enhanced. This study provided two candidate genes that could be used to genetic engineering rotenoid biosynthesis in M. himalaica and an alternative method to produce rotenoid using transgenic hairy root cultures.

Identification and expression of a stearoyl-ACP desaturase gene responsible for oleic acid accumulation in Xanthoceras sorbifolia seeds.

Xanthoceras sorbifolia Bunge is an oilseed tree that grows well on barren lands in dry climate. Its seeds contain a large amount of oil rich in oleic acid (18:1(Δ9)) and linoleic acid (18:2(Δ9, 12)). However, the molecular regulation of oil biosynthesis in X. sorbifolia seeds is poorly understood. Stearoyl-ACP desaturase (SAD, EC 1.14.99.6) is a plastid-localized soluble desaturase that catalyzes the conversion of stearic acid (18:0) to oleic acid, which plays a key role in determining the ratio of saturated to unsaturated fatty acids. In this study, a full-length cDNA of XsSAD was isolated from developing X. sorbifolia embryos. The XsSAD open reading frame had 1194-bp, encoding a polypeptide of 397 amino acids. XsSAD expression in Escherichia coli cells resulted in increased 18:1(Δ9) level, confirming the biological activity of the enzyme encoded by XsSAD. XsSAD expression in Arabidopsis ssi2 mutants partially restored the morphological phenotype and effectively increased the 18:1(Δ9) level. The levels of other unsaturated fatty acids synthesized with 18:1(Δ9) as the substrate also increased to some degree. XsSAD in X. sorbifolia had a much higher expression in embryos than in leaves and petals. XsSAD expression also correlated well with the oleic acid, unsaturated fatty acid, and total fatty acid levels in developing embryos. These data suggested that XsSAD determined the synthesis of oleic acid and contributed to the accumulation of unsaturated fatty acid and total oil in X. sorbifolia seeds. A preliminary tobacco rattle virus-based virus-induced gene silencing system established in X. sorbifolia can also be helpful for further analyzing the functions of XsSAD and other oil synthesis-related genes in woody plants.

XsFAD2 gene encodes the enzyme responsible for the high linoleic acid content in oil accumulated in Xanthoceras sorbifolia seeds.

BACKGROUND: Xanthoceras sorbifolia Bunge is a valuable oilseed tree that has linoleic acid-rich seed oil. Microsomal oleate desaturase (FAD2; EC 1.3.1.35) is responsible for the conversion of oleic acid to linoleic acid during fatty acid synthesis. In this study, XsFAD2 was cloned from developing embryos of X. sorbifolia. RESULTS: XsFAD2 contained three histidine boxes, a C-terminal endoplasmic reticulum retrieval motif, and five putative transmembrane domains representing the characteristics of membrane-bound fatty acid desaturase. XsFAD2 expression in yeast cells resulted in linoleic acid (18:2) and palmitolinoleic acid (16:2) production, confirming the biological activity of the enzyme encoded by XsFAD2. These fatty acids are not normally present in wild-type yeast. Phylogenetic analysis indicated that XsFAD2 is located in a subgroup of FAD2 enzymes specifically or highly expressed in developing seeds. The expression level of XsFAD2 in seeds was much higher than those in leaves and petals. Furthermore, XsFAD2 expression pattern correlated well with linoleic acid accumulated in seeds. CONCLUSION: Results suggested that XsFAD2 is responsible for the high linoleic acid content in X. sorbifolia seed oil. This study provides insight on the regulation mechanism of fatty acid synthesis in X. sorbifolia seeds and a valuable gene for improving the oil quality in oilseed trees.

Expression profiling and functional characterization of a DREB2-type gene from Populus euphratica.

A novel DREB (dehydration responsive element binding) gene, designated PeDREB2, was isolated from the desert-grown tree, Populus euphratica. Based on multiple sequence alignment and phylogenetic characterization, PeDREB2 was classified as an A-2 group member of the DREB family. Expression of PeDREB2 was induced by cold, drought, and high salinity, but not by abscisic acid (ABA) treatment. PeDREB2 could bind specifically to DRE elements and was targeted to the nucleus when transiently expressed in onion epidermis cells. 35S promoter-driven expression of PeDREB2 improved salt tolerance in transgenic tobacco and did not cause growth retardation. The results indicate that PeDREB2 functions as a novel transcription factor involved in the response of salt stress and might be useful in improving salt tolerance in transgenic plants.

[Cloning and structure analysis of zinc finger protein gene in Populus euphratica Oliv].

Zinc finger proteins belong to a family of nuclear transcription factors which function is to regulate gene expression in both prokaryotic and eukaryotic cells. A pair of primers was designed after analyzing the conservation of salt-tolerant zinc protein Alfin-1 in such diverse plants as alfalfa and Arabidopsis. The zinc finger protein gene is isolated from total RNA with RT-PCR in aquaculture leaves of Populus euphratica . Its full cDNA length is 924bp. Analysis of its amino acid sequence showed it has a typical Cys(2)/His(2) zinc finger structure and a G-rich promoter binding site GTGGGG, starting from position 556. Since transcrptional factors which have the same function show conservation in structure and amino acid sequence of DNA binding region, the structure analysis in this paper indicates the cloned zinc finger protein gene may have functional correlation to Alfin-1.