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Scientia Horticulturae 129 (2011) 159–166

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l homepage: www.e lsev ier .com/ locate /sc ihor t i

itric oxide accumulation and actin distribution during auxin-induceddventitious root development in sunflower

unita Yadav, Anisha David, Satish C. Bhatla ∗

aboratory of Plant Physiology and Biochemistry, Department of Botany, University of Delhi, North Campus, Delhi 110007, India

r t i c l e i n f o

rticle history:eceived 3 January 2011eceived in revised form 17 March 2011ccepted 20 March 2011

eywords:ctindventitious rootinguxinitric oxideitric oxide synthaseunflower hypocotyls

a b s t r a c t

Auxin-mediated adventitious root (AR) formation from the basal ends of hypocotyl segments in sun-flower (Helianthus annuus L., cv. Morden) is associated with gradual subcellular and anatomical changes.Pre-mitotic events (induction phase) are evident within 1 d (24 h) in the interfascicular parenchyma. Evi-dence for nitric oxide (NO) accumulation in the interfascicular cells 2 d after indole-3-acetic acid (IAA)treatment, highlights the involvement of NO specifically during root initiation, following AR inductionphase. Thus, adventitious root induction and initiation can be distinguished as NO-independent andNO-dependent phases. Treatment of hypocotyl explants with 10 �M 1-napthylphthlamic acid (NPA), aninhibitor of polar auxin transport in plant cells, leads to complete abolition of NO-associated fluorescence,detected upon incubation of hypocotyl sections with 4,5-diaminofluorescein diacetate (DAF-2DA). Thisindicates a signaling link between auxin transport and NO accumulation in the target cells. Latrunculin B(Lat B), an actin depolymerizing agent, leads to substantial inhibition of IAA-induced AR response and NO

accumulation in the responding cells. Aminoguanidine [an inhibitor of inducible form of nitric oxide syn-thase (iNOS)] brings about a reduction in the tissue NO content, indicating a significant activity of putativeNOS leading to endogenous NO accumulation. Confocal laser scanning microscope (CLSM) imaging hasrevealed bundling of actin with NPA and Lat B treatments. A probable interaction is thus evident betweenactin and NO during auxin-mediated AR formation in the initiation and extension phase. Present work,thus, provides novel observations on an interaction among endogenous NO, IAA and actin at specific stages of AR formation.

. Introduction

Adventitious root (AR) formation begins with redifferentiationf predetermined interfascicular parenchymatous cells in the basalegion of the stem after the removal of primary root system. Thisesponse is known to be evoked by endogenous auxins, princi-ally indole-3-acetic acid (Taiz and Zeiger, 2011). Although genetic

pproaches have clarified some discrete aspects of auxin trans-ort and related signal transduction events, not much is knownbout the molecular mechanisms associated with AR formation.ndogenous auxin concentrations are known to alter during the

Abbreviations: AR, adventitious roots; AtNOA1, Arabidopsis thaliana nitric oxidessociated 1; CLSM, confocal laser scanning microscope; CsA, cyclosporin A; DAF-DA, 4,5-diaminofluorescein diacetate; GDC, glycine decarboxylase; GEF, guanineucleotide exchange factor; GMP, guanine mononucleotide phosphate; IAA, indole--acetic acid; IBA, indole-3-butyric acid; iNOS, inducible nitric oxide synthase; Lat B,

atrunculin B; NBP, NPA binding receptor protein; Ni–NOR, nitrite–NO oxidoreduc-ase; NPA, 1-napthylphthalamic acid; NO, nitric oxide; NOS, nitric oxide synthase;MSF, phenylmethylsulfonyl fluoride.∗ Corresponding author. Tel.: +91 11 27666744; fax: +91 11 27666744.

E-mail address: bhatlasc@gmail.com (S.C. Bhatla).

304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.scienta.2011.03.030

© 2011 Elsevier B.V. All rights reserved.

three phases of AR i.e., induction, initiation and extension (Naget al., 2001). Using various pharmacological agents, a probablesignaling cascade (linking NO, cyclic GMP and mitogen-activatedprotein kinases) for auxin-induced adventitious root formation hasbeen proposed in cucumber seedlings (Pagnussat et al., 2002, 2003,2004). These observations are, however, devoid of any extensiveanalysis of the modulation of endogenous NO distribution. Themode of auxin interaction with NO, resulting in adventitious rootformation, still remains unknown.

Recent work in the author’s laboratory has demonstrated thatdifferent phases of auxin-induced AR in sunflower hypocotylsare either NO-independent (induction phase) or NO-dependent(initiation and extension phases). These observations indicated apossible interaction between auxin efflux transporters (PIN) andactin during adventitious rooting (Yadav et al., 2010). Based onour present understanding of the signaling events associated withadventitious rooting, the temporal accumulation of endogenous

NO in the adventitious root differentiating zone at various stagesof development, has been monitored by fluorescence microscopy,using DAF-2DA for detection of NO, in presence of various phar-macological agents. These observations highlight the effects ofvarious promoters and inhibitors of AR on the spatial distribu-

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ion of NO in the responding cells. Estimation of total NO contentnd NO content due to putative NOS activity has been undertakenn the hypocotyl explants subjected to pharmacological treat-

ents (known to stimulate or inhibit AR). Lastly, a comparativeonfocal microscopic image analysis of the bundling of actin inresence of auxin efflux blocker (NPA) and nitric oxide scavenger2-phenyl-4,4,5,5-tetramethyllimidazoline-1-oxyl-3-oxide (PTIO)]as highlighted the common features of their molecular action dur-

ng AR formation.

. Materials and methods

.1. Plant material and explant treatments

Raising of hypocotyl explants from sunflower seedlingsHelianthus annuus L. cv. Morden) and their treatment with variousharmacological agents was undertaken according to Yadav et al.2010). Briefly, the hypocotyls from light-grown, 4 d old seedlingsith similar growth rate, were excised up to 6 cm below the cotyle-onary node. Cotyledons were carefully excised, retaining the shooteristem. The resulting explants were used for adventitious root-

ng experiments. Freshly harvested explants were put upright inlass vials with their cut ends dipped in 1 ml of various test solu-ions in 5 ml corning glass vials. Explants were maintained in darkuring the course of experiments (i.e., till 4 d). The test solutionsf IAA (10 �M), NPA (10 �M), Lat B (100 nM), cyclosporin A (CsA,0 �M) and PTIO (1.5 mM) were used either alone or in combina-ions, as described in Section 3. Hypocotyl explants incubated inistilled water for 4 d served as control.

.2. Anatomical changes and epifluorescence microscopicisualization of fluorescence due to endogenous NO distributionsing DAF-2DA

Hypocotyl explants subjected to various pharmacologicalgents (for a period of 4 d) were used for cutting (using razorlade) fine sections from the basal 2–4 mm regions and they werebserved for various stages of AR formation, using a photomicro-cope. Digital images were taken using AxioCam digital cameraZeiss, Germany). Cross sections obtained from the basal 2–4 mmegions of various explants were incubated for 40 min in 10 �M ofAF-2DA prepared in 10 mM Tris–HCl buffer set at pH 7.4 (accord-

ng to Corpas et al., 2006). Sections were then washed twice formin each with the same buffer (without DAF-2DA) and observed

or fluorescence due to DAF-2DA binding with NO (ex 485 nm; em15 nm), using epifluorescence microscope (Axioskop from Zeiss,ermany). In order to check the specificity of fluorescence due toO (control), sections were preincubated for 1 h with 1 mM PTIO in0 mM Tris–HCl buffer (pH 7.4), subsequently incubated for 40 minith 10 �M DAF-2DA in presence of 1 mM PTIO and then examined

or fluorescence quenching microscopically.

.3. Estimation of NO production due to putative NOS activity

Putative NOS activity was estimated spectrofluorometrically,ccording to David et al. (2010). Briefly, 1 g tissue from the basalmm hypocotyl segments (pooled from several explants subjected

o specified treatments till 4 d) was homogenized in the extractionuffer (50 mM Tris–HCl, 250 mM sucrose and 1 mM EDTA, pH 7.4)ontaining 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluo-

ide (PMSF), 1 mM benzamidine, 2 �g ml−1 aprotinin, 25 �g ml−1

oybean trypsin inhibitor and 10 �l g−1 protein inhibitor cock-ails procured from Sigma (USA). The homogenate was frozen andhawed with liquid nitrogen thrice and centrifuged at 10,000 g for0 min at 4 ◦C, according to Butt et al. (2003). The supernatant was

urae 129 (2011) 159–166

collected and stored at −20 ◦C. Protein concentration was deter-mined by Bradford assay (Bradford, 1976). Total NO content wasestimated in triplicate for each sample in a reaction buffer contain-ing 1 mM l-arginine and 1 �M �-NADPH. After addition of 10 �MDAF-2DA, the reaction mixture was incubated at 25 ◦C for 30 min,according to Corpas et al. (2004). In order to determine putativeNOS activity, samples were preincubated with 10 mM aminoguani-dine (iNOS inhibitor) in the reaction buffer for 30 min, followed bythe addition of 1 mM l-arginine, 1 �M �-NADPH and 10 �M DAF-2DA (Valderrama et al., 2007). The reaction mixture was incubatedat 25 ◦C for 30 min. Reaction mixture without protein served as con-trol for the respective sample. Fluorescence was measured usinga spectrofluorometer (Model: FS920; Edinburgh Instruments, UK)at the excitation and emission wavelengths of 485 and 515 nm,respectively, according to Kojima et al. (1998). Data was presentedas fluorescence units (counts per second).

2.4. Localization of actin

Actin was visualized according to Blancaflor (2000), using AlexaFluor phalloidin (Molecular Probes Inc., USA) as the fluorescentprobe. Fine sections obtained by razor blade from the basal 4 mmregions of control explants and those treated with Lat B, IAA, NPAand PTIO till 4 d, were immersed for 30 min in 50 mM PIPES buffer(pH 6.9) containing 4 mM MgSO4 and 10 mM EGTA. Sections werethen incubated for 10 min in the same buffer containing 0.1 �MAlexa Fluor phalloidin, 0.3 M mannitol and 2% glycerol. Sections,subsequently washed once with PIPES buffer, were mounted on aglass slide and immediately observed for fluorescence under CLSMusing argon laser.

All experiments were performed at least thrice and statisticallyanalyzed wherever necessary.

3. Results and discussion

3.1. Pre-mitotic events (induction phase) leading to AR formationare evident in the interfascicular parenchyma within 1 d (24 h)

Auxin-mediated AR formation in the hypocotyl explants isassociated with gradual anatomical changes in the interfascicularparenchyma. During the induction phase (1 d), predetermined cellsbecame cytoplasmically dense. Initiation of cell division becameevident after 2 d (Fig. 1I). After 3 d of IAA treatment (Fig. 1M), tiersof cells became prominent in this region. Few cells of the inner tierexhibited characteristic cell wall thickenings, which proliferatedto a greater extent by day 4. These presumably differentiate intovasculature in the developing root primordium. Root primordium,thus initiated after 4 d of IAA treatment, showed a well developedmeristem (m) and procambial tissues (pc) (Fig. 1Q). AR formation isstimulated as a result of root excision, which possibly reduces thesink size for basipetally translocating substances, leading to theirbuild up in the hypocotyl base. The view gains support from our pre-liminary experiments, where explants with intact apical meristemexhibited AR formation but those with decapitated apical meris-tem did not form AR. Root excision could also probably remove theinhibitors of AR being synthesized in roots.

3.2. Significant NO accumulation occurs in the interfasicular cellswithin 2 d (48 h)

Within the plant cells, NO is not as mobile as expected because

of its trapping in the hydrophobic core of the membranes, aswell as due to its chemical reactivity with lipids and/or proteins(Stöhr, 2007). The diffusion coefficient of NO is highly dependenton membrane composition. Cell membranes exhibit varying lipidcomposition in different regions. Thus, they are not homogenous

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Fig. 1. Fluorescence microscopic visualization of NO signal in hypocotyl sections. Gradual changes in endogenous NO distribution in parenchyma cells between the neigh-bouring vascular bundles in hypocotyl explants accompanying adventitious rooting, upon treatment with 10 �M IAA. Sections were observed for fluorescence in the absenceof DAF-2DA, in presence of DAF-2DA and in presence of DAF-2DA (10 �M) + PTIO (1 mM). Arrowhead in ‘S’ indicates intense fluorescence in the epidermal cells of a youngprimordium. m represents meristem and pc procambial tissue. Magnification: 200×.

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ipid layers. Fluid phase membranes have been observed to act astrong barriers to NO transport. Based on these facts, it is technicallyossible to visualize NO accumulation in viable cells by fluores-ence microscopy. DAF-2DA, a widely used fluorescent probe forO, is employed in plant and animal systems for endogenous NOisualization (Kojima et al., 1998). As also reported earlier in mungean (Huang et al., 2007), coinciding with AR differentiation inunflower hypocotyl explants (present work), a gradual increasen fluorescence due to endogenously localized NO was observedn the interfascicular parenchyma with increasing period of IAAreatment. Distribution of NO during lateral root formation haslso been investigated earlier in tomato, supporting the involve-ent of NO in the auxin signaling pathway (Correa-Aragunde et al.,

004). NO signal did not exhibit any significant difference up tod after IAA treatment (Fig. 1C and G). After 2 d of treatment,

ntense green symplastic and apoplastic fluorescence was observedn the interfascicular region (Fig. 1K). Our previous report (Yadavt al., 2010) on the demarcation of auxin-mediated induction phasend IAA–NO interaction-mediated initiation phase, is strengthenedy the present observations. These results implicate the possi-le involvement of IAA alone during the induction phase (1 d). A

ag phase (when IAA probably leads to NO synthesis) is, however,ssential for the transition from induction to initiation phase (2 dtage) and thereafter. NO generation in cells exhibiting AR initiationppears to accompany mitotic activity during the initiation phase.urther increase in fluorescence intensity was observed after 3 d ofAA treatment (Fig. 1O). Interestingly, appreciably intense fluores-ence due to NO was visualized in the root primordium after 4 d,t being maximum in the apical meristematic cells (Fig. 1S). This

ay be attributed to the possible involvement of NO during cellivision and differentiation. In the root primordium, NO signal wasniform and symplastic in the epidermal layer of the primordium.reatment with NO specific scavenger (PTIO) leads to a significanteduction in fluorescence at all stages of AR development, indicat-ng that the green fluorescence is, in fact, due to NO. The persistentuorescence contained in the lignified tissue (xylem) in controlnd PTIO-treated sections can be attributed to autofluorescence ofignin at the excitation wavelength (ex. 485 nm) being employedor detecting fluorescence due to DAF-2DA and NO interaction. Athis point, it will be worthwhile to mention that while evaluatingO signal in plant cells by DAF treatment, care must be taken to

ake note of such autofluorescence from lignified cells and it maye pointed out that this lignin autofluorescence is not quenchabley PTIO treatment. Thus, it can be distinguished from fluorescenceue to NO. Evidence for NO generation in the interfascicular cellsd after IAA treatment highlights the involvement of NO in thedventitious rooting process accompanying root initiation (afterhe auxin-mediated induction phase).

.3. A correlation between IAA, NO and actin distribution isvident during AR formation

Auxin (principally IAA) synthesized in the apical tissues movesownward through its characteristic polar transport mechanism.he central feature of this transport is that IAA-efflux is directedy polar localization of some efflux carriers (mainly PIN proteins)hich determine polar intracellular transport of IAA. Polarity of PINistribution on the plasma membrane of a cell involves movementf newly synthesized PIN proteins through the endomembraneecretory system. PIN is rapidly cycled between the plasma mem-rane at the base of the cell and an unidentified endosomal

ompartment, by an actin-dependent mechanism (Morris, 2000).PA, an inhibitor of polar auxin transport, has been widely used astool to investigate the mechanism and regulation of polar auxin

ransport (Muday and Murphy, 2002). NPA-binding receptor pro-ein (NBP) is functionally associated with PIN proteins. The link

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between NBP and the actin cytoskeleton suggests its involvementin directing the polar distribution of PIN, which has been shown tocycle along actin filaments (Geldner et al., 2001; Taiz and Zeiger,2011).

Treatment of hypocotyl explants with NPA (10 �M) leads tocomplete abolition of NO-associated fluorescence detected by DAF-2DA (Fig. 2C), establishing that NO acts downstream of auxin duringAR formation. In the non-inductive conditions of NPA + IAA, somefluorescence was observed (Fig. 2G) which was quenchable by PTIO(Fig. 2H).

Although previous reports indicated a role of NO in mediat-ing auxin response during AR formation in cucumber (Pagnussatet al., 2002, 2003), a cross talk between IAA and NO still remainsto be elucidated. In order to further explore IAA–NO interactionduring AR formation in sunflower, Latrunculin B (Lat B), an actindepolymerizing agent was employed. Lat B has been widely usedto investigate the function of actin in gymnosperms, monocotyle-donous and dicotyledonous plants. It induces profound changes inthe organization of microfilaments while leaving the organizationof microtubules unaltered. Lat B associates with actin monomersin 1:1 ratio, thereby preventing their repolymerization into actinfilaments (Morton et al., 2000). In the present work, Lat B leadsto a negative impact on NO accumulation in the responding cells(Fig. 2K). Actin depolymerization using Lat B would supposedly leadto substantial inhibition of IAA transport, thus resulting in reducedaccumulation of IAA in the basal responding region of the hypocotylsegments. This might explain the observed significant reduction inNO accumulation. While NO fluorescence was evident in Lat B + IAAtreatment, it was relatively less as compared to treatment with IAAonly (Fig. 2O). The interfascicular NO fluorescence, quenchable byPTIO, authenticates NO-associated DAF-2DA fluorescence.

Cyclophilins constitute a family of ubiquitous proteins known tobind immunosuppressant drugs. These proteins belong to the clus-ter of immunophilin proteins and catalyze cis–trans isomerizationof nascent proteins, thereby assisting protein folding. Cyclophilins(which are known to bind with cyclosporin) have recently beenreported to be a component in auxin regulation of plant growth anddevelopment (Oh et al., 2006). They interact with the N-terminalregion of the GNOM protein, a guanine nucleotide exchange factor(GEF) required for the proper localization of the auxin efflux car-rier PIN1 (Geldner et al., 2003). Cyclosporin A (CsA) inhibits NOSactivity in animal systems (Diaz-Ruiz et al., 2005). The cyclophilininhibitor, CsA, inhibits auxin-induced adventitious root initiationin tomato hypocotyl sections and also reduces the expressionof auxin-induced early auxin response genes (Oh et al., 2006).Compared to the IAA-induced NO fluorescence, intensity of NOfluorescence in root initials of CsA-treated explants was faint. More-over, the intense fluorescence due to NO from epidermis in the rootinitials of CsA-treated explants was not detectable (Fig. 2S). Inter-estingly, CsA + IAA treatment markedly reduced the intensity ofNO-associated symplastic fluorescence in the dividing cells of inter-fascicular region and the developing root initials. Thus, a potentialinvolvement of cyclophilins in AR formation is apparent from thepresent work. Further investigations in this direction would pro-vide evidence for cyclophilins as potential signaling intermediatesduring AR response.

3.4. Differences in total NO content and NO due to putative NOSactivity are evident in hypocotyl explants subjected to ARinducing and inhibitory treatments

In higher plants, both enzymatic and non-enzymatic (Bethkeet al., 2004) sources of NO have been documented. Enzymaticsources mainly involve nitrate reductase (NR) (Neill et al., 2008),as described in auxin-induced formation of lateral root initials(Kolbert et al., 2008); plasma membrane-associated nitrite–NO

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Fig. 2. NO visualization in 4 d hypocotyl explants in presence of various pharmacological agents. Distribution of endogenous NO content in the parenchyma cells between theneighbouring vascular bundles of 4 d old hypocotyl explants treated with NPA (10 �M), Lat B (100 �M) and CsA (10 �M), alone or in combination with IAA (10 �M). Sectionswere observed for fluorescence in the absence of DAF-2DA (10 �M), in presence of DAF-2DA (10 �M) and in presence of DAF-2DA (10 �M) + PTIO (1 mM). Magnification:200×.

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ig. 3. Spectrofluorometric estimation of NO content. Effect of various treatmentd hypocotyl explants, accompanying expression/suppression of AR. Data represe

eplicates.

xidoreductase (Ni–NOR) (Stöhr and Stremlau, 2006) and a NOS-ike enzyme, AtNOA1 (Guo et al., 2003; Crawford et al., 2006).ecently, surveying NADPH-diaphorase activity (marker for NOSctivity in animal systems), Huang et al. (2007) have indicatedhe involvement of putative NOS in NO biosynthesis during IBA-nduced adventitious rooting. In order to further investigate therobable involvement of putative NOS in AR formation, tissueomogenates (10,000 g supernatant) from the basal regions of sun-ower hypocotyl explants were assayed for total NO content inhe present work (Fig. 3). Employing aminoguanidine, NO produc-ion by putative NOS activity was also estimated. NO synthesisn explants subjected to distilled water and IAA treatment waslmost equal. Endogenous IAA accumulation at the base of thexplants is expected to be high due to reduced sink size and thisould account for the above observation. The optimum NO concen-ration required for adventitious rooting at this stage is probablychieved by this endogenous IAA concentration. NO content in IAAnd distilled water treated explants is almost three times more thanhat obtained with treatments inhibitory for AR formation (NPA,at B and their combinations with IAA). NO contribution by puta-ive NOS activity also showed a similar pattern. It is, thus, evidenthat NOS-associated NO production is significantly more in auxin-reated explants. NPA alone, and in conjugation with IAA, lead todrastic reduction in NO production that appears to be primarily

ontributed by a decrease in putative NOS activity. Similar trend forO production and NO contribution by putative NOS was evident inther AR inhibitory treatments as well. Marked reduction in NOS-ike activity by NPA and Lat B brings forth the probable involvementf auxin and actin during NO synthesis by putative NOS activity.inimum NO content was estimated in CsA treatment, with no

vidence for NOS activity.NOS is known to be active only when dephosphorylated.

yclosporin–cyclophilin A dimer is known to bind calcineurina class PP2B phosphatase), inhibiting its phosphatase activityRomano et al., 2005). The observed inhibition in NO synthesis dueo putative NOS activity in CsA-treated explants may be explaineds a probable result of CsA-mediated maintenance of phospho-

tal NO content and NO content due to putative NOS activity in the basal part ofean and standard errors of fluorescence intensity (counts per second) from three

rylated, inactive state of putative NOS. Similar reduction in NOSactivity in rat spinal cord cells on treatment with CsA, has also beenreported earlier (Diaz-Ruiz et al., 2005). It is further suggested thatbinding of CsA to cyclophilin D could block mitochondrial transitionpore permeability, thus decreasing intracellular calcium concentra-tion, resulting in a reduction in the activity of calcium-dependentNOS. A complete inhibition of putative NOS activity in presence ofCsA throws light on the presumption about the presence of a NOS-like enzyme. A reduction in NO content and NO due to putative NOSevident in NPA, Lat B and CsA treatments, indicates the involve-ment of the three components of AR signaling (NO, IAA and actin)and their interaction. Thus, the inhibitory effect of aminoguanidinesuggests that the generation of endogenous NO may be catalyzedby a NOS-like enzyme contributing to NO accumulation in the rootinitiation region, resulting in the observed morphological response.

3.5. Actin organization associated with AR formation exhibitsmarked differences in AR inducing and inhibitory treatments

Any signal (like IAA) leading to a polarized developmental event,such as AR formation, would very likely affect cytoskeleton orga-nization, in particular actin (Xuan et al., 2008). Auxin promotesthe localization of cycling PIN proteins on the plasma membrane(Paciorek et al., 2005). Recently, Nick et al. (2009) have observedauxin-induced transformation of actin bundles into finer strandsand have proposed a self-regulating circuit between polar auxintransport and actin organization. Confocal laser scanning micro-scopic (CLSM) imaging of fluorescence due to actin upon treatmentwith Alexa Fluor phalloidin (present work) shows contrasting dif-ferences between auxin-treated hypocotyl segments and thosetreated with NPA and Lat B (present work). The basal regionof IAA-treated hypocotyl segments responding by exhibiting AR

formation, shows enhanced and well distributed actin in the inter-fascicular cells after 4 d of incubation (Fig. 4A). This is in accordancewith the findings of Nick et al. (2009) that auxin availability isa requisite for the maintenance of actin cytoskeleton. Enhancedexpression of actin in presence of auxin has also been observed in

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ig. 4. Bundling and debundling of actin cytoskeleton in interfascicular cells of hypoundles of 4 d hypocotyl explants treated with IAA (a), NPA/Lat B (b) and PTIO (c) u

he hypocotyl cuttings from loblolly pine (Diaz-Sala et al., 1996). Inhe present work on sunflower hypocotyls, Lat B treatment lead toundling of actin and markedly reduced fluorescence due to actin.imilar pattern of actin disintegration was also visualized in NPA-reated hypocotyl explants (Fig. 4B). Actin disintegration by NPApresent work) seems contradictory to the reports of Petrásek et al.2003) who reported that inhibitory action of NPA on auxin effluxad no observable effect on the arrangement of actin filaments.ut, NPA leads to inhibition of polar auxin transport, thereby dras-ically reducing the cellular auxin concentration and consequentlyeading to observed actin disintegration. Interestingly, PTIO treat-

ent (Fig. 4C) also lead to a drastic actin cytoskeleton breakdown,ppearing as a thick fluorescent layer below the cell membrane andundling of actin, visible as spots of intense fluorescence. As NOppears downstream of auxin in the signaling pathway (Pagnussatt al., 2003), auxin-mediated actin cytoskeleton maintenance couldossibly be modulated by NO. Recently, NO has been reported toodulate actin cytoskeleton in the root apices of maize seedlings

Kasprowicz et al., 2009).

. Conclusions

To sum up, present work provides evidence for changes inemporal and spatial expression of endogenous NO in the interfas-icular cells of hypocotyl segments, accompanying auxin-induceddventitious rooting. NO generation during AR initiation and exten-ion (and not AR induction) has been demonstrated. EndogenousO accumulation in the AR differentiating zone has been shown

o be largely due to the enhanced activity of putative NOS. A pos-ible correlation between NO, IAA and actin has become evident.vidence has also been provided for a self-regulatory role of IAA inctin-mediated PIN recycling, thus altering auxin transport to thearget sites. Involvement of NO in auxin-mediated actin cytoskele-on maintenance is also apparent during the differentiation ofdventitious roots. Present findings using cyclosporin indicate therobable involvement of cyclophilins in PIN localization, therebypening a new front for further investigations with regard to bio-hemical events associated with AR formation. Specifically, thempact of cyclosporin on the phosphorylation status of putativeOS holds promise for newer and significant findings.

cknowledgements

Confocal microscopic analysis was undertaken with the help of

r. M.K. Raheja, using the CLSM facility established in the Depart-ent of Botany, University of Delhi, under FIST programme of DST,ew Delhi, India. The fluorescence photomicroscopy work wasndertaken on the microscope provided by Alexander von Hum-oldt Foundation to SCB. Thanks are due to Dr. Souvik Maiti at the

segments. Visualization of actin in parenchyma cells between the adjacent vascularLSM using Alexa Fluor phalloidin as the fluorescent probe. Magnification: 1000×.

Institute of Genomics and Integrative Biology, Delhi, for spectroflu-orometric analysis. This work was supported by CSIR, New Delhi inthe form of Junior Research Fellowship to SY and AD.

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