Combining Ionizing Irradiation and Ultrasound Technologies: Effect on Beans Hydration and Germination.

The present work studied, for the first time, the effect of two technologies on the hydration and germination process of beans: ionizing irradiation and ultrasound. In addition, this work proposed a possible biological effect of ultrasound to describe its effect on grain hydration. Carioca beans were irradiated at doses of 2.3 and 3.8 kGy using γ-rays, establishing different metabolic activities for the beans. Then, they were hydrated with and without the ultrasound technology (91 W/L and 25 kHz) at 25 °C. Both the hydration and germination kinetics were evaluated considering the application of both technologies alone and in combination. As results, irradiation did not significantly affect (P > 0.05) the hydration rate, the equilibrium moisture, or the lag phase time, but ultrasound affected them, reducing 50% of the processing time. On the other hand, only the equilibrium moisture was slightly reduced by the interactive effect of irradiation and ultrasound technology, which was discussed based on different metabolism levels. Furthermore, the germination process of carioca bean was hindered by both the studied variables, reducing its germination capacity to 0% in some cases. Therefore, by studying the effect of ultrasound on beans with different active metabolism (due to different irradiation doses), it was suggested that both physical and biological mechanisms are involved during hydration process and that both can be affected by ultrasound. Further, irradiation could be used on carioca beans with objectives such as avoid germination, insects, and microorganism's growth, without an important effect on the hydration kinetics. PRACTICAL APPLICATION: Ultrasound technology has demonstrated to accelerate the soaking process of several legume grains. However, sometimes, grains are irradiated for disinfestation purpose to improve their shelf life. Indeed, irradiation can change the grain properties as the cooking time, hydration time, and germination capacity. Therefore, this work verified if irradiation changes the desirable effect of ultrasound on grain processing: soaking and germination. Moreover, this work novelty is also based on describing the phenomena: by proposing a possible biological effect, further approaches to improve the grain processing can be achieved.

Gas exchange and chlorophyll a fluorescence measurements as proxies of X-ray resistance in Phaseolus vulgaris L.

Phaseolus vulgaris L. plants were irradiated with different doses (0.3, 10, 50 and 100 Gy) of X-rays in order to obtain a reference curve of response to ionizing radiations for this species. Growth analysis, gas exchange and chlorophyll a fluorescence measurements were performed to estimate the radio-resistance of bean plants. Specifically, there was a negative influence of X-rays on the net photosynthesis rate at 50 and 100 Gy, already on the day of irradiation. Experimental data showed a recovery over time in the gas exchange while the theoretical maximum photochemical efficiency of the photosystem II (Fv/Fm) was fairly constant throughout the period of measurements (20 days) and for all the experimental conditions. On the other hand, the quantum yield of PSII linear electron transport (ΦPSII) and non-photochemical quenching (NPQ) were deeply influenced over time by X-ray dose, suggesting a decrease in the functionality of the photosynthetic apparatus at the highest radiation doses. The growth was affected only at the highest doses of radiation with a significant and severe reduction of leaf expansion and number of leaves per plant. Despite the arrest in growth, X-ray exposure seems to trigger an increased photochemical activity probably signifying that P. vulgaris plants have a fairly elevated resistance to this kind of ionizing radiation. Our current results will provide a complete analysis of the photosystem II (PSII) response of P. vulgaris to different doses (0.3, 10, 50 and 100 Gy) of X-rays, providing sound references for both space-oriented and radioecology questions.

Study on the Relationship between Seed Absorbed Dose and Seed Composition of 252Cf Neutron Source Irradiated Bean Seed.

This study aims to further identify the biological effects of neutron-irradiated plants and provides insights into the mutation breeding of such plants. In this study, the neutron irradiation device designed by our institute was used to analyze the relationship between the seed components in different legume crops and their neutron absorption dose rate, fission gamma absorption dose rate, and induced gamma absorption dose rate. The results show that the effect sizes of the components on the neutron absorbed dose rate are as follows: ash > fat > moisture > carbohydrate > protein. The effect sizes of the components on the absorbed dose rate of fission gamma are as follows: ash > moisture > fat > carbohydrate > protein. There is a positive correlation between fission gamma absorbed dose rate and the weight of ash, water and fat, while a negative correlation with carbohydrate and protein. However, the linear relationship between each component and the absorbed dose rate of induced gamma is not significant, this needs to be identified by further researches. Based on the results of the present study, we conclude that the neutron absorbed dose can be calculated without taking into account the fat composition of bean crop seeds (except for soybean seeds) in the process of mutation breeding induced by radiation. In special cases where the accuracy requirement of the dose rate is not high, it is possible to use protein instead of legume crop seeds for neutron absorption dose calculations.

On the relevance of thermophysical characterization in the microwave treatment of legumes.

This study is focused on the characterization of the thermal behavior and physical properties of the most consumed legumes in the daily diet such as beans, lentils and chickpeas. Because of a lack of information in the literature about the effect of microwave treatments on legumes, characterization protocols have been applied before and after subjecting them to microwave irradiation suitable for pest disinfestation. The effects of two different radiative treatments, one suitable for inactivating the infesting fauna and the other simulating uncontrolled treatments, characterized by very high temperatures, were tested. The impacts of microwave treatments on legumes, in terms of thermal behavior, germination capability, tannin and total polyphenol composition and other physical properties (water uptake capability, texture change, mineral losses), after typical soaking cooking processes, are also studied. The thermal properties of the examined legumes were found to be comparable for all samples. Similarly, no significant differences in antinutritional factors, polyphenol and tannin content among all samples were detected. From the structural point of view, samples exposed to high temperatures showed texture degradation and in turn, loss of mineral nutrients during soaking processes. Moreover, their germination capability was drastically reduced. These latter results highlighted why it is important to correctly perform the radiative microwave process in order to both ensure effective and safe disinfestation and avoid nutritional value loss and the worsening of physical properties.

INDUCED CYTOMICTIC VARIATIONS AND SYNCYTE FORMATION DURING MICROSPOROGENESIS IN PHASEOLUS VULGARIS L.

The intercellular translocation of chromatin material along with other cytoplasmic contents among the proximate meiocytes lying in close contact with each other commonly referred as cytomixis was reported during microsporogenesis in Phaseolus vulgaris L., a member of the family Fabaceae. The phenomenon of cytomixis was observed at three administered doses of gamma rays viz. 100, 200, 300 Gy respectively in the diploid plants of Phaseolus vulgaris L. The gamma rays irradiated plants showed the characteristic feature of inter-meiocyte chromatin/chromosomes transmigration through various means.such as channel formation, beak formation or by direct adhesion between the PMC's (Pollen mother cells). The present study also reports the first instance of syncyte formation induced via cytomictic transmigration in Phaseolus vulgaris L. Though the frequency of syncyteformation was rather low yet these could play a significant role in plant evolution. It is speculated that syncyte enhances the ploidy level of plants by forming 2n gametes and may lead to the production ofpolyploid plants. The phenomenon of cytomixis shows a gradual inclination along with the increasing treatment doses of gamma rays. The preponderance of cytomixis was more frequent during meiosis I as compared to meiosis II. An interesting feature noticed during the present study was the channel formation among the microspores and fusion among the tetrads due to cell wall dissolution. The impact of this phenomenon is also visible on the development of post-meiotic products. The formation of heterosized pollen grains; a deviation from the normal pollen grains has also been reported. The production of gametes with unbalanced chromosomes is of utmost importance and should be given more attention in future studies as they possess the capability of inducing variations at the genomic level and can be further utilized in the improvement of germplasm.

Friend or Foe-Light Availability Determines the Relationship between Mycorrhizal Fungi, Rhizobia and Lima Bean (Phaseolus lunatus L.).

Plant associations with root microbes represent some of the most important symbioses on earth. While often critically promoting plant fitness, nitrogen-fixing rhizobia and arbuscular mycorrhizal fungi (AMF) also demand significant carbohydrate allocation in exchange for key nutrients. Though plants may often compensate for carbon loss, constraints may arise under light limitation when plants cannot extensively increase photosynthesis. Under such conditions, costs for maintaining symbioses may outweigh benefits, turning mutualist microbes into parasites, resulting in reduced plant growth and reproduction. In natural systems plants commonly grow with different symbionts simultaneously which again may interact with each other. This might add complexity to the responses of such multipartite relationships. We experimented with lima bean (Phaseolus lunatus), which efficiently forms associations with both types of root symbionts. We applied full light and low-light to each of four treatments of microbial inoculation. After an incubation period of 14 weeks, we quantified vegetative aboveground and belowground biomass and number and viability of seeds to determine effects of combined inoculant and light treatment on plant fitness. Under light-limited conditions, vegetative and reproductive traits were inhibited in AMF and rhizobia inoculated lima bean plants relative to controls (un-colonized plants). Strikingly, reductions in seed production were most critical in combined treatments with rhizobia x AMF. Our findings suggest microbial root symbionts create additive costs resulting in decreased plant fitness under light-limited conditions.

Response of two cultivars of Phaseolus vulgaris L. (French beans) plants exposed to enhanced UV-B radiation under mountain ecosystem.

Enhanced solar ultraviolet-B (UV-B) irradiance resulting from depletion in the ozone layer has the potential to cause detrimental effects on plants. Higher altitudes tend to receive higher doses of ambient UV-B radiation. The present study was carried out to assess the effects of enhanced UV-B (ambient + 10.2 kJ m(-2) day(-1)) radiation on two cultivars of Phaseolus vulgaris L. (cv. Pusa Himlata and Pusa Parvati) at growth, physiological, and biochemical levels grown under mountain ecosystem. The magnitudes of negative effects of enhanced UV-B radiation were found more in Pusa Parvati as compared to Pusa Himlata. Non-enzymatic (total phenolics and flavonoids content) and enzymatic antioxidants (superoxide dismutase, catalase, and peroxidase) were increased in both cultivars at both the ages of growth but increase was found more in Pusa Himlata as compared to Pusa Parvati. The study further showed that the economic yield of Pusa Himlata and Pusa Parvati was decreased by 14 and 44%, respectively, due to enhanced UV-B radiation. The higher decrease in the economic yield of Pusa Parvati depicted that increased amounts of total flavonoids content and stimulation of their antioxidant defense mechanism via increasing the activities of enzymatic antioxidants were not able to completely detoxify the produced reactive oxygen species under enhanced UV-B radiation and made it more sensitive to applied stress. From the present study, it can be concluded that enhanced UV-B radiation in the mountain areas of the Indian Himalayan Regions could be one of the environmental causes for lower yields of agricultural crops. Cultivation of P. vulgaris L. cv. Pusa Himlata should be promoted at higher altitudes of the Indian Himalayan Regions.

Effects of acute O3 stress on PSII and PSI photochemistry of sensitive and resistant snap bean genotypes (Phaseolus vulgaris L.), probed by prompt chlorophyll "a" fluorescence and 820 nm modulated reflectance.

The response of PSII and PSI photochemistry to acute ozone (O3) stress was tested in a "model plant system", namely the O3 sensitive (S156) and O3 resistant (R123) genotype pairs of Phaseolus vulgaris L., during a phenological phase of higher O3 sensitivity (pod formation). The modulation of the photosynthetic activity during O3 stress was analysed by measuring gas exchanges, Prompt Fluorescence (PF, JIP-test) and 820 nm Modulated Reflectance (MR), a novel techniques which specifically detects the changes in the redox state of P700 and plastocyanin. The results showed that, coherently with genotypic-specific O3 sensitivity, the response of the two snap bean genotypes differed for the intensity and time of onset of the considered physiological changes. In fact, despite leaf injury and gas exchanges reduction appeared concurrently in both genotypes, S156 showed a PSII down regulation already after the first day of fumigation (DOF), and an enhancement of Cyclic Electron Flow of PSI after the second DOF, whereas R123 showed only slight adjustments until the third DOF, when the activity of both photosystems was down-regulated. Despite these differences, it is possible to distinguish in both genotypes an early O3 response of the photochemical apparatus, involving PSII only, and a following response, in which PSI activity and content are also modulated. The measurement of the MR signal, performed simultaneously with the PF measurements and the JIP-test analysis, has allowed a better understanding of the role that PSI plays in the O3 stress response of the S156/R123 model plant system.

Secondary metabolite perturbations in Phaseolus vulgaris leaves due to gamma radiation.

Oxidative stress is a condition in which the balance between the production and elimination of reactive oxygen species (ROS) is disturbed. However, plants have developed a very sophisticated mechanism to mitigate the effect of ROS by constantly adjusting the concentration thereof to acceptable levels. Electromagnetic radiation is one of the factors which results in oxidative stress. In the current study, ionizing gamma radiation generated from a Cobalt-60 source was used to induce oxidative stress in Phaseolus vulgaris seedlings. Plants were irradiated with several radiation doses, with 2 kGy found to be the optimal, non-lethal dose. Metabolite distribution patterns from irradiated and non-irradiated plants were analyzed using UHPLC-qTOF-MS and multivariate data models such as principal component analysis (PCA) and orthogonal projection to latent structures discriminate analysis (OPLS-DA). Metabolites such as hydroxycinnamic phenolic acids, flavonoids, terpenes, and a novel chalcone were found to be perturbed in P. vulgaris seedlings treated with the aforementioned conditions. The results suggest that there is a compensatory link between constitutive protectants and inducible responses to injury as well as defense against oxidative stress induced by ionizing radiation. The current study is also the first to illustrate the power of a metabolomics approach to decipher the effect of gamma radiation on crop plants.

Effect of illumination on the content of melatonin, phenolic compounds, and antioxidant activity during germination of lentils (Lens culinaris L.) and kidney beans (Phaseolus vulgaris L.).

This study reports the effects of two different illumination conditions during germination (12 h light/12 h dark vs 24 h dark) in lentils (Lens culinaris L.) and kidney beans (Phaseolus vulgaris L.) on the content of melatonin and phenolic compounds, as well as the antioxidant activity. Germination led to relative increase in melatonin content and significant antioxidant activity, while the content of phenolic compounds decreased. The highest melatonin content was obtained after 6 days of germination under 24 h dark for both legumes. These germinated legume seeds with improved levels of melatonin might play a protective role against free radicals. Thus, considering the potent antioxidant activity of melatonin, these sprouts can be consumed as direct foods and be offered as preventive food strategies in combating chronic diseases through the diet.

Impact of ambient and supplemental ultraviolet-B stress on kidney bean plants: an insight into oxidative stress management.

In the present study, the response of kidney bean (Phaseolus vulgaris L. cv. Pusa Komal) plants was evaluated under three different levels of ultraviolet-B (UV-B), i.e., excluded UV-B (eUV-B), ambient UV-B (aUV-B; 5.8 kJ m(-2) day(-1)), and supplemental UV-B (sUV-B; 280-315 nm; ambient + 7.2 kJ m(-2) day(-1)), under near-natural conditions. eUV-B treatment clearly demonstrated that both aUV-B and sUV-B are capable of causing significant changes in the plant's growth, metabolism, economic yield, genome template stability, total protein, and antioxidative enzyme profiles. The experimental findings showed maximum plant height at eUV-B, but biomass accumulation was minimum. Significant reductions in quantum yield (Fv/Fm) were observed under both aUV-B and sUV-B, as compared to eUV-B. UV-B-absorbing flavonoids increased under higher UV-B exposures with consequent increments in phenylalanine ammonia lyase (PAL) activities. The final yield was significantly higher in plants grown under eUV-B, compared to those under aUV-B and sUV-B. Total protein profile through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analysis of isoenzymes, like superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), and glutathione reductase (GR), through native PAGE revealed major changes in the leaf proteome under aUV-B and sUV-B, depicting induction of some major stress-related proteins. The random amplified polymorphic DNA (RAPD) profile of genomic DNA also indicated a significant reduction of genome template stability under UV-B exposure. Thus, it can be inferred that more energy is diverted for inducing protection mechanisms rather than utilizing it for growth under high UV-B level.

Anatomical alterations of Phaseolus vulgaris L. mature leaves irradiated with X-rays.

The cultivation of higher plants in Space involves not only the development of new agro-technologies for the design of ecologically closed Space greenhouses, but also understanding of the effects of Space factors on biological systems. Among Space factors, ionising radiation is one of the main constraints to the growth of organisms. In this paper, we analyse the effect of low-LET radiation on leaf histology and cytology in Phaseolus vulgaris L. plants subjected to increasing doses of X-rays (0.3, 10, 50, 100 Gy). Leaves irradiated at tissue maturity were compared with not-irradiated controls. Semi-thin sections of leaves were analysed through light and epi-fluorescence microscopy. Digital image analysis was applied to quantify anatomical parameters, with a specific focus on the occurrence of signs of structural damage as well as alterations at subcellular level, such as the accumulation of phenolic compounds and chloroplast size. Results showed that even at high levels of radiation, general anatomical structure was not severely perturbed. Slight changes in mesophyll density and cell enlargement were detected at the highest level of radiation. However, at 100 Gy, higher levels of phenolic compounds accumulated along chloroplast membranes: this accompanied an increase in number of chloroplasts. The reduced content of chlorophylls at high levels of radiation was associated with reduced size of the chloroplasts. All data are discussed in terms of the possible role of cellular modifications in the maintenance of high radioresistance and photosynthetic efficiency.

Microwave and micronization treatments affect dehulling characteristics and bioactive contents of dry beans (Phaseolus vulgaris L.).

BACKGROUND: Heat pretreatment is considered the first step in grain milling. This study therefore evaluated microwave and micronization heat treatments in improving the dehulling characteristics, phenolic composition and antioxidant and α-amylase activities of bean cultivars from three market classes. RESULTS: Heat treatments improved dehulling characteristics (hull yield, rate coefficient and reduced abrasive hardness index) depending on bean cultivar, whereas treatment effects increased with dehulling time. Micronization increased minor phenolic components (tartaric esters, flavonols and anthocyanins) of all beans but had variable effects on total phenolic content depending on market class. Microwave treatment increased α-amylase inhibitor concentration, activity and potency, which were strongly correlated (r² = 0.71, P < 0.0001) with the flavonol content of beans. Heat treatment had variable effects on the phenolic composition of bean hulls obtained by abrasive dehulling without significantly altering the antioxidant activity of black and pinto bean hulls. Principal component analysis on 22 constituents analyzed in this study demonstrated the differences in dehulling characteristics and phenolic components of beans and hulls as major factors in segregating the beneficial heat treatment effects. CONCLUSION: Heat treatment may be useful in developing novel dietary fibers from beans with variable composition and bioactivity with a considerable range of applications as functional food ingredients.

Growth alteration and leaf biochemical responses in Phaseolus vulgaris exposed to different doses of ionising radiation.

Ionising radiation may have different effects on plant metabolism, growth and reproduction, depending on radiation dose, plant species, developmental stage and physiological traits. In this study, exposure of dwarf bean plants to different doses of X-rays (0.3, 10, 50, 100 Gy) was investigated with a multidisciplinary approach consisting of morphological, ecophysiological and biochemical analysis. Both mature and young leaves still growing during the X-rays exposure were compared with non-irradiated control leaves. In particular, leaf expansion, leaf anatomy and functional traits, as well as photosynthetic pigment content and Rubisco expression were analysed. Moreover, the activity of poly(ADP-ribose) polymerase (PARP) was also measured as an indicator of radiation-induced DNA damage. Our data showed that leaf growth is affected by high levels of radiation and demonstrate that mature leaves are more radio-resistant than young leaves, which experience severe dose-dependent changes in leaf functional traits. In particular, young leaves exhibited a reduction of area and an increase in specific mass and dry matter content, as well as a decline in Rubisco activity. Moreover, they showed elevated PARP activity and an increase in phenolic compounds in wall cells if compared with mature leaves. Both of these strategies have been interpreted as a way to help developing leaves withstand irradiation.

[Effect of the increasing levels of soil radioactive pollution on the biochemical composition of plants].

The study was undertaken to study a relationship between the changes of some parameters of the biochemical and mineral composition of different plants, such as rape, pods, and lentil, and the levels of soil radiation pollution, by using the conventional methods. Radioactive pollution of dark-grey forest soils was found to cause a change in the biochemical composition of plant seeds even at the level of cesium 137 (137Cs) within the present temporary permissible levels (TPL) (600 Bq/kg): there were elevated concentrations of salts of potassium, phosphorus, ammonia nitrogen, catechols, sucrose, and some amino acids. With the radioactive cesium level exceeding the TPL, biochemical changes in the seeds depended on the species of the plants: in the rape seeds, the additional formation of sucrose and amino acids continued, but less intensively than with its lower radiation; the pod beans were significantly positively correlated with the increasing amounts of catechols.

Photoelectrocatalytic decolorization and degradation of textile effluent using ZnO thin films.

Zinc oxide (ZnO) thin films have been successfully deposited onto fluorine doped tin oxide coated glass at substrate temperature of 400 °C and used as electrode in photoelectrocatalytic reactor. The untreated textile effluent was circulated through photoelectrocatalytic reactor under UVA illumination for the decolorization and degradation. Textile effluent was decolorized by 93% within 3h at room temperature with significant reduction in COD (69%). High performance liquid chromatography (HPLC) and Fourier transform infrared spectroscopy (FTIR) analysis of samples before and after decolorization confirmed the degradation of dyes molecules from textile effluent into simpler oxidizable products. Phytotoxicity study revealed reduction in toxic nature of textile effluent after treatment.

Light at night resynchronizes the evening-phased rhythms of TOC1 and ELF4 in Phaseolus vulgaris.

Circadian clocks regulate the adaptation of the organisms' physiology to the environmental light-dark cycles. Photic resetting of the clock differs among plant species. In Arabidopsis thaliana, morning-phased genes are not responsive to light signals at night, while in Phaseolus vulgaris, morning-phased genes are responsive to light at trough phases that are reached during the night. In order to explore this further, in this work we investigated the light-responsiveness at night of two P. vulgaris evening phased genes, the orthologs of TOC1 and ELF4. Our results demonstrate that the oscillation of their expression is symphasic under all applied photic conditions. Thus, under photoperiod peak phases are obtained in the evening (LD 12:12) or early at night (LD 6:18). Light application at the beginning of the night under LD 6:18 results in a phase shift of the PvTOC1 and PvELF4 oscillation, while at the end of the night the phase remains unchanged. Moreover, when light is applied at the narrow time window of the peak phase, a significant transient increase in the expression of both PvTOC1 and PvELF4 is caused. These results indicate that, depending on the plant species, evening-phased genes may also participate in the resetting of the circadian clock machinery by light.

Novel vitamin E forms in leaves of Kalanchoe daigremontiana and Phaseolus coccineus.

In the present study, we isolated novel tocochromanols from green leaves of Kalanchoe daigremontiana and primary leaves of etiolated seedlings of Phaseolus coccineus that were identified as ß-, γ-, and δ-tocomonoenols with unsaturation at the terminal isoprene unit of the side chain. The content of γ-tocomonoenol in leaves of etiolated bean increased gradually with the age of seedlings, reaching 50% of the γ-tocopherol level in 40-day-old plants. The content of this compound in leaves was increased by short illumination of etiolated plants and by addition of homogentisic acid, a biosynthetic precursor of tocopherols. These data indicated that γ-tocomonoenol is synthesized de novo from homogentisic acid and tetrahydro-geranylgeraniol diphosphate, a phytol precursor. Based on these results, a biosynthetic pathway of tocomonoenols is proposed.

Acceleration of germination and early growth of wheat and bean seedlings grown under various magnetic field and osmotic conditions.

Magnetic field (MF) can have different effects on plant metabolism depending on its application style, intensity, and environmental conditions. This study reports the effects of different intensities of static MF (4 or 7 mT) on seed germination and seedling growth of bean or wheat seeds in different media having 0, 2, 6, and 10 atmosphere (atm) osmotic pressure prepared with sucrose or salt. The germination percentages of the treated seeds were compared with untreated seeds germinated in different osmotic pressure during 7 days of incubation. The application of both MFs promoted the germination ratios of bean and wheat seeds, regardless of increasing osmotic pressure of sucrose or salt. Growth data measured on the 7th day showed that the treated plants grew faster than control. After 7 days of incubation, the mean length of treated seedlings was statistically higher than control plants in all the media. The greatest germination and growth rates in both plants were from the test groups exposed to 7 mT MF. Strikingly, effects of static MF on germination and growth increased positively with increasing osmotic pressure or salt stress compared to their respective controls. On the other hand, MF application caused an increase in dry biomass accumulation of root and shoots of both seedlings; however, this effect was found statistically important in all the conditions for wheat but not for bean, in general.

On the resilience of nitrogen assimilation by intact roots under starvation, as revealed by isotopic and metabolomic techniques.

The response of root metabolism to variations in carbon source availability is critical for whole-plant nitrogen (N) assimilation and growth. However, the effect of changes in the carbohydrate input to intact roots is currently not well understood and, for example, both smaller and larger values of root:shoot ratios or root N uptake have been observed so far under elevated CO(2). In addition, previous studies on sugar starvation mainly focused on senescent or excised organs while an increasing body of data suggests that intact roots may behave differently with, for example, little protein remobilization. Here, we investigated the carbon and nitrogen primary metabolism in intact roots of French bean (Phaseolus vulgaris L.) plants maintained under continuous darkness for 4 days. We combined natural isotopic (15)N/(14)N measurements, metabolomic and (13)C-labeling data and show that intact roots continued nitrate assimilation to glutamate for at least 3 days while the respiration rate decreased. The activity of the tricarboxylic acid cycle diminished so that glutamate synthesis was sustained by the anaplerotic phosphoenolpyruvate carboxylase fixation. Presumably, the pentose phosphate pathway contributed to provide reducing power for nitrate reduction. All the biosynthetic metabolic fluxes were nevertheless down-regulated and, consequently, the concentration of all amino acids decreased. This is the case of asparagine, strongly suggesting that, as opposed to excised root tips, protein remobilization in intact roots remained very low for 3 days in spite of the restriction of respiratory substrates.