Indoleamines regulate a variety of physiological functions during the growth, morphogenesis and stress-induced responses in plants. Present investigations report the effect of NaCl stress on endogenous serotonin and melatonin accumulation and their differential spatial distribution in sunflower (Helianthus annuus) seedling roots and cotyledons using HPLC and immunohistochemical techniques, respectively. Exogenous serotonin and melatonin treatments lead to variable effect on hypocotyl elongation and root growth under NaCl stress. NaCl stress for 48 h increases endogenous serotonin and melatonin content in roots and cotyledons, thus indicating their involvement in salt-induced long distance signaling from roots to cotyledons. Salt stress-induced accumulation of serotonin and melatonin exhibits differential distribution in the vascular bundles and cortex in the differentiating zones of the primary roots, suggesting their compartmentalization in the growing region of roots. Serotonin and melatonin accumulation in oil body rich cells of salt-treated seedling cotyledons correlates with longer retention of oil bodies in the cotyledons. Present investigations indicate the possible role of serotonin and melatonin in regulating root growth during salt stress in sunflower. Effect of exogenous serotonin and melatonin treatments (15 μM) on sunflower seedlings grown in the absence or presence of 120 mM NaCl substantiates their role on seedling growth. Auxin and serotonin biosynthesis are coupled to the common precursor tryptophan. Salt stress-induced root growth inhibition, thus pertains to partial impairment of auxin functions caused by increased serotonin biosynthesis. In seedling cotyledons, NaCl stress modulates the activity of N-acetylserotonin O-methyltransferase (HIOMT; EC 2.1.1.4), the enzyme responsible for melatonin biosynthesis from N-acetylserotonin.

Present work highlights the involvement of endogenous nitric oxide (NO) in sodium chloride (NaCl)-induced biochemical regulation of seedling growth in sunflower (Helianthus annuus L., cv. Morden). The growth response is dependent on NaCl concentration to which seedlings are exposed, they being tolerant to 40 mM NaCl and showing a reduction in extension growth at 120 mM NaCl. NaCl sensitivity of sunflower seedlings accompanies a fourfold increase in Na(+) /K(+) ratio in roots (as compared to that in cotyledons) and rapid transport of Na(+) to the cotyledons, thereby enhancing Na(+) /K(+) ratio in cotyledons as well. A transient increase in endogenous NO content, primarily contributed by putative NOS activity in roots of 4-day-old seedlings subjected to NaCl stress and the relative reduction in Na(+) /K(+) ratio after 4 days, indicates that NO regulates Na(+) accumulation, probably by affecting the associated transporter proteins. Root tips exhibit an early and transient enhanced expression of 4,5-diaminofluorescein diacetate (DAF-2DA) positive NO signal in the presence of 120 mM NaCl. Oil bodies from 2-day-old seedling cotyledons exhibit enhanced localization of NO signal in response to 120 mM NaCl treatment, coinciding with a greater retention of the principal oil body membrane proteins, i.e. oleosins. Abolition of DAF positive fluorescence by the application of specific NO scavenger [2-phenyl-4,4,5,5-tetramethyllimidazoline-1-oxyl-3-oxide (PTIO)] authenticates the presence of endogenous NO. These novel findings provide evidence for a possible protective role of NO during proteolytic degradation of oleosins prior to/accompanying lipolysis.

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.

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Detection of nitric oxide (NO) in plant cells is mostly undertaken using diaminofluorescein (DAF) dyes. Serious drawbacks and limitations have been identified in methods using DAF as a probe for NO detection. The present work reporting an alternative fluorescent probe for NO detection is thus proposed for varied applications in plant systems for physiological investigations. This method involves a simple, two-step synthesis, characterization, and application of MNIP-Cu {Copper derivative of [4-methoxy-2-(1H-napthol[2,3-d]imidazol-2-yl)phenol]} for specific and rapid binding with NO, leading to its detection in plant cells by epifluorescence microscopy and confocal laser scanning microscopy (CLSM). Using sunflower (Helianthus annuus L.) whole seedlings, hypocotyl segments, stigmas from capitulum, protoplasts, and isolated oil bodies, present investigations demonstrate the versatile nature of MNIP-Cu in applications for NO localization studies. MNIP-Cu can detect NO in vivo without any time lag (ex. 330-385 nm; em. 420-500 nm). It exhibits fluorescence both under anoxic and oxygen-rich conditions. This probe is specific to NO, which enhances its fluorescence due to MNIP-Cu complexing with NO and treatment with PTIO leads to quenching of fluorescence. It is relatively nontoxic when used at a concentration of up to 50 μM.

This study evaluated the rooting and sprouting responses of four ornamental Mussaendas species (Flag bush) stem cuttings to treatment with varying concentrations of 1-naphthalene acetic acid (NAA). Species evaluated include Mussaenda afzelii (wild), M. erythrophylla, M. philippica and Pseudomussaenda flava. Different concentrations of NAA phytohormone were applied to the cuttings grown in mixed river sand and saw dust (1:1; v/v); and laid out in a 4 x 4 factorial experiment in completely randomized design (CRD; r=4). Results showed that increasing concentrations of NAA application slowed down emerging shoot bud in M. afzelii, P. flava, M. erythrophylla and M. philippica. While other species responded positively at some point to increased concentrations of the NAA applications, the P. flava showed retarding effect of phytohormone treatment on its number of leaves. However, M. afzelii, M. erythrophylla and M. philippica, showed marked boost in their number of roots (NR) with the NAA increased application. The 0.2% NAA treated cuttings gave highest mean NR (4.6 roots) per stem cutting followed by the cuttings that received 0.4% NAA treatment which gave 4.3 mean NR, all of which were significantly different (p≤0.05). In terms of species response to the phytohormone positive effect, M. philippica gave highest mean NR (6.1 roots), followed by M. afzelii and M. erythrophylla which had 3.8 and 3.4 roots per cutting respectively. Evidently, the study has contributed to the conservation and propagation of ornamental Mussaenda collections in addition to providing vital information towards domestication of the wild indigenous species Mussaenda afzelii.