Nitric oxide and iron modulate heme oxygenase activity as a long distance signaling response to salt stress in sunflower seedling cotyledons

Nitric oxide is a significant component of iron signaling in plants. Heme is one of the iron sensors in plants. Free heme is highly toxic and can cause cell damage as it catalyzes the formation of reactive oxygen species (ROS). Its catabolism is carried out by heme oxygenase (HOs; EC 1.14.99.3) which uses heme both as a prosthetic group and as a substrate. Two significant events, which accompany adaptation to salt stress in sunflower seedlings, are accumulation of ROS and enhanced production of nitric oxide (NO) in roots and cotyledons. Present investigations on the immunolocalization of heme oxygenase distribution in sunflower seedling cotyledons by confocal laser scanning microscopic (CLSM) imaging provide new information on the differential spatial distribution of the inducible form of HO (HO-1) as a long distance in response to NaCl stress. The enzyme is abundantly distributed in the specialized cells around the secretory canals (SCs) in seedling cotyledons. Abundance of tyrosine nitrated proteins has also been observed in the specialized cells around the secretory canals in cotyledons derived from salt stressed seedlings. The spatial distribution of tyrosine nitrated proteins and HO-1 expression further correlates with the abundance of mitochondria in these cells. Present findings, thus, highlight a link among distribution of HO-1 expression, abundance of tyrosine nitrated proteins and mitochondria in specialized cells around the secretory canal as a long distance mechanism of salt stress tolerance in sunflower seedlings. Enhanced spatial distribution of HO-1 in response to NaCl stress in seedling cotyledons is in congruence with the observed increase in specific activity of HO-1 in NaCl stressed conditions. The enzyme activity is further enhanced by hemin (HO-1 inducer) both in the absence or presence of NaCl stress and inhibited by zinc protoporphyrin. Western blot analysis of cotyledon homogenates using anti-HO-1 polyclonal antibody shows one major band (29 kDa) of HO-1. NaCl-modulated HO-1 activity correlates with endogenous NO content in the cotyledons. Increased NO accumulation by hemin treatment also correlates with enhanced activity of HO-1 in both control and NaCl stress conditions. Present work indicates that NO positively modulates HO-1 activity in sunflower seedling cotyledons. NaCl stress tends to antagonize NO action on HO-1 activity. NO (from sodium nitroprusside; SNP) is probably positively modulating HO-1 activity by way of its interaction/binding with heme group. Present work also shows enhanced NO accumulation in seedling cotyledons both in the absence or presence of iron in the growth medium, in response to NaCl stress. Thus, a probable link between endogenous NO, NaCl stress and iron-homeostasis by way of modulation of HO-1 activity at early stage of sunflower seedling growth has been proposed.