Barley Leaf Tissue Research Articles

A secreted fungal histidine‐ and alanine‐rich protein regulates metal ion homeostasis and oxidative stress

Like pathogens, beneficial endophytic fungi secrete effector proteins to promote plant colonization, for example, through perturbation of host immunity. The genome of the root endophyte Serendipita indica encodes a novel family of highly similar, small alanine- and histidine-rich proteins, whose functions remain unknown. Members of this protein family carry an N-terminal signal peptide and a conserved C-terminal DELD motif. Here we report on the functional characterization of the plant-responsive DELD family protein Dld1 using a combination of structural, biochemical, biophysical and cytological analyses. The crystal structure of Dld1 shows an unusual, monomeric histidine zipper consisting of two antiparallel coiled-coil helices. Similar to other histidine-rich proteins, Dld1 displays varying affinity to different transition metal ions and undergoes metal ion- and pH-dependent unfolding. Transient expression of mCherry-tagged Dld1 in barley leaf and root tissue suggests that Dld1 localizes to the plant cell wall and accumulates at cell wall appositions during fungal penetration. Moreover, recombinant Dld1 enhances barley root colonization by S.indica, and inhibits H2 O2 -mediated radical polymerization of 3,3'-diaminobenzidine. Our data suggest that Dld1 has the potential to enhance micronutrient accessibility for the fungus and to interfere with oxidative stress and reactive oxygen species homeostasis to facilitate host colonization.

Visualization of spatial gene expression in plants by modified RNAscope fluorescent in situ hybridization

BackgroundIn situ analysis of biomarkers such as DNA, RNA and proteins are important for research and diagnostic purposes. At the RNA level, plant gene expression studies rely on qPCR, RNAseq and probe-based in situ hybridization (ISH). However, for ISH experiments poor stability of RNA and RNA based probes commonly results in poor detection or poor reproducibility. Recently, the development and availability of the RNAscope RNA-ISH method addressed these problems by novel signal amplification and background suppression. This method is capable of simultaneous detection of multiple target RNAs down to the single molecule level in individual cells, allowing researchers to study spatio-temporal patterning of gene expression. However, this method has not been optimized thus poorly utilized for plant specific gene expression studies which would allow for fluorescent multiplex detection. Here we provide a step-by-step method for sample collection and pretreatment optimization to perform the RNAscope assay in the leaf tissues of model monocot plant barley. We have shown the spatial distribution pattern of HvGAPDH and the low expressed disease resistance gene Rpg1 in leaf tissue sections of barley and discuss precautions that should be followed during image analysis.ResultsWe have shown the ubiquitous HvGAPH and predominantly stomatal guard cell associated subsidiary cell expressed Rpg1 expression pattern in barley leaf sections and described the improve RNAscope methodology suitable for plant tissues using confocal laser microscope. By addressing the problems in the sample collection and incorporating additional sample backing steps we have significantly reduced the section detachment and experiment failure problems. Further, by reducing the time of protease treatment, we minimized the sample disintegration due to over digestion of barley tissues.ConclusionsRNAscope multiplex fluorescent RNA-ISH detection is well described and adapted for animal tissue samples, however due to morphological and structural differences in the plant tissues the standard protocol is deficient and required optimization. Utilizing barley specific HvGAPDH and Rpg1 RNA probes we report an optimized method which can be used for RNAscope detection to determine the spatial expression and semi-quantification of target RNAs. This optimized method will be immensely useful in other plant species such as the widely utilized Arabidopsis.

MLO Differentially Regulates Barley Root Colonization by Beneficial Endophytic and Mycorrhizal Fungi.

Loss-of-function alleles of MLO (Mildew Resistance Locus O) confer broad-spectrum resistance to foliar infections by powdery mildew pathogens. Like pathogens, microbes that establish mutually beneficial relationships with their plant hosts, trigger the induction of some defense responses. Initially, barley colonization by the root endophyte Serendipita indica (syn. Piriformospora indica) is associated with enhanced defense gene expression and the formation of papillae at sites of hyphal penetration attempts. This phenotype is reminiscent of mlo-conditioned immunity in barley leaf tissue and raises the question whether MLO plays a regulatory role in the establishment of beneficial interactions. Here we show that S. indica colonization was significantly reduced in plants carrying mlo mutations compared to wild type controls. The reduction in fungal biomass was associated with the enhanced formation of papillae. Moreover, epidermal cells of S. indica-treated mlo plants displayed an early accumulation of iron in the epidermal layer suggesting increased basal defense activation in the barley mutant background. Correspondingly, the induction of host cell death during later colonization stages was impaired in mlo colonized plants, highlighting the importance of the early biotrophic growth phase for S. indica root colonization. In contrast, the arbuscular mycorrhizal fungus Funneliformis mosseae displayed a similar colonization morphology on mutant and wild type plants. However, the frequency of mycorrhization and number of arbuscules was higher in mlo-5 mutants. These findings suggest that MLO differentially regulates root colonization by endophytic and AM fungi.

Influence of PEG induced drought stress on molecular and biochemical constituents and seedling growth of Egyptian barley cultivars

In order to investigate the effects of drought stress on germination components of barley cultivars, a laboratory experiment was conducted in a factorial randomized complete design with four replications. The controlled experiment included ten of Egyptian barley cultivars namely; (Giza 123, 124, 125, 126, 127, 129, 130, 134, 135 and 2000) as first factor. The second factor included 4 levels of drought stress inducer by applying 0, 5, 10 and 20% of polyethylene glycol-6000 (PEG) which is equivalent to four osmotic potential levels including −0.001, −0.27, −0.54 and −1.09 MPa, respectively. The results showed that, the highest reduction was related to the drought level of 20% PEG among the barley cultivars. The best cultivars in terms of germination traits were Giza 134, Giza 127, and Giza 126 this indicate their tolerance to drought stress and Giza 130, 135, 2000 cultivars was moderately tolerance and remaining is less tolerance. The protein band 27 kDa and 78 kDa showed high intensity after stress in almost all cultivars. Those two protein bands their exciting was very clear in treated barley leaf tissue. It could be related to dehydrine and oxygen evolving enhancer protein 2 (OEE2) which involved in drought stress tolerance response. Cultivars Giza 127, 130 and 134 showed highest tolerance response under drought stress. The antioxidant enzymes PAGE pattern of Peroxidase (POX), Sodium dismutase (SOD) and Ascorbate peroxidase (APX) for Barley cultivars under drought stress revealed a high activities for Giza 126, 127, 134, 136 and 2000 under −0.5 MPa osmotic stress by PEG in most of their isoforms. Based on similarity coefficient values the highest values were 1.0 with 100% similarly between tolerant cultivars Giza 130 and Giza 127. Similarly between the susceptible cultivars 125 and Giza 129 was 60%.These data confirmed by the growth parameters which we ranked as tolerant to drought stress.

Colonization of Barley by the Broad-Host Hemibiotrophic Pathogen Phytophthora palmivora Uncovers a Leaf Development-Dependent Involvement of Mlo.

The discovery of barley Mlo demonstrated that filamentous pathogens rely on plant genes to achieve entry and lifecycle completion in barley leaves. While having a dramatic effect on foliar pathogens, it is unclear whether overlapping or distinct mechanisms affect filamentous pathogen infection of roots. To remove the bias connected with using different pathogens to understand colonization mechanisms in different tissues, we have utilized the aggressive hemibiotrophic oomycete pathogen Phytophthora palmivora. P. palmivora colonizes root as well as leaf tissues of barley (Hordeum vulgare). The infection is characterized by a transient biotrophy phase with formation of haustoria. Barley accessions varied in degree of susceptibility, with some accessions fully resistant to leaf infection. Notably, there was no overall correlation between degree of susceptibility in roots compared with leaves, suggesting that variation in different genes influences host susceptibility above and below ground. In addition, a developmental gradient influenced infection, with more extensive colonization observed in mature leaf sectors. The mlo5 mutation attenuates P. palmivora infection but only in young leaf tissues. The barley-P. palmivora interaction represents a simple system to identify and compare genetic components governing quantitative colonization in diverse barley tissue types.

Changes in Physiological and Agronomical Parameters of Barley (Hordeum vulgare) Exposed to Cerium and Titanium Dioxide Nanoparticles.

The aims of our experiment were to evaluate the uptake and translocation of cerium and titanium oxide nanoparticles and to verify their effects on the growth cycle of barley (Hordeum vulgare L.). Barley plants were grown to physiological maturity in soil enriched with either 0, 500 or 1000 mg·kg−1 cerium oxide nanoparticles (nCeO2) or titanium oxide nanoparticles (nTiO2) and their combination. The growth cycle of nCeO2 and nTiO2 treated plants was about 10 days longer than the controls. In nCeO2 treated plants the number of tillers, leaf area and the number of spikes per plant were reduced respectively by 35.5%, 28.3% and 30% (p ≤ 0.05). nTiO2 stimulated plant growth and compensated for the adverse effects of nCeO2. Concentrations of Ce and Ti in aboveground plant fractions were minute. The fate of nanomaterials within the plant tissues was different. Crystalline nTiO2 aggregates were detected within the leaf tissues of barley, whereas nCeO2 was not present in the form of nanoclusters.

A transient gene expression system using barley protoplasts to evaluate microRNAs for post-transcriptional regulation of their target genes

Transient gene expression assays using protoplasts have been frequently used in high-throughput screening and functional characterization of plant genes. In barley, however, very few studies have explored the use of protoplasts isolated from green tissues. In this study, a reliable and efficient transient gene expression system has been established using barley green tissue protoplasts. Due to the importance of osmolarity in maintaining protoplast viability and transfection efficiency, different mannitol concentrations were tested to determine the optimal osmolarity suitable for barley protoplast preparation. The method and conditions were also described for efficient isolation of protoplasts from barley leaf and stem tissues and transient expression of exogenous gene constructs. This transient expression system has been successfully demonstrated for protein immunoblot analysis, subcellular protein localization and quantitative analysis of gene expression. Furthermore, a simplified method has been described to quickly evaluate microRNAs for post-transcriptional regulation of their target genes in barley protoplasts.

Members of the barley NAC transcription factor gene family show differential co-regulation with senescence-associated genes during senescence of flag leaves.

The senescence process of plants is important for the completion of their life cycle, particularly for crop plants, it is essential for efficient nutrient remobilization during seed filling. It is a highly regulated process, and in order to address the regulatory aspect, the role of genes in the NAC transcription factor family during senescence of barley flag leaves was studied. Several members of the NAC transcription factor gene family were up-regulated during senescence in a microarray experiment, together with a large range of senescence-associated genes, reflecting the coordinated activation of degradation processes in senescing barley leaf tissues. This picture was confirmed in a detailed quantitative reverse transcription-PCR (qRT-PCR) experiment, which also showed distinct gene expression patterns for different members of the NAC gene family, suggesting a group of ~15 out of the 47 studied NAC genes to be important for signalling processes and for the execution of degradation processes during leaf senescence in barley. Seven models for DNA-binding motifs for NAC transcription factors were designed based on published motifs, and available promoter sequences of barley genes were screened for the motifs. Genes up-regulated during senescence showed a significant over-representation of the motifs, suggesting regulation by the NAC transcription factors. Furthermore, co-regulation studies showed that genes possessing the motifs in the promoter in general were highly co-expressed with members of the NAC gene family. In conclusion, a list of up to 15 NAC genes from barley that are strong candidates for being regulatory factors of importance for senescence and biotic stress-related traits affecting the productivity of cereal crop plants has been generated. Furthermore, a list of 71 senescence-associated genes that are potential target genes for these NAC transcription factors is presented.

Cell surface and cell outline imaging in plant tissues using the backscattered electron detector in a variable pressure scanning electron microscope.

BackgroundScanning electron microscopy (SEM) has been used for high-resolution imaging of plant cell surfaces for many decades. Most SEM imaging employs the secondary electron detector under high vacuum to provide pseudo-3D images of plant organs and especially of surface structures such as trichomes and stomatal guard cells; these samples generally have to be metal-coated to avoid charging artefacts. Variable pressure-SEM allows examination of uncoated tissues, and provides a flexible range of options for imaging, either with a secondary electron detector or backscattered electron detector. In one application, we used the backscattered electron detector under low vacuum conditions to collect images of uncoated barley leaf tissue followed by simple quantification of cell areas.ResultsHere, we outline methods for backscattered electron imaging of a variety of plant tissues with particular focus on collecting images for quantification of cell size and shape. We demonstrate the advantages of this technique over other methods to obtain high contrast cell outlines, and define a set of parameters for imaging Arabidopsis thaliana leaf epidermal cells together with a simple image analysis protocol. We also show how to vary parameters such as accelerating voltage and chamber pressure to optimise imaging in a range of other plant tissues.ConclusionsBackscattered electron imaging of uncoated plant tissue allows acquisition of images showing details of plant morphology together with images of high contrast cell outlines suitable for semi-automated image analysis. The method is easily adaptable to many types of tissue and suitable for any laboratory with standard SEM preparation equipment and a variable-pressure-SEM or tabletop SEM.

Broad-Spectrum Acquired Resistance in Barley Induced by the Pseudomonas Pathosystem Shares Transcriptional Components with Arabidopsis Systemic Acquired Resistance

Inducible resistance responses play a central role in the defense of plants against pathogen attack. Acquired resistance (AR) is induced alongside defense toward primary attack, providing broad-spectrum protection against subsequent pathogen challenge. The localization and molecular basis of AR in cereals is poorly understood, in contrast with the well-characterized systemic acquired resistance (SAR) response in Arabidopsis. Here, we use Pseudomonas syringae as a biological inducer of AR in barley, providing a clear frame of reference to the Arabidopsis-P. syringae pathosystem. Inoculation of barley leaf tissue with the nonadapted P. syringae pv. tomato avrRpm1 (PstavrRpm1) induced an active local defense response. Furthermore, inoculation of barley with PstavrRpm1 resulted in the induction of broad-spectrum AR at a distance from the local lesion, "adjacent" AR, effective against compatible isolates of P. syringae and Magnaporthe oryzae. Global transcriptional profiling of this adjacent AR revealed similarities with the transcriptional profile of SAR in Arabidopsis, as well as transcripts previously associated with chemically induced AR in cereals, suggesting that AR in barley and SAR in Arabidopsis may be mediated by analogous pathways.

Plasma membrane H+‐ATPase gene expression, protein level and activity in growing and non‐growing regions of barley (Hordeum vulgare) leaves

Plasma membrane proton ATPase (PM-H⁺-ATPase) is the key means through which plant cells energize nutrient uptake and acidify the apoplast. Both of these processes aid cell elongation; yet, it is not known how such a suspected role of the PM-H⁺-ATPase in growth is reflected through changes in its transcript level and activity in grass leaves. In the present study on leaf three of barley, the elongation zone and the emerged blade, which contained fully expanded cells were analyzed. Plasma membranes were isolated and used to assay the activity (ATPase assay) and abundance (western blotting) of PM-H⁺-ATPase protein. Expression of mRNA was quantified using real-time polymerase chain reaction (qPCR). PM-H⁺-ATPase transcript and protein level and activity differed little between growing and non-growing leaf regions when values were related to unit extracted total RNA and cell number, respectively. However, when values were related to unit surface area of plasma membrane, they were more than twice as high in growing compared with non-growing leaf tissue. It is concluded that this higher surface density of PM-H⁺-ATPase activity in growing barley leaf tissue aids apoplast acidification and cell expansion.

Characterization of barley leaf tissue using direct and indirect desorption electrospray ionization imaging mass spectrometry

Chemical profiling of barley (Hordeum vulgare) leaves was demonstrated using direct and indirect desorption electrospray ionization (DESI) imaging mass spectrometry. Direct DESI analysis of the untreated leaves was not possible despite a significant content of hydroxynitrile glucosides known to reside in the epidermis of the leaves. Instead, the epidermis was stripped off the leaves, thus allowing direct DESI imaging to be performed on the back of the epidermis. Furthermore, indirect DESI imaging was performed by making imprints in porous Teflon of the intact leaves as well as of the stripped epidermis. The DESI images reveal accumulation of hydroxynitrile glucosides in the leaf epidermis, homogeneously distributed throughout the surface. The indirect DESI approach enables relative quantitation, confirming variations of hydroxynitrile glucosides content in primary leaves of three different cultivars of barley seedlings. The study presents an example of how to overcome the morphological barriers from the plant surface and perform rapid and repeatable DESI imaging. In addition, a comparison is made of direct and indirect DESI imaging, contributing to the characterization of the recently developed method of indirect DESI imaging of plant material via porous Teflon imprints.

Identification of Pyrenophora teres f. maculata, Causal Agent of Spot Type Net Blotch of Barley in North Dakota.

Net blotch of barley (Hordeum vulgare L.) caused by the fungus Pyrenophora teres (anamorph Drechslera teres) is found in two forms, net form net blotch (NFNB) and spot form net blotch (SFNB). When inoculated on susceptible varieties, P. teres f. teres produces lesions with a characteristic net-like pattern surrounded by necrosis or chlorosis (NFNB), whereas P. teres f. maculata produces lesions consisting of spots surrounded by necrosis or chlorosis (SFNB). Recently, epidemics of SFNB have occurred throughout the world (4). Currently, net blotch is a significant foliar disease of barley in the North Dakota-Northwestern Minnesota agricultural region, a leading barley-production area. Diseased barley leaf tissue was collected annually from 2004 to 2008 in Fargo and Langdon, ND. Diseased leaves were incubated to promote sporulation. Ten single-spore isolates of P. teres collected from each location each year were tested for virulence by inoculation on 20 commonly used barley net blotch differential lines. Among the 100 isolates collected, one isolate collected in Fargo in 2006 (FGOH06Pt-8) and one isolate collected in Langdon in 2008 (LDNH08Pt-4) were identified as P. teres f. maculata due to their induction of spot-type lesions across the differential set. Conidial morphology of the two isolates was similar to P. teres f. teres isolates. A pathogenicity test of all isolates was performed on regional barley cvs. Tradition, Robust, and Lacey as well as barley lines Rika and Kombar (1) as previously described (3). The net form isolate 0-1 and spot form isolate DEN2.6 (obtained from B. Steffenson, University of Minnesota) were used as controls. The P. teres f. teres isolate 0-1 produced typical net type symptoms on all barley lines except the resistant line Rika, in which only small, dark spots were observed. DEN2.6 produced pin-point spot-like lesions with an extensive yellow halo on Robust, Lacey, Rika, and Kombar, but without chlorosis on Tradition. The two newly identified isolates induced elliptical spot-type lesions measuring 3 × 6 mm, larger than those produced by P. teres f. maculata isolate DEN 2.6, suggesting a higher level of virulence. We constructed a neighbor-joining phylogenetic tree using ClustalW2 ( http://www.ebi.ac.uk/ ) based on sequence identity of the internal transcribed spacer (ITS) region from 0-1 (GenBank No. GU014819), DEN2.6 (GenBank No. GU014820), FGOH06Pt-8 (GenBank No. GU014821), and LDNH08Pt-4 (GenBank No. GU014822) as well as P. teres f. maculata, P. teres f. teres, and P. tritici-repentis (causal agent of tan spot of wheat) accessions obtained from GenBank (2). All P. teres isolates clustered together and were clearly separated from the P. tritici-repentis cluster. Isolates FGOH06Pt-8 and LDNH08Pt-4 had identical ITS sequences and differed from DEN2.6 by only a single nucleotide. To our knowledge, this is the first report of P. teres f. maculata in North Dakota. Resistance to SFNB should now be considered in local barley breeding programs and cultivar releases. Reference: (1) M. Abu Qamar. Theor. Appl. Genet. 117:1261, 2008. (2) R. M. Andrie et al. Fungal Genet. Biol. 45:363, 2008. (3) Z. Lai et al. Fungal Genet. Biol. 44:323, 2007. (4) M. S. McLean et al. Crop Pasture Sci. 60:303, 2009.

Effects of Deoxynivalenol on Content of Chloroplast Pigments in Barley Leaf Tissues

To understand further the role of deoxynivalenol (DON) in development of Fusarium head blight (FHB), we investigated effects of the toxin on uninfected barley tissues. Leaf segments, 1 to 1.2 cm long, partially stripped of epidermis were floated with exposed mesophyll in contact with DON solutions. In initial experiments with the leaf segments incubated in light, DON at 30 to 90 ppm turned portions of stripped tissues white after 48 to 96 h. The bleaching effect was greatly enhanced by addition of 1 to 10 mM Ca(2+), so that DON at 10 to 30 ppm turned virtually all stripped tissues white within 48 h. Content of chlorophylls a and b and of total carotenoid pigment was reduced. Loss of electrolytes and uptake of Evans blue indicated that DON had a toxic effect, damaging plasmalemmas in treated tissues before chloroplasts began to lose pigment. When incubated in the dark, leaf segments also lost electrolytes, indicating DON was toxic although the tissues remained green. Thus, loss of chlorophyll in light was due to photobleaching and was a secondary effect of DON, not required for toxicity. In contrast to bleaching effects, some DON treatments that were not toxic kept tissues green without bleaching or other signs of injury, indicating senescence was delayed compared with slow yellowing of untreated leaf segments. Cycloheximide, which like DON, inhibits protein synthesis, also bleached some tissues and delayed senescence of others. Thus, the effects of DON probably relate to its ability to inhibit protein synthesis. With respect to FHB, the results suggest DON may have multiple roles in host cells of infected head tissues, including delayed senescence in early stages of infection and contributing to bleaching and death of cells in later stages.

A Single Binding Site Mediates Resistance- and Disease-Associated Activities of the Effector Protein NIP1 from the Barley Pathogen Rhynchosporium secalis

The effector protein NIP1 from the barley (Hordeum vulgare) pathogen Rhynchosporium secalis specifically induces the synthesis of defense-related proteins in cultivars of barley expressing the complementary resistance gene, Rrs1. In addition, it stimulates the activity of the barley plasma membrane H(+)-ATPase in a genotype-unspecific manner and it induces necrotic lesions in leaf tissues of barley and other cereal plant species. NIP1 variants type I and II, which display quantitative differences in their activities as elicitor and H(+)-ATPase stimulator, and the inactive mutant variants type III* and type IV*, were produced in Escherichia coli. Binding studies using (125)I-NIP1 type I revealed a single class of binding sites with identical binding characteristics in microsomes from near-isogenic resistant (Rrs1) and susceptible (rrs1) barley. Binding was specific, reversible, and saturable, and saturation ligand-binding experiments yielded a K(d) of 5.6 nm. A binding site was also found in rye (Secale cereale) and the nonhost species wheat (Triticum aestivum), oat (Avena sativa), and maize (Zea mays), but not in Arabidopsis (Arabidopsis thaliana). For NIP1 types I and II, equilibrium competition-binding experiments revealed a correlation between the difference in their affinities to the binding site and the differences in their elicitor activity and H(+)-ATPase stimulation, indicating a single target molecule to mediate both activities. In contrast, the inactive proteins type III* and type IV* are both characterized by high affinities similar to type I, suggesting that binding of NIP1 to this target is not sufficient for its activities.

Water permeability differs between growing and non-growing barley leaf tissues

A pressure probe technique and an osmotic swelling assay were used to compare water transport properties between growing and non-growing tissues of leaf three of barley. The epidermis was analysed in planta by pressure probe, whereas (predominantly) mesophyll protoplasts were analysed by osmotic swelling. Hydraulic conductivity (Lp) and, by implication, water permeability (Pf) of epidermal cells was 31% higher in the leaf elongation zone (Lp=0.5+/-0.2 microm s-1 MPa-1; Pf=65+/-25 microm s-1; means+/-SD of n=17 cells) than in the, non-growing, emerged leaf zone (Lp=0.4+/-0.1 microm s-1 MPa-1; Pf=50+/-15 microm s-1; n=24; P<0.05). Similarly, water permeability of mesophyll protoplasts was by 55% higher in the elongation compared with emerged leaf zone (Pf=13+/-1 microm s-1 compared with 8+/-1 microm s-1; n=57 and 36 protoplasts, respectively; P<0.01). Within the leaf elongation zone, a small population of larger-sized protoplasts could be distinguished. These protoplasts, which originated most likely from parenchymateous bundle sheath or midrib parenchyma cells, had a three-fold higher water permeability (P<0.001) as mesophyll protoplasts. The effect on Lp and Pf of known aquaporin inhibitors was tested with the pressure probe (Au+, Ag+, Hg2+, phloretin) and the osmotic swelling assay (phloretin). Only phloretin, when applied to protoplasts in the swelling assay caused an average decrease in Pf, but the effect varied between isolations. Technical approaches and cell-type and growth-specific differences in water transport properties are discussed.

Barley transcript profiles under dehydration shock and drought stress treatments: a comparative analysis

A microarray including 1654 cDNAs, mainly derived from dehydration-shocked barley leaf tissues, was utilized to monitor expression changes in leaves of barley plants subjected to slow drying conditions (7 d and 11 d: 7d-WS and 11d-WS) in soil and after rehydration. The results were compared with those obtained under shock-like conditions imposed with a 6 h dehydration treatment. A total number of 173 transcripts (approximately 10% of all transcripts profiled) were declared up- or down-regulated in at least one of the conditions tested. The majority of the transcripts were regulated by only one of the drought treatments, with 57% of the differentially expressed transcripts exclusively affected in the dehydration shock treatment, 6% at 7d-WS, 14% at 11d-WS, and 6% after rehydration. Irrespective of the low percentage of transcripts (10%) with similar expression changes between shock and slow stress treatments, a sizeable portion of these transcripts shared a common expression trend under the different drought treatment conditions, as evidenced by low but significant correlations between the fast occurring and the 7d-WS and 11d-WS treatments (r=0.32 and 0.41, P=0.001, respectively). These results are discussed with respect to the merit of different dehydration treatments in the investigation of the changes in transcript profiling.

Rapid and tissue-specific accumulation of solutes in the growth zone of barley leaves in response to salinity.

The aim of the present study was to test whether rapid accumulation of solutes in response to salinity in leaf tissues of barley (Hordeum vulgare L.) contributes to recovery and maintenance of residual elongation growth. Addition of 100 mM NaCl to the root medium caused an immediate reduction close to zero in elongation velocity of the growing leaf 3. After 20-30 min, elongation velocity recovered suddenly, to 40-50% of the pre-stress level. Bulk osmolality increased first, after 60 min, significantly in the proximal half of the elongation zone. Over the following 3 days, osmolality increases became significant in the distal half of the elongation zone, the adjacent, enclosed non-elongation zone and finally in the emerged portion of the blade. The developmental gradient and time course in osmolality increase along the growing leaf was reflected in the pattern of solute (Cl, Na and K) accumulation in bulk tissue and epidermal cells. The partitioning of newly accumulated solutes between epidermis and bulk tissue changed with time. Even though solute accumulation does not contribute to the sudden and partial growth recovery 20-30 min after exposure to salt, it does facilitate residual growth from 1 h onwards. This is due to a high sink strength for solutes of the proximal part of the growth zone and its ability to accumulate solutes rapidly and at high rates.

Applied jasmonates accumulate extracellularly in tomato, but intracellularly in barley

Jasmonic acid (JA) and its derivatives are well-characterized signaling molecules in plant defense and development, but the site of their localization within plant tissue is entirely unknown. To address the question whether applied JA accumulates extracellularly or intracellularly, leaves of tomato and barley were fed with 14C-labeled JA and the label was localized in cryofixed and lyophilized leaf tissues by microautoradiography. In tomato the radioactivity was detectable within the apoplast, but no label was found within the mesophyll cells. By contrast, in barley leaf tissues, radioactivity was detected within the mesophyll cells suggesting a cellular uptake of exogenously applied JA. JA, applied to leaves of both plants as in the labeling experiments, led in all leaf cells to the expression of JA-inducible genes indicating that the perception is completed by JA signal transduction.

Differential Defense Reactions in Leaf Tissues of Barley in Response to Infection by Rhynchosporium secalis and to Treatment with a Fungal Avirulence Gene Product

Expression of defense-associated genes was analyzed in leaf tissues of near-isogenic resistant and susceptible barley cultivars upon infection by Rhynchosporium secalis. The genes encoding pathogenesis-related (PR) proteins PR-1, PR-5, and PR-9 are specifically expressed in the mesophyll of resistant plants, whereas a germin-like protein (OxOLP) is synthesized in the epidermis irrespective of the resistance genotype. Restriction-mediated differential display was employed to identify additional epidermis-specific genes. This resulted in the detection of another PR gene, PR-10, along with a lipoxygenase gene, LoxA, and a gene of unknown function, pI2-4, which are specifically induced in the epidermis of resistant plants. The gene encoding a putative protease inhibitor, SD10, is preferentially but not exclusively expressed in the epidermis. The fungal avirulence gene product NIP1 triggers the induction of the four PR genes only. At least two additional elicitors, therefore, must be postulated, one for the unspecific induction of OxOLP and one for the resistance-specific induction of LoxA, pI2-4, and SD10. PR-10 expression can be assumed to be the consequence of NIP1 perception by epidermis cells. In contrast, gene expression in the mesophyll is likely to be triggered by an as yet unknown signal that appears to originate in the epidermis and that is strongly amplified in the mesophyll.