The role of cytokinins,ethephon, and chlorocholine chloride in the native proteolytic activity of forest soils

Purpose

Plant growth-promoting rhizobacteria (PGPRs) synthesizes and exports phytohormones which are called plant growth regulators (PGRs). These PGRs may play a regulatory role in plant growth and development. PGRs are organic substances that influence physiological processes of plants at extremely low concentrations. The objective of this study was to find out whether three PGRs, cytokinins, ethephon, and chlorocholine, have a stimulatory or inhibitory effect on the activity of native proteases in soil.

Materials and methods

A revised methodology was used to determine soil protease activity, where TRIS-HCl buffer was replaced with demineralized water. This is described as native protease activity as its activity is not affected by chemicals. The aim was to approximate the protease response to cytokinins in the native soil environment. The native soil proteolytic activity was determined spectrophotometrically.

Results and discussion

The present paper shows that cytokinin, ethephon, and chlorocholine chloride negatively affect the native proteolytic activity of forest soils with the exceptions of the organic and organomineral horizons of European beech (Fagus sylvatica L.) on a rendzic Leptosol. In addition, 6-benzylaminopurine stimulates the native proteolytic activity of the organic horizon of pedunculate oak (Quercus robur L.). A negative effect of cytokinins on the soil proteolytic activity can decrease the rate of organic matter decomposition. The results provide soil biochemists with an insight into the roles of rhizospheric substances on soil microbial activity.

Conclusions

This work has shown that cytokinins and PGRs inhibit the activity of native soil proteases in most of the studied forest sites. Results describe the effect of rhizospheric compounds on the activity of soil microorganisms, with potentially significant implications for the nitrogen cycle in forest soils.

     

Effect of auxins on the native proteolytic activity of forest soils

The soil proteolytic complex plays an important role within mineral nutrient cycling in ecosystems. Both auxins and proteases are produced by soil microbiota, and reciprocally, their activities affect the metabolic role of biota in soil. The objective of this study was to find out whether auxins (indole‐3‐acetic acid, indole‐3‐butyric acid, 1‐naphthaleneacetic acid and 2‐naphthoxyacetic acid) have a stimulatory or inhibitory effect on the activity of native proteases in soil. The results presented here demonstrate the positive effect of auxins on the native proteolytic forest soil activity. For example, in organic Oe horizons synthetically produced auxins stimulated native proteolytic activity in the European beech stands, where NOA led to the highest increase in L‐tyrosine production (from 564.02 µg at 0 µg auxin to 645.19 µg at the auxin dose of 100 µg). Comparing organic and organomineral horizons, auxins promote a higher stimulation of protease activity in the organic horizons. Comparing the different forest stands, auxins promote a high stimulation of protease activity in European beech stands. In the organomineral soil horizons, auxins promote a high stimulation of protease activity in Leptosols in European beech stands and Cambisols in pedunculate oak stands. The results confirmed the interrelationships between auxins and the native proteolytic forest soil activity from the viewpoint of dead organic matter decomposition. Furthermore, the general importance of substances produced in the rhizosphere for nitrogen cycling was verified.     

A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction

ABSTRACT: BACKGROUND: We have developed a new analytical approach for isolation and quantification of cytokinins (CK) in minute amounts of fresh plant material, which combines a simple one-step purification with ultra-high pressure liquid chromatography-fast scanning tandem mass spectrometry. RESULTS: Plant tissue samples (1-5 mg FW) were purified by stop-and-go-microextraction (StageTip purification), which previously has only been applied for clean-up and pre-concentration of peptides. We found that a combination of two reverse phases and one cation-exchange phase, was the best tool, giving a total extraction recovery higher than 80%. The process was completed by a single chromatographic analysis of a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides) in 24.5 minutes using an analytical column packed with sub-2-microne particles. In multiple reaction monitoring mode, the detection limits ranged from 0.05 to 5 fmol and the linear ranges for most cytokinins were at least five orders of magnitude. The StageTip purification was validated and optimized using samples of Arabidopsis thaliana seedlings, roots and shoots where eighteen cytokinins were successfully determined. CONCLUSIONS: The combination of microextraction with one-step high-throughput purification provides fast, effective and cheap sample preparation prior to qualitative and quantitative measurements. Our procedure can be used after modification also for other phytohormones, depending on selectivity, affinity and capacity of the selected sorbents.