Comparative physiological, antioxidant and proteomic investigation reveal robust response to cold stress in Digitalis purpurea L.

BACKGROUND OF THE STUDY: Digitalis purpurea (L) is an important medicinal plant growing at Alpine region of Himalayas and withstands low temperatures and harsh climatic conditions existing at high altitude. It serves as an ideal plant system to decipher the tolerance to cold stress (CS) in plants from high altitudes. METHODS AND RESULTS: To understand the complexity of plant response to CS, we performed a comparative physiological and biochemical study complemented with proteomics in one-month-old D. purpurea grown at 25 °C (control) and 4 °C (CS). We observed an enhanced accumulation of different osmo-protectants (glycine betaine, soluble sugar and proline) and higher transcription (mRNA levels) of various antioxidant enzymes with an increased antioxidant enzyme activity in D. purpurea when exposed to CS. Furthermore, higher concentrations of non-enzymatic antioxidants (flavonoids, phenolics) was also associated with the response to CS. Differential proteomic analysis revealed the role of various proteins primarily involved in redox reactions, protein stabilization, quinone and sterol metabolism involved in CS response in D. purpurea.. CONCLUSION: Our results provide a framework for better understanding the physiological and molecular mechanism of CS response in D. purpurea at high altitudes.

Overexpression and RNAi-mediated Knockdown of Two 3ß-hydroxy-Δ5-steroid dehydrogenase Genes in Digitalis lanata Shoot Cultures Reveal Their Role in Cardenolide Biosynthesis.

3ß-hydroxy-Δ5-steroid dehydrogenases (3ßHSDs) are supposed to be involved in 5ß-cardenolide biosynthesis. Here, a novel 3ßHSD (Dl3ßHSD2) was isolated from Digitalis lanata shoot cultures and expressed in E. coli. Recombinant Dl3ßHSD1 and Dl3ßHSD2 shared 70% amino acid identity, reduced various 3-oxopregnanes and oxidised 3-hydroxypregnanes, but only rDl3ßHSD2 converted small ketones and secondary alcohols efficiently. To explain these differences in substrate specificity, we established homology models using borneol dehydrogenase of Salvia rosmarinus (6zyz) as the template. Hydrophobicity and amino acid residues in the binding pocket may explain the difference in enzyme activities and substrate preferences. Compared to Dl3ßHSD1, Dl3ßHSD2 is weakly expressed in D. lanata shoots. High constitutive expression of Dl3ßHSDs was realised by Agrobacterium-mediated transfer of Dl3ßHSD genes fused to the CaMV-35S promotor into the genome of D. lanata wild type shoot cultures. Transformed shoots (35S:Dl3ßHSD1 and 35S:Dl3ßHSD2) accumulated less cardenolides than controls. The levels of reduced glutathione (GSH), which is known to inhibit cardenolide formation, were higher in the 35S:Dl3ßHSD1 lines than in the controls. In the 35S:Dl3ßHSD1 lines cardenolide levels were restored after adding of the substrate pregnane-3,20-dione in combination with buthionine-sulfoximine (BSO), an inhibitor of GSH formation. RNAi-mediated knockdown of the Dl3ßHSD1 yielded several shoot culture lines with strongly reduced cardenolide levels. In these lines, cardenolide biosynthesis was fully restored after addition of the downstream precursor pregnan-3ß-ol-20-one, whereas upstream precursors such as progesterone had no effect, indicating that no shunt pathway could overcome the Dl3ßHSD1 knockdown. These results can be taken as the first direct proof that Dl3ßHSD1 is indeed involved in 5ß-cardenolide biosynthesis.

Identification of key genes involved in secondary metabolite biosynthesis in Digitalis purpurea.

The medicinal plant Digitalis purpurea produces cardiac glycosides that are useful in the pharmaceutical industry. These bioactive compounds are in high demand due to ethnobotany's application to therapeutic procedures. Recent studies have investigated the role of integrative analysis of multi-omics data in understanding cellular metabolic status through systems metabolic engineering approach, as well as its application to genetically engineering metabolic pathways. In spite of numerous omics experiments, most molecular mechanisms involved in metabolic pathways biosynthesis in D. purpurea remain unclear. Using R Package Weighted Gene Co-expression Network Analysis, co-expression analysis was performed on the transcriptome and metabolome data. As a result of our study, we identified transcription factors, transcriptional regulators, protein kinases, transporters, non-coding RNAs, and hub genes that are involved in the production of secondary metabolites. Since jasmonates are involved in the biosynthesis of cardiac glycosides, the candidate genes for Scarecrow-Like Protein 14 (SCL14), Delta24-sterol reductase (DWF1), HYDRA1 (HYD1), and Jasmonate-ZIM domain3 (JAZ3) were validated under methyl jasmonate treatment (MeJA, 100 µM). Despite early induction of JAZ3, which affected downstream genes, it was dramatically suppressed after 48 hours. SCL14, which targets DWF1, and HYD1, which induces cholesterol and cardiac glycoside biosynthesis, were both promoted. The correlation between key genes and main metabolites and validation of expression patterns provide a unique insight into the biosynthesis mechanisms of cardiac glycosides in D. purpurea.

Cardenolide Increase in Foxglove after 2,1,3-Benzothiadiazole Treatment Reveals a Potential Link between Cardenolide and Phytosterol Biosynthesis.

Cardenolides are steroidal metabolites in Digitalis lanata with potent cardioactive effects on animals. In plants, cardenolides are likely involved in various stress responses. However, the molecular mechanism of cardenolide increase during stresses is mostly unknown. Additionally, cardenolides are proposed to arise from cholesterol, but indirect results show that phytosterols may also be substrates for cardenolide biosynthesis. Here, we show that cardenolides increased after methyl jasmonate (MJ), sorbitol, potassium chloride (KCl) and salicylic acid analog [2,1,3-benzothiadiazole (BTH)] treatments. However, the expression of three known genes for cardenolide biosynthesis did not correlate well with these increases. Specifically, the expression of progesterone-5ß-reductases (P5ßR and P5ßR2) did not correlate with the cardenolide increase. The expression of 3ß-hydroxysteroid dehydrogenase (3ßHSD) correlated with changes in cardenolide levels only during the BTH treatment. Mining the D. lanata transcriptome identified genes involved in cholesterol and phytosterol biosynthesis: C24 sterol sidechain reductase 1 (SSR1), C4 sterol methyl oxidase 1, and 3 (SMO1 and SMO3). Surprisingly, the expression of all three genes correlated well with the cardenolide increase after the BTH treatment. Phylogenetic analysis showed that SSR1 is likely involved in both cholesterol and phytosterol biosynthesis. In addition, SMO1 is likely specific to phytosterol biosynthesis, and SMO3 is specific to cholesterol biosynthesis. These results suggest that stress-induced increase of cardenolides in foxglove may correlate with cholesterol and phytosterol biosynthesis. In summary, this work shows that cardenolides are important for stress responses in D. lanata and reveals a potential link between phytosterol and cardenolide biosynthesis.

Ploidy Status, Nuclear DNA Content and Start Codon Targeted (SCoT) Genetic Homogeneity Assessment in Digitalis purpurea L., Regenerated In Vitro.

Digitalis purpurea L. is a therapeutically important plant that synthesizes important cardiotonics such as digitoxin and digoxin. The present work reports a detailed and efficient propagation protocol for D. purpurea by optimizing various PGR concentrations in Murashige and Skoog (MS) medium. The genetic homogeneity of in vitro regenerants was assessed by the flow cytometric method (FCM) and Start Codon Targeted (SCoT) marker technique. Firstly, the seeds inoculated in full MS medium added with 0.5 mg/L GA3 produced seedlings. Different parts such as hypocotyl, nodes, leaves and apical shoots were used as explants. The compact calli were obtained on BAP alone or in combinations with 2, 4-D/NAA. The hypocotyl-derived callus induced somatic embryos which proliferated and germinated best in 0.75 mg/L BAP-fortified MS medium. Scanning electron microscopic (SEM) images confirmed the presence of various developmental stages of somatic embryos. Shoot regeneration was obtained in which BAP at 1.0 mg/L and 2.0 mg/L BAP + 0.5 mg/L 2,4-D proved to be the best treatments of PGRs in inducing direct and indirect shoot buds. The regenerated shoots showed the highest rooting percentage (87.5%) with 24.7 ± 1.9 numbers of roots/shoot in 1.0 mg/L IBA augmented medium. The rooted plantlets were acclimatized in a greenhouse at a survival rate of 85-90%. The genome size and the 2C nuclear DNA content of field-grown, somatic embryo-regenerated and organogenic-derived plants were estimated and noted to be 3.1, 3.2 and 3.0 picogram (pg), respectively; there is no alteration in ploidy status and the DNA content, validating genetic uniformity. Six SCoT primers unveiled 94.3%-95.13% monomorphic bands across all the plant samples analyzed, further indicating genetic stability among in vitro clones and mother plants. This study describes for the first time successful induction of somatic embryos from hypocotyl callus; and flow cytometry and SCoT marker confirmed the genetic homogeneity of regenerated plants.

Isolation of the 3ß-HSD promoter from Digitalis ferruginea subsp. ferruginea and its functional characterization in Arabidopsis thaliana.

BACKGROUND: Although members of the SDR gene family (short chain dehydrogenase) are distributed in kingdom of life, they have diverse roles in stress tolerance mechanism or secondary metabolite biosynthesis. Nevertheless, their precise roles in gene expression or regulation under stress are yet to be understood. METHODS: As a case study, we isolated, sequenced and functionally characterized the 3ß-HSD promoter from Digitalis ferruginea subsp. ferruginea in Arabidopsis thaliana. RESULTS: The promoter fragment contained light and stress response elements such as Box-4, G-Box, TCT-motif, LAMP element, ABRE, ARE, WUN-motif, MYB, MYC, W box, STRE and Box S. The functional analysis of the 3ß-HSD promoter in transgenic Arabidopsis seedlings showed that the promoter was expressed in cotyledon and root elongation zone in 2 days' seedlings. However, this expression was extended to hypocotyl and complete root in 6 days' seedlings. In 20 days-old seedlings, promoter expression was distributed to the whole seedling including hydathodes aperture, vascular bundle, shoot apical meristem, trichomes, midrib, leaf primordia, hypocotyl and xylem tissues. Further, expression of the promoter was enhanced or remained stable under the different abiotic stress conditions like osmotic, heat, cold, cadmium or low pH. In addition, the promoter also showed response to methyl jasmonate (MeJA) application. The expression could not be induced in wounded cotyledon most likely due to lack of interacting elements in the promoter fragment. CONCLUSIONS: Taken together, the 3ß-HSD promoter could be a candidate for the development of transgenic plants especially under changing environmental conditions.

Plastidial Expression of 3ß-Hydroxysteroid Dehydrogenase and Progesterone 5ß-Reductase Genes Confer Enhanced Salt Tolerance in Tobacco.

The short-chain dehydrogenase/reductase (SDR) gene family is widely distributed in all kingdoms of life. The SDR genes, 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and progesterone 5-ß-reductases (P5ßR1, P5ßR2) play a crucial role in cardenolide biosynthesis pathway in the Digitalis species. However, their role in plant stress, especially in salinity stress management, remains unexplored. In the present study, transplastomic tobacco plants were developed by inserting the 3ß-HSD, P5ßR1 and P5ßR2 genes. The integration of transgenes in plastomes, copy number and transgene expression at transcript and protein level in transplastomic plants were confirmed by PCR, end-to-end PCR, qRT-PCR and Western blot analysis, respectively. Subcellular localization analysis showed that 3ß-HSD and P5ßR1 are cytoplasmic, and P5ßR2 is tonoplast-localized. Transplastomic lines showed enhanced growth in terms of biomass and chlorophyll content compared to wild type (WT) under 300 mM salt stress. Under salt stress, transplastomic lines remained greener without negative impact on shoot or root growth compared to the WT. The salt-tolerant transplastomic lines exhibited enhanced levels of a series of metabolites (sucrose, glutamate, glutamine and proline) under control and NaCl stress. Furthermore, a lower Na+/K+ ratio in transplastomic lines was also observed. The salt tolerance, mediated by plastidial expression of the 3ß-HSD, P5ßR1 and P5ßR2 genes, could be due to the involvement in the upregulation of nitrogen assimilation, osmolytes as well as lower Na+/K+ ratio. Taken together, the plastid-based expression of the SDR genes leading to enhanced salt tolerance, which opens a window for developing saline-tolerant plants via plastid genetic engineering.

RNAi-mediated gene knockdown of progesterone 5ß-reductases in Digitalis lanata reduces 5ß-cardenolide content.

KEY MESSAGE: Studying RNAi-mediated DlP5ßR1 and DlP5ßR2 knockdown shoot culture lines of Digitalis lanata, we here provide direct evidence for the participation of PRISEs (progesterone 5ß-reductase/iridoid synthase-like enzymes) in 5ß-cardenolide formation. Progesterone 5ß-reductases (P5ßR) are assumed to catalyze the reduction of progesterone to 5ß-pregnane-3,20-dione, which is a crucial step in the biosynthesis of the 5ß-cardenolides. P5ßRs are encoded by VEP1-like genes occurring ubiquitously in embryophytes. P5ßRs are substrate-promiscuous enone-1,4-reductases recently termed PRISEs (progesterone 5ß-reductase/iridoid synthase-like enzymes). Two PRISE genes, termed DlP5ßR1 (AY585867.1) and DlP5ßR2 (HM210089.1) were isolated from Digitalis lanata. To give experimental evidence for the participation of PRISEs in 5ß-cardenolide formation, we here established several RNAi-mediated DlP5ßR1 and DlP5ßR2 knockdown shoot culture lines of D. lanata. Cardenolide contents were lower in D. lanata P5ßR-RNAi lines than in wild-type shoots. We considered that the gene knockdowns may have had pleiotropic effects such as an increase in glutathione (GSH) which is known to inhibit cardenolide formation. GSH levels and expression of glutathione reductase (GR) were measured. Both were higher in the Dl P5ßR-RNAi lines than in the wild-type shoots. Cardenolide biosynthesis was restored by buthionine sulfoximine (BSO) treatment in Dl P5ßR2-RNAi lines but not in Dl P5ßR1-RNAi lines. Since progesterone is a precursor of cardenolides but can also act as a reactive electrophile species (RES), we here discriminated between these by comparing the effects of progesterone and methyl vinyl ketone, a small RES but not a precursor of cardenolides. To the best of our knowledge, we here demonstrated for the first time that P5ßR1 is involved in cardenolide formation. We also provide further evidence that PRISEs are also important for plants dealing with stress by detoxifying reactive electrophile species (RES).

Sequencing, de novo assembly and annotation of Digitalis ferruginea subsp. schischkinii transcriptome.

There is an increasing demand for elucidating the biosynthetic pathway of medicinal plants, which are capable of producing several metabolites with great potentials for industrial drug production. Digitalis species are important medicinal plants for the production of cardenolide compounds. Advancement on culture techniques is strictly related to our understanding of the genomic background of species. There are a limited number of genomic studies on Digitalis species. The goal of this study is to contribute to the genomic data of Digitalis ferruginea subsp. schischkinii by presenting transcriptome annotation. Digitalis ferruginea subsp. schischkinii has a limited distribution in Turkey and Transcaucasia, and has a high level of lanatoside C, an important cardenolide. In the study, we sequenced the cDNA library prepared from RNA pools of D. ferruginea subsp. schischkinii tissues treated with various stress conditions. Comprehensive bioinformatics approaches were used for de novo assembly and functional annotation of D. ferruginea subsp. schischkinii transcriptome sequence data along with TF families predictions and phylogenetic analysis. In the study, 58,369 unigenes were predicted and unigenes were annotated by analyzing the sequence data in the non-redundant (NR) protein database, the non-redundant nucleotide (NT) database, Gene Orthology (GO), EuKaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG), SwissProt, and InterPro databases. This study is the first transcriptome data for D. ferruginea subsp. schischkinii.

Differential expression of 3ß-HSD and mlncRNAs in response to abiotic stresses in Digitalis nervosa.

Digitalis nervosa is an important medicinal plant species belonging to the family of Scrophulariaceae that has the potential to be used for heart failure. 3ß-hydroxysteroid dehydrogenase (3ß-HSD) is a key gene in the biosynthesis of cardenolides for making digitalis effective compounds, hence identification of this gene is important for genetic engineering purposes towards increasing the yield of cardiac glycosides. In addition, mRNA-like non-coding RNAs (mlncRNAs), a class of long non coding RNAs, play key roles in various biological processes and may affect cardenolides pathway in digitalis plants.  In the present work, full sequence of 3ß-HSD was isolated from Digitalis nervosa. Gene expression patterns of 3ß-HSD along with three mlncRNAs including mlncRNA23, mlncRNA28 and mlncRNA30 were studied and the results indicated that they are differentially expressed in different tissues including roots, stems and leaves, with the highest expression levels in leaves.  Moreover, the transcript levels of these genes affected by the cold and drought stresses. The results obtained from the present study is important in order to understand the potential role of mlncRNAs in digitalis plants, especially in response to abiotic stresses.

The Foxgloves (Digitalis) Revisited.

This review provides a renewed look at the genus Digitalis. Emphasis will be put on those issues that attracted the most attention or even went through paradigmatic changes since the turn of the millennium. PubMed and Google Scholar were used ("Digitalis" and "Foxglove" were the key words) to identify research from 2000 till 2017 containing data relevant enough to be presented here. Intriguing new results emerged from studies related to the phylogeny and taxonomy of the genus as well as to the biosynthesis and potential medicinal uses of the key active compounds, the cardiac glycosides. Several Eastern and Western Foxgloves were studied with respect to their propagation in vitro. In this context, molecular biology tools were applied and phytochemical analyses were conducted. Structure elucidation and analytical methods, which have experienced less exciting progress, will not be considered here in great detail.

Biotechnological Approaches for Biomass and Cardenolide Production in Digitalis purpurea L.

Digitalis purpurea L. is one of the main economically viable sources of cardenolides (cardiac glycosides) for the pharmaceutical industry. Nevertheless, production of cardenolides in plants grown by traditional agriculture is not always an efficient process and can be affected by biotic and abiotic factors. This chapter provides two biotechnology strategies for biomass and cardenolide production in D. purpurea. Firstly, we report biomass production using a temporary immersion system (TIS), combined with cardenolide extraction and quantification. Secondly, an efficient protocol for genetic transformation via Agrobacterium tumefaciens is provided. These strategies can be used independently or combined in order to increase the content of cardiac glycosides in D. purpurea and to unravel biosynthetic pathways associated to cardiac glycoside production.

[Plants regeneration from genetically transformed root and callus cultures of periwinkle Vinca minor L. and foxglove purple Digitalis purpurea L].

Plants regenerated from hairy roots and calluses of foxglove purple and periwinkle have been obtained. It was found that organogenesis in hairy root culture occurs spontaneously on hormone-free medium but with different efficiencies. The frequency of direct shoot formation from root cultures was up to 60% in Digitalis and 3.7% in Vinca. Addition of 1 mg/l BA, 0.1 mg/l NAA and 5% sucrose to B5 medium increased regenerative capacity of Vinca roots up to 19.1%. Regenerated plants showed morphological features typically seen in Ri-transgenic plants. They include growth and plagiotropism of the root system, increased shoot formation, changed leaf morphology and short internodes.

Comprehensive analysis of alternative splicing in Digitalis purpurea by strand-specific RNA-Seq.

Digitalis purpurea (D. purpurea) is one of the most important medicinal plants and is well known in the treatment of heart failure because of the cardiac glycosides that are its main active compounds. However, in the absence of strand specific sequencing information, the post-transcriptional mechanism of gene regulation in D. purpurea thus far remains unknown. In this study, a strand-specific RNA-Seq library was constructed and sequenced using Illumina HiSeq platforms to characterize the transcriptome of D. purpurea with a focus on alternative splicing (AS) events and the effect of AS on protein domains. De novo RNA-Seq assembly resulted in 48,475 genes. Based on the assembled transcripts, we reported a list of 3,265 AS genes, including 5,408 AS events in D. purpurea. Interestingly, both glycosyltransferases and monooxygenase, which were involved in the biosynthesis of cardiac glycosides, are regulated by AS. A total of 2,422 AS events occurred in coding regions, and 959 AS events were located in the regions of 882 unique protein domains, which could affect protein function. This D. purpurea transcriptome study substantially increased the expressed sequence resource and presented a better understanding of post-transcriptional regulation to further facilitate the medicinal applications of D. purpurea for human health.

A stable and efficient Agrobacterium tumefaciens-mediated genetic transformation of the medicinal plant Digitalis purpurea L.

In this study, we developed a rapid and efficient method for in vitro propagation and Agrobacterium tumefaciens-mediated transformation of Digitalis purpurea L. (syn. foxglove), an important medicinal plant. Mature leaf explants of D. purpurea were used for 100 % adventitious shoot regeneration on Murashige and Skoog (MS) medium supplemented with 1 mg L(-1) thidiazuron (TDZ) (a cytokine) and 0.1 mg L(-1) 1-naphthaleneacetic acid (NAA) (an auxin). Transformation was achieved by inoculating leaf explants with the A. tumefaciens strains GV2260/pBI121 or GV3101/pBI121. The binary vector pBI121 contained the reporter ß-glucuronidase gene (GUS) and kanamycin selection marker nptII. Kanamycin-resistant shoots were regenerated directly on the selection medium 4-6 weeks after co-cultivation. Approximately, 52.2 and 60 % of kanamycin-resistant shoots transformed with Agrobacterium strains GV2260 and GV3101, respectively, showed strong GUS staining by histochemical assay. Furthermore, PCR and Southern blot analysis confirmed the presence of nptII and GUS on the chromosome of the transformed D. purpurea plants, and stable GUS expression was detected in the transformants by RT-PCR analysis. This efficient method of shoot regeneration and genetic transformation of D. purpurea will provide a powerful tool to increase and produce valuable components such as digitoxin, digoxin, and digoxigenin in D. purpurea through improved secondary metabolic pathways via a biotechnological approach.

Calcium and magnesium elimination enhances accumulation of cardenolides in callus cultures of endemic Digitalis species of Turkey.

Elimination of calcium (Ca), magnesium (Mg) or both from the medium of callus cultures of Digitalis davisiana Heywood, Digitalis lamarckii Ivanina, Digitalis trojana Ivanina and Digitalis cariensis Boiss. ex Jaub. et Spach increased cardenolides production. Callus was induced from hypocotyl segments from one-month old seedlings were cultured on MS medium containing 0.5 µg ml(-1) thidiazuron (TDZ) and 0.25 µg ml(-1) indole acetic acid (IAA). After 30 days of culture, callus was transferred in hormone-free MS medium (MSO) as well as Ca or Mg or both were completely eliminated from same medium. The amount of five cardenolides from D. davisiana Heywood, D. lamarckii Ivanina, D. trojana Ivanina and D. cariensis Boiss. ex Jaub. et Spach were compared. Higher amounts of five cardenolides and total cardenolides were obtained when callus of four Digitalis species were incubated on MS medium lacking both Ca and Mg. The mean contents of total cardenolides obtained were in the order of D. lamarckii (2017.97 µg g(-1))>D. trojana (1385.75 µg g(-1))>D. cariensis (1038.65 µg g(-1))>D. davisiana (899.86 µg g(-1)) when both Ca and Mg were eliminated from the medium, respectively. This protocol is useful for development of new strategies for the large-scale production of cardenolides.

Inferring species trees directly from biallelic genetic markers: bypassing gene trees in a full coalescent analysis.

The multispecies coalescent provides an elegant theoretical framework for estimating species trees and species demographics from genetic markers. However, practical applications of the multispecies coalescent model are limited by the need to integrate or sample over all gene trees possible for each genetic marker. Here we describe a polynomial-time algorithm that computes the likelihood of a species tree directly from the markers under a finite-sites model of mutation effectively integrating over all possible gene trees. The method applies to independent (unlinked) biallelic markers such as well-spaced single nucleotide polymorphisms, and we have implemented it in SNAPP, a Markov chain Monte Carlo sampler for inferring species trees, divergence dates, and population sizes. We report results from simulation experiments and from an analysis of 1997 amplified fragment length polymorphism loci in 69 individuals sampled from six species of Ourisia (New Zealand native foxglove).

Vein Patterning 1-encoded progesterone 5ß-reductase: activity-guided improvement of catalytic efficiency.

Progesterone 5ß-reductases (P5ßR; EC 1.3.99.6) encoded by Vein Patterning 1 (VEP1) genes are capable of reducing the CC double-bond of a variety of enones enantioselectively. Sequence and activity data of orthologous P5ßRs were used to define a set of residues possibly responsible for the large differences in enzyme activity seen between rAtSt5ßR and rDlP5ßR, recombinant forms of P5ßRs from Arabidopsis thaliana and Digitalis lanata, respectively. Tyrosine-156, asparagine-205 and serine-248 were identified as hot spots in the rDlP5ßR responsible for its low catalytic efficiency. These positions were individually substituted for amino acids found in the strong rAtSt5ßR in the corresponding sites. Kinetic constants were determined for rDlP5ßR and its mutants as well as for rAtSt5ßR using progesterone and 2-cyclohexen-1-one as substrates. Enzyme mutants in which asparagine-205 was substituted for methionine or alanine showed considerably lower km and higher K(cat)/k(m) values than the wild-type DlP5ßR, approaching the catalytic efficiency of strong P5ßRs. The introduced mutations not only lead to an improved capability to reduce progesterone but also to altered substrate preference. Our findings provided structural insights into the differences seen among the natural P5ßRs with regard to their substrate preferences and catalytic efficiencies.

Comprehensive transcriptome analysis reveals novel genes involved in cardiac glycoside biosynthesis and mlncRNAs associated with secondary metabolism and stress response in Digitalis purpurea.

BACKGROUND: Digitalis purpurea is an important ornamental and medicinal plant. There is considerable interest in exploring its transcriptome. RESULTS: Through high-throughput 454 sequencing and subsequent assembly, we obtained 23532 genes, of which 15626 encode conserved proteins. We determined 140 unigenes to be candidates involved in cardiac glycoside biosynthesis. It could be grouped into 30 families, of which 29 were identified for the first time in D. purpurea. We identified 2660 mRNA-like npcRNA (mlncRNA) candidates, an emerging class of regulators, using a computational mlncRNA identification pipeline and 13 microRNA-producing unigenes based on sequence conservation and hairpin structure-forming capability. Twenty five protein-coding unigenes were predicted to be targets of these microRNAs. Among the mlncRNA candidates, only 320 could be grouped into 140 families with at least two members in a family. The majority of D. purpurea mlncRNAs were species-specific and many of them showed tissue-specific expression and responded to cold and dehydration stresses. We identified 417 protein-coding genes with regions significantly homologous or complementary to 375 mlncRNAs. It includes five genes involved in secondary metabolism. A positive correlation was found in gene expression between protein-coding genes and the homologous mlncRNAs in response to cold and dehydration stresses, while the correlation was negative when protein-coding genes and mlncRNAs were complementary to each other. CONCLUSIONS: Through comprehensive transcriptome analysis, we not only identified 29 novel gene families potentially involved in the biosynthesis of cardiac glycosides but also characterized a large number of mlncRNAs. Our results suggest the importance of mlncRNAs in secondary metabolism and stress response in D. purpurea.

Expression of 3beta-HSD and P5betaR, genes respectively coding for Delta5-3beta-hydroxysteroid dehydrogenase and progesterone 5beta-reductase, in leaves and cell cultures of Digitalis lanata EHRH.

Plants of the genus Digitalis produce 5 beta-cardenolides that are used in the therapy of cardiac insufficiency in humans. 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and progesterone 5 beta-reductase (P5 betaR) are both supposed to be important enzymes in the biosynthesis of these natural products. Activity and gene expression were demonstrated for both enzymes in cardenolide-accumulating leaves of Digitalis lanata but also in cardenolide-free permanent cell suspension cultures initiated from D. lanata leaf tissue. Enzyme activities were determined and quantified by HPLC and GC-MS methods. Expression of the respective genes, namely AY585867.1 (P5betaR gene) and DQ466890.1 (3beta-HSD gene), was made evident by real-time polymerase chain reaction (qPCR) analysis. We demonstrate for the first time that the P5betaR gene, encoding an enzyme described as a key enzyme in cardenolide biosynthesis, is also expressed in cardenolide-free tissues of cardenolide-containing plants.