Possible direct uptake of organic nitrogen from soil by chingensai
(
Brassica campestris
L.) and carrot (
Daucus carota
L.)
Shingo Matsumoto
a,*, Noriharu Ae
b, Makoto Yamagata
ca
Shimane Agricultural Experiment Station, Izumo 693-0035, Japan b
National Institute of Agro-Environmental Sciences, Tsukuba 305-8604, Japan c
Hokkaido National Agricultural Experiment Station, Memuro 082-0071, Japan
Received 8 April 1999; received in revised form 8 September 1999; accepted 16 February 2000
Abstract
On comparing the nitrogen uptake of four dierent kinds of vegetables, i.e., pimento, leaf lettuce, chingensai (a kind of Chinese cabbage), and carrot, from soil to which rapeseed cake (RC) or ammonium sulfate (AS) were applied at the same N concentration, dierent N uptake responses were observed. Chingensai and carrot took up more N from the soil with applied RC than with applied AS. A smaller amount of inorganic N was detected in the soil with applied RC than the one with applied AS. On the other hand, pimento and leaf lettuce grew better on the soil with applied AS than on that with applied RC. A possible explanation for the superior N uptake by chingensai and carrot in the soil with applied RC could be the direct uptake of organic N, especially a protein-like N compound with a uniform MW of 8000±9000 Da, that accumulated in the soil with applied RC. In order to support this hypothesis, two typical vegetables were examined: chingensai, which responds better to organic N, and pimento, which responds better to inorganic N. Xylem sap was collected from these plants and analyzed using size-exclusion high pressure liquid chromatography (HPLC). In xylem sap of chingensai grown in the soil with applied RC, a peak similar to that found in the soil solutions extracted with phosphate buer was detected on the chromatogram, while this peak was absent from the chromatograms of chingensai grown in inorganic nutrients culture solution. In contrast, there were no similar peaks for the xylem sap of pimento grown in the soil with applied RC. Further, when chingensai, carrot, and pimento were cultivated in an N-free medium under aseptic conditions, the N uptake of chingensai and carrot increased with the addition of a soil solution extracted by phosphate buer, while that of pimento did not increase. These results strongly suggest that the superior N uptake response to the application of organic material in chingensai and carrot might be related to the direct uptake of organic N from the soil.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Chingensai; Pimento; Size-exclusion HPLC; Protein-like N compound; Xylem sap
1. Introduction
The utilisation of large amounts of nitrogen fertilizer has become a common agricultural practice for obtain-ing the high vegetable yields. However, such a practice has raised many environmental concerns, especially regarding groundwater pollution due to the leaching of excess nitrate-N (Singh and Sekhon, 1979; Ritter,
1989). From the viewpoint of the ecient utilization and management of natural resources, organic veg-etable farming using wastes derived from agriculture and the food industry is becoming popular (Jong and Kim, 1995). The in¯uence of the addition of organic materials on N mineralization and dynamics has been studied over a long period. Most of the studies are based on the premise that crops take up only inorganic N released from the organic material. Some authors, however, have suggested an alternate mechanism. For example, Mattingly (1973) showed that potato and sugar beet absorbed N from organic N sources more
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* Corresponding author. Fax: +81-853-24-3342.
eciently than barley and wheat. Yamagata et al. (1996) reported that upland rice took up more N than maize and soyabean, when a mixture of organic ma-terials (rice bran and straw in a 4:1 ratio) with a high C-to-N ratio of 20 was applied. Chapin et al. (1993) reported that sedge grass in tundra soils preferentially took up amino acid N rather than inorganic N com-pared with barley. Likewise, Nasholm et al. (1998) showed that boreal forest plants could take up organic N by a method in which 13C- and 15N-labeled amino acids were injected into the organic layer of an old successional boreal coniferous forest. In our previous work, we observed that spinach grown on soil supplied with organic N in the form of rapeseed cake (RC) took up more N, than when grown on a soil supplied with inorganic N in the form of ammonium sulfate (AS), (Matsumoto et al., 1999). All these reports indi-cate that the N uptake by some crops could not be explained solely by the amount of inorganic N in the soil. One of our objectives is, therefore, to compare the N uptake responses amongst several kinds of veg-etable crops.
In a previous study, we applied organic material to soil and the N status was then examined using size-exclusion HPLC and SDS-PAGE (Matsumoto et al., 2000). Results showed that the organic material was decomposed by soil microorganisms, and the microbial biomass increased. Some of this microbial biomass was converted into a decomposition-resistant material with a uniform molecular weight (8000±9000 Da) and exhibited some of the characteristics of a protein. This protein-like N compound was deposited in the soil in the same manner as when any type of organic material was applied to any type of soil. This protein-like N compound existed as a major fraction of the source of gradually mineralized N and it originated from the remains of soil microbes (Kai et al., 1973; Marumoto et al., 1982; Chantigny et al., 1997). Higuchi (1983) showed that the amount of inorganic N released from the soil strongly correlated with the amount of a pro-tein-like N compound extracted with 1/15 M phos-phate buer at pH 7.0. If a plant performs better and takes up more N in soil amended with organic ma-terial, this plant may take up such a protein-like N compound. Therefore, we attempted to analyze the re-lationship between the protein-like N compound extracted from soil with a phosphate buer and the xylem sap of crops grown on a soil with applied RC or that of crops grown on a solution culture with inor-ganic nutrient only, using size-exclusion HPLC. Further, the crops were cultivated on a medium con-taining the protein-like N compound extracted from soil as N sources under aseptic conditions in order to eliminate the microbial rhizosphere eects, such as transportation of these organic N compounds by sym-biotic mycorrhizal fungi to the plants or the microbial
mineralization of the protein-like N compound in rhi-zosphere. In this paper, we discuss the possible direct uptake of the protein-like N compound by plants.
2. Materials and methods
2.1. Soil culture
Surface soil (0±20 cm) from a ®eld at the National Institute of Agro-Environmental Sciences (volcanic ash soil, Tsukuba, Japan) was collected for this exper-iment. A 4:1 mixture of vermiculite and the Tsukuba soil (41.7 g C kgÿ1, 3.4 g N kgÿ1dry soil) adjusted to
pH 6.0 with CaCO3, was used for crop cultivation to
minimize the initial N content. Organic N was applied at 100 mg N kgÿ1 soil in the form of rapeseed cake
(RC, 50.0 g N kgÿ1, C-to-N ratio: 7.0). Inorganic N
was applied as AS at 100 mg N kgÿ1soil. The control
did not receive additional N. Single superphosphate (150 mg P kgÿ1soil) and K
2SO4 (100 mg K kgÿ1 soil)
were also supplied to all soils. These soils were incu-bated at room temperature for 14 days at 60% of their maximum water-holding capacity. Pimento (Capsicum annum L. cv. Kyo-midori), leaf lettuce (Lactuca sativa
L. cv. Red ®re), carrot (Daucus carota L. cv. Asubeni-gosun), and chingensai (Brassica campestrisL. cv. cho-yo No. 2) were germinated on perlite without the ad-dition of any nutrients. Seedlings were transplanted on 11 August 1997 into 500 ml polyethylene pots ®lled with 400 ml of the incubated soil and grown in a glass-house. Plants were sampled at 28 days after transplant-ing (DAT), and the unplanted soil was also collected for the evaluation of N status at 1, 7, 14, 21, and 28 DAT with three replications. The xylem sap of these plants was collected at 28 DAT.
2.2. N uptake by crops
Plant samples were dried at 708C for 3 days, weighed, and ground in a bowl mill for a chemical analysis. The N concentration was determined using an automatic NC analyzer (Model NC-80, Sumitomo Chemicals).
2.3. Inorganic N, amino acids, and protein in unplanted soils
Inorganic N (NH4+ and NO3ÿ) in the soil was
extracted with 2 M KCl and determined by colori-metric procedures (Auto Analyzer, Technicon Instru-ments, New York, USA). Amino acids in the soil were extracted with 0.2 M H2SO4, and the concentration
and the concentration was determined by the Lowry method using egg albumin as a standard (Rej, 1974).
2.4. HPLC analysis of soil solution extracted by 1/15 M phosphate buer
Ten grams of the soil sample with 40 ml of 1/15 M phosphate buer (pH 7.0) was shaken for an hour to extract the protein-like N compound. These extracts were analyzed by both size-exclusion and ion-exchange HPLC techniques. The operating conditions for the size-exclusion HPLC (Shimadzu, LC-6A) were as fol-lows: column, Shimpack Diol 150 (Shimadzu); elution; 50 mM phosphate buer containing 0.3 M NaCl at pH 7.0; ¯ow rate, 2.0 ml minÿ1; sample size, 20 ml;
detector, UV-280 nm. The operating conditions for the ion-exchange HPLC were as follows: column, IEC DEAE-825 (Shodex); elution, A: 20 mM Tris±HCl buer at pH 8.2, B: A including 0.5 M NaCl; linear gradient A to B; ¯ow rate, 1.0 ml minÿ1; sample size:
20ml; detector, UV-280 nm.
2.5. Solution culture
Chingensai and pimento seedlings grown for 21 days under the above conditions described were trans-planted into a 50 l plastic container ®lled with Hoag-land solution (Aiello and Graves, 1997) and grown for 14 days, the xylem saps of these plants were then col-lected. This culture solution was continually aerated, and the pH was adjusted on a daily basis to 6.5.
2.6. Xylem sap
Plant stems were cut at 10 mm above the ground level. The cutting section was sterilized by wiping with 50% ethanol and then attached to a 1.5 ml plastic tube with absorbent cotton washed with ethanol. These procedures were performed at 9:00 h and contin-ued for 8 h, the plastic tube was then removed at 17:00 h. Xylem saps were extracted from absorbent cotton and analyzed by size-exclusion HPLC under the same conditions as described above. The elution of the xylem sap of chingensai from the HPLC analysis was collected every 20 s, and the N concentration of each fraction was determined by a micro-nitrogen analyzer (Mitsubishi Chemical Model TN-05).
2.7. In vitro culture under aseptic conditions
The seeds of pimento, carrot, and chingensai were surface sterilized for 25 min in 100 ml of sodium hydrochlorite solution (5%) and washed three times with sterilized water. Seeds were germinated on steri-lized vermiculite. Seedlings were transplanted into 300 ml ¯asks containing 25 g of sterilized vermiculite
including 50 ml N-free MS liquid medium (Murashige and Skoog, 1962). Of the MS media added, the control was N-free MS medium without soil extract, and soil extract at 10 or 20% in the MS liquid medium on the basis of volume was contained in the other two media. The soil extracts were prepared as follows: 200 g of dry soil was added to glucose (1.6 g) and AS (0.16 g) in a 500 ml plastic bottle and incubated for 14 days at 60% maximum water-holding capacity. The incubated soil with 400 ml of 1/15 M phosphate buer at pH 7.0 was shaken for 1 h. The soil extract was passed through the No. 6 ®lter paper, excluding molecular weights under 3500 Da by dialysis using a cellophane tube (Spectrapor 3, Wako) in distilled water at 48C. The dialysis was run, until amino acids and inorganic N were not detected by the above methods for 3 days.
Protein-N concentration of the soil extract was 88.2 mg lÿ1 after dialysis. The puri®ed soil extract was
added to the ¯ask through a membrane ®lter (0.2 mm Steradisk, Kurabo).
Seedlings were grown for 28 days under a 16 h photoperiod at 3000 lx supplied by the ¯uorescent lamps.
3. Results
3.1. Changes in the concentrations of each N form in unplanted soil
Inorganic N varied between 28 and 50 mg kgÿ1 in
the control that received no additional N, and between 91 and 133 mg kgÿ1 in the AS treatment. Inorganic N
(41.0±82.5 mg kgÿ1) in the RC treatment increased
gradually with time but failed to equal the amount of N in the AS treatment (Fig. 1A). The concentration of amino acids-N in the RC treatment was much higher than in the AS treatment, although the concentrations of the amino acids-N were low even in the RC treat-ment (Fig. 1B). The concentration of protein-N in the RC treatment was considerably higher than that in the AS treatment and in the control during the cultivation period (Fig. 1C).
3.2. N uptake by the crops in soil culture
N uptake by pimento and leaf lettuce was the high-est in the AS treatment followed by the RC treatment
and the lowest in the control at 28 DAT. These results correlated well with the concentration of inorganic N in the soils undergoing treatment. On the other hand, despite the lower amount of inorganic N in the soil, N uptake by carrot and chingensai in the RC treatment at 28 DAT were 34.3% and 46.3%, respectively, higher than in the AS treatment (Fig. 2).
3.3. HPLC analysis of the soil extract with phosphate buer
Fig. 3 shows the chromatograms of the extracts from the unplanted soils of the AS and RC treatments at 28 DAT. Both those treatments displayed the same peak with a retention time of 8.4 min under size-exclu-sion HPLC and with a retention time of 2.8 min under ion-exchange HPLC. These results agree with our pre-vious results (Matsumoto et al., 2000). The peak heights and areas for both HPLC analyses were higher with the RC than with the AS treatments. This order corresponds to the concentration of the soil protein fraction extracted with the phosphate buer. The mol-ecular weight of this peak was estimated to be about 8000±9000 Da on the basis of the retention time and standard molecular weight compounds (Gel-®ltration compounds, Bio-rad).
3.4. HPLC analysis of xylem sap
Xylem sap collected from chingensai grown in the RC treatment showed distinctive major peaks at reten-tion times of 8.4, 8.9, 9.5, 10.6, 11.4 and 12.2 min on
the size-exclusion HPLC chromatogram (Fig. 4A). Out of these peaks, the one at 8.4 min appeared to be iden-tical to the major peak (8.4 min) detected in the soil extracts. Xylem sap collected from chingensai grown in Hoagland culture solution containing only inorganic nutrients did not show the peak at 8.4 min, although it did show the familiar peaks at 8.9, 9.5, 10.1, 10.6, 11.4 and 12.2 min. No peak at a retention time of 8.4 min was detected in pimento under the growth conditions of soil culture or solution culture (Fig. 4B).
Fractions of xylem sap of chingensai grown with the RC treatment were collected every 20 s from the elution of size-exclusion HPLC, and the N concen-tration in each fraction was examined (Fig. 5). The
fraction including the peak at a retention time of 8.4 min was detected as N, so it was concluded that the 8.4 min peak corresponded to a nitrogenous substance with a molecular weight of 8000±9000 Da.
3.5. N uptake by crops under aseptic conditions
The growth of chingensai and carrot under aseptic conditions was improved by the addition of the soil extract. On the other hand, the addition of the soil extract appeared to have no eect on pimento (Fig. 6). The N uptake of chingensai and carrot increased with increases in the amount of soil extract added, while that of pimento did not increase (Table 1).
Fig. 4. Size-exclusion HPLC chromatograms of the xylem sap collected from chingensai (A) and pimento (B) grown in the soil applied with rape-seed cake and Hoagland solution.
4. Discussion
Under the soil culture conditions, N uptake by pimento and leaf lettuce was the highest in the AS treatment, followed by the RC treatment, and lowest in the control. This tendency corresponded well with
the inorganic N concentration in these soils. In con-trast, N uptake by carrot and chingensai in the RC treatment was higher than in the AS treatment, despite the lower inorganic N concentration in the RC treat-ment.
Two dierent hypotheses are proposed for superior N uptake by carrot and chingensai with the appli-cation of RC. (1). These crops, when compared with pimento and leaf lettuce, may accelerate N mineraliz-ation from organic N in rhizosphere soils through the activity of protease or other enzymes. However, Hayano (1983, 1986, 1995) reported signi®cant dier-ences in the activity of phosphatase, phosphomonoes-terase, phosphodiesterase and b-glucosidase between soil volumes with or without root systems, hereafter referred to as `rhizosphere and non-rhizosphere soils', but the dierences in protease activity appeared to be negligible. Kanazawa et al. (1988) also detected no dierence in the protease activity between rhizosphere and non-rhizosphere soils for several crops. (2) Another possibility is that carrot and chingensai may take up organic N even more eciently than other crops. Yamagata et al. (1997a), by examining the dierences in the response of cereals to organic N (in the form of rice bran), demonstrated that upland rice took up more organic N than maize and soyabean. However, this was not due to a higher protease activity in the rhizosphere because the protease activity in upland rice was lower than that in maize and soya-bean. Also, Yamagata et al. (1997b) reported that the
15
N concentration of upland rice was higher than that of maize and soyabean, when 15N-labeled rice bran was applied to soils. Inorganic N uptake would reduce
15
N concentration in a crop since inorganic-15N de-rived from rice bran would be diluted by sucient inorganic N pool in the soil.
Therefore, they suggested that the higher 15N con-centration in upland rice might be caused by uptake of organic N before mineralization of rice bran. This sug-gestion was supported by the fact that 15N concen-tration within crops showed no dierence when 15 N-labeled ammonium sulfate was applied to soils. Mori (1986) reported that upland rice and barley absorbed proteins, such as albumin and hemoglobin in solution culture. These results imply that plants utilize not only inorganic N, but also organic N, including molecules which are bigger than amino acids. Our observation (Fig. 2) suggested that the preferential uptake of or-ganic N by carrot and chingensai might be related to their ability to utilize organic N in a soil.
There were larger amino acids-N and protein-N con-centrations in the RC treatment than in the AS treat-ment (Fig. 1B and C). The amount of amino acids-N in the RC treatment was in the range of 0.37±0.59 mg N kgÿ1, and that in the AS treatment was 0.27±0.40
mg N kgÿ1during the growth period (Fig. 1B). These
amounts were extremely low as compared to inor-ganic-N and protein-N. Nemeth et al. (1988) also reported that amino acids accounted for only about 3% of soil organic N extracted by electro-ultra®ltra-tion, because those were easily mineralized. If plants take up amino acids from soil, there must be consider-able competition with microorganisms. Yamamuro et al. (1999) reported that 13C- and 15N-labeled aspartic acid, glutamic acid, serine and leucine applied to soil as tracers degraded to inorganic N after 2 or 3 days, and the proportion of direct uptake of the amino acids was only about 0.4±1.9% in the tomato plants. There-fore, we considered that the amounts of amino acids in our study were not enough, if, carrot and chingensai were only utilizing amino acids preferentially. On the other hand, we focused on soil organic N, especially the soil protein fraction, which was the dominant form of N in the RC-amended soil. It was clear that there was enough organic N to explain the N absorbed by chingensai and carrot (34.6±55.9 mg N kg ÿ1 in the
RC treatment, and 18.9±31.0 mg kgÿ1in the AS
treat-ment, Fig. 1C).
When the soil extracts at 28 DAT were examined by size-exclusion and ion-exchange HPLC at 280 nm, both HPLC chromatograms detected only one major peak (Fig. 3). Regarding the appearance of a single peak in the soil extract, we have already reported that the original peaks of the organic material disappeared rapidly and formed a major peak at 8.4 min, and when an antibiotic (chloramphenicol) and organic ma-terial were applied simultaneously, the original peaks remained and the peak at 8.4 min appeared much later (Matsumoto et al., 2000).
Xylem sap collected from chingensai, which showed a good response to organic N with the RC treatment had a peak with the same retention time (8.4 min), as the soil extract in the size-exclusion HPLC chromato-gram (Fig. 4A). Xylem sap from chingensai and soil extract were mixed, and the solution mixture was ana-lyzed using size-exclusion HPLC. Chromatogram peaks of both soil extract and xylem sap matched com-pletely (data not shown). The N concentration of the xylem sap of chingensai grown in the RC treatment was then analyzed. N was detected in the peak with a
retention time of 8.4 min, and this was assumed to be a protein-like N compound (Fig. 5). This peak was absent from the chromatogram of xylem sap from chingensai grown in a solution with only inorganic nutrients, while common peaks at 8.9, 9.5, 10.1, 10.6, 11.4 and 12.2 min were present (Fig. 4A). The com-mon peaks might represent amino acids and peptides that form complexes with polyvalent heavy metal cat-ions during xylem transport (Catald et al., 1988). Therefore, we believe that this peak with a retention time of 8.4 min was not produced by chingensai itself. Carrot, which also showed a good response to organic N grown in a soil with RC, also produced such a peak in the chromatogram of xylem sap (data not shown). In contrast, when pimento was grown in the RC treat-ment or in culture solution, no such peak was detected in the xylem sap (Fig. 4B). Pimento showed a poor re-sponse to organic N (Fig. 2).
The mineral-containing water in soil is taken into the root through the surface of the root epidermis. One of the major routes for transfer of materials between organs is the vascular bundle, which is com-posed of xylem and phloem. The xylem consists mainly of xylem vessels, which form a kind of apoplastic space, in which the xylem sap ¯ows from the roots to the shoots. The vital activities of organs depend on the supply of inorganic and organic compounds from the xylem sap, which are produced and blended in the root and transported via xylem vessels (Nooden and Mauk, 1987). Satoh et al. (1992) showed that several proteins (i.e., 75,000, 40,000, 32,000, 19,000 and 14,000 Da) were transported into xylem sap in squash seed-lings. The proteins might be secreted into the cell wall or apoplastic space of the cells in the central cylinder according to their signal sequences and then apoplasti-cally transported to the xylem vessels with the move-ment of water. Sakuta et al. (1998) reported that proteins estimated to be 30,000 Da were transported through the xylem from the root to above ground organs, such as leaves and shoots in cucumber. Further, Cleve et al. (1991) reported that poplar sto-rage protein (32,000 Da) could be detected in xylem sap during the dormant period and especially during budbreak, and a long distance transport of not only
Table 1
Nitrogen uptake of vegetable crop species grown on N-free MS medium with or without a soil extract supplement under in vitro conditionsa
sugars and amino acids but also protein molecule may be possible. Though further experiments are needed to con®rm that the 8.4 min peak (8000±9000 Da) of xylem sap from chingensai and carrot and that of soil extract with phosphate buer are identical, according to above reports, our observation suggest that such a high molecular substance can be taken up by chingen-sai and carrot, but not by pimento.
Chen et al. (1999) reported that arbuscular mycorri-zal fungi (AM) promoted the N uptake of crops. Amongst those crop species, which showed increased N uptake on the RC treatment, carrot was considered to form a bene®cial association with AM fungi, while chingensai, belonging to Brassica failed to associate with mycorrhizal fungi (Tompson, 1991). Thus, it is not clear whether N uptake was stimulated by these crop species in the RC treatment or by their symbiosis with AM fungi. So, we cultivated two typical crops under aseptic conditions in order to eliminate a mi-crobial eect, that is, mimi-crobial decomposition of pro-tein-like N compounds extracted from a soil and the direct transport of these substances into these plants through the hyphae of AM fungi. Under these aseptic conditions, there was no symbiosis with AM fungi and the protein-like N compound added to the medium, as an N source was not mineralized.
The growth and N uptake of chingensai and carrot (whose xylem saps showed the same peak as the tein-like N compound extracted from soil) were pro-moted by the addition of soil extract under aseptic conditions. While those of pimento (whose xylem sap did not show the same peak as the protein-like N com-pound in soil) were not promoted (Fig. 6, Table 1). These results strongly suggest that chingensai and car-rot have an ability to take up organic N directly from the soil extract, while pimento does not have this abil-ity. N uptake response by crop species to organic amendments seemed to be dependent on this ability.
Two steps must be involved in the absorption of a protein-like N compound from the soil by chingensai and carrot. First, chingensai and carrot should have the ability to solubilize the protein-like N compound adsorbed by the soil colloid. Hayashi and Harada (1969) suggested that the soil proteins which resisted microbial attack were adsorbed by inorganic or or-ganic soil colloids. The action mechanism is not yet understood; it might, however, be related to root exu-dates with chelating eects such as organic acids (Cha-ney et al., 1972; Ae et al., 1990). Secondly, protein-like N compounds from soil must be able to penetrate into a cell through the cell wall and plasma membrane. These mechanisms have already been demonstrated in solution culture with applied haemoglobin. The hae-moglobin molecules were taken up through cell mem-branes by invaginations of the plasmalemma, and they then moved to vacuoles, where they were digested
(Nishizawa and Mori, 1980). The molecule of the pro-tein like-N compound that we have investigated is much smaller than haemoglobin, and this phenomenon may possibly occur in carrot and chingensai. Further studies of these two mechanisms and how the protein-like N compound is assimilated by plants are surely required.
Acknowledgements
We thank Dr. Ancha Srinivasan and Dr. Renfang Shen for their valuable suggestions and assistance in preparing this manuscript.
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