Research on ways to improve crop productivity through the regulation of rhizosphere environments

ABSTRACT

In this mini review, the importance of rhizosphere is focused. As the rhizosphere is underneath the soil, the analytical approach is still required from the viewpoints of understanding the interaction among root, soil and its interface. For this purpose, multi omics approach has been carried out with the effort to visualize the active rhizosphere area.

KEY WORDS:

• Ionomics
• plant soil interaction
• root exudate proteomics
• metabolomics
• soil meta genomics

1. Introduction

Rhizosphere is known to differ from surrounding soils physically, chemically and biologically. These differences result from how the roots directly affect the soil and microorganisms and from considerable changes in nutrient concentrations due to plant root uptake. Such changes in mineral and organic compound concentrations also drive changes in biological activity in rhizosphere. It is thus demonstrated that biotic (plant and microorganism) and abiotic (soil) entities directly and indirectly develop close mutual relationships within rhizospheres. These relationships render the rhizosphere distinctive (Figure 1). However, it is difficult to specify an area of soil as a rhizosphere. Most related work has been carried out on areas close to root surfaces.

Figure 1. Image of material flow in the rhizosphere.

2. Comparisons of gramineae and fabaceae

Levels of dry matter production per absorbed nitrogen are higher for Gramineae than for Fabaceae (Osaki, Shinano, and Tadano 1992). This difference is attributed to higher respiratory rates observed in Fabaceae (Shinano et al. 1993; Shinano, Osaki, and Tadano 1993). As higher respiratory rates are linked to metabolism, mainly through nitrogenous compounds, the reconstruction of nitrogenous compounds in plants is considered to be a central reason for higher rates of respiration observed in Fabaceae in isotope studies (Shinano, Osaki, and Tadano 1994). Based on these findings, we reintroduce the concept of growth efficiency to explain differences in productivity observed between Gramineae and Fabaceae (Osaki et al. 1996). The low levels of productivity observed cannot be attributed to strong energy requirements for the synthesis of proteins and/or lipids in Fabaceae, and differences are identified even during the vegetative growth stage when the chemical compositions of Gramenae and Fabacease are similar (Shinano, Osaki, and Tadano 1995). Thus, we suggest that the reconstruction of carbon and nitrogen along with retranslocation appear to be important. However, these phenomena indicate that Fabaceae plants require more carbon to produce the same amount of dry matter, and similar tendencies have been observed for other elements. Roots absorb nutrients from the soil in different ways, and for Gramineae, root lengths are important, while for Fabaceae, nutrient uptake levels per unit of root length (or root surface) are critical. Root activity has been thoroughly studied in relation to phosphorus uptake (i.e. in relation to the exudation of organic acid (Li, Shinano, and Tadano 1997; Luo et al. 1999)), and enzyme-acid phosphatase exudation (Wasaki et al. 1999, 2000, 2003a) plays an important role in the solubilization of less soluble phosphorus compounds in soil. In terms of nutrient use efficiency, it is also important to consider internal phosphorus utilization efficiency levels in relation to the nutrient efficiency of an entire plant (Shinano et al. 2001, 2005; Nanamori et al. 2004; Dissanayaka et al. 2018, Nishida et al. 2019).

3. Applying omics to the rhizosphere

In addition to conducting a transcriptome analysis on plant root gene expression (Wasaki et al. 2003b, 2003c), we perform a comprehensive analysis of root exudates. A proteome analysis of root exuding proteins was carried out using rice, as rice genome information has become available. Rice was aseptically grown, and then, protein was collected from the bathing solution; after concentration, a proteome analysis was carried out via nano-LC MS/MS (Nanoscale liquid chromatography coupled to tandem mass spectrometry). More than 100 proteins were detected and identified. Most include a signal peptide involved in secretion and are considered to serve as pathogenesis-related (PR) proteins (Shinano et al. 2011, 2013). As the expression patterns of some proteins are not known, we identify the rhizosphere as an important area in which plant selectively expresses proteins. While aseptically culturing itself can apply stress to a plant, we suggest that various PR proteins are constantly expressed in the rhizosphere before a pathogen attack. Further research on how these PR proteins fight against soil pathogens is needed.

A metabolite analysis was carried out by applying methods developed for plant metabolomes (Okazaki et al. 2008, 2009, 2010, 2012) and root exudates (Suzuki et al. 2009) using GC-MS (Gas chromatography-mass spectrometry). To improve the sensitivity of our analysis, CE-MS (Capillary electrophoresis-mass spectrometry) was introduced and responses to phosphorus nutrition were measured (Tawaraya et al. 2013, 2014, 2018). Root exudate is known to play an important role in solubilizing minerals (essential and nonessential elements) from clay and organic materials.

Plant uptake nutrients in the soil, and sometimes nonessential element are also absorbed. The uptake and accumulation of elements is carried out by transporters and through ion homeostasis in the body. To determine how plants regulate ion homeostasis, an ionomics approach was applied. Lotus japonicus has been used as a model plant for Fabaceae, and EMS (ethyl methane sulfonate)-treated seeds with several mutants have been obtained to examine varied behaviors of element accumulation (Chen et al. 2009a, 2009b). One mutant includes very low molybdenum concentrations with molybdenum transporter mutations (Duan et al. 2017). Ionome is also useful for investigating the effects of material application in soils on plant nutrient concentrations, especially for trace elements. The effects of manure application (Sha et al. 2012) and temperature on the accumulation of elements have been examined (Quadir et al. 2011).

4. Clarifying the role of rhizosphere microorganisms

Though Rhizobium and mycorrhiza are known to develop a close symbiotic relationship with plants, a large number of studies have reported on the role of various microorganisms in the rhizosphere without obvious relationships. Most of such research has been carried out to screen for beneficial microorganisms based on their specific roles as part of an index. Otherwise, microorganism diversity levels have been used by applying DGGE (Denaturing gradient gel electrophoresis) methods and so on. We have also used protease and phosphatase genes of microorganisms as indexes and have applied PCR (Polymerase chain reaction)-DGGE to investigate how soil microorganism behavior changes with the application of organic materials to the soil (Sakurai et al. 2007, 2008). The activity of such enzymes is enhanced with an increase in diversity and especially in the rhizosphere.

Furthermore, the importance of utilizing unavailable phosphorus resources in soil must also be considered. Phytate is the main organic phosphorus compound and plants cannot decompose and utilize phosphorus from phytate in the soil. Soil microorganisms seem to alter the phytate status of soil (Unno et al. 2005; Unno and Shinano 2013). When phytate is applied to the soil, in most cases, plants are not able to utilize this compound because plants do not exudate phytase to the rhizosphere. However, sometimes, when soil is used as a medium, plants can grow better with the addition of phytate (Unno et al. 2005). It has been speculated the introduction of soil microorganisms may decompose phytate and release phosphorus (mainly using phytate as a carbon source), and several isolates have been shown to exhibit this capacity. For a gnotobiotic environment (when Lotus japonicas and an isolate exhibiting phytase and phosphorus releasing activity are the only organisms in the system), this isolate promotes plant growth with an increase in phosphorus uptake, but this was not confirmed for soil conditions. This means that under actual soil conditions, non-culturable microorganisms and/or microbial complexes may play a role. To determine the role of rhizosphere microorganisms in the utilization of phytate, a metagenome analysis was applied. While several microorganisms have been categorized, differences in phytate utilization have not been determined. We analyzed functional gene categorizations and their relative existence in phytate based on the status of plants. When plant growth was promoted through the application of phytate, rhizosphere soil from the plant contributed more to citrate synthase and alkaline phosphatase (Unno and Shinano 2013). As phytic acid binds to metal ions (Al, Fe, etc.) and organic compounds and forms phytate in the soil, complexes such as citric acid are required to solubilize phytate. Furthermore, phytase, as one an alkaline phosphatase that enhances alkaline phosphatase genes, may contribute to the decomposition of phytate in rhizosphere soil. The improvement of information on soil microbial functional genes will further our understanding of the rhizosphere’s biological functions.

Hence, functional analyzes of rhizosphere microorganisms will prove useful. Microorganisms exhibit strong abilities, and it is not necessary for microbial communities to be composed of the same members. It is possible to form a metabolic pathway through the combination of functions. Thus, we must analyze rhizosphere soil and develop technical methods for regulate it from this perspective.

However, though the definition of the rhizosphere is clear (the sphere affected by root activity), it is difficult to precisely define rhizosphere soil. Most related research has been carried out based on the distance from the root surface or from physical strength of attachments to roots. The effects of the root on the soil occur more or less physically, chemically and biologically, but it is not possible to evaluate root activity (by active exudation) in the rhizosphere. Part of a photosynthate is known to translocate from the shoot to the root and then to the rhizosphere. Several trials have used ¹⁴CO₂ to evaluate the distribution of photosynthate to roots. To specify the area of the rhizosphere, we choose ¹¹C as radioactive carbon with a short half-life (20.334 minutes) to visualize the movement of photosynthate to roots and subsequent distribution to the soil. In examining plant species with strong root exuding capacities, we obtain a clear account of rhizosphere soil of high radioactivity, and we in turn analyze degrees of microbial diversity across different levels of radioactivity. By linking microbial functions, mineral element dynamics in soil and subsequent uptake by roots, we can better define the role of the rhizosphere (Wasaki et al. 2018).

5. Links to countermeasures of radioactive cesium

From recent studies developing countermeasures for radio cesium contamination in soil, we are interested in how potassium in clay is solubilized through plant activity. To mitigate radioactive cesium transport from soil to plants, potassium is typically applied (as fertilizer and/or potassium containing resources) in the field (Eguchi et al. 2015; Kubo et al. 2015, 2017; Yamamura et al. 2018). Though the mechanism to explain the role fo potassium on ¹³⁷Cs has been thoroghly studied especially from the viewpoint of soil mineral characteristics (e.g. Kurokawa et al. 2019; Nakao et al. 2008; Yamaguchi et al. 2017; Ogasawara, Nakao, and Yanai 2017), it is stil unclear how the plant root interact with the dynamics of ¹³⁷Cs in the rhizosphere. However, a remaining issue to address in coping with radioactivity in agricultural is high levels of Fabaceae transference. It is known that Lupine has the highest transfer factor, which is roughly 2 to 4 times higher than that for soybeans under the same conditions. The strong exudation capacities of Lupine have been speculated to play a role. As exudation is especially accelerated under phosphorus deficient conditions, we must determine to what extent and how roots exudate and affect soil to release radioactive cesium even under different soil conditions. These mechanisms may facilitate the development of new technologies that regulate radioactive cesium transfer from soils to plants.

Acknowledgments

This research was initiated under the guidance of the late Dr. Tanaka, Dr. Tadano and former Professor of Hokkaido University, Dr. Osaki.

Disclosure statement

No potential conflict of interest was reported by the author.

Additional information

Funding

This work was supported by the KAKENHI [19H01169];