Position of the fluorescent label is a crucial factor determining signal intensity in microarray hybridizations

A key issue in applications of short oligonucleotide-based microarrays is how to design specific probes with high sensitivity. Some details of the factors affecting microarray hybridization remain unclear, hampering a reliable quantification of target nucleic acids. We have evaluated the effect of the position of the fluorescent label [position of label (POL)] relative to the probe-target duplex on the signal output of oligonucleotide microarrays. End-labelled single-stranded DNA targets of different lengths were used for hybridization with perfect-match oligonucleotide probe sets targeting different positions of the same molecule. Hybridization results illustrated that probes targeting the labelled terminus of the target showed significantly higher signals than probes targeting other regions. This effect was independent of the target gene, the fluorophore and the slide surface chemistry. Comparison of microarray signal patterns of fluorescently end-labelled, fluorescently internally random-labelled and radioactively end-labelled target-DNAs with the same set of oligonucleotide probes identified POL as a critical factor affecting signal intensity rather than binding efficiency. Our observations define a novel determinant for large differences of signal intensities. Application of the POL effect may contribute to better probe design and data interpretation in microarray applications

Global characterization of the root transcriptome of a wild species of rice, Oryza longistaminata, by deep sequencing

<p>Abstract</p> <p>Background</p> <p><it>Oryza longistaminata</it>, an AA genome type (2 n = 24), originates from Africa and is closely related to Asian cultivated rice (<it>O. sativa L</it>.). It contains various valuable traits with respect to tolerance to biotic and abiotic stress, QTLs with agronomically important traits and high ability to use nitrogen efficiently (NUE). However, only limited genomic or transcriptomic data of <it>O. longistaminata </it>are currently available.</p> <p>Results</p> <p>In this study we present the first comprehensive characterization of the <it>O. longistaminata </it>root transcriptome using 454 pyrosequencing. One sequencing run using a normalized cDNA library from <it>O. longistaminata </it>roots adapted to low N conditions generated 337,830 reads, which assembled into 41,189 contigs and 30,178 singletons. By similarity search against protein databases, putative functions were assigned to over 34,510 uni-ESTs. Comparison with ESTs derived from cultivated rice collections revealed expressed genes across different plant species, however 16.7% of the <it>O. longistaminata </it>ESTs had not been detected as expressed in <it>O. sativa</it>. Additionally, 15.7% had no significant similarity to known sequences. RT-PCR and Southern blot analyses confirmed the expression of selected novel transcripts in <it>O. longistaminata</it>.</p> <p>Conclusion</p> <p>Our results show that one run using a Genome Sequencer FLX from 454 Life Science/Roche generates sufficient genomic information for adequate de novo assembly of a large number of transcripts in a wild rice species, <it>O. longistaminata</it>. The generated sequence data are publicly available and will facilitate gene discovery in <it>O. longistaminata </it>and rice functional genomic studies. The large number of abundant of novel ESTs suggests different metabolic activity in <it>O. longistaminata </it>roots in comparison to <it>O. sativa </it>roots.</p

Catch crop diversity increases rhizosphere carbon input and soil microbial biomass

Catch crops increase plant species richness in crop rotations, but are most often grown as pure stands. Here, we investigate the impacts of increasing plant diversity in catch crop rotations on rhizosphere C input and microbial utilization. Mustard (Sinapis alba L.) planted as a single cultivar was compared to diversified catch crop mixtures of four (Mix4) or 12 species (Mix12). We traced the C transfer from shoots to roots towards the soil microbial community and the soil respiration in a 13C pulse labelling field experiment. Net CO2-C uptake from the atmosphere increased by two times in mix 4 and more than three times in mix 12. Higher net ecosystem C production was linked to increasing catch crop diversity and increased belowground transfer rates of recently fixed photoassimilates. The higher rhizosphere C input stimulated the growth and activity of the soil microbiome, which was investigated by phospholipid fatty acid (PLFA) analyses. Total microbial biomass increased from 14 to 22 g m−2 as compared to the fallow and was 18 and 8% higher for mix 12 and mix 4 as compared to mustard. In particular, the fungal and actinobacterial communities profited the most from the higher belowground C input and their biomass increased by 3.4 and 1.3 times as compared to the fallow. The residence time of the 13C pulse, traced in the CO2 flux from the soil environment, increased with plant diversity by up to 1.8 times. The results of this study suggest positive impacts of plant diversity on C cycling by higher atmospheric C uptake, higher transport rates towards the rhizosphere, higher microbial incorporation and prolonged residence time in the soil environment. We conclude that diversified catch crop mixtures improve the efficiency of C cycling in cropping systems and provide a promising tool for sustainable soil management

Seasonal variation in the biocontrol efficiency of bacterial wilt is driven by temperature-mediated changes in bacterial competitive interactions

Microbe-based biocontrol applications hold the potential to become an efficient way to control plant pathogen disease outbreaks in the future. However, their efficiency is still very variable, which could be due to their sensitivity to the abiotic environmental conditions. Here, we assessed how environmental temperature variation correlates with ability of Ralstonia pickettii, an endophytic bacterial biocontrol agent, to suppress the Ralstonia solanacearum pathogen during different tomato crop seasons in China. We found that suppression of the pathogen was highest when the seasonal mean temperatures were around 20 °C and rapidly decreased with increasing mean crop season temperatures. Interestingly, low levels of disease incidence did not correlate with low pathogen or high biocontrol agent absolute densities. Instead, the biocontrol to pathogen density ratio was a more important predictor of disease incidence levels between different crop seasons. To understand this mechanistically, we measured the growth and strength of competition between the biocontrol agent and the pathogen over a naturally occurring temperature gradient in vitro. We found that the biocontrol strain grew relatively faster at low temperature ranges, and the pathogen at high temperature ranges, and that similar to field experiments, pathogen suppression peaked at 20 °C. Together, our results suggest that temperature-mediated changes in the strength of bacterial competition could potentially explain the variable R. solanacearum biocontrol outcomes between different crop seasons in China. Synthesis and applications. Our results suggest that abiotic environmental conditions, such as temperature, can affect the efficacy of biocontrol applications. Thus, in order to develop more consistent biocontrol applications in the future, we might need to find and isolate bacterial strains that can retain their functionality regardless of the changing environmental conditions

Induction of complex intracytoplasmic membranes related to nitrogen fixation in Azoarcus sp. BH72

We report the discovery of novel subcellular structures related to bacterial nitrogen fixation in the strictly respiratory diazotrophic bacterium Azoarcus sp, BH72, which was isolated as an endophyte from Kallar grass, Nitrogenase is derepressed under microaerobic conditions at O-2 concentrations in the micromolar range. With increasing O-2 deprivation, bacteria can develop into a hyperinduced state, which is characterized by high specific rates of respiration and efficient nitrogen fixation at approximately 30 nM O-2 Ultrastructural analysis of cells in the course of hyperinduction revealed that complex intracytoplasmic membrane systems are formed, which consist of stacks of membranes and which are absent under standard nitrogen-fixing conditions, The iron protein of nitrogenase was highly enriched on these membranes, as evidenced by immunohistochemical studies, Membrane deficiency in NifH/K- mutants, a deletion mutant in the nifK gene and the character of NH4+-grown cells suggested, in concert with the membrane localization of nitrogenase, that these structures are specialized membranes related to nitrogen fixation. We propose the term 'diazosomes' for them, Development of intracytoplasmic membranes coincides with the appearance of a high-molecular-mass form of the iron protein of nitrogenase, which was detectable in membrane fractions, Mutational analysis, and determination of the N-terminal amino acid sequence indicate that the nifH gene product is covalently modified by a mechanism probably different from adenosine diphosphoribosylation. Development of diazosomes in nitrogen-fixing cells can be induced in pure cultures and in co-culture with a fungus isolated from the rhizosphere of Kallar grass