Galactosides in the rhizosphere: utilization by Sinorhizobium meliloti and development of a biosensor.

Identifying the types and distributions of organic substrates that support microbial activities around plant roots is essential for a full understanding of plant-microbe interactions and rhizosphere ecology. We have constructed a strain of the soil bacterium Sinorhizobium meliloti containing a gfp gene fused to the melA promoter which is induced on exposure to galactose and galactosides. We used the fusion strain as a biosensor to determine that galactosides are released from the seeds of several different legume species during germination and are also released from roots of alfalfa seedlings growing on artificial medium. Galactoside presence in seed wash and sterile root washes was confirmed by HPLC. Experiments examining microbial growth on alpha-galactosides in seed wash suggested that alpha-galactoside utilization could play an important role in supporting growth of S. meliloti near germinating seeds of alfalfa. When inoculated into microcosms containing legumes or grasses, the biosensor allowed us to visualize the localized presence of galactosides on and around roots in unsterilized soil, as well as the grazing of fluorescent bacteria by protozoa. Galactosides were present in patches around zones of lateral root initiation and around roots hairs, but not around root tips. Such biosensors can reveal intriguing aspects of the environment and the physiology of the free-living soil S. meliloti before and during the establishment of nodulation, and they provide a nondestructive, spatially explicit method for examining rhizosphere soil chemical composition.

Photosynthetic Electron Transport in Single Guard Cells as Measured by Scanning Electrochemical Microscopy.

Scanning electrochemical microscopy (SECM) is a powerful new tool for studying chemical and biological processes. It records changes in faradaic current as a microelectrode ([less than equal]7 [mu]m in diameter) is moved across the surface of a sample. The current varies as a function of both distance from the surface and the surface's chemical and electrical properties. We used SECM to examine in vivo topography and photosynthetic electron transport of individual guard cells in Tradescantia fluminensis, to our knowledge the first such analysis for an intact plant. We measured surface topography at the micrometer level and concentration profiles of O2 evolved in photosynthetic electron transport. Comparison of topography and oxygen profiles above single stomatal complexes clearly showed photosynthetic electron transport in guard cells, as indicated by induction of O2 evolution by photosynthetically active radiation. SECM is unique in its ability to measure topography and chemical fluxes, combining some of the attributes of patch clamping with scanning tunneling microscopy. In this paper we suggest several questions in plant physiology that it might address.

Dependence of the Extent and Direction of Average Stomatal Response in Zea mays L. and Phaseolus vulgaris L. on the Frequency of Fluctuations in Environmental Stimuli.

Stomatal responses to fluctuating light and CO2 were investigated in Zea mays and Phaseolus vulgaris. Slow-moving stomata can affect carbon gain and water loss by plants during light flecks, under dynamic cloud cover, during alternating windy and calm air conditions (which influence CO2 concentrations and humidity immediately around leaves in plant canopies), at natural CO2 vents, or in growth chambers with imperfect CO2 control. It was found that the frequency of constant-amplitude fluctuations in light and CO2 dramatically affected the time-averaged stomatal conductance in both Zea and Phaseolus. During oscillations in light, average stomatal conductance was driven either above or below that observed at steady state at the average light level, depending on the frequency of the oscillations. Under oscillating CO2, the departure of average stomatal conductance away from that observed at steady state at the average CO2 level was also frequency dependent in both species. Upon cessation of oscillations and return of light or CO2 to the stable median level, stomatal conductance also returned to a steady state, matching that before oscillations were initiated. This work shows that fluctuations in light and CO2, and equally important, their frequency, can be critical in determining time-averaged stomatal conductance under unstable environmental conditions.

Effects of O(2) and CO(2) Concentration on the Steady-State Fluorescence Yield of Single Guard Cell Pairs in Intact Leaf Discs of Tradescantia albiflora: Evidence for Rubisco-Mediated CO(2) Fixation and Photorespiration in Guard Cells.

A procedure for following changes in the steady-state yield of chlorophyll a fluorescence (F(s)) from single guard cell pairs in variegated leaves of Tradescantia albiflora is described. As an indicator of photosynthetic electron transport, F(s) is a very sensitive indirect measure of the balance of adenosine 5'-triphosphate (ATP) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), producing reactions with the sink reactions that utilize those light-generated products. We found that F(s) under constant light is sensitive to manipulation of ambient CO(2) concentrations, as would be expected if either phosphoenolpyruvate carboxylase or ribulose-1, 5 bisphosphate carboxylase/oxygenase (Rubisco)-dependent CO(2) fixation is the sink for photosynthetic ATP and NADPH in guard cells. However, we also found that changing O(2) concentration had a strong effect on fluorescence yield, and that O(2) sensitivity was only evident when the concentration of CO(2) was low. This finding provides evidence that both O(2) and CO(2) can serve as sinks for ATP and NADPH produced by photosynthetic electron transport in guard cell chloroplasts. Identical responses were observed with mesophyll cell chloroplasts in intact leaves. This finding is difficult to reconcile with the view that guard cell chloroplasts have fundamentally different pathways of photosynthetic metabolism from other chloroplasts in C(3) plants. Indeed, Rubisco has been detected at low levels in guard cell chloroplasts, and our studies indicate that it is active in the pathways for photosynthetic carbon reduction and photorespiration in guard cells.

Evidence that Ribulose 1,5-Bisphosphate (RuBP) Binds to Inactive Sites of RuBP Carboxylase in Vivo and an Estimate of the Rate Constant for Dissociation.

The binding of ribulose 1,5-bisphosphate (RuBP) to inactive (noncarbamylated) sites of the enzyme RuBP carboxylase in vivo was investigated in Spinacia oleracea and Helianthus annuus. The concentrations of RuBP and inactive sites were determined in leaf tissue as a function of time after a change to darkness. RuBP concentrations fell rapidly after the change to darkness and were approximately equal to the concentration of inactive sites after 60 s. Variations in the concentration of inactive sites, which were induced by differences in the light intensity before the light-dark transition, correlated with the concentration of RuBP between 60 and 120 s after the change to darkness. These data are discussed as evidence that RuBP binds to inactive sites of RuBP carboxylase in vivo. After the concentration of RuBP fell below that of inactive sites (at times longer than 60 s of darkness), the decline in RuBP was logarithmic with time. This would be expected if the dissociation of RuBP from inactive sites controlled the decline in RuBP concentration. These data were used to estimate the rate constant for dissociation of RuBP from inactive sites in vivo.