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Apparent cross-talk of two signaling pathways that regulate Zea mays coleoptilegrowth

Aparente entrecruzamiento de dos vas de sealizacin que regulan el crecimiento del coleoptilo

en Zea mays

Buentello Volante1

B, F Daz de Len-Snchez1

, F Rivera-Cabrera1

, R Aguilar Caballero2

,M Ponce-Valadez1, E Snchez de Jimnez2, LJ Prez-Flores1

Abstract. Auxin and insulin promote Zea maysembryo growth,induce S6 ribosomal protein (S6rp) phosphorylation, and promotespecic protein synthesis. Te objective o this research was to testa possible cross-talk between insulin and auxin transduction path-ways in Z. mays coleoptiles, typical auxin target tissues. Auxin andinsulin produced diferential quantitative and qualitative stimulationo cytoplasmic and ribosomal protein phosphorylation, and specicpatterns o de novo synthesized cytoplasmic proteins. In addition,insulin induced S6rp phosphorylation was strongly inhibited by

rapamycin, indicating target o rapamycin (OR) kinase participa-tion; auxin-induced S6rp phosphorylation was insensitive to thisinhibitor. Phosphatidic acid (PA), a second messenger o OR inmetazoan, was also tested. It produced similar results to insulin andrapamycin sensitiveness, supporting the existence o OR pathwayin plants and the participation o PA as an intermediate o insulinaction. Tese results seem to imply that auxin and insulin induce Z.mayscoleoptile growth through two independent signal transductionpathways.

Keywords: Auxins; Insulin; Phosphatidic acid; ZmS6K; OR;Plant growth.

Resumen. Las auxinas y la insulina promueven el crecimiento deembriones de Zea mays, inducen la osorilacin de la protena ribo-somal S6 (prS6) y promueven la sntesis de protenas especcas. Elobjetivo de esta investigacin ue probar el posible entrecruzamientoentre las vas de transduccin de insulina y auxinas en coleoptilos deZ. mays, tejidos blanco tpicos de auxinas. Las auxinas y la insulinaprodujeron una estimulacin dierencial cuantitativa y cualitativa dela osorilacin de protenas citoplsmicas y ribosomales, as comopatrones especcos de protenas citoplsmicas sintetizadas de novo.

Adems, la induccin de la osorilacin de prS6 por insulina ueuertemente inhibida por rapamicina, indicando la participacin dela cinasa blanco de rapamicina (OR), mientras que la osorilacinde prS6 inducida por auxinas ue insensible a este inhibidor. Se pro-b tambin cido osatdico (PA), un segundo mensajero de ORen metazoarios. Esto produjo resultados y sensibilidad a rapamicinasimilares a los de insulina, apoyando la existencia de la va OR enplantas y la participacin de PA como un intermediario de accin deinsulina. Estos datos parecen implicar que las auxinas y la insulinainducen el crecimiento de coleoptilos de Z. maysa travs de dos vasde transduccin de seales independientes.

Palabras clave: Auxinas; Insulina; cido osatdico; ZmS6K;OR; Crecimiento vegetal.

1 Departamento de Ciencias de la Salud Universidad Autnoma Metropolitana-Iztapalapa, Av. San Raael Atlixco No. 186 Col. Vicentina Iztapalapa 09270 Mxico, D.F.2 Departamento de Bioqumica, Facultad de Qumica, Universidad Nacional Autnoma de Mxico. Circuito Institutos, Ciudad Universitaria 04510 Mxico, D.F.Address Correspondence to: Dra. Laura Josena Prez-Flores, email: ljp@xanum.uam.mx; ax 052-55-58044727, phone 052-55-58046481 and E. Snchez de Jimnez,email: estelas@servidor.unam.mx; Fax 052-55-56225329, phone 052-55-56225278.Recibido / Received 24.I.2010. Aceptado / Accepted 7.V.2010.

REVISTA INTERNACIONAL DE BOTNICA EXPERIMENTALINTERNATIONAL JOURNAL OF EXPERIMENTAL BOTANY

FUNDACION ROMULO RAGGIOGaspar Campos 861, 1638 Vicente Lpez (BA), Argentina

www.revistaphyton.fund-romuloraggio.org.ar

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INRODUCION

Auxins regulate many processes such as growth, cell prolier-ation and plant development and dierentiation (Dharmasiri &

Estelle, 2004; Weijers & Jrgens, 2004; Overvoorde et al., 2005;Benjamins & Scheres, 2008). However, the auxin-induced sig-naling pathways involved in these processes are at present notully understood. Te search or auxin receptors has led to theisolation o auxin-binding proteins, the best characterized beingthe ABP1 (Chen, 2001; Delker et al., 2008). Overexpression oABP1 in plant leaves has resulted in cell number reduction butincreased cell size, indicating that auxins can regulate both cellelongation and cell division through dierent pathways (Hob-bie et al., 2000). Moreover, two dierent ABP1 auxin receptorshave been identied inN. tabacum,suggesting that they corre-spond to each o the two modes o auxin-induced cell growth,

elongation or cellular division (Campanoni & Nick, 2005).Another auxin-receptor has also been reported, the transportinhibitor response 1 protein (IR1), a member o the plantSCF complex o the proteasome. Tis receptor mediates auxin-induced transcriptional responses, and thereater regulates theexpression o auxin-regulated genes (Kepinski & Leyser, 2005;Badescu & Napier, 2006; Mockaitis & Estelle, 2008). All theseresponses seem to be tissue and developmental stage dependentevents (Dharmasiri & Estelle, 2004).

On the other hand, cell growth and cell prolieration areregulated by insulin/IGFs (insulin-like growth actors) inmetazoans (Meyuhas & Hornstein, 2000; Hannan et al.,2003; Oldham & Haen, 2003). Tese eectors induce a sig-nal transduction pathway known as the PI3K-OR pathway,that targets the translational apparatus selectively inducingprotein synthesis (mainly proteins o the translational ap-paratus) (Ruvinsky & Meyuhas, 2006; Patursky et al., 2009).A central role in this pathway is played by OR (target orapamycin) kinase, a highly conserved cell growth controllerwhich regulates the downstream signals initiated by the eec-tor (Hay & Soneneberg, 2004; Lorberg & Hall, 2004). Re-cently, PA has been demonstrated as a critical intermediate omOR pathway. Mitogenic stimulation o mammalian cellsled to a phospholipase D-dependent accumulation o cellularPA (250% at 5 min o treatment), which was required or ac-

tivation o mOR downstream eectors (Fang et al., 2001).In plants, PA has been identied as an important signalingmolecule, triggered in response to various biotic and abioticstress actors; it has also been implicated in seed germination.In general, PA signal production is ast (minutes) and tran-sient (Munnik, 2001; esterink & Munnik, 2005; Carman &Henry, 2007).

Among the best known targets o mOR pathway is thephosphorylation o S6rp by activation o specic S6 kinase(S6K). In mammals, S6K can be phosphorylated by at least twodierent protein kinases, OR and 3-phosphoinositide depen-dent kinase 1 (PDK1). Tese kinases phosphorylate dierent

residues o S6K; OR commonly accepted targets are S411and 389 (Dennis et al., 2001; Ruvinsky & Meyuhas, 2006),whereas or PDK1 are 229, 252 and 412 (empleton, 2001;Rebholz et al., 2006). Moreover, the S6K1 phosphorylation at

S411 is required or the rapamycin-sensitive phosphorylation o389 and the subsequent activation o S6K1 (Hou et al., 2007).Recently, it has been shown that this pathway is also unctionalin plants. OR and S6K plant ortholog enzymes have beenreported or Arabidopsis thaliana(Menand et al., 2002; urcket al., 2004) and Z. mays(Reyes de la Cruz et al., 2004); theydemonstrated to perorm similar roles and regulatory eects asin other non-photosynthetic eukaryotes (Mahouz et al., 2006;Dinkova et al., 2007). InArabidopsisthaliana, a PDK1 orthologhas been identied; this enzyme phosphorylates AtS6K2 andcan be regulated by their association with dierent proteins(Otterhag et al., 2006).

In Z. mays, an insulin like growth actor (ZmIGF) has beenisolated (Garca et al., 2001) and proved to stimulate ORand S6K, and phosphorylate S6rp (Dinkova et al., 2007), themain S6K substrate. Tis pathway is also responsive to insulinin Z. mays(Garca et al., 2001; Beltrn et al., 2002).

Interestingly, recent reports have shown that auxin also in-duces S6rp phosphorylation in Z. mays(Beltrn et al., 2002),as well as inA. thaliana(urck et al., 2004). Tis suggests thatS6rp phosphorylation in Z. mays, and likely in other plants,might be regulated by two dierent eectors -auxins and in-sulin/ZmIGF. Tis opens the question o a possible cross-talk between these two cell growth signaling pathways. Tisquestion is very interesting since peptide-induced signal trans-duction pathways are at present not widely known in plants(Ryan et al., 2002). Te term cross-talk has been proposedto reer to situations where dierent signaling pathways shareone or more intermediates/components or have some com-mon outputs. However, signaling pathways sharing commoncomponents may not necessarily cross-talk i the commoncomponents are scaolded into distinct protein complexes(Park et al., 2003; Chinnusamy et al., 2004).

Te main objective o this research was to test whether in-sulin and auxin induce signaling pathways cross-talk at com-mon steps, to understand the meaning that this phenomenonmight have in regulating plant cell growth. Te participation

o PA as an intermediate o OR pathway in plants was alsoanalyzed. Since Z.mayscoleoptiles are typical targets o auxinresponsive tissues (Woodward & Bartel, 2005), they werechosen as the model system to pursue this investigation. Teresults rom the present study demonstrated that PA is trig-gered by both eectors (auxin and insulin) and that auxin,insulin or PA conuence at the phosphorylation o S6rp.However, insulin induced S6rp phosphorylation was stronglyinhibited by rapamycin, supporting the involvement o ORkinase. At the same time, S6rp phosphorylation induced byauxin was insensitive to this inhibitor, suggesting a OR ki-nase independent pathway. Te rapamycin inhibition o PA

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induced S6rp phosphorylation supports the participation oPA as an intermediary o insulin pathway. Furthermore, theoutput events induced ater S6rp phosphorylation by eithereector were not biochemically equivalent.

MAERIALS AND MEHODS

Biological material and treatments. Seeds rom Z. maysL. var. Chalqueo were disinected with 10% (v/v) sodium hy-pochlorite solution and imbibed in sterile water or 22 h indarkness at 25 C. Embryonic axes were manually dissectedand incubated or 2 h at 25 C in Murashige Skoog mediumcontaining any o the ollowing eectors: 200 mol/L auxin(indole acetic acid, IAA), 200 U insulin or 100 mol/L PA.Rapamycin (100 nmol/L) was also added or some experi-ments. At the end o the treatment, coleoptiles were excised,

rozen in liquid nitrogen and stored at -70 C until used. Aux-in and insulin concentrations and time o treatments have beenpreviously demonstrated to be eective in inducing coleoptilegrowth (Beltrn et al., 1995; Reyes de la Cruz et al., 2004).Due to its availability, insulin was used to stimulate Z. mayscoleoptiles. Te concentration o PA has been used in mam-malian cells to stimulate OR pathway (Fang et al., 2001).

Quantifcation o PA concentration. Phospholipids wereisolated and separated according to Munnik et al. (1995). Em-bryonic axes rom 22 h germinated seeds were excised and thenlabeled with 3.7 x 106 Bq [32P]-orthophosphate. Tey were thenincubated with either o the above mentioned eectors and 1%(v/v) butanol or 5, 10, 15 and 30 minutes. Ater this incubationperiod, coleoptiles (200 mg) were dissected, pulverized with liq-uid nitrogen, and rozen powder was homogenized with 6 mLo extraction buer (250 mmol/L sucrose; 3 mmol/L EDA;2 mmol/L EGA; 14 mmol/L 2-mercaptoethanol; 2 mmol/LD; 30 mmol/L ris-HCl, pH 7.4). Te homogenate wascentriuged at 100000 g at 4 C or 1 h. Te pellet was resus-pended in Hepes 50 mmol/L, and the lipids were extractedwith 1.5 mL o CHCl

3/ methanol (1:2 v/v), 0.5 mL o HCl

2.4 mol/L and 0.5 mL o CHCl3, vortexing or 30 s. A two-

phase system was observed by adding 2 mL o HCl 1 mol/L /methanol (1:1 v/v). Organic phase was recovered and dried un-der nitrogen atmosphere. [32P]-Phospholipids were separated

on heat-activated silica 60 thin layer chromatography (LC)plates (20 cm x 20 cm; Merck) with a mixture o ethyl acetate/ iso-octane / ormic acid / water (13:2:3:10 v/v). Radiolabeledphospholipids were visualized by autoradiography and bandscorresponding to PA or PA-BuOH were recovered and quanti-ed by scintillation counting. PA can be produced by two ways:(1) phospholipase C (PLC) in combination with diacylglycerolkinase (DAGK) and (2) phospholipase D (PLD).

Isolation and analysis o phosphorylated ribosomal andcytoplasmic proteins. Embryonic axes rom 22 h germinatedseeds were excised and then labeled in vivo with 7.4 x 106 Bq[32P]-orthophosphate and incubated with either o the above

mentioned eectors or 2 h. For some experiments, 100 nmol/Lrapamycin was also included in the incubation medium. Aterthis incubation period, the embryonic axes were dissected andtheir coleoptiles separated. Te coleoptiles (200 mg) were ho-

mogenized in liquid nitrogen,and the rozen powder was mixedwith three volumes o extraction buer (50 mmol/L ris-aceticacid (pH 8.2); 50 mmol/L KCl; 5 mmol/L magnesium acetate;250 mmol/L sucrose; 5 mmol/L 2-mercaptoethanol; 5 mmol/LNaF, and 1 mmol/L PMSF). Te ribosomal and cytoplasmicproteins were obtained as reported by Beltrn et al. (1995). Tehomogenate was centriuged at 27000 g or 30 min. Te super-natant was centriuged during 4 h at 280000 g in 1.6 mol/Lsucrose cushion to isolate the ribosomal raction. Te ribosomalpellet was resuspended in 1 mL buer (20 mmol/L HEPES;20 mmol/L KOH; 5 mmol/L magnesium acetate; 125 mmol/Lpotassium acetate, and 6 mmol/L 2-mercaptoethanol). Ribo-somal proteins were extracted with acetic acid-magnesium ac-etate precipitated with acetone and centriuged at 3000 g. Pel-lets were dried at room temperature and resuspended in water.Te supernatant o the 240000 g centriugation was treatedwith cold CA [(10% (w/v) nal concentration]. Precipitatedcytoplasmic proteins were centriuged at 3000 g; washed againwith cold CA [10% (w/v)] recentriuged at 3000 g; the pelletwas resuspended in water, and neutralized with 1mol/L KOHor urther analysis. Protein concentration was determined ac-cording to Bradord (1976), using bovine serum albumin asstandard. Scintillation counting was used to determine [32P] in-corporation. Labeled ribosomal proteins were resolved on onedimensional 12% acrylamide gels; cytoplasmic proteins were

separated by 2-D gel electrophoresis using IPG ReadyStripwith 5 to 8 pH gradient (BIO-RAD) and 12% SDS polyacryl-amide gels. Labeled proteins were analyzed by Phosphorimager(BIO-RAD GS-525).

2-D gel electrophoresis o newly synthesized proteins.Embryonic axes rom 22 h germinated seeds were excised andincubated with 1.48 x 107 Bq [35S] methionine or 2 h, andexposed to the eectors as mentioned above. Ater incubationat 25 C, the coleoptiles were separated. Tereater, cytoplas-mic proteins were isolated as described above, and analyzedby 2-D gel electrophoresis as indicated. Labeled proteins wereanalyzed by Phosphorimager (BIO-RAD GS-525).

Statistical analysis. All experiments were repeated at leastthree times using extracts rom dierent biological samples.[32P]-Orthophosphate and [35S]-methionine incorporationvalues were analyzed by ANOVA and ukeys mean compari-sons (p

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Foster, 2007; oschi et al., 2009). Moreover, PA has beenimplicated in the regulation o seed germination and as anintermediary in the response to several stresses (Munnik,2001; esterink & Munnik, 2005). Tereore, the levels o

this phospholipid were quantied in Z. mays coleoptiles ex-posed to IAA or insulin or dierent time periods. Incubationwith butanol allowed the identication o the PA originatedrom PLD activity, since this phospholipid binds to the alco-hol, while when it is originated rom the combined actions oPLC and DAGK this binding does not occur. Results o thepresent research indicate a higher contribution (almost 80%)o PLD in PA biosynthesis. A signicant transient increasein total PA levels was observed at 5 and 10 min with IAAor insulin treatments (p

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Ribosomal protein phosphorylation ater auxin, insulinor PA induction. Auxin and insulin are known to induce phos-phorylation o the S6rp in Z. maysandA. thaliana(Beltrn etal., 2002; urck et al., 2004), a protein known to be targeted by

activation o the PI3K-OR pathway in mammals. Tus, thepossibility o cross-talk at this step by the auxin- or insulin-in-duced signaling pathways was analyzed. With this purpose, theribosomal raction rom Z. maysaxes [32P] labeled and stimu-lated by either auxin, insulin or PA was isolated, and the ribo-somal proteins were obtained. For some samples rapamycin, aninhibitor o OR kinase, was applied to the tissues in combi-

Control

IAA

PA

Insulin

pH 5kD 86645

3424

18

12

66453424

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12

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3424

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Fig. 2. Autoradiography o 2D-gel electrophoresis o phosphorylated

cytoplasmic proteins induced by auxin, insulin or PA onZea mays co-

leoptiles. The experiment was independently reproduced at least three

times with similar results. The arrow heads indicate phosphorylated

cytoplasmic proteins induced by auxin; the arrows with closed heads

those phosphorylated by insulin, and the arrows represent phospho-rylated proteins by PA.Fig. 2. Autoradiograa de electrooresis en geles 2D de protenas citop-

lsmicas osoriladas inducidas por auxinas, insulina o PA en coleoptilos de

Zea mays. El experimento se reprodujo al menos tres veces en orma inde-

pendiente con resultados similares. Las puntas de fecha indican protenas

citoplsmicas osoriladas inducidas por auxinas; las fechas con puntas

negras, indican las osoriladas por insulina y las fechas representan las

protenas osoriladas por PA.

Auxin and insulin growth induction

A) B)43210

C IAA I PA43210

C IAA I PA

Arbitraryunits

+ Rapamycin

Arbitraryunits

C IAA

I PA

51

42

32S6RP

51

42

32S6RP

kD

45

34

24

18

14

45

34

24

18

14

Fig.3. Analysis o phosphorylated ribosomal proteins, induced by

auxin, insulin, or PA on Zea mays coleoptiles. A) Autoradiography o

phosphorylated ribosomal proteins resolved by SDS-PAGE. B) Den-

sitometric analysis o phosphorylated S6rp. The experiments were re-

produced independently at least three times with similar results.Fig. 3. Anlisis de las protenas ribosomales osoriladas, inducidas por

auxinas, insulina o PA en coleoptilos deZea mays. A) Autoradiograa de

las protenas ribosomales osoriladas resueltas por SDS-PAGE. B) Anlisis

densitomtrico de prS6 osorilada. El experimento se reprodujo al menos

tres veces en orma independiente con resultados similares.

nation with the eector. [32P] incorporation was determinedas mentioned above. Signicantly higher [32P] incorporation(approximately 400% with respect to the control) was ound inribosomal proteins rom insulin or PA-stimulated coleoptiles

than or the auxin-treated coleoptiles (approximately 225%with respect to the control) (able 1).

Te phosphorylated ribosomal proteins were urther re-solved by SDS-PAGE (Fig. 3A). Te correspondent autora-diographs showed again three dierent patterns, one or eacheector (auxin, insulin or PA). A phosphorylated 32 kDa bandwas observed in all samples, which corresponded to S6rp iden-tied by Western blot (data not shown). Te densitometricanalysis o this protein indicated a signicantly higher level o[32P]-incorporation (approximately 250%) in the insulin or PA-stimulated samples, whereas the stimulation was signicantlylower (150%) in the auxin-induced ones, relative to the control

(Fig. 3B). Regarding the rest o the phosphorylated ribosomalproteins, important qualitative dierences were also observed.Insulin or PA induced phosphorylation o 42 kDa and 51 kDaribosomal proteins. Auxin induced phosphorylation o a ribo-somal protein o approximately 24 kDa, and o a smaller one(14 kDa), which possibly corresponded to the so called acidicribosomal phosphoproteins (Montoya et al., 2002; Santos et al.,2004) (Fig. 3A). Addition o rapamycin to the axes resulted ina dramatic decrease o the insulin or PA-induced S6rp phos-phorylation, but almost no eect was observed in the S6rpphosphorylated protein induced by auxin (Fig. 3A and 3B).Tis suggests that OR pathway is involved on insulin and PAaction, and that PA is an intermediary o the insulin eect.

Auxin, insulin and PA eect on protein synthesis oZ.mayscoleoptiles. OR activation o S6K by insulin is knownto target the translational apparatus regulating de novo syn-

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thesis o proteins in the stimulated cells. o test whetherauxin and insulin dierentially aected the pattern o pro-tein synthesis in Z. mays coleoptiles, the next step was toanalyze the proteins synthesized ater coleoptile stimulation

either by insulin, PA or auxin. Excised embryonic axes rom24 h germinating seeds were incubated with [35S]-methionineand stimulated by either eector. Te total amount o [ 35S]incorporated into cytoplasmic proteins was determined andresolved by 2D gel electrophoresis.

Slight quantitative changes were ound on the [35S]-me-thionine incorporated in the cytoplasmic protein raction, byeither IAA, insulin or PA induction. Te incorporation rangedwithin 25% to 31%, as compared to the control. Tese pro-

teins, urther resolved by two-dimensional gel electrophoresis,showed specic patterns or each eector in the correspondentautoradiographs. IAA samples showed changes in the patterno synthesized cytoplasmic proteins compared to the control

(arrow heads), and were dierent rom those induced by insu-lin (arrows with closed head) or PA (arrows). It is important toemphasize that PA induced the synthesis o an extra proteinwith respect to insulin, and inhibited the synthesis o a proteinwith respect to the control (Fig. 4). Tese data clearly indicatedthat each eector (auxin, insulin or PA) caused a dierent bio-chemical output ater coleoptile stimulation.

DISCUSSION

Plants have two dierent modes o growth. Plant growthollows two dierent modes in response to various stimuli: cell

elongation and cell division. Te balance between these twogrowth modes depends on the stimulus, the type o target tis-sue and its developmental stage (Eckardt, 2005). Our resultsseem to be consistent with this proposal, since two growthregulators produced dierent biochemical events ater target-ing the same tissue in Z. mays, even when these eectors havesome intermediates in common. Coleoptile elongation is wellknown as an auxin eect (Benjamins & Scheres, 2008). On theother hand, insulin as well as insulin-like growth actors suchas ZmIGF have been recognized to exert mitogenic activityin plant tissues, inducing DNA synthesis in Z. maysseedlings,and cell division in both Daucus carotaand Z. maysin vitro cellcultures (Garca et al., 2001; Yamazaki et al., 2003). Te lit-erature support the proposal that auxins might be consideredgrowth stimulators by promoting cell elongation, whereas in-sulin might do so by preerentially stimulating cell division.

Biochemical dierences between two signaling path-ways. In general terms, the data presented here support amodel or coleoptile growth through two dierent pathwaysinduced either by auxin or by insulin. Te interpretation otwo dierent pathways induced by auxin or insulin is con-sistent with the dierent phosphorylation patterns o cyto-plasmic proteins ound ater stimulation by each eector (Fig.2). Furthermore, although both eectors stimulate S6rp phos-phorylation, a dierent set o other specic phosphorylated

products were identied or auxin and insulin (Fig. 3). Moreimportant and consistent with these dierences are the spe-cic rapamycin inhibition observed or insulin-induced S6rpphosphorylation, which was not observed when this phos-phorylation was auxin-induced (Fig. 3). Tis indicates that inthe rst case the OR pathway was activated (Fang et al.,2001; Hay & Sonenberg, 2004), whereas induction by auxindid not ollow this path. An explanation or this dierencemight be the activation o two dierent ZmS6 kinase iso-orms responsible or S6rp phosphorylation by each eector(able 1, Fig. 4). Tis possibility is supported by the literature,indicating the presence o two S6K isoorms in many organ-

Control

IAA

PA

Insulin

6645

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453424

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Fig. 4. Auxin, insulin and PA eect on de novo synthesis o cytoplasmic

proteins romZea mays coleoptiles. Autoradiography o 2D-gel electro-

phoretic patterns ode novo synthesized cytoplasmic proteins. The ex-

periments were reproduced at least three times with similar results. The

arrow heads indicate de novo synthesized cytoplasmic proteins inducedby auxin; the arrows with closed heads those synthesized by insulin,

and the arrows represent those induced by PA.Fig. 4. Eecto de auxinas, insulina y PA en la sntesis de novo de protenas

citoplsmicas de coleoptilos deZea mays. Autoradiograa de los patrones

electroorticos en geles 2D de las protenas citoplsmicas sintetizadas

de novo. Los experimentos se reprodujeron al menos tres veces con re-

sultados similares. Las puntas de fecha indican protenas citoplsmicas

sintetizadas de novo inducidas por auxinas; las fechas con puntas ne-

gras indican aquellas sintetizadas por insulina, y las fechas representan

aquellas inducidas por PA.

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isms, one o which could be a nuclear enzyme (Panasyuk et al.,2006). Te dierent S6rp phosphorylated products obtainedater stimulation by either eector, as well as the dierent ra-pamycin sensitivity o S6rp phosphorylation observed in this

research are consistent with this explanation (Fig. 3). In addi-tion, there are reports indicating that S6K might also be acti-vated by another kinase, closely related to the OR pathway,namely PDK1, which is Ca2+ dependent activated (Bgre etal., 2003). Since it is known that auxin induces a cytoplasmicCa2+ increase, ater cell stimulation (Macdonald, 1997), thesedata suggest that auxin-induced S6 kinase activation in Z.mayscoleoptile might be due to this second pathway. On theother hand, our results indicate that insulin-activated ZmS6Kwas mostly phosphorylated via OR activation, since S6rpphosphorylation showed to be sensitive to rapamycin inhibi-tion (Fig. 3). Previous results have also demonstrated that aOR-S6K signaling pathway is unctional in Z. maysduring

germination, targeting the translational apparatus through aOR kinase that is inhibited by rapamycin (Dinkova, 2007).Interestingly, auxin, but not insulin or PA, induced phospho-rylation o low molecular weight ribosomal proteins (Fig. 3A,14 kDa approximately), which most likely correspond to acid-ic phosphoribosomal proteins (ARP) (Montoya et al., 2002;Santos et al., 2004). Tese proteins are known to play a centralrole in the regulation o translation due to their interactionwith elongation actors (Vard et al., 1997; Santos et al., 2004).Tese data are consistent with the dierential unctional pro-teomics observed on the 2D-gels ode novo synthesized pro-teins induced by each eector: auxin or insulin (Fig. 4), whichinduced distinctive sets o synthesized proteins.

Auxin, as well as insulin induced an increase o PA levels.However, insulin stimulation was higher than that producedby auxin (Fig. 1). In addition, insulin and PA eects on cyto-solic protein phosphorylation, de novo protein synthesis andribosomal protein phosphorylation (among them S6rp) weremore similar between them than to those produced by auxin.Moreover, rapamycin sensitivity o PA-induced S6rp phos-phorylation supports the participation o PA as a second mes-senger o insulin action involving the OR pathway in plants,as it was demonstrated to occur in metazoans (Fang et al.,2001). However, PA and insulin responses were not identical,possibly indicating the participation o PA as an intermedi-

ary o other pathways induced by other hormones or stresses.PA participation as an intermediary o auxin action requiresurther studies.

Overall, the present research contributes to the understand-ing o the possible mechanisms involved in plant growth reg-ulation. Te data support the proposal that auxin and insulintriggered Z. mayscoleoptile growth by distinctive mechanisms,through two independent signal transduction pathways. Evenwhen both eectors share some intermediates, they do not seemto share neither the same target products nor induce the samedownstream biochemical events, particularly on the synthesiso proteins.

ACKNOWLEDGEMENS

We thank Dr. . Hernndez, Dr. L. Brito, Dr. H. Estrada orhelp in establishment o methodologies; Dr. H. Gonzalez, Dr. J. L.Gmez, and Dr. H. Reyes de la Cruz or their technical assistance.Tis work is part o the BBV PhD dissertation. Tis research wasunded by Universidad Autnoma Metropolitana, CONACY(Grant No.2006-1-61424) SEP-PROMEP (Grant No. UAM-I-CA-26), DGAPA UNAM (Grant No. IN207903). BBV holdsa ellowship rom CONACY (No. 153010) during her studiesin the Experimental Biology PhD Program.

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