Plant–pathogen interactions have been widely studied, but mostly from the site of the plant secondary defense. Less is known about the effects of pathogen infection on plant primary metabolism. The ...possibility to transform a fluorescing protein into prokaryotes is a promising phenotyping tool to follow a bacterial infection in plants in a noninvasive manner. In the present study, virulent and avirulent
Pseudomonas syringae
strains were transformed with green fluorescent protein (GFP) to follow the spread of bacteria
in vivo
by imaging Pulse-Amplitude-Modulation (PAM) fluorescence and conventional binocular microscopy. The combination of various wavelengths and filters allowed simultaneous detection of GFP-transformed bacteria, PAM chlorophyll fluorescence, and phenolic fluorescence from pathogen-infected plant leaves. The results show that fluorescence imaging allows spatiotemporal monitoring of pathogen spread as well as phenolic and chlorophyll fluorescence
in situ
, thus providing a novel means to study complex plant–pathogen interactions and relate the responses of primary and secondary metabolism to pathogen spread and multiplication. The study establishes a deeper understanding of imaging data and their implementation into disease screening.
Reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anions significantly accumulate during biotic and abiotic stress and cause oxidative damage and eventually cell death. There is ...accumulating evidence that ROS are also involved in regulating beneficial plant-microbe interactions, signal transduction and plant growth and development. Due to the relevance of ROS throughout the life cycle and for interaction with the multifactorial environment, the physiological phenotyping of the mechanisms controlling ROS homeostasis is of general importance.
In this study, we have developed a robust and resource-efficient experimental platform that allows the determination of the activities of the nine key ROS scavenging enzymes from a single extraction that integrates posttranscriptional and posttranslational regulations. The assays were optimized and adapted for a semi-high throughput 96-well assay format. In a case study, we have analyzed tobacco leaves challenged by pathogen infection, drought and salt stress. The three stress factors resulted in distinct activity signatures with differential temporal dynamics.
This experimental platform proved to be suitable to determine the antioxidant enzyme activity signature in different tissues of monocotyledonous and dicotyledonous model and crop plants. The universal enzymatic extraction procedure combined with the 96-well assay format demonstrated to be a simple, fast and semi-high throughput experimental platform for the precise and robust fingerprinting of nine key antioxidant enzymatic activities in plants.
is a major Christmas tree species in Europe, but their uneven and prolonged growth slows down their production. By a 16S and 18S rRNA gene amplicon sequencing approach, we performed a ...characterization of root-associated bacterial and fungal communities for three-year-old
plants collected from two nurseries in Denmark and Germany and displaying different growth patterns (small versus tall plants). Proteobacteria had the highest relative abundance at both sampling sites and plant sizes, and Ascomycota was the most abundant fungal phylum. At the order level, Acidobacteriales, Actinomycetales, Burkholderiales, Rhizobiales, and Xanthomonadales represented the bacterial core microbiome of
, independently of the sampling site or plant size, while the fungal core microbiome included members of the Agaricales, Hypocreales, and Pezizales. Principal Coordinate Analysis indicated that both bacterial and fungal communities clustered according to the sampling site pointing to the significance of soil characteristics and climatic conditions for the composition of root-associated microbial communities. Major differences between communities from tall and small plants were a dominance of the potential pathogen
(Hypocreales) in the small plants from Germany, while Agaricales, that includes reported beneficial ectomycorrhizal fungi, dominated in the tall plants. An evaluation of plant root antioxidative enzyme profiles showed higher levels of the antioxidative enzymes ascorbate peroxidase, peroxidase, and superoxide dismutase in small plants compared to tall plants. We suggest that the higher antioxidative enzyme activities combined with the growth arrest phenotype indicate higher oxidative stress levels in the small plants. Additionally, the correlations between the relative abundances of specific taxa of the microbiome with the plant antioxidative enzyme profiles were established. The main result was that many more bacterial taxa correlated positively than negatively with one or more antioxidative enzyme activity. This may suggest that the ability of bacteria to increase plant antioxidative enzyme defenses is widespread.
Endophytic fungi that colonize the plant root live in an environment with relative high concentrations of different sugars. Analyses of genome sequences indicate that such endophytes can secrete ...carbohydrate-related enzymes to compete for these sugars with the surrounding plant cells. We hypothesized that typical plant sugars can be used as carbon source by root endophytes and that these sugars also serve as signals to induce the expression and secretion of glycolytic enzymes. The plant-growth-promoting endophytes
and
were selected to first determine which sugars promote their growth and biomass formation. Secondly, particular sugars were added to liquid cultures of the fungi to induce intracellular and extracellular enzymatic activities which were measured in mycelia and culture supernatants. The results showed that both fungi cannot feed on melibiose and lactose, but instead use glucose, fructose, sucrose, mannose, arabinose, galactose and xylose as carbohydrate sources. These sugars regulated the cytoplasmic activity of glycolytic enzymes and also their secretion. The levels of induction or repression depended on the type of sugars added to the cultures and differed between the two fungi. Since no conventional signal peptide could be detected in most of the genome sequences encoding the glycolytic enzymes, a non-conventional protein secretory pathway is assumed. The results of the study suggest that root endophytic fungi translocate glycolytic activities into the root, and this process is regulated by the availability of particular plant sugars.
Nitrogen (N) efficiency of winter oilseed rape (Brassica napus L.) line-cultivars (cvs.), defined as high grain yield under N limitation, has been primarily attributed to maintained N uptake during ...reproductive growth (N uptake efficiency) in combination with delayed senescence of the older leaves accompanied with maintained photosynthetic capacity (functional stay-green). However, it is not clear whether genotypic variation in N starvation-induced leaf senescence is due to leaf-inherent factors and/or governed by root-mediated signals. Therefore, the N-efficient and stay-green cvs. NPZ-1 and Apex were reciprocally grafted with the N-inefficient and early-senescing cvs. NPZ-2 and Capitol, respectively and grown in hydroponics. The senescence status of older leaves after 12 days of N starvation assessed by SPAD, photosynthesis and the expression of the senescence-specific cysteine protease gene SAG12-1 revealed that the stay-green phenotype of the cvs. NPZ-1 and Apex under N starvation was primarily under the control of leaf-inherent factors. The same four cultivars were submitted to N starvation for up to 12 days in a time-course experiment. The specific leaf contents of biologically active and inactive cytokinins (CKs) and the expression of genes involved in CK homeostasis revealed that under N starvation leaves of early-senescing cultivars were characterized by inactivation of biologically active CKs, whereas in stay-green cultivars synthesis, activation, binding of and response to biologically active CKs were favoured. These results suggest that the homeostasis of biologically active CKs was the predominant leaf-inherent factor for cultivar differences in N starvation-induced leaf senescence and thus N efficiency.
Cytokinins and cell wall invertase are positive players in regulating fruit sink strength, growth, and yield under salinity as components of the same signalling cascade establishing and developing ...sink organs.
Salinization of water and soil has a negative impact on tomato (Solanum lycopersicum L.) productivity by reducing growth of sink organs and by inducing senescence in source leaves. It has been hypothesized that yield stability implies the maintenance or increase of sink activity in the reproductive structures, thus contributing to the transport of assimilates from the source leaves through changes in sucrolytic enzymes and their regulation by phytohormones. In this study, classical and functional physiological approaches have been integrated to study the influence of metabolic and hormonal factors on tomato fruit sink activity, growth, and yield: (i) exogenous hormones were applied to plants, and (ii) transgenic plants overexpressing the cell wall invertase (cwInv) gene CIN1 in the fruits and de novo cytokinin (CK) biosynthesis gene IPT in the roots were constructed. Although salinity reduces fruit growth, sink activity, and trans-zeatin (tZ) concentrations, it increases the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) during the actively growing period (25 days after anthesis). Indeed, exogenous application of the CK analogue kinetin to salinized actively growing fruits recovered sucrolytic activities (mainly cwInv and sucrose synthase), sink strength, and fruit weight, whereas the ethylene-releasing compound ethephon had a negative effect in equivalent non-stressed fruits. Fruit yield was increased by both the constitutive expression of CIN1 in the fruits (up to 4-fold) or IPT in the root (up to 30%), owing to an increase in the fruit number (lower flower abortion) and in fruit weight. This is possibly related to a recovery of sink activity in reproductive tissues due to both (i) increase in sucrolytic activities (cwInv, sucrose synthase, and vacuolar and cytoplasmic invertases) and tZ concentration, and (ii) a decrease in the ACC levels and the activity of the invertase inhibitor. This study provides new functional evidences about the role of metabolic and hormonal inter-regulation of local sink processes in controlling tomato fruit sink activity, growth, and yield under salinity.
Abies nordmanniana
is used for Christmas tree production but poor seed germination and slow growth represent challenges for the growers. We addressed the plant growth promoting potential of ...root-associated bacteria isolated from
A. nordmanniana
. Laboratory screenings of a bacterial strain collection yielded several
Bacillus
and
Paenibacillus
strains that improved seed germination and produced indole-3-acetic acid. The impact of three of these strains on seed germination, plant growth and growth-related physiological parameters was then determined in greenhouse and field trials after seed inoculation, and their persistence was assessed by 16S rRNA gene-targeted bacterial community analysis. Two strains showed distinct and significant effects.
Bacillus
sp. s50 enhanced seed germination in the greenhouse but did not promote shoot or root growth. In accordance, this strain did not increase the level of soluble hexoses needed for plant growth but increased the level of storage carbohydrates. Moreover, strain s50 increased glutathione reductase and glutathione-S-transferase activities in the plant, which may indicate induction of systemic resistance during the early phase of plant development, as the strain showed poor persistence in the root samples (rhizosphere soil plus root tissue).
Paenibacillus
sp. s37 increased plant root growth, especially by inducing secondary root formation, under in greenhouse conditions, where it showed high persistence in the root samples. Under these conditions, it further it increased the level of soluble carbohydrates in shoots, and the levels of starch and non-structural carbohydrates in roots, stem and shoots. Moreover, it increased the chlorophyll level in the field trial. These findings indicate that this strain improves plant growth and vigor through effects on photosynthesis and plant carbohydrate reservoirs. The current results show that the two strains s37 and s50 could be considered for growth promotion programs of
A. nordmanniana
in greenhouse nurseries, and even under field conditions.
Five cyanobacterial strains, Anabaena sp. Ck1, Oscillatoria sp. Ck2, Phormidium sp. Ck3, Chroococcidiopsis sp. Ck4, and Synechosystis sp. Ck5 were selected for their positive cytokinins-like activity ...using cucumber cotyledon bioassay and GUS assay in Arabidopsis ARR5::GUS. Classical cucumber cotyledon bioassay was modified for direct screening of cyanobacteria avoiding need for extraction and purification. Cytokinins from cyanobacteria were absorbed onto filter paper which was then assayed for cytokinins-like activity. A rapid chromatographic method was developed for the simultaneous determination of cytokinins and indole-3-acetic acid (IAA). Cyanobacterial biomass (50-100 mg) and cell-free culture filtrate were extracted in Bieleski buffer and purified by solid-phase extraction. The extract was used to determine phytohormones by ultra performance liquid chromatography and electrospray ionization-tandem mass spectrometry in positive and negative modes, respectively, with multiple reactions monitoring. Stable isotope-labeled cytokinins and IAA standards were added in the samples to follow recovery of the compounds and method validation. Five cytokinins determined in the selected strains were Zeatin (cis and trans isomers), Zeatin riboside, Dihydrozeatin riboside, and zeatin-o-glucoside. The strains were shown to accumulate as well as release the phytohormones.
Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly ...show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family.
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