Most of the variation for in situ exudates was explained by differences between exudates collected in clay, compared with other conditions, which is evident from a principal component analysis . Similarly, in pairwise comparisons, clay-collected exudates showed the most distinct metabolites , followed by 250 µm sand-collected exudates with 5%–18% distinct metabolites . In contrast to in situ exudates, in vitro exudates exhibited similar metabolite profiles when analyzed with a principal component analysis , and fewer metabolites had statistically significant abundances in pairwise comparisons . Notably, in vitro exudates of clay-grown plants showed a comparable number of distinct metabolites in pairwise comparisons with plants grown in other substrates, suggesting that the in situ differences observed between exudate profiles of clay grown plants and plants grown in other conditions resulted from the presence of the clay, and not from an altered plant metabolism.To further investigate differences in in situ-collected exudates, exudate profiles of the groups by their particle sizes and “big beads,” “small beads,” “big sand,” and “small sand” were compared . In a principal component analysis, exudate profiles of hydroponic and “big beads” exudates overlapped, whereas “big beads” versus “small beads” and “big sand” versus “small sand” separated . Pairwise comparisons showed thirteen distinct metabolites between “big beads” and “small beads,”vertical indoor farming and three distinct metabolites between “big sand” and “small sand.” Two thirds of metabolites were more abundant in “big beads” than “small beads,” among them nucleobases and derivatives, as well as organic acids. Four phenolic acids were more abundant in “small beads” versus “big beads.” In “small sand,” two nitrogenous compounds were higher abundance than in “big sand,” a nucleobase, , and an amino acid derivative .
To further examine the differences between clay-grown and hydroponically grown in situ exudates, a multi-variant test was used to compare metabolite abundances between the two conditions. Most of these metabolites were nitrogenous, with more than half containing a heterocyclic nitrogen group. Among these metabolites were nucleic bases, nucleosides and derivatives with acidic groups, amino acids with acidic and/or basic groups, and linear as well as phenolic organic acids. Two nitrogenous metabolites, an organic acid and choline-O-sulfate with an acidic and a basic group, were more abundant in clay-collected exudates . These compounds were not detected in exudates of hydroponically grown plants, in in vitro-collected exudates of clay-grown plants, or in clay control samples without plants , which suggests that these compounds were released from clay only in the presence of plants.Since clay particles were found to strongly sorb exudate metabolites, we wondered whether the sorbed metabolites were accessible to a plant-associated bacterium, supporting microbial growth. Thus, we first determined the desorption rate of metabolites from the various substrates by determining the metabolite recovery rate from glass beads, sand, and clay incubated with defined medium. As shown previously , the metabolite recovery rate was comparable between the no substrate control and glass beads , lower in sand , and the lowest for clay . The metabolite recovery from washes was 4%–14% for all substrates, indicating all substrates had similar desorption rates. Growth of the rhizobacterium Pseudomonas fluorescens on sand or glass beads pre-incubated with defined medium resulted in the same optical density change as growth on particles pre-incubated with water , indicating that these substrates did not retain metabolites supporting growth. Incubation of the bacterium with clay pre-incubated with defined medium however did result in bacterial growth. As the control incubation of clay with defined medium also showed a small increase in OD, presumably as a result of fine particles, the data presented in Figure 6 are normalized by no-bacterial clay control samples .
These data show an increase in OD of bacteria grown on clay pre-incubated with defined medium, indicating that the bacteria are able to utilize the sorbed metabolites for growth. As an additional control experiment, the pre-incubated clay was incubated with water for three days under sterile conditions , allowing for desorption of metabolites from clay particles. The supernatant was subsequently pipetted into a new well, and bacteria were added and allowed to grow for another three days. This experiment resulted in no-bacterial growth , suggesting that bacterial presence is needed to desorb metabolites from clay particles. We conclude that this particular rhizobacterium is capable of desorbing exudate metabolites from clay to support growth.Growth of B. distachyon in particles with different sizes resulted in various morphological changes. A decrease in particle size resulted in decreased root weight, total root length, and in total root number, although the last parameter correlated less strongly . Root weight correlated positively with shoot weight, total root length, and total root number, indicating a dependency of the different parameters. Notably, the morphology of B. distachyon grown in glass beads or sand was not directly comparable: Plants grown in 5-µm sand had higher root weight and total root length than plants grown in 0.5-mm glass beads. The three-dimensional particle arrangement and other differences between the substrates, such as texture, might account for root morphological differences observed between glass bead and sand-grown plants. For glass bead-grown plants, the reduction in total root length was caused by a reduction in second-order root length, whereas the primary order root length was reduced in all sizes smaller than 3 mm. These trends for reduced root weight and root length but not root number are in line with observations made for maize grown in 1 mm versus hydroponic conditions . However, these previous studies noted an even larger decrease in shoot than in root weight, whereas B. distachyon shoot weight did not change significantly in our experimental conditions. Similar results were found for lettuce grown in three different soils, where root fresh weight and morphology changed, but shoot weight was not affected . The constant B. distachyon shoot weight might indicate sufficient nutrient uptake even by smaller root systems in the environments investigated. Thus, in future studies, it might be interesting to evaluate how the different root systems as generated herewith different substrate sizes further respond to altered nutrient levels.
One could expect an additional change in root morphology with different nutrient starvation conditions, for example, root systems optimized for phosphate scavenging form numerous, short lateral roots, whereas roots optimized for nitrogen uptake exhibit fewer, but long lateral roots . Phosphate movement is hindered by particles with a charged substrate, whereas nitrate movement is less affected by soil chemistry . Thus, changes in root morphology of plants grown in phosphate-limited clay might be distinct from plants grown in phosphate-limited sand or glass beads. Plants grown in soil may exhibit additional changes in root morphology and metabolism, as shown for B. distachyon grown in a sterile soil extract, which showed reduced root length, and elongated root hairs and which depleted a variety of metabolites from soil extract . Root systems further respond to local alterations in soil structure, such as to the presence of micro- or macropores, or to air pockets . Investigations of local root morphology responses in heterogeneous settings with multiple, defined substrate sizes and chemistries will thus shed more light onto how plants respond to soil physiochemistry on a spatial and time scale. Multiple systems exist in which such experiments could be attempted, ranging from EcoFAB model systems to rhizotron designs .B. distachyon exhibited significantly altered root morphology when grown in particles with various sizes, with root weight, and root lengths differing between conditions. The exudate profile however was very similar for these plants when collected in vitro , and exudate extraction volumes were normalized by root fresh weight before measurement. Thus indicating that exudation per root fresh weight is constant. As root weight correlated with both, total root length and with total root number, an additional method was needed to determine whether the number of roots or the root length was important for exudation. In the literature, root tips are often mentioned as predominant sites of exudation for several reasons: a) cell wall suberization of this young tissue is still low , b) exudates have been imaged around root tips , and c) more microbes associate with tips compared with other root sections . Few studies exist investigating spatial patterning of exudation, but some examples suggest that other tissues besides root tips might be involved in exudation. For example, vertical growing towers the localization of the malate transporter ALMT1 in Arabidopsis is confined to the root tip in untreated roots, but expands to the entire root system when treated with an activator, aluminum . This suggests differential malate exudation from different parts of the root, depending on the environment. Similarly, strigolactone exudation is environment-dependent, with its transporter PDR1 expressed in single cells along most of the roots . In addition, microbes do not only colonize root tips, but also prominently sites of lateral root emergence, and are found throughout the root system of plants . Distinct microbial populations, associated with B. distachyon seminal and nodal roots, as well as for nodal root tips versus nodal root bases , could be influenced by differential exudation by these organs. We used mass spectrometry imaging to investigate exudation across roots. These data cannot directly be compared to the root morphology and LC/MS data for technical reasons and the fact that the exudates were collected from three-week-old plants, whereas the imaging experiment was performed with seedlings due to technical limitations. Some ions were observed to be most abundant around the root tip, whereas others were also found in the root elongation and maturation zone, or all along the root axis. In addition, some ions were detected on the root itself, which could mean that they are part of the cell wall, or that they have a low diffusion speed.
Despite these limitations, our data suggest that root exudation is a spatially complex process. Exudation might take place in different ways: Root tip-exuded metabolites might diffuse, due to the absence of Casparian strips or secondary cell walls, or might be actively transported. Metabolites exuded from older root tissues are more likely to be transported, either by channels facilitating diffusion, or by active transport proteins. Future studies are needed to investigate the role of various root zones in exudation to determine which tissues are involved in exudation of various compounds and if exudation differs between root types.Root exudate metabolite profiles were unaltered when plants were grown in different particle sizes. As the root weight, root number, and root length correlated and the exudation of compounds was spatially complex, we conclude that exudation profiles are similar across different root morphologies. However, these investigations were limited and may be better informed by comparison of exudation profiles in plants with more radically altered root morphologies, in plants without secondary roots or root hairs. Exudation profiles were also comparable between plants grown in clay, sand, or glass beads, when collected in vitro. This suggests that the physiochemical environment does not alter plant metabolism, as long as other factors such as nutrient levels, light intensity, and humidity, remain unchanged. However, the exudate metabolite profile of clay- versus sand- or glass bead-grown plants was clearly different for in situ exudates . A recent study found differences in sorghum exudates of plants grown in clay, sand, and soil . In this study, exudates were collected from roots with rhizosphere substrate still attached. The largest difference in this dataset was observed between soilgrown and sand- or clay-grown plants, which might be explained by soil-derived metabolites co-extracted with root exudates . The authors showed some ions to be specifically up- or down-regulated in exudates of clay- versus sand grown plants, but their effect was not strong enough to separate the two conditions in a principal component analysis . This may be explained by their exudate collection method, which was a mixture between the in situ and in vitro conditions utilized here. Recently, it was suggested that root tips might detect the concentration of rhizosphere metabolites, altering root morphology and exudation accordingly . Thus, clay-grown plants should exhibit an altered root morphology compared to hydroponically grown plants, as clay sorbs a significant amount of exudates, changing the metabolite concentration around the root tip. However, the root morphology of clay-grown plants is statistically not different from hydroponically grown plants .