Identifikation und Lokalisation der seneszenzassoziierten Cysteinprotease HvPAP14 in Gerste

Während der Blattseneszenz werden die Proteine des Photosyntheseapparates in den Chloroplasten abgebaut und der enthaltene Stickstoff remobilisiert. Um Proteasen zu identifizieren, die entscheidend am Proteinabbau während der Seneszenz von Gerstenblättern beteiligt sind, wurden zwei Ansätze verfolgt. Zum einen wurde die Genexpression während der Seneszenz an Fahnenblättern der Gerste unter Freilandbedingungen untersucht. Zum anderen wurde ein biochemischer Ansatz zur Identifizierung aktiver Cysteinproteasen in seneszierenden Primärblättern der Gerste durchgeführt. Die Genexpressionsuntersuchungen erfolgten mit Blattmaterial der Hochleistungssorte cv. Lomerit. Durch eine Microarray-Analyse wurden Gene identifiziert, die während der Seneszenz in Blättern der Sorte Lomerit verstärkt exprimiert werden. Zur Unterscheidung von Genen, die entweder eine Rolle in der Stickstoffremobilisierung oder in entwicklungsabhängigen Prozessen spielen, wurde die Genexpression in unterschiedlich stark mit Stickstoff gedüngten Pflanzen verglichen. Das entwicklungsabhängig verstärkt exprimierte Gen HvPAP14 war unabhängig von der Düngung eines der am stärksten während der Seneszenz aufregulierten Gene. Mittels einer Affinitätsaufreinigung über DCG-04 und anschließender Massenspektrometrie wurde HvPAP14 unter den während der Blattseneszenz proteolytisch aktiven Cysteinproteasen identifiziert. Mit Hilfe von zwei Antikörpern gegen unterschiedliche Bereiche von HvPAP14 konnte eine Proform in Microbodies und in Chloroplasten nachgewiesen werden. In den Chloroplasten wurde die Proform sowohl in der Thylakoidmembranfraktion als auch im Lumen und die potentiell aktive Form nur in der Thylakoidmembranfraktion detektiert. Diese Ergebnisse zeigen, dass die Cysteinprotease HvPAP14 aus dem ER in die Chloroplasten transportiert wird und dort vermutlich am Abbau von Proteinen der Thylakoidmembran beteiligt ist.During leaf senescence proteins of the photosynthetic apparatus are degraded in chloroplasts and nitrogen is remobilized. To identify proteases involved in protein degradation during barley leaf senescence, two approaches were used. First, gene expression of barley flag leaves grown under field conditions was analysed. Second, a biochemical approach was used to identify active cysteine proteases in senescing primary foliage leaves. For gene expression studies leaves of the high-yield barley variety cv. Lomerit were used. By microarray analyses senescence-associated genes were identified in barley flag leaves collected from field plots with standard or high nitrogen supply. Due to the different amounts of nitrogen supply it was possible to distinguish between genes that play a role in nitrogen remobilization or in development-dependent processes. Independent of the nitrogen supply, HvPAP14 was identified as one of the most up- regulated genes in senescing flag leaves. By an affinity approach based on the probe DCG-04 and subsequent mass spectrometry, HvPAP14 was identified as one of the proteolytic active cysteine proteases during leaf senescence. Using two antibodies against different regions of HvPAP14 the subcellular localisations of these different forms were analysed. The proform was detected in micro- bodies as well as in chloroplasts in association with thylakoid membranes and in the lumen. The potentially proteolytic active form of the enzyme could be detected exclusively in the thylakoid membranes. The results show, that the cysteine protease HvPAP14 is transported from the ER into the chloroplasts and is presumably involved in the degradation of thylakoid membrane proteins

Getting ready for host invasion: elevated expression and action of xyloglucan endotransglucosylases/hydrolases in developing haustoria of the holoparasitic angiospermcuscuta

Changes in cell walls have been previously observed in the mature infection organ, or haustorium, of the parasitic angiosperm Cuscuta, but are not equally well charted in young haustoria. In this study, we focused on the molecular processes in the early stages of developing haustoria; that is, before the parasite engages in a physiological contact with its host. We describe first the identification of differentially expressed genes in young haustoria whose development was induced by far-red light and tactile stimuli in the absence of a host plant by suppression subtractive hybridization. To improve sequence information and to aid in the identification of the obtained candidates, reference transcriptomes derived from two species of Cuscuta, C. gronovii and C. reflexa, were generated. Subsequent quantitative gene expression analysis with different tissues of C. reflexa revealed that among the genes that were up-regulated in young haustoria, two xyloglucan endotransglucosylase/hydrolase (XTH) genes were highly expressed almost exclusively at the onset of haustorium development. The same expression pattern was also found for the closest XTH homologues from C. gronovii. In situ assays for XTH-specific action suggested that xyloglucan endotransglucosylation was most pronounced in the cell walls of the swelling area of the haustorium facing the host plant, but was also detectable in later stages of haustoriogenesis. We propose that xyloglucan remodelling by Cuscuta XTHs prepares the parasite for host infection and possibly aids the invasive growth of the haustorium

Getting ready for host invasion: elevated expression and action of xyloglucan endotransglucosylases/hydrolases in developing haustoria of the holoparasitic angiospermcuscuta

Changes in cell walls have been previously observed in the mature infection organ, or haustorium, of the parasitic angiosperm Cuscuta, but are not equally well charted in young haustoria. In this study, we focused on the molecular processes in the early stages of developing haustoria; that is, before the parasite engages in a physiological contact with its host. We describe first the identification of differentially expressed genes in young haustoria whose development was induced by far-red light and tactile stimuli in the absence of a host plant by suppression subtractive hybridization. To improve sequence information and to aid in the identification of the obtained candidates, reference transcriptomes derived from two species of Cuscuta, C. gronovii and C. reflexa, were generated. Subsequent quantitative gene expression analysis with different tissues of C. reflexa revealed that among the genes that were up-regulated in young haustoria, two xyloglucan endotransglucosylase/hydrolase (XTH) genes were highly expressed almost exclusively at the onset of haustorium development. The same expression pattern was also found for the closest XTH homologues from C. gronovii. In situ assays for XTH-specific action suggested that xyloglucan endotransglucosylation was most pronounced in the cell walls of the swelling area of the haustorium facing the host plant, but was also detectable in later stages of haustoriogenesis. We propose that xyloglucan remodelling by Cuscuta XTHs prepares the parasite for host infection and possibly aids the invasive growth of the haustorium

Conserved <i>cis</i>-regulatory elements putatively implicated in ABA signalling pathways.

<p>Regions 1 kb upstream of the predicted start codon of HvHK1, HvHP2 and HvRR1 were screened for known <i>cis</i>-elements and interacting transcription factors by using PlantPAN (<a href="http://plantpan.mbc.nctu.edu.tw" target="_blank">http://plantpan.mbc.nctu.edu.tw</a>) web-based interface under the default settings (1.0 - core similarity, 0.75 - matrix similarity; detailed results <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041867#pone.0041867.s006" target="_blank">Table S5</a>).</p>*<p>statistical analysis of co-occurence of transcription factor binding sites in the promoter regions (−1000 bp) of the three genes was conducted by using the PlantPAN tool ‘Gene Group Analysis’ with distance constraint.</p

Phylogenetic relationship of barley TCS elements and counterparts of <i>Arabidopsis thaliana</i> and <i>Oryza sativa</i>.

<p>Phylogenetic trees are based on inferred amino acid sequences from complemented barley 454 contigs (see information in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041867#pone-0041867-t002" target="_blank">Table 2</a>) and full-length amino acid sequences adopted from different databases (TAIR release 9, Rice Genome Annotation project and NCBI Protein database). Multiple alignments were performed using ClustalW algorithm with BLOSUM protein weight matrix (DNAstar software). Barley sequences are highlighted by blue boxes. Elements from other species are identified by chromosome loci or GenBank accession number. (A) Histidine kinases, colours indicate different subgroups. Arabidopsis PDK was used as outgroup. (B) HPt elements, protein sequences of <i>Zea mays</i> and <i>Triticum aestivum</i> were additionally included in the alignment. Yeast HPt protein YPD1 was used as outgroup. (C–E) Type-A, -B, -C response regulators, amino acid sequences of selected maize response regulators were included in the alignment. For reasons of simplicity not all Arabidopsis and rice elements were included in the phylogenetic trees.</p

Comparison of 454 sequences with public barley sequence information.

<p>Venn diagram displays the number of overlapping sequences between ETC-454 contigs and sequences of the HarvEST assembly 35 (BlastN <1E-20). Commonalities using tBlastX searches with different stringencies are given at the bottom.</p