Month: October 2022

In Arabidopsis, a tonoplast-localized proton-pumping pyrophosphatase AVP1 was shown to be the key enzyme for cytosolic PPi metabolism in different cell types of various plants. This enzyme activity has been correlated with the important function that AVP1 plays in many physiological processes. Arabidopsis fugu5 mutants lacking functional AVP1 show elevated levels of cytosolic PPi and display heterotrophic growth defects resulting from the inhibition of gluconeogenesis. This important role in controlling PPi level in plant cells is reinforced by a recent study showing that higher-order mutants defective in both tonoplast and cytosolic pyrophosphatases display much severe phenotypes including plant dwarfism, ectopic starch accumulation, decreased cellulose and callose levels, and structural cell wall defects. Moreover, the tonoplast-localized H+ -PPase AVP1 appears to be a predominant contributor to the regulation of cellular PPi levels because the quadruple knockout mutant lacking cytosolic PPase isoforms ppa1 ppa2 ppa4 ppa5 showed no obvious phenotypes. Interestingly, in companion cells of the phloem, AVP1 was also shown to be localized to the plasma membrane and function as a PPi synthase that contribute to phloem loading, photosynthate partitioning, and energy metabolism. On the other hand, AVP1 is also believed to contribute to the establishment of electrochemical potential across the vacuole membrane, which is important for subsequent vacuolar secondary transport and ion sequestration. Constitutive over expression of AVP1 improves the growth and yield of diverse transgenic plants under various abiotic stress conditions—including drought, salinity, as well as phosphorus and nitrogen deficiency—although the mechanism remains to be fully understood. Taken together, AVP1 serves as a multi-functional protein involved a variety of physiological processes in plants, some of which await to be fully understood. Magnesium is an essential macro-nutrient for plant growth and development, functioning in numerous biological processes and cellular functions,nft vertical farming including chlorophyll biosynthesis and carbon fixation. Either deficiency or excess of Mg in the soil could be detrimental to plant growth and therefore plants have evolved multiple adaptive mechanisms to maintain cellular Mg concentration within an optimal range.

In higher plants, the most well-documented Mg2+ transporters belong to homologues of bacterial CorA super family and are also called “MRS2” based on their similarity to yeast Mitocondrial RNA splicing 2 protein. Several members of the MGT family mediate Mg2+ transport in bacteria or yeast as indicated by functional complementation as well as 63Ni tracer assay. In plants, they have been shown to play vital roles in Mg2+ uptake, translocation, and homeostasis associated with their different sub-cellular localizations and diverse tissue-specific expression patterns. For instance, MGT2 and MGT3 are tonoplast localized and possibly involved in Mg2+ partitioning into mesophyll vacuoles; MGT4, MGT5, and MGT9 are strongly expressed in mature anthers and play a crucial role in pollen development and male fertility. MGT6 and MGT7 are shown to be most directly involved in Mg homeostasis because knocking-down or knocking-out either of the genes leads to hypersensitivity to low Mg conditions. MGT6 encodes a plasma membrane-localized high-affinity Mg2+ transporter and mediates Mg2+ uptake in root hairs, particularly under Mg-limited conditions. MGT7 is also preferentially expressed in roots and loss-of-function of MGT7 caused poor seed germination and severe growth retardation under low-Mg conditions. Double mutant of mgt6 and mgt7 displayed a stronger phenotype than single mutants, suggesting that MGT6 and MGT7 may be synergistic in controlling Mg homeostasis in low-Mg environment conditions. In contrast to considerable research on Mg transport and homeostasis under Mg deficient conditions, the regulatory mechanisms required for adaptation to excessive external Mg remain poorly understood. Recent studies suggested that MGT6 and MGT7 are essential for plants to adapt to both normal and high Mg conditions. The mgt6 mutant displayed dramatic growth defects with a decrease in cellular Mg content in the shoot, when grown under high Mg2+. Grafting experiments further suggested a shoot-based mechanism for Mg2+ detoxification although the exact role of MGT6 in this process is still not clear. More importantly, a core regulatory pathway consisting of two calcineurin B-like Ca sensors partnering with four CBL-interacting protein kinases has been established that allows plant cells to sequester Mg2+ into plant vacuoles, thereby protecting plant cells from high Mg2+ toxicity. In this study, we identified the tonoplast pyrophosphatase, AVP1, as an important component in high Mg2+ tolerance in Arabidopsis.

Furthermore, by analyzing the avp1-4 mgt6 double mutant and avp1-4 cbl2 cbl3 triple mutant, we showed that the role of AVP1 in high-Mg tolerance was independent of previously reported MGT6 or CBL/CIPK-mediated pathway. Instead, our results suggested a novel link between high Mg2+ stress and PPi homeostasis in plants. The originally reported T-DNA insertional mutant avp1-1 contains an additional T-DNA insertion causing phenotypes unrelated to AVP1 mutation. We thus characterized another T-DNA insertion line avp1-4for this study. The avp1-4 mutant carried a T-DNA insertion in the third exon of AVP1 as further confirmed by PCR analysis and DNA sequencing . The avp1-4 homozygous mutants lacked detectable AVP1 transcripts , and its tonoplast PPi hydrolysis activity was considerably diminished, to only 10% of wild type . Compared with wild-type plants , avp1-4 mutants exhibited no obvious phenotypic changes during the life cycle including vegetative and reproductive periods , which is quite different from avp1-1, because pleiotropic phenotypes observed in avp1-1 are caused by mutation in the GNOMgene. We examined the phenotype of avp1-4 plants under multiple ionic stress conditions and found that avp1-4 mutant and wild-type seedlings grew similarly on the MS medium and did not show hypersensitive response to most of the ionic stresses such as 60 mM Na+ , 60 mM K+ , 40 mM Ca2+, 100 µM Zn2+, 40 µM Cu2+, or 100 µM Fe3+ . However, the growth of avp1-4 seedlings were severely impaired when 20 mM MgCl2 was supplemented . To validate the hypersensitivity of avp1-4 to MgCl2, we grew the seedlings of the mutant together with the wild-type plants on the 1/6 MS medium containing various levels of Mg2+, the avp1-4 mutant plants were clearly stunted as compared with Col-0 , although the primary root length of avp1 was comparable to that of Col-0 . In addition, we also studied one more mutant allele of AVP1 gene in the Wassilewskija background, designated as avp1-3, and another three mutant alleles of AVP1, fugu5-1, fugu5-2, and fugu5-3 in the Col-0 background. Measurements of seedling fresh weight confirmed a severe growth inhibition by 8 mM MgCl2 in both avp1-4 and avp1-3 mutants, as compared with their respective wild-type counterparts . Consistently, we also found that high-Mg sensitivity phenotypes in the three fugu5 mutants were comparable to those in avp1-4 . Together, these results suggested that AVP1 is required for Mg2+ tolerance in Arabidopsis. To verify that the observed phenotypes in the avp1 mutants are caused by a defect in AVP1, we conducted a complementation test in avp1-4 background. A coding sequence fragment of AVP1 was introduced into the avp1-4 mutant, and several homozygous transgenic lines were obtained . Phenotypic analysis of two representative lines showed that oblong-shaped cotyledons of avp1-4 when germinated on MS media containing low sucrose or in soil were fully restored to normal shape .

In addition, seedling growth defects of avp1-4 under high-Mg conditions were also completely rescued . Root length and shoot fresh weight of the transgenic lines under high Mg conditions were similar to those of the wild type . These data further confirmed that loss-of-function in AVP1 was indeed the causal mutation for the high-Mg hypersensitive phenotype of avp1-4.Reducing the PPi concentration in the cytoplasm and increasing the acidification of vacuoles represent the two main biochemical functions of AVP1. In order to dissect if both activities are required in this specific high Mg2+-associated process, we resorted to the transgenic line expressing yeast IPP1 gene under the control of the AVP1 promoter in the fugu5-1 mutant background. IPP1 is a cytosolic soluble protein which is not capable of translocating H+ , thus decoupling the hydrolysis and proton pump activities. Interestingly, our results showed that the severely retarded growth of fugu5-1 mutant plants under high-Mg conditions was completely recovered by expression of the IPP1 gene .To extend the phenotypic analysis of the avp1 mutants in mature plants, we examined the phenotype of avp1 mutants using hydroponic culture system. Consistent with the patterns of plant growth on agar plates,indoor vertical farming the mutant plants exhibited a pronounced growth defect than wild-type plants in the hydroponic solutions supplemented with 15 mM external Mg2+, as revealed bymuch lower fresh weight and lower chlorophyll content . The IPP1 transgenic line also behaved like wild-type plants but not avp1 mutant under this condition, suggesting that PPi hydrolysis is the key function that AVP1 plays in high-Mg adaptation. To address the contribution of PPi hydrolysis activity to high-Mg tolerance, we directly measured V-PPase activity and PPi content under normal and high-Mg conditions. Under normal conditions, PPi hydrolysis activity of two avp1 mutant alleles was reduced by ∼85%, whereas activity from two complementary lines was comparable to the wild-type control . Consistently, the amount of PPi from both mutants was increased by ∼50% . After grown for three days on 15 mM Mg2+, all the plants displayed reduced PPi hydrolysis activity and higher PPi content. However, thePPi elevation of mutant plants during high Mg2+ stress was significantly higher than that of wild type . Altogether, these results strongly indicate that the dampened hydrolysis of cytosolic PPi is the major reason for the increased Mg sensitivity in the avp1 mutants.To assess whether increased Mg2+ sensitivity in the avp1 mutant is associated with Mg2+ homeostasis, we measured the Mg content in wild-type and mutant plants using ICP-MS. When 8 mM Mg2+ was added to the growth medium, Mg content in either shoot or root in all the plants was strikingly elevated, but no significant difference between wild-type and mutant plants in Mg content was observed. . Considering Ca and Mg often affect each other in their uptake and transport, we also measured the Ca content in the same plants.

Consistent with Mg-Ca antagonism, the Ca content in both wild-type and avp1 mutant plants was evidently lower when plants were grown under high external Mg2+ conditions, but Ca content in the shoots and roots in avp1 mutants was similar to that in wild-type plants . These data suggest that both Mg and Ca homeostasis are not altered in the avp1 mutants, which are consistent with the earlier conclusion that PPi hydrolysis rather than vacuolar acidification is responsible for AVP1 function under high-Mg stress.In Arabidopsis, the magnesium transporter MGT6 is important for controlling plant Mg2+ homeostasis and adaptation to both low- and high-Mg conditions. To investigate the functional interaction between AVP1 and MGT6, we created a double mutant that lacks both AVP1 and MGT6transcripts . We next tested the sensitivity of avp1-4 mgt6 double mutant to high external Mg conditions. When grown on the 1/6 MS medium containing 0.25 mM Mg2+, the mgt6 and avp1-4 mgt6 plants showed obvious growth retardation compared with Col-0 and avp1-4 seedlings, resulting from mgt6 mutation that renders plants hypersensitive to low Mg2+ . When the medium Mg2+ levels reached 1 mM, the growth of mgt6 and avp1-4 mgt6 mutants appeared comparable to that of wild-type . Notably, in the presence of high Mg levels such as 4 mM and 6 mM Mg2+ , avp1-4 mgt6 double mutant exhibited more severe inhibition of shoot growth with significantly lower fresh weight and more reduced chlorophyll content as compared to either mgt6 or avp1 single mutant. The enhanced sensitivity of the avp1-4 mgt6 double mutant suggest that AVP1 and MGT6 may represent two independent functions that are required for plant tolerance to high Mg2+ stresses. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 15 resulting from mgt6 mutation that renders plants hypersensitive to low Mg2+ . When the medium Mg2+ levels reached 1 mM, the growth of mgt6 and avp1-4 mgt6 mutants appeared comparable to that of wild-type . Notably, in the presence of high Mg levels such as 4 mM and 6 mM Mg2+ , avp1-4 mgt6 double mutant exhibited more severe inhibition of shoot growth with significantly lower fresh weight and more reduced chlorophyll content as compared to either mgt6 or avp1 single mutant. Although Mg is an essential macro-nutrient required for plant growth, high concentrations of environmental Mg2+ could be detrimental, and the targets underlying toxic effect of high-Mg are not well understood.

Among plant species analyzed, P. vulgaris was the only one that showed a binding-site penalty score lower than 5, corresponding to a score recommended to consider a small RNA-target binding as probably functional. For other species, the high penalty scores, ranging from 7.5 to 9, indicate a very low probability for the existence of a functional miR1511/ALS3 regulatory node .A previous study about the response of different Andean and Mesoamerican common-bean cultivars to AlT showed that Andean genotypes are more tolerant to this abiotic stress, as compared to Mesoamerican genotypes . Our phylogenetic analysis revealed that all the Andean genotypes present a deleted version of the MIR1511 that would result in the absence of functional mature miR1511 . Previous work from our group showed that common-bean miR1511 expression responds to AlT stress . Here we analyzed the regulation of miR1511 and ALS3, as well as the early effects of AlT in roots of common-bean plants from the Mesoamerican BAT93 genotype vs. Andean G19833 genotype, with a deleted MIR1511 . Common-bean plantlets from BAT93 and G19833 genotypes were grown in hydroponic conditions either in control or AlT treatments, for up to 48 hrs. First, we performed the expression analysis of miR1511 and ALS3 target gene, using qRT-PCR . In AlT-stressed BAT93 plants, the transcript accumulation level of mature miR1511 gradually decreased, reaching more than half at 24 hours post-treatment ,vertical grow while at 48 hpt it returned to values close to those of time 0 . As expected, G19833 plants did not express mature miR1511 . The transcript level of ALS3 target gene increased in AlT treatment. The ALS3 transcript accumulation was significantly higher in G19833 roots, which lack miR1511, compared to BAT93 roots .

At 6 hpt, ALS3 expression in G19833 roots almost doubled and remained unchanged up to 48 hpt, when transcript accumulation in BAT93 and G1988 roots reached similar levels . To further study the role of miR1511/ALS3 in the physiological reaction of common-bean plants to high Al levels, we aimed to over express the miR1511 precursor in transgenic roots. As long as stable transformation of Phaseolus vulgaris plants is, to date, nearly impossible, we chose to use BAT93 and G19833 composite plants -with untransformed aerial organs and transgenic roots . As long as common bean is recalcitrant to stable transformation, this method is an alternative to demonstrate miRNA functionality . The miR1511-overexpressing composite plants as well as control plants, transformed with empty vector , were grown in AlT and control treatments. The expression level of miR1511 and ALS3 were determined by qRT-PCR in roots from composite plants harvested at 48 hpt . A two-fold accumulation of miR1511 transcript was observed in BAT93 OE1511 roots from plants grown in either treatment, compared to EV . In G19833 EV roots, the absence of miR1511 was confirmed, however a significant accumulation of miR1511 mature transcript was observed in OE1511 roots, albeit at lower levels than the level from BAT93 OEmiR1511 roots . In control treatment, both genotypes showed lower expression level of ALS3 in OEmiR1511 vs. EV roots. In addition, increased ALS3 transcript levels were observed in AlT stressed roots from both genotypes, as compared to control treatment . The primary and earliest symptoms of plants subjected to AlT stress is an inhibition of lateral root formation and root growth due to the alteration of root cell expansion and elongation, inhibiting cell division . On this basis, we analyzed the root architecture phenotype of BAT93 and G19833 OEmiR1511 and EV plants, grown under AlT or control treatments for 48 h . The growth rate of root length, width and area as well as the number of lateral roots, was calculated from the difference of each value at 48 hpt vs. time 0. The BAT93 EV plants under AlT showed decreased rates of each root parameter , thus indicating the drastic effect of high Al on root development. In contrast, G19833 EV plants showed higher tolerance to AlT evidenced by similar rate of the root length, area, width and lateral root number in stress vs control treatments . These results are in agreement with those previously reported indicating a higher tolerance to AlT of Andean common-bean genotypes compared to Mesoamerican genotypes . Surprisingly, in G19833 plants genetically engineered for the expression of mature miR1511, the effect of root phenotype was evident.

The rate of root length, area, width and lateral root number of G19833 OEmiR1511 AlT-stressed plants significantly decreased as compared to EV plants, showing reduced levels similar to those from BAT93 stressed plants . In A. thaliana, primary root growth inhibition under phosphate limitation or AlT is mediated by ALS3 and LPR1, a ferroxidase . LPR1 acts downstream of ALS3 and its expression is epistatic to ALS3 expression . To determine if LPR1 could be involved in the different response to AlT of BAT93 vs. G19833 plants, we measured the accumulation of LPR1 transcripts in similar AlT conditions as those from Figure 4. The transcript level of LPR1 gene decreased in AlT treatment. In AlT BAT93 roots, the transcript level of LPR1 gradually decreased reaching half of the initial expression at 48 hpt. In AlT G19833 roots, the LPR1 expression was significantly lower compared to BAT93 roots from 6 hpt to 24 hpt . At 48 hpt, LPR1 transcript reached similar levels in roots from both genotypes . The LPR1 expression pattern was opposite to the ALS3 expression profile in AlT-stressed roots , indicating an epistatic relation between these two genes and the possible participation of LPR1 together with ALS3 in the control of common-bean root growth under AlT. In order to determine if miR1511 indirectly controls LPR1 expression via the regulation of ALS3 transcript, we evaluated the LPR1 transcripts accumulation in transgenic roots from OEmiR1511 and EV composite plants, growing in Alt vs control conditions. In both BAT93 and G19833 roots, a significant increase of LPR1 transcript accumulation was observed in OEmiR1511 roots from plants grown in either treatment, compared to EV roots . In AlT treatment, roots from both genotypes showed significant lower LPR1 transcript level compared to roots from control condition. Again, LPR1 expression pattern was the opposite compared to that of ALS3 in the same transgenic root samples , thus indicating the probable epistatic relationship between these two genes and the indirect regulation of miR1511 on LPR1 expression.In plants, microRNA genes have a higher birth and death rates than protein-coding genes . For various authors, the miRNAs’ evolution rate generates a reservoir of adaptability for gene regulation . Due to this high evolutionary turnover rate, new miRNA families and members emerge, while others lose their regulatory role and disappear from genomes of phenotypically close species or genotypes. In soybean, MIR1511 is subjected to this mechanism. Htwe et al.,reported two altered versions of MIR1511 alleles in some annual wild soybean genotypes, whereas no deletion was found in G. max and perennial wild soybean MIR1511. Here, we report a similar phenomenon for P. vulgaris MIR1511 genotypic variations. Only part of the MW1 subgroup of P. vulgaris Mesoamerican genotypes and all the Andean genotypes analyzed displayed a 58 bp-deletion in the miR1511 precursor gene compared to the corresponding sequence of P. dumosus, P. coccineus, the PhI gene pool and the rest of P. vulgaris Mesoamerican genotypes .

As MIR1511 is present in non-legume species, the most parsimonious hypothesis to explain the evolution process associated with this event is to consider a deletion of part of miR1511 precursor sequence. In contrast to soybean, where probably two deletion events were required for the generation of two alternative MIR1511 alleles, our results suggest a single deletion event in the common ancestor of a part of MW1 Mesoamerican subgroup and the Andean genotypes for the generation of a different allele of miR1511 precursor gene. This single MIR1511 deletion event hypothesis supports the Mesoamerican model proposed by Ariani and colleagues where the Mesoamerican gene pool is the ancestral population from which the other gene pools have derived. The fact that the PhI gene pool contains an untruncated version, as do the other closely-related Phaseolus species included in this analysis, further confirms the sister-species status of the PhI gene pool, now known as P. debouckii . P. debouckii also contains ancestral, i.e., non-derived, sequences for phaseolin seed protein and chloroplast DNA . Based on the MIR1511 phylogenetic history presented here , we propose an addendum to this model where AW gene pool genotypes derived from one, or more,indoor growers member of the MW1 Mesoamerican subgroup. A clear distinct geographical distribution pattern was observed among the P. vulgaris genotypes featuring the MIR1511 deletion and the ones with an unaltered allele . MIR1511 deletion occurred in genotypes originating from the northern and southern extreme limits of the common-bean distribution in Latin American area. Such distribution pattern correlates with the annual precipitation pattern reported for the American continent , indicating that genotypes with MIR1511 deletion originated from areas with significantly less precipitation as compared to areas where genotypes with unaltered MIR1511 originated . Drought makes soil not suitable for agriculture; it tends to increase soil concentration of different compounds that would result in plant toxicity, including aluminum toxicity, which is an important plant growth-limiting factor . The harsh soil conditions of areas where P. vulgaris genotypes lacking of MIR1511 originated probably forced these common-bean populations to adapt and favored selection of genotypes with higher AlT tolerance. In this work, we showed the experimental validation of a target gene for P. vulgaris miR1511. We validated the miR1511-induce cleavage of ALS3 transcript, an ABC transporter participating in Al detoxification in plants . However, additional action of miR1511 by translation repression of ALS3 cannot be excluded. Other proposed target genes for P. vulgaris miR1511 are not related to plants AlT response and show high binding-site penalty score, thus improbable to be considered as functional in the AlT response.We interpret that the MIR1511 deletion resulting in lack of mature miR1511 allowed common-bean adaptation to high Al in the soils by eliminating the negative regulation of ALS3 transcript and the accumulation of LPR1, in the first 48 hpt, thus increasing its tolerance to AlT and favoring plant growth. Interestingly, similar characteristics hold for the soybean MIR1511-deletion case where the origin of soybean genotypes featuring a MIR1511-altered allele is geographically correlated with areas susceptible to high Al concentration in soil due to presence of drought in these regions .

High aluminum levels in soil mainly affect plant roots; aluminum can be allocated to different sub-cellular structures thus altering the growth of the principal root and the number of lateral roots . In this sense, it has been observed that AlT-stressed plants favor the transport of chelated Al to vacuoles and from roots, through the vasculature, to aerial tissues that are less sensitive to Al accumulation . In Arabidopsis and other plants, ALS1 and ALS3, from the ATP-binding cassette transporter family, are involved in Al detoxification and enhance tolerance to AlT. ALS3 is located in the tonoplast, the plasma membrane of root cortex epidermal cells, and in phloem cells throughout the plant . It has been shown that Arabidopsis als3 mutants are more sensitive to AlT exhibiting extreme root growth inhibition, compared to wild type plants . Recent studies on the role of Arabidopsis ALS3 in root growth inhibition revealed its regulation via the inhibition of the STOP1-ALMT1 and LPR1 pathways, which indirectly control ROS accumulation in roots via the modulation of Fe accumulation . Furthermore, Arabidopsis ALS3 expression is induced by excess Al , a phenomenon we observed in common-bean plants as well . Common-bean ALS3 expression doubled after 6 hours under AlT in roots from G19833 plants, while in stressed roots from BAT93 plants a similar level was reached only after 48 h of treatment . The opposite expression profile was found for the ALS3-epistatic gene LPR1, in the same samples . Our data on the different ALS3 and LPR1 expression level from both genotypes indicate that the absence of the negative regulator miR1511 in G19833 plants allows a faster response to AlT. Although the level of mature miR1511 decreased in BAT93 roots up to 24 h of after exposure to high Al, this level seems sufficient to induce degradation of ALS3 transcript, which showed reduced levels compared to G19833 roots, and an increase of LPR1 expression . Our analysis of root architecture in common-bean plants showed that G19833 Andean genotype plants are more tolerant to AlT as compared to Mesoamerican BAT93 plants .

Deficiency of B has been identified as a serious agricultural issue in more than 100 crops in 80 countries . Limitation of B impairs growth of young tissues and seed set, which results in depressed quality and quantity of agricultural products. In rice, B content is up to 10-time lower than those of dicot plants . And thus rice young seedling is relatively resistant to B limited condition compared to dicot plants such as Arabidopsis . However, the effect of B limitation until the reproductive phase is little known in rice. In this study, we evaluate the growth and yield of rice subjected to B deficient condition by hydroponic experiments. We previously reported that a boron efflux transporter OsBOR1 plays a crucial role in efficient root-to-shoot translocation in rice . Over-expression of AtBOR1 improved growth and seed fertility in Arabidopsis plants under B deficient condition . But this strategy, up-regulation of native B transporter to achieve the tolerance to B deficiency, has not been applied for the crop so far. We herein generated several independent lines of transgenic rice plants over expressing rice BOR1 and characterised the phenotypes of these transgenic rice plants under B deficient condition. Rice cultivar Nipponbare was used. Growth experiments were carried out in a green house under natural light condition . After germination on the agar medium containing 0.5 mM CaCl2, seedlings were transferred to a Kimura B hydroponic solution containing 2 mM MES with different B levels: Concentration of B in the solution was 18 µM for B sufficient condition, and 18, 0.18, 0.03 µM for B deficient treatments. Seedlings were grown until grain ripening stage and plant height was measured every other week. At harvest, stackable planters yield components were measured. Concentration of B in flag leaf and husked grain were determined by ICP-MS after digestion using nitric acid and hydrogen peroxide.

Difference in plant height among the B treatments was not clear until 4 weeks after treatments . However, when subjected to lower than 0.18 µM B, reduced plant height was clearly observed according to the growth periods . Visible symptom on leaf blades such as chlorosis was not observed in B deficient-treated plants. On the other hand, the grain yield was greatly reduced by B deficient treatments lower than 0.18 µM B, which was mainly because of decreased numbers of spikelets . Under 0.03 µM B, seedlings showed dwarf phenotype and panicle formation was severely inhibited . Concentration of B in flag leaf and brown rice was also decreased according to the level of B supply . These results suggest that in rice, young seedlings were relatively tolerant to B limited condition, however, continuous low B supply clearly impairs the vegetative and reproductive growth, leading to decreased yields. RT-PCR analysis of 11 independent transgenic lines demonstrated the expression and its variation of introduced gene in transgenic plants . We selected 9 lines with sufficient numbers of seeds and tested the growth under moderate B deficient condition for 2 weeks. The growth of shoots and roots did not differ among 8 transgenic lines and NT . However, among transgenic lines over-expressing introduced OsBOR1, B concentrations in xylem sap of several lines were higher compared to those of NT and transformants with weak expression of introduced gene . These results suggest that increased expression of the rice boron transporter BOR1 enhanced root-to-shoot translocation of boron, which might be resulted in improved B acquisition and further growth under boron deficient condition. Deltas are the pinnacles of life: they provide resources for a diverse array species and it is therefore critical that we protect them. After describing the current issues facing the Sacramento San Joaquin River Delta, CA, US, this report will describe a case study of a water management and agricultural diversification system at Sherman Island which can serve as a demonstration project for future application to deltas around the globe. The magnitude and diversity of California’s agricultural, environmental, industrial, recreational, and urban interests in the Sacramento-San Joaquin Delta emphasize the importance of protecting the Delta infrastructure. Protected by 1,100 miles of levees are over 538,000 acres of farming, 64,000 acres of cities and towns, and 75,000 acres of undeveloped land from flooding and saltwater intrusion the Delta is home to nearly 515,000 people living in seven counties, 500 different plant and animal species, including 20 that are endangered and major transportation and utility infrastructure. 

The Sacramento, San Joaquin, Mokelumne, Cosumnes, Calaveras Rivers and their tributaries flow into the Delta and provide water to over 22 million Californians – over two-thirds of the population. Sherman Island sits on the western edge of the Sacramento-San Joaquin River Delta and is one of the key geographic features protecting the Delta as a water resource. The island is located northeast of the city of Antioch, California, and lies within the jurisdiction of Sacramento County. The Sacramento and San Joaquin rivers meet at its western boundary, which is bordered to the northeast by Three-Mile Slough. Levee instability caused by continued subsidence in the region is a severe risk to cause catastrophic failure. If Sherman Island’s levees were to fail water quality for the entire Delta and most of California, would be compromised, most likely on a timescale of years to decades. Because residents rely on the Delta for their drinking water numerous health issues would result from the Delta being compromised. In addition, the livelihoods of those who utilize the freshwater, such as farmers and industry works, could deteriorate. Delta Bearier Engineering estimates the cost to recover lost water supply and to repair levees, infrastructure, and damaged homes in the event of levee failures to be as high as $2.2 trillion . The increased salinity due to saltwater intrusion will also likely destroy populations of wildlife species. The loss of species has the ladder effect on the diet and shelter of other species; thus, whole ecosystems are susceptible to deterioration. Global climate change is a daunting but undeniable reality, the impacts of which must be considered. Engineered systems must be designed with these effects in mind if they are to remain resilient over the entire life span of the project. In considering just how climate change will affect the Sacramento-San Joaquin Delta, it is important to evaluate the following: Sea-Level Rise Numerous scenarios and global climate models have been developed to predict the effect of climate change on global mean sea level . The Intergovernmental Panel on Climate Change gives an estimated increase in GMSL of 0.1 m – 0.9 m between the year 1990 and 2100 . Similarly, a more recent study suggests sea level will rise by as much as 0.5 m to 1.4 m over the course of the next century . There are several implications for Sherman Island and the Delta given these predictions. Governed by significant tidal inflows, the Delta is very susceptible to increased salinity in inland waterways due to rising sea level . This poses a great threat to the viability of Sherman Island and the Delta as a water resource for the State of California. Saltwater intrusion into the lower Delta could compromise water quality, resulting in reduced agricultural yields and greater stresses on alternative water sources. Additionally, a higher mean sea level also means increased pressure on the already fragile levee system.

Levee heights would need to be drastically increased, or else the combination of increased sea level and major storm events would pose an even greater risk to the system. While sea level rise has greater impact on long term water level variations, it is changes in river flow that have the biggest effect on a short-timescale for the Delta . Most predictions indicate that there will be increased flows during the winter months and reduced flows in spring and summer. From a water resource perspective, this implies that less water will be available as the state approaches times of warmer temperature and increasing agricultural demand. Furthermore, reduced spring flows will invite saltwater intrusion further inland into the Delta at a time when even less water will be available to flush the system and maintain the integrity of water supplies. While it is difficult to apply global climate change models to smaller scale regions such as the Delta, it can be inferred from the available studies that wind velocity and intensity will increase in the area. The serious implications of this matter are that wind velocities in the Delta region determine wind and wave action, two factors which have a significant impact on levee erosion . Consequently, levees weakened by wind and waves are subject to greater risk of failure in the event of high water or severe storm. Changes in average temperature and the amount and type of precipitation are also expected as part of global climate change. The amount and timing of annual runoff is one of the biggest impacts, stackable flower pots as precipitation normally falling in the form of snow will turn to rain, reducing the amount of water available for spring flows from snow melt . Increased temperature will have significant effect on the temperature gradient between the San Francisco Bay Area and the Central Valley, further increasing the intensity of wind velocities . Warmer temperatures will also lead to earlier melting of snow, resulting in reduced water availability for an agriculture-dependent state already plagued by drought. Delta system must consider the needs of global climate change including: flood control, agricultural development, water quality, and environmental sustainability. Consequently, efforts must be made to reverse subsidence, stabilize the fragile levee system, increase economic productivity, and protect vital water and environmental resources. The Aquaponics Water Management System combines hydroponics , aquaculture , and restored wetlands enclosed in flood storage zone. The aquaponics system is a bio-integrated system in which waste byproducts from aquaculture are used as nutrients for plant growth in the hydroponics components. Each aquaponics system consists of fish rearing tanks, solids settling and removal tanks, a bio-filter, the hydroponics rafts and a sump. The first step of the aquaponics cycle is fish eat food and excrete ammonia rich effluent. The effluent is sent through the settling tanks to reduce the amount of suspended organic matter. Next the ammonia is removed and bacteria convert ammonia and nitrites to nitrates in the bio-filter. The nitrate rich water is then pumped to the hydroponics component where plants’ roots hang into the pipes and absorb the nutrients from the water. Once water has reached the end of the hydroponics component, it is collected in a sump and then returned back to the rearing tanks. 

The water flow through the aquaculture and hydroponics systems is shown in Figure 1. The aquaculture components sit outside of the flood management zone while the hydroponics and wetland systems dwell within. The flood storage zone spans 800 acres and is able to store 12,000 to 16,000 acre-feet of water. Figure 2 displays the approximate layout of the system which consists of floating hydroponics rafts that can move up and down with the variations of the water levels but are anchored to prevent lateral movement. In addition, the flood storage zone provides wetland acreage for wildlife habitat restoration, recreation, carbon sequestration, and subsidence reversal. Levees and Siphons function to enclose the flood storage zone, transport and store water during high river water levels as shown in Figure 3.Levees construction and upgrades are the primary infrastructure needed for the flood storage zone. Levees currently bound the northern, southern, and western edges of the Aquaponics Water Management System. The western and southern edges are bounded by Army Corp Levees protecting the Island from Sacramento and San Joaquin rivers, respectively. The northern edge is bounded by Mayberry Slough levees which are not engineered project levees. Therefore, there is little boring data and high uncertainty of the construction materials and stability. Therefore, a sandy berm will be constructed to filter, and buttress the levees to provide support. The largest infrastructure component to construct is the internal cutoff levee to enclose the flood storage zone by bounding the eastern side. This levee will be constructed 1,160 ft west of the Antioch Bridge, encompassing Scour Lake while maintaining a buffer zone between the system and the maintenance setbacks for the Antioch bridge.

To evaluate whether the observed metal accumulation phenotypes were due to impaired expression of the OPT3 gene, the coding sequence of OPT3 was expressed in opt3- 2 under the control of the Cauliflower mosaic virus 35S promoter. Over expression of OPT3 in four independent lines was confirmed by qPCR . opt3-2 contains a T-DNA insertion in the 5’ UTR of OPT3 . Therefore, the residual OPT3 transcript observed in opt3-2 is expected in this knockdown line. OPT3 complementation lines were grown on heavy metal-containing soil, and the metal concentration of their seeds was determined by ICP–OES. Cd accumulation in seeds of the four complemented lines was reduced to wild-type levels . Over expression also rescued the seedling sensitivity of opt3-2 to Cd , indicating that ectopic expression of OPT3 is sufficient to complement the sensitivity and metal accumulation phenotypes of opt3-2.Previous GUS staining experiments have shown that OPT3 is expressed throughout the vasculature; however, localization at a higher resolution has not been evaluated . To identify where in the vasculature OPT3 is preferentially expressed, β-glucuronidase was expressed under the control of the native OPT3 promoter. Under standard growth conditions, GUS staining was negligible; however, under Fe-limiting conditions , staining was clearly observed in the phloem, but not in the pith or endodermis . Consistent with our findings, cell-type-specific microarray data sets show the highest intensity values of OPT3 in the phloem, comparable to the phloem sucrose transporter SUC2 . Thus, two independent approaches show preferential expression of OPT3 in the phloem. To gain insight into the subcellular localization of OPT3, an N-terminal YFP–OPT3 translational fusion was infiltrated into Nicotiana benthamiana leaves. Fluorescence was detected along the cell periphery, indicative of plasma membrane localization . A weaker perinuclear fluorescence and transvacuolar strands were also observed in some cells , indicating that a fraction of the YFP–OPT3 localizes to the endoplasmic reticulum . The ER fluorescence pattern, however, was not present in all cells. Furthermore, Hechtian strands were clearly present connecting the cell wall to the plasma membrane of plasmolyzed leaf cells .These results suggest that OPT3 is a plasma membrane transporter preferentially expressed in the phloem.The Arabidopsis mutant opt3-2 shows a constitutive Fe-deficiency response in roots including the up-regulation of the Fe/Zn/Mn transporter IRT1 . Despite this Fe-deficiency response, Fe sensing in shoots remains intact . The molecular mechanisms mediating shoot-to-root signaling of iron status in plants remain largely unknown.

The impaired iron sensing in roots but not shoots of opt3-2, in conjunction with phloem localization, suggests a possible role of OPT3 in shoot-to-root transport of a signal reporting metal status. To test this hypothesis, the OPT3 coding sequence was expressed in opt3-2 under the control of the shoot-specific chlorophyll a/b binding protein promoter . Shoot specificity of the CAB2 promoter was determined by GUS staining . RT–PCR analyses confirmed that OPT3 is preferentially expressed in the shoots of three independent transgenic lines . The residual OPT3 transcript in opt3-2 roots expressing CAB2pro:OPT3 plants is consistent with the knockdown nature of the opt3-2 allele. Thus, the low level of OPT3 transcript in roots is not sufficient to properly regulate metal homeostasis in roots . Two of the major phenotypes described in opt3-2 are the constitutive iron-deficiency response in roots, as illustrated by high IRT1 expression , and the over-accumulation of Cd in seeds . Thus, we tested whether shoot-specific expression of OPT3 was able to complement both phenotypes. As shown by RT– PCR, IRT1 transcript levels were greatly reduced in the roots of CAB2pro:OPT3-expressing plants compared to the opt3-2 mutant . These results show that shoot-specific expression of OPT3 is sufficient for proper regulation of metal homeostasis, including communication between leaves and roots. Furthermore, the Cd accumulation in CAB2pro:OPT3 seeds was reduced to wild-type levels . Seedling hypersensitivity to Cd was also rescued in the three independent CAB2pro:OPT3– expressing lines . Collectively, these results demonstrate that shoot-specific OPT3 expression is sufficient to complement opt3-2 root phenotypes, suggesting that OPT3 may mediate the long-distance transport of a signaling molecule from leaves to relay information about metal status, thus contributing to whole-plant metal homeostasis.To test whether OPT3 functions in the mobilization of Fe or other molecules, we first assessed the capacity of wild-type and opt3-2 to remobilize Fe from one leaf to other leaves using the radiotracer 59Fe. In these experiments, 59Fe was loaded into a mature leaf as Fe2+ at a slightly acidic pH to resemble the apoplastic pH. The addition of ascorbic acid was used to reduce Fe3+ to Fe2+ and maintain it in the reduced form. Figure 7A shows that Fe can be re-mobilized from one leaf to adjacent leaves in wild-type. In contrast,aeroponic tower garden system opt3-2 shows negligible movement of 59Fe between leaves. Figure 7B shows the 59Fe activity in the four leaves adjacent to the leaf where the 59Fe was originally applied.

Compared to wild-type, opt3-2 shows a severe reduction in the quantity of 59Fe mobilized from one leaf to the adjacent leaves , suggesting that OPT3 is required for the reallocation of Fe between plant tissues. In fact, opt3-2 plants over-accumulate Fe in mature leaves compared to wild-type, as visualized by Perls’ staining . Interestingly, over-accumulation of Fe in opt3-2 occurs only in mature leaves but not in young leaves . Moreover, accumulation of Fe in opt3-2 is more evident at the base of the trichomes and near the vasculature in the minor veins but not in the main vasculature, suggesting that, in opt3-2, the reallocation of Fe between leaves is impaired, particularly at advanced stages of leaf development . To test whether OPT3 functions as a Fe2+ transporter similarly to IRT1, we expressed OPT3 in a yeast strain deficient in Fe2+ uptake . As previously shown, IRT1 expression in yeast allows the fet3fet4 strain to grow on minimal media without the addition of extra Fe . OPT3 was unable to rescue the fet3fet4 strain , suggesting that, in yeast, OPT3 does not mediate the uptake of Fe2+ like IRT1. Subcellular localization studies, however, show that OPT3–YFP protein fusions do not localize to the plasma membrane in yeast in contrast to in planta . This mislocalization of OPT3 in yeast precluded further characterization of OPT3 using yeast as a heterologous system. Note that, if fet3fet4 yeast cells were not sufficiently pre-starved of iron, growth of the fet3fet4 mutant was observed, and therefore long-term starvation of yeast was required for these complementation tests. We attempted complementation with different yeast promoters, starting with the strong GAL promoter . Using the phosphoglycerate kinase yeast promoter, OPT3 also did not complement the pre-iron-starved fet3fet4 yeast mutant, consistently with previous studies showing that OPT3 does not complement this yeast mutant . Nevertheless, 59Fe re-mobilization studies suggest that OPT3 is essential for the remobilization of Fe within plant tissues; whether this transport occurs as Fe2+ or as a Fe-ligand complex remains to be determined. OPT3 is a member of the oligopeptide transporter family and some members of this family have been found to have broad substrate specificity for peptides of different length and amino acid composition . To test whether OPT3 mediates the long-distance transport of GSH in planta, we pursued radiotracer experiments to assess the movement of 35S-GSH from one leaf to adjacent leaves . No differences were found between wild-type and opt3-2, suggesting that OPT3 does not participate in the mobilization of GSH between plant tissues. Interestingly, opt3- 2 rosette leaves from plants exposed to 20 μM CdCl2 and supplemented with 0.5mM GSH accumulated Cd, but no other metals, to wild-type levels. These results suggest that GSH is required for Cd retention in leaves. On the other hand, GSH supplemented to the roots reduced Cd in the leaves of both opt3-2 and wild-type plants likely because GSH trapped Cd in roots of both opt3-2 and wild-type plants .

Glutathione has recently been shown to play a critical role in Fe signaling in yeast by stabilizing FeS clusters in the cytosol . In Arabidopsis, GSH is also important to maintain proper homeostasis and crosstalk between Zn and Fe metabolism . To test whether long-distance transport of GSH is important for proper shoot-to-root signaling and homeostasis of trace metals in roots, we measured the constitutive high activity of the root ferric reductase in opt3-2 after foliar application of GSH . In all cases, including the application of foliar GSH, GSH applied to roots, or foliar application of Fe, the activity of the root ferric reductase remained constitutively high in opt3-2. We also tested whether iron applied directly to roots or complexed with GSH, citrate, or nicotianamine is sufficient to repress the high activity of the Fe chelate reductase in opt3-2 roots. Supplemental Figure 6B shows that iron alone , or in complex with GSH, nicotianamine, or citrate, cannot down-regulate the constitutive iron-deficiency response in opt3-2 back to wild-type levels.We have identified an Arabidopsis mutant, opt3-2, that over-accumulates Cd in seeds and roots but, unexpectedly, under-accumulates Cd in leaves . Cadmium distribution throughout the plant is an orchestrated process dictated by root uptake, root-to-shoot translocation through the xylem, dutch buckets for sale and redistribution of Cd from leaves to sink tissues via the phloem. opt3-2 displays constitutive up-regulation of IRT1, a root transporter with broad specificity for heavy metals including Cd . Over-accumulation of Cd, Zn, Fe, and Mn in roots may be explained by the constitutively high expression of IRT1. However, under-accumulation of Cd in leaves and over-accumulation of Cd in seeds, which is different from essential metals , is inconsistent with the high expression of IRT1 . Nutrients, water, and heavy metals are mobilized from leaves into seeds through the phloem . Accumulation of metabolites in sink tissues and under-accumulation in source tissues is best described as an increased redistribution process, likely through the phloem . Notably, of the analyzed metals, only Cd under-accumulates in leaves . These results suggest that, in contrast to the broad specificity of heavy metal uptake at the root level, metal-specific mechanisms mediate the remobilization of heavy metals from leaves to sink tissues. In addition to the altered distribution of heavy metals within the plant leading to over-accumulation of Cd in seeds, opt3-2 also shows hypersensitivity to Cd at the seedling stage . Both the increased accumulation of Cd in seeds and the Cd hypersensitivity of seedling growth are restored to wild-type levels by ectopically expressing OPT3, demonstrating that the altered redistribution of Cd through the plant is the result of the reduced expression of OPT3 in opt3-2 .The opt3-2 mutant displays a constitutive iron-deficiency response in roots, while the leaves properly respond to iron levels as indicated by wild-type levels of ferritin expression , suggesting that the iron status response is mainly disrupted in roots. In plants, the root iron-deficiency response is regulated by local signals within the root and also by unknown systemic signals originating from aerial tissues . OPT3 is a plasma membrane transporter preferentially expressed in phloem cells during iron starvation . Cell-specific micro-arrays and OPT3pro:GUS analysis under Fe-limiting conditions show preferential expression of OPT3 in phloem cells, suggesting a role of OPT3 in long-distance transport processes. Notably, shoot specific expression of OPT3 in the opt3-2 background rescued the constitutively high expression of IRT1 in roots, the seed Cd over-accumulation phenotype, and the seedling sensitivity to Cd . These results suggest that the impaired metal homeostasis in opt3-2 roots is caused by a disruption of the shoot-to-root signaling of the leaf metal status. Thus, OPT3 is the first shoot-expressed gene required for proper communication from leaves to roots to maintain metal homeostasis at the whole-plant level. Several Arabidopsis and tomato mutants displaying an Fe-deficiency response in roots can be rescued by foliar application of Fe ; these experiments suggest that shoot-to-root Fe signaling plays an important role in Fe homeostasis which in turn could also impact the uptake and accumulation of other transition metals such as Zn, Mn, and Cd, as seen in opt3-2 . Foliar application of Fe does not repress the Fe-deficiency response in opt3-2 roots to wild-type levels , suggesting that source-to-sink transport of Fe, or a molecule mediating Fe signaling, is impaired in opt3-2. Radiotracer experiments using 59Fe demonstrate that the movement of Fe between leaves is impaired in opt3-2 ; whether this leaf-to-leaf transport occurs as Fe2+ or as an Fe–ligand complex remains to be determined.

They found that reading the final sentence of expository texts relative to narrative texts elicited a greater increase in N400 amplitude, and they concluded that expository texts required more demanding semantic processing. Eason et al. also reported differences between genres, showing that expository texts placed higher demands on executive function than narrative texts, particularly inferencing and planning/organizing information. EF is thought to be essential to the process of building a coherent text representation because it enables readers to store previously read text ideas as they simultaneously read new ideas and integrate them into their mental representation . While behavioral data certainly support the theoretical significance of EF to reading comprehension in general , Eason et al.’s findings of higher demands on EF for expository text suggest that for this particular text genre, which is critical for acquiring new information, EF is particularly salient.One hallmark of successful reading comprehension is that the reader can distinguish between ideas that are important, or central, to the overall meaning of the text, and those that are less important, or peripheral.The ideas and their connections form a network in the reader’s mind . Some ideas are causally or logically connected to a great number of other ideas and as a result emerge as being important, or central, to the overall meaning of the text, while others have relatively few connections and fall out as being peripheral, or unimportant . A robust finding in the comprehension literature is that skilled readers are more likely to recognize and recall an idea the more central it is to the overall meaning of the text . This finding holds for both narrative and expository texts . van den Broek et al. propose that a reader’s ability to distinguish a text’s central and peripheral ideas, or their sensitivity to structural centrality,hydroponic fodder system is an important indicator of their comprehension ability.

For example, adults show greater sensitivity to structural centrality than do children ; typically developing children show greater sensitivity to centrality than do children with reading disability as well as those with Attention Deficit Hyperactivity Disorder ; and readers show greater sensitivity to centrality when reading in their native compared to foreign language . Importantly, studies suggest that centrality tends to emerge as a feature of the developing text representation. van den Broek used eye-tracking equipment to show that skilled readers fixate more frequently and spend more time reading central ideas than they do peripheral ones. This suggests that centrality is a dynamic construct that emerges as the reader processes a text, consistent with the idea that readers form connections among semantically related text ideas as they read. In theory, the ideas that are most important stand out because they are the ones that have the most connections and are consequently the ones most likely to be recalled. To date, although sensitivity to centrality has been investigated behaviorally, the neural mechanisms remain unknown. Understanding the neural mechanisms underlying sensitivity to centrality may allow for a more specific understanding of normal and disrupted comprehension processes.The current study sought first to identify the neural correlates specific to expository text comprehension, looking both at regions which overlap with single word processing and those which are specific to discourse-level processing. Once the systems for expository text comprehension were identified, we employed temporal analysis techniques to examine how these systems change over the course of building and maintaining a coherent mental representation of the text. We hypothesized that when isolating discourse-level comprehension from word-level comprehension, we would see regions that have previously been implicated by sentence and narrative comprehension, particularly those associated with discourse-level language processing separate from social cognition [bilateral TP, angular gyrus , and PCC] . We predicted that the other traditional language regions, such as left-lateralized inferior frontal gyrus , middle temporal gyrus , and anterior superior temporal sulcus , would most likely be shared by both word and passage tasks, but that these multi-functional regions would behave differently over the temporal course of passage comprehension compared to single-word comprehension .

Additionally, due to prevalence of information organization in expository comprehension, we expected to see activations in the dorsal attention network [dorsolateral prefrontal cortex , intraparietal sulcus , inferior parietal lobule ], which has been associated with the kind of updating, integrating, and immediate planning of information that has been behaviorally described in previous studies on expository comprehension . We consequently hypothesized that over the course of passages alone, semantic control areas shared by words and passages at the mean-level would become increasingly responsive over time in passage comprehension alone due to the increased semantic demands associated with integrating and maintaining new information in a global text representation. Given Yarkoni et al.’s study showing that the parietal visuospatial attention regions are involved in the construction of text , while classic language areas are reflective of the maintenance of readers’ mental models, we hypothesized that along with the emergence of a greater reliance on perisylvian regions over time, in passages we would see a decrease over time of posterior parietal regions. The second goal of the current study was to examine the patterns of neural activation that are uniquely associated with processing central vs. peripheral ideas. While behavioral measures clearly indicate that readers distinguish central from peripheral ideas, both online and when recalling the text, the neurobiological processes that support this fundamental aspect of text comprehension have yet to be explored. Gaining insight into processes that promote a reader’s sensitivity to centrality advances current comprehension models. More focally, it allows for isolating the underlying neural mechanisms supporting processes that may be disrupted in individuals with poor sensitivity to centrality. Such knowledge may eventually inform ways to individualize intervention for problematic reading comprehension. Given previous behavioral findings, we expected there to be unique semantic and integrative regions that differentiate central ideas from peripheral ideas. Finally, we predicted that with the temporal progression of the text, there are changes in the cognitive demands required in differentiating central and peripheral information and integrating those units into the mental model, resulting in temporally dynamic neural systems for different types of textual information.

To accomplish the goals of the study, an fMRI passage comprehension task was designed in which participants viewed three types of stimuli: coherent expository passages and scrambled words and nonalphanumeric symbols . Within the Passages condition, we delineated the text’s central and peripheral ideas. To examine differences between central vs. peripheral ideas, as well as overall patterns of activation associated with text, we employed a typical general linear model . To examine the emergence of a mental representation of the text, or dynamic changes taking place over time, an approach sensitive to temporal features was taken , whereby examination of neural activation that emerged or diminished over time for various conditions was revealed.Seventeen adults participated in the study. All participants met the following inclusion criteria: native English speakers; normal hearing and vision; no history of major psychiatric illness; no traumatic brain injury/epilepsy; no history of a developmental disability; and no contraindication to MRI. Each participant gave written consent at the beginning of the study, with procedures carried out in accordance with Vanderbilt University’s Institutional Review Board. All participants had a standard score within the average range on a composite of standardized reading tests or had no history of difficulty with reading. Participants received $25 as compensation for a 2-h testing session.Coh-Metrix 2.0 was used to create 8 passages that were equivalent across measures of word concreteness, syntactic simplicity, referential cohesion, causal cohesion, and narrativity. Passages were matched on descriptive factors, including: number of words, average sentence length, and all passages measured a Flesch-Kincaid grade-level between 4.0 and 4.9. To insure that passages were equivalent in difficulty, each of the 8 passages was isolated and compared to the mean of the remaining 7 passages. Passages were considered equivalent when measures were within a 90% confidence interval of the mean of the group of remaining passages. At the end of this process, fodder system the passages were equal across 23 measures of descriptive statistics, vocabulary frequency, word concreteness, syntactic simplicity, referential cohesion, causal cohesion, and narrativity . Four of these passages were used for the Passages condition and four were used for the Words condition , which included words from the passages in randomized order. All passages were 150 words in length. Each sentence was no longer than 13 words. The passages were all expository and included the following topics: Hang Gliding, Wrasses, Velvet Worms, and Hydroponics. Each passage consisted of two paragraphs, the first of which served to introduce the topic while the second elaborated on a particular detail of the subject matter.Using imaging technology to explore the neural correlates of reading comprehension is a challenging task due to the temporal nature of discourse processing. Previous studies have presented the entire paragraph on one screen , but this procedure prohibits comparing how readers process specific aspects of the passage, such as central vs. peripheral ideas, because the block contains both types of information.The most temporally precise presentation method is to present the story one word at a time, and several studies have employed this procedure . When piloting passages using this approach, participants reported that it created an uncomfortable, artificial reading experience, likely in part because readers typically process words up to 14–15 letters to the right of their fixation , and using a single word-by-word presentation prevents this.

The moving window procedure is an alternative method that allows examination of the processing associated with single words. The advantage of this procedure is that the word immediately preceding and following the word under fixation are also visible. Although this allows for a more naturalistic reading experience, the approach was undesirable for this study because it requires a self-paced design, and temporal consistency in the presentation of stimuli is required for group comparisons. To avoid both the above confounds, we presented our passages one meaningful phrase at a time. This procedure enabled us to compare activation related to processing central and peripheral ideas, yet decreased the artificial demands imposed by a word-byword presentation. Each phrase was presented on a separate trial. The phrases included noun phrases, verb phrases, and prepositional phrases, and they ranged from 1 to 6 words in length. The number and type of words presented together determined the phrases’ presentation duration. We allowed 550 ms for each content word and 275 ms for each function word. For timing purposes, we presented no more than three content words per slide and randomized the time between phrases to allow comparison across phrases. The Words condition followed the same presentation format as the Passages condition. The baseline condition was presented between paragraph 1 and paragraph 2 of both the Passages and Words conditions. The purpose of this design was to allow participants’ activation to return to baseline after reading each block . The presentation sequence was: Passage condition, Paragraph 1; Baseline condition; Passage condition, Paragraph 2; Baseline condition; Words condition; Baseline condition. The mean time for the passages block was 78.54 ; Baseline mean = 47.69 ; and Words mean = 82.45 . In all three conditions, 8% of the stimuli were repeated on two consecutive screens. To monitor whether participants attended to the stimuli, participants pressed a button with their right thumb when they detected a phrase repetition or a symbol configuration repetition. Mean percentage correct response was very high .Imaging was performed on a research-dedicated Philips Achieva 3T MR scanner with a 32-channel head coil. Functional images were acquired using a gradient echo planar imaging sequence with 40 slices with no gap and consisted of 4 runs, each 7 min . Other relevant imaging parameters for the functional images are TE = 30 ms , FOV 240 × 240 mm, slice thickness = 3 mm with 0 mm gaps, 75◦ flip angle, TR = 2200 ms, and a matrix size 80 × 80 , implying 3 mm3 isotropic voxels. All functional data were analyzed using MATLAB and SPM8 . The functional data for each participant were slice-timing corrected, aligned to the mean functional image, normalized to MNI space, and spatially smoothed with a 8 mm FWHM Gaussian filter.

The sprouted stem fragments rested on a floating plastic mesh supported by a ring of plastic pipe, on the surface of each trash can’s nutrient solution. A sheet of opaque white plastic was wrapped around and over each trash can to block out sunlight preventing algae growth and high temperatures in the nutrient solution. The nutrient solutions in the containers were monitored two times per week during a 48-week growth period. Each check consisted of the following: addition of enough deionized water to bring the can’s nutrient solution level up to a 100-L mark, determination of the can’s electrical conductivity in full volume. A concentrated Hoagland solution was added to re-establish the conductivity of the nutrient solution to its original value. The pH was adjusted to 5.7. The harvest dates were partially determined by the growth of the plants as the experiment progressed. Harvested plants were separated into apical meristems, green leaf blades, brown leaf blades , green leaf sheaths, brown leaf sheaths , stems, rhizomes, and roots. Plant parts were dried to a constant weight at 60 C. Biomass of the tissue was determined and sub-samples were ground in a Wiley mill to pass a 0.5 mm mesh screen . The nitrogen and carbon contents of the tissues were determined using an organic elemental analyzer . Stem fragments with meristems can root and regenerate new Arundo plants , as has been reported earlier by . There were significant patterns in rooting success of meristems on Arundo stems throughout the growing season. In the winter months of November through January, rooting is low, and success percentage lies below 20%, with the exception of 28 ± 12% rooting for meristems from hanging stems in January . Nearly all meristems rooted from March through September. The speed with which meristems rooted showed a related pattern through the growing season . In the period with the lowest rooting success, t50 had the highest values,dutch buckets system indicating the slowest rooting.

Rooting was most rapid in the months of May through July, a time window that was more narrow that the period in which rooting is most successful.There are no significant differences between the results in plain water and half-Hoagland nutrient solution . The rooting success and speed pattern is similar in soil, but the single replicate meristems do not allow for inclusion in the two-way ANOVA. When compared within each sampling, the rooting success of meristems from hanging stems was significantly higher than that of meristems from upright stems . When split for rooting medium, there was no significant different in rooting success between hanging and upright meristems over time in plain water , but the differences remain significant in the nutrient solution and in soil . Like with rooting success, rooting speed of hanging meristems was significantly faster when compared to that of the upright meristems of the same sampling date . When separated among rooting media, the difference in the speed with which rooting occurs was most pronounced in plain water , but still exists in the nutrient solution and soil . Though these differences in rooting success and the speed of rooting may be statistically significant, they generally are too small to be ecologically significant. Stem diameter at the node where the meristem is placed is an indicator of relative height on the stem, and the age of the meristem. Within stems, there was no relation of rooting success or speed with the diameter of the stem at the point of the meristem, so the older meristems on an Arundo stem do not root better or faster than the younger meristems on Arundo stems.When the temperature of the rooting environment was controlled at 28/15 °C for 14/10 h during the entire growing season the seasonal patterns of rooting success and speed remained, and differences between the seasonal rooting patterns of the fragments from hanging and upright stems emerged . The overall patterns differed slightly from those in the greenhouse experiment, with the lowest rooting by both stem type fragments in February through March. The rooting percentages increased in April, and the highest rooting rates occurred from July through September at 80 – 92% for both stem types.

In October the rooting of the upright stem fragments decreased more than that of those from hanging stems. The lowest rooting rates of the stem fragments from upright stems were 0 – 10% in February and March, while the rooting rates of the hanging stem fragments only decreased to 30% . The positive influence of the seasonal effect in the months of July – September on the rooting rates of both stem types masked the difference that emerged in the Winter and Spring months. The rooting by meristems from upright stems benefits more from this seasonal effect that of meristems from hanging stems. The seasonal effect on the rooting rates of the stem types could be related to a number of environmental factors that change during the growing season, such as temperature, light intensity, and daylength. determined that stem fragments sampled at the same time, but stored at different temperatures, displayed different sprouting percentages when potted and regenerated at the same temperature in a single greenhouse. We hypothesize that the different ambient temperatures prior to sampling in our experiment was an ecophysiological equivalent of the experimental factor “storage temperature” in the Boose and Holt study, and one of the factors involved in the seasonal pattern of A. donax stem fragment rooting.The seasonal differences in the rooting percentages and speed between meristems from upright and hanging stems that was striking under controlled temperature conditions had been much less pronounced in the greenhouse rooting experiment. The results of the greenhouse rooting experiment show that the temperature at the time of rooting influence the effects of the seasonal factor. Environmental effects, such as temperature and inundation, are known to affect the success of invasive plants with either negatively or positively In the greenhouse experiment, the temperatures of the rooting medium varied with the ambient temperatures and solar irradiation, while the temperature of the rooting environment in the growth chamber experiment was the same throughout the growing season. The greenhouse, the temperature of the rooting media in the winter ranged from 0.5 – 2 °C at night to 19 –21 °C during the day. In the spring and summer, solar irradiation increased these temperatures to 16 -18 and 28 – 34 °C, respectively. To test the effect of the temperature at rooting, we tested the rooting of fragments of hanging A. donax stems at different temperatures in April and May, a period that in the year-round temperature controlled experiment the success rates were 45 ± 10% in 1998, and 45 ± 21% in 1999. In this test using constant temperatures, no rooting occurred at 10 °C during the 40 days of the experiment .

At 15 °C, rooting was better than at 10 °C , but significantly less than at 17.5, 20, and 22.5 °C . In the greenhouse experiment, the seasonal pattern of rooting success was present, but the inherent advantage of fragments of the hanging stems in the winter months was masked by the negative effect of the lower temperatures of the rooting media. The temperatures chosen for the year-round temperature controlled experiment were selected to reflect the temperature conditions in the habitats invaded by A. donax in Southern California in the months of April and May. From the results of this April constant temperature experiment, it appears that the lower night temperature in the 28/15 °C for 14/10 h experiment led to a reduction in rooting success from the maximum possible in that month. This reemphasizes the effect of in situ temperature on the success of stem fragment meristem rooting, and the ecological danger of the floating stem fragments in shallow waters. The inherent seasonal pattern observed in the year round temperature controlled experiment may have resulted from cycles in the concentrations of the plant growth regulators that play a role in the growth of the side shoots, and the apical dominance of the top of the main stems. One of the growth regulators that plays a major role in the regulation of apical dominance is indole-3-acetic acid. The effect of IAA on the rooting of axillary bud on A. donax stem fragments throughout the growing season was tested through the use of exogenous IAA in the rooting medium,dutch buckets and the determination of endogenous IAA levels in the shoots that grew from the axillary buds. When the stem fragments and their axillary buds were exposed to 5 and 10 µM IAA in the rooting medium, the difference between the hanging and the upright stems disappeared. The main effect of the exogenous IAA was a significant improvement of the rooting percentage and speed of the upright stem fragments in the winter and spring periods, so that the difference between the two stem types was minimized. The exogenous IAA had little effect on the rooting success and speed of the hanging stem fragments . At 20 µM exogenous IAA, the highest concentration applied, the success rate and the speed of upright stem fragment rooting decreased from the optimum observed at 5 and 10 µM, almost down to the percentages and t50 observed in the absence of the hormone . The IAA in the rooting medium may have reached the axillary bud through the vascular bundles of the main stem piece, directly through the cuticle of the bud itself, which was positioned immediately below the rooting medium surface, or both. In early studies into the effect of IAA onplant growth, the growth regulator was sometimes applied to the leaf tissues, and the position of the axillary bud in the rooting medium could have resulted in a similar situation. There is a striking similarity between the seasonal pattern of the endogenous IAA levels in the shoots that grow from the hanging and upright stem fragments and their seasonal rooting patterns. The lowest IAA levels for both stem types occurred in the spring, and the highest levels in late summer . At the time that the endogenous IAA levels are low, levels in the shoots from the upright stem fragments are significantly lower than in those from the hanging stems. As the IAA levels in the shoots increase with the progression of the growing season, the levels in the shoots from the upright stem fragments increase more than in those from the hanging stems, and the difference between the shoots of the two stem types disappears . The role of this IAA is unclear. Although studies have also shown more complicated mechanisms , IAA produced in the main stem apex plays an important role in the apical dominance, and the growth of side shoots. A difference between the rooting and the endogenous IAA patterns is that the seasonal pattern of endogenous IAA concentrations in the new shoots runs approximately one month behind that of the rooting success. This makes it less likely that the IAA concentrations in these new shoots, of which the growth is initiated prior to root growth, is the direct cause of the rooting of the stem fragments. It is possible that both the rooting pattern and the endogenous IAA pattern result from seasonal variation in another factor, possibly the concentrations of another plant growth regulator. Preliminary enzyme kinetics results have shown higher NADP-dependent indole- 3-acetatealdehyde oxidase activity in the new shoots that developed from the hanging stems in April than in those from the upright stems . This indicates that the IAA measured in the new shoots may be the product of de novo synthesis in these shoots, rather than a trace of the IAA that may have been stored in the main stem fragment that was used in the experiment. IAA plays an important role in tracheary cell differentiation and xylem regeneration . The support for the formation of the vascular system by IAA and its promotion of the formation of adventitious roots may be related, but no causal relationship was determined in this study.

Plant and rhizosphere metabolomics thus provide a snapshot of the entire plant-associated metabolome, that can be directly correlated with real-time functional footprint of the cellular state. The need for measuring metabolite levels in plants in response to ENMs was realized early on by the nanotoxicology community. However, most studies evaluated the total content of classes of metabolites such as sugars, phenolics, flavonoids, chlorophylls, non-enzymatic antioxidants, lignin, etc. using less sensitive biochemical assays . In some studies, profiling of specific classes of compounds like carbohydrates, fatty acids, and amino acids was also performed . However, based on the evaluation of a few metabolic parameters, a comprehensive understanding of the underlying mechanisms of ENM transport and effects is rather limited and biased by author’s interpretations. To address these challenges and identify the crucial role of the affected metabolites in response to ENMs, it is important to increase the analytical coverage of the plant metabolome.Metabolomic analysis can be categorized as untargeted or targeted, the choice of which depends on the scope of the study . Key factors that separate untargeted from targeted metabolomics are: extent of sample preparation required; number of metabolites detected; level of quantification; and extent of data processing post acquisition. Untargeted metabolomics is a discovery-based approach that screens the entire pool of metabolites in an organism. In nanotoxicity studies, untargeted metabolomics can be used to discover unknown metabolites of significance and identify markers in order to generate hypothesis on biological pathways involved in response to ENM exposures . Metabolite identification is based on available literature or in house experimental database,grow strawberry in containers and the quantitation is either relative or semi-quantitative, aided with extensive data processing . However, untargeted metabolomics also suffers from practical challenges.

Plant metabolites span over a broad range of composition and physio-chemical properties, which makes it challenging to extract and identify all of them simultaneously with acceptable recovery using a single analytical procedure. In addition, low molecular weight plant metabolites differ by several orders of magnitude, ranging from femto- or picomolar to millimolar concentrations , which present sensitivity and accuracy challenges for the detection of less abundant metabolites . Targeted metabolomics, on the other hand, focus on selected classes of chemically characterized and biologically annotated metabolites. Targeted metabolomics is hypothesis-driven, in which a defined set of known metabolites are analyzed with significantly higher selectivity and sensitivity, focusing on a specific biochemical question. Calibration and isotopically-labelled internal standards are used for absolute quantitation of the metabolites under investigation. Metabolomic analysis demands careful attention to details at each analytical step including sampling, metabolite extraction, storage, instrumental analysis, data processing, and interpretation for factual representation and reproducibility.Hence, a robust experimental design, randomization, efficient reporting of experimental details as per the Metabolomics Standards Initiative guidelines, and submission of results in data repositories are highly advisable, to ensure reproducibility and consistency of the metadata obtained from metabolomic analysis . To compensate for the qualitative and quantitative variations among plant samples, biological replicates are essential, which results in more powerful statistical analysis. However, in case of high variability and limited sample availability, sample pooling is a common procedure to represent the population, which nonetheless should be reported and considered during data analysis . Although these requirements are quintessential for any metabolomic experiment, nanotoxicity studies should also consider ENM stability in the exposure media throughout the experiment duration, environmentally or agriculturally relevant dosing, ambient fluctuations, use of appropriate positive and negative controls, and comparison with ionic and bulk-particle controls to identify nano-specific effects.

Representative techniques and key challenges in each step of the metabolomic analysis in ENM-plant interaction research are summarized below .In a metabolomic study, a robust sampling protocol is critical in order to obtain maximum information from the plants exposed to ENMs. Some of the factors considered during sampling are: scope of the study, route of exposure , kinetic of ENM transport in the species under investigation, growth stage of the plant and time of the day when harvesting is done. Different plant organs such as root, leaf or fruit have distinct metabolic footprint owing to varying cellular organization. Thus, to prevent loss of information about chemical signaling across organs, it is necessary to analyze them separately. The metabolite profile of a tissue also varies significantly depending on the growth stage of the plant; hence, results from an ENM exposure study in a germinating seedling cannot be extended to compare or interpret metabolic events in the plant in its vegetative or matured stage, or vice versa. Tissues collected from untreated and ENM-treated plants should be immediately quenched in liquid nitrogen or freeze-dried to capture the metabolic state of the plant at the desired moment. Prior to freezing, tissues may be washed with water at room temperature, if necessary; cold or hot solvents must be avoided to prevent leakage of intracellular metabolites. Frozen tissues are ground to homogenous dry powder using precooled homogenizing apparatus, and appropriate extraction methods must be used depending on the analytical technique used. To prevent loss of unstable metabolites and to maximize analytical recovery through all the steps of extraction, adequate sample storage and handling is important. In untargeted analysis, the aim is to broaden the coverage of metabolites and maximize identification. The most common strategy is to extract a wide range of metabolites in solvent mixtures, such as methanol/ methyl tert-butyl ether/water or methanol/chloroform/water, followed by separation of the polar and non-polar metabolites by biphasic partitioning . Each phase is collected for analysis using suitable analytical platforms. In targeted analysis, specific extraction protocols are optimized to retain the metabolites of interest. Additional sample processing such as filtration or solid phase extraction are also used to reduce matrix effects and remove undesired metabolites.However, GC-MS is best suitable for volatile and thermally stable metabolites, and requires derivatization/ chemical modification of polar metabolites such as sugars, amino acids and organic acids .

In contrast, LC-MS allows analysis of polar and high molecular weight metabolites without any derivatization. For untargeted analysis, LC is hyphenated with high resolution MS like time-of-flight or Orbitrap mass analyzers with electrospray ionization . Nevertheless, LC-MS also suffers from drawbacks due to ion suppression, differential adduct formation and retention time shifts due to matrix effects. Different analytical approaches are however complementary, and can be used together to obtain a comprehensive coverage of metabolites with high confidence. Extraction of plant metabolites in solvent mixtures results in an immediate loss of inter- and sub-cellular resolution. Advances in MS imaging techniques have made it possible to map metabolites in intact plant tissues at sub-cellular level and connect the spatial complexity of the plant molecular organization to phenotypical features. Some techniques that have been hyphenated with MSI for imaging metabolites in plant specimens are matrix-assisted laser desorption/ionization and laser ablation electrospray ionization . However, these approaches have not yet been utilized in ENM plant interaction studies.In targeted metabolomics, low resolution MS such as linear ion traps or triple quadrupole are predominantly used . Advances in QqQ in single reaction monitoring mode allows robust absolute quantification of metabolites with high sensitivity and selectivity, even at trace levels with relatively high throughput. Column chemistry and its retention mechanism are extremely critical for targeted analysis of metabolites using LC-MS; and hence, reverse phase and hydrophilic interaction liquid chromatography are used to cover broad range of metabolites with contrasting polarity . Targeted analysis is particularly useful as a follow up from untargeted analysis, in order to focus on specific metabolic pathways or testing application of ENMs in pathogen defense, nutrient acquisition, photosynthetic efficiency or productivity.Data processing in metabolomics comprises baseline correction, feature extraction, spectral alignment across samples, identification and interpretation . Post data-acquisition, baseline correction is performed on the datasets to remove low frequency artifacts and aberrations across multiple samples that could be generated by experimental or instrumental variation. As chromatographic retention time may vary between samples, spectral alignment is performed across the dataset, which may precede or follow feature extraction process. In MS-based studies, peak-based algorithms are used to extract and quantify all relevant chromatographic and spectral information for all known and unknown metabolites in each sample. Upon detection, related ions indicative of a single-component chromatographic peak are identified and grouped . Several open source and commercial MS reference and MS/MS library matching databases are available; but the databases on plant metabolites is limited and they are not fully optimized for all analytical platforms. Electron ionization fragmentation patterns and indexed retention times for an extensive array of compounds using GC-MS and NMR spectral library are available; however, LC-MS data lack standardization in the available resources . However, due to an increasing number of studies in plant metabolism, high quality spectral and chromatographic data are continuously being added to and curated within these spectral libraries, which will eventually improve the routine peak identification in non-model plants. Software such as XCMS, PRIMe, MeltDB etc. are used for data processing including feature detection, retention time correction, alignment, annotation, statistical analysis, and data visualization. Another online tool, Metabo Analyst,hydroponic nft system is used to process identified metabolites for data normalization, statistic alanalysis, and visualization . Statistical analysis of the metabolomics data helps to identify features that are differentially regulated compared to control samples. The identified markers are then projected on to the available metabolic networks, which can be used in a phenotypic context to generate mechanistic hypothesis.Kocide-3000 is a commercial micron-sized pesticide, composed of copper hydroxide 2) nanosheets, which can be applied using aerial or ground spray in the field. Zhao et al. investigated the metabolomes of lettuce, cucumber, maize and spinach plants chronically sprayed with Kocide .

Twenty four-days-old lettuce plants were foliar sprayed with 1050, 1550, 2100 mg/l Kocide twice a week for four weeks . Although the chronic exposure did not induce overt response, metabolomic analysis of the leaves revealed differential accumulation of 50 metabolites ultimately affecting six biological pathways, including Gly/Se/Thr metabolism, Ala/Asp/Glu metabolism, TCA cycle, pantothenate and CoA biosynthesis, glycolysis or gluconeogenesis, and pyruvate metabolism. Chronic exposure to Kocide deteriorated the antioxidant defense mechanism in the lettuce plants expressed by decreasing levels of phenolic compounds like cis-caffeic acid, chlorogenic acid, 3,4- hydroxycinnamic acid. Dehydroascorbic acid, its oxidation products and GABA levels were also diminished at high exposure concentrations, suggesting impairment of the antioxidant system in lettuce. A chronic exposure of spinach leaves for 7 days to 1000 mg/l Kocide also resulted in depreciated levels of antioxidant or defense associated metabolites, including ascorbic acid, a-tocopherol, GABA, threonic acid, b-sitosterol, 4-hydroxybutyric acid, and ferulic acid, resulting in the perturbation of phenylpropanoid and ascorbate/aldarate pathways . However, the decreased levels of antioxidants and activation of antioxidant defense system in spinach leaves were driven by Cu ion release. In addition to amino acids , accumulation of phytol, a chlorophyll degradation product, was also decreased in Kocide-exposed spinach leaves, similar to Kocide-exposed maize and cucumber leaves . Metabolomic response in artificial soil-grown three-weeks old cucumber and maize plants exposed to 100 and 1000 mg/lKocide by foliar spray for a week was compared; however it is to be noted that the final dose in maize plants were higher than the cucumber plants due to hydrophobicity of maize leaves . In maize leaves, Kocide treatment induced accumulation of glycolysis and TCA intermediates thereby enhancing energy metabolism and enhance branched chain amino acids accumulation, which play important role in oxidative phosphorylation energy source. Kocide resulted in increased levels of polyphenols such as 4-hydroxycinnamic acid, 1,2,4-benezenetiol, and their amino acid precursors , thereby activating shikimate phenylpropanoid pathway in maize leaves suggesting induction of antioxidative processes. In cucumber plants, Kocide significantly affected the Arg/Pro pathway intermediates . In both plants, Kocide exposure resulted in reduced levels of unsaturated fatty acids and increased saturated fatty acid indicating an alteration of plasma membrane homeostasis.Metabolomic studies suggest that the toxicity effects in plants exposed to nAg is primarily attributed to the released Agþ ions . In 7-day old Arabidopsis thaliana plants, a 24 hexposure to 1 mg/L of citrate-nAg, polyvinylpyrrolidone -nAg and AgNO3 via roots resulted in alteration in six, three, and nine metabolites respectively, which were identified as fatty acid derivates.

Over the 6 years of study, except perhaps for 2013 when we focused on other study priorities, we never experienced ideal conditions to adequately test the flood extension concept. We were either in a severe drought, during which the Yolo Bypass did not flood from the river, or we experienced severe and sustained flooding, which made it impossible to contain flood waters within study fields. Based on these experiences, studying the concept of flood extension appears to depend on the occurrence of moderate flood events at the right time of year , provided fields are appropriately designed to hold water and allow efficient immigration and emigration of potentially large numbers of juvenile salmon. However, significant outreach and communication is necessary with landowners to maintain floodwaters on their fields during the natural drainage period. Because these events cannot be predicted well ahead of time, these communications—and availability of robust infrastructure—need to be constantly maintained even outside the flood extension period. As suggested in the previous section, such potential actions would need to be taken in a way that maintains hydrologic connectivity and salmon access, so that salmon can successfully locate potential managed habitats, use them for rearing, and then successfully emigrate from them at the appropriate time. Timing of such potential manipulations is critical because previous sampling has shown that salmon quickly emigrate from the floodplain during large scale drainage events , leaving relatively low densities of salmon in remaining ponded areas to potentially benefit from flood extension. Although our use of hatchery salmon gave us more experimental options during drought conditions, the use of these fish resulted in additional challenges. Our approach relied on a non-traditional use of hatchery salmon, which required a suite of permits and approvals to execute the project. As noted above, the project coincided with a major drought, so access to hatchery salmon was limited as a result of low salmon population levels. In addition, use of hatchery salmon affected the time-period when we could conduct experimental work. We were unable to test salmon response to early season flooding ,ebb and flow tray because the hatchery salmon were too small to receive coded-wire tags as required under our permit conditions.

Similarly, the timing of our work was affected by the availability of holding tanks at our partner hatchery , and by the availability of transport staff and vehicles to move salmon to our study site. While we were able to assess many important biological metrics in our study, direct measurement of the population-level effect of floodplain rearing on agricultural habitats proved elusive. A traditional approach to addressing this question involves inserting CWTs into very large numbers of experimental salmon and estimating the population response from expanded CWT recaptures in the ocean fisheries. Recoveries of CWTs in adult salmon from experimental releases made in the Yolo Bypass have generally been very low , making it difficult to get a high level of resolution with which to reliably compare survival rates, including with values in the literature. Although CWT recoveries could potentially be improved by increasing the number of tagged salmon, the effort required even to collect a single data point would be substantial and is limited by the availability of surplus hatchery salmon. A related issue is that it is difficult to design a survival experiment that provides a useful comparison to other management strategies or migration corridors. For example, it is challenging to assess the incremental survival value of flooded agricultural habitat versus adjacent perennial channels . Telemetry can partially address this issue, but current acoustic tagging technology does not allow estimates of survival once smolts emigrate from the estuary, and is also limited in the size of salmon that can be tagged. Ultimately, addressing the question of population-level effects will likely depend on a combination of measured field data, incorporating new methodologies for assessing survival to adulthood , and fish population models.Our observations must be considered within the constraints of the infrastructure in the Yolo Bypass during the time of our field work. In the intervening years since our field studies, there has been a substantial amount of progress in improving Yolo Bypass infrastructure to support native fishes. During 2017-2018, an inflatable dam fish barrier and collection facility was constructed at Wallace Weir at Knights Landing Ridge Cut . This facility can enhance potential water distribution options for managed flooding studies under relatively low flow conditions when only Yolo Bypass tributary flows are available, including sources from Colusa Basin, which may not always have suitable water quality for juvenile salmonids.

It is important to note, however, that these local water sources are not useful unless there is improved connectivity with the Sacramento River, allowing wild juvenile salmon to access seasonal habitat throughout the Yolo Bypass. To that end, the joint Environmental Impact Statement/Report was finalized in 2019 for a project that will improve connectivity between the Sacramento River and Yolo Bypass with a proposed notch in Fremont Weir . This proposed facility would allow managed flows at lower Sacramento River stages than the current weir structure, thereby increasing the frequency and duration of seasonal inundation, and providing improved access to the floodplain from the Sacramento River fish migration corridor. This upgrade is required as a condition of the 2009 Biological Opinion for Salmonids for long term operation of the federal and state water projects . Our study did not specifically address these new facilities or their operations, and how the concept of managed agricultural floodplain habitat can be integrated into the primary purposes of these improvements. Hence, potential use of flooded agricultural fields as juvenile salmon rearing habitat should be evaluated in light of both a modified hydrology and local land use and infrastructure changes. Additional research is needed to address the efficacy and suitability of different potential water sources, hydrology timing, connectivity with the Sacramento River, and related issues, such as the effects of operations on land use and other species or life stages .Using conservation easements to protect farmland from urbanization is a relatively new application of a technique that has been used in land preservation programs in the United States for about a century. Landowners who voluntarily sell easements or donate them for tax benefits, in effect give up development rights on their land in perpetuity while still retaining basic ownership . Only in the past 20 years in California, and slightly longer in several other states, have state and local programs employed easements for the express purpose of keeping farmland in production. The more traditional uses have been for preserving land with natural resource or recreational values, such as riparian areas, wetlands, habitat and trails. Thousands of agricultural acres — primarily grazing land — have been covered by environmentally oriented easements over the years. In part this is because farmland is a form of open space, a passive environmental amenity. But protecting agricultural production with easements intended to protect more specific natural resources, such as habitat and riparian areas, is more difficult for much of California’s farmland, because orchards, vineyards, vegetable cultivation and other intensive crop production constitute a “working” rather than “natural” landscape. While sustainable agriculture offers considerable promise for minimizing the impacts of farming on natural resources, intensive crop production still generally involves chemical applications, the use of heavy machinery and other industrial like activities.

We address it only in the context of conservation easements, by examining the agendas of local organizations with land conservation objectives. We identify 34 California organizations that emphasize farmland protection to varying degrees, and examine the mix of agricultural and other conservation purposes in their missions, drawing primarily from open-ended phone and personal interviews with program managers and from mission statements.The 34 California programs that fit our standards for having an interest in farmland protection include 30 nonprofit land trusts and four open space districts. Land trusts are nonprofit, community organizations founded and run by volunteers and small staffs, while open space districts are local government entities governed by publicly elected boards . All four of the state’s open space districts are included on this list. But the 30 land trusts are only a small portion of the more than 130 land trusts that operate throughout California, according to the 1998 directory of the national Land Trust Alliance . Among 20 different types of resource conservation purposes noted in the directory for individual California trusts , farmland/ranches ranked eighth in the frequency of mention. The top mentions were watersheds/water quality, rare species habitat, scenic views, wetlands, river corridors, trails and forests/timberlands. To select 30 farmland-oriented land trusts, we first identified 37 organizations that cited a farmland or ranch purpose in the LTA directory. Next, we added about 10 other trusts identified as having an agricultural orientation by other sources such as state and foundation programs, the American Farmland Trust and our own files. Finally, after speaking with program managers and reviewing mission statements, we narrowed the list down to 30 trusts. The final list does not include land trusts that acquired easements on grazing acres or other farmland primarily for environmental,4×8 flood tray rather than agricultural purposes. The two essential selection criteria were an expressed interest in the preservation of farmland as an agricultural resource and the intention to use conservation easements. For our purposes the farmland criterion encompasses both cropland and rangeland. The few California trusts that have forestry or growing of trees as their only “agricultural” activity were not included. Otherwise, we defined “farmland” and “agriculture” broadly. For example, one manager for a coastal land trust described the scope of his agricultural program as including, “all agricultural uses in accordance with sound and generally accepted agricultural management practices, such as breeding, raising, pasturing and grazing of livestock; production of food and fiber; breeding, raising and boarding horses, bees, poultry and other fowl; and planting, raising, harvesting other agricultural, horticultural and forestry crops.” In applying the second selection standard, use of easements, we emphasized intention rather than accomplishment. It was not necessary for an organization to have actually acquired specific easements. Rather, we deemed it sufficient to express an intention to acquire easements for farmland protection in the future. One justification for this liberal approach was recognition of how difficult it is for a new land trust to complete its first easement transaction, primarily due to funding limitations and landowner resistance . The trusts we identified are located in 29 of California’s 58 counties .

A few operate in more than one county, and the California Rangeland Trust has a statewide orientation with an emphasis on the Sierra foothills and Central Coast hillsides. Twenty-one of the 34 programs are concentrated in coastal areas, with 14 in the Bay Area alone . The Central Valley has nine and Sierra foothill counties have two. Least represented in relation to area and population, Southern California has only two organizations on our list, in Ventura and San Bernardino counties. No such programs are located in the major counties of Los Angeles, Orange, Riverside and San Diego.Both nonprofit organizations and public agencies are legally able to acquire and hold conservation easements on private properties that restrict their future use. The key is their noncommercial and public interest character, which under federal law allows them to accept contributions of money or land interests as tax deductions. Nonprofit land trusts specifically qualify under section 501 of the Internal Revenue Code. But the California land trusts and public agencies have distinctly different forms and operational procedures. Land trusts are independent, nongovernmental entities with no formal accountability to the community or its political bodies. Volunteer boards of directors, who fill their own vacancies, govern them and a number have small staffs and membership or donor rolls. Because of their grassroots origins, flexibility, citizen participation and conservation credentials, land trusts are an attractive alternative to local government as the major mechanism for acquiring and holding easements . No California county and city governments directly operate agricultural easement programs, although they frequently require the donation of easements as mitigation for approving development projects. The open space districts and several regional conservancies organized by state government are the only public agencies in California with active easement programs. Unlike the nonprofits, the districts are accountable to their communities via separately elected governing boards or are extensions of county government and the elected board of supervisors.

The data came from 661 respondents to a statewide survey fielded between May and July 2016, which achieved an overall response rate of 28% . In addition to UC Cooperative Extension professionals, the survey included people from organizations that are part of the knowledge network engaged in outreach, education and communication: producer groups, nongovernmental organizations, consultants, resource conservation districts, government agencies and others. While some respondents did manage farms, we were not targeting farmers but rather those who develop and deliver information to farmers. It would be useful for future research to extend the survey to farmer populations, specific consultant groups such as pest control advisors, and agricultural knowledge networks in other countries and U.S. states. Using the framework of the diffusion of innovation theory, our analysis tested the following hypotheses: extension professionals who perceive a greater relative advantage are more likely to adopt ICT; less likely to adopt ICT are extension professionals who perceive ICT as incompatible with their values and social norms, extension professionals who perceive ICT as too complex or time consuming and extension professionals who are uncertain about how to measure ICT effectiveness or strategically target audiences. In addition, we tested how demographic factors may be linked to ICT adoption, with the expectation that the patterns would be similar to the patterns in the general population. To pursue the possibility of an extension gap,ebb and flow table we tested whether UC employees had a lower ICT adoption rate relative to other types of extension professionals.We tested our hypotheses by first constructing dependent variables for the number of ICT platforms used and the frequency of social media use. The survey assessed ICT adoption by asking respondents if they used blogs, websites, email, mobile applications, Facebook, Twitter, Pinterest, Instagram or LinkedIn to communicate or learn about sustainable agriculture. We constructed a yes/no variable for each ICT platform. To zero in on the frequency of use for just the social media platforms , we followed the Pew Internet Survey in establishing the following categories of use: several times a day, once a day, a few days a week, every few weeks, or less often.

Importantly, the focus was on using social media for professional communication about sustainable agriculture, not personal use of social media. To analyze how perceptions about the attributes of ICT are related to ICT behavior, we constructed a social media frequency scale that calculated the average frequency of social media use across all five platforms, plus an “other social media” category. The scale ranged from 1 = do not use any social media to 6 = use all social media several times per day, with mean = 2.01. To calculate the number of total ICT platforms used, not just the social media platforms, we summed the number of platforms respondents checked; the numbers ranged from 0 to 9, with mean = 3.81. We then estimated multivariate models with social media frequency and total number of ICT platforms as dependent variables, and the four attributes of innovations as independent variables . Respondents’ perception of social media’s relative advantage was measured by averaging their responses to six statements related to its capacity to reach audiences and help extension professionals coordinate professional activities. These statements form a reliable scale ranging from 1 = strongly disagree on all statements to 5 = strongly agree on all statements, with mean = 3.71. Respondents’ perception of compatibility was measured in their responses to four statements: social media risks spreading fake news, there are positive incentives for its use, most colleagues use it, and it involves too much interaction with trolls. The response options did not form a reliable scale, so we included each statement as a separate variable in the analysis. Perception of social media complexity was measured in responses to these four statements: it takes too much time, it’s technically difficult to use, best practices are well known, and the large number of platforms is confusing. Again, the response options did not form a reliable scale, so we included each statement as a separate variable in the analysis. Lastly, respondents’ perception of the measurability of social media’s effectiveness was assessed. One statement suggested it was easy to measure effectiveness; the other statement suggested it was easy to identify appropriate audiences for social media. Responses were averaged into a reliable scale that ranged from 1 = strongly disagree on both statements to 5 = strongly agree on both statements, with mean = 2.32. To test the effect on ICT adoption of users’ demographic characteristics, we used the same dependent variables as described above, and we estimated the same models using the following demographic independent variables: sustainability attitude , age , income , UC system , male and education .

Figure 1 reports the overall adoption rates for the ICT platforms. In decreasing order of use, email was used by 92% of the respondents, followed by websites and Facebook , with Instagram and Pinterest having the lowest adoption rates. While the results for the most popular platforms echo the results for the general population, Twitter and LinkedIn were used relatively more by extension professionals because they are specifically intended for information dissemination and professional networking. Figure 2 reports the average temporal frequency indicated by each respondent for using just social media. For the general population, Pew reports that 55% of Facebook users and 23% of Twitter users access their accounts several times per day. In contrast, our sustainable agriculture stakeholders in California access Twitter and Facebook at the lower rates of once a day or a few days per week. The lower frequency of use for LinkedIn most likely reflects that the content changes more slowly than the events communicated on Twitter and Facebook. Figure 3 plots the coefficients from an ordinary least squares regression model for social media frequency and a Poisson model for number of ICT platforms , with the variables arranged in order of decreasing magnitude from the social media frequency model. Relative advantage had the strongest positive relationship with both the frequency of social media use and number of ICT platforms. Respondents who thought most of their colleagues used social media also used more ICT platforms, more frequently.Interestingly, the attributes of innovations were less important for the number of ICT platforms used than for the frequency of social media use, where the capacity to measure effectiveness and identify the audience had a positive influence and lack of time and concern about fake news had strong negative effects. The existence of professional incentives, confusion about the number of platforms and knowledge about best practices were unimportant in both models. Figure 4 suggests some demographic variables behaved in ways consistent with the general population: ICT and social media use was higher among female, younger and wealthier respondents. The results also corroborated the technology gap described earlier: UC employees used fewer ICT platforms and used social media less frequently than other respondents. More educated respondents also used social media less frequently, which contrasts to the general population, where educational levels are positively correlated with social media use. In the context of professional extension activities, UC system employees or those with advanced degrees may be stressed for time, perceive social media as incompatible with norms of scientific knowledge sharing or lack professional incentives.

The breakdown of UC respondents was 35% campus faculty, 13% Cooperative Extension specialist, 22% Cooperative Extension county advisor, 6% other academic title, 10% student/ postdoctoral scholar, 11% staff and 3% other. Sustainable agriculture stakeholders in both developed and developing countries are quickly catching up to the information revolution that has transformed society in the 21st century. Our results confirmed the usefulness of diffusion of information theory, which frames the debate about the benefits and risks of ICT in terms of innovation attributes related to relative advantage, compatibility, complexity, observability and trialability. Extension professionals clearly recognized the relative advantages for ICT in terms of quickly communicating with a more diverse and distant audience, but with less potential to coordinate on-the-ground activities. Extension professionals are more likely to capitalize on these relative advantages if their colleagues are also using ICT, and they have good tools for measuring effectiveness. The most important barriers for widespread adoption of ICT were time constraints, technical complexity and incompatibility between norms of scientific discourse and the reality of trolls and misinformation on the internet. These results support some concrete recommendations for organizations seeking to increase the use of ICT and make it more effective for extension professionals. Resources could be invested in developing a community of practice for aspiring ICT users interested in using ICT for outreach, with leadership from extension professionals with an established reputation for successful innovation. Communities of practice are one of the organizational concepts in e-eXtension, and are defined as informal networks of professionals with a common goal who regularly interact to share information and expertise . They can help creatively solve problems, transfer knowledge and develop professional skills and are effective where the network of individuals is distributed across many administrative units or system components, as is the case with extension professionals experimenting with ICT. A sponsoring organization can help foster a community of practice by identifying potential members,flood table providing organizational infrastructure for interaction and measuring effectiveness with appropriate metrics. Such a community of practice should document the potential advantages of ICT and provide information about best practices. It would increase awareness about how many extension professionals are using ICT, which would help create a community norm. The community of practice should include a diverse set of stakeholders, including digital technology specialists from outside of agriculture who are knowledgeable about different types of tools, altmetrics and social media strategies that are effective in digital communication. It is also important for agricultural extension organizations, including land-grant universities, to establish clear guidelines for recognizing the value of ICT as an extension tool that complements traditional communication strategies and ways of extending knowledge. If extension professionals know what counts in terms of documenting their professional activities for job performance evaluation, they are less likely to be confused and view ICT as a risky endeavor. Developing such guidelines would benefit from consultation by outside specialists with expertise in digital tools and measurement. The risks of misinformation and credibility may be some of the most important for extension organizations to address at a strategic level.

Such organizations typically desire to be perceived as impartial providers of evidence-based information. Social media platforms recognize that legitimate knowledge exists outside of Cooperative Extension but also provides a gateway for misinformation. At the individual level, the risk is not so much that a particular extension professional may make a mistake in communicating their own research, but rather they may unintentionally spread misinformation from others and be required to invest additional resources in sorting accurate social media information from misinformation. At the institutional level, social media’s democratization of information creates the fear of messages being corrupted, misinterpreted or simply lost in the wash of real information and misinformation. In both cases, it is important to avoid damaging the reputation of providing high-quality science communication. However, Bastos et al. provide some evidence that may mitigate these fears. Examining the topology of Twitter networks connected with UC Agriculture and Natural Resources Twitter users, Bastos et al. found that communities focused on specialized agricultural topics formed centralized networks in which a relatively small number of expert nodes collected and broadcast information to a large audience. In other words, relative to more general users and nonspecialists, technical experts become more central in the online networks and serve as important information hubs for specialized and technical issues. This suggests that social media communication is not completely incompatible with the traditional extension goal of providing hubs of expertise. Further research is needed to increase confidence in our results and recommendations. More systematic comparison between extension professionals and their clientele would corroborate the extent and nature of the technology gap. While our findings are relevant for extension professionals involved in sustainable agriculture, it would be important to generalize the research to other types of agricultural sectors, compare different commodity groups, directly survey farmers and extend the research to natural resource managers. Using big data approaches to monitor the dynamics and effectiveness of communication is also an important effort going forward.

The factors to which models respond vary among models and evolve as modelers attempt to make them more comprehensive and universally applicable. In contrast, some researchers who want to apply them do not have all needed inputs, or they may want to embed a crop model into economic or other models for analyzing responses across scales. Some researchers have used more comprehensive crop models to create reduced form crop models that have much fewer input requirements, run fast, and produce responses needed for specific applications . For example, Chikowo et al. used the APSIM cropping system model to generate parameters and variables needed to operate a much simpler field scale crop model . The reduced form summary model responds to nitrogen and phosphorus levels for different soil characteristics and management inputs. Dzotsi et al. used a similar approach, showing that reduced maize, peanut, and cotton models parameterized from the DSSAT CSM model accurately reproduced DSSAT results across time and space. Reduced form crop models allow researchers to produce situation specific summary models that approximate the responses of a more comprehensive model for use in broader scale analyses that may involve socioeconomic, livestock, and environmental sustainability components. Reduced-form crop models can be interpreted as the “production function” that is the foundation of economic production models , and can be linked to economic models to create “hybrid” models for policy analysis and impact assessment . Keating et al. demonstrated a similar process of summary model development, building from the foundation of a comprehensive set of crop soil and management system simulations. They developed a summary model from thousands of APSIM simulations with three key parameters that captured 88% of the variation in space and time of key water balance variables.A central element driving production, profitability, and efficiency in livestock systems is animal performance. Hence, the most commonly used livestock models are those that predict animal meat and milk productivity. Precursors to performance models have existed since the 1940s when the first feed requirements for livestock were developed . Since then,grow lights many have been built and refined regularly across the US and Europe . Nutrient requirements models are the workhorse of the feed industry for ration formulation and for recommending changes in feed management to farm advisors. These models are often based on a mixture of statistical regressions derived from experimental data plus mechanistic principles of the energetics and protein metabolism of mammals.

They also need an estimate of feed intake, perhaps the most important parameter. While these models are good for calculating feed requirements, dynamic models of digestion are more accurate at predicting the nutrient supply to animals under a wide range of conditions from the high-yielding dairy cow to the smallholder goat , because they predict intake more accurately, and they can deal with more complex diets and their interactions. Some models also predict methane production by ruminants and manure quantity and quality, which are important for estimating GHG emissions and the role of livestock in nutrient cycles. These models are typically used to answer ‘what if’ questions around the impacts of different feeding practices or changes of animal types on animal performance . Herd dynamics models follow herd evolution over time in terms of animal numbers and herd structure. Herd dynamics models usually start by splitting a herd into cohorts of different ages or weight, and sex. These cohorts are specified with different mortality, reproductive,selling and replacement rates. Adult females produce offspring at specified reproductive rates, which grow or die, become part of the next cohort, and get sold, and the cycle continues. The best of these models include interactions between animal nutrition and reproduction to drive reproductive and mortality parameters stochastically. This feature is important as feed availability or supplementation strategies have significant impacts on herd reproduction and performance. Some applications of herd dynamics models include estimating optimal stocking rates and carrying capacities, assessing the impacts of reproductive technologies and/or reductions in mortality, and predicting removal of biomass from crop or pasture systems. These models are also widely used by livestock epidemiologists for estimating impacts of diseases on herd mortality and morbidity. They have also been used with dynamic programming for optimizing replacement decisions in commercial dairy herds , or in linear programming applications for studying optimal sales policies, herd sizes, etc. Biological simulation models are sometimes used as input output coefficient generators for linear programming models to aid in the selection of management strategies in livestock systems .These models represent whole livestock farms and their key components . The complexity of some livestock systems justifies the need to build whole-system models using simulation and optimization techniques to represent different components and their interactions . For example, grazing management strategies cannot be defined without also considering herd and nutritional management, since herd dynamics or feed supplementation practices determine the grazing intensity, use of forage, and subsequently animal performance.

Thus, simulations of the biology of livestock enterprises include flexible models representing pasture growth, structure and quality; individual animal performance to test nutritional strategies; and population dynamics describing management practices at herd or flock level , which subsequently determine animal numbers and their age or physiological state classes .Biologists have been building mathematical models to describe the population dynamics of agricultural weeds, pests and diseases for more than a hundred years. Recent progress in modeling these components is discussed by Donatelli et al. ; here we focus more on broad concepts and general state of progress. The diversity of modeling approaches that constitute the current state of science can be categorized in different ways. The first and most obvious is by production type and threat. Thus there are models that describe the dynamics of weeds, diseases and pests that are threats to arable crops, the diseases of livestock, and the diseases of fish used in aquaculture. While threats such as pests and diseases have been recognized since pre-history, the complexities of the microbial communities on the crop surface and in the soil around plants, and in the gut and rumen, are only just becoming more fully understood. Models of the mixture of beneficial and pathogenic organisms that these systems contain have not yet been developed. A broad distinction can be made between mechanistic and non-process-based pest and disease models. The former include explicit biology while the latter use a purely statistical approach. The choice of modeling approach depends greatly on the intended application. For example, a farm manager may want to know when to apply a prophylactic insecticide against a common insect pest. For this purpose, future insect population density may be best predicted by a statistical model containing independent biological variables such as crop stage, and dependent weather variables such as temperature and rainfall. In some cases, information about the pest itself may be included in the model, for example from pheromone or other traps monitored by the farmer or in the case of mobile insects from publicly-operated monitoring networks. A different statistical application is the use of climate-matching models to predict future pest problems. The current distribution of an organism is modeled using a set of predictors including climate. The distribution of the organism after climate change is then estimated by mapping the “climate envelope” using scenarios developed from global climate models. There is now a broad literature on the strengths and weaknesses of this approach, particularly challenging the assumption that organisms are able to move easily to track climate. Important recent advances in statistical models of pest dynamics have included the application of modern spline and neural net estimation techniques, and in the use of personal computers and mobile devices. Mechanistic models incorporate at least some information about the biology of the crop and pest species concerned. The models may be highly abstract – summarizing, for example, a pest population by a single state variable such as density – or, alternatively, highly complex with individual pests each represented by numerous attributes. The simplest models sacrifice realism for mathematical tractability and general insights, while models of intermediate complexity include more biological detail but are constructed in such a way that simpler analytical models can be recovered as limiting cases to help interpretation. Pest and disease models also vary in the degree to which they explicitly incorporate stochastic processes and in whether they treat a population as homogeneous or spatially variable. An important area is the coupling of pest and disease models with crop models . Donatelli et al. review issues involved and existing major projects that have attempted to bridge this gap.

They also propose a road map to improve pest and disease modeling focusing on improving the data resources available for parameterization and validation, bettering the coupling of crop to antagonist models,led grow lights and creating a community of researchers that can collaborate to share expertise and produce community tools.Mechanistic models can be used to predict near-future pest and disease threats in similar ways to statistical models. As was discussed with crop models, they may be more successful than statistical models if biological insights can substitute for missing data or if they can aid prediction by suggesting a model structure that simple statistical fitting would miss. Consider, for example, the response of an insect to daily temperature. Higher temperatures may elevate growth and reproduction, and thus result in more pests, a pattern that could be derived with sufficient weather and population data. Alternatively, the physiological response of the insect could be modeled, which might improve the model’s predictive power or allow insect dynamics to be predicted in data-poor systems . Several schools of physiological modeling exist. However, we are not aware of any formal comparison of different process and statistical approaches to the same problem. An area where biological insights have proven fruitful has been in disease spread through commercial livestock populations. An understanding of how animals interact, and more importantly how they are moved around, can provide critical advice to policy-makers. Current state-of-the-art livestock models incorporate data on movements of animals between individual farms coupled with modern Bayesian parameter estimation. However, the type of data needed for such approaches is prohibitively expensive to obtain or politically unacceptable for many countries to collect . Modeling has also proved valuable in assessing possible pest risks and in guiding general policy development. The basic epidemiological number is the number of secondary cases of a disease that are expected to happen when a primary case occurs in a susceptible population. Calculation of R0 for prevalent human diseases has proved useful in prioritizing investment in control strategies and vaccine development. Today, sophisticated mathematical tools are available for calculating R0 for complex structured populations, for spatially extended populations, and in the presence of stochastic effects . Probably the most sophisticated applications of population genetics to weed, pest and disease issues in agriculture are models of the evolution of resistance to pesticides, and of the dynamics of plant diseases. Evolutionary models can be broadly categorized as genetic or phenotypic. Although phenotypic models have been explored in agriculture , the vast majority of evolutionary models have been genetic. Based on theoretical analyses, areas of fields have been set aside unsprayed or not planted with modified crops that express an insecticide in order to slow the rate of spread of resistance . The genetic basis of plant-pathogen interactions have been resolved for a number of major systems, which has allowed detailed analysis of strain dynamics and how disease spread may be slowed by judicious use of a range of different crop varieties. State-of-the-art work in genetic models of weeds, pests and diseases includes using the avalanche of data that modern high-throughput DNA measurement technologies are providing, and modeling how novel genetic interventions may be used to suppress pest populations. Some of the most sophisticated pest monitoring software now includes specific economic variables with parameters such as commodity prices that can be updated dynamically. The farmer may make different decisions about pest management depending on current market conditions. More generally, a goal of many people working to increase the sustainability of agriculture is to reduce chemical inputs by practicing “integrated pest management”. The models required to support such work are challenging to construct, but some of the most advanced incorporate economic elements as well as various biological processes.Linear economic optimization models of farm systems, developed in the 1950–60s, provide a basis for prescriptive farm management advice .