Rapid auxin-induced nitric oxide accumulation and subsequent tyrosine nitration of proteins during adventitious root formation in sunflower hypocotyls

Abstract

Using NO specific probe (MNIP-Cu), rapid nitric oxide (NO) accumulation as a response to auxin (IAA) treatment has been observed in the protoplasts from the hypocotyls of sunflower seedlings (Helianthus annuus L.). Incubation of protoplasts in presence of NPA (auxin efflux blocker) and PTIO (NO scavenger) leads to significant reduction in NO accumulation, indicating that NO signals represent an early signaling event during auxin-induced response. A surge in NO production has also been demonstrated in whole hypocotyl explants showing adventitious root (AR) development. Evidence of tyrosine nitration of cytosolic proteins as a consequence of NO accumulation has been provided by western blot analysis and immunolocalization in the sections of AR producing hypocotyl segments. Most abundant anti-nitrotyrosine labeling is evident in proteins ranging from 25-80 kDa. Tyrosine nitration of a particular protein (25 kDa) is completely absent in presence of NPA (which suppresses AR formation). Similar lack of tyrosine nitration of this protein is also evident in other conditions which do not allow AR differentiation. Immunofluorescent localization experiments have revealed that non-inductive treatments (such as PTIO) for AR develpoment from hypocotyl segments coincide with symplastic and apoplastic localization of tyrosine nitrated proteins in the xylem elements, in contrast with negligible (and mainly apoplastic) nitration of proteins in the interfascicular cells and phloem elements. Application of NPA does not affect tyrosine nitration of proteins even in the presence of an external source of NO (SNP). Tyrosine nitrated proteins are abundant around the nuclei in the actively dividing cells of the root primordium. Thus, NO-modulated rapid response to IAA treatment through differential distribution of tyrosine nitrated proteins is evident as an inherent aspect of the AR development.

Keywords: adventitious rooting; auxin; immunolocalization; nitric oxide; tyrosine nitration of proteins.

Figure 1. NO distribution in hypocotyl explants at different stages of adventitious rooting. Hypocotyl explants as visualized with MNIP-Cu treatment after 4 (A), 5 (B) and 7 (C) days of incubation in 10 µM of IAA in dark. Scale bar represents 2 mm.

Figure 2. A. Effect of various physiological treatments on NO distribution in protoplasts isolated from the hypocotyls of 4 d old, light grown seedlings. (A−D) represent control protoplasts, (E−H) are treated with auxin (10 µM IAA). (I−L) are treated with auxin influx inhibitor (10 µM NPA) and (M−P) are treated with NO donor (100 µM SNP). After incubation in each treatment for 30 min, NO was localized using 25 µM MNIP-Cu and visualized at ex. 365 nm (em. 420 nm) (B, F, J and N). Co-incubation with 1 mM PTIO (D, H, L and P) confirmed the fluorescence due to NO. All the observations were taken at 630×. B. Relative fluorescence units data from protoplasts subjected to MNIP-Cu treatment for NO localization. Each datum represents mean value and standard errors from ten protoplasts subjected to a particular treatment.

Figure 3. Localization of tyrosine nitrated proteins by CLSM imaging using anti-nitrotyrosine antibody. Visible and fluorescence micrographs of 10 µM thick hypocotyls sections from the basal regions of the explants subjected to treatment of PTIO (1.5 mM) (A-D), SNP (100 µM) + NPA (10 µM) (E-H), SNP (100 µM) + NPA (10 µM) (I-L) and SNP (100 µM) (M-P) in the presence of anti-nitrotyrosine antibody. (C, D, G, H, K, L, O and P): Maginified views (400×) of (A, B, E, F, I, J, M and N) (100×) respectively, show the differential distribution of tyrosine nitrated proteins in the vascular bundles.

Figure 4. western blot analyses of anti-nitrotyrosine labeled proteins (A) and Coomassie stained gel images (B) of hypocotyl segments incubated in IAA (10 µM), NPA (10 µM), SNP (100 µM), SNP+NPA, PTIO (1 mM) and PTIO+IAA for seven days. Other details as in “Materials and Methods.” M, marker.