Dipodal Silanes Greatly Stabilize Glass Surface Functionalization for DNA Microarray Synthesis and High-Throughput Biological Assays.

Glass is by far the most common substrate for biomolecular arrays, including high-throughput sequencing flow cells and microarrays. The native glass hydroxyl surface is modified by using silane chemistry to provide appropriate functional groups and reactivities for either in situ synthesis or surface immobilization of biologically or chemically synthesized biomolecules. These arrays, typically of oligonucleotides or peptides, are then subjected to long incubation times in warm aqueous buffers prior to fluorescence readout. Under these conditions, the siloxy bonds to the glass are susceptible to hydrolysis, resulting in significant loss of biomolecules and concomitant loss of signal from the assay. Here, we demonstrate that functionalization of glass surfaces with dipodal silanes results in greatly improved stability compared to equivalent functionalization with standard monopodal silanes. Using photolithographic in situ synthesis of DNA, we show that dipodal silanes are compatible with phosphoramidite chemistry and that hybridization performed on the resulting arrays provides greatly improved signal and signal-to-noise ratios compared with surfaces functionalized with monopodal silanes.

Global transcriptome profiling reveals root- and leaf-specific responses of barley (Hordeum vulgare L.) to H2O2.

In cereal crops, such as barley (Hordeum vulgare L.), the ability to appropriately respond to environmental cues is an important factor for yield stability and thus for agricultural production. Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), are key components of signal transduction cascades involved in plant adaptation to changing environmental conditions. H2O2-mediated stress responses include the modulation of expression of stress-responsive genes required to cope with different abiotic and biotic stresses. Despite its importance, knowledge of the effects of H2O2 on the barley transcriptome is still scarce. In this study, we identified global transcriptomic changes induced after application of 10 mM H2O2 to five-day-old barley plants. In total, 1883 and 1001 differentially expressed genes (DEGs) were identified in roots and leaves, respectively. Most of these DEGs were organ-specific, with only 209 DEGs commonly regulated and 37 counter-regulated between both plant parts. A GO term analysis further confirmed that different processes were affected in roots and leaves. It revealed that DEGs in leaves mostly comprised genes associated with hormone signaling, response to H2O2 and abiotic stresses. This includes many transcriptions factors and small heat shock proteins. DEGs in roots mostly comprised genes linked to crucial aspects of H2O2 catabolism and oxidant detoxification, glutathione metabolism, as well as cell wall modulation. These categories include many peroxidases and glutathione transferases. As with leaves, the H2O2 response category in roots contains small heat shock proteins, however, mostly different members of this family were affected and they were all regulated in the opposite direction in the two plant parts. Validation of the expression of the selected commonly regulated DEGs by qRT-PCR was consistent with the RNA-seq data. The data obtained in this study provide an insight into the molecular mechanisms of oxidative stress responses in barley, which might also play a role upon other stresses that induce oxidative bursts.

Comparative analysis of stress-induced calcium signals in the crop species barley and the model plant Arabidopsis thaliana.

BACKGROUND: Plants are continuously exposed to changing environmental conditions and biotic attacks that affect plant growth. In crops, the inability to respond appropriately to stress has strong detrimental effects on agricultural production and yield. Ca2+ signalling plays a fundamental role in the response of plants to most abiotic and biotic stresses. However, research on stimulus-specific Ca2+ signals has mostly been pursued in Arabidopsis thaliana, while in other species these events are little investigated . RESULTS: In this study, we introduced the Ca2+ reporter-encoding gene APOAEQUORIN into the crop species barley (Hordeum vulgare). Measurements of the dynamic changes in [Ca2+]cyt in response to various stimuli such as NaCl, mannitol, H2O2, and flagellin 22 (flg22) revealed the occurrence of dose- as well as tissue-dependent [Ca2+]cyt transients. Moreover, the Ca2+ signatures were unique for each stimulus, suggesting the involvement of different Ca2+ signalling components in the corresponding stress response. Alongside, the barley Ca2+ signatures were compared to those produced by the phylogenetically distant model plant Arabidopsis. Notable differences in temporal kinetics and dose responses were observed, implying species-specific differences in stress response mechanisms. The plasma membrane Ca2+ channel blocker La3+ strongly inhibited the [Ca2+]cyt response to all tested stimuli, indicating a critical role of extracellular Ca2+ in the induction of stress-associated Ca2+ signatures in barley. Moreover, by analysing spatio-temporal dynamics of the [Ca2+]cyt transients along the developmental gradient of the barley leaf blade we demonstrate that different parts of the barley leaf show quantitative differences in [Ca2+]cyt transients in response to NaCl and H2O2. There were only marginal differences in the response to flg22, indicative of developmental stage-dependent Ca2+ responses specifically to NaCl and H2O2. CONCLUSION: This study reveals tissue-specific Ca2+ signals with stimulus-specific kinetics in the crop species barley, as well as quantitative differences along the barley leaf blade. A number of notable differences to the model plants Arabidopsis may be linked to different stimulus sensitivity. These transgenic barley reporter lines thus present a valuable tool to further analyse mechanisms of Ca2+ signalling in this crop and to gain insights into the variation of Ca2+-dependent stress responses between stress-susceptible and -resistant species.

Organellar calcium signaling in plants: An update.

Calcium ion (Ca2+) is a versatile signaling transducer in all eukaryotic organisms. In plants, intracellular changes in free Ca2+ levels act as regulators in many growth and developmental processes. Ca2+ also mediates the cellular responses to environmental stimuli and thus plays an important role in providing stress tolerance to plants. Ca2+ signals are decoded by a tool kit of various families of Ca2+-binding proteins and their downstream targets, which mediate the transformation of the Ca2+ signal into appropriate cellular response. Early interest and research on Ca2+ signaling focused on its function in the cytosol, however it has become evident that this important regulatory pathway also exists in organelles such as nucleus, chloroplast, mitochondria, peroxisomes and the endomembrane system. In this review, we give an overview on the knowledge about organellar Ca2+ signaling with a focus on recent advances and developments.