Tag: greenhouse vertical farming

The foliar fertilizer product “CleanStart,” “Kick-Off,” and “GroZyme” were obtained from Ag Spectrum Company.0.1% Silwet L-77was added in each solution.The solutions were applied to the leaves of sunflower 8 h before darkness , and all plant Thissues except the sprayed leaf were covered to prevent inadvertent spray application.The petioles of all sprayed leaves were carefully protected by coating the leaf base petiole junction with lanolinand Teflon membranes.Four plants were treated as one replication, with three replications for each treatment.The foliar application of the fertilizers was replicated one time after 7 days, and then plants were harvested 7 days later.GroZyme is a microbial fermentation product derived from a proprietary mix of organic cereal grains inoculated with specific bacterial cultures and fermented.The fermentation process occurs under controlled environmental conditions until a specific metabolic profile is achieved at which time the live bacterium are liaised and the material is filtered to remove large particles.This concentrate is then extended and stabilized to make the final product.Soil applications of GroZyme have been reported to alter soil microbial activity and nitrogen transformations.The metabolic basis for the biological activity of foliar applied GroZyme is not known.However, field observations suggest that GroZyme functions to enhance plant growth by enhancing K metabolism and sugar transport.CleanStart is derived from ammonia, urea, orthophosphoric acid and potassium hydroxide,ebb flow tray and Kick-Off is a micro-nutrient mix of Fe, Mn, Cu, Zn predominantly derived from nitrate sources with additional surfactants and stabilizers.The elemental composition of all spray applications is provided in Table 1.Mid-sections of leaf petioles were cut from the leaves treated with different foliar treatments.Leaf cross-sections were cut with a cryotomeat a temperature of −20◦C.

Single sections of each treatment were selected under light microscopy for their ultra structural integrity and then freeze-dried under −20◦C for 3 days prior to μ-XRF analysis.Since μ-XRF analysis is time consuming and expensive only single samples from each treatment could be analyzed.Given that true replicate analyses could not be performed additional steps were taken to avoid the potential for experimental artifacts and to avoid any sample selection or analysis bias.All treatments were carefully controlled such that treatment conditions and experimental duration were identical; petioles were then taken from the four replicate plants and multiple sections from each petiole were prepared as described above.All sections were then assessed by light microscopy for ultra structural integrity and a single section was then selected and transported to the Stanford Synchrotron Radiation Laboratory for μ-XRF analysis.Samples selected in this fashion, therefore represent unbiased examples of treatment effects.To investigate the effects of different fertilizers on retranslocation of nutrients in the leaves of sunflower, μ-XRF mapping was performed.Cross sections of petioles were cut from the sun- flower plants at approximately 1.0 cm below the leaves and imaged under a light microscope prior to utilization for μ-XRF imaging.The cross section of petiole was composed of epidermis, parenchyma, vascular bundle containing xylem, phloem, and surrounding collenchyma.The microscope image in Figure 1 shows that the phloem within the vascular bundle exists as a discrete layer of cells on the abaxialside of the vascular bundle with xylem on the adaxial or upper side of the petiole.The entire vascular bundle is enclosed in Thissue that is likely collenchyma.Integrated intensity for Zn and other elements were calculated from the spectrum and normalized by the intensity of the Compton scattering peak.Elemental mapping for the measurement area was obtained from the normalized intensity for each element.The elemental distribution maps of Zn , Ca , and Kin the petioles collected from sunflowers plants with different treatments are presented in Figures 2A–F, together with corresponding photographs taken using an optical microscope.

The quantification of the fluorescence yields was normalized by I0 and the dwell time.The normalized X-ray fluorescence intensities were scaled to different color brightness for individual elements, with the brightest spots corresponding to the highest elemental fluorescence.Each map indicates the relative distribution of the three elements, and the scale of fluorescence counts for individual elements is the same for each map.Very slight signals of Znwas noted in the petioles collected from the control and CleanStart treated plants of sunflower.At the resolution used in these experiments it is not possible to determine if the deposition of Zn in regions other than the phloem was resent in xylem or in collenchyma immediately adjacent to the xylem.A modest increase in the K signal in petioles was also observed in the Clean Start treatment.Foliar application of “Kick-Off,” a product containing Co, S, Fe, Cu, Mn, Mo, and Zn chelated with EDTAto sunflower leaves resulted in a marked increase in the concentration of Zn detected in the petioles with a notable deposition in a narrow band corresponding to the phloem Thissues of the petiole and a more diffuse band in the xylem/collenchyma region , while no such preferential localization to phloem Thissues was noted for control, CleanStart or ZnSO4 treatments and no phloem specific accumulation of other elements was observed.Application of ZnSO4 at the same Zn levels as used for all Zn treatments to the sunflower leaves also increased phloem Zn in the petiolesas compared with the controls , but the effect is much less pronounced than that of “Kick-Off.” The combined foliar application of “Kick-Off”with “CleanStart” resulted in a similar enhancement in Zn uptake and preferential distribution of Zn to phloem Thissues and xylem/collenchyma.The addition of the bio-stimulant product GroZyme resulted in a much more concentrated enrichment of Zn in the phloem and xylem/collenchyma region of the petiole vascular bundle.Low spatial resolution μ-XRF imaging provides only semiquantitative data.To further investigate the effects of “CleanStart” and “GroZyme” on phloem mobility of foliar applied Zn, microXRF scanning at higher resolution was performed, focusing on the vascular Thissues of the treated leaf veins.The Zn concentration in the petiole was also determined along a single scan line that transected the petiole and passed through the vascular system.Both “CleanStart” and “GroZyme,” which do not contain Zn, clearly increased the FIGURE 5 | Concentrations of Znin the petioles collected from sunflower plants treated with different foliar fertilizers.Full expanded leaves of sunflower were treated with control, CleanStart, Kick-Off, CleanStart + Kick-Off, CleanStart + Kick-Off + GroZyme, and ZnSO4, and the Zn concentration of leaf veins were analyzed by ICP-MS.

Compositions of the nutrients in different treatments were shown in Table 1.Data points and error bars represent means and SEs of three replicates.concentration of Zn following Kick-Off application.The patterns of Zn deposition in the “Kick-Off” + “CleanStart” and “KickOff” + “CleanStart” + “GroZyme” were far less diffuse and more intensely located in the phloem region and xylem/collenchyma than the pattern of Zn in petioles from leaves provided with “Kick-Off” or ZnSO4 alone.Intensity analysis across a single scan line through the vascular system of the petiole demonstrated that the peak of Zn densities in the phloem Thissues and xylem/collenchyma was markedly increased with addition of the biostimulant “GroZyme” to “Kick-Off”+“CleanStart” treatments.Total concentrations of Zn and other elements including Fe, K, Cu, Ca, and Mn etc.were determined by ICP-MS for the petioles collected from the sunflowers treated with different foliar fertilizers.The results showed that Zn concentrations in the leaf veins of sunflower ranged from 29.2 to 36.7 mg kg−1 DW.While the overall pattern of Zn concentration differences analyzed by ICPMS corresponded with the μ-XRF analysis results the total Zn concentration was not significantly different between treatments.Similarly, no difference in Fe, K, Cu, Ca, and Mn was observed among any treatments.The apparently greater sensitivity of μ-XRF is primarily a consequence of the ability of μ-XRF to analyze specifically within phloem and closely associated vascular organs while ICPMS provides analysis of the total petiole.Since vascular Thissue represents only a very small proportion of the petiole as a whole, and as the petile was fully mature at the time of treatment, changes in vascular Thissue element concentration may not be seen against the background of the bulk of petiole Thissue in which Zn was not increased.Efficacy of foliar applied nutrients depends not only on the absorption of the nutrients but also on the transport of these nutrients to other plant parts.It has been suggested that even a relatively small transport of foliar nutrients out of treated leaves and Thissues may have a short-term,flood and drain tray critical benefit to the plant.Knowledge of the ability of an element to be transported from the site of application is critical to provide insight into the longevity and potential nutritional impact of foliar application on non-sprayed Thissues.Analysis by μ-XRF in the present study shows clearly enhanced transport and localization of Zn in the vascular system of the sunflower petiole 7 days after application of “Kick-Off,” which contains Zn-EDTA , while Zn was not detectable in the petiole of the control plants and was very low in the petiole of ZnSO4 sprayed leaves.This demonstrates clearly that Zn is phloem mobile in sunflower and that the use of Zn-EDTA results in greater phloem Zn transport than ZnSO4 alone.While it has been demonstrated that Zn-EDTA is superior to ZnSO4 under some circumstances, it has not been demonstrated that the EDTA molecule can penetrate the leaf cuticle.It cannot be determined from this current research if the superiority of the EDTA containing Kick-Off material is a consequence of enhanced cuticular penetration or enhanced transport of the Zn once it enters the leaf.The ‘Kick-Off’ material also contains the micro-elements including Fe, Cu, Mn, and Mo and this may also enhance Zn uptake as has been observed previously.The addition of “CleanStart” derived from ammonia, urea, orthophosphoric acid and potassium hydroxide, significantly increased the phloem transport and xylem/collenchyma deposition of Zn when co-applied with ‘Kick-Off’.Addition of Urea to foliar Zn sprays, for example, is known to enhance Zn uptake and efficacy and the N status of cereals is known promote Zn retranslocation.While it is clear that addition of the multi-elements present in the Clean Start enhanced Zn retranslocation into sunflower petioles, the mechanism of this effect remains uncertain.

The chemical form in which a foliar nutrient is applied will influence plant nutrient uptake by altering the point of deliquescence of the applied foliar fertilizer thereby altering its solubility on the leaf surface, or by altering the charge on the ion of interest to facilitate its movement through the cuticle and cell wall.There is no direct evidence, however, to suggest that the formulation of a fertilizer spray can directly influence the transport of the absorbed nutrient from the site of application.The addition of the bio-stimulant “GroZyme” clearly enhanced Zn translocation when co-applied with “Kick-Off” and “CleanStart”.Grozyme is a non-living microbial fermentation product derived from a proprietary mix of organic cereal grains inoculated with specific bacterial cultures and fermented.The specific functional metabolite in GroZyme has not been identified.However, extensive field trials and research published in this issuehave demonstrated positive growth effects and enhanced translocation of K and other nutrient elements.Previous research utilizing soil applications of GroZyme has also shown that this product was able to alter microbial populations in a soil environment and improve N mobilization and uptake of soil nutrients especially organic nitrogen.The benefit of bacterial source metabolites on efficacy of foliar fertilizers has been demonstrated previously and it is plausible that the microbial extracts present in GroZyme have the capability to form metal complexes that enhance Zn uptake or mobility.Many putative bio-stimulants also contain plant growth hormones or plant signaling molecules that may alter plant metabolic processes and stimulate growth and indirectly influence the movement of substrates, including minerals, within the plant.In these experiment, the high spatial resolution and direct imaging capability of μ-XRF was useful in distinguishing differences in Zn transport through the vascular system of sunflower that could not be detected by ICP-MS.XRF provides a powerful strategy to trace foliar applied microelements within the plants with high sensitivity, a result that is consistent with our previous studies.This technique will be useful to facilitate the development of foliar fertilizers and application techniques that optimize transport of nutrients from site of application, which is one of the most important challenges to the foliar fertilizer industry.Heavy metal contamination, such as cadmiumand lead , in various environmental mediaposes a severe threat to ecological and human health as long as they are bio-available.Although there are natural sources of these elements, anthropogenic releases from activities such as metal mining and smelting, coal combustion, trace levels in fertilizers and even some wastewater sludge and biosolids, can increase concentrations to high levels in soils and sediment beds of lakes and rivers.

Two soil experiments were performed. In the first experiment, four cotton plants were grown for four months. For these experiments, the plants were positioned with the root crown approximately 8 cm deep . In the second experiment, a pregerminated maize seed was planted 3 cm deep and then grown for four months .The iCSD results showed that the current leakage occurred in the very proximal regions of the root systems in both soil and hydroponic conditions. The proximal leakage was observed despite the return electrode being placed at the bottom of the rhizotron to allow deep current pathways. Nonetheless, the expected influence of the return electrode position was observed in the laboratory test with metallic roots and motivates the use of this electrode configuration in future laboratory experiments. Our results are consistent with the early studies on maize root electrical properties , and corroborate the recent works that questioned the assumptions of the BIA methods . The high resistivity of the top layer of soil is expected to induce root suberization : our results would support the physiological hypothesis of water and nutrient absorption through older and possibly suberized roots . It is worth noticing that the absence of current leakage along the section of the cotton stems in the top dry soil supports the assumption that the electrical structure of roots controls their current conduction behavior. Suberized epidermal cells can affect the movement of ions and, consequently,ebb and flow bench the current conduction in roots . Therefore, it is feasible for current to be conducted along deeper portions of the more woody roots with minor leakage.

The BIA experimental results that have observed positive correlations between electrical signals and root area are likely a result of physiological correlations between the root regions that contribute to the current flow and hair roots, which contributes most to functional root surface area . While correlations between BIA electric signals and investigated root traits appear to be indirect, the correlations observed across experimental platforms and species continue to validate its value for in-situ root phenotyping . In their study on field grapevines, Mary et al. concluded that the CSD could be used to infer the root depth distribution. Because of the significant suberization of major roots in grapevines and orange trees, the electric current could penetrate deep into the root system before significant leakage occured. The deeper current penetration allowed the iCSD method to access and phenotype the root system. On the contrary, limited current results in lower sensitivity of the iCSD to distal and younger parts of the root system that are likely dominated by less suberized, finer roots. We attribute differences in current penetration among root systems of maize, cotton, grapevine, and orange tree to the differences in physiological traits such as the extent of suberization and lignification. If on one hand the sensitivity to the root physiological traitsis a promising opportunity, on the other hand it has to be accounted for when phenotyping more herbaceous roots.To assess the involvement of the MsLEC1 and MsLEC2 genes in nodulation of alfalfa, we examined the responses of rooted cuttings of transgenic vector control plants, plants expressing the antisense transgene for MsLEC2 , and plants expressing the antisense transgene for MsLEC1to inoculation with -glucuronidase – or green fluorescent protein -marked strains of S. meliloti Rm1021. Because legume lectins have been associated with facilitation of nodulation, reduced nodulation of lectin loss-of function plants was predicted. However, contrary to our expectations, all the transgenic plants, including the controls, were nodulated 7 days post inoculation .

By this time, the LEC1AS plant lines had already developed abnormally large numbers of nodules . The colonized nodules, as evidenced by the presence of GUS- or GFP-marked rhizobia, were frequently adjacent to each other or directly opposite one another on the root. Infection thread development in root hairs, as viewed by fluorescent microscopy of GFP-marked Rm1021, was not impaired in the LEC1AS roots, although some of the nodules appeared to be uninfected . Occasionally, uninfected nodules also developed on the roots of LEC2AS plants , but generally, the LEC2AS root nodules contained the marked strains . With one exception, line 49b, the LEC2AS roots developed markedly separate rather than clustered nodules. By 12 to 14 dpi, many of the LEC1AS nodules were already beginning to show signs of senescence, as indicated by the reduction in overall staining in a nodule 13 dpi with a GUSmarked strain and by the decrease in Rm1021 GFP fluorescence in the center part of a 2-week-old nodule . In the Rm1021 GFP nodules, there was a concomitant accumulation of autofluorescent compounds, presumably flavonoids, in the central and proximal parts of the nodule . Sections of senescent nodules demonstrated that the rhizobia had senesced from the inside outward . In contrast, the vector control and LEC2AS nodules as well as the plant lines containing the cognate sense transgenes did not show any symptoms of senescence until many weeks after inoculation. All nodules were colonized by rhizobia . Nodules formed on vector control plants grown in potting soil appeared identical, i.e. pink and elongated, to nodules formed by nontransgenic, wild type alfalfa . Similarly, nodules formed on the LEC2AS plants were elongated and pink in color . In contrast, although some LEC1AS root nodules appeared morphologically normal, many LEC1AS nodules were large, multilobed structures that showed signs of senescence, including loss of pink color due to breakdown of leghemoglobin, whereas others showed arrested development at early stages of nodule formation .

Nodulation was also examined in an inert root medium . The Turface grown plants exhibited an identical nodulation phenotype . The LEC1AS lines displaying the most severe developmental and reproductive abnormalities had the highest proportion of abnormal nodules. We also examined plants that expressed the sense transgene for MsLEC1 and found that these roots also developed some large, albeit pink, multilobed nodules , suggesting that cosuppression might be the cause. We did not pursue this analysis further. In contrast, the LEC2ST plants produced nodules that were identical to those of the vector control .When grown hydroponically, the differences between inoculated versus uninoculated plants became very obvious. The shoots of the uninoculated plants were paler than the nodulated plants and, of the three types of transgenic plants, the LEC1AS plants were the most chlorotic . The differences between the vector control, LEC2AS, and LEC1AS plants 35 dpi, were striking . The LEC1AS plants were much less robust than either the vector control or the LEC2AS plants; the plants were consistently small and chlorotic, with a poorly developed root system. Nevertheless, a number of large, prominent nodules were observed on the roots of the LEC1AS plants , as well as small, senescent nodules . The abnormal nodulation phenotype in LEC1AS plants was evident 15 to 20 dpi under hydroponic conditions. In contrast, nodulated vector control and LEC2AS plants appeared normal, and many fewer nodules formed on the root system . To express these findings in a quantitative way, the nodules were removed from the roots of the hydroponically grown plants and were separated into pink and senescent categories. Table 1 shows that the mean number of pink nodules was significantly lower for the LEC1AS plants than for the vector control and the LEC2AS plants. In contrast, the mean number of senescent nodules was significantly higher for the LEC1AS plants compared with the vector control andLEC2AS plants . The mean for the total number of nodules for the three plant groups, which included pink and senescent nodules,4x8ft rolling benches did not differ significantly . However, when the mean total nodule number was normalized to grams of root dry weight, the value was significantly higher for LEC1AS plants compared with vector control and LEC2AS plants; the vector control and LEC2AS plants did not differ significantly from each other . These results demonstrate that the LEC1AS plants produced more nodules, even though their overall root mass was less than the vector control and LEC2AS plants. We reasoned that if the nodules produced on LEC1AS plants senesced faster than vector control and LEC2AS plants, then more rhizobial colony-forming units would appear on agar medium after squashing the LEC1AS nodules. Moreover, we anticipated that the large pink nodules would also have an increased number of CFU. Nodules were collected, weighed, surface-sterilized, and crushed, and the resulting extracts were diluted and plated on RDM culture plates. LEC2AS, LEC2ST, and vector control plants all had similar numbers of culturable S. meliloti cells per mg of nodule tissue . In contrast, there were large mean levels of culturable S. meliloti cells in LEC1ST and especially in LEC1AS nodules. These results suggest that the excessive proliferation of the nodule tissue on LEC1ST and especially on the LEC1AS plants was accompanied by an extensive increase in the numbers of culturable bacteria. Both pink and senescent nodules contained large numbers of viable bacteria. RNA from LEC1AS roots with nodules contained detectable levels of the antisense-MsLEC1 transgene ; no endogenous sense mRNA was observed. The nodulated roots from the different LEC1AS plants showed considerable variability in the amount of accumulation of this transgenic RNA. We had found earlier that there was a correlation between plants that demonstrated moderate to severe developmental and reproductive abnormalities and those with low accumulation of MsLEC1-antisense RNA in nodulated roots .

Similarly, low levels of MsLEC1-antisense RNA were detected in nodules using Northern analysis. The transgenic plants were stably transformed, and different lines contained varying numbers and positions of transgene insertions , which may have contributed to variability in transgenic and endogenous lectin mRNA expression . Transcripts hybridizing to MsLEC1-sense mRNA were not detected in RNA isolated from nodules of LEC1AS plants . In situ hybridization analysis. Because of the difficulty in detecting MsLEC1 transcripts in nodules using Northern blot analysis, we performed in situ hybridization experiments on nontransgenic alfalfa nodules to get a better idea of the spatial expression pattern of this gene. Transcripts hybridizing to MsLEC1 were detected in alfalfa nodule meristems and adjacent cells of the invasion zone , whereas no transcripts were observed in the comparable cells of the sense controls . We also examined MsLEC1 expression in alfalfa roots and found that this lectin gene was expressed in the root apical meristem and also in cells of the elongation zone ; no transcripts were observed in the sense controls . It was difficult to evaluate the difference in the extent of MsLEC1 expression in individual uninoculated versus inoculated roots. The two sets of roots looked almost identical. For MsLEC2, essentially the same pattern of transcript localization was observed. Figure 6J illustrates an entire alfalfa nodule primordium 7 dpi; MsLEC2 mRNA was detected throughout the developing nodule using the WISH method. As the nodule matured, the signal became more concentrated in the cells of zones I and II, the nodule meristem and the invasion zone, respectively . More mature nodules showed the same pattern of MsLEC2 mRNA localization . MsLEC2 transcripts were also detected in the root meristems and adjacent regions and lateral root tips . Similar to the MsLEC1 results, there was no obvious difference in the amount of transcript observed in inoculated versus uninoculated roots. There was no signal detected in the nodule hybridized with the sense probe or in the comparable control for the root . To help confirm the spatial expression pattern of the soluble lectin genes in indeterminate nodules, we examined white sweetclover roots and nodules. This legume appears to have only a single copy of a putative seed lectin gene, termed MaLEC . This contrasts with alfalfa and Medicago truncatula, each of which has three different LEC genes . In white sweetclover nodules, MaLEC mRNA was detected in a 21-day-old nodule, in the cells of zones I and II, the nodule meristem and the invasion zone, respectively , in a pattern that was identical to that observed for alfalfa nodules . The nodule hybridized with the sense probe is depicted in Figure 6L; no signal was detected. Transcripts hybridizing to MaLEC were also detected in the main root tip and in lateral root meristems of both inoculated and uninoculated roots ; no transcripts were observed in the roots hybridized with the sense control .

Free-living nematodes are ubiquitous in the soil. They are beneficial to the plants by playing a role in nutrient cycling and in defense against insects and microbial infections through signaling interactions with the roots . Conversely, infections by parasitic nematodes in the roots increase the plant’s susceptibility to stress and other pathogenic bacteria, fungi, and viruses creating major losses in crop productivity . With an impending rise in nematode infections due to climate change, understanding nematode behavior and interactions in the rhizosphere becomes important to develop appropriate bio-control methods to ensure long term food security .However, these substrates do not accurately emulate the physical textures and heterogeneity of soil and create homogenous solute and temperature gradients which could impact nematode behavior and interactions with the roots . Indeed, nematode motility speed and dispersal decreased in substrates more closely mimicking sand . On the other hand, studying nematode behavior in the soil is a difficult endeavor as its near-transparent body and small size makes it almost indistinguishable from soil particles. Cross-sectioning and staining infected roots make it possible for nematode visualization but they are destructive and provide only static snapshots of cellular changes or nematode behavior during infections . On the other hand,mobile grow system microscopy rhizosphere chambers provide non-invasive detection and observation of nematode activity in the rhizosphere .

The roots in these chambers grow between a glass slide and a nylon membrane . The membrane restricts movement of roots except root hairs into the soil while the transparent glass enables microscopy of the roots at high resolution . Coupled with fluorescently stained nematodes, microscopy rhizosphere chambers allowed for non-destructive in situ observations of nematode infection in its host species over the entire life of the parasite . Nonetheless, staining nematodes is an additional challenge as nematode cuticles are impermeable to stains . This can, however, be alleviated by using advanced imaging technologies which eliminates the need for staining. A recent study demonstrated live screening of nematode-root interactions in a transparent soil-like substrate through the use of label-free light sheet imaging termed Bio-speckle Selective Plane Illumination Microscopy coupled with Confocal Laser Scanning Microscopy . Using this set up, researchers were able to monitor roots for nematode activity at high resolution and suggest its possible use in rapid testing of chemical control agents against parasitic nematodes in soil-like conditions . Fungal communities in the rhizosphere are involved in the degradation of organic matter in the soil and subsequent nutrient turnover affecting plant health as well as the microbial community . Fungal biomass often reaches a third of total microbial biomass carbon and almost all terrestrial plants are able to form symbiotic associations with mycorrhizal fungi . The majority of these associations are with arbuscular mycorrhiza fungi which penetrate into root cortex cells to form highly branched structures . The investment of photosynthetic carbon by plants to AMF is rewarded with increased nutrient availability made possible by the extended hyphal network in the soil. For instance, up to 90% of phosphorus uptake in plants can be contributed by symbiosis with AMF .

AMF networks in the soil also  influence water retention and soil aggregation further impacting plant growth . Moreover, next-generation sequencing technologies and advances in imaging techniques have greatly improved our knowledge on the taxonomical and functional properties of fungal communities in the rhizosphere . However, these methods are optimized for fine scale analysis and are not capable of assessing the foraging capabilities of hyphal networks which can span across centimeter to meter scales. Toward this end, several researchers have used compartment setups with physical barriers created by 20–37 µm nylon membranes which restrict movement of roots but not mycorrhizal fungi. This separation creates root-free and plant free soil compartments connected only by mycorrhizal fungi to examine the transport of various compounds across these compartments. Using this set up, the importance of mycorrhizal fungi in the flow of different elements such as carbon , nitrogen and phosphorus between plants, soil and microbes over centimeter distances have been validated. Repeated disruption of the hyphal connections also led to a decreased resistance in plants to drought stress . The membranes can also be placed horizontally to create different depth gradients to investigate hyphal contributions to water uptake . In some studies, an additional 1.5–3 mm air gap is created between two membranes with a wire net to restrict solute movement between two chambers . A common feature of these set ups is the size-exclusion membranes which proved to be critical in distinguishing fungal hyphae processes in the rhizosphere soil. In addition to AMF interactions, a split root set up, which separates the roots of one plant into halves, can be introduced to investigate the systemic response of plants . In essence, the split-root system directs the growth of the roots to generally two different growth conditions and enables the investigation of whether a local stimuli have a local or global response which can be observed at the root or shoot level . Split-root systems are widely studied and have been adapted to rhizoboxes as well as to pots and tubes .

In the rhizosphere, plants host a wide diversity of bacteria on the surface of the root as well as within roots in the vascular tissue . Due to its abundance and importance, the bacterial community in the rhizosphere is perhaps the most widely studied among other microbial members in the rhizosphere ecosystem. While the study of endophytic bacteria requires inevitable destructive sampling due to its localization, several non-destructive approaches have been developed to study microbes inhabiting the rhizoplane. One of the most widely studied plant-microbe interactions in the rhizosphere is that of the symbiotic relationship between legumes and rhizobia . Once a potential nodule forming bacteria is isolated, it is often required to authenticate its nodule forming phenotype by inoculating on host plants. However, conventional methods such as the use of soil pouches do not allow long term incubation, while “Leonard jars,” consisting of two stacked glass jars forming the top soil layer and the bottom nutrient solution layer, can be expensive and time consuming . A recent study challenges this by describing the use of clear plastic CD cases as minirhizotrons with potential for use in phenotyping root traits such as legume formation, and demonstrated innovation that democratizes research opportunities in rhizosphere research . Other microbial interactions in the rhizosphere, however, may not result in visible changes to the root system and often rely on next-generation omics technologies. As such, physical separation of the rhizosphere from the bulk soil becomes paramount in elucidating changes to microbial community and interactions. One approach to this end is the use of nylon bags with differing pore sizes . The nylon bag restricts the movement of roots and the soil inside the bag is then regarded as the rhizosphere soil to compare against the surrounding root free bulk soil . Developing further on this concept, Wei et al. designed a specialized rhizobox that allowed repeated non-destructive sampling by adding individual nylon bags of root-free soil surrounding the root compartment which are then used as a proxy for the rhizosphere . These methods allowed easy distinction of the rhizosphere and the bulk soil but, we now know that the rhizosphere community is not only distinct from the bulk soil but also varies with type, part and age of the root,mobile vertical rack largely as a consequence of varying root exudation patterns . Studying this phenomenon in situ in the soil requires separation of desired roots from others without disturbance to plant growth or soil. To address this, researchers have used a modified rhizobox design with a side compartment to regulate root growth and quarantine specific roots from the main plant chamber . This additionally creates easy distinction between old and new roots and allows testing on specific quarantined roots despite plant age. A study using this set up showed specific microbial chemotaxis toward different exudates on an individual root whereas another showed spatial and temporal regulation of niche differentiation in microbial rhizosphere guilds . Similar physical perturbations to regulate root growth in response to microbial stimuli have also been applied in the micro-scale and are explored in the next section. Our assessment of the major growth chambers showed that most of the systems applied share similarities in basic structural components such as in the use of two parallel sheets in rhizoboxbased devices. While these growth chambers brought many of the rhizosphere processes to light, limitations do exist. One limitation is with the scale of applicability. Most of these growth systems are micro scale and can easily reproduce pot scale studies but may not be easily translatable to interactions occurring at the micro-scale nor recapitulate processes occurring at field-relevant scale. The next section describes advances in technology resulting in a new wave of unique devices making use of microfluidic processes and fabricated ecosystems which are specifically made to investigate specific rhizosphere processes.

A complex web of biochemical processes and interactions occur in microscale dimensions in the rhizosphere. Having the ability to interrogate and manipulate these microscale processes and environmental conditions with high spatio temporal resolution will elucidate mechanistic understanding of the processes. Microfluidics has proven to be a powerful approach to minimize reagent usage and to automate the often-repetitive steps. The micro-scale of the channels also allows precise control of reproducible conditions utilizing the laminar flow and automated fluidic operations . In addition, the micro-fluidic devices are well integrated with conventional imaging techniques by using a glass slide or cover slip as a substrate bonded with polydimethylsiloxane . These characteristics, as well as the ability to rapidly prototype and reproducibly manufacture using soft lithography technique, have enabled new ways of interrogating and studying the rhizosphere environment in a reproducible manner. Many of the microfluidic devices used for studying the rhizosphere share a similar design concept . They have an opening port, sometimes with pipette tips inserted into the PDMS body where the seed of the seedling rests and a micro-channel where the primary root grows into. The dimension of the channel depends on the type and age of the plant. For example, an Arabidopsis thaliana’s seedling is typically grown in a microfluidic device up to 10 days, with chamber dimension around 150 to 200 µm in height, whereas the Brachypodium distachyon seedling chamber is 1 mm in height due to its thicker roots . Media and/or inoculation of the micro-biome is achieved through additional channels to the main chamber. The PDMS body with the channels is typically bonded on a 50 mm by 75 mm microscope slide, and is made to accommodate multiple plants to increase throughput. Automated control offers the ability for continuous imaging and manipulation of media conditions with high temporal resolution. One notable example of a microfluidic device for rhizosphere studies is the RootChip, which uses the micro-valves in a PDMS device to control the fluidics . The first study using the RootChip grew 8 Arabidopsis plants on a single device with micro-valves but by the second iteration, the throughput has been doubled indicating rapid technological advances in the field. In addition, all these studies demonstrated spatiotemporal imaging at single-cell resolution and dynamic control of the abiotic environments in the rhizosphere. Another microfluidics-specific application to rhizosphere study is to use the laminar flow to generate the spatially precise and distinct microenvironment to a section of the root as demonstrated by Meier et al. . A young Arabidopsis’ seedling was sandwiched and clamped between two layers of PDMS slabs with micro-channel features to tightly control synthetic plant hormone flow with 10 to 800 µm resolution to the root tip area, enabling observations of root tissues’ response to the hormones. As many root bacteria produce auxin to stimulate the interactions with the root, this study showed the possible mechanism of microbiome inducing the interaction by stimulating root hair growth. Another application of laminar flow utilized the Root Chip architecture by adding the two flanking input channels to generate two co-laminar flows in the root chamber, subjecting a root to two different environmental conditions along the axial direction to study root cells adaptation to the micro-environment at a local level . These studies revealed locally asymmetrical growth and gene pattern regulations in Arabidopsis root in response to different environmental stimuli.

Chlorpyrifos application data for the simulated watershed were obtained from California Department of Pesticide Regulation database . Each individual report of agricultural use includes a unique grower identification code, the grower’s identification of the treated field , the geographic location of the treated field to within a square mile , the county, the name of the crop, the number of acres of the planted crop, the date and time of application, the number of acres treated, the pounds of active ingredient applied, the name of the pesticide product and the pounds of product applied. Using ArcGIS 9.1 software , Orestimba Creek watershed boundaries were intersected with the state Public Land Survey System map layer to obtain which townships and sections remain within the watershed boundaries. The monthly chlorpyrifos application data for the period of 2004 to 2010 was then summed for these sections. The average values were used as inputs for the model .Model calibration was performed by adjusting model coefficients within reasonable ranges to improve the match between model predictions and observed data. The observed data were retrieved from the U.S. Geological Survey stations along the San Joaquin River and embedded in the model. The model predictions and observed data were plotted and the differences between the predicted and observed values were calculated in terms of relative error,dutch bucket hydroponic absolute error, root mean square error and coefficient of determination. The correlation coefficient is often used to ensure that the simulation results predict measured data.

With a time series, however, the correlation coefficient ignores errors in time. For example, a one-day error in the prediction of a peak flow causes a low correlation coefficient even if the magnitude of the flow peak is predicted correctly . It is also possible to have a high correlation coefficient if the simulation results are a consistent multiple of the measured data, so it is very important to use other measures of error. Two types of error commonly used when calibrating WARMF include relative error and absolute error , where xs is the simulated value, xo is the observed value and n is the number of observations.Relative error represents the average difference between each simulated value and the observed value for the corresponding location and time. Since positive and negative errors cancel out, this is a measure of model accuracy or bias. Absolute error determines the average magnitude of the difference between simulated and observed values and is thus a measure of model precision. Absolute error reflects the expected error of an individual output value, while relative error reflects the expected error over an entire time series. A reasonable goal for calibration is to have the relative error within 10% of the average observed value, although this criterion might not be easily met for constituents with observed data near the detection limit . With very low measured concentrations, the error in observed data is often too large for precise calibration. Realistic expectations for absolute error vary by parameter and watershed.

According to Herr and Chen 2012, a good absolute error is generally less than 20% for flow and conservative chemical constituents, less than 30% for nutrients, and less than 50% for phytoplankton and total suspended sediment . Hydrological calibration and calibration for total suspended sediments were performed first, because an accurate flow and suspended sediments simulation is a pre-requisite for accurate water quality simulations.Flow was calibrated using data from 2005 to 2009. Boundary river inflows were checked for their accuracy and evapotranspiration coefficients, field capacity, saturated moisture and hydraulic conductivity were then adjusted so that the simulated agricultural return flow andgroundwater accretion can account for flow changes between the monitoring stations. There are three levels of hydrologic calibration: global, seasonal, and event-based . “Global” means that the simulated annual volume of water passing a gauge is the same as the volume measured. “Seasonal means” that the simulated seasonal variation of hydrology follows the same pattern of measured hydrograph. The measured hydrograph typically has a high flow during the rainfall season and a recession to base flow during the dry season. Event means that the simulated peak flows match the observed peaks during precipitation events. Key parameters adjusted during hydrology calibration are listed in Table 4-3. There are seven gaging stations along the San Joaquin River where simulated flow can be compared against observed data. Comparison of simulated and observed hydrographs for Orestimba Creek and SJR at Vernalis are shown in Figure 4-3 through Figure 4-6. Table 4-5 provides the summary statistics of model errors, assuming the measured flows are accurate. The relative error at Vernalis station was slightly bigger than the calibration target . The model slightly over-predicted the peak storm events observed in winter months . This was also reflected in the absolute error , which was slightly bigger than the calibration target for flow . At the Orestimba Creek, both the relative error and the absolute error were higher than the target of 10% for relative error and the target of 20% for flow .

That was partly due to the over-prediction of peak storm events and very low flow measurements approaching zero at that site . As mentioned previously, the error in observed data is often too large for precise calibration with very low measured concentrations. A visual inspection of figures 4-3 and 4-5 shows, however, that the model was able to capture trends in flow fluctuations most of the time. Therefore, the flow calibration was considered satisfactory for our purposes.Figure 4-7 through Figure 4-10 show the simulated and observed time series of total suspended sediment at two stations along the San Joaquin River. The stations presented here were selected based on the availability of the monitoring data for the calibration and validation period. Sediment load to the San Joaquin River came from boundary river inflows and overland flows from land catchments. Once in the river, sediment could settle out or be scoured from the river bed. The settling velocity for clay was set at 0.0003 m/d for most of the river segments due to turbulence. The monitoring data from the San Joaquin River had more total suspended sediment than can be explained by boundary river inflows alone. The monitoring data suggested the likelihood of scouring in the river bed that was predicted by the model using the default settling rate, scouring shear stress and velocity embedded in the model database. The observed seasonal variations of total suspended sediment were simulated by the model with good precision in general. However, the model simulated high peak concentrations resulting from storm runoff which were not observed in biweekly monitoring data at Orestimba Creek in 2005 and 2006 . The relative error at Vernalis station was higher than the calibration target due to two peak total suspended sediment concentrations during storm events in 2006 and 2008, which were under-predicted by the model. . The absolute error at Vernalis station was barely within the target for total suspended sediment calibration . At the Orestimba Creek, both the relative error and the absolute error were within the calibration targets of 10% and 50% respectively . Based on these results, the calibration for total suspended sediments was considered satisfactory.Key parameters adjusted for calibration of nitrate were nitrification and denitrification rates at catchments and rivers, adsorption coefficients, vegetation composition and initial pore water concentrations. Figure 4-11 through Figure 4-14 compare the time series of simulated and observed nitrate at two stations along the San Joaquin River. The stations presented here were selected based on the availability of the monitoring data for the calibration and validation period. The relative error at Vernalis station was within the calibration target of 10%, but the absolute error was higher than the calibration target for nutrients . This is partly due to the several extreme nitrate concentrations observed during 2008,dutch buckets system which were not simulated by the model . It is also evident from Figure 4-13 that the number of observed data at the Vernalis station dramatically increases after October 2007, with multiple data points accommodating one single day. It was not clear whether these data were hourly monitoring data or data from multiple sources embedded in the model input. Since this effect cancels out during relative error calculation, the relative error remained low; however, this effect was reflected in the absolute error . At the Orestimba Creek, the relative error was within the calibration target of 10%, but the absolute error was higher than the calibration target for nutrients . This is partly due to the several peak nitrate concentrations during storm events simulated by the model which did not match with the observed data . Another reason for the high absolute error might be the fact that only a few observed data points were available at this station. There were big data gaps especially for winter months and for the period between 2007 and 2009 . The accuracy of the monitoring data at some monitoring stations should also be verified. However, a deeper analysis of the model calibration and fine-tuning of the model was beyond the scope of this study.

There were several other monitoring stations along the San Joaquin River, such as Crows Landing, Stevinson, Patterson, and Maze Road, which performed better during nitrate calibration . Based on these results, nitrate calibration was considered satisfactory for our purposes.Key parameters adjusted for chlorpyrifos calibration were chlorpyrifos decay rates in water and bed sediments, adsorption coefficients, and initial pore water concentrations. Figures 4-15 and 4-16 compare the time series of simulated and observed chlorpyrifos at Orestimba Creek, the site selected for chlorpyrifos simulations. Table 4-5 provides the summary statistics of model errors for chlorpyrifos. Chlorpyrifos calibration was limited by the scarcity of the number of the observed data resulting in very high relative and absolute errors. The errors were also partly due to the presence of two abnormally high observed chlorpyrifos concentrations, which were not simulated by the model. Another limitation for the chlorpyrifos calibration was the fact that a portion of the observed data was at or near the method detection limit. However, the visual inspection of Figure 4-15 indicates that the model was able to follow the seasonal patterns and magnitude of chlorpyrifos peak concentrations most of the time. Therefore, chlorpyrifos calibration was considered satisfactory for our purposes.California has committed to cutting greenhouse gas emissions by 40% of 1990 levels by 2030. As a sector, agriculture is responsible for 8% of state emissions. Approximately two-thirds of that is from livestock production ; 20% from fertilizer use and soil management associated with crop production; and 13% from fuel use associated with agricultural activities . California plays an essential role in the nutritional quality of our national food system, accounting for, by value, roughly two-thirds of U.S. fruit and nut production, half of U.S. vegetable production and 20% of U.S. dairy production. Assembly Bill 32, California’s primary climate policy law, adopted in 2006, has spurred research into practices and technologies that could assist in reducing emissions and sequestering carbon. Here we report onmore than 50 California-based studies prompted by this landmark legislation. We note that the California Department of Food and Agriculture, California Air Resources Board, California Energy Commission and California Department of Water Resources have been critical to funding much of the science reviewed here. This article grew out of conversations with state agencies concerning the need for a review of the current evidence base to inform emissions-reduction modeling and revisions to the state Climate Change Scoping Plan , which specifies net emissions reduction targets for each major sector of the California economy . It is important to note that the Scoping Plan states that work will continue through 2017 to estimate the range of potential sequestration benefits from natural and working lands . With over 76,000 farm and ranch operations in California, covering about 30 million acres , there are no one size fits all solutions. But as we outline below, there are numerous opportunities to both reduce GHG emissions and sequester carbon across diverse agricultural operations — small to large, organic and conventional, crop and livestock. Perhaps most importantly, many of these practices have cobenefits for water conservation, restoration and conservation of natural lands, or farm economics.

DAB polymerizes in the presence of H2O2 and endogenous peroxidase to form a brownish-red precipitate that can be easily visualized using bright-field microscopy. After staining, trimmed sheath segments were mounted in 50% glycerol. Images were acquired digitally and further processed with the Olympus analySIS cell^F software.To assess the ISR-triggering capacity of S. plymuthica IC1270, susceptible rice plants were grown in soil containing IC1270 bacteria, and subsequently challenged with several fungal pathogens exhibiting different modes of infection. In these ISR bio-assays, the resistance-inducing potential of IC1270 was compared to that of P. aeruginosa 7NSK2, a well-studied PGPR strain which we previously uncovered as a potent activator of induced resistance responses in rice. We first tested whether root colonization by S. plymuthica IC1270 exerts a protective effect against infection by the hemibiotrophic ascomycete M. oryzae, causal agent of the devastating rice blast disease and a major threat to food security worldwide. By 4 days post-inoculation , leaves of control, non-induced plants displayed typical water-soaked, diamond-shaped lesions, developing conidia at the center of each lesion by 6 dpi. In contrast, IC1270-colonized plants exhibited a marked reduction in the number of these susceptible-type lesions, producing a resistance phenotype mimicking that of quantitative trait loci-governed intermediate resistance . This resistance type is characterized by the abundance of small necrotic non-sporulating lesions,flood and drain table less than 2 mm in diameter, 60 to 72 h post-inoculation . Consistent with our previous findings, treatment with P. aeruginosa 7NSK2 resulted in a substantial reduction of disease as well.

No significant differences in the number of susceptible-type lesions could be observed between IC1270- and 7NSK2-treated plants, indicating that IC1270 and 7NSK2 are equally effective in suppressing M. oryzae. Because IC1270 clearly inhibited the growth of M. oryzae in dual culture experiments , possible systemic plant colonization by the rhizobacteria was checked. However, in all bio-assays performed, IC1270 bacteria were absent from sheaths or leaves of root induced plants, indicating that bacterial colonization remained confined to the root zone . Although such spatial separation does not rule out the possibility that IC1270-conferred protection might result from long-distance translocation of bacteria-produced allelochemicals to systemic leaves, the latter is rather unlikely as pilot experiments aimed at elucidating the bacterial traits underpinning IC1270-ISR revealed that mutants defective in the global response regulator protein GacA, which controls the synthesis of various anti-fungal metabolites , were as effective as wild-type IC1270 in reducing rice blast disease severity . The cumulative data therefore strongly suggest that the beneficial protective activity exerted by S. plymuthica IC1270 is based on activation of the plant’s defensive repertoire, rather then being caused by microbial antagonism. To test the spectrum of effectiveness of this IC1270-mediated ISR, we next assayed for induction of resistance against the sheath blight pathogen, Rhizoctonia solani, and the brown spot pathogen, Cochliobolus miyabeanus, both of which are considered necrotrophic fungi. In contrast to M. oryzae, which sequentially invades living cells, R. solani and B. oryzae kill host cells at very early stages in the infection, leading to extensive tissue damage. As shown in Fig 1B, both IC1270 and 7NSK2 failed to reduce disease caused by R. solani.

This impaired ISR response was not due to insufficient root colonization as bacterial counts in the rhizosphere of treated rice seedlings were comparable to those obtained in the M. oryzae bio-assays . Interestingly, in all four independent experiments, IC1270 pretreatment favored subsequent infection by R. solani, causing an average 39.6% increase in disease severity relative to non-induced controls. A similar trend was observed when challenging with C. miyabeanus, with IC1270 consistently promoting vulnerability to the latter pathogen . Root colonization by 7NSK2, however, yielded variable results. No significant differences between control and 7NSK2- treated plants could be observed in three bio-assays, whereas in the two remaining assays, root treatment with7NSK2 rendered rice seedlings substantially more susceptible to brown spot. In all experiments, mock-inoculated control plants remained healthy, and no apparent differences in appearance, size, or weight of control, 7NSK2 or IC1270-treated plants were observed prior to challenge infection . Thus, under the experimental conditions used in this study, root treatment with the ISR-inducing bacteria did not lead to detectable effects on plant growth that could have affected the growth or development of the respective pathogens. Collectively, these findings demonstrate that S. plymuthica IC1270 plays an ambivalent role in the rice induced resistance network, acting as a potent elicitor of resistance to the hemibiotroph M. oryzae while promoting susceptibility to the necrotrophs C. miyabeanus and R. solani.To begin to unravel the defense mechanism underpinning IC1270-mediated ISR, we analyzed the cytological alterations associated with restriction of M. oryzae in IC1270-induced plants using the intact leaf sheath method designed by Koga and associates. In this system, intact leaf sheaths of control, non-induced and IC1270-treated plants of the highly susceptible rice variety CO39 were routinely inoculated by injecting a conidial suspension of the virulent blast isolate VT7.

For comparison with R gene-mediated ETI, we also included the VT7- resistant variety C101LAC, the latter being a near-isogenic line of CO39 carrying the blast resistance genes Pi-1 and Pi-33.Similarly, quantitative recording of attempted blast infections revealed no significant differences in the number of unsuccessful penetration events, indicating that both IC1270-mediated ISR and R-gene-conditioned ETI are unlikely to impede pre-penetration development by M. oryzae . On the other hand, epidermal cells were found to respond to fungal ingress through various cellular reaction types depicted at 48 hpi in Fig 2A. A susceptible reaction was manifested as a type 1 phenotype in which extensively branched invasive hyphae vigorously invaded living epidermal cells with little or no visible host response. Interaction phenotype 2, on the other hand, was characterized by prompt arrest of fungal growth in the first-invaded epidermal cell, a phenomenon associated with enhanced vesicular activity and browning of the anti-clinal cell walls, while a type 3 reaction represented infection sites in which fungal invasion was curtailed shortly after penetration due to development of HR-like cell death, as indicated by the characteristic aggregation of the cytoplasm and a bright autofluorescence of the anticlinal cell walls. As expected, sheath cells of noninduced, susceptible CO39 plants inoculated with virulent VT7 predominantly mounted a type 1 reaction, whereas HR was the prevailing plant response in the incompatible interaction between VT7 and C101LAC. Most conspicuously, IC1270-induced CO39 sheath cells displayed an interaction profile resembling that observed in VT7-invaded sheaths of genetically resistant C101LAC, with type 3 reactions accounting for approximately 60% of all interactions by 48 hpi . At later stages of infection, M. oryzae had massively colonized the epidermis and mesophyll of CO39 sheaths causing extensive host damage as evidenced by the ubiquitous presence of cellular debris and fragmented remnants of host cell walls around invasive hyphae in the mesophyll . By contrast, in resistant C101LAC, as well as in IC1270-induced CO39, invading hyphae were largely trapped within hypersensitively dying cells in the epidermal layer, preventing fungal passage to the underlying tissue. Because rapid accumulation of phenolic compounds is a hallmark of rice defense against M. oryzae, we also examined the effect of IC1270 pre-treatment on the level of autofluorescence. Autofluorescence was detectable as early as 18 hpi, irrespective of IC1270 treatment or the level of resistance of the cultivars used . However, similar to what was observed in resistant C101LAC,rolling bench root treatment of CO39 with IC1270 caused the frequency of autofluorescent appressorial sites to increase rapidly from 18 hpi onward, reaching a level of 60 and 100% of all interactions by 24 and 36 hpi, respectively . By contrast, in non-induced CO39 cells, less than 6% of the appressorial sites showed autofluorescence 24 hpi, indicating that root colonization by IC1270 primes rice sheath cells for accelerated deposition of autofluorescent phenolic compounds at sites of attempted pathogen invasion. Along with the high frequency of hypersensitively reacting cells, these observations suggest that IC1270- mediated ISR and R-gene-conditioned ETI act, at least in part, through a similar set of defense reactions.There is ample evidence demonstrating the active involvement of reactive oxygen species , and H2O2 in particular, in the induction, signaling and execution of blast resistance in rice.

Furthermore, in the course of previous studies, we demonstrated that pyocyanininduced H2O2 micro-bursts are primordial for the onset of P. aeruginosa 7NSK2-mediated ISR against M. oryzae. Taking these facts into account, we sought to extend ourcytological analysis of ISR elicited by IC1270 by monitoring the spatiotemporal patterns of pathogenesis-related H2O2 production. In planta accumulation of H2O2 was visualized using an endogenous peroxidase-dependent staining procedure with 3,3′-diaminobenzidine . In these DAB assays, reddish-brown precipitates are deposited at the sites of H2O2 accumulation. No DAB accumulation was observed in mock-inoculated controls, regardless of IC1270 treatment or the inherent level of resistance of the cultivars used. However, comparative analysis of H2O2 production in pathogen-inoculated seedlings revealed the occurrence of a wide range of distinct DAB staining patterns that could be grouped into five categories . The first type comprised interaction sites in which DAB accumulation was not detectable despite massive fungal colonization of both penetrated and neighboring epidermal cells. Conversely, interaction sites displaying H2O2 accumulation in the primary invaded epidermal cell following spread of the invasive hyphae into neighboring cells were classified as a type II reaction. Type III interaction sites were characterized by the ubiquitous occurrence of DAB-positive vesicle-like bodies targeted to the invading hyphae. A type IV reaction referred to intracellular DAB staining tightly associated with the characteristic cytoplasmic aggregates of HR-expressing cells , while interaction sites displaying wholecell DAB accumulation were scored as a type V reaction. Importantly, when the DAB solution was supplemented with ascorbate, staining was abolished, indicating that the staining was due to H2O2 . Leaf sheath cells of susceptible CO39 were characterized by the high ratio of H2O2-negative type I reactions, accounting for 78% and 67% of all interaction sites by 36 and 48 hpi, respectively . In some incidences , H2O2accumulated in the initially penetrated epidermal cell following the formation of an extensively branched mycelium in the neighboring cells. Yet, this type II reaction seemingly occurred too late to effectively stall the pathogen. IC1270-induced CO39 cells, on the other hand, exhibited a strikingly different set of responses in that type I reactions, reaching a level of 33% at 36 hpi, were no longer discernible by 48 hpi. The rapid decline in the frequency of type I reactions from 36 hpi onward corresponded to an approximately 15% increase in the frequency of both type III and type V reactions. HR-like cell death of attacked epidermal cells, seen at approximately 52% of all interaction sites, was always associated with H2O2 accumulation in the cytoplasmic aggregates, beginning 32 hpi. Although not identical, by 48 hpi the H2O2 signature of IC1270-treated CO39 plants showed substantial similarity to that observed in the incompatible interaction between C101LAC and VT7, thereby further emphasizing the possible mechanistic similarities between IC1270-mediated ISR and R-protein-dictated ETI. Starting 50 hpi, a strong accumulation of H2O2 was found in CO39 mesophyll cells that appeared to collapse, whereas in samples from IC1270-induced CO39 or C101LAC sheaths, DAB staining in the mesophyll layer was seldom observed . However, at these late infection stages, massive H2O2 accumulation is most likely a consequence of progressive cellular destruction and overtaxed anti-oxidative capacities, and hence, a chaotic reaction associated with susceptibility, rather than a controlled defense response restricting cellular accessibility for M. oryzae. Together these results clearly demonstrate the potential of IC1270 to prime rice for augmented generation of epidermis-localized H2O2.In light of the well-documented ability of ROS to serve multiple defense-related signaling functions, sometimes with opposite effects in different contexts, we asked whether the ability of IC1270 to boost pathogenesis-related H2O2 generation might account for the differential effectiveness of IC1270-mediated ISR against M.oryzae, R. solani and C. miyabeanus. To address this question, we examined the effect of manipulating the oxidative stress in pathogen-inoculated leaves on subsequent disease development. To artificially raise the level of ROS in inoculated leaves, detached leaves were pressure-infiltrated with mixtures of glucose plus glucose oxidaseand xanthine plus xanthine oxidase .

Similar results were observed in LUX ARRHYTHMO mutants in diploid wheat, another component of the evening complex . These results suggest that in the temperate cereals, the evening complex of the circadian clock acts as a transcriptional repressor of PPD1 . Interestingly, two LUX-binding sites are present in the PPD1 promoter, including one in the region deleted in the Ppd-A1a allele. In barley, changes in photoperiod have been shown to have rapid effects on the expression of circadian clock genes . However, we did not observe significant changes in the expression profiles ofany of the core circadian clock genes after 21 NBs, suggesting that changes in the clock played a limited role in the induction of flowering by NBs . Moreover, the strong delay in heading time observed in the Kronos and Paragon ppd1-null mutants under NBmax demonstrated that PPD1 is the major driver of the acceleration of heading time by NBs. This does not rule out the possibility that the circadian clock may play an important role in the regulation of the intermediate steps between PPD1 and FT1 induction or in the PPD1-independent photoperiod pathway in the temperate grasses.In this study, we show that while a single NB as short as 15 min in duration is sufficient to induce PPD1, the peak of expression is not observed until 3 h after the NB . This timeline of events suggests that additional molecular steps may be involved in the transcriptional activation of PPD1 following the initial short exposure to white light. NB responses have previously been shown to be rapid, hydroponic equipment and red-light NBs of 2 min were shown to be sufficient to accelerate flowering . The short length of the light pulse required to trigger the NB response is consistent with a role of the phytochromes in the initial steps of the NB response. In Arabidopsis, conversion of phytochromes from Pfr to Pr forms occurs within 5 min of exposure to high radiance R light, and 2 min of R light treatment is sufficient to initiate the phosphorylation of PIFs, which are direct targets of activated phytochromes .

Phosphorylated PIFs are targeted for degradation by the 26S proteasome, triggering downstream transcriptional responses within 15 min of the light signals . The time lag between the light application and the up-regulation of PPD1 transcript levels suggests the existence of intermediate molecular steps. Based on the involvement of wheat PHYB and PHYC in the light activation of PPD1 transcription and the known interactions between phytochromes and PIFs in Arabidopsis, we hypothesize that the degradation of one or more PIFs acting as PPD1 transcriptional repressors may be involved in the light activation of this gene. A putative PIF binding site is present within the region of the PPD1 promoter that is deleted in the Ppd-A1a allele . According to this hypothesis, the application of FR after NB reduces Pfr levels and limits the degradation of this putative PIF, thereby maintaining some transcriptional repression of PPD1 . Although NBs do not perfectly mimic the LD response, there are several similarities between the two processes, particularly in the PPD1-dependent photoperiodic response. Both processes are dependent on the PHYB/PHYC-mediated light activation of PPD1, both processes require multiple inductive cycles to accelerate flowering, and in both NBs and in plants carrying the Ppd-A1a allele, expression of PPD1 during the night is associated with accelerated flowering. Based on these similarities and on previous studies, we propose a tentative working model for the PPD1-dependent photoperiodic regulation of flowering in wheat . According to this model, flowering is accelerated only when the light-induced expression of PPD1 coincides with the expression and/or activity of one or more circadian-regulated factor required for the induction of FT1. Under LD, but not under SD, PPD1 expression coincides with the putative additional factor, inducing FT1 expression .

When NBs are applied in the middle of the night, light-induced PPD1 expression coincides with a peak of the putative additional factor, resulting in maximal activation of FT1 and early flowering . Although NBs applied earlier or later than this point still result in the induction of PPD1, these NBs no longer coincide with a peak of the putative circadian-regulated factor required for the activation of FT1. In Arabidopsis, the sensitivity of the flowering response to the induction of FT expression is most effective when FT is artificially induced during the evening and early night , suggesting that the timing of FT induction can also carry information relevant to the acceleration of flowering. Other studies support the hypothesis that the timing of PPD1 induction is critical for flowering. In wheat plants carrying the Ppd-A1a allele conferring reduced sensitivity to photoperiod, PPD1 is expressed during darkness . Therefore, even in non-inductive SD photoperiods, PPD1 expression coincides with the peak activity of the putative circadian-regulated factor required for the activation of FT1 and the induction of flowering . This last result suggests that no light stimuli are required to induce FT1 and flowering when PPD1 is mis-expressed during the night. However, in both the phyB-null and phyC-null mutants, the relatively high transcript levels of PPD-A1a were insufficient to induce FT1. A possible explanation for this observation is that PHYB and PHYC are important for some of the intermediate molecular steps required for the FT1 up-regulation by PPD1.The putative additional factor required for FT1 induction is likely to be regulated by the circadian clock, with its expression or activity peaking between 6 and 10 h after dusk under a SD photoperiod of 16 h of darkness. This putative factor could function to stabilize or activate the PPD1 protein or be an additional factor that acts either in a complex with PPD1 or downstream of PPD1 to activate FT1. Alternatively, PPD1 may activate a protein that degrades a repressor of FT1 or induce epigenetic changes in FT1 or other intermediate genes.

The identification of this clock-regulated putative factor involved in the PPD1 activation of FT1 is an outstanding question of the PPD1-mediated photoperiodic response in wheat.Plant-based pharmaceutical production is appealing given its inexpensive facility and production cost, linear scale-up, the absence of animal pathogens, and capability to produce complex proteins and perform post-translational modifications, which overcomes one or more drawbacks of traditional recombinant protein expression systems such as animal cell culture and bacterial fermentation.Much work has been carried out using stably transformed plants, but the significantly reduced development and production timeline makes transient expression of proteins in whole plants a particularly attractive option, cutting the time to bring critical medications to the market during a pandemic. Vacuum agroinfiltration is the most widely used method for uniformly introducing agrobacterium harboring an expression cassette containing a gene of interest into plant tissue given its natural ability to transfer T-DNA into plant cells, which is ideal for transient protein production in plants. Although a plant-based recombinant protein production system provides distinct advantages over traditional systems, the differences between N-glycosylation of proteins produced in plants and humans could limit the use of plant systems for the production of glycoprotein-based pharmaceuticals. In higher eukaryotes, the initial steps of N-glycosylation processing are well conserved between plants and human, resulting in oligomannose-type N-glycosylation. However, late N-glycosylation maturation in the Golgi apparatus is kingdom-specific, and thus results in different N-glycosylation on proteins produced in plants compared with human. These plant-specific glycans may lead to potential safety issues such as hypersensitivity or allergy, as plant-specific α-fucose and β-xylose are known to be important IgE binding determinants of plant allergens. Thus, if these plant-specific glycans are present in an injected pharmaceutical product, the glycoprotein could trigger immunological response, or at least, result in a short circulation half-life. N-glycans of proteins produced from mammals are often terminated in β-galactose and sialic acid; sialic acid is particularly important as it typically increases the circulation half-life of proteins. There are a number of ways to avoid incorporating plant-specific glycans in the product such as adding a signal sequence at the C-terminus of the target protein to retain it within ER, or RNAi-mediated knock-down of α-fucose and β-xylose. These methods require modification to either the protein sequence or to the expression system, which can potentially affect protein structure and require a long developmental time. As an alternative, the use of small molecule inhibitors of intracellular glycosidases is a highly flexible bio-processing approach for controlling protein N-glycosylation patterns in transient agroinfiltration processes, and it is the approach investigated in this study. Here, we report an easy and fast way to modify N-glycosylation of recombinant proteins produced transiently in N. benthamiana through the addition of kifunensine in the agrobacterium suspension prior to vacuum agroinfiltration,vertical grow table which avoids modification to protein sequence or expression system while producing recombinant protein with oligomannose-type N-glycans that are similar between plant and human. Oligomannose-type N-glycan is preferred for the HIV-1 viral vaccine development as a vast majority of broad and potent neutralizing antibody responses during HIV-1 infection target mannose-glycan-dependent epitopes. In addition, monoclonal antibodies with oligomannose N-glycans show increased ADCC activity and affinity for FcγRIIIA. Protein N-glycosylation starts in the endoplasmic reticulum , where N-glycan precursors Glc3Man9GlcNAc2 are first synthesized, followed by the removal of terminal Glc residues, resulting in Man9GlcNAc2 structures. Then, a single α1,2 linked mannose is removed by ER class I α-mannosidase, producing Man8GlcNAc2 structures. The trimming of α1-2 mannose residues continues with the action of Golgi class I α-mannosidases in cis-Golgi to give Man5 structures. Kifunensine is a highly selective inhibitor of class I α-mannosidases in both plants and animals, and it has been used in cell cultures to produce recombinant proteins with oligomannose-type N-glycans. Although the general effects of kifunensine and other alkaloid-like processing glycosidases inhibitors are well understood, for the most part this information comes from cell culture system studies. Meanwhile, the study of kifunensine on whole-plant transient protein expression through agroinfiltration is new.

There are only two published papers on whole-plant kifunensine treatment, where kifunensine was supplied hydroponically throughout the whole incubation period, which requires larger quantities of kifunensine, a more expensive hydroponic system and constant monitoring especially at large scale as compared to our method. In addition, it was also shown that hydroponic kifunensine treatment resulted in dramatic decrease of protein expression level which was not observed with our method. In this study, Fc-fused capillary morphogenesis gene-2 , an anthrax decoy protein, served as a model protein, which contains single N-glycosylation site within its Fc domain . CMG2-Fc is a potent anthrax decoy protein as shown previously, where the CMG2 domain binds to anthrax protective antigen and prevents the anthrax toxin from entering the cell. Meanwhile, the presence of Fc domain significantly increases the serum half-life, which prolongs therapeutic activity owing the slower renal clearance for larger sized molecules and interaction with the salvage neonatal Fc-receptor. CMG2-Fc thus can be used as potent anthrax therapeutic and prophylactic without frequent redosing. The expression levels of CMG2-Fc produced transiently in wild type N. benthamiana under kifunensine treated and untreated conditions were measured with a sandwich ELISA, and protein N-glycosylation profiles were evaluated with mass spectrometry for kifunensine treated and untreated conditions. The findings in this study can be applied for N-glycosylation modification of other plant recombinant proteins when oligomannose-type N-glycans lacking core fucose are preferred, without the need to modify protein sequence and/or subcellular targeting.The CMG2-Fc expression levels in crude leaf extract were quantified through a sandwich ELISA to confirm the expression of CMG2-Fc, and to evaluate the effect of kifunensine on protein expression. The ELISA relies on binding of CMG2-Fc through the Fc region to protein A coated on a 96-well plate. A secondary anti-Fc polyclonal antibody linked to a horseradish peroxidase enzyme binds to the CMG2-Fc allowing colorimetric detection. The potential interference of plant host cell proteins and nonspecific binding were determined to be negligible. Twenty wild-type 5–6-week old N. benthamiana plants were divided equally into experimental and control groups, agro-infiltrated and incubated for 6 days, then whole leaves were extracted under identical conditions to determine protein expression. Kifunensine at a concentration of 5.4 µM was included in the agrobacterium suspension in the Kifunensine group. This kifunensine concentration was chosen as a starting point by taking the average of concentrations used in a previous CHO cell culture study, as no reference concentration is available for vacuum infiltration of kifunensine.

Excess Cd uptake in plants normally induces the accumulation of reactive oxygen species in plants and has severe consequences, such as chromosome aberrations, protein inactivation, membrane damage, and and further leading to leaf chlorosis and root growth inhibition. Furthermore, accumulation of Cd in crops enhances the risk of Cd poisoning in humans and animals. Brassica species have been identified as Cd hyperaccumulators. Brassica parachinensis L.H. Bailey is a leafy vegetable widely consumed in China, Europe, and other regions of the world. Thus, elucidating the molecular mechanisms of Cd accumulation in this plant is essential for developing effective strategies to control Cd accumulation in the plant’s edible parts. Cd accumulation in plant tissues generally involves a three-step process: absorption and accumulation of Cd in roots from the soil, translocation of Cd to the shoot via vascular tissue, and Cd storage in leaves. The HMA , ZIP , and Nramp families are among the transporter families that have been identified as being involved in these processes. Our previous transcriptome analyses of B. parachinensis also showed that differentially expressed genes enriched in the gene ontogeny terms ‘transmembrane transport’ and ‘metal ion transport’ may be involved in response to Cd, including genes encoding members of some transporter families, such as the subfamily C of ATP-binding cassette proteins and HMAs. HMAs,growing strawberries vertically which belong to the P1B subfamily of the P-type ATPase super family, have been extensively investigated in the model plant Arabidopsis as well as in some crop plants, and the main focus of these studies has been on their functions.

For example, eight members of HMAs have been identified in Arabidopsis thaliana, and among these, AtHMA1–AtHMA4 are thought to specifically transport divalent cations, such as Zn2+, Cd2+, Co2+, and Pb2+ [10]. AtHMA2 is generally regarded as a Zn2+- ATPase. It contains a conserved short metal binding domain in the N-terminus and a long metal binding domain in the C-terminal end; Zn2+-binding affinity was detected in both domains, and Cd2+- and Cu+-binding affinity was detected in the Nterminal domain. Some studies showed that AtHMA2 functioned as an efflux to drive the outward transport of metals from the cell cytoplasm and responsible for cytoplasmic Zn2+ homeostasis and Cd detoxification. Some researchers proposed that AtHMA2 together with AtHMA4 played key roles in the long-distance root to-shoot transport of Zn2+ and Cd2+ by loading these ions into the xylem. Similar results were also reported in wheat TaHMA2. However, it seems that OsHMA2 in rice has a different role. The enhanced sensitivity to Cd and tolerance to zinc deprivation afforded by heterologous expression of OsHMA2 in yeast cells suggest that OsHMA2 functions as a Cd influx transporter. These studies showed that HMA2 and its subfamily members in different plants may function differently. There is a lack of thorough knowledge of the role of BrpHMA2 in Cd hyper accumulation in the leafy vegetable B. parachinensis. The function of BrpHMA2 and the mechanisms that regulate its expression must be elucidated. Previous studies have indicated that plants employ a universal and conserved approach to regulate the transcription of heavy metal uptake and tolerance genes. For example, in a bean , PvMTF-1 , which could be induced by PvERF15 , may regulate the expression of the stress-related gene PvSR2 and confer Cd tolerance to the plant.

In Arabidopsis, two basic helix–loop–helix transcription factors , FIT and PYE , modulate iron deficiency responses by regulating the expression of IRT1 and FRO2, whereas the bHLH TFs IAA-leucine resistant 3 and bHLH104 can form heterodimers and bind to specific elements in the promoter of PYE to regulate PYE. NAC TFs are members of the most prominent TF families in plants. These TFs play essential roles in diverse biological processes, such as growth, development, senescence, and morphogenesis, and are widely involved in various signaling pathways in response to different phytohormones and multiple abiotic and biotic stresses. For example, NAC019, NAC055, and NAC072 negatively regulate drought stress-responsive signaling. NAC096 is associated with drought stress. It could exert its function via a mechanism like that of basic leucine zipper protein -type TFs to bind specifically to abscisic acid – responsive elements in the promoters of several drought stress-responsive genes. This finding implies that NAC096 and bZIP-type TFs can sometimes regulate the same target genes. Studies have also shown that the core DNA-binding sequences of NACRE and ABRE are PyCACG and PyACGTGG/TC , respectively. In a previous study, we identified a few NAC and AREB TFs triggered by Cd stress in B. parachinensis. However, their functions remain unclear. To clarify the molecular mechanisms of Cd accumulation in B. parachinensis, the function of a Cd-responsive metal ion transporter gene BrpHMA2 and the coregulation of BrpHMA2 transcription by two TFs were examined in this study. The findings reveal a precise regulatory mechanism in B. parachinensis in response to Cd stress.We previously analyzed the Cd-induced mRNA transcriptome of B. parachinensis and found that several HMA homologs were substantially expressed under Cd stress. We cloned one of the HMA2 homologs and constructed the phylogenetic tree of this HMA2 homolog with other HMAs in A. thaliana, Oryza sativa, Zea mays, and Alfred stone crop by the neighbor-joining method using MEGA5. The results revealed that the sequence of this HMA2 homolog is closer to that of the AtHMA2 gene , and thus it was named BrpHMA2.

The transcript level of BrpHMA2 in seedlings grown hydroponically was examined using reverse transcription–quantitative PCR to investigate the expression pattern of BrpHMA2 in B. parachinensis. According to the results, BrpHMA2 was expressed at higher levels in leaves than in roots. Cd stress may increase BrpHMA2 expression in leaves and roots, although BrpHMA2 expression in leaves fluctuates owing to developmental regulation . The GUS gene was transformed and expressed in Arabidopsis using the promoter of BrpHMA2 to corroborate the expression pattern, and histochemical assays were performed. Instant β-glucuronidase staining for 0.5 hours showed that the GUS signal was visible in the vascular bundles of the leaves and roots of the plants treated with 50 μM Cd 2 for 2 days, but not in vascular bundles ofseedlings that were not treated with Cd . Results from an examination of transcripts of the GUS gene in the reporter line were also consistent with these findings . This showed that BrpHMA2 could be induced by Cd stress. However, when the pBrpHMA2::GUS transgenic seedlings were subjected to GUS staining for 3 hours, a strong GUS signal could be observed in the vascular bundles of the cotyledons, true leaves, stems, petals, filaments, and the carpopodium of the seeds in young siliques. The blue GUS signal was particularly strong in the tissue junction regions where the vascular bundles were clustered . These results indicate that BrpHMA2 may function primarily in transport in vascular tissues. The fluorescent signal of BrpHMA-GFP was detected at the plasma membrane by transient expression analysis in protoplasts of B. parachinensis leaf cells , indicating that BrpHMA2 is localized at the plasma membrane.To further analyze the function of BrpHMA2, BrpHMA2 fused with the galactose-inducible promoter was transformed into a Cd-hypersensitive yeast mutant, ycf. In the presence of the transcriptional inducer galactose, Cd2+ considerably inhibited the growth of yeast cells with heterologous expression of BrpHMA2 compared with that of cells transformed with the empty vector . However, when gene expression was suppressed by the presence of glucose, no growth differences were detected between the cells transformed with BrpHMA2 and those transformed with the empty vector. The Cd content in the heterologous transgenic cells grown in liquid medium was higher than that in the control cells . These results indicate that BrpHMA2 functions as an affluxtype Cd transporter.To determine the TFs responsible for BrpHMA2 expression in B. parachinensis, a cis-element analysis of 2000 bp of the BrpHMA2 promoter was performed. In the promoter region, three ABRE cis elements were identified,vertical farming equipment all of which contain the G-box family core sequence ACGT . The NAC recognition site CGTG is likewise present in these ABREs. In the promoter of BrpHMA2, two additional NAC recognition motifs, CDBS and CACG, were found. Three ABREs , four NACRESs, and four CDBS cis elements were found in the promoter of BrpHMA2 . These findings suggest that certain transcription factors, such as NACs or AREBs, may control BrpHMA2 in B. parachinensis via these cis elements. To confirm this deduction and identify the regulatory pathways involved in the response to Cd stress, the transcriptome of B. parachinensis as mentioned above was used to collect data for the NAC and AREB genes that showed differential expression following Cd stress. Eighteen NAC genes and 11 AREB genes were selected to create a heat map, and three NAC TFs and three AREB TFs were identified as Cd-induced TFs . Their transcription levels were further analyzed by RT–qPCR. The results showed that the NAC TF genes BraA03000895, BraA010004584, and BraA10002796 were upregulated in the roots of the plants exposed to Cd for 1 day . After 4 days of Cd exposure, the AREB TF gene BraA01000449 was induced in roots, and BraA05001227, BraA01000449, and BraA01003678 were induced in leaves . Similar to the findings for BrpHMA2, our results suggest that these TF genes may respond to Cd. The coding sequences of the three NAC TFs and three AREB TFs listed above were cloned and submitted to the NCBI database. The last three or four numbers of each gene’s full name was used as the gene name. MEGA5 was used to create a phylogenetic tree of these NAC TF or AREB TF genes and Arabidopsis NAC or AREB genes using the neighbor-joining method.

The results revealed that the BrpNAC4584 and BrpNAC895 sequences were closer to those of Arabidopsis ANAC046 and ANAC087, respectively ; in addition, the BrpABI227 and BrpABI678 sequences were closer to that of AtABF4, and the BrpABI449 sequence was more comparable to that of AtABF3 .Electrophoretic mobility shift assays were conducted to investigate whether the BrpNAC895 protein directly binds to the promoter of BrpHMA2. Three probes containing NACRES and CBDS motifs on the BrpHMA2 promoter were designed and used for the EMSA. The results revealed that the BrpNAC895-MBP fusion protein could bind to the three probes in vitro . A chromatin immuno precipitation assay was performed using an anti-GFP antibody to precipitate BrpNAC895-GFP fusion proteins expressed in B. parachinensis protoplasts, and three fragments covering the NACRES and CBDS motifs on the BrpHMA2 promoter were designed and used for PCR. Moreover, there is only one base interval between the last two NACRES cis elements, so they were considered as one fragment . Approximately 1.5- to 2-fold enrichment of fragments pF1, pF2, and pF3 was detected compared with those found in the control . The results demonstrate that BrpNAC895 can promote the expression of BrpHMA2 by binding directly to the NACRES and CBDS motifs of its promoter.To investigate the mechanism of BrpHMA2 coregulation by BrpNAC895 and BrpABI449, a ChIP assay was performed by expressing BrpABI449-GFP in B. parachinensis protoplasts to analyze the binding affinity of BrpABI449 with the promoter of BrpHMA2. A qPCR analysis revealed that the BrpABI449 protein was enriched with fragments containing pF2 and pF3 of the BrpHMA2 promoter . We further performed an EMSA to confirm the binding of BrpABI449 to ABRE motifs in the promoter of BrpHMA2. The results proved that BrpABI449 could bind directly to the probes containing ABRE cis elements in the pF2 and pF3 regions of the BrpHMA2 promoter .The roles of BrpNAC895-binding loci in BrpHAM2 transcriptional regulation were investigated by constructing a series of BrpHMA2 promoter mutants by changing CACG/CGTG in the NACRES or CBDS to AAAA . A dual LUC assay was performed using the effector p35S::BrpNAC895 vector and the reporter vector was cotransformed into B. parachinensis protoplasts. Compared with pBrpHMA2::LUC, the cotransformation of p35S::BrpNAC895 with pMUT1::LUC or pMUT3::LUC resulted in much reduced LUC activity, but the cotransformation of the p35S::BrpNAC895 effector with pMUT2::LUC resulted in considerably higher LUC activity . Among the cotransformations of the promoter of BrpHMA2 with two or more mutations, substantially weaker LUC activity could only be seen in the transformations with promoters mutated at both locus 1 and locus 3 . These findings indicate that the mutation in the first and third NACRES motifs reduced the BrpNAC895-activated transcription of BrpHMA2, and these two binding loci may play central roles in the BrpNAC895-activated transcription of BrpHMA2.To elucidate the relationship between NAC and AREB TFs, a bimolecular fluorescence complementation approach was used.

Only one particle size of clay was used here, and diffusion rates were lower in this environment. In systems with low diffusion rates, exudate concentration is likely higher around the roots, which might lead to higher exudate re-uptake than in systems with larger particle sizes . Clays with different particle sizes might provoke a root morphology and exudation profile distinct from glass bead-grown plants and is worth further investigation. Further, substrate particle size might be a factor defining the amount of exudates present in soils.The largest difference in exudate profiles observed was between in situ clay-grown plants and other in situ conditions. Notably, the distinct exudation of clay-grown plants disappeared when exudates were collected in vitro, indicating that the differences observed resulted from the presence of clay, and not from an altered exudation of compounds by B. distachyon. About 20% of compounds were distinct between hydroponic and clay exudates, and most of these compounds were reduced in abundance in the presence of clay. Among these compounds were organic acids, amino acids, and nucleosides. When clay was incubated with a defined medium, 75% of compounds were reduced in abundance,container vertical farming among them negatively and positively charged compounds, as well as neutral compounds. The higher metabolite retention by clay in the defined medium experiment compared with the plant experiment might be due to several factors: The clay was incubated for two hours with the defined medium, but for three weeks with plants producing exudates.

Although exudates were also collected for two hours in the plant experiment, the clay was likely already saturated to some degree with exudates. The quantification of exudate amounts at different plant developmental stages in future studies would enable a better estimation of the total amount of compounds exuded and would correct for the difference in the two experimental setups. The reduction of metabolite abundance in the presence of clay is most likely due to its high ion exchange capacity, compared with quartz-based particles such as sand or glass beads . Previous studies investigating sorption of bacterial lysates to ferrihydrite found a depletion of more than half of the metabolites . Similarly, incubation of bacterial lysates with a soil consisting of 51% sand, 28% silt, and 21% clay resulted in low metabolite recovery rates . These findings are consistent with our data. Interestingly, two nitrogenous metabolites were higher in abundance in exudates of in situ clay-grown plants, . These compounds were not detected in clay negative controls, or in in vitro exudates of clay-grown plants, making it likely that the presence of plants leads to the release of these compounds from clay. Multiple examples exist in literature that describe a release of compounds from minerals by specific exudates. For example, plant-derived organic acids such as malate and citrate solubilize mineral-bound phosphate , and plant-derived oxalate releases organic compounds bound to minerals, making them available to microbial metabolism . Altered exudation depending on the growth substrate was also described for tomato, cucumber, and sweet pepper growing in stone wool, with higher exuded levels of organic acids and sugars compared with glass bead-grown plants .

The authors suggest that the presence of aluminum ions in stone wool might be responsible for the altered exudation observed. As the authors did not investigate in vitro-collected exudates of stone wool grown plants, it is unclear to which degree the observed effect was due to changes in plant metabolism or due to the presence of stone wool. In soils, metabolite sorption to minerals can lower decomposition rates . Also, the amount of clay in soil is correlated with retention of labeled carbon in soils . In clay-dominated soils, the size of clay particles shapes how much carbon can be retained: large clay aggregates were found to adsorb more carbon than smaller aggregates . Here, we only investigated one size of clay particles. Thus, it would be prudent to investigate the sorption behavior of clays with different particle sizes, and the ability of microbes to subsequently desorb these compounds. In natural systems, the presence of large amounts of clay with a specific particle size likely results in the sorption of plant-derived compounds to particles, changing the direct availability of these compounds to heterotroph organisms and, thus, altering soil processes.Microbes can release sorbed compounds from minerals, and they likely preferentially colonize minerals that are associated with compounds missing from the environment . The rhizobacterium Pseudomonas fluorescens utilized in this study was indeed able to desorb metabolites from clay, utilizing them as a carbon source for growth . In soils, root exudation creates zones with high metabolite concentrations.Although P. fluorescens was able to grow on particles conditioned with exudates, it did not grow on the effluent of the washed particles.

This suggests that the organism is able to release mineral-bound metabolites as an additional source for growth—a trait that supports competitiveness and survival in the rhizosphere. Root-associated bacteria have distinct exudate substrate preferences from bulk soil bacteria , which might also define the kind of compounds bacteria are able to release from minerals . Our results are further evidence that minerals play an important role in plant–microbe interactions by sorbing root exudates, which can later be solubilized by microbes for growth. We conclude that alteration in particle size affects root morphology in B. distachyon. Root exudation was constant per root fresh weight, and the exudate metabolite profiles were similar across root morphologies. Mass spectrometry imaging detected ion abundances across various regions of the root system, suggesting involvement of different tissues in exudation. Exudates were strongly sorbed by clay, significantly reducing the availability of free metabolites. Some of the clay-bound metabolites however could be utilized by a rhizobacterium for growth. Soil clay content thus is likely an important factor to consider when investigating root exudates or plant–microbe interactions in natural environments.Substrates with various particle sizes and surface chemistries were chosen as experimental systems to assess changes in root morphology and exudation. The particle sizes used correspond to large soil particles , intermediary particles , and small particles . Glass beads constitute an inert experimental system, for which the diameter of the spheres is defined, and the particles have a defined mineral composition . The sand and clay substrates constitute more natural environments than glass beads . The sand and clay mineral composition was either defined by the manufacturer or determined here. To assess the chemical properties of the substrates, the sorption of metabolites to the substrates was assessed by incubating them with a defined medium . The defined medium consisted of amino acids, organic acids, sugars, nucleobases, nucleosides, and others, see Table S2. The various substrates were sterilized, and the defined medium was prepared as a sterile, 20 µM equimolar solution. The substrates were fully submerged in the defined medium in sterile conditions and incubated at 24°C for 8 hr. The sterility of the system was confirmed by plating an aliquot on LB plates, followed by a 3-day incubation. The defined medium was removed by pipetting. The recovered volume was recorded; for substrates with smaller particle sizes, the entire volume could not be reclaimed. Samples were filtered through a 0.45-µm filter and frozen at −80°C. See “Liquid chromatography–mass spectrometry sample preparation” for sample processing.The frozen samples were lyophilized , resuspended in 3 ml LC/MS grade methanol , vortexed three times for 10 s, sonicated for 20 min in a water bath at 24°C, and incubated at 4°C for 16 hr for salt precipitation. Samples were then centrifuged for 5 min at 5,000 g and 4°C, and supernatants were transferred to new micro-centrifuge tubes and evaporated at 24°C under vacuum until dry. The dried extracts were resuspended in 500 µl LC/MS grade methanol,hydroponic vertical garden and the above procedure centrifugation, drying and resuspension procedure were repeated. Finally, samples were resuspended in 100% LC/MS grade methanol with 15 µM internal standards , with the solvent volume being proportional to the root biomass .In the various experimental systems used here, exudation rates could be limited by diffusion. To determine the diffusion rates of various substrates, sterilized substrates were added to pipettes with a 50 ml volume. The pipettes were sealed at the bottom with parafilm, placed vertically, 50 ml of substrate was added, and approximately 25 ml of sterilized 0.5× MS was added to fully immerse submerse the substrate . The experimental setup was sterile, but the experiment was conducted in non-sterile conditions.

Congo red 4B was solubilized in water at a concentration of 20 mg/ ml, and 250 µl of the dye was added simultaneously to pipettes containing the various substrates. The front of the dye was followed recorded over time up to 4.5 hr. Initially, the movement of the dye front was supported by mass flow .Brachypodium distachyon Bd21-3 seeds were dehusked and sterilized in 70% v/v ethanol for 30 s, and in 6% v/v NaOCl with 0.1% v/v Triton X-100 for 5 min, followed by five wash steps in water. Seedlings were germinated on 0.5× Murashige & Skoog plates in a 150 µmol/ m2 s −1 16-hr light/8-hr dark regime at 24°C for three days. Weck jars were rinsed five times with MilliQ water, sprayed with 70% v/v ethanol, treated with UV light for 1 hr in a laminar flow hood, and dried over night. The jars were filled with 150 ml of the respective substrate, and 50 ml of 0.5× MS basal salts liquid medium. Three seedlings were transferred into each jar, with the roots buried in the substrate. As a control, jars without substrate were prepared: PTFE mesh was cut to fit the size of the jar, and autoclaved. Three openings were cut into the mesh to hold the seedlings. The mesh was transferred to jars with 50 ml 0.5× MS medium. For each condition, an experimental negative control was prepared containing substrate, but no seedlings. The experimental control jars were treated the same as the jars containing plants. To enable gas exchange, two strips of micropore tape were placed across the jar opening, and the lid was set on top and wrapped with micropore tape to ensure sterility. Plants were grown in a 16-hr light/8-hr dark regime at 24°C with 150 µmol/m2 s −1 illumination, and the growth medium was replaced weekly: The old medium was removed by pipetting, and new 0.5× MS was added. Sterility of the jars was tested in week 3 by plating 50 µl of medium on Luria-Bertani plates, following by three days incubation at 24°C. Plants were grown for 21 days before exudate collection and root morphology determination.Mass spectrometry imaging was used to investigate spatial patterning of root exudation across the root system. Brachypodium distachyon seeds were sterilized and germinated on 0.5 MS plates as described above. A stainless steel MALDI plate was cleaned with 100% v/v ethanol, and a 7 × 7 cm square of aluminum foil was affixed to the plate with double-sided scotch tape. The foil was overlayed with 4 ml 0.1% ultrapure agarose to create a thin layer of agarose. Four-day-old seedlings were transferred to the agarose layer, and gentle pressure was applied with a spatula to embed the roots in the agarose. The stainless steel plate was transferred into a petri dish plate to keep humidity constant and incubated for 6 hr in a growth chamber with 150 µmol/m2 s −1 illumination and 24°C. MALDI matrix was prepared as follows: 10 mg/ml a-cyano-4-hydroxycinnamic acid and 10 mg/ml Super-DHB were dissolved in 75% v/v methanol, 24.9% LCMS-grade water, and 0.1% formic acid. The plate with the seedlings was removed from the growth chamber, leaves were cut to ensure flatness of the sample, and the sample was sprayed with MALDI matrix, which simultaneously desiccated the tissue. The plate was incubated for 24 hr in a vacuum desiccator until completely dry. Mass Spectrometry Imaging was performed using a 5,800 MALDI TOF/TOF in positive reflector MS mode with an Nd:YAG laser acquiring spectra over a range of 50−2000 Da and accumulating 20 shots/spot. The 4,800 Imaging Tool software was used to raster across the sample and record spectra in x-y step-sizes of 75 × 75 μm. Data viewing and image reconstruction were performed using OpenMSI .Ion chromatograms corresponding to metabolites represented within our in-house standard library were extracted from LC/MS data with Metabolite Atlas.