Download THE POTENTIAL ROLE OF PEON (PHOSPHATE BUFFER

THE POTENTIAL ROLE OF PEON (PHOSPHATE BUFFER-EXTRACTABLE ORGANIC
NITROGEN) IN THE SOIL FOR PLANT NUTRITION AND ITS IMPLICATION FOR
ORGANIC FARMING
Noriharu? AE1 , Shingo MATSUMOTO2 , Yoh-ichi KOYAMA 3 and Katsumasa IIJIMA 3
1 (Kobe University, Biological and Environmental Science, Rokkodai-cho, Nada, Kobe, 078-8501, Japan)
2 (Shimane University, Education and Research Center for Biological Resources, kamihonjo, Matsue,
Shimane, 690-1102, Japan)
3 (Nippi Research Institute of Biomatrix, Senju-Midoricho, Adachi, Tokyo, 120-8601, Japan)
Abstract
Organic farming has become popular due to consumer concern with human health and the environment.
However, the defined features of organic manure remain vague. Stimulated growth in some crops,
irrespective of lower amounts of inorganic nitrogen (N), after organic matter was applied has been
observed. In this paper, we show that a ubiquitous organic nitrogen (N) called PEON (Protein -like N:
Phosphate-buffer Extractable Organic Nitrogen; Matsumoto et al. 2000a) is produced by amendment of
manure in a soil and could be a nitrogen as well as an inorganic nitrogen source. PEON consisting of
sugars and amino acids has a molecular weight of about 8,000 Da. Spinach (Spinacia oleracea L.) is a
plant species that grows better with application of organic matter as opposed to chemical fertilizer. We
could detect the existence of a PEON-like peak in xylem sap of spinach grown on a field applied with
manure. Furthermore, the anti-PEON antibody reacted with xylem sap collected from spinach grown on a
field applied with manure. However it did not react with xylem sap from spinach grown in a culture
solution without organic nitrogen. These results suggest the potential role of PEON in the nutritional
mechanisms of plants and may contribute to the management of organic farming.
Introduction
Modern agricultura l production, including “organic farming”, is based on Liebig’s theory (1840) that
crops take up only inorganic nitrogen (N) released from organic materials and/or soils. This theory
contributes to chemical fertilizer development and stable and high yields in crop production. But in
agricultural practices, compulsory use of organic matter is common not just for organic farming but also to
achieve high production, especially among sugar beet, spinach and carrot cultivating farmers in Hokkaido,
northern Japan. Long-term field experiments conducted at Rothamsted in the United Kingdom have
reported the same phenomenon. Mattingley (1973) reported that potato and sugar beet absorbed N from
organic sources more efficiently than barley and wheat. Coock (1977) described that an increase in yield
of these two crops was not due to the improvement of soil physical condition by application of manure,
but by organically-provided N that behaved in ways not easily imitated by fertilizer N (Figure 1). In this
context, there may be two types of crop species, one responding to organic N and another that does not.
Response of crop species to organic matter
Sedge plants preferentially took up amino acids more in Arctic tundra where mineralization of N was
suppressed under low temperature, acid and moist conditions, compared to wheat (Chapin et al. 1993).
Also boreal forest plants were reported to take up amino acid N rather than inorganic N (Nasholm et al.
1998). These reports suggest that amino acids in a soil can be better N sources for specific plants,
compared to inorganic N. Can amino acids in arable soil become a sufficient N source for crops by
replacing inorganic N? Nemeth et al. (1988) reported that the amount of amino acids in a soil was
extremely low compared to inorganic N.
?
Keywords: organic matter, manure, available nitrogen, xyle m sap, spinach, organic farming
1
International Workshop on Sustained Management of the Soil-Rhizosphere System for Efficient Crop Production and Fertilizer Use
16 – 20 October 2006
Land Development Department, Bangkok 10900 Thailand
We conducted a pot experiment using rapeseed cake (C/N ratio 7.0) as organic N and ammonium sulphate
containing the same amount of N as rapeseed cake. Five kinds of vegetable crops (Pimento: Capsicum L.;
lettuce: Lactuca sativa L.; carrot: Daucus carota L.; chingensai: a kind of chinese cabbage, Braccica
campestris L.; and spinach: Spinacia oleracea L.) were grown for 28 days. During the growth period, N
status in the soil of unplanted pots was monitored for evaluating N supply to these crops. For N status in
the soil, inorganic N (ammonium and nitrate N), amino acids and protein-like N contents were measured.
Protein-like N was extracted with a 1/15M phosphate buffer at pH 7.0 and it was considered to be easily
mineralizable and/or decomposable N for estimating inorganic N supply to a plant. Inorganic N levels in
unplanted fallow soil with rapeseed cake were higher than in the control soil without any N source but
lower than in the soil with ammonium sulphate during the 28 days of the growth period (Table 1). In
contrast, the content of organic N such as amino acid and protein was higher in rapeseed cake-amended
soil than the control and the ammonium sulphate-amended soil. Interestingly, there were very few amino
acids compared to the amount of inorganic N and protein like N.
The N uptake by chingensai, spinach and carrot was higher in soil amended with rapeseed cake than in
soil amended with ammonium sulphate, irrespective of lower inorganic N in the plot receiving rapeseed
cake. However, pimento and leaf lettuce responded well to inorganic N levels in a soil and their growth
was lower in rapeseed cake amended soil (Figure 2). These facts imply that chingensai, carrot and spinach
preferentially took up organic nitrogen such as amino acids and protein. However, the content of amino
acids in the unplanted plot with rapeseed cake soil was only 0.6 mg N/kg dry soil at the most. Growth and
N uptake of carrot, spinach, and chingensai increased 30 to 40% with application of rapeseed cake
compared to chemical fertilizer. This increase in N uptake cannot be explained by the amount of amino
acids in the soil, but could be explained by the amount of protein-like N (Matsumoto et al. 2000b).
Protein-like N: PEON
Protein-like N was developed for evaluating available or mineralizable organic N in a soil, and could be
extracted with a 1/15 M phosphate buffer (Higuchi 1983). We called this protein-like N PEON
(Phosphate-buffer Extractable Organic Nitrogen). Although several extraction methods (excepting this
method) for available N or mineralizable N have been reported, organic N showed protein-like
characteristics, similar amino acid composition and C/N ratios irrespective of extraction methods (Senwo
& Tabatabai 1998; Marumoto et al. 1974; Ogiuchi et al. 2000; Higuchi 1982; Michrina et al. 1982). The
amount of PEON in the soil was comparatively high, and it increased markedly when organic matter was
applied (Table 1). We extracted PEON with a 1/15M phosphate buffer (pH 7.0) from 25 soils including
paddy, upland and forest soils in Japan, grazing fields in Brazil and fertilized fields in Niger. These
extracts were analyzed by size-exclusion HPLC (at 280nm). Interestingly, only one major peak of about
8,000 Da was detected in extracts from 25 types of soil irrespective of soil types, soil management and
locations in the world (Figure 3).
In order to know the structure and origin of PEON, a part of the major peak detected in the extract from
the Andosol (see first soil sample in Figure 4) was purified using gel chromatography. PEON was
hydrolyzed to analyze the composition of amino acid, amino-sugar and neutral sugar. Amino acid
enantiometric ratios provide useful information about the origin of nitrogenous materials (Kvenvolden
1975) because the principal source of D-amino acid is peptideglycans, the main structural component of
bacteria l cell walls. In bacterial cell walls, the D-enantiometric forms of alanine, glutamic acid and
aspartic acid are prevalent, and D-alanine is the most abundant D-amino acid (Rogers 1983). In the
present study, three D-amino acids were detected in PEON: D-alanine (12% D/L amino acid ratio), Darginine (25%) and D-glutamic acid (15%) (Table 2). These results imply the possibility that PEON
derives, at least in part, from bacterial cell walls. However, muramic acid in PEON was small in amount.
This suggests that PEON may not be derived solely from pepitideglycans of cell wall components of soil
bacteria. Although element analysis showed PEON contained 19% of ash (data not shown, probably
minerals like Fe and Al) and sugar content was abundant, chemical structure is needed for characterization
2
International Workshop on Sustained Management of the Soil-Rhizosphere System for Efficient Crop Production and Fertilizer Use
16 – 20 October 2006
Land Development Department, Bangkok 10900 Thailand
(Table 2). PEON has UV absorption qualities and reacts positively to ninhydrin staining (Wenzel 1990)
and the Bradford method (Michirina 1982). These characteristics may imply that PEON which is at least
in stable form in a soil, is composed of peptidegylcan and humic and/or polyphenol (Robert et al. 1995)
derived from soil organic matter.
Evidence for direct uptake of PEON by spinach
If increase in growth and yield of sugar beet and spinach due to uptake of PEON is much more direct
under field conditions applied with organic matter, it may be possible to detect traces of PEON and/or its
derivatives in xylem sap collected from these crop species.
We collected two samples of xylem sap from spinach grown on solution culture containing ammonium
sulphate as the only N source, and grown on a soil applied with farmyard manure (cattle feces with
sawdust). We analyzed two samples of xylem sap and soil extract (PEON) using size-exclusion HPLC.
PEON showed only one peak at 8.6 mins of the chromatogram shown in Figures 3 and 4 and a higher and
sharper peak at the same retention time (near 8.6 mins) was present in xylem sap from spinach grown on
soil. However, xylem sap from the solution culture did not peak at the same retention times as PEON.
This suggests direct uptake of PEON by spinach. For strong evidence to prove direct uptake of PEON, an
anti-PEON Ig antibody (polyclonal antibody) was produced from a rabbit injected with purified PEON.
The immunological relationship between PEON and the xylem sap of spinach was examined using the
anti-PEON antibody. A rabbit was immunized with purified PEON conjugated with a carrier protein.
Western blot analysis of PEON, which had been frozen and dried to a concentrate prior to Western
blotting, showed a ladder-like staining pattern of PEON with a smaller band. No specific reaction was
observed with the pre-immune antibody (Figure 5). These results suggest that a specific antibody could be
raised against PEON and that PEON may polymerize during freezing and drying processes. Using this
anti-PEON antibody, we examined whether a PEON-like antigen was present in xylem sap. As shown in
Figure 5, a positive reaction for the anti-PEON antibody was observed in xylem sap of spinach grown on a
soil in the presence of organic matter (Figure 5, lane 2). In contrast, no positive reaction was observed in
xylem sap when spinach was grown in culture solution without organic matter (Figure 5, lane 1). No
positive reaction was observed when xylem sap from maize grown on a soil amended with rapeseed cake
was tested with anti-PEON antibody (data not shown). Considering that maize does not respond to organic
matter (Yamagata et al. 2002), these results suggest close association of the presence of a PEON-like
antigen in xylem sap and the responsiveness of a crop to organic N.
PEON uptake mechanisms
Spinach (Matsumoto et al. 1999) and sugar beet (Beta vulgaris) (Mattingley 1974) respond to organic
matter, which increases their yields. These plants belong to Chenopodiaceae and they are non-mycorrizhal
plants. This means that soil micro-organism association does not relate in PEON uptake mechanisms.
When rice plants (Oryza sativa L. cv. Honenwase) were water-cultured with hemoglobin, ovalbumine,
bovine serum albumin or peptone as sole N sources, many plasma membrane invaginations were
observed. Endocytosed extracellular molecules are degraded in the lytic compartment within cells
(Nishizawa & Mori 2001). Although uptake mechanisms of macro-molecule s include heterophagy and/or
endocytosis, it is still unclear how endocytosis works on PEON uptake by spinach.
Role of PEON in organic matter application and its implication for soil N fertility
Relative abundances of N isotopes( d15N) are useful in assessing direct organic N and inorganic N
absorption by plants. Variation in leafd15N of arctic plant species suggested differentiation in N source,
and possibly absorbed soil N (Schulze et al. 1994; Michelsen et al. 1996). PEON, which some plants can
use, is a universal substance in the soil. The preferential uptake rate of PEON depends on the plant
species. Okamoto et al. (2003) observed that sorghum (Sorghum bicolor (L.) Moench) grew better in a
3
International Workshop on Sustained Management of the Soil-Rhizosphere System for Efficient Crop Production and Fertilizer Use
16 – 20 October 2006
Land Development Department, Bangkok 10900 Thailand
plot applied with a mixture of incorporated rice bran and rice straw, compared to a plot with chemical
fertilizer only. But maize showed no response to the organic matter. It is necessary to re-evaluate the
growth response of crop species to organic matter application.
Evaluation of N availability in a soil is defined by mineralizable organic N released from a soil. However,
if PEON is accepted as the direct source of N, we are in a better position to re-consider the evaluation
method for soil N fertility.
Bibliography
Chapin III, F.S., Moilanen, L. & Kieland, K. 1993. Preferential use of organic nitrogen for growth by a
non-mycorrhizal arctic sedge. Nature, 361, 150-153.
Coock, G.W. 1977. The role of organic manures and organic matter in managing soils for higher crop
yields, -A review of the experimental evidence-, In “Proceeding of the International Seminar on
Soil Environment and Fertility Management in Intensive Agriculture, p.53-64, Japanese Society of
Soil Science and Plant Nutrition, Tokyo.
Higuchi, M. 1982. Characterization of soil organic nitrogen extracted with phosphate buffer. Japanese
Soil Science and Plant Nutrition, 53, 433-437. (In Japanese.)
Higuchi, M. 1983. Immobilization-remineralization of nitrogen in soil following addition of inorganic
nitrogen and organic substances. In “Bulletin of National Institute of Agricultural Science, Series
B”, 34, 1-84. (In Japanese.)
Kvenvolden, K.A. 1975. Advances in the geochemistry of amino acids. In Fred A. Donath (ed.), Annual
Review of Earth Planet. Science., 3, 183-212.
Liebig, L. 1840. Chemistry in its applications to agriculture and physiology. British Association for the
Advancement of Science. London.
Matsumoto, S., Ae, N. & Yamagata, M. 2000a. Extraction of mineralizable organic nitrogen from soils
by a neutral phosphate buffer solution. Soil Biology and Biochemistry, 32, 1293-1299.
Matsumoto, S., Ae, N. & Yamagata, M. 2000b. Possible direct uptake of organic nitrogen from soil by
chingensai (Brassica campestris L.) and carrot (Daucus carota L.). Soil Biology and Biochemistry.
1301-1310.
Matsumoto, S., Ae, N. & Yamagata, M. 1999. Nitrogen uptake response of vegetable crops to organic
materials. Soil Scence and Plant Nutrition. 45, 269-278.
Marumoto, T., Furukawa, K. & Yoshida, T. 1974. Contribution of microorganisms and cell wall to
easily decomposable soil organic matter. Japanese Soil Science and Plant Nutrition, 45, 23-28 (In
Japanese.)
Mattingly, G.E.G. 1974. The Woburn organic manuring experiment. II. Design, crop yields and nutrient
balance, 1964-1972. In “Report for 1973, Part 2”. p.98-133. Rothamsted Experimental Station.
Michrina, B.P., Fox, R.H. & Piekielek, W.P. 1982. Chemical characterization of two extracts used in the
determination of available soil nitrogen, Plant and Soil, 64, 331-341.
Michelsen, A., Schmidt, I.K., Jonasson, S., Quarmby, G. & Sleep, D. 1996. Leaf 15N abundance of
subarctic plants provide fields evidence that ericoid, ectomycorrhizal and non- and arbuscular
mycorrhizal species access different sources of soil nitrogen. Oecologia, 105, 53-63.
Nasholm, T., Ekblad, A., Nordin, A., Giesler, R., Hogberg, M. & Hogberg, P. 1998. Boreal forest
plants take up organic nitrogen. Nature, 392, 914-916.
Nemeth, K., Bartels, M., Vogel, M. & Mengel, K. 1988. Organic nitrogen compounds extracted arable
and forest soils by electro-ultrafiltration and recovery rates and amino acids. Biology and Fertility
of Soils, 5, 271-275.
Nishizawa, N.K. & Mori, S. 2001. Direct uptake of macro organic molecules. In “Plant Nutrient
Acquisition, new perspectives” Ae, N., J. Arihara, K. Okada and A. Srinivasan. eds, pp. 421-444.
Springer-Verlag, Tokyo.
Ogiuchi, K., Nakajima, N., Ae, N. & Matsumoto, S. 2000. Amino acid composition of soil organic
nitrogen extracted with phosphate buffer solution, Japanese Soil Science and Plant Nutrition, 60,
160-163. (In Japanese.)
4
International Workshop on Sustained Management of the Soil-Rhizosphere System for Efficient Crop Production and Fertilizer Use
16 – 20 October 2006
Land Development Department, Bangkok 10900 Thailand
Okamoto, M., Okada, K., Watanabe , T. & Ae, N. 2003. Growth responses of cereal crops to organic
nitrogen application in the field. Soil Science and Plant Nutritio , 49, 445-452.
Robert, R.R., Yu Zengshou, Randy, A.D. & Kristina, A.V. 1995. Polyphenol control of nitrogen
release from pine litter. Nature, 377, 227-229.
Rogers, H.J. 1983. In “Aspect of Microbiology”, Vol. 7, pp.6-25, Van Nostrand Reinhold, Workingham,
UK.
Schulze, E-D., Chapin III, F.S. & Gebauer, G. 1994. Nitrogen nutrition and isotope differences among
life forms at the northern treeline of Alaska. Oecologia , 100, 406-412.
Senwo, Z.N. & Tabatabai, M.A. 1998. Amino acid composition of soil organic matter, Biology and
Fertility of Soils, 26, 235-242.
Wenzel, W. 1990. Extraktion und Charakterisierung von stickstoffreichen Nichtuminsoffen einer
Braunerde. VDLUFA-Schriftenreihe, 32, 337-344.
Yamagata, M., Matsumoto, S. & Ae, N. 2002. Possibility of direct acquisition of organic nitrogen by
crops. In “Plant Nutrient Acquisition, new perspectives” Ae, N., J. Arihara, K. Okada and A. Srinivasan
eds, pp.399-420. Springer-Verlag, Tokyo.
5
International Workshop on Sustained Management of the Soil-Rhizosphere System for Efficient Crop Production and Fertilizer Use
16 – 20 October 2006
Land Development Department, Bangkok 10900 Thailand
Table and Figure
Table 1: Inorganic, amino acid, and protein-like N in an Andosol applied with ammonium sulfate,
rapeseed cake, and no N source over a period of 28 days.
Table 2: Amino acid and sugar contents of PEON
Fig. 1: Yield of sugar beet in the case of manure application (from Coock 1977)
Fig. 2: N uptake by pimento, leaf lettuce, carrot, chingensai, and spinach supplied with ammonium sulfate
and rapeseed cake and with no N source at 28 days after planting. Bars, indicate SE (n=3)
Fig. 3: Chromatograms (at 280 nm) of 25 soil extracts with 1/15 M phosphate buffer at pH 7.0.Each soil
extract from 25 soil samples were analyzed by size-exclusion HPLC. Blue line in Figure 1-a shows
a chromatogram of standard sample of mixture of Thyoglobulin (MW;670,000 ), Bovine-?globulin (MW;158,000), Chicken ovalbumin (MW;44,000), Myoglobin (MW;17,000 ) and
Vitamin B12 (MW;1,300 ).
Fig. 4: Chromatograms of size-exclusion HPLC of xylem saps collected from spinach grown on solution
culture with inorganic nitrogen and on Andosol with rapeseed cake as nitrogen.
Fig. 5: Western blotting of PEON (A) and xylem saps (B) collected from spinach grown in solution
culture with inorganic nitrogen as the N source and in soil culture with farmyard manure with saw
dust as N source.
6
International Workshop on Sustained Management of the Soil-Rhizosphere System for Efficient Crop Production and Fertilizer Use
16 – 20 October 2006
Land Development Department, Bangkok 10900 Thailand
Table 1. Inorganic, amino acid, and protein-like N in an Andosol applied with
ammonium sulfate, rapeseed cake, and no N source over a period of 28 days
Applied N
Inorganic N
(mg N/kg)
(mg N/kg)
Protein-like N
(mg N/kg)
50.0
0.1 ~
0.2
18.7 ~
34.7
Chemical Fertilizer 90.5 ~ 133.0
(Ammonium sulfate)
0.3 ~
0.4
18.9 ~
31.0
0.4 ~
0.6
34.6 ~
55.9
Control
(Without nitrogen)
Rapeseed cake
27.5 ~
Amino acid N
41.0 ~
82.5
7
International Workshop on Sustained Management of the Soil-Rhizosphere System for Efficient Crop Production and Fertilizer Use
16 – 20 October 2006
Land Development Department, Bangkok 10900 Thailand
Table 2. Amino acid and sugar contents of PEON
8
International Workshop on Sustained Management of the Soil-Rhizosphere System for Efficient Crop Production and Fertilizer Use
16 – 20 October 2006
Land Development Department, Bangkok 10900 Thailand
Sugar production (t ha -1)
Manure applied
P and K Fertilizer applied
corresponding to the manure
0
50
100
150
200
Nitrogen as chemical fertilizer (kg N ha-1)
Figure 1. Yield of sugar beet in the case of manure application (from Coock
1977)
Figure 2. N uptake by pimento, leaf lettuce, carrot, chingensai, and spinach
supplied with ammonium sulphate and rapeseed cake and with no N source at
28 DAP. Columns indicate SE (n=3)
9
International Workshop on Sustained Management of the Soil-Rhizosphere System for Efficient Crop Production and Fertilizer Use
16 – 20 October 2006
Land Development Department, Bangkok 10900 Thailand
Fig. 3
Chromatograms (at 280 nm) of 25 soil extracts with 1/15 M phosphate buffer at pH
Figure 3. Chromatograms (at 280 nm) of 25 soil extracts with 1/15 M phosphate buffer at pH
7.0. Each soil extract from 25 soil samples was analyzed by size -exclusion HPLC. The blue
line in Figure 3-a shows a chromatogram of standard sample of mixture of
Thyoglobulin (MW;670,000 ), Bovine -?-globulin (MW;158,000), chicken ovalbumin
(MW;44,000), Myoglobin (MW;17,000 ) and Vitamin B12 (MW;1,300 )
Figure 4. Chromatograms of size -exclusion HPLC of xylem saps collected
from spinach grown on solution culture with inorganic N and on an Andosol
with rapeseed cake as N input
10
International Workshop on Sustained Management of the Soil-Rhizosphere System for Efficient Crop Production and Fertilizer Use
16 – 20 October 2006
Land Development Department, Bangkok 10900 Thailand
Technical Session V
Dr. Chin-Hua
MA from spinach
Figure 5. Western blotting Chairperson:
of PEON (A) and xylem
saps (B) collected
grown in solution culture with inorganic N as the N source and in soil culture with
Wednesday
18 October 2006
farmyard manure with sawdust
as the N source
10.40 – 12.00
11
International Workshop on Sustained Management of the Soil-Rhizosphere System for Efficient Crop Production and Fertilizer Use
16 – 20 October 2006
Land Development Department, Bangkok 10900 Thailand