Suitable transfection methods for single particle tracing in plant suspension cells

Background

Besides classical utilization of wood and paper, lignocellulosic biomass has become increasingly important with regard to biorefinery, biofuel production and novel biomaterials. For these new applications the macromolecular assembly of cell walls is of utmost importance and therefore further insights into the arrangement of the molecules on the nanolevel have to be gained. Cell wall recalcitrance against enzymatic degradation is one of the key issues, since an efficient degradation of lignocellulosic plant material is probably the most crucial step in plant conversion to energy. A limiting factor for in-depth analysis is that high resolution characterization techniques provide structural but hardly chemical information (e.g. Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM)), while chemical characterization leads to a disassembly of the cell wall components or does not reach the required nanoscale resolution (Fourier Tranform Infrared Spectroscopy (FT-IR), Raman Spectroscopy).

Results

Here we use for the first time Scanning Near-Field Optical Microscopy (SNOM in reflection mode) on secondary plant cell walls and reveal a segmented circumferential nanostructure. This pattern in the 100 nm range was found in the secondary cell walls of a softwood (spruce), a hardwood (beech) and a grass (bamboo) and is thus concluded to be consistent among various plant species. As the nanostructural pattern is not visible in classical AFM height and phase images it is proven that the contrast is not due to changes in surfaces topography, but due to differences in the molecular structure.

Conclusions

Comparative analysis of model substances of casted cellulose nanocrystals and spin coated lignin indicate, that the SNOM signal is clearly influenced by changes in lignin distribution or composition. Therefore and based on the known interaction of lignin and visible light (e.g. fluorescence and resonance effects), we assume the elucidated nanoscale structure to reflect variations in lignification within the secondary cell wall.     

A rapid biosensor-based method for quantification of free and glucose-conjugated salicylic acid

Background  

Salicylic acid (SA) is an important signalling molecule in plant defenses against biotrophic pathogens. It is also involved in several other processes such as heat production, flowering, and germination. SA exists in the plant as free SA and as an inert glucose conjugate (salicylic acid 2-O-β-D-glucoside or SAG). Recently, Huang et al. developed a bacterial biosensor that responds to free SA but not SAG, designated as Acinetobacter sp. ADPWH_lux. In this paper we describe an improved methodology for Acinetobacter sp. ADPWH_lux-based free SA quantification, enabling high-throughput analysis, and present an approach for the quantification of SAG from crude plant extracts.     

Study of the structure and biosynthetic pathway of lignin in stone cells of pear

Pear stone cell content is one of key determinants of fruit quality. Stone cells form by the deposition of lignin on primary cell walls, following secondary thickening of cell walls. Studies of the structure and metabolic pathway of pear lignin are rare. Stone cell and lignin content in the pulp of Pyrus bretschneideri cv. Dangshan Su were determined during fruit development. Lignin was extracted, purified and analyzed by ultraviolet (UV) light, Fourier-transform infrared (FTIR) spectroscopy and ¹H nuclear magnetic resonance (¹H NMR) spectroscopy. Intermediates of lignin biosynthesis were detected by high-performance liquid chromatography (HPLC). The results show that lignin content increases initially and decreases afterwards during fruit development, peaking twice at day 47 and day 63 after flowering. The lignin peaks precede both peaks of stone cell content (day 51 after flowering) and at the point at which sclereids reach their maximum diameter (day 67 after flowering). The milled wood lignin in pulp was identified as guaiacyl-syringyl-lignin by spectroscopic analyses. In the FTIR spectra, the peak intensity ratio of A₁₂₆₉/A₁₂₂₇ was 1.25. HPLC analyses detected cinnamic acid and p-coumaric acid, but not caffeic acid or ferulic acid. In conclusion, during pear fruit development, lignin is first biosynthesized, and subsequently deposited on cell walls to form stone cells and sclereids. The biosynthesis of guaiacyl-syringyl- lignin in pear pulp may involve a phenylpropane-type metabolic pathway from phenylalanine to cinnamic acid and acyl-CoA ester, as pear pulp lignin contains more guaiacyl units than syringyl units.     

Species absence in developed landscapes: an experimental evaluation

Context

Conversion of landscapes is widely associated with loss of biodiversity. While there are several competing hypotheses for the local extinction of species in developed landscapes, experimental approaches are seldom applied to elucidating mechanisms.

Objectives

In this study, we focus on the habitat degradation hypothesis and predict that poor quality of relictual wetlands in developed landscapes contributes to the absence of wood frogs (Rana sylvatica = Lithobates sylvaticus) by decreasing their performance.

Methods

In a translocation experiment, we reared wood frog larvae within enclosures in seven ponds where they naturally occur and in five ponds in developed landscapes where they are absent. Premature pond drying precluded assessing performance in one present pond and one absent pond.

Results

Absent ponds were surrounded by upland buffers dominated by developed land covers while ponds with wood frog breeding populations were surrounded primarily by intact forest. Ponds were largely similar in their attributes. Survival and growth rate did not differ between pond types. Development tended to be slightly more rapid in some absent ponds perhaps related to higher water temperatures.

Conclusions

Despite the highly altered landscapes surrounding them, we find no evidence that absent wetlands provide inferior habitat for wood frog larval recruitment. Performance in absent ponds matched or exceeded that observed in present ponds implying that absence of this species may stem from influences mediated by the upland landscape. These results provide a caution to the typically unexamined presumption that relictual habitats in developed landscapes are degraded in their utility for wildlife.

     

Wood fibre value simulation model: a new tool to assist measuring changes in forest landscapes by evaluating forest inventory

Context

A challenging issue in landscape ecology is the evaluation of changes in a forest landscape following a disturbance. This evaluation usually entails examining changes in the forest inventory, which represents the best information available for a given forest region.

Objectives

Our aim was to extend existing methods used to evaluate forest inventory to include additional variables, such as value-based forest product options, wood fibre attributes, and ecosystem services. Inclusion of such variables in forest inventory evaluations would allow research results to be presented from an economic perspective, which is often required for policy development and forest management decision-making.

Methods

We developed a value-based framework to evaluate forest inventory and implemented it in the wood fibre value simulation model. We then used a local data set from Manitoba, Canada, to show how the model can be applied to the mapping of new inventory layers to facilitate the evaluation of landscape changes.

Results

Five new inventory layers are mapped including bioenergy and heating value that can be directly used for evaluating landscape changes, and wood density, fibre length, and pulp yield, which can be combined with total wood volume to derive new variables or indices to express changes in landscape conditions.

Conclusions

Our model can contribute to the assessment of landscape changes by indicating the values a forest can have when it is used for different conservation or utilization purposes. The model can also support improved decision-making with respect to the management of forest resources.

     

A novel fluorescent pH probe for expression in plants

Background  

The pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av-GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt-GFP) and compare its properties with pHluorins for expression and use in plants.     

Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> and oat

Background  

Gene silencing vectors based on Barley stripe mosaic virus (BSMV) are used extensively in cereals to study gene function, but nearly all studies have been limited to genes expressed in leaves of barley and wheat. However since many important aspects of plant biology are based on root-expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created a need for tools to study gene function in these species.     

Isolation of a strong <Emphasis Type="Italic">Arabidopsis</Emphasis> guard cell promoter and its potential as a research tool

Background  

A common limitation in guard cell signaling research is that it is difficult to obtain consistent high expression of transgenes of interest in Arabidopsis guard cells using known guard cell promoters or the constitutive 35S cauliflower mosaic virus promoter. An additional drawback of the 35S promoter is that ectopically expressing a gene throughout the organism could cause pleiotropic effects. To improve available methods for targeted gene expression in guard cells, we isolated strong guard cell promoter candidates based on new guard cell-specific microarray analyses of 23,000 genes that are made available together with this report.     

Rapid metabolic profiling of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> defence responses against <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> using direct infrared laser desorption ionization mass spectrometry and principal component analysis

Background  

Successful defence of tobacco plants against attack from the oomycete Phytophthora nicotianae includes a type of local programmed cell death called the hypersensitive response. Complex and not completely understood signaling processes are required to mediate the development of this defence in the infected tissue. Here, we demonstrate that different families of metabolites can be monitored in small pieces of infected, mechanically-stressed, and healthy tobacco leaves using direct infrared laser desorption ionization orthogonal time-of-flight mass spectrometry. The defence response was monitored for 1 - 9 hours post infection.     

A high-throughput method for isolation of salicylic acid metabolic mutants

Background  

Salicylic acid (SA) is a key defense signal molecule against biotrophic pathogens in plants. Quantification of SA levels in plants is critical for dissecting the SA-mediated immune response. Although HPLC and GC/MS are routinely used to determine SA concentrations, they are expensive and time-consuming. We recently described a rapid method for a bacterial biosensor Acinetobacter sp. ADPWH_lux-based SA quantification, which enables high-throughput analysis. In this study we describe an improved method for fast sample preparation, and present a high-throughput strategy for isolation of SA metabolic mutants.     

A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation

Background  

The floral dip method of transformation by immersion of inflorescences in a suspension of Agrobacterium is the method of choice for Arabidopsis transformation. The presence of a marker, usually antibiotic- or herbicide-resistance, allows identification of transformed seedlings from untransformed seedlings. Seedling selection is a lengthy process which does not always lead to easily identifiable transformants. Selection for kanamycin-, phosphinothricin- and hygromycin B-resistance commonly takes 7–10 d and high seedling density and fungal contamination may result in failure to recover transformants.     

Cell type-specific characterization of nuclear DNA contents within complex tissues and organs

Background  

Eukaryotic organisms are defined by the presence of a nucleus, which encloses the chromosomal DNA, and is characterized by its DNA content (C-value). Complex eukaryotic organisms contain organs and tissues that comprise interspersions of different cell types, within which polysomaty, endoreduplication, and cell cycle arrest is frequently observed. Little is known about the distribution of C-values across different cell types within these organs and tissues.     

The Anatomy and Chemistry Of “Rubbery” Wood in Apple Var. Lord Lambourne

The anatomy of a one-year-old vertical normal stem of apple var. Lord Lamboume was compared with that of a comparable stem infected with rubbery wood virus and with that of a normal stem caused to undergo an anatomical georeaction by bending it at right angles to the vertical. The stem anatomy of 5-year-old orchard trees showing rubbery wood symptoms was compared with that of comparable normal trees. Staining reactions and investigations using light, polarizing and ultra-violet microscopes, and X-ray diffraction techniques established the anatomical identity of rubbery and georeaction wood.

Rubbery wood resembled georeaction wood in having a lignin content less than that of normal wood. It differed from georeaction wood in that oxidation of its lignin produced a molar ratio of syringaldehyde to vanillin which was less than that from normal wood lignin. Rubbery wood gave a yield of milled wood lignin greater than that of normal wood. This suggests that the lignin of rubbery wood is less firmly bound to the polysaccharide framework. This could account for the greater flexibility of rubbery wood.     

Isolation of intact sub-dermal secretory cavities from <Emphasis Type="Italic">Eucalyptus</Emphasis>

Background  

The biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Eucalyptus with cavities that are relatively large and rich in essential oils.     

Coincidences between different landscape ecological zones and growth forms of Cembran pine (Pinus cembra L.) in subalpine habitats of the Central Alps

The forgotten depots of the European Nutcracker (Nucifraga c. caryocatactes) often lead to the development of tufts of Pinus cembra. In many cases the other individuals of such tufts are not suppressed by the fittest one, rather there is an intraspecific coexistence up to the senescent stage of the trees. There are fusions of separate trunks, and so frequently the individual history of older trees can only be reconstructed by studying sutures, crown structures or trunk cross sections. Different types of trunk fusions are worked out. By means of transect counting the occurrence of these multiple trunk trees is documented quantitatively in different landscape ecological zones of the Engadin region (the Grisons, Switzerland). The data base is 3024 counted microsites of Pinus cembra individuals arising from seeds, including 5272 living individuals. These multiple trunk trees significantly play an important role in the landscape ecological zones of recent glacier recession and at the alpine timberline. Their growth forms have a higher biomechanical stability.     

In planta transient expression as a system for genetic and biochemical analyses of chlorophyll biosynthesis

Background  

Mg chelatase is a multi-subunit enzyme that catalyses the first committed step of chlorophyll biosynthesis. Studies in higher plants and algae indicate that the Mg chelatase reaction product, Mg-protoporphyrin IX plays an essential role in nuclear-plastid interactions. A number of Mg chelatase mutants have been isolated from higher plants, including semi-dominant alleles of ChlI, the gene encoding the I subunit of the enzyme. To investigate the function of higher plant CHLI, bacterial orthologues have been engineered to carry analogous amino acid substitutions to the higher plant mutations and the phenotypes examined through in vitro characterization of heterologously produced proteins. Here, we demonstrate the utility of a transient expression system in Nicotiana benthamiana for rapidly assaying mutant variants of the maize CHLI protein in vivo.     

Kinome profiling of Arabidopsis using arrays of kinase consensus substrates

Background  

Kinome profiling aims at the parallel analysis of kinase activities in a cell. Novel developed arrays containing consensus substrates for kinases are used to assess those kinase activities. The arrays described in this paper were already used to determine kinase activities in mammalian systems, but since substrates from many organisms are present we decided to test these arrays for the determination of kinase activities in the model plant species Arabidopsis thaliana.     

High-throughput retrotransposon-based fluorescent markers: improved information content and allele discrimination

Background  

Dense genetic maps, together with the efficiency and accuracy of their construction, are integral to genetic studies and marker assisted selection for plant breeding. High-throughput multiplex markers that are robust and reproducible can contribute to both efficiency and accuracy. Multiplex markers are often dominant and so have low information content, this coupled with the pressure to find alternatives to radio-labelling, has led us to adapt the SSAP (sequence specific amplified polymorphism) marker method from a ³³P labelling procedure to fluorescently tagged markers analysed from an automated ABI 3730 xl platform. This method is illustrated for multiplexed SSAP markers based on retrotransposon insertions of pea and is applicable for the rapid and efficient generation of markers from genomes where repetitive element sequence information is available for primer design. We cross-reference SSAP markers previously generated using the ³³P manual PAGE system to fluorescent peaks, and use these high-throughput fluorescent SSAP markers for further genetic studies in Pisum.     

Protocol: a simple gel-free method for SNP genotyping using allele-specific primers in rice and other plant species

Background  

Genotype analysis using multiple single nucleotide polymorphisms (SNPs) is a useful but labor-intensive or high-cost procedure in plant research. Here we describe an alternative genotyping method that is suited to multi-sample or multi-locus SNP genotyping and does not require electrophoresis or specialized equipment.