Plant materials and growth conditions
Tobacco seeds (Nicotiana tabacum var. Turkish) were purchased from Altin Tohumculuk Company (Konak, Izmir, Turkey). Two week-old seedlings of 5 cm height and the same number of leaves were carefully removed from the soil and transferred to a hydroponic system-Hoagland’s nutrient solution- (Caisson Laboratories Inc., North Logan, UT, USA), in a growth chamber (Bionex, model VS-3DM, Bucheon, Korea) at a16-h photoperiod, a photosynthetic photon flux density of 250 to 300 μmol m− 2 s− 1, day/night temperatures of 30/23 °C, and relative humidity of 60/30%. TEM-checked (Fe3O4) NP of three different sizes (5, 10, and 20 nm) from Sigma-Aldrich (St. Louis, Missouri, USA) were prepared in three different concentrations 3, 10, and 30 mg/L for each size. ImageJ measurements were taken to confirm the internalization of these NPs inside the treated plants (Additional file 1: Figure S1). The experimental design was a randomized complete block, with three treatments and three replicates per treatment.
Shoot heights and root lengths were measured using a metric ruler. Leaf area (cm2) was measured for all control and iron oxide-treated plants using a Licor leaf area device (Model 3100 Area Meter, Lincoln, Nebraska, USA).
Five leaf discs (1.0 cm diameter) were excised from two mature leaves avoiding mid-ribs. Discs were carefully ground with a pestle in 5 ml 80% acetone on ice. Then, extracts were refrigerated in the dark for 2 h. The extracts were transferred to a centrifuge tube. An additional 5 mL of 80% aqueous acetone solution was used to wash the pestle and added to the extract. Extracts were centrifuged for 20 min at high speed (approximately 500x g). The supernatant solution was decanted and the volume brought to 10 mL with 80% aqueous acetone. Spectrophotometric analyses of all samples were determined at 645 nm and 663 nm (UV-M51 UV-VIS, BEL, BEL Engineering, Monza, Italy) and used for calculations of chlorophyll concentration.
An infrared gas analyzer (IRGA) (CI-340, CID Bio-Science Inc., Camas, WA, USA) was used to measure the net photosynthetic rate (PN), stomatal conductance (gs), transpiration rate (E). Briefly, the third and fourth leaf from the top of each plant were selected for the measurment. All measurements were taken at PPFD of 1300 μmol m− 2 s− 1.
Sugar and protein content
Anthron method was used to determine the sugar content in control and NP-treated leaves [27, 28]. Briefly, 100 mg of control and NP-treated leaf samples was ground in liquid nitrogen. Then, the powder was extracted in 5 cm3 of 80% (v/v) ethanol, and centrifuged at 1500 rpm for 10 min (a low speed centrifuge TD6, Pingfan Instruments, Changsha, China). A water bath at 80 °C was used to evaporate the supernatant. Then, 1 cm3 of distilled water was added and after 3 min, 4 cm3 of a freshly prepared 0.2% (m/v) anthron solution [0.2 g of anthron dissolved in 100 cm3 of chilled 75% (m/m) H2SO4] was added and stirred well by a vortex. The mixture was heated in a water bath at 95 °C for 15 min and then rapidly cooled. The absorbance of the solution was measured at 630 nm using a spectrophotometer (UV-M51 UV-VIS, BEL, BEL Engineering, Monza, Italy). Distilled water was used as blank, and as reference, D-glucose solutions were used for a calibration curve.
On the other hand, Bradford method was used to estimate the protein content in control and NP-treated leaves . For controls and all NP-treated plants, 1 gram of ground leaf tissue sample (ground in liquid nitrogen) was placed in a tube. Then, 3 ml of phosphate buffered saline (PBS) (PH 7.4) was added to each tube, and the tubes were incubated overnight at 4 °C. Then, all tubes were centrifuged (5000 rpm (1118 g), 10 min, 4 °C), and from each tube a 20 μl aliquot of the supernatant was transferred to a 2 ml Eppendorf tube. Then, 980 μl of PBS and 1 ml of Coomassie Brilliant Blue G-250 were added. The absorbance of the solution was measured at 595 nm using a spectrophotometer (UV-M51 UV-VIS, BEL, BEL Engineering, Monza, Italy). The concentrations of protein for the control and NP- treated plants were determined by plotting a standard curve using bovine serum albumin (BSA) as a standard.
Light and transmission electron microscopy
Mature root and leaf samples (0.5–1.0 cm) from control and NP -treated plants were prepared for light and transmission microscopy following the same protocol as described by Alkhatib et al. . Briefly, samples were fixed in 2.5% (m/v) glutaraldehyde and post-fixed in osmium tetroxide (EMS, Hatfield, PA, USA). After fixing, the samples were dehydrated in an ascending ethanol series (50, 70, 80, 95, and 100%, v/v) for 10–15 min each, and embedded in a freshly prepared Araldite resin. Light microscopy samples were examined using a light microscope (Micros MCX50, Austria) equipped with a digital camera (Amscope, MU100, China). For transmission electron microscopy, ultrathin sections (70 nm) of root and leaf samples were cut with a diamond knife (Diatome Ltd., Bienne, Switzerland) using a Leica ultramicrotome (Leica, Switzerland), stained with uranyl acetate  then with lead citrate , and examined using a transmission electron microscope (TEM) running at 80 KV (FEI Morgagni 268, FEI, Netherland) equipped with a Mega View III soft imaging system.
Statistical analyses for all parameters (shoot height, root length, leaf area, chlorophyll content, net photosynthetic rate, stomatal conductance, transpiration rate, sugar content, and protein content) were conducted using PC SAS (v. 9.2; SAS Institute, Cary, NC, USA). One-way and two-way ANOVA were used to assess significant differences in sizes, concentrations, and interaction between size and concentration for each parameter tested at α = 0.05. A post hoc Tukey test was used to estimate pairwise comparisons for significant results from the 2-way ANOVA. Graphs of physiological parameters were made using Graph pad Prism 5.