PfERF106, a novel key transcription factor regulating the biosynthesis of floral terpenoids in Primula forbesii Franch.

BACKGROUND: Flowers can be a source of essential oils used in the manufacture of substances with high economic value. The ethylene response factor (ERF) gene family plays a key role in regulating secondary metabolite biosynthesis in plants. However, until now, little has been known about the involvement of ERF transcription factors (TFs) in floral terpenoid biosynthesis. RESULTS: In this study, an aromatic plant, Primula forbesii Franch., was used as research material to explore the key regulatory effects of PfERF106 on the biosynthesis of terpenoids. PfERF106, which encodes an IXb group ERF transcription factor, exhibited a consistent expression trend in the flowers of P. forbesii and was transcriptionally induced by exogenous ethylene. Transient silencing of PfERF106 in P. forbesii significantly decreased the relative contents of key floral terpenes, including (z)-ß-ocimene, sabinene, ß-pinene, γ-terpinene, linalool, eremophilene, α-ionone, and α-terpineol. In contrast, constitutive overexpression of PfERF106 in transgenic tobacco significantly increased the relative contents of key floral terpenes, including cis-3-hexen-1-ol, linalool, caryophyllene, cembrene, and sclareol. RNA sequencing of petals of PfERF106-silenced plants and empty-vector control plants revealed 52,711 expressed unigenes and 9,060 differentially expressed genes (DEGs). KEGG annotation analysis revealed that the DEGs were enriched for involvement in secondary metabolic biosynthetic pathways, including monoterpene and diterpene synthesis. Notably, 10 downregulated DEGs were determined to be the downstream target genes of PfERF106 affecting the biosynthesis of terpenoids in P. forbesii. CONCLUSION: This study characterized the key positive regulatory effects of PfERF106 on the biosynthesis of terpenoids, indicating high-quality genetic resources for aroma improvement in P. forbesii. Thus, this study advances the artificial and precise directional regulation of metabolic engineering of aromatic substances.

Transcriptome analysis reveals plasticity in gene regulation due to environmental cues in Primula sikkimensis, a high altitude plant species.

BACKGROUND: Studying plasticity in gene expression in natural systems is crucial, for predicting and managing the effects of climate change on plant species. To understand the contribution of gene expression level variations to abiotic stress compensation in a Himalaya plant (Primula sikkimensis), we carried out a transplant experiment within (Ambient), and beyond (Below Ambient and Above Ambient) the altitudinal range limit of species. We sequenced nine transcriptomes (three each from each altitudinal range condition) using Illumina sequencing technology. We compared the fitness variation of transplants among three transplant conditions. RESULTS: A large number of significantly differentially expressed genes (DEGs) between below ambient versus ambient (109) and above ambient versus ambient (85) were identified. Transcripts involved in plant growth and development were mostly up-regulated in below ambient conditions. Transcripts involved in signalling, defence, and membrane transport were mostly up-regulated in above ambient condition. Pathway analysis revealed that most of the genes involved in metabolic processes, secondary metabolism, and flavonoid biosynthesis were differentially expressed in below ambient conditions, whereas most of the genes involved in photosynthesis and plant hormone signalling were differentially expressed in above ambient conditions. In addition, we observed higher reproductive fitness in transplant individuals at below ambient condition compared to above ambient conditions; contrary to what we expect from the cold adaptive P. sikkimensis plants. CONCLUSIONS: We reveal P. sikkimensis's capacity for rapid adaptation to climate change through transcriptome variation, which may facilitate the phenotypic plasticity observed in morphological and life history traits. The genes and pathways identified provide a genetic resource for understanding the temperature stress (both the hot and cold stress) tolerance mechanism of P. sikkimensis in their natural environment.

Effect-directed analysis by high-performance thin-layer chromatography for bioactive metabolites tracking in Primula veris flower and Primula boveana leaf extracts.

The genus Primula (Primulaceae) comprises species with high medicinal as well as ornamental values. Plants belonging to this genus are well recognized for their richness in bioactive constituents. The huge variety of secondary metabolites make their complete analysis impossible. In order to cope with this challenge, effect-directed analysis (EDA) via HPTLC coupled to structure elucidation techniques was applied on Primula species for the first time. As straightforward non-target bioanalytical technique, HPTLC-UV/Vis/FLD-EDA-ESI-HRMS hyphenates three different orthogonal dimensions, i.e. chromatography with spectrometric detection, biological/enzymatic assays and HRMS. The bioactive metabolites were determined in the middle polar extracts of two Primula species, P. veris (flower) and P. boveana (leaf). The bioactivity profiling comprised the antibacterial activity against Aliivibrio fischeri and Bacillus subtilis bacterial strains and acetyl-/butyrylcholinesterase (AChE/BChE) inhibition assays. The compounds were characterized and identified via their recorded spectral data (HRMS and 1H NMR). The results showed that linoleic and linolenic acids were the principle bioactive compounds present in the studied P. veris flower extract. In the P. boveana leaf extract, flavone, 2'-methoxy-, 2'-hydroxy- and 5,6,2',6'-tetramethoxyflavone (zapotin) were determined as active metabolites. The identification of zapotin, which was previously undescribed in the investigated plant, was considered as the strength of the straightforward non-target bioanalytical technique. Flavone turned out to be the highest potent metabolite, and at the same time, a multipotent compound referring to its various bioactivities discovered. An equivalency calculation of the HPTLC-AChE inhibition by flavone was performed with reference to the well-known inhibitor rivastigmine. As a result, the amount of flavone contained in 10.0 µg dry powder of P. boveana (corresponding to 0.1 µL extract) inhibited as strong as 4.5 µg rivastigmine. In other words, the flavone contained in P. boveana leaf extract powder turned out to be half as strong as the well-known AChE inhibitor rivastigmine.

Comparative Metabolite Profiling of Triterpenoid Saponins and Flavonoids in Flower Color Mutations of Primula veris L.

Primula veris L. is an important medicinal plant with documented use for the treatment of gout, headache and migraine reaching back to the Middle Ages. Triterpenoid saponins from roots and flowers are used in up-to-date phytotherapeutic treatment of bronchitis and colds due to their expectorant and secretolytic effects. In addition to the wild type plants with yellow petals, a red variant and an intermediate orange form of Primula veris L. have recently been found in a natural habitat. The secondary metabolite profiles of roots, leaves and flowers of these rare variants were investigated and compared with the wild type metabolome. Two flavonoids, six flavonoid glycosides, four novel methylated flavonoid glycosides, five anthocyanins and three triterpenoid saponins were identified in alcoholic extracts from the petals, leaves and roots of the three variants by high performance liquid chromatography (HPLC)-diode array detection (DAD)/mass spectrometry (MSn) analyses. Anthocyanins were detected in the petals of the red and orange variety, but not in the wild type. No other effects on the metabolite profiles of the three varieties have been observed. The possibility is discussed that a regulatory step of the anthocyanin biosynthetic pathway may have been affected by mutation thus triggering color polymorphism in the petals.

Plasticity in response to phosphorus and light availability in four forest herbs.

The differential ability of forest herbs to colonize secondary forests on former agricultural land is generally attributed to different rates of dispersal. After propagule arrival, however, establishing individuals still have to cope with abiotic soil legacies from former agricultural land use. We focused on the plastic responses of forest herbs to increased phosphorus availability, as phosphorus is commonly found to be persistently bioavailable in post-agricultural forest soils. In a pot experiment performed under field conditions, we applied three P levels to four forest herbs with contrasting colonization capacities: Anemone nemorosa, Primula elatior, Circaea lutetiana and Geum urbanum. To test interactions with light availability, half of the replicas were covered with shade cloths. After two growing seasons, we measured aboveground P uptake as well as vegetative and regenerative performance. We hypothesized that fast-colonizing species respond the most opportunistically to increased P availability, and that a low light availability can mask the effects of P on performance. All species showed a significant increase in P uptake in the aboveground biomass. The addition of P had a positive effect on the vegetative performances of two of the species, although this was unrelated to their colonization capacities. The regenerative performance was affected by light availability (not by P addition) and was related to the species' phenology. Forest herbs can obviously benefit from the increased availability of P in post-agricultural forests, but not all species respond in the same way. Such differential patterns of plasticity may be important in community dynamics, as they affect the interactions among species.

Characteristics of leaf photosynthesis and simulated individual carbon budget in Primula nutans under contrasting light and temperature conditions.

Primula nutans Georgi is widely distributed in hummock-and-hollow wetlands on the Qinghai-Tibetan Plateau. To assess the ecophysiology of this species in responding to microenvironments, we examined the photosynthetic characteristics and individual carbon gain of plants growing in different microsites from a hummock-and-hollow wetland on the Qinghai-Tibetan Plateau and under laboratory conditions. Plants from wetland hummock microsites showed significantly higher light-saturated photosynthetic CO(2) uptake (A (max)) than those from microsites in hollows at a controlled temperature of 15 degrees C in leaf chamber. Leaf dark respiration rate (R) was only significantly higher in plants from hummocks than hollows at the measuring temperature of 35 degrees C. Optimum temperature for A (max) was 15 degrees C for all plants in the field despite different microsites. In plants growing under laboratory conditions differing in light and temperature, both A (max) and R were significantly higher under higher growth light (photosynthetic photon flux density, PPFD: 800 or 400 micromol m(-2) s(-1)) than low light of 90 micromol m(-2) s(-1). No statistically significant differences in A (max) and R existed in plants differing in growing temperatures. Estimates derived from the photosynthetic parameters of field plants, and microsite environmental measures including PPFD, air temperature and soil temperature showed that the optimum mean daily temperature for net daily carbon gain was around 10 degrees C and the net daily carbon gain was largely limited under lower daily total PPFD. These results suggest that the differences in A (max) and R in P. nutans are strongly affected by growing light regimes but not by temperature regimes.

Identification and characterization of pin and thrum alleles of two genes that co-segregate with the Primula S locus.

The study of heteromorphy in Primula over the past 140 years has established the reproductive significance of this breeding system. Plants produce either thrum or pin flowers that demonstrate reciprocal herkogamy. Thrums have short styles and produce large pollen from anthers at the mouth of the flower; pins have long styles and produce small pollen from anthers located within the corolla tube. The control of heteromorphy is orchestrated by the S locus with dominant (S) and recessive (s) alleles that comprise a co-adapted linkage group of genes. Thrum plants are heterozygous (Ss) and pin plants are homozygous (ss). Reciprocal crosses between the two forms are required for fertilization; within-morph crosses are impeded by a sporophytic self-incompatibility system. Rare recombination events within the S locus produce self-fertile homostyles. As a first step towards identifying genes located at the S locus, we used fluorescent differential display to screen for differential gene expression in pin and thrum flowers. Rather than only detecting differentially regulated genes, we identified two S locus linked genes by virtue of allelic variation between pin and thrum transcripts. Analysis of pin and thrum plants together with homostyle recombinant reveals that one gene flanks the locus, whereas the other shows complete linkage. One gene is related to Arabidopsis flower-timing genes Col9 and Col10; the other encodes a small predicted membrane protein of unknown function. Notwithstanding the diallelic behaviour of the Primula S locus, analysis of pin and thrum plants reveal three alleles for each gene: two pin and one thrum.

Reactive oxygen species-independent G1 arrest induced by evening primrose extract in Ehrlich ascites tumor cells.

We previously demonstrated that evening primrose extract (EPE) induced apoptosis in Ehrlich ascites tumor cells (EATC), and this effect was specific on tumor cells. Furthermore, our results demonstrated that EPE exposure elicited a rapid increase in the activity of superoxide dismutase and intracellular peroxides levels. These changes caused translocation of Bax to mitochondria and a subsequent release of mitochondrial cytochrome c. However, no activation of caspase-3 was observed in EPE-treated EATC. On the other hand, apoptosis-inducing factor (AIF) was translocated from mitochondria to nuclei. The EPE-induced translocation of AIF was suppressed with the addition of catalase, suggesting that the rapid intracellular peroxide levels after addition of EPE triggers off induction of apoptosis, which is AIF-mediated and caspase-independent. In this study, we have shown that EPE elicited a dose-dependent accumulation of cells in the G1 phase and inhibited DNA synthesis. Our results also demonstrated that cell cycle arrest and inhibition of proliferation in EATC by EPE are associated with decreased Rb phosphorylation. Furthermore, inhibitions of Rb phosphorylation and DNA synthesis by EPE were not suppressed with the addition of catalase. The present study suggests that intracellular peroxides, which trigger off induction of apoptosis, are not the trigger of EPE-induced G1 arrest in cell cycle.