The appropriate transporters can give strategy I plants the advantage by giving them the ability to take up FeIII-PS complexes originally secreted from nearby strategy II plants. For example, iron efficient oat produces PS that are taken up by iron inefficient muskmelon and T3238fer tomato when they are hydroponically co-cultivated . If a strategy I plant is unable to take advantage of the FeIII-PS complexes in this manner, they are at a comparative disadvantage: soybeans which were unable to take up these compounds in the same studies suffered from increased iron-deficiency chlorosis as compared to plants grown alone. Another source of biotic complexity is found in the microbiome. Microbes take up iron and other essential nutrients using siderophores, molecules that are similar in structure to PS and which are taken up using similar transporters; the negatively charged groups of microbial cell walls also adsorb cations like FeII, thus removing them from the soil solution . While there is general interchange between plant and microbial siderophores , PS also serve as attractive carbon sources for microbes in the soil . Microbial metabolisms can act to decrease the soil pH, particularly in FeII poor environments ; in conditions where the necessary cations are freely available, however, the microbiome can act to increase the pH of the soil . The impact of the soil microbiome on iron uptake strategies that do not depend on PS can therefore be acute, as FeII availability depends heavily upon soil pH. Those lines classified as maxima tend to produce only one or two large, pendulous panicles, and do not generally tiller. Indica lines appear to be an intermediate between the other two races in terms of tiller number and panicle habit . While maxima lines conform best to the expected domestication syndrome,vertical aereponic tower garden there exist elite cultivars that are considered to be of the moharian race.
The tripartite morphological division is not universally accepted: Li et al.apply a fourth designation, nana, to the cultivars that most closely match the phenotype of S. italica subsp. viridis. In their designatory scheme, moharia has more domesticated characteristics than does nana, with relatively fewer tillers and shorter culms. There is a strong correlation between morphotype and location of cultivation: moharian lines are grown in Europe and southwest Asia, maxima in transcaucasian Russia and eastern Asia, and indica lines in India and southeast Asia. Li et al. claim that nana is found in Lebanon, Iran, and Afghanistan. It is apparent that, though race, degree of domestication, and geographic origin are inextricably intertwined, these factors do operate independently of each other to an extent, largely due to intensive breeding and cultivation in the United States, China, and Europe. Because maxima is the most suitable for modern cultivation practices, it is likely that the cultivation of this race has been preferred in regions where intensive breeding of S. italica has been carried out. The regional associations discussed in this paragraph were largely replicated in a RFLP analysis of the ribosomal DNA intergenic spacer sequence, with the exception of the nana race, which was not clearly present in their analysis. Additionally, the RFLP analysis suggests that there is recent shared parentage between most African and Indian lines. The known variation in iron content in S. italica combined with the likelihood that one or more of the factors described above suggest that there exists unexplored variation for this phenotype in the Setaria species complex. A study was therefore devised to determine if sufficient variation existed for improvement of the iron content in the species. A hydroponic system was developed to assay the impact of varying concentrations of iron on the morphology and ionome of numerous accessions of S. italica collected from around the globe. All three races were represented in these accessions. A hydroponic system was used to assay the ionic content, as these systems are excellent tools in plant nutritional studies .
As a complement to the hydroponic system, the accessions were also grown in soil and analyzed in the same manner as the hydroponic system. Unsupervised machine learning methods were leveraged to group morphological and ionomic observations. These data revealed three distinct morphological groups segregated along geopolitical boundaries and two distinct ionomic groups containing accessions of mixed geopolitical origins, thus indicating that morphology does not define the S. italica ionome. Additionally, valuable data were collected on the influence of morphotype on robustness and response to hydroponic growth. The insights gained through this study demonstrate that sufficient natural variation in Fe homeostasis exists in the S. italica pangenome for use in biofortification.Two other iron media were also used: a solution containing 1 mM FeEDTA and a solution containing 0.06 mM FeEDTA. The high iron concentration was selected based on Santana et al. 2014, which illustrated that leaf bronzing, which is a symptom of iron toxicity, can be visible at 1mM FeEDTA in Paspalum urvillei, a grass that can tolerate high iron soils. In the same study, Setaria parviflora, a hyper accumulating species, showed bronzing at 2 mM FeEDTA. The intent of this experiment was to supply the plants with a stressful concentration of iron without killing them. Because most species are, by definition, neither hyper accumulators nor hypertolerant of excess iron, the highest concentration of iron that did not visibly stress S. parviflora was used. The low iron concentration was selected based on the interactions between iron and potassium in maize that were elucidated in Celik et al. 2010. When exposed to conditions of potassium ranging from 1-8 mM and 0.03 – 0.12 mM FeEDDHA in an otherwise standard nutrient medium, the dry weights of maize roots and shoots reached their peaks at 4-6 mM potassium and 0.12 mM FeEDDHA. At 0.06 mM FeEDDHA, the dry root weight was decreased significantly in all potassium regimes, and the dry shoot weight was decreased significantly in all but the 8 mM potassium treatment. This concentration of ferric chelate was therefore used in an attempt to produce chlorotic plants that still set seed.
All media types were produced in 20x solutions,vertical gardening in greenhouse which were adjusted to a pH of 6 using NaOH and glacial acetic acid where necessary. Germinated seeds were rooted in a fully hydrated mixture containing 70% perlite and 30% vermiculite when their radicles had reached approximately 1 cm in length. Seedlings were blocked by experimental treatment. Each block was supplied with 50% nutrient medium the day following planting through bottom watering. Every three days thereafter, seedlings were supplied with the appropriate nutrient medium at full strength. When the seedlings had reached the four or five leaf stage, they were transferred to a hydroponic system . All S. italica varieties were placed in the system on the same day; S. macrostachya and S. sphacelata reached the appropriate stage and were placed in the system two weeks after the other varieties.HU containing plants of the same genotype were placed in physical proximity in an attempt to control for the effects of micro-environments. Once in the system, plants were supplied with fresh nutrient medium every three days. Leaf number, shoot height, tiller number, and panicle number were assessed on those days. Within the first ten days, 24/90 plants had died; by 13 days post hydroponic placement , that number had increased to 40/90. Examination of the system revealed that the most likely cause of this phenomenon was the physical position of a plant’s HU. Plants placed in the two rows closest to the western windows had a much greater likelihood of dying than their neighbors. These plants had HU that were hot to the touch by the late afternoon. Several steps were taken in response to this issue. First, HU positions were altered every three days. All units were transferred to positions previously occupied by another unit in their treatment group in order to control for physical position and to ensure that all plants occupied the same positions in the course of the experiment. Secondly, all plants were separated into individual HU on 8/24/2016 in order to remove the discrepancy in treatment between plants with living neighbors and plants without. Plants were harvested when they had set seed. Due to the broad range of domestication traits in the cultivars assessed, the date of collection varied widely; while White Wonder produced only two panicles per plant, some produced upwards of 100. In spite of the short life span of most Setaria species in soil, many of the cultivars assayed in the hydroponic system appeared to thrive in the context of the hydroponic system, with new tillers and panicles emerging until the date of harvest, six months post germination. These indeterminate plants were harvested simultaneously. Plants were dried for one week before collection of final morphological data, including dry root and shoot weights. Material was collected from the flag leaf on the main axis of the plant and sent to the Baxter lab at the Donald Danforth Plant Science Center for mass spectroscopic ionomic analysis . The Baxter lab has a documented, standardized pipeline for elemental analysis .
Flag leaf samples collected from each plant were dried, weighed, and digested in 2.5 mL of concentrated nitric acid with internal standard added . They were then diluted to 10 mL using ultra pure water from a Milli-Q system . Concentrations of the elements B, Na, Mg, Al, P, S, K, Ca, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Rb, Sr, Mo and Cd were measured in each sample using an Elan 6000 DRC-e mass spectrometer connected to a PFA micro-flow nebulizer and Apex HF desolvator . Nitrogen concentration was not considered, as this technique does not allow for its measurement. A calibration curve was produced before each run by analyzing six dilutions of a stock solution that was produced by mixing multiple single element standards .To reduce interference due to the presence of polyatomic and double charged species, the lens voltage and nebulizer gas flow rate of the ICP-MS were optimized for maximum Indium signal intensity and low CeO+/Ce+ and Ba++/Ba+ ratios. Machine drift within and between runs was corrected for by the inclusion of a control solution after every ten samples. This control sample was the result of mixing the remaining samples from the second dilution. The control therefore reflected any drift in the sample matrix. This same control was used in each run, so that inter-run variation could be corrected.K fold cross-validation shows that Fe was the most important feature for the designation of ionomic groups using DBSCAN. The importance of Fe in determining these groups suggests that there is sufficient variation in the homeostasis of Fe to differentiate between groups. Additionally, Fe homeostasis is clearly associated with genotype in Setaria italica: Group 1 consists of all assayed African accessions and one Indian accession, and Group 2 consists of two elite cultivars and the lab strain of Setaria viridis known as A10. Though the accessions in Group 2 are not morphologically similar, they have been cultivated in similar environments for the past two decades ; it is possible that the consistent supply of fertilizer that both lab specimens and elite varieties enjoy has shaped their ionome. A similar phenomenon has occurred in Arabidopsis thaliana: though this species only became a common model organism in the 1980s , by 1997 both the Col-0 and Ler genotypes had lost secondary seed dormancy . This sort of unconscious domestication is common, having occurred in fruit flies, bacteria, and the green alga Chlamydomonas, among others . Group 2 accumulated more than twice the amount of iron as did Group 1, which may be a result of the prolonged, heavy use of fertilizer on the accessions in Group 2. Fertilizers containing high levels of phosphorus are applied regularly to both lab strains and elite varieties cultivated in the United States and Europe, with application levels since 1980 ranging from 0.5 grams of phosphorus per meter squared per year to triple that amount . In contrast, phosphate fertilizer use in Africa has held steady at less than 0.5 g P/m2/yr in the same time period.