A sterile hydroponic system for characterising root exudates from specific root types and whole-root systems of large crop plants

Abstract

Background: Plant roots release a variety of organic compounds into the soil which alter the physical, chemical and biological properties of the rhizosphere. Root exudates are technically challenging to measure in soil because roots are difficult to access and exudates can be bound by minerals or consumed by microorganisms. Exudates are easier to measure with hydroponically-grown plants but, even here, simple compounds such as sugars and organic acids can be rapidly assimilated by microorganisms. Sterile hydroponic systems avoid this shortcoming but it is very difficult to maintain sterility for long periods especially for larger crop species. As a consequence, studies often use small model species such as Arabidopsis to measure exudates or use seedlings of crop plants which only have immature roots systems.

Results: We developed a simple hydroponic system for cultivating large crop plants in sterile conditions for more than 30 days. Using this system wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) plants were grown in sterile conditions for 30 days by which time they had reached the six-leaf stage and developed mature root systems with seminal, nodal and lateral roots. To demonstrate the utility of this system we characterized the aluminium-activated exudation of malate from the major types of wheat roots for the first time. We found that all root types measured released malate but the amounts were two-fold greater from the seminal and nodal axile roots compared with the lateral roots. Additionally, we showed that this sterile growth system could be used to collect exudates from intact whole root systems of barley.

Conclusions: We developed a simple hydroponic system that enables cereal plants to be grown in sterile conditions for longer periods than previously recorded. Using this system we measured, for the first time, the aluminium-activated efflux of malate from the major types of wheat roots. We showed the system can also be used for collecting exudates from intact root systems of 30-day-old barley plants. This hydroponic system can be modified for various purposes. Importantly it enables the study of exudates from crop species with mature root systems.

Keywords: Aluminium-tolerance; Barley; Hydroponic; Malate; Organic anion; Root exudates; Sterile system; TaALMT1; Wheat.

Fig. 1

A photograph and a schematic drawing of the hydroponic system. The system is mainly comprised of an upper chamber that contains aerial part of the plant and a lower chamber that contains the nutrient solution and the plant root system. The plant is held between the two compartments with a plant holder insert. Numbers in bracket represent the part numbers listed in Table 2

Fig. 2

Wheat growth stage and root diameter in the hydroponic system. Box plots showing a the leaf number (leaf stage), number of tillers and the number of nodal roots developed on the hydroponic plants after 30 days of cultivation, and b the diameter of each root type of the hydroponically-grown wheat plants (n = 10). Different lowercase letters above the plots indicate significant differences as determined by a one-way ANOVA with Holm–Sidak post hoc test (P < 0.001)

Fig. 3

Biomass of hydroponically grown plants. Box plots showing the final shoot and root dry weight of a wheat (n = 10) and b barley (n = 8) after 30 days of cultivation in the sterile hydroponic system

Fig. 4

Malate efflux from the apices of various root types. Data are presented as a malate efflux per root apex, or b malate efflux normalized to the tissue surface area. Data are mean ± SE (n = 3–10). Lowercase letters above the bars indicate significant differences (P < 0.05) between the root types within the control or +Al³⁺ treatments, and asterisks above the +Al³⁺ bars indicate significantly more malate exudation (P < 0.05) than the counterpart root type in the control treatment. Equal variance tests failed so data were natural log-transformed prior to two-way ANOVA with Bonferroni post hoc test

Fig. 5

TaALMT1 expression in various root types. Relative TaALMT1 expression in the various roots of wheat were measured using GAPDH or α-tubulin as reference genes. Data from the control and +Al³⁺ treatments were combined. Data are mean ± SE (n = 6). Different lowercase letters indicate significant differences in the gene expression between the root types (P < 0.05) within each reference gene used. Equal variance test failed so data were natural log-transformed prior to one-way ANOVA with Holm–Sidak post hoc test