Research article
Degradation of proteins by enzymes exuded by Allium porrum roots–A potentially important strategy for acquiring organic nitrogen by plants

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Abstract

Nitrogen is one of the crucial elements that regulate plant growth and development. It is well-established that plants can acquire nitrogen from soil in the form of low-molecular-mass compounds, namely nitrate and ammonium, but also as amino acids. Nevertheless, nitrogen in the soil occurs mainly as proteins or proteins complexed with other organic compounds. Proteins are believed not to be available to plants. However, there is increasing evidence to suggest that plants can actively participate in proteolysis by exudation of proteases by roots and can obtain nitrogen from digested proteins. To gain insight into the process of organic nitrogen acquisition from proteins by leek roots (Allium porrum L. cv. Bartek), casein, bovine serum albumin and oxidized B-chain of insulin were used; their degradation products, after exposure to plant culture medium, were studied using liquid chromatography–mass spectrometry (LC–MS). Casein was degraded to a great extent, but the level of degradation of bovine serum albumin and the B-chain of insulin was lower. Proteases exuded by roots cleaved proteins, releasing low-molecular-mass peptides that can be taken up by roots. Various peptide fragments produced by digestion of the oxidized B-chain of insulin suggested that endopeptidase, but also exopeptidase activity was present. After identification, proteases were similar to cysteine protease from Arabidopsis thaliana. In conclusion, proteases exuded by roots may have great potential in the plant nitrogen nutrition.

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.

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