Walnut trees have been shown to be very sensitive to abiotic stresses, especially to soil hypoxia consequence of soil flooding or waterlogging. The aim of study was to search for correlation between polyphenols and tolerance to flooding stress. Two-three years old walnut trees, Juglans regia, J. nigra and interspecific hybrids, grown in pots were subjected to soil flooding in summer at full leaves expansion. In high sensitive J. regia trees, when flooded, the net CO2 assimilation rapidly decreased and 3-4 days of flooding were enough to block the recovery when soils was drained. In walnut hybrid (J. nigra x J. regia) and less sensitive J. regia genotypes, a longer resistance was observed; after 13 days of treatment leaves culd still be green and trees can recovered till to the normal photosynthesis when drained. J. nigra when flooded reduced net photosynthetic rate but maintained leaves without damages and with the capacity to recover CO2 assimilation. The data showed resistance of J. nigra and hybrid trees and of some J. regia genotypes to the stress. The HPLC analyses of polyphenols showed a modification of the patterns during the stress. The plants less tolerant to hypoxia have higher content of polyphenols distributed in a lot of compounds. Trees more tolerant, J. nigra, showed a very simple HPLC pattern. All samples contained juglone, more in less tolerant genotypes. Hydroxy juglone glucoside was detectable in all genotypes, but only in low quantity, it increased in trees with high resistance to hypoxia and decreased in J. regia. Less flooded tolerant J. regia genotypes have higher polyphenols and juglone content. The metabolism of hydroxyl juglone glucoside could be involved in a mechanism of juglone detoxification during the hypoxia to give stress tolerance.

Central Asia where walnut was grown since Oligocene was recognized as a center of its origin and distribution. Now in Uzbekistan, J. regia stands occupy three areas geographically isolated from each other by more than 250 km: Western Tien-Shan, Nurata and Hissar. Survey expeditions were performed mainly in natural thickets, half-wild plantations and single tree in "dehkan" gardens. Morphological descriptors, biochemical and genetic markers were used to evaluate the biodiversity of walnut for conservation and developmental use. Trees from 20-30 years up to over-matured (300-500 years old) were considered. A variety in nuts was recorded: as to weight (5 up to 25 g) and other morphological and biochemical attributes. Lipid in kernels contain PUFA for more of 80%. Vitamin E was also extracted in the range 0.1-1.5 mg/g of kernel. Using 14 SSR primers, DNA fragments of different size were obtained with variable number of alleles in the walnut trees sampled. Spatial genetic structure analysis divided the Uzbek trees into three groups that partially corresponded to the growing area of trees. Studies in progress preliminary indicated that the genetic structure of Uzbek walnut partially overlapped to trees from outside the country. The ancestors of Uzbek trees could share the origin with the nearby forests, now belonging to other countries, but in the past together identified as Turkestan, and focal point of the Silk Roads for East-West trade and migrations.

The natural area of walnut in Central Asia includes the territory of Kyrgyzstan, Tajikistan and Uzbekistan. There the walnut has been grown since Oligocene times. The walnut forests in these countries are the centre of origin of walnut around the world. Walnut forests in Uzbekistan are widespread in an area of about 4000 hectares. They occupy areas with optimum forest growing conditions on slopes of mountains of Western Tien-Shan and Pamir-Alai at a height from 800 up to 2300 m above sea level. The Bostanlyk District of Tashkent Region is related to the zone of the densest concentration of walnut plantations. Surveys carried out in 2007-2009 in Uzbekistan walnut forests in a part of Western Tien-Shan territory showed that Juglans regia is widespread on ranges as separate, small populations on small river inflow and slopes of Ugam, Pskem, Chatkal. From the complex of investigated biomorphological attributes of trees, forms can be classified as related to breeding categories. From the study area selected trees in Charvak and Chatkal plots deserve special attention. Fruits of the surveyed trees showed a wide variability in size and shape as well as in the nutrient content. Oil, fatty acids, proteins, vitamin E were analysed. Several forms can be added as potential candidates for reproduction and establishment of a collection-mother production plantations and to become named cultivars.

In the current climate change scenarios, the choice of seed sources is one of the main factors affecting the establishment and productivity of plantations of forest trees. Juglans regia L. (walnut) is one of the more valuable hardwood species since it could provide high quality timber and fruits. The aim of this study was to search peculiar Italian walnut provenances as putative biodiversity sources for seed orchards and to establish new hardwood plantations. A multidisciplinary approach, integrating molecular markers (ISSR), seed morphological traits (equatorial and polar diameter, shape, dry weight) and fruit composition (total oil, fatty acids, tocopherol) was applied to analyze samples collected in ten sites (three in Campania and seven in Abruzzo regions) and samples of four varieties (two from Southern and two from Northern Italy). Eleven selected ISSR primers exhibited a strong ability to discriminate walnut provenances.

Ferredoxin-dependent glutamate synthase (Fd-Gogat; EC 1.4.7.1) in leaf and root plastids is the last enzyme involved in the pathway of nitrate assimilation in higher plants. Arabidopsis thaliana expresses two different genes: the first, light regulated, specific of green tissues and the second expressed in other tissues. In this work, we investigated whether in our clone, OsGog2 AC Y12595, this gene is up-regulated by light or it is expressed under darkness. Fd-Gogat specific activity, protein and mRNA increased after light treatment in rice shoots. In roots, the activity and the protein content remained constant, whereas the mRNA is repressed by light treatment. The results obtained using a specific probe, situated in the 3′ untranslated region of the OsGog2 cDNA, indicated that OsGog2 gene is up-regulated by light and that its expression is tissue specific and suggested that a dark expressed Fd-Gogat gene could be present in rice similarly as in Arabidopsis.

In the seed, arginine, glutamate and alanine dominated cotyledonary free amino acid pool, whereas alanine was the main component of the embryo free amino acid pool. Seed germination resulted in marked changes in the metabolism of free amino acids present in cotyledons. In one month of seedlings subjected to nitrogen autotrophic growth, cotyledons were characterized by high concentrations of free arginine and citrulline. The relative high percentage of arginine and citrulline characterized not only the cotyledonary tissue, but all the tissues of one-month-old seedlings, especially the embryonic axis, tap root and stem. In these tissues, which have a nutrient storage function, the concentrations of citrulline and arginine made up about 80% of the total free amino acid, whereas in fine roots and leaves, characterized more as sink tissues, the relative percentages of citrulline and arginine were less than the 30%. In young walnut trees (2 years old), at spring regrowth, the concentration of free amino acids and in particular the concentrations of citrulline, arginine and proline present in woody tissues were dependent on the availability of nitrogen in soil, while the protein content of these tissues did not show significantly dependence. The xylem transport of free amino acids during a nitrogen autotrophic growth (germination) involved mainly citrulline. Its relative concentration in the xylem sap was greater than 50% of the total free amino acid translocated. Citrulline functions as a preferential compound by which nitrogen (amino acids) is translocated also during spring regrowth or after stress recovery; i.e. at all physiological stages where mobilization of nitrogen stored compounds occurs.

Anoxic stress induces a strong change in sugar, protein, and amino acid metabolism in higher plants. Sugars are rapidly consumed through the anaerobic glycolysis to sustain energy production. Protein degradation under anoxia is a mechanism to release free amino acids contributing in this way to maintaining the osmotic potential of the tissue under stress. Among free amino acids, a particular role is played by glutamic acid, being a precursor of some characteristic compounds of the anaerobic metabolism (alanine, γ-aminobutyric acid, and putrescine). The glutamine synthetase/glutamate synthase cycle contributes to ammonia reassimilation and primary assimilation of nitrate, and resynthesizes constantly glutamate for the synthesis of other compounds. Some polypeptides involved in these pathways are expressed under anoxia. The importance of amino acid metabolism for the response to anaerobic stress is discussed.

We investigated the composition of free amino acids in walnut (Juglans regia L.) seeds (embryo and cotyledons). We also examined xylem transport of free amino acids in young seedlings grown in the absence of external nutrients. A relatively high concentration of free alanine was found in seed tissue, whereas a relatively high concentration of citrulline was detected in young seedlings. Citrulline was the main free amino acid transported in the xylem to stem and leaves. The negligible presence of citrulline, a non-protein amino acid, in the kernel and its presence in high concentrations in all of the tissues of young seedlings, including cotyledons, embryonic axis, taproot and stem, suggest that citrulline is synthesized during walnut germination. We conclude that citrulline plays an important role in nitrogen translocation during walnut germination.

Few data on amino acid presence and composition in xylem sap of trees have been reported; the studies were mainly focused on the seasonal variation and the phenological stages of the trees. The aim of the present work has been to study the influence of water stresses, either drought and flooding, during the vegetative stage of walnut tree on the composition of amino acids in leaf sap. During the all vegetative season, July-September, in control trees well watered and with well drained soil, the amino acids composition remain without significant difference. In the leaf sap the concentration of amino acids ranged between 130-200 muM and it is 0.3 % only of total free amino acids in leaves. Glutamine account for about the 40-50% of the total amino acids. Glutamine has been reported to be the dominant one in the xylem of willow, poplar and black locust trees. During stress condition increase in concentration to the mM level for drought trees and over 10 mM for flooded one is the first evident effect. Glutamine remains one of the main component but the % increase in aspartate and glutamate in drought and of GABA in soil hypoxia stress are the main changes. After removing the stresses condition, the citrulline become one of main amino acids. The increase in leaf sap of free amino acids during the stress may be consequence of different causes: protein hydrolysis, synthesis or conversion. However, considering that few amino acids accounting for over 80% of the total, or more simple the release of free amino acids present in the cells, can occur.

An enhanced efficiency of absorption and utilization of nitrogen fertilizers by maize cultivars should be an important goal in view of a sustainable agriculture respecting the environment. In this study, we evaluated the variability in traits of nitrate absorption and assimilation (Km, Vmax, glutamine synthetase (GS) and ferredoxin-dependent glutamate synthase (FdGOGAT) activities) and the associa- tion among them using 40 inbred lines of maize. Our results showed that wide differences exist for all the traits examined. Some statistically significant associations between the biochemical traits were deter- mined. The best inbred lines for uptake of nitrate (higher Vmax/Km ratio) and enzyme activities were identified. In particular, some inbred lines belonging to the Lancaster group or selected from ancient Italian maize populations exhibited high Vmax/Km ratio, whereas Stiff Stalk Synthetic inbred lines showed elevated enzyme activities for GS and Fd-GOGAT. Additional index terms: glutamate synthase, glutamine syntetase, inbred lines, nitrate uptake, nitrogen assimilation.

Anaerobic nitrate assimilation was studied in rice seedlings. Incorporation of (NO3)-N-15 into amino acids was detected both in 3-day-old aerobic rice seedlings subjected for 24 h to anaerobic conditions and in anaerobically germinated seedlings. The combined incorporation of N-15 into glutamate, glutamine, and alanine accounted for 89% and 85% of its total incorporation into the coleoptile and root, respectively, in aerobic seedlings transferred to anoxia, while other amino acids were heavily labeled in anaerobically germinated seedlings. The specific activities of the enzymes involved in the nitrate assimilation (nitrate reductase, NR; nitrite reductase, NiR;,glutamine synthetase, GS; ferredoxin-dependent glutamate synthase, Fd-GOGAT; Fd-NADP oxidoreductase, FNR) were found in the anaerobic coleoptile. All these activities, with the exception of GS, increased in the presence of exogenous nitrate (5 mM). The Northern blot analysis revealed the presence of mRNAs for NR, NiR, cytosolic GS, Fd-GOGAT, and root FNR. Nitrate did not exert any effect on the transcript level of cytosolic GS. These data indicate that, in rice, the nitrate assimilation pathway is operative under anoxia.

Woody species vary in their ability to tolerate flooding. In sensitive plants when soil is flooded, the reduction of root nutrient and water uptake, due to root mortality or hypoxic root metabolism, are clearly observable on the foliage. The flood tolerance in woody species has been correlated with the capacity of the plants to produce adventitious roots to compensate the decay of original roots.Walnut (Juglans regia) tree is known to be very sensitive either to drought then to flooding. Of interest is the search of characters and parameters useful to select genotypes and/or phenotypes with the ability to survive in unfavorable conditions.Walnut trees, J. regia of different provenience, and interspecific hybrids J. regia x J. nigra and J. regia x J. major were subjected to waterlogging when full expansion leaves were reached. Physicochemical measures gave indication of decrease of net CO2 assimilation after 1 day of waterlogging in all genotypes tested. After 3 days, some J. regia varieties trees showed rapid desiccation and lost of all leaves and some plants clearly died. On other hand trees of a selected progeny of J. regia and of hybrids did not shown any visible apparent damaging symptoms till 10-12 days of anoxia stress treatment.Biochemical analyses of indole-acetic acid, abscisic acid, ethylene and ethanol content in leaves gave no significant difference between control and stress treated plants for the first two compounds, ethylene showed a rise of production that opposite the CO? assimilation. Ethanol, present in control plants, increased approaching the time of appearance of chlorosis and wilting of leaves.The data can confirm previous work that walnut trees have a low capacity to survive to soil anaerobiosis, but differences trough J. regia provenance and hybrid plants indicate the potentiality to select genotypes with more stress resistance capacity.

Rice, one of the few plant species adapted to growth in wetland conditions, is able to germinate in waterlogged soils promoting only the growth of a white coleoptile in order to reach the surface of the water, contact the atmosphere, and transfer oxygen to the seed, allowing subsequent growth of the radicle and leaf. In the anoxic cells of rice coleoptiles, an efficient alcoholic fermentation allows an elevated energy charge to be maintained. Significant RNA and protein syntheses including phosphorylation and glycosylation occur too. The cytoplasmic pH is maintained at a level far from acidosis. The anoxic growth of rice coleoptiles, essentially an elongation growth, is sustained by a high turgor pressure, with free amino acids and potassium as main components. Among the metabolic processes involved in the regulation of the elongation of rice coleoptiles, a crucial role is played by amino acid metabolism and the accumulation of putrescine, which is able to stimulate plasmalemma ATPase activity. Anaerobic elongation is also stimulated in the presence of 20% CO2 in the growth medium, inhibited by light and abscisic acid, unaffected by ethylene, and slightly promoted by auxin. The role of both metabolites and hormones along with environmental factors in maintaining cellular homeostasis and coleoptile elongation are reconsidered and discussed in Light of new data.

The pretreatment of rice roots for Ih in aerobic conditions with GTP gamma S or AlF4- induced a higher gamma-aminobutyrate (GABA) accumulation during 3 h of anoxia, while GDP beta S or GDP slightly inhibited this process. Pretreatment with the Ca2+-channel blocker ruthenium red (RR) or the CaM-antagonist trifluoperazine inhibited the GTP gamma S- or the AlF4--stimulated anaerobic GABA accumulation. At elevated RR concentrations, GABA accumulation was completely inhibited both in the absence and presence of AlF4-. Furthermore, western immunodetection with anti-a common antibody revealed a higher amount of G alpha-like proteins (30 and 28 kDa) in the presence of GTP gamma S or AlF4-. These data are discussed in relation to an involvement of G proteins in the transduction af an anaerobic signal.

This paper describes the synthesis and the expression of the enzymes assimilating nitrogen and their regulation by exogenous nitrate in the anaerobic rice (Oryza sativa L.) coleoptile. Two nitrate reductases (NR, EC 1.6.6.1), nitrite reductase (NiR, EC 1.7.7.1), cytosolic and plastidic glutamine synthetase (GS, EC 6.3.1.2) and ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) were detected after immunoprecipitation with specific antibodies followed by SDS-PAGE. The Northern blot analysis revealed the presence of mRNA for NIR, cytosolic GS and Fd-GOGAT. The addition of exogenous nitrate increased the synthesis and the expression of all these proteins with the exception of the GS polypeptides. The role of these proteins in the rice coleoptile during the anaerobic germination is discussed.

Ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) is the last enzyme involved in the pathway of nitrate assimilation in higher plants. This paper describes the synthesis and expression of the enzyme in anaerobic coleoptiles of rice (Oryza sativa L.) and its regulation by exogenous nitrate. The activity of Fd-GOGAT was strongly inhibited by cycloheximide between 4 and 9 d of anaerobic germination. The addition of nitrate slightly increased, in the first 5 h, the specific activity of Fd-GOGAT as well as the amount of a 160-kDa protein specifically immunoprecipitated with anti-Fd-GOGAT serum. Northern blot analysis, performed with a specific riboprobe, showed the presence of mRNA of the expected size and the inductive effect of nitrate. The role of Fd-GOGAT is discussed in relation to the anaerobic assimilation of nitrate by rice coleoptiles.

The coleoptiles of rice seedlings germinated under water bubbled with nitrogen (anaerobic conditions) and placed to grow in a horizontal position by turning the growth jar 90 degrees, showed a fast gravitropic responsiveness. This process was inhibited by IAA at 0.1 mM. The decrease in the osmotic potential induced in rice coleoptiles by exogenous auxin and the role of auxin in the anaerobic elongation and gravitropic response of rice coleoptiles are discussed.