Protease-Dead Separase Is Dominant Negative in the C. elegans Embryo

Separase is a protease that promotes chromosome segregation at anaphase by cleaving cohesin. Several non-proteolytic functions of separase have been identified in other organisms. We created a transgenic C. elegans line that expresses protease-dead separase in embryos to further characterize separase function. We find that expression of protease-dead separase is dominant-negative in C. elegans embryos, not previously reported in other systems. The C. elegans embryo is an ideal system to study developmental processes in a genetically tractable system. However, a major limitation is the lack of an inducible gene expression system for the embryo. We have developed two methods that allow for the propagation of lines carrying dominant-negative transgenes and have applied them to characterize expression of protease-dead separase in embryos. Using these methods, we show that protease-dead separase causes embryo lethality, and that protease-dead separase cannot rescue separase mutants. These data suggest that protease-dead separase interferes with endogenous separase function, possibly by binding substrates and protecting them from cleavage

Barley plasma membrane intrinsic proteins (PIP aquaporins) as water and CO2 transporters

We identified barley aquaporins and demonstrated that one, HvPIP2;1, transports water and CO2. Regarding water homeostasis in plants, regulations of aquaporin expression were observed in many plants under several environmental stresses. Under salt stress, a number of plasma membrane-type aquaporins were down-regulated, which can prevent continuous dehydration resulting in cell death. The leaves of transgenic rice plants that expressed the largest amount of HvPIP2;1 showed a 40% increase in internal CO2 conductance compared with leaves of wild-type rice plants. The rate of CO2 assimilation also increased in the transgenic plants. The goal of our plant aquaporin research is to determine the key aquaporin species responsible for water and CO2 transport, and to improve plant water relations, stress tolerance, CO2 uptake or assimilation, and plant productivity via molecular breeding of aquaporins.</p

Effect of nitrate supply and mycorrhizal inoculation on characteristics of tobacco root plasma membrane vesicles

Plant plasma membrane (pm) vesicles from mycorrhizal tobacco (Nicotiana tabacum cv. Samsun) roots were isolated with negligible fungal contamination by the aqueous two-phase partitioning technique as proven by fatty acid analysis. Palmitvaccenic acid became apparent as an appropriate indicator for fungal membranes in root pm preparations. The pm vesicles had a low specific activity of the vanadate-sensitive ATPase and probably originated from non-infected root cells. In a phosphate-limited tobacco culture system, root colonisation by the vesicular arbuscular mycorrhizal fungus, Glomus mosseae, is inhibited by external nitrate in a dose-dependent way. However, detrimental high concentrations of 25 mM nitrate lead to the highest colonisation rate observed, indicating that the defence system of the plant is impaired. Nitric oxide formation by the pm-bound nitrite:NO reductase increased in parallel with external nitrate supply in mycorrhizal roots in comparison to the control plants, but decreased under excess nitrate. Mycorrhizal pm vesicles had roughly a twofold higher specific activity as the non-infected control plants when supplied with 10–15 mM nitrate

Adaptations to Endosymbiosis in a Cnidarian-Dinoflagellate Association: Differential Gene Expression and Specific Gene Duplications

Trophic endosymbiosis between anthozoans and photosynthetic dinoflagellates forms the key foundation of reef ecosystems. Dysfunction and collapse of symbiosis lead to bleaching (symbiont expulsion), which is responsible for the severe worldwide decline of coral reefs. Molecular signals are central to the stability of this partnership and are therefore closely related to coral health. To decipher inter-partner signaling, we developed genomic resources (cDNA library and microarrays) from the symbiotic sea anemone Anemonia viridis. Here we describe differential expression between symbiotic (also called zooxanthellate anemones) or aposymbiotic (also called bleached) A. viridis specimens, using microarray hybridizations and qPCR experiments. We mapped, for the first time, transcript abundance separately in the epidermal cell layer and the gastrodermal cells that host photosynthetic symbionts. Transcriptomic profiles showed large inter-individual variability, indicating that aposymbiosis could be induced by different pathways. We defined a restricted subset of 39 common genes that are characteristic of the symbiotic or aposymbiotic states. We demonstrated that transcription of many genes belonging to this set is specifically enhanced in the symbiotic cells (gastroderm). A model is proposed where the aposymbiotic and therefore heterotrophic state triggers vesicular trafficking, whereas the symbiotic and therefore autotrophic state favors metabolic exchanges between host and symbiont. Several genetic pathways were investigated in more detail: i) a key vitamin K–dependant process involved in the dinoflagellate-cnidarian recognition; ii) two cnidarian tissue-specific carbonic anhydrases involved in the carbon transfer from the environment to the intracellular symbionts; iii) host collagen synthesis, mostly supported by the symbiotic tissue. Further, we identified specific gene duplications and showed that the cnidarian-specific isoform was also up-regulated both in the symbiotic state and in the gastroderm. Our results thus offer new insight into the inter-partner signaling required for the physiological mechanisms of the symbiosis that is crucial for coral health

Gas-tight triblock-copolymer membranes are converted to CO2 permeable by insertion of plant aquaporins

We demonstrate that membranes consisting of certain triblock-copolymers were tight for CO2. Using a novel approach, we provide evidence for aquaporin facilitated CO2 diffusion. Plant aquaporins obtained from heterologous expression were inserted into triblock copolymer membranes. These were employed to separate a chamber with a solution maintaining high CO2 concentrations from one with depleted CO2 concentrations. CO2 diffusion was detected by measuring the pH change resulting from membrane CO2 diffusion from one chamber to the other. An up to 21 fold increase in diffusion rate was determined. Besides the supply of this proof of principle, we could provide additional arguments in favour of protein facilitated CO2 diffusion to the vivid on-going debate about the principles of membrane gas diffusion in living cells

Plant Aquaporins and CO2

Aquaporins in plants show more abundant and greater diversity than aquaporins in bacteria and animals. This unique characteristic provided versatile tool boxes for plants, dealing with environmental changes, which overcome the disadvantage of immobility. Aquaporins were first known for their function as water channel proteins. Later on, more and more studies showed that other small solutes, i.e., ammonia, glycerol, urea, hydrogen peroxide and metalloids, can also pass through the channel of various aquaporins. Moreover, the function of aquaporins as CO2 gas channels was studied by several groups (Nakhoul et al. Am J Physiol Cell Physiol 43(2): C543-C548, 1998; Yang et al. J Biol Chem 275(4): 26862692, 2000; Tholen and Zhu Plant Physiol 156(1): 90-105, 2011). In parallel, studies on model reconstituted membranes claim that no such type of channel would be needed due to the high permeability of those model membranes (Missner et al. Proc Natl Acad Sci USA 105(52): E123, 2008a; J Biol Chem 283(37): 25340-25347, 2008b). However, experimental data showed the physiological significance of CO2-conducting channels, particularly in plants. It is generally accepted that plant science presented the first evidence for the physiological relevance and importance of aquaporins as CO2 transport facilitators (Boron Exp Physiol 95(12): 1107-1130, 2010; Terashima and Ono Plant Cell Physiol 43(1): 70-78, 2002; Uehlein et al. Nature 425 (6959): 734-737, 2003; Heckwolf et al. Plant J 67 (5): 795-804, 2011; Uehlein et al. Plant Cell 20(3): 648-657, 2008). In this chapter, we discuss the CO2 diffusion across membranes and the role of plant aquaporins during this process

PIP1 and PIP2 aquaporins are differentially expressed during tobacco

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Protease-Dead Separase Is Dominant Negative in the <i>C. elegans</i> Embryo

<div><p>Separase is a protease that promotes chromosome segregation at anaphase by cleaving cohesin. Several non-proteolytic functions of separase have been identified in other organisms. We created a transgenic <i>C. elegans</i> line that expresses protease-dead separase in embryos to further characterize separase function. We find that expression of protease-dead separase is dominant-negative in <i>C. elegans</i> embryos, not previously reported in other systems. The <i>C. elegans</i> embryo is an ideal system to study developmental processes in a genetically tractable system. However, a major limitation is the lack of an inducible gene expression system for the embryo. We have developed two methods that allow for the propagation of lines carrying dominant-negative transgenes and have applied them to characterize expression of protease-dead separase in embryos. Using these methods, we show that protease-dead separase causes embryo lethality, and that protease-dead separase cannot rescue separase mutants. These data suggest that protease-dead separase interferes with endogenous separase function, possibly by binding substrates and protecting them from cleavage.</p></div