Use of endogenous signal sequences for transient production and efficient secretion by moss (Physcomitrella patens) cells

BACKGROUND: Efficient targeting to appropriate cell organelles is one of the bottlenecks for the production of recombinant proteins in plant systems. A common practice is to use the native secretory signal peptide of the heterologous protein to be produced. Though general features of secretion signals are conserved between plants and animals, the broad sequence variability among signal peptides suggests differing efficiency of signal peptide recognition. RESULTS: Aiming to improve secretion in moss bioreactors, we quantitatively compared the efficiency of two human signal peptides and six signals from recently isolated moss (Physcomitrella patens) proteins. We therefore used fusions of the different signals to heterologous reporter sequences for transient transfection of moss cells and measured the extra- and intracellular accumulation of the recombinant proteins rhVEGF and GST, respectively. Our data demonstrates an up to fivefold higher secretion efficiency with endogenous moss signals compared to the two utilised human signal peptides. CONCLUSION: From the distribution of extra- and intracellular recombinant proteins, we suggest translational inhibition during the signal recognition particle-cycle (SRP-cycle) as the most probable of several possible explanations for the decreased extracellular accumulation with the human signals. In this work, we report on the supremacy of moss secretion signals over the utilised heterologous ones within the moss-bioreactor system. Though the molecular details of this effect remain to be elucidated, our results will contribute to the improvement of molecular farming systems

ABA-Induced Vegetative Diaspore Formation in Physcomitrella patens

The phytohormone abscisic acid (ABA) is a pivotal regulator of gene expression in response to various environmental stresses such as desiccation, salt and cold causing major changes in plant development and physiology. Here we show that in the moss Physcomitrella patens exogenous application of ABA triggers the formation of vegetative diaspores (brachycytes or brood cells) that enable plant survival in unfavorable environmental conditions. Such diaspores are round-shaped cells characterized by the loss of the central vacuole, due to an increased starch and lipid storage preparing these cells for growth upon suitable environmental conditions. To gain insights into the gene regulation underlying these developmental and physiological changes, we analyzed early transcriptome changes after 30, 60, and 180 min of ABA application and identified 1,030 differentially expressed genes. Among these, several groups can be linked to specific morphological and physiological changes during diaspore formation, such as genes involved in cell wall modifications. Furthermore, almost all members of ABA-dependent signaling and regulation were transcriptionally induced. Network analysis of transcription-associated genes revealed a large overlap of our study with ABA-dependent regulation in response to dehydration, cold stress, and UV-B light, indicating a fundamental function of ABA in diverse stress responses in moss. We also studied the evolutionary conservation of ABA-dependent regulation between moss and the seed plant Arabidopsis thaliana pointing to an early evolution of ABA-mediated stress adaptation during the conquest of the terrestrial habitat by plants

Peroxidases identified in a subtractive cDNA library approach show tissue-specific transcript abundance and enzyme activity during seed germination of Lepidium sativum

The micropylar endosperm is a major regulator of seed germination in endospermic species, to which the close Brassicaceae relatives Arabidopsis thaliana and Lepidium sativum (cress) belong. Cress seeds are about 20 times larger than the seeds of Arabidopsis. This advantage was used to construct a tissue-specific subtractive cDNA library of transcripts that are up-regulated late in the germination process specifically in the micropylar endosperm of cress seeds. The library showed that a number of transcripts known to be up-regulated late during germination are up-regulated in the micropylar endosperm cap. Detailed germination kinetics of SALK lines carrying insertions in genes present in our library showed that the identified transcripts do indeed play roles during germination. Three peroxidases were present in the library. These peroxidases were identified as orthologues of Arabidopsis AtAPX01, AtPrx16, and AtPrxIIE. The corresponding SALK lines displayed significant germination phenotypes. Their transcripts were quantified in specific cress seed tissues during germination in the presence and absence of ABA and they were found to be regulated in a tissue-specific manner. Peroxidase activity, and particularly its regulation by ABA, also differed between radicles and micropylar endosperm caps. Possible implications of this tissue-specificity are discussed

Increased natural mortality at low abundance can generate a demographic Allee effect

Negative density-dependent regulation of population dynamics promotes population growth at low abundance and is therefore vital for recovery following depletion. Inversely, any process that reduces the compensatory density-dependence of population growth can negatively affect recovery. Here, we show that increased adult mortality at low abundance can reverse compensatory population dynamics into its opposite—a demographic Allee effect. Northwest Atlantic cod (Gadus morhua) stocks collapsed dramatically in the early 1990s and have since shown little sign of recovery. Many experienced dramatic increases in natural mortality, ostensibly attributable in some populations to increased predation by seals. Our findings show that increased natural mortality of a magnitude observed for overfished cod stocks has been more than sufficient to fundamentally alter the dynamics of density-dependent population regulation. The demographic Allee effect generated by these changes can slow down or even impede the recovery of depleted populations even in the absence of fishing

Use of endogenous signal sequences for transient production and efficient secretion by moss () cells-1

<p><b>Copyright information:</b></p><p>Taken from "Use of endogenous signal sequences for transient production and efficient secretion by moss () cells"</p><p>BMC Biotechnology 2005;5():30-30.</p><p>Published online 7 Nov 2005</p><p>PMCID:PMC1291358.</p><p>Copyright © 2005 Schaaf et al; licensee BioMed Central Ltd.</p>s. After 5 days the concentrations of secreted and intracellularly-retained recombinant protein were measured by ELISA. Additionally, GST was affinity-purified from both medium and supernatant and detected by western blot. Culture medium of two transfections was pooled. Mean values of eight transfections are given. Protoplasts of eight transfections were pooled and GST was affinity-purified from the crude extracts. Values reflect the means of the two measurements. Control: mock-transfected protoplasts

Use of endogenous signal sequences for transient production and efficient secretion by moss () cells-5

<p><b>Copyright information:</b></p><p>Taken from "Use of endogenous signal sequences for transient production and efficient secretion by moss () cells"</p><p>BMC Biotechnology 2005;5():30-30.</p><p>Published online 7 Nov 2005</p><p>PMCID:PMC1291358.</p><p>Copyright © 2005 Schaaf et al; licensee BioMed Central Ltd.</p>pFLP, PpLTP, PpPME1, PpXTH1, PpCALP, and VEGF are shown as predicted by SignalP. Letters with yellow background represent hydrophobic amino acid residues (V, L, I, W, F, M), orange letters positive (K, R) and green letters small and neutral residues (A, C, G, N, P, S, T, V). The predicted cleavage sites are indicated as well as the positions adjacent to this site. In the right part of the figure probabilities for each signal peptide and the cleavage site given by the SignalP-HMM prediction as well as the length of the signal peptides are shown

Use of endogenous signal sequences for transient production and efficient secretion by moss () cells-2

<p><b>Copyright information:</b></p><p>Taken from "Use of endogenous signal sequences for transient production and efficient secretion by moss () cells"</p><p>BMC Biotechnology 2005;5():30-30.</p><p>Published online 7 Nov 2005</p><p>PMCID:PMC1291358.</p><p>Copyright © 2005 Schaaf et al; licensee BioMed Central Ltd.</p> and seven (pASP-VEGF) independent transfections. After 5 days the concentrations of secreted and intracellularly retained recombinant protein were measured by ELISA. mean values of concentrations of extra- and intracellular rhVEGF measured by ELISA. Western blot with culture medium of transiently rhVEGF-producing cell. Control: untransfected moss protoplasts