Understanding the Role of Populus ECERIFERUM2-Likes in the Biosynthesis of Very-Long-Chain Fatty Acids for Cuticular Waxes.

Cuticular waxes are derived from very-long-chain fatty acid (VLCFA) precursors made by the concerted action of four enzymes that form the fatty acid (FA) elongation complex. The condensing enzyme of the complex confers specificity to substrates of different chain lengths, yet on its own cannot account for the biosynthesis of VLCFAs longer than 28 carbons (C28). Recent evidence from Arabidopsis thaliana points to a synergistic role of clade II BAHD acyltransferases and condensing enzymes in the elongation of VLCFAs beyond C28. In Populus trichocarpa, clade II is composed of seven uncharacterized paralogous genes (PtCER2-like1-7). In the present study, five of these genes were heterologously expressed in yeast and their respective FA profiles were determined. PtCER2-likes differentially altered the accumulation of C28 and C30 FAs when expressed in the presence of the condensing enzyme AtCER6. Among these, PtCER2-like5 produced the highest levels of C28 FAs in yeast and its expression was localized to the epidermis in ß-glucuronidase-reporter poplar lines, consistent with a role in cuticular wax biosynthesis. Complementation of the A. thaliana cer2-5 mutant with PtCER2-like5 increased the levels of C28-derived cuticular waxes at the expense of C30-derived components. Together, these results demonstrate that the role of CER2-likes in cuticular wax biosynthesis is conserved in Populus clade II BAHD acyltransferases.

In-vitro cytotoxicity in relation to chemotypic diversity in diploid and tetraploid populations of Gentiana kurroo Royle.

ETHNOPHARMACOLOGICAL RELEVANCE: Gentiana kurroo is a multipurpose critically endangered medicinal herb prescribed as medicine in Ayurveda in India and exhibits various pharmacological properties including anti-cancer activity. The species is rich repository of pharmacologically active secondary metabolites together with secoiridoidal glycosides. AIM OF THE STUDY: The study aimed to investigate the chemical diversity in different populations/cytotypes prevailing in G. kurroo to identify elite genetic stocks in terms of optimum accumulation/biosynthesis of desired metabolites and having higher in-vitro cytotoxicity potential in relation to chemotypic diversity. MATERIAL AND METHODS: The wild plants of the species were collected from different ranges of altitudes from the Kashmir Himalayas. For cytological evaluation, the standard meiotic analysis was performed. The standard LC-MS/MS technique was employed for phytochemical analysis based on different marker compounds viz. sweroside, swertiamarin, and gentiopicroside. Different tissues such as root-stock, aerial parts, and flowers were used for chemo-profiling. Further, the methanolic extracts of diploid and tetraploid cytotypes were assessed for cytotoxic activity by using MTT assay against four different human cancer cell lines. RESULTS: The quantification of major bioactive compounds based on tissue- and location-specific comparison, as well as in-vitro cytotoxic potential among extant cytotypes, was evaluated. The comprehensive cytomorphological studies of the populations from NW Himalayas revealed the occurrence of different chromosomal races viz. n = 13, 26. The tetraploid cytotype was hitherto unreported. The tissue-specific chemo-profiling revealed relative dominance of different phytoconstituents in root-stock. There was a noticeable increase in the quantity of the analyzed compounds in relation to increasing ploidy status along the increasing altitudes. The MTT assay of methanolic extracts of diploid and tetraploid cytotypes displayed significant cytotoxicity potential in tetraploids. The root-stock extracts of tetraploids were highly active extracts with IC50 value ranges from 5.65 to 8.53 µg/mL against HCT-116 colon cancer. CONCLUSION: The chemical evaluation of major bioactive compounds in diverse cytotypes from different plant parts along different altitudes presented an appreciable variability in sweroside, swertiamarin, and gentiopicroside contents. Additionally, the concentrations of these phytoconstituents varied for cytotoxicity potential among different screened cytotypes. This quantitative difference of active bio-constituents was in correspondence with the growth inhibition percentage of different tested cancer cell lines. Thus, the present investigation strongly alludes towards a prognostic approach for the identification of elite cytotypes/chemotypes with significant pharmacological potential.

Flourensia fiebrigii S.F. blake: A medicinal plant from the Argentinean highlands with potential use as anti-rheumatic and anti-inflammatory.

ETHNOPHARMACOLOGICAL RELEVANCE: Flourensia fiebrigii is a plant used in traditional medicine in the Argentine Calchaquí Valley as purgative, expectorant, anti-rheumatic and anti-inflammatory. AIM OF THE STUDY: The aim of this study was to analyze the macroscopic and microscopic characteristics of F. fiebrigii leaf and stem, the phytochemical composition of leaves ethanolic extracts and to validate its traditional use as anti-rheumatic and anti-inflammatory. MATERIALS AND METHODS: The macroscopic and microscopic description of F. fiebrigii leaf and stem was carried out. Two extracts (immersions and tinctures) from leaves were obtained. The phytochemical analysis and UHPLC-OT-MS metabolome fingerprinting of both extracts were performed. The anti-rheumatic and anti-inflammatory activities of both extracts were determined using enzymatic inhibition assays of xanthine-oxidase (XOD), secretory phospholipase A2 (sPLA2) and lipoxygenase (LOX). RESULTS: The macroscopic and micrographic characters of F. fiebrigii were described to allow the botanical characterization of the plant species. The leaves extracts showed a high level of phenolic compounds with similar chromatographic patterns. Forty-five compounds were identified based on UHPLC-OT-MS including several sesquiterpenes, chalcones, flavonoids, isoflavonoids, a lignan and phenylpropanoids phenolic acids that have been identified for the first time in this plant species. F. fiebrigii extracts were able to inhibit the XOD activity and, consequently, the formation of uric acid and reactive oxygen species, primary cause of diseases, such as gouty arthritis (IC50 values of 1.10-2.12 µg/mL). Pro-inflammatory enzymes like sPLA2 and LOX were also inhibited by F. fiebrigii extracts (IC50 values of 22.00-2.20 µg/mL) decreasing the production of inflammation mediators. CONCLUSIONS: The present work validates the traditional medicinal use of F. fiebrigii as anti-rheumatic and anti-inflammatory through the use of enzymatic assays. The presence of several chemical compounds with demonstrated anti-rheumatic and anti-inflammatory properties also supports the bioactivity of the F. fiebrigii.

Soil PAHs contamination effect on the cellular and subcellular organelle changes of Phragmites australis Cav.

The concentrations of ∑16 priority polycyclic aromatic hydrocarbons (PAHs) for soils, roots, and above-ground parts of reed (Phragmites australis Cav.) were determined on different monitoring plots located near the city of Kamensk-Shakhtinsky, southern Russia, where historically received industrial sewage and sludge. The total PAHs concentration in monitoring soil plots was significantly higher than those in the background site which situated at the distance of 2 km from the contamination source. Accordingly, the maximum accumulation was found for phenanthrene and chrysene among the 16 priority PAHs in most of the plant samples collected in the impact zone. The effects of PAHs' pollution on changes of Phragmites australis Cav. cellular and subcellular organelles in the studied monitoring sites were also determined using optical and electron microscopy, respectively. The obtained data showed that increasing of PAHs contamination negatively affected the ultrastructural changes of the studied plants. Phragmites australis Cav. showed a high level of adaptation to the effect of stressors by using tissue and cell levels. In general, the detected alterations under the PAHs effect were possibly connected to changes in biochemical and histochemical parameters as a response for reactive oxygen species and as a protective response against oxidative stress. The obtained results introduce innovative findings of cellular and subcellular changes in plants exposed to ∑16 priority PAHs as very persistent and toxic contaminants.

Pharmacognostic standardization and preliminary phytochemical studies of Gaultheria trichophylla.

CONTEXT: Gaultheria trichophylla Royle (Ericaceae) has long been used for various ailments in traditional systems of medicines; most importantly it is used against pain and inflammation. AIMS: This study determines various pharmacognostic and phytochemical standards helpful to ensure the purity, safety, and efficacy of medicinal plant G. trichophylla. MATERIAL AND METHODS: Intact aerial parts, powdered materials, and extracts were examined macro- and microscopically and pharmacognostic standardization parameters were determined in accordance with the guidelines given by the World Health Organization (WHO). Parameters including extractive values, ash values, and loss on drying were determined. Preliminary phytochemical tests, fluorescence analysis, and chromatographic profiling were performed for the identification and standardization of G. trichophylla. RESULTS: The shape, size, color, odor, and surface characteristics were noted for intact drug and powdered drug material of G. trichophylla. Light and scanning electron microscope images of cross section of leaf and powdered microscopy revealed useful diagnostic features. Histochemical, phytochemical, physicochemical, and fluorescence analysis proved useful tools to differentiate the powdered drug material. High-performance liquid chromatography (HPLC) analysis showed the presence of important phytoconstituents such as gallic acid, rutin, and quercetin. DISCUSSION AND CONCLUSIONS: The data generated from the present study help to authenticate the medicinally important plant G. trichophylla. Qualitative and quantitative microscopic features may be helpful for establishing the pharmacopeia standards. Morphology as well as various pharmacognostic aspects of different parts of the plant were studied and described along with phytochemical and physicochemical parameters, which could be helpful in further isolation and purification of medicinally important compounds.

Study of the spatial distribution of mercury in roots of vetiver grass (Chrysopogon zizanioides) by micro-pixe spectrometry.

Localization of Hg in root tissues of vetivergrass (Chrysopogon zizanioides) was investigated by micro-Proton Induced X-ray Emission (PIXE) spectrometry to gain a better understanding of Hg uptake and its translocation to the aerial plant parts. Tillers of C. zizanioides were grown in a hydroponic culture for 3 weeks under controlled conditions and then exposed to Hg for 10 days with or without the addition of the chelators (NH(4))(2)S(2)O(3) or KI. These treatments were used to study the effects of these chelators on localization of Hg in the root tissues to allow better understanding of Hg uptake during its assisted-phytoextraction. Qualitative elemental micro-PIXE analysis revealed that Hg was mainly localized in the root epidermis and exodermis, tissues containing suberin in all Hg treatments. Hg at trace levels was localized in the vascular bundle when plants were treated with a mercury solution only. However, higher Hg concentrations were found when the solution also contained (NH(4))(2)S(2)O(3) or KI. This finding is consistent with the observed increase in Hg translocation to the aerial parts of the plants in the case of chemically induced Hg phytoextraction.

Plant single-cell and single-cell-type metabolomics.

In conjunction with genomics, transcriptomics, and proteomics, plant metabolomics is providing large data sets that are paving the way towards a comprehensive and holistic understanding of plant growth, development, defense, and productivity. However, dilution effects from organ- and tissue-based sampling of metabolomes have limited our understanding of the intricate regulation of metabolic pathways and networks at the cellular level. Recent advances in metabolomics methodologies, along with the post-genomic expansion of bioinformatics knowledge and functional genomics tools, have allowed the gathering of enriched information on individual cells and single cell types. Here we review progress, current status, opportunities, and challenges presented by single cell-based metabolomics research in plants.

Uptake and cellular distribution, in four plant species, of fluorescently labeled mesoporous silica nanoparticles.

KEY MESSAGE: We report the uptake of MSNs into the roots and their movement to the aerial parts of four plant species and their quantification using fluorescence, TEM and proton-induced x - ray emission (micro - PIXE) elemental analysis. Monodispersed mesoporous silica nanoparticles (MSNs) of optimal size and configuration were synthesized for uptake by plant organs, tissues and cells. These monodispersed nanoparticles have a size of 20 nm with interconnected pores with an approximate diameter of 2.58 nm. There were no negative effects of MSNs on seed germination or when transported to different organs of the four plant species tested in this study. Most importantly, for the first time, a combination of confocal laser scanning microscopy, transmission electron microscopy and proton-induced X-ray emission (micro-PIXE) elemental analysis allowed the location and quantification MSNs in tissues and in cellular and sub-cellular locations. Our results show that MSNs penetrated into the roots via symplastic and apoplastic pathways and then via the conducting tissues of the xylem to the aerial parts of the plants including the stems and leaves. The translocation and widescale distribution of MSNs in plants will enable them to be used as a new delivery means for the transport of different sized biomolecules into plants.

Comparative analysis of Arabidopsis UGT74 glucosyltransferases reveals a special role of UGT74C1 in glucosinolate biosynthesis.

The study of glucosinolates and their regulation has provided a powerful framework for the exploration of fundamental questions about the function, evolution, and ecological significance of plant natural products, but uncertainties about their metabolism remain. Previous work has identified one thiohydroximate S-glucosyltransferase, UGT74B1, with an important role in the core pathway, but also made clear that this enzyme functions redundantly and cannot be the sole UDP-glucose dependent glucosyltransferase (UGT) in glucosinolate synthesis. Here, we present the results of a nearly comprehensive in vitro activity screen of recombinant Arabidopsis Family 1 UGTs, which implicate other members of the UGT74 clade as candidate glucosinolate biosynthetic enzymes. Systematic genetic analysis of this clade indicates that UGT74C1 plays a special role in the synthesis of aliphatic glucosinolates, a conclusion strongly supported by phylogenetic and gene expression analyses. Finally, the ability of UGT74C1 to complement phenotypes and chemotypes of the ugt74b1-2 knockout mutant and to express thiohydroximate UGT activity in planta provides conclusive evidence for UGT74C1 being an accessory enzyme in glucosinolate biosynthesis with a potential function during plant adaptation to environmental challenge.

Cardamine hirsuta: a versatile genetic system for comparative studies.

A major goal in biology is to identify the genetic basis for phenotypic diversity. This goal underpins research in areas as diverse as evolutionary biology, plant breeding and human genetics. A limitation for this research is no longer the availability of sequence information but the development of functional genetic tools to understand the link between changes in sequence and phenotype. Here we describe Cardamine hirsuta, a close relative of the reference plant Arabidopsis thaliana, as an experimental system in which genetic and transgenic approaches can be deployed effectively for comparative studies. We present high-resolution genetic and cytogenetic maps for C. hirsuta and show that the genome structure of C. hirsuta closely resembles the eight chromosomes of the ancestral crucifer karyotype and provides a good reference point for comparative genome studies across the Brassicaceae. We compared morphological and physiological traits between C. hirsuta and A. thaliana and analysed natural variation in stamen number in which lateral stamen loss is a species characteristic of C. hirsuta. We constructed a set of recombinant inbred lines and detected eight quantitative trait loci that can explain stamen number variation in this population. We found clear phylogeographic structure to the genetic variation in C. hirsuta, thus providing a context within which to address questions about evolutionary changes that link genotype with phenotype and the environment.

A plasmodesmal glycosyltransferase-like protein.

Plasmodesmata (Pd) are plant intercellular connections that represent cytoplasmic conduits for a wide spectrum of cellular transport cargoes, from ions to house-keeping proteins to transcription factors and RNA silencing signals; furthermore, Pd are also utilized by most plant viruses for their spread between host cells. Despite this central role of Pd in the plant life cycle, their structural and functional composition remains poorly characterized. In this study, we used a known Pd-associated calreticulin protein AtCRT1 as bait to isolate other Pd associated proteins in Arabidopsis thaliana. These experiments identified a beta-1,6-N-acetylglucosaminyl transferase-like enzyme (AtGnTL). Subcellular localization studies using confocal microscopy observed AtGnTL at Pd within living plant cells and demonstrated colocalization with a Pd callose-binding protein (AtPDCB1). That AtGnTL is resident in Pd was consistent with its localization within the plant cell wall following plasmolysis. Initial characterization of an Arabidopsis T-DNA insertional mutant in the AtGnTL gene revealed defects in seed germination and delayed plant growth.

The Arabidopsis cer26 mutant, like the cer2 mutant, is specifically affected in the very long chain fatty acid elongation process.

Plant aerial organs are covered by cuticular waxes, which form a hydrophobic crystal layer that mainly serves as a waterproof barrier. Cuticular wax is a complex mixture of very long chain lipids deriving from fatty acids, predominantly of chain lengths from 26 to 34 carbons, which result from acyl-CoA elongase activity. The biochemical mechanism of elongation is well characterized; however, little is known about the specific proteins involved in the elongation of compounds with more than 26 carbons available as precursors of wax synthesis. In this context, we characterized the three Arabidopsis genes of the CER2-like family: CER2, CER26 and CER26-like . Expression pattern analysis showed that the three genes are differentially expressed in an organ- and tissue-specific manner. Using individual T-DNA insertion mutants, together with a cer2 cer26 double mutant, we characterized the specific impact of the inactivation of the different genes on cuticular waxes. In particular, whereas the cer2 mutation impaired the production of wax components longer than 28 carbons, the cer26 mutant was found to be affected in the production of wax components longer than 30 carbons. The analysis of the acyl-CoA pool in the respective transgenic lines confirmed that inactivation of both genes specifically affects the fatty acid elongation process beyond 26 carbons. Furthermore, ectopic expression of CER26 in transgenic plants demonstrates that CER26 facilitates the elongation of the very long chain fatty acids of 30 carbons or more, with high tissular and substrate specificity.

Characterization of cell wall components of wheat straw following hydrothermal pretreatment and fractionation.

Thermophysical pretreatment enhances the enzymatic hydrolysis of lignocellulose. However, its impact on cell wall chemistry is still poorly understood. This paper reports the effects of hydrothermal pretreatment on the degradation and alkali-extractability of wheat straw cell wall polymers. Pretreatment resulted in loss and/or solubilization of arabinoxylans (by 53%), ferulic and diferulic acids which are important cross-linking agents accompanied by concomitant increases in cellulose (up to 43%) and lignin (29%). The remaining water-insoluble hemicelluloses were more readily extractable in alkali and were reduced in molecular weight indicating substantial thermochemical depolymerization. They were also associated with smaller but significant amounts of (cellulose-derived) glucose. The alkali-insoluble residues consisted predominantly of cellulosic glucose and lignin and contained p-coumaric acid. The depolymerization of hemicelluloses, reduction in cinnamic acids and partial degradation of cellulose is likely to contribute significantly to the accessibility of cellulases during subsequent enzymolysis.

[Macroscopic and microscopic identification of Ligularia przewalskii].

OBJECTIVE: To provide macroscopic and microscopic identification basis for Ligularia przewalskii. METHODS: Macroscopic and microscopic identification of roots, stems and leaves of Ligularia przewalskii were carried out with the methods of paraffin section, leaves epidermal section and powder transdermal section. RESULTS: The microscopic characteristics included: Open collateral vascular bundles in stem were not in the same size and arranged in two rings; Lots of fiber bundles scattered in the column parts; There were two vascular bundles in principal vein of leaf; Anticlinal wall of upper epidermis cells was thickened like moniliform, lower epidermis were like waves with irregular; The type of stoma was anomocytic; Calcium oxalate acicular crystal could be seen in the powder. CONCLUSION: These features can provide references for identification of Ligularia przewalskii.

Colonization of spinach by Verticillium dahliae and effects of pathogen localization on the efficacy of seed treatments.

Verticillium wilt on spinach (Spinacia oleracea) is caused by the soilborne fungus Verticillium dahliae. The pathogen is seedborne and transmission through seed is a major concern because of the dispersal of the pathogen to areas where fresh and processing spinach crops are grown in rotation with susceptible crops. Reduction in seedborne inoculum minimizes pathogen spread; therefore, knowledge of pathogen localization in seed is critical to develop methods to reduce seedborne inoculum. Spinach seedlings were inoculated with conidial suspensions of a green fluorescent protein-tagged strain of V. dahliae and colonization events were followed through seed production by confocal laser-scanning microscopy. Between 24 to 96 h postinoculation (PI), conidia germinated and formed hyphal colonies on root tips and in root elongation zones. Hyphae colonized root cortical tissues both intra and intercellularly by 2 weeks, and colonized the taproot xylem with abundant mycelia and conidia that led to vascular discoloration coincident with foliar symptom expression by 8 weeks PI. At 10 weeks PI, the xylem of the upper stem, inflorescence, and spinach seed parts, including the pericarp, seed coat, cotyledons, and radicle, had been colonized by the pathogen but not the perisperm (the diploid maternal tissue). Maximum concentration of the fungus was in the seed coat, the outermost layer of the vasculature. Infection of V. dahliae in spinach seed was systemic and transmissible to developing seedlings. Additional analyses indicated that fungicide and steam seed treatments reduced detectable levels of the pathogen but did not eliminate the pathogen from the seed. This information will assist in the development of seed treatments that will reduce the seedborne inoculum transmission to crop production fields.

Nuclear ribosome biogenesis mediated by the DIM1A rRNA dimethylase is required for organized root growth and epidermal patterning in Arabidopsis.

Position-dependent patterning of hair and non-hair cells in the Arabidopsis thaliana root epidermis is a powerful system to study the molecular basis of cell fate specification. Here, we report an epidermal patterning mutant affecting the ADENOSINE DIMETHYL TRANSFERASE 1A (DIM1A) rRNA dimethylase gene, predicted to participate in rRNA posttranscriptional processing and base modification. Consistent with a role in ribosome biogenesis, DIM1A is preferentially expressed in regions of rapid growth, and its product is nuclear localized with nucleolus enrichment. Furthermore, DIM1A preferentially accumulates in the developing hair cells, and the dim1A point mutant alters the cell-specific expression of the transcriptional regulators GLABRA2, CAPRICE, and WEREWOLF. Together, these findings suggest that establishment of cell-specific gene expression during root epidermis development is dependent upon proper ribosome biogenesis, possibly due to the sensitivity of the cell fate decision to relatively small differences in gene regulatory activities. Consistent with its effect on the predicted S-adenosyl-l-Met binding site, dim1A plants lack the two 18S rRNA base modifications but exhibit normal pre-rRNA processing. In addition to root epidermal defects, the dim1A mutant exhibits abnormal root meristem division, leaf development, and trichome branching. Together, these findings provide new insights into the importance of rRNA base modifications and translation regulation for plant growth and development.

Rice RNA-dependent RNA polymerase 6 acts in small RNA biogenesis and spikelet development.

Higher plants have evolved multiple RNA-dependent RNA polymerases (RDRs), which work with Dicer-like (DCL) proteins to produce different classes of small RNAs with specialized molecular functions. Here we report that OsRDR6, the rice (Oryza sativa L.) homolog of Arabidopsis RDR6, acts in the biogenesis of various types and sizes of small RNAs. We isolated a rice osrdr6-1 mutant, which was temperature sensitive and showed spikelet defects. This mutant displays reduced accumulation of tasiR-ARFs, the conserved trans-acting siRNAs (tasiRNAs) derived from the TAS3 locus, and ectopic expression of tasiR-ARF target genes, the Auxin Response Factors (including ARF2 and ARF3/ETTIN). The loss of tasiR-mediated repression of ARFs in osrdr6-1 can explain its morphological defects, as expression of two non-targeted ARF3 gene constructs (ARF3muts) in a wild-type background mimics the osrdr6 and osdcl4-1 mutant phenotypes. Small RNA high-throughput sequencing also reveals that besides tasiRNAs, 21-nucleotide (nt) phased small RNAs are also largely dependent on OsRDR6. Unexpectedly, we found that osrdr6-1 has a strong impact on the accumulation of 24-nt phased small RNAs, but not on unphased ones. Our work uncovers the key roles of OsRDR6 in small RNA biogenesis and directly illustrates the crucial functions of tasiR-ARFs in rice development.

Epidermal cell differentiation in cotton mediated by the homeodomain leucine zipper gene, GhHD-1.

Gossypium hirsutum L. (cotton) fibres are specialized trichomes a few centimetres in length that grow from the seed coat. Few genes directly involved in the differentiation of these epidermal cells have been identified. These include GhMYB25-like and GhMYB25, two related MYB transcription factors that regulate fibre cell initiation and expansion. We have also identified a putative homeodomain leucine zipper (HD-ZIP) transcription factor, GhHD-1, expressed in trichomes and early fibres that might play a role in cotton fibre initiation. Here, we characterize GhHD-1 homoeologues from tetraploid G. hirsutum and show, using reporter constructs and quantitative real-time PCR (qRT-PCR), that they are expressed predominantly in epidermal tissues during early fibre development, and in other tissues bearing epidermal trichomes. Silencing of GhHD-1 reduced trichome formation and delayed the timing of fibre initiation. Constitutive overexpression of GhHD-1 increased the number of fibres initiating on the seed, but did not affect leaf trichomes. Expression of GhHD-1 in cotton silenced for different fibre MYBs suggest that in ovules it acts downstream of GhMYB25-like, but is unaffected in GhMYB25- or GhMYB109-silenced plants. Microarray analysis of silencing and overexpression lines of GhHD-1 indicated that it potentially regulates the levels of ethylene and reactive oxidation species (ROS) through a WRKY transcription factor and calcium-signalling pathway genes to activate downstream genes necessary for cell expansion and elongation.

Trichome isolation with and without fixation using laser microdissection and pressure catapulting followed by RNA amplification: expression of genes of terpene metabolism in apical and sub-apical trichome cells of Artemisia annua L.

The aim of this project was to evaluate the effect of fixation on plant material prior to Laser Microdissection and Pressure Catapulting (LMPC) and to identify an appropriate method for preserving good RNA quality after cell isolation. Therefore, flower buds from Artemisia annua L. were exposed to either the fixative formaldehyde or a non-fixative buffer prior to cell isolation by LMPC. Proteinase K was used after cell isolation from fixed plant tissue, in an attempt to improve the RNA yield. The ability to detect gene expression using real-time quantitative PCR with or without previous amplification of RNA from cells isolated by LMPC was also evaluated. Conclusively, we describe a new technique, without fixation, enabling complete isolation of intact glandular secretory trichomes and specific single trichome cells of A. annua. This method is based on LMPC and preserves good RNA quality for subsequent RNA expression studies of both whole trichomes, apical and sub-apical cells from trichomes of A. annua. Using this method, expression of genes of terpene metabolism was studied by real-time quantitative PCR. Expression of genes involved in artemisinin biosynthesis was observed in both apical and sub-apical cells.

Differential responses of microsomal proteins and metabolites in two contrasting cadmium (Cd)-accumulating soybean cultivars under Cd stress.

While there are significant genotypic differences in cadmium (Cd) uptake and distribution in soybean cultivars, little attention has been paid to the underlying molecular mechanisms. We adopted a comparative proteomic approach coupled with metabolite analysis to examine Cd uptake and translocation in two contrasting Cd-accumulating soybean cultivars, Enrei and Harosoy, which accumulate higher amount of Cd in the roots and aerial parts, respectively. Proteins extracted from the root microsomal fraction were evaluated by immunoblot analysis using different subcellular marker proteins. Analysis of control and Cd-exposed samples by two-dimensional gel electrophoresis coupled with mass spectrometry revealed a total of 13 and 11 differentially expressed proteins in the Enrei and Harosoy cultivars, respectively. Metabolome profiling identified a total of 32 metabolites, the expression of 18 of which was significantly altered in at least in one cultivar in response to Cd stress. Analysis of the combined proteomic and metabolomic results revealed that proteins and amino acids associate with Cd-chelating pathways are highly active in the Enrei cultivar. In addition, proteins associated with lignin biosynthesis are significantly upregulated in the Enrei cultivar under Cd stress. Our results indicate that in the Enrei cultivar, Cd-chelating agents may bind excess free Cd ion and that translocation of Cd from the roots to the aerial parts might be prevented by increased xylem lignification.