Research articleDegradation of proteins by enzymes exuded by Allium porrum roots–A potentially important strategy for acquiring organic nitrogen by plants
Introduction
Nitrogen is one of the most essential macronutrients for plant growth. Plant roots are able to take up the inorganic form of nitrogen as well as intact amino acids. It was shown that plant roots are able to take up considerable amounts of amino acids in laboratory conditions (i.e. [12], [29], [32]) but also in field conditions [24], [25], [26], [27]. According to Weigelt et al. [39], some plants may even prefer amino acids over inorganic forms of nitrogen. In addition, plants can take up not only individual amino acids but also dipeptides, tripeptides or even larger oligopeptides (i.e. [35], [38]). However, it is still unclear how well plant roots compete with soil microorganisms for soil organic nitrogen in natural conditions (see for review – [13], [14], [19]).
Soil amino acids are present mainly as proteins [14], [34]. Amino acids are released from soil proteins by degradation via proteolytic enzymes exuded by microbes (i.e. [7], [23]). According to previous results, such proteases could be exuded not only by microorganisms but also by plant roots; it has been shown that numerous wild and agricultural plant species secrete proteases from the roots, such exudation being species- and cultivar-specific. These root-secreted proteases have optimum activity in neutral pH and belong to cysteine protease family [8]. In addition, it has been shown that casein can compensate for lack of inorganic nitrogen in the culture medium – compared with full Murashige Skoog medium (MS), Triticum aestivum seedlings grew better on MS medium without inorganic nitrogen but with addition of casein [1].
To further understand how plants acquire nitrogen from proteins, the degradation products of casein, bovine serum albumin and the B-chain of insulin, after exposure to purified plant culture medium, were studied using liquid chromatography–mass spectrometry (LC–MS). Allium porrum was chosen for this study, because of the relatively high proteolytic activity of the culture medium of leek [8]. As a model protein/peptide for digestion, three substrates were used: i) casein, which in previous studies was easily degraded by proteases [1], [8], ii) bovine serum albumin because of the popularity of this substrate in proteolysis studies, and iii) oxidized B-chain of insulin because of its short and well-known amino acid sequence. In addition, protease was identified using the sequence tags technique.
Section snippets
Plant material
Seeds of A. porrum L. cv. Bartek were obtained from a commercial plant distributor (Torseed, Poland). Plant cultivation and sterility control were performed as described elsewhere [1], [8]. Briefly, seeds were surface sterilized with 70% ethyl alcohol and with 10% sodium hypochlorite; the seeds were germinated on Petri dishes (seven days), and the seedlings cultivated separately in tubes (for 2 weeks). The seedlings were separately placed into small tubes with 10 ml of autoclaved, deionised
Enzyme purification
Fractions collected after Sephadex G-100 chromatography showed a single broad peak of proteolytic activity (fractions number 20–25) and 3 peaks of total protein concentration (Fig. 1).
Main degradation products of casein
Incubation of casein with the purified culture medium resulted in gradual protein fragmentation (Fig. 2). After 1 h of incubation 11 main peaks were obtained (Fig. 2B), the highest peak representing peptide with a molecular mass of 1.5 kDa. After 2 h of incubation, the highest absorbance was obtained for peak nr 1,
Discussion
Soil organic matter contains nitrogen mainly in the form of proteins alone or complexed with other organic compounds. Proteins are considered to be a source of nitrogen exclusive for microbes and animals [14]. According to a traditional paradigm, to access that pool of nitrogen plants need assistance from microorganisms. However, it has been shown that some plants are capable of exuding proteases from roots and suggested that by using such exuded proteases, plants were able to obtain nitrogen
Acknowledgments
We are grateful to Pauli Karpinen for technical help with LC-MS and to Dr Joann von Weissenberg for checking the English language of this paper. In addition, we thank Minna Salonen, Michel Schmidt and Matti Vihakas for valuable technical suggestions and also Zhang Zhongqi and Brian Munro for help with bioinformatics software. This work was supported by the Academy of Finland.
References (42)
- et al.
Nitrogen mobilization from protein–polyphenol complex by ericoid and ectomycorrhizal fungi
Soil Biol. Biochem.
(1996) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding
Anal. Biochem
(1976)- et al.
Plasma membrane transporters: a machinery for uptake of organic solutes and stress resistance
Plant Sci.
(2001) - et al.
Influence of various forms of green manure amendment on soil microbial community composition, enzyme activity and nutrient levels in leek
Appl. Soil Ecol.
(2007) - et al.
The ability of plants to secrete proteases by roots
Plant Physiol. Biochem.
(2007) - et al.
Are microorganisms more effective than plants at competing for nitrogen?
Trends Plant Sci.
(2000) - et al.
Plant N capture and microfaunal dynamics from decomposing grass and earthworm residues in soil
Soil Biol. Biochem.
(2000) - et al.
Dissolved organic nitrogen uptake by plants – an important N uptake pathway?
Soil Biol. Biochem.
(2005) - et al.
Competition for nitrogen between plants and soil microorganisms
Trends Ecol. Evol.
(1997) - et al.
Structure and expression of two genes that encode distinct drought-inducible cysteine proteinases in Arabidopsis thaliana
Gene
(1993)