Nitrogen use plasticity in response to light intensity in neotropical tree species of distinct functional groups.

Environmental conditions influence the use of different nitrogen (N) sources by plants. We hypothesized that an increase in light intensity favors the use of nitrate (NO3 - ) relative to ammonium (NH4 + ) by seedlings of neotropical tree species from different functional groups, that is, Cecropia pachystachya (a shade-intolerant species), Cariniana estrellensis (a shade-tolerant canopy species), and Guarea kunthiana (a shade-tolerant understory species). We analyzed the growth and N metabolism in seedlings simultaneously provided with NH4 + and NO3 - , under lower (LL) and higher (HL) light intensity. 15 N incorporation into amino acids was monitored after incubation with 15 N-labeled NH4 + or NO3 - . Under HL, all species showed decreased leaf area ratio, and increased growth, nitrate reductase activity and assimilated N content. Cecropia pachystachya increased the use of both N sources under HL, with substantial increases in 15 N-amino acids derived from 15 NO3 - (12.5- and 4.0-fold in roots and leaves, respectively) and 15 NH4 + (4.5- and 3.0-fold in roots and leaves, respectively). Guarea kunthiana showed the greatest plasticity in N use, as the assimilation of 15 NO3 - in roots and leaves increased substantially under HL (11.2- and 17.0-fold, respectively). Cariniana estrellensis increased the assimilation of 15 NH4 + in roots and 15 NO3 - in leaves under HL. Therefore, the responses of N use strategies to light intensity varied with the species according to their ecological characteristics.

The sugarcane ShMYB78 transcription factor activates suberin biosynthesis in Nicotiana benthamiana.

KEY MESSAGE: A sugarcane MYB present in the culm induces suberin biosynthesis and is involved both with fatty acid and phenolics metabolism. Few transcription factors have been described as regulators of cell wall polymers deposition in C4 grasses. Particularly, regulation of suberin biosynthesis in this group of plants remains poorly understood. Here, we showed that the sugarcane MYB transcription factor ShMYB78 is an activator of suberin biosynthesis and deposition. ShMYB78 was identified upon screening genes whose expression was upregulated in sugarcane internodes undergoing suberization during culm development or triggered by wounding. Agrobacterium-mediated transient expression of ShMYB78 in Nicotiana benthamiana leaves induced the ectopic deposition of suberin and its aliphatic and aromatic monomers. Further, the expression of suberin-related genes was induced by ShMYB78 heterologous expression in Nicotiana benthamiana leaves. ShMYB78 was shown to be a nuclear protein based on its presence in sugarcane internode nuclear protein extracts, and protoplast transactivation assays demonstrated that ShMYB78 activates the promoters of the sugarcane suberin biosynthetic genes ß-ketoacyl-CoA synthase (ShKCS20) and caffeic acid-O-methyltransferase (ShCOMT). Our results suggest that ShMYB78 may be involved in the transcriptional regulation of suberin deposition, from fatty acid metabolism to phenylpropanoid biosynthesis, in sugarcane internodes.

Enhanced Nitric Oxide Synthesis Through Nitrate Supply Improves Drought Tolerance of Sugarcane Plants.

Nitric oxide (NO) is an important signaling molecule associated with many biochemical and physiological processes in plants under stressful conditions. Nitrate reductase (NR) not only mediates the reduction of NO3 - to NO2 - but also reduces NO2 - to NO, a relevant pathway for NO production in higher plants. Herein, we hypothesized that sugarcane plants supplied with more NO3 - as a source of N would produce more NO under water deficit. Such NO would reduce oxidative damage and favor photosynthetic metabolism and growth under water limiting conditions. Sugarcane plants were grown in nutrient solution and received the same amount of nitrogen, with varying nitrate:ammonium ratios (100:0 and 70:30). Plants were then grown under well-watered or water deficit conditions. Under water deficit, plants exhibited higher root [NO3 -] and [NO2 -] when supplied with 100% NO3 -. Accordingly, the same plants also showed higher root NR activity and root NO production. We also found higher photosynthetic rates and stomatal conductance in plants supplied with more NO3 -, which was associated with increased root growth. ROS accumulation was reduced due to increases in the activity of catalase in leaves and superoxide dismutase and ascorbate peroxidase in roots of plants supplied with 100% NO3 - and facing water deficit. Such positive responses to water deficit were offset when a NO scavenger was supplied to the plants, thus confirming that increases in leaf gas exchange and plant growth were induced by NO. Concluding, NO3 - supply is an interesting strategy for alleviating the negative effects of water deficit on sugarcane plants, increasing drought tolerance through enhanced NO production. Our data also provide insights on how plant nutrition could improve crop tolerance against abiotic stresses, such as drought.

Products of anaerobic metabolism in waterlogged roots of soybean are exported in the xylem.

Waterlogging leads to hypoxia of the root system. Metabolic changes occur that enable the plant to tolerate the hypoxic stress. We investigated the export of organic acids, products of anaerobic metabolism, via xylem of waterlogged soybean (Glycine max) plants. Organic acids were quantified by GC-MS and their formation via aspartate metabolism investigated using [4-13C]aspartate. Elevated levels of malate were found together with variable amounts of other organic acids, notably lactate and succinate. Addition of [4-13C]aspartate to the medium led to isotopic enrichment of several organic acids in the xylem sap. Quantitatively, malate carried the highest amount of label among the organic acids. Labelling of succinate indicates its formation by reversal of the TCA-cycle from oxaloacetate. Since aspartate was a prominent amino acid of the phloem sap, it is suggested that this is an important source of malate exported in the xylem. The export of these organic acids will play the role of removing electrons from the hypoxic roots, representing an additional mechanism in the metabolic response to root hypoxia. Malate, normally considered an intermediate in succinate formation, is definitively a product of anaerobic metabolism.

Are phloem-derived amino acids the origin of the elevated malate concentration in the xylem sap following mineral N starvation in soybean?

MAIN CONCLUSION: A substantial increase in malate in the xylem sap of soybean subjected to mineral N starvation originates mainly from aspartate, a prominent amino acid of the phloem. A substantial increase in xylem malate was found when non-nodulated soybean plants were transferred to a N-free medium. Nodulated plants growing in the absence of mineral N and, therefore, dependent on symbiotic N2 fixation also contained elevated concentrations of malate in the xylem sap. When either nitrate or ammonium was supplied, malate concentrations in the xylem sap were low, both for nodulated and non-nodulated plants. Evidence was obtained that the elevated malate concentration of the xylem was derived from amino acids supplied by the phloem. Aspartate was a prominent component of the phloem sap amino acids and, therefore, a potential source of malate. Supplying the roots of intact plants with 13C-aspartate revealed that malate of the xylem sap was readily labelled under N starvation. A hypothetical scheme is proposed whereby aspartate supplied by the phloem is metabolised in the roots and the products of this metabolism cycled back to the shoot. Under N starvation, aspartate metabolism is diverted from asparagine synthesis to supply N for the synthesis of other amino acids via transaminase activity. The by-product of aspartate transaminase activity, oxaloacetate, is transformed to malate and its export accounts for much of the elevated concentration of malate found in the xylem sap. This mechanism represents a new additional role for malate during mineral N starvation of soybean, beyond that of charge balance.

Flooding of the root system in soybean: biochemical and molecular aspects of N metabolism in the nodule during stress and recovery.

Nitrogen fixation of the nodule of soybean is highly sensitive to oxygen deficiency such as provoked by waterlogging of the root system. This study aimed to evaluate the effects of flooding on N metabolism in nodules of soybean. Flooding resulted in a marked decrease of asparagine (the most abundant amino acid) and a concomitant accumulation of γ-aminobutyric acid (GABA). Flooding also resulted in a strong reduction of the incorporation of (15)N2 in amino acids. Nodule amino acids labelled before flooding rapidly lost (15)N during flooding, except for GABA, which initially increased and declined slowly thereafter. Both nitrogenase activity and the expression of nifH and nifD genes were strongly decreased on flooding. Expression of the asparagine synthetase genes SAS1 and SAS2 was reduced, especially the former. Expression of genes encoding the enzyme glutamic acid decarboxylase (GAD1, GAD4, GAD5) was also strongly suppressed except for GAD2 which increased. Almost all changes observed during flooding were reversible after draining. Possible changes in asparagine and GABA metabolism that may explain the marked fluctuations of these amino acids during flooding are discussed. It is suggested that the accumulation of GABA has a storage role during flooding stress.

Utilization of (15)NO3 (-) by nodulated soybean plants under conditions of root hypoxia.

Waterlogging of soils is common in nature. The low availability of oxygen under these conditions leads to hypoxia of the root system impairing the development and productivity of the plant. The presence of nitrate under flooding conditions is regarded as being beneficial towards tolerance to this stress. However, it is not known how nodulated soybean plants, cultivated in the absence of nitrate and therefore not metabolically adapted to this compound, would respond to nitrate under root hypoxia in comparison with non-nodulated plants grown on nitrate. A study was conducted with (15)N labelled nitrate supplied on waterlogging for a period of 48 h using both nodulated and non-nodulated plants of different physiological ages. Enrichment of N was found in roots and leaves with incorporation of the isotope in amino acids, although to a much smaller degree under hypoxia than normoxia. This demonstrates that nitrate is taken up under hypoxic conditions and assimilated into amino acids, although to a much lesser extent than for normoxia. The similar response obtained with nodulated and non-nodulated plants indicates the rapid metabolic adaptation of nodulated plants to the presence of nitrate under hypoxia. Enrichment of N in nodules was very much weaker with a distinct enrichment pattern of amino acids (especially asparagine) suggesting that labelling arose from a tissue source external to the nodule rather than through assimilation in the nodule itself.

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.

Nitrite decreases ethanol production by intact soybean roots submitted to oxygen deficiency: a role for mitochondrial nitric oxide synthesis?

Nitrate increases the tolerance of plants to hypoxia, although the mechanisms related to this beneficial effect are still unclear. Recently, we observed that cultivation of soybean plants with nitrate reduced hypoxic accumulation of fermentation end products by isolated root segments compared with the ammonium treatment. Interestingly, the same decrease in the intensity of fermentation was detected when ammonium-grown root segments were incubated with nitrite, suggesting the involvement of this anion in the nitrate-mediated modulation of fermentative metabolism. Here we extended these experiments to intact plants subjected to root hypoxia and observed similar effects of nitrate and nitrite in reducing root ethanol production, which indicates the physiological relevance of the in vitro results. In both experimental systems, nitrite stimulated nitric oxide emission by ammonium-grown roots to levels similar to that of nitrate-cultivated ones. The involvement of mitochondrial reduction of nitrite to nitric oxide in the root response to hypoxia is suggested.

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

Plants submitted to O(2) deficiency present a series of biochemical modifications, affecting overall root metabolism. Here, the effect of hypoxia on the metabolic fate of (15)N derived from (15)NO(3)(-), (15)NO(2)(-) and (15)NH(4)(+) 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. O(2) deficiency decreased the uptake of the nitrogen sources from the solution, as ratified by the lower (15)NO(3)(-) and (15)NH(4)(+) enrichment in the root segments. Moreover, analysis of endogenous NO(2)(-) and (15)NH(4)(+) levels suggested a slower metabolism of these ions under hypoxia. Accordingly, regardless of the nitrogen source, hypoxia reduced total (15)N incorporation into amino acids. Analysis of (15)N 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 (15)NH(4)(+), (15)NO(2)(-) assimilation into amino acids was more strongly affected by hypoxia and NO(2)(-) accumulated in root segments during this stress, indicating that nitrite reductase may be an additional limiting step. NO(2)(-) accumulation was associated with a higher nitric oxide emission. (15)NO(3)(-) led to much lower (15)N incorporation in both O(2) 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.

Recovery of nitrogen fixation after short-term flooding of the nodulated root system of soybean.

Nitrogen fixation of terrestrial legumes is strongly and rapidly diminished under flooding. Although recovery is possible with the formation of aerenchyma, information is scarce regarding recovery after draining following short-term flooding, before the appearance of morphological adaptations. This study used soybean (Glycine max) plants nodulated with Bradyrhizobium elkanii to determine xylem sap glutamine as an indication of nitrogen fixation activity during recovery from different periods of flooding. Xylem glutamine levels showed rapid recovery (within 90 min) following periods of flooding up to 4 h. Recovery was progressively slower after longer periods of flooding. After 48 h flooding very little recovery could be observed within the first 120 min after draining but recovery was possible within 48 h. Consistent with the changes in xylem glutamine, direct measurements of apparent nitrogenase activity carried out immediately on draining revealed rapid recovery after flooding for 1 h and slow recovery following 48 h of flooding. In the latter case, nitrogenase activity largely recovered 24 h after draining. Experiments with (15)N(2) incorporation into amino acids exported in the xylem sap revealed that glutamine was by far the most highly labelled amino acid in sap collected over the first 30 min of exposure to the isotope. This is conclusive evidence that xylem sap glutamine is an immediate product of N(2) fixation and export. The changes in xylem sap glutamine seen on flooding (decline) and after draining (recovery) can therefore be attributed to changes in nitrogenase activity. The data show that xylem sap glutamine is a useful means for assessing changes in nitrogenase activity, especially when the root system is submersed in water and activity cannot be measured directly.

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.

24-epibrassinolide restores nitrogen metabolism of pigeon pea under saline stress.

BACKGROUND: Several studies have shown that brassinosteroids attenuate the effects of salt stress. However, nothing is known about their effects on amino acid transport, nor the effects of these hormones on nitrate uptake under saline conditions. This study set out to determine the effects of 24-epibrassinolide, at concentrations of 10-7 M and 0.5 × 10-9 M, and clotrimazole (inhibitor of brassinosteroid synthesis), at 10-4 M, on nitrate uptake and metabolism in plants of C. cajan (L.) Millsp, cultivar C11, growing under salinity. The following aspects were analyzed: levels of proteins, amino acids, nitrate, nitrate reductase of roots and the composition of xylem sap amino acids. RESULTS: Salinity reduced the proportion of N-transport amino acids ASN (the major component), GLU, ASP and GLN. The effect of the hormone in reducing the adverse effects of salt was related to the reestablishment (totally or partially) of the proportions of GLU, ASN and GLN, transported in the xylem and to the small but significant increase in uptake of nitrate. Increased nitrate uptake, induced by 24- epibrassinolide, was associated with a higher activity of nitrate reductase together with greater levels of free amino acids and soluble proteins in roots of plants cultivated under saline conditions. CONCLUSION: The decline in several components of nitrogen metabolism, induced by salt, was attenuated by 24-epibrassinolide application and accentuated by clotrimazole, indicating the importance of brassinosteroid synthesis for plants growing under salinity.

Effect of salt on the growth and metabolism of Glycine max

Soybean plants cultivated with 50, 100 and 200 mM of NaCl, revealed that root growth was less affected by salinity than shoots. Salinity led to a reduction in leaf area and an increase in water content of the roots. These factors could contribute to the adaptation of the plant, improving its hydration. Although nitrate and free amino acid levels were reduced by salt treatment in roots, protein content of leaves was not altered. Salinity led to alterations in xylem amino acid composition, with increases in Ser, Ala, Gaba and Pro and a decrease in Asn. Similar changes were seen for Asn and Ser in roots together with a much stronger increase in Gaba. It is suggested that the decline in Asn reflects its conversion to Ala and Gaba (via Glu) in the roots while the increase in Pro and Gaba could be related to the adaptation of the plant to salinity.

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.

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.

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.

Allantoin has a limited role as nitrogen source in cultured coffee cells.

In plants the ureides allantoin (ALN) and allantoic acid (ALA) are formed in purine metabolism, and in some legumes both compounds play an important role as nitrogen (N) sources. In coffee plants, ALN and ALA are catabolites of caffeine degradation. Caffeine is found throughout the coffee plant and in some parts this alkaloid can accumulate up to 4% dry basis. Therefore, caffeine degradation via ureides may make an important contribution to N metabolism of the plant. Using coffee cell suspension as a model we investigated the contribution of ALN as a source of N in coffee. ALN was incorporated in the liquid medium and after 20 d of cultivation, cell mass, NO(3), NH(4), amino acids, soluble proteins, ALN and caffeine were determined in the cells. The activity of glutamine synthetase was also studied. The results showed that despite being taken up by cells ALN does not contribute significantly as a source of N in coffee cells. Compared with mineral N sources, cells grown with ALN-N accumulated much less mass. The inclusion of ALN in the medium caused significant alterations in the content of some N compounds indicating a stress condition.