Seawater salt-trapped Pseudomonas aeruginosa survives for years and gets primed for salinity tolerance

Background In nature, microorganisms have to adapt to long-term stressful conditions often with growth limitations. However, little is known about the evolution of the adaptability of new bacteria to such environments. Pseudomonas aeruginosa, an opportunistic pathogen, after natural evaporation of seawater, was shown to be trapped in laboratory-grown halite crystals and to remain viable after entrapment for years. However, how this bacterium persists and survives in such hypersaline conditions is not understood. Results In this study, we aimed to understand the basis of survival, and to characterise the physiological changes required to develop salt tolerance using P. aeruginosa as a model. Several clones of P. aeruginosa were rescued after 14 years in naturally evaporated marine salt crystals. Incubation of samples in nutrient-rich broth allowed re-growth and subsequent plating yielded observable colonies. Whole genome sequencing of the P. aeruginosa isolates confirmed the recovery of the original strain. The re-grown strains, however, showed a new phenotype consisting of an enhanced growth in growing salt concentration compared to the ancestor strain. The intracellular accumulation of K+ was elicited by high concentration of Na+ in the external medium to maintain the homeostasis. Whole transcriptomic analysis by microarray indicated that 78 genes had differential expression between the parental strain and its derivative clones. Sixty-one transcripts were up-regulated, while 17 were down-regulated. Based on a collection of single-gene knockout mutants and gene ontology analysis, we suggest that the adaptive response in P. aeruginosa to hyper-salinity relies on multiple gene product interactions. Conclusions The individual gene contributions build up the observed phenotype, but do not ease the identification of salinity-related metabolic pathways. The long-term inclusion of P. aeruginosa in salt crystals primes the bacteria, mediating a readjustment of the bacterial physiology to growth in higher salt concentrations. Our findings provide a starting point to understand how P. aeruginosa, a relevant environmental and pathogenic bacterium, survives to long-term salt stress

Regulation of seed germination and seedling growth by an Arabidopsis phytocystatin isoform, AtCYS6

Phytocystatins are cysteine proteinase inhibitors in plants that are implicated in the endogenous regulation of protein turnover and defense mechanisms against insects and pathogens. A cDNA encoding a phytocystatin called AtCYS6 (Arabidopsis thaliana phytocystatin6) has been isolated. We show that AtCYS6 is highly expressed in dry seeds and seedlings and that it also accumulates in flowers. The persistence of AtCYS6 protein expression in seedlings was promoted by abscisic acid (ABA), a seed germination and post-germination inhibitory phytohormone. This finding was made in transgenic plants bearing an AtCYS6 promoter–β-glucuronidase (GUS) reporter construct, where we found that expression from the AtCYS6 promoter persisted after ABA treatment but was reduced under control conditions and by gibberellin4+7 (GA4+7) treatment during the germination and post-germinative periods. In addition, constitutive over-expression of AtCYS6 retarded germination and seedling growth, whereas these were enhanced in an AtCYS6 knock-out mutant (cys6-2). Additionally, cysteine proteinase activities stored in seeds were inhibited by AtCYS6 in transgenic Arabidopsis. From these data, we propose that AtCYS6 expression is enhanced by the germination inhibitory phytohormone ABA and that it participates in the control of germination rate and seedling growth by inhibiting the activity of stored cysteine proteinases

Distinct expression patterns of two Arabidopsis phytocystatin genes, AtCYS1 and AtCYS2, during development and abiotic stresses

The phytocystatins of plants are members of the cystatin superfamily of proteins, which are potent inhibitors of cysteine proteases. The Arabidopsis genome encodes seven phytocystatin isoforms (AtCYSs) in two distantly related AtCYS gene clusters. We selected AtCYS1 and AtCYS2 as representatives for each cluster and then generated transgenic plants expressing the GUS reporter gene under the control of each gene promoter. These plants were used to examine AtCYS expression at various stages of plant development and in response to abiotic stresses. Histochemical analysis of AtCYS1 promoter- and AtCYS2 promoter-GUS transgenic plants revealed that these genes have similar but distinct spatial and temporal expression patterns during normal development. In particular, AtCYS1 was preferentially expressed in the vascular tissue of all organs, whereas AtCYS2 was expressed in trichomes and guard cells in young leaves, caps of roots, and in connecting regions of the immature anthers and filaments and the style and stigma in flowers. In addition, each AtCYS gene has a unique expression profile during abiotic stresses. High temperature and wounding stress enhanced the expression of both AtCYS1 and AtCYS2, but the temporal and spatial patterns of induction differed. From these data, we propose that these two AtCYS genes play important, but distinct, roles in plant development and stress responses

Expression of two barley proteinase inhibitors in tomato promotes endogenous defensive response and enhances resistance to Tuta absoluta

[EN] Background: For as long as 350 million years, plants and insects have coexisted and developed a set of relationships which affect both organisms at different levels. Plants have evolved various morphological and biochemical adaptations to cope with herbivores attacks. However, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) has become the major pest threatening tomato crops worldwide and without the appropriated management it can cause production losses between 80 to 100%. Results: The aim of this study was to investigate the in vivo effect of a serine proteinase inhibitor (BTI-CMe) and a cysteine proteinase inhibitor (Hv-CPI2) from barley on this insect and to examine the effect their expression has on tomato defensive response. We found that larvae fed on the double transgenic plants showed a notable reduction in weight. Moreover, only 56% of the larvae reached the adult stage. The emerged adults showed wings deformities and reduced fertility. We also investigated the effect of proteinase inhibitors ingestion on the insect digestive enzymes. Our results showed a decrease in larval trypsin activity. Transgenes expression had no harmful effect on Nesidiocoris tenuis (Reuter) (Heteroptera: Miridae), a predator of Tuta absoluta, despite transgenic tomato plants attracted the mirid. We also found that barley cystatin expression promoted plant defense by inducing the expression of the tomato endogenous wound inducible Proteinase inhibitor 2 (Pin2) gene, increasing the production of glandular trichomes and altering the emission of volatile organic compounds. Conclusion: Our results demonstrate the usefulness of the co-expression of different proteinase inhibitors for the enhancement of plant resistance to Tuta absoluta.This work was partly supported by grants BIO2013-40747-R and AGL2014-55616-C3 from the Spanish Ministry of Economy and Competitiveness (MINECO)Hamza, R.; Pérez-Hedo, M.; Urbaneja, A.; Rambla Nebot, JL.; Granell Richart, A.; Gaddour, K.; Beltran Porter, JP.... (2018). Expression of two barley proteinase inhibitors in tomato promotes endogenous defensive response and enhances resistance to Tuta absoluta. BMC Plant Biology. 18. https://doi.org/10.1186/s12870-018-1240-6S18Oerke EC. Crop losses to pests. J Agric Sci. 2005;144(01):31.Jouanin L, Bonadé-Bottino M, Girard C, Morrot G, Giband M. Transgenic plants for insect resistance. Plant Sci. 1998;131(1):1–11.Markwick NP, Docherty LC, Phung MM, Lester MT, Murray C, Yao JL, Mitra DS, Cohen D, Beuning LL, Kutty-Amma S, et al. 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Effects on milk production in F1 crossbred of Alpine goat breed (♂) and Albanian goat breed (♀)

About 950,000 goats, farmed mostly in hilly and mountainous areas of Albania, contribute about 8% of the country’s total milk production. In order to increase milk production, farmers are currently using crosses of the local goat breed with exotic breeds, mainly the Alpine breed from France. This study examines milk production data of first lactation from 45 goats of the local breed, 82 goats of the Alpine breed and 58 F1 crosses (♂Alpine breed x ♀local breed). The goats were kept on small-scale farms according to the traditional Albanian system. Milking was carried out in the morning and evening. Kids were weaned at 65 days of age after which milking started. Milk yield was recorded twice with a 15-day interval between the two readings. Total milk yield was calculated using the Fleischmann method. The F1 goats produced 37.8 kg more milk than local breed goats although the lactation length (P<0.05) of F1 goats was six days shorter compared to that of local breed goats (P<0.05). Analysis of variance showed a highly significant effect (P<0.01) of the genotype factor on milk production. The average Cappio-Borlino curves of three genotypes indicated that the lactation curves of local breed and F1 crosses were similar. Although the F1 cross goats had 50% of their genomes from a genetically improved breed they were still able to deal with the difficult conditions that characterize the traditional extensive farming systems in Albania. Breeding pure Alpine breed or its crosses with the local goat breed improved milk production in an extensive traditional system

Barley trypsin inhibitor CMe confers insect resistance to wheat

Proteinase inhibitors have been proposed to be involved in the defence response against herbivorous pests (Hoffmann et al. 1992). The efficacy of a specific inhibitor depends on the structural compatibility of its reactive site with the substrate-binding site of the targeted proteinase. For example, trypsin-inhibitors include either an arginyl or lysyl residue, which is recognized by trypsin-like enzymes. Barley trypsin inhibitor CMe (BTI-CMe), an abundant protein in barley endosperm, is one of the best characterized members of the cereal multigene family of trypsin/a-amylase inhibitors (Carbonero and Gracia-Olmedo, 1998) and was first purified from barley flour as a protein of 14 KDa that was specifically active in vitro against trypsin. Recently BTI-CMe was shown to inhibit specifically the trypsin-like proteases of the gut extracts of the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) (Alfonso et al. 1997), whereas the BTICMe inhibitor is rapidly degraded in the digestive tract of mammals (unpublished data, Carbonero et al.). We have transformed wheat (Triticum aestivum L.) with the Itrl gene encoding the BTICMe (Altpeter et al. 1998), in order to evaluate its potential for improvement of resistance against a major storage pest in many developing countries: The Angoumois Grain Moth (Sitotroga cerealella, Lepidoptera: Gelechiidae)

Induced new mutation of D1 serine-268 in soybean photosynthetic cell cultures produced atrazine resistance, increased stability of S2QB- and S3QB- states, and increased sensitivity to light stress.

We have isolated several herbicide-resistant cell lines from photosynthetic cell suspensions of soybean (Glycine max) that possessed different levels of herbicide resistance, photosystem II activity, and chlorophyll a/b ratio. We have further studied the STR7 mutant, which showed the highest level of resistance to atrazine as well as a cross-resistance to 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (50- and 3-fold, respectively, compared with the wild type). Sequencing of the psbA gene (coding for the D1 polypeptide of photosystem II) from this mutant revealed a single change, serine-268 to proline, in the D1 protein. To our knowledge, this substitution has not previously been described in any photosynthetic organism. In addition to affecting atrazine resistance, this single amino acid change caused a decrease in the electron transfer rate between the secondary acceptors QA and QB and a stabilization of the S2QB- and S3QB- states. The mutant also showed a larger antenna size, an increase in non-QB-reducing centers, and a higher sensitivity to light stress. The unusual stability of the S2QB- and S3QB- states indicates that STR7 belongs to a new class of QB-site mutants

Barley trypsin inhibitor CMe confers insect resistance to wheat

Proteinase inhibitors have been proposed to be involved in the defence response against herbivorous pests (Hoffmann et al. 1992). The efficacy of a specific inhibitor depends on the structural compatibility of its reactive site with the substrate-binding site of the targeted proteinase. For example, trypsin-inhibitors include either an arginyl or lysyl residue, which is recognized by trypsin-like enzymes. Barley trypsin inhibitor CMe (BTI-CMe), an abundant protein in barley endosperm, is one of the best characterized members of the cereal multigene family of trypsin/a-amylase inhibitors (Carbonero and Gracia-Olmedo, 1998) and was first purified from barley flour as a protein of 14 KDa that was specifically active in vitro against trypsin. Recently BTI-CMe was shown to inhibit specifically the trypsin-like proteases of the gut extracts of the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) (Alfonso et al. 1997), whereas the BTICMe inhibitor is rapidly degraded in the digestive tract of mammals (unpublished data, Carbonero et al.). We have transformed wheat (Triticum aestivum L.) with the Itrl gene encoding the BTICMe (Altpeter et al. 1998), in order to evaluate its potential for improvement of resistance against a major storage pest in many developing countries: The Angoumois Grain Moth (Sitotroga cerealella, Lepidoptera: Gelechiidae)

Increased insect resistance in transgenic wheat stably expressing trypsin inhibitor CMe

Proteinase inhibitors have been proposed to function as plant defence agents against herbivorous pests. We have introduced the barley trypsin inhibitor CMe (BTI-CMe) into wheat (Triticum aestivum L.) by biolistic bombardment of cultured immature embryos. Of the 30 independent transgenic wheat lines selected, 16 expressed BTI-CMe. BTI-CMe was properly transcribed and translated as indicated by northern and western blot, with a level of expression in transgenic wheat seeds up to 1.1% of total extracted protein. No expression was detected in untransformed wheat seeds. Functional integrity of BTI-CMe was confirmed by trypsin inhibitor activity assay. The significant reduction of the survival rate of the Angoumois grain moth (Sitotroga cerealella, Lepidoptera: Gelechiidae), reared on transgenic wheat seeds expressing the trypsin inhibitor BTI-CMe, compared to the untransformed control confirmed the potential of BTI-CMe for the increase of insect resistance. However, only early-instar larvae were inhibited in transgenic seeds and expression of BTI-CMe protein in transgenic leaves did not have a significant protective effect against leaf-feeding insects