Trifluoperazine (TFP)-mediated fluorescence imaging approach reveals a probable calmodulin (CaM)-independent calcium signaling accompanying differential protein phosphorylation in NaCl-stressed sunflower seedlings (Helianthus annuus L. var. KBSH44)

Abstract

The impact of calcium (Ca2+) under abiotic stress is manifested by both calmodulin (CaM)-dependent and calmodulin-independent pathways of signaling mechanisms in plants. Present work demonstrates the impact of NaCl (120 mM) stress on Ca2+-calmodulin activity and its spatial distribution in roots of 2d old sunflower seedlings (Helianthus annuus). NaCl stress significantly increases [Ca2+]cyt in roots (69%) which corresponds with 8% increase in activated calmodulin (Ca2+-CaM) levels. Thus, NaCl stress induced Ca2+ signaling in roots is likely to majorly operate through CaM-independent routes. Trifluoperazine-mediated fluorescence imaging reveals differential distribution of Ca2+-CaM complex in the elongation and differentiation zone of seedling roots which indicate spatial regulation of Ca2+-CaM signaling operative in the presence of NaCl stress. NaCl stress-induced CaM-independent Ca2+ signaling is further accompanied by differential protein phoshorylation of cytosolic proteins in seedling roots and cotyledons. Phosphoproteins exhibiting qualitative differences in roots in response to salt-stress were of both low and high molecular weight range (76–17.5 kDa) unlike in cotyledons where only high molecular weight (>76 kDa) phosphoproteins exhibited major differential expressions in response to 120 mM NaCl stress. Among the various polypeptides in the fraction of OB membrane proteins two polypeptides of 48 and >76 kDa exhibited increased protein phosphorylation in the presence of NaCl stress. NaCl stress for 48 h induces differences in the expression of phosphoproteins in roots and cotyledons, suggesting the long distance signaling of Na+ stress from roots to cotyledons. Roots seem to exhibit much stringent regulation of phosphoproteins in comparison with cotyledons which have lesser qualitative differences. Thus, present work provides evidence on the modulation of Ca2+-CaM signaling accompanying differential protein phosphorylation in sunflower seedling roots and cotyledons under NaCl stress.