Category: hydroponic agriculture

Salinity stress is a major abiotic stress that affects plant growth, resulting in a loss of crop yield, especially rice, which is one of the most salt-sensitive plants in comparison to other cereals.Salt stress affects plants via both osmotic and ionic effects.Osmotic effects result in a reduction of water absorption ability such that the effects are similar to drought stress.Ionic stress causes Na+ toxicity, which disrupts photosynthesis, protein synthesis, and enzyme activity.Numerous reports have shown negative effects of salt stress on rice growth and productivity based on the total chlorophyll content, protein concentration, photosynthetic CO2 fixation, stomatal conductance, transpiration, shoot dry weight, tiller number per plant, spikelets per panicle, and grain yield.Ca2+ is a crucial second messenger consisting of a transient elevation of cytosolic [Ca2+].The Ca2+ signals are transduced and decoded via Ca2+ binding protein, and then the information is relayed to downstream responses.The signals are mainly transduced through kinases mediating the phosphorylation cascade, resulting in downstream response regulation, including changes in gene expression through the regulation of transcription factors.Calcium signaling is used to respond to environmental stimuli, as well as to coordinate growth and development in plants.In the plant calcium signal transduction process, calcium sensors, including calmodulin ,hydroponic nft channel calcineurin B-likeprotein and Ca2+-dependent protein kinase , play important roles in the transduction of various stimuli.CaM is a protein that contains characteristic EF-hand motifs that bind Ca2+ ions with high affinity and specificity.CaM binding to Ca2+ leads to the exposure of hydrophobic regions on the molecule surface and subsequent interactions with target proteins or nucleic acids.

Rice carries 5 CaM-encoding genes: OsCam1–1, OsCam1–2, OsCam1–3, OsCam2 and OsCam3.The expression of OsCam1–1 increases to a great extent in response to NaCl, mannitol and wounding treatment.Several lines of evidence have revealed that calcium sensors are involved with an enhanced abiotic tolerance capacity in plants.Evidence has shown that the constitutive expression of bovine calmodulin in tobacco results in a shortened germination time of transgenic tobacco seeds under salt stress.Arabidopsis over expressing GmCaM4exhibit increased expression of AtMYB2-regulated genes, including proline-synthesizing enzymes, suggesting that this feature confers salt tolerance to the transgenic Arabidopsis by enabling the accumulation of proline.Our previous report have shown that transgenic rice over-expressing OsCam1–1 grow better under salt stress than wild type.Wu H.and colleagues have found that the biphasic Ca2+ signal and enhancement of OsCam1–1 expression in rice cause heat stress-mediated expression of downstream heat shock-related genes, and OsCam1–1 over expression Arabidopsis are more tolerant to heat stress than its wild type.In another report, AtCam3 knockout mutant Arabidopsis showed a clear reduction of thermotolerance after heat treatment at 45 °C, and when AtCam3 was over expressed in mutant and wild type Arabidopsis, the thermotolerant ability was rescued and increased, respectively.Moreover, co-expression of some heat shock protein genes with AtCaM3 suggested that AtCam3 plays a key role in the Ca2+-CaM heat shock transduction pathway.The versatile functions of CaM are interesting, especially the role in the regulation of gene expression.CaM proteins directly modulate transcription factors , and some of these TFs have been verified to play roles in stress signaling pathways; however, the Ca2+ and Ca2+/CaM-regulating TF mechanisms remain incompletely understood and require further investigation.The aim of the present study was to investigate the downstream effects of OsCam1–1 over expression on gene expression regulation in rice under salt stress using a transcriptomic approach and to identify the interacting proteins to elucidate the role of OsCam1–1 in the salt stress response mechanism.

CaM is a multifunctional protein that regulates the activities of numerous target proteins.Genome-wide analysis techniques such as transcriptome profiling are particularly suitable for identifying the downstream components that are potentially regulated by CaM.In our previous report, rice over expressing OsCam1– 1 showed a significantly higher relative growth rate than wild type when grown under salt stress.Here, transcriptome profiling of the 3-week-old rice leaves of transgenic rice over-expressing OsCam1–1and its wild type under normal condition and salt stress conditions for 4 h was conducted.More than 185 million reads from eight libraries from single-end RNA-Seq by Illumina Hi-Seq 2000 were obtained, with a total read of each library between 22 and 25 million reads.The reads were processed by POPE, which provided a total clean read per library of more than 99% of the total reads.At least 93% of the clean reads were mapped to the rice genome reference, Michigan State University rice annotation project’s MSU7 and less than 11% of the clean reads were multiple alignment reads.To compare the transcriptome profiles of the rice, differential gene expression analysis of the transcriptome data using DESeq was carried out, which provided the number of differentially expressed genes summarized in Table 2.Analysis of the wild type identified 12,184 DEGs between the transcriptome profile under normal and salt stress conditions , in which 5842 and 6342 genes were up-regulated and down-regulated, respectively.For transgenic rice over-expressing OsCam1–1, comparisons between normal and salt stress conditions revealed a total of 13,259 DEGs with 6434 and 6825 up-regulated and down-regulated genes, respectively.Furthermore, the transcriptome profiles of the transgenic rice were compared with those of the wild type.Under normal conditions , 2022 DEGs were identified, with 892 and 1130 DEGs expressed at higher or lower levels in the transgenic rice, respectively.Under salt stress, comparisons of transgenic rice with wild type rice revealed 1677 DEGs, with 957 and 720 DEGs expressed at higher or lower levels in the transgenic rice, respectively.The scatter plots showed quantitative overview of the four transcriptome profile comparisons.

OsCam1–1 was found to be highly expressed in transgenic rice under both normal and stress condition, with an average RPKM of 1758.67 and 1644.62, while the average RPKM of wild type under normal and stress conditions was 91.94 and 97.84, respectively.The expression of OsCam1–1 in the wild type was not induced at 4 h after salt stress , in good agreement with a previous study.According to a gene expression study conducted by Chinpongpanich et al., the transcript level of OsCam1–1 determined by qRT-PCR was highly induced at 1 h after 150 mM NaCl treatment and then sharply decreased after 1 h.This result validated the over expression of OsCam1–1 in transgenic rice with an approximately 18-fold change in RPKM compared with wild type.Based on a differential transcriptome analysis, the gene expression levels of those 2022 and 1677 DEGs were thus likely affected by OsCam1–1 over expression.In our previous report, OsCam1–1-overexpressing lines showed a significantly higher relative growth rate than wild type when grown under salt stress.Based on the genes identified herein, among which several were involved in central energy pathways, sucrose and starch levels were determined in the three independent lines under normal and salt stress conditions at day 3 and 5 after treatment.Salt stress led to a significant reduction of the starch level and slightly decreased sucrose levels in both wild type and transgenic rice lines.Noticeably, at day 3, the transgenic lines could maintain the sucrose and starch levels better than the wild type under salt stress conditions.At day 5, the trends observed for sucrose and starch levels in transgenic rice under salt stress conditions were similar to those in wild type.In addition, the photosynthesis rate , stomatal conductance , intercellular carbon dioxide and transpiration rate were examined in the transgenic rice over-expressing OsCam1–1.Under salt stress, Pn, gs and E decreased at both day 3 and day 5, while Ci decreased slightly at day 3 of treatment.Interestingly, transgenic rice had slightly lower Pn values than wild type rice at both day 3 and day 5 and tended to have lower gs and E values at day 5 of salt stress treatment.In contrast, the Ci measurements did not reveal significant difference between the transgenic and wild type.For FV′/FM′, which reflects the maximum efficiency of photosystem II, no change was observed under the given salt stress conditions,nft growing system and the transgenic rice did not exhibit difference either under normal or salt stress conditions compared with the wild type.In a previous study, the northern blot results showed the highest expression levels of OsCam1–1 in transgenic rice line L1 among the three transgenic rice lines.Under both normal and salt stress conditions, the sucrose and starch content correlated with the expression level of OsCam1–1 in those transgenic rice lines.CaM does not possess functional domains other than EF hand motifs, so it functions by binding to and altering the activities of various interacting proteins.To understand how CaM1 mediates Ca2+-signal responses, its specific interacting proteins were identified using a cDNA expression library with 35S-labeled rOsCaM1 protein as the probe.The purity of the prepared 35S-labeled rOsCaM1 protein was examined by SDS-PAGE.To test its specificity, PVDF membrane spotted with various amounts of CaMKII peptide, calcineurin, and BSA was incubated with the probe.The autoradiograph showed that the probe only interacted with CaMKII peptide and calcineurin but not BSA, and the intensity of the signal on the X-ray film was dose-dependent.The results indicated that the 35S-labeled rOsCaM1 protein could specifically bind to well-known target proteins in the presence of Ca2+.After screening the cDNA library, the purified clones from the tertiary screening were titered before performing single-clone excision.As a result, 10 distinct positive cDNA clones were obtained.All unique pBlue script SK plasmids obtained from the single-clone excision were sequenced to determine the cloned cDNA insert sequences.The resulting sequences were BLAST searched against the Rice Genome Annotation Project and the Rice Annotation Project databases.The functions of 8 OsCaM1 targets were identified , which were diverse and potentially involved in various cellular processes, including metabolism, transcription, movement of organelles and vesicles, membrane transport, and signal transduction.Four known CaM-binding proteins previously identified in other plants were obtained from this screening, which included a cyclic nucleotide-gated ion channel , a glutamate decarboxylase, a CaM-binding transcription activator, and a kinesin motor domain-containing protein.The six identified putative novel CaM1-binding proteins comprised a transferase family protein , a response regulator receiver domain-containing protein , a lipin , a myosin heavy chain-containing protein , and two proteins with unknown function, LOC_Os08g34060 and LOC_Os02g13060.Interaction of eight putative target proteins with OsCaM1 was confirmed by protein blot analysis.

The mitogen-activated protein kinase cascade is a highly conserved central regulator of diverse cellular processes.CaM plays role in the MAPK/MPK cascade by binding to mitogen-activated protein kinase and/or mitogen-activated protein kinase phosphatase.A rice MAPK, BWMK1 encoded by an HT salt-responsive DEG, could phosphorylate the OsEREBP1 transcription factor for binding to the GCC box element , which is a basic component of several pathogenesis-related gene promoters.Inositol 1,3,4-trisphosphate 5/6-kinaseencoded by two HT salt-responsive DEGs, phosphorylates inositol 1,3,4-trisphosphate to form inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4,6, tetrakisphosphate, which are ultimately converted to inositol hexaphosphate and play roles in plant growth and development.In rice, the T-DNA mutant of an IPTK gene showed reduced osmolyte accumulation and growth under drought conditions, and some genes involved in osmotic adjustment and reactive oxygen species scavenging were down-regulated.In addition, over expression of DSM3resulted in a decrease in inositol trisphosphate , and the phenotypes were similar to the mutant under salt and drought stress conditions.These findings suggested that DSM3 might play a role in fine-tune balancing the inositol phosphate level when plants are exposed to stress or during development.Diacylglycerol kinaseen coded by an HT salt-responsive DEG, catalyzes the conversion of diacylglycerolto phosphatidic acid, and PA plays a role in the stress signaling pathway, including the MAPK/MPK cascade.A report has shown that the expression of OsBIDK1 encoding rice DGK is induced by benzothiadiazole and fungal infection.Moreover,transgenic tobacco constitutively expressing OsBIDK1 was more tolerant to plant pathogenic virus and fungi.These findings suggest that several genes in the signaling process might be enhanced by OsCam1–1 under salt stress.Interestingly, a universal stress protein gene was identified as an HT salt-responsive DEG.Evidence has shown that the expression of tomato USPis induced by drought, salt, oxidative stress and ABA, and over expression of spUSP improves tomato drought tolerance via interactions with annexin, leading to the accumulation of ABA.In addition, a xylanase inhibitor protein gene , was highly expressed and induced by salt stress and OsCam1–1 over expression.A previous report has shown that OsXIP can be induced by methyl jasmonate and wounding, so it was suggested that OsXIP may play a role in pathogen defense.As many OsCam1–1 and/or salt stress affecting DEGs involve both biotic and abiotic stresses, OsCam1–1 may be a component that mediates the crosstalk of biotic stress and abiotic stress responses.

Developing detailed spatial below-ground information about both plant and microbial location was considered to be both a priority and a major hurdle by many attendees.Current approaches include infrared, X-ray, chemical sensing, and acoustic imaging although all have limitations.The EcoPOD provides an opportunity to collaborate with teams who are developing these methods, such as those at the Danforth Center and the ARPA-E TERRA program, to test existing methods over a range of soil types and conditions such as temperature and soil saturation, as well as develop new methods.The EcoPOD user has access to the full depth of the soil column.This gives an opportunity to place sensors through the column.Many sensors exist, but may benefit from miniaturization, as space is still relatively limited in the EcoPOD quadrants.This is a good opportunity to develop collaborations with both academia and industry partners for the engineering expertise needed to miniaturize sensors.The group noted that above ground phenotyping and sensing is relatively advanced, primarily due to the physical ease of access.As a result, there are many more field derived datasets to compare to experiments performed in the EcoPOD.This will be useful for bench marking.In particular, multiple participants raised concerns about whether the artificial lighting was sufficient.Previous Ecotron efforts have often been criticized for the poor quality lighting but recent advances in LED lighting may resolve much of this and the EcoFAB team has recently found that they can simulate field lighting conditions using new PHYTOFY RL LED lights which are tunable in 6 different spectra from UVA to far red that could also be used in EcoPODs.Collecting sensor and imaging data on aerial growth of plants under a set of standard conditions in a defined field soil should be a priority.

There has been much recent interest in predictive phenotyping, where data on young plants is predictive of mature traits such as yield and grain quality.This has been developed in field systems,ebb and flow trays including via Advanced Research Projects Agency–Energy funded projects such as Transportation Energy Resources from Renewable Agriculture , but the EcoPOD would allow further interrogation of these models, in particular the response to individual climatic parameters e.g., drought vs.temperature.The addition of EcoPOD capabilities to bridge the gap between small-scale EcoPOD and large scale field capabilities will enable scientists to address critical DOE missions in energy and the environment.For example these capabilities will enable elucidation of molecular mechanisms by which microbial communities and abiotic constraints control key geochemical processes such as soil carbon cycling.They will also enable rapid development and translation of beneficial microbial communities from benchtop to field applications to support efforts in sustainable bio-energy and bio-products.Importantly, the containment and control afforded by EcoPODs and EcoFABs will enable pioneering studies in secure bio-systems design to provide key insights into the persistence, fate, and control of engineered microorganisms within soil micro-environments.Finally, there are opportunities to collaborate with other fabricated ecosystem projects at various scales across the globe.There was a lot of enthusiasm from the participants about using the EcoPODs for experiments that are challenging to do in the field due to regulatory or safety concerns.Examples include understanding the persistence and fate of engineered microbes within contained and controlled environments to identify risks and effective containment strategies.It was also noted that it’s an opportunity to monitor the effects of potentially environmentally hazardous materials such as plastic microfibers or carbon nanotubes.There was also interest from National Labs with secure facilities who could adapt technologies such as the EcoBOT, a robot that enables automated EcoFAB experiments, and EcoPOD, once they are derisked and developed further, for work which requires secure facilities that are not available at Berkeley Lab.

Due to the breadth of scientific background among our workshop participants, many great experimental ideas were discussed including topics such as carbon sequestration experiments, for which the EcoPOD’s semi-closed system can facilitate mass balance calculations easier than in field experiments.Furthermore, deep soil processes were discussed as most data from soil experiments does not exceed the top 10 cm.Additionally, soil atmosphere gas exchange was mentioned, which we will keep in mind as we are aiming to develop future prototypes that can provide gas-tight conditions.Similarly, climate change simulations that include warming or elevated CO2 concentrations are a future experimental goal that will require prototype updates that are achievable.All participants were excited about the possibility of separating individual environmental parameters to observe their effect on the plant-microbe-soil atmosphere ecosystem.This includes being able to better predict the phenotype of engineered bio-energy crops under different environmental conditions, reducing the need for expensive and complex multi-site field trials.The EcoFAB team has made access to the units purchased by Berkeley Lab a priority.For example, detailed protocols on how to fabricate them have been published , and TEAMS, a project funded by DOE, is dedicated towards the dissemination of EcoFAB supplies and protocols including model micro-biomes to foster interlaboratory science and experimental standards.Through these efforts, laboratories across the globe are now using Berkeley Lab-designed EcoFABs as well as developing their own iterations, with a new EcoFAB ring-trial study with B.distachyon and a synthetic microbial community planned for early 2021.As discussed above, EcoFABs and EcoPODs are complementary technologies with different strengths and weaknesses.One way Berkeley Lab is leveraging this fact is through the use of higher throughput EcoFABs experiments to assess important parameters that can later be implemented within EcoPODs.

The EcoBOT being developed at Berkeley Lab through the Trial Ecosystems for the Advancement of Microbiome Science project will support remote, high throughput EcoFAB experiments to improve to improve turnaround, standardization, and reproducibility for EcoFAB experiments.ESM analyses are typically performed to evaluate and optimize space mission payloads to minimize launch costs as a function of mass, volume, power, cooling, and crew time needs.NASA’s exploration medical system trade study tools, which includes a systems engineering model and a medical risk analysis model, have the potential to serve as a foundation for this analysis.There are many obstacles ahead before making pharmaceutical foundries in space a reality.What has not been thoroughly discussed in this review is the downstream processing of a molecular medical foundry, which will depend on the purity needed for the pharmaceutical formulation, delivery method, production host, etc.Downstream processing, the purification of the target molecule from the production host, is a resource-intensive aspect of bio-pharmaceutical production across all platforms.There is a lack of downstream processing technology that translates well from Earth-based constraints to those of space, as they often require a high quantity of consumables, raw materials, equipment, and cleaning.This bottleneck will need to be addressed for pharmaceutical foundries in space to succeed.One approach is to conduct research on novel drug delivery modalities to reduce the need for downstream processing, and another is to diminish the resource demands of the processing itself.A growing emphasis on distributed and just-in-time pharmaceutical production for healthcare on Earth is already driving solutions to these downstream challenges.The other major hurdle is in regulatory compliance.Production and administration of pharmaceuticals in space will require extensive quality control; manufacturing a small molecule might have 50 critical tests, while manufacturing a biologic may have over 250 tests.Here, the advent of personalized medicine on Earth will illuminate a path forward.The shift from mass produced to individualized patient-specific medicine hinges on re-structuring the path to regulatory approval and quality control.While there are many challenges ahead that need to be addressed to pave the way for Earth-independent life support, the rewards of this pursuit will include great insights into supporting life on Earth and beyond.Understanding this value, we aim to highlight the critical importance of developing Earth-independent systems in the future of human exploration.We illustrate that molecular pharming provides a diverse production tool set that could be used to establish a robust molecular medical foundry subsisting on a small fraction of food crop needs.In addition to advocating for molecular pharming as a synergistic asset of space life support systems,4×8 flood tray we focus on the need for multi-faceted utilization of resources in limited environments such as space and extraterrestrial bodies.Sunflower is an oil seed crop of great importance worldwide, due to the excellent quality of the oil extracted from its seeds that are consumed in various ways.Cultivation of the sunflower is becoming increasingly significant globally.In 2008–2009, the world sunflower seed production was about 33 million tones, around 8.5% of the world’s total oil seed production , the leading producers of which are the EU, Russia, Ukraine, Argentina, USA,China, India, and Turkey.Foliar fertilization is an increasingly popular practice with particular importance for the production of high value crops such as sunflowers with many examples of positive responses to foliar application of micro-nutrients, including zinc , iron ,boron , manganese , and molybdenum , on the seed yield and seed quality of sunflower.Foliar fertilization has particular value in overcoming nutrient deficit resulting from stress conditions, such as salinity and drought, which often compromise root growth and decrease root absorption capacity.Foliar application of Zn resulted in a greater improvement in Zn densities in rice and wheat grain when compared with soil applied Zn.

Foliar fertilization is theoretically more immediate and target oriented than soil fertilization since nutrients can be directly delivered to plant.Thissues during critical stages of plant growth.Optimizing the efficacy of the foliar applied nutrients is therefore of great importance from an economic, agronomic and environmental point of view.Our understanding of the factors that determine the ultimate efficacy of foliar applications remains poor and the response of plants to foliar Zn applications is highly variable.Many reports indicate that foliar application of Zn may significantly increase the concentrations of Zn in the applied leaves but may have little effect on foliar concentrations in non-sprayed Thissues or Thissue that develop subsequent to the foliar application.The factors that control the uptake and subsequent translocation of foliar applied nutrients out of the leaf, and the effect of spray formulation on these processes are poorly understood.While various approaches have been used to determine the efficacy of foliar applied nutrients using stable and radioactive isotopic labeling , it remains challenging to determine the pathways of mobilization from leaf to shoot and to monitor the influence of foliar formulation on phloem loading and micro-nutrient transport.To address the inherently low efficiency of many foliar Zn formulations, a wide range of commercial products have been developed and marketed.Recently, there has been much interest in the incorporation of organic moleculesor bio-stimulants into foliar fertilizers with the rationalization that these additives will enhance the uptake, or subsequent mobility of the applied nutrient.The term ‘bio-stimulant’ is used to describe a substance or material, with the exception of nutrients and pesticides, which when applied to plants has the capacity to beneficially modify plant growth.Currently there is very little scientific evidence that bio-stimulants can specifically enhance the uptake and utilization of foliar applied fertilizer materials.The technique of X-ray fluorescencehas been widely used in the research of elemental distribution in plant Thissues, and has proved to be a promising tool to study in vivo localization of metals in plants due to its high-resolution and sensitivity.XRF analyses can be performed to visualize cellular and subcellular distribution of elements in plants without significant pretreatment of the samples.We have previously applied this technique to characterize the location and to monitor changes in concentration and distribution of Zn during plant development or following foliar applications.In this current study, we will utilize μ- XRF to obtain high spatial quantification of elemental distribution and transport following the application of various Zn formulations with the aim of:increasing our understanding of the processes that govern the localization and transport of foliar applied nutrients with emphasis on Zn, and to determine if the formulation of the foliar applied Zn, with addition of macro-nutrients or bio-stimulant, alters the mobility of the element following its absorption by the leaf of sunflower.Because of the complex nature of the commercial products used, it was not possible to prepare a control spray treatment that contained equivalent amounts of all nutrient elements present in Kick-Off or CleanStart.At the rate used here, GroZyme contains negligible concentrations of all essential plant elements.To avoid the possibility that the effect of the foliar spray was a consequence of alleviation of secondary nutrient deficiency, all plants were grown with continuous and abundant soil nutrient.Leaf analysis was conducted and all nutrients were found to be present at adequate levels and plants showed no sign of nutrient deficiency.

The viewing surfaces were covered with opaque material to stop the light from affecting the development of the roots. The back viewing surface was removable, allowing homogeneous soil packing for the plant experiments and convenient access to the electrodes. Besides the top opening, the rhizotrons were waterproof to enable hydroponic experiments and controlled evapotranspiration conditions during the soil experiments and plant growth. All the experiments were performed in a growth chamber equipped with automatic growth lights and controlled temperature and humidity. The temperature varied with a day/night temperature regime of 25/20 °C. The humidity ranged from 45 to 60%. For both ERT and MALM methods, the electrical potential field is characterized by a set of potential differences measured between pairs of electrodes. It is important to properly arrange the electrodes on the rhizotron viewing surface and design a suitable acquisition sequence to obtain a good sensitivity coverage of the investigated system . This is particularly true for the iCSD, as both ERT and MALM acquisitions affect its result. The 64 electrodes were arranged in a 8 by 8 grid on the back viewing surface of the rhizotron, leaving the front surface clear for the observation . For the ERT, the designed arrangement of the electrodes offers a good compromise between a high coverage on the central part of the rhizotron, which encompasses the root zone,flood table and a sufficient coverage on the rhizotron sides to avoid an excessive ERT inversion smoothness. For the MALM, the arrangement of the electrodes is highly sensitive to the position of the investigated current sources. Because of their central positions, the electrodes are closer to the expected sources of current and thus in the region of maximum potential gradient. Hence, this electrode configuration maximizes the changes in both magnitude and sign of the measured ΔV associated with a change in the CSD distribution.

The electrode diameter was 1.5 mm. The penetration of the electrodes into the rhizotron was 4 mm ± 1 mm. To evaluate the possible distorting effects of the densely populated electrodes on the potential field distribution, a test was performed with low conductivity water . The test showed no resistivity anomalies, which may be caused by the presence of the electrodes . Therefore, while rhizotron setups with electrodes only on the sides were successfully adopted , we found that the current setup represents a better solution for iCSD experiments . Data were acquired with a MTP DAS-1 resistivity meter with 8 potential channels. For the ERT acquisition over the 2D grid of electrodes, we chose a dipole-dipole skip 2 configuration. For each skip 2-couple of injection electrodes the remaining skip-2 couples of electrodes were used as potential dipoles . The associated complete set of reciprocals was also acquired, the resulting acquisition sequence contained 3904 data points . Following the ERT data acquisition, the MALM data acquisition required little setup adjustments and time. As the two current electrodes are fixed, the use of a multichannel resistivity meter significantly reduced the acquisition time and, consequently, supported the acquisition of more robust data sets. Electrode 1 was used to inject the current into the plant stem, while electrode 64 was used as a return electrode in the growing medium . The remaining 62 electrodes were used to map the resulting potential field. A sequence with 204 ΔVs was used. Considering the grid in Fig. 2a, the sequence included the vertical, horizontal, and diagonal ΔVs between adjacent electrodes. While 61 ΔVs would provide all the independent differences, the 204 ΔV sequence was preferred because of its redundancy and consequent lower sensitivity to acquisition errors. The acquisition time remained relatively short as the multichannel instrument was optimized with fixed current electrodes that allowed 8 ΔVs to be measured at once.

The iCSD inversion that we developed was based on the physical principles of a bounded system in which linearity and charge conservation were applied to decompose the investigated CSD distribution into the sum of point current sources. This provided a discrete representation of the root system portions where the current leaks from the roots into the surrounding medium. Because of the linearity of the problem, the collective potential field from multiple current sources is the linear combination of their individual potential fields. As such, the measured ΔV can be viewed as and decomposed into the sum of multiple ΔVs from a set of possible current sources. These possible current sources are named ViRTual electrodes . As purely numerical electrodes, they are simulated by mesh nodes representing possible current sources, but with no direct correlation with the real electrodes used during data acquisition. Basically, the VRTe were distributed to represent a grid over which the true CSD distribution is discretized. In order to account for any possible CSD, a 2D grid of 306 VRTe was arranged to cover the entire rhizotron . The charge conservation law implies that the sum of the current fractions associated with the VRTe has to be equal to the overall injected current, which is provided by the resistivity meter. If we normalize the injected current to be equal to 1, the sum of the VRTe weights has to be 1 as well. Briefly, for Ohm’s law, normalizing the current to 1 is equivalent to calculating the resistance, R, from ΔV. Then, the use of R simplifies the presentation of the numerical problem. Once the VRTe nodes are added to the ERT-based ρmed structure, the potential field associated with each of the VRTes is simulated with BERT. From these simulated potential fields, the same sequence of 204 R is extracted, each corresponding to a single VRTe. Each extracted sequence contains the resistances that would be measured in the laboratory if all the current sources were concentrated at the VRTe point .Once the VRTe weights are estimated and associated with the respective VRTe coordinates, they provide a discrete visualization of the investigated CSD.The linear problem formulation is conducive to the inversion optimization during the calculation of the pareto front.

The calculation time of the Pareto front can be further reduced by code optimization as the calculations that do not depend on the regulation weights can separated from the inversion routine and performed only once during the initialization of the linear problem. The initialization phase includes the processing of the MALM experimental data, forward calculation of the VRTe responses for the given ρmed, inclusion of the continuity constraint, and construction of the matrices. Continuity constraint, bounded-value constraint, and first-order spatial regularization stabilize the inversion while limiting the impact of the spatial regularization strategy. The impact of the spatial regularization was evaluated by monitoring the relative components of the misfit and the resulting distribution of the current source. In both synthetic and laboratory tests, as well as in plant experiments the iCSD results are often limited to few current sources .Synthetic numerical and laboratory experimental tests were performed in order to evaluate the capabilities of the setup and inversion routine to couple the ERT and MALM approaches for the iCSD. In the numerical tests both the true source response and VRTe responses were calculated with BERT. Figure 3 shows an explanatory numerical test with inversion of a point source, and the associated Pareto front that was used to select the optimum regularization strength. As this first experiment was performed to specifically test the inversion routine, a homogeneous ρmed was used in order to avoid influence from the baseline resistivity distribution complexity. For the second experiment, the laboratory tests were conducted. Because of the ρmed heterogeneity of any experimental system, these laboratory tests need to include the ERT inversion,rolling bench and the use of the obtained ρmed as input in the iCSD. The true current sources were obtained using insulated metallic wires inserted into the rhizotron . The insulating plastic cover was removed at the tips of the metallic wires to obtain the desired current sources. Six experimental tests were performed using different numbers and positions of these current sources. The rhizotron was filled with tap water and left to equilibrate to achieve steady state conditions of water temperature and salinity, thus minimizing ρmed heterogeneity and changes during the experiment. Changes in ρmed during the ERT and MALM acquisition periods would make the ERT-based ρmed less accurate and compromise the iCSD. To make sure ρmed was stable, a second ERT was performed after the MALM acquisition and compared with the initial measurement. The conductivity of the solution was also measured in several locations of the rhizotron with a conductivity meter to validate the ρmed obtained from the ERT inversion. This setup allowed the acquisition of good quality data sets since less than 5% of the data were discharged during the data processing. Because of the controlled laboratory conditions, the ρmed obtained with the ERT was stable and consistent with the direct conductivity measurements. The quality of the ERT inversion was also confirmed by comparing the model responses with the acquired data . Similarly, the acquired iCSD data were plotted against the resistances calculated with the CSD distribution obtained from the iCSD. The tests also allowed a more informed definition of the VRTe grid. For our setup, a spacing of 3 cm provided a good compromise between resolution, stability, and duration of the iCSD routine. The 3-cm spacing also agrees with the ERT resolution, which would not support a higher iCSD resolution. Successive numerical tests were based on the 8- source laboratory tests shown in Fig. 4. These tests aimed to 1) link laboratory and numerical tests to evaluate the influence of the numerical iCSD routine and laboratory setup on the overall iCSD stability and resolution; 2) account for a more complex CSD, given by the 8 wire-tip sources that were used to simulate distal current pathways; and 3) account for possible ρmed heterogeneity. To address goals 1 and 2, the position of the 8 sources was replicated in the numerical tests and a test with homogeneous ρmed was included to simulate the water resistivity of the laboratory tests.

To address goal 3, heterogeneous ρmed were tested.In order to account for the heterogeneous ρmed the following modeling steps were carried out. First, a true ρmed was assigned to the mesh cells of the rhizotron ERT model. We included homogeneous, linear, and quadratic resistivity profiles in the y direction, see Fig. 5. Second, the ERT acquisition was simulated with the ERT laboratory sequence and 3% of Gaussian error, in line with reciprocal and stacking errors observed in the laboratory data sets. Third, the forwarded ERT data sets were inverted following the exact laboratory procedure. A refined and different mesh was used for forward and inverse problems to, respectively, increase the simulation accuracy and avoid the inverse crime . The ERT forward calculation was then repeated over the inverted ρmed. The obtained inverted responses were compared with the responses of the true models. As for ERT, we compared true and inverted MALM responses. First, the true response was simulated with the 8 current sources overt the true ρmed. Second, a MALM response was calculated over the inverted ρmed and inverted to obtained the inverted CSD. Third, the obtained inverted CSD was used to forward calculate the inverted MALM response over the inverted ρmed. True and inverted MALM responses were then compared.e performed hydroponic and soil experiments using maize and cotton plants. In all the plant experiments, the injection electrode was positioned in the plant stem at a height of 1 cm from the surface of the growth media. For the hydroponic experiments, the plants were first grown in columns with aerated nutrient solution . They were then moved to the rhizotron for the experiments. As in the metallic roots test, the rhizotron was filled 1 day before the experiment to reach stable and homogeneous temperature and salinity conditions. The plant was positioned at the center of the rhizotron with soft rubber supports. The plants were submerged at the same level as in the growing column to avoid discrepancies caused by the plant tissue adaptation to the submerged and aerated conditions, as discussed above with regard to the growing conditions. Consequently, the root crown was approximately 3 cm below the water surface. For the soil experiments, seedlings were grown directly in the rhizotron to avoid damaging the roots and altering the root-soil interface. The soil was prepared by mixing equal volumes of sandy and clay natural soils acquired from an agricultural study site run by U.C. Davis, CA .

Understanding these intricate rhizosphere relationships is vital in devising strategies to increase plant productivity and comprehend localized biogeochemical processes. In many rhizosphere studies, the use of pots and containers is predominant as it allows the plants to be cultivated under controlled conditions and at low cost. Compared to field studies, growth of plants in defined spaces also offers advantages in ease of handling, monitoring and sampling . Much of what we know of the rhizosphere microbiome has resulted from such pot-grown plants. However, since the rhizosphere and roots are still out of view in the soil, destructive sampling of the root is required prior to analysis. Destructive sampling may result in the loss of three-dimensional spatial information on rhizosphere processes over time, which is increasingly being recognized as a critical parameter. On the other hand, soil free techniques such as hydroponics and aeroponics can provide visual access to the rhizosphere circumventing the need for destructive sampling. Other alternatives are gel-based substrates which can maintain rhizosphere transparency as well as the 3D architecture of roots and have been applied successfully in high throughput imaging, phenotyping and trait mapping platforms . Nonetheless, the root phenotype and traits of plants grown under soil-free conditions are known to differ from those of soil-grown plants . These soil substitutes do not also accurately simulate the heterogeneous nature of soil aggregates, thus complicating extrapolations for field relevance. Sophisticated imaging approaches such as magnetic resonance imaging and X-ray computer tomography can be used to analyze root systems in the soil with minimal disturbance but they are low throughput,led grow lights expensive and may not be easily accessible . It is apparent that structural changes in design catered to solving specific challenges in the rhizosphere are indeed necessary. To overcome these challenges relating to the rhizosphere in soil, specialized plant growth chamber systems have been designed, and successful implementation has led to multiple variations of similar designs.

These specialized systems often have a visible rhizosphere which enables coupling with other technologies thereby increasing the breadth of experimental techniques applicable to the rhizosphere system. This review discusses representative growth chamber systems designed to study major rhizosphere processes and interactions in soil. Growth platforms resembling conventional containers such as pots and tubes are not covered. specifically, the reviewed growth systems are selected based on the following criteria: the growth chamber is amenable for use with soil/soil-like substrates and therefore, hydroponics, aeroponics and agar/gel-based systems are not discussed except in microfluidic-based platforms, it is built with the intention to maintain growth of the plant and has architectural features distinct from conventional pots, and lastly it is able to be set up in a laboratory; i.e., field measurement systems and observation platforms are not included. For instance, a minirhizotron, consisting of a camera mounted in a glass tube submerged in the soil which provides non-destructive root imaging over time will not be discussed as it is out of the scope of this review. Through our assessment of lab-based chamber systems, we identify unique advantages and challenges associated with each system . We hope that future fabrication designs can benefit and improve on designs that work well. Lastly, we offer our perspectives on areas in which technological advances are needed to fill current knowledge gaps.In studying rhizosphere processes, the myriad of complex interactions among members of the rhizosphere are often dissected to two interacting variables such as root-and-soil or root-and-microbes, etc. Each of these interactions inherently operates under distinct parameters and requires specifically designed platforms to effectively answer different research questions. This review is structured in a way that first describes each rhizosphere process briefly and then reports on the specific growth chamber systems designed to facilitate experiments for answering related research questions.

The major rhizosphere processes discussed below include root system architecture, physicochemical gradients in the soil, exudation patterns by the roots and interactions between roots and nematodes, fungi or bacteria. Root system architecture encompasses structural features that provide spatial configuration such as root length, width, spread and number and is an important rhizosphere parameter in regulating soil porosity, and nutrient and water uptake efficiency by plants . Plants have been observed to “sense” and direct root growth toward nutrient sources in soil, and the RSA of a plant exhibits great malleability in response to environmental stimuli which in turn, influences microbial communities . For instance, bean plants grew deeper roots under drought conditions to enhance water foraging capabilities while low phosphate conditions stimulated the formation of dense lateral roots involved in P uptake from upper soil layers . Given that most soils are heterogenous, understanding the RSA of plants becomes critical in improving resource use efficiency and agricultural yields . Often, RSA in pot-grown plants is investigated by excising the roots via mechanical means such as root washing or blowing with compressed air . These methods are, however, time-consuming, cause inevitable damage of fine root hairs and result in loss of spatial and temporal information . An appealing alternative for studying RSA is the use of rhizotrons. Rhizotrons were initially constructed as underground facilities designed for viewing and measuring roots in the field . In the lab, the rhizotron implies a chamber constructed using two vertical sheets with at least one or both of the sheets being transparent and/or removable . This allows repeated visual inspections of individual roots; a feature unachievable with destructive sampling. In some cases, the word “rhizobox” is used for a similar set up although this was first introduced in as compartmentalized systems to separate the root and soil compartments . Rhizotrons/rhizoboxes are often constructed with PVC or acrylic materials and come in many sizes to accommodate different plants with soil or soil-less substrates .

Root growth and morphology in the rhizotron can be tracked by a variety of methods ranging from manual tracing onto a plastic sheet, using handheld or flatbed scanners to fully automated time-lapse imaging camera systems .Data can be subsequently analyzed with a wide range of software packages . Affordable and robust RSA imaging platforms using rhizotrons have also been developed for increased accessibility in low-income countries . The versatile construction of a rhizotron design for RSA studies has inspired many variations. For instance, ara-rhizotrons were designed to enable the study of 3D canopy competition with simultaneous root growth observation in an Arabidopsis plant population . The horizontal and radial design of HorhizotronTM and mini-Horhizotron consisting of transparent quadrants attached to a central chamber were developed to study lateral growth of roots in a semi-3D space and to perform post-transplant assessment . The separated quadrants can also be used with different soil substrates simultaneously to study substrate effects on root growth . A rhizotron fitted with water-tight gasket seals has also been used successfully to investigate the RSA of plants under water-logged conditions . Despite the continuous real-time visual read-out, most rhizotron designs suffer from inevitable loss of information from roots occluded by soil particles. The GLO-Roots system overcomes this by imaging from both sides of the rhizotron while using bioluminescent roots to create higher contrast against the soil, enabling quantitative studies on RSA . Following advances in engineering and device fabrication, more rhizotron variants adapted to specific plant growth conditions can be envisioned. In a typical topsoil, approximately half is composed of solid minerals and organic matter while the rest is a fluctuating composition of water and gas filled spaces influenced by environmental conditions and uptake/release of solutes from plants . Changes in gaseous and hydrologic parameters, such as ions, O2 and moisture among others, create a spatially complex environment that influences microbial communities and overall plant health. These physicochemical fluxes are heterogeneously distributed along roots and vary with root types and zones . Often, they exist as gradients in the rhizosphere , thus emphasizing the need for non-destructive sampling in order to accurately capture processes occurring at biologically relevant times and scales. Rhizotron chambers with a visually accessible rhizosphere allows in situ and continuous mapping of these gradients in the soil through the use of different types of imaging methods. For instance, photo luminescence-based optical sensors enable in situ,vertical grow system repeated detection of small molecule analytes in addition to pH , O2 and NH4 . Methods like zymography to detect enzyme activity and diffusive gradients in thin film can be used to map solute concentrations in the soil down to sub-mm scales with high spatial resolution more realistically than traditional destructive approaches.

For example, transport and distribution of water in the rhizosphere soil has been imaged on both 2D and 3D planes by coupling a rhizotron with neutron radiography and tomography, respectively and showed varying moisture gradients along the root system with higher water uptake at the rhizosphere compared to bulk soil. On the other hand, if the rhizotron slabs are thin enough , even simple imaging solutions based on light transmission can be set up to capture water uptake by roots in sand . Despite trade-offs in method sensitivity between these two studies, a rhizotron set up is critical in both designs and illustrates its adaptability to multiple equipment.Roots exude a substantial amount of photosynthetically fixed organic carbon into the soil consisting of a wide variety of compounds such as sugars, organic acids, and primary and secondary metabolites . Together with mucilage and border cells , root exudates provide a major source of nutrients for the rhizosphere microbiome . Root exudation is regulated under genetic control as well as in response to environmental conditions in the soil such as nutrient limitations or increase in toxicity.As a central player in the rhizosphere ecosystem, it is imperative to understand root exudation patterns to unravel subsequent impacts to the surrounding soil and microbial community. Improvements in analytical instrumentation have made it possible to move from targeted to untargeted explorations with mass spectrometry to create root exudate fingerprints in its entire complexity . Regardless, the impact of such techniques relies partly on our exudate sampling techniques. Detection of exudates in real-time is difficult due to rapid biotransformation and sorption to the soil matrix. As such, common collection methods rely on root washing in hydroponic systems to overcome complications in the soil matrix and preserve native exudation profiles. However, a comparison between a soil-based collection method and hydroponic methods showed varied responses particularly in amino acid exudation although the underlying cause was not elucidated . It is possible that the differing growth conditions between hydroponics and soil, which include differences in gas concentrations, mechanical impedance and microbial spatial composition, can elicit differing root exudation responses to the same environmental stimuli. Rhizoboxes offer the advantage of localized sampling in soil using sorption media such as paper and membrane filters, compound specific ion exchange binding resin or micro-suction cups placed closed to root zones of interest to collect exudates . Moreover, in a rhizobox fitted at the bottom with a porous root impenetrable membrane, a root mat is allowed to be formed which is then further transferred onto a collection compartment . The collection compartment containing soil could then be cut into thin slices parallel to the membrane to represent differing distances from the rhizosphere . While this approach can be used to investigate exudate release and sorption under soil conditions, the root mat growth generalizes exudate production in terms of the whole root system and occludes spatial exudation patterns. In a hybrid set up by Oburger et al. , the rhizobox is transplanted to a second specialized rhizobox for continued vertical root growth. This specialized rhizobox consists of a nylon membrane close to the transparent side to restrict root growth into the soil except for root hairs . This creates a vertical flat root mat onto which localized exudate samples can be collected. A comparison of this novel set up to conventional collection methods showed that amino acid exudation rates were most varied among the different methods , further highlighting the need for specialized chambers. Nonetheless, successful implementation of these chambers is still limited to fast-growing plants which can form active root mats. The high density of root mats could also lead to unnatural root exudate levels and an overestimation of rhizosphere effects. In addition, care has to be given to the choice of membrane as selective sorption of certain root exudates onto the membrane may also occur .

Previous studies reported photolysis half-lives of 10-20 days in soil surface under continuous UV radiation . Under field conditions however, there won’t be continuous exposure to sunlight, and photolytic degradation will probably be much slower than reported values, especially in winter months. Another factor that potentially affected the observed degradation rates was pesticide exposure histories at the study sites. According to data from the California Department of Pesticide Regulation , the Hospital Creek watershed had a higher use of chlorpyrifos than the other study sites and chlorpyrifos use in this watershed consistently increased between 2007 to 2010 . Although the SJRNWR site is downstream, at the confluence of Hospital Creek and Ingram Creek, it is less susceptible to chlorpyrifos exposure because the compound is deposited to sediments upstream. When soils are repeatedly exposed to chlorpyrifos, some microorganisms may gain an enhanced capability to degrade the compound, a phenomenon called enhanced biodegradation . For example, Singh et al. observed rapid degradation of both chlorpyrifos and the transformation product TCP in an Australian soil collected from a site where chlorpyrifos had been used continuously for more than 14 years. It has also been reported in some other studies, however,hydroponic dutch buckets that chlorpyrifos was resistant to enhanced biodegradation . Additional research is needed to assess the differences in microbial communities among the study sites and their link to the pesticide exposure history. The functionality of the study sites as riparian buffers was assessed using the California Rapid Assessment Method for Wetlands . Results of the CRAM analysis indicate that these riparian areas range from very low functionality , to high functionality.

The finding of higher chlorpyrifos degradation capacity of sites with low functionality may be related to the fact that healthy wetlands often contain large areas of anaerobic sediments that may not be conducive to microbial communities that rapidly degrade chlorpyrifos. As a result, strategies intended to increase wetland functionality alone may not be conducive to pesticide degradation. The hydrolysis of the P-O linkage of organophosphate insecticides is catalyzed by phosphotriesterase , a zinc metalloenzyme capable of hydrolyzing organophosphate compounds including agricultural pesticides and chemical warfare agents . This enzyme reportedly has been responsible for the biodegradation of organophosphate insecticides in previous studies. Phosphomonoesterase and phosphodiesterase enzymes were involved in chlorpyrifos mineralization making phosphorus available for uptake by microorganisms . To investigate the relationship between different types of phosphoesterase enzymes and observed chlorpyrifos degradation rates, enzyme activities were measured and compared to the biodegradation rates. A positive correlation between PTE enzyme activities and chlorpyrifos degradation rates was observed, however, the relationship differed for each site . These results suggest that the PTE enzyme assay may be useful as a tool for assessing temporal variations at individual sites after PTE enzyme activities are calibrated to the site-specific degradation rates. No correlation was observed between chlorpyrifos degradation rates and phosphomonoesterase and phosphodiesterase activities .The engineering module simulates the hydrology and water quality for the landscape of a river basin. A river basin has tributary lands, rivers, and reservoirs. To capture the spatial variations within a river basin, the WARMF model divides the basin into a hydrologic network of land catchments, stream segments, and lake layers.

Physical dimensions of the hydrologic components are prescribed by digital elevation map data, which can be downloaded from the website of US Geological Survey . By selecting a specific land catchment, stream segment, or reservoir on the basin map, dialog boxes for physical, chemical, and biological characteristics of a location can be displayed with the description of variable names and their units. Values for model coefficients can be modified by the user during model calibration. The model allows the user to include atmospheric deposition from precipitation and dry dust, point source discharges and fertilizers applied to farm lands. Hydrology, non-point load and water quality are simulated in all sections of the waterway. Simulated parameters include flow, water depth, and an array of water quality parameters including pH, temperature, dissolved oxygen, ammonia, nitrate, phosphate, suspended sediments, fecal coliforms, major cations and anions, and pesticides. The time step of simulations is typically one day. The model’s database contains default input data and data to evaluate simulation results. The model also uses input including digital elevation maps, land use, fertilizer application, air quality, meteorology, and point source discharges. A variety of model outputs are automatically saved for graphical, tabular or GIS displays. The sub-models embedded in WARMF are adapted from many well established algorithms, such as sediment erosion and pollutant transport algorithms in ANSWERS , and pollutant accumulation and wash off algorithms in SWMM .To simulate the dynamics of a river basin, WARMF uses a number of time series data sets in modeling. Meteorology, air quality, precipitation quality, point source discharge, and reservoir flow release data are used to drive the model. Hydrology and water quality data are used to check model results. The data module allows users to review the input data sets and make changes. The time series data sets are stored in ASCII text files, one file per monitoring station . Each row of a data file contains a date and a series of numerical values for monitored parameters in separate columns. As a part of continuous planning process, new data may be collected after the planning is completed.

When there is a need to extend the time period of old data sets to include the new data, the data module allows users to enter new data as they become available. The knowledge module can include reservoir operation rules, water quality standards, rate coefficients, and other items. The reservoir operation rules are replaced by an input file that contains the specified flow releases from various outlets. Water quality standards are included in the water quality criteria for various designated uses. The knowledge module can be used to store files used to process input data for a site-specific application, spreadsheets used to calculate the cost of best management practices, references, applicable laws, and relevant case studies.Consensus and TMDL modules are road maps that provide guidance for stakeholders during the decision-making process. Through the TMDL Module, calculations are made for a series of control points throughout the river basin. A road map is provided for the step-by-step procedure. An iterative set of simulations is performed to calculate various combinations of point and non-point loads that the water body can accept and meet the water quality criteria of the designated uses. The water quality criteria can be specified for multiple parameters and based on percent compliance. The consensus module is an application tool embedded in WARMF. The purpose of the module is to guide stakeholders to a general agreement on a watershed management plan. It relies on the engineering models to furnish technical information for stakeholders to make decisions.The San Joaquin River Model Interface is a WARMF version developed to simulate the San Joaquin River and its watershed . The section of the San Joaquin River within the watershed was divided into 93 river segments. The irrigated lands were divided into 17 land catchments. The model simulates natural storm water runoff, irrigation return flow, groundwater table of land catchments, and groundwater lateral flow from land catchments to the receiving river segments. There are gauge stations that provide measured inflows as inputs to the model. For example, there are three gauges for the three major east-side tributaries . For the agricultural lands, the model inputs includes daily diversions, locations of diversions, and areas upon which the irrigation water was applied. Based on the locations of diversions, the model uses the water quality of the source water when applying that water for irrigation.The model simulates the hydrological processes of snow pack accumulation, snow melt, canopy interception, through fall, evaporation and transpiration, infiltration, percolation, groundwater lateral flow, and surface runoff. These processes are simulated based on water balance and physics of flow . Precipitation and irrigation water can percolate into the soil. Within the soil, water increases the moisture level in each soil layer. After the field capacity is exceeded, water percolates down to the water table,bato bucket where it flows laterally out of the land catchment according to Darcy’s Law. Water on the soil or within the soil is subject to evapotranspiration, which is calculated based on temperature, humidity, and season. The amount of water entering and leaving each soil layer is tracked by the model. If the amount of water entering the soil layer is greater than the amount leaving the soil layer, the water table rises. If the water table reaches the surface, the soil is saturated and overland flow occurs, which is calculated by Manning’s equation . Rivers accept the subsurface and overland flow from linked catchments. They also receive point source discharges and flow from upstream river segments. Diversion flows are removed from river segments. The remaining water in the river is routed downstream using the kinematic wave algorithm. The channel geometry, Manning’s roughness coefficient, and bed slope are used to calculate depth, velocity, and flow. The velocity is a measure of the travel time down the river, which in turn affects the water quality simulation.The fundamental principle which guides the model’s simulation of water quality is heat and mass balance. Heat is transferred to the soil via precipitation and irrigation. Heat exchange occurs between catchments and the atmosphere based on the thermal conductivity of the soil. Temperature is then calculated by heat balance throughout the model. There are various ways by which chemical constituents can enter the model domain.

They can enter the land surface via irrigation water, land application, atmospheric deposition or point source discharge. Chemical species move with water by percolation between soil layers, groundwater lateral flow to rivers, and surface runoff. Each soil layer is modeled as a completely mixed reactor, as is the land surface within each land use . Competitive cation exchange between the major cations are simulated on the available soil exchange sites. Anions and optional metals partition between dissolved and adsorbed phases in the soil based on an adsorption isotherm. Nutrient cycling between soil and vegetation is simulated for each land use. The model simulates the soil erosion of sand, clay, and silts from the land surface, and sedimentation and resuspension of particles in streams. A dynamic equilibrium is maintained between dissolved and adsorbed phases of each ion. The dissolved oxygen concentration is tracked during the simulations, and anoxic reactions take place as DO is depleted. When overland flow takes place, sediment is eroded from the catchment surface according to the modified universal soil loss equation. Adsorbed ions are carried on soil particles to the river. Each river segment is modeled as a completely mixed reactor. Sediment can settle into the river bed and is scoured from the river bed when velocity is high enough. Chemical reactions take place in the canopy, soil surface, soil layers, and surface waters. Reaction rates are based on first-order decay with reaction-specific rates. .The model requires six categories of input data: geometric dimensions of land catchments and river segments and their elevations, soil characteristics of the watersheds, model coefficients, land uses, meteorological conditions, and operating conditions. The first 4 categories of data are time invariant variables, which remain constant during the model simulation. Their input values are set only once during model setup. The model coefficients include reaction rates and their temperature correction factors. The model allows for land use changes, which can occur once every few years. In that case, the model uses a “warm start” procedure to run the simulation in sequence. In this procedure, the model uses a set of land use data to perform a simulation for a period of a few years. It saves the results at the end of the simulation and uses them as the initial condition to start the simulation for the next few years with the new land use data. The last two categories of data vary with time. These are sometimes referred to as “driving variables” . The meteorology affects the annual and seasonal variations of hydrology and water quality . The operating conditions include human activities such as fertilizer applications, reservoir releases, diversions, irrigation and waste discharges, which can be modified by management alternatives to improve water quality.

The translocation of the short arm of rye chromosome 1 from the cultivar Petkus into the long arm of wheat chromosome 1B confers improved tolerance to several abiotic and biotic stresses. Although several genes for resistance to biotic stresses are no longer effective, the1RS.1BL translocation is still widely used because of its beneficial effects on grain yield and improved abiotic stress tolerance . We have previously shown that the presence of a short segment of wheat 1BS chromosome from cultivar Pavon in the distal region of the 1RS translocation was associated with reduced grain yield, biomass, and canopy water status relative to near-isogenic lines carrying the complete 1RS chromosome arm . Carbon isotope discrimination data showed that the lines with the complete 1RS chromosome arm achieve higher yields and better water status through increased access to water throughout the season, rather than through water conservation . A subsequent field study showed that the improved water status of the isogenic lines with the 1RS chromosome was associated with enhanced root density below 20 cm relative to the lines with the 1RSRW chromosome . Changes in root architecture in the field were correlated with drastic changes in root development in hydroponic growth systems, where the 1RSRW line showed a regulated arrest of the seminal root apical meristem ∼2 wk after germination. By the same time, the 1RSRW plants displayed altered gradients of reactive oxygen species in the root tips and emergence of lateral roots close to the RAM . In this study, we performed exome captures for 1RS, 1RSRW, ebb flow tray and its parental lines T-9 and 1B+40 . We show that, as a result of a distal inversion between 1RS and 1BS chromosome arms, T-9 and 1B+40 have duplicated 1BS and 1RS orthologous regions in opposite orientations and that a crossover between these chromosomes resulted in a duplicated 1RS region colinear to the inserted 1BS segment in 1RSRW.

Using these genetic stocks, we demonstrate that the dosage of the genes in the duplicated region plays an important role in the regulation of the seminal root growth. We also describe a radiation mutant with a deletion in the inserted 1BS segment and the adjacent 1RS region that restored long roots, confirming the importance of the dosage of the genes in this region on root development. Finally, we identified 38 genes within this deletion and used published RNA-sequencing data and annotation to discuss their potential as candidates for the genes regulating seminal root elongation in wheat.The genetic stocks including the 1RS and 1RSRW chromosome arms were initially generated in the cultivar Pavon 76 , a spring wheat developed at the International Maize and Wheat Improvement Center . The 1RS chromosome arm translocation in Pavon was introgressed from the CIMMYT cultivar Genaro, which, in turn, received the translocation from the cultivar Kavkaz . The donor of the 1RS arm in Kavkaz was the rye cultivar Petkus, one of the leading rye cultivars in the 20th century.To name the different chromosome constitutions we used two superscripts, with the first superscript indicating the proximal position and the second superscript the distal position. The 1RSRW chromosome arm was generated by homologous crossover in overlapping wheat segments of the primary 1BS–1RS recombinant T-9, which possessed a distal wheat 1BS segment, and 1B+40, which possessed a distal 1RS segment . The 1RSWR arm was generated by a crossover in overlapping wheat segments in primary 1BS–1RS recombinants T-38, which possessed a large distal wheat 1BS segment, and 1B+44, which possessed a long distal 1RS segment . The 1RSWW chromosome was generated by a crossover between 1RSRW and 1RSWR chromosomes and was designated as chromosome MA1 in Lukaszewski . The lines carrying the 1RSRW, 1RSWR, and 1RSWW chromosomes were previously back crossed into the CIMMYT common wheat cultivar Hahn, which has the 1RS.1BL translocation, with 1RS also originating from cultivar Kavkaz, the same as in Pavon-1RS.

The introgressions involved six marker-assisted back crosses, resulting in near-isogenic lines that were deposited in the National Small Grains Collection as accessions PI 672839 , PI 672838 , and PI 672837 . We have previously shown that the 1RSRW chromosome results in short roots in the Hahn background but not in the Pavon background. Therefore, to analyze the effects of different 1RS/1BS recombinant chromosomes on root length, we back crossed primary recombinants with varying lengths of wheat and rye segments—T-9, T-18, T-21, and 1B+40 —four times into Hahn. Line T-21 is identical to T-9 and line T-18 carries a large distal 1BS segment on its 1RS arm and was used as 1BS reference in the calculation of ratios for copy number determination. Line 1B+37, which carries a large distal 1RS segment on its 1BS arm , was used as 1RS reference in the exome capture comparisons but was not used in the hydroponic experiments.To dissect the chromosome region affecting root length, we irradiated 5,000 wheat F2 seeds from the cross between Hahn × Hahn-1RSWW with 300 Gy . This mutant population was established in 1RSWW before we knew which wheat segment was affecting root length. The objective of mutagenizing F2 plants rather than homozygous plants was to detect deletion mutants in the heterozygous plants of the first generation without having to wait for progeny tests. We extracted DNA from the 2,200 mutagenized plants that survived and used a dominant wheat marker and a dominant rye molecular marker to eliminate plants that were homozygous for the 1RS or 1BS segments. We identified 907 plants that were heterozygous for the proximal segment, of which, we expected the majority to also be heterozygous for the distal 1BS segment. We then screened the selected plants with multiple markers for the distal 1BS insertion and identified one mutant . From the progeny of this plant, we selected two sister homozygous plants, designated hereafter as 1RSWW-del-8 and 1RSWW-del-10. We then back crossed these two deletions independently to Hahn-1RSRW and to Hahn four times to reduce background mutations and to test the effect of the deletion on the root length in both backgrounds. Although the two lines carry the same deletion, independent back crosses increase chances of eliminating different background mutations, and they served as biological replicates in the root length experiments.We performed two exome capture experiments using different platforms. In the wheat exome capture using the assay developed by Arbor Bio-sciences, we included lines T-9, T- 18, T-21, 1B+37, 1B+40, and 1RSRW ethyl methanesulfonate mutant lines RW_M4_43_11 and RW_M4_47_12 . In the wheat exome capture using the assay developed by NimbleGen , we included lines 1RS, 1RSRW, and deletion lines 1RSWW-del-8 and 1RSWW-del-10. Based on the average similarity between the wheat and rye genes and the hybridization conditions used in the capture, we expect most of the rye genes to be captured with both wheat exome capture assays. The exome captures were sequenced using the Illumina platform and 150 bp paired-end reads at the University of California, Genome Center.

The sequencing reads were preprocessed to trim adapters with Trimmomatic v0.39 . Since the capture included both wheat and rye reads, we mapped the reads to a combined reference including wheat Chinese Spring RefSeq v1.0 chromosome 1B and the rye chromosome arm 1RSAK58 from the 1RS.1BL translocation in cultivar Aikang58 . To minimize off-target mapping, we mapped the reads at high-stringency with ‘bwa aln’ v0.7.16a-r1181 , allowing only perfectly mapped reads . Alignments were sorted by using samtools v1.7 , and duplicate reads were removed with Picard tools v2.7.1 . We normalized the number of mapped reads so that all lines have the same total number of reads mapped to the chromosome arm 1BL. We selected the 1BL arm as reference because 1RS/1BS recombinant lines differ in their short arm constitutions, but all share identical 1BL arms. We then calculated normalized read number ratios using a common reference line . We generated heat maps for these ratios and visually determined the borders of duplication, recombination, and deletion events. We then validated these borders using t tests of the ratios at both sides of the border . For these analyses we excluded genes with less than six reads in the accessions used as denominator for normalization. We report wheat gene coordinates using CS RefSeq v1.1 and rye gene coordinates using the 1RSAK58 genome as references , which is almost identical to our 1RS sequence. The other available genome reference for rye inbred line Lo7 is less similar to the 1RS sequences from Hahn 1RS.1BL translocation.Hydroponic experiments were performed in growth chambers at 22–23 ˚C with a photoperiod of 16 h light vs. 8 h dark . In all experiments, grains were imbibed at 4 ˚C for 4 d and then placed at room temperature. Once the coleoptiles emerged, seedlings were floated on a mesh to develop roots for 4 d. After removing the grain, seedlings were wrapped at the crown with foam and inserted in holes precut in a foam core board placed on top of the solution. The detail protocols and solutions are described in our previous paper . As in our previous study,flood and drain tray experiments in this study were performed in two different laboratories in Argentina and the United States using tanks of 0.35 and 13 L, respectively. As a result of the different conditions, final root lengths differed across experiments. However, differences among genotypes were consistent across experiments, and all statistical comparisons among genotypes were performed within experiment or using experiments as blocks. In experiments performed in 13-L tanks, we changed nutrient solution every 3 d and we included all genotypes in each tank. When necessary, we used multiple tanks as blocks. In experiments performed in 0.35- L tanks, we changed nutrient solution every 2 d, and a single genotype was included per pot, with multiple pots used as replications. To determine the effect of the 1RSWW-del-8 and 1RSWW-del-10 deletions on root development, we evaluated the segregating plants in the BC2F2 and BC4F2 generations to account for potential random effects of residual deletions in other chromosomes.To define the borders of the inserted 1BS region, we used the Arbor Biosciences exome capture to characterize the 1RSRW line and its two parental lines 1B+40 and T-9 .

We also included line T- 21 that appears to be identical to T-9 , line T-18 that has a distal 1BS segment longer than T-9/T-21 and was used as a wheat reference, and line 1B+37 that has a longer distal 1RS segment than 1B+40 and was used as a rye reference. We mapped the reads of each capture to a combined reference without allowing any SNP and then normalized the counts to a similar number of mapped reads per capture in the 1BL arm.The recent sequencing of the 1RS arm revealed the presence of a large inversion between the distal region of chromosome arms 1RS and 1BS , which suggests that lines with breakpoints within this region, such as T-9, T-21 and 1B+40, may be more complex than originally thought. The 1RSRW line was generated by a crossover of the primary recombinant lines T-9 and 1B+40 , and the previous results indicate that 1RSRW has retained the 1RS-1BS breakpoints of T-9 and 1B+40 . The 1RSRW chromosome arm also has the same strong telomeric C-band as 1RS and 1B+40, indicating that it has retained the complete 1RS segment present in 1B+40 . We initially assumed that the 1BS segment in 1RSRW replaced the orthologous rye genes and that the loss of these genes could be responsible for the shorter roots of Hahn- 1RSRW. However, the codominant marker THdw11 has both the 1RS and 1BS bands in T-9, 1B+40, and 1RSRW but not in T-18 or 1B+37 , suggesting a duplication rather than a replacement in the lines with distal crossover events. To investigate the extent of this duplication, we first identified 14 orthologous 1BS-1RS gene pairs including high-confidence wheat genes located within the 1BS insertion and rye 1RSAK58 genes that were at least 90% identical with an aligned region covering >90% of the gene . Surprisingly, all 14 ryeorthologues were present in the exome capture of T-9, 1B+40, and 1RSRW , which indicated that the complete rye region orthologous to the 1BS insertion was present in these lines. Since no 1RS gene was missing in the 1BS orthologous region, we rejected the hypothesis that lost rye genes were responsible for the differences in root length between Hahn- 1RS and Hahn-1RSRW isogenic lines.

Plants have evolved a powerful immune system to resist their potential colonization by microbial pathogens and parasites. Over the past decade, it has become increasingly clear that this innate immunity is, in essence, composed of two interconnected branches, termed PAMP-triggered immunity and effector-triggered immunity. PTI is triggered by recognition of pathogen- or microbial-associated molecular patterns , which are conserved molecular signatures decorating many classes of microbes, including non-pathogens. Perception of MAMPs by pattern recognition receptorsat the cell surface activates a battery of host defense responses leading to a basal level of resistance. As a result of the evolutionary arms-race between plants and their intruders, many microbial pathogens acquired the ability to dodge PTI-based host surveillance via secretion of effector molecules that intercept MAMP triggered defense signals. In turn, plants have adapted to produce cognate R- proteins by which they recognize, either directly or indirectly, these pathogen specific effector proteins, resulting in a superimposed layer of defense variably termed effector-triggered immunity , gene-for-gene resistance or R-gene-dependent resistance. In many cases, effector recognition culminates in the programmed suicide of a limited number of challenged host cells, clearly delimited from the surrounding healthy tissue. This hypersensitive responseis thought to benefit the plant by restricting pathogen access to water and nutrients and is correlated with an integrated set of physiological and metabolic alterations that are instrumental in impeding further pathogen ingress, among which a burst of oxidative metabolism leading to the massive generation of reactive oxygen species. Apart from local immune responses, ETI-associated HR formation also mounts a long-distance immune response termed systemic acquired resistance , in which naïve tissues become resistant to a broad spectrum of otherwise virulent pathogens.

It should be noted, however, that PTI, when activated by PAMPs that activate the SA signaling pathway,ebb and flow bench can trigger SAR as well. An archetypal inducible plant defense response, SAR requires endogenous accumulation of the signal molecule salicylic acidand is marked by the transcriptional reprogramming of a battery of SA-inducible genes encoding pathogenesis-related proteins. By contrast, there is ample evidence for induced disease resistance conditioned by molecules other than SA, as illustrated by rhizobacteria-mediated induced systemic resistance [ISR; [9]]. ISR, which delivers systemic protection without the customary pathogenesis-related protein induction, is a resistance activated upon root colonization by specific strains of plant growth-promoting rhizobacteria. In a series of seminal studies using the reference strain Pseudomonas fluorescens WCS417r, Pieterse and associates demonstrated that, at least in Arabidopsis, ISR functions independently of SA, but requires components of the jasmonic acidand ethylene response pathways. Even though colonization of the roots by ISR-triggering bacteria leads to a heightened level of resistance against a diverse set of intruders, often no defense mechanisms are activated in above ground plant tissues upon perception of the resistance-inducing signal. Rather, these tissues are sensitized to express basal defense responses faster and/or more strongly in response to pathogen attack, a phenomenon known as priming. As demonstrated recently, priming of the plant’s innate immune system confers broad-spectrum resistance with minimal impact on seed set and plant growth. Hence, priming offers a cost-efficient resistance strategy, enabling the plant to react more effectively to any invader encountered by boosting infection-induced cellular defense responses. In contrast to the overwhelming amount of information on inducible defenses in dicotyledonous plant species, our understanding of the molecular mechanisms underpinning induced disease resistance in rice and other cereals is still in its infancy.

Evidence demonstrating that central components of the induced resistance circuitry, including the master regulatory protein NPR1, are conserved in rice has only recently been presented. Moreover, reports on SAR-like phenomena in rice are scarce. Most tellingly in this regard, a 17- year-old report of systemically enhanced resistance against the rice blast pathogen M. oryzae triggered by a localized infection with the non-rice pathogen P. syringae pv. syringae remains one of the most compelling examples of a SAR-like response in rice to date. In contrast, there is a sizeable body of evidence demonstrating systemic protection against various rice pathogens resulting from ISR elicited by, amongst others, Pseudomonas, Bacillus and Serratia strains. However, in most if not all cases, still very little is known about the basic mechanisms governing this ISR response. In a previous report, we demonstrated that rice plants of which the roots were colonized by the fluorescent pseudomonad P. aeruginosa 7NSK2 developed an enhanced defensive capacity against infection with M. oryzae. Bacterial mutant analysis revealed that this 7NSK2-mediated ISR is based on secretion of the redox-active pigment pyocyanin. Perception of pyocyanin by the plant roots was shown to cue the formation of reiterative micro-oxidative bursts in naïve leaves, thereby priming these leaves for accelerated expression of HR-like cell death upon pathogen attack. Aiming to gain further insight into themolecular mechanisms underpinning rhizobacteria-modulated ISR in rice, we tested the ability of the biocontrol agent Serratia plymuthica IC1270 to induce systemic resistance against various rice pathogens with different modes of infection. Originally isolated from the rhizosphere of grapes,S. plymuthica IC1270 is a well-characterized PGPR strain producing a broad palette of antimicrobial compounds. In addition to its potential as a direct antagonist of a wide array of plant pathogens, preliminary experiments in bean and tomato revealed that IC1270 is equally capable of reducing disease through activation of a plant-mediated defense response. Here, we demonstrate that colonization of rice roots by IC1270 renders foliar tissues more resistant to M. oryzae.

Using a combined cytological and pharmacological approach, evidence is provided that IC1270 locks plants into a pathogen-inducible program of boosted ROS formation, culminating in the prompt execution of HR cell death at sites of attempted pathogen entry. Similar, yet even more pronounced, phenotypes of hypersensitively dying cells in the vicinity of fungal hyphae were observed in a genetically incompatible rice-M. oryzae interaction, suggesting that IC1270-mediated ISR and R-gene-mediated ETI involve similar defense mechanisms. Bacterial strains used in this study were Serratia plymuthica IC1270, which was originally described as Enterobacter agglomerans, and Pseudomonas aeruginosa 7NSK2. For inoculation experiments, IC1270 and 7NSK2 were grown on iron-limiting King’s B medium [KB; [34]] for 24 h at 28°C and 37°C, respectively. Bacterial cells were scraped off the plates and suspended in sterile saline . Densities of the bacterial suspensions were adjusted to the desired concentration based on their optical density at 620 nm. Magnaporthe oryzae isolate VT7, a field isolate from rice in Vietnam, was grown at 28°C on half-strength oatmeal agar . Seven-day-old mycelium was flattened onto the medium using a sterile spoon and exposed to blue light for seven days to induce sporulation. Conidia were harvested as described in De Vleesschauwer et al., and inoculum concentration was adjusted to a final density of 1 × 104 spores ml-1 in 0.5% gelatin .Rhizoctonia solani isolate MAN-86, belonging to anastomosis group AG-1 IA, was maintained on potato dextrose agar . Inoculum was obtained according to Rodrigues et al. with minor modifications. After autoclaving, 15 toothpicks, 1 cm in length, and five agar plugs , obtained from the margin of an actively growing colony of R. solani, were transferred to PDA plates. These plates were then incubated for 8 days at 28°C so R. solani could colonize the toothpicks. Cochliobolus miyabeanus strain 988, obtained from diseased rice in field plots at the International Rice Research Institute , was grown for sporulation at 28°C on PDA. Seven-day-old mycelium was flattened onto the medium using a sterile spoon and exposed to blue light for three days under the same conditions mentioned above. Upon sporulation, conidia were harvested exactly as stated in Thuan et al. and re-suspended in 0.5% gelatin to a final density of 1 × 104 conidia ml-1.Four-week-old rice seedlings were challenge inoculated with Magnaporthe oryzae as described in De Vleesschauwer et al.. Six days after inoculation, disease severity on the fourth leaves of each plant was rated by counting the number of elliptical to round-shaped lesions with a sporulating gray center, and expressed relative to non-induced control plants. R. solani bioassays were performed essentially as described in Rodrigues et al.. Plants were challenged when four weeks old by placing a 1-cm toothpick colonized by R. solani inside the sheath of the second youngest fully expanded leaf. Inoculated plants were maintained inside humid inoculation chambersfor 72 h, and, thereafter,4x8ft rolling benches transferred to greenhouse conditions. Four days after challenge infection, disease severity was assessed by measuring the length of the water-soaked lesions. C. miyabeanus bio-assays were performed as described in Ahn et al. with minor modifications. Five-week-old seedlings were misted with a C. miyabeanus spore suspension containing 1 × 104 conidia ml-1 in 0.5% gelatin. Inoculated plants were kept in a dew chamber for 18 h to facilitate fungal penetration, and subsequently transferred to greenhouse conditions for disease development. Disease symptoms were scored at four days after inoculation for about 48 leaves per treatment. Disease ratings were expressed on the basis of diseased leaf area and lesion type: I, no infection or less than 2% of leaf area infected with small brown specs less than 1 mm in diameter; II,less than 10% of leaf area infected with brown spot lesions with gray to white center, about 1–3 mm in diameter; III, average of about 25% of leaf area infected with brown spot lesions with gray to white center, about 1–3 mm in diameter; IV, average of about 50% of leaf area infected with typical spindle-shaped lesions, 3 mm or longer with necrotic gray center and water-soaked or reddish brown margins, little or no coalescence of lesions; V, more than 75% of leaf area infected with coalescing spindle-shaped lesions.Induced systemic resistance assays were performed as described in De Vleesschauwer et al. with minor modifications.

Briefly, rice plants were grown under greenhouse conditions in commercial potting soil that had been autoclaved twice on alternate days for 21 min. Rice seeds first were surface sterilized with 1% sodium hypochlorite for two min, rinsed three times with sterile, demineralized water and incubated for five days on a wet sterile filter paper in sealed Petri dishes at 28°C. Prior to sowing in perforated plastic trays , roots of germinated seeds were dipped in a bacterial suspension of the ISR-inducing strains [5 × 107 colony-forming unitsml-1] for 10 min. The auto claved soil was thoroughly mixed with bacterial inoculum to a final density of 5 × 107 cfu ml-1. To ensure consistent root colonization by the eliciting bacteria, rice plants were soil-drenched a second time with bacterial inoculumat ten days after sowing. In control treatments, soil and rice plants were treated with equal volumes of sterilized saline. For experiments in which purified pyocyanin was applied to the roots of rice seedlings, plants were grown in a hydroponic gnotobiotic system as described before. In this system, plants were fed with various concentrations of pyocyanin and ascorbate 4 days before challenge inoculation by adding the desired concentration to the half-strength Hoagland nutrient solution. Pyocyanin extraction, quantification and application were performed exactly as stated in De Vleesschauwer et al..To gain more insight into the nature of IC1270-mediated ISR against M. oryzae, cytological studies were performed at sites of pathogen entry. To this purpose, we adopted the intact leaf sheath assay previously described by Koga et al.. Briefly, leaf sheaths of the fifth leaf of rice plants at the 5.5 leaf stage were peeled off with leaf blades and roots. The leaf sheath was laid horizontally on a support in plastic trays containing wet filter paper, and the hollow space enclosed by the sides of the leaf sheaths above the mid vein was filled with a suspension of sporesof M. oryzae. Inoculated leaf sheaths were then incubated at 25°C with a 16-h photoperiod. When ready for microscopy, the sheaths were hand-trimmed to remove the sides and expose the epidermal layer above the mid vein. At least five trimmed sheath tissue sections originating from different control and IC1270-treated plants were used for each sampling point. Phenolic compounds were visualized as autofluorescence under blue light epifluorescence . To detect H2O2 accumulation, staining was performed according to the protocol of Thordal-Christensen et al. with minor modifications. Six hours before each time point, trimmed sheath segments were vacuum-infiltrated with an aqueous solution of 1 mg ml-1 3,3′-diaminobenzidine-HCLfor 30 min. Thereafter, infiltrated segments were incubated in fresh DAB solution until sampling.

To determine the importance of PPD1 for the NB response, we compared wild-type and ppd1-null mutant lines lacking all functional copies of PPD1 in the photoperiod-sensitive hexaploid variety Paragon and in the tetraploid line Kronos-PS . We first measured heading date in these lines when grown in SD or LD conditions since germination. Under SD conditions, neither the wild type nor the ppd1-null mutants of either variety flowered within 150 d, when the experiment was terminated . Under LD conditions, the ppd1-null mutants headed 60 d and 34 d later than the wild type Kronos-PS and Paragon-PS lines respectively . In a separate experiment using slightly different conditions , we compared the effect of NBmax in photoperiod-sensitive and ppd1-null mutant lines. Kronos-PS and Paragon-PS plants headed on average at 73 d and 91 d under NB, respectively, but neither ppd1-null line flowered within 150 d, when the experiment was terminated . These results demonstrated that PPD1 plays a major role in the effect of NB and LD on heading time. We next assayed PPD-B1 and FT1 transcript levels in Kronos-PS and ppd1-null plants at four time points, including dusk, when these flowering time genes are normally expressed at high levels under LD . Plants were grown in SDs for 4 weeks, then either maintained in SD conditions or moved to NBmax conditions for 6 weeks. In Kronos-PS plants, PPD-B1 transcript levels were upregulated 1 h and 3 h after the start of the NB and to even higher levels at dusk . In the Kronos-PS plants kept under SD, PPD-B1 transcript levels were not upregulated during the night but showed an increase at dusk, although the levels were significantly lower than in plants that were exposed to multiple NBs . As expected, PPD-B1 transcripts in the Kronos ppd1-null mutants were not detected in either SD or NB conditions, confirming the specificity of the qRT-PCR primers used in this assay .

Consistent with previous results, FT1 transcripts were undetected in Kronos-PS plants under SD but were highly upregulated in NB conditions at all time points . However, in the ppd1-null mutant, FT1 transcripts were not detected in any sampled time points, including dusk,indoor garden under either SD or NB conditions.We next tested the effect of the timing of the NB on PPD-B1 induction by exposing SD-grown plants to a single NB at different times of the night. We hypothesized that maximal induction of PPD-B1 would coincide with the strongest acceleration of heading date . This hypothesis proved to be incorrect and, instead, we found that PPD-B1 was induced to progressively higher levels in accordance with the duration of the dark period preceding the NB . We first thought that the gradual accumulation of inactive Pr phytochromes in the nucleus resulting from dark reversion could explain the increased PPD-B1 induction with longer periods of darkness. However, plants treated with FR light immediately before a NB applied after 2 h of darkness did not exhibit increased PPD-B1 expression . This result suggested that the accumulation of Pr phytochromes in the nucleus was not responsible for the progressive induction of PPD-B1 with extended dark periods. We then thought that PPD-B1 induction could be associated with the de novo synthesis of phytochromes or other intermediate proteins during darkness. To test this hypothesis, we grew Kronos-PS plants in hydroponic solution and treated half of them with cycloheximide to block protein synthesis and left half of the plants untreated as a control. Consistent with previous results, control plants maintained in darkness showed noinduction of PPD-B1, whereas those exposed to a single NB exhibited strong up-regulation of PPD-B1 expression 2 h after the NB . The induction of PPD-B1 in response to NB was abolished in plants treated with cycloheximide , which demonstrates that the expression of PPD1 in response to light requires active protein synthesis during darkness. This experiment was performed twice with identical results.Many studies using NBs to characterize the effects of changing photoperiods on flowering time focused on SD plants, mainly because the inhibition of flowering by NB was found to be a simpler system of study than the acceleration of flowering by NB in LD plants .

Our characterization of the NB response in wheat highlights some of the similarities and differences between these two systems. In many SD plants, flowering is inhibited by NB and in rice; this effect is associated with the suppression of Hd3a transcription . When rice plants are moved from NB back to inductive SD photoperiods, this inhibition is lost and Hd3a expression returns to high levels. In wheat, NBs also affect the expression of FT1 and flowering time, although these responses are reversed. These results suggest SD and LD plants both respond to NB through regulatory mechanisms acting on FT expression. The opposite effect of NB on FT expression and flowering in rice and wheat is likely determined by the opposite roles of PPD1 in different grass species. In LD grasses, such as wheat and barley, PPD1 induces FT1 and accelerates flowering , whereas in SD grasses, such as sorghum and rice, PRR37 suppresses FT-like genes and delays flowering .In some SD plants, the suppression of flowering by a single R light NB is completely reversible by immediate exposure to FR light . In wheat, we found that a single FR exposure after NB had a limited effect on heading time . One-minute pulses of FR after 1-min pulses of white light were more effective , but did not completely abolish the acceleration of heading by NB . The partial effect of FR light on the NB acceleration of flowering is consistent with previous results in the LD grass barley . Finally, rice and wheat differ in the role of PHYC in the NB response. In rice, the NB response is completely abolished in plants carrying PHYB loss-of-function mutations but is unaffected by similar mutations in PHYC . By contrast, the NB response in wheat is abolished in both the phyB-null and phyC-null mutants . The different roles of PHYC on NB parallel the different roles played by this phytochrome in the photoperiodic response in wheat and rice. PHYC is a positive regulator of flowering time in some temperate grasses such as wheat, barley, and Brachypodium distachyon but has limited or no effect on flowering time in rice and Arabidopsis . These results suggest PHYC plays a more critical role in the photoperiod and NB response in the LD temperate grasses than in other plant species.Whereas a single NB is sufficient to repress flowering in rice and promote flowering in Lolium temulentum cv Ceres , multiple LDs are required to accelerate flowering in many temperate grasses .

Most temperate grasses show some acceleration of flowering after being exposed to 4 to 8 LD photoperiods, but full saturation of this response requires 12 to 16 d of exposure to LD . These results are consistent with our observations for wheat, where 6 to 10 LDs induced a mild acceleration in flowering, but the greatest acceleration in flowering was seen in plants exposed to 12 or more LDs . The acceleration in heading time in response to increasing numbers of NBs was similar to that observed in response to increasing numbers of LDs,but the effects were smaller and at least 15 NBs were required to initiate the acceleration of flowering . These results are consistent with the existence of a PPD1- independent photoperiod pathway, which may be more responsive to LDs than to NBs. In Arabidopsis, the induction of the transition from the vegetative to the reproductive apex also requires cycles of FT induction repeated over several days. However, while 4 to 5 LDs are sufficient to saturate the acceleration of flowering in Arabidopsis more than 20 LDs are required in wheat . Possible explanations for the requirement of multiple NBs or LDs to induce FT1 in wheat include a gradual accumulation of a flowering promoter, a gradual reduction of a flowering repressor, or a gradual change in epigenetic marks in some of the involved genes. No correlation was detected between the number of NBs and transcript levels of ZCCT2 , suggesting that this gene is not critical for the observed changes in FT1 in this genetic background . Similarly,hydroponic farming PPD1 transcript levels did not increase in response to multiple NBs, indicating that the putative accumulating factor is unlikely to be a regulator of PPD1 transcription. However, it is still possible that the number of NBs affect the levels of active PPD1 protein. To test this hypothesis, we have initiated the generation of transgenic wheat plants expressing an HA-tagged PPD1 protein. It is also possible that proteins other than PPD1 also play a role in the regulation of FT1 in response to multiple NBs.Interestingly, we found that the magnitude of PPD-B1 induction by NBs was proportional to the length of darkness preceding the NB. This phenomenon appears to be unrelated to the accumulation of Pr phytochrome protein arising from dark reversion, since exposure to FR light prior to NBs had no effect on the subsequent induction of PPD1 by light . Instead, we found that treating plants with cycloheximide during the night abolished the NB up-regulation of PPD1 , which suggests that the induction of PPD1 by light is dependent on active protein synthesis during darkness.

One possibility is that the de novo synthesis of Pr isoforms of PHYB and/or PHYC during darkness is correlated with the strength of PPD1 induction. During longer periods of darkness, newly synthesized PHYB and PHYC proteins would accumulate to higher levels, so that subsequent light signals would result in stronger induction of PPD1. An alternative possibility is the de novo synthesis and dark accumulation of a PHYB/PHYC-induced transcription factor required for the activation of PPD1. This may include one or more PIFs, which have been shown to act as coactivators of light-induced genes in some cases . Additional experiments will be required to test these hypotheses and to identify the darksynthesized protein responsible for the increased activation of PPD1 with longer periods of darkness.Despite the stronger NB induction of PPD1 following longer periods of darkness, PPD1 transcript levels were not directly correlated with heading date. The greatest effect of NB on heading date was observed when the NB was timed to coincide with the middle of the night, even though PPD1 transcript levels were lower at this point than after NBs applied later in the night. This dependence on the time of the night suggests that PPD1 activity may be gated by circadian clock-regulated genes. The existence of a gating mechanism is also supported by the fact that although PPD1 transcription is induced during the light phases of both SD and LD, FT1 transcription is only observed under LD photoperiods . Furthermore, rhythmic sensitivities for NB-induced flowering have been observed in other LD grasses . L. temulentum cv Ceres plants, which are induced to flower by a single LD cycle, showed two phases of high sensitivity to NB when SD-grown plants were moved to constant darkness. The first phase occurred between 4 and 8 h from the start of the darkness period, and the second one was approximately 20 to 24 h later, suggesting the involvement of a circadian rhythm in the control of flowering in L. temulentum . Similar experiments would be challenging to perform in wheat because of the requirement for multiple NBs to induce flowering. It is tempting to speculate that the regulation of FT1 expression by PPD1 may function in a manner analogous to the regulation of FT by CO in Arabidopsis. In Arabidopsis, FT is induced only in LD conditions when the transcriptional peak of CO coincides with light, which is required to stabilize the CO protein . In wheat, FT1 induction and flowering may be determined by the coincidence of an external signal with an internal rhythm mediated by the circadian clock. In addition to this putative role in gating the effect of PPD1, the circadian clock is known to be involved in the regulation of PPD1 expression . Plants carrying loss-of function mutations in EARLY FLOWERING3 exhibited elevated expression of PPD1 and earlier flowering under both LD and SD .

Extensive literature is available documenting many aspects of wastewater treatment using aquatic plants as sum marized, e.g., by Moshiri , Heavy metal uptake be wetland plants was documented by Simmers et al. , Jamil et at, , Lyngby , Shutes et al. , etc. Examples of storm water runoff treatment using wetlands are less numerous. Among the successful ones we can cite an artificial marsh constructed to remove suspended solids and nutrients from storm water runoff from the city of Tallahassee, FL . Many communities outside California already use constructed wetlands for treatment of storm water effluent. Wetland systems are used for a number of reasons, including low maintenance costs and the potential to combine water treatment with habitat creation. However, regulatory agencies that would permit or comment on the establishment of these constructed wetlands are concerned about the potential for bio-accumulation of urban runoff pollutants. If wetlands are to be used for the treatment of urban runoff, an understanding of nutrient and pollutant cycling within wetland plants during the year is crucial. It will help wetland managers to design proper harvesting schemes for removing pollutants from the wetland environment. While many states and communities have experimented with urban effluent and wetlands to determine the most appropriate plant species and management, little work has been completed in California. Results obtained outside of the state can only partially assist in designing California storm water wetlands as plants, weather,indoor vertical farming and soil conditions differ significantly from those of other states. Consequently, significant interest has been generated in developing a useable information base which would be available to both communities and the state regulatory agencies when making decisions regarding storm water wetland management and design. Another important aspect of this problem is that urban runoff currently drains into existing wetlands, which are protected as waters of the United States by the Clean Water Act.

It is important to know the effects and potential impacts that storm water is or may have on naturally occurring wetlands and to what extent pollutants are being concentrated in parts of plants that will be consumed by migratory or resident animals in the wetland environment. In our project we focused primarily on obtaining data on seasonal dynamics of growth and resource allocation including heavy metals allocation in selected wetland plant species grown under conditions of elevated heavy metals. The obtained results provide a basis for using these species for wetland construction. Hydrocotyle verticillata, Ludwigia pep/oides, Nasturtium aquaticum, Sagitta ria latitolia and Polygonum hydropiperoides, all representing a group of soft and/or creeping emergent macrophytes, and Scirpus ecutus, S. celitomicus. S. robustus, Typha letitolie, T. domingensis and Phragmites australis, representing a group of erect emergent macrophytes. These species were selected because they are native to California with the exception of Nasturtium aquaticum, which is naturalized. According to our previous research and the literature data, they are robust primary producers and posses excellent reproductive capabilities. To obtain data on biomass production, the representative species were sampled at various location in the Central Valley. The above ground biomass was harvested from a 50 x 50 cm quadrat, dried and weighed. The rhizome propagation requires close monitoring of soil water as the rhizome cuttings are highly susceptible to both desiccation and drowning. A greenhouse experiment was carried out to determine the dependence of growth characteristics on water level. Three species, Hydrocotyle verticil/ata, Ludwigia pep/aides and Nasturtium aquaticum were tested. Five different water depth in relation to soil surface were used: -10cm, -Scm, Oem, -scm, and +10 cm, each in five replicates. Individual plants were planted in pots with sand and placed in a large metal container with nutrient solution. Nitrogen concentration in the solution was checked at four day intervals and readjusted to 50ppm. The experiment lasted four weeks, at the end plants were harvested, divided into leaves, shoots and roots, dried and weighed.

To determine the dependence of growth of Hydrocotyle verticil/ata and Nasturtium aquaticum and its tissue nitrogen concentration on the concentration of nitrogen in water, we planted individual plants in 500 ml Erlenmeyer flasks or plastic holders placed in buckets in 1/2-strength Hoagland nutrient solution with 1.4, 7, 14, 35, 70, and 140 ppm of nitrogen added as CaN03′ The nutrient solution was changed every second day. The experiment lasted 25 days.In the second year of the project, four wetlands receiving urban runoff and two control sites were selected for analyses. Two of the runoff retention basins, Octo Inn and Rancho Solano , are located in Fairfield, California, two, the North Pond and West Pond are in Davis, California. A small natural wetland in the Cosumnes River Preserve , together with a marsh at Calhoun Slough at the Jepson Prairie Reserve represent the non-polluted habitats. Plant and sediments were collected in the fall and winter. Water was sampled in January, February and March, 1992, following the rain events. All samples were analyzed for lead, zinc, and copper; sediment samples were also analyzed for molybdenum and cadmium. Metals analysis was conducted using Ionized Coupled Plasma Atomic Emission Spectroscopy . During winter and spring of 1992 a wetland cultivation facility was built in the Putah Creek Reserve area adjacent to the Institute of Ecology, UCD. It includes three sets of fiberglass or plastic containers. The first set consists of 24 round fiberglass tanks . Each tank is divided into four compartments by plywood partitions and filled with soil. These tanks were used for studying the effect of different water levels on the growth of wetland plants. The second set includes 80 small plastic containers that were used for experiments assessing the effect of elevated concentrations of heavy metals on plant growth. Propagules of five wetland plant species, native to California’s Central Valley, were collected in the winter of 1991-1992. Scirpus acutus , S. calffornicus , and Typha domfngensis , were collected and propagated from rhizomes. Ludwigia pep/oideswas propagated from stem cuttings and Sagittaria latifoliawas propagated by tubers. Each plant propagule was planted individually into a #6 nursery pOL Washed sand was used as a substrate. A pot with each species was then put into a large tub and the water level was raised to saturation .

Once the plants had sprouted and established themselves, Hoagland’s nutrient solution, without micro-nutrients, was added and nitrate levels were kept as close to 40 ppm as possible. This was to ensure good plant growth at the time of exposure to the metals. The tubs were then flooded with another 10 L of water to ensure flooded conditions. A randomized complete block design was used for the layout. There were five blocks, each with twelve tubs. Treatments were randomly assigned to the twelve tubs. The four metal salts, mentioned above , were administered at three treatment levels: 0.1, 1.0 and 10.0 parts per million .. There were ten control tubs that had no metals added to the water. Prior to treatment, all plants were measured for length or leaf area. The tubs were treated as batch reactors, according to the design layout. The plants were allowed to grow for an additional two weeks after metal treatment. After this time, the plants were harvested, remeasured, separated into leaves, rhizomes, roots,best indoor vertical garden system adventitious roots, tubers and the original propagule stock. Each part was dried at 80° C for 48 hours and then weighed. Once weighed, the samples were ground and submitted to the University of California’s Department of Agriculture and Natural Resources Laboratory for metals analysis. Metals analysis was conducted using Ionized Coupled Plasma Atomic Emission Spectroscopy . Growth, as a function of biomass increase was determined by using correlations of shoot length to dry weight biomass for Scirpus acutus, S. californicus and Typha domingensis, Stem length and number of branches over 10 cm were used to get relationships for biomass in Ludwigia peploides, and leaf area was used for biomass correlations in Sagitta ria latifalia. These correlation relationships were obtained by destructive sampling in accordance with Vymazal et al. 1993.Evaluating the effect of water level on the growth and biomass allocation of eight species of emergent macrophytes was conducted in the 24 round fiberglass tanks . Each tank was divided into four compartments by plywood partitions and filled with soil. Eight plant species, Scirpus californicus, S. acutus, S. robustus, Typha domingensis, Phragmites australis, Ludwigia pep/oides, Po/ygonum hydropiperoides and Sagittaria latifolia were planted in the late summer of 1992. They were initially kept at the same water level until established, after which, four different flooding regimes, +30, +10, 0, and -20 cm, each in three replicates, were initiated by the end of June 1993. Plants in 40 cm x 40 cm grid placed in each compartment were measured biweekly and the allometric correlations established previously were used for assessing the biomass changes. In September, all the above ground biomass was collected from the grid, separated into leaves, stems, adventitious roots and litter. These plant parts were dried and weighed. In one tank from each replicate, the below ground biomass was collected from the following layers: 0 to -5cm, -5 to -tocrn, -10 to -15 em, -15 to – 25cm, -25 to -35 cm and -35 to -50cm . Below ground harvest turned out to be much more time consuming than expected and teams of people were working on it almost non-stop for 12 weeks. To better quantify standing biomass, obvious features such as stem length or plant height were measured on individual harvested plants and related to the dry weight of each plant respectively. Field recorded measurements of plant height and density can then give estimates of standing biomass over larger areas. We used simple stem length to determine biomass for Phragmites australis, Scirpus acutus, S. californicus and S. robustus. The biomass of Typha spp. was best correlated with a “leaf length index.” This index was calculated as the average length of the four tallest leaves of Typha multiplied by the total number of leaves. Biomass of leaves of Sagittaria fatifolia was determined by measuring the leaf length and width and correlating the index CxD with weight. CxD index showed very close correlastion with the leaf area mesured by the L1COR-L13000A leaf area meter.

The biomass of petioles estimated from the regression of petiole length on its weight was added to leaf biomass. Due to the creeping nature of Ludwigia pep/oides and Po/ygonum sp. allometric correlations were not very accurate. Table 1 presents examples of the biomass and tissue nitrogen ranges for several types of wetland macrophytes from natural habitats. The time course of decomposition for of some of these species is shown in Fig. 4. The survey of heavy metals in water from several runoff retention basins sampled in January, February and March, 1992, following rain events did, not reveal any increased metal concentrations. Except for Zn, no detectable levels of heavy metals were found in water samples. Of the plant samples from polluted sites, Nasturtium aquaticum was found to contain the highest levels of Zn . Surprisingly, Sagittaria latifolia from Cosumnes Preserve, our control site, also contained high levels of Zn. If we divide all the plant species tested into two groups based on their growth form, t.e., erect emergent macrophytes and creeping emergent macrophytes, the group of creeping macrophytes, represented by species such as Nasturtium aquaticum, have significantly higher levels of Cu and Zn in their tissues than does the group of erect emergents . Heavy metal concentrations in sediments were not as high as we expected; samples from Octo Inn Basin showed the highest levels of plant available Zn, Pb and Cu among the “polluted” sites. Sediments from the Cosumnes Preserve had the overall highest metals concentrations . However, the levels of lead were generally quite low at all sites. The heavy metal content in plant biomass was not correlated with the concentrations of individual elements in sediments. While the Cosumnes Preserve sediments had the highest concentrations of metals, the highest metal concentrations in plants were found at Rancho Solano. Cadmium is one of the major environmental pollutants and a potential hazard to worldwide agriculture.

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 .