Root uptake under mismatched distributions of water and nutrients in the root zone

Most plants derive their water and nutrient needs from soils, where the resources are often scarce, patchy, and ephemeral. In natural environments, it is not uncommon for plant roots to encounter mismatched patches of water-rich and nutrient-rich regions. Such an uneven distribution of resources necessitates plants to rely on strategies that allow them to explore and acquire nutrients from relatively dry patches. We conducted a laboratory study to provide a mechanistic understanding of the biophysical factors that enable this adaptation. We grew plants in split-root pots that permitted precisely controlled spatial 5 distributions of resources. The results demonstrated that spatial mismatch of water and nutrient availability does not cost plant productivity compared to matched distributions. Specifically, we showed that nutrient uptake is not reduced by overall soil dryness, provided that the whole plant has access to sufficient water elsewhere in the root zone. Essential strategies include extensive root proliferation towards nutrient-rich dry soil patches that allows rapid nutrient capture from brief pulses. Using high-frequency water potential measurements, we also observed nocturnal water release by roots that inhabit dry and nutrient10 rich soil patches. Soil water potential gradient is the primary driver of this transfer of water from wet to dry soil parts of the root zone, which is commonly known as hydraulic redistribution (HR). The occurrence of HR prevents the soil drying from approaching the permanent wilting point, and thus supports root functions and enhance nutrient availability. Our results indicate that roots facilitate HR by increasing root-hair density and length and deposition of organic coatings that alter water retention. Therefore, we conclude that biologically-controlled root adaptation involves multiple strategies that compensate for 15 nutrient acquisition under mismatched resource distributions. Based on our findings, we proposed a nature-inspired nutrient management strategy for significantly curtailing water pollution from intensive agricultural systems.

Rhizosheath, i.e. the combination of root tissues and sand-covered on the root surfaces, from another replicate of each treatment was preserved with minimum agitation, for microscopic analysis. Confocal images were obtained using a Zess LSM 880 Airyscan confocal microscope and EC Plan-Neofluar 10x/0.30NA objective lens (Carl Zeiss Microscopy LLC, White Plains, NY). 405 nm and 488 nm lasers were used to excite and acquire autofluorescent compounds from the roots. T-PMT detec-90 tor was used to acquire transmitted light images. SEM images were taken at 3 kV after coating with gold (E5000 Sputter Coater, Quorum Technologies Ltd, East Sussex, UK) using ZEISS GeminiSEM 500 scanning electron microscope (Carl Zeiss Microscopy LLC, White Plains, NY). Image analysis and processing was done using ImageJ (Schneider et al., 2012).

Statistical and data analysis
Plant physiological indicators were compared across treatments using a Welch's analysis of variance (ANOVA) (Welch, 1947) 95 and posthoc Games-Howell test for multiple comparison from R (Games and Howell, 1976).
The water retention curve of silica sand mixed with nutrient solution (520 mgN/L, a concentration that is consistent with the pore water in the dry compartment of treatment D) was determined by water potentiometer (WP4C of Meter, Pullman, 100 WA). Rhizosphere wetting was calculated by subtracting minimum daily rhizosphere water content from the subsequent daily maximum values. More detail of the calculation is provided in supplemental information.

Plant physiological characteristics
In the primary treatment of our experiment (labeled D) ≈ 90% of the irrigation water was applied to one compartment of the 105 pot, while the other received 100% of the nutrient supply delivered along with the remaining 10% water. In a control experiment (labeled C1), we added 100% of the nutrients to the wet compartment of the split-root pots, while the dry compartment received the remaining 10% of the water without nutrients. In a second control experiment (labeled C2), both compartments received equal amounts of water and nutrients (See Figure S1). We used nutrient-free sand as a growing medium in order to fully constrain nutrient availability to the targeted region. 110 We measured a series of indicators to assess whether mismatched resource distributions influenced plant performance. Critical indicators of above-ground plant performance, including total above-ground biomass, fruit mass, number of flowers, N uptake by total biomass and fruits, and N use efficiency (NUE) are reported in Figure 1. Additional measures of plant performance, including N distribution and leaf greenness within individual plant canopy are reported in Figure S2. The latter was evaluated in terms of normalized difference vegetation index (NDVI) captured by hyperspectral analyses of leaf samples.

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Considerable variability in N concentration and NDVI was observed within each plant, with younger leaves at the top of the canopy having higher N and NDVI than the oldest leaves at the base of the canopy ( Figure S2). However, there were no signif-4 https://doi.org/10.5194/bg-2020-109 Preprint. Discussion started: 4 May 2020 c Author(s) 2020. CC BY 4.0 License. icant differences at the whole-plant scale in all the indicators we measured (p > 0.05; Table A1). These findings unequivocally demonstrated that mismatch of spatial distributions of water and nutrients does not have a measurable effect on the above ground measures of performance, provided that both resources are available in equal amounts.

Plant root distribution and rhizosphere characteristics
The pattern of root proliferation is often associated with localized root feedbacks to the spatial distributions of water or nutrients within soil profile (Robbins and Dinneny, 2018;Fan et al., 2017;Boyer et al., 2010;Orosa-Puente et al., 2018). However, how and to what extent mismatched water and nutrient distributions affect root architecture is mostly unknown. In Figure 2 (a), we provide a visual demonstration of the root proliferation during a growth period when 100% of the nutrients are isolated 125 from 90% of the irrigation water (treatment D). It is remarkable that the density of the roots in the wet and dry compartments are indistinguishable, despite the vast disparity in water availability. The soil that surrounded the roots was carefully extracted using a pipette tip connected to a vacuum. Therefore, the three-dimensional root architecture is not apparent in Figure 2 (a) Quantitative comparison of the root mass distribution between the two compartments and among the treatments is provided in Figure 2 (f-h) and Table A2. The soil in both compartments of selected replicates was excavated at depth intervals of 2 cm, drained moisture at the base of the compartment. In contrast, the roots grown in the nutrient-rich dry compartment were concentrated in the mid-section, coinciding with the depth at which nutrient solution was supplied using a subsurface injector.
Overall, the nutrient-rich dry compartment accounted for 60% of the total root biomass.

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The root density in the nutrient-rich wet compartment of the first control treatment C1 increased with depth, with a notable accumulation of root biomass at the base. This accumulation suggests that in addition to the accumulation of drained water, Replicates are shown in different colors.
leaching must also have resulted in a substantial accumulation of plant-available nutrients at the base of the compartment. It is important to recall that had this been an open profile, a portion of the nutrient supply could have leached below the rooting depth. The root growth in the nutrient-free dry compartment was stunted and accounted for only 20% of the total root biomass.

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There was no difference in root density and distribution between the two compartments of the second control treatment C2, where water and nutrient were supplied equally to both compartments.
The roots in all the treatments exhibited the formation of rhizosheath, which is often associated with soil binding by either root exudates or root-hairs and is credited for the facilitation of water and nutrient extraction (Pang et al., 2017;Watt et al., 1994;Albalasmeh and Ghezzehei, 2014). There were no visual differences in the appearance and abundance between the rhizosheaths 145 formed in the wet and dry compartments or among treatments. However, closer inspection under Scanning Electron Microscope (SEM) revealed significantly denser root hairs in the dry compartment of treatment D. This observation is consistent with the emerging consensus on the importance of nutrients in regulating the growth and development of root hairs (Zhang et al., 2018).

Plant water and nutrient uptake dynamics
The above observations show that the adaptation of the root characteristics ( Figure 2) explains how plants respond to mis-150 matched water and nutrient distributions without incurring performance loss ( Figure 1). However, understanding the mechanism of nutrient uptake from dry soil patches requires a closer look at the dynamics of soil water content and water potential in the wet and dry compartments. In Figure 3, a snapshot of typical soil water content dynamics over one week is shown (see Figure S3 for complete dataset). The wet compartments of treatments D and C1, as well as both compartments of treatment C2 remained comparably wet because of the frequent irrigation. However, there was a considerable difference in water content 155 dynamics between the dry compartments of treatments D and C1. Specifically, in the presence of nutrients, the water content in the dry compartment of treatment D was depleted within one day after each application of nutrient solution (weekly to bi-weekly interval). Whereas the water content of the nutrient-free dry compartment of treatment C1 declined slowly over a  week. This difference in water uptake rate is consistent with the root density differences between the dry compartments shown in Figure 2 (f) and 2 (g).

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The soil water potential data (Figure 4 (a)) was converted to rhizosphere water content dynamics (Figure 4 (b); see Figure   S5 for complete dataset) using a soil water retention curve ( Figure S4 and Table S1). The effects on soil water potential from increasing nutrient concentrations were considered by using pore water with appropriate nutrient concentrations. Close inspection of the HR water content dynamics reveals that HR does not significantly contribute to transpiration at the wholeplant scale, given that the small volume of water is reversibly taken up the roots. However, it can be essential in enhancing root 165 survival and growth (Boyer et al., 2010;Bauerle et al., 2008), as well as serving as a critical carrier for nutrient acquisition from dry soil patches. Furthermore, the preferential proliferation of root hairs in the nutrient-rich dry compartment suggests that HR was an essential factor in creating a habitable environment.
The water content dynamics data can be utilized to infer the quantity of water that is released during each episode of HR.
In Figure 4 (c) we show the increase soil moisture across all five working sensors (reported as an equivalent soil moisture depth in the 50 mm soil intervals where maximum root density was observed). The magnitude of HR remained consistent for the most part of the study, with slight increases observed in the first few days after each injection of nutrient solution. These data can also be used to analyze the role of rhizosphere water status on the magnitude of HR (Meinzer et al., 2004;Prieto et al., 2010;Scholz et al., 2008). We observed that the magnitude of HR outflows to be inversely correlated with rhizosphere water potential (p < 0.05 in Figure 4 (d)). This demonstrates that the rhizosphere soil under drier conditions exerts the larger appears that the elevated HR dynamics we observed were actively facilitated by root activities.

Discussion
Plant responses to water and nutrient stresses have been the subject of extensive research in ecological and agricultural settings (Robinson, 1994;Jackson et al., 1990). Typically, these stresses are considered individually or as compounded factors.

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However, how plants cope with mismatched distribution of water and nutrients in the soil profile is rarely examined. We did not observe a measurable difference in reproductive success (number of flowers and fruits) and nutrient acquisition between unstressed plants and plants that experienced severe partial-root nutrient and water stresses. While rapid nutrient capture from brief pulses, i.e. intermittent wetting, could have been essential in plant performance, it is important to note that the plants subjected to mismatched resource allocation derived all their nutrient uptake from a soil patch that persistently remained dry at 190 −900 to −500 kPa (85% of growing time), which corresponds to residual moisture status for the coarse sandy soil used in this experiment. Thus, we can confidently conclude that plant performance is less sensitive to localized water and nutrient stresses provided that both resources are available in sufficient quantities within the rooting zone.
Therefore, we can safely assert that plants subjected to mismatched resource distribution employed strategies that are distinct from plants grown under uniform resource availability. Specifically, we suggest a three-part mechanism that appears to be at 195 play, which are schematically illustrated in Figure 5.
First, roots proliferated in dry soil patches provided that the available nutrients are constrained in the dry patches and water is available in sufficient quantity elsewhere (Figure 2 (f)). The root proliferation is the prerequisite for the rapid nutrient capture in such a short time window, i.e. 15% of the growing period. Moreover, multi-scale signaling and feedbacks appear to be involved. The marked differences in root allocations between the two compartments of the three treatments, despite having Second, roots appear to rely on hydraulic redistribution (HR) to maintain substantial root biomass in dry soil patches (Boyer et al., 2010) as well as to facilitate transport and uptake of nutrients (Figure 4 (a-c)). The roots grown in dry nutrient-rich 205 patches also appear to be more vigorous than roots grown in nutrient-free dry patches as evidenced by the marked difference in drying after intermittent wetting events (compare Figure 3 (a) and 3 (b), respectively). However, despite the fast initial decline in water content, HR prevented the water potential from ever approaching permanent wilting point (−1500 kPa) as shown in 10 https://doi.org/10.5194/bg-2020-109 Preprint. Discussion started: 4 May 2020 c Author(s) 2020. CC BY 4.0 License. Figure 4 (a). This suggests that HR helps maintain and rejuvenate root activities until conditions of nutrient uptake become favorable (Bauerle et al., 2008).

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Third, HR appears to be induced or accelerated by the action of roots. Absence of HR in treatment C1 given identical partitioning of water between the two compartments supports the suggestion that HR is not as previously suggested a "sweet accident" passively regulated by physical conditions of the environments, but a biologically-mediated feedback process triggered by mismatched distribution of water and nutrients (Matimati et al., 2014). Primarily, drying of the rhizosphere soil in the dry compartment by root uptake provides the necessary water potential gradient to pull water from the wet compartment.

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However, drying also counters HR by dropping hydraulic conductivity (van Genuchten, 1980), which restricts the ability of HR to be transported away from root surfaces. Therefore, rhizosphere hydraulic conductivity plays the dominant role in controlling HR, as evidenced by the positive correlation between water potential and HR shown in Figure 4 (d) (Meinzer et al., 2004;Prieto et al., 2010;Scholz et al., 2008).
In this study, two possible pathways allow roots to modify rhizosphere hydraulic properties. One is through emerging root 220 hairs that alters the soil porosity and connectivity at the root-soil interfaces. The other is through localized elevation of water retention around active roots by root-secreted organic materials (Carminati et al., 2010;Moradi et al., 2011;Albalasmeh and Ghezzehei, 2014). Therefore, we suggest that root morphological adaptation and exudation in nutrient-rich dry patches may play an integral part in HR-assisted sustenance of roots and nutrient acquisition. Fluorescence images of sand particles from the nutrient-rich dry compartments exhibited evidence of organic coatings that support this hypothesis ( Figure S6).

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It should be noted that the current study considered an ideal condition with a single plant species grown in the homogenous texture of sandy soils. Such conditions nearly never occur in natural systems due to the complexity of environmental factors and response variation between different plant species. Therefore, extensive studies beyond the controlled system involving different plant species and soil types are required for further mechanistic understanding.

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Our findings demonstrated that plants could utilize heterogeneously distributed resources without adverse impact on their performance, provided that these resources are present in sufficient quantities. Specifically, we showed the ability of plants to acquire 100% of their nutrient needs under the extreme mismatch of water and nutrient distributions. We provided multiple lines of evidence that suggest a successful adaptation to such an environment involves coordination between components of the root system that inhabit environments with contrasting resource availability. Critical to this mechanism is a reliance 235 on multiple strategies, including extensive root proliferation that allows rapid nutrient capture from immediate widows of availability under favorable moisture conditions, and sustained HR to support an active root system and facilitate nutrient transport under unfavorable or drought stress conditions. It appears that HR is biologically regulated when its occurrence supports the acquisition of nutrients. Regulating mechanisms include the enhancement of rhizosphere water retention but root exudation and development of dense and thick roothairs.

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The findings of this study, which was carried out under a highly controlled laboratory environment, sheds light on how plants thrive in regions where surface soil drying is typical, and water and nutrient availabilities are often spatially mismatched. As illustrated in Figure 5 (b), actively controlled HR employed by plant roots can support active, thriving root biomass in the shallow nutrient-rich soils. Furthermore, HR induced wetting can support microbial communities that sustain vigorous nutrient cycling in the otherwise dry soil layers. Facilitation mechanisms could include regulation of root exudation on microbial 245 activities (Williams and de Vries, 2020). This hypothesis is consistent with the frequent occurrence of HR in deep-rooted shrubs of arid and semi-arid regions (Kizito et al., 2007;Bogie et al., 2018). The fact that we observed HR in a shallow-rooted herbaceous plant (tomato) suggests that the mismatch of resources rather than climate and plant types are the primary drivers of HR.
The lessons gleaned from the above effective resource utilization strategies can serve as templates for highly efficient, 250 nature-inspired agricultural systems. The use of synthetic fertilizers, particularly N, played a significant role in boosting crop production (Tilman et al., 2002). While the NUE in many industrialized countries has been increasing at a modest rate, the yield gains achieved in most developing countries over the past half-century came at a significant decline in NUE (Zhang et al., 2015) and environmental and ecological degradations, including air and water pollution and the accumulation of potent greenhouse gases (Bowles et al., 2018). To meet the 2050 global food demand while safeguarding environmental quality would require 255 harvested N to increase by 45% while NUE to increase from 40% to 70% (Zhang et al., 2015). Therefore, economically affordable approaches to reducing N loss from agriculture are critical to achieving this goal. Our findings suggest that the colocation of nutrients and water, which is the main driver for N loss by leaching and volatilization, is not necessary to maintain productivity. Thus, spatially isolating the bulk of irrigation water from the applied N can be effective in drastically cutting N losses. We suggest one such approach, illustrated in Figure 5 (c), that uses existing technologies and minimal investment. This 260 approach involves applying irrigation water and fertilizers to alternating rows and capitalizing on the roots' ability to acquire both resources effectively.
Code and data availability. The data sets and R code were uploaded in Dryad with a doi:10.6071/M39M2T. The unpublished dataset and code for review was shared using this temporary link: click for data sets and code for review.