Dynamics of adaptation of stomatal behaviour to moderate or high relative air humidity in Tradescantia virginiana

Chlorophyll fluorescence imaging was used to measure stomatalclosure in response to desiccation of Tradescantia virginianaleaves grown under high (90%) and moderate (55%) relative humidities(RHs), or transferred between these humidities. Stomata in leavesgrown at high RH were less responsive to desiccation than thoseof leaves grown at moderate RH. Stomata of plants transferredfrom moderate RH conditions to high RH showed the same diminishedclosure in response to desiccation as did stomata that developedat high RH. This response was found both when the leaves werefully expanded and when still actively expanding during themoderate RH pre-treatment. Four days of exposure to high RHwas the minimal exposure time to induce the diminished closureresponse. When leaves were grown in high RH prior to a 10 dmoderate RH treatment, the reduced stomatal closure responseto desiccation was only reversed in leaves (regions) which wereactively expanding during moderate RH treatment. This indicatesthat with respect to stomatal responses to desiccation, highRH leaf regions have a limited capacity to adapt to moderateRH conditions. The decrease in responsiveness to desiccationof the stomata, induced by long-term exposure to high RH, wasnot due to osmotic adjustment in the leaves. Within 1 d aftertransferring moderate RH-grown plants to a high RH, the abscisicacid (ABA) concentration of their leaves decreased to the lowlevel of ABA found in high RH-grown leaves. The closure responsein leaves exposed to high RH for 5 d, however, could not befully restored by the application of ABA. Transferring plantsfrom high to moderate RH resulted in increased ABA levels within2 d without a recovery of the stomatal closing response. Itis discussed that the diminished stomatal closure in plantsexposed to high RH could be due to changes in the signallingpathway for ABA-related closure of stomata or to an increasedsequestration of ABA by mesophyll tissue or the symplast inthe epidermis, induced by a longer period (several days) ofa low ABA level. Key words: Abscisic acid, desiccation, PSII efficiency, relative water content, stomatal closure, vapour pressure deficit, water potential Received 8 October 2007; Revised 5 November 2007 Accepted 9 November 2007     

Doubled haploid plants following colchicine treatment of microspore-derived embryos of oilseed rape (<Emphasis Type="Italic">Brassica napus L.</Emphasis>)

An efficient method for producing doubled haploid plants of oilseed rape (Brassica napus L.) was established using in vitro colchicine treatment of haploid embryos. Haploid embryos in the cotyledonary stage were treated with one of four colchicine concentrations (125, 250, 500 and 1,000 mg/L); for one of three treatment durations (12, 24 and 36 h) at one of the two temperatures (8 and 25°C) and were compared to control embryos (without colchicine treatment). The number of chromosomes, seed recovery, size and density of leaf stomata, and pollen grain size from regenerated plants were determined. No doubled haploid plants were regenerated from control embryos; however, the doubled haploid plants were regenerated from colchicine-treated embryos. A high doubling efficiency, 64.29 and 66.66% of regenerated plants, was obtained from 250 mg/L colchicine treatment for 24 h and 500 mg/L colchicine treatment for 36 h, respectively, at 8°C. Following 500 mg/L colchicine treatment for 36 h, a few plants regenerated (9 plants). At the higher colchicine concentration (1,000 mg/L), no plant regenerated. These results indicate that the colchicine treatment of embryos derived from microspores can induce efficient chromosome doubling for the production of doubled haploid lines of oilseed rape.     

Polo Kinase Interacts with RacGAP50C and Is Required to Localize the Cytokinesis Initiation Complex

The assembly and constriction of an actomyosin contractile ring in cytokinesis is dependent on the activation of Rho at the equatorial cortex by a complex, here termed the cytokinesis initiation complex, between a microtubule-associated kinesin-like protein (KLP), a member of the RacGAP family, and the RhoGEF Pebble. Recently, the activity of the mammalian Polo kinase ortholog Plk1 has been implicated in the formation of this complex. We show here that Polo kinase interacts directly with the cytokinesis initiation complex by binding RacGAP50C. We find that a new domain of Polo kinase, termed the intermediate domain, interacts directly with RacGAP50C and that Polo kinase is essential for localization of the KLP-RacGAP centralspindlin complex to the cell equator and spindle midzone. In the absence of Polo kinase, RacGAP50C and Pav-KLP fail to localize normally, instead decorating microtubules along their length. Our results indicate that Polo kinase directly binds the conserved cytokinesis initiation complex and is required to trigger centralspindlin localization as a first step in cytokinesis.     

Reassessment of inclusion body-based production as a versatile opportunity for difficult-to-express recombinant proteins

The production of recombinant proteins in the microbial host Escherichia coli often results in the formation of cytoplasmic protein inclusion bodies (IBs). Proteins forming IBs are often branded as difficult-to-express, neglecting that IBs can be an opportunity for their production. IBs are resistant to proteolytic degradation and contain up to 90% pure recombinant protein, which does not interfere with the host metabolism. This is especially advantageous for host-toxic proteins like antimicrobial peptides (AMPs). IBs can be easily isolated by cell disruption followed by filtration and/or centrifugation, but conventional techniques for the recovery of soluble proteins from IBs are laborious. New approaches therefore simplify protein recovery by optimizing the production process conditions, and often include mild resolubilization methods that either increase the yield after refolding or avoid the necessity of refolding all together. For the AMP production, the IB-based approach is ideal, because these peptides often have simple structures and are easy to refold. The intentional IB production of almost every protein can be achieved by fusing recombinant proteins to pull-down tags. This review discusses the techniques available for IB-based protein production before considering technical approaches for the isolation of IBs from E. coli lysates followed by efficient protein resolubilization which ideally omits further refolding. The techniques are evaluated in terms of their suitability for the process-scale production and downstream processing of recombinant proteins and are discussed for AMP production as an example.     

Genetic diversification and population structure of <Emphasis Type="Italic">Barbus cyri</Emphasis> De Filippi, 1865 (Teleostei: Cyprinidae) in northern Iran inferred from the mitochondrial D-loop gene sequence

A genetic survey of Barbus cyri populations from two biogeographical endorheic basins (Caspian Sea and Urmia Lake) was carried out using a mitochondrial marker (partial D-loop) in order to ascertain intra- and inter-population genetic diversity, population demography and to address their genetic structure which is the key to conservation action planning. Analyses were conducted on sequences obtained from 68 individuals collected from 10 sampling sites, from two basins. By means of morphological characteristics all specimens collected from the Caspian Sea basin were ascribed to Barbus cyri. Genetic diversity values (h and π) of sampling groups were all different from 0 (in Babolrud River population) to 0.857 (in Kalibar River population). Population connectivity and colonization patterns of the studied area were inferred from an analysis of molecular variance distribution and evolutionary relationships among haplotypes. The results point to different levels of isolation among sampling groups due to ecological and anthropogenic factors and the effect of an artificial barrier on genetic variability and conservation status of the population. Finally, this study confirms the uncertainty associated with systematic classification of Barbus spp. based on morphological characters due to the phenotypic plasticity of the species.