Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro

Abstract

Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved. Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved. Tobacco mature pollen represents an extremely resistant structure filled with a desiccated cytoplasm that is surrounded by an extremely tough cell wall. This metabolically quiescent stage of male gametophyte has to reach stigma tissue in a viable state. After pollination, the rehydration and metabolic activation of a pollen grain starts. The pollen activation is represented by a time period when there is no pollen tube growth, and only metabolic processes within the original volume of cytoplasm occur together with cytoplasm hydration (1.Vogler F. Konrad S.S.A. Sprunck S. Knockin' on pollen's door: live cell imaging of early polarization events in germinating Arabidopsis pollen.Front. Plant Sci. 2015; 6: 246Crossref PubMed Scopus (18) Google Scholar). Within this period, the pollen aperture later used for pollen tube outgrowth is selected. After that, a rapid pollen tube tip growth starts in order to deliver the genetic information carried by two sperm cells to the ovaries. Desiccated mature pollen of many angiosperm species can be also rehydrated and activated in vitro (2.Mascarenhas J.P. Molecular mechanisms of pollen tube growth and differentiation.Plant Cell. 1993; 5: 1303-1314Crossref PubMed Scopus (363) Google Scholar). Here we aim to elucidate the regulation processes of pollen grain re-hydration and activation mediated by protein phosphorylation. Protein phosphorylation, representing one of the most frequent regulatory mechanisms, was shown to control a number of cellular processes, such as signal transduction, regulation of transcription and translation, regulation of cytoskeleton dynamics, cell regulation, regulation, regulation of protein and protein in PubMed Scopus Google The cell Scopus Google control of PubMed Scopus Google Scholar). to pollen the rehydration of was accompanied by changes in protein phosphorylation F. of with a PubMed Scopus Google Scholar). of a group to the the of a protein to more of Scopus Google Scholar). changes of protein within a and Scopus Google F. phosphorylation by the Cell. PubMed Scopus Google Scholar). In of phosphorylation activity by the active of the as was for for of of PubMed Scopus Google Scholar). In order to be to it is to some of the enrichment in PubMed Scopus Google for phosphopeptide enrichment to by mass Google because of are are by the of protein is in many and contains many phosphorylation sites phosphorylation sites are only only a of the protein be a position the majority be The of phosphopeptides by mass is the of The enrichment can be two The first is to the of a protein the on the enrichment of of the protein of are for for as as have been in PubMed Scopus Google Scholar). In order to the a first the and also the one was of in in Arabidopsis of Cell. PubMed Scopus Google for of PubMed Scopus Google Scholar). The first angiosperm pollen was that of Arabidopsis S. of the of mature Arabidopsis PubMed Scopus Google which the pollen data because that, Arabidopsis pollen data by the S. of mature pollen of Arabidopsis 5: PubMed Scopus Google S. of the Arabidopsis mature pollen PubMed Scopus Google and functional of proteins in Arabidopsis mature Plant Scopus Google and one protein for data into Arabidopsis pollen and PubMed Scopus Google were and identified many number of which in regulation of and protein function, protein signal transduction, and cellular Many were identified in the data that these were to phosphorylation, for protein protein and protein S. of the of mature Arabidopsis PubMed Scopus Google Scholar). The pollen was by a only S. protein in early and pollen to pollen tube Cell. PubMed Scopus Google Scholar). In this study, and the of male gametophyte to pollen the first pollen the was S. in pollen activated in PubMed Scopus Google Scholar). In order to in mature pollen and pollen activated in vitro for phosphoprotein enrichment an was carried F. S. of proteins and 5: PubMed Scopus Google Scholar). This to the of only one phosphorylation that titanium used protein protein protein in protein protein mass activation with enrichment was more phosphorylation sites in the proteins mature proteins were for and metabolic proteins and and not many proteins were identified in this The male gametophyte to was that of a the of this species was the of growth and to and were identified and of pollen PubMed Scopus Google Scholar). titanium protein protein protein in protein protein mass activation protein with The is a of male gametophyte we phosphopeptide enrichment by with the of phosphopeptides and titanium PubMed Scopus Google on of male this time two of activated pollen and as as mature Collectively, 471 phosphopeptides 432 phosphorylation sites have been identified in the of male These phosphorylation sites to 301 that were into 13 functional with transcription, protein synthesis, destination and storage, as as signal the functional phosphorylation with a and one with a data to the of which were into seven based on regulatory the Tobacco were in a to were and were the and on a mature pollen was by a and The of germinating pollen 6: Scopus Google it was The pollen represented groups of that were in of the These were to as the pollen was activated in vitro as a for and stage in as in with and growth of in pollen tube Plant PubMed Scopus Google Scholar). The activated pollen was by on a and in The as mature pollen represents an structure with a desiccated Upon activated pollen becomes with a one pollen aperture is for pollen tube outgrowth The proteins were the by TCA/acetone protein with in Google with S. in pollen activated in PubMed Scopus Google in as for In mature activated pollen was by a in a The was in of in with After in an the were in for and After the of the the were by with for in and for After the the the was the was and The protein were in and with as Protein In in vitro PubMed Scopus Google and were for of the were in with and subjected to phosphopeptide enrichment by were to the in order to the of the The phosphopeptides to were and as S. in pollen activated in PubMed Scopus Google F. The the the the mass of PubMed Scopus Google Scholar). The were by on an mass to an were on a and on a and a of a to in were in the a of the as The most were subjected to and of and an activation time of and were in the were to for and for The were the for Tobacco with and phosphorylation were the and and phosphorylation PubMed Scopus Google Scholar). were with the trypsin as a with two sites, as and together with phosphorylation as the results were subjected to the of mass and to a on the The of the of the was the results were were different were Of identified only the that showed a of the the of the same and that were identified in of the were in the only phosphopeptides with an assigned phosphorylation with a were data and results have been in F. J.P. S. data and PubMed Scopus Google with the of the trypsin was by to the same as was also as phosphorylation as proteins the were in of for had to be that of two in were as The and were by the identified in the were the Arabidopsis many of the the into function, and cellular together with the were assigned to the Arabidopsis some of the Arabidopsis in the the was not the were to protein and to S. S. S. S. S. of of of Arabidopsis PubMed Scopus Google to of the In a protein had more it was to the phosphopeptides were for the phosphorylation by Scopus Google to the of protein phosphorylation data PubMed Scopus Google Scholar). were one and the one for were not because there was only one in the phosphopeptide data The of a phosphorylation was to 13 the was into the number of to and to a data of was The phosphopeptides were divided into seven to regulatory trends. The was not on the phosphopeptide data because it only a number of The of the in the was by the F. a for Scopus Google Scholar). In this study, 471 phosphopeptides were identified with an assigned position of the phosphorylation and The majority of the identified phosphopeptides was only a was S. S. S. S. S. of of of Arabidopsis PubMed Scopus Google (2.Mascarenhas J.P. Molecular mechanisms of pollen tube growth and differentiation.Plant Cell. 1993; 5: 1303-1314Crossref PubMed Scopus (363) Google These 471 identified phosphopeptides 432 phosphorylation sites. The number of phosphorylation sites is the number of identified in phosphopeptides because some of the identified phosphorylation sites were a was for in of of where one of which was the carried one trypsin represented by the and protein and an represents the phosphorylation in of two one of which was on a the was not in that and protein and protein an represents phosphorylation and represents an were only were and The was with phosphorylation sites by represented by phosphorylation sites one phosphorylation to was on a it the in the data The 471 identified phosphopeptides 432 phosphorylation sites were assigned to 301 proteins as proteins more one phosphorylation protein was the as a identified with a number with a of The of was in of one assigned to two more the and the and some were assigned to more one as an number in with and and a in represents a the phosphopeptides to one phosphoprotein are put together into one The proteins were to the original protein many of these were not in of some phosphoproteins, the was the by in the The proteins were to The was by the results not an to protein function, the was into the to S. S. S. S. S. of of of Arabidopsis PubMed Scopus Google Scholar). protein was into one In one protein had more it was into the with the The and was as the proteins with a only to some with an were as the proteins a functional were as The protein are by a in The one of the was represented by species with one of proteins into two protein synthesis, and protein destination and was with also to transcription and exactly to signal transduction. structure and The were represented only by a cell and the are in Protein phosphorylation on on of these can be can cellular regulatory in more The phosphorylation in data with the based on the protein were identified by were one on and the one The was not subjected to this because only one was in the data The phosphorylation had to be in the data to be The most and the only were represented by the phosphorylation that was by a and phosphorylation is by an and the position that can be by is shown as The with a was a was only The were two and two The were represented by a by a an by two In the was the was carried by The were of a by a one and a by one with two The was the was in phosphopeptides The that such are to more in in the In order to changes on the of protein the different time were on the The of the this as a and the of the phosphopeptides was with this of the in with the changes of the The of these are shown in in The of such because of the of the proteins as a of the phosphorylation the not the changes of the proteins and showed showed a the phosphopeptide not on the of the The of such proteins is shown in in changes in phosphopeptide that were not by the of the protein are to be by protein phosphorylation proteins were based on two to a with phosphopeptide the was to for because of the complexity of the the in two of was The proteins to be of a different had to two we these proteins and the phosphopeptides that to we which were into seven regulatory groups and The first groups phosphopeptides that were identified in one of the The number of into the for mature phosphopeptides phosphopeptides were identified in and activated pollen The groups that were in two of were in and activated pollen and phosphopeptides were in mature pollen and activated pollen phosphopeptides into the group that was in activated pollen The regulation group was represented by the phosphopeptides to represented by seven phosphopeptides The protein where the phosphopeptides to were transcription, translation, and protein and to the in These for one to one of phosphopeptides in the regulation were also phosphopeptides with that for a of the phosphopeptides in the regulatory groups to of the and and in the group with the regulation to the and the activated it represented a in the group that were activated In the regulation group to the functional was with which represented a of identified and group represented the phosphopeptides that were in mature pollen, and were dephosphorylated pollen activation. in mature pollen, changes of which were not by changes on the protein were represented for by protein and a of proteins with such as protein and protein The that were by activated pollen and mature pollen were for in and protein protein and pollen tube The groups and were of phosphorylation sites that only pollen activation. were for protein protein protein protein The most regulatory was by group of which showed phosphorylation in mature pollen, pollen activation and a later during pollen activation a regulation was in these transcription protein proteins and transcription group the proteins that were in and showed changes the These species were for represented by and protein protein can phosphorylation sites that different regulatory activity is by phosphorylation PubMed Scopus Google of phosphorylation sites in the and in PubMed Scopus Google Scholar). of such proteins identified in were and protein The is by phosphorylation sites that showed regulatory and The phosphopeptides and were identified in mature pollen the phosphopeptide was in mature pollen and activated We can that the first in mature pollen, in activated pollen, and in activated pollen the the in mature pollen later on was it is that in mature pollen phosphorylation and a a different regulatory The protein is by seven phosphorylation sites regulatory and The phosphopeptides and in mature pollen, the phosphopeptide was only in mature pollen and activated is that phosphorylation of these in mature pollen, it in activated pollen, and that two of the phosphorylation sites of pollen activation. The of activation be to pollen the the phosphopeptides and were identified in activated pollen, the phosphopeptide was also in mature the in mature pollen, and in activated pollen the two only of pollen activation. results showed phosphorylation for a number of proteins in the early processes during pollen activation. In the data 471 phosphopeptides have been identified in of male gametophyte pollen, pollen activated for and pollen activated for which carried 432 phosphorylation sites. The was by of one of which was and the one was by trypsin such as These 432 phosphorylation sites have been assigned to 301 The number of phosphorylation sites identified represents a in to male gametophyte that identified phosphorylation sites S. in pollen activated in PubMed Scopus Google Scholar). In that we for phosphoprotein enrichment the of only one phosphorylation and phosphopeptide enrichment for the of phosphorylation sites of proteins mature in the number of phosphorylation sites phosphopeptide enrichment in the with the phosphoprotein enrichment in the is in with where phosphoprotein enrichment only a number of phosphorylation sites F. of with a PubMed Scopus Google of the Arabidopsis PubMed Scopus Google Arabidopsis protein a protein with a in PubMed Scopus Google Scholar). a first for and trypsin also for phosphopeptides was shown of in in Arabidopsis of Cell. PubMed Scopus Google for of PubMed Scopus Google Scholar). The to the of and because to a in its is is during the enrichment and during The and of of PubMed Scopus Google Scholar). In most phosphoproteomics the is with by and In study, we the of that was to the Arabidopsis mature pollen with a of S. of the of mature Arabidopsis PubMed Scopus Google Scholar). In of cell of were F. in and phosphorylation dynamics in PubMed Scopus Google S. of phosphopeptides mass Sci. PubMed Scopus Google of cell Sci. PubMed Scopus Google Scholar). there was in in cell the was on cell that have a phosphorylation The in Arabidopsis cell phosphorylation of Arabidopsis proteins by mass and PubMed Scopus Google to S. phosphoproteomics of early in Cell. 6: PubMed Scopus Google Scholar). the a to the such as phosphorylation the of phosphorylation in Scopus Google and phosphoproteomics phosphorylation sites in PubMed Scopus Google in Arabidopsis cell and in a cell phosphoproteomics phosphorylation sites in PubMed Scopus Google Scholar). the of in the data it is that it phosphorylation in is Protein phosphorylation in more Plant Sci. PubMed Scopus Google on signal and PubMed Scopus Google Scholar). the of phosphorylation and to the pollen its growth the dynamics of cytoskeleton protein phosphorylation pollen and pollen tube growth the PubMed Scopus Google Scholar). the position of phosphorylation in pollen proteins to be as as of phosphorylation during pollen tube was shown to be carried by proteins an the of a such as phosphorylation of the as a of in Sci. PubMed Scopus Google proteins in phosphorylation the activity of PubMed Scopus Google Scholar). The only in data was identified in the of the the identified phosphoproteins, the functional was with it for one of the identified is that some of these proteins are pollen-specific with and that is In it was clearly shown that specific proteins of that were not by and were not as as in and the of the S. protein in early and pollen to pollen tube Cell. PubMed Scopus Google Scholar). of these into the that represented of pollen-specific proteins by and S. protein in early and pollen to pollen tube Cell. PubMed Scopus Google Scholar). this of data is with the male gametophyte a of the identified have a in in protein synthesis, in protein destination and Tobacco pollen activation and pollen tube growth was shown to be on of transcription Protein in pollen of species of Plant Scopus Google Scholar). a number of during pollen tube growth of S. of pollen in cell and during Plant PubMed Scopus Google S. pollen tube PubMed Google many of the in the desiccated mature pollen are in These of and together with J.P. The pollen-specific is in during pollen Plant Scopus Google in male gametophyte are with and are in protein synthesis, and PubMed Scopus Google and the of starts pollen activation. was shown to be by protein phosphorylation of and regulatory proteins control of PubMed Scopus Google the of the such as of and in data The of proteins during cellular processes is also by to which proteins by are Protein is to have a during male gametophyte it was demonstrated that in protein was for pollen tube of is in pollen and PubMed Scopus Google Scholar). We a of in transcription in the there was no phosphoprotein to transcription in data S. in pollen activated in PubMed Scopus Google because of and the of protein a fact was demonstrated for Arabidopsis where phosphopeptide enrichment to the of phosphorylation sites that were not identified by the of the Arabidopsis PubMed Scopus Google Scholar). active transcription in activated pollen grain as as of pollen tube growth has been shown S. of pollen in cell and during Plant PubMed Scopus Google S. pollen tube PubMed Google Scholar). we identified transcription most of which a of was shown to be by two protein and of a transcription Arabidopsis as a of PubMed Scopus Google Scholar). most of transcription showed also phosphorylation of to phosphorylation sites with PubMed Scopus Google Scholar). were identified in male gametophyte S. protein in early and pollen to pollen tube Cell. PubMed Scopus Google during pollen Plant Scopus Google activated by hydration in Cell. PubMed Scopus Google Scholar). data these to have to be with the data S. in pollen activated in PubMed Scopus Google we identified a number of proteins with in this to phosphoprotein of the are of a and the of the phosphoprotein data the pollen-specific be the an in a tip cell growth of pollen and activity is by the the and to the of this activity was shown to be for pollen tube growth and cell growth in pollen PubMed Scopus Google Scholar). The proteins data were protein and was because the regulation the the metabolically quiescent pollen grain to the pollen tube is to the activity of and phosphorylation of which was shown to activity of the regulatory phosphorylation sites in protein PubMed Scopus Google Scholar). Many of the identified showed to the in the the of the This be by the fact that represented pollen-specific of which were in Many protein phosphorylation phosphorylation In order to we the data be that the information a to a phosphorylation is in and the information is to a group of signal PubMed Scopus Google Scholar). were for for was shown to be in phosphorylation the position of only one phosphorylation The first to be is the phosphorylation, a by a of the of The phosphorylation is for two groups of protein protein and protein to phosphorylation sites with PubMed Scopus Google Scholar). these were identified in the male gametophyte S. protein in early and pollen to pollen tube Cell. PubMed Scopus Google a regulatory in many processes and pollen hydration activated by hydration in Cell. PubMed Scopus Google Scholar). were shown to cell and activity in male gametophyte was because pollen are S. of pollen in cell and during Plant PubMed Scopus Google Scholar). The of is for the regulation of of in pollen tube that cell is with the to and pollen in Cell. PubMed Scopus Google Scholar). phosphorylation and are by protein to phosphorylation sites with PubMed Scopus Google Scholar). with two one of which to and the to was shown to be in male gametophyte of and regulation of the for protein in PubMed Scopus Google Scholar). in pollen cell and to in the an to that the of this to and that the events during this is in also by the protein of protein S. The Arabidopsis of protein PubMed Scopus Google Scholar). of this were identified in male gametophyte and S. protein in early and pollen to pollen tube Cell. PubMed Scopus Google Scholar). not we identified two with a and in to the that is by to phosphorylation sites with PubMed Scopus Google Scholar). was shown to be activated by in activity is in early events of activation by in PubMed Scopus Google and two were identified in male gametophyte and S. protein in early and pollen to pollen tube Cell. PubMed Scopus Google Scholar). We have to that the were identified in data it with the the and which of the is for a phosphorylation were seven groups the phosphopeptides to regulatory trends. the groups and phosphopeptides that were in mature the phosphopeptides in group and group in pollen we that the of phosphorylation is in mature pollen represents the The were protein and protein destination and storage, represented by many proteins for by protein was also identified protein that the activity of that are for tip growth of pollen tube and cell growth in pollen PubMed Scopus Google Scholar). to the of its phosphorylation was not to it can be that its activity is on by of the phosphopeptide in this regulatory group because the were to the protein in mature pollen and the of the only phosphopeptide in group in pollen activated in vitro for The specific to mature pollen was were to pollen rehydration activated by hydration in Cell. PubMed Scopus Google this phosphorylation be the for pollen grain activation. The groups and proteins pollen activation. for the of a protein and Protein is to be pollen activation in order to the proteins and to with the in Arabidopsis was to its only phosphorylation of its S. S. S. of Arabidopsis is for and the of PubMed Scopus Google Scholar). the identified in data also phosphorylation, we that this phosphorylation represents an activation phosphorylation. The only protein phosphorylation. we only phosphorylation activity a in cytoskeleton dynamics and it was shown to be on a S. phosphorylation of the of the PubMed Scopus Google Scholar). The of phosphorylation during pollen tube growth was the pollen tube by phosphorylation that pollen and pollen protein phosphorylation pollen and pollen tube growth the PubMed Scopus Google Scholar). the pollen showed a different of it be that not only also proteins as have to be The most regulation was shown for the group This phosphopeptides that were in mature pollen, dephosphorylated in activated pollen and later activation of the proteins were put to this not phosphopeptides to group of transcription protein protein and The had phosphopeptides also in regulation These phosphorylation sites that with the signal during pollen tube growth control the rapid in growth of pollen Plant Scopus Google Scholar). we not have the phosphoproteomics data of pollen tube growth in is the of this comprised the phosphopeptides that in were only proteins that with phosphopeptides into this and The proteins were identified by that not only into this group also in one group and Collectively, we and identified phosphopeptides mature pollen, activated pollen, and activated pollen, the an early of male gametophyte activation. This the first phosphoproteomics data angiosperm activated pollen the dynamics of early phosphorylation events during pollen re-hydration and activation activated The only pollen were these of a and of pollen PubMed Scopus Google Scholar). We identified 471 phosphopeptides 432 phosphorylation sites that were assigned to 301 the quantitative data highlighted the dynamics of protein phosphorylation during pollen activation and the regulation of phosphopeptides of the same phosphoprotein pinpointed the complexity of protein phosphorylation in its functional context. of proteins also represents a for the of the most for the of phosphorylation and its into the processes pollen tube growth and this into the activation of pollen because highlighted the proteins that are which in the regulation and processes of pollen tube activation.