Methodology and Significance of Microsensor-based Oxygen Mapping in Plant Seeds – an Overview
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paper_title: Embryogeny of Phaseolus: Developmental pattern of lactate and alcohol dehydrogenases

paper_content:

Abstract Alcohol and lactate dehydrogenase activity and their electrophoretic isoenzymes were determined in developing Phaseolus vulgaris embryos, seed coat
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paper_title: Studies on the Mechanism of Differences in Photosynthesis among Species : III. Influence of low oxygen concentration on dry matter production and grain fertility of rice plant

paper_content:

It has been clarified that the rates of apparent photosynthesis in C3 plant such as rice and wheat, which evolve a large quantity of carbon dioxide in the light, become higher as the oxygen concentration in the air decreases. However, the drastic change in oxygen concentration from 21 per cent to a few per cent may inhibit some of the physiological functions of plant which have adapted to the present atmosphere. The present experiment was conducted to evaluate the effect of low oxygen concentration for a long period on dry matter production and growth of rice plant. The plant was kept in the assimilation chamber in both control and low oxygen plots. The plant in low oxygen plot was treated with the air containing 3 per cent oxygen and the plant in control plot was aerated with ordinary outdoor air. The gas treatment was conducted only in the daytime for 20 days from the heading stage to the milky dough stage. The following results were obtained. 1. The increased weight of dry matter during the treated period was significantly higher in the low oxygen plot than in the control. This increase was mainly due to the reduced photorespiration rate in low oxygen concentration. 2. The space between tillers was extremely expanded after 20 hours from the begining of low oxygen treatment. 3. When the rice plant was exposed to low oxygen concentration at flowering time, remarkable sterility occurred. Such phenomenon was also observed when only the panicle was kept in low oxygen concentration. Thus, the sterility observed here seems to be caused, not by the translocated substances from the leaf and stem, but by the direct damage of low oxygen to fertilization. 4. The grain fertility of Cyperus microiria Steudel, on the other hand, was hardly affected by oxygen concentration of 3 per cent at which the rice plant showed remarkable sterility. This fact suggests that the influence of low oxygen on fertility of plant differs with species. 5. It follows from these findings that the lowering of oxygen in the air is effective in increasing the dry matter production of rice plant, while deteriorate the physiological functions relating to fertility and growth.
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paper_title: The oxygen status of the developing seed.

paper_content:

Recent applications of oxygen-sensitive microsensors have demonstrated steep oxygen gradients in developing seeds of various crops. Here, we present an overview on oxygen distribution, major determinants of the oxygen status in the developing seed and implications for seed physiology. The steady-state oxygen concentration in different seed tissues depends on developmental parameters, and is determined to a large extent by environmental factors. Photosynthetic activity of the seed significantly diminishes hypoxic constraints, and can even cause transient, local hyperoxia. Changes in oxygen availability cause rapid adjustments in mitochondrial respiration and global metabolism. We argue that nitric oxide (NO) is a key player in the oxygen balancing process in seeds, avoiding fermentation and anoxia in vivo. Molecular approaches aiming to increase oxygen availability within the seed are discussed.
---
paper_title: Oxygen Control of Reproductive Growth: Possible Mediation via Dark Respiration

paper_content:

The rates of dark respiration of Glycine max leaves, pods, and seeds, are shown to be positively related to O2 concentration ([O2]) between 2 and 21 per cent. It is suggested that the reported depression of seed growth, due to subatmospheric concentrations of O2 is caused simply by depression of respiration and that it is not necessary to postulate a new control mechanism mediated by [O2].
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paper_title: Reproductive Growth and Dry Matter Production of Glycine max (L.) Merr. in Response to Oxygen Concentration

paper_content:

Reproductive as well as vegetative parameters of mature soybean ( Glycine max [L.] Merr. cv. Wye) plants grown in chambers in which the aerial portion was exposed to altered p O 2 during all or part of the growth cycle were measured. Oxygen concentration was found to be a key factor controlling all phases of reproductive development. Exposure to 5% O 2 from early seedling stage to senescence increased leaf, stem, and root dry weights and reduced seed yields when compared to 21% O 2 ; exposure to low O 2 during the vegetative growth stage from early seedling to mid-flowering arrested pod but not seed development; exposure from mid-flowering to mid-pod filling almost completely arrested seed but not pod development; exposure from mid-pod filling to senescence arrested seed development at the mid-filling stage. Exposures to 5% O 2 initiated at mid-flowering for 1, 2, 3, 5, 10, and 15 days had no effect on seed development when the exposure was up to 3 days and produced almost total arrest when the exposure was 10 or more days, suggesting reversibility. The requirement for O 2 in seed development is independent of CO 2 concentration with similar results produced by subambient O 2 combined with ambient CO 2 , elevated CO 2 up to 2000 μl/l or depressed levels of CO 2 with the CO 2 /O 2 ratio as in air. An elevated O 2 atmosphere containing 40% O 2 and ambient or elevated CO 2 inhibited total growth but did not affect the balance of vegetative to reproductive growth. We conclude that an unknown reaction or process requiring at least atmospheric concentrations of O 2 but independent of CO 2 in contrast to photorespiration is necessary for optimization of all phases of reproductive growth and the effect is reversible for exposures of up to 3 days but not for exposures of 10 days or more. We propose that this O 2 phenomenon may be the result of a unique physical process or chemical reaction associated with translocation and accumulation of assimilates in reproductive structures.
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paper_title: Fiber‐optic oxygen microsensors, a new tool in aquatic biology

paper_content:

A new fiber-optic oxygen microsensor (microoptrode) based on dynamic fluorescence quenching has been developed to measure oxygen gradients in marine sediments and microbial mats. The microoptrodes are fabricated by immobilizing an oxygen-quenchable fluorophore at the tapered tip of an optical fiber. A special optoelectronic system has been designed to measure oxygen with these microoptrodes. It is based on small and cheap optical components and can easily be miniaturized for field applications. In contrast to oxygen microelectrodes, the new oxygen microoptrodes are easy to make, do not consume oxygen, and show no stirring dependence of the signal. In addition, they show excellent long-term stability and storage stability. Hydrogen sulfide, carbon dioxide, and other relevant chemical parameters do not interfere with the measurement. Oxygen profiles in marine sediments obtained from measurements with microoptrodes show good correlation to profiles measured with oxygen microelectrodes.
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paper_title: Microelectrodes: Their Use in Microbial Ecology

paper_content:

Among the fundamental goals of microbial ecology is the development of methods that will enable the identification and counting of the important microorganisms in nature, the determination of their physical and chemical microenvironment, and the analysis of their metabolic processes and interactions. Due to the small size of the organisms, much effort has been devoted to the development of high-resolution techniques for the observation and understanding of the world of bacteria on a microscale. Scanning and transmission electron microscopy and fluorescent staining, immunofluorescence and other techniques for light microscopy have been the most successful in terms of reaching a high spatial resolution. With respect to our understanding of the microbial microenvironments and of the nature of the microorganisms that carry out the measured metabolic activities, there is still a long way to go. Most chemical and radiotracer techniques in use today operate on a centimeter or at best on a millimeter scale and in most cases their results cannot be directly related to the relevant microorganisms. One notable exception to this is the combined use of autoradiography and fluorescence microscopy on microbial communities.
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paper_title: Hypoxia in Static and Dynamic 3D Culture Systems for Tissue Engineering of Bone

paper_content:

Tissue engineering of sizeable cell-scaffold constructs is limited by gradients in tissue quality from the periphery toward the center. Because homogenous delivery of oxygen to three-dimensional (3D) cell cultures remains an unsolved challenge, we hypothesized that uneven oxygen supply may impede uniform cellular growth on scaffolds. In this study we challenged static and dynamic 3D culture systems designed for bone tissue engineering applications with a well-growing subclone of MC3T3-E1 preosteoblasts and continuously measured the oxygen concentrations in the center of cell-seeded scaffolds and in the surrounding medium. After as little as 5 days in static culture, central oxygen concentrations dropped to 0%. Subsequently, cells died in central regions of the scaffold but not in its periphery, where oxygen levels were ∼4%. The use of perfusion bioreactors successfully prevented cell death, yet central oxygen concentrations did not rise above 4%. We conclude that 3D culture in vitro is associated with rel...
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paper_title: Measurement of a gradient of oxygen partial pressure across the intact root

paper_content:

Oxygen partial pressures in the vacuolar sap of root cells have been measured using oxygen sensitive polarographic microelectrodes. A significant gradient in oxygen partial pressure was observed across intact sunflower roots growing in air saturated culture solution. The relevance of this finding to the mechanism of salt transfer across the root and in particular to the Crafts-Broyer theory is discussed.
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paper_title: Positional cues for the starch/lipid balance in maize kernels and resource partitioning to the embryo

paper_content:

This study tests the hypotheses that in vivo oxygen levels inside developing maize grains locally affect assimilate partitioning and ATP distribution within the kernel. These questions were addressed through combined topographical analysis (O2- and ATP-mapping), metabolite profiling, and isotope flux analysis. Internal and external oxygen levels were also experimentally altered. Under ambient conditions, mean O2 concentration immediately inside starchy endosperm dropped to only 1.4% of atmospheric saturation (approximately 3.8 microm), but was 10-fold higher in the oil-storing embryo. Increasing the O2 supply to intact kernels stimulated their O2 demand, shifted ATP localization within the kernel, and elevated their ATP/ADP ratio. Enhanced O2 availability also increased steady-state levels of glycolytic intermediates and those of the citric acid cycle, as well as some related pools of free amino acids. Subsequent analyses indicated that starch formation within endosperm, but not lipid biosynthesis within embryo, was adapted to the endogenous low oxygen. Increasing the O2 supply did not change ADP-glucose levels, activity of ADP-glucose pyrophosphorylase, 13C-labeling of ADP-glucose, or flux of 14C-sucrose into starch. In contrast, enhanced O2 availability increased 14C-label uptake into the embryo, 13C-labeling of acetyl-coenzyme A, and finally 14C-incorporation into lipids. Lipid accumulation in embryo appeared highest in regions with higher ATP. Consistent with labeling data, a decrease in O2 supply most strongly affected the embryo, whereas rising O2 levels expanded ATP-rich zones toward the starch-storing endosperm and the scutellar part of embryo. The latter might be responsible for higher 14C-label uptake into the embryo and flux toward lipid. Collectively, data indicate that the in vivo oxygen distribution in maize kernels markedly affects ATP gradients, metabolite levels, and favors assimilate partitioning toward starch within the O2-depleted endosperm. Clear advantages are thus evident for peripheral localization of the protein and lipid storing structures in maize kernels.
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paper_title: Oxygen-depleted zones inside reproductive structures of Brassicaceae: implications for oxygen control of seed development.

paper_content:

Growth of Arabidopsis thaliana (L.) Heynh. in decreasing oxygen partial pressures revealed a linear decrease in seed production below 15 kPa, with a complete absence of seed production at 2.5 kPa oxygen. This control of plant reproduction by oxygen had previously been attributed to an oxygen effect on the partitioning between vegetative and reproductive growth. However, plants grown in a series of decreasing oxygen concentrations produced progressively smaller embryos that had stopped developing at progressively younger stages, suggesting instead that their growth is limited by oxygen. Internal oxygen concentrations of buds, pistils, and developing siliques of Brassica rapa L. and siliques of Arabidopsis were measured using a small-diameter glass electrode that was moved into the structures using a micromanipulator. Oxygen partial pressures were found to be lowest in the developing perianth (11.1 kPa) and pistils (15.2 kPa) of the unopened buds. Pollination reduced oxygen concentration inside the pistils by 3 kPa after just 24 h. Inside Brassica silique locules, partial pressures of oxygen averaged 12.2 kPa in darkness, and increased linearly with increasing light levels to 16.2 kPa. Measurements inside Arabidopsis siliques averaged 6.1 kPa in the dark and rose to 12.2 kPa with light. Hypoxia in these microenvironments is postulated to be the point of control of plant reproduction by oxygen.
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paper_title: Oxygen Distribution in Wetland Plant Roots and Permeability Barriers to Gas-exchange with the Rhizosphere: a Microelectrode and Modelling Study with Phragmites australis

paper_content:

Abstract Adventitious roots of intact Phragmites plantlets were secured horizontally 2–3 mm below the surface of an oxygen-depleted fluid agar across which oxygen-free nitrogen was gently streamed to create a constant oxygen sink; the leafy shoot was fully exposed to air. Radial oxygen profiles through rhizosphere and root at different distances from the apex were obtained polarographically using Clark-type bevelled microelectrodes servo-driven in steps of 10 μm (root) or 10–50 μm (rhizosphere). The pattern of radial oxygen loss (ROL) typical of wetland plants, viz. high at the apex and declining sharply sub-apically, was related to synergism between ROL, and oxygen consumption and increasing impedance to diffusion within the epidermal/hypodermal cylinder rather than to a surface resistance. The smallest oxygen deficit (2 kPa) to develop across the 80 μm thick epidermal/hypodermal cylinder was within the apical 10 mm and was consistent with tissue oxygen diffusivities similar to water. At 100 mm from the apex, consumption and impedance had increased the deficit to about 15 kPa and reduced ROL almost to zero. The developing impedance within the epidermal/hypodermal cylinder was least in cell layers immediately adjoining the cortex and increased most in the hypodermal cell layer abutting the epidermis. The sub-apical decline in ROL appeared to coincide with the appearance of aerenchyma in the cortex but thin walled ‘passage areas’ (windows) in the hypodermal/epidermal cylinder persisted locally and remained leaky to oxygen to some degree. It is through these windows that lateral roots emerge and the cortex in line with the windows remains non-aerenchymatous. The radial and longitudinal oxygen profiles were consistent with modelling predictions. The shapes of the stelar oxygen profiles were consistent with a higher oxygen demand in the outer region (viz. pericycle, phloem, protoxylem and early metaxylem cylinder) than in the inner core (late metaxylem cylinder and medulla), but the deficits were relatively small (⩽3 kPa) and consistent with minimal wall thickening in the endodermis and narrowness of stele. The possible relevance of the results to entry of methane and other products from the rhizosphere into root and to the mechanism of aerenchyma formation are discussed.
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paper_title: Energy state and its control on seed development: starch accumulation is associated with high ATP and steep oxygen gradients within barley grains.

paper_content:

The role of oxygen and energy state in development and storage activity of cereal grains is an important issue, but has remained largely uninvestigated due to the lack of appropriate analytical methods. Metabolic profiling, bioluminescence-based in situ imaging of ATP, and oxygen-sensitive microsensors were combined here to investigate barley seed development. For the first time temporal and spatial maps of O2 and ATP distribution in cereal grains were determined and related to the differentiation pattern. Steep O2 gradients were demonstrated and strongly hypoxic regions were detected within the caryopsis (<0.1% of atmospheric saturation). Growing lateral and peripheral regions of endosperm remained well-supplied with O2 due to pericarp photosynthesis. ATP distribution in the developing grain was coupled to endosperm differentiation. High ATP concentrations were associated with the local onset of starch storage within endosperm, while low ATP overlapped with the hypoxic regions. Temporally, the building of steep gradients in ATP coincided with overall elevating metabolite levels, specific changes in the metabolite profiles (glycolysis and citrate cycle), and channelling of metabolic fluxes towards storage (increase of starch accumulation rate). These findings implicate an inhomogenous spatial arrangement of metabolic activity within the caryopsis. It is suggested that the local onset of starch storage is coupled with the accumulation of ATP and elevated metabolic activity. Thus, the ATP level reflects the metabolic state of storage tissue. On the basis of these findings, a hypothetical model for the regulation of starch storage in barley seeds is proposed.
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paper_title: Legume embryos develop in a hypoxic environment

paper_content:

Specific morphological and biochemical characteristics of seeds can cause oxygen deficiency within maternal and embryonic tissues. In this study, optical sensors were used to measure O 2 profiles across developing seeds of Vicia faba and Pisum sativum and developmental and environmental modulations of internal O 2 levels were studied. In addition, the metabolic state of developing embryos was analysed by monitoring adenylate energy charge, adenylate nucleotides and the levels of nucleotide sugars. Within the seed coat O 2 concentration decreased sharply to 3% towards the inner border. Lowest O 2 levels were detected within the endospermal cavity between the seed coat and embryo. It is probable that low seed coat permeability provides an hypoxic environment for legume embryo development. The O 2 concentration in embryonic tissue changed during development with the lowest levels in the early stages. Measured in darkness, the levels were below 3%, but increased upon illumination indicating that photosynthesis significantly contributes to internal O 2 levels. Only in very young embryos were ATP levels and energy charge low. Otherwise they were maintained at a constant higher value. ADP-glucose and UDP-glucose did not show large fluctuations. Throughout embryo development fermentative activity did not play a major role. Obviously, specific mechanisms prevent seed tissues from becoming anoxic during development. The possible role of low oxygen on seed metabolism and on the control of seed development in legumes is discussed.
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paper_title: Temperature-dependent endogenous oxygen concentration regulates microsomal oleate desaturase in developing sunflower seeds

paper_content:

Oleoyl-phosphatidylcholine desaturase (FAD2) is a key enzyme involved in fatty acid desaturation in oilseeds, which is affected by environmental temperature. The results of this study show that FAD2 is regulated in vivo via temperature-dependent endogenous oxygen concentrations in developing sunflower (Helianthus annuus L.) seeds. By combining in vivo oxygen profiling, in situ hybridization of FAD2 genes, an assay of energy status, fatty acid analysis, and an in vitro FAD2 enzyme activity assay, it is shown that: (i) the oil-storing embryo is characterized by a very low oxygen level that is developmentally regulated. Oxygen supply is mainly limited by the thin seed coat. (ii) Elevations of external oxygen supply raised the energy status of seed and produced a dramatic increase of the FAD2 enzyme activity as well as the linoleic acid content. (iii) A clear negative correlation exists between temperature and internal oxygen concentration. The changes occurred almost instantly and the effect was fully reversible. The results indicate that the internal oxygen level acts as a key regulator for the activity of the FAD2 enzyme. It is concluded that a major mechanism by which temperature modifies the unsaturation degree of the sunflower oil is through its effect on dissolved oxygen levels in the developing seed.
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paper_title: Microelectrode and Modelling Study of Oxygen Distribution in Roots

paper_content:

Abstract The use of polarographic microelectrodes to explore the radial distribution of oxygen in maize roots aerated chiefly via the shoot is described. Also given is a brief description of a mathematical model of root aeration which introduces two simultaneous oxygen sources (the shoot and the rooting medium), and mimicks more realistically than hitherto the diffusive characteristics of the non-porous epidermal/hypodermal shell. In keeping with modelling predictions, steep radial diffusion gradients were a characteristic of the non-porous epidermal/hypodermal shell and stele, while shallow profiles generally characterised the cortex. Stelar anoxia, previously forecast by earlier mathematical modelling, was inducible by manipulating the oxygen concentrations around the shoot. The stelar diffusion gradients always indicated an inward diffusion from the cortex. However, two-way gradients were sometimes evident in the epidermal/hypodermal annulus, and were also found in the sheathing extensions of the root cap. These indicated diffusion into these annuli simultaneously from both the cortex and the rooting medium. Elsewhere the gradients in the epidermis/hypodermal cylinder were either solely positive or negative depending upon the oxygen concentrations within the root cortex and surrounding agar. Examples of two-way and one-way diffusion in the epidermal/hypodermal cylinder were created satisfactorily by the model. Finally, the results indicate that with roots in stagnant anaerobic media, the bulk of the root cap proper is likely to be anoxic, and that some anoxia may be the norm within the basipetal extensions of the cap which ensheath the root apex.
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paper_title: Energy Status and Its Control on Embryogenesis of Legumes. Embryo Photosynthesis Contributes to Oxygen Supply and Is Coupled to Biosynthetic Fluxes

paper_content:

Legume seeds are heterotrophic and dependent on mitochondrial respiration. Due to the limited diffusional gas exchange, embryos grow in an environment of low oxygen. O(2) levels within embryo tissues were measured using microsensors and are lowest in early stages and during night, up to 0.4% of atmospheric O(2) concentration (1.1 micro M). Embryo respiration was more strongly inhibited by low O(2) during earlier than later stages. ATP content and adenylate energy charge were lowest in young embryos, whereas ethanol emission and alcohol dehydrogenase activity were high, indicating restricted ATP synthesis and fermentative metabolism. In vitro and in vivo experiments further revealed that embryo metabolism is O(2) limited. During maturation, ATP levels increased and fermentative metabolism disappeared. This indicates that embryos become adapted to the low O(2) and can adjust its energy state on a higher level. Embryos become green and photosynthetically active during differentiation. Photosynthetic O(2) production elevated the internal level up to approximately 50% of atmospheric O(2) concentration (135 micro M). Upon light conditions, embryos partitioned approximately 3-fold more [(14)C]sucrose into starch. The light-dependent increase of starch synthesis was developmentally regulated. However, steady-state levels of nucleotides, free amino acids, sugars, and glycolytic intermediates did not change upon light or dark conditions. Maturing embryos responded to low O(2) supply by adjusting metabolic fluxes rather than the steady-state levels of metabolites. We conclude that embryogenic photosynthesis increases biosynthetic fluxes probably by providing O(2) and energy that is readily used for biosynthesis and respiration.
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paper_title: Phloem import and storage metabolism are highly coordinated by the low oxygen concentrations within developing wheat seeds

paper_content:

We studied the influence of the internal oxygen concentration in seeds of wheat (Triticum aestivum) on storage metabolism and its relation to phloem import of nutrients. Wheat seeds that were developing at ambient oxygen (21%) were found to be hypoxic (2.1%). Altering the oxygen supply by decreasing or increasing the external oxygen concentration induced parallel changes in the internal oxygen tension. However, the decrease in internal concentration was proportionally less than the reduction in external oxygen. This indicates that decreasing the oxygen supply induces short-term adaptive responses to reduce oxygen consumption of the seeds. When external oxygen was decreased to 8%, internal oxygen decreased to approximately 0.5% leading to a decrease in energy production via respiration. Conversely, increasing the external oxygen concentration above ambient levels increased the oxygen content as well as the energy status of the seeds, indicating that under normal conditions the oxygen supply is strongly limiting for energy metabolism in developing wheat seeds. The intermediate metabolites of seed storage metabolism were not substantially affected when oxygen was either increased or decreased. However, at subambient external oxygen concentrations (8%) the metabolic flux of carbon into starch and protein, measured by injecting 14C-Suc into the seeds, was reduced by 17% and 32%, respectively, whereas no significant effect was observed at superambient (40%) oxygen. The observed decrease in biosynthetic fluxes to storage compounds is suggested to be part of an adaptive response to reduce energy consumption preventing excessive oxygen consumption when oxygen supply is limited. Phloem transport toward ears exposed to low (8%) oxygen was significantly reduced within 1 h, whereas exposing ears to elevated oxygen (40%) had no significant effect. This contrasts with the situation where the distribution of assimilates has been modified by removing the lower source leaves from the plant, resulting in less assimilates transported to the ear in favor of transport to the lower parts of the plant. Under these conditions, with two strongly competing sinks, elevated oxygen (40%) did lead to a strong increase in phloem transport to the ear. The results show that sink metabolism is affected by the prevailing low oxygen concentrations in developing wheat seeds, determining the import rate of assimilates via the phloem.
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paper_title: The oxygen status of the developing seed.

paper_content:

Recent applications of oxygen-sensitive microsensors have demonstrated steep oxygen gradients in developing seeds of various crops. Here, we present an overview on oxygen distribution, major determinants of the oxygen status in the developing seed and implications for seed physiology. The steady-state oxygen concentration in different seed tissues depends on developmental parameters, and is determined to a large extent by environmental factors. Photosynthetic activity of the seed significantly diminishes hypoxic constraints, and can even cause transient, local hyperoxia. Changes in oxygen availability cause rapid adjustments in mitochondrial respiration and global metabolism. We argue that nitric oxide (NO) is a key player in the oxygen balancing process in seeds, avoiding fermentation and anoxia in vivo. Molecular approaches aiming to increase oxygen availability within the seed are discussed.
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paper_title: Evidence of a key role for photosynthetic oxygen release in oil storage in developing soybean seeds

paper_content:

Based on the topographical analysis of photosynthesis and oil storage, we propose in a companion paper that photosynthetic oxygen release plays a key role in the local energy state, storage metabolism and flux toward lipid biosynthesis in developing soybean seeds. To test this hypothesis, we combined topographical analysis of ATP gradients across tissues, microsensor quantifications of internal O2 levels, assays of energy balance, metabolite profiles and isotope-labelling studies. Seeds show a marked degree of oxygen starvation in vivo (minimum O2 levels 0.1 kPa, approximately 1.3 microm), affecting ATP gradients, overall energy state, metabolite pools and storage activity. Despite the low light availability, photosynthesis supplies significant amounts of oxygen to the hypoxic seed tissue. This is followed by an increase in local ATP levels, most prominently within the lipid-synthesizing (inner) regions of the embryo. Concomitantly, partitioning of 14C-sucrose to lipids is increased, suggesting higher rates of lipid biosynthesis. It is concluded that both respiratory and biosynthetic fluxes are dynamically adjusted to photosynthetic oxygen supply.
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paper_title: Positional cues for the starch/lipid balance in maize kernels and resource partitioning to the embryo

paper_content:

This study tests the hypotheses that in vivo oxygen levels inside developing maize grains locally affect assimilate partitioning and ATP distribution within the kernel. These questions were addressed through combined topographical analysis (O2- and ATP-mapping), metabolite profiling, and isotope flux analysis. Internal and external oxygen levels were also experimentally altered. Under ambient conditions, mean O2 concentration immediately inside starchy endosperm dropped to only 1.4% of atmospheric saturation (approximately 3.8 microm), but was 10-fold higher in the oil-storing embryo. Increasing the O2 supply to intact kernels stimulated their O2 demand, shifted ATP localization within the kernel, and elevated their ATP/ADP ratio. Enhanced O2 availability also increased steady-state levels of glycolytic intermediates and those of the citric acid cycle, as well as some related pools of free amino acids. Subsequent analyses indicated that starch formation within endosperm, but not lipid biosynthesis within embryo, was adapted to the endogenous low oxygen. Increasing the O2 supply did not change ADP-glucose levels, activity of ADP-glucose pyrophosphorylase, 13C-labeling of ADP-glucose, or flux of 14C-sucrose into starch. In contrast, enhanced O2 availability increased 14C-label uptake into the embryo, 13C-labeling of acetyl-coenzyme A, and finally 14C-incorporation into lipids. Lipid accumulation in embryo appeared highest in regions with higher ATP. Consistent with labeling data, a decrease in O2 supply most strongly affected the embryo, whereas rising O2 levels expanded ATP-rich zones toward the starch-storing endosperm and the scutellar part of embryo. The latter might be responsible for higher 14C-label uptake into the embryo and flux toward lipid. Collectively, data indicate that the in vivo oxygen distribution in maize kernels markedly affects ATP gradients, metabolite levels, and favors assimilate partitioning toward starch within the O2-depleted endosperm. Clear advantages are thus evident for peripheral localization of the protein and lipid storing structures in maize kernels.
---
paper_title: Energy state and its control on seed development: starch accumulation is associated with high ATP and steep oxygen gradients within barley grains.

paper_content:

The role of oxygen and energy state in development and storage activity of cereal grains is an important issue, but has remained largely uninvestigated due to the lack of appropriate analytical methods. Metabolic profiling, bioluminescence-based in situ imaging of ATP, and oxygen-sensitive microsensors were combined here to investigate barley seed development. For the first time temporal and spatial maps of O2 and ATP distribution in cereal grains were determined and related to the differentiation pattern. Steep O2 gradients were demonstrated and strongly hypoxic regions were detected within the caryopsis (<0.1% of atmospheric saturation). Growing lateral and peripheral regions of endosperm remained well-supplied with O2 due to pericarp photosynthesis. ATP distribution in the developing grain was coupled to endosperm differentiation. High ATP concentrations were associated with the local onset of starch storage within endosperm, while low ATP overlapped with the hypoxic regions. Temporally, the building of steep gradients in ATP coincided with overall elevating metabolite levels, specific changes in the metabolite profiles (glycolysis and citrate cycle), and channelling of metabolic fluxes towards storage (increase of starch accumulation rate). These findings implicate an inhomogenous spatial arrangement of metabolic activity within the caryopsis. It is suggested that the local onset of starch storage is coupled with the accumulation of ATP and elevated metabolic activity. Thus, the ATP level reflects the metabolic state of storage tissue. On the basis of these findings, a hypothetical model for the regulation of starch storage in barley seeds is proposed.
---
paper_title: Embryogeny of Phaseolus: Developmental pattern of lactate and alcohol dehydrogenases

paper_content:

Abstract Alcohol and lactate dehydrogenase activity and their electrophoretic isoenzymes were determined in developing Phaseolus vulgaris embryos, seed coat
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paper_title: Studies on the Mechanism of Differences in Photosynthesis among Species : III. Influence of low oxygen concentration on dry matter production and grain fertility of rice plant

paper_content:

It has been clarified that the rates of apparent photosynthesis in C3 plant such as rice and wheat, which evolve a large quantity of carbon dioxide in the light, become higher as the oxygen concentration in the air decreases. However, the drastic change in oxygen concentration from 21 per cent to a few per cent may inhibit some of the physiological functions of plant which have adapted to the present atmosphere. The present experiment was conducted to evaluate the effect of low oxygen concentration for a long period on dry matter production and growth of rice plant. The plant was kept in the assimilation chamber in both control and low oxygen plots. The plant in low oxygen plot was treated with the air containing 3 per cent oxygen and the plant in control plot was aerated with ordinary outdoor air. The gas treatment was conducted only in the daytime for 20 days from the heading stage to the milky dough stage. The following results were obtained. 1. The increased weight of dry matter during the treated period was significantly higher in the low oxygen plot than in the control. This increase was mainly due to the reduced photorespiration rate in low oxygen concentration. 2. The space between tillers was extremely expanded after 20 hours from the begining of low oxygen treatment. 3. When the rice plant was exposed to low oxygen concentration at flowering time, remarkable sterility occurred. Such phenomenon was also observed when only the panicle was kept in low oxygen concentration. Thus, the sterility observed here seems to be caused, not by the translocated substances from the leaf and stem, but by the direct damage of low oxygen to fertilization. 4. The grain fertility of Cyperus microiria Steudel, on the other hand, was hardly affected by oxygen concentration of 3 per cent at which the rice plant showed remarkable sterility. This fact suggests that the influence of low oxygen on fertility of plant differs with species. 5. It follows from these findings that the lowering of oxygen in the air is effective in increasing the dry matter production of rice plant, while deteriorate the physiological functions relating to fertility and growth.
---
paper_title: The oxygen status of the developing seed.

paper_content:

Recent applications of oxygen-sensitive microsensors have demonstrated steep oxygen gradients in developing seeds of various crops. Here, we present an overview on oxygen distribution, major determinants of the oxygen status in the developing seed and implications for seed physiology. The steady-state oxygen concentration in different seed tissues depends on developmental parameters, and is determined to a large extent by environmental factors. Photosynthetic activity of the seed significantly diminishes hypoxic constraints, and can even cause transient, local hyperoxia. Changes in oxygen availability cause rapid adjustments in mitochondrial respiration and global metabolism. We argue that nitric oxide (NO) is a key player in the oxygen balancing process in seeds, avoiding fermentation and anoxia in vivo. Molecular approaches aiming to increase oxygen availability within the seed are discussed.
---
paper_title: Oxygen Control of Reproductive Growth: Possible Mediation via Dark Respiration

paper_content:

The rates of dark respiration of Glycine max leaves, pods, and seeds, are shown to be positively related to O2 concentration ([O2]) between 2 and 21 per cent. It is suggested that the reported depression of seed growth, due to subatmospheric concentrations of O2 is caused simply by depression of respiration and that it is not necessary to postulate a new control mechanism mediated by [O2].
---
paper_title: Reproductive Growth and Dry Matter Production of Glycine max (L.) Merr. in Response to Oxygen Concentration

paper_content:

Reproductive as well as vegetative parameters of mature soybean ( Glycine max [L.] Merr. cv. Wye) plants grown in chambers in which the aerial portion was exposed to altered p O 2 during all or part of the growth cycle were measured. Oxygen concentration was found to be a key factor controlling all phases of reproductive development. Exposure to 5% O 2 from early seedling stage to senescence increased leaf, stem, and root dry weights and reduced seed yields when compared to 21% O 2 ; exposure to low O 2 during the vegetative growth stage from early seedling to mid-flowering arrested pod but not seed development; exposure from mid-flowering to mid-pod filling almost completely arrested seed but not pod development; exposure from mid-pod filling to senescence arrested seed development at the mid-filling stage. Exposures to 5% O 2 initiated at mid-flowering for 1, 2, 3, 5, 10, and 15 days had no effect on seed development when the exposure was up to 3 days and produced almost total arrest when the exposure was 10 or more days, suggesting reversibility. The requirement for O 2 in seed development is independent of CO 2 concentration with similar results produced by subambient O 2 combined with ambient CO 2 , elevated CO 2 up to 2000 μl/l or depressed levels of CO 2 with the CO 2 /O 2 ratio as in air. An elevated O 2 atmosphere containing 40% O 2 and ambient or elevated CO 2 inhibited total growth but did not affect the balance of vegetative to reproductive growth. We conclude that an unknown reaction or process requiring at least atmospheric concentrations of O 2 but independent of CO 2 in contrast to photorespiration is necessary for optimization of all phases of reproductive growth and the effect is reversible for exposures of up to 3 days but not for exposures of 10 days or more. We propose that this O 2 phenomenon may be the result of a unique physical process or chemical reaction associated with translocation and accumulation of assimilates in reproductive structures.
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paper_title: Redistribution of intracellular oxygen in hypoxia by nitric oxide: effect on HIF1alpha.

paper_content:

Cells exposed to low oxygen concentrations respond by initiating defense mechanisms, including the stabilization of hypoxia-inducible factor (HIF) 1alpha, a transcription factor that upregulates genes such as those involved in glycolysis and angiogenesis. Nitric oxide and other inhibitors of mitochondrial respiration prevent the stabilization of HIF1alpha during hypoxia. In studies of cultured cells, we show that this effect is a result of an increase in prolyl hydroxylase-dependent degradation of HIF1alpha. With the use of Renilla luciferase to detect intracellular oxygen concentrations, we also demonstrate that, upon inhibition of mitochondrial respiration in hypoxia, oxygen is redistributed toward nonrespiratory oxygen-dependent targets such as prolyl hydroxylases so that they do not register hypoxia. Thus, the signaling consequences of hypoxia may be profoundly modified by nitric oxide.
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paper_title: Does nitric oxide modulate mitochondrial energy generation and apoptosis?

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The physiological role of nitric oxide (NO) in the maintenance of vascular tone, in synaptic transmission and in cellular defence is now firmly established. Recent evidence indicates that NO can also affect mitochondrial function. Here, we review findings indicating that NO — through its interaction with components of the electron-transport chain — might function not only as a physiological regulator of cell respiration, but also to augment the generation of reactive oxygen species by mitochondria, and thereby trigger mechanisms of cell survival or death.
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paper_title: Expression of a stress-induced hemoglobin affects NO levels produced by alfalfa root cultures under hypoxic stress

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Transgenic alfalfa root cultures expressing sense and antisense barley hemoglobin transcripts were examined under varying levels of atmospheric oxygen. Root cultures overexpressing the hemoglobin gene (Hb + ) maintained root growth when placed under 3% oxygen, whereas control cultures or cultures underexpressing hemoglobin (Hb - ) experienced 30-70% declines in growth under the same conditions. ATP levels and ATP/ADP ratios for Hb + lines did not significantly differ in 40 and 3% oxygen, whereas the ATP levels and ATP/ADP ratios in control and Hb - lines were significantly lower under 3% oxygen. Large increases in the production of nitric oxide (NO) were measured in root cultures grown under hypoxic conditions compared to aerobic conditions. The amount of NO accumulated in an Hb - line was 2.5-fold higher than that in the Hb + line. Treatment of transgenic root lines under 40% oxygen with NO resulted in significant declines in the ATP levels and ATP/ADP ratio of an Hb - line and the control line, with no significant change in an Hb + line. The root cell structure of an Hb - line showed evidence of cell breakdown under hypoxic growth, whereas an Hb + line had no evidence of cell breakdown under similar growth conditions. These results lead us to hypothesize that NO is involved in the response of plants to hypoxia and that hemoglobin modulates the levels of NO in the hypoxic cell.
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paper_title: Low oxygen sensing and balancing in plant seeds: a role for nitric oxide

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Summary • Storage product accumulation in seeds of major crop species is limited by their low internal oxygen concentration. Adjustment of energy and storage metabolism to oxygen deficiency (hypoxia) in seeds is highly relevant for agriculture and biotechnology. However, the mechanisms of low-oxygen sensing and balancing remain a mystery. • Here, it is shown that normal hypoxia in seeds of soybean ( Glycine max ) and pea ( Pisum sativum ) triggers a nitrite-dependent increase in endogenous nitric oxide (NO) concentrations. NO, in turn, reduces the oxygen consumption of seeds, generating a localized decrease in both ATP availability and biosynthetic activity. Increasing oxygen availability reduces endogenous NO concentrations, thereby abolishing mitochondrial and metabolic inhibition. • This auto-regulatory and reversible oxygen balancing, via NO, avoids seed anoxia and suggests a key role for NO in regulating storage activity. This hypothesis is reinforced by changes in energy status (ATP:ADP ratio), steady-state metabolite concentrations and biosynthetic fluxes under NO treatment. • The proposed mechanism of low-oxygen sensing and balancing in plants offers the prospect of a new field of study in crop biotechnology.
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paper_title: Inhibitory effects of nitric oxide on oxidative phosphorylation in plant mitochondria.

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Plant nitrate reductase (NR) produces nitric oxide (NO) when nitrite is provided as the substrate in the presence of NADH [H. Yamasaki and Y. Sakihama (2000) FEBS Lett. 468, 89-92]. Using a NR-dependent NO producing system, we investigated the effects of NO on the energy transduction system in plant mitochondria isolated from mung bean (Vigna radiata). Plant mitochondria are known to possess two respiratory electron transport pathways-the cytochrome and alternative pathways. When the alternative pathway was inhibited by n-propyl gallate, the addition of NR strongly suppressed respiratory O(2) consumption driven by the cytochrome pathway. In contrast, the alternative pathway measured in the presence of antimycin A was not affected by NO. The extent of the steady-state membrane potential (Deltapsi) generated by respiratory electron transport rapidly declined in response to NO production. The addition of bovine hemoglobin, a quencher of NO, resulted in the recovery of Deltapsi to the uninhibited level. Consistent with its inhibition of Deltapsi, NO produced by NR strongly suppressed ATP synthesis in the mitochondria. These results provide substantial evidence to confirm that the plant alternative pathway is resistant to NO and support the idea that the alternative pathway may lower respiration-dependent production of active oxygens under conditions where NO is overproduced.
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paper_title: Carbon Dioxide Fixation in the Carbon Economy of Developing Seeds of Lupinus albus (L.)

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The effects of CO(2) concentration and illumination on net gas exchange and the pathway of (14)CO(2) fixation in detached seeds from developing fruits of Lupinus albus (L.) have been studied.Increasing the CO(2) concentration in the surrounding atmosphere (from 0.03 to 3.0% [v/v] in air) decreased CO(2) efflux by detached seeds either exposed to the light flux equivalent to that transmitted by the pod wall (500 to 600 micro-Einsteins per square meter per second) in full sunlight or held in darkness. Above 1% CO(2) detached seeds made a net gain of CO(2) in the light (up to 0.4 milligrams of CO(2) fixed per gram fresh weight per hour) but (14)CO(2) injected into the gas space of intact fruits (containing around 1.5% CO(2) naturally) was fixed mainly by the pod and little by the seeds.Throughout development seeds contained ribulose-1,5-bisphosphate carboxylase activity (EC 4.1.1.39), especially in the embryo (up to 99 micromoles of CO(2) fixed per gram fresh weight per hour) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) in both testa (up to 280 micromoles of CO(2) fixed per gram fresh weight per hour) and embryo (up to 355 micromoles of CO(2) fixed per gram fresh weight per hour).In kinetic experiments the most significant early formed product of (14)CO(2) fixation in both light and dark was malate but in the light phosphoglyceric acid and sugar phosphates were also rapidly labeled. (14)CO(2) fixation in the light was linked to the synthesis of sugars and amino acids but in the dark labeled sugars were not formed.
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paper_title: Evidence of a key role for photosynthetic oxygen release in oil storage in developing soybean seeds

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Based on the topographical analysis of photosynthesis and oil storage, we propose in a companion paper that photosynthetic oxygen release plays a key role in the local energy state, storage metabolism and flux toward lipid biosynthesis in developing soybean seeds. To test this hypothesis, we combined topographical analysis of ATP gradients across tissues, microsensor quantifications of internal O2 levels, assays of energy balance, metabolite profiles and isotope-labelling studies. Seeds show a marked degree of oxygen starvation in vivo (minimum O2 levels 0.1 kPa, approximately 1.3 microm), affecting ATP gradients, overall energy state, metabolite pools and storage activity. Despite the low light availability, photosynthesis supplies significant amounts of oxygen to the hypoxic seed tissue. This is followed by an increase in local ATP levels, most prominently within the lipid-synthesizing (inner) regions of the embryo. Concomitantly, partitioning of 14C-sucrose to lipids is increased, suggesting higher rates of lipid biosynthesis. It is concluded that both respiratory and biosynthetic fluxes are dynamically adjusted to photosynthetic oxygen supply.
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paper_title: Arabidopsis Seed Development and Germination Is Associated with Temporally Distinct Metabolic Switches

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While the metabolic networks in developing seeds during the period of reserve accumulation have been extensively characterized, much less is known about those present during seed desiccation and subsequent germination. Here we utilized metabolite profiling, in conjunction with selective mRNA and physiological profiling to characterize Arabidopsis (Arabidopsis thaliana) seeds throughout development and germination. Seed maturation was associated with a significant reduction of most sugars, organic acids, and amino acids, suggesting their efficient incorporation into storage reserves. The transition from reserve accumulation to seed desiccation was associated with a major metabolic switch, resulting in the accumulation of distinct sugars, organic acids, nitrogen-rich amino acids, and shikimate-derived metabolites. In contrast, seed vernalization was associated with a decrease in the content of several of the metabolic intermediates accumulated during seed desiccation, implying that these intermediates might support the metabolic reorganization needed for seed germination. Concomitantly, the levels of other metabolites significantly increased during vernalization and were boosted further during germination sensu stricto, implying their importance for germination and seedling establishment. The metabolic switches during seed maturation and germination were also associated with distinct patterns of expression of genes encoding metabolism-associated gene products, as determined by semiquantitative reverse transcription-polymerase chain reaction and analysis of publicly available microarray data. When taken together our results provide a comprehensive picture of the coordinated changes in primary metabolism that underlie seed development and germination in Arabidopsis. They furthermore imply that the metabolic preparation for germination and efficient seedling establishment initiates already during seed desiccation and continues by additional distinct metabolic switches during vernalization and early germination.
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paper_title: Nitrite-nitric oxide control of mitochondrial respiration at the frontier of anoxia.

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Actively respiring animal and plant tissues experience hypoxia because of mitochondrial O(2) consumption. Controlling oxygen balance is a critical issue that involves in mammals hypoxia-inducible factor (HIF) mediated transcriptional regulation, cytochrome oxidase (COX) subunit adjustment and nitric oxide (NO) as a mediator in vasodilatation and oxygen homeostasis. In plants, NO, mainly derived from nitrite, is also an important signalling molecule. We describe here a mechanism by which mitochondrial respiration is adjusted to prevent a tissue to reach anoxia. During pea seed germination, the internal atmosphere was strongly hypoxic due to very active mitochondrial respiration. There was no sign of fermentation, suggesting a down-regulation of O(2) consumption near anoxia. Mitochondria were found to finely regulate their surrounding O(2) level through a nitrite-dependent NO production, which was ascertained using electron paramagnetic resonance (EPR) spin trapping of NO within membranes. At low O(2), nitrite is reduced into NO, likely at complex III, and in turn reversibly inhibits COX, provoking a rise to a higher steady state level of oxygen. Since NO can be re-oxidized into nitrite chemically or by COX, a nitrite-NO pool is maintained, preventing mitochondrial anoxia. Such an evolutionarily conserved mechanism should have an important role for oxygen homeostasis in tissues undergoing hypoxia.
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paper_title: Germination, Respiration, and Adenylate Energy Charge of Seeds at Various Oxygen Partial Pressures

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The effect of O2 partial pressure on the germination and the respiration of 12 cultivated species was studied. The reciprocal of the time necessary to observe rootlet emergence in 50% of the seeds was used to approach the germination rate. The maximum germination and respiration rates were reached in most seeds at O2 pressures close to that of air. Decreasing the O2 pressure produced a gradual decrease of the germination rate. The seeds could be classed in two groups according to their response to low O2 pressures. Group I includes lettuce, sunflower, radish, turnip, cabbage, flax, and soybean: at O2 pressures close to 2 kilopascals, the germination in this group was stopped and the adenylate energy charge was lower than 0.6. Group II includes rice, wheat, maize, sorghum, and pea. The germination rate of these seeds was also gradually decreased by lowering the O2 partial pressure but germination still occured, very slowly, at 0.1 kilopascal; the adenylate energy charge remained higher than 0.6. These differences in the germination rates and adenylate energy charge values could not be explained by differences in the sensitivity of respiration to O2.
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paper_title: ATP Production by Respiration and Fermentation, and Energy Charge during Aerobiosis and Anaerobiosis in Twelve Fatty and Starchy Germinating Seeds

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The respiration and fermentation rates were compared in germinating seeds of 12 different cultivated species from five families. In air, fermentation contributes significantly to the energy metabolism only in some species (pea, maize), but is generally negligible when compared to respiration. The fermentation rate under anoxia was related either to the metabolic activity under air or to the adenine nucleotide content of the seeds: it was generally higher in seeds which contain starchy reserves (rice, maize, sorghum, pea), than in seeds which do not contain starch (lettuce, sunflower, radish, turnip, cabbage, flax); however, it was similar in wheat, sorghum (starchy seeds), and soya (nonstarchy seeds). The value of the energy charge of all the seeds was lower under anoxia than in air: after 24 hours under anoxia, it was higher than 0.5 in the starchy seeds and in soya and it was around 0.25 in the other fatty seeds.
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paper_title: Barley Grain Maturation and Germination: Metabolic Pathway and Regulatory Network Commonalities and Differences Highlighted by New MapMan/PageMan Profiling Tools

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Plant seeds prepare for germination already during seed maturation. We performed a detailed transcriptome analysis of barley (Hordeum vulgare) grain maturation, desiccation, and germination in two tissue fractions (starchy endosperm/aleurone and embryo/scutellum) using the Affymetrix Barley1 GeneChip. To aid data evaluation, Arabidopsis thaliana MapMan and PageMan tools were adapted to barley. The analyses allow a number of conclusions: (1) Cluster analysis revealed a smooth transition in transcription programs between late seed maturation and germination within embryo tissues, but not in the endosperm/aleurone. (2) More than 12,000 transcripts are stored in the embryo of dry barley grains, many of which are presumably activated during germination. (3) Transcriptional activation of storage reserve mobilization events occurs at an early stage of germination, well before radicle protrusion. (4) Key genes of gibberellin (GA) biosynthesis are already active during grain maturation at a time when abscisic acid peaks suggesting the formation of an endogenous store of GA in the aleurone. This GA probably acts later during germination in addition to newly synthesized GA. (5) Beside the well-known role of GA in gene activation during germination spatiotemporal expression data and cis-element searches in homologous rice promoters confirm an equally important gene-activating role of abscisic acid during this developmental period. The respective regulatory webs are linked to auxin and ethylene controlled networks. In summary, new bioinformatics PageMan and MapMan tools developed in barley have been successfully used to investigate in detail the transcriptome relationships between seed maturation and germination in an important crop plant.
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paper_title: Gradients of lipid storage, photosynthesis and plastid differentiation in developing soybean seeds

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This study establishes a topographical framework for functional investigations on the regulation of lipid biosynthesis and its interaction with embryo photosynthesis in developing soybean seed. Structural observations, combined with molecular and functional parameters, revealed the gradual transformation of chloroplasts into storage organelles, starting from inner regions going outwards. This is evidenced by electron microscopy, confocal laser scanning microscopy, in situ hybridization and histochemical/biochemical data. As a consequence of plastid differentiation, photosynthesis becomes distributed along a gradient within the developing embryo. Electron transport rate, effective quantum yield and O2 production rate are maximal in the embryo periphery, as documented by imaging pulse-amplitude-modulated fluorescence and O2 release via microsensors. The gradual loss of photosynthetic capacity was accompanied by a similarly gradual accumulation of starch and lipids. Noninvasive nuclear magnetic resonance spectroscopy of mature seeds revealed steep gradients in lipid deposition, with the highest concentrations in inner regions. The inverse relationship between photosynthesis and lipid biosynthesis argues against a direct metabolic involvement of photosynthesis in lipid biosynthesis during the late storage stage, but points to a role for photosynthetic oxygen release. This hypothesis is verified in a companion paper.
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Title: Methodology and Significance of Microsensor-based Oxygen Mapping in Plant Seeds – an Overview

Section 1: Introduction
Description 1: Provide background information on the importance of seed development, the impact of oxygen levels on seed physiology, and the role of microsensors in measuring oxygen distribution within seeds.

Section 2: Short Overview on Microsensors
Description 2: Discuss the types of oxygen-sensitive microsensors, their principles of operation, advantages, and limitations, specifically comparing electrochemical oxygen microsensors and optical oxygen microsensors.

Section 3: Oxygen Mapping in Plant Seeds
Description 3: Explain the procedures and methodologies used for oxygen profiling in seeds, including the types of plant species studied and the outcomes obtained from generating oxygen maps.

Section 4: Monitoring Environmental Effects on Steady-state Oxygen Concentrations within the Seed
Description 4: Describe how microsensor-based measurements are used to assess the effects of environmental factors, such as light availability and nitric oxide injection, on the steady-state oxygen concentration within seeds.

Section 5: The Establishment of Hypoxic Conditions during Seed Germination
Description 5: Explore the dynamics of oxygen concentration changes during seed germination and early seedling growth, including experiments on imbibition and how microsensors track these changes.

Section 6: Conclusions
Description 6: Summarize the significance of microsensor-based oxygen mapping in seed science, the insights gained regarding oxygen balancing mechanisms in seeds, and the future prospects for improved spatial resolution and combined sensor applications.