Xylem sap nitrogen compounds of some Crotalaria species

Compostos nitrogenados da seiva do xilema de algumas espécies de Crotalaria

Xylem sap nitrogen compounds of some Crotalaria species

Angela Pierre Vitória; Ladaslav Sodek*

Depto. de Fisiologia Vegetal - IB/UNICAMP, C.P. 6109 - CEP: 13083-970 - Campinas, SP.

*e-mail: [e-mail]

ABSTRACT: Thirteen species of Crotalaria were analysed for nitrogen compounds in the xylem root bleeding sap. Amino acids were the main form of organic nitrogen found, but only traces of ureides were present. Of the four species analysed for amino acid composition, asparagine was found to be the major amino acid, accounting for over 68% of the nitrogen transported. No striking deviations from this general pattern was found between species, between vegetative and floral stages of development, or between nodulated and non-nodulated plants. It was concluded that the Crotalaria species studied here have an asparagine-based nitrogen metabolism, consistent with many other non-ureide-producing legume species.

Key words:Crotalaria, transport, nitrogen, amino acids, asparagine

Compostos nitrogenados da seiva do xilema de algumas espécies de Crotalaria

RESUMO: Treze espécies de Crotalaria foram analisadas quanto aos compostos nitrogenados presentes na seiva do xilema. Os aminoácidos foram os principais compostos nitrogenados encontrados, e apenas traços de ureídeos estavam presentes. Uma análise da composição de aminoácidos realizada para quatro espécies revelou que a asparagina é o aminoácido predominante, representando mais de 68% do nitrogênio transportado. Nenhum desvio marcante deste padrão foi encontrado entre espécies, entre plantas noduladas e não-noduladas e nem entre estádios florais e vegetativos. Chegou-se a conclusão de que as espécies aqui estudadas possuem metabolismo de nitrogênio baseado na asparagina, concordando com dados da literatura para muitas outras espécies de leguminosas que pertencem à categoria de não-produtoras de ureídeos.

Palavras-chave:Crotalaria, transporte, nitrogênio, aminoácidos, asparagina

INTRODUCTION

The genus Crotalaria L. (Leguminosae) consists of over 500 species, spread over tropical and subtropical areas (Pilbean & Bell, 1979). Despite being considered weeds, Crotalaria does have some economic importance, in view of their use in nematode control (Miranda, 1981), as forage (Rizzini & Mors, 1995), as a source of fibres, as green manure and in the control of soil erosion (Miller, 1967).

Little is known about the nitrogen metabolism of Crotalaria. One comprehensive study was made of the amino acid composition of Crotalaria seeds in relation to toxic properties associated with many seeds of this genus (Pilbean & Bell, 1979). Other legumes, especially those of greater economic importance, have received more attention with regard to nitrogen metabolism. The picture that emerges is that nitrogen metabolism of legumes is centred around asparagine. Asparagine is the principal or one of the principal forms of nitrogen transport (Schubert, 1986) . In those cases where asparagine does not predominate, it usually represents the second most abundant compound. For example, in nodulated soybean, the ureides allantoin and allantoic acid are responsible for the vast majority (over 80%) of nitrogen transported, but asparagine predominates the amino acid fraction (Sawazaki et al., 1987). Furthermore, in non-nodulated soybean, where ureides are reduced to trace levels, asparagine takes over as the principal form of nitrogen transported (Sawazaki et al., 1987).

The aim of this study was to determine which organic compounds characterise the transport of nitrogen in the xylem sap of Crotalaria species.

MATERIAL AND METHODS

Plant Material - The following Crotalaria species were used in this study: C. grantiana Harv., C. juncea L., C. paulina Scharank, C. retusa L., C. spectabilis Roth., C. striata D.C., C. usaramoensis Bak., C. anagyroides Kunth, C. estipularia Desv., C. incana L., C. lanceolota E. Mey, C. mucronata Desv., C. zanzibarica Benth.

Growth Conditions - Seeds were germinated in 3-litre pots with either vermiculite for non-nodulated plants, or a mixture of vermiculite with soil (3:1 v/v), when nodulated plants were required. The plants were cultivated in a green-house under natural temperature, light and relative humidity conditions. Pots were irrigated twice a week with 250 ml of full-strength (NO₃=15 mM) nutrient solution (Hoagland & Arnon, 1950) and water as necessary. The nutrient solution applied to nodulating plants was the same except for the omission of mineral nitrogen.

Xylem sap collection - The plants were prepared for xylem sap collection after 4 months of growth when some plants had flowered. First, the pots were saturated with water and the stems severed at about 5 cm from the base. After rinsing the cut surface with distilled water and removing excess moisture with filter-paper, the sap exudate was collected with a glass capillary. During the 3 hour morning collection period, the sap was kept in vials on ice, and subsequently frozen until analysis.

Ureide determination - The colorimetric method described by Vogels & Van der Drift (1970) was used for total ureide determination in the xylem sap, using allantoin as standard.

Amino acid determination - The sap amino acid concentration was determined by the ninhydrin method of Yemm & Cocking (1955), using leucine as standard.

Amino acid chromatography - Individual free amino acids of the sap were separated and quantified by reverse-phase HPLC of their OPA (o-phthaldialdyde) and FMOC (9-fluorenylmethoxycarbonyl chloride) derivatives. The FMOC analysis was used mainly for proline and the confirmation of any doubtful peeks after OPA analysis. Details of the method are as described previously (Marur et al., 1994).

RESULTS

Considerable variation was found for exudation rates of the plants used in this study. In general, very low rates of exudation were obtained. This hindered the collection of xylem sap and consequently limited the number of analyses possible. Even for those plants where collection was possible, a 3 hour collection period was necessary in order to obtain sufficient material for analysis. In some cases, the amount of sap collected was sufficient for only one analysis. Although no systematic study was made of this phenomenon, it was apparently related to species and stage of development, since the problem was recurrent in repeat experiments.

The analysis of xylem exudates for ureides revealed a very low level of these compounds for all Crotalaria species studied (TABLE 1). In all but one case, the range found lay between 0.06 and 0.14 mmol/ml of exudate, values close to the detection limit of the method. Plants at both flowering and vegetative stages presented low levels of ureide in the xylem sap. Analyses were also made for three species after nodulation, but again only trace levels were found (TABLE 2).

The level of ninhydrin-positive amino compounds, on the other hand, was found to be relatively high for all 13 species studied (TABLE 1). The sap concentration varied from about 1.8 to 6.9 mmol/ml for most species, but two species presented levels above 9 mmol/ml. Both flowering and vegetative plants presented a similar range of levels (not different statistically by Student's t-test, where each case was considered as a replicate), although a direct comparison of these developmental stages within the same species was only possible for two of them, in view of the problems in obtaining sufficient exudate. When the amino acid levels of nodulated and non-nodulated plants were compared (TABLE 2), no striking differences were found except in one case. One species, C. mucronata, did present a significantly higher level of amino acid for the non-nodulated plant, but this could be a reflection of a more concentrated exudate since the ureide level was also relatively much higher.

A more detailed analysis of the amino acid fraction for 4 selected species of Crotalaria revealed the predominance of asparagine in the xylem sap (TABLE 3). This amino acid alone, on a molar basis, accounted for between 52 and 70% of the total free amino acids. The other amide, glutamine, was generally very low. Aspartic acid was the only other amino acid of note, generally present at near 20% in most cases. All other amino acids were present in small amounts, many at the limit of detection. Although only a reduced number of cases were analysed, there was no evidence that the stage of development of the plant (flowering versus vegetative) or nodulation had any striking effect on amino acid composition. There was an appreciable variability in the level of asparagine itself among the species and developmental stages analysed, but no clear relationship was apparent between this variability and the plant developmental condition.

DISCUSSION

Asparagine is one of the principal and frequently the most abundant amino acids involved in the transport of nitrogen, a characteristic of the legume species studied so far (Schubert, 1986). Some variability can be found in the case of glutamine, which can also be important in some legumes, but not all. In this case, the Crotalaria species studied here were no exception in that asparagine clearly predominates in the xylem exudate, but with respect to glutamine, they were typically low. Unlike soybean (Puiatti, 1997) and cowpea (Pate et al., 1980), the level of glutamine in Crotalaria does not appear to be related to symbiotically effective plants, since nodulated plants did not show any increase in glutamine transport. Possibly, glutamine is more important in ureide-producing legumes (Pate et al., 1980), to which Crotalaria evidently does not belong according to our data.

Taking into account the presence of 2 nitrogen atoms per molecule of asparagine, as opposed to one for the majority of the remaining amino acids, one can estimate that some 68 to 82% of the total nitrogen is transported in the form of asparagine. This is comparable to the range of nitrogen transported as asparagine in the xylem of Lupinus albus (Pate et al., 1979).

Some legumes, notably those of the Phaseoleae tribe, are characterised by the presence of high levels of ureides (allantoin and allantoic acid) in the transport stream (Schubert, 1986). These ureides appear to be specific products of nitrogen fixation since they are reduced to trace levels when the plants are nodule-free. Here, asparagine generally takes over as the main form of nitrogen transport, though it is noteworthy that even in the nodulated plants, where ureides predominate, asparagine was still the principal amino acid present. In the case of Crotalaria, only very low levels of ureides were detected in the xylem sap of the 13 species studied here. The levels encountered were close to the limits of detection of the method. Soybean, for example may present over 20 mmol/ml of ureides in the xylem sap of nodulated plants (Sodek & Silva, 1996). Even the "trace" levels found in non-nodulated soybean plants, of the order of 0.4 mmol/ml (Sodek & Silva, 1996), are higher than those found in Crotalaria plants, nodulated or not. Levels of total amino acids in the xylem sap, on the other hand, were of the same order of magnitude as those found in soybean (Sodek & Silva, 1996).

CONCLUSION

The data show that the nitrogen metabolism of the Crotalaria species studied was asparagine-based, in view of the predominance of this amino acid in the organic nitrogen fraction of the xylem transport stream.

Received March 10, 1998

Accepted July 20, 1998

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Publication Dates

• Publication in this collection
  17 Sept 1999
• Date of issue
  July 1999

History

• Accepted
  20 July 1998
• Received
  10 Mar 1998