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.

Effects of salicylic acid on thermotolerance and cardenolide accumulation under high temperature stress in Digitalis trojana Ivanina.

Long periods of high temperature or transitory increased temperature, a widespread agricultural problem, may lead to a drastic reduction in economic yield, affecting plant growth and development in many areas of the world. Heat stress causes many anatomical and physiological changes in plants. Its unfavorable effects can be alleviated by thermotolerance induced by exogenous application of plant growth regulators and osmoprotectants or by gradual application of temperature stress. Digitalis trojana Ivanina is an important medicinal plant species well known mainly for its cardenolides. The production of cardenolides via traditional agriculture is commercially inadequate. In this study, elicitation strategies were employed for improving crop thermotolerance and accumulation of cardenolides. For these purposes, the effects of salicylic acid (SA) and/or high temperature treatments in inducing cardenolide accumulation and thermotolerance were tested in callus cultures of D. trojana. Considerable increases in the production of cardenolides (up to 472.28 µg.g(-1) dry weight, dw) and induction of thermotolerance capacity were observed when callus cultures were exposed to high temperature for 2 h after pretreating with SA. High temperature treatments (2 h and 4 h) caused a marked reduction in superoxide dismutase (SOD; EC 1.15.1.1) and catalase (CAT; EC 1.11.1.6) activities, while SA pretreatment increased their activities. High temperature and/or SA appeared to increase the levels of proline, total phenolic, and flavonoid content. Elevated phenolic accumulation could be associated with increased stress protection. These results indicated that SA treatments induced synthesis of antioxidants and cardenolides, which may play a significant role in resistance to high temperature stress.

Nectar yeasts of two southern Spanish plants: the roles of immigration and physiological traits in community assembly.

Recent studies have shown that dense yeast populations often occurring in floral nectar are numerically dominated by a few species from the flower-insect interface specialized genus Metschnikowia, while generalist yeast species commonly occurring on leaf surfaces, soil, freshwater, and air were rarely isolated from nectar samples. This study was designed to understand the main factors responsible for the assembly of nectar yeast communities, by combining field experiments with laboratory tests characterizing the physiological abilities of all yeast species forming the pool of potential colonizers for two Spanish flowering plants (Digitalis obscura and Atropa baetica). Yeast frequency and species richness were assessed in external sources (bee glossae, air, plant phylloplane) as well as in pollinator rewards (pollen, nectar). Yeasts were most frequent in external sources (air, flower-visiting insects), less so in the proximate floral environment (phylloplane), and least in pollen and nectar. Nectar communities appeared to be considerably impoverished versions of those in insect glossae and phylloplane. Nectar, pollen, and insect yeast assemblages differed in physiological characteristics from those in other substrates. Nectarivorous Metschnikowia were not more resistant than other yeast species to plant secondary compounds and high sugar concentrations typical of nectar, but their higher growth rates may be decisive for their dominance in ephemeral nectar communities.

Stress-induced expression of cyclophilins in proembryonic masses of Digitalis lanata does not protect against freezing/thawing stress.

Using proembryonic masses (PEMs) of Digitalis lanata Erh., it was demonstrated that cold, hormonal or osmotic stress, which increased freezing tolerance during cryopreservation, induced an increasing level of two peptidyl-prolyl-cis/transisomerases (PPIases). The difference in pI (9.2 +/- 0.2 and 9.5 +/- 0.2, +/- SD; n = 3) allowed the separation of the two enzymes by free-flow isoelectrophoresis. Both were inhibited by cyclosporin A and thus belong to the cyclophilin family of PPIases. The enzymes differed slightly in their substrate specificity and their relative molecular masses of 18038 +/- 4 Da (D. lanataCyp18.0) and 18132 +/- 3 Da (D. lanataCyp18.1). Both cyclophilins were blocked N-terminally. Partial internal amino acid sequences from the two cyclophilins, with a length of 34 amino acids, displayed 82% sequence identity to each other. Pretreatment of PEMs with abscisic acid, sorbitol or a combination of both substances led to a 270 +/- 30% elevation of the total cytosolic cyclophilin concentration determined with a cyclophylin affinity sensor. During the first 4 d of pretreatment, the total PPIase activity was enhanced up to 230 +/- SD% compared with the control culture. The lag phase between maximal PPIase concentration after 4 d of pretreatment and maximal effect of freezing tolerance after 10 d of pretreatment indicated that increasing levels of cytosolic PPIases may be necessary to overcome the stress induced by hormones and osmotica during pretreatment but not to protect against freezing/thawing stress.

Cultivation and breeding of Digitalis lanata in the Netherlands.

After the second world war Marshall Aid funds were used to establish a cooperative organisation for growing, drying, and selling Digitalis lanata (and other medicinal, aromatic, and culinary herbs) in the Netherlands. The crop is sown in mid April and the fully mechanised harvest of the leaves takes place from September to late November. The leaves are dried for 10-12 hours at 50 degrees C maximum. The aim of breeding trials is to improve leaf production, erect leaf attitude, resistance to Septoria leaf spot and to bolting, and a higher dry matter and digoxin content.