.8 0.4 0.Shoota ansShootNa+/K+ Ratio8 6 4a a Shootb c nsa c cb c nsb0.06 0.ControlNaClControlNaClControlNaClGP = 0.8801 P = 1.010-8 P = three.050-HP = 0.-0.0.0 0.two 0.4 0.6 K+ net uptake price (mmol g DW-1root d-1)0.2 3 four 5 six Na+ net uptake rate (mmol g DW-1root d-1)Fig. 3. OsCYB5-2 improves salt tolerance in rice by regulating OsHAK21-mediated K+ transport. (A ) Phenotypes of OsCYB5-2-overexpressed lines in WT (Nipponbare) and oshak21 backgrounds. Rice seedlings were hydroponically grown with or with out 150 mM NaCl for 12 d. Representative photographs of plants (A), total chlorophyll in shoots (B), and fresh weight (C) are shown. The transformed empty vector (pCM1307) seedlings have been applied as adverse controls. (D ) Effects of OsCYB5-2-overexpression on Na+ and K+ μ Opioid Receptor/MOR Purity & Documentation accumulation in shoots below salt pressure. Seedlings have been treated as in a, and the shoots were harvested for Na+ and K+ content assay. DW, dry weight. Information are shown as suggests SD (B and C, n = 12; D , n = five biologically independent seedlings for every transgenic rice lines). Lowercase letters above the bars in B indicate significant differences among suggests (P value = 0.05, Kruskal allis bilateral test). ns indicates nonsubstantial differences at that level of significance. (G and H) K+ and Na+ net uptake prices in rice seedlings throughout ten d with the treatment with 150 mM NaCl. Data in G and H are shown as signifies SD (n = 5). Statistically considerable differences had been determined by the two-tailed Student’s t test.constructed and tested inside the yeast split-ubiquitin method (Fig. 5A). The cytoplasmic C-terminal fragment of OsHAK21 didn’t bind OsCYB5-2 (Fig. 5A). The C-terminal deletions up to 183 mino acid (aa) residues did not considerably affect OsCYB5-2 binding (Fig. 5A), suggesting that the OsCYB5-2 binding domain resides inside the first 183-aa residues. ToSong et al. + An endoplasmic reticulum ocalized cytochrome b5 regulates high-affinity K transport in response to salt anxiety in riceestablish the vital residues for OsCYB5-2 binding within the initial 183 residues, site mutations were produced. In yeast systems, leucine (L) residues are thought to become essential for the binding of sugar (and sorbitol) transport proteins with MdCYB5 from apple plants (29). We therefore performed site-directed mutagenesis to separately replace each and every on the 10 L residues (withinPNAS j 5 of 12 doi.org/10.1073/pnas.PLANT BIOLOGYControlNaClP = three.390-P = eight.720-P= two.170-P = 2.380-A i ii iii B0.6 0.5 0.2u 35S 2u 35S 2u 35SFLAG Tag CFP NosT 35S 35SHA Tag YFP OsCYB5-2 NosTEK+ content (mmol g DW-1)0.5 0.four 0.3 0.2 0.1 0.0 30 60 90 120 OsHAK21+OsCYB5-2 P = three.130-6 OsHAK21 OsCYB5-2 P = 6.920-4 Empty PPARγ Gene ID vectorP = 0.0187 P = 0.0357 P = 0.OsHAKOsHAK21 NosT OsHAK21 NosTOsCYB5-2 NosTiv35SOsCYB5-2 NosTBufferTreatmentFRET EfficiencyNaCl MannitolTime (min)Na+ content (mmol g DW-1)0.3 0.2 0.1 0.0 0 200 400 600 800 1000F0.7 0.6 0.5 0.four 0.3 0.two 0.1 0.0.OsHAK21 Relative Expression0.five 1.1.Time (s)CNaClHigh300 s 400 s 500 s 600 s 700 s 800 sP = 9.63-P = 8.720-MannitolTime (min)300 s 400 s 500 s 600 s 700 s 800 sLowG50.0 0.5 1.0 1.five 2.0 2.5 three.0 3.OsCYB5-2 Relative ExpressionD100 mM NaCl Time (min): 0 OsHAK21-FC Input(-FLAG)KDa -135 -100 -Na+/K+ Ratio3 2 1P = 0.P = 8.510-YH-OsCYB5-(-HA)OutputIB: HARelative value: 1.0 1.14 1.46 two.53 two.-P = 0.IP: FLAGTime (min)Fig. four. The interaction among OsHAK21 and OsCYB5-2 is triggered by salt remedy. (A) Schematic diagram in the coexpression proteins integrated into a vector. The vectors (i and ii)