A laboratory high-throughput glass chamber using dynamic headspace TD-GC/MS method for the analysis of whole Brassica napus L. plantlet volatiles under cadmium-related abiotic stress.

INTRODUCTION: The dynamic headspace sampling technique using thermal desorption, gas chromatography-mass spectrometry (TD-GC/MS) is a powerful method for analysing plant emissions of volatile organic compounds (VOCs), and experiments performed in sterile and controlled conditions can be useful for VOC metabolism investigations. OBJECTIVE: The main purpose of this study was to set up a laboratory high-throughput glass chamber for whole plant volatiles analysis. Brassica napus L. plantlets were tested with the developed system to better understand the relationship between low emission of induced terpene and cadmium (Cd)-related abiotic stress. METHODOLOGY: VOCs emitted by 28-day-old Brassica napus L. plantlets cultivated in vitro were trapped with our device using adsorbent cartridges that were desorbed with a thermal desorption unit before cryofocusing with a cooled injection system and programmable temperature vaporising inlet into an HP-5 ms GC column. Terpene detection and quantitation from chromatogram profiles were acquired using selected ion monitoring (SIM) mode during full scan analysis and mass spectra were obtained with a quadrupole-type mass spectrometer. RESULTS: The new trapping method produced reliable qualitative profiles of oilseed rape VOCs. Typical emissions of monoterpenes (myrcene, limonene) and sesquiterpenes (ß-elemene, (E,E)-α-farnesene) were found for the different concentrations tested. One-way analysis of variance for quantitative results of (E,E)-α-farnesene emission rates showed a Cd concentration effect. CONCLUSION: This inexpensive glass chamber has potential for wide application in laboratory sterile approach and replicated research. Moreover, the non-invasive dynamic sampling technique could also be used to analyse volatiles under both abiotic and biotic stresses.

Comprehensive comparison of the chemical and structural characterization of landfill leachate and leonardite humic fractions.

Humic substances (HS) are complex and heterogeneous mixtures of organic compounds that occur everywhere in the environment. They represent most of the dissolved organic matter in soils, sediments (fossil), water, and landfills. The exact structure of HS macromolecules has not yet been determined because of their complexity and heterogeneity. Various descriptions of HS are used depending on specific environments of origin and research interests. In order to improve the understanding of the structure of HS extracted from landfill leachate (LHS) and commercial HS from leonardite (HHS), this study sought to compare the composition and characterization of the structure of LHS and HHS using elemental composition, chromatographic (high-performance liquid chromatography (HPLC)), and spectroscopic techniques (UV-vis, FTIR, NMR, and MALDI-TOF). The results showed that LHS molecules have a lower molecular weight and less aromatic structure than HHS molecules. The characteristics of functional groups of both LHS and HHS, however, were basically similar, but there was some differences in absorbance intensity. There were also less aliphatic and acidic functional groups and more aromatic and polyphenolic compounds in the humic acid (HA) fraction than in the fulvic acid (FA) and other molecules (OM) fractions of both origins. The differences between LHS and HHS might be due to the time course of humification. Combining the results obtained from these analytical techniques cold improve our understanding of the structure of HS of different origins and thus enhance their potential use.

Expression of PiABP19, Picdc2 and PiSERK3 during induction of somatic embryogenesis in leaflets of Prunus incisa (Thunb.).

Somatic embryogenesis is a useful tool of plant breeding. In this context, a procedure for inducing somatic embryogenesis in Prunus incisa leaf explants had been previously developed. The original in vitro protocol relies on picloram treatments and exposure to darkness as inductive conditions, the best frequency of embryogenesis being obtained on the second leaf (F(2)) exposed to 4 µM picloram during 30 days. The morphological and biochemical changes observed during somatic embryogenesis occur in response to alterations in gene expression regulation patterns. A molecular study was conducted in order to provide deeper insight into the fundamental biological factors involved in the induction of this process using a gene candidate strategy and semi-quantitative reverse transcription polymerase chain reaction analysis. So far, no sequence data related to somatic embryogenesis has been available in cherry. In the present study, we cloned and sequenced cDNA fragments of putative genes encoding auxin-binding protein, cell cycle regulator and somatic embryogenesis receptor kinase. Time-course differential transcript accumulations were observed for all investigated genes in leaves or derived callus tissues during the observation period (first month of culture). Their possible involvement in the sequential steps of the embryogenic pathway (dedifferentiation, cell proliferation, differentiation through somatic embryogenesis) is presented and discussed.

Epoxiconazole exposure affects terpenoid profiles of oilseed rape plantlets based on a targeted metabolomic approach.

Epoxiconazole is a broad-spectrum fungicide described as highly persistent in soil and as such can be considered as an abiotic agent like other problematic agrochemicals. Furthermore, the plant phenotyping tool involving non-invasive monitoring of plant-emitted volatile organic compounds (VOCs) may be useful in the identification of metabolic markers for abiotic stress. We therefore decided to profile the VOCs from secondary metabolism of oilseed rape through a dose-response experiment under several epoxiconazole concentrations (0, 0.01, 0.1 and 1 mg L-1). VOC collections of 35-day-old whole plantlets were performed through a dynamic headspace sampling technique under defined and controlled conditions. The plantlets grew freely within a home-made, laboratory and high-throughput glass chamber without any disturbance. Putative metabolic markers were analysed using a targeted metabolomic approach based on TD-GC-MS method coupled with data acquisition in SIM mode in order to focus on terpenes and sulphur-containing volatiles. Chromatograms of emitted terpenes were achieved accurately for the 35-day-old oilseed rape plantlets. We also analysed the presence of sulphur-containing volatiles in samples of shoot and root tissues using an innovative DHS-TD-GC-MS method, but no difference was found between qualitative profiles. Nevertheless, we demonstrated through this experiment that sesquiterpenes such as ß-elemene and (E,E)-α-farnesene are involved in epoxiconazole dose-response. In particular, (E,E)-α-farnesene could serve as a metabolic marker of fungicide exposure for oilseed rape plantlets.

Stress regulated expression of the GUS-marker gene (uidA) under the control of plant calmodulin and viral 35S promoters in a model fruit tree rootstock: Prunus incisa x serrula.

The fact that calmodulin genes (CaM) are tightly associated with the Ca(2+) regulatory pathway, as well as their putative role in plant defence against pathogens, indicate a potential use of alternative plant promoters to express genes of interest in specific tissues or developmental stages. To study the expression level of the apple CaM promoter, 981 bp sequences upstream were fused to the uidA gene, introduced into cherry and compared with a 35S-GUS construct. Transgene copy number and transgenic expression levels were analysed using Southern blot, Western blot and RT-PCR techniques. Transcription levels were assessed by GUS fluorometry, histochemistry and real-time PCR techniques in leaves of plantlets grown in vitro under various abiotic stresses like low- and high-temperature, salicylic acid and wounding, harvested after 0, 0.5, 1, 2, 4, 10, 24 and 72 h. Histochemical analyses showed staining only in veins and petioles of CaM-GUS lines, while in 35S-GUS plants staining extended to the entire leaf. Furthermore, real-time qPCR data indicate that both promoters are differently regulated by various stresses. Obtained results suggest that the selected apple CaM promoter responsible for the expression of a gene in vascular tissues may offer interesting perspectives for plant defense programs.

Change in ATP-binding cassette B1/19, glutamine synthetase and alcohol dehydrogenase gene expression during root elongation in Betula pendula Roth and Alnus glutinosa L. Gaertn in response to leachate and leonardite humic substances.

Humic substances (HS) are complex and heterogeneous compounds of humified organic matter resulting from the chemical and microbiological decomposition of organic residues. HS have a positive effect on plant growth and development by improving soil structure and fertility. They have long been recognized as plant growth-promoting substances, particularly with regard to influencing nutrient uptake, root growth and architecture. The biochemical and molecular mechanisms through which HS influence plant physiology are not well understood. This study evaluated the bioactivity of landfill leachate and leonardite HS on alder (Alnus glutinosa L. Gaertn) and birch (Betula pendula Roth) during root elongation in vitro. Changes in root development were studied in relation to auxin, carbon and nitrogen metabolisms, as well as to the stress adaptive response. The cDNA fragments of putative genes encoding two ATP-binding cassette (ABC) transporters (ABCB1 and ABCB19) belonging to the B subfamily of plant ABC auxin transporters were cloned and sequenced. Molecular data indicate that HS and their humic acid (HA) fractions induce root growth by influencing polar auxin transport (PAT), as illustrated by the modulation of the ABCB transporter transcript levels (ABCB1 and ABCB19). There were also changes in alcohol dehydrogenase (ADH) and glutamine synthetase (GS) gene transcript levels in response to HS exposure. These findings confirmed that humic matter affects plant growth and development through various metabolic pathways, including hormonal, carbon and nitrogen metabolisms and stress response or signalization.

Micropropagation of Prunus species relevant to cherry fruit production.

Cherry tree micropropagation is limited to the production of healthy cultivars of Prunus avium and Prunus cerasus, and their rootstocks; mainly the dwarfing ones. By using meristem-tip (0.1 mm long) or healthy shoot tips/nodes, four successive steps are needed to obtain whole plants capable of growing in the nursery: multiplication by axillary branching, shoot elongation, rooting, and plantlet acclimation. Along this process, several parameters have to be adjusted for each phase of the culture, including media composition, environmental culture conditions and plant handling. These parameters vary depending on genotypic response and specific vulnerability to physiological disorders such as hyperhydricity, apex necrosis, unstable propagation, and rooting rates. Based on a 40 year-long experience of study and application of culture conditions to large-scale plant production, this document summarizes the main problems (variability of the propagation rate, hyperhydricity, apex necrosis, plant re-growth) and solutions encountered to solve them, with means validated on many mericlones.