Horticulture

The first group includes four genes annotated as defense genes, a function that is likely not closely related with the observed phenotypes. This group includes TraesCS1B02G017500 and TraesCS1B02G0017600 , which encode proteins with NB-ARC and LRR domains characteristic of plant disease-resistance proteins involved in pathogen recognition and activation of immune responses. It also includes TraesCS1B02G017700and TraesCS1B02G0018100 , which are both annotated as defensins, a family of small plant antimicrobial peptides that serve to defend plants against pathogens. A second group includes three genes annotated as having enzymatic or housekeeping functions, which may not be compatible with the developmental nature of the observed changes in the roots of 1RSRW. The first gene in this group, TraesCS1B02G017800, encodes a methionine Smethyltransferase that has been implicated in the volatilization of selenium and in the biosynthesis of S-methylmethionine, a compound that is important in the transport of sulfur . The last two genes in this group encode proteins with chaperon functions. TraesCS1B02G019200 is a tubulin-folding cofactor E involved in the second step of the tubulin folding pathway. TraesCS1B02G019300 encodes a chaperone protein DnaJ,hydroponic gutter which stimulates the heat-shock protein Hsp70’s ATPase activity, stabilizing its interaction with client proteins.

These chaperon proteins play important roles under plant stress but are unlikely to play an important role in the phenotypic differences we observed under optimal hydroponic conditions. The third group includes genes involved in regulatory processes or in cell growth or division, processes more likely to be involved in the observed developmental changes in root growth . TraesCS1B02G017900 encodes an E3 ubiquitin-protein ligase CHIP-like protein that ubiquinate heat shock misfolded client proteins, targeting them for proteasomal degradation. Since E3 ubiquit inprotein ligases can ubiquitinate and regulate multiple targets, we could not rule it out as a potential candidate gene. We also included in this group the genes TraesCS1B02G018900 and TraesCS1B02G0019100, which encode 64% similar small GTP-binding proteins from the RAB family. These conserved proteins serve as molecular switches in signal transduction and play important roles in intracellular membrane trafficking, cross-talk with plant hormones and regulation of organogenesis, polar growth, and cell division , all functions that seem relevant to the observed differences in root development. TraesCS1B02G018700, TraesCS1B02G019700, and TraesCS1B02G019800 encode 12-oxophytodienoate reductase-like proteins involved in the biosynthesis of jasmonic acid. Since hormones can affect multiple developmental traits, these are also strong candidate genes. Finally, TraesCS1B02G020200 encodes a wall associated receptor kinase . These serine–threonine kinases are involved in signaling and cell expansion, making it an interesting candidate for the differences in root length observed in 1RSRW.

For the Cd-sensitive cultivar , addition of Cd significantly decreased SOD activities in roots compared with the control, which was intensified with increasing Cd concentrations . The activity of SOD was increased by 47.3%, 12.0% and 9.6% in the plants treated with Cd plus Si compared with the corresponding Cd treatments without Si, respectively . For the Cd-tolerant cultivar , very similar changes were noted in SOD activity in the Cd treatments with or without Si added, with an exception that no significant differences in SOD were found between the Cd1 treatment alone and the control . For the sensitive cultivar , CAT activity in the Cd treatment significantly decreased with increasing Cd concentrations compared with the control. Addition of Si significantly increased CAT activity in Cd-stressed pakchoi roots compared with Cd treatment alone throughout the whole experiment . For example, addition of Si increased CAT activities by 3.7%, 28.4% and 25.7%, respectively, at 0, 0.5 and 5.0 mg L-1 Cd, compared with the corresponding Cd treatments alone. For the Cd-tolerant cultivar , very similar results were obtained of CAT activities in the Cd treatments with or without Si, with an exception that addition of Si did not result in significant differences in CAT activities between the lower and the higher Cd treatments . For the Cd-sensitive cultivar, addition of Si significantly increased APX activities in roots by 55.1% compared with the control. The activity of APX was 16.7% higher in the Cd1 plus Si treatment than in the Cd1 treatment alone, compared to 11.4% at the Cd2 level . For the Cd-tolerant cultivar, very similar changes were observed in APX activities in the Cd treatments with or without Si, with an exception that significant increases in APX activity were found between the Cd plus Si treatment and the Cd treatment alone .Engineered nanoparticles have attracted great interests in commercial applications due to their unique physical and chemical properties. Increased usage of ENPs has raised concerns in the probability of nanoparticles exposure to environment and entry to food chain.

Plants are essential components of ecosystems and they not only provide organic molecules for energy but they can also filter air and water, removing certain contaminants. Definitively, plants play a very important role in uptake and transport of ENPs in the environment. Once ENPs are uptaken by plants and translocated to the food chains, they could accumulate in organisms and even cause toxicity and bio magnification. Nanoparticles are known to interact with plants and some of those interaction have been studied to understand their potential health and environmental impact, including quantum dots, zinc oxide, cerium oxide, iron oxide, carbon nanotubes , among others. The uptake of various ENPs by different plants was summarized in Table 1. Nanoparticles are known to stimulate morphological and physiological changes in several edible plants. Hawthorne et al. noted that the mass of Zucchini’s male flowers were reduced by exposed to CeO2 NPs. Quah et al. observed the browner roots and less healthy leaves of soybean treated by AgNPs, but less effects on wheat treated under same condition. Qi et al. reported that the photosynthesis in tomato leaves could be improved by treated with TiO2 NPs at appropriate concentration. Yttrium oxide ENPs have been broadly used in optics, electrics and biological applications due to their favorable thermal stability and mechanical and chemical durability.One of the most common commercial applications is employed as phosphors imparting red color in TV picture tubes. The environmental effects of yttria ENPs have not been reported. Even though the effects of certain NPs have been studied on several plants, the uptake, translocation and bio-accumulation of yttria NPs in edible cabbage have not been addressed until this study. This plant species was chosen and tested as part of a closed hydroponic system designed to study nanoparticles movement and distribution in a sub-strateplant-pest system as a model of a simple and controlled environment. The final test “substrate” used was plain distilled water , in which the tested NPs were mixed.

In order to observe the translocation and distribution of ENPs in plants, transmission electron microscopy has been one of the most commonly used techniques to identify the localization at cellular scale in two-dimensions , because it can be used to observe all kinds of ENPs. On the other hand, ENPs with special properties, such as up conversion NPs and quantum dots with a particular band gap can be studied with a confocal microscope with alternative excitation wavelengths to trace the ENPs. Several synchrotron radiation imaging techniques exploiting high energy X-ray have become widely used in plant science, which can measure both spatial and chemical information simultaneously, like micro X-ray fluorescence and computed tomography. In this research, we use synchrotron X-ray microtomography with K-edge subtraction to investigate the interaction of yttria NPs with edible cabbage. By using the KES technique, the µ-XCT can not only detect the chemical and spatial information in 3D, but also analyze the concentration of target NPs. The uptake,hydroponic nft channel accumulation, and distribution mapping of yttria NPs in both micro scale and relatively full view of cabbage roots and stem were investigated. We found that yttria NPs were absorbed and accumulated in the root but not readily transferred to the cabbage stem. Compared with yttria NPs, other minerals were observed along the xylem in both cabbage roots and stem. To the best of our knowledge, few reports have studied the impact of yttria NPs on cabbage plants. In addition, by using µ-XCT with KES technique, the distribution and concentration mapping of nanoparticles in full view of plant root have not been previously reported.The µ-XCT was carried out at Beamline 8.3.2 at the advanced light source, Lawrence Berkley National Laboratory. From scanning energies of 16.5 to 17.2 keV, below and above yttrium K-edge, the X-ray attenuation coefficient sharply increases by a factor of 5. Other elements decrease slightly in their attenuation coefficients over this energy range. The localization of yttria NPs can be identified by the subtraction between two reconstructed image datasets , shown in Fig. 2. The slices collected above and below the K-edge were set with same brightness and contrast settings to fairly compare with each other. These are inorganic elements which support the growth of cabbage. Some biological structures suffered radiation damage during scanning, resulting in a small amount of shrinkage. The bright regions circled in Fig. 2c were caused by such shrinkage, resulting in a registration mismatch between the images above and below the edge. To identify and map the distribution of yttria NPs, an image segmentation protocol was employed that could highlight regions with yttria without finding these regions corresponding to sample shrinkage. The detailed segmentation process is given in the “Method” section.By using K-edge subtracted image technique with Monochromatic X-ray tomography, the translocation and distribution of NPs in the cabbage root is clear . Figure 3a and b were constructed by 17.2 keV and 16.5 keV reconstructed slice datasets, respectively. Their color maps were based on the transverse slice pixel values/absorption coefficients over the range from 0.2 to 17.8 cm−1 . An obvious difference between 17.2 and 16.5 keV visualization in absorption coefficient of yttria NPs was observed. The distribution of yttria NPs in root was segmented and colored in red . A large amount of NPs were found aggregated at left bottom of the root. Since yttria NPs were not water-soluble, the water that contained them was kept in constant movement with an air pump working 24/7. However, it seems that the dense roots formed a web-like structure that made the suspended NPs to accumulate and aggregate among the roots.

Uptake of NPs by the root has been observed at primary and lateral root junction as well according to the transverse slice. Figure 2a is one transverse slice localized at the arrow in Fig. 3c showing the junction between primary root and lateral root. We found that the yttria NPs were absorbed by the lateral roots, and particulates began to accumulate along the outer epidermis of primary roots with limited entrance into the vascular tissue of the primary root. It might happen that endodermal cell walls were blocking the entrance of aggregated yttria NPs into vascular tissue. This is shown in the upper section of the 3D visualization where no yttria NPs were observed above the root system. Besides the full view of the translocation in the cabbage root system, the distribution of yttria NPs at the micro-scale within a lateral root was detected and investigated . Figure 4a shows the localization of the micro-scale lateral root visualization. The 3D visualization of micro-scale was built by the segmented transverse reconstructed slices, and the red regions were localized yttria NPs . It is clear that roots are able to uptake the yttria NPs in ground tissue , which appear to accumulate in the root with limited entrance of yttria NPs into vascular tissue being transported through the xylem. Xylem vessels are small with diameters usually smaller than 1 μm in vegetables like cabbage plants to over 100 μm in vessels found in trunks of large trees. Vessels allow nutrients contained in water to be distributed throughout the plant. For NPs, however, if they aggregate, the blockage is expected, that is what we have observed in this study. Long term studies might show that yttria NPs might provide more negative than positive effects on plant growth and development as found with other NPs. Using K-edge subtraction image technique with dualenergy X-ray scanning, the concentration of target NPs can be calculated. This method has been discussed elsewhere. For the cabbage shoot, no yttria NPs were observed , which means that no yttria NPs transported from roots to shoots. As we found no yttria NPs entering vascular tissues of primary root, the yttria NPs accumulated making it difficult to be transported by xylem from the root to the rest of the plant.

Similar to what has been observed in other plant species, supplying rice leaves with G/GO resulted in the sustained production of H2O2within the apoplast , whereas a mixture of xanthine and xanthine oxidase was found to generate both superoxide and H2O2, the latter by dismutation. Treatment with either compoundor with the enzymes alone had no significant effect on disease development compared to buffer-treated control leaves . However, infiltration of G/GO or X/XO dramatically reduced the size of the necrotic lesions incited by M. oryzae infection . By contrast, pre-treatment with G/GO or X/XO mixtures strongly stimulated necrosis induced by R. solani . By 60 hours after infection, the majority of ROS-treated and Rhizoctonia-inoculated leaves showed extensive necrosis and were almost completely deteriorated . Enhanced ROS generation also greatly enhanced lesion formation by C. miyabeanus, suggesting a common pathogenicity mechanism for both these necrotrophs .Although exogenous catalase did not significantly alter lesion development, infiltration of rice leaves with a specific catalase inhibitor, 3-aminotriazole,grow table hydroponic prior to inoculation, was indistinguishable from the G/GO- or X/XO-treated leaves. No lesions were detected in leaves infiltrated with ROS-producing mixtures, catalase or 3-AT alone, as previously reported.

Building on our earlier work with respect to 7NSK2-mediated ISR, we sought to extend our analysis of the proposed dual role of ROS in rice defense by feeding the pro-oxidative pigment pyocyanin to hydroponically grown rice plants and observe any effects on plant resistance. Opposite to the enhanced resistance observed against M. oryzae, pyocyanin feeding favored subsequent infection by both C. miyabeanus and R. solani . Amending the pyocyanin solution with ascorbate, which has long been recognized as a major antioxidant buffer and free-radical scavenger, severely attenuated the pyocyanin-provoked resistance or susceptibility, corroborating our previous findings. Taken together, these results clearly demonstrate that enhanced ROS levels in inoculated leaves positively influence resistance to M. oryzae, while exerting a negative effect on rice defense to C. miyabeanus and R. solani.Despite the emergence of rice as a pivotal model for molecular genetic studies of disease resistance in cereal crops, molecular information regarding chemically and biologically induced defenses is still largely missing. In an effort to broaden our understanding of the rice induced resistance machinery, we analyzed the host defense responses underpinning ISR triggered by the biocontrol agent S. plymuthica IC1270. The results presented in this study demonstrate that root colonization by IC1270 predisposes rice to undergo a massive oxidative burst and related HR-like cell death at sites of attempted pathogen invasion, a process culminating in heightened resistance to the hemibiotrophic blast pathogen, M. oryzae.

The same treatment, however, rendered plants more susceptible to attack by the necrotrophic pathogens R. solani and C. miyabeanus. Besides tagging ROS and HR-like cell death as two-faced players in the rice defense response, these findings strengthen the argument that rice requires distinct mechanisms for defense against M. oryzae and the necrotrophs R. solani and C. miyabeanus. Mounting evidence indicates that generation of systemic resistance does not necessarily require direct activation of defense mechanisms, but can also result from a faster and stronger activation of basal defenses in response to pathogen attack. For instance, unlike pathogen-induced SAR, classic rhizobacteria-mediated ISR in Arabidopsis is not associated with a direct induction of defense mechanisms, but with priming for augmented defense activation upon challenge inoculation. Other ISR-inducing PGPRs also have been found to enhance the plant’s defensive capacity by hyper-activating pathogen-activated defenses, suggesting that priming for enhanced defense is a common mechanism in PGPR-mediated ISR. The results presented in this study add further support to this concept as root colonization by IC1270 did not cause a strong constitutive resistance phenotype, but ratherprimed plants to hyper-respond to subsequently inoculated pathogens, resulting in excessive defense activation and enhanced resistance to M. oryzae.

This priming effect of IC1270 was borne out by the observation that challenge inoculation of IC1270-colonized plants with M. oryzae entailed a rapid accumulation of autofluorogenic phenolic compounds in and around epidermal cells displaying dense cytoplasmic granulation , two features that are considered as hallmarks of an ETIassociated HR. Comparative profiling of pathogenesis-related H2O2 accumulation in blast susceptible, yet ISR-expressing, and genetically resistant leaf sheath cells, further strengthened the parallels between R protein-mediated ETI and IC1270- triggered ISR priming . Hence, IC1270 appears to protect rice from M. oryzae by reprogramming pathogen attacked epidermal cells to undergo a rapid HR-like response, thereby providing a possible functional interface between rhizobacteria-mediated ISR and avirulent pathogen-induced ETI. Such mechanistic similarities between ISR and ETI are compatible with the idea that defense signals from multiple ‘entry points’ can converge and target overlapping sets of defense effectors. Particularly relevant in this regard is the substantial overlap between gene expression changes and alterations in SA content induced during an avirulent pathogen-triggered ETI response, and those induced by treatment with flg22, an 22-amino-acid epitope of the archetypal MAMP elicitor flagellin. Although unequivocal evidence is still lacking, the striking homologies with the sensitive perception mechanisms for pathogen-derived MAMPs that function in PTI suggest that ISR-triggering rhizobacteria are recognized in a similar manner. In this perspective, it is not inconceivable that the mechanistic parallels between IC1270-mediated ISR and ETI can be traced to converging MAMP- and R-protein-induced defense responses. Furthermore, consistent with the view of ETI as an accelerated and amplified PTI response, such MAMP-orchestrated ISR elicitation may also explain the partial nature of the IC1270-induced resistance against M. oryzae. Apart from S. plymuthica IC1270, several other biological and chemical agents have been shown to be capable of inducing resistance to M. oryzae, among which the SA analog BTH and the redox-active pigment pyocyanin, key determinant of ISR induced by P. aeruginosa 7NSK2. Interestingly, both these resistance inducers appear to mimic IC1270 in that they produce a similar resistance phenotype, characterized by hypersensitively dying cells in the vicinity of fungal hyphae. Although it does not follow that the signaling conduit governing IC1270-mediated ISR is necessarily the same as that leading to pyocyanin- or BTH-inducible blast resistance, such commonalities apparent at the level of defense mobilization suggest that these elicitors may feed into related, if not identical, resistance pathways. Further supporting this hypothesis is the overlap manifest at the level of resistance to attackers, with IC1270, BTH and pyocyanin all being ineffective or even increasing vulnerability to C. miyabeanus and R. solani. Intriguingly, induction of ISR by the PGPR strain P. fluorescens WCS374r appears to rely on a different resistance strategy and was found to be associated with priming for a diverse set of HR-independent cellular defenses, the prompt elaboration of invading hyphaeembedding tubules being a prominent component. Considering this apparent plasticity in the molecular processes leading to induced resistance against M. oryzae, it is tempting to speculate that rice is endowed with multiple blast-effective induced resistance pathways.

The rapid production of ROS during the so-called oxidative burst is a hallmark of the plant’s defense response. Although ROS are generally viewed as initiating agents in the disease resistance network ,grow table accumulating evidence indicates that ROS formation can cascade either to the detriment or benefit of the plant depending on the lifestyle and parasitic habits of the invading pathogen. Hence, ROS can play a dual role in pathogen defense, acting as key players in resistance to biotrophic pathogens on the one hand, while weakening necrotroph resistance by assisting pathogen-induced host cell death on the other. Taking these facts into account, we propose that priming for enhanced ROS generation may likewise function in IC1270-mediated ISR, thereby accounting for the differential effectiveness of this resistance against hemibiotrophic and necrotrophic pathogen assault. Critical to the formation of a hypothesis of primed ROS generation as a key event in ISR by IC1270 was the observation that artificially increased H2O2 levels, either resulting from infiltration of ROS-generating mixtures, inhibition of endogenous catalase activity or hydroponic feeding of pro-oxidative pyocyanin, faithfully mimicked IC1270 in conditioning resistance to M. oryzae but susceptibility to C. miyabeanus and R. solani. Although we are aware that final proof for primed ROS generation as the causal resistance mechanism underpinning IC1270-mediated ISR requires the use of inhibitor compounds able to abrogate the oxidative burst , such scavenger experiments could not be performed since detached leaves, needed for effective infiltration of chemicals in rice, somehow failed to develop ISR. Therefore, we can not rule out the possibility that the altered pathogen response of IC1270-induced plants may result in part from ROS-independent processes. Nonetheless, the involvement of boosted ROS generation in the establishment of IC1270-mediated ISR is apparent. In accordance with previous studies, continuous generation of H2O2 in situ by infiltration of G/GO or 3-AT did not induce any detectable cell death per se, indicating that additional pathogen-induced signals are needed for expression of HR-like cell death. Indeed, current concepts suggest that death of host cells during the HR requires the poised production of nitric oxideand ROS, coupled to simultaneous suppression of the plant’s antioxidant machinery. In view of these data, it could be reasoned that IC1270-mediated priming for potentiated ROS generation might lower the threshold for activation of programmed cell death, thereby blocking the hemibiotroph M. oryzae in its initial biotrophic phase. In line with this concept, there is ample evidence demonstrating that early-produced H2O2 is a central signal leading to the elicitation of a wide range of blast-effective defenses, among which programmed cell death. Most tellingly, Kachroo and associates [84] reported a fungal glucose oxidase gene to sequentially induce H2O2 generation, rapid HR-like cell death and enhanced resistance against M. oryzae when ectopically expressed in young rice plants. On the other hand, it is not inconceivable that IC1270-mediated priming for H2O2 may tilt the ROS-controlled cellular life-or death balance toward death, thereby facilitating subsequent tissue colonization by the necrotrophs R. solani and C. miyabeanus.

This notion is corroborated by recent observations demonstrating that IC1270 pretreatment has no marked impact on the early infection events in C. miyabeanus– or R. solani-challenged plants except for a substantial increase in the number of dying cells preceding the fungal growth front . However, given the myriad defense related plant responses modulated by ROS, other yet unidentified mechanisms also may play a role.Parasitic plants directly invade and rob nutrients from host plants. The consequences can be devastating to the host plant and some of the world’s most pernicious agricultural pests are parasitic weeds. The number of parasitic angiosperms is surprisingly large with over four thousand parasitic species identified in nineteen different plant families. Parasitic plants have a wide diversity of growth habits ranging from the tiny flowered mistletoes that live in the tops of trees to the enormously flowered and rootless Rafflesia whose entire vegetative body is endophytic. The degree to which parasites rely on hostresources also varies. Some obligate parasites, like Rafflesia, have lost photosynthetic capabilities and are fully heterotrophic. Others, like Triphysaria, are facultative parasites that can mature without a host plant but will parasitize neighboring plants when available. The single feature shared by all parasitic plants is the ability to invade host tissues via a haustorium. Haustoria of parasitic plants fulfill multiple functions including host attachment, penetration, and translocation of resources from host to parasite. Interestingly, the competence to develop haustoria has originated in autotrophic ancestors multiple times during the evolution of angiosperms. There are two general hypotheses for the evolutionary origins of haustoria. One hypothesis suggests that the genes encoding haustorium development are derived from nonplant organisms, such as bacteria or fungi, that are endophytic or which have transferred a set of genes required for haustorium formation into the parasite genome. The second is that genes encoding haustorium developmentare derived from those present in autotrophic angiosperms where they fulfill functions unrelated to parasitism. The identification of genes associated with haustorium development will provide insights into the evolutionary origins of plant parasitism. These genes will also elucidate the degree to which haustoria in different parasitic families are encoded by convergent or homologous genetic pathways. Parasitic plants in the Orobanchaceae develop haustoria on their roots in response to contact with host roots. Several molecules, typically products of the phenylpropanoid pathway, have been identified that induce haustorium development when applied to Orobanchaceae roots in vitro.

The average mass of CMG2-Fc per kg leaf fresh weight was 717 and 874 mg/kg leaf FW for the Kifunensine and Kifunensine samples, respectively . The control group expression level was consistent with previous results. These data suggest that the addition of kifunensine in the agro-infiltration process was not detrimental to transient protein production, and in this case, it resulted in a 22% increase in CMG2-Fc yield. This allows the use of kifunensine for modification of glycosylation profiles without compromising protein expression. Total soluble protein content of whole leaf extract of Kifunensine and Kifunensine groups were similar as shown in Figure 1, which indicates that kifunensine does not have significant impact on plant protein synthesis in general.In this study, the influences of one-time kifunensine vacuum infiltration on the expression level, N-glycan profile of a recombinant protein, mobile vertical grow tables namely CMG2-Fc, produced transiently in N. benthamiana plants were evaluated in both whole-leaf extract and AWF. We found that kifunensine had a positive impact on protein production when supplied in the agroinfiltration solution; specifically, we observed a 22% increase of protein expression with kifunensine treatment condition, presumably owing to its suppressing effects on ER-associated degradation pathway.

This finding is consistent with previous observations in multiple mammalian cell culture systems, and there is no reason to suspect that this will not be the case for other eukaryotic systems, including plant systems. Plants were monitored visually throughout the incubation period, and there were no significant phenotypical differences between kifunensine-treated and control groups.In the case of a whole-plant study, Roychowdhury et al. have shown that the yield of recombinant cholera toxin B sub-unit dropped by 30% and 75% when kifunensine was supplied at 5 µM hydroponically for 3 days and 5 days post agroinfiltration, respectively. In contrast, we observed slight increase in protein yield when kifunensine was infiltrated in leaf tissue instead of being supplied hydroponically. Thus, the lower protein yield they observed may have resulted from continued application in the hydroponic solution, and it is eliminated when kifunensine was supplied directly to leaf tissue through vacuum infiltration. In addition, in the hydroponic study, the target protein was retained in ER, while the model protein in our study and other cell culture studies were targeted for secretion. This difference in protein targeting may also play a role in protein yield changes upon kifunensine treatment. Kifunensine is known to inhibit enzymatic activity of class I α-mannosidases, and thus should stop mannose trimming in the first place to yield single Man9 N-glycan structures. However, we observed multiple oligomannose-type N-glycans with mannose residues ranging from 3 to 9, although the most abundant structure was Man9.

This observation is consistent with cell culture kifunensine studies, where multiple oligomannose-type N-glycans were detected under kifunensine treatment. This could potentially due to the difference in inhibition efficacy of kifunensine towards class I α-mannosidases isoforms, which results in an incomplete inhibition of mannose trimming from Man9 structure. Also taking enzyme kinetics into consideration, depending on the ER concentrations of Man9 glycoprotein substrate, class I α-mannosidases, and kifunensine, enzymatic mannose trimming from Man9 could take place even if the amount of active ER α-mannosidase I is low. Although mannose trimming was not completely inhibited at Man9 structure, this method still showed the ability to significantly modify glycosylation using a simple bio-processing approach. It is likely that Man9 abundance can be further increased if treated with higher concentration of kifunensine, but it is not necessary if the goal is to eliminate the production of plant-specific complex N-glycans. Although in this case, CMG2-Fc can be purified easily from whole-leaf extracts through a one-step purification with Protein A chromatography, in many cases, multiple steps of chromatography are required to purify a target protein from a large pool of host native proteins when a highly selective affinity tag is not present. This could result in low protein yield and difficulties to achieve high purity, which is typically required for therapeutic recombinant protein products. Targeting proteins to the apoplast allows the collection of target protein in AWF, which contains much lower levels of plant native proteins than whole-leaf extract since only secreted proteins are collected, thus lowers the downstream process complexity. In this case, CMG2-Fc purity and concentration increased by 3.9-folds and 4.4-folds, respectively, when collected in AWF versus in whole-leaf extract.

A similar trend was observed in kifunensine-treated samples, which confirms that kifunensine does not affect protein secretion, allowing secretion of CMG2-Fc with oligomannose-type glycoforms. The increase in purity and concentration was consistent with a previous study on harvesting a target protein from plant AWF. Hence, AWF collection is a feasible method for recombinant protein harvesting, which avoids contamination with intracellular host cell proteins, and is particularly valuable when target protein is hard to purify. Together with kifunensine treatment, apoplast-targeted recombinant protein without any plant-specific glycoforms can be transiently produced in N. benthamiana, and likely in other plants as well. Products can be collected at high concentration and purity from AWF, containing predominantly oligomannose-type N-glycans. Further studies should focus on determining how long the inhibition effect of kifunensine lasts after the one-time vacuum infiltration by monitoring the protein glycoform profile at multiple time points after vacuum infiltration, and the threshold concentration of kifunensine that results in a complete N-glycan shift from plant complex-type to oligomannose-type for other glycoproteins, particularly those with more N-linked glycosylation sites. In addition, the protein expression kinetics should be compared between kifunensine-treated and untreated groups to maximize target protein yield. Depending on the desired glycoform, this method can also be applied to other N-glycan processing inhibitors such as castanospermine, deoxynojirimycin, and swainsonine.The transient expression of CMG2-Fc is achieved by whole-plant vacuum infiltration of N. benthamiana with Agrobacterium tumefaciens EHA 105 containing binary vector for CMG2-Fc expression and Agrobacterium tumefaciens containing the pBIN binary vector for expression of the RNA silencing suppressor P19 from Tomato bushy stunt virus. Each A. tumefaciens strain was cultured separately in 20 mL of Luria-Bertani media with selection antibiotics for 18 h in the dark at 28 ◦C, on an orbital shaker at 250 rpm. Then, 8 mL of each culture is transferred to 250 mL LB media and incubated for 18 h in the dark at 28 ◦C at 250 rpm shaking to further amplify the cell population. The A. tumefaciens cells were collected by centrifugation at 1800× g for 30 min at 15 ◦C, and the cell density was quantified through absorbance measurement at 600 nm. Both A. tumefaciens strains were resuspended into infiltration buffer with a final cell density of 0.4 for each strain . Six-week old N. benthamiana plants were turned upside down and submerged into the agrobacteria suspension, followed with vacuum infiltration for 1 min after vacuum pressure reached 20 inches Hg. Infiltrated plants were then incubated in a growth chamber at 20 ◦C and 90% humidity for 6 days. All leaves were cut from the petioles were used for AWF collection or stored at −80 ◦C for whole leaf protein extraction.Microplate wells were coated with unlabeled Protein A at a concentration of 0.05 mg/mL in phosphate-buffered saline at 37 ◦C for 1 h, mobile vertical farm and then blocked with 5% nonfat dry milk in PBS buffer. Crude plant extract and purified CMG2-Fc standards were added to wells and incubated at 37 ◦C for 1 h. CMG2-Fc bound to Protein A on the plate was detected by adding 50 µL of horseradish peroxidase -labeled goat anti-human IgG at concentration of 0.4 µg/mL and incubated for 1 h at 37 ◦C.

Between each of these steps, microplates were washed with PBS with 0.05% v/v of Tween-20. Finally, the protein concentration was quantified by adding 100 µL of TMB substrate . Plates were incubated at room temperature for 10 min, followed by addition of 100 µL 1N HCl to stop the reaction. The TMB substrate reacts with HRP, allowing colorimetric detection of CMG2-Fc levels.Site-specific N-linked glycosylation analysis including measurement of the glycopeptide relative abundance of the protein was analyzed with mass spectrometry. Samples were first reduced with 2 µL of 550 mM dithiothreitol at 65 ◦C for 50 min to break the disulfide bond between cysteine amino acids. Then 4 µL of 450 mM iodoacetamide was added as an alkylation reagent for 25 min. Samples were placed in the dark environment to prevent the loss of IAA efficacy. After denaturing, proteins were digested with 1 µg of trypsin for 18 h in a 37 ◦C water bath. The digestion process was stopped by placing samples at −20 ◦C for around one hour. An Agilent 1290 infinity ultra-high-pressure liquid chromatography system coupled to an Agilent 6495 triple quadrupole mass spectrometer was used for N-glycosylation analysis. For sample separation, the analysis column used on the UHPLC system was an Agilent Eclipse plus C18 column . To protect the analysis column, an Agilent Eclipse plus C18 trap column was used to trap samples first. The mass spectrometer was operated using the dynamic multiple reaction monitoring mode which is a targeted tandem MS mode and Agilent MassHunter Quantitative Analysis B.05.02 software was used for data analysis. In the MRM method, glycopeptides were quantified with the glycopeptide mass used as the precursor ion and the common oxonium fragments with m/z 204.08 and 366.14 used as product ion. The concentration of glycopeptide in ion counts was normalized to the total ion counts of glycopeptides in the sample for the relative abundance calculation. The rhizosphere is the area around the plant root in soil where microorganisms are densely populated and dynamic interactions between the plants and microorganisms occur. These complex interactions and the assemblage of microorganisms are established, shaped, and maintained by the plant root. Plants exude photosynthetically fixed carbons as nutrient sources, secondary metabolites, and signaling molecules to populate root ecological niches with beneficial microorganisms. This “rhizosphere effect” has important implications for plant growth and protections against biotic and abiotic stressors, and for geochemical carbon cycling in soil environments. With the expected rise of severe, climate-change-related weather conditions such as drought and the growing need to increase plant productivity with sustainable agricultural practices, it is vital to gain a mechanistic understanding of the rhizosphere effect and engineer the rhizosphere to improve plant growth and resilience. To understand the mechanisms of the highly dynamic processes occurring in the rhizosphere, in situ interrogation of the system with high spatiotemporal resolution is necessary. However, due to the underground nature of the root system and the sheer complexity of the microbiome in the highly heterogeneous soil environment, studying root and microbial interactions has been challenging.

For example, in a typical rhizosphere analysis experiment, the plant is uprooted from soil in the field or a pot at a defined time point, and the microbiome and other relevant chemicals are sampled in a destructive manner. This practice of uprooting the plant is limiting because the spatial information on microbial community members is lost and the same plant generally cannot be sampled over time. Further, there are many confounding variables, such as chemical reactions with minerals in soil that complicate the analysis and make the studies less reproducible and relevant across different locations and environments. To overcome these experimental hurdles, researchers have designed various types of specialized devices such as rhizotrons and microfluidics devices to improve specific aspects of sampling, analytics, and manipulation of the rhizosphere system, albeit often deviating substantially from the natural system. Fluorescent microscopy is a promising tool for noninvasive in situ imaging of the microbial and root interactions with high spatiotemporal resolution. Developing in situ rhizosphere imaging methods is made especially more relevant as the microbial community colonization of the root and the persistence and succession patterns are likely dynamic and dependent on the developmental stage of the plant. Better knowledge in this can help guide synthetic microbial community inoculation protocols to optimize plant productivity. A notable development of in situ imaging devices is a microfluidic root chip by Massalha et al., in which real-time imaging of the microbial colonization of Arabidopsis thaliana seedlings was captured by high-resolution fluorescent confocal microscopy.

Metabolites were identified following the Metabolomics Standards Initiative conventions, using the highest confidence level , which is identified as at least two orthogonal measures versus authentic chemical standards . Three orthogonal measures were used to compare samples with authentic chemical reference standards: retention time , fragmentation spectra , and accurate mass . Peak height and retention time consistency for the LC/MS run were ascertained by analyzing quality control samples that were included at the beginning, during, and at the end of the run. Internal standards were used to assess sample-to sample consistency for peak area and retention times. Metabolite peak heights were scaled relative to the maximum peak height in any sample within an experiment to allow for relative comparison of peak heights between samples , but not for absolute metabolite level quantification.Chemical classes were assigned to metabolites with the ClassyFire compound classification system.To explore the variation between experimental conditions,vertical vegetable tower the metabolite profiles were PCA-ordinated, and the 95% confidence level was displayed as ellipses for each treatment. Hierarchical clustering analysis with a Bray–Curtis Dissimilarity Matrix was performed with the python 2.7 Seaborn package. Metabolite significance levels were analyzed with the Python SciPy ANOVA test coupled to a python Tukey’s honestly significant difference test with alpha = .05 corresponding to a 95% confidence level.

To test whether metabolites sorbed to clay were accessible for a plant-associated bacterium, the desorption rates of metabolites from different substrates were tested in a first experiment, and the growth rate of Pseudomonas fluorescens WCS415 on various substrates pre-incubated with metabolites was tested in a second experiment. The desorption rate of metabolites from substrates was determined for glass beads , sand , and clay . The substrates were incubated with 50 times concentrated defined medium or with 0× DM for 6 hr at 23°C. The substrates were subsequently washed three times with water, to remove soluble metabolites. The recovered metabolites of all three steps were analyzed by LC/MS, as described above. Substrates were added to a 12-well plate , and 2 ml of 0× DM was added to each well. A Pseudomonas fluorescens WCS415 preculture was grown in 5 ml 20× DM for 16 hr at 30°C, 200 rpm. The culture was pelleted at 4,000 g, 23°C for 5 min, and resuspended in 0× DM. The wells were inoculated with an initial optical density of 0.05 in triplicates. The plates were incubated at 30°C for 3 d , 1 ml of the supernatant was removed to determine OD at 600 nm. Positive growth controls were P. fluorescens grown in the same experimental setup in 50×, 20×, 10×, and 0× DM, but without substrate. A set of negative controls was prepared to account for different variables in the experiment: Substrates incubated with 0× DM with bacteria were set up as a growth control, accounting for metabolites already adsorbed to clay. Substrates incubated with 50× and 0× DM but without bacteria were used to control for changes in optical density of clay caused by DM.

The metabolite desorption experiment was performed by adding 2 cm3 of clay pre-incubated with 50× or 0× DM to a 12-well plate in triplicates, followed by the addition of 2 ml of 0× DM. The plate was incubated for 3 d at 30°C. Subsequently, 1.5 ml of the supernatant was removed by pipetting and placed in a new 12-well plate. Half of the wells were inoculated with P. fluorescens, the other half served as negative controls. OD at 600 nm was determined after 3 d of growth at 30°C.Global climate change has resulted in shifts in precipitation patterns, causing stress on freshwater resources, especially in arid and semi-arid regions . In many of these areas, demand for water has led to increasing use of municipally treated wastewater . Agriculture has been one of the primary targets for TWW reuse with water districts and governments promoting the adoption of recycled water for irrigation . However, the use of TWW for irrigation may come with potential risks, as TWW is known to contain a wide variety of human pharmaceuticals . The use of pharmaceutical compounds has increased with population growth and economic development, resulting in over 1500 compounds currently in circulation . Their widespread consumption has led to their occurrence in TWW as well as in TWW impacted surface water . For many of these pharmaceuticals, there is limited knowledge about their potential chronic effects in the environment . Further, many of these compounds can transform in the environment, resulting in the formation of transient or recalcitrant transformation products, many with unknown fates and effects in environmental compartments .

Diazepam belongs to the class of psychoactive compounds known as benzodiazepines, one of the most prescribed classes of pharmaceuticals . Diazepam is one of the most commonly detected pharmaceuticals in TWW, with concentration ranging from ng L−1 to low μg L−1 . This is likely due to its extensive use and low removal efficiency during secondary wastewater treatment . In humans, diazepam is primarily metabolized via phase I oxidative metabolism by demethylation to nordiazepam , or hydroxylation to temazepam , and then further oxidized to oxazepam . Oxazepam undergoes phase II metabolism via rapid glucuronidation and then excretion via urine . The three primary metabolites of diazepam are psychoactive compounds, and each is a prescribed pharmaceutical for treating psychological conditions and alcohol withdrawal symptoms . Both oxazepam and nordiazepam have been commonly detected in TWW, often at μg L−1 levels . However, there is little knowledge about the occurrence, formation, and fate of such metabolites outside the wastewater treatment systems . Several studies have focused on the uptake and accumulation of pharmaceuticals in agricultural plants as a result of TWW irrigation . These studies have demonstrated the capacity of higher plants to take up these compounds; however, until recently, relatively little consideration has been given to their metabolism in plants . Recent studies have shown that higher plants can metabolize xenobiotics similarly to humans with phase I modification reactions followed by phase II conjugation reactions using detoxification enzymes that function as a ‘green liver’ . In higher plants, phase I and phase II reactions are followed by a phase III sequestration, resulting in the formation of bound residues . Many of these studies have also highlighted a chemical-specific and species-specific nature of plant metabolism of pharmaceuticals. In this study, we examined the uptake and bio-transformation of diazepam in higher plants.Cucumber and radish seedlings were then used under hydroponic conditions to understand metabolism of diazepam and its effect on selected metabolic enzymes in whole plants.PSB-D A. thaliana cell line was purchased from the Arabidopsis Biological Resource Center at Ohio State University and cultured in a liquid culture suspension at 25 °C and 130 rpm in the dark.

Cell cultures were maintained in accordance with the ARBC maintenance protocol . The A. thaliana seed culture was produced by inoculating 7 mL of cell culture into 43 mL fresh growth media, followed by 96 h cultivation at 25 °C on a rotary shaker in the dark. After 96 h, 3 mL of the seed culture was inoculated into 27 mL fresh growth media to create an approximate initial cell density of 3.3 g . Flasks were spiked with 30 μL of a stock solution of diazepam and 10 μL of a 14Cdiazepam stock solution to yield an initial concentration of 1 μg mL−1 and a specific radioactivity of 7.4 × 103 dpm mL−1 with an initial methanol content of 0.13% . Simultaneously, control treatments were prepared by auto-claving cell suspension flasks before chemical spiking , flasks containing diazepam without cells , and flasks containing living cells without diazepam . Control treatments were used to determine adsorption,vertical farming equipments abiotic degradation, and potential toxicity to cells. Flasks were incubated for 120 h in triplicate and sacrificed at 0, 6, 12, 24, 48 and 96 h for sampling and analysis. At each sampling time point, samples were collected and centrifuged at 13,000g for 15 min in 50 mL polypropylene tubes. The supernatant was collected and stored at −20 °C until further analysis. Cells were immediately stored at −80 °C and then freeze-dried for 72 h. After drying, each sample was spiked with 50 μL of 10 mg L−1 diazepam-d5 as a surrogate for extraction-recovery calibration and extracted using a method from Wu et al. , with minor modifications. Briefly, cells were sonicated for 20 min with 20 mL methyl tert-butyl ether and then 20 mL of acetonitrile and centrifuged at 13,000g for 15 min. The supernatants were combined and concentrated to near dryness under nitrogen at 35 °C and then reconstituted in 1 mL of methanol. The cells were then extracted with 20 mL acidified deionized water and the supernatant was combined with the methanol extract for cleanup. Prior to clean-up, 100 μL of cell material extract and growth media were combined with 5 mL liquid scintillation cocktail I to measure the radioactivity in the extractable form on a Beckman LS500TD Liquid Scintillation Counter . Clean-up was carried out using solid phase extraction with 150 mg Waters Oasis© HLB cartridges that were preconditioned with 7 mL methanol and 14 mL deionized water. Samples were loaded onto cartridges and then eluted with 20 mL methanol under gravity. The eluate was dried under nitrogen and further recovered in 1.5 mL methanol:water . After re-suspension extracts were transferred to micro-centrifuge tubes and centrifuged at 12,000g in a tabletop d2012 Micro-Centrifuge . Samples were further filtered through a 0.22-μm polytetrafluoroethylene membrane into 2 mL glass vials and stored at −20 °Cbefore analysis. Extraction of growth media was done after adjusting the solution to pH 3 using HCl, and followed by SPE with Waters HLB cartridges, as described above. The extraction recoveries for the tissues and media were 88 ± 7% and 80 ± 14%, respectively. After extraction, the cell matter was air dried, and a 10-mg sub-sample was removed and combusted on a Biological Oxidizer OX-500 to determine the radioactivity in the non-extractable form. The evolved 14CO2 was captured in 15 mL Harvey Carbon-14 Cocktail II and analyzed on a LSC.Hydroponic cultivations were carried out using cucumber and radish seedlings.

Seeds were purchased from Lowes and germinated in a commercially labeled organic potting soil in a growth chamber . After the appearance of the first true leaf, uniform seedlings were selected, rinsed with distilled water, and individually placed in amber jars containing 900 mL hydroponic solution . After 3 d of adaption, plants were exposed to diazepam by spiking with 100 μL of the above stock solutions to reach a nominal concentration of 1 mg L−1 and an initial specific radioactivity of 2.5 × 103 dpm L−1 . The cultivation lasted for 7 d. A parallel treatment with an initial diazepam concentration at 1 μg L−1 and a specific radioactivity of 2.5 × 102 dpm was included to simulate more realistic exposure levels and to validate the high level treatments. The cultivation lasted for 28 d with the culture solution renewed every 3 d. Plant blanks and treatment blanks were placed alongside the treatment jars. At the end of 7 d or 28 d cultivation, the seedlings were removed from the jars. Before sample preparation, roots were rinsed thoroughly with distilled water. Harvested plants were separated into below ground biomass and above ground biomass . Flowering buds from cucumbers were also separated to observe any potential for accumulation in fruits. Tissues were freeze-dried and then stored at −80 °C until analysis. A 0.2-g aliquot of the dried plant tissue was ground to a fine powder using a mortar and pestle. Samples were extracted and prepared as described above. A six-point TCP standard calibration curve was used to determine activity.Active plant metabolism of diazepam was validated using a range of controls. No diazepam was detected in the non-treated media or the cell blanks, and there was no significant degradation of diazepam in the cell free media, suggesting no contamination or significant abiotic transformation. Moreover, no significant difference was seen in cell mass between the chemical-free control and the treatments, indicating that diazepam did not inhibit the growth of A. thaliana. Furthermore, no significant amount of diazepam was adsorbed to the cell matter in the non-viable cell control. In contrast, diazepam dissipated appreciably from the media containing viable cells, with the average concentration decreasing from 698 ± 41.5 to 563 ± 8.93 ng mL−1 after 120 h of incubation, a decrease of nearly 20% .

In the face of the potentially negative consequences of climate change on agriculture, all avenues of mitigation must be examined, and even small improvements may prove worthwhile.Bio-fuel crops have been developed as an alternative, carbonneutral energy source, among which the perennial C4 grass, Panicum virgatum L. , native to North America, can adapt to a wide range of environments, including those with marginal soils and low water input. However, in order to better manage and optimize this crop for bio-fuel production, it is important to understand the mechanisms that enable its adaptivity, and how nutrient-poor environments impact chemical composition, biomass yield and feed stock quality. A long-standing barrier to this mechanistic understanding lies in the difficulty in characterizing plant chemical composition and quantifying plant-available nutrients at the rhizosphere. Phosphorus is a critical nutrient, and poor P management poses a global risk for environmental sustainability and food security. P limitation severely restricts photosynthesis and reduces CO2 fixation, but upregulates pathways associated with organic acid/ carboxylate exudation. P limitation can also be associated with increasing biosynthesis of defense metabolites, such as increased lignification of cell walls,hydroponic vertical farming systems suggesting that changes in plant carbon allocation in response to P limitation may alter both the yield and the chemical composition of bio-fuel feed stocks, and therefore productivity.

In this light, it may be beneficial to monitor plant available P concentration and plant chemical composition during the growth season, with the goal of improving biomass production and optimizing the chemical composition for improving feed stock quality through active land management, especially when growing in marginal soils. Quantifying soil P available to plants is challenging, especially if attempting to do this dynamically during a growing season. Typical chemical extraction methods quantify only a fraction of the inorganic P pool and are typically measured in top soils prior to planting. Although the P concentration data obtained with these methods have been widely used in the literature to represent total P availability, they are not an accurate measure of P available for plant growth. Perennial grasses such as switch grass produce deep roots that explore and obtain nutrients and water from distinct locations deep into the soil, and these locations vary across the growing season and lifetime of a plant. Further, plants have developed a number of strategies to access P from different types of soil, including the adaptive secretion of compounds such as organic acids, enzymes and side rophores which either mobilize soil P directly, or indirectly through their stimulation of the rhizosphere microbiome and symbiotic fungi. Combined, dynamic growth of roots through a soil profile with distinct concentrations and chemical forms of P, an adaptive allocation of photosynthate below ground, and a micro-biome with typically unknown capacity for P mobilization, makes predicting plant available P a highly complex task.

Meanwhile, there has been renewed interest and some success in predicting plant nutrient levels using spectroscopic methods for remote sensing with the help of machine intelligence. A variety of machine learning tools have been utilized to achieve satisfactory results with independent variables obtained, for example, by visible to near-infrared spectroscopy, to predict nutrient levels in shoots in agricultural crops, total nitrogen content of soils and plant adaptive responses to stress. Most of these tools are linear models such as partial least squares regression, principal component analysis, or support vector machines often with a nonlinear kernel, likely due to their inherent robustness and reduced chance of over fitting.Thus, it is conceivable that a machine learning approach could predict nutrient availability by monitoring the biochemical signatures of plant shoots. However, to our knowledge, this aspect has not been well explored. In this paper, we use a molecular spectroscopic method to determine and quantify the organic P and inorganic P in leaf tissue. This approach also provides important information on overall plant tissue biochemistry that can be used as multi-plex signatures of a plant’s response to environmental conditions. P speciation can be quantified dynamically and feed stock quality for bio-fuel production can be inferred. We then use this tissue biochemical information to infer and evaluate plant-available P using a machine-learning model trained using a dataset from a controlled laboratory experiment. Building off this approach, we used the model to interpret plant spectral data from two field locations where contrasting available P was expected.A series of experiments in sand cultures were performed to evaluate the dose-response of plant tissue chemistry to varying N and P. The chemical signatures in plant leaves varied substantially with P and N availability in the growth media, as shown in Fig. 1. Note that the absorbance data were normalized to the maximum to show the relative concentration changes on the same scale. We observed higher cellulose, lower lignin, lower lipids, and higher organic and inorganic phosphate concentrations in the leaves of plants grown in solution with closer to optimal P concentration, and increased lipid and amide concentrations in solution with closer to optimal N concentration.

The cellulose/ lignin ratio was very sensitive to P concentration, showing a 3-fold increase from <150 μM to 500 μM P, but was not consistently sensitive to N concentration. Note that the P/N concentration likely fluctuated during plant growth and between the fluid replenishment, thus the values here refer to the average concentration through the growth. Since the C/L ratio is an important metric for bio-fuel production, we suggest that higher P concentrations would produce a higher C/L ratio for potentially increased bio-fuel yield. The higher relative amide concentration in plants grown in the lowest concentration of P compared to those in intermediate P concentrations likely reflected severe P-stress in these plants, as soluble nitrogenous compounds including amino-acids and amides accumulate in other species under P-deficiency, and consistent with our other observations. P deficiency was also associated with higher lipid content, as indicated by the increased signal from carbonyl bonds, possibly related to the production of triacylglycerides as storage compounds under P limitation. Note that while lignin concentrations increased gradually with decreasing P , cellulose concentrations showed a threshold effect with a large increase between 30 and 150 μM P, and these opposing responses manifested in a cellulose/lignin ratio that is highly sensitive to P deficiency, but not to N deficiency. The increase in lignin concentration under P deficiency was possibly related to induction of defense genes and defense metabolites and the overall shift to lower cellulose and more lignin may represent a more pathogen-resistant, rigid cell wall.Because of the strong dependence of feed stock chemical composition on soil phosphorus concentration in the controlled growth experiments, we evaluated switch grass growth at two field locations contrasting in soil P availability. The soil texture of these locations differs with the RR site being a sandy loam and 3rd Stbeing a silt loam. Chemical characterization of bulk soil samples indicated a significantly higher Mehlich-III extractable P concentration in RR soils relative to 3rd St , with no significant seasonality observed . In general, plants grew taller in the RR plot than at 3rd St, reaching maximum heights at T4. Our leaf-tissue measurements showed that leaf Pi concentration increased over the course of the growing season in both field experiments. The Pi concentration of leaf tissue collected at RR was higher than that at 3rd St , consistent with the bulk soil characterization,vertical grow rack although the plant Pi as determined by FTIR was more similar than the extractable soil P data might have suggested. This may be expected due to P homeostasis and overall biomass difference. Pi concentration showed an earlier increase in plants at Red River , presumably reflecting greater uptake early on due to higher concentrations of plant-available phosphate in the soil. Further increase in the concentration of Pi, especially in the late season along with the decrease of Po at RR may reflect mobilization of Pi for translocation elsewhere in the plant, including storage tissues that support regrow in the next growing season. The trend of organic phosphates in the lower panel of Fig. 3 shows that, contrary to that of Pi, the concentration of Po plateaued in the later stages of growth at around T4, when the plant reached maximal biomass as indicated by the maximal plant heights . Po concentration decreased significantly during senescence in plants grown on the higher P soils at Red River. This explains the transient sharp increase in Pi described above and the large decrease of Po and total P . Seasonal increase of total P content in the shoots of switch grass has been observed before35, but our spectroscopic method enabled us to dissect P speciation during the growth season.

Meanwhile, we observed a similar trend in concentration of lipid signature in the late growth stage . Since the leaves we collected tended to be younger leaves to be consistent with our sand-based experiments, the maximum P concentration at T4 may reflect a combination of P uptake over the growth period, plus reallocation from old to younger leaves, resulting in higher concentrations of major P-containing molecular classes like phospholipids and/or ribosomal RNA.Because of the critical role of P in the growth of switch grass and its strong correlation with biochemical composition for this bio-fuel species, we believe the seasonal characterization of plant available P in the rhizosphere and P speciation may be beneficial for crop management and improved environmental outcomes. We demonstrate here that a machine-learning model can be used to quantify P availability using the plant leaves themselves as sensors. Since the nutrient concentration in the rhizosphere in a hydroponic substrate is relatively well controlled, this experiment allowed us to develop training data for an ML model. We achieved a principal component regression model with a high R2 of ~1 , which allows us to predict plant-available P concentrations based on the spectral data collected on the leaf tissue from field-grown plants. The predicted P concentrations are shown in Fig. 4a. Note that in a more traditional approach, the model prediction would be further validated by another independent method to evaluate the model’s accuracy. However, such a method for accurate estimation of bio-available P concentration through the soil profile over time does not yet exist in practice. We believe that our model contains an accurate statistical description of the correlation between the P concentrations in the growth media and all the spectral features in younger leaf samples, given the high accuracy achieved with large concentration range and the high affinity of P uptake; thus this model can be used for prediction of the P concentration available to each plant within the rhizosphere. The predicted P concentration available to the plants shows a gradual increase and then a sharp dip in T4 when the plant reached maximal biomass, reflecting an increase in P uptake at T4 and a quick decrease in P uptake at T5, when the shoot senesces. As a perennial plant, switch grass remobilizes and stores P in roots to support the subsequent year’s growth, consistent with previous observations with P remobilization efficiency ranging from 31% to 65% in different ecotypes. The increase in Pi in the tissue at the later stage of growth, the strong correlation of cellulose content with total P concentration, and the large reduction of plant available P in the rhizosphere provided us with a clear picture of the interaction of P availability and tissue composition during the life cycle of switch grass. This may be a consideration in the timing of harvest to achieve optimal bio-fuel yield and reserve P in the root for the optimal growth in the next year. The total P concentration in the roots followed a similar seasonal trend, showing a reduction of total P concentration near the period of maximal growth and at least a partial recovery in T5. The P concentrations measured in roots by ICP-MS at T5, a point at which plants had begun senescing, were similar across the two field locations , with a mean value of 729 ppm at 3rd St and 703 ppm at RR, respectively. The similar root P concentrations at T5 may be reflective of reduced plant P demand during senescence as well as plant P re-allocation and is described further below.

Differences in total root length, surface and volume density, average root diameter, and root tips and fork densities were analyzed using a split-plot design with genotypes as main plots and depth as subplot. This is a conservative statistical analysis because it reduces the df for genotype from 3 to 1. Therefore, we also compared the two same pairs of genotypes using statistical contrasts in an ANOVA including all four genotypes. To account for the inability to randomize depths, we used a conservative estimate of the df for subplots and for the interaction between subplot and main plot. Conservative df were calculated by dividing their df by the number of subplots. This strategy is similar to that used for repeated measures in time and does not affect comparisons among main plots , which are the main objective of this study. Homogeneity of variance and normality of the residuals was confirmed for all the individual ANOVAs performed at each depth for all parameters. When necessary, data was transformed using power transformations to satisfy ANOVA assumptions.The experiments were performed in 13 L hydroponic tanks containing the nutrient solution. Twenty-four seedlings were placed in each tank in a six by four pattern. All genotypes were included in one tank and, if necessary, multiple tanks were used as replications. In the experiments to study the effect of different nitrogen sources and concentrations ,vertical growing systems seedlings were grown in normal growth solution for seven days and then transferred to four separate tanks with each of the four nitrogen sources for 10 days .

Roots were measured at 22 DAG. Each tank included 6 replications of each of the four genotypes organized in a completely randomized design. The results were analyzed in a 2 x 2 factorial ANOVA with distal and proximal 1RS regions as factors and wheat or rye chromosome segments as levels. For the analysis of distances between the first lateral roots and the RAM, the four genotypes were grown in a tank with normal nitrogen conditions in a completely randomized design . Lines carrying distal rye or wheat segments were compared using a t-test. Chascomús, Buenos Aires Argentina: The CaCl2 from the germination tank was replaced by nutrient solution on the 4th day. On the 5 th day, plants were transferred to 350 mL pots containing nutrient solution, with each pot being a replicate. Pots were rotated every two days to ensure that they occupied different positions within the growth chamber. For the root elongation time course, the length of the second longest seminal root was measured daily four hours after the start of the light period, starting 6 DAG. Within each experiment, data was analyzed as repeated measures . A combined ANOVA was performed using experiments as blocks.The NBT staining experiments were performed using 5 cm root sections excised from the second longest root of 1RS and 1RSRW plants 17 DAG. This root segment was placed for 90 min in a 0.1 mg mL-1 NBT solution dissolved in 200 mM potassium phosphate buffer, pH 7.6, in darkness. For the DCF-DA staining similar root segments were placed in the same buffer supplemented with 10 µM DCF-DA for 60 min, in darkness. Roots segments stained with NBT or DCF-DA were washed in the same buffer for 30 min and placed on a slide. Roots were observed using a Zeiss Discovery.V20 stereo microscope equipped with a coaxial fluorescence mechanism. Pictures were obtained with an Axiocam 512 color . The images were processed with the ImageJ software obtaining a longitudinal profile of color and fluorescence intensities.

These experiments were performed in Chascomús. Previous studies have shown an association between the introgression of the rye 1RS arm in wheat and improved resistance to water stress . In three of these studies, the 1RS.1BL lines showed increased root biomass compared to the non-1RS control lines in large pot or sand-tube experiments. However, these differences were not validated in the field. In this study we showed that differences in grain yield and biomass between plants carrying a complete 1RS translocation and NILs with an introgressed distal wheat chromosome segment are associated with differences in total root length density and average root diameter in the field. Field excavations of the four different 1RS NILs provided an opportunity to visualize the differences in their root systems and to quantify these differences using horizontal soil cores at consistent depths. This experiment confirmed the hypothesis that the 1RSxR lines have a higher root density throughout the soil profile, with roots that reach deeper in the soil than the 1RSxW lines . The more extensive root system of the 1RSxR lines relative to the 1RSxW lines may have contributed to their better tolerance to drought and water logging conditions in the experiments presented in this study , and to the higher carbon isotope discrimination and increased stomatal conductance values detected in a previous study . Through their deeper root system, the 1RSxR plants can access more stored soil moisture and nutrients, keep their stomata open longer, and generate additional photosynthetic products and biomass than the 1RSxW plants. However, we cannot rule out the possibility that the genes in the distal 1BS introgression may have a more direct effect on aerial biomass or on other anatomical and/or physiological root differences known to impact tolerance to water logging and drought . The differences in root depth observed between the Hahn 1RSxR and 1RSxW NILs in the field were paralleled by drastic changes in seminal root length in hydroponic cultures . These differences were robust across experiments and were detected with different nitrogen sources and concentrations .

We hypothesize that these early differences in seminal root length may have contributed to the observed differences in total root length density observed in the deepest soil core samples in the field . The early and consistent differences in root growth under controlled conditions provided the opportunity to study the process in detail. During the first week of development, root growth occurred at the same rate for both genotypes, suggesting that the differences were not primarily associated with embryonically determined differences in root elongation. Instead, differences in root growth consistently manifested during the second week across multiple experiments. The growth rate of the seminal roots of the 1RSxW plants gradually decreased during the second and third week, to come close to zero by the end of the third week, whereas growth continued in the 1RSxR plants . The consistent timing of these events suggests that these changes are developmentally regulated. The growth arrest of the seminal roots in the 1RSxW plants was accompanied by the proliferation of lateral roots in close proximity to the RAM, suggesting important changes in the RAM. The RAM consists of a quiescent center surrounded by stem cells that generate new daughter cells, which undergo additional divisions in the proximal region of the meristem and differentiatein the transition zone . At a cellular level, a balance between cell proliferation and cell elongation/differentiation determines root growth rate . The arrest of the growth of seminal roots in 1RSxW plants suggests a modification in cell proliferation and/or cell elongation/differentiation. Additional studies will be required to determine if this arrest involves changes in the QC and/or modifications in the root regions adjacent to the meristem. In any case,outdoor vertical plant stands the dramatic reduction in seminal root growth and increased lateral root proliferation close to the RAM argues for an early developmental program switch in the regulation of the RAM in the 1RSxW plants. The transition from cell proliferation to cell elongation and differentiation and the subsequent development of lateral roots depends on the distribution of ROS along the root axis, specifically on the opposing gradients of superoxide and hydrogen peroxide.

Superoxide is predominant in dividing cells in the meristematic zone, while hydrogen peroxide is predominant in elongated cells in the differentiation zone . The balance between these ROS modulates the transition between root proliferation and differentiation zones. Seventeen days after germination, the apical region of 1RS seminal roots showed opposing gradients of superoxide and hydrogen peroxide characteristic of elongating roots . A different ROS distribution was detected in the arrested 1RSxW roots, where superoxide was restricted to the distal ~700 m and increased levels of DCF-DA fluorescence were detected between 250-950 m in the cell proliferation zone . The contrasting patterns of ROS distribution reflect the major developmental changes that differentiate the seminal roots of the 1RS and 1RSxW genotypes. Studies in Arabidopsis have shown that changes in ROS distribution can be triggered by the altered expression of major genes that control the size of the meristematic zone. These genes include UPBEAT1 , a basic helix-loop-helix transcription factor that regulates the meristematic zone size by restricting H2O2 distribution in the elongation zone . In addition, ROOT MERISTEM GROWTH FACTOR 1and the transcription factor RGF1 INDUCIBLE TRANSCRIPTION FACTOR 1that mediates RGF1 signaling can modulate the distribution of ROS along the root developmental zones leading to enhanced stability of PLETHORA2 . Reduced expression of PLETHORA in the root apical region or changes in its distribution have been associated with impaired root growth. To test if these Arabidopsis results are applicable to wheat, we are initiating expression studies of these genes in the 1RSxR and 1RSxW lines. It remains unknown if the differential pattern of ROS distribution in the roots of the 1RSxW plants is the result of changes in the wheat homologs of these central developmental genes or a more direct effect on genes affecting the redox balance in different developmental root zones. The differences in superoxide and hydrogen peroxide distribution between the seminal roots of the 1RSRW and 1RS plants were measured after the arrest in root growth . Therefore, we currently do not know if the changes in ROS distribution are a cause or consequence of the changes observed in root growth and lateral root proliferation close to the RAM. Strawberry frigoplants cv. Elsanta were cultivated in hydroponics. The variation in N-form started when the plants switched over from the vegetative to the generative growth phase. During the vegetative phase, all plants were fed with NO3-N. After changing to the generative stage, plants were divided into three different nutrition treatments, containing two ammonium treatments and one nitrate treatment. The solution pH was stabilized to pH 3.5 and 5.5 by pH-stat. The nutrient solution was changed every 3 d.The concentrations of the main organic acids in the fruits were measured using high performance liquid chromatography . Seeds of E. camaldulensis were germinated on acid-washed and sterilized sand in a growth chamber . Seedlings were watered daily with a nutrient solution and grown for 3 to 4 months. After seedlings were cultured hydroponically for 5 days, roots of seedlings were treated with 0.35 mM CaCl2 solution containing 0 or 1 mM AlCl3 for 24 h. The 5 mm apical portions of roots were excised, freeze-thawed, and centrifuged on an ultrafilter to obtain soluble compounds from root-tip cells. Al-binding compounds which were soluble in the form of complex with Al were separated by gel-filtration chromatography. The cellular extract added with Al was subjected to a gel-filtration column to separate the complexes of Al-binding compounds with Al from free Al. The Al concentration of each fraction was determined with an inductively coupled plasma atomic emission spectrometer . In addition, Al-binding compounds which became insoluble in the form of complex with Al were separated by HPLC equipped with a reversed-phase column. The cellular extract added with or without Al was subjected to HPLC analysis. Chromatographic peaks whose area was decreased by the addition of Al were judged to be those of Al-binding compound. When Al was added to the cellular extract from roots cultivated without Al and the extract was subjected to the gel-filtration column, one peak was observed by monitoring the Al concentration in each fraction. Two peaks were found when the roots were treated with 1 mM Al. These results indicate that roots of E. camaldulensis contain Al-binding compounds which are soluble in the form of complex with Al, and the compounds increase with Al treatment. We also measured organic acids in each fraction, and found that the peak of citrate corresponded to that of the Al-complex. The increase of Al-binding compound can be explained partly by the increase of citrate.

Interestingly, plants can be divided into four categories based on the quantity of B required: 1) Lactifers, contain the highest amount of B ; 2) Cole crops have the second highest B concentrations; 3) Legumes and the lily family of monocots are in the third group and 4) Graminaceous plants contain the least amount of B . When graminaceous plants flower, their B requirements increase. Now we know that, except for the lactifers, the B content of plants is closely aligned with the amount of pectin in their cell walls . Cell wall scientists have discovered that the RGII fraction of cell wall pectin contains B, and that cell wall structures in plants differ among species.Cell walls of grasses are much lower in pectin, and therefore these plants contain less B . Since each carbon fixed in photosynthesis is released to the Calvin cycle in bundle sheath cells by Mn-activated NAD-malic enzyme in this sub-type of C4 plant, perhaps the Mn requirement of these plants would be higher than that of C3 or NADP-malic enzyme plants . We tested this hypothesis by using hydroponic solutions where Mn concentrations could be carefully controlled. A survey of plant nutrient solution recipes indicated that most nutrient solutions contain around 2µM Mn. In this experiment, we compared the growth and photosynthetic rates of two NAD-malic enzyme C4 plants, Pearl millet and amaranthus, with two NADP-malic enzyme C4 plants,vertical hydroponic corn and sorghum, and two C3 plants, squash and wheat. Corn, sorghum, squash and wheat produced maximum biomass with the normal 2 µM Mn concentration in the hydroponic medium.

On the other hand, NAD-malic enzyme C4 plants, Pearl millet and amaranthus, produced maximum biomass with ~50 µM Mn in the nutrient solution . Photosynthetic rate responses of each species to nutrient solution Mn concentration were similar to their biomass responses. These results clearly show that when all of the carbon going into photosynthesis goes through a single Mn-activated enzyme, plant growth response is dependent on high levels of available Mn. Legumes are some of the highest protein crops grown, and they utilize N from the atmosphere rather that relying on N fertilizer to produce this protein. There are two major types of leguminous plants when it comes to root nodules and forms of N transported from these nodules to leaves and developing pods . There are determinate nodules, which tend to be round, with life spans of about 35 days. These are nodules formed on roots of warm season legumes and contain bacteroids that fix atmospheric N and use the fixed N to synthesize the ureide molecule, allantoate, for transport in xylem to leaves and developing pods. Allantoate contains 4N’s and 4C’s, and is a very efficient molecule for transporting N. Cool season legumes have indeterminate nodules that are elongated and often form a Y-shape. Bacteroids in these nodules fix N and, in general, synthesize the amide, asparagine, for transport in xylem to leaves and pods. Asparagine contains 2N’s and 4N’s. The fixed-N is released in leaves and developing pods of ureide-transporting legumes by an enzyme called allantoate amidohydrolase, and one interesting feature of this enzyme is that it is activated by Mn .

Therefore, according to the soil N status, a large proportion of the total N, mostly protein-N in the harvested legume, comes through this Mn-activated enzyme. As with the Mn-activated NADmalic enzyme plants, perhaps ureide-utilizing leguminous plants, like soybean, cowpea and lespedeza, will require higher Mn nutritional levels than asparagine-transporting legumes, like alfalfa and clover or all legumes grown on nitrate-N. To my knowledge, the Mn-requirements of ureide-transporting, amide-transporting and nitrate-fed leguminous plants have not be directly compared. In addition to a Mn-ureide metabolism connection, there is a Mn-bacteroid connection inside the root nodule. Bacteroids depend on their host legume for a source of energy to support the nitrogen fixation process. Although plants usually transport sucrose via the phloem from leaves to root nodules, root nodule cells metabolize the sucrose and provide bacteroids organic acids, like malate, as an energy source. Bacteroids in nodules of some species, like soybean, use the Mn-activated NAD-malic enzyme in the initial step of malate utilization . Therefore, a Mn-enzyme plays a central role in root nodule/legume N metabolism! The knowledge gained on structure and metabolism of a wide range of plant species over the past few years allows us to predict special nutrient needs. High protein plants require large quantities of K because components involved in protein synthesis must be bathed in high K concentrations in order to maintain the proper configurations. Plus, K+ is used to balance the negative charges of asparagine and glutamine in proteins produced. Plants with pectin-rich cell walls have high B contents, and thus plants with low pectin cell walls have low B contents. NAD-malic enzyme sub-type C4 plants have high Mn requirements for maximum growth and photosynthesis rates since every C fixed is released in bundle sheath cells by this Mn-activated enzyme. Based on their ureide metabolism with the Mn-activated enzyme, allantoate amidohydrolase, and with malate as the primary C source for bacteroids via NAD-malic enzyme, N-fixing soybeans may have a higher Mn requirement than nitrate-N grown plants.

In addition to these examples, there are other specific nutrient requirements that can be predicted based on our knowledge of plant structure and metabolism. Both Casparian strips and suberin lamellae, two extracellular hydrophobic barriers located in the wall of endodermal cells of the root, are thought to play important roles in restricting the free difusion of solutes and water . Casparian strips act as apoplastic barriers not only to block solutes moving into the xylem through the free space between cells, but also to prevent their back fow from the stele to the apoplast of the cortex. Suberin lamellae, due to their deposition between the endodermal plasma membrane and secondary cell wall, do not block aploplastic transport but rather limit transcellular transport of nutrients and possibly water at the endodermis. Cross talk between the Casparian strip and suberin lamellae exists, with suberin being deposited in response to disruption of Casparian strips.These extracellular barriers are therefore at a cross-road between control of mineral nutrient and water uptake. However, the mechanisms that allow plants to integrate both these barrier functions to enable the simultaneous uptake of sufcient water and mineral nutrients remain under explored. Te dirigent-like protein Enhanced Suberin1 functions in the correct formation of Casparian strips by allowing the lignin, deposited at the Casparian Strip Domain through the action of Peroxidase 64 and the Respiratory Burst Oxidase Homolog F, to form into a continuous ring. In the absence of this dirigent-like protein defective Casparian strips are formed along with enhanced and early deposition of suberin in the endodermis. A similar pattern of Casparian strip disruption and response is also observed when the Casparian Strip Domain is disrupted through the loss of Casparian Strip Domain Proteins. Tese changes lead to systematic alterations in the profle of mineral nutrients and trace elements accumulating in leaves, and this phenotype provided the frst tool for identification of genes involved in Casparian strip development. Detection of the difusible vasculature-derived peptides CASPARIAN STRIP INTEGRITY FACTORS 1 & 2 through interaction with the SCHENGEN3 receptor-like kinase is what drives this endodermal response to loss of Casparian strip integrity. Here, we report that detection of a loss of Casparian strip integrity at the root endodermis by the CIFs/SGN3 pathway leads to an integrated local and long-distance response. This response rebalances water and mineral nutrient uptake,what is vertical farming compensating for breakage of the Casparian strip apoplastic seal between the stele and the cortex. This rebalancing involves both a reduction in root hydraulic conductivity driven by deactivation of aquaporins, and limitation of ion leakage through deposition of suberin in endodermal cell walls. This local root-based response is also coupled to a reduction in water demand in the shoot driven by ABA-mediated stomatal closure.The dirigent-like protein Enhanced Suberin1 functions in the formation of Casparian strips by allowing the correct deposition of lignin at the Casparian strip domain. The enhanced deposition of suberin in the esb1-1 mutant with disrupted Casparian strips can clearly be observed using the lipophilic stain Fluorol Yellow 088 close to the root tip , and this can be quantifed by counting the number of endodermal cells afer the onset of cell expansion to the frst appearance of yellow fuorescence . This early deposition of suberin is also verifed by the clear correspondence of FY 088 staining with enhanced promoter activity of known suberin biosynthetic genes, including GPAT5 monitored through both GUS staining and GFP fuorescence , and also others through GUS staining . This is further reinforced by the enhanced expression of known suberin biosynthetic genes in esb1-1 relative to wild-type . To better understand the causal link between Casparian strip integrity and enhanced deposition of suberin, we performed a reciprocal grafing experiment that revealed that the esb1-1 mutation is only required in the root to drive enhanced deposition of suberin at the endodermis, placing the function of ESB1 and the driver for increased suberin in the same tissue . To determine the cause and effect relationship between damaged Casparian strips and enhanced suberin we carefully monitored the first appearance of both Casparian strips and enhanced suberin in esb1-1. Using lignin staining in the Casparian strip marker line pCASP1::CASP1::GFP, we are able to observe that damaged Casparian strips are visible 2.5 days after sowing .

This is at least 12hr before the first indication of enhanced suberin biosynthesis, which we monitor using promoter activity of suberin biosynthetic genes GPAT5, FAR4, FAR1 and FAR5 . This was also verified by the direct observation of suberin deposition with FY 088 . The observation that treatment with the CIF2 peptide, normally leaked from the stele through loss of Casparian strip integrity, can enhance suberin deposition in wild-type plants supports our interpretation that enhanced suberin deposition is a response to loss of integrity of the Casparian strip-based apoplastic diffusion barrier. Furthermore, loss-of-function of the receptor-like kinase SGN3, required for sensing of CIFs, blocks the enhanced deposition of suberin in esb1-1 and casp1-1casp3-1 based on a chemical analysis of suberin in esb1-1 , and also on FY 088 staining. We conclude that Casparian strip defects sensed by the CIFs/SGN3 surveillance system lead to enhanced deposition of suberin in the endodermis.Te observation that enhanced suberin is deposited as a response to loss of integrity of the endodermal-based diffusion barrier between stele and cortex, raises the question, what is the function of this increased suberin deposition? Previously, the extent of endodermal suberin has been shown to be part of the response to nutrient status. We therefore tested the selectivity to solutes σNaCl, in roots varying in the extent of suberin deposition and the functionality of Casparian strips. For this, we measured solute leakage into xylem sap of pressurized roots at increasing sodium chloride concentrations in the solution bathing the roots. Taken individually, σNaCl of roots of esb1-1, sgn3-3 and wild-type were not significantly diferent from one another , which is surprising given the disruption of the Casparian strip-based apoplastic diffusion barrier in both mutants. However, removal of suberin in esb1-1, by endodermal-specific ectopic expression of a cutinase, caused a signifcant decrease in σNaCl compared to wild-type plants , and a similar tendency when compared to esb1-1 . This supports the notion that enhanced suberin deposition at the endodermis helps prevent passive solute leakage caused by defects in the Casparian strips of the esb1-1 mutant. We also observed a significant decrease in σNaCl in the double mutant esb1-1sgn3-3 compared to both wild-type and sgn3-3 . It is known that SGN3 is required for the enhanced deposition of suberin that occurs at the endodermis in esb1-1. Our observation that removal of this enhanced suberin in esb1-1sgn3-3 decreases σNaCl further supports our conclusion that the role of this increased suberin deposition is to limit solute leakage where Casparian strip barriers are disrupted.It has also been suggested that endodermal suberin may impact water permeability, though how is still unclear. To further address the role of enhanced endodermal suberin, we investigated root hydraulic conductivity of esb1-1 and observed a significant reduction by 62% with respect to wild-type.Importantly, this difference in esb1-1 Lpr originates mainly from a reduction in an aquaporin-mediated water transport pathway.

The BEST program also sponsored a phytoremediation workshop for BEST investigators and students that was attended by more than 60 participants. Additional workshops are planned for the coming year. In this report, the research is organized by subject area, and two-page briefs are presented for each of 28 BEST projects. The projects presented provide a good representation of the state-of-the-science research being done with students in the BEST program – the best of BEST.Over the next 75 years, the U.S. government will undertake what has been called the largest civil works project in world history to restore the environment damaged by previous activities at federal sites, e.g., Department of Defense military bases and Department of Energy nuclear facilities. Legislative action, resulting from concern over the accumulating hazards, has mandated pollution control measures and environmental restoration of hazardous waste at all sites. Estimates of total cleanup costs range from $230 billion to more than half a trillion dollars. Given the trend of diminishing budgets throughout the federal government,vertical home farming future generations could inherit both an environmental and budgetary disaster. The imprecision of the cost estimates results from the lack of knowledge of how “clean” the contaminated sites will need to be. Some of the environmental damage is permanent—cleanup technologies either do not exist or are incapable of remediating the contamination.

For DoD bases being closed by the Base Realignment and Closure Program, all toxic sites must be remediated before the site is returned to public use. The projected costs of site restoration using existing technologies are staggering: the estimated cleanup cost is at least $24.5 billion for the 7,313 identified U.S. sites . The pollutants at these sites include chlorinated hydrocarbons, metals, petroleum products, explosives, mixed waste and other organics. DOE also has substantial remediation costs—estimated to be from $90 billion to $200 billion . The domestic private sector presents yet another huge set of remediation problems, dwarfed only by the international problems in Eastern Europe and Russia . There is clearly a need for new cost-effective treatment technologies. Bio-remediation, the use of microorganisms to detoxify hazardous waste, promises to provide economical and ecologically sound clean-up strategies. An Office of Technology Assessment analysis concluded that the U.S. does not possess a sufficient pool of qualified environmental professionals, i.e., the trained scientific personnel required to support this rapidly developing multidisciplinary field. In response to these national environmental needs, the Bio-remediation Education, Science and Technology Program, funded by DoD, was established in 1996. In a few short years, BEST has pioneered a new and successful model for environmental science and education. This partnership has a highly integrated programmatic focus on the scientific and workforce needs of DoD. Since the inception of the BEST program, a significant number of major milestones and deliverables have been achieved. They are described below. The BEST program has made these dramatic accomplishments by using an approach that combines a training-education element with an integrated research project, described later in this introduction.

Successful restoration of DoD hazardous waste sites and the growth of the bio-remediation industry is dependent on a cadre of trained scientists, engineers and technicians. By the year 2005, thousands of trained professionals will be required to meet DoD and national environmental needs. The BEST partnership continues to build upon accomplishments and successful programs developed with DoD support. The training-educational element continues to provide career development opportunities for underrepresented groups with bio-remediation curricula, courses and fellowships. The training-educational experience is personalized to provide students with a meaningful bio-remediation curriculum, financial support, an extensive mentorship network, and research and field training. The shared resources of the BEST partnership institutions aid faculty in the development of curricula, courses and environmental research projects. The training-education programs span the continuum from community college outreach to faculty development, but focuses on undergraduate and graduate students. Innovative features of the BEST program are the use of distributed learning technologies and a Rotating Scholar Program, integrated with a coordinated academic and video seminar curriculum.The primary objective of the BEST program is to provide hands-on training in bio-remediation, phytoremediation and ecotox/risk assessment for underrepresented minorities. Students will obtain the necessary skills and knowledge to enable them to either enter the environmental science workforce with a bachelor’s degree or enter graduate school at the master’s or doctoral level. Another objective is to ensure a continued supply of skilled workers to address the diverse environmental restoration needs at DoD sites.

DoD sites throughout the United States contain highly contaminated soils, groundwater and sediments. These properties pose direct and indirect exposure hazards to humans and wildlife. Conventional remedial solutions for contaminated soils and sediments or groundwater are slow and expensive, increase inputs to hazardous waste disposal sites, and can increase human exposure to contaminants. Bio-remediation — the use of microo ganisms to destroy hazardous contaminants or to convert them to harmless forms — is an emerging treatment technology that can in many instances restore contaminated environments more quickly, at lower cost and at lower human risk than alternative remediation technologies. Bio-remediation can operate in either an in situ mode where contaminants are treated in place, or in an ex situ mode where contaminants are removed from a contaminated zone for treatment . In situ bio-remediation can be used when excavation is impractical — under buildings, highways, runways, etc. In situ bio-remediation can simultaneously treat soil and groundwater in one step, without the generation of hazardous waste products. In situ contaminant degradation can be achieved by either intrinsic or enhanced bio-remediation. Intrinsic bio-remediation exploits the innate capabilities of indigenous microbial communities to degrade pollutants. Enhanced bio-remediation seeks to accelerate in situ microbial activity by isolating and controlling the contaminated site so that the microbial environment can be purposely manipulated to correct nutritional or gas phase limitations. Ex situ treatment seeks to further control the remedial environment by placing the contaminants in an engineered treatment system. Phytoremediation, a process in which plants and associated microbial communities are used for contaminant biodegradation or bioimmobilization, is an important and rapidly developing mode of bio-remediation. To realize the full potential benefits of plant and microbial treatment systems at DoD sites, these bio-technologies must be developed and optimized for remediation of DoD priority contaminants by an expanded pool of qualified professionals. It was in response to these DoD environmental needs that the BEST partnership of institutions was established.

In order to determine whether plants can stimulate the degradation of PAHs in soil, plant species found in literature on phytoremediation of metal-contaminated sites were selected to measure the removal of PAHs in artificially contaminated soil over a period of 62 days. The plant species used for this experiment were alfalfa , barley , tall fescue and orchard grass . The PAHs were phenanthrene and anthracene, in a mixture of 600 ppm each. As shown in Figures 1 and 2,vertical growers phenanthrene and anthracene were removed from the soils with plants after 62 days. More than 98% of the phenanthrene was removed during that period while the anthracene removal was found to be between 70 and 90%. The results suggest that the rate of disappearance of phenanthrene in soil was greater than anthracene under the same conditions. From the results, it is also indicated that the disappearance of PAHs in soil depends on the bio-availability of the compounds. Because phenanthrene is approximately 10 times more soluble in water than anthracene, it was expected to be more readily available to microbial degradation than anthracene. Plant-assisted degradation of PAHs is thought to be more effective on PAHs with a higher number of rings and higher molecular weights, such as benzopyrene. Anthracene removal in the soil planted with alfalfa was greater than in the soil without plants, while all the other plants have minimal to no effect on anthracene removal compared to the control soil. Phenanthrene was removed to a greater extent in the soil with alfalfa and tall fescue compared to the control without plants . However, both barley and orchard grass showed no effects of the removal of phenanthrene during that period when compared to the soil without plants. Overall, plants had minimum effect on phenanthrene degradation while anthracene degradation was more dependent on plant species. In order to determine the effect of PAH degradation by plants on bacterial numbers in soil, bacteria were counted in soil during the course of the experiment.The result suggests that the degradation of PAHs by plants is not affected by differences in bacterial biomass in the soil. Sterile controls will be used in the future in order to assess the role of bacteria in the degradation process.Wheat is a crop of major importance and together with other staple cereals supply the bulk of calories and nutrients in the diets of a large proportion of the world population . Cereals are inherently low in protein and mineral micro-nutrients such as Fe and Zn . A major focus of wheat breeders has been grain protein concentration as it affects bread- and pasta-making quality, but micro-nutrient improvement has received less attention. Approximately half of the world’s population suffers from Fe and/or Zn deficiencies and millions of children suffer from protein-energy malnutrition . As such, the improvement of nutritional quality of wheat could benefit the nutritional status of millions of people. A common agronomic practice to increase grain protein concentration is the use of N fertilization. However, this practice is expensive and excess fertilizer run-off is a potential environmental contaminant . A substantial percentage of the N in wheat grain is supplied by amino acids remobilized from vegetative tissue .

Much of this N content is derived from proteins that are disassembled and recycled during the leaf senescence stage of development . Likewise, Fe and Zn have been shown to be remobilized from vegetative tissues in several plants , although the specific sources are unknown. Zinc fertilization has been a successful strategy to improve wheat grain Zn concentration , and improvement in the partitioning or remobilization of Zn to grain could make fertilization efforts more efficient. Wheat grain with higher Zn concentration has been demonstrated to produce more vigorous crops . Thus, breeding or transgenic approaches that result in plants with increased partitioning of minerals to grain could be useful for both nutritional bio-fortification and reduced fertilizer application. Chromosome 6B from wild emmer wheat was identified as a potential source of genetic variation for grain protein , Zn, and Fe concentration . A quantitative trait locus for grain protein concentration was mapped on chromosome arm 6BS and later mapped as a single Mendelian locus, Gpc-B1 . In near-isogenic lines of this locus, increased grain protein was associated with the increased remobilization of amino acids from the flag leaf , higher grain Fe and Zn concentrations , and accelerated leaf yellowing, indicating accelerated senescence . A NAC transcription factor, NAM-B1, was identified as the causal gene for Gpc-B1 by positional cloning . Other members of the NAC family are known to regulate developmental processes , including leaf senescence . In transgenic wheat NAM RNA interference lines in which NAM-B1 and its homeologous genes had decreased expression, leaf yellowing was delayed, and grain protein, Fe, and Zn concentrations were greatly decreased . These results, together with higher N, Fe, and Zn concentrations in RNAi line flag leaves at maturity, suggested a role for NAM-B1 homeologues in the remobilization of N compounds, Fe, and Zn. However, without taking organ mass, nutrient concentrations at prior time points, and total nutrient accumulation of other organs into account, this model could not be confirmed. In addition, the body of literature does not contain sufficient data regarding sources of grain minerals to support the idea that remobilization alone could account for the differences observed. Because a whole-plant partitioning profile has not been undertaken in plants differing in NAM-B1 expression, it is currently unclear whether this gene directly affects remobilization , alters partitioning of nutrients within the plant, alters total plant uptake of these nutrients, or influences a combination of these processes. The current study uses multiple time point sampling of an expanded profile of mineral concentrations and contents of all shoot organs in NAM knockdown and control lines. This sampling allows the quantification of N and mineral remobilization as contributors to final grain protein and mineral content, and provides a better understanding of the physiological effects of the NAM genes.

Arabidopsis is known to synthesize four GDP-linked sugars: GDP-L-fucose, GDP-L-galactose, GDP-D-glucose and GDP-D-mannose. GDP-mannose for the glycosylation of glycosylinositolphosphorylceramides is transported into the Golgi by GONST1 , whereas GDP-fucose is transported by GONST4, which has been renamed GDPfucose transporter1 . No Golgi-localized GDP-L-galactose transporters have been identified to date. GDP-L-galactose is synthesized from GDP-mannose in the cytosol by GDP-mannose epimerase . Most GDP-L-galactose is then converted, via L-galactose, into L-ascorbate , which is important for maintaining redox balance in the cell, particularly under abiotic or biotic stress . However, some GDP-Lgalactose is required for cell wall polysaccharide synthesis since L-galactose is present in side chain A of RG-II, in the side-chains of xyloglucan from a limited number of plant species  and in corn bran glucuronoarabinoxylan . Here we provide evidence that GONST3 probably encodes a Golgi-localized GDP-L-galactose transporter, which we rename Golgi GDP-L-galactose transporter1 . We used RNA interference to suppress GGLT1 expression in Arabidopsis, since complete loss of GGLT1 is lethal. Plants with decreased GGLT1 expression have growth defects, which are rescued by increasing the amount of borate in their growth medium. Chemical analysis of the cell walls of GGLT1 knock-down plants revealed a substantial reduction in the L-galactose decoration of RG-II,vertical gardening in greenhouse which is correlated with a decrease in the proportion of RG-II dimer in the wall and a decrease in the stability of the cross link. Our results underscore the importance of RG-II to plant survival, and highlight an unexpectedly critical role for L-galactose in borate cross linking of this unusual pectic polysaccharide.

Publicly available gene expression data reveal that GGLT1 is a ubiquitously expressed gene, with a level of expression that is slightly lower than GONST1 and GFT1 . In an earlier study, the subcellular localization of GGLT1 was not determined because tagged GGLT1 could not be expressed in vivo . To overcome this issue, the full-length GGLT1 coding sequence tagged with a fluorescent protein was introduced into onion epidermal cells by biolistic transformation. Confocal imaging revealed that the fluorescently tagged GGLT1 gave a punctate signal that co-localized with a Golgi marker .No Arabidopsis lines carrying a T-DNA insertion in the GGLT1 open reading frame have been reported. A singleT-DNA line , with an insertion 841 bp upstream of the start of transcription was obtained, but we were unable to identify any plants homozygous for the TDNA insertion despite screening at least 30 different seedlings. Therefore, we took a targeted gene-knockdown approach and generated RNAi transgenic lines with a hairpin RNA construct, which specifically targeted GGLT1. Forty independent hpGGLT1 transformants were screened, and four were selected for characterization . These lines all had rosettes which were smaller than the empty vector control . Quantitative real-time PCR showed that in the rosette leaves of the hpRNAi lines 1–3 the levels of GGLT1 silencing were similar . These data, together with the lack of T-DNA lines, suggest that stronger suppression of GGLT1 or null mutants will produce plants that are not viable. The expression of GFT1, the closest homolog of GGLT1, was not affected in lines 3 and 4 but was decreased by up to 50% in lines 1 and 2 . The monosaccharide compositions of the walls, including fucose, were not significantly altered in any of the hpRNAi lines , indicating that their phenotypes do not result from altered fucosylation of cell wall glycans and are thus a consequence of GGLT1 silencing.

Moreover, the shortened petiole phenotype that is characteristic of silenced GFT1 plants as well as mur1 plants, which also have cell walls with reduced fucose , was not observed in our hpGGLT1 knock-down lines .L-Galactose replaces L-fucose in the xyloglucan formed by mur1 and GFT1-silenced plants where GDP-fucose synthesis or transport is perturbed . Since GGLT1 and GFT1 are closely related NSTs, we first determined if xyloglucan fucosylation is altered in hpGGLT1 . No differences were discernible in the matrix-assisted laser desorption–ionization time-of-flight mass spectra of the oligosaccharides generated by enzymatic fragmentation of the xyloglucan from hpGGLT1 and EV control lines . The presence of fucosylated side chains, together with no substantial increase in the abundance of galactosylated side-chains in the hpGGLT1 lines, supports our assertion that GDP-fucose transport is unaffected in the silenced plants. GGLT1 is in the same NST subclade as GONST1, which provides GDP-mannose specifically for GIPC glycosylation, as opposed to polysaccharide biosynthesis . Although glycosylation of GIPCs is still poorly understood, it is possible that other GDP-sugars, in addition to GDP-mannose are required. Therefore, we used thin layer chromatography and LC-MS to determine the GIPC glycan composition of hpGGLT1. No major differences were discernible between hpGGLT1 and EV GIPCs. The overall sphingolipidomic composition was also unchanged . Together, the combined results of these studies show that GGLT1 does not encode a Golgi-localized protein involved in the transport of GDP-L-fucose or GDP-D-mannose. Thus, we next investigated if the L-galactose content of the wall was altered in the GGLT1 suppressed lines.No significant differences were detected in the wall monosaccharide compositions of leaves from soil-grown EV and hpGGLT1 lines . This is not surprising since in primary cell walls D-galactose is far more abundant than L-galactose . Moreover, D-galactose and L-galactose are not separated when the monosaccharide composition of the cell wall is determined by high-performance anion exchange chromatography with pulsed amperometric detection . Rhamnogalacturonan-II is the only known L-galactose containing polysaccharide present in wild-type Arabidopsis cell walls, so we next determined whether the structure of RG-II differed in hpGGLT1 and EV plants. Material enriched in pectic polysaccharides, including RG-II, was obtained by extracting hpGGLT1 and EV leaf alcohol-insoluble residue with ammonium oxalate, a calcium chelator. This material was then treated with endopolygalacturonase and the products separated by size-exclusion chromatography . This separates RG-II from RG-I and oligogalacturonides, and also separates the RG-II monomer and dimer. In EV control plants the dimer accounts for 77% of the total RG-II isolated from the wall. Somewhat unexpectedly, the dimer accounts for only 49% of the hpGGLT1 RG-II, and makes up only 6% of the RG-II in mur1-1 .

This led us to suspect that the ability of hpGGLT1 RG-II to form dimers or the stability of those dimers had been altered.Under these conditions, in the absence of a chelating agent, the dimer accounted for 97% of the RG-II in the EV control plants , 87% of the RG-II in the hpGGLT1 lines and 70% of the mur1-1 . These results, together with data showing that calcium chelators partially convert the RG-II dimer to the monomer , strongly suggest that both the extent of formation and the stability of the borate cross-link in RG-II are affected in the hpGGLT1 lines. The differences in dimer abundance in the EPG and oxalate fractions were most pronounced with mur1-1 plants. This mutant produces RG-II that lacks L-galactose because its A side-chain is truncated , which led us to suspect that the L-galactose content of side-chain A of the RG-II from the hpGGLT1 lines may also be reduced. To determine if RG-II structure is indeed altered in the hpGGLT1 lines we isolated the total RG-II from the silenced and EV plants. Glycosyl residue composition analyses showed that D/L-galactose was reduced by about 35% in the most strongly affected hpGGLT1 lines . We then treated the RG-II with warm trifluoroacetic acid  to release side chains A and B. The MALDI-TOF MS analysis showed that a substantial portion of side-chain A from hpGGLT1 RG-II existed as a heptasaccharide whereas virtually all the A chain from the EV control was present as an octasaccharide . The A side-chains produced by hpGGLT1 and EV plants differ in mass by 162 Da, corresponding to a hexose residue, which we consider likely to be L-galactose. The side-chain B of RG-II contains a D-galactose residue . However, no differences were discernible in the structures of this side-chain from RG-II of hpGGLT1 and EV plants . Our structural data provide compelling evidence that the abundance of terminal L-galactose present on the A side-chain of RG-II is specifically affected in hpGGLT1 plants. To confirm the identity of the missing hexose in sidechain A,greenhouse vertical farming the RG-II monomers generated from the hpGGLT1 and EV plants were treated with a recently identified a-Lgalactosidase 95 from Bacteroides thetaiotaomicron that specifically removes the terminal L-galactose from side-chain A of RGII . Galactose was the only monosaccharide detected by HPAEC-PAD following hydrolysis of EV control RG-II with the a-L-galactosidase . Less galactose was released from the RG-II of the hpGGLT1- silenced lines relative to the control . The MALDI-TOF MS analysis of side-chain A, released by mild TFA hydrolysis following a-L-galactosidase treatment of RG-II monomer, revealed that the predominant oligosaccharides in the EV control plants correspond to side-chain A lacking L-galactose . The L-galactose was almost completely removed as only low-intensity signals corresponding to L-galactosylated A side-chains were discernible . The mass spectra of side-chain A from both hpGGLT1 silenced lines are similar to that of the EV control , demonstrating that the mass difference of 162 Da between the EV control and hpGGLT1 lines in Figure 3 is due to the specific loss of L-galactose. It has been proposed that pectin domains may be linked covalently to each other or to other cell wall components . To investigate whether the altered RG-II structure in the hpGGLT1 silenced lines had affected other pectic domains, the oxalate cell wall fraction was used to perform immune dotblots with a panel of antibodies raided against different pectin epitopes .

However, no difference was observed between the EV control and the silenced lines. In combination with the monosaccharide composition data and the xyloglucan data we conclude that the reduction in GGLT1 expression does not affect non-RG-II polymers. These data provide strong evidence that silencing of GGLT1 leads to a reduction in the abundance of L-galactose on side-chain A of RG-II, and provides additional evidence that the absence of this sugar leads to a decrease in the ability of the RG-II monomer to self-assemble into a borate cross-linked dimer. Moreover, this L-galactosedepleted dimer is less stable in the presence of calcium chelators than its wild-type counterpart, a result consistent with the notion that interactions of borate and calcium with RG-II are important for plant growth .Several growth phenotypes, including the dwarf phenotype of mur1, that have been attributed to defects in RG-II structure and cross-linking have been reported to be rescued by supplementing the growth medium with additional borate . To further explore the observed growth phenotypes of the hpGGLT1 lines , plants were grown hydroponically to control the availability of all macro- and micro-nutrients, including borate. In low-borate media the hpGGLT1 lines are severely stressed, and their rosette diameter is about 70% smaller than that of EV control plants . However, this phenotype is not observed when the silenced plants are grown in high-borate media . The amount of borate in the growth medium did not affect GGLT1 expression, thereby excluding a potential effect of borate deficiency or supplementation on transgene expression and silencing strength . Therefore, we conclude that partial loss of the RG-II L-galactose decoration in hpGGLT1 reduces the rate of RG-II borate-dependent dimerization, directly affecting plant development.Since hpGGLT1 plants grown in the presence of 1 mM boric acid or no added boric acid had different phenotypes,we were curious to know if altering the structure and dimerization of RG-II in hpGGLT1 led to changes in other cell wall components. Therefore we determined the monosaccharide composition of destarched leaf AIR from plants grown under different borate concentrations . No significant visible differences were discernible in hpGGLT1 and EV plants grown with 1 mM borate . However, we saw increases in the abundance of several neutral monosaccharides, in particular glucose , in the walls of plants grown with no added borate. No differences in aniline blue staining of the walls of EV and hpGGLT1 lines were observed, suggesting that the increase in non-cellulosic glucose in plants which appear severely stressed is not due to callose deposition . Finally, we performed Saeman hydrolysis of the TFA-resistant AIR to determine the amount of glucose derived from crystalline cellulose. A substantial increase in cellulose-derived glucose was detected in the hpGGLT1 lines grown with no added borate but not in plants grown under high-borate conditions .