Exogenous auxins supplementation plays a central role in the formation of adventitious roots (AR) for several plant species. However, the molecular mechanisms underlying the process of adventitious ...rooting are still not completely understood and many plants with economic value, including several olive cultivars, exhibit a recalcitrant behavior towards cutting propagation, which limits its availability in plant nurseries. PIN-formed proteins are auxin efflux transporters that have been widely characterized in several plant species due to their involvement in many developmental processes including root formation. The present study profiled the expression of the
,
,
,
, and
gene members during indole-3-butyric acid (IBA)-induced in vitro adventitious rooting using the olive cultivar 'Galega vulgar'. Gene expression analysis by quantitative real time PCR (RT-qPCR) showed drastic downregulation of most transcripts, just a few hours after explant inoculation, in both nontreated and IBA-treated microcuttings, albeit gene downregulation was less pronounced in IBA-treated stems. In contrast,
showed a distinct expression pattern being upregulated in both conditions, and
was highly upregulated in IBA-induced stems. All transcripts, except
, showed different expression profiles between nontreated and IBA-treated explants throughout the rooting experiment. Additionally, high levels of reactive oxygen species (ROS) were observed soon after explant preparation, decreasing a few hours after inoculation. Altogether, the results suggest that wounding-related ROS production, associated with explant preparation for rooting, may have an impact on auxin transport and distribution via changes in
gene expression. Moreover, the application of exogenous auxin may modulate auxin homeostasis through regulation of those genes, leading to auxin redistribution throughout the stem-base tissue, which may ultimately play an important role in AR formation.
Peptidoglycan (PG) is a ubiquitous structural polysaccharide of the bacterial cell wall, essential in preserving cell integrity by withstanding turgor pressure. Any change that affects its ...biosynthesis or degradation will disturb cell viability, therefore PG is one of the main targets of antimicrobial drugs. Considering its major role in cell structure and integrity, the study of PG is of utmost relevance, with prospective ramifications to several disciplines such as microbiology, pharmacology, agriculture, and pathogenesis. Traditionally, high-performance liquid chromatography (HPLC) has been the workhorse of PG analysis. In recent years, technological and bioinformatic developments have upgraded this seminal technique, making analysis more sensitive and efficient than ever before. Here we describe a set of analytical tools for the study of PG structure (from composition to 3D architecture), identify the most recent trends, and discuss future challenges in the field.
PG is a major bacterial cell wall component, crucial for cell integrity, and strongly related to antibiotic sensitivity.Methods to analyze PG structure have evolved over time, and new and improved methods are currently replacing previous tedious and long procedures.Current tools, ranging from chromatography–mass spectrometry methods to imaging techniques, allow characterizing PG from a structural and architectural perspective.
Plant hormones, and especially auxins, are low molecular weight compounds highly involved in the control of plant growth and development. Auxins are also broadly used in horticulture, as part of ...vegetative plant propagation protocols, allowing the cloning of genotypes of interest. Over the years, large efforts have been put in the development of more sensitive and precise methods of analysis and quantification of plant hormone levels in plant tissues. Although analytical techniques have evolved, and new methods have been implemented, sample preparation is still the limiting step of auxin analysis. In this review, the current methods of auxin analysis are discussed. Sample preparation procedures, including extraction, purification and derivatization, are reviewed and compared. The different analytical techniques, ranging from chromatographic and mass spectrometry methods to immunoassays and electrokinetic methods, as well as other types of detection are also discussed. Considering that auxin analysis mirrors the evolution in analytical chemistry, the number of publications describing new and/or improved methods is always increasing and we considered appropriate to update the available information. For that reason, this article aims to review the current advances in auxin analysis, and thus only reports from the past 15 years will be covered.
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•Recent developments in auxin analysis are covered.•Critical review of sample preparation methods is presented.•Extraction, purification and derivatization strategies are discussed.•Main analytical techniques are critically compared and debated.
•Olive may become a model species for studies on adventitious rooting.•Fundamental research on adventitious root formation is scarce.•Analytical methods are difficult to establish in woody ...perennials.•Studies must be redirected to specific tissues and cells.•Biochemical analyses are inefficient without concomitant genetic evaluation.
Olive (Olea europaea) is one of the most important fruit species in the Mediterranean basin, where 95% of the world’s olive orchards are planted, and it has become an economically valuable crop worldwide, due to an increasing interest in olive oil for human consumption. New olive orchards are being planted outside the Mediterranean, calling for an effort to identify the genotypes best adapted to the new conditions. However, some olive cultivars remain difficult to propagate, which significantly reduces the capacity to use the full genetic diversity of the species. Improving rooting ability in cuttings from recalcitrant olive cultivars has become a critical topic, which implies fundamental research on the anatomy, physiology, biochemistry and genetics of the adventitious root formation process. Besides, the existence of different rooting behaviors among olive cultivars also makes the species a candidate model plant for these studies. Olive propagation techniques evolved through time from field- or nursery-planted hardwood cuttings, to semi-hardwood cuttings in greenhouses under mist, and, more recently, to in vitro culture techniques. Nevertheless, research about adventitious root formation carried on each propagation method was mostly based on trial and error approaches. Researchers have mainly investigated different factors involved in the process of adventitious rooting by testing their effect in the rooting capacity of different cultivars, leading to a high dispersion and fragmentation of the available information. The goal of this review is to present the most relevant results achieved on adventitious root formation in olive cuttings, aiming to provide an integrated perspective of the current knowledge.
Abstract
Human breast milk is an incredibly rich and complex biofluid composed of proteins, lipids and complex carbohydrates, including a diverse repertoire of free human milk oligosaccharides ...(HMOs). Strikingly, HMOs are not digested by the infant but function as prebiotics for bacterial strains associated with numerous benefits. Considering the broad variety of beneficial effects of HMOs, and the vast number of factors that affect breast milk composition, the analysis of HMO diversity and complexity is of utmost relevance. Using human milk samples from a cohort of Bangladeshi mothers participating in a study on malnutrition and stunting in children, we have characterized breast milk oligosaccharide composition by means of permethylation followed by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-MS/MS) analysis. This approach identified over 100 different glycoforms and showed a wide diversity of milk composition, with a predominance of fucosylated and sialylated HMOs over nonmodified HMOs. We observed that these samples contain on average 80 HMOs, with the highest permethylated masses detected being >5000 mass units. Here we report an easily implemented method developed for the separation, characterization and relative quantitation of large arrays of HMOs, including higher molecular weight sialylated HMOs. Our ultimate goal is to create a simple, high-throughput method, which can be used for full characterization of sialylated and/or fucosylated HMOs. These results demonstrate how current analytical techniques can be applied to characterize human milk composition, providing new tools to help the scientific community shed new light on the impact of HMOs during infant development.
•DFG-5 α-1,6-mannase associates with cell wall glycoproteins. This association is dependent upon N-linked galactomannans.•DFG-5 recognizes the α-1,6-backbone of the N-linked galactomannans and has ...enzymatic activity.•DFG-5 discriminates between cell wall glycoproteins and secreted glycoproteins.•By cleaving the N-linked galactomannans on cell wall glycoproteins, DFG-5 targets them for incorporation into the cell wall.•Secreted glycoproteins are not recognized by DFG-5 and are released into the growth medium.
The formation of a cell wall is vital for the survival and growth of a fungal cell. Fungi express members of the GH76 family of α-1,6-mannanases which play an important role in cell wall biogenesis. In this report we characterize the Neurospora crassa DFG-5 α-1,6-mannanase and demonstrate that it binds to the α-1,6-mannose backbone of an N-linked galactomannan found on cell wall glycoproteins. We show that DFG-5 has an enzymatic activity and provide evidence that it processes the α-1,6-mannose backbone of the N-linked galactomannan. Site-directed mutagenesis and complementation experiments show that D116 and D117 are located at the DFG-5 active site. D76 and E130, which are located in a groove on the opposite side of the protein, are also important for enzyme function. Cell wall glycoproteins co-purify with DFG-5 demonstrating a specific association between DFG-5 and cell wall glycoproteins. DFG-5 is able to discriminate between cell wall and secreted glycoproteins, and does not bind to the N-linked galactomannans present on secreted glycoproteins. DFG-5 plays a key role in targeting extracellular glycoproteins to their final destinations. By processing the galactomannans on cell wall proteins, DFG-5 targets them for cell wall incorporation by lichenin transferases. The N-linked galactomannans on secreted proteins are not processed by DFG-5, which targets these proteins for release into the extracellular medium.
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•Rooting ability is likely related with IBA-IAA conversion and oxidative enzymes activity.•Olive microcuttings and semi-hardwood cuttings present similar rooting behavior.•Molecular ...pathways potentially involved in olive adventitious rooting are described.•The potential involvement of AOX genes in rooting is exploited.•SHAM may be a good choice to obtain negative controls in rooting assays.
The activity of oxidative enzymes and the levels of free auxins were determined during adventitious root formation in olive explants. Rooting trials were performed both with in vitro-cultured microshoots of the cultivar ‘Galega Vulgar’, treated with indole-3-butyric acid (IBA) and with salicylhydroxamic acid (SHAM)+IBA, as well as with semi-hardwood cuttings of the cultivars ‘Galega Vulgar’ (difficult-to-root) and ‘Cobrançosa’ (easy-to-root), treated with IBA. The auxin (IBA) was used in all experiments as a rooting promoter, while SHAM was used in micropropagation trials as rooting inhibitor, providing a negative control. Free indole-3-acetic acid (IAA) and IBA concentrations were determined in microshoots, as well as in semi-hardwood cuttings, throughout the rooting period at pre-established time-points. At the same time-points, the enzymatic activity of polyphenol oxidases (PPO), peroxidases (POX), and IAA oxidase (IAAox) was evaluated in the microshoots. Microshoots treated with SHAM+IBA revealed higher POX and IAAox activity, as well as lower PPO activity, than those treated only with IBA. IAA levels were higher in IBA-treated microshoots during induction phase, but lower during early initiation phase. In contrast, free IBA levels were higher in microshoots treated with SHAM+IBA during induction, but lower during initiation. A similar pattern of free auxin levels was observed in semi-hardwood cuttings of the two contrasting cultivars under evaluation. The similarities found on the auxin patterns of microshoots treated with SHAM and those of semi-hardwood cuttings of the difficult-to-root olive cultivar allow considering SHAM a reliable control for when simulation of a difficult-to-root behavior is necessary. The inhibitory effect of SHAM in root formation could be related with 1) the inhibition of alternative oxidase (AOX), leading to a downregulation of phenylpropanoid biosynthetic pathways, which would decrease the concentration of phenolic substrates for PPO; 2) an increase in IAAox activity resulting in lower free IAA levels or; 3) a defective conversion of IBA into IAA.
Several studies have suggested that differences in the natural rooting ability of plant cuttings could be attributed to differences in endogenous auxin levels. Hence, during rooting experiments, it ...is important to be able to routinely monitor the evolution of endogenous levels of plant hormones. This work reports the development of a new method for the quantification of free auxins in auxin-treated Olea europaea (L.) explants, using dispersive liquid–liquid microextraction (DLLME) and microwave assisted derivatization (MAD) followed by gas chromatography/mass spectrometry (GC/MS) analysis. Linear ranges of 0.5–500 ng mL −1 and 1–500 μg mL −1 were used for the quantification of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), respectively. Determined by serial dilutions, the limits of detection (LOD) and quantification (LOQ) were 0.05 ng mL −1 and 0.25 ng mL −1 , respectively for both compounds. When using the calibration curve for determination, the LOQ corresponded to 0.5 ng mL −1 (IAA) and 0.5 μg mL −1 (IBA). The proposed method proved to be substantially faster than other alternatives, and allowed free auxin quantification in real samples of semi-hardwood cuttings and microshoots of two olive cultivars. The concentrations found in the analyzed samples are in the range of 0.131–0.342 μg g −1 (IAA) and 20–264 μg g −1 (IBA).
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