In vitro plant regeneration of Aristolochia indica through axillary shoot multiplication and organogenesis

Protocols for in vitro plant regeneration via axillary and adventitious shoot regeneration were established in an important medicinal plant, Aristolochia indica L. (Aristolochiaceae). Basal Murashige and Skoog's (MS) medium supplemented with 0.54 μM α-naphthaleneacetic acid (NAA) and 13.31 μM benzyladenine (BA) induced the maximum number of shoots (45-50) from shoot tip and nodal segment cultures. Phenolic accumulation in leaf and internodal stem derived callus cultured in MS medium containing NAA or 2,4-dichlorophenoxyacetic acid and BA or kinetin was controlled by the addition of 1.0 mg l^-1 phloroglucinol (PG) to the callus induction medium. Basal medium supplemented with 2.69 μM NAA, 13.31 μM BA and 1.0 mg l^-1 PG induced the best results in terms of shoot bud regeneration from leaf derived callus. Direct de novo development of shoots from leaf segments was achieved using 13.31 μM BA along with 50 mg l^-1 activated charcoal. The microshoots were rooted in White's medium supplemented with 2.46 μM indolebutyric acid. More than 85% of rooted plants survived in the soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

ORGANIZATION OF THE nif GENES OF THE NONHETEROCYSTOUS CYANOBACTERIUM TRICHODESMIUM SP. IMS101

An approximately 16-kb fragment of the Trichodesmium sp. IMS101 (a nonheterocystous filamentous cyanobacterium) "conventional" nif gene cluster was cloned and sequenced. The gene organization of the Trichodesmium and Anabaena variabilis vegetative ( nif 2 ) nitrogenase gene clusters spanning the region from nif B to nif W are similar except for the absence of two open reading frames (ORF3 and ORF1) in Trichodesmium . The Trichodesmium nif EN genes encode a fused Nif EN polypeptide that does not appear to be processed into individual Nif E and Nif N polypeptides. Fused nif  EN genes were previously found in the A. variabilis nif 2 genes, but we have found that fused nif EN genes are widespread in the nonheterocystous cyanobacteria. Although the gene organization of the nonheterocystous filamentous Trichodesmium nif gene cluster is very similar to that of the A. variabilis vegetative nif 2 gene cluster, phylogenetic analysis of nif sequences do not support close relatedness of Trichodesmium and A. variabilis vegetative ( nif 2 ) nitrogenase genes.     

Ecophysiological and morphological parameters related to survival in grass species exposed to an extreme climatic event

An experiment was performed to elucidate interspecific differences in survival time of grass species subjected to an extreme climatic event. We exposed eight grass species to a simulated heat wave in the field ('free air' temperature increase at 11°C above ambient) combined with drought. We determined whether interspecific differences in survival time were related to the responses of the species to the imposed stress or could be explained by their ecophysiological or morphological characteristics in unstressed conditions. Surprisingly, there was no effect of specific leaf area, but species with a higher total leaf area survived longer. This may arise from a greater water reserve in the plant as a whole, which could delay the desiccation of the meristem, or from reduced evaporation due to a higher leaf area index. Species in which the decrease in light-saturated stomatal conductance ( g _s ) and photosynthetic CO₂ uptake rate ( A _max ) was strongly related to the decrease in soil water availability (measured as soil relative water content and stress duration) survived longer than species in which g _s and A _max likewise declined but responded more to daily fluctuations in irradiance, temperature, and vapor pressure deficit during the heat wave. We, therefore, hypothesize that interspecific differences in stress survival time might be related to the extent to which stomata react to changes in soil water conditions relatively to changes in other environmental and physiological factors. The results suggest that resistance to extremes is governed by other mechanisms than resistance to moderate drought.     

Speciation,phenotypic plasticity,or ontogeny,the case of the genus Galkinius (Pyrgomatidae,Cirripedia, Crustacea)

Barnacles of the genus Galkinius occupy a large spectrum of host corals, making it one of the least host‐specific genera within the Pyrgomatidae. Molecular analyses show that within the genus Galkinius there are highly supported clades, suggesting that the genus Galkinius is a complex of evolutionarily significant units (ESUs). The morphology of the opercular valves has been used as the basis for the separation of species of Galkinius. In this study, morphological variability was found both between specimens within ESUs extracted from different host species and between specimens extracted from the same colony. Identifications based on the opercular valves cannot therefore be assigned to different species despite being genetically distinguishable. It is proposed that in many cases the differences between valve morphology of different species of Galkinius are the outcome of ontogeny. Allometric growth of the valves has resulted in differences in the proportions of the parts of the valve. © 2015 The Linnean Society of London     

The Pacific White Shrimp β-actin Promoter: Functional Properties and the Potential Application for Transduction System Using Recombinant Baculovirus

A newly isolated Pacific white shrimp (Litopenaeus vannamei) beta-actin promoter SbaP and its derivative compact construct SbaP (ENX) have recently been demonstrated to promote ectopic gene expression in vitro and in vivo. To further explore the potential transduction application, this newly isolated shrimp promoter SbaP was comparatively tested with cytomegalovirus (CMV), simian virus 40 (SV40), polyhedrin (Polh), and white spot syndrome virus immediate early gene 1 (WSSV ie1) four constitutive promoters and a beta-actin promoter (TbaP) from tilapia fish to characterize its promoting function in eight different cell lines. Luciferase quantitation assays revealed that SbaP can drive luciferase gene expression in all eight cell lines including sf21 (insect), PAC2 (zebrafish), EPC (carp), CHSE-214 (chinook salmon), GSTEF (green sea turtle), MS-1 (monk seal), 293T (human), and HeLa (human), but at different levels. Comparative analysis revealed that the promoting activity of SbaP was lower (≤10-fold) than CMV but higher (2–20 folds) than Polh in most of these cell lines tested. Whereas, SbaP mediated luciferase expression in sf21 cells was over 20-fold higher than CMV, SV40, Polh, and TbaP promoter. Compared to the SbaP, SbaP (ENX), which was constructed on the basis of SbaP by deletion of two “negative” regulatory elements, exhibited no significant change of promoting activity in EPC and PAC2 cells, but a 5 and 16 % lower promoting effect in 293T and HeLa cells, respectively. Additionally, a recombinant baculovirus was constructed under the control of SbaP (ENX), and efficient promoter activity of newly generated baculoviral vector was detected both in vitro of infected sf21 cells and in vivo of injected indicator shrimp. These results warrant the potential application of SbaP, particularly SbaP (ENX) in ectopic gene expression in future.     

Repression of isoperoxidase formation in excised tobacco pith by exogenous, auxin-controlled RNA

Previous work has shown that tobacco pith tissue contains two constitutive isoperoxidases migrating toward the anode at pH 9·0. Within 24 hr of aseptic culture on basal medium, such tissue develops five new isoperoxidases, three cathodic and two anodic. The appearance of the new isoperoxidases involves de novo protein formation; it is inhibited by anaerobic conditions, by such inhibitors as Actinomycin D, and by the plant hormone indole-3-acetic acid (IAA). We now find that phenol RNA extracted from parent pith and injected or vacuum infiltrated into cultured pith explants prevents the appearance of the new isozymes; RNA from cultured pith has no such effect. Hydrolysis with 0·3 N KOH, ribonuclease or proteolytic enzymes partially destroys this activity, while treatment with both ribonuclease and proteolytic enzymes completely destroys it. Fractionation of the RNA indicates that part of the repressor activity is associated with an mRNA-like fraction.     

Inhibition of oxidative catabolism and oxy free radical production as a possible mode of cytokinin control of foliar senescence

Increment of levels of both lipoxygenase and superoxide-dismutase which typically increase with age or upon light deprivation is significantly lowered by cytokinin treatment. It is suggested that cytokinin on the one hand by means of inhibition of polyunsaturated fatty acid catabolism prevents incipient formation of free radicals while on the other serves as a scavenger of radicals already formed.     

Involvement of Calcium and Calmodulin in Membrane Deterioration during Senescence of Pea Foliage

The prospect that Ca(2+) promotes senescence by activating calmodulin has been examined using cut pea (Pisum sativum co Alaska) foliage as a model system. Senescence was induced by severing 17-day-old plants from their roots and maintaining them in aqueous test solutions in the dark for an additional 4 days. Treatment of the foliage with the Ca(2+) ionophore (A23187) during the senescence-induction period promoted a lateral phase separation of the bulk lipids in microsomal membranes indicating that internalization of Ca(2+) facilitates membrane deterioration. In addition, microsomal membranes from ionophore-treated tissue displayed an increased capacity to convert 1-aminocyclopropane-1-carboxylic acid to ethylene and an increased propensity to produce the superoxide anion (O(2) (tau)). Treatment of the tissue with fluphenazine during the senescence-induction period, which prevents binding of the Ca:Calmodulin complex to enzymes, delayed membrane deterioration as measured by these criteria. It also proved possible to simulate these in situ effects of the Ca(2+) ionophore on ethylene production and O(2) (tau) formation by treating microsomal membranes isolated from young tissue with phospholipase A(2) in the presence of Ca(2+) and calmodulin, and these effects of phospholipase A(2) and Ca:calmodulin were inhibited by calmodulin antagonists. The observations collectively suggest that internalized Ca(2+) promotes senescence by activating calmodulin, which in turn mediates the action of phospholipase A(2) on membranes.     

Membrane phospholipid catabolism and Ca2+ activity in control of senescence

Leshem, Y. Y. 1987. Membrane phospholipid catabolism and Ca²⁺ activity in control of senescence. A key role in the regulation of plant development and senescence appears to be a finely balanced equilibrium between membrane phospholipid catabolism on the one hand, and synthesis and remodelling on the other. In the catabolic “phosphatidyl-linoleyl(-enyl) cascade”, entering of Ca²⁺ into the cytosol triggers the catabolic process by binding to calmodulin and activating phospholipase A₂, (EC 3.1.1.4). The latter proceeds to release linoleic or linolenic acid from the sn-2 (stereospecific numbering) location of intact phospholipid, thus providing substrate for lipoxygenase (EC 1.13.11.12). The action of lipoxygenase then generates a series of oxy-free radicals, ethylene, endogenous Ca²⁺ ionophores, malondialdehyde and jasmonic acid. These may recycle to the membrane, causing the entry of more Ca²⁺ and induction of a further, identical catabolic cycle. With increased cycling, membranes become progressively senescent and undergo biophysical changes altering microviscosity, fluidity, phase configurations of membrane phospholipids and transition temperatures. The cascade does not appear to be specific for the phospholipid substrate, and it is envisaged that besides phospholipase A₂, both phospholipase B (EC 3.1.1.5) and lipolytic acylhydrolase could participate in the process. A parallel process counteracting the above, is membrane remodelling and turnover, proceeding initially by the same Ca²⁺- and possibly calmodulin-triggering, but leading via phospholipase C (EC 3.1.4.10) action and diacylglycerol formation to protein kinase activation and proton pump recharging. It is speculated that auxin and cytoki-nin, albeit by different pathways, induce this route, for which membrane phospho-inositides may be the preferred membrane-associated phospholipid substrate.