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Mutational approaches using the model plant species Arabidopsis have become an important complement to these studies

Outside of organic acid release as a form of Al tolerance or resistance, a logical but unproven mechanism of Al tolerance is root-mediated alkalinization of the rhizosphere, since Al toxicity is dependent on the pH of the growth environment. Despite the appeal of this process as an Al tolerance mechanism, there is only one report that clearly demonstrates this role in Al tolerance. An Al tolerant mutant in Arabidopsis was shown to release similar amounts of organic acids to wild-type seedlings, indicating that this mutant has a different mechanism of Al tolerance. It was then found that its mechanism of Al tolerance was correlated with an Alactivated root apical H+ influx. This H+ influx resulted in an increase in rhizosphere pH at the surface of the root tip, which was significant enough to decrease Al activity around the root tip . In addition to the various resistance mechanisms described, there has been limited evidence that modification of extra- and intracellular anionic sites can have a positive impact on Al resistance. Polyamines are small aliphatic polycationic molecules that could compete with Al ions for binding sites at the cell wall and membrane to prevent Al from entering the cell . Plant polyamines are detected in actively growing tissues and under stress conditions. They also have been connected to the control of cell division, embryogenesis, root formation, fruit development and ripening, and responses to biotic and abiotic stresses .There have been two reports that describe the relationship between Al and polyamines; one report has demonstrated the positive effects of polyamines on Al tolerance, while another has discussed the effects of Al on cellular polyamines. In saffron, 1mM polyamines were able to reduce Al toxicity. Polyamines were also able to decrease H2O2 content in the presence of Al, as well as decrease Al accumulation at the roots . In cell cultures of a woody plant Catharanthus roseus, it was observed that polyamine levels increase upon Al exposure. Spermine levels increased by two- to three-fold after 24 and 48 hours of exposure to Al, and putrescine levels slightly increased after four hours of exposure,black flower buckets but was surprisingly followed by a sharp decrease . This suggests that even non-chelating molecules can have a significant impact on Al tolerance in plants. It has been argued based on these studies that Al exclusion must be a rapid response to minimize Al uptake and subsequent Al-dependent stress.

Interestingly, much of the findings on Al exclusion mechanisms have arisen from studies that move roots from a no Al environment to one that has highly toxic levels with the research focused on the immediate responses to Al. It is hard to imagine a real world situation in which roots go from an environment with little to no Al to one that has highly inhibitory concentrations. Therefore, it is arguable whether the approach of studying immediate responses to Al is necessarily relevant to Al toxicity in soils since stoppage of root growth in such an environment is likely due to chronic long-term exposure to Al. Consequently, it is of critical importance to determine the toxic effects of Al as it accumulates within plant tissue.It has been very difficult to determine other mechanisms of Al tolerance and resistance in agriculturally relevant plants due to issues such as genome size, availability of knockout lines, generation time, and difficulty in creating transgenic lines. The use of Arabidopsis thaliana has evaded this issue by utilizing the model plant that has a smaller diploid genome, an extensive library of knockout lines, and short generation time . Additionally Arabidopsis has similar sensitivity to Al in comparison to other crop plants and shows classic signs of Al toxicity, making Arabidopsis a suitable plant model for Al toxicity for crop species . The mechanisms of Al resistance have been intensively studied on crop species using natural genetic variation within and across species, such as wheat and maize. While clearly an insightful approach that has given extensive knowledge on Al exclusion mechanisms, this work is limited based on currently existing variability with regard to growth in the presence of Al. Beyond the obvious advantages of using a model species, Arabidopsis has a similar Al toxic threshold to many agriculturally relevant crop species making it a valuable system for investigating how plants sense and respond to Al through the identification of mutants with altered growth capabilities in the presence of Al . This has been particularly true with regard to identification ofAl sensitiveArabidopsis mutants, which have reduced root growth in the presence of Al likely due to deffects in mechanisms required for Al exclusion, Al detoxification, or response to Al-dependent damage.

By screening for Al sensitive Arabidopsis mutants, eight complementation groups were identified indicating that Al toxicity is complex, which is to be expected considering the likely number of factors involved in mechanisms of Al resistance and tolerance . Most importantly, as will be discussed later, identification of these als mutants has allowed for use of a suppressor mutagenesis approach that has resulted in identification of factors that are important for Al-dependent stoppage of root growth. In order to find factors that are required for Al stress response, a genetic approach was taken to identify Arabidopsis mutants that exhibit increased Al sensitivity. Mutant lines were generated through ethyl methanesulfonate mutagenesis of Arabidopsis thaliana ecotype Col-0 wt. M2 seedlings were subsequently screened for their response to Al by identifying seedlings with normal growth in the absence of Al, but restricted growth on low levels of Al. The seedlings were grown on a two-layer gel system, with the upper layer consisting of nutrient medium with no added Al and the lower layer consisting of the same nutrient medium equilibrated with a subtoxic level of AlCl3. Any seedlings that could grow normally through the upper layer but could not penetrate the lower layer were isolated. Each of these Al sensitivemutants likely represents deffects in genes that are required for mechanisms for Al resistance or tolerance. Two of the identified mutants, als1-1 and als3-1 have been studied in depth. Both of these mutations represent a recessive loss of function mutations resulting in greater than wild type root growth inhibition in the presence of sub-threshold levels of AlCl3. The first mutant characterized was als3-1 due to its extreme sensitivity to Al at low levels. When grown on Al asl3-1 shows complete arrest of growth of the primary root and shoot meristems. The roots of als3-1 are stunted and have a swollen club shaped root apex, with root hairs at or near the root tip when grown on Al media. Also, als3-1 roots do not initiate any lateral roots, but can produce secondary roots from the base of the hypocotyl. Growth of lateral roots can reinitiate if als3-1 plants are removed from Al media, but the primary root is irreversibly inhibited by the Al treatment. This response differs from the roots of wild type plants, which are able to fully recover from the Al treatment. Following Al treatment the shoot phenotype of als3-1 shows reduced cotyledon expansion. als3-1 shoots show a delayed growth recovery in comparison to the root, which does not recover at all. Leaf expansion is blocked for several days after the Al recovery.

In addition, the first true leaves of als3-1 develop abnormally following Al treatment. They are severely stunted with very few trichomes, poor leaf expansion,french flower bucket and irregular shaped epidermal cells with a rough leaf surface. The leaves that developed after Al treatment also do not expand but became a disorganized cluster of leaf pegs that eventually expand without petiole development. Eight days after removal from Al, a second shoot apex forms that develops relatively normally except for a greater number of rosette leaves and some fused inflorescences. This shoot phenotype is found to only in als3-1 plants that were challenged with Al and has not been described for als mutants in general. Since this mutation is completely Al-dependent, it was hypothesized that als3-1 represents a factor required for Al-tolerance or resistance. This factor was found to be specific for Al tolerance since als3-1 did not show increased sensitivity to other metals such as copper, nickel, cadmium or lanthanum and does not display any other growth deffects in low pH without Al. Staining of als3-1 roots with hematoxylin and morin, two stains that bind to Al, resulted in similar intensity of staining to wild type, suggesting that als3-1 mutation does not alter the amount of Al uptake . This was later confirmed using ICP-OES . Although there was no difference in the amount of Al uptake, when wild type and als3-1 roots were stained with hematoxylin, wild type plants showed a diffuse pattern of surface bound Al extending from the root apex to the mature region of the root, while als3-1 roots displayed intense staining just proximal to the root tip . Map-based cloning of als3-1 showed that it represents a defect in a gene that encodes an ABC-like transporter homologous to bacterial ybbM, which is a metal resistance protein from Escherichia coli. Based on this similarity and the localization pattern of ALS3, which shows it predominantly at the plasma membrane of root cortical cells and cells of the vasculature, it was proposed that it redistributes Al away from the most sensitive plant tissues in order to maintain cell division . Loss of ALS3 as in the als3-1 mutant would result in inappropriate accumulation of Al in vulnerable areas such as the root tip and would consequently cause growth arrest at levels of Al that have no measurable effect on wild type. GUS staining of plants harboring the ALS3 promoter fused with GUS indicates that ALS3 expression is localized primarily to the sieve tube elements of the phloem in all plant organs, and trichoblast cell files and immature root hairs. GUS activity was also found in the epithem tissue of the hydathodes but not in the actual water pore of the hydathode. Since Northern analysis determined that ALS3 is Al inducible, GUS analysis of Al treated lines resulted in a shift of expression from the root epidermis to the root cortex.

From these results, ALS3 was hypothesized to mediate Al transport within the plant, transporting Al from sensitive tissues from the plant such as the root apical meristem in order to sequester it in less sensitive tissues or also to the hydathodes for excretion by guttation. It is hypothesized that by disrupting this partial ABC transporter, there is inappropriate accumulation of Al in the root, leading to severe symptoms of Al toxicity . Consistent with the importance of ALS3 to Al tolerance, an ALS3 homolog was identified in rice, called STAR2. Although both ALS3 and STAR2 are required for plant Al tolerance, the expression patterns and cellular localization differ. STAR2 is only expressed in roots upon Al treatment and is located in all cell types except for the epidermal cells in the mature root zone . In contrast, ALS3 is expressed at a basal level in the vasculature throughout the plant and its expression is dramatically increased in the Arabidopsis root tip following exposure to Al . Similar to ALS3, STAR2 contains several transmembrane domains that likely form a pore or channel that is involved in substrate movement. Both ALS3 and STAR2 lack an ATPase domain, making them unusual with regard to ABC transporters that often have the transmembrane domains and ATPase domain all as part of one protein. While a separate ATPase domain containing protein partner has not been found for ALS3 , rice STAR2 was shown to interact with another protein, STAR1, which contains an ATPase domain . The STAR1/STAR2 complex functions together as a bacterial-type ABC transporter that is speculated to transport UDP-glucose, although it is currently unclear as to how the transport of UDP-glucose by STAR1/2 is responsible for rice Al tolerance . Other Arabidopsis mutants that have been identified with altered responses to Al include als1 and als7. Both als1 and als7 were identified in the original screen for Arabidopsis mutants with Al hypersensitivity. als1-1 was also identified by mapped based cloning and subsequently characterized. Similarly to als3- 1, als1-1 has an extreme increase in Al sensitivity.

Data-driven statistical approaches can provide complementary insight into these questions

Additionally, studies have found statistically significant negative associations between living in proximity to agriculture and adverse outcomes , but not with pesticide metabolite levels directly. Similarly counter intuitive results have illustrated that specific chemicals such as methyl bromide or OP pesticides have negative associations with some birth outcomes, but also unexpected positive associations for others.Large samples provide a powerful opportunity to control for various different demographic and environmental characteristics that may be obscuring the relationship between agricultural pesticide exposure and adverse birth outcomes in surrounding communities. Here we revisit the relationship between pesticide exposure and birth outcomes using a large sample of births , which includes individual-level data on maternal and birth characteristics, and pesticide exposure at a small geographical scale. We concentrate on the agriculturally dominated San JoaquinValley, California. California is the most populous state in the United States with roughly 12% of annual births. It is also the greatest user of pesticides with over 85 million kg applied annually, an amount equivalent to roughly 30% of the cumulative active ingredients applied to US agriculture. The San Joaquin Valley is the state’s most productive agricultural region, growing an abundance of high value, high chemical input, and labor-intensive fruit, vegetable, and nut crops. We evaluate pesticide exposure by summing active ingredients of agricultural pesticides applied over gestation, by trimester,round flower buckets and by grouped the United States Environmental Protection Agency’s acute toxicity categories, along with several additional robustness checks. For outcomes, we focus on birth weight, gestational age, and birth abnormalities.

Our sample of over 500 000 individual birth observations and fine-scale data on the timing and amount of pesticide applied allow us to detect statistically significant negative effects of pesticide exposure for all birth outcomes, but generally only for pregnancies exposed to the very highest levels of pesticides .To explore if either inaccuracies in geocoding or spillover of pesticides from surrounding areas contaminated our results we excluded births for mothers living within 200 m of a PLS Section boundary. We found a similar overall pattern of statistical significance as in the larger sample. Although the magnitude of the coefficients increased, the effects on birth weight and gestational length remained <1%, and the effects on the probability of low birth weight, preterm birth, and abnormalities were at most 13% higher for the high exposure group relative to the low exposure group . We also estimated the trimester model including pesticide use in the “fourth trimester” . As anticipated, exposure during the three months following birth did not have a significant effect on any outcomes observed at birth . This “placebo” analysis indicates that our empirical results are unlikely to be caused by omitted trends or factors that are correlated with both pesticide applications and infant health. To further ensure the robustness of our results and inference, we checked different exposure cutoffs as well as a continuous measure of exposure . The magnitude of effects was small and generally non-significant with the 75th percentile cutoff. Being in the top one percent of pesticide exposure led to an 11% increased probability of preterm birth, 20% increased probability of low birth weight, and ~30 g decrease in birth weight relative to lower exposure . We also evaluated models with different location fixed effects, different assumptions about clustering the standard errors to address spatial and temporal error correlation, different sample exclusion restrictions on gestational age and different calculations of trimester, as well as models with other environmental contaminants that can affect in utero infant health .

Although the exact magnitude and patterns of significance did change with these different models, all models consistently reported similar effect sizes. Overall, we report over 100 coefficients in the main text, of which 19 are significant. It is noteworthy that in all these tests, only a single significant coefficient in one model has the opposite sign from that expected. The fact that only one of roughly 20 statistically significant coefficients has the wrong sign is consistent with the notion that our empirical estimates are not plagued by omitted variable bias. Further, since we do not adjust p-values for multiple comparisons, the number of significant effects we report is an upper bound on the “true” number of significant effects. Applying a Bonferroni correction for multiple comparisons that accounts for five outcomes and up to five covariates of interest , the α-level for statistical significance would change from 0.05 to as small as 0.002 . The only three coefficients that remained statistically significant with this Bonferroni correction were those associated with a single covariate of interest, total pesticide exposure over the gestation . Of these, two were associated with preterm birth and one with log gestation .Concerns about the effects of harmful environmental exposure on birth outcomes have existed for decades. Great advances have been made in understanding the effects of smoking and air pollution, among others, yet research on the effects of pesticides has remained inconclusive. While environmental contaminants generally share the ethical and legal problems of evaluating the health consequences of exposure in a controlled setting and the difficulties associated with rare outcomes, pesticides present an additional challenge. Unlike smoking, which is observable, or even air pollution, for which there exists a robust network of monitors, publicly available pesticide use data are lacking for most of the world. As a result, studies have typically been either highly correlative at coarse resolutions or have included a small number of subjects. Both constraints make it difficult to assess whether residential agricultural pesticide exposure has no effector whether logistical and analytical barriers have obfuscated the identification of important effects. Our study bridges the gap between detail and scale by leveraging vast pesticide and birth data for the San Joaquin Valley, CA. Our study has far stronger statistical power to identify effects than previous studies owing to over a hundred thousand birth observations, individual maternal and birth characteristics, and the inclusion of fine-scale regional and temporal fixed effects .

As a result of our statistical design, we have the analytical power to identify extremely small, but statistically significant negative effects of pesticide exposure on several birth outcomes, if they occur. Furthermore, our study design and extensive pesticide data enable us to evaluate many details of the nature of pesticide exposure. For example, we can evaluate whether pesticide exposure in different trimesters or pesticides of different toxicity levels affected birth outcomes in different ways. Fetal susceptibility to environmental exposure varies through development. Similarly, different chemical toxicity can have different expected health outcomes. Here we focused on aggregate chemicals grouped into high and low toxicity pesticides by their EPA Signal Word, which reflects acute toxicity. Acute toxicity does not necessarily indicate impacts from long-term exposure. As such, chemicals suspected to cause negative birth outcomes, such as organophosphates or atrazine would be classified as low toxicity. Nevertheless, we consistently find effects of less than a 10% increase in adverse outcomes for individuals in the top 5% of exposure regardless of timing or toxicity of exposure, even though which effects are statistically significant depends on the model. Pesticide exposure has a highly skewed distribution in the San Joaquin Valley, where over half of births received no pesticides,plastic flower buckets wholesale the top quarter received about 250 kg and the top 5% received over 16 times that amount. Further, exposure to the top 25% levels had virtually no detectable effect whereas exposure to the top 1% had effects that were up to double the magnitude of effects observed for the top 5% of exposure. In other words, for most births, there is no statistically identifiable impact of pesticide exposure on birth outcome. Yet, for individuals in the top 5 percent of exposure, pesticide exposure led to 5–9% increases in adverse outcomes. The magnitude of effects were further enlarged for the top 1%, where these extreme exposures led to an 11% increased probability of preterm birth, 20% increased probability of low birth weight, and ~30 g decrease in birth weight. For perspective, other environmental conditions such as air pollution and extreme heat generally report a 5–10% increase in adverse birth outcomes, but from less extreme exposure. Similar magnitudes of effects are also observed for other, non-exposure conditions of pregnancy. For example, stress during pregnancy may increase the probability of low birth weight by ~6%, while enrollment in supplemental nutrition programs is estimated to reduce the probability of low birth weight by a similar amount. The significance of the negative effects of extreme pesticide exposure on birth outcomes is heightened by the fact that birth outcomes are persistent and costly. Reducing the incidence of adverse birth outcomes has obvious benefits for individuals, but also for society.

Healthier babies require less intensive care as infants, have better long term health and are higher achieving in terms of earnings and employment. Thus, even small reductions in adverse outcomes can economically offset societal investment in programs such as supplemental nutrition programs offered to millions of low-income women. Due to the concentration of negative outcomes at the very highest pesticide exposures, policies, and interventions that target the extreme right tail of the pesticide exposure distribution could largely eliminate the adverse birth outcomes associated with agricultural pesticide exposure documented in this study. As such, valuable and pressing future directions for research should focus on identifying the extreme pesticide users near human development and on the underlying causes for their extreme quantities of use. These insights are critical to designing appropriate and adaptive interventions for the population living nearby. For instance, crops vary dramatically in their average pesticide use. Commodities such as grapes receive nearly 50 kg ha−1 per year of insecticides alone in the San Joaquin Valley region, while other high value crops such as pistachios receive barely on third of that amount. Within these broad differences, there are also relevant differences among crops with regard to the chemical composition and seasonal timing of pesticide application. Finally, not all agricultural fields are in proximity to human settlement. Rather, as we illustrate, areas with consistent births and pesticides are a small fraction of the San Joaquin Valley. Thus, if extreme pesticide areas and vulnerable populations could be identified, strategies or interventions could be developed to mitigate the likelihood of extreme exposures. One further difficulty is isolating the roles of individual chemicals and their mixtures in driving the negative outcomes. Doing so is extremely challenging, because many chemicals are used in conjunction or in close spatial or temporal windows. Using a large scale data-driven approach could provide a starting point from which individual or community based studies could be built. For example, statewide birth certificate data could enable the identification of potential hot spots of negative birth outcomes while the Pesticide Use Reports provide a large sample of different pesticide mixtures. This could yield valuable information for targeting more detailed studies of individual exposures and difficult to observe outcomes towards regions and months of the highest concern. There are some important limitations to our study. As with other environmental contaminants, controlled experiments evaluating the effects of pesticide exposure on birth outcomes are impossible due to clear ethical and legal constraints. This presents challenges both for interpretation and estimation. With regard to interpretation, we cannot observe all individual adaptive responses to pesticide use, such as staying indoors to avoid exposure to pesticide. Further, we can only observe the effects on live births. As a result, our estimates reflect both the direct effect of exposure on live births and the mitigating effects of avoidance behaviors. With regard to identification and estimation, establishing causality without random assignment into pesticide exposure relies on quasi-experimental approaches, such as the panel data models used here with observational data. While there is no way to formally test if our methods have eliminated all sources of bias that preclude causal interpretation of the regression coefficients, our results are robust to multiple modeling approaches, including controlling for other environmental contaminants such as ambient concentration of pollutants and extreme temperatures. Similarly, we find no significant placebo effects of exposure in the 3 months following birth.Birth records do not fully capture adverse outcomes such as abnormalities that are difficult to observe at birth nor are they comprehensive with regards to socio-demographics. Measurement error on the outcome variable would not bias our estimates of the effects of pesticide exposure unless it was somehow correlated with pesticide use, yet it could reduce our precision and thus the likelihood of finding statistical significance.