Since the deletion mutants showed similar root length as the 1RS line, we decided to focus on the 14 genes expressed in roots that were deleted in both the 1RS duplication and the adjacent and orthologous 1BS insertion . Although the annotated functions of these genes based on conserved domains and homology will require further experimental validation, the list is useful to summarize their inferred functions and to provide a preliminary idea of potential candidate genes. The first group includes four genes annotated as defense genes, a function that is likely not closely related with the observed phenotypes. This group includes TraesCS1B02G017500 and TraesCS1B02G0017600 , which encode proteins with NB-ARC and LRR domains characteristic of plant disease-resistance proteins involved in pathogen recognition and activation of immune responses. It also includes TraesCS1B02G017700and TraesCS1B02G0018100 , which are both annotated as defensins, a family of small plant antimicrobial peptides that serve to defend plants against pathogens. A second group includes three genes annotated as having enzymatic or housekeeping functions, which may not be compatible with the developmental nature of the observed changes in the roots of 1RSRW. The first gene in this group, TraesCS1B02G017800, encodes a methionine Smethyltransferase that has been implicated in the volatilization of selenium and in the biosynthesis of S-methylmethionine,indoor vertical farming a compound that is important in the transport of sulfur .
The last two genes in this group encode proteins with chaperon functions. TraesCS1B02G019200 is a tubulin-folding cofactor E involved in the second step of the tubulin folding pathway. TraesCS1B02G019300 encodes a chaperone protein DnaJ, which stimulates the heat-shock protein Hsp70’s ATPase activity, stabilizing its interaction with client proteins. These chaperon proteins play important roles under plant stress but are unlikely to play an important role in the phenotypic differences we observed under optimal hydroponic conditions. The third group includes genes involved in regulatory processes or in cell growth or division, processes more likely to be involved in the observed developmental changes in root growth . TraesCS1B02G017900 encodes an E3 ubiquitin-protein ligase CHIP-like protein that ubiquinate heat shock misfolded client proteins, targeting them for proteasomal degradation. Since E3 ubiquitinprotein ligases can ubiquitinate and regulate multiple targets, we could not rule it out as a potential candidate gene. We also included in this group the genes TraesCS1B02G018900 and TraesCS1B02G0019100, which encode 64% similar small GTP-binding proteins from the RAB family. These conserved proteins serve as molecular switches in signal transduction and play important roles in intracellular membrane trafficking, cross-talk with plant hormones and regulation of organogenesis, polar growth, and cell division , all functions that seem relevant to the observed differences in root development.
TraesCS1B02G018700, TraesCS1B02G019700, and TraesCS1B02G019800 encode 12-oxophytodienoate reductase-like proteins involved in the biosynthesis of jasmonic acid. Since hormones can affect multiple developmental traits, these are also strong candidate genes. Finally, TraesCS1B02G020200 encodes a wall associated receptor kinase . These serine–threonine kinases are involved in signaling and cell expansion, making it an interesting candidate for the differences in root length observed in 1RSRW.Nanoparticles are materials with at least two dimensions between 1 and 100 nm. The small size and large surface area provide NPs with different physical strength, chemical reactivity, electrical conductivity, magnetism, and optical effects, compared to bulk materials. These special properties allow NP utilization in electronics, engineering, energy production, catalysis, pharmaceutics, cosmetics, textiles, food industry, and agricultural products, among others.Recent statistics indicate that 1317 products containing NPs were on the market in 2010, and it has been estimated that there was a global release of 22 000–80 400, 1100–29 200, and 590–4800 metric tons of engineered nanomaterials into soil, water and atmosphere, respectively. The increase in NP utilization has raised concerns about their release into the environment and possible impacts on living organisms.Very few studies have reported the effects of these two Cu-based NPs to terrestrial plants.Shah and Belozerova15 found that the shoot/ root ratio in lettuce plants treated for 15 days with 0.013% nCu in soil was 2.7, while in control plants the ratio was 1.4. This suggests nCu affects lettuce growth. Another report indicates that, compared to control, 1000 mg nCu/L reduced by 77% the root length and by 90% the biomass of Zucchini grown in hydroponics.
Similarly, Musante and White16 observed that nCu not only affected root length and biomass of zucchini, but also reduced the transpiration volume by 51% in plants exposed to 100 mg nCu/L and 61% in plants treated with500 mg nCu/L. Lee et al. reported that the seedlings’ length of Mung bean and wheat were reduced by 60% and 75%, respectively, when exposed to 1000 mg nCu/L. They also observed less reduction in shoot growth compared to root, which could be associated with low translocation of NPs from roots to shoots.nCuO has also shown to affect plants in different ways. Reports indicate that nCuO did not affect seed germination in zucchini10 and maize ;however, other reports mentioned root length reduction in wheat and duckweed ,and DNA damage in radish , perennial ryegrass , and annual ryegrass .The activity of antioxidant enzymes such as catalase and ascorbate peroxidase can be affected in Cu exposed plants. These enzymes, which are overproduced under abiotic or biotic stress, protect plants from reactive oxygen species damage. Excess ROS can damage proteins, lipids, and DNA.Hou et al.reported that Cu2+, up to 10 mg L1 , increased CAT activity in duckweed in a concentration-dependent manner; however, a reduction in CAT activity was observed when Cu2+ was higher than 10 mg L 1 . A similar trend was observed in APX activity on duckweed treated with copper sulfate.25 nCuO were also found to increase CAT activity in cucumber and wheat.Besides the effects of nCuO on seedlings growth and CAT and APX activity, no reports were found about the effects of Cu NPs/compounds on nutrient uptake by crop plants. Recent literature has shown that nanoparticulate forms of Cu are more effective against pathogenic fungi than the corresponding bulk forms.Thus, very likely, in the near future, there will be an intensive use of nCu and nCuO in agricultural practices. In addition, CuPRO 2005 and kocide 3000 2- based materials have been cataloged as nanoparticulate Cu species. These compounds are intensely used in agricultural production due to their fungicidal properties.Thus,hydroponic vertical farming the possibility of Cu-based NPs contamination of food supply and entrance into the food chain is increasing. However, it is not well understood yet if their effects are different from those of Cu bulk materials. Thus, three Cu-based NPs and other Cu compounds were evaluated in lettuce and alfalfa . The plants were selected because they are intensely produced and exposed to pesticides. Alfalfa is one of the most important forage crops worldwide and lettuce is cultivated worldwide and eaten in a raw form by many people. In this study, root and shoot elongations were measured with a ruler, macro and micro-nutrients’ uptake by using inductively couple plasma-optical emission spectroscopy , and enzyme activity with UV-Vis.The Cu particles used in this study include nanoparticulate CuO , micron-sized Cu and CuO , nanoparticulate Cu , Kocide 3000 , and CuPRO 2005 . Reagent grade CuCl2 salt was obtained from Sigma Aldrich. The size and surface charge of particles at pH 7 were determined by measuring hydrodynamic diameter and zeta potential using a Zetasizer Nano-ZS90 . A previous study showed that phosphate buffer, at the concentration used, had only minimal effects on zeta potential measurements. Primary particle size and morphology were determined via scanning electron microscopy equipped with an Oxford INCA energy-dispersive X-ray spectroscopy probe. Copper content of each particle was determined via ICP-AES .The main copper phase and crystal structure of particles were determined via X-ray diffraction . Cu NPs/compounds’ suspensions/solutions were prepared at 0, 5, 10, and 20 mg L 1 in modified Hoagland’s nutrient solution36 and homogenized by sonication in a water bath at 25 C for 30 min.
There were four replicates per treatment.Seeds of alfalfa Mesa variety and black seeded Simpson lettuce were stirred in 4% ClONa solution for 30 min, rinsed with deionized water three times and kept in DI for 24 h. Subsequently, seeds were rolled in auto claved wet germination paper towels, as described by Carrillo-Casta˜ neda et al.Ten drops of antimycotic/antibiotic solution were added to the seeds before the paper was rolled. The rolls were put into Mason jars containing approximately 10 ml of DI, incubated in the dark for four days and exposed to light for one day. After that, the young plants were transferred into magenta boxes containing 300 mL of modified Hoagland’s nutrient solution. All the boxes and lids were covered with aluminum foil to prevent algae growth. Aquarium pumps were used to aerate boxes, which were put in an Environmental Growth Chamber with light intensity of 300 mmol m2 s 1 , 25/ 20C day/night temperature, and 65% relative humidity. After 10 days of growth in the nutrient solution, seedlings were transferred to magenta boxes containing the Cu NPs/ compounds suspended in nutrient solution and were cultivated for 15 days. Subsequently, plants were removed from the growth medium, washed with tap water and rinsed with DI. The length of the primary root and shoot for each seedling was measured with a ruler and the samples were saved for further analyses. There were 20 young plants per replicate.Major physicochemical properties of the particles are presented in Table S1. nCuO has particles between 10–100 nm, but much larger particles were found in nCu . A reasonable fraction of bulk CuO particles were less than 1 mm but bulk Cu particles were much larger and diverse in morphology. Bulk Cu contains dendritic, plate-like, and irregularly-shaped particles . Kocide 3000 and CuPRO 2005 mainly consist of micron-sized spherical particles, and the main copper phase in them are orthorhombic Cu2. While bulk Cu only contained Cu as the main phase, the presence of Cu2O was observed in nCu, possibly due to higher reactivity of the nanoparticles which may lead to slight oxidation during synthesis and/or storage. All the particles were negatively charged at pH 7 as confirmed by zeta potential measurement. The presence of a small amount of carbon was observed in the nanoparticles, possibly from a surfactant used by the manufacturer to stabilize the NPs. In addition to carbon, Kocide 3000 and CuPRO 2005 also contain oxygen, sodium, aluminum, and silicon .The size of roots of alfalfa and lettuce plants treated for 15 days with Cu NPs/compounds is shown in Fig. S1. After 15 days of treatment, the roots of plants treated with Cu NPs/compounds, showed brown color compared to controls. As seen in Fig. S1,at all concentrations, all Cu NPs/compounds significantly reduced root length in both plant species. The shortest root in lettuce and alfalfa occurred in plants treated with 20 mg L 1 nCuO and nCu, respectively. The reduction of root length was 49.5% 7.8% in lettuce and 47.6% 1.0% in alfalfa. Similar results have been reported for Mung bean, zucchini, and wheat.10,16,17 Lee et al.17 reported that nCu at 200 mg L 1 affected the roots of wheat, while only at 800 mg L1 reduced wheat shoot length. Another report indicates that 10 mg nCuO/L reduced the length of root and shoot in radish seedlings by 46% and 4%, respectively.21 Moreover, 1000 mg nCuO/L, decreased the root and shoot length of radish by 97% and 79%, respectively. In our study, although the Cu concentration was high in alfalfa shoots, compared to lettuce, there was no shoot length reduction; however, bulk Cu, CuPRO 2005, Kocide 3000, and CuCl2 at 20 mg L 1 , significantly reduced lettuce shoot length . This suggests a species specific response to Cu toxicity. A previous report indicated that in Vigna unguiculata, Cu caused a reduction in shoot growth because it also caused a reduction in Fe.In our study, almost all the Cu NPs/compounds reduced Fe in tissues of lettuce and alfalfa . We hypothesize that differences on Cu accumulation were responsible for the different effects of Cu NPs/compounds on plant growth in both plant species. Copper in tissues triggers direct production of ROS via Fenton and induces root growth inhibition.All Cu compounds, at all concentrations, reduced P uptake in roots of both plant species .