Activity of cefepime, carbapenems and new ß-lactam/ß-lactamase inhibitor combinations on Enterobacter cloacae complex and Klebsiella aerogenes in Spain (SMART 2016-2022).

Objectives: To analyse the susceptibility profile to cefepime, carbapenems and new ß-lactam/ß-lactamase inhibitor combinations in Enterobacter cloacae complex and Klebsiella aerogenes isolated from intra-abdominal, urinary, respiratory and bloodstream infections in the SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study in Spain. Methods: The susceptibilities of 759 isolates (473 E. cloacae complex and 286 K. aerogenes) collected in 11 Spanish hospitals from 2016 to 2022 were analysed following the EUCAST 2023 criteria. Molecular characterization looking for ß-lactamase genes was performed through PCR and DNA sequencing analysis. Results: E. cloacae complex showed resistance to third-generation cephalosporins in 25% of the cases, whereas K. aerogenes was resistant in 35%. Regarding cefepime, resistance in E. cloacae was higher (10%) than in K. aerogenes (2%). Carbapenems showed >85% activity in both microorganisms. Ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam had good activity against these microorganisms (>95%). In contrast, the activity of ceftolozane/tazobactam was lower (80%). A high proportion of the isolates resistant to new ß-lactam/ß-lactamase inhibitor combinations carried a carbapenemase, mainly OXA-48-like and VIM-1. Conclusions: Ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam show high activity against both E. cloacae complex and K. aerogenes isolates recovered in the SMART-Spain study. In contrast, differences have been found in the case of cefepime, showing more activity against K. aerogenes than E. cloacae complex. These results are useful for antimicrobial stewardship programmes and for the implementation of local and national guidelines.

Salt Tolerance of Sea Flax (Linum maritimum L.), a Rare Species with Conservation Interest in Eastern Spain.

Seldom found in saltmarshes, Linum maritimum is a halophyte of great conservation interest in the eastern Iberian Peninsula. Although the species has been reported in different plant communities, there is no information on its range of salinity tolerance or mechanisms of response to environmental stress factors. In this study, L. maritimum plants were subjected to increasing salt concentrations in controlled conditions in a greenhouse. After six months of watering with salt solutions, only plants from the control, 50 mM and 100 mM NaCl treatment groups survived, but seeds were produced only in the first two. Significant differences were found between the plants from the various treatment groups in terms of their growth parameters, such as plant height, fresh weight, and the quantity of flowers and fruits. The main mechanism of salt tolerance is probably related to the species' ability to activate K+ uptake and transport to shoots to partly counteract the accumulation of toxic Na+ ions. A biochemical analysis showed significant increases in glycine betaine, flavonoids and total phenolic compounds, highlighting the importance of osmotic regulation and antioxidant compounds in the salt tolerance of Linum maritimum. These findings have implications for the conservation of the species, especially under changing climatic conditions that may lead to increased soil salinity in its Mediterranean distribution area.

The Highly Embryogenic Brassica napus DH4079 Line Is Recalcitrant to Agrobacterium-Mediated Genetic Transformation.

Brassica napus is a species of high agronomic interest, used as a model to study different processes, including microspore embryogenesis. The DH4079 and DH12075 lines show high and low embryogenic response, respectively, which makes them ideal to study the basic mechanisms controlling embryogenesis induction. Therefore, the availability of protocols for genetic transformation of these two backgrounds would help to generate tools to better understand this process. There are some reports in the literature showing the stable transformation of DH12075. However, no equivalent studies in DH4079 have been reported to date. We explored the ability of DH4079 plants to be genetically transformed. As a reference to compare with, we used the same protocols to transform DH12075. We used three different protocols previously reported as successful for B. napus stable transformation with Agrobacterium tumefaciens and analyzed the response of plants. Whereas DH12075 plants responded to genetic transformation, DH4079 plants were completely recalcitrant, not producing any single regenerant out of the 1784 explants transformed and cultured. Additionally, an Agrobacterium rhizogenes transient transformation assay was performed on both lines, and only DH12075, but no DH4079 seedlings, responded to A. rhizogenes infection. Therefore, we propose that the DH4079 line is recalcitrant to Agrobacterium-mediated transformation.

Calcium dynamics and modulation in carrot somatic embryogenesis.

Free calcium (Ca2+) is a pivotal player in different in vivo and in vitro morphogenic processes. In the induction of somatic embryogenesis, its role has been demonstrated in different species. In carrot, however, this role has been more controversial. In this work, we developed carrot lines expressing cameleon Ca2+ sensors. With them, Ca2+ levels and distribution in the different embryogenic structures formed during the induction and development of somatic embryos were analyzed by FRET. We also used different chemicals to modulate intracellular Ca2+ levels (CaCl2, ionophore A23187, EGTA), to inhibit calmodulin (W-7) and to inhibit callose synthesis (2-deoxy-D-glucose) at different times, principally during the first stages of embryo induction. Our results showed that high Ca2+ levels and the development of a callose layer are markers of cells induced to embryogenesis, which are the precursors of somatic embryos. Disorganized calli and embryogenic masses have different Ca2+ patterns associated to their embryogenic competence, with higher levels in embryogenic cells than in callus cells. The efficiency of somatic embryogenesis in carrot can be effectively modulated by allowing, within a range, more Ca2+ to enter the cell to act as a second messenger to trigger embryogenesis induction. Once induced, Ca2+-calmodulin signaling seems related with the transcriptional remodeling needed for embryo progression, and alterations of Ca2+ or calmodulin levels negatively affect the efficiency of the process.

Calcium Dynamics, WUSCHEL Expression and Callose Deposition during Somatic Embryogenesis in Arabidopsis thaliana Immature Zygotic Embryos.

In this work, we studied the induction of somatic embryogenesis in Arabidopsis using IZEs as explants. We characterized the process at the light and scanning electron microscope level and studied several specific aspects such as WUS expression, callose deposition, and principally Ca2+ dynamics during the first stages of the process of embryogenesis induction, by confocal FRET analysis with an Arabidopsis line expressing a cameleon calcium sensor. We also performed a pharmacological study with a series of chemicals know to alter calcium homeostasis (CaCl2, inositol 1,4,5-trisphosphate, ionophore A23187, EGTA), the calcium-calmodulin interaction (chlorpromazine, W-7), and callose deposition (2-deoxy-D-glucose). We showed that, after determination of the cotiledonary protrusions as embryogenic regions, a finger-like appendix may emerge from the shoot apical region and somatic embryos are produced from the WUS-expressing cells of the appendix tip. Ca2+ levels increase and callose is deposited in the cells of the regions where somatic embryos will be formed, thereby constituting early markers of the embryogenic regions. We also found that Ca2+ homeostasis in this system is strictly maintained and cannot be altered to modulate embryo production, as shown for other systems. Together, these results contribute to a better knowledge and understanding of the process of induction of somatic embryos in this system.

Constitutive and Adaptive Traits of Environmental Stress Tolerance in the Threatened Halophyte Limonium angustebracteatum Erben (Plumbaginaceae).

Limonium angustebracteatum is a halophyte endemic to the E and SE Iberian Peninsula with interest in conservation. Salt glands represent an important adaptive trait in recretohalophytes like this and other Limonium species, as they allow the excretion of excess salts, reducing the concentration of toxic ions in foliar tissues. This study included the analysis of the salt gland structure, composed of 12 cells, 4 secretory and 8 accessory. Several anatomical, physiological and biochemical responses to stress were also analysed in adult plants subjected to one month of water stress, complete lack of irrigation, and salt stress, by watering with aqueous solutions of 200, 400, 600 and 800 mM NaCl. Plant growth was inhibited by the severe water deficit and, to a lesser extent, by high NaCl concentrations. A variation in the anatomical structure of the leaves was detected under conditions of salt and water stress; plants from the salt stress treatment showed salt glands sunken between epidermal cells, bordered by very large epidermal cells, whereas in those from the water stress treatment, the epidermal cells were heterogeneous in shape and size. In both, the palisade structure of the leaves was altered. Salt excretion is usually accompanied by the accumulation of salts in the foliar tissue. This was also found in L. angustebracteatum, in which the concentration of all ions analysed was higher in the leaves than in the roots. The increase of K+ in the roots of plants subjected to water stress was also remarkable. The multivariate analysis indicated differences in water and salt stress responses, such as the accumulation of Na and Cl, or proline, but K+ homeostasis played a relevant role in the mechanism of tolerance to both stressful conditions.

Cell Wall Composition and Structure Define the Developmental Fate of Embryogenic Microspores in Brassica napus.

Microspore cultures generate a heterogeneous population of embryogenic structures that can be grouped into highly embryogenic structures [exine-enclosed (EE) and loose bicellular structures (LBS)] and barely embryogenic structures [compact callus (CC) and loose callus (LC) structures]. Little is known about the factors behind these different responses. In this study we performed a comparative analysis of the composition and architecture of the cell walls of each structure by confocal and quantitative electron microscopy. Each structure presented specific cell wall characteristics that defined their developmental fate. EE and LBS structures, which are responsible for most of the viable embryos, showed a specific profile with thin walls rich in arabinogalactan proteins (AGPs), highly and low methyl-esterified pectin and callose, and a callose-rich subintinal layer not necessarily thick, but with a remarkably high callose concentration. The different profiles of EE and LBS walls support the development as suspensorless and suspensor-bearing embryos, respectively. Conversely, less viable embryogenic structures (LC) presented the thickest walls and the lowest values for almost all of the studied cell wall components. These cell wall properties would be the less favorable for cell proliferation and embryo progression. High levels of highly methyl-esterified pectin are necessary for wall flexibility and growth of highly embryogenic structures. AGPs seem to play a role in cell wall stiffness, possibly due to their putative role as calcium capacitors, explaining the positive relationship between embryogenic potential and calcium levels.

Doubled Haploids in Eggplant.

Eggplant is a solanaceous crop cultivated worldwide for its edible fruit. Eggplant breeding programs are mainly aimed to the generation of F1 hybrids by crossing two highly homozygous, pure lines, which are traditionally obtained upon several self crossing generations, which is an expensive and time consuming process. Alternatively, fully homozygous, doubled haploid (DH) individuals can be induced from haploid cells of the germ line in a single generation. Several attempts have been made to develop protocols to produce eggplant DHs principally using anther culture and isolated microspore culture. Eggplant could be considered a moderately recalcitrant species in terms of ability for DH production. Anther culture stands nowadays as the most valuable technology to obtain eggplant DHs. However, the theoretical possibility of having plants regenerated from somatic tissues of the anther walls cannot be ruled out. For this reason, the use of isolated microspores is recommended when possible. This approach still has room for improvement, but it is largely genotype-dependent. In this review, we compile the most relevant advances made in DH production in eggplant, their application to breeding programs, and the future perspectives for the development of other, less genotype-dependent, DH technologies.

Doubled Haploid Production in High- and Low-Response Genotypes of Rapeseed (Brassica napus) Through Isolated Microspore Culture.

Rapeseed (Brassica napus) is one of the most important oilseed crops worldwide. It is also a model system to study the process of microspore embryogenesis, due to the high response of some B. napus lines, and to the refinements of the protocols. This chapter presents a protocol for the induction of haploid and DH embryos in B. napus through isolated microspore culture in two specific backgrounds widely used in DH research, the high response DH4079 line and the low response DH12075 line. We also present methods to identify the best phenological window to identify buds with microspores/pollen at the right developmental stage to induce this process. Methods to determine microspore/pollen viability and to check the ploidy by flow cytometry are also described.

Species with Haploid or Doubled Haploid Protocols.

In this chapter, we present a list of species (and few interspecific hybrids) where haploids and/or doubled haploids have been published, including the method by which they were obtained and the corresponding references. This list is an update of the compilation work of Maluszynski et al. published in 2003, including new species for which protocols were not available at that time, and also novel methodologies developed during these years. The list includes 383 different backgrounds. In this book, we present full protocols to produce DHs in 43 of the species included in this list. In addition, this book includes a chapter for one species not included in the list. This makes a total of 384 species where haploids and/or DHs have been reported up to date.

Division Plane Orientation Defects Revealed by a Synthetic Double Mutant Phenotype.

TANGLED1 (TAN1) and AUXIN-INDUCED-IN-ROOTS9 (AIR9) are microtubule-binding proteins that localize to the division site in plants. Their function in Arabidopsis (Arabidopsis thaliana) remained unclear because neither tan1 nor air9 single mutants have a strong phenotype. We show that tan1 air9 double mutants have a synthetic phenotype consisting of short, twisted roots with disordered cortical microtubule arrays that are hypersensitive to a microtubule-depolymerizing drug. The tan1 air9 double mutants have significant defects in division plane orientation due to failures in placing the new cell wall at the correct division site. Full-length TAN1 fused to yellow fluorescent protein, TAN1-YFP, and several deletion constructs were transformed into the double mutant to assess which regions of TAN1 are required for its function in root growth, root twisting, and division plane orientation. TAN1-YFP expressed in tan1 air9 significantly rescued the double mutant phenotype in all three respects. Interestingly, TAN1 missing the first 126 amino acids, TAN1-ΔI-YFP, failed to rescue the double mutant phenotype, while TAN1 missing a conserved middle region, TAN1-ΔII-YFP, significantly rescued the mutant phenotype in terms of root growth and division plane orientation but not root twisting. We use the tan1 air9 double mutant to discover new functions for TAN1 and AIR9 during phragmoplast guidance and root morphogenesis.

A DII Domain-Based Auxin Reporter Uncovers Low Auxin Signaling during Telophase and Early G1.

A sensitive and dynamically responsive auxin signaling reporter based on the DII domain of the INDOLE-3-ACETIC ACID28 (IAA28, DII) protein from Arabidopsis (Arabidopsis thaliana) was modified for use in maize (Zea mays). The DII domain was fused to a yellow fluorescent protein and a nuclear localization sequence to simplify quantitative nuclear fluorescence signal. DII degradation dynamics provide an estimate of input signal into the auxin signaling pathway that is influenced by both auxin accumulation and F-box coreceptor concentration. In maize, the DII-based marker responded rapidly and in a dose-dependent manner to exogenous auxin via proteasome-mediated degradation. Low levels of DII-specific fluorescence corresponding to high endogenous auxin signaling occurred near vasculature tissue and the outer layer and glume primordia of spikelet pair meristems and floral meristems, respectively. In addition, high DII levels were observed in cells during telophase and early G1, suggesting that low auxin signaling at these stages may be important for cell cycle progression.

Pathogen and circadian controlled 1 (PCC1) protein is anchored to the plasma membrane and interacts with subunit 5 of COP9 signalosome in Arabidopsis.

The Pathogen and Circadian Controlled 1 (PCC1) gene, previously identified and further characterized as involved in defense to pathogens and stress-induced flowering, codes for an 81-amino acid protein with a cysteine-rich C-terminal domain. This domain is essential for homodimerization and anchoring to the plasma membrane. Transgenic plants with the ß-glucuronidase (GUS) reporter gene under the control of 1.1 kb promoter sequence of PCC1 gene display a dual pattern of expression. At early post-germination, PCC1 is expressed only in the root vasculature and in the stomata guard cells of cotyledons. During the transition from vegetative to reproductive development, PCC1 is strongly expressed in the vascular tissue of petioles and basal part of the leaf, and it further spreads to the whole limb in fully expanded leaves. This developmental pattern of expression together with the late flowering phenotype of long-day grown RNA interference (iPCC1) plants with reduced PCC1 expression pointed to a regulatory role of PCC1 in the photoperiod-dependent flowering pathway. iPCC1 plants are defective in light perception and signaling but are not impaired in the function of the core CO-FT module of the photoperiod-dependent pathway. The regulatory effect exerted by PCC1 on the transition to flowering as well as on other reported phenotypes might be explained by a mechanism involving the interaction with the subunit 5 of the COP9 signalosome (CSN).

Diverse functional interactions between nitric oxide and abscisic acid in plant development and responses to stress.

The extensive support for abscisic acid (ABA) involvement in the complex regulatory networks controlling stress responses and development in plants contrasts with the relatively recent role assigned to nitric oxide (NO). Because treatment with exogenous ABA leads to enhanced production of NO, it has been widely considered that NO participates downstream of ABA in controlling processes such as stomata movement, seed dormancy, and germination. However, data on leaf senescence and responses to stress suggest that the functional interaction between ABA and NO is more complex than previously thought, including not only cooperation but also antagonism. The functional relationship is probably determined by several factors including the time- and place-dependent pattern of accumulation of both molecules, the threshold levels, and the regulatory factors important for perception. These factors will determine the actions exerted by each regulator. Here, several examples of well-documented functional interactions between NO and ABA are analysed in light of the most recent reported data on seed dormancy and germination, stomata movements, leaf senescence, and responses to abiotic and biotic stresses.

Pathogen and Circadian Controlled 1 (PCC1) regulates polar lipid content, ABA-related responses, and pathogen defence in Arabidopsis thaliana.

Pathogen and Circadian Controlled 1 (PCC1) was previously characterized as a regulator of defence against pathogens and stress-activated transition to flowering. Plants expressing an RNA interference construct for the PCC1 gene (iPCC1 plants) showed a pleiotropic phenotype. They were hypersensitive to abscisic acid (ABA) as shown by reduced germination potential and seedling establishment, as well as reduced stomatal aperture and main root length in ABA-supplemented media. In addition, iPCC1 plants displayed alterations in polar lipid contents and their corresponding fatty acids. Importantly, a significant reduction in the content of phosphatidylinositol (PI) was observed in iPCC1 leaves when compared with wild-type plants. A trend in reduced levels of 18:0 and increased levels of 18:2 and particularly 18:3 was also detected in several classes of polar lipids. The enhanced ABA-mediated responses and the reduced content of PI might be responsible for iPCC1 plants displaying a complex pattern of defence against pathogens of different lifestyles. iPCC1 plants were more susceptible to the hemi-biotrophic oomycete pathogen Phytophthora brassicae and more resistant to the necrotrophic fungal pathogen Botrytis cinerea compared with wild-type plants.

A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunity.

Wounded leaves of Arabidopsis thaliana show transient immunity to Botrytis cinerea, the causal agent of grey mould. Using a fluorescent probe, histological staining and a luminol assay, we now show that reactive oxygen species (ROS), including H(2)O(2) and O(2) (-), are produced within minutes after wounding. ROS are formed in the absence of the enzymes Atrboh D and F and can be prevented by diphenylene iodonium (DPI) or catalase. H(2)O(2) was shown to protect plants upon exogenous application. ROS accumulation and resistance to B. cinerea were abolished when wounded leaves were incubated under dry conditions, an effect that was found to depend on abscisic acid (ABA). Accordingly, ABA biosynthesis mutants (aba2 and aba3) were still fully resistant under dry conditions even without wounding. Under dry conditions, wounded plants contained higher ABA levels and displayed enhanced expression of ABA-dependent and ABA-reporter genes. Mutants impaired in cutin synthesis such as bdg and lacs2.3 are already known to display a high level of resistance to B. cinerea and were found to produce ROS even when leaves were not wounded. An increased permeability of the cuticle and enhanced ROS production were detected in aba2 and aba3 mutants as described for bdg and lacs2.3. Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is strongly linked with ROS formation and resistance to B. cinerea. The amount of oxalic acid, an inhibitor of ROS secreted by B. cinerea could be reduced using plants over expressing a fungal oxalate decarboxylase of Trametes versicolor. Infection of such plants resulted in a faster ROS accumulation and resistance to B. cinerea than that observed in untransformed controls, demonstrating the importance of fungal suppression of ROS formation by oxalic acid. Thus, changes in the diffusive properties of the cuticle are linked with the induction ROS and attending innate defenses.

Genome-wide analyses of the transcriptomes of salicylic acid-deficient versus wild-type plants uncover Pathogen and Circadian Controlled 1 (PCC1) as a regulator of flowering time in Arabidopsis.

Salicylic acid (SA) has been characterized as an activator of pathogen-triggered resistance of plants. SA also regulates developmental processes such as thermogenesis in floral organs and stress-induced flowering. To deepen our knowledge of the mechanism underlying SA regulation of flowering time in Arabidopsis, we compared the transcriptomes of SA-deficient late flowering genotypes with wild-type plants. Down- or up-regulated genes in SA-deficient plants were screened for responsiveness to ultraviolet (UV)-C light, which accelerates flowering in Arabidopsis. Among them, only Pathogen and Circadian Controlled 1 (PCC1) was up-regulated by UV-C light through a SA-dependent process. Moreover, UV-C light-activated expression of PCC1 was also dependent on the flowering activator CONSTANS (CO). PCC1 gene has a circadian-regulated developmental pattern of expression with low transcript levels after germination that increased abruptly by day 10. RNAi plants with very low expression of PCC1 gene were late flowering, defective in UV-C light acceleration of flowering and contained FLOWERING LOCUS T (FT) transcript levels below 5% of that detected in wild-type plants. Although PCC1 seems to function between CO and FT in the photoperiod-dependent flowering pathway, transgenic plants overexpressing a Glucocorticoid Receptor (GR)-fused version of CO strongly activated FT but not PCC1 after dexamethasone treatment.