Hydroponic isotope labeling of entire plants (HILEP) for quantitative plant proteomics

Quantitative analysis by mass spectrometry (MS) is a major challenge in proteomics as the correlation between analyte concentration and signal intensity is often poor due to varying ionisation efficiencies in the presence of molecular competitors. However, relative quantitation methods that utilise differential stable isotope labelling and mass spectrometric detection are available. Many drawbacks inherent to chemical labelling methods (ICAT, iTRAQ) can be overcome by metabolic labelling with amino acids containing stable isotopes (e.g. 13C and/or 15N) in methods such as Stable Isotope Labelling with Amino acids in Cell culture (SILAC). SILAC has also been used for labelling of proteins in plant cell cultures (1) but is not suitable for whole plant labelling. Plants are usually autotrophic (fixing carbon from atmospheric CO2) and, thus, labelling with carbon isotopes becomes impractical. In addition, SILAC is expensive. Recently, Arabidopsis cell cultures were labelled with 15N in a medium containing nitrate as sole nitrogen source. This was shown to be suitable for quantifying proteins and nitrogen-containing metabolites from this cell culture (2,3). Labelling whole plants, however, offers the advantage of studying quantitatively the response to stimulation or disease of a whole multicellular organism or multi-organism systems at the molecular level. Furthermore, plant metabolism enables the use of inexpensive labelling media without introducing additional stress to the organism. And finally, hydroponics is ideal to undertake metabolic labelling under extremely well-controlled conditions. We demonstrate the suitability of metabolic 15N hydroponic isotope labelling of entire plants (HILEP) for relative quantitative proteomic analysis by mass spectrometry. To evaluate this methodology, Arabidopsis plants were grown hydroponically in 14N and 15N media and subjected to oxidative stress.

Progressive erosion of genetic and epigenetic variation in callus-derived cocoa (Theobroma cacao) plants

Relatively little is known about the timing of genetic and epigenetic forms of somaclonal variation arising from callus growth. We surveyed for both types of change in cocoa (Theobroma cacao) plants regenerated from calli of various ages, and also between tissues from the source trees. For genetic change, we used 15 single sequence repeat (SSR) markers from four source trees and from 233 regenerated plants. For epigenetic change, we used 386 methylation-sensitive amplified polymorphism (MSAP) markers on leaf and explant (staminode) DNA from two source trees and on leaf DNA from 114 regenerants. Genetic variation within source trees was limited to one slippage mutation in one leaf. Regenerants were far more variable, with 35% exhibiting at least one mutation. Genetic variation initially accumulated with culture age but subsequently declined. MSAP (epigenetic) profiles diverged between leaf and staminode samples from source trees. Multivariate analysis revealed that leaves from regenerants occupied intermediate eigenspace between leaves and staminodes of source plants but became progressively more similar to source tree leaves with culture age. Statistical analysis confirmed this rather counterintuitive finding that leaves of ‘late regenerants’ exhibited significantly less genetic and epigenetic divergence from source leaves than those exposed to short periods of callus growth.

Optical sensors for monitoring water uptake in plants

The monitoring of water uptake in plants is becoming increasingly important. Optical sensors offer considerable advantages over conventional methods and several sensors have been developed including an optical potometer that monitors water uptake from individual roots, the detection of xylem cavitation using audio acoustic emissions with an interferometric force feedback microphone, and an optical fiber displacement transducer that detects changes in leaf thickness in relation to leaf-water potential.

Reconstruction of historical pollination rates reveals linked declines of pollinators and plants

Widespread reports of low pollination rates suggest a recent anthropogenic decline in pollination that could threaten natural and agricultural ecosystems. Nevertheless, unequivocal evidence for a decline in pollination over time has remained elusive because it was not possible to determine historical pollination rates. Here we demonstrate a widely applicable method for reconstructing historical pollination rates, thus allowing comparison with contemporary rates from the same sites. We focused on the relationship between the oil-collecting bee Rediviva peringueyi (Melittidae) and the guild of oil-secreting orchid species (Coryciinae) that depends on it for pollination. The guild is distributed across the highly transformed and fragmented lowlands of the Cape Region of South Africa. We show that rehydrated herbarium specimens of Pterygodium catholicum, the most abundant member of the guild, contain a record of past pollinator activity in the form of pollinarium removal rates. Analysis of a pollination time series showed a recent decline in pollination on Signal Hill, a small urban conservation area. The same herbaria contain historical species occurrence data. We analyzed this data and found that there has been a contemporaneous shift in orchid guild composition in urban areas due to the local extirpation of the non-clonal species, consistent with their greater dependence on seeds and pollination for population persistence.

DNA barcodes for Mexican Cactaceae, plants under pressure from wild collecting

DNA barcodes could be a useful tool for plant conservation. Of particular importance is the ability to identify unknown plant material, such as from customs seizures of illegally collected specimens. Mexican cacti are an example of a threatened group, under pressure because of wild collection for the xeriscaping trade and private collectors. Mexican cacti also provide a taxonomically and geographically coherent group with which to test DNA barcodes. Here, we sample the matK barcode for 528 species of Cactaceae including approximately 75% of Mexican species and test the utility of the matK region for species-level identification. We find that the matK DNA barcode can be used to identify uniquely 77% of species sampled, and 79-87% of species of particular conservation importance. However, this is far below the desired rate of 95% and there are significant issues for PCR amplification because of the variability of primer sites. Additionally, we test the nuclear ITS regions for the cactus subfamily Opuntioideae and for the genus Ariocarpus (subfamily Cactoideae). We observed higher rates of variation for ITS (86% unique for Opuntioideae sampled) but a much lower PCR success, encountering significant intra-individual polymorphism in Ariocarpus precluding the use of this marker in this taxon. We conclude that the matK region should provide useful information as a DNA barcode for Cactaceae if the problems with primers can be addressed, but matK alone is not sufficiently variable to achieve species-level identification. Additional complementary regions should be investigated as ITS is shown to be unsuitable

Sugars in crop plants

We review current knowledge of the most abundant sugars, sucrose, maltose, glucose and fructose, in the world's major crop plants. The sucrose-accumulating crops, sugar beet and sugar cane, are included, but the main focus of the review is potato and the major cereal crops. The production of sucrose in photosynthesis and the inter-relationships of sucrose, glucose, fructose and other metabolites in primary carbon metabolism are described, as well as the synthesis of starch, fructan and cell wall polysaccharides and the breakdown of starch to produce maltose. The importance of sugars as hormone-like signalling molecules is discussed, including the role of another sugar, trehalose, and the trehalose biosynthetic pathway. The Maillard reaction, which occurs between reducing sugars and amino acids during thermal processing, is described because of its importance for colour and flavour in cooked foods. This reaction also leads to the formation of potentially harmful compounds, such as acrylamide, and is attracting increasing attention as food producers and regulators seek to reduce the levels of acrylamide in cooked food. Genetic and environmental factors affecting sugar concentrations are described.

Patents for plants: context and current status

One of the important themes in any discussion concerning the application of genetic transformation technology in horticulture or elsewhere is the role of Intellectual Property Rights (IPR). This term covers both the content of patents and the confidential expertise, usually related to methodology and referred to as “Trade Secrets”. This review will explain the concepts behind patent protection, and will discuss the wide-ranging scope of existing patents that cover novel genotypes of plants as well as all aspects of transgenic technology, from selectable markers and novel promoters to methods of gene introduction. Although few of these patents have any significant commercial value there are a small number of key patents that may restrict the “freedom to operate” of any company seeking to exploit the methods in the production of transgenic varieties. Over the last twenty years, these restrictions have forced extensive cross-licensing between ag-biotech companies and have been one of the driving forces behind the consolidation of these companies. Although such issues may have limited relevance in the horticultural sector, and are often considered to be of little interest to the academic scientist working in the public sector, they are of great importance in any debate about the role of “public-good breeding” and of the relationship between the public and private sectors.

Sub-irrigation of Petunia: benefits in dry summers

With the increasing frequency and magnitude of warmer days during the summer in the UK, bedding plants which were a traditional part of the urban green landscape are perceived as unsustainable and water-demanding. During recent summers when bans on irrigation have been imposed, use and sales of bedding plants have dropped dramatically having a negative financial impact on the nursery industry. Retaining bedding species as a feature in public and even private spaces in future may be conditional on them being managed in a manner that minimises their water use. Using Petunia x hybrida ‘Hurrah White’ we aimed to discover which irrigation approach was the most efficient for maintaining plants’ ornamental quality (flower numbers, size and longevity), shoot and root growth under water deficit and periods of complete water withdrawal. Plants were grown from plugs for 51 days in wooden rhizotrons (0.35 m (h) x 0.1 m (w) x 0.065 m (d)); the rhizotrons’ front comprised clear Perspex which enabled us to monitor root growth closely. Irrigation treatments were: 1. watering with the amount which constitutes 50% of container capacity by conventional surface drip-irrigation (‘50% TOP’); 2. 50% as sub-irrigation at 10 cm depth (‘50% SUB’); 3. ‘split’ irrigation: 25% as surface drip- and 25% as sub-irrigation at 15 cm depth (‘25/25 SPLIT’); 4. 25% as conventional surface drip-irrigation (‘25% TOP’). Plants were irrigated daily at 18:00 apart from days 34-36 (inclusive) when water was withdrawn for all the treatments. Plants in ‘50% SUB’ had the most flowers and their size was comparable to that of ‘50% TOP’. Differences between treatments in other ‘quality’ parameters (height, shoot number) were biologically small. There was less root growth at deeper soil surface levels for ‘50% TOP’ which indicated that irrigation methods like ‘50% SUB’ and ‘25/25 SPLIT’ and stronger water deficits encouraged deeper root growth. It is suggested that sub-irrigation at 10 cm depth with water amounts of 50% container capacity would result in the most root growth with the maximum flowering for Petunia. Leaf stomatal conductance appeared to be most sensitive to the changes in substrate moisture content in the deepest part of the soil profile, where most roots were situated.

Manipulating resource allocation in plants

The distribution of nutrients and assimilates in different organs and tissues is in a constant state of flux throughout the growth and development of a plant. At key stages during the life cycle profound changes occur, and perhaps one of the most critical of these is during seed filling. By restricting the competition for reserves in Arabidopsis plants, the ability to manipulate seed size, seed weight, or seed content has been explored. Removal of secondary inflorescences and lateral branches resulted in a stimulation of elongation of the primary inflorescence and an increase in the distance between siliques. The pruning treatment also led to the development of longer and larger siliques that contained fewer, bigger seeds. This seems to be a consequence of a reduction in the number of ovules that develop and an increase in the fatty acid content of the seeds that mature. The data show that shoot architecture could have a substantial impact on the partitioning of reserves between vegetative and reproductive tissues and could be an important trait for selection in rapid phenotyping screens to optimize crop performance.

Extracellular release of a heterologous phytase from roots of transgenic plants: does manipulation of rhizosphere biochemistry impact microbial community structure?

To maintain the sustainability of agriculture, it is imperative that the reliance of crops on inorganic phosphorus (P) fertilizers is reduced. One approach is to improve the ability of crop plants to acquire P from organic sources. Transgenic plants that produce microbial phytases have been suggested as a possible means to achieve this goal. However, neither the impact of heterologous expression of phytase on the ecology of microorganisms in the rhizosphere nor the impact of rhizosphere microorganisms on the efficacy of phytases in the rhizosphere of transgenic plants has been tested. In this paper, we demonstrate that the presence of rhizosphere microorganisms reduced the dependence of plants oil extracellular secretion of phytase from roots when grown in a P-deficient soil. Despite this, the expression of phytase in transgenic plants had little or no impact on the microbial community structure as compared with control plant lines, whereas soil treatments, such as the addition of inorganic P, had large effects. The results demonstrate that soil microorganisms are explicitly involved in the availability of P to plants and that the microbial community in the rhizosphere appears to be resistant to the impacts of single-gene changes in plants designed to alter rhizosphere biochemistry and nutrient cycling.

High prevalence of multidrug-tolerant bacteria and associated antimicrobial resistance genes isolated from ornamental fish and their carriage water

Background: Antimicrobials are used to directly control bacterial infections in pet (ornamental) fish and are routinely added to the water these fish are shipped in to suppress the growth of potential pathogens during transport. Methodology/Principal Findings: To assess the potential effects of this sustained selection pressure, 127 Aeromonas spp. isolated from warm and cold water ornamental fish species were screened for tolerance to 34 antimicrobials. Representative isolates were also examined for the presence of 54 resistance genes by a combination of miniaturized microarray and conventional PCR. Forty-seven of 94 Aeromonas spp. isolates recovered from tropical ornamental fish and their carriage water were tolerant to >= 15 antibiotics, representing seven or more different classes of antimicrobial. The quinolone and fluoroquinolone resistance gene, qnrS2, was detected at high frequency (37% tested recent isolates were positive by PCR). Class 1 integrons, IncA/C broad host range plasmids and a range of other antibiotic resistance genes, including floR, blaTEM21, tet(A), tet(D), tet(E), qacE2, sul1, and a number of different dihydrofolate reductase and aminoglycoside transferase coding genes were also detected in carriage water samples and bacterial isolates. Conclusions: These data suggest that ornamental fish and their carriage water act as a reservoir for both multi-resistant bacteria and resistance genes.