Advertisement

Phosphoproteome Dynamics Upon Changes in Plant Water Status Reveal Early Events Associated With Rapid Growth Adjustment in Maize Leaves*

  • Ludovic Bonhomme
    Correspondence
    To whom correspondence should be addressed: INRA/University Paris-Sud/CNRS/AgroParisTech, UMR 0320/UMR 8120 Génétique Végétale, Gif-sur-Yvette, 91190, France.
    Affiliations
    INRA/University Paris-Sud/CNRS/AgroParisTech, UMR 0320/UMR 8120 Génétique Végétale, Gif-sur-Yvette, 91190, France;
    Search for articles by this author
  • Benoît Valot
    Affiliations
    INRA, Plateforme d'Analyse Protéomique de Paris Sud Ouest, PAPPSO, Gif-sur-Yvette, 91190, France;
    Search for articles by this author
  • François Tardieu
    Affiliations
    INRA, Laboratoire d'Ecophysiologiedes Plantes sous Stress Environnementaux, LEPSE, Montpellier, 34060, France
    Search for articles by this author
  • Michel Zivy
    Correspondence
    To whom correspondence should be addressed: INRA/University Paris-Sud/CNRS/AgroParisTech, UMR 0320/UMR 8120 Génétique Végétale, Gif-sur-Yvette, 91190, France.
    Affiliations
    INRA/University Paris-Sud/CNRS/AgroParisTech, UMR 0320/UMR 8120 Génétique Végétale, Gif-sur-Yvette, 91190, France;
    Search for articles by this author
  • Author Footnotes
    * This research work was supported by a grant from the Agence National de la Recherche (DROMADAiR project, 08-GENM-003).
    This article contains supplemental Figs. S1 to S6 and Tables S1 to S3.
Open AccessPublished:July 10, 2012DOI:https://doi.org/10.1074/mcp.M111.015867
      Plant growth adjustment during water deficit is a crucial adaptive response. The rapid fine-tuned control achieved at the post-translational level is believed to be of considerable importance for regulating early changes in plant growth reprogramming. Aiming at a better understanding of early responses to contrasting plant water statuses, we carried out a survey of the protein phosphorylation events in the growing zone of maize leaves upon a range of water regimes. In this study, the impact of mild and severe water deficits were evaluated in comparison with constant optimal watering and with recovery periods lasting 5, 10, 20, 30, 45, and 60 min. Using four biological replicates per treatment and a robust quantitative phosphoproteomic methodology based on stable-isotope labeling, we identified 3664 unique phosphorylation sites on 2496 proteins. The abundance of nearly 1250 phosphorylated peptides was reproducibly quantified and profiled with high confidence among treatments. A total of 138 phosphopeptides displayed highly significant changes according to water regimes and enabled to identify specific patterns of response to changing plant water statuses. Further quantification of protein amounts emphasized that most phosphorylation changes did not reflect protein abundance variation. During water deficit and recovery, extensive changes in phosphorylation status occurred in critical regulators directly or indirectly involved in plant growth and development. These included proteins influencing epigenetic control, gene expression, cell cycle-dependent processes and phytohormone-mediated responses. Some of the changes depended on stress intensity whereas others depended on rehydration duration, including rapid recoveries that occurred as early as 5 or 10 mins after rewatering. By combining a physiological approach and a quantitative phosphoproteomic analysis, this work provides new insights into the in vivo early phosphorylation events triggered by rapid changes in plant water status, and their possible involvement in plant growth-related processes.
      The maintenance of crop productivity upon reduced water resources will be a challenging issue to ensure food production under future climate conditions. Water deficit induces a wide range of responses at the whole plant, cellular and molecular levels (
      • Chaves M.M.
      • Maroco J.P.
      • Pereira J.S.
      Understanding plant responses to drought - from genes to the whole plant.
      ). Among adjustments triggered by water deficit, leaf growth is one of the earliest and most sensitive processes that occurs independently of photosynthetic rates and plant carbon status (
      • Saab I.N.
      • Sharp R.E.
      Non-hydraulic signals from maize roots in drying soil: inhibition of leaf elongation but not stomatal conductance.
      ,
      • Westgate M.E.
      • Boyer J.S.
      Osmotic adjustment and the inhibition of leaf, root, stem and silk growth at low water potentials in maize.
      ), which tends to show that growth reduction is a crucial adaptive response (
      • Tardieu F.
      • Granier C.
      Water deficit and growth. Co-ordinating processes without an orchestrator?.
      ). A reduced leaf growth limits water losses by transpiration via a decreased leaf area but also limits the potential light interception. This reduces biomass accumulation and further, leads to substantial yield losses. Thus, leaf growth reduction has both a positive effect on plant stress avoidance and a negative effect on final crop yields. The optimization balance between the control of water losses and the potential carbon assimilation can differ according to climatic scenarios and increasing knowledge on its determinism may allow future progress in the maintenance of crop production on changing plant water statuses. In maize, leaf growth is restricted to the base of the leaf in a 6 to 8 cm-long fragment, named the growing zone (
      • Granier C.
      • Tardieu F.
      Water deficit and spatial pattern of leaf development. Variability in responses can be simulated using a simple model of leaf development.
      ,
      • Ben-Haj-Salah H.
      • Tardieu F.
      Temperature affects expansion rate of maize leaves without change in spatial distribution of cell length (Analysis of the coordination between cell division and cell expansion).
      ). This growing zone includes tissues in which both cell division and cell expansion happen, partly overlapping in time and space (
      • Ben-Haj-Salah H.
      • Tardieu F.
      Temperature affects expansion rate of maize leaves without change in spatial distribution of cell length (Analysis of the coordination between cell division and cell expansion).
      ). Cells are produced near the leaf base in the meristematic region, pushing forward the expanding cells into the distal regions of the leaf growing zone. Upon water deficit, leaf growth reprogramming is achieved by modulating both cell division and cell expansion (
      • Skirycz A.
      • Inzé D.
      More from less: plant growth under limited water.
      ,
      • Schuppler U.
      • He P.H.
      • John P.C.
      • Munns R.
      Effect of water stress on cell division and cell-division-cycle 2-like cell-cycle kinase activity in wheat leaves.
      ,
      • Aguirrezabal L.
      • Bouchier-Combaud S.
      • Radziejwoski A.
      • Dauzat M.
      • Cookson S.J.
      • Granier C.
      Plasticity to soil water deficit in Arabidopsis thaliana: dissection of leaf development into underlying growth dynamic and cellular variables reveals invisible phenotypes.
      ). Previous analyses have already reported that altered plant growth occurs concomitantly with changes in cyclin-dependent kinase activity, in cell wall expansion genes (such as expansin genes), in turgor or in cell wall properties (
      • Granier C.
      • Tardieu F.
      Water deficit and spatial pattern of leaf development. Variability in responses can be simulated using a simple model of leaf development.
      ,
      • Granier C.
      • Inzé D.
      • Tardieu F.
      Spatial distribution of cell division rate can be deduced from that of p34(cdc2) kinase activity in maize leaves grown at contrasting temperatures and soil water conditions.
      ,
      • Cosgrove D.J.
      Growth of the plant cell wall.
      ,
      • Vincent D.
      • Lapierre C.
      • Pollet B.
      • Cornic G.
      • Negroni L.
      • Zivy M.
      Water deficits affect caffeate O-methyltransferase, lignification, and related enzymes in maize leaves. A proteomic investigation.
      ,
      • Muller B.
      • Bourdais G.
      • Reidy B.
      • Bencivenni C.
      • Massonneau A.
      • Condamine P.
      • Rolland G.
      • Conéjéro G.
      • Rogowsky P.
      • Tardieu F.
      Association of specific expansins with growth in maize leaves is maintained under environmental, genetic, and developmental sources of variation.
      ,
      • Harb A.
      • Krishnan A.
      • Ambavaram M.M.
      • Pereira A.
      Molecular and physiological analysis of drought stress in Arabidopsis reveals early responses leading to acclimation in plant growth.
      ). In a recent paper, pioneer efforts in identifying the earliest molecular events controlling cell division upon osmotic stress have evidenced that ethylene signaling acts upstream on the cell cycle arrest and on cyclin-dependent kinase A activity, independently to a transcriptional control (
      • Skirycz A.
      • Claeys H.
      • De Bodt S.
      • Oikawa A.
      • Shinoda S.
      • Andriankaja M.
      • Maleux K.
      • Eloy N.B.
      • Coppens F.
      • Yoo S.D.
      • Saito K.
      • Inzé D.
      Pause-and-stop: the effects of osmotic stress on cell proliferation during early leaf development in Arabidopsis and a role for ethylene signaling in cell cycle arrest.
      ). However, the molecular mechanisms involved in the rapid plant growth adjustment in water stress responses is still fragmentary.
      Phosphorylation of proteins is of considerable importance for regulating plant growth (
      • Novak B.
      • Kapuy O.
      • Domingo-Sananes M.R.
      • Tyson J.J.
      Regulated protein kinases and phosphatases in cell cycle decisions.
      ). As a rapid and transient post-translational modification, protein phosphorylation achieves a fine-tuned regulation of protein function in a wide array of cellular processes during development or in response to environmental cues, from signaling cascades to gene expression (
      • Schulze W.X.
      Proteomics approaches to understand protein phosphorylation in pathway modulation.
      ). Therefore, early protein phosphorylation and dephosphorylation events could play a pivotal role in the rapid growth adjustment occurring in plants facing water limitation. Studying phosphoproteome dynamics has already proven to be a useful strategy to decipher sucrose-induced responses in cell cultures (
      • Niittylä T.
      • Fuglsang A.T.
      • Palmgren M.G.
      • Frommer W.B.
      • Schulze W.X.
      Temporal analysis of sucrose-induced phosphorylation changes in plasma membrane proteins of Arabidopsis.
      ) or ABA
      The abbreviations used are:
      ABA
      abscisic acid
      MWD
      mild water deficit
      SWD
      severe water deficit
      RH5
      RH10, RH20, RH30, RH45, and RH60, rewatering duration of 5, 10, 20, 30, 45 and 60 min, respectively
      SCX
      strong cation exchange chromatography
      FDR
      false discovery rate
      XIC
      extracted ion chromatogram
      SOTA
      self organizing tree algorithm
      CL
      SOTA cluster.
      1The abbreviations used are:ABA
      abscisic acid
      MWD
      mild water deficit
      SWD
      severe water deficit
      RH5
      RH10, RH20, RH30, RH45, and RH60, rewatering duration of 5, 10, 20, 30, 45 and 60 min, respectively
      SCX
      strong cation exchange chromatography
      FDR
      false discovery rate
      XIC
      extracted ion chromatogram
      SOTA
      self organizing tree algorithm
      CL
      SOTA cluster.
      -dependent changes in planta (
      • Kline K.G.
      • Barrett-Wilt G.A.
      • Sussman M.R.
      In planta changes in protein phosphorylation induced by the plant hormone abscisic acid.
      ) for instance, but early responses to changing plant water status at the level of a growing tissue have not yet been well documented. To our knowledge, data dealing with water deficit-induced changes in protein phosphorylation have been mainly obtained from long-term stressed plants (
      • Ke Y.
      • Han G.
      • He H.
      • Li J.
      Differential regulation of proteins and phosphoproteins in rice under drought stress.
      ,
      • Röhrig H.
      • Schmidt J.
      • Colby T.
      • Bräutigam A.
      • Hufnagel P.
      • Bartels D.
      Desiccation of the resurrection plant Craterostigma plantagineum induces dynamic changes in protein phosphorylation.
      ), and no time-course analysis of rapid in vivo changes has been explored.
      To investigate relations between early phosphorylation events and growth, the present exploratory study was carried out in planta on the phosphoproteome of the leaf growing zone of maize plants submitted to contrasting water statuses. Quantitative changes in phosphorylation status were assessed in two scenarios of water stress, an early and mild water deficit as well as a more severe one (MWD and SWD, respectively). Because water deficit is usually fluctuating in the fields with dehydration and rewatering events, we also analyzed a sequence of rehydration that causes rapid recovery of leaf elongation rate in maize. In maize, the full recovery of leaf growth occurs 40 min after rehydration, which is even faster than the recovery of leaf water potential (
      • Parent B.
      • Hachez C
      • Redondo E.
      • Simonneau T.
      • Chaumont F.
      • Tardieu F.
      Drought and abscisic acid effects on aquaporin content translate into changes in hydraulic conductivity and leaf growth rate: a trans-scale approach.
      ). In addition, rewatering is instantaneous and enables to accurately define the response time to the changes in plant water status.

      DISCUSSION

      In this study, we report the first analysis of the phosphorylation dynamics in the growing zone of leaves that resolves an in vivo time course of dehydration and rehydration. Quantitative changes of protein phosphorylation status were evaluated during an early and mild water deficit (MWD), and a severe water deficit (SWD) both impacting plant growth in comparison with constant optimal watering at the same ontogenic stage (CTRL1 and CTRL2, respectively). Phosphoproteome changes were also analyzed in comparison with SWD, over a time course of recovery periods lasting 5, 10, 20, 30, 45, and 60 min (RH5, RH10, RH20, RH30, RH45, and RH60, respectively) that induced partial recovery of the midday leaf water potential. As leaf elongation rate recovers faster than leaf water potential (
      • Parent B.
      • Hachez C
      • Redondo E.
      • Simonneau T.
      • Chaumont F.
      • Tardieu F.
      Drought and abscisic acid effects on aquaporin content translate into changes in hydraulic conductivity and leaf growth rate: a trans-scale approach.
      ), reactivation of plant growth processes was likely to occur within the duration of the studied rehydration kinetics.

      Phosphoproteomics of the Growing Zone of Leaves Evidences In Vivo Covariations Between Protein Phosphorylation Events and Plant Water Status

      In the growing zone of maize leaves, the length of the zone with cell division is close to 70 mm in the sixth leaf and lengthens in younger leaves (
      • Granier C.
      • Inzé D.
      • Tardieu F.
      Spatial distribution of cell division rate can be deduced from that of p34(cdc2) kinase activity in maize leaves grown at contrasting temperatures and soil water conditions.
      ). Within this range, cell division rate is maximum in the first 20 mm and decreases along with the distance to the leaf insertion point, overlapping in space with expanding cells. This means that our analysis, carried out on a 5 cm-long leaf fragment, provides a picture of phosphorylation events occurring in tissues where both cell division and expansion happen. Upon water deficit, cell division rate is affected all along the growing zone, and cell expansion is altered approximately to the same extent (
      • Tardieu F.
      • Reymond M.
      • Hamard P.
      • Granier C.
      • Muller B.
      Spatial distributions of expansion rate, cell division rate and cell size in maize leaves: a synthesis of the effects of soil water status, evaporative demand and temperature.
      ). Thus, phosphopeptide variations observed between controls and WD samples should not be primarily related to the variation of the ratio between dividing and expanding cells. The variation of this ratio is even less expected to occur during the short period of rehydration.
      Using quantitative phosphoproteomics, we identified 138 phosphorylation sites that were altered upon changing plant water statuses in the leaf growing zone. Interestingly, 75% of the changes referring to proteins of known functions, belonged to five main processes directly or indirectly involved in the regulation of plant development, either through epigenetic and transcriptional regulations, cell cycle-related changes, hormone-mediated responses or carbohydrate metabolism adjustments (Fig. 7).
      Figure thumbnail gr7
      Fig. 7Overview of the main processes involving proteins whose phosphorylation status was rapidly altered in response to changing plant water statuses in the growing zone of maize leaves. The five main processes identified in this study are shown. Graphs refer to the SOTA clusters shown in , only the average profile is depicted in red. Individual profiles are shown in . Proteins displaying at least one phosphopeptide belonging to any SOTA clusters are indicated. Underlined proteins show that mild water deficit (MWD) induced at least half of the change in phosphopeptide abundance that was detected during severe water deficit (SWD). Proteins depicted in red show that 5, 10, 20, 30, 45, or 60 min rehydration (RH5, RH10, RH20, RH30, RH45, or RH60, respectively) enabled at least a half recovery and * indicates when the linear regression of phosphopeptide abundances versus duration of the rewatering events, was significant (p value < 0.05). Arrows refer to functional links between processes. Abbreviations: ABA, abscisic acid; IAA, auxin; JA, jasmonic acid; ABC tsp, MDR-like ABC transporter; PPi2, protein phosphatase inhibitor 2; GCK, GCK-like kinase MIK; MAPK3, Map3k delta-1 protein kinase; NINJA, novel Interactor of JAZ-family proteins; Aux Rp, auxin-repressed protein; SAPK10, serine/threonine-protein kinase SAPK10; Ste 20, Ste-20 related kinase; RHG1, receptor-like kinase RHG1; SnRK, sucrose nonfermenting related kinase; CTR1, serine/threonine-protein kinase ctr1; SIPK, salt-inducible protein kinase; S/t PP, Serine/threonine-protein phosphatase; G11A, protein kinase G11A; PBS1, Serine/threonine-protein kinase PBS1; NGO, naringenin,2-oxoglutarate 3-dioxygenase; HMG, high mobility group protein; DMT, DNA (cytosine-5)-methyltransferase 1; H5, histone H5; HDT2, histone deacetylase HDT2; DEK, protein DEK; MBD105/115, methyl-binding domain protein 105/115; HDA2b, Histone deacetylase 2b; HemK, methyltransferase family member 2; P3d, plus-3 domain containing protein; SANT, SANT DNA-binding protein; R3h, R3h domain containing protein; Mpt5, pumilio/Mpt5 family RNA-binding protein; Bdp, Bromodomain-containing protein; BAH, bromo-adjacent homology (BAH) domain-containing protein; Carbohydrate tsp, carbohydrate transporter/sugar porter/transporter; SPS A, sucrose phosphate synthase A; PGM, phosphoglucomutase; SuSy 3, sucrose synthase 3; Citrate tsp, citrate transporter family protein; 6 PFK, 6-phosphofructokinase; GM-like, galactose mutarotase-like; GIF2, GRF-interacting factor GIF2; TfC, protein TIME FOR COFFEE; HY5, LONG HYPOCOTYL 5 bZIP transcription factor; DNL Zn, DNL zinc finger family protein; RSG, bZIP transcriptional activator RSG; RING 126, RING finger protein 126; CIP7, COP1-interacting protein 7; SF3, pollen-specific protein SF3; AIR9, microtubule-associated protein AIR9; Arf, ADP-ribosylation factor GTPase-activating protein; CeSy, cellulose synthase-2; USP, universal stress protein; PrCW, proline-rich cell wall protein; SNARE 12, golgi SNARE 12 protein; MAP65, microtubule-associated protein MAP65–1a; S3GT, sterol 3-beta-glucosyltransferase; VPS26, vacuolar protein sorting 26; CLIP1, CLIP-associating protein 1; Vill 3, villin-3; NMCP1, nuclear matrix constituent protein 1; Cal bp, calmodulin binding protein.
      In this study, MWD exemplified the first stage of the growth decrease when leaf water potential was not altered yet. Therefore, the similar changes in phosphorylation status observed in both MWD and SWD could correspond to early responses which were maintained over the establishment of water deficit, whereas the changes of lower magnitude suggest that part of the variations could depend on the duration and/or on the intensity of water shortage. On the contrary, the changes of higher magnitude or opposite to those detected during SWD, reveal specific responses of the early stage of water stress response. Our findings also emphasize rapid events in protein phosphorylation occurring in vivo within minutes, during early changes in plant water status. The picture of these rapid phosphorylation events, that were basically observed during the rehydration kinetics, evidenced that the earliest phosphorylation events mainly concerned cell cycle-related proteins. This emphasizes that the regulation of the cell cycle-related processes are among the primary targets of the phosphorylation and dephosphorylation machinery. Early changes in the phosphorylation status of cell cycle-related proteins could sustain the rapid recovery of leaf growth occurring within minutes in maize (
      • Parent B.
      • Hachez C
      • Redondo E.
      • Simonneau T.
      • Chaumont F.
      • Tardieu F.
      Drought and abscisic acid effects on aquaporin content translate into changes in hydraulic conductivity and leaf growth rate: a trans-scale approach.
      ).
      Transitory phosphorylation/dephosphorylation events in the range of 10 to 30 min after rewatering were mainly observed for kinases and phosphatases, including hormone-related proteins (Fig. 7). This could be a consequence of the recovery of water fluxes and turgor in the leaf, which was likely to occur concomitantly with the recovery of leaf water potential. These phosphorylation events detected on kinase and phosphatase proteins could act within signaling cascades, in an upstream control of further changes detected later during the rehydration time-course. On the contrary, no change was detected in kinases or phosphatases within 5 min rehydration although signaling events are rather expected at an early stage upon changing environments, and despite the identification, as early as 5 min after watering, of changing phosphorylation statuses of proteins involved in other functional processes. Inherently to exploratory phosphoproteomics approaches, some fine-tuned events were probably not detected in this study.
      Fully or partially reversible events were also widely observed in the phosphorylation status of proteins related to epigenetic and transcriptional regulation (Fig. 7), which is consistent with the fact that the cell cycle-related changes require extensive epigenetic and transcriptional regulations for achieving cell cycle transitions, as already reported in growing organs of Arabidopsis thaliana plants (
      • Beemster G.T.
      • De Veylder L.
      • Vercruysse S.
      • West G.
      • Rombaut D.
      • Van Hummelen P.
      • Galichet A.
      • Gruissem W.
      • Inzé D.
      • Vuylsteke M.
      Genome-wide analysis of gene expression profiles associated with cell cycle transitions in growing organs of Arabidopsis.
      ,
      • Desvoyes B.
      • Sanchez M.P.
      • Ramirez-Parra E.
      • Gutierrez C.
      Impact of nucleosome dynamics and histone modifications on cell proliferation during Arabidopsis development.
      ). In contrast, the phosphorylation events occurring in carbohydrate metabolism-related proteins, appeared rather as sustainable changes over the whole rehydration time course (Fig. 7).
      As a whole, the dependence on stress intensity, the significant correlations with plant water status as well as the rapid recoveries observed during rehydration, collectively suggest that a substantial part of the observed phosphorylation dynamics are of functional relevance in the response of maize leaves to contrasting water regimes.

      Earliest Changes in Plant Water Status Involve Cytokinesis- and Cell Expansion-related Proteins

      The cell cycle is likely to encounter substantial changes in response to adverse environmental conditions (
      • Schuppler U.
      • He P.H.
      • John P.C.
      • Munns R.
      Effect of water stress on cell division and cell-division-cycle 2-like cell-cycle kinase activity in wheat leaves.
      ,
      • Aguirrezabal L.
      • Bouchier-Combaud S.
      • Radziejwoski A.
      • Dauzat M.
      • Cookson S.J.
      • Granier C.
      Plasticity to soil water deficit in Arabidopsis thaliana: dissection of leaf development into underlying growth dynamic and cellular variables reveals invisible phenotypes.
      ,
      • Granier C.
      • Inzé D.
      • Tardieu F.
      Spatial distribution of cell division rate can be deduced from that of p34(cdc2) kinase activity in maize leaves grown at contrasting temperatures and soil water conditions.
      ). In many plant organs, including leaves, roots and seeds, water deficit rapidly decreases cell division rate (
      • Tardieu F.
      • Granier C.
      Water deficit and growth. Co-ordinating processes without an orchestrator?.
      ,
      • Skirycz A.
      • Inzé D.
      More from less: plant growth under limited water.
      ,
      • Stals H.
      • Inzé D.
      When plant cells decide to divide.
      ). In this study, 19 changes in phosphorylation status involved proteins acting in cell cycle-related processes (Fig. 7). These proteins are likely involved in a general control of the cell cycle (i.e. USP family proteins, calmodulin binding protein and calmodulin), in the phragmoplast and the nuclear envelope assembly (i.e. vacuolar protein sorting 26, nuclear matrix constituent protein 1, pollen-specific protein, villin-3, MAP65–1a microtubule-associated protein, CLIP-associating protein 1, and kinesin), and in the cell plate initiation and maturation (i.e. Arf-GTPase, epsin, adaptin, Golgi-SNARE 12 protein, and AIR9). Although previous studies mainly reported the impact of water deficit in delaying the G1-to-S transition as a result of a reduced cyclin-dependent kinase activity (
      • Granier C.
      • Tardieu F.
      Water deficit and spatial pattern of leaf development. Variability in responses can be simulated using a simple model of leaf development.
      ,
      • Inzé D.
      • De Veylder L.
      Cell cycle regulation in plant development.
      ), here we identified quantitative changes of phosphoproteins involved in the mitosis phase and in cytokinesis.
      Another evidence of changes in growth-related processes was the identification of phosphoproteins involved in cell wall biogenesis and remodeling, because both processes of cytokinesis and cell expansion require the addition of new cell wall components (
      • Backues S.K.
      • Konopka C.A.
      • McMichael C.M.
      • Bednarek S.Y.
      Bridging the divide between cytokinesis and cell expansion.
      ,
      • Jürgens G.
      Cytokinesis in higher plants.
      ). Here, we identified three altered phosphosites belonging to a cellulose synthase, a proline-rich cell wall protein (two proteins involved in cell wall building), and a sterol glycosyltransferase providing sterolglycosides which can act as precursors for cellulose biosynthesis. Another change was observed in a sucrose synthase which could be involved in the synthesis of cell wall components by providing UDP-glucose directly to the cellulose synthases and/or callose synthases (
      • Amor Y.
      • Haigler C.H.
      • Johnson S.
      • Wainscott M.
      • Delmer D.P.
      A membrane-associated form of sucrose synthase and its potential role in synthesis of cellulose and callose in plants.
      ).

      Recovery of Leaf Water Potential Mainly Matches With Hormone-mediated Responses and Cell Signaling Events

      Plant hormones are major regulators of growth and development, and they rapidly affect many aspects of plant biology during responses to biotic and abiotic stresses (
      • Santner A.
      • Calderon-Villalobos L.I.
      • Estelle M.
      Plant hormones are versatile chemical regulators of plant growth.
      ,
      • Wasternack C.
      Jasmonates: an update on biosynthesis, signal transduction and action in plant stress response, growth and development.
      ). For instance, abscisic acid, jasmonate, and ethylene have already been reported as critical regulators of the early plant growth adjustments during drought or osmotic stress (
      • Harb A.
      • Krishnan A.
      • Ambavaram M.M.
      • Pereira A.
      Molecular and physiological analysis of drought stress in Arabidopsis reveals early responses leading to acclimation in plant growth.
      ,
      • Skirycz A.
      • Claeys H.
      • De Bodt S.
      • Oikawa A.
      • Shinoda S.
      • Andriankaja M.
      • Maleux K.
      • Eloy N.B.
      • Coppens F.
      • Yoo S.D.
      • Saito K.
      • Inzé D.
      Pause-and-stop: the effects of osmotic stress on cell proliferation during early leaf development in Arabidopsis and a role for ethylene signaling in cell cycle arrest.
      ). Here, we identified ten sites with significant changes in phosphorylation status belonging to proteins involved in either ABA-, ethylene-, auxin- or jasmonate-related responses. Our study evidenced single sites in two kinase proteins, SAPK10 and SnRK2.4, both belonging to the SnRK2 family proteins which play a pivotal role in regulating sucrose metabolism, several ion channels and ABA-induced gene expression (
      • Christmann A.
      • Moes D.
      • Himmelbach A.
      • Yang Y.
      • Tang Y.
      • Grill E.
      Integration of abscisic acid signalling into plant responses.
      ,
      • Zheng Z.
      • Xiaoping X.
      • Crosley R.A.
      • Greenwalt S.A.
      • Sun Y.
      • Blakeslee B.
      • Wang L.
      • Ni W.
      • Sopko M.S.
      • Yao C.
      • Yau K.
      • Burton S.
      • Zhuang M.
      • McCaskill D.G.
      • Gachotte D.
      • Thompson M.
      • Greene T.W.
      The protein kinase SnRK2.6 mediates the regulation of sucrose metabolism and plant growth in Arabidopsis.
      ). Interestingly, the phosphorylation status of SnRK2.4 transiently recovered to the control level in the range of 10 to 20 min after rewatering. A rapid phosphorylation increase has already been observed in SnRK2 family proteins following 30-min ABA treatment (
      • Kline K.G.
      • Barrett-Wilt G.A.
      • Sussman M.R.
      In planta changes in protein phosphorylation induced by the plant hormone abscisic acid.
      ). A slight and transitory recovery was also identified during 10 to 20 min rewatering for a phosphopeptide of Ctr1, a Raf-like protein kinase acting as the main negative regulator of the ethylene signal transduction chain. This is an interesting finding because ethylene has recently been reported as an early upstream regulator of the cell cycle machinery (
      • Skirycz A.
      • Claeys H.
      • De Bodt S.
      • Oikawa A.
      • Shinoda S.
      • Andriankaja M.
      • Maleux K.
      • Eloy N.B.
      • Coppens F.
      • Yoo S.D.
      • Saito K.
      • Inzé D.
      Pause-and-stop: the effects of osmotic stress on cell proliferation during early leaf development in Arabidopsis and a role for ethylene signaling in cell cycle arrest.
      ). Regarding auxin-related proteins, two drought-induced phosphorylation changes recovered in two auxin-repressed proteins (one recovered after 60 min rewatering whereas the other one was correlated with the recovery of plant water status). Finally, two increasing phosphorylated forms were detected in NINJA-family proteins 5 and 6 (Novel Interactor of JAZ-family proteins). NINJA is a member of the ABI five binding proteins that interact with the Jasmonate ZIM-domain proteins (JAZ proteins) for repressing jasmonate response genes (
      • Pauwels L.
      • Barbero G.F.
      • Geerinck J.
      • Tilleman S.
      • Grunewald W.
      • Pérez A.C.
      • Chico J.M.
      • Bossche R.V.
      • Sewell J.
      • Gil E.
      • García-Casado G.
      • Witters E.
      • Inzé D.
      • Long J.A.
      • De Jaeger G.
      • Solano R.
      • Goossens A.
      NINJA connects the co-repressor TOPLESS to jasmonate signalling.
      ).
      Furthermore, we evidenced phosphorylation events in seven additional protein kinases and two phosphatases, named G11A, PBS1, Ste-20 related kinase, Map3k delta-1, GCK-like kinase MIK, salt-inducible protein kinase (SIPK), receptor-like kinase RHG1, protein phosphatase inhibitor 2 containing protein, and a serine/threonine-protein phosphatase, although little information is available for most of them. GCK MIK is believed to play a role in plant growth and development. Belonging to the GCK subgroup of MAP4Ks, it interacts with the maize atypical receptor kinase (MARK), a receptor expressed during embryogenesis and in the meristems of maize (
      • Llompart B.
      • Castells E.
      • Río A.
      • Roca R.
      • Ferrando A.
      • Stiefel V.
      • Puigdomenech P.
      • Casacuberta J.M.
      The direct activation of MIK, a germinal center kinase (GCK) -like kinase, by MARK, a maize atypical receptor kinase, suggests a new mechanism for signaling through kinase-dead receptors.
      ). Noteworthy, phosphorylation changes in the Ste-20 related kinase, in the Map3k delta-1 protein kinase, in SIPK and in RHG1 displayed transient recoveries after 20 min rewatering.

      Changes in Plant Water Status Induce Reversible Modifications Preparing Epigenetic and Transcriptional Regulations

      Up to 25% of the phosphorylation sites displaying significant changes in response to water regimes belonged to proteins involved in an upstream control of gene expression, mainly influencing chromatin structure and transcriptional regulation. Here, significant changes were observed in 25 phosphorylation sites belonging to proteins directly or indirectly involved in histone modification and DNA methylation. In response to environmental cues, gene expression and plant development are influenced by extensive chromatin remodeling, mainly achieved through histone post-translational modifications or DNA methylation patterns (
      • Chinnusamy V.
      • Zhu J.K.
      Epigenetic regulation of stress responses in plants.
      ). Likewise, specific chromatin modifications occurring at both histone and DNA levels are also a prerequisite to sustain the transcriptional regulation involved in the cell cycle progression (
      • Beemster G.T.
      • De Veylder L.
      • Vercruysse S.
      • West G.
      • Rombaut D.
      • Van Hummelen P.
      • Galichet A.
      • Gruissem W.
      • Inzé D.
      • Vuylsteke M.
      Genome-wide analysis of gene expression profiles associated with cell cycle transitions in growing organs of Arabidopsis.
      ,
      • Sanchez
      • Mde L.
      • Caro E.
      • Desvoyes B.
      • Ramirez-Parra E.
      • Gutierrez C.
      Chromatin dynamics during the plant cell cycle.
      ). In this study, decreased phosphorylated forms in response to dehydration were detected in 12 phosphorylation sites belonging to proteins acting on chromatin structure or in DNA methylation (Fig. 7). Partial or full recoveries were observed for five of them, including a small chromatin-associated proteins (HMG I/Y-2), a DEK protein (a nuclear chromatin-associated phosphoprotein (
      • Kappes F.
      • Scholten I.
      • Richter N.
      • Gruss C.
      Functional domains of the ubiquitous chromatin protein DEK.
      ), a SANT domain- (a histone-interaction module (
      • Boyer L.
      • Latek R.
      The SANT domain: a unique histone-tail-binding module?.
      )), a plus-3 domain-containing proteins, and a HemK protein (a class of methyl transferase proteins). Furthermore, increased phosphorylation upon SWD was evidenced in 11 phosphorylation sites belonging to seven proteins. Partial or full recoveries were observed for six of them, including two sites of a Pumilio/MPT5 proteins and single sites belonging to a MBD105 proteins, a MBD115 protein, a bromodomain- and a BAH domain- containing proteins. BAH and bromodomains are structural motifs commonly found in chromatin-associated proteins that recognize methylated and acetylated lysine residues, respectively. Their phosphorylation changes were concomitant to those observed on DNA methylation or histone deacetylation-related proteins.
      Along with these nuclear architectural events changing the overall DNA accessibility, significant modifications of five critical regulators of plant growth and development were evidenced, including single sites in a GRF-interacting factor GIF2, in a bZIP transcriptional activator RSG (for repression of shoot growth), in a COP1-interacting protein 7 (CIP7), in a LONG HYPOCOTYL 5 bZIP transcription factor (HY5) and two sites in a TIME FOR COFFEE protein (TIC). GIF genes act in organ size control through a possible role in cell proliferation by positively regulating cell-cycle gene expression (
      • Kim J.H.
      • Choi D.
      • Kende H.
      The AtGRF family of putative transcription factors is involved in leaf and cotyledon growth in Arabidopsis.
      ,
      • Lee B.H.
      • Ko J.H.
      • Lee S.
      • Lee Y.
      • Pak J.H.
      • Kim J.H.
      The Arabidopsis GRF-INTERACTING FACTOR gene family performs an overlapping function in determining organ size as well as multiple developmental properties.
      ) and RSG is involved in cell elongation by controlling the endogenous amounts of gibberellins (
      • Fukazawa J.
      • Sakai T.
      • Ishida S.
      • Yamaguchi I.
      • Kamiya Y.
      • Takahashi Y.
      Repression of shoot growth, a bZIP transcriptional activator, regulates cell elongation by controlling the level of gibberellins.
      ). The function of RSG is known to be negatively regulated by 14–3-3 proteins that bind to RSG depending on its phosphorylation status, thereby sequestering RSG in the cytoplasm and making it unable to regulate its targets in the nucleus (
      • Igarashi D.
      • Ishida S.
      • Fukazawa J.
      • Takahashi Y.
      14-3-3 proteins regulate intracellular localization of the bZIP transcriptional activator RSG.
      ). These results suggest that the above processes may trigger further changes in cell division and expansion under water deficit. Likewise, TIC is a nuclear regulator of the circadian clock and could control various aspects of hormone-related processes involved in plant growth and development (
      • Ding Z.
      • Millar A.J.
      • Davis A.M.
      • Davis S.J.
      TIME FOR COFFEE encodes a nuclear regulator in the Arabidopsis thaliana circadian clock.
      ). CIP7 and HY5 are pivotal regulators of plant photomorphogenic development and both are interactors of COP1 (
      • Yi C.
      • Deng X.W.
      COP1 – from plant photomorphogenesis to mammalian tumorigenesis.
      ). Being required for hypocotyl inhibition in all light conditions, HY5 is linked to a wide range of processes involving auxin, jasmonate, abscissic acid, ethylene and gibberellins pathways (
      • Nemhauser J.L.
      Dawning of a new era: photomorphogenesis as an integrated molecular network.
      ) which is in agreement with the hormone-mediated responses that have already been suggested above. CIP7 is a negative regulator of COP1 whereas this latter is a direct repressor of HY5 by inducing its proteasome-mediated degradation, resulting in the reduced expression of photomorphogenic genes (
      • Nemhauser J.L.
      Dawning of a new era: photomorphogenesis as an integrated molecular network.
      ,
      • Lee J.
      • He K.
      • Stolc V.
      • Lee H.
      • Figueroa P.
      • Gao Y.
      • Tongprasit W.
      • Zhao H.
      • Lee I.
      • Deng X.W.
      Analysis of transcription factor HY5 genomic binding sites revealed its hierarchical role in light regulation of development.
      ). Previous experiments suggested a role of HY5 phosphorylation in changing COP1-HY5 interaction.

      Phosphorylation Changes Related to Carbohydrate Metabolism Refer to Long-term Adjustments

      Phosphorylation changes occurring in proteins involved in carbohydrate metabolism accounted for a minor part of the whole detected variations, although sugars are major metabolites involved in stress responses. Glucose and especially sucrose can act as signaling molecules influencing the expression of a wide spectrum of genes and as the most suitable source of carbon and energy for many aspects of plant growth and development (
      • Ramon M.
      • Rolland F.
      • Sheen J.
      Sugar sensing and signaling.
      ). In response to contrasting water regimes, two phosphopeptides of a sucrose phosphate synthase (SPS) and single phosphopeptides in a sucrose synthase (SuSy), in a phosphoglucomutase (PGM), in a phosphofructokinase (PFK), in a carbohydrate transporter and in a citrate transporter displayed significant changes in relative abundance. SPS phosphorylation occurs on multiple seryl residues in vivo and involves SnRK protein kinases at least (
      • Zheng Z.
      • Xiaoping X.
      • Crosley R.A.
      • Greenwalt S.A.
      • Sun Y.
      • Blakeslee B.
      • Wang L.
      • Ni W.
      • Sopko M.S.
      • Yao C.
      • Yau K.
      • Burton S.
      • Zhuang M.
      • McCaskill D.G.
      • Gachotte D.
      • Thompson M.
      • Greene T.W.
      The protein kinase SnRK2.6 mediates the regulation of sucrose metabolism and plant growth in Arabidopsis.
      ,
      • Toroser D.
      • Huber S.C.
      Protein phosphorylation as a mechanism for osmotic-stress activation of sucrose-phosphate synthase in spinach leaves.
      ), which is in agreement with the rapid changes in the two SnRK2 proteins, described above. Changes in SPS activity during osmotic stress have already been observed as a consequence of phosphorylation events that differently occur on different sites (
      • Toroser D.
      • Huber S.C.
      Protein phosphorylation as a mechanism for osmotic-stress activation of sucrose-phosphate synthase in spinach leaves.
      ). Here, except for the site identified in the carbohydrate transporter, detected phosphorylation sites were overall increased during SWD, with a partial recovery during rehydration for PFK only. Our results suggest long-term changes in the sucrose supply through phosphorylation events in both SPS and SuSy, but also in PGM and PFK which provide substrates for sucrose synthesis.
      In conclusion, by combining physiological characterizations with phosphoproteome analysis, we reported here the first phosphoproteome dynamics occurring in vivo in the growing zone of maize leaves upon rapid changes in plant water status. Severe water deficit deeply modified the phosphorylation status of proteins, and part of these modifications was observed early, i.e. at a time when leaf water potential and plant growth were only slightly affected. During rehydration, that enables leaf growth resumption, this work enabled to identify transitory phosphorylation events as well as rapid in vivo changes occurring within five to 10 min after watering, independently to protein abundance variations. This exploratory approach emphasizes a comprehensive picture of the molecular plasticity of the phosphoproteome of leaf growing tissues upon dehydration/rehydration, and reveals a number early protein targets of the phosphorylation/dephosphorylation process potentially involved in an upstream control of the rapid leaf growth adjustment. This provides first insights into the in vivo dynamic behavior of individual protein phosphorylation sites upon changing plant water statuses. These findings are meant to guide future research on early plant water stress responses and on the understanding of processes involved in the rapid growth adjustment occurring upon fluctuating plant water status.

      Acknowledgments

      We thank Edlira Nano for her excellent technical assistance in the use of the MassChroQ software and to Olivier Langella for making LC-MS/MS data available in the PROTICdb database. We gratefully acknowledge Delphine Pflieger for her careful reading and editing of this manuscript. We also acknowledge Attila Csordas, from the PRIDE staff, for his excellent technical assistance in MS data submission.

      REFERENCES

        • Chaves M.M.
        • Maroco J.P.
        • Pereira J.S.
        Understanding plant responses to drought - from genes to the whole plant.
        Func. Plant Biol. 2003; 30: 239-264
        • Saab I.N.
        • Sharp R.E.
        Non-hydraulic signals from maize roots in drying soil: inhibition of leaf elongation but not stomatal conductance.
        Planta. 1989; 179: 466-474
        • Westgate M.E.
        • Boyer J.S.
        Osmotic adjustment and the inhibition of leaf, root, stem and silk growth at low water potentials in maize.
        Planta. 1985; 164: 540-549
        • Tardieu F.
        • Granier C.
        Water deficit and growth. Co-ordinating processes without an orchestrator?.
        Curr. Opin. Plant Biol. 2011; 14: 283-289
        • Granier C.
        • Tardieu F.
        Water deficit and spatial pattern of leaf development. Variability in responses can be simulated using a simple model of leaf development.
        Plant Physiol. 1999; 119: 609-620
        • Ben-Haj-Salah H.
        • Tardieu F.
        Temperature affects expansion rate of maize leaves without change in spatial distribution of cell length (Analysis of the coordination between cell division and cell expansion).
        Plant Physiol. 1995; 109: 861-870
        • Skirycz A.
        • Inzé D.
        More from less: plant growth under limited water.
        Curr. Opin. Biotech. 2010; 21: 197-203
        • Schuppler U.
        • He P.H.
        • John P.C.
        • Munns R.
        Effect of water stress on cell division and cell-division-cycle 2-like cell-cycle kinase activity in wheat leaves.
        Plant Physiol. 1998; 117: 667-678
        • Aguirrezabal L.
        • Bouchier-Combaud S.
        • Radziejwoski A.
        • Dauzat M.
        • Cookson S.J.
        • Granier C.
        Plasticity to soil water deficit in Arabidopsis thaliana: dissection of leaf development into underlying growth dynamic and cellular variables reveals invisible phenotypes.
        Plant Cell Environ. 2006; 29: 2216-2227
        • Granier C.
        • Inzé D.
        • Tardieu F.
        Spatial distribution of cell division rate can be deduced from that of p34(cdc2) kinase activity in maize leaves grown at contrasting temperatures and soil water conditions.
        Plant Physiol. 2000; 124: 1393-1402
        • Cosgrove D.J.
        Growth of the plant cell wall.
        Nat. Rev. Mol. Cell Biol. 2005; 6: 850-861
        • Vincent D.
        • Lapierre C.
        • Pollet B.
        • Cornic G.
        • Negroni L.
        • Zivy M.
        Water deficits affect caffeate O-methyltransferase, lignification, and related enzymes in maize leaves. A proteomic investigation.
        Plant Physiol. 2005; 137: 949-960
        • Muller B.
        • Bourdais G.
        • Reidy B.
        • Bencivenni C.
        • Massonneau A.
        • Condamine P.
        • Rolland G.
        • Conéjéro G.
        • Rogowsky P.
        • Tardieu F.
        Association of specific expansins with growth in maize leaves is maintained under environmental, genetic, and developmental sources of variation.
        Plant Physiol. 2007; 143: 278-290
        • Harb A.
        • Krishnan A.
        • Ambavaram M.M.
        • Pereira A.
        Molecular and physiological analysis of drought stress in Arabidopsis reveals early responses leading to acclimation in plant growth.
        Plant Physiol. 2010; 154: 1254-1271
        • Skirycz A.
        • Claeys H.
        • De Bodt S.
        • Oikawa A.
        • Shinoda S.
        • Andriankaja M.
        • Maleux K.
        • Eloy N.B.
        • Coppens F.
        • Yoo S.D.
        • Saito K.
        • Inzé D.
        Pause-and-stop: the effects of osmotic stress on cell proliferation during early leaf development in Arabidopsis and a role for ethylene signaling in cell cycle arrest.
        Plant Cell. 2011; 23: 1876-1888
        • Novak B.
        • Kapuy O.
        • Domingo-Sananes M.R.
        • Tyson J.J.
        Regulated protein kinases and phosphatases in cell cycle decisions.
        Curr. Opin. Cell Biol. 2010; 22: 801-808
        • Schulze W.X.
        Proteomics approaches to understand protein phosphorylation in pathway modulation.
        Curr. Opin. Plant Biol. 2010; 13: 279-286
        • Niittylä T.
        • Fuglsang A.T.
        • Palmgren M.G.
        • Frommer W.B.
        • Schulze W.X.
        Temporal analysis of sucrose-induced phosphorylation changes in plasma membrane proteins of Arabidopsis.
        Mol. Cell. Proteomics. 2007; 6: 1711-1726
        • Kline K.G.
        • Barrett-Wilt G.A.
        • Sussman M.R.
        In planta changes in protein phosphorylation induced by the plant hormone abscisic acid.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 15986-15991
        • Ke Y.
        • Han G.
        • He H.
        • Li J.
        Differential regulation of proteins and phosphoproteins in rice under drought stress.
        Biochem. Biophys. Res. Commun. 2009; 379: 133-138
        • Röhrig H.
        • Schmidt J.
        • Colby T.
        • Bräutigam A.
        • Hufnagel P.
        • Bartels D.
        Desiccation of the resurrection plant Craterostigma plantagineum induces dynamic changes in protein phosphorylation.
        Plant Cell Environ. 2006; 29: 1606-1617
        • Parent B.
        • Hachez C
        • Redondo E.
        • Simonneau T.
        • Chaumont F.
        • Tardieu F.
        Drought and abscisic acid effects on aquaporin content translate into changes in hydraulic conductivity and leaf growth rate: a trans-scale approach.
        Plant Physiol. 2009; 149: 2000-2012
        • Méchin V.
        • Damerval C.
        • Zivy M.
        Plant Proteomics: Methods and Protocols.
        Humana Press, Totowa, New Jersey, U.S.A2007: 1-8
        • Boersema P.J.
        • Raijmakers R.
        • Lemeer S.
        • Mohammed S.
        • Heck A.J.R.
        Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics.
        Nat. Protoc. 2009; 4: 484-494
        • Nühse T.S.
        • Bottrill A.R.
        • Jones A.M.
        • Peck S.C.
        Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses.
        Plant J. 2007; 51: 931-940
        • Valot B.
        • Langella O.
        • Nano E.
        • Zivy M.
        MassChroQ: A versatile tool for mass spectrometry quantification.
        Proteomics. 2011; 17: 3572-3577
        • Ferry-Dumazet H.
        • Houel G.
        • Montalent P.
        • Moreau L.
        • Langella O.
        • Negroni L.
        • Vincent D.
        • Lalanne C.
        • de Daruvar A.
        • Plomion C.
        • Zivy M.
        • Joets J.
        PROTICdb: a web-based application to store, track, query, and compare plant proteome data.
        Proteomics. 2005; 5: 2069-2081
        • Storey J.D.
        • Tibshirani R.
        Statistical significance for genomewide studies.
        Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 9440-9445
        • Sperry J.S.
        • Hacke U.G.
        • Oren R.
        • Comstock J.P.
        Water deficits and hydraulic limits to leaf water supply.
        Plant Cell Environ. 2002; 25: 251-263
        • Boersema P.J.
        • Foong L.Y.
        • Ding V.M.
        • Lemeer S.
        • van Breukelen B.
        • Philp R.
        • Boekhorst J.
        • Snel B.
        • den Hertog J.
        • Choo A.B.
        • Heck A.J.R.
        In-depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immunoaffinity purification and stable isotope dimethyl labeling.
        Mol. Cell. Proteomics. 2010; 9: 84-99
        • Olsen J.V.
        • Blagoev B.
        • Gnad F.
        • Macek B.
        • Kumar C.
        • Mortensen P.
        • Mann M.
        Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.
        Cell. 2006; 127: 635-648
        • Tardieu F.
        • Reymond M.
        • Hamard P.
        • Granier C.
        • Muller B.
        Spatial distributions of expansion rate, cell division rate and cell size in maize leaves: a synthesis of the effects of soil water status, evaporative demand and temperature.
        J. Exp. Bot. 2000; 51: 1505-1514
        • Beemster G.T.
        • De Veylder L.
        • Vercruysse S.
        • West G.
        • Rombaut D.
        • Van Hummelen P.
        • Galichet A.
        • Gruissem W.
        • Inzé D.
        • Vuylsteke M.
        Genome-wide analysis of gene expression profiles associated with cell cycle transitions in growing organs of Arabidopsis.
        Plant Physiol. 2005; 138: 734-743
        • Desvoyes B.
        • Sanchez M.P.
        • Ramirez-Parra E.
        • Gutierrez C.
        Impact of nucleosome dynamics and histone modifications on cell proliferation during Arabidopsis development.
        Heredity. 2010; 105: 80-91
        • Stals H.
        • Inzé D.
        When plant cells decide to divide.
        Trends Plant Sci. 2001; 6: 359-364
        • Inzé D.
        • De Veylder L.
        Cell cycle regulation in plant development.
        Annu. Rev. Genet. 2006; 40: 77-105
        • Backues S.K.
        • Konopka C.A.
        • McMichael C.M.
        • Bednarek S.Y.
        Bridging the divide between cytokinesis and cell expansion.
        Curr. Opin. Plant Biol. 2007; 10: 607-615
        • Jürgens G.
        Cytokinesis in higher plants.
        Annu. Rev. Plant Biol. 2005; 56: 281-299
        • Amor Y.
        • Haigler C.H.
        • Johnson S.
        • Wainscott M.
        • Delmer D.P.
        A membrane-associated form of sucrose synthase and its potential role in synthesis of cellulose and callose in plants.
        Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 9353-9357
        • Santner A.
        • Calderon-Villalobos L.I.
        • Estelle M.
        Plant hormones are versatile chemical regulators of plant growth.
        Nat. Chem. Biol. 2009; 5: 301-307
        • Wasternack C.
        Jasmonates: an update on biosynthesis, signal transduction and action in plant stress response, growth and development.
        Ann. Bot. 2007; 100: 681-697
        • Christmann A.
        • Moes D.
        • Himmelbach A.
        • Yang Y.
        • Tang Y.
        • Grill E.
        Integration of abscisic acid signalling into plant responses.
        Plant Biol. 2006; 8: 314-325
        • Zheng Z.
        • Xiaoping X.
        • Crosley R.A.
        • Greenwalt S.A.
        • Sun Y.
        • Blakeslee B.
        • Wang L.
        • Ni W.
        • Sopko M.S.
        • Yao C.
        • Yau K.
        • Burton S.
        • Zhuang M.
        • McCaskill D.G.
        • Gachotte D.
        • Thompson M.
        • Greene T.W.
        The protein kinase SnRK2.6 mediates the regulation of sucrose metabolism and plant growth in Arabidopsis.
        Plant Physiol. 2010; 153: 99-113
        • Pauwels L.
        • Barbero G.F.
        • Geerinck J.
        • Tilleman S.
        • Grunewald W.
        • Pérez A.C.
        • Chico J.M.
        • Bossche R.V.
        • Sewell J.
        • Gil E.
        • García-Casado G.
        • Witters E.
        • Inzé D.
        • Long J.A.
        • De Jaeger G.
        • Solano R.
        • Goossens A.
        NINJA connects the co-repressor TOPLESS to jasmonate signalling.
        Nature. 2010; 464: 788-791
        • Llompart B.
        • Castells E.
        • Río A.
        • Roca R.
        • Ferrando A.
        • Stiefel V.
        • Puigdomenech P.
        • Casacuberta J.M.
        The direct activation of MIK, a germinal center kinase (GCK) -like kinase, by MARK, a maize atypical receptor kinase, suggests a new mechanism for signaling through kinase-dead receptors.
        J. Biol. Chem. 2003; 278: 48105-48111
        • Chinnusamy V.
        • Zhu J.K.
        Epigenetic regulation of stress responses in plants.
        Curr. Opin. Plant Biol. 2009; 12: 133-139
        • Sanchez
        • Mde L.
        • Caro E.
        • Desvoyes B.
        • Ramirez-Parra E.
        • Gutierrez C.
        Chromatin dynamics during the plant cell cycle.
        Semin. Cell. Dev. Biol. 2008; 19: 537-546
        • Kappes F.
        • Scholten I.
        • Richter N.
        • Gruss C.
        Functional domains of the ubiquitous chromatin protein DEK.
        Mol. Cell. Biol. 2004; 24: 6000-6010
        • Boyer L.
        • Latek R.
        The SANT domain: a unique histone-tail-binding module?.
        Nat. Rev. Mol. Cell. 2004; 5: 1-6
        • Kim J.H.
        • Choi D.
        • Kende H.
        The AtGRF family of putative transcription factors is involved in leaf and cotyledon growth in Arabidopsis.
        Plant J. 2003; 36: 94-104
        • Lee B.H.
        • Ko J.H.
        • Lee S.
        • Lee Y.
        • Pak J.H.
        • Kim J.H.
        The Arabidopsis GRF-INTERACTING FACTOR gene family performs an overlapping function in determining organ size as well as multiple developmental properties.
        Plant Physiol. 2009; 151: 655-668
        • Fukazawa J.
        • Sakai T.
        • Ishida S.
        • Yamaguchi I.
        • Kamiya Y.
        • Takahashi Y.
        Repression of shoot growth, a bZIP transcriptional activator, regulates cell elongation by controlling the level of gibberellins.
        Plant Cell. 2000; 12: 901-915
        • Igarashi D.
        • Ishida S.
        • Fukazawa J.
        • Takahashi Y.
        14-3-3 proteins regulate intracellular localization of the bZIP transcriptional activator RSG.
        Plant Cell. 2001; 13: 2483-2497
        • Ding Z.
        • Millar A.J.
        • Davis A.M.
        • Davis S.J.
        TIME FOR COFFEE encodes a nuclear regulator in the Arabidopsis thaliana circadian clock.
        Plant Cell. 2007; 19: 1522-1536
        • Yi C.
        • Deng X.W.
        COP1 – from plant photomorphogenesis to mammalian tumorigenesis.
        Trends Cell Biol. 2005; 15: 618-625
        • Nemhauser J.L.
        Dawning of a new era: photomorphogenesis as an integrated molecular network.
        Curr. Opin. Plant Biol. 2008; 11: 4-8
        • Lee J.
        • He K.
        • Stolc V.
        • Lee H.
        • Figueroa P.
        • Gao Y.
        • Tongprasit W.
        • Zhao H.
        • Lee I.
        • Deng X.W.
        Analysis of transcription factor HY5 genomic binding sites revealed its hierarchical role in light regulation of development.
        Plant Cell. 2007; 19: 731-749
        • Ramon M.
        • Rolland F.
        • Sheen J.
        Sugar sensing and signaling.
        The Arabidopsis Book. 2008; 6: 1-22
        • Toroser D.
        • Huber S.C.
        Protein phosphorylation as a mechanism for osmotic-stress activation of sucrose-phosphate synthase in spinach leaves.
        Plant Physiol. 1997; 114: 947-955