Ladaslav Sodek

Glycolysis and the tricarboxylic acid cycle are linked by alanine aminotransferase during hypoxia induced by waterlogging of Lotus japonicus.

The role of nitrogen metabolism in the survival of prolonged periods of waterlogging was investigated in highly flood-tolerant, nodulated Lotus japonicus plants. Alanine production revealed to be a critical hypoxic pathway. Alanine is the only amino acid whose biosynthesis is not inhibited by nitrogen deficiency resulting from RNA interference silencing of nodular leghemoglobin. The metabolic changes that were induced following waterlogging can be best explained by the activation of alanine metabolism in combination with the modular operation of a split tricarboxylic acid pathway. The sum result of this metabolic scenario is the accumulation of alanine and succinate and the production of extra ATP under hypoxia. The importance of alanine metabolism is discussed with respect to its ability to regulate the level of pyruvate, and this and all other changes are discussed in the context of current models concerning the regulation of plant metabolism.

Alanine metabolism and alanine aminotransferase activity in soybean (Glycine max) during hypoxia of the root system and subsequent return to normoxia

Abstract Alanine is one of the main products of anaerobic metabolism in plants and its formation believed to involve alanine aminotransferase (AlaAT), an enzyme induced under such conditions. Effects of hypoxia on roots of soybean ( Glycine max ) and the subsequent return to normoxia were studied with regard to alanine metabolism in the roots and its transport in the xylem, together with the role of AlaAT in this process. Non-nodulated plants were grown in a hydroponic system with nutrient solution containing nitrate as source of N. Subjecting the root system to hypoxia for up to 120 h led to large increases in alanine in both roots (4–44 mol%) and xylem sap (0.5–45 mol%). The increase in the roots preceded that of the xylem, consistent with alanine formation in the roots being the source of the increase in the xylem. On return to normoxia, following 120 h under hypoxia, the levels of alanine returned rapidly to pre-hypoxic levels. AlaAT activity of the roots more than doubled over the 120 h period of hypoxia. However, the bulk of this increase in activity took place after 48 h of hypoxia, long after the large increases in alanine had initiated. It is suggested that induction of AlaAT activity in soybean roots under hypoxia has only a limited role in the increase of alanine formation but may be responsible for the rapid recovery of alanine to pre-hypoxic levels on return to normoxia.

Waterlogging affects nitrogen transport in the xylem of soybean

Abstract Transfer of nodulated and non-nodulated plants grown in vermiculite to hydroponic culture without soil was used to study waterlogging and nitrogen transport in the xylem of soybean. Non-aeration, aeration or aeration with nitrogen gas were used to obtain different levels of oxygen in the culture solutions. Ureides, the principal form of nitrogen transport in nodulated plants, were considerably reduced in waterlogged plants or after transfer to water-culture, especially when not aerated or aerated with nitrogen gas. Aeration of the water-culture following a period of non-aeration allowed some recovery of ureides, as did the return of plants to drained vermiculite. Although smaller changes in the total amino acid fraction were observed for the different treatments, marked changes occurred in the composition depending on the treatment imposed. A high proportion of asparagine and low glutamine characterised non-nodulated plants grown on nitrate, or nodulated plants subsequently fed nitrate. A higher level of glutamine and lower level of asparagine characterised nodulated plants dependent on nitrogen fixation. High levels of aspartic acid characterised plants transferred to water-culture with aeration, especially in N-deficient solution, while alanine and serine were very prominent in non-aerated or hypoxic water-culture. These changes also occurred in non-nodulated plants and plants kept in vermiculite in a flooded condition. Some of the changes in transport were accompanied by similar changes in the free amino acid fraction of the roots. It is suggested that an alteration in asparagine metabolism may underlie the changes in amino acid transport in the xylem associated with waterlogging.

Analysis of alanine aminotransferase in various organs of soybean (Glycine max) and in dependence of different nitrogen fertilisers during hypoxic stress

Alanine aminotransferase (AlaAT) catalyses the reversible conversion of pyruvate and glutamate into alanine and oxoglutarate. In soybean, two subclasses were identified, each represented by two highly similar members. To investigate the role of AlaAT during hypoxic stress in soybean, changes in transcript level of both subclasses were analysed together with the enzyme activity and alanine content of the tissue. Moreover, the dependency of AlaAT activity and gene expression was investigated in relation to the source of nitrogen supplied to the plants. Using semi-quantitative PCR, GmAlaAT genes were determined to be highest expressed in roots and nodules. Under normal growth conditions, enzyme activity of AlaAT was detected in all organs tested, with lowest activity in the roots. Upon waterlogging-induced hypoxia, AlaAT activity increased strongly. Concomitantly, alanine accumulated. During re-oxygenation, AlaAT activity remained high, but the transcript level and the alanine content decreased. Our results show a role for AlaAT in the catabolism of alanine during the initial period of re-oxygenation following hypoxia. GmAlaAT also responded to nitrogen availability in the solution during waterlogging. Ammonium as nitrogen source induced both gene expression and enzyme activity of AlaAT more than when nitrate was supplied in the nutrient solution. The work presented here indicates that AlaAT might not only be important during hypoxia, but also during the recovery phase after waterlogging, when oxygen is available to the tissue again.

N-stress alters aspartate and asparagine levels of xylem sap in soybean

Abstract The influence of N-stress on the transport of amino acids in the xylem sap was studied for nodulated and non-nodulated soybean plants. The transfer of non-nodulated plants cultivated with nitrate or of nodulated plants dependent exclusively on N 2 fixation to hydroponics with N-free nutrient solution (N-stress), led to a pronounced increase in aspartate and fall in asparagine (Asn) in the xylem sap. Such a change was also observed during the ‘N-hunger’ phase of the growth cycle of nodulated plants, characterized by the transient yellowing of leaves and coinciding with the initial stages of nodule development. In this case, aspartate increased to around 68% of the xylem amino acids while Asn, normally the most abundant amino acid, fell to 10%. The changes were reversible on recovery of plants from N-stress. In the case of nodulated plants, the fall in xylem Asn during N-stress and its subsequent rise during recovery closely followed Asn synthetase activity in the nodule, while aspartate produced an inverse relationship. Aspartate was prominent in the phloem, independent of the treatment. The possibility that the phenomenon is related to the metabolism of aspartate recycled through the root system is discussed.

Inoculação de Azospirillum amazonense em dois genótipos de milho sob diferentes regimes de nitrogênio

The adaptability of maize genotypes to environments where nutrients are not readily available may be related to an adaptation to the predominance of the soil nitric and ammoniacal forms of N and to the association with beneficial microorganisms such as diazotrohpic bacteria and/or plant growth promoters. The objective of this study was to evaluate the response of two maize intervarietal hybrids to different nitrogen doses and forms as well as the effect of inoculation with A. amazonense. The experiment was carried out in a greenhouse with pots filled with vermiculite and Hoagland nutrient solution. A three-factor randomized complete block design was used with treatments arranged in a factorial scheme represented by: H1 (Carioca x Eldorado) and H2 (Palha Roxa ES x Sol da Manha) intervarietal hybrids; plants inoculated or not with A. amazonense; and three nitrogen proportions and doses: 126 mg week-1 of N (75 % NH4+ : 25 % NO3-); 126 mg week-1 of N (25 % NH4+ : 75 % NO3-); and 12.6 mg week-1 of N (50 % NH4+ : 50 % NO3-). After 25 days of growth the plant roots and shoots were separated for the determination of dry matter production, total N and P content, nitrate reductase and glutamine synthetase activity and total soluble sugars. The H1 hybrid, considered more efficient in preliminary field evaluations, produced greater dry matter and was more efficient in N and P utilization. In plants that received the highest N dose, independently of the predominance of the ammoniacal or nitric forms, the N (roots and shoots) and P (shoots) accumulation as well as the P utilization index were higher. Under the predominance of the ammonium-N plants grew more and the glutamine synthetase activity was increased, while the concentration of root total soluble sugars was lowered. The inoculation with A. amazonense resulted in higher root dry matter production and N accumulation.

Nitrogen metabolism and translocation in soybean plants subjected to root oxygen deficiency

Although nitrate (NO3(-)) but not ammonium (NH4(+)) improves plant tolerance to oxygen deficiency, the mechanisms involved in this phenomenon are just beginning to be understood. By using gas chromatography-mass spectrometry, we investigated the metabolic fate of (15)NO3(-) and (15)NH4(+) in soybean plants (Glycine max L. Merril cv. IAC-23) subjected to root hypoxia. This stress reduced the uptake of (15)NO3(-) and (15)NH4(+) from the medium and decreased the overall assimilation of these nitrogen sources into amino acids in roots and leaves. Root (15)NO3(-) assimilation was more affected by hypoxia than that of (15)NH4(+), resulting in enhanced nitrite and nitric oxide release in the solution. However, (15)NO3(-) was translocated in substantial amounts by xylem sap and considerable (15)NO3(-) assimilation into amino acids also occurred in the leaves, both under hypoxia and normoxia. By contrast, (15)NH4(+) assimilation occurred predominantly in roots, resulting in accumulation of mainly (15)N-alanine in this tissue during hypoxia. Analysis of lactate levels suggested higher fermentation in roots from NH4(+)-treated plants compared to the NO3(-) treatment. Thus, foliar NO3(-) assimilation may be relevant to plant tolerance to oxygen deficiency, since it would economize energy expenditure by hypoxic roots. Additionally, the involvement of nitric oxide synthesis from nitrite in the beneficial effect of NO3(-) is discussed.

Nitrogen stress and the expression of asparagine synthetase in roots and nodules of soybean (Glycine max)

The difficulty of assaying asparagine synthetase (AS) (EC 6.3.5.4) activity in roots of soybean has been circumvented by measuring expression of the AS genes. Expression of three soybean asparagine synthetase (SAS) genes (SAS1, SAS2 and SAS3) was observed in roots of non-nodulated soybean plants cultivated on nitrate. Expression of these genes was reduced to very low levels within days after submitting the plants to a N-free medium. The subsequent return to a complete medium (containing nitrate) restored expression of all three AS genes. Roots of nodulated plants, where symbiotic nitrogen fixation was the exclusive source of N (no nitrate present), showed very weak expression of all three AS genes, but on transfer to a nitrate-containing medium, strong expression of these genes was observed within 24 h. In nodules, all three genes were expressed in the absence of nitrate. Under conditions that impair nitrogen fixation (nodules submerged in aerated hydroponics), only SAS1 expression was reduced. However, in the presence of nitrate, an inhibitor of N(2) fixation, SAS1 expression was maintained. High and low expressions of AS genes in the roots were associated with high and low ratios of Asn/Asp transported to the shoot through xylem. It is concluded that nitrate (or one of its assimilatory products) leads to the induction of AS in roots of soybean and that this underlies the variations found in xylem sap Asn/Asp ratios. Regulation of nodule AS expression is quite different from that of the root, but nodule SAS1, at least, appears to involve a product of N assimilation rather than nitrate itself.

Involvement of nitrite in the nitrate-mediated modulation of fermentative metabolism and nitric oxide production of soybean roots during hypoxia.

It is widely accepted that nitrate but not ammonium improves tolerance of plants to hypoxic stress, although the mechanisms related to this beneficial effect are not well understood. Recently, nitrite derived from nitrate reduction has emerged as the major substrate for the synthesis of nitric oxide (NO), an important signaling molecule in plants. Here, we analyzed the effect of different nitrogen sources (nitrate, nitrite and ammonium) on the metabolic response and NO production of soybean roots under hypoxia. Organic acid analysis showed that root segments isolated from nitrate-cultivated plants presented a lower accumulation of lactate and succinate in response to oxygen deficiency in relation to those from ammonium-cultivated plants. The more pronounced lactate accumulation by root segments of ammonium-grown plants was followed by a higher ethanol release in the medium, evidencing a more intense fermentation under oxygen deficiency than those from nitrate-grown plants. As expected, root segments from nitrate-cultivated plants produced higher amounts of nitrite and NO during hypoxia compared to ammonium cultivation. Exogenous nitrite supplied during hypoxia reduced both ethanol and lactate production and stimulated cyanide-sensitive NO emission by root segments from ammonium-cultivated plants, independent of nitrate. On the other hand, treatments with a NO donor or a NO scavenger did not affect the intensity of fermentation of soybean roots. Overall, these results indicate that nitrite participates in the nitrate-mediated modulation of the fermentative metabolism of soybean roots during oxygen deficiency. The involvement of mitochondrial reduction of nitrite to NO in this mechanism is discussed.

Effect of oxygen deficiency on nitrogen assimilation and amino acid metabolism of soybean root segments.

Plants submitted to O2 deficiency present a series of biochemical modifications, affecting overall root metabolism. Here, the effect of hypoxia on the metabolic fate of 15N derived from 15NO3−, 15NO2− and 15NH4+ in isolated soybean root segments was followed by gas chromatography–mass spectrometry, to provide a detailed analysis of nitrogen assimilation and amino acid biosynthesis under hypoxia. O2 deficiency decreased the uptake of the nitrogen sources from the solution, as ratified by the lower 15NO3− and 15NH4+ enrichment in the root segments. Moreover, analysis of endogenous NO2− and 15NH4+ levels suggested a slower metabolism of these ions under hypoxia. Accordingly, regardless of the nitrogen source, hypoxia reduced total 15N incorporation into amino acids. Analysis of 15N enrichment patterns and amino acid levels suggest a redirecting of amino acid metabolism to alanine and γ-aminobutyric acid synthesis under hypoxia and a differential sensitivity of individual amino acid pathways to this stress. Moreover, the role of glutamine synthetase in nitrogen assimilation both under normoxia and hypoxia was ratified. In comparison with 15NH4+, 15NO2− assimilation into amino acids was more strongly affected by hypoxia and NO2− accumulated in root segments during this stress, indicating that nitrite reductase may be an additional limiting step. NO2− accumulation was associated with a higher nitric oxide emission. 15NO3− led to much lower 15N incorporation in both O2 conditions, probably due to the limited nitrate reductase activity of the root segments. Overall, the present work shows that profound alterations of root nitrogen metabolism occur during hypoxic stress.