DNA endoreduplication does not appear to affect this overall growth rate but may be required to sustain it beyond a critical cell size, giving rise to the robust continued growth of optoBem1 cells. It has been shown in other organisms, for example, that DNA endoreduplication enables large increases in cell size. One possibility by which our findings can be reconciled with prior observations of exponential growth in wild type budding yeast is that cells become surface area-limited at sizes just above that of wild type cells, thereby inducing a shift from volume proportional growth to surface area-proportional growth.Cell size control pathways exist to correct for deviations from a set-point size, yet most previously-identified size control pathways specifically operate on cells that are born too small, delaying cell cycle progression to enable further growth to occur. Because the light and temperature-shift stimuli with which we prepared ‘giant’ yeast are fully reversible, we reasoned that we could monitor the return to a steady-state size distribution after releasing giant cells from their block. We prepared giant optoBem1 cells by incubating them in red light for 8 h and monitored them by live-cell microscopy after releasing them into infrared light. Strikingly, we found that cell populations rapidly returned to their unperturbed state , with individual daughter cells reaching the set-point volume in as few as three rounds of division .Return to the set-point size is not driven by cell shrinking, as giant mothers maintained their maximum volume over multiple rounds of budding . Instead, the giant mothers are eventually diluted out as successive generations are born, an effect that is especially prominent in cell populations at least 10 h post-Bem1 release .

In these populations,hydroponic bucket size distributions have a single mode near the set-point volume but exhibit long tails towards larger volumes . Our observation that cell size recovers after only a few generations strongly supports the existence of size control acting on large cells and demonstrates that size homeostasis across a cell population is robust even to extreme increases in cell volume.Quantitatively monitoring cell growth in yeast—as well bacterial, archaeal, and mammalian cells—has shown that the behavior of many organisms is consistent with an adder that monitors size across an entire cell cycle to correct for deviations in cell size and maintain size homeostasis in the population. However, a recent study argued that in budding yeast, the adder behavior could arise from independent regulation of pre- and post-Start events, without a cell needing to keep track of its added volume across all cell cycle phases, and may fail under various perturbations. To test whether adder-based mechanisms could account for size control in giant yeast, we quantified inter division volume change in successive cell division cycles after releasing optoBem1 cells into infrared light. For this experiment we prepared optoBem1 cells that also expressed fluorescently-labeled septin rings, which enabled us to time both bud emergence and cytokinesis and thus separate pre-Start and post Start size regulation . The ‘adder’ model predicts that the cell volume at division should be proportional to cell volume at birth with a slope of 1. Indeed, for unperturbed cells, we found that cell volume at division was linearly related to volume at birth with a slope of 1.19 . However, we found that the adder model poorly explained the cell size relationships in our giant cells, where the volume at division was related to volume at birth with a slope of 1.73 . This relationship was also evident when individual cells were tracked over time: the interdivision volume change, Δ, was positively correlated with the volume at birth . This size-dependent volume change occurred entirely during S/G2/M phase, as cells added a minimal volume during G1 that did not vary with cell size . We also performed analogous experiments in cdk1-ts giant cells that were shifted back to the permissive temperature. These experiments revealed a similar relationship: large cells grew more than small cells, exhibiting a linear relationship between volume at division and volume at birth with a slope of 1.70 .

These results are broadly consistent with recent work showing that although size control in unperturbed cells resembles an adder-based mechanism, no mechanistic adder regulates volume addition across the entire cell cycle. Our data also suggest that any size regulation limiting the growth of large cells is likely a consequence of regulation in S/G2/M, as growth during G1 is negligible.If an adder is unable to explain size homeostasis in giant yeast, what regulatory mechanisms or growth laws might operate on the daughters of giant cells during S/G2/M? Two possibilities include a bud ‘sizer’, where bud growth would be restricted after reaching a critical size; and a bud ‘timer’ in which cytokinesis would occur at a fixed duration following the beginning of S/G2/M . Such ‘sizers’ and ‘timers’ have been proposed to operate in a variety of biological systems. To distinguish between these possibilities, we tracked the timing of bud emergence and cytokinesis by septin ring appearance and disappearance, respectively, following reactivation of Bem1 in giant optoBem1 cells . Daughter volume strongly correlated with mother volume , inconsistent with a bud sizer mechanism. Our prior observation that the inter division volume change scales positively with cell birth size further argues against a bud sizer for cell volume control. In contrast, our data were consistent with a timer specifying the duration of S/G2/M: the time from bud emergence to cytokinesis did not vary as a function of mother cell volume and took average 95 min across cells of all volumes .Similar experiments performed using cdk1-ts cells were consistent with our observations in optoBem1 cells, revealing a size-independent duration of budding. However, we observed one notable difference: the duration of the size-invariant bud timer in giant cdk1-ts cells was substantially longer than that of giant optoBem1 cells . As Cdk1 is a key driver of mitosis in eukaryotes, the increased duration of the bud timer in cdk1-ts cells may arise from the need to refold or synthesize new Cdk1 molecules to complete S/G2/M following a shift from the restrictive to permissive temperature. Furthermore, even when grown at the permissive temperature, the doubling time of cdk1-ts cells is longer than an isogenic wild type strain , suggesting that cdk1-ts may not be able to fully complement CDK1. In summary, we find that a timer specifying a constant budding duration describes how a cell population founded by ‘giant’ cells returns to their set-point volume.

Although mother and daughter sizes are correlated across a broad size range, daughters are always born smaller than mother cells. After cytokinesis, daughter cells remaining larger than the set-point volume exhibit a G1 phase with virtually no growth and bud rapidly, leading to a geometric shrinking in successive generations . Indeed, a back-of-the-envelope calculation demonstrates that if newly-budded daughters are each 50% smaller than their mothers, a 32-fold decrease in cell volume can be achieved in 5 generations . Assuming a 100 min doubling time , a return to the set-point size would take ~8 h. A fixed budding time, even in the absence of active molecular size sensors in S/G2/M, is sufficient to buffer against persistence of abnormally large cell sizes in the population. We also note that the bud duration timer we describe is quite complementary to G1-phase size sensors such as Whi5, which compensate for a small size at birth by elongating G1 phase.Our conclusions are derived from cells prepared using two independent perturbations: optogenetic inactivation of the Bem1 polarity factor and a temperature-sensitive cdk1 allele. Importantly,stackable planters each of these perturbations targets distinct cellular processes and thus produces distinct physiological defects. Cells lacking Bem1 activity exhibit weakened cells walls and undergo successive rounds of DNA endoreduplication following their initial arrest in G1 . In contrast, loss of Cdk1 does not produce such defects but its disruption requires incubating cells at 37˚C, which may broadly activate environmental stress response pathways. Furthermore, cdk1-ts may not fully complement CDK1, even at the permissive temperature . That each of these perturbations reveals similar mother-daughter size correlations as well as a size-invariant bud timer strongly supports the generality of our conclusions. The bud timer we describe here is consistent with prior work suggesting that the duration of budding tends to be invariant to changes in growth rates. However, such a timer need not be a dedicated biochemical circuit to sense budding duration, compare it to a set-point, and dictate the transition to cytokinesis. Its existence could simply arise due to the time required by independent cellular processes that coincide with bud growth, such as the combined duration of S-phase or mitosis. Nevertheless, one observation suggests more complex regulation: the duration of the size-invariant bud timer is markedly longer in enlarged cdk1-ts vs. optoBem1 cells , yet mother-daughter sizes are nearly identical in these two backgrounds . These data suggest that the duration of the bud timer may be inter-related to Cdk1 activity and cells’ growth rate during S/G2/M. Recent work has found that mitosis and bud growth rate are closely coordinated and that cells may extend the duration of mitosis to compensate for slow growth that occurs under poor nutrient conditions. Dissecting the dependencies between growth rate, Cdk1 activity and the duration of post-Start events presents a promising direction for future study.All yeast strains used are isogenic to an ‘optoBem1’ strain which was created in the w303 genetic background and contained exogenous PhyB-mCherry-Tom7 with endogenous Bem1 C-terminally tagged with mCherry-PIF, as previously described. The cdc28-13strain was a kind gift from David Morgan. A pACT1-CDC10-eGFP expression vector was created by Gibson assembly, with the CDC10 expression cassette inserted between the NotI and XmaI sites of the pRS316 vector.

For the experiments described in Figs 3 and 4; Fig D, E, F, and G in S1 Fig; and S2 Fig; the indicated vector was transformed into our optoBem1 or cdk1-ts strain and selection was maintained by growing yeast in synthetic complete media lacking uracil . For all other experiments, yeast were cultured in synthetic complete media .Preparation of yeast prior to optogenetic experiments was performed, in general, as previously described. Yeast undergoing exponential growth in synthetic media were treated with 31.25 μM phycocyanobilin and incubated in foil-wrapped tubes at 30˚C for a minimum of 2 h. For all microscopy experiments, yeast were spun onto glass-bottom 96-well plates coated with Concanavalin A and washed once with fresh PCB-containing media to remove floating cells. Cells remained approximately spherical following this procedure, as assessed by Concanavalin A staining .Imaging was performed atroom temperature. For experiments where isotropic growth was measured , yeast were plated and imaged immediately following PCB treatment. For experiments where growth following Bem1 reactivation was examined , PCB-treated yeast were first placed in clear culture tubes and incubated at room temperature for >6 h while undergoing constant illumination with a red LED panel . Cells were then plated and imaged. For experiments involving the cdk1-ts strain, cells were maintained in liquid cultures of synthetic complete media at 25˚C for at least 24 h and plated as described for the optoBem1 strain. Imaging was performed at 37˚C for experiments where isotropic growth during G1 was measured . For experiments where size control was assessed , cells were incubated at 37˚C for 8 hr, then shifted to 25˚C 30 min prior to imaging.For isotropic growth experiments, samples were imaged on a Nikon Eclipse Ti inverted microscope equipped with a motorized stage , a Lamba XL Broad Spectrum Light Source , a 60x 1.4 NA Plan Apo objective , and a Clara interline CCD camera . Samples were imaged by bright-field microscopy every 10 min for 12 h. Throughout experiments involving optoBem1 cells, a red LED panel was carefully balanced against the motorized stage and microscope body to provide oblique illumination to the cells and ensure that Bem1 remained deactivated. Generous amounts of lab tape were applied to the LED panel and scope to prevent slippage during image acquisition and stage movement. For the remaining experiments, samples were imaged on one of two spinning disk confocal microscopes, both of which were Nikon Eclipse Ti inverted microscopes with motorized stages . The first microscope was equipped with a Diskovery 50-μm pinhole spinning disk , a laser merge module with 405, 440, 488, 514, and 561-nm laser lines, a 60x 1.49 NA TIRF Apo objective , and a Zyla sCMOS camera . The second microscope was equipped with CSU-X1 spinning disk , a MLC400B monolithic laser combiner with 405, 488, 561, and 640-nm laser lines, a 60x 1.4 NA Plan Apo objective , and a Clara interline CCD camera.

As AtNLP7 has been reported to be regulated by nitrate via a nuclear retention mechanism , we next tested the subcellular localization of ZmNLP6 and ZmNLP8 proteins under nitrate starvation and nitrate re-addition conditions. Both ZmNLP6 and ZmNLP8 were found to be localized in cytosol after nitrate starvation while in the nucleus when nitrate was resupplied . Thus, ZmNLP6 and ZmNLP8 are mainly localized in the nucleus and slightly in cytosol in the presence of nitrate while both proteins are localized in cytosol when nitrate is absent. Previous studies have shown that nitrate assimilation is impaired and the nitrate content is increased in nlp7 mutants . To test if ZmNLP6 and ZmNLP8 affect nitrate assimilation, we measured nitrate content in transgenic lines and found that the increased nitrate content in nlp7-4 was recovered to WT levels . To investigate if this recovery is associated with nitrate reduction, we examined the NR activity and found that this activity was restored in the transgenic lines . Furthermore, the deficiency of amino acid in nlp7-4 mutant was completely rescued when ZmNLP6 and ZmNLP8 were over expressed in nlp7-4 mutant . Taken together, these data indicate that ZmNLP6 and ZmNLP8 are involved in regulating nitrate assimilation when over expressed in Arabidopsis. In order to further explore the underling mechanism whereby ZmNLP6 and ZmNLP8 affect nitrate content, we examined nitrate accumulation and the expression of several genes involved in nitrate assimilation. Plants were grown on 2.5 mM ammonium succinate for 7 days and then treated with 5 mM KNO3 for 0, 0.25, 0.5, 1, 2, hydroponic nft system and 4 h in the presence of 2.5 mM ammonium succinate. The nitrate content in whole seedlings was investigated and the results showed that no difference was found in nitrate accumulation among the WT, nlp7-4, and transgenic lines . However, the expression of Gln1.1, Gln1.3, NIA2, and NiR in transgenic lines was recovered to the levels even higher than in WT .

These findings suggest that ZmNLP6 and ZmNLP8 can regulate the nitrate assimilation more strongly than nitrate accumulation. It has been found that Arabidopsis NLP proteins can bind nitrate regulatory elements to regulate the nitrate responsive genes . To investigate whether ZmNLP6 and ZmNLP8 can bind NREs in maize, Y1H assay was performed. Firstly, we searched for NREs with a module from the 50 and 30 franking sequences of nitrate uptake and assimilation genes. Nine putative NREs were obtained as shown in Supplementary Table S2. Then, the candidate NREs were used for testing the binding activity of ZmNLP6 and ZmNLP8. The results showed that ZmNLP6 and ZmNLP8 could bind the NRE-like motifs of ZmNRT1.2 and ZmNiR2 , respectively . But no binding activity was found between ZmNLP6/ZmNLP8 and other NREs . These data suggest that the ZmNLP6 and ZmNLP8 proteins can bind putative NREs similar to what was reported for Arabidopsis. Low NUE in agriculture system is a global problem and therefore we assessed the potential of both genes to affect root architecture and NUE of in Arabidopsis. We first examined the primary root length and lateral root number in plants grown vertically on the media with different nitrate concentrations . The results showed that the length of primary roots and number of lateral roots were higher in ZmNLP6/nlp7-4 and ZmNLP8/nlp7-4 transgenic lines than in WT and nlp7-4 mutant under these three nitrate conditions . To determine whether ZmNLP6 and ZmNLP8 can enhance NUE in plants, we investigated the biomass of WT, nlp7-4, and ZmNLP6/nlp7- 4 and ZmNLP8/nlp7-4 transgenic lines under different nitrate concentrations. The results showed that the transgenic seedlings grew bigger than WT and nlp7-4 and the biomass of the whole seedlings increased by 15, 35, and 40% more than WT under 0.2, 2.5, and 5 mM KNO3 conditions , indicating that the ZmNLP6 and ZmNLP8 can rescue the defificient growth phenotype of nlp7-4 mutant and promote plant growth under both low and high nitrate conditions. Seed yield is an important trait for agricultural production and also for assessing the NUE of plants. Thus, we investigated the seed yield of ZmNLP6/nlp7-4 and ZmNLP8/nlp7-4 transgenic lines grown under both high and low nitrate conditions.

The results showed that the yield per plant was higher in ZmNLP6/nlp7-4 and ZmNLP8/nlp7-4 transgenic lines by 44 and 45%, respectively, than in WT under low nitrate conditions . However, no significant difference was found between the transgenic lines and WT when grown under high nitrate conditions . These data suggest that ZmNLP6 and ZmNLP8 may improve plant NUE under low nitrate conditions. It has been reported that Arabidopsis NLP genes are involved in nitrate regulation, but the functions of maize NLP genes remain unknown. As maize is one of the main crops of the world, identifying the genes associated with nitrate signaling and deciphering the corresponding gene networks are of great importance for improving NUE and reducing environmental pollution. In this study, we identified nine ZmNLP genes containing RWP-RK and PB1 domains by genome-wide analysis in maize. The RWP-RK super family includes NLP and RKD families, both of which contain RWP-RK domain . NLP family is conserved in the land plants we searched, especially in maize , sorghum , rice , and Arabidopsis . This family can be divided into three subfamilies and each subfamily shows different gene structure characteristics from each other . A previous study also reported a similar subfamily division in Arabidopsis, rice, and Lotus japonicus . We found two novel NLP motifs among these searched 34 species: GAF motif and GSL motif . The GAF motif exists in the N-terminus of NLP protein, a region involved in receiving the nitrate signal . The most conserved signature structure in GAF motif is the first serine, the fifth phenylalanine, and GLPGR.The GSL motif, located in the front of the RWP-RK domain, is conserved only in the NLP proteins but not in RDK proteins. The expression profiles of ZmNLP genes showed that ZmNLP6 and ZmNLP8 exhibited the highest expression levels among the whole gene family, especially in R1 in roots and V13 in leaves . As roots of R1 and the leaves of V13 are important for absorbing and remobilizing nitrate to pool organs and critical for yield of maize , ZmNLP6 and ZmNLP8 may be important for nutrient uptake and translocation. The nitrate induction was tested for all ZmNLP genes and the results showed that only the expression of ZmNLP3 and ZmNLP4 were induced by low nitrate, and induced poorly or not at all by high nitrate , implying that a post-transcriptional regulation response to nitrate may exist.

Under different nitrate conditions, ZmNLP1, ZmNLP2, ZmNLP3, and ZmNLP7 exhibited relatively higher expression in roots under nitrogen starvation and they mainly belongs to ZmNLP Group I, implying that the function of Group I may be involved in nitrogen starvation . Moreover, ZmNLP6 and ZmNLP8 showed the highest expression levels under higher concentrations of nitrate in shoots while the highest levels on 2.5 mM nitrate condition in roots and may participated in nitrate absorb and allocation. The expression patterns of ZmNLPs implicate that functional redundancy of ZmNLP family members in nitrate regulation may exist,nft channel and these NLP genes may play important roles in nitrate regulation in different stages and organs under various nitrate conditions. In Arabidopsis, several nitrate regulatory genes have been identified and these genes can modulate genes involved in nitrate transport, assimilation, and response. But so far, no nitrate regulators have been reported in maize. We investigated the function of maize NLP genes and found that over expression of ZmNLP6 and ZmNLP8 in Arabidopsis nlp7-4 mutant could recover the YFP fluorescence from the NRP-YFP transgene product and the induction of nitrate responsive genes to WT levels , indicating that ZmNLP6 and ZmNLP8 can restore the nitrate signaling in nlp7-4 mutant. Previous studies have reported that NRE works as an important nitrateresponsive cis-acting element and can be bound by NLPs in Arabidopsis . Our Y1H results showed that ZmNLP6 and ZmNLP8 proteins can bind potential NREs of ZmNRT1.2 and ZmNiR2 in vitro , suggesting a direct regulation of ZmNRT1.2 and ZmNiR2 by ZmNLP6 and ZmNLP8 may exist in maize. In Arabidopsis, NLP7 has a profound influence on nitrate responsive genes at the transcriptional level and some target genes of NLP7 are regulated by binding to their NREs . Thus, the ZmNLPs may bind NRE to regulate nitrate signaling in maize similar to that in Arabidopsis, and this regulation mechanism may be conserved in monocots and dicots. The subcellular localization of ZmNLP6 and ZmNLP8 is regulated by nitrate , similar to a mechanism controlling AtNLP7 localization in Arabidopsis. Our physiological and molecular analyses further revealed that the nitrate reduction process could be recovered in the ZmNLP6/nlp7-4 and ZmNLP8/nlp7-4 transgenic lines, indicating that both ZmNLP6 and ZmNLP8 can modulate nitrate assimilation when constitutively over expressed in Arabidopsis.

It has been reported that over expression AtNLP7 in Arabidopsis can increase fresh weight and modify root architecture under low and high nitrate conditions. In our study, over expression of ZmNLP6 and ZmNLP8 in nlp7-4 mutant can also enhance the biomass and root development. However, the nitrate content, amino acid content, and NR activity were increased in AtNLP7 over expression lines while restored to WT levels in ZmNLP transgenic lines. In addition, we found an increase in seed yield in ZmNLP6 and ZmNLP8 transgenic Arabidopsis lines under low nitrate conditions. It remains to be further investigated if the seed yield can be increased in AtNLP7 over expression lines. Results shown in this study suggest the function of the group III NLPs in Arabidopsis and maize may be partially conserved in nitrate regulation. Improving NUE of crops is of great importance for sustainable agriculture. Several nitrate-related genes have been implicating in improving NUE. OsDEP1, encoding a highly cysteine – rich G protein γ subunit, has been reported to increase rice harvest index and grain yield under moderate levels of nitrogen fertilization . OsNRT1.1B-indica variation has been identified to enhance the ability of nitrate uptake and root to-shoot transport to improve NUE in rice . In addition, over expression of OsNRT2.3b can improve grain yield and NUE by increasing the capacity of pH-buffering and uptake of N, Fe, and P in rice . In Arabidopsis, over expression of AtNLP7 can improve plant growth under both nitrogen-limiting and -sufficient conditions . In this paper, our results showed that ZmNLP6 and ZmNLP8 could promote plant growth under both low and high nitrate conditions, and increase seed yield under low nitrate conditions . Therefore, both ZmNLP6 and ZmNLP8 genes may be of great potential in improving NUE of maize. It would be also interesting to assess the role of other NLP members in promoting NUE of maize in the near future.Plastids originate from a single endosymbiontic event involving a cyanobacterium-related organism. In the course of endosymbiosis a massive gene transfer occurred, during which most plastidic genes were transferred to the host cell nucleus. Consequently, today the majority of plastidic proteins must be post-translationally imported back into the organelle. So far, two protein translocation complexes have been characterized in the outer and inner envelope membrane: Toc and Tic. After passing the outer membrane via the Toc translocon, the Tic complex catalyses import across the IE membrane. So far, seven components have been unambiguously described as Tic subunits: Tic110, Tic62, Tic55, Tic40, Tic32, Tic22 and Tic20 . Tic110 is the largest, most abundant and best studied Tic component. It contains two hydrophobic transmembrane-helices at its N-terminus, anchoring the protein in the membrane, and four amphipathic a-helices in the large C-terminal domain that are responsible for channel formation. At the intermembrane space side, Tic110 contacts the Toc machinery and recognizes preproteins. Moreover, loops facing the stroma provide a transit peptide docking site and recruit chaperones such as Cpn60, Hsp93 and Hsp70. Tic110 is expressed in flowers, leaves, stems and root tissues, indicating a role in import in all types of plastids. It is essential for chloroplast bio-genesis and embryo development. Heterozygous knockout plants are clearly affected: they have a pale green phenotype, exhibit defects in plant growth, display strongly reduced amounts of thylakoid membranes and starch granules in chloroplasts, coupled with impaired protein translocation across the IE membrane.

Apparently, there are some yet unknown pathways by which aneuploids can be generated.In most crops where androgenesis is used, regular meiotic pairing provides for a chromosome constitution of the microspores. In the absence of structural chromosome differences between the parents of a hybrid, any deviation from the standard karyotype among the regenerants can be attributed to chromosome instability while in culture . Still, in crops with regular meiosis such as wheat and rice, 11.1 and 10.2% aneuploids among regenerants have been detected, respectively . In triticale, with its inherent tendency to univalency, especially in F1 hybrids , there is an ample supply of aberrant microspores, and at least some data presented here can be interpreted as indicative of selection for such microspores at some stage in the process of androgenesis. While chromosomal abnormalities are noted in numerous crops to which androgenesis has been successfully applied , its frequency in triticale is such that any population of the DH lines must be produced considerably larger than the minimum required, to compensate for the aneuploids. Unfortunately, as experience here shows, aneuploidy appears to be the most frequent in the most recalcitrant combinations. This is perhaps because it eliminates, with some frequency, chromosomes that carry genetic loci that prevent the switch from the gametophytic to the sporophytic microspore. While aneuploids generated by androgenesis could potentially be used for some genetic experiments such as marker allocation to chromosomes,ebb flow table by and large they are an additional burden on the already cumbersome method and may tip the scale toward unprofitability.

The Berkeley Prize is the centerpiece of an endowment established in 1996 in the Department of Architecture at the College of Environmental Design at the University of California, Berkeley. Its activities are overseen by a group of interested academics and professionals who form a loosely knit Berkeley Prize Committee. Each year this group formulates a question, posted online, that asks students to put their thoughts and experiences with regard to an important social issue into words and selects a jury to review entries. In 2004 the prize jury included Marco Casagrande, architect and environmental artist, Finland; Beth Gali, urban planner and landscape architect, Spain; Peter Prangnell, architect, critic, and author, Canada; and Minja Yang, Culture Sector, UNESCO, France. In past years, the prize questions have asked students to think about such issues as the equitable use of public places; the appropriateness of institutional care for the elderly; the street as mediator between public and private selves; the search for lasting values in architecture; and the exploration of meaning in social architecture. All of these topics have been presented in ways that provoke consideration of the role architects may play in identifying and redressing complex social issues. In response and encouragingly, there is throughout the submitted essays an implicit idealism about the possibilities of an architecture that addresses social ills through good design. The 2004 prize cycle was no exception:When the general public is questioned about the problem of homelessness in their community, the answers are always the same. “They chose this lifestyle” or “if they wanted to change they would.” As we wait for someone else to initiate a solution, the number of people on the street grows steadily. Sure, we feel compassion when the nostalgia of the holiday’s set in, or on those cold winter nights when the thermometer plummets below freezing and we can’t imagine how anyone will survive on the street. Where is our concern through the remainder of the year as we hurry along ignoring the voices asking for spare change or even crossing the road so we won’t have to deal with this nuisance?We need a general reawakening of common sense and empathy towards our fellow citizen. Schools can start the process by integrating a social conscience into the education system. Young architects need to learn financial and social accountability when developing design concepts. To be told this isn’t important now reinforces the notion we are designing only for those with power and money.

Integrating all levels of income needs to play an important role in the design education of an architect.As in previous years, in 2004 students were not only asked to think about the chosen topic but to propose solutions. This is where the responses often become most ingenious. In the 2004 competition most entrants find fault with the conventional solution of the “shelter,” as they believed it to be conceived. Essentially, they argued that, after a brief reconnaissance, many displaced people find shelters stigmatizing and/or dangerous, and prefer the street. As an alternative, some students argue for respecting what the homeless do for themselves to solve their problems. Others argue for this respect, but also, simultaneously, for resources from the outside to augment their efforts. In essays from Singapore and Austria two students discuss racism as a factor underlying their cities’ reluctance to effectively assist outsiders. In one case, people considered different were seen as an urban blemish; in the other, it was people of color. As architects, and students of architecture, we should want to know if and how our professional skills can be of use to local governments, institutions, private groups, and individuals in meeting specific social needs — such as those posed by disenfranchised populations studied by entrants in the 2004 prize competition. To find out, we must first understand the issues directly, “on the ground.” Second, we must recognize that solutions to these problems are, by definition, interdisciplinary. Both ideas are an integral part of how students are asked to research and respond to the prize questions. By stressing essay writing, the prize deliberately forces students into unfamiliar territory where they must grapple with a form of communication not normally stressed in architecture education. Architects generally feel most comfortable communicating by way of drawn or modeled images of one sort or another. But they must also be able to communicate their research findings and design proposals effectively in verbal and written form to the public, to clients, and to colleagues. The Berkeley Prize asks students to create images with words, much like they might create images with drawings in the design studio. When they succeed in describing their ideas in words, they can not help but realize that, as future professionals, they have begun to train themselves in another way to advocate their thoughts to a general population that do not understand abstract drawings or even ordinary plans.

Similarly, as a result of this process, it is hoped that students might also begin to realize that it is actually through language that the images of intended building forms and the context in which they are embedded are conceived. At its heart, the Berkeley Prize seeks to challenge undergraduate students to use language to engage with and communicate ideas which are typically more sophisticated than those they might project through hand drawings or conjure up on their computers. Through essay writing the Berkeley Prize attempts to educate architects-in-training that the smallest act of building has global implications: that design can and does play a major role in the social,hydroponic grow table cultural and psychological life of both the individual and society.The success of the Berkeley Prize as a vehicle to encourage the study of social architecture will not ultimately be judged by the level or quality of the rhetoric of the competitors — although fine writing is a prize in itself. It will be successful because it has been able to encourage writing as a means by which awareness of social issues is explored and magnified. It will be successful because it demonstrates the potential for architects to become proponents of better policies and designs through words as well as images. The emphasis on encouraging students to view the public as their audience and to learn new ways to communicate with them has one final practical benefit. By learning to talk to the public, competitors should also discover that they can be persuasive advocates of social architecture among those for whom such advocacy is most important: those who use architecture, rather than those who make it.Students enrolled in any undergraduate architecture program throughout the world are invited to submit a 500-word essay proposal responding to the question. From this pool of essays, approximately 25 are selected by the prize committee as particularly promising. These semifinalists are then asked to submit a 2,500-word essay expanding on their proposals. A group of readers, composed of committee members and invited colleagues, selects five to eight of the best essays and sends these finalists on to a jury of international academics and architects to select the winners. The prize is announced, papers submitted, and reader and jury-reviewed all online. The most recent winner was awarded $3,500 from a $5,000 prize pool. During the past six years, hundreds of students have submitted proposals and essays, representing dozens of schools of architecture from nearly forty countries. In recognition of these efforts, the prize was the recipient of the 2002 American Institute of Architects’ Education Honor Award. The Berkeley Prize has also garnered international acclaim, not the least reason for which is its complete embracing of digital technology. In partial recognition of this outreach, the 2003 Berkeley Prize competition was named a special event of “World Heritage in the Digital Age,” a virtual congress helping to commemorate the thirtieth anniversary of the UNESCO World Heritage Convention.

Organized by the UNESCO World Heritage Center, the virtual congress was one of a series of events scheduled to highlight the far-reaching goals of the World Heritage Convention to “maintain, increase, and diffuse knowledge, by assuring the conservation and protection of the world’s heritage.” The 2004 prize competition attracted 97 entries from students representing 29 countries and 43 undergraduate architecture programs on six continents. Twelve of these entries were collaborative efforts. The 2004 competition also established the Berkeley Prize Travel Fellowship. This new prize recognizes the vital role that exposure to other cultures and environments plays in helping to demonstrate the reality and importance of the social art of architecture. All finalists for the essay competition are invited to submit proposals demonstrating how they would use a two-week, expenses-paid, trip to an architecturally-significant destination selected by the prize committee. The 2004 Berkeley Prize honored its continuing association with UNESCO’s World Heritage Center by enabling the travel fellowship winner to visit Barcelona for two The Berkeley Prize: How it Works weeks and attend Forum Barcelona 2004, a six month-long, city-wide, international cultural event partially sponsored by UNESCO . The travel fellowship was also seen as potentially enabling the student to take part in the international conference, “Arquitectura 3000: the Architecture of Indifference”, sponsored by Escola Técnica Superior d´Arquitectura de Barcelona at the Universitat Politècnica de Catalunya in Barcelona in the summer of 2004 .The over consumption of nutritive sugars continues to be a major dietary problem in different parts of the world. A recent report indicates than an average American consumes about 17 teaspoons of added sugar daily, which is nearly twice the amounts of the 6 and 9 teaspoons, recommended for women and men, respectively. This dietary behavior is linked to various adverse health effects such as increased risk of diabetes, obesity, high blood pressure and cardiovascular diseases. Hence, there are worldwide efforts to reduce sugar consumption. For instance, the World Health Organization made a conditional recommendation to reduce sugar consumption to less than 5% of the total caloric intake, along with a strong recommendation to keep sugar consumption to less than 10% of the total caloric intake for both adults and children. Currently, added sugar consumption accounts for approximately 11–13% of the total energy intake of Canadian adults, is greater than 13% in the US population, and is as high as 17% in US children and adolescents, the latter principally from sugar-sweetened beverages . Consequently, taxes on SSB have been proposed as an incentive to change individuals’ behavior to reduce obesity and improve health. Notably, the city of Berkeley, CA, USA successfully accomplished a 21% decrease in SSBs consumption within a year of implementation. Therefore, it is expected that more states and cities will adopt this policy. On the regulatory level, the U.S. Food and Drug Administration updated the Nutrition Facts label requirement on packaged foods and beverages, starting 1 January 2020, to declare the amount of added sugars in grams and show a percent daily value for added sugar per serving.

Few signals from pectin and xyloglucan were detected in these experiments, due to both their relatively high mobility and low abundance in the secondary cell wall. Since the glucan chains in cellulose fibrils are polymorphic in structure, the two domains of cellulose were detected in at least three identified environments each. The three distinguished environments for both amorphous and crystalline cellulose may arise from differences in hydrogen bonding patterns between glucan chains, slight changes in glucan chain conformation, variations in bond geometries, and changes and inconsistencies in neighboring chain environments within the microfibrils different environments as follows: superscript [C] or [A] represents the. Here, for each carbon in a glucosyl unit from cellulose we represent these crystalline or amorphous cellulose domain respectively, and superscript represents the three distinguished environments of cellulose in each domain. The major chemical shift differences between the two domains of cellulose are present in the cellulose carbon-4 at ~89 ppm and ~84 ppm , and C6 at ~65 ppm and ~62 ppm . The signals from xylosyl units of three-fold screw xylan backbone were identified , but there were a lack of signals from xylosyl units of two-fold screw xylan . In addition, high intensity signals from arabinosyl units decorating the xylan backbone were identified, which is consistent with our monosaccharide analysis on the non-cellulosic components from stem internodes . Lower intensity signals from carbons 1 and 2 of glucuronic acid were also detected due to the lower abundance of glucuronic acid substitutions on the xylan backbone, as compared to arabinose. A DP-INADEQUATE experiment with relatively short recycle delay, 2 s,ebb and flow bench was also performed on the sorghum stem internode samples to enhance the detection of the relatively mobile components in the secondary cell walls, and spectrum was labeled and shown in Supplementary Fig. 3.

Two-fold screw xylan with a flat-ribbon shape has been reported as the dominant conformation detected by CP in the secondary cell walls of dicot plants and softwoods, which facilitates the binding with the cellulose fibrils on their hydrophilic surface via hydrogen bonding. However, our CP-INADEQUATE result indicated a significantly higher fraction of xylan was present in the three-fold screw conformation than in the two-fold screw conformation in the sorghum secondary cell wall. According to the intensities of cross peaks from the Xn4 and Xn5 of the xylosyl units from both two- and three-fold screw xylan backbone, the immobile three-fold screw xylan was the dominant conformation. This could be due to the large quantity of arabinosyl substitutions on the sorghum xylan backbone. Dupree et al. previously demonstrated that the spacing between the substitutions is critical for the formation of two-fold screw xylan due to the steric hindrance effect. For instance, xylan in dicots has little or no arabinosyl substitution, and acetylations and glucuronic acid substitutions are evenly spaced on a major fraction of the xylan backbone, which allows the formation of two-fold screw conformation. Similarly, although softwood xylan is substituted by both glucuronic acid and arabinose, substitutions are evenly spaced on every six and two xylosyl units respectively on the xylan backbone. Hence, we speculate that a large number of closely spaced substitutions on sorghum xylan would likely disrupt any pattern of regular spacing on a major fraction of the xylan. The result of this would be more xylan in the three-fold screw conformation in sorghum secondary cell walls. This spacing pattern has yet to be confirmed, due to a lack of a glycosyl hydrolase with the required specificity for cleaving at an arabinosyl substitution, akin to the glucuronic aciddependent xylanase XynC used to determine the spacing pattern in Arabidopsis. However, since CP experiments emphasize the signals from relatively immobile components of the cell wall, the large amount of three-fold screw xylan detected suggests that this confirmation could be important for xylan-cellulose interactions in sorghum.

In contrast, in recent work by Kang et al., two-fold screw xylan was detected by CP-INADEQUATE as the dominant conformation present in maize, a grass closely related to sorghum. However, their CP-INADEQUATE data did not detect arabinose signals, which is surprising given the reported structure of maize xylan. We hypothesized that this could be due to the additional lyophilization step they performed on the plant samples prior to analysis, which may alter the native wall structure. To test this, we lyophilized the sorghum stem tissue and rehydrated the sample according to the procedure that Kang et al. described in. A CP-INADEQUATE experiment was conducted on the rehydrated sample and compared with our previously collected CP-INADEQUATE spectrum on the untreated sample . We found that the previously strong signals from arabinosyl units were lost in the spectrum of lyophilized-rehydrated sample . The intensities of 3fXn4 and 3fXn5 also decreased in the lyophilizedrehydrated sample, suggesting that lyophilization may disrupt the immobile three-fold screw xylan interactions with other wall components. Rehydration of the sample did not restore these interactions, but instead, this free three-fold xylan became more mobile in the rehydrated water, and therefore no longer detectable by the CP. In addition, we also collected DP-INADEQUATE spectrum on the lyophilized-rehydrated sample and compared it with the previously collected DP-INADEQUATE spectrum from the untreated sample . The spectra show that there is a significant enhancement of signals from arabinosyl and xylosyl units of the three-fold screw xylan in the lyophilizedrehydrated sample, which indicates a significant amount of arabinosyl and xylosyl units from three-fold screw xylan have become more mobile after lyophilization and rehydration. On the other hand, the cross peak intensity from crystalline cellulose and two-fold screw xylan was enhanced in the lyophilized-rehydrated sample. Two-fold screw xylan has a flat-ribbon shape that can form a crystalline structure that is similar to cellulose.

Lyophilization of the sample increases the crystallinity of these structures. Although rehydration of the sample could reduce crystallinity to a certain extent, the cell wall architecture will remain altered permanently as compared to native cell walls. Hence, the increased rigidity of twofold screw xylan and cellulose led to enhanced CP signals for the lyophilized-rehydrated sample. For comparison, we also performed additional CPINADEQUATE experiments on never-dried sorghum leaf and root samples of sorghum, which are richer in primary cell walls, for comparison with the stem internode samples. The leaf samples show that the majority of xylan detected by CPINADEQUATE are in a three-fold screw conformation, but to a lower extent compared to the stem . However, analysis of the root material indicates that there is almost no immobile xylan in either conformation, as detected by CP-INADEQUATE .Xylan in the intact plant cell wall can populate both mobile and immobile states. The states can be distinguished based on the extent of their interactions with the immobile cellulose, which are mediated via either hydrogen bonding or Van der Waals forces: the cellulose-bound fraction of xylan is immobile, whereas the fraction of xylan that fills the inter-microfibril space is highly mobile. In contrast to reported data for dicots, we found that the three-fold screw xylan, not two-fold screw xylan, was the dominant conformation of xylan in the sorghum secondary cell wall,4x8ft rolling benches in both immobile and mobile forms . To investigate how this impacts xylan-cellulose interactions, 13C-13C proton-driven spin diffusion experiments were performed with CP with three different mixing times . Due to the CP transfer used in the experiment, these measurements report on the immobile fraction of xylan in the sample characterized by limited larger-scale molecular motions about an average structure with motional timescales on the order of µs to ms. This is in contrast to the mobile fraction of xylan, which is characterized by much faster molecular reorientations with motional timescales on the order of ns to µs. PDSD experiments provide information on carbons in close spatial proximity. The longer the mixing time, the longer the distance observed on the spectra. The short-mixing time CP-PDSD experiment is dominated by intramolecular peaks, such as carbons from glucan chains with six identified allomorphs in cellulose microfibrils, xylosyl units in the xylan backbone in both two- and three-fold screw conformations, and arabinosyl units from xylan .In addition, some intermolecular cross peaks between cellulose and xylan are observed. There are no cross peaks representing the interaction between the two-fold screw xylan and either the crystalline or amorphous domain of cellulose, likely due to the limited amount of two-fold screw xylan in the sorghum secondary cell wall. Instead, cross peaks, such as 3fXn4-2AC4 , 3fXn3-2AC4 , 3fXn3-1AC6 , 3fXn3-3AC6 , 3f,AXn3-2AC4 , and 3f,AXn3-3AC6 , all indicate that the xylosyl units from xylan in a three-fold screw conformation is closely interacting with amorphous cellulose. Additionally, carbons from the xylosyl units of three-fold screw xylan, 3fXn2 to 3fXn5, show cross peaks with the C1 from cellulose , which derives from cellulose in multiple environments. However, since we observed no cross peaks between carbons from three-fold screw xylan and CC2 to CC6, we conclude that the 3fXn2- to 3fXn5-C1 cross peaks were primarily contributed by the C1 from amorphous cellulose. Furthermore, arabinosyl units from xylan show close interactions with the cellulose C1, as indicated by two cross peaks, A2-C1 and A4-C1 . Since a low abundance of two-fold screw xylan was observed , the arabinose signals are likely from three-fold screw xylan. The close interactions between the arabinosyl units and the cellulose imply that the less ordered amorphous cellulose is able to bind with helical three-fold screw xylan. In the CP-PDSD experiment with 100 ms mixing time, the spectrum showed similar intermolecular cross peaks to the 30 ms mixing time, but with enhanced intensities . Together, these data suggest the xylan-cellulose interaction is dominated by an immobile xylan with three-fold screw conformation and amorphous cellulose across short distances.

It remains unclear to us what type of forces are facilitating such interactions, but we speculate it involves both Van der Waals contacts and some hydrogen bonds. The less ordered amorphous cellulose may have a distorted flat-ribbon shape and therefore create more surface space to occasionally enable the formation of hydrogen bonds with the three-fold screw xylan on the hydrophilic side. Interactions with Van der Waals forces are mainly from the hydrophobic surface of cellulose fibrils. Hence, the xylan-cellulose interactions in sorghum secondary cell walls are significantly weaker than those in dicot plants and softwoods which are dominated by hydrogen bonds between two-fold screw xylan and cellulose fibrils on the hydrophilic surface. To further explore the interaction between three-fold screw xylan and amorphous cellulose, we measured the spin-lattice relaxation times at various chemical shifts representing different components . The higher T1 indicates slower molecular dynamics of the cell wall component. The results show that carbons from crystalline cellulose, such as C1, 1CC6, and 2CC6, have the highest T1 values, ~9 s, and the carbons from amorphous cellulose, such as 1AC3/5, 1AC6, and 2AC6, have similar T1 values as the carbons from arabinosyl and xylosyl units in relatively immobile three-fold screw xylan, ~5 s. T1 measurements of the cell wall components further demonstrate that amorphous cellulose shares similar molecular dynamics with the relatively immobile fraction of the three-fold screw xylan, while crystalline cellulose has significantly reduced molecular motion. This supports our interpretation that amorphous cellulose and threefold screw xylan are closely interacting with each other. One-dimensional spectra were extracted at seven chemical shifts from the F1 plane of the CP-PDSD spectra with both 30 and 1500 ms mixing times and compared in Supplementary Fig. 8. No interaction between the three-fold screw xylan and the crystalline cellulose was detected in the short-mixing time . This is consistent with previous work reported by Dupree et al. using Arabidopsis, which showed that the flat-ribbon shape of two-fold screw xylan with even pattern of substitutions is required for binding on the highly ordered crystalline cellulose hydrophilic surface. In addition to the intramolecular interactions, many more intermolecular interactions were also detected with long mixing time . Although cellulose domains in different environments were expected to be close to each other, cross peaks between crystalline and amorphous cellulose were only detected in the long-mixing time CP-PDSD experiment .We interpret this as being due to the relatively low ratio of crystalline to amorphous cellulose in the sorghum cell wall, as described in the following experiments, and that crystalline cellulose is spatially further away from the amorphous cellulose than the three-fold screw xylan.

Additional byproducts present in the ZmCPS4 product profile represent manoyl oxide as based on comparison to a manoyl oxide standard produced from the coupled reaction of G. robusta GrTPS1 and MvELS from M. vulgare , and a closely related unidentified diterpenoid . These products are likely resulting from rearrangement of the ZmCPS4 products 8,13-CPP and LPP after dephosphorylation by endogenous E. coli phosphatases, as has been described for various related class II diTPSs . To test for possible downstream products of 8,13-CPP and LPP, ZmCPS4 was co-expressed with the currently known maize class I diTPS functions, including the ent-kaurene synthase ZmKSL3 and the dolabradiene synthase ZmKSL4 . No new products were detected when co-expressing ZmCPS4 and ZmKSL3 as compared to the expression of ZmCPS4 alone . When combining ZmCPS4 with ZmKSL4, compound identified as manoyl oxide was significantly increased and, albeit at low abundance, an additional product was formed that predictably represents a labdane diterpene olefin as based on characteristic mass fragments of m/z 272 and 257 . To further investigate the functions of ZmCPS3 and ZmCPS4, we next examined their gene expression patterns using publicly available transcriptome and proteome data across various organs, tissues, and developmental stages of unstressed maize plants . With the exception of 5-days-old primary root and 2 cm tassels, 1–2 mm anthers, and mature pollen , all samples were from the B73 genotype . Detectable transcripts of ZmCPS3 were distributed across all organs and tissues with highest abundance included germinating kernels . Notably,hydroponic indoor growing system when specifically analyzing gene expression in roots, transcript of ZmCPS3 was observed to be significantly more abundant with expression levels 5–481 times higher as compared to ZmAN1, ZmAN2, and ZmCPS4 in the same tissue .

Transcript of ZmCPS4 was detected at only trace levels and present exclusively in primary and secondary root and root cortex tissues . Consistent with observed transcript abundance, query of public proteome data showed that protein levels of ZmCPS3 were highest in root tissues .Previous studies demonstrated that ZmAN2 gene expression is induced under both pathogen and oxidative stress in above- and below-ground tissues . In the context of these findings, the relatively higher transcript abundance of ZmCPS3 in roots and the low but possibly root-specific expression of ZmCPS4 suggested a role of these enzymes in below ground stress responses. To investigate this hypothesis, we analyzed transcript abundance of ZmCPS3 and ZmCPS4 using a previously reported set of samples of 53-days-old maize Mo17 roots incision inoculated with fungal spores of F.v. and F.g. and harvested 7 days later . Plants treated by incision-wounding only were used as controls. QPCR analysis showed no significant fold change in ZmCPS3 transcript abundance in roots exposed to F.v. or F.g. as compared to wounded controls . On average, gene expression of ZmCPS4 was significantly decreased in response to F.v. and F.g. elicitation as compared to control plants. To examine ZmCPS3 and ZmCPS4 gene expression in response to abiotic stress, qPCR analyses were performed on two week-old roots of maize plants that were exposed to 1 mM CuSO4 treatment , previously shown to induce diter penoid biosynthesis . All treatments were compared to a water treated control at each time point using the 2−11 Ct method in which the control has a fold change of 1. Transcript levels of ZmCPS3 in CuSO4-treated roots did not differ significantly from those observed in the roots of water-treated control plants at 2 and 4 h post treatment, but were significantly reduced at the 24 h post treatment time point Conversely, ZmCPS4 showed increased transcript abundance in CuSO4-treated roots as early as 2 h post treatment and with an up to sixfold change at 4 h, before decreasing again after 12 h, with a peak of sixfold increase in transcript abundance .

Rapidly increasing biotic and abiotic pressures can overcome the natural defense systems of plants, leading to substantial harvest losses in major food crops . Given the proven protective properties of crop-specific diterpenoid arsenals, a deeper knowledge of their biosynthesis and biological functions may aid new solutions to optimize crop stress resilience traits and mitigate yield loss . Elucidation of the enzyme activities of ZmCPS3 and ZmCPS4 completes the functional range of the maize class II diTPS family, which controls the early committed reactions responsible for the diversity of maize diterpenoid pathways. Advances in the discovery and mechanistic analysis of diTPSs across the plant kingdom increasingly enable the prediction of diTPS functions , as exemplified here for ZmCPS3 and ZmCPS4. However, accurate computational annotation of diTPS activities remains to be constrained by the vast sequence and functional space of the enzyme family, thus necessitating biochemical characterization. Modular co-expression assays with an expanding catalog of diTPSs of known substrate/product-specificity can be leveraged for efficient diTPS functional analysis, and were applied in this study for the identification of ZmCPS3 as a -CPP synthase and ZmCPS4 as an 8,13-CPP synthase . While absent in rice , a -CPP synthase has been identified in wheat, where -CPP can be further converted by class I diTPSs to form labdane structures, such as pimara- 8,15-diene, abietadiene, and isopimara-7,15-diene . While corresponding end-products and physiological functions have yet to be discovered in maize and wheat, the wide distribution of -CPP-derived diterpenoids in both angiosperm and gymnosperm species suggests roles in stress defense . Similar to the recently demonstrated accumulation and predicted defensive functions of dolabralexins in maize roots , a possible function of ZmCPS3 in below ground stress responses could be hypothesized based on the higher transcript abundance in roots as compared to ZmCPS4, ZmAN1, and ZmAN2. However, the observed lack of elicited ZmCPS3 expression in maize roots in response to Fusarium infection or oxidative stress did not support an inducible defense role for ZmCPS3-derived pathways and metabolites.

Although ZmCPS3 and its protein product were more highly expressed in roots as compared to other healthy tissues, its broad tissue- and development-wide presence may suggest a possibly more constitutive function. Tissue-wide expression levels of ZmCPS3 suggest a conceptual parallel to the high constitutive levels of benzoxazinoid pathway enzymes and metabolites in maize seedlings, which have been shown to be important for the maize biotic and abiotic stress responses . The largely constitutive and moderately inducible role is supported by a separate yet related study, where ZmCPS3 transcript levels displayed an approximately twofold accumulation in the leaves of a resistant maize recombinant inbred line two weeks after challenge with gray leaf spot . In addition to the -CPP synthase ZmCPS3, characterization of ZmCPS4 as an 8,13-CPP synthase adds an uncommon scaffold to the diterpenoid network of maize . Mechanistically, the proposed ZmCPS4-catalyzed reaction will proceed through terminal deprotonation of the common -labda-13E-en-8-yl+ diphosphate intermediate at C-9 to yield 8,13-CPP, as opposed to the more typical deprotonation of the carbocation at the exocyclic C-17 methyl to form the -CPP or ent-CPP isomers . ZmCPS4-catalyzed formation of LPP as a minor product will require hydroxylation at C-8 of the -labda-13E-en-8-yl+ diphosphate intermediate . Dual product activity is rarely observed in class II diTPSs . Lacking available ZmCPS4 maize mutants to enable in planta gene function studies, it can only be speculated if LPP represents a native ZmCPS4 product or results from a possibly reduced enzyme activity in vitro that could cause a slower conversion of the intermediary carbocation and thus facilitate water quenching toward formation of LPP. Notably, an 8,13-CPP synthase was also recently discovered in switch grass,vertical rack system where the enzyme was characterized as a single product class II diTPS . By contrast, an 8,13-CPP synthase function is absent in rice and wheat . Although a geneloss in these species cannot be excluded, this selective presence of 8,13-CPP synthases indicates the independent evolution of this function in maize and switch grass. Similar to ZmCPS3, no pathogen-elicited gene expression was observed for ZmCPS4; in fact, transcript abundance was significantly decreased in response to Fusarium infection . This contrasts the well-established pathogen-elicited gene expression of ZmAN2 , and may suggest that ZmAN2-derived pathways are the predominant inducible mediators of pathogen defense responses .

Conversely, CuSO4-induced expression of ZmCPS4 in roots points to a possible role in below ground plant-environment interactions as also proposed for kauralexins and dolabralexins . The identity of predominant pathway end-products derived from -CPP, 8,13-CPP, and possibly LPP, as well as their corresponding roles in plant-environmental adaptation will require further investigation and ultimately the generation and analysis of defined genetic mutants in future studies. Currently, the biochemical characterization of ZmCPS3 and ZmCPS4 expands the known chemical landscape surrounding maize diterpenoid metabolism and completes the characterization of predicted class II diTPSs in the maize genome. Modular pathway networks composed of class II and class I diTPSs are likely operating in maize to convert the ZmCPS3 and/or ZmCPS4 products into a broader spectrum of specialized diterpenoids. In vitro formation of several labdane-type diterpenoids through the sequential activity of ZmKSL4, but not the ent-kaurene synthase ZmKSL3, with ZmCPS3 and ZmCPS4 support this hypothesis. However, no -CPP, 8,13-CPP or LPP derivatives have yet been demonstrated in maize and the biological role of ZmCPS3/4 pathway branches has to be demonstrated in planta. Nevertheless, functional knowledge of all maize class II diTPSswill now enable a detailed investigation of modular diterpenoidmetabolic pathway branches in maize that are formed by class II diTPSs and known or thus far uncharacterized class I diTPSs as the biochemical foundation for maize diterpenoid diversity. Such insight can be of substantial value to elucidate and ultimately harness the genetic basis of crop stress resilience . The use of reclaimed wastewater for irrigation and biosolids or animal wastes as fertilizers in agriculture is on the rise worldwide . There are many benefits from reuse of these waste materials, such as augmenting water supply, increasing soil nutrient content and improving crop yields . However, concerns remain about the safety of such practices , as they introduce a multitude of trace contaminants, including pharmaceuticals and personal care products , into the agroeco systems . In recent years, the fate of PPCPs in the soil plant continuum has been extensively studied . Furthermore, several studies have considered uptake and accumulation of subsets of PPCPs by different crop species . Although many PPCPs are inherently bioactive substances, their toxicity to plants is comparatively less understood. A few studies showed that exposure to PPCPs affected plant development and physiological functions . For example, root growth and development were markedly reduced when pinto beans were exposed to chlortetracycline antibiotics . Tetracyclines and sulfonamides were shown to negatively affect seed germination , and the influence varied among plant species and the different PPCPs considered in the study . Although such studies have shown various phytotoxic effects from PPCPs, the corresponding physiological and molecular mechanisms contributing to the toxicity were not adequately explored. Once taken up by the plant root, PPCPs may be metabolized, leading to their detoxification, inactivation and sequestration . Another nodal point in the response of plant cells to xenobiotics is reactive oxygen species generation, ultimately imposing oxidative stress to fundamental plant bio-molecules . Recent studies suggested that ROS overproduction-triggered oxidative damage may be the cause of the longer-term visual phytotoxic responses , such as root growth inhibition and seed germination reduction. For instance, phenanthrene-induced oxidative stress in Arabidopsis was responsible for the observed reductions in germination and root growth and the damage to organelle structures . On the other hand, plants have developed sophisticated antioxidant mechanisms to protect their cellular components from oxidative damage . However, to our knowledge, so far little information is available on the potential impacts of PPCPs on ROS metabolism in higher plants, such as ROS production, oxidative damage and antioxidant system responses. When exposed to single compounds of PPCPs, the observed toxic effects to plants were generally low . However, PPCPs always enter agroecosystems as mixtures of many compounds. Comparison between individual and mixtures of PPCPs in aquatic organisms suggested that exposure to single PPCPs underestimated the actual environmental effect, and did not allow prediction of the risk of mixtures at environmentally relevant doses . This study was designed to evaluate PPCP accumulation and potential effects on ROS production and oxidative damage. Cucumber seedlings were exposed hydroponically to a mixture of 17 PPCPs at incremental levels covering environmentally relevant occurrence .

Quantification analysis of H2O2 production Fig. 8B, measured as relative fluorescence as a percentage of control, showed that H2O2 levels were slightly, but not significantly, higher in the Cd-treated HE protoplasts compared with controls. In NHE protoplasts H2O2 formation occurred on a rapid timescale when under Cd stress and was significantly higher than that of controls at 30 min and 60 min .In hyper accumulators, the enhanced ability to protect roots from metal toxicity is facilitated partially through efficient shuttling of metals to the shoots . Our previous studies clearly showed a greatly enhanced rate of root-toshoot translocation was pivotal in the Cd hyper accumulator S. alfredii . The results in the present study confirm the highly enhanced transport of Cd in shoots of this plant species, as indicated by the marked enrichment of Cd within the vascular bundles of its stems and leaves after Cd exposure . The presence of metals within vascular systems is generally associated primarily with a xylem mode of transport and delivery of metals to aerial parts . The distribution patterns of Cd in stem tissues showed a larger proportion of Cd localized to the stem vascular bundles of HEs after treatments with 10 μM or 100 μM Cd for 7 d . This was not observed in NHEs . Based upon Cd signal intensity, the Cd content of stem vascular tissues of HEs was more than 10-fold higher than that of NHEs. This suggests that a considerable amount of Cd has been transported via the xylem vessels of HE S. alfredii,hydroponic grow kit probably as a result of its highly efficient xylem loading of Cd in its roots . The efficacy of Cd transport within xylem tissues and into shoots is further confirmed by the enrichment of Cd within the vascular tissues of young stems collected from HE S. alfredii plants treated with 100 μM Cd for 30 d . Tissue- and age-dependent variations in Cd distribution patterns in stems of HE S. alfredii were also identified in this study, similar to that previously reported for Zn in this plant species .

Vascular-enriched Cd was observed in stems treated with Cd for 7 d and young stems treated with Cd for 30 d, while Cd localization to the pith tissue and cortex layer was observed in the stems of HE S. alfredii exposed to Cd for 14 d or 30 d . This is consistent with previously reported results . Cd signal intensity in stem parenchyma cells of HE S. alfredii is more than 10-fold higher than those in vascular tissues of old stems after 30 d of Cd exposure, implying that sequestration of Cd is probably an active process in these tissues. This result is consistent with our previous work, which indicated that Cd is highly accumulated in the pith and cortex of stems and the mesophyll of leaves . The age- and tissue-dependent variation of Cd-enriched sites in stems implies efficient movement of Cd from vascular bundles into the pith and cortex during its translocation in shoots of HE S. alfredii. The parenchyma cells in these tissues, as well as leaf mesophyll , may serve specifically as terminal storage sites for Cd in this hyper accumulator plant species. This result is quite different from that reported for the other hyper accumulators, such as Noccaea Caerulescensand Arabidopsis halleri, where non-photosynthetic cells of the epidermis or trichomes accumulated the highest levels of Cd and there was a higher uptake rate of Cd into epidermal storage cells when compared with mesophyll cells . A possible explanation for Cd hyper accumulation in HE S. alfredii is that parenchyma cells in its shoot may have enhanced storage capacity for Cd sequestration. To evaluate the characteristics of Cd uptake by parenchyma cells in the shoots of HE S.alfredii, mesophyll protoplasts were isolated from the leaves of these plants and compared with those from NHE leaves. The results suggested metabolically dependent Cd uptake by mesophyll protoplasts of the two ecotypes, without significant differences in Cd accumulation. Transport of metal ions is generally an active process, which requires an energy supply as a driving force and selective binding sites . Furthermore, metabolically dependent uptake of metals by plants is essentially inhibited at low temperatures .

The significant inhibition of Cd uptake at low temperatures in both the time and concentration-dependent kinetics experiments suggests Cd uptake into the mesophyll protoplasts of both S.alfredii ecotypes is metabolically dependent. However, there was little difference in the time- and concentration dependent accumulation of Cd into mesophyll protoplasts between the two ecotypes . This suggests that the high capacity of Cd sequestration and tolerance in shoots of HE S. alfredii, at least for leaves, cannot be solely explained by its rapid cellular uptake rates. Similar results were reported for Cd uptake by leaf protoplasts of the hyper accumulators N. caerulescens and A. halleri , showing the absence of constitutively high transport capacities for Cd at the level of leaf protoplasts. A significant aspect of the differences between the two S. alfredii ecotypes is Cd transport into the vacuoles of mesophyll protoplasts. In most hyper accumulators, the metal is sequestered preferentially into compartments where they cannot impair metabolic processes . It makes sense for plants to store metals in their vacuoles since this organelle only contains enzymes such as phosphatases, lipases, and proteinases, which have not been identified as targets of heavy metal toxicity . It has been reported in the hyper accumulator N.caerulescens that Cd accumulated in the cytoplasm a few minutes after its addition and was then transported into vacuoles within its leaf cells . This is strongly supported by our observations of Cd distribution patterns in protoplasts of HE S. alfredii imaged using LeadmiumTM Green AM dye. Cadmium ion transport into vacuoles of protoplasts was not observed in NHEs, while Cd sequestration into vacuoles was consistently observed in protoplasts of HEs after 90–120 min exposure to Cd . This suggests that Cd ions were rapidly transported into vacuoles for storage, providing an efficient form of protection for the functional mesophyll cells in shoots of HE S. alfredii. This is supported by the results of viability and membrane integrity experiments , and by H2O2 imaging of Cd-treated mesophyll cells from the two ecotypes, which showed significantly higher Cd tolerance in HE mesophyll protoplasts than those of NHEs.

Here μ -XRF images of Cd in stems of HE S. alfredii , together with our previous studies , indicate that large amounts of Cd are stored in the pith and cortex cells after long-term Cd exposure. The essentially uniform distribution patterns of Cd in the pith and cortex cells of young stems shown by high resolution images further implied that considerable amounts of Cd are efficiently localized to the vacuoles of these parenchyma cells. It should be noted that freeze drying of the plant samples may have shifted dissolved Cd from the vacuole to the nearest available surface, hence localization of Cd in cell walls of the young stems in Fig. 2 is probably an artifact of sample preparation. As a succulent plant, S. alfredii has exceptionally large vacuoles in its parenchyma cells, making Cd storage there safer than it would be in regular sized mesophyll cells of other hyper accumulators . It is therefore logical that the efficacy of vacuolar sequestration by the parenchyma cells in shoots of HE S. alfredii is an important aspect of metal homeostasis and tolerance in this Cd hyper accumulator plant species. Taken together,hydroponic indoor growing system the results in the present study clearly demonstrate that one of the primary factors responsible for high Cd accumulation in HE S. alfredii is highly efficient root-to-shoot translocation, as suggested by the much enhanced Cd signal in the vascular bundles of its young stems. Furthermore, the efficient storage of Cd in vacuoles of the parenchyma cells, as opposed to its rapid transport into protoplasts, may represent a pivotal process in metal homeostasis and tolerance of cells in shoots of the Cd hyper accumulator HE S.alfredii. Combined with the idea that cellular uptake and sequestration of Cd are active processes within the terminal storage sites of HE S. alfredii, this study suggests that efficient transport across the tonoplast membranes within the parenchyma cells is the driving force for Cd hyper accumulation. This provides insights into specific translocation and storage strategies for Cd hyper accumulation in plants, particularly succulents that have large vacuoles in the thickened and fleshy leaf mesophyll.It is estimated that agriculture contributes 80% of anthropogenic nitrous oxide emissions , of which most are ultimately derived from nitrogen applied as N-fertilizers and manure-N . These agricultural N2O emissions account for about 5.5% of global annual anthropogenic GHG emissions . The N2O emitted originates from microbial N pathways, the balance of which is affected by the application of N fertilizers or by irrigation regime, aside from natural factors. Application of N fertilizers through micro-irrigation systems presents certain advantages over dry fertilization, especially in allowing more precise administration of N in coordination with crop demand . It has special relevance in arid and Mediterranean systems, where many high value tree crops are irrigated. Insufficient work has been done to describe the N2O emissions from N fertigation and to describe strategies of mitigation. Reports from fertigated tree crops have so far been lower than global expectations of N2O-N from mineral fertilizer applications, sometimes estimated as 0.9% of the total applied N , with a default Tier 1 value of 1% used by the IPCC . With various motives, renewed attention is being paid to “split applications” of nitrogen fertilizers, now usually called “high frequency” in the fertigation context. “High-frequency” N-fertigation in tree crops currently refers to at least 7 applications over the course of a growing season , and can reach up to daily applications during certain stages of development .

Monitored systems which adjust water and nutrient delivery to tree crops on an hourly basis are best described as “open hydroponics” . Increasing crop N-use efficiency may be the primary goal among growers who adopt high-frequency fertigation. Positive effects have been seen with daily application on low-OM, sandy soils with pomegranate , table grape and tomato . In Australia, high frequency N fertigation is becoming standard in citrus production, although yield differences in citrus have been elusive . Further crop-specific research should allow finer tuning for greater synchronicity with demand, as is sought by almond growers in California . Risks of nitrate leaching below the crop root zone accompany N fertigation, a problem with both productive and ecosystemic facets. Mediterranean trees such as almond can have strong fall root flushes with corresponding N uptake that aids against these losses. They can also have deep root systems, but they are not necessarily developed in such a way by farming practices, especially in drier climates . The utility of high-frequency fertigation in minimizing nitrate leaching is widely recognized particularly in sandy soils, or in soils with low cation exchange capacity . Physically, high-frequency reduces the peak soil solution N concentrations seen after fertigations and maintains root zones with more consistent levels of soluble nutrients . A larger fraction of applied N may be adsorbed on mineral or organic surfaces. And root physiological responses may increase N uptake efficiency. For similar reasons, high-frequency N-fertigation may also meet a third priority: reducing N2O emissions. At lower concentrations, N-processing soil microbes, whose rates are limited by a number of factors, are likely to transform a larger proportion of the applied N while the soil remains wet and conditions favorable. Among denitrifiers, with lower levels of available NO3, a lower fraction of this pool has been emitted as N2O , possibly in part because of inhibition of N2O reduction with greater NO3. The only study we are aware of that has compared N2O from high-frequency and standard Nfertigations was carried out under laboratory conditions , where it was found that splitting an application of KNO3 into 5 doses decreased total emissions. This result, too, would reflect the capacities of denitrifiers. Research at the field level is needed, since fertigation typically affects a large volume of soil, with relevant spatial variations in water-filled pore space, O2 and substrate concentrations. Field studies must also explore whether frequency of mineral N applications modifies soil microbial populations over time scales greater than the interval between applications, increasing the rates of certain transformations involved in nitrification and denitrification.

Natural metal oxides such as clays are known to strongly and preferentially sorb phosphate over other organic and inorganic ligands,and research has shown that metal oxide ENMs can also sorb phosphorus and thereby potentially affect its bioavailability in soils and other environmental media.Fourth, we hypothesized that higher light and lower nutrient conditions would be more physiologically stressful forClarkia plants, and that highly stressed plants would be most vulnerable to ENM toxicity. Additionally, since TiO2 and CeO2 are photoactive and produce ROS when exposed to light,we predicted that they would have the greatest effect in plants grown under high illumination by interfering with photosynthesis in leaves.Metals from ENMs were taken up into all tissues in all treatments, although the amounts depended on ENM type, soil ENM concentrations, growth condition , high light limited nutrient , low light excess nutrient , and low light limited nutrient, and tissue type. Mean tissue metal concentrations can be seen in Figure 1, and results from multiple regressions can be seen in Figures 2 4 and Table S1. In general, Ce and Ti were found in highest concentration in roots , while Cu was primarily found in leaves , although relatively high concentrations of Ti were also seen in stems . Background concentrations of Ti and Cu were found in all three tissues, while background Ce was only found in roots. Among plants in the Control group , it is likely that Ce was not found in stems or leaves because it was not present in the soil at concentrations as high as Ti , nor is it an essential micro-nutrient as is Cu. Of the three ENMs to which plants were exposed, those exposed to CeO2 and TiO2 followed the pattern of distribution described in our first hypothesis, with concentrations being consistently highest in the roots followed by leaves then stems . In Cu2-exposed plants, however, Cu concentrations were roughly an order of magnitude higher in leaves than in roots . Plants from all groups showed statistically significant positive correlations between exposure concentration and metal concentration in roots and,growing hydroponically with a few exceptions, tended to have the highest metal concentrations at the highest exposure level in all tissues. The most notable exceptions to this trend are the variable Ce and Ti content of leaves from plants grown under high light, excess nutrient and high light, limited nutrient conditions .

This reflects the high inter-leaf metal content variability for Ce and Ti and may be due to a randomized or patchy accumulation of these nanoparticles between leaves. There were no significant associations between leaf metal content and leaf node number, which is analogous to order of production . Since C. unguiculata leaves are produced in a temporal sequence along the height of the plant and are also larger lower on the plant, this indicates that ENM uptake into leaves was independent of both stage of growth and leaf size. Growth conditions also played a role in ENM uptake, with plants grown under high light accumulating more Ce and Ti in their leaves than those grown in low light and HL leaves accumulating more Ti than HE . Along with the increased transpiration rates seen in plants grown under high light , these findings validate our second hypothesis that high light plants would exhibit elevated uptake of ENMs to leaves due to increased transpiration. However, increased uptake of Cu into leaves and roots was found under low light conditions . These differences among ENM types in uptake and distribution are also likely to be due to differences in particle characteristics, particularly morphology and surface charge. The CeO2 ENMs we used had a moderately high aspect ratio and thus had a smaller minimum dimension, which may allow them to pass through narrow vascular tissues in the stem more easily than the spherical TiO2. Due to this physical size limitation, TiO2 may also aggregate in the conductive tissues of the stems at higher concentrations, causing the buildup seen in Figure 1.Table S2 shows that all three ENMs used here had a weak negative charge in potting soil pore solution, although this was likely due to the high ionic strength and organic content of this soil shielding the particle surfaces and not a result of a direct alteration of the ENM crystal surface. Wang et al.and Zhu et al.found that under hydroponic conditions, well-dispersed particles coated with positively charged polymers are more readily taken up into plant roots compared to those coated with negatively charged polymers , which had higher accumulation in leaves. The results seen here provide confirmation of the importance of surface charge in ENM uptake and distribution in plants under more environmentally relevant conditions, i.e., in soil and with poly disperse ENMs. In addition to its surface charge, the tendency of Cu2 to dissolve at low pH,such as is found in the soil used in this study , likely also contributes to its uptake behavior. Rhizosphere pH tends to be more acidic than the surrounding soil due to the release of protons by roots to stimulate and counterbalance the uptake of ions from the soil; one effect of this acidity may be to dissolve a portion of the Cu2.

Dissolved Cu would, in turn, encounter less size exclusion than ENMs and be retained less in the roots and stems in addition to being actively transported to the leaves, consequently making Cu uptake and translocation less dependent on plant transpiration than CeO2 or TiO2. Although Cu is an essential component of several enzymes and other compounds in chloroplasts and mitochondria,it can be toxic at higher concentrations.34 Last, although we predicted that P would be correlated with metal content in tissues due to physiochemical sorption of phosphate to the ENMs, it was only in root tissue of HL plants exposed to CeO2 ENMs that we found a relationship. At root Ce concentrations below 100 μg g 1 , P was positively associated with Ce , but this trend plateaued at higher concentrations. One possible explanation for this is that CeO2 ENMs adsorbed P from the soil and were then sorbed into/onto the plant roots, but at higher exposure concentrations, the soil was depleted of readily available P for the ENMs to adsorb. Previous studies using hydroponic systems have shown increased P uptake in maize exposed to ZnO ENMs and in spinach exposed to nZVI,although these results were due to the uptake of dissolved metal/phosphate complexes rather than ENM-sorbed P. Rui et al.observed the partial transformation of CeO2 ENMs into particulate CePO4 that were then taken up into hydroponically grown cucumber seedlings, although the general lack of correlation between tissue Ce and P concentrations suggests this process was not occurring to a significant extent in this study. Overall, our results did not indicate a significant relationship for C. unguiculata between ENM exposure and P bio-availability.We found that the physiological effects of ENM exposure on our test plants were strongly dependent on the environmental conditions under which plants were grown, namely, high light excess nutrient , high light limited nutrient , low light excess nutrient , and low light limited nutrient . By comparing photosyntheticrates and other physiological parameters of the zero concentration groups across growth conditions, we can establish baseline levels of stress for each condition, which can be used to explain the trends seen in ENM-exposed plants. On the basis of the responses to growing conditions of A, transpiration rate , intracellular CO2 , and quantum yield of CO2 assimilation in zero concentration groups , the relative rankings from most to least stressful growth condition appear to be HL > HE > LL ≈ LE. This ranking aligns with our hypothesis that higher light and lower nutrient conditions are the most stressful conditions imposed in this experiment. For plants exposed to these ENMs, few significant correlations between the physiological parameters measured and ENM exposure concentration were seen at the second or sixth week of exposure, and by the eighth week, all high light plants had reached the end of their life cycle and ceased photosynthesizing. However, at the fourth week of exposure we found that in HE plants exposed to CeO2 and TiO2, A and ΦCO2 decreased significantly and Ci increased significantly with increasing exposure concentration .

This supports our final hypothesis and indicates that these two photoactive ENMs reduce photosynthetic rate by interfering with the assimilation of CO2 required for photosynthesis,growing strawberries hydroponically which results in a buildup of CO2 within leaf cells. Additionally, there were no changes in ΦPSII in these plants, and this lack of correlation between ΦPSII and ΦCO2 could indicate that energy transfer from photosystem II to the Calvin cycle is being disrupted by the ENMs. This effect appears to be light-driven since no impacts of exposure concentration on any physiological parameters were seen in low light plants. High light conditions had the 2-fold impact of increasing particle uptake to leaves by increasing transpiration rates and possibly stimulating greater photo activity of TiO2 and CeO2. The disruption of energy transfer observed may be due to the absorption of electrons from photo system II by the ENM upon the creation of an e /hþ pair after excitation by a photon, or alternately through reactions with ROS produced by the ENM. Exposure to CeO2 had slightly weaker effects on physiological parameters than TiO2, and if the latter scenario is correct, this could be due to the lower relative ROS production rate of CeO2 compared to TiO2. Barhoumi et al. saw an inhibition of PSII and a corresponding increase in ROS inLemna gibba exposed to iron oxide ENMs, so similar phenomena may be occurring here. ROS production by TiO2 and CeO2 ENMs may also explain why no physiological effects were seen in HL plants, since plants upregulate antioxidant production at higher stress levels40 that may counteract ROS produced by these ENMs. Additionally, interference with photosynthetic mechanisms implies that CeO2 and TiO2 ENMs are able to penetrate or be actively transported not only into the leaf cells but also into the chloroplasts as well, and are able to intercalate themselves between thylakoid stacks to intercept electrons from PSII. Given that interthylakoid gaps can be on the order of 50 250 nm,individual particles or small aggregates would not necessarily be excluded based on size alone. White side et al.found uptake of NH2-coated quantum dots <15 nm in diameter into bluegrass chloroplasts, so it is plausible that at least primary particles of TiO2 and CeO2 were able to enter the chloroplasts of our model plant. Both of these ENMs have limited dissolution and have been shown to be taken up into plant tissues as nanoparticles,making it unlikely that any effects on photosynthesis are due to ionic Ti or Ce. A similar decrease in A was seen in HL plants after 4 weeks of exposure to Cu2, but without a corresponding change in ΦCO2 or Ci . By further decreasing the already low photosynthetic rate of HL plants, Cu2 had a larger relative impact than in HE, LE, or LL plants. This suggests that Cu2 may affect photosynthesis through a different mechanism than TiO2 and CeO2. Additionally, we found that the fraction of oxidized PSII reaction centers increased significantly with increasing exposure concentration . In healthy plants, qL is typically positively associated with photosynthetic production,38 but since we found a negative correlation between Cu2 exposure and photosynthesis , the increases in qL we observed were likely due to interference with the oxidation of the primary PSII quinine acceptor by light rather than increased photosynthetic efficiency. Others have found similar oxidation of PSII reaction centers in plants exposed to ionic copper due to interference with the photon antennae of PSII,which may indicate Cu2 toxicity seen in this study is due to Cu ions released from the Cu2 ENMs. In our system, Cu2 could be dissolved either in the rhizosphere and taken up as ionic Cu or be taken up into the plant in particle form and dissolve within the plant tissues. However, since these Cu2 particles has been shown to have increased dissolution at acidic pH and lower dissolution at basic pH,the majority of dissolution probably occurs in the soil rather than in the neutral or slightly basic conditions of cell or chloroplast interiors.Linear growth rates , maximum height, leaf production rate, leaf loss rate , maximum number of leaves, and week of maximum leaf production were calculated from physical measurements and are shown in Figures S1 S6. Few effects due to ENM exposure were seen under any growth condition, although LE plants exposed to Cu- 2 had reduced growth rates, leaf production rates, and maximum number of leaves with increasing exposure concentrations . Cu is an essential plant micro-nutrient but at high concentrations such as those observed in this experiment, Cu can decrease the uptake of other nutrients from the soil and disrupt nitrogen metabolism.Nutrient limitation caused by the presence of Cu2 may have been responsible for limiting the growth of LE plants . The lack of a growth response in HE plants exposed to CeO2 and TiO2 may be because, under high light conditions, reductions in CO2 assimilation have been shown to have minimal impacts on C gain.

Historical interrelations among manufacturing, industrialization, and patents has resulted in a distinct “thingliness” , though business models, construction processes, chemical formulas, cartographic systems, methods of manufacturing, and other “nonthings” also have a long history of patent innovation.Things and non-things alike may be granted the protection of a utility patent, given that the nature of their claims is non-obvious, innovative, and discloses the function and configuration of a specific “art.” The hybridizing of geographical studies with patent innovation studies suggests a scale, scope, and orientation for intellectual property claims that verge of the infrastructural, ecological, and environmental. Landscapes are not things, cities are not things, and coastal zones are not things, yet each is subject to the iterative and often deterministic forces of human ingenuity. In the following texts and images, I investigate site specific patents that function at landscape and regional scales but with drawings and diagrams that are siteless and scaleless. We know of each patent’s site specificity through the inclusion of geographical terminology and reference to specific places and regions within the patent text, but the scale and impact of the proposed intervention remains open to interpretation. In one drawing per patent, I adapt claims and technical specifications to the geographical location described in the text, synthesizing historical research and maps with the “new” innovation disclosed in the patent. The texts and images presented here are, in their simplest form, ruminations on the intersections of place and intellectual property. They provide geographical context to patents that may have radically altered the American landscape,vertical growing systems transcending the object-oriented history of patents to suggest a new hybrid at the intersection of technology and environmental geography of innovation.

Fluoroquinolone antibiotics, an important class of synthetic antibacterial agents, were developed through structural modifications of the nonfluorinated quinolone nalidixic acid to act against both Gramnegative and Gram-positive pathogenic bacteria . Due to this broad-spectrum antibacterial activity, the FQs are widely prescribed in both human and veterinary medicine. However, as a result of incomplete metabolism and the relative ineffectiveness of conventional water treatment technologies in removing them , they are now detected frequently in both receiving and surface waters . Although FQs were developed to have specific mechanisms of action against pathogens through the inhibition of bacterial DNA replication and repair , they can exhibit unintended toxicological effects on indigenous bacteria and other sensitive organisms when released into the environment . Of special interest are some recent aquatic microcosm studies indicating that photosynthetic species are perturbed when exposed to FQs . For instance, ciprofloxacin , the dominant FQ antibiotic in current use, has been shown to affect both the structure and richness of algal communities exposed to environmentally relevant concentrations . The ability of Euglena gracilis, a facultative photosynthetic protozoan, to form green colonies was vitiated as a result of exposure to Naldx , which also was shown to be a specific inhibitor of chloroplast DNA synthesis. In a key study of seedlings of the higher plant Arabidopsis thaliana, grown in media containing Cipro, Wall et al. reported that DNA gyrase is a chloroplast-specific enzyme in addition to being a bacterial enzyme, thus revealing a specific target of FQs in photosynthetic organisms that is similar to their bacterial target. The FQs may have other chloroplast-specific targets. For instance, exposure to Naldx led to an inhibition of photosynthetic electron transport that was an order of magnitude greater than the inhibition of chloroplast DNA synthesis and replication in isolated pea chloroplasts and to a reduction in the production of both ATP and NADPH in carrot cell cultures.

These important findings have motivated the proposal that Naldx may be acting as a significant inhibitor of photosynthesis, interfering with the generation of reduced electron carriers, which then can impede the production of both ATP and NADH . In agreement with the hypothesis of Mills et al. , in vitro exposure of spinach chloroplasts to nonfluorinated quinolone-containing compounds resulted in the inhibition of photosystem II and the cytochrome b6f complexes, which are key enzymes involved in PET. In sum, these disconcerting results suggest that quinolones, the moieties upon which the broad-spectrum antibacterial activity of FQs largely depends , may have a toxicological impact on PET similar to that of herbicides . However, there appear to be no published investigations of the most widely used FQs as to their specific targets in PET.In the present study, we employed a combination of modeling and experimental techniques in an effort to probe how FQs may have secondary toxic effects on photosynthetic organisms by interfering with the functioning of PET. We identified the structural components of FQs that may be responsible for photosynthetic inhibition and the possible targeted enzymes by performing a structure-activity relationship analysis using well-known protein substrates in and inhibitors of PET. This was followed by molecular modeling of the interactions of Naldx and Cipro with the most likely site of PET inhibition as predicted by the SAR analysis. Guided by these modeling outcomes, we used intact thylakoids isolated from spinach chloroplasts to characterize the effects of Cipro on PET following both in vitro and in vivo exposure at Cipro concentrations up to 50 µM, corresponding to the levels that may be found near industrial effluents .Quinones, and in particular proteins containing quinone sites , play an important role in mediating electron transport within the bio-energetic membranes of photosynthetic bacteria, mitochondria, and chloroplasts. To evaluate FQ antibiotics as potential Q site antagonists in PET, 50 known Q site inhibitors which target 8 different proteins involved in photosynthetic and mitochondrial electron transport were subjected to SAR analysis using the Lead Scope software package.

This software package employs a database of molecular structures that are typical in medicinal chemistry to build correlations between important chemical substructures in a selected set of compounds and the biological effects of the compounds. Validation studies of the software to ensure structure-activity correlations specific to each protein target were performed before using it for SAR analysis of the FQs. The principal result of the SAR analysis was that four unique chemical substructures were identified in about 80% of the compounds that target reaction center II , the pheophytin-quinone-type center present in PS-II, wherein photoexcited chlorophyll molecules facilitate the flow of electrons from an electron donor chlorophyll to a pheophytin molecule, which then transfers electrons to a primary quinone site to reduce subsequently mobile quinone molecules at a secondary quinone site . Two of the four chemical substructures, namely, secondary amine and halide aryl, are found in the FQ antibiotics pipemidic acid, norfloxacin, enoxacin, lomefloxaicn, and ciprofloxacin. This structural correlation encouraged us to apply molecular docking simulations to explore in more detail the interactions of Naldx and Cipro with the QB site in RC-II, a common target of PS-II inhibitors. The quinolone antibiotic Naldx was used for comparison because, as previously noted, it exhibits herbicidal activity by inhibiting PET and possesses fragments of two of the substructures targeting RC-II that are not found in the FQs. The software package Autodock4 was used to generate favorable binding configurations of Naldx and Cipro at RC-II in Rheudopseudomonas viridis PDB ID 2prc,outdoor vertical plant stands which is homologous to RC-II systems found in other photosynthetic organisms . The docking procedure employs a grid map of a three-dimensional lattice to store both van der Waals and electrostatic potential energies that would result from the interaction of each atom of the antibiotic with the atoms of the R. viridis RC-II located in a specified region considered to be a possible target site of the antibiotic. Accordingly, the L and M subunits of the R. viridis RC-II , which respectively house the binding sites for the protein bound quinone menaquinone and the soluble quinone ubiquinone , were considered in the docking simulations . A simulation cell having 20 Å sides centered on the QB site served to isolate the atoms of the receptor to be included in the grid map. Docking was optimized by allowing the ligand 6 degrees of freedom within the cell and by taking into account rotatable bonds of the ligand to include torsional degrees of freedom. Thermo dynamically favorable binding conformations were identified by sequentially implementing random changes in each of the degrees of freedom mentioned above, calculating the intermolecular interaction energies, and subjecting each configuration to an annealing step to remove those that are energetically unfavorable.Simulations of the structure of R. viridis RC-II cocrystallized with QB were performed to validate the docking procedure; the conformation of the docked ligand predicted by the simulation was in good agreement with that determined experimentally from crystal structure analysis.

Docking simulations of Naldx and Cipro within the L and M subunits were then performed similarly, with a 20 Å×20 Å cell centered on the QB site; the resulting lowest energy docking realizations were analyzed in detail.n vitro exposure to Cipro was not inhibited , a distinct contrast from previous results obtained with Naldx , further suggesting that Cipro may have a mode of action different from that of Naldx. This finding is consistent with our modeling results that Cipro may not exhibit a strong binding competition with the secondary quinone at its PS-II target site. Since Cipro was shown to be structurally similar to known inhibitors of the oxidizing site of PS-II , which can induce disruption of the redox states required in PS-II for PET, we monitored the redox state of the primary quinone in the presence of Cipro. Moreover, a statistically significant increase in the Fo/Fm values indicates a corresponding increase in the amount of photoexcited Chmolecules that are not translating to photo reduction of the quinone molecules. In accordance with this phenomenon, the kinetics of QA photo reduction were affected significantly as a function of [Cipro] . The calculated time to reach half the maximum value of Fv, i.e., the time to produce half the total amount of photo reduced QA, was 0.081 50 µM, i.e., a major delay in the kinetics of QA photo reduction under our experimental conditions. Taken collectively, these in vitro results led us to propose that Cipro interferes with PET indirectly, by decreasing the rate of energy transfer from antenna Chl molecules to the reaction center in the spinach thylakoids. The above conclusions were strengthened by results obtained following in vivo exposure of spinach leaves to Cipro. After in vivo exposure, the maximal fluorescence yield did not change appreciably, but there were pronounced changes in the relative yields of Fo andFv . Increasing concentrations of Cipro caused an increase in the parameter Fo, representing the amount of excited Chl that is blocked from or otherwise incapable of transferring energy to the PSII reaction center . Conversely, the fluorescence parameter Fv decreased systematically with increasing [Cipro] , reflecting the lower levels of excitation energy reaching the PSII reaction center molecule from the antenna chlorophyll. We should point out that this in vivo biochemical effect was observed after a short 8 day exposure to Cipro in the growth media, wherein the plants still appeared to be healthy, with no observable decrease in the green pigment of the leaves, in the amount of newly synthesized leaves, or in the growth of the plant roots. More adverse effects may result from longer exposures. Following growth of the spinach plants in Cipro-containing nutrient solution for 26 days, we observed stunted growth as evidenced by a significant decrease in the number of newly synthesized leaves and the length of the roots . Similarly, the synthesis of new chloroplasts and mitochondria in both seedlings and cell cultures of A. thaliana was severely impaired as a result of exposure to Cipro-containing growth media, with Cipro shown to target DNA gyrase , just as it does in its antibacterial action. A comparable mechanism may be responsible for the longer term adverse effects of Cipro on the morphology of the spinach plants investigated in the present study; the observable decrease in the amount of both new leaves and the green pigment of the leaves 50 µM is in agreement with this mechanism . Both in vitro and in vivo effects of Cipro on QA photo reduction underscore that the observed Cipro-induced decrease in the fraction of photo reduced QA is not likely due to direct chemical inhibition at the quinone binding site, but instead is a downstream effect of Cipro toxicity in the PS-II units characterized by an increase in inactive antenna Chl molecules.

Conversely, the growth of the aggregates of MoO3 and Cu2 NPs observed in the presence of RE correlated with higher dissolution rates and may reflect stronger attractive forces between NPs even in the presence of the organic ligands, or possible bridging between organic molecules.Nevertheless, the change in size distributions observed with spICP-MS are in line with the Zetasizer observations. Higher dissolution and nonaggregation of Cu-based NP have been reported for wheat plants in saturated paste extracts,as well as in wheat RE.In both studies, the influence of electrolytes 2) and organic matter content , contributed to the dissolution and nonaggregation of Cu based NPs. This is also in line with previous finding that support that organic matter in soil solution highly correlated with dissolved Cu.Since in our experiment the Cu-based NPs were in contact with low concentration of both electrolyte and organic acids, that can also explain the formation of aggregates and low dissolution rate. While the characterization of NPs in DI water is useful for understanding their general behavior, it can lead to significant misconceptions of how the NPs will actually behave in realistic conditions. Here we demonstrated that the surface charge, aggregation state, size distribution, and amount of metal ion release differed considerably depending on the composition of the aqueous medium into which the NPs are placed. In DI water, all the NPs aggregated, and that influenced the size distribution and the release of metal ions. However,equipment for vertical farming when placed in RE or soil leachate, the aggregation behavior differed substantially from that in DI water, with considerable reversal and disaggregation of all NPs in soil leachate and the CeO2 and Mn3O4 NPs in RE.

This was further confirmed by spICP-MS,which demonstrated the change in particle size distribution, even after just a few hours of exposure of the CeO2 and Mn3O4 NPs to soybean RE. While the rate of release of metal ions in different media did not vary as much for some NPs , it was very important for Cu2NPs, which would result in lower concentrations of ionic or complexed Cu in RE and soil leachate. Therefore, if the dissolution behavior in DI water was used to estimate the exposure concentration of the metal ion in a soil or hydroponic experiment, it could lead to a substantial error. These results highlight the importance of characterizing the NPs in the exposure medium to be used in subsequent plant exposure experiments, as well as for the design of better delivery mechanisms for the NPs or the active ingredients that they will release. Characterization should include extensive analyses of the main components of an agricultural setting, such as soil and exudates, in terms of the electrolytes, organic matter concentration, identification of main organic molecules in organic matter, pH, and soil cation exchange capacity. A better understanding of the role these factors play will enhance the effectiveness of NP delivery systems at the root-soil interface.Patents have operated as an invisible landscape-of-power in the built environment since the Italian Renaissance, when the world’s first patent was issued to the eminent architect Filippo Brunelleschi in 1421 for a “machine or ship” and method of transporting materials for his Duomo of Florence, establishing seminal legal and architectural precedents.Brunelleschi’s patent protected his invention of a new machine and method for transporting heavy loads by water, solving one of three major engineering problems associated with his novel dome construction processes.Although the patent’s legalese and the dome’s structure operated independently on discrete legal and structural principles, they formed together a highly interdependent and deterministic mechanism governing the form of the built environment. In this manner, the patent—western civilization’s oldest legal and institutional mechanism for incentivized innovation—has long mirrored, defined, and shaped the built environment, yet failed to represent it eidetically in a way that is commonly recalled.

In his book The New Architecture and the Bauhaus , the modernist architect and theorist Walter Gropius foretold the transformation of architecture and design through industrial process, and, true to form, he and his business partner Konrad Wachsmann secured a U.S. Patent for a “Prefabricated Building System” in 1942, applying Bauhaus principles to contemporary housing problems.Just a few years earlier, in 1938, Stanley Hart White, a professor of landscape architecture at the University of Illinois, unified new steel structural principles with advances in hydroponic technology to create a vertical garden model called the “Vegetation Bearing Architectonic Structure and System.” Correlating modern landscape theory to U.S. Patent claims, White’s invention was a truly modern accomplishment in the context of academic Beaux Arts.This coevolution of patent development and the built environment can also be traced through other complex infrastructural and natural systems, such as rivers, coasts, cities, buildings, and designed landscapes.A patent is, in essence, a representation of a specific invention. U.S. patents have been accompanied by models, drawings, and textual descriptions since the Patent Act of 1790, which established American patent law and pertinent representational standards.The Patent Act states that grantees shall deliver to the Secretary of State, Secretary of War, and Attorney General “a specification in writing, containing a description, accompanied with drafts or models, and explanations and models of the thing or things, by him or them invented or discovered.” If the invention was found to be new and valuable by the cabinet secretaries and the Attorney General, the patent was granted and signed, bearing ultimately the “teste” of the President himself. In that manner, the government and inventors coevolved the technological substrate of “the arts” towards unforeseen ends.

Patent law places no restriction on what may be invented or what might be deemed useful or valuable among the arts, opening up a world of possibilities limited only by the ingenuity of the citizenry and the representational standards of the patent, which today is global, territorial, nanoscale, atmospheric, and even astronomical in reach . Most patents related to landscapes, rivers, cities, regions, coastlines, and other complex environmental systems are intentionally site-less, distancing intellectual property claims from any specific locations. Patents of this sort typically use diagrammatic or typological drawings to disclose inventions and protect the widest possible scope of intellectual property claims while maintaining ambiguity as to where the patent might be applied . Those drawings cover a range of design thinking and processes— describing workflows, evaluative methods, detailed material configurations, gadgets of one kind or another, and a dizzying array of objects—ultimately representing the environment as a series of typological conditions, tectonic assemblages, data sets, and operations often contingent on specific spatial or physical conditions yet, in essence, without specific sites. The siteless quality of environmental patent documents does not diminish their potential impact on large-scale complex systems. Consider, for example, the design and construction of Eads’ Jetties at the South Pass of the Mississippi River, near Fort Jackson, a patented system realized between 1875 and 1879 and credited with saving the Port of New Orleans by sustaining commercial activities along the Mississippi . James Buchannan Eads and his business partner James Andrews prototyped and tested their jetty system at full scale for four years before receiving their fee for the maintenance of a navigable channel at the mouth of the Mississippi,vertical farming systems radically altering the fluvial geomorphology and ecology at the Head of Passes.The patent granted to Eads and Andrews was designed to suit the unique conditions at the Heads of Passes, yet the document itself makes no mention of this specific location, referencing only environmental conditions common to deltaic landscapes and a method of construction. We know of the patent’s use through Eads’ petitions to Congress and detailed histories of the jetties, but the patent itself makes no reference to a known geographical location. Eads’ patent may be siteless, but its imprint on a specific landscape is bound to the fabric of culture and remains legible today in the morphology of the Mississippi River.The anomaly of site-specificity in patents weaves a distinct narrative through geographies of the American landscape dating back to the earliest days of the Patent Office. In this nascent area of environmental innovation studies, I propose Thomas Paine as the first person to submit site-specific works to the patent office, though we may never know for sure about that precedence as most of the earliest American patents were destroyed in a fire in 1836.

Paine never built a steel bridge in America, contrary to what was suggested in correspondence with Thomas Jefferson. He did, however, propose bridges in New York, New Jersey, and Pennsylvania a short time after his book Common Sense helped catalyze the American Revolution. Models of Paine’s designs for bridges spanning the Schuylkill and Delaware Rivers were exhibited in France and England prior to being sent to the U.S. Patent Office for dissemination and safekeeping, establishing the earliest known precedent for site-specific works curated by the patent office.Although the models mentioned in Paine’s writings were probably destroyed in one of several conflagrations of the Patent Office, we can reflect on the confounding intersection of intellectual property and place, or real property, and trace a lineage to the environmental challenges of today. Paine’s submission of bridge models to the U.S. Patent Office was not an isolated instance of site specificity within the annals of patent history. In fact, many site-specific works have been premised on intellectual property of one sort or another. These proposals range in scale and scope from design patents that protect the form and appearance of specific buildings, such as architect Wallace Harrison’s patent for models of the Trylon and Perisphere and Apple Inc.’s patent for its store on Fifth Avenue in New York City , to utility patents for systems that aim to reconfigure the function and performance of cities, regions, and ecosystems. Speaking generally, the siteless quality of patents has obscured an intimate relationship between known places and specific technologies. One may easily miss the relationship between patent and place when surveying millions of documents, which at first glance appear as a treasure trove of things—gadgets, machines, and objects—but not of the environment as a whole, a place, or any known geography. Cartographic forms of representation within patent documents quickly reorient the mind to the potential intersections of intellectual property and environment through the familiar imagery of maps . Although patent cartographies usually lack the scale and graticule of conventional mapping, known locations are sometimes clearly demarcated with labels and identifiable boundaries. Not only can those places be recalled, known, or visited in the real world; they are also sites of technological innovation. As representations, the maps range in specificity from systems diagrams that situate an invention within a known location to detailed bathymetries that show the resultant geomorphology of a specific intervention. Examples include proposals for the removal of ice from New York Harbor and the East River, a passive dredge system for Galveston Bay, a hydroelectric plant for Niagara Falls that preserves scenery and produces power, and even current infrastructure/ecology hybrids designed to reinforce and cultivate mangrove ecosystems in Florida and around the world.What is the relationship between patent cartographies and known geographical locations? Site specificity within patents raises important questions about the extents and jurisdiction of patent law, in addition to challenging commonly accepted models for innovation in complex environmental systems. Take, for example, the life work of Lewis M. Haupt , a professor of civil engineering at the University of Pennsylvania and, before that, a patent examiner at the USPTO.Haupt’s theories on the “Physical Phenomena of Harbor Entrances” earned him a Magellanic Premium award from the American Philosophical Society in 1887, and, on the same day that he accepted that award, he was granted a U.S. Patent for a “Dike or Breakwater,” which linked his design theories to known environmental conditions and specific locations.Following in the footsteps of Eads and others advancing American infrastructure through public/private partnerships, the “Reaction Breakwater,” as Haupt’s invention was popularly known, was to be prototyped at Aransas Pass, Texas, by the Reaction Breakwater Company using the specification of his patent . After a revision to the contract, however, the Federal Government ultimately awarded the bid for construction to another company, which intended to build the breakwater per Haupt’s specifications. During this process, Haupt’s patent was assigned to the U.S.

While all three plant species differ in their leaf structure and topography, the first two were shown to support significant levels of Salmonella internalization. In contrast, nearly no internalization was shown in tomato leaves by confocal laser microscopy , making these leaves an ideal control system for assessing potential misinterpretation when using surface sterilization and viable count.Green-fluorescent protein -labeled S. enterica serovar Typhimurium SL1334 strain was used throughout the study. Bacterial culture was prepared and stored in Lysogeny broth supplemented with glycerol at −70°C, as described . For each experiment, fresh culture was prepared by plating the bacteria on a new LB plate supplemented with 100mg/ml streptomycin and 10mg/ml gentamicin for 24h at 37°C. Two to three single colonies were as inoculated into LB broth devoid of NaCl and grown at 37°C with shaking for 18–20h. Cultures were washed twice with sterile saline by centrifugation at 2,700 g for 10min, and the final pellet was resuspended in sterile saline. Bacterial concentration was determined by plating × 10-fold serial dilutions on LB agar supplemented with the two antibiotics.Inoculation of leaves was performed, essentially as described before , except for the incubation temperature. Briefly, a single tomato leaflet, Arabidopsis leaf, or lettuce piece were each submerged in a single 50-ml sterile polypropylene tube containing 30-ml saline. The leaves were illuminated for 20min under a light intensity of 150-μE m−2 s−1 at room temperature, and then,lettuce vertical farming the saline was removed and replaced with a bacterial suspension containing ca. 108 SalmonellaCFU/ml saline. While this high inoculum does not represent real-life conditions, such high inocula were previously used to study Salmonella internalization in vivo and in vitro .

The incubation proceeded for 2h at 40°C, a temperature that increases stomatal openings in multiple species to facilitate Salmonella internalization. The leaf samples were washed twice by dipping in fresh sterile saline for 1min each time to remove unattached bacteria. Salmonella attachment to the leaf surface and internalization was analyzed by confocal microscopy and viable count, as described below. Each experiment included three leaves of the same plant, each in a single tube and the three plants species were processed on the same day.Surface disinfection was performed using one of the three disinfectants, 1% sodium hypochlorite , 1% silver nitrate , and 70% ethanol . Briefly, whole leaves of Arabidopsis and tomato or lettuce leaf samples were submerged in 20-ml disinfectant solution with gentle agitation for 7min. Leaf samples were taken out after 1, 3, 5, and 7min and washed extensively by dipping the leaves four times in 20-ml sterile double-distilled water to remove the residual disinfectant solution. In order to avoid interference by bacteria that may enter through the cut tissues, an internal leaf disks were excised from the three leaves using a sterile corkborer. The leaf disks were aseptically cut into two identical pieces with a sterile scalpel, one was taken for bacterial extraction and viable count, and the other was taken for confocal microscopy. A high-speed benchtop homogenizer Fast Prep®-24 was used for the homogenization of the leaf samples in 2-ml micro-tubes containing glass beads and 500μl of buffer peptone water . Homogenization conditions were 4,000 rpm for 40 s at room temperature. Homogenate samples and 10× serial dilutions were spread plated into XyloseLysine-Desoxycolate agar supplemented with streptomycin and gentamicin in order to enumerate internalized Salmonella cells that presumably survived the disinfection treatment.

Inoculated leaves suspended for up to 7min in SDDW without disinfection and then washed in fresh SDDW served as non-treated control to determine the initial number of leaf-associated bacteria. Salmonella counts of control and treated samples were converted to log CFU/cm2 .Human pathogens can colonize plants and persist on and sometimes within various plant’s tissues, and upon consumption may cause foodborne diseases . Accordingly, accurate determination of the localization of human pathogen on or within leaves is vital for basic science as well as for developing new strategies for preventing and intervening to address the problem of fresh produce contamination. The determination of bacterial internalization in a plant is a function of, among others, the method used to assess bacterial localization . Ultimately, surface sterilization should completely inactivate external bacteria while leaving internalized bacteria intact. Still, only a few studies have systematically validated the efficacy of surface sterilization to kill surface-attached bacteria. In one such study, 13 disinfection conditions/methods were compared for their effectiveness in killing GFP-tagged E. coli O157:H7 on lettuce leaf surfaces using leaf imprints on agar media. Dipping in 80% ethanol for 10 s followed by immersion in 0.1% HgCl2 for 10min was reported to be the most effective disinfection method for inactivating both E. coli and Salmonella strains . However, no confocal microscopy study corroborated the results. Many studies have adopted previously reported protocols to inactivate surface-attached bacteria, even when utilizing different plants and/or bacterial strains . Bacteria may vary in their intrinsic tolerance to disinfectants and may preferentially reside at unique leaf-specific microsites , which may facilitate the protection of the colonized bacteria against disinfection . Consequently, a disinfection protocol developed for inactivating a specific Salmonella strain on the leaves of a particular plant cultivar may not fit all. Evidently, when a partial inactivation is achieved, some surface residing bacteria may be mis-classified as internal bacteria, while truly internalized bacteria killed due to permeation of the disinfectant into the intact leaf tissue may be mistakenly regarded as surface-attached bacteria. The present study provides data from a systematic comparison of leaf internalization through stomata by a GFP-tagged Salmonella Typhimurium strain in the leaves of the three plant species using surface sterilization and plate-count technique.

The study did not compare leaf internalization among plants but rather the effect of the various disinfection protocols on leaf internalization in each plant species. We used three disinfectants , commonly applied, alone or in combination with others, for sterilizing plant surfaces . To simplify the comparison between the protocols, we used a single concentration of the disinfectants, each time, and compared the effect of the sterilization time on quantifying viable bacteria, apparently representing internalized Salmonella cells. In parallel to the viable counts measurements, we utilized confocal microscopy to accurately assess bacterial localization on or within the leaf tissue. We initially examined leaf internalization in iceberg lettuce leaves previously shown by confocal microscopy to support Salmonella internalization . Indeed,vertical grow shelf confocal microscopy confirmed a high incidence of internalization in non-disinfected leaves; however, surface disinfection with all three agents resulted in reducing fluorescence, suggesting that the disinfectants seemingly penetrated the leaf tissues to some degree and injured the cells . A substantial decrease in the number of apparent internalized bacteria was observed using the plate-count method following 1 to 7 min treatment . The determination of leaf internalization by surface disinfection and viable count showed wide variations in the number of apparently internalized bacteria in leaves of each plant species, depending upon the type of the disinfectant and the treatment duration. These differences are likely attributed to the increased killing of leaf-associated bacteria with time or to the transition of a portion of the Salmonella population to the viable but non-culturable state . The observation of fluorescent cells on the leaf surface does not provide a clear indication regarding the presence of disinfection-tolerant bacteria, since the Salmonella strain carried a stable GFP , which may continue to emit fluorescence in VBNC bacteria, as well as in dead cells with intact GFP. Further studies using methods that can discriminate between live and dead bacteria are needed to determine the physiological status of the treated fluorescent bacteria on the leaves’ surface. Still, the possible entry of Salmonella into a VBNC state in the plant environment may lead to underestimation of both attachment and internalization when using the viable count assay alone. Based on the confocal microscopy studies, Salmonella displays a lower incidence of leaf internalization in Arabidopsis than in lettuce . Likewise, the viable count method demonstrated lower numbers of viable bacteria during all treatment times . All three agents displayed comparable surface disinfection effectiveness; however, they varied significantly in the apparent internalization . A 7-min treatment with 1% NaHClO or 1% AgNO3 resulted in the complete loss of fluorescent cells inside the leaf, suggesting that they efficiently penetrated the leaves and injured the internalized bacteria. In a previous report, we were not able to show internalization of the same Salmonella strain in tomato leaves . Consequently, the assessment of tomato leaf internalization, side by side, by the two methodologies provided a unique opportunity to assess the suitability of the tested disinfection conditions inactivate bacteria in the leaf surface. Evaluation of Salmonella internalization by confocal microscopy, with no surface sterilization, confirmed our inability to demonstrate the internalization of Salmonella in these tomato leaves with the techniques used. Usage of 1% NaHClO for 1 to 7min resulted in different numbers of apparent internalized bacteria, ranging from 4 logs CFU/cm2 to 0, respectively. Parallel confocal microscopy analysis of the treated leaf samples confirmed the lack of detection of leaf internalization, suggesting that only 7-min treatment resulted in sufficient killing of external bacteria in this model system. The use of 70% ethanol as a sole disinfectant for up to 7min failed to inactivate all external bacteria, as determined by viable counts, thus mistakenly suggesting the internalization of about 3 log CFU/cm2 .

Treatment with 1% AgNO3 resulted in substantial inactivation of surface attached bacteria in 1 and 3min treatment, while treatment duration of 5 and 7min was sufficient to kill all external bacteria, hence providing results comparable to those obtained by confocal microscopy. These findings indicate that non-validated surface sterilization conditions may lead to misinterpretation of the actual number of internalized bacterial cells. Notably, the apparent lack of leaf internalization of the tested S. typhimurium strain in the tomato cultivar used in this study , as well as in S. lycopersicon cv. MP1, tested previously , calls for further research. It is particularly interesting to examine whether the two cultivars are naturally resistant to leaf internalization of other Salmonella serovars and strains under more natural tomato growing conditions. Elucidation of the mechanisms involved in the inhibition of leaf internalization might prove important for understanding human pathogen plant interactions and developing new mitigation strategies for Salmonella internalization. Surface disinfection by treatment with 1% AgNO3 was less effective in lettuce compared to tomato leaves. These differences are likely correlate to specific leaf features, such as surface morphology and/or physico-chemical properties known to impact leaf colonization . Previous studies have already noted that the attachment of bacteria to specific micro-environments on the leaf, such as cavities and crevices on the leaf surface, may favor the persistence of surface-attached bacteria following disinfection . Altogether, this is the first time a systematic study reported a comparison of three surface sterilization protocols in leaves of three plants, side by side, with a confocal microscopy study. While the selection of an optimal disinfection protocol for each of the three plants was beyond the scope of this study, we have demonstrated the dependency of the apparent bacterial internalization on the disinfection conditions and shown the impact of the quantification method on the extent of leaf internalization. It should be noted that entry of bacterial pathogens into the leaf tissue might occur through stomata, hydathodes, and injured tissues or by transport through the roots and stem . In the present study, we utilized specific in vitro inoculation and experimental conditions to compare the effect of three surface disinfection protocols on Salmonella internalization through stomata. The study was not designed to investigate other factors that might affect bacterial internalization nor the different mode of leaf internalization. Therefore, we suggest interpretation of our results with caution, especially when comparing to other studies that used different inoculation models and surface disinfection protocols. Whole leaves or leaflets were used for inoculation in the case of Arabidopsis and tomato, respectively; however, in the case of lettuce, square leaf pieces were used, which potentially may enable direct access of bacteria into the apoplast through the injured tissue. However, previous confocal microscopy observations showed a limited penetration of Salmonella through the cut tissues , which did not affect the internal leaf tissue used for bacterial enumeration.