An in Vivo Imaging Assay Detects Spatial Variability in Glucose Release from Plant Roots.

Plants secrete a plethora of metabolites into the rhizosphere that allow them to obtain nutrients necessary for growth and modify microbial communities around the roots. Plants release considerable amounts of photosynthetically fixed carbon into the rhizosphere; hence, it is important to understand how carbon moves from the roots into the rhizosphere. Approaches used previously to address this question involved radioactive tracers, fluorescent probes, and biosensors to study sugar movement in the roots and into the rhizosphere. Although quite effective for studying sugar movement, it has been challenging to obtain data on spatial and temporal variability in sugar exudation using these techniques. In this study, we developed a gel-based enzyme-coupled colorimetric and fluorometric assay to image glucose (Glc) in vivo and used this assay to show that there is spatial variability in Glc release from plant roots. We found that the primary roots of maize (Zea mays) released more Glc from the base of the root than from the root tip and that the Glc release rate is reduced in response to water stress. These findings were confirmed independently by quantifying Glc release in well-watered and water-stressed maize primary roots using high-performance anion-exchange chromatography. Additionally, we demonstrated differential patterns of Glc exudation in different monocot and eudicot plant species. These findings and their implications on root-rhizosphere interactions are discussed.

Enzyme-Less Growth in Chara and Terrestrial Plants.

Enzyme-less chemistry appears to control the growth rate of the green alga Chara corallina. The chemistry occurs in the wall where a calcium pectate cycle determines both the rate of wall enlargement and the rate of pectate deposition into the wall. The process is the first to indicate that a wall polymer can control how a plant cell enlarges after exocytosis releases the polymer to the wall. This raises the question of whether other species use a similar mechanism. Chara is one of the closest relatives of the progenitors of terrestrial plants and during the course of evolution, new wall features evolved while pectate remained one of the most conserved components. In addition, charophytes contain auxin which affects Chara in ways resembling its action in terrestrial plants. Therefore, this review considers whether more recently acquired wall features require different mechanisms to explain cell expansion.

Why small fluxes matter: the case and approaches for improving measurements of photosynthesis and (photo)respiration.

Since its inception, the Farquhar et al. (1980) model of photosynthesis has been a mainstay for relating biochemistry to environmental conditions from chloroplast to global levels in terrestrial plants. Many variables could be assigned from basic enzyme kinetics, but the model also required measurements of maximum rates of photosynthetic electron transport (J max ), carbon assimilation (Vcmax ), conductance of CO2 into (g s ) and through (g m ) the leaf, and the rate of respiration during the day (R d ). This review focuses on improving the accuracy of these measurements, especially fluxes from photorespiratory CO2, CO2 in the transpiration stream, and through the leaf epidermis and cuticle. These fluxes, though small, affect the accuracy of all methods of estimating mesophyll conductance and several other photosynthetic parameters because they all require knowledge of CO2 concentrations in the intercellular spaces. This review highlights modified methods that may help to reduce some of the uncertainties. The approaches are increasingly important when leaves are stressed or when fluxes are inferred at scales larger than the leaf.

Impact of cuticle on calculations of the CO2 concentration inside leaves.

MAIN CONCLUSION: Water vapor over-estimates the CO 2 entering leaves during photosynthesis because the cuticle and epidermis transmit more water vapor than CO 2 . Direct measurements of internal CO 2 concentrations may be preferred. The CO2 concentration inside leaves (c i) is typically calculated from the relationship between water vapor diffusing out while CO2 diffuses in. Diffusion through the cuticle/epidermis is usually not considered. This study was undertaken to determine how much the calculations would be affected by including cuticle properties. Previous studies indicate that measurable amounts of CO2 and water vapor move through the cuticle, although much less CO2 than water vapor. The present experiments were conducted with sunflower (Helianthus annuus L) leaves in a gas exchange apparatus designed to directly measure c i, while simultaneously calculating c i. Results showed that, in normal air, calculated c i were always higher than directly measured ones, especially when abscisic acid was fed to the leaves to close the stomata and cause gas exchange to be dominated by the cuticle. The effect was attributed mostly to the reliance on the gas phase for the calculations without taking cuticle properties into account. Because cuticle properties are usually unknown and vary with the turgor of the leaf, which can stretch the waxes, it is difficult to include cuticle properties in the calculation. It was concluded that direct measurement of c i may be preferable to the calculations.

Turgor and the transport of CO2 and water across the cuticle (epidermis) of leaves.

Leaf photosynthesis relies on CO2 diffusing in while water vapour diffuses out. When stomata close, cuticle waxes on the epidermal tissues increasingly affect this diffusion. Also, changes in turgor can shrink or swell a leaf, varying the cuticle size. In this study, the properties of the cuticle were investigated while turgor varied in intact leaves of hypo stomatous grape (Vitis vinifera L.) or amphistomatous sunflower (Helianthus annuus L.). For grape, stomata on the abaxial surface were sealed and high CO2 concentrations outside the leaf were used to maximize diffusion through the adaxial, stoma-free cuticle. For sunflower, stomata were closed in the dark or with abscisic acid to maximize the cuticle contribution to the path. In both species, the internal CO2 concentration was measured directly and continuously while other variables were determined to establish the cuticle properties. The results indicated that stomatal closure diminished the diffusion of both gases in both species, but for CO2 more than for water vapour. Decreasing the turgor diminished the movement of both gases through the cuticle of both species. Because this turgor effect was observed in the adaxial surface of grape, which had no stomata, it could only be attributed to cuticle tightening. Comparing calculated and measured concentrations of CO2 in leaves revealed differences that became large as stomata began to close. These differences in transport, together with turgor effects, suggest calculations of the CO2 concentration inside leaves need to be viewed with caution when stomata begin to close.

A UV-blocking polymer shell prevents one-photon photoreactions while allowing multi-photon processes in encapsulated upconverting nanoparticles.

Sun block for nanoparticles: Unintentional photorelease triggered by UV light is a problem in photodynamic therapy. Encapsulating upconverting nanoparticles containing photoswitches in a UV-blocking amphiphilic polymer shuts down the one-photon process and only allows two-photon-driven photochemistry. Thus, UV light is blocked while NIR light can reach the nanoparticle core and trigger photorelease.

Differences in membrane selectivity drive phloem transport to the apoplast from which maize florets develop.

BACKGROUND AND AIMS: Floral development depends on photosynthetic products delivered by the phloem. Previous work suggested the path to the flower involved either the apoplast or the symplast. The objective of the present work was to determine the path and its mechanism of operation. METHODS: Maize (Zea mays) plants were grown until pollination. For simplicity, florets were harvested before fertilization to ensure that all tissues were of maternal origin. Because sucrose from phloem is hydrolysed to glucose on its way to the floret, the tissues were imaged and analysed for glucose using an enzyme-based assay. Also, carboxyfluorescein diacetate was fed to the stems and similarly imaged and analysed. KEY RESULTS: The images of live sections revealed that phloem contents were released to the pedicel apoplast below the nucellus of the florets. Glucose or carboxyfluorescein were detected and could be washed out. For carboxyfluorescein, the plasma membranes of the phloem parenchyma appeared to control the release. After release, the nucellus absorbed apoplast glucose selectively, rejecting carboxyfluorescein. CONCLUSIONS: Despite the absence of an embryo, the apoplast below the nucellus was a depot for phloem contents, and the strictly symplast path is rejected. Because glucose and carboxyfluorescein were released non-selectively, the path to the floret resembled the one later when an embryo is present. The non-selective release indicates that turgor at phloem termini cannot balance the full osmotic potential of the phloem contents and would create a downward pressure gradient driving bulk flow toward the sink. Such a gradient was previously measured by Fisher and Cash-Clark in wheat. At the same time, selective absorption from the apoplast by the nucellar membranes would support full turgor in this tissue, isolating the embryo sac from the maternal plant. The isolation should continue later when an embryo develops.

Near infrared light triggered release of biomacromolecules from hydrogels loaded with upconversion nanoparticles.

Using a photosensitive hybrid hydrogel loaded with upconversion nanoparticles (UCNPs), we show that continuous-wave near-infrared (NIR) light (980 nm) can be used to induce the gel-sol transition and release large, inactive biomacromolecules (protein and enzyme) entrapped in the hydrogel into aqueous solution "on demand", where their bioactivity is recovered. This study is a new demonstration and development in harnessing the unique multiphoton effect of UCNPs for photosensitive materials of biomedical interest.

Pectate chemistry links cell expansion to wall deposition in Chara corallina.

Pectate (polygalacturonic acid) acts as a chelator to bind calcium and form cross-links that hold adjacent pectate polymers and thus plant cell walls together. When under tension from turgor pressure in the cell, the cross-links appear to distort and weaken. New pectate supplied by the cytoplasm is undistorted and removes wall calcium preferentially from the weakened bonds, loosening the wall and accelerating cell expansion. The new pectate now containing the removed calcium can bind to the wall, strengthening it and linking expansion to wall deposition. But new calcium needs to be added as well to replenish the calcium lost from the vacated wall pectate.  A recent report demonstrated that growth was disrupted if new calcium was unavailable.  The present addendum highlights this conclusion by reviewing an experiment from before the chelation chemistry was understood. Using cell wall labeling, a direct link appeared between wall expansion and wall deposition. Together, these experiments support the concept that newly supplied pectate has growth activity on its way to deposition in the wall. Growth rate is thus controlled by signals affecting the rate of pectate release. After release, the coordination of expansion and deposition arises naturally from chelation chemistry when polymers are under tension from turgor pressure. 

Multimodal fluorescence modulation using molecular photoswitches and upconverting nanoparticles.

The intensity and colour of the light emitted from upconverting nanoparticles is controlled by the state of photoresponsive dithienylethene ligands decorated onto the surface of the nanoparticles. By selectively activating one or both ligands in a mixed, 3-component system, a multimodal read-out of the emitted light is achieved.

Calcium deprivation disrupts enlargement of Chara corallina cells: further evidence for the calcium pectate cycle.

Pectin is a normal constituent of cell walls of green plants. When supplied externally to live cells or walls isolated from the large-celled green alga Chara corallina, pectin removes calcium from load-bearing cross-links in the wall, loosening the structure and allowing it to deform more rapidly under the action of turgor pressure. New Ca(2+) enters the vacated positions in the wall and the externally supplied pectin binds to the wall, depositing new wall material that strengthens the wall. A calcium pectate cycle has been proposed for these sub-reactions. In the present work, the cycle was tested in C. corallina by depriving the wall of external Ca(2+) while allowing the cycle to run. The prediction is that growth would eventually be disrupted by a lack of adequate deposition of new wall. The test involved adding pectate or the calcium chelator EGTA to the Ca(2+)-containing culture medium to bind the calcium while the cycle ran in live cells. After growth accelerated, turgor and growth eventually decreased, followed by an abrupt turgor loss and growth cessation. The same experiment with isolated walls suggested the walls of live cells became unable to support the plasma membrane. If instead the pectate or EGTA was replaced with fresh Ca(2+)-containing culture medium during the initial acceleration in live cells, growth was not disrupted and returned to the original rates. The operation of the cycle was thus confirmed, providing further evidence that growth rates and wall biosynthesis are controlled by these sub-reactions in plant cell walls.

Near-infrared light-triggered dissociation of block copolymer micelles using upconverting nanoparticles.

We demonstrate a novel strategy enabling the use of a continuous-wave diode near-infrared (NIR) laser to disrupt block copolymer (BCP) micelles and trigger the release of their "payloads". By encapsulating NaYF(4):TmYb upconverting nanoparticles (UCNPs) inside micelles of poly(ethylene oxide)-block-poly(4,5-dimethoxy-2-nitrobenzyl methacrylate) and exposing the micellar solution to 980 nm light, photons in the UV region are emitted by the UCNPs, which in turn are absorbed by o-nitrobenzyl groups on the micelle core-forming block, activating the photocleavage reaction and leading to the dissociation of BCP micelles and release of co-loaded hydrophobic species. Our strategy of using UCNPs as an internal UV or visible light source upon NIR light excitation represents a general and efficient method to circumvent the need for UV or visible light excitation that is a common drawback for light-responsive polymeric systems developed for potential biomedical applications.

Two-way photoswitching using one type of near-infrared light, upconverting nanoparticles, and changing only the light intensity.

Only one type of lanthanide-doped upconverting nanoparticle (UCNP) is needed to reversibly toggle photoresponsive organic compounds between their two unique optical, electronic, and structural states by modulating merely the intensity of the 980 nm excitation light. This reversible "remote-control" photoswitching employs an excitation wavelength not directly absorbed by the organic chromophores and takes advantage of the fact that designer core-shell-shell NaYF(4) nanoparticles containing Er(3+)/Yb(3+) and Tm(3+)/Yb(3+) ions doped into separate layers change the type of light they emit when the power density of the near-infrared light is increased or decreased. At high power densities, the dominant emissions are ultraviolet and are appropriate to drive the ring-closing, forward reactions of dithienylethene (DTE) photoswitches. The visible light generated from the same core-shell-shell UCNPs at low power densities triggers the reverse, ring-opening reactions and regenerates the original photoisomers. The "remote-control" photoswitching using NIR light is as equally effective as the direct switching with UV and visible light, albeit the reaction rates are slower. This technology offers a highly convenient and versatile method to spatially and temporally regulate photochemical reactions using a single light source and changing either its power or its focal point.

Absolute quantum yield measurements of colloidal NaYF4: Er3+, Yb3+ upconverting nanoparticles.

In this communication we describe a technique for measuring the absolute quantum yields (QYs) of upconverting nanomaterials based on the use of a commercially available fluorimeter and an integrating sphere. Using this setup, we have successfully acquired luminescence efficiency data (pump laser, absorbed pump, and visible emitted intensities) for lanthanide-doped upconverting nanoparticles. QYs in the range of 0.005% to 0.3% were measured for several NaYF(4): 2% Er(3+), 20% Yb(3+) nanoparticles with particle sizes ranging from 10 to 100 nm while a QY of 3% was measured for a bulk sample.

Sugar input, metabolism, and signaling mediated by invertase: roles in development, yield potential, and response to drought and heat.

Invertase (INV) hydrolyzes sucrose into glucose and fructose, thereby playing key roles in primary metabolism and plant development. Based on their pH optima and sub-cellular locations, INVs are categorized into cell wall, cytoplasmic, and vacuolar subgroups, abbreviated as CWIN, CIN, and VIN, respectively. The broad importance and implications of INVs in plant development and crop productivity have attracted enormous interest to examine INV function and regulation from multiple perspectives. Here, we review some exciting advances in this area over the last two decades, focusing on (1) new or emerging roles of INV in plant development and regulation at the post-translational level through interaction with inhibitors, (2) cross-talk between INV-mediated sugar signaling and hormonal control of development, and (3) sugar- and INV-mediated responses to drought and heat stresses and their impact on seed and fruit set. Finally, we discuss major questions arising from this new progress and outline future directions for unraveling mechanisms underlying INV-mediated plant development and their potential applications in plant biotechnology and agriculture.

Historical evolution of medical quality assurance in the Department of Defense.

The Department of Defense (DoD) Military Health System (MHS) embodies decades of health care practice that has evolved in scope and complexity to meet the demands for quality care to which its beneficiaries are entitled. War, Base Realignment and Closure (BRAC), and other dynamic forces require the ongoing review and revision of health care policy and practice in military hospitals as well as the expanded network of civilian providers who care for our nation's soldiers, sailors, airmen, and marines and their families. The result has been an incrementally constructed quality assurance (QA) program with emphasis on organizational structures, programs, and systems, and the use of robust data sources and standard measures to analyze and improve processes, manage disease, assess patient perceptions of care, and ensure that a uniform health care benefit and high quality health care is accessible to all MHS beneficiaries.