Proteomic and Morphological Analyses of Post-Flooding Recovery in Soybean Root Exposed to Aluminum Oxide Nanoparticles ○Farhat YASMEEN 1, 2, Naveed Iqbal RAJA 1, Ghazala MUSTAFA 2, Setsuko KOMATSU 2 1 PMAS-Arid Agriculture University, Rawalpindi 46000, Pakistan 2 National Institute of Crop Science, NARO, Tsukuba 305-8518, Japan 40 80 Nanoparticles are playing an influential role in various fields of life. Among them, aluminum oxide (Al2O3) nanoparticles have been produced and used in various commercial and agricultural products. Soybean, which is a flooding intolerant legume, exhibits severe reduction in growth resulting in economical loss. To elucidate Al2O3 nanoparticles role in recovery of soybean under flooding stress, a gel-free proteomic technique was used. Flooding exposure for 2 and 4 days reduced seedling weight; however, soybean unveiled recovery at 50 ppm after stress removal. Fresh weight of root including hypocotyl was improved at 50 and 500 ppm, but tremendous recovery was at 50 ppm. Similarly, length of root including hypocotyl exhibited remarkable improvement at 50 ppm as compared to other concentrations after removal of flooding stress. Recovery on 50 ppm significantly improved morphology. Based on morphological results, soybeans were flooded without or with 50 ppm Al2O3 nanoparticles for proteomic analysis. A total of 211 common proteins that significantly changed in abundance under flooding without or with Al2O3 nanoparticles were mainly related to proteins, stress, cell wall, and signaling. Hierarchical clustering divided identified proteins into three clusters. Cluster II exhibited greatest change in abundance of proteins related to protein synthesis, transport, and development under flooding with Al2O3 nanoparticles. These results suggest that Al2O3 nanoparticles might be helpful to soybean in recovering from flooding stress by regulating its metabolic processes.  Abstract   68 211 165 Flooding 279 proteins Flooding + Al2O3 NPs 376 proteins (-) (-) (-) Fig. 2. Functional categorization of identified proteins in soybean flooded without or with Al2O3 NPs. Two-day-old soybeans were flooded without or with Al2O3 NPs. Proteins were extracted from root including hypocotyl, and identified using gel-free proteomic technique. The identified proteins for flooding without or with Al2O3 NPs were 279 and 376 respectively. MapMan bin codes were used for functional categorization of identified proteins. Soybean flooded with Al2O3 NPs exhibited significant increase in proteins associated with proteins, stress, cell wall, and signaling. Introduction Nanoparticles, having one dimension less then 100 nm in size and being unique in their characteristics, are becoming area of research interest (Nowack and Bucheli, 2007). They have exhibited the potential to develop tolerance in plants against various stresses. Among abiotic stresses, flooding is a major abiotic stressor, resulted in growth reduction and death in maladapted species (Bailey-Serres and Voesenek, 2008). Al2O3 nanoparticles are most profusely used in various military and commercial applications (Handy et al., 2008). They have promoted the growth of soybean seedlings under flooding stress by affecting protein abundance involved in oxidation reduction, stress signaling, and hormonal pathways related to growth and development (Hossain et al., 2015). They have regulated energy metabolism and cell death in soybean at germination stage under flooding stress (Mustafa et al., 2015). Wide discussion on plant responses to flooding stress have been done but few reports are on consequent processes involved in post flooding recovery. Predominant effect on cell wall metabolism and cytoskeleton reorganization (Salavati et al., 2012) and an increase in peroxidases in soybean root during recovery has been reported (Khan et al., 2014). Keeping in view the positive effect of nanoparticles on plants and proteomic variations under flooding stress including post flooding recovery, a proteomic study was planned to elucidate role of nanoparticles in recovery from flooding stress. To investigate the role of Al2O3 nanoparticles in recovery of soybean, morphological analysis was performed under flooding without and with Al2O3 nanoparticles. Based on morphological analysis and survival percentage, 50 ppm Al2O3 nanoparticles were used for gel-free proteomic analysis.     Flooding Flooding + Al2O3 NPs Log2 FC -1 -2 -3 0 1 2 3 Fig. 3. Abundance of proteins identified in soybean flooded without or with Al2O3 NPs. The 279 and 376 significantly changed proteins were visualized using MapMan software in soybeans flooded without or with Al2O3 NPs respectively after 4 days of stress, were used. Each square and color indicates the log2FC value of differentially changed proteins. The analysis identified significantly changed proteins were related to cell vesicle, minor CHO, and nucleotides under flooding with Al2O3 NPs. Materials and Methods Soybean (G. max cv. Enrei) Al2O3 NPs (30-60 nm) (0, 5, 50, 500 ppm) Recovery Sowing Water removal NPs Flooding Days after sowing (Days after treatment) 2(0) 4(2) 6(4) 8(4) 10(4) 12(4)   2(0) 4(2)F 6(4)F 8(4)F 10(4)F 4(2)NF 8(4)NF 10(4)NF 6(4)NF II III I Sample collection Fig. 4. Cluster analysis of significantly changed proteins in soybean flooded without or with Al2O3 NPs. Abundance patterns of individual proteins are indicated based on the color legend for flooded without or with Al2O3 NPs at 2, 4 days of exposure with subsequent recovery for 2, and 4 days (left to right). Time-based expression profile of the 211 commonly changed proteins that were commonly identified in soybeans flooded without and with Al2O3 NPs were used to group the proteins into three clusters. According to cluster analysis, Proteins related to protein, major CHO metabolism, amino acid metabolism, and cell wall exhibited significant change under flooding without or with Al2O3 nanoparticles. Proteomic Analysis Morphological Analysis Root weight Protein extraction Seedling weight 37 proteins Root length Protein purification digestion LC MS/MS analysis Data analysis a ab abc bc b   cd cd cd d cd cd cd d cd d cd 2(0) cd bcd bcd 162 proteins bcd abc d ab 4(2) a a a 6(4) a ab bc d 12 proteins Al2O3 Ratio 8(4) Al2O3 -3 0 3 Al2O3   ` Conclusions Morphology of soybeans was improved under flooding with 50 ppm Al2O3 nanoparticles compared to flooding stress. Flooding with Al2O3 nanoparticles mainly targeted the proteins related to protein synthesis/ degradation, cell vesicles, amino acid metabolism, lipid metabolism, and nitrogen metabolism. Significant change in protein abundance related to cell vesicles, minor CHO metabolism, and nucleotide metabolism under flooding with Al2O3 nanoparticles. According to cluster analysis, proteins related to protein, major CHO metabolism, amino acid metabolism, and cell wall exhibited significant change under flooding with Al2O3 nanoparticles. b 10(4) ab ab a a a a a a a a a a a a a a a a a a (1cm) 12(4) Fig.1 . Morphological response of soybean under flooding without or with Al2O3 NPs. Survival percentage, seedling weight, and weight/ length of root including hypocotyl were measured under flooding without or with Al2O3 nanoparticles. Fifty ppm Al2O3 nanoparticles improved the morphology and survival percentage of soybean after stress removal. Al2O3 nanoparticles might be helpful to soybean in recovering from flooding stress by regulating its amino acid, lipid, and nitrogen metabolisms.