Optimized fluorescent proteins for the rhizosphere-associated bacterium Bacillus mycoides with endophytic and biocontrol agent potential.

Tracking of fluorescent protein (FP)-labelled rhizobacteria is a key prerequisite to gain insights into plant-bacteria interaction mechanisms. However, the performance of FPs mostly has to be optimized for the bacterial host and for the environment of intended application. We report on the construction of mutational libraries of the superfolder green fluorescent protein sfGFP and the red fluorescent protein mKate2 in the bacterium B. mycoides, which next to its potential as plant-biocontrol agent occasionally enters an endophytic lifestyle. By fluorescence-activated cell sorting and comparison of signal intensities at the colony and single-cell level, the variants sfGFP(SPS6) and mKate (KPS12) with significantly increased brightness were isolated. Their high applicability for plant-bacteria interaction studies was shown by confocal laser scanning microscopy tracking of FP-tagged B. mycoides strains after inoculation to Chinese cabbage plants in a hydroponic system. During the process of colonization, strain EC18 rapidly attached to plant roots and formed a multicellular matrix, especially at the branching regions of the root hair, which probably constitute entrance sites to establish an endophytic lifestyle. The universal applicability of the novels FPs was proven by expression from a weak promoter, dual-labelling of B. mycoides, and by excellent expression and detectability in additional soil- and rhizosphere-associated Bacillus species.

Functional optics of glossy buttercup flowers.

Buttercup (Ranunculus spp.) flowers are exceptional because they feature a distinct gloss (mirror-like reflection) in addition to their matte-yellow coloration. We investigated the optical properties of yellow petals of several Ranunculus and related species using (micro)spectrophotometry and anatomical methods. The contribution of different petal structures to the overall visual signal was quantified using a recently developed optical model. We show that the coloration of glossy buttercup flowers is due to a rare combination of structural and pigmentary coloration. A very flat, pigment-filled upper epidermis acts as a thin-film reflector yielding the gloss, and additionally serves as a filter for light backscattered by the strongly scattering starch and mesophyll layers, which yields the matte-yellow colour. We discuss the evolution of the gloss and its two likely functions: it provides a strong visual signal to insect pollinators and increases the reflection of sunlight to the centre of the flower in order to heat the reproductive organs.

Roots Withstanding their Environment: Exploiting Root System Architecture Responses to Abiotic Stress to Improve Crop Tolerance.

To face future challenges in crop production dictated by global climate changes, breeders and plant researchers collaborate to develop productive crops that are able to withstand a wide range of biotic and abiotic stresses. However, crop selection is often focused on shoot performance alone, as observation of root properties is more complex and asks for artificial and extensive phenotyping platforms. In addition, most root research focuses on development, while a direct link to the functionality of plasticity in root development for tolerance is often lacking. In this paper we review the currently known root system architecture (RSA) responses in Arabidopsis and a number of crop species to a range of abiotic stresses, including nutrient limitation, drought, salinity, flooding, and extreme temperatures. For each of these stresses, the key molecular and cellular mechanisms underlying the RSA response are highlighted. To explore the relevance for crop selection, we especially review and discuss studies linking root architectural responses to stress tolerance. This will provide a first step toward understanding the relevance of adaptive root development for a plant's response to its environment. We suggest that functional evidence on the role of root plasticity will support breeders in their efforts to include root properties in their current selection pipeline for abiotic stress tolerance, aimed to improve the robustness of crops.

How to colour a flower: on the optical principles of flower coloration.

The coloration of flowers is due to the wavelength-selective absorption by pigments of light backscattered by structures inside the petals. We investigated the optical properties of flowers using (micro)spectrophotometry and anatomical methods. To assess the contribution of different structures to the overall visual signal of flowers, we used an optical model, where a petal is considered as a stack of differently pigmented and structured layers and we interpreted the visual signals of the model petals with insect vision models. We show that the reflectance depends, in addition to the pigmentation, on the petal's thickness and the inhomogeneity of its interior. We find large between-species differences in floral pigments, pigment concentration and localization, as well as floral interior structure. The fractions of reflected and transmitted light are remarkably similar between the studied species, suggesting common selective pressures of pollinator visual systems. Our optical model highlights that pigment localization crucially determines the efficiency of pigmentary filtering and thereby the chromatic contrast and saturation of the visual signal. The strongest visual signal occurs with deposition of pigments only on the side of viewing. Our systematic approach and optical modelling open new perspectives on the virtues of flower colour.

Draft Genome Sequence of Bacillus mycoides M2E15, a Strain Isolated from the Endosphere of Potato.

We present the draft genome sequence of Bacillus mycoides M2E15, a bacterium isolated from potato endosphere. Analysis of the 6.08-Mbp draft genome sequence identified 6,386 protein-encoding sequences, including potential plant growth promoting genes. Specifically, genes for proteins involved in phosphate utilization, iron acquisition, and bacteriocin production were identified.

Iridescent flowers? Contribution of surface structures to optical signaling.

The color of natural objects depends on how they are structured and pigmented. In flowers, both the surface structure of the petals and the pigments they contain determine coloration. The aim of the present study was to assess the contribution of structural coloration, including iridescence, to overall floral coloration. We studied the reflection characteristics of flower petals of various plant species with an imaging scatterometer, which allows direct visualization of the angle dependence of the reflected light in the hemisphere above the petal. To separate the light reflected by the flower surface from the light backscattered by the components inside (e.g. the vacuoles), we also investigated surface casts. A survey among angiosperms revealed three different types of floral surface structure, each with distinct reflections. Petals with a smooth and very flat surface had mirror-like reflections and petal surfaces with cones yielded diffuse reflections. Petals with striations yielded diffraction patterns when single cells were illuminated. The iridescent signal, however, vanished when illumination similar to that found in natural conditions was applied. Pigmentary rather than structural coloration determines the optical appearance of flowers. Therefore, the hypothesized signaling by flowers with striated surfaces to attract potential pollinators presently seems untenable.

Light quality-mediated petiole elongation in Arabidopsis during shade avoidance involves cell wall modification by xyloglucan endotransglucosylase/hydrolases.

Some plants can avoid shaded conditions via rapid shoot elongation, thus growing into better lit areas in a canopy. Cell wall-modifying mechanisms promoting this elongation response, therefore, are important regulatory points during shade avoidance. Two major cell wall-modifying protein families are expansins and xyloglucan endotransglucosylase/hydrolases (XTHs). The role of these proteins during shade avoidance was studied in Arabidopsis (Arabidopsis thaliana). In response to two shade cues, low red to far-red light (implying neighbor proximity) and green shade (mimicking dense canopy conditions), Arabidopsis showed classic shade avoidance features: petiole elongation and leaf hyponasty. Measurement of the apoplastic proton flux in green shade-treated petioles revealed a rapid efflux of protons into the apoplast within minutes, unlike white light controls. This apoplastic acidification probably provides the acidic pH required for the optimal activity of cell wall-modifying proteins like expansins and XTHs. Acid-induced extension, expansin susceptibility, and extractable expansin activity were similar in petioles from white light- and shade-treated plants. XTH activity, however, was high in petioles exposed to shade treatments. Five XTH genes (XTH9, -15, -16, -17, and -19) were positively regulated by low red to far-red light conditions, while the latter four and XTH22 showed a significant up-regulation also in response to green shade. Consistently, knockout mutants for two of these XTH genes also had reduced or absent shade avoidance responses to these light signals. These results point toward the cell wall as a vital regulatory point during shade avoidance.

Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca-associated opening of plasma membrane anion channels.

The perception of microbes by plants involves highly conserved molecular signatures that are absent from the host and that are collectively referred to as microbe-associated molecular patterns (MAMPs). The Arabidopsis pattern recognition receptors FLAGELLIN-SENSING 2 (FLS2) and EF-Tu receptor (EFR) represent genetically well studied paradigms that mediate defense against bacterial pathogens. Stimulation of these receptors through their cognate ligands, bacterial flagellin or bacterial elongation factor Tu, leads to a defense response and ultimately to increased resistance. However, little is known about the early signaling pathway of these receptors. Here, we characterize this early response in situ, using an electrophysiological approach. In line with a release of negatively charged molecules, voltage recordings of microelectrode-impaled mesophyll cells and root hairs of Col-0 Arabidopsis plants revealed rapid, dose-dependent membrane potential depolarizations in response to either flg22 or elf18. Using ion-selective microelectrodes, pronounced anion currents were recorded upon application of flg22 and elf18, indicating that the signaling cascades initiated by each of the two receptors converge on the same plasma membrane ion channels. Combined calcium imaging and electrophysiological measurements revealed that the depolarization was superimposed by an increase in cytosolic calcium that was indispensable for depolarization. NADPH oxidase mutants were still depolarized upon elicitor stimulation, suggesting a reactive oxygen species-independent membrane potential response. Furthermore, electrical signaling in response to either flg22 or elf 18 critically depends on the activity of the FLS2-associated receptor-like kinase BAK1, suggesting that activation of FLS2 and EFR lead to BAK1-dependent, calcium-associated plasma membrane anion channel opening as an initial step in the pathogen defense pathway.

Micro-electrode flux estimation confirms that the Solanum pimpinellifolium cu3 mutant still responds to systemin.

In this study, we introduce the Micro-Electrode Ion Flux Estimation technique as a sensitive and accurate technique to study systemin-induced changes in ion fluxes from isolated nearly intact plant tissues. Our results demonstrate the effectiveness and value of the Micro-Electrode Ion Flux Estimation technique to monitor and characterize those elicitor-induced ion flux changes from intact tissues. We used the method to monitor the systemin-induced changes in ion fluxes from leaf tissue of various plant species, including wild-type and cu3 mutant tomato (Solanum pimpinellifolium) plants, and confirm previous observations, but now in intact leaf tissue. Upon exposure of leaf tissue of plant species from the subtribe solaneae to systemin, the H(+) influx and K(+) efflux were transiently strongly increased. Plant species of other clades did not show a response upon systemin exposure. Although it has been reported that the gene containing the cu3 null mutation is identical to the SR160/tBRI1 gene, which encodes the systemin/brassinosteroid receptor and is essential in systemin and brassinosteroid perception, we observed no differences in the response of H(+) and K(+) fluxes from both wild-type and mutant leaf tissue to systemin. Also, the effects of various pharmacological effectors on systemin-induced flux changes were similar. Moreover, a SR160/tBRI1 transgene-containing tobacco (Nicotiana tabacum) line was insensitive to systemin, whereas both this line and its wild-type predecessor were responsive to the elicitor flg22. Our results support the conclusion that the Cu3 receptor of tomato is not the systemin receptor, and, hence, another receptor is the principal systemin receptor.

Signal Integration by ABA in the Blue Light-Induced Acidification of Leaf Pavement Cells in Pea (Pisum sativum L. var. Argenteum).

Leaf pavement cell expansion in light depends on apoplastic acidification by a plasma membrane proton-pumping ATPase, modifying cell wall extensibility and providing the driving force for uptake of osmotically active solutes generating turgor. This paper shows that the plant hormone ABA inhibits light-induced leaf disk growth as well as the blue light-induced pavement cell growth in pea (Pisum sativum L.). In the phytochrome chromophore-deficient mutant pcd2, the effect of ABA on the blue light-induced apoplastic acidification response, which exhibits a high fluence phase via phytochrome and a low fluence phase via an unknown blue light receptor, is still present, indicating an interaction of ABA with the blue light receptor pathway. Furthermore, it is shown that ABA inhibits the blue light-induced apoplastic acidification reversibly. These results indicate that the effect of ABA on apoplastic acidification can provide a mechanism for short term, reversible adjustment of leaf growth rate to environmental change.

NaCl salinity affects lateral root development in Plantago maritima.

Root growth and morphology were assessed weekly in hydroponically-grown seedlings of the halophyte Plantago maritima L. during exposure to 0, 50, 100 and 200 mm NaCl for 21 d. Relative growth rate was reduced by 25% at 200 mm NaCl. The lower NaCl treatments did not affect relative growth rates. Primary and lateral roots responded differently to NaCl. While primary-root length increased at all NaCl concentrations, total lateral-root length increased at 50 and was not affected at 100 mm but was considerably reduced at 200 mm NaCl. NaCl concentrations of 50 and 100 mm, which had no effect on relative growth rate or total lateral-root length, severely affected root branching pattern in that the number of first, second and third order laterals was reduced. At 200 mm NaCl third order laterals were not formed at all. However, mean lateral-root length was increased at all NaCl concentrations and was highest at 200 mm NaCl. We conclude that the increase in total lateral-root length in plants at 50 and 100 mm NaCl was mainly caused by increased length growth, while the decrease in total lateral-root length at 200 mm was the consequence of inhibition of lateral root primordia and / or the activation of apical meristems rather than reduced length growth.

Spatial patterns of radial oxygen loss and nitrate net flux along adventitious roots of rice raised in aerated or stagnant solution.

Roots of rice (Oryza sativa L.) grown in stagnant de-oxygenated solution contain a 'tight' barrier to radial oxygen loss (ROL) in basal zones, whereas roots of plants grown in aerated solution do not. It is generally accepted that the barrier to ROL involves anatomical modifications in the apoplast of cell layers exterior to the aerenchyma. A possible drawback of this adaptation is a reduced capacity for nutrient uptake. Whether or not induction of a barrier to ROL influences the capacity of adventitious roots of rice to take up NO3- was determined in the present study, using NO3--selective microelectrodes. When transferred into O2-free root medium, ROL from positions at 30-50 mm behind the tip of adventitious roots of plants raised in stagnant solution was only 4-6% of the rate from roots of plants raised in aerated solution, indicating the barrier to ROL was induced by growth in stagnant solution. For plants transferred into aerobic nutrient solution containing 0.1 mM NO3-, net NO3- uptake by these root zones, with or without a barrier to ROL, was the same. It is concluded that induction of a barrier to ROL had no effect on the capacity of adventitious roots of rice to take up NO3- from aerobic solution.