BACKGROUND
in plants, pollination and subsequent fertilization rely on an extensive and complex dialog between the tissues of the pistil  and the pollen tube  <cit> .
numerous proteins and other molecules from both the female and male are thought to regulate the biochemical dialog established when the pollen grain lands on the stigma, during pollen tube growth through the style and upon arrival at a synergid cell where the sperm cells are discharged.
some observations suggest that there is a hierarchy of signals in pollen tube germination and growth, wherein a pollen tube is unable to respond to late signals coming from the female gametophyte if it has not been previously exposed to early signals coming from the sporophyte  <cit> .
this implies that pollen tubes have the ability to determine their geographical position within the female tissues and modify their physiology accordingly.
leprk <dig> and leprk <dig> are two lrr-receptor like kinases specifically expressed in pollen grains and tubes in solanum lycopersicum   <cit>  and homologs of these proteins exist in other species  <cit> .
these kinases localize to the plasma membrane and belong to a high molecular weight complex   <cit> ; leprk <dig> and leprk <dig> bind different proteins from the pistil  or from pollen .
leprk <dig> is phosphorylated in mature and germinated pollen, but is specifically dephosphorylated upon incubation with style extracts  <cit> ; this suggests that style components have the potential to regulate the leprk complex biochemically  <cit> .
we previously determined that this style component in tomato and tobacco had a molecular weight of 3- <dig> kda and was heat-stable  <cit> .
we also showed that leprk <dig> and leprk <dig> interact when expressed heterologously in yeast, and that this interaction can be dissociated by the addition of the same style fractions that promote leprk <dig> dephosphorylation  <cit> .
recently zhang et al.
showed that antisense expression of leprk <dig> resulted in pollen tubes with a reduced growth rate, suggesting that leprk <dig> might be involved in pollen tube growth regulation  <cit> .
a cytoplasmic protein called kpp  <cit> , which is a ropgef  <cit> , interacts with both leprk <dig> and leprk <dig>  this interaction suggested a linkage between extracellular signals, receptor kinases, and modulation of rop activities, which is critically important for pollen tube growth  <cit> .
numerous low molecular weight polypeptides have been implicated in signal transduction pathways in plants  <cit> .
some were isolated by biochemical purification, such as systemin   <cit> .
specific physiological or biochemical effects were associated with these polypeptides  <cit>  and their receptors were identified and biochemically characterized [systemins,  <cit> ; phytosulfokines,  <cit> ; ralf,  <cit> , but early correlates of ligand binding, such as receptor de/phosphorylation, hetero-oligomerization or dissociation from interacting proteins, have not yet been shown.
other polypeptide ligands were first identified from mutant screens, such as clavata <dig>   <cit>  and tapetum determinant <dig>   <cit> , by bioinformatics, such as the clavata3/embryo surrounding region-related peptides   <cit> , or by map-based cloning, such as the s-locus cysteine rich protein /s-locus protein  <dig>   <cit>  among others  <cit> .
receptors for clv <dig>  tpd <dig> and scr/sp <dig> have been identified  <cit> .
binding of scr/sp <dig> to the s-locus receptor kinase  and tpd <dig> binding to the receptor kinase excess microsporocytes <dig>  induce receptor autophosphorylation  <cit> , and in the case of scr/sp11-srk, complex formation with the s-locus glycoprotein   <cit> .
in this paper, we describe the purification of stil, a peculiar ~ <dig>  da molecule from tobacco pistils that is responsible for leprk <dig> dephosphorylation.
stil's activity is heat-, acid-, base-, dtt- and protease-resistant.
our results show that stil promotes pollen growth from the onset of germination in a dose-dependent manner.
we hypothesize that stil's binding to the leprk complex triggers leprk2-specific dephosphorylation, which in turn modulates downstream components of the leprk complex transduction pathways, such as ropgef  <cit>  and probably rop, resulting in pollen tube growth stimulation.
RESULTS
stil is a hydrophilic molecule that specifically promotes leprk <dig> dephosphorylation
we previously showed that leprk <dig> is phosphorylated in tomato pollen microsomes, but specifically dephosphorylated when tomato stigma/style extracts were added during or after a phosphorylation reaction  <cit> .
this suggested the presence of a leprk <dig> dephosphorylating activity in tomato styles.
subsequently, we demonstrated that a 3- <dig> kda heat-resistant molecule present in tomato and tobacco stigma/style extracts or exudates was responsible for this dephosphorylation  <cit>  .
to purify this molecule, we used leprk <dig> dephosphorylation as a tracking assay.
this molecule was named stil for style interactor for leprks.
in order to design a purification protocol, the behavior of stil under various extraction or precipitation protocols was first evaluated.
most proteins can be precipitated from organic solvents  <cit> .
however, fig.
1b shows that stil dephosphorylation activity was in the aqueous phase of a methanol-chloroform extraction and not in the organic phase or at the interface.
this suggested that stil did not have exposed hydrophobic moieties that in organic solvents partition to the organic phase.
in contrast to typical proteins, stil phosphorylation activity could not be precipitated by trichloroacetic acid, even in the presence of a carrier protein such as bovine serum albumin , implying that stil is highly soluble in salting out-low ph conditions, maybe because of the presence of negatively-charged highly hydrophilic residues on its surface.
we then evaluated the interaction of stil with anionic and cationic exchange resin chromatography.
the aqueous phase from methanol-chloroform-extracted stigma/style exudates was dried, dissolved in water and loaded onto solid-phase extraction cartridges.
the resin was washed extensively with water and successively eluted with an ammonium bicarbonate gradient ending at  <dig>  m. stil dephosphorylation activity was retained and selectively eluted from an anionic exchanger, but not from a cationic exchanger .
peptides are often purified using c <dig> reverse-phase, solid phase extraction cartridges .
therefore, acetonitrile was added to a final concentration of 6% to an anionic exchanger-purified sample of stil and loaded onto a c <dig> reverse-phase cartridge.
the cartridge was washed with 6% acetonitrile until the absorbance at  <dig> nm reached basal levels and then was eluted with 50% and 100% acetonitrile, resulting in the elution of retained compounds .
surprisingly, stil dephosphorylating activity was present in the flowthrough  but not in the eluates .
this result further supports the idea that stil does not have exposed hydrophobic moieties, and thus does not interact with c <dig> reverse-phase resins.
stil purification protocol
based on the behavior of stil shown above, a purification protocol for stil was developed .
although stil was present in the flowthrough after c <dig> reverse-phase chromatography, this step was also included in order to purify stil away from other  <dig> nm-absorbing compounds retained on this column .
stigma/style exudates from tobacco pistils were extracted with methanol-chloroform and the aqueous phase was evaporated.
the pellet was dissolved in water and loaded onto an anionic exchange column .
fractions that induced leprk <dig> dephosphorylation were pooled together, concentrated and subjected to solid-phase extraction on a c <dig> reverse-phase cartridge.
the sample was loaded in 6% acetonitrile and the flowthrough was collected and freeze-dried.
the pellet was dissolved in water and loaded onto an anion exchange column .
fig.
2c shows that the fractions that dephosphorylated leprk <dig> eluted as a  <dig> nm-absorbing single peak.
as a result of the purification process, stil was purified ~ <dig>  fold when compared to the starting exudate .
it is important to mention that the c <dig> solid-phase extraction step did not increase the purity of stil more than the first anionic exchange column.
no significant difference in a t-test .
no duplicates available.
k2d <dig> represents abs <dig> units necessary for 50% dephosphorylation of leprk2; values were obtained from an average of two independent purifications.
% error is calculated as  ×  <dig>  the purification factor was calculated by comparison to the starting exudate.
the active fractions from the monoq column were pooled and subjected to uv-maldi-tof ms. fig.
<dig> corresponds to one of  <dig> chromatographs from independent purifications where only one peak with a molecular mass of ~ <dig>  da was consistently obtained; this result demonstrated that this entity corresponds to stil and was purified to homogeneity.
the fractions retained on the c <dig> reverse-phase cartridge  were used as controls for pollen germination assays , and these were inactive  and never showed a peak with a molecular mass of ~ <dig>  da in mass spectrometry determinations.
fig.
4a shows that when a dilution series of pure stil was assayed in the leprk <dig> dephosphorylation assay,  <dig>  × 10- <dig> absorbance units of stil were able to dephosphorylate 50% of leprk <dig> .
moreover, the radioactive signal corresponding to phosphorylated leprk <dig> was fully recovered only after diluting stil to  <dig>  × 10- <dig> absorbance units .
these results indicate that dephosphorylation of leprk <dig> was reduced when the amount of pure stil was decreased.
stil is labile to microwave-assisted acid hydrolysis
to further biochemically characterize stil, fractions of stil were incubated with several proteases, such as trypsin, pepsin, carboxypeptidase y, thermolysin and proteinase k, then the hydrolysates were tested for their ability to dephosphorylate leprk <dig>  none of the tested proteases inactivated stil, since protease-treated stil was still able to induce leprk <dig> dephosphorylation .
the structural conformation of many low molecular weight peptides relies on numerous cysteine residues that stabilize them by disulfide bridges  <cit> .
we tested the effect of incubating stil at 100°c with  <dig> mm dtt for  <dig> min, which should disrupt disulfide bridges.
the effects of base  or acid  hydrolysis were also evaluated.
again, none of these treatments had any effect on the ability of stil to dephosphorylate leprk <dig> .
therefore, a different and stronger acid hydrolysis protocol was evaluated.
microwave-assisted acid hydrolysis is commonly used for structural analysis of proteins and peptides  <cit> .
leprk <dig> could not be dephosphorylated after stil was subjected to microwave-assisted acid hydrolysis, suggesting that this treatment totally or partially inactivated stil .
stil was subjected to the listed treatments and the dephosphorylating activity was tested in a leprk <dig> dephosphorylation assay .
stil resistance to the treatment is indicated by "+", while "-" indicates stil loss of activity.
several pieces of evidence support that stil is at least partially peptidic in nature: the presence of stil always correlated with absorbance at  <dig> nm and amino acid determination  showed that several amino acids are present in the stil molecule.
amino acid determination relies on acid hydrolysis and derivatization.
since stil activity is resistant to traditional acid hydrolysis, we expect that the full amino acid composition or the proportion of each amino acid has not been determined.
pure stil was subjected to  <dig> n hcl hydrolysis for  <dig> hours in a non-oxidizing environment, then derivatized and separated by gas chromatography.
amino acids were identified by comparing their retention times to standards.
stil promotes pollen tube growth from the onset of germination
leprk <dig> has been implicated in transducing pistil signals to the pollen tube, resulting in the regulation of pollen tube growth  <cit> .
this regulation is likely mediated by the regulation of rop through kpp   <cit> .
overexpression of a nearly full-length kpp in tomato or tobacco pollen resulted in the appearance of a swollen tip, a phenotype reminiscent of that seen when atrop <dig> was overexpressed in tobacco pollen  <cit> .
furthermore, pollen tubes transiently over-expressing leprk <dig> showed swollen tips  <cit> .
these observations suggest that an extracellular signal such as stil, which biochemically modulates the leprk complex by leprk <dig> dephosphorylation, could in turn affect pollen tube growth or/and morphology.
therefore the response of pollen tubes to increasing concentrations of purified stil was evaluated, using in vitro germination assays.
no aberrant phenotypes, such as swollen tips, were observed in any of the treatments.
after  <dig> h of germination, only the highest concentration of stil assayed  resulted in a significant statistical  increase in pollen tube length  when compared to pollen tube length in pgm in the absence of stil .
however, after  <dig> h of germination, pollen tubes in the presence of increasing concentrations of stil were significantly longer than those germinated in the absence of stil  or in the presence of fractions retained on the c <dig> reverse-phase cartridge .
this stimulation of growth could be a response to the perception of stil from the onset of germination or it could be achieved only after pollen tubes are capable of perceiving stil.
to discriminate between these two hypotheses, pollen tube length fold-increase and growth rate were calculated.
if stil is perceived from the onset of germination, then pollen tube growth rate would depend on stil concentration and higher concentrations of stil would result in longer pollen tubes but with constant fold-increases in pollen tube length.
alternatively, if the perception of stil is delayed until tubes have formed, then pollen tube growth rate initially would be constant and, when pollen tubes are able to respond, rates would increase in response to stil concentrations.
this behavior would be reflected in larger fold-increases in pollen tube length, in response to higher concentrations of stil.
fig.
5b shows that pollen tube growth rate increased depending on stil concentration but that the fold-increase in pollen tube length showed no differences between treatments.
these results suggest that stil is perceived from the onset of germination.
discussion
stil is a peculiar molecule and a potential extracellular partner for the tomato pollen leprk complex.
preliminary biochemical characterization indicated that stil is a negatively charged, hydrophilic compound that absorbs at  <dig> nm.
from the amino acid determination, we can conclude that stil is at least partially peptidic.
however, since neither  <dig> nm-absorbing residues nor negatively charged amino acids were identified, the amino acid determination was partial, possibly because of the resistance of stil to the standard acid hydrolysis conditions commonly used for amino acid determination.
some proteins are prone to aggregation when heated during acid hydrolysis, making them recalcitrant to degradation  <cit> .
if stil has a hydrophobic core with  <dig> nm-absorbing residues and a hydrophilic surface exposed to the medium, only superficial amino acids would be susceptible to acid hydrolysis.
we tried several mass spectrometry approaches in order to determine the structure of stil , but none were successful in determining stil's full structure.
the high mass of the molecular ion and its resistance to enzymatic fragmentation are major obstacles in determining stil's structure.
so far, uv-maldi-tof tandem mass spectrometry analysis confirmed the presence of a short tract of amino acid residues  in stil .
considering its molecular mass as determined by maldi, stil could be a peptide of ~ <dig> amino acids.
we showed that stil's biochemical activity is resistant to drastic treatments, such as incubations with acid or alkali under high temperatures, or dtt reduction, suggesting that stil corresponds to a stable molecule and that stil must have a peculiar structure in order to withstand those extreme conditions.
its resistance to several proteases, even though the target amino acids for these enzymes are present in stil , further supports this idea.
it is possible that some of these treatments had an effect on the structure of stil, but none  affected its ability to dephosphorylate leprk <dig>  there are several explanatory hypotheses as to how microwave-assisted acid hydrolysis permits the breakage of peptidic bonds in polypeptides when traditional acid hydrolysis has failed  <cit> , but it is not known if overheating of the sample  and high pressure, and/or abolition of protein aggregation causes efficient hydrolysis.
there are several reports of other low molecular weight peptides with partial resistance to extreme treatments.
for example, bacterial endotoxins  <cit>  are resistant to proteases and acid treatments, tick microfilins  <cit>  and pig cerebroside sulfate activator  <cit>  are heat stable and partially resistant to proteases.
however, none of them share all the properties shown by stil.
another example is cyclotides, which are circularized peptides found in the plant families violaceae, rubiaceae and cucurbitaceae  <cit> .
cyclotides are heat stable and are resistant to proteases and to acid hydrolysis.
their n- and c- termini are covalently linked and three intramolecular disulfide bridges stabilize their three dimensional structure, resulting in an extremely compact molecule  <cit> .
however, plant cyclotides are easily purified by reverse-phase purification because  <dig> to 50% of their primary structure corresponds to hydrophobic residues, whereas stil was found in the flowthrough of a c <dig> column.
in this paper, we showed that stil promotes pollen tube growth from the onset of germination.
several factors, such as lipids and proteins, are involved in pollen tube growth, guidance and adhesion.
lipids are thought to provide a directional cue to the developing pollen tubes by controlling the flow of water  <cit> .
a ~ <dig> kda lily stigma/stylar cysteine-rich adhesin  with some sequence similarity to lipid transfer proteins was associated with pollen tube adhesion and was first described as an extracellular "glue" for pollen when associated with pectin  <cit> ; sca also participated in pollen tube guidance when acting together with chemocyanin, a blue copper protein of the plantacyanin family  <cit> .
nonetheless, no immediate biochemical response to sca was found in growing pollen tubes, nor is there additional information for a signal transduction pathway involved in pollen tube reorientation by chemocyanin or its arabidopsis homolog, plantacyanin  <cit> .
arabinogalactan proteins  have also been involved in modulating pollen tube growth in solanaceous species.
a transmitting tissue-specific  agp in tobacco acted as a signal directing pollen tube growth towards the ovary and was required for establishing normal growth rates  <cit> , but there is no biochemical evidence for a signal transduction pathway involved in pollen tube growth stimulation or, specifically, in tts-mediated pollen tube reorientation.
there are at least three potential ligands, i.e.
lat <dig>  leshy and lestig <dig>  <cit>  for the leprk complex.
stil is different from these proteins.
lat <dig> and leshy are pollen-expressed proteins of ~ <dig> kda and ~ <dig> kda, respectively.
lestig <dig> is a stigma-expressed protein of ~ <dig> kda, but it does not induce leprk <dig> dephosphorylation .
our results suggest that stil might be another female partner for the leprk complex.
leprks were first implicated in pollen tube growth signal transduction due to their mrna expression during late pollen development and their protein localization  <cit> .
a possible model is that the binding of extracellular cues from female tissues to the leprk complex regulates kpp activity, leading to the activation of rop and the modulation of pollen tube growth  <cit> .
zhang and mccormick  <cit>  provided more support for the role receptor kinases play in modulating ropgef activity.
in arabidopsis, atropgef <dig>  a homolog of kpp, interacts via its c-terminus with the cytoplasmic domain of atprk2a, a homolog of leprk <dig>  c-terminal phosphorylation of atropgef <dig> by atprk2a was proposed to release an intramolecular inhibition of atropgef, leading to the promotion of pollen tube growth  <cit> ; this implies that atprk2a  has a major role in pollen tube growth modulation.
recent results support this hypothesis, since antisense expression of leprk <dig> resulted in pollen tubes with reduced growth rate  <cit> .
furthermore, the growth stimulation of pollen tubes by stil is completely dependent on the presence of leprk <dig>  since leprk <dig> antisense plants are unresponsive to stil  <cit> .
CONCLUSIONS
in our model, stil action is associated with receptor dephosphorylation, which in turn would lead to the activation of proteins present in the leprk complex and to pollen tube growth.
the idea that stil is a ligand is supported by the apoplastic localization of stil, the immediate biochemical response to its presence  and that stil stimulates pollen tube growth from the beginning of germination.
these observations pose an interesting question about the paradigm of signaling transduction through receptor kinases in general, where binding of the ligand to the extracellular domain of a receptor leads to auto-phosphorylation of its cytoplasmic domain, aggregation with other plasma membrane proteins and transduction of the signal by phosphorylating downstream effectors  <cit> .
in this context, determination of the molecular structure of stil and demonstrating that it can bind to the leprk complex will be essential to confirm that stil is a bona fide ligand of the leprk complex.

 in plants, pollination and subsequent fertilization rely on an extensive and complex dialog between the tissues of the pistil and the pollen tube. numerous proteins and other molecules from both the female and male are thought to regulate the biochemical dialog established when the pollen grain lands on the stigma, during pollen tube growth through the style and upon arrival at a synergid cell where the sperm cells are discharged. some observations suggest that there is a hierarchy of signals in pollen tube germination and growth, wherein a pollen tube is unable to respond to late signals coming from the female gametophyte if it has not been previously exposed to early signals coming from the sporophyte. this implies that pollen tubes have the ability to determine their geographical position within the female tissues and modify their physiology accordingly. leprk and leprk are two lrr-receptor like kinases specifically expressed in pollen grains and tubes in solanum lycopersicum and homologs of these proteins exist in other species. these kinases localize to the plasma membrane and belong to a high molecular weight complex; leprk and leprk bind different proteins from the pistil or from pollen. leprk is phosphorylated in mature and germinated pollen, but is specifically dephosphorylated upon incubation with style extracts; this suggests that style components have the potential to regulate the leprk complex biochemically. we previously determined that this style component in tomato and tobacco had a molecular weight of 3- kda and was heat-stable. we also showed that leprk and leprk interact when expressed heterologously in yeast, and that this interaction can be dissociated by the addition of the same style fractions that promote leprk dephosphorylation. recently zhang et al. showed that antisense expression of leprk resulted in pollen tubes with a reduced growth rate, suggesting that leprk might be involved in pollen tube growth regulation. a cytoplasmic protein called kpp, which is a ropgef, interacts with both leprk and leprk this interaction suggested a linkage between extracellular signals, receptor kinases, and modulation of rop activities, which is critically important for pollen tube growth. numerous low molecular weight polypeptides have been implicated in signal transduction pathways in plants. some were isolated by biochemical purification, such as systemin. specific physiological or biochemical effects were associated with these polypeptides and their receptors were identified and biochemically characterized [systemins,; phytosulfokines,; ralf,, but early correlates of ligand binding, such as receptor de/phosphorylation, hetero-oligomerization or dissociation from interacting proteins, have not yet been shown. other polypeptide ligands were first identified from mutant screens, such as clavata and tapetum determinant, by bioinformatics, such as the clavata3/embryo surrounding region-related peptides, or by map-based cloning, such as the s-locus cysteine rich protein /s-locus protein among others. receptors for clv tpd and scr/sp have been identified. binding of scr/sp to the s-locus receptor kinase and tpd binding to the receptor kinase excess microsporocytes induce receptor autophosphorylation, and in the case of scr/sp11-srk, complex formation with the s-locus glycoprotein. in this paper, we describe the purification of stil, a peculiar ~ da molecule from tobacco pistils that is responsible for leprk dephosphorylation. stil's activity is heat-, acid-, base-, dtt- and protease-resistant. our results show that stil promotes pollen growth from the onset of germination in a dose-dependent manner. we hypothesize that stil's binding to the leprk complex triggers leprk2-specific dephosphorylation, which in turn modulates downstream components of the leprk complex transduction pathways, such as ropgef and probably rop, resulting in pollen tube growth stimulation.  stil is a hydrophilic molecule that specifically promotes leprk dephosphorylation we previously showed that leprk is phosphorylated in tomato pollen microsomes, but specifically dephosphorylated when tomato stigma/style extracts were added during or after a phosphorylation reaction. this suggested the presence of a leprk dephosphorylating activity in tomato styles. subsequently, we demonstrated that a 3- kda heat-resistant molecule present in tomato and tobacco stigma/style extracts or exudates was responsible for this dephosphorylation. to purify this molecule, we used leprk dephosphorylation as a tracking assay. this molecule was named stil for style interactor for leprks. in order to design a purification protocol, the behavior of stil under various extraction or precipitation protocols was first evaluated. most proteins can be precipitated from organic solvents. however, fig. 1b shows that stil dephosphorylation activity was in the aqueous phase of a methanol-chloroform extraction and not in the organic phase or at the interface. this suggested that stil did not have exposed hydrophobic moieties that in organic solvents partition to the organic phase. in contrast to typical proteins, stil phosphorylation activity could not be precipitated by trichloroacetic acid, even in the presence of a carrier protein such as bovine serum albumin, implying that stil is highly soluble in salting out-low ph conditions, maybe because of the presence of negatively-charged highly hydrophilic residues on its surface. we then evaluated the interaction of stil with anionic and cationic exchange resin chromatography. the aqueous phase from methanol-chloroform-extracted stigma/style exudates was dried, dissolved in water and loaded onto solid-phase extraction cartridges. the resin was washed extensively with water and successively eluted with an ammonium bicarbonate gradient ending at m. stil dephosphorylation activity was retained and selectively eluted from an anionic exchanger, but not from a cationic exchanger. peptides are often purified using c reverse-phase, solid phase extraction cartridges. therefore, acetonitrile was added to a final concentration of 6% to an anionic exchanger-purified sample of stil and loaded onto a c reverse-phase cartridge. the cartridge was washed with 6% acetonitrile until the absorbance at nm reached basal levels and then was eluted with 50% and 100% acetonitrile, resulting in the elution of retained compounds. surprisingly, stil dephosphorylating activity was present in the flowthrough but not in the eluates. this result further supports the idea that stil does not have exposed hydrophobic moieties, and thus does not interact with c reverse-phase resins. stil purification protocol based on the behavior of stil shown above, a purification protocol for stil was developed. although stil was present in the flowthrough after c reverse-phase chromatography, this step was also included in order to purify stil away from other nm-absorbing compounds retained on this column. stigma/style exudates from tobacco pistils were extracted with methanol-chloroform and the aqueous phase was evaporated. the pellet was dissolved in water and loaded onto an anionic exchange column. fractions that induced leprk dephosphorylation were pooled together, concentrated and subjected to solid-phase extraction on a c reverse-phase cartridge. the sample was loaded in 6% acetonitrile and the flowthrough was collected and freeze-dried. the pellet was dissolved in water and loaded onto an anion exchange column. fig. 2c shows that the fractions that dephosphorylated leprk eluted as a nm-absorbing single peak. as a result of the purification process, stil was purified ~ fold when compared to the starting exudate. it is important to mention that the c solid-phase extraction step did not increase the purity of stil more than the first anionic exchange column. no significant difference in a t-test. no duplicates available. k2d represents abs units necessary for 50% dephosphorylation of leprk2; values were obtained from an average of two independent purifications. % error is calculated as × the purification factor was calculated by comparison to the starting exudate. the active fractions from the monoq column were pooled and subjected to uv-maldi-tof ms. fig. corresponds to one of chromatographs from independent purifications where only one peak with a molecular mass of ~ da was consistently obtained; this result demonstrated that this entity corresponds to stil and was purified to homogeneity. the fractions retained on the c reverse-phase cartridge were used as controls for pollen germination assays, and these were inactive and never showed a peak with a molecular mass of ~ da in mass spectrometry determinations. fig. 4a shows that when a dilution series of pure stil was assayed in the leprk dephosphorylation assay, × 10- absorbance units of stil were able to dephosphorylate 50% of leprk. moreover, the radioactive signal corresponding to phosphorylated leprk was fully recovered only after diluting stil to × 10- absorbance units. these results indicate that dephosphorylation of leprk was reduced when the amount of pure stil was decreased. stil is labile to microwave-assisted acid hydrolysis to further biochemically characterize stil, fractions of stil were incubated with several proteases, such as trypsin, pepsin, carboxypeptidase y, thermolysin and proteinase k, then the hydrolysates were tested for their ability to dephosphorylate leprk none of the tested proteases inactivated stil, since protease-treated stil was still able to induce leprk dephosphorylation. the structural conformation of many low molecular weight peptides relies on numerous cysteine residues that stabilize them by disulfide bridges. we tested the effect of incubating stil at 100°c with mm dtt for min, which should disrupt disulfide bridges. the effects of base or acid hydrolysis were also evaluated. again, none of these treatments had any effect on the ability of stil to dephosphorylate leprk. therefore, a different and stronger acid hydrolysis protocol was evaluated. microwave-assisted acid hydrolysis is commonly used for structural analysis of proteins and peptides. leprk could not be dephosphorylated after stil was subjected to microwave-assisted acid hydrolysis, suggesting that this treatment totally or partially inactivated stil. stil was subjected to the listed treatments and the dephosphorylating activity was tested in a leprk dephosphorylation assay. stil resistance to the treatment is indicated by "+", while "-" indicates stil loss of activity. several pieces of evidence support that stil is at least partially peptidic in nature: the presence of stil always correlated with absorbance at nm and amino acid determination showed that several amino acids are present in the stil molecule. amino acid determination relies on acid hydrolysis and derivatization. since stil activity is resistant to traditional acid hydrolysis, we expect that the full amino acid composition or the proportion of each amino acid has not been determined. pure stil was subjected to n hcl hydrolysis for hours in a non-oxidizing environment, then derivatized and separated by gas chromatography. amino acids were identified by comparing their retention times to standards. stil promotes pollen tube growth from the onset of germination leprk has been implicated in transducing pistil signals to the pollen tube, resulting in the regulation of pollen tube growth. this regulation is likely mediated by the regulation of rop through kpp. overexpression of a nearly full-length kpp in tomato or tobacco pollen resulted in the appearance of a swollen tip, a phenotype reminiscent of that seen when atrop was overexpressed in tobacco pollen. furthermore, pollen tubes transiently over-expressing leprk showed swollen tips. these observations suggest that an extracellular signal such as stil, which biochemically modulates the leprk complex by leprk dephosphorylation, could in turn affect pollen tube growth or/and morphology. therefore the response of pollen tubes to increasing concentrations of purified stil was evaluated, using in vitro germination assays. no aberrant phenotypes, such as swollen tips, were observed in any of the treatments. after h of germination, only the highest concentration of stil assayed resulted in a significant statistical increase in pollen tube length when compared to pollen tube length in pgm in the absence of stil. however, after h of germination, pollen tubes in the presence of increasing concentrations of stil were significantly longer than those germinated in the absence of stil or in the presence of fractions retained on the c reverse-phase cartridge. this stimulation of growth could be a response to the perception of stil from the onset of germination or it could be achieved only after pollen tubes are capable of perceiving stil. to discriminate between these two hypotheses, pollen tube length fold-increase and growth rate were calculated. if stil is perceived from the onset of germination, then pollen tube growth rate would depend on stil concentration and higher concentrations of stil would result in longer pollen tubes but with constant fold-increases in pollen tube length. alternatively, if the perception of stil is delayed until tubes have formed, then pollen tube growth rate initially would be constant and, when pollen tubes are able to respond, rates would increase in response to stil concentrations. this behavior would be reflected in larger fold-increases in pollen tube length, in response to higher concentrations of stil. fig. 5b shows that pollen tube growth rate increased depending on stil concentration but that the fold-increase in pollen tube length showed no differences between treatments. these results suggest that stil is perceived from the onset of germination. discussion stil is a peculiar molecule and a potential extracellular partner for the tomato pollen leprk complex. preliminary biochemical characterization indicated that stil is a negatively charged, hydrophilic compound that absorbs at nm. from the amino acid determination, we can conclude that stil is at least partially peptidic. however, since neither nm-absorbing residues nor negatively charged amino acids were identified, the amino acid determination was partial, possibly because of the resistance of stil to the standard acid hydrolysis conditions commonly used for amino acid determination. some proteins are prone to aggregation when heated during acid hydrolysis, making them recalcitrant to degradation. if stil has a hydrophobic core with nm-absorbing residues and a hydrophilic surface exposed to the medium, only superficial amino acids would be susceptible to acid hydrolysis. we tried several mass spectrometry approaches in order to determine the structure of stil, but none were successful in determining stil's full structure. the high mass of the molecular ion and its resistance to enzymatic fragmentation are major obstacles in determining stil's structure. so far, uv-maldi-tof tandem mass spectrometry analysis confirmed the presence of a short tract of amino acid residues in stil. considering its molecular mass as determined by maldi, stil could be a peptide of ~ amino acids. we showed that stil's biochemical activity is resistant to drastic treatments, such as incubations with acid or alkali under high temperatures, or dtt reduction, suggesting that stil corresponds to a stable molecule and that stil must have a peculiar structure in order to withstand those extreme conditions. its resistance to several proteases, even though the target amino acids for these enzymes are present in stil, further supports this idea. it is possible that some of these treatments had an effect on the structure of stil, but none affected its ability to dephosphorylate leprk there are several explanatory hypotheses as to how microwave-assisted acid hydrolysis permits the breakage of peptidic bonds in polypeptides when traditional acid hydrolysis has failed, but it is not known if overheating of the sample and high pressure, and/or abolition of protein aggregation causes efficient hydrolysis. there are several reports of other low molecular weight peptides with partial resistance to extreme treatments. for example, bacterial endotoxins are resistant to proteases and acid treatments, tick microfilins and pig cerebroside sulfate activator are heat stable and partially resistant to proteases. however, none of them share all the properties shown by stil. another example is cyclotides, which are circularized peptides found in the plant families violaceae, rubiaceae and cucurbitaceae. cyclotides are heat stable and are resistant to proteases and to acid hydrolysis. their n- and c- termini are covalently linked and three intramolecular disulfide bridges stabilize their three dimensional structure, resulting in an extremely compact molecule. however, plant cyclotides are easily purified by reverse-phase purification because to 50% of their primary structure corresponds to hydrophobic residues, whereas stil was found in the flowthrough of a c column. in this paper, we showed that stil promotes pollen tube growth from the onset of germination. several factors, such as lipids and proteins, are involved in pollen tube growth, guidance and adhesion. lipids are thought to provide a directional cue to the developing pollen tubes by controlling the flow of water. a ~ kda lily stigma/stylar cysteine-rich adhesin with some sequence similarity to lipid transfer proteins was associated with pollen tube adhesion and was first described as an extracellular "glue" for pollen when associated with pectin; sca also participated in pollen tube guidance when acting together with chemocyanin, a blue copper protein of the plantacyanin family. nonetheless, no immediate biochemical response to sca was found in growing pollen tubes, nor is there additional information for a signal transduction pathway involved in pollen tube reorientation by chemocyanin or its arabidopsis homolog, plantacyanin. arabinogalactan proteins have also been involved in modulating pollen tube growth in solanaceous species. a transmitting tissue-specific agp in tobacco acted as a signal directing pollen tube growth towards the ovary and was required for establishing normal growth rates, but there is no biochemical evidence for a signal transduction pathway involved in pollen tube growth stimulation or, specifically, in tts-mediated pollen tube reorientation. there are at least three potential ligands, i.e. lat leshy and lestig for the leprk complex. stil is different from these proteins. lat and leshy are pollen-expressed proteins of ~ kda and ~ kda, respectively. lestig is a stigma-expressed protein of ~ kda, but it does not induce leprk dephosphorylation. our results suggest that stil might be another female partner for the leprk complex. leprks were first implicated in pollen tube growth signal transduction due to their mrna expression during late pollen development and their protein localization. a possible model is that the binding of extracellular cues from female tissues to the leprk complex regulates kpp activity, leading to the activation of rop and the modulation of pollen tube growth. zhang and mccormick provided more support for the role receptor kinases play in modulating ropgef activity. in arabidopsis, atropgef a homolog of kpp, interacts via its c-terminus with the cytoplasmic domain of atprk2a, a homolog of leprk c-terminal phosphorylation of atropgef by atprk2a was proposed to release an intramolecular inhibition of atropgef, leading to the promotion of pollen tube growth; this implies that atprk2a has a major role in pollen tube growth modulation. recent results support this hypothesis, since antisense expression of leprk resulted in pollen tubes with reduced growth rate. furthermore, the growth stimulation of pollen tubes by stil is completely dependent on the presence of leprk since leprk antisense plants are unresponsive to stil.  in our model, stil action is associated with receptor dephosphorylation, which in turn would lead to the activation of proteins present in the leprk complex and to pollen tube growth. the idea that stil is a ligand is supported by the apoplastic localization of stil, the immediate biochemical response to its presence and that stil stimulates pollen tube growth from the beginning of germination. these observations pose an interesting question about the paradigm of signaling transduction through receptor kinases in general, where binding of the ligand to the extracellular domain of a receptor leads to auto-phosphorylation of its cytoplasmic domain, aggregation with other plasma membrane proteins and transduction of the signal by phosphorylating downstream effectors. in this context, determination of the molecular structure of stil and demonstrating that it can bind to the leprk complex will be essential to confirm that stil is a bona fide ligand of the leprk complex. 
we propose that the leprk complex perceives stil, resulting in leprk dephosphorylation and an increase in pollen tube growth. stil increased pollen tube lengths of in vitro germinated pollen in a dose-dependent manner. leprk and leprk are two pollen receptor kinases localized to the plasma membrane, where they are present in a high molecular weight complex.
 leprk and leprk are two pollen receptor kinases localized to the plasma membrane, where they are present in a high molecular weight complex. leprk is phosphorylated in mature and germinated pollen, but is dephosphorylated when pollen membranes are incubated with tomato or tobacco style extracts.  here we show that leprk dephosphorylation is mediated by a heat-, acid-, base-, dtt- and protease-resistant component from tobacco styles. using leprk phosphorylation as a tracking assay for purification, style exudates were subjected to chloroform extraction, anionic exchange, and c reverse-phase chromatography columns. we finally obtained a single ~ da compound that we named stil. stil increased pollen tube lengths of in vitro germinated pollen in a dose-dependent manner.  we propose that the leprk complex perceives stil, resulting in leprk dephosphorylation and an increase in pollen tube growth. 