JXB Advance Access originally published online on September 5, 2008
Journal of Experimental Botany 2008 59(13):3705-3719; doi:10.1093/jxb/ern220
RESEARCH PAPER |
Cytokinin-induced photomorphogenesis in dark-grown Arabidopsis: a proteomic analysis
k Zdráhal2
ná2
árka Koukalová2,3
í Malbeck4emysl Souek1,3etislav Brzobohat
1,3,*
1Institute of Biophysics AS CR, v.v.i., Královopolská 135, CZ-61265, Brno, Czech Republic
2Department of Functional Genomics and Proteomics, Institute of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5, CZ-62500 Brno, Czech Republic
3Department of Molecular Biology and Radiobiology, Faculty of Agronomy, Mendel University of Agriculture and Forestry, Zemdlská 1, CZ-61300, Brno, Czech Republic
4Institute of Experimental Botany AS CR, v.v.i., Rozvojová 135, CZ-16502 Prague, Czech Republic
* To whom correspondence should be addressed. E-mail: brzoboha{at}ibp.cz
Received 19 June 2008; Revised 19 June 2008 Accepted 31 July 2008
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Abstract |
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High concentrations of cytokinins (CKs) in the cultivation medium can induce partial photomorphogenesis in dark-grown Arabidopsis seedlings. However, no significant increases in endogenous CK levels have been found in de-etiolated mutants, suggesting that either parallel pathways are involved in the light and CK responses, or changes in the sensitivity to CKs occur during photomorphogenesis. Here it is shown that even modest increases in endogenous CK levels induced by transgenic expression of the CK biosynthetic gene, ipt, can lead to many typical features of light-induced de-etiolation, including inhibition of hypocotyl elongation and partial cotyledon opening. In addition, significant changes in expression of 37 proteins (mostly related to chloroplast biogenesis, a major element of light-induced photomorphogenesis) were detected by image and mass spectrometric analysis of two-dimensionally separated proteins. The identified chloroplast proteins were all up-regulated in response to increased CKs, and more than half are up-regulated at the transcript level during light-induced photomorphogenesis according to previously published transcriptomic data. Four of the up-regulated chloroplast proteins identified here have also been shown to be up-regulated during light-induced photomorphogenesis in previous proteomic analyses. In contrast, all differentially regulated mitochondrial proteins (the second largest group of differentially expressed proteins) were down-regulated. Changes in the levels of several tubulins are consistent with the observed morphological alterations. Further, 10 out of the 37 differentially expressed proteins detected have not been linked to either photomorphogenesis or CK action in light-grown Arabidopsis seedlings in previously published transcriptomic or proteomic analyses.
Key words: Arabidopsis thaliana, cytokinin, 2D electrophoresis, photomorphogenesis, proteome
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
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The perception of light signals and appropriate growth responses are vital for seedling survival following germination. If a seedling is buried and, consequently, light is limited, it will undergo skotomorphogenesis (etiolated growth), characterized by elongation of the hypocotyl, tightly closed, underdeveloped cotyledons, and limited root development. Once it emerges into the light, de-etiolation will occur as it switches from skotomorphogenesis to photomorphogenesis, and inter alia the hypocotyl thickens while the cotyledons open and expand. Further, shoot apical meristem (SAM) function is initiated, giving rise to true leaves, and the root system proliferates. Etiolation represents an adaptation to germination below the soil surface that is largely restricted to angiosperm seedlings. Genetic studies in Arabidopsis have revealed that etiolation is a consequence of the inhibition of photomorphogenesis, and various Arabidopsis mutants have been identified that display photomorphogenesis when grown in continuous darkness. This phenotype is described either as de-etiolated (det) or constitutively photomorphogenic (cop). Thus, in wild-type plants, the DET and COP genes suppress photomorphogenesis in dark-grown seedlings. Additional mutants with the cop/det phenotype, called fusca (fus), have been recovered in screens for purple seedlings. Some of the COP/DET/FUS genes (including at least COP8, COP9, FUS5, and FUS6) encode components of the multisubunit protein complex COP9 (reviewed by Wei and Deng, 1999). The best understood constituent of the COP9 complex is COP1, an E3 ubiquitin ligase (Ma et al., 2002) that targets, among others, the transcription factor ELONGATED HYPOCOTYL5 (HY5) for accelerated ubiquitination (Osterlund et al., 2000). The process of light-induced de-etiolation has been investigated at the proteomic level by examining changes in the chloroplast proteomes of etiolated and light-grown maize (Lonosky et al., 2004) and dark-grown Arabidopsis seedlings exposed to light (Wang et al., 2006).
Several recent studies have identified convergence points in the signal transduction pathways of light and various hormones, most directly for auxins, gibberellins, and brassinosteroids (Kamiya and Garcia-Martinez, 1999; Neff et al., 1999; Reed, 2001). The possibility that cytokinins (CKs) may also be involved in photomorphogenesis was first raised by their ability to promote de-etiolation in the absence of light (Chory et al., 1991). Dark-grown seedlings exposed to exogenous cytokinins exhibit short hypocotyls, expanded cotyledons, leaves with partially developed chloroplasts, and activated light-regulated promoters. In addition, a CK-insensitive mutant, cin4, has been found to be allelic to cop10, providing further evidence for CK involvement in light perception (Vogel et al., 1998). Moreover, the amp mutant, which has elevated CK levels, displays various features of de-etiolation when grown in the dark (Chin-Atkins et al., 1996). In contrast, no changes in CK levels have been detected in wild-type or det1 plants exposed to different light regimes, suggesting either that parallel pathways are involved in the light and CK responses, or that changes in sensitivity to CKs occur during photomorphogenesis (reviewed by Neff et al., 2000).
Currently, the only reliably characterized CK signal transduction system is a two-component system involving histidine–aspartate phosphorelays, in which a histidine-containing phosphotransmitter (HPt) mediates signal transmission from a sensory histidine kinase to a response regulator. Genetic screens, genetic studies in His kinase-deficient yeast, and in vitro CK binding assays have demonstrated that the histidine kinases AHK2, AHK3, and AHK4/CRE1/WOL act as CK receptors (Choi and Hwang, 2007, and references therein). The phenotypes of plants carrying single and multiple mutations in CK receptors have shown no clear indications that CK signalling is involved in photomorphogenesis via the two-component system (Riefler et al., 2006). However, AHK3 in combination with either AHK2 or AHK4/CRE1 has been shown to mediate de-etiolation triggered by exogenous cytokinins in dark-grown Arabidopsis seedlings (Riefler et al., 2006). Furthermore, these findings do not exclude the possibility that photomorphogenesis may be regulated to some degree by an alternative CK signalling system recently postulated to explain the formation of a basic vegetative body plan in a triple mutant lacking the AHK2, AHK3, and AHK4/CRE1/WOL receptors (Riefler et al., 2006).
A clear molecular link for light–CK interactions appears to be provided by AtpC, a gene that is regulated by both light and CKs (via the same cis-element in its promoter, probably by regulating the same trans-acting factor), and the developmental stage of plastids (Kusnetsov et al., 1999). The cited study was not designed to distinguish between two signalling pathways acting in parallel that converge on the same promoter and direct interactions between light and CK. However, strong indications have been presented recently that CK and light signalling pathways operate independently and converge at regulation of the stability of HY5, a key transcription factor promoting photomorphogenesis (Vandenbussche et al., 2007).
Characterization of further genes that may be involved in CK-mediated photomorphogenesis in dark-grown Arabidopsis seedlings may contribute to understanding of the role(s) of CKs in this process. Towards this goal, two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) was used to identify proteins that are differentially expressed in response to increased levels of endogenous CKs in 7-day-old, dark-grown wild-type and transgenic Arabidopsis seedlings. Endogenous CK levels were increased by activating a CK biosynthetic gene, ipt, throughout the cultivation period using the dexamethasone (DEX)-inducible pOp/LhGR transcription activation system (Craft et al., 2005; ámalová et al., 2005). The results are consistent with the hypothesis that increased CK levels induce a partial transition from skotomorphogenesis to photomorphogenesis. Several proteins that were previously not known to respond to CK in dark-grown seedlings were identified in addition to those known to be CK regulated.
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Materials and methods |
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Plant material and growth conditions
Plants representing transgenic CaMV35S>GR>ipt lines pOpBK-ipt line 11 and pOpBK-ipt line 13 (Craft et al., 2005) and the corresponding wild-type Arabidopsis thaliana (ecotype Columbia 0) were used in this work. Seeds (
64 per plate) were grown on solid Murashige–Skoog (MS) medium supplemented with 1% sucrose, adjusted to pH 5.7 prior to autoclaving, and solidified with 1% agar (Duchefa, The Netherlands) in square plastic Petri plates (12.5x12.5 cm). To synchronize germination, plates with seeds were kept in darkness at 4 °C for 72 h, and subsequently transferred to a growth chamber (AR-36L Percival). Germination was induced by illuminating the plates with fluorescent white light (100 µmol photons m–2 s–1) for 24 h at 21 °C and then the plates were wrapped in several layers of aluminium foil and kept in darkness at 19 °C for 7 d, after which the seedlings were harvested, photographed, and frozen at –80 °C. The lengths of the hypocotyls of 30 individuals per line were measured from the acquired images using Analysis software (Olympus), and the significance of differences in their lengths was statistically evaluated by paired t-tests.
Dexamethasone treatment
To induce ipt expression in the transgenic seedlings, MS medium was supplemented with water-soluble DEX (Sigma-Aldrich) to a final concentration of 0.25 µM. Since the water-soluble DEX preparation contains 2-hydroxypropyl-β-cyclodextrin (hpcd), two types of control media were used—MS medium without any additional supplements and MS medium supplemented with hpcd (Fluka) at a final concentration of 1.1 µM, equivalent to the hpcd concentration in MS medium supplemented with 0.25 µM water-soluble DEX.
Quantitative RT-PCR and data analysis
To quantify the expression levels of the ipt gene, quantitative real-time PCR (RT-qPCR) was used, as follows. Total RNA was isolated from sampled plants using a NucleoSpin® RNA Plant kit (Macherey-Nagel) according to the manufacturer's instructions, and treated with TURBO DNA-free (Ambion) to exclude DNA contamination. The efficiency of the DNase treatment was checked by PCR amplification of ACTIN2 and 8 using non-transcribed RNA as a template. First-strand cDNA was prepared using SuperScript II reverse transcriptase (Invitrogen) and oligo(dT) primer (Souek et al., 2007) according to the manufacturer's instructions. The subsequent RT-qPCR was performed using a RotorGene 3000 (Corbett Research) with SYBR Green I as a fluorescent dye to monitor DNA contents. To amplify gene-specific products, the following primers were used: ACTfwd (5'-GGT GAT GGT GTG TCT-3'), ACTrev (5'-ACT GAG CAC AAT GTT AC-3') (Szyroki et al, 2001), fIPTrt (5'-ATC CTC CCT CAA GAA TAA GC-3'), and rIPTrt (5'-CTG AAA GGA ACG ACG C-3'). The steady-state levels of ipt transcripts were quantified by standard curve quantitation, and expression levels were normalized to ACTIN2 and 8. To enable statistical indications of the variations in ipt expression levels to be obtained, two fully independent real-time RT-qPCR experiments (starting with seedling cultivation) were performed and each RT-qPCR sample was run in triplicate. The significance of differences between non-activated and activated plants was evaluated by one-way analysis of variance (ANOVA; P < 0.05).
Extraction and purification of CKs
The procedures used to extract, purify, and quantitatively analyse CKs have been previously described (Lexa et al., 2003; Kiran et al, 2006). Quantification was performed using a multilevel calibration graph with deuterated CKs as internal standards. Since standards of cis-zeatin-glucosides and cis-zeatin-9-riboside-O-glucoside were not available, the amounts of these compounds were estimated from the calibration graphs of the corresponding trans-isomers. Results were obtained for two biological replicates, each representing an independent cultivation and extraction experiment.
Preparation of total protein extracts
Total protein was isolated from 7-day-old, dark-grown seedlings using the acetone precipitation procedure described by Tsugita and Kamo (1999). Each resulting dried protein pellet was resuspended by vortexing for 1 h at 37 °C in sample buffer containing 7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 60 mM dithiothreitol (DTT), 0.5% (w/v) SDS, and 0.8% Bio-Lyte 3/10 Ampholyte (Bio-Rad). Insoluble matter was removed by centrifugation for 10 min at 15 000 g, and the protein concentration was determined using an RC DC Protein Assay (Bio-Rad).
Two-dimensional gel electrophoresis and image analysis
To separate proteins in extracts of the plants, portions containing 120 µg of protein were initially subjected to isoelectric focusing in 18 cm gel strips (Bio-Rad) with an immobilized, non-linear pH gradient (3–10) formed by rehydrating the strips overnight at room temperature in buffer consisting of 7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 60 mM (DTT), 0.8% Bio-Lyte 3/10 Ampholyte (Bio-Rad), and the extract. In the focusing, voltages of 150, 300, 600, 1500, and 3500 V were sequentially applied for 30 min, 1, 1, 3, and 5 h, respectively, and finally 5000 V up to 90 000 Vh, at 20 °C in a PROTEAN IEF Cell unit (Bio-Rad). The strips were then equilibrated for 15 min at room temperature with equilibration buffer I [50 mM TRIS-HCl, pH 8.8, 6 M urea, 2% (w/v) SDS, 30% (v/v) glycerol, 1% DTT], transferred to equilibration buffer II [50 mM TRIS-HCl, pH 8.8, 6 M urea, 2% (w/v) SDS, 30% (v/v) glycerol, 3% iodoacetamide] and then embedded on top of an 11% homogenous SDS–polyacrylamide gel for second-dimension, electrophoretic separation. For this, an Ettan DALTsix Electrophoresis System (Amersham Biosciences) was used for the wild type and line 11 samples, while a PROTEAN Plus Dodeca Cell (Bio-Rad) was used for line 13 samples. However, for both electrophoretic units, the same running conditions were used: 50 V for 2 h followed by 100 V for 16 h. Gels were stained with SYPRO Ruby, for which a linear quantitation range of over three orders of magnitude has been documented (Berggren et al., 2000), and the linearity of the relationship between spot density and protein content in the range 0.02–5.0 µg was verified. Gels were imaged using a Storm 840 phosphorimager (Applied Biosystems), and the images were analysed using PD-Quest 7.3.0 software (Bio-Rad). Each plant line was evaluated separately and each biological sample was analysed in triplicate. For normalization, the Total Density in Gel Image procedure was used, in which the raw quantity of each spot in a member gel is divided by the total intensity value of all the pixels in the image. An assumption of the model applied in this procedure is that the total density of an image is relatively consistent from gel to gel (PD-Quest User Guide for version 7.3, chapter 6.7.b). Between-sample differences in normalized expression levels 
2-fold were regarded as significant, and the significance of differences in expression levels was assessed using Student's t-tests, setting the significance threshold at P <0.05.
Mass spectrometric analysis and data processing
Selected protein spots were subjected to digestion with trypsin, MALDI-MS, and liquid chromatography (LC)-MS/MS analysis, then the MS and MS/MS data were searched by MASCOT against the MSDB protein database (Release 20060831), all following procedures described by Bouchal et al. (2006).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
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Phenotypic and molecular analyses of the CaMV35S>GR>ipt seedlings
The binary pOp-ipt/LhGR system of DEX-inducible ipt expression was used to increase endogenous CK levels of dark-grown Arabidopsis seedlings, then the associated morphological and proteomic changes were examined. No clearly consistent morphological alterations compared with wild-type seedlings were observed in pOpBK-ipt lines 11 and 13 grown in darkness on MS medium for 7 d. However, when the transgenic seedlings were cultivated on MS medium supplemented with 0.1, 0.25, 0.5, and 1 µM DEX, strong dose-dependent inhibition of hypocotyl elongation was observed, while only slight reductions in hypocotyl length were observed at the highest DEX concentrations in wild-type seedlings (not shown). Based on these results, a working DEX concentration of 0.25 µM was chosen for subsequent experiments, at which the reduction in hypocotyl length was maximal in line 11, still in the linear range of the dose–response curve of line 13 seedlings, and there was no significant effect on hypocotyls in wild-type seedlings (Fig. 1A, B). In addition, the cotyledons had begun to open in the transgenic seedlings.
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Ipt transcript and CK levels were determined by RT-qPCR and LC-MS/MS, respectively, to examine their correlations with the inhibition of hypocotyl elongation. No significant differences were observed in CK levels between wild-type seedlings grown on MS in the presence and absence of DEX (total CK contents, <100 pmol g–1 FW in each case) and the transgenic seedlings grown in the absence of DEX (Fig. 1D). When grown on MS supplemented with 0.25 µM DEX, lines 11 and 13 displayed <2-fold and
5-fold higher levels of biologically active and total CKs, respectively, compared with controls, and the proportion of active CKs was lower in line 13 (the active/total CK ratio was 1.22±0.07-fold higher in line 11 than in line 13). In accordance with the extent of inhibition of hypocotyl length and content of biologically active CKs, DEX-activated lines 11 and 13 showed significantly higher (P <0.05) levels of ipt transcripts than controls; 4.6-fold and 1.4-fold higher, respectively (Fig. 1C).
Proteomic responses to increased CK levels
Total proteins were extracted from dark-grown transgenic (lines 11 and 13) and wild-type seedlings and separated by 2-DE (Fig. 2; see Materials and methods for details). Image analysis revealed >800 reproducibly resolved spots in gels over pI and molecular mass ranges of 3–10 and 10–120 kDa, respectively, and the proteome patterns of activated and non-activated seedlings of lines 11 and 13 were compared both qualitatively and quantitatively. No significant differences were detected for most (>90%) of the protein spots, but significant differences (P < 0.05) were found for 36 out of 626 matched spots in line 11 and 61 out of 810 matched spots in line 13. Furthermore, 14 of these protein spots were differentially expressed in both lines, and showed similar expression patterns in them. Altogether, 37 of the changed proteins were successfully analysed by MALDI-TOF MS and/or LC-MS/MS. From the intersection group, 12 proteins were identified. The remaining proteins could not be identified because the amounts obtained were below the detection limits of both methods. Identified protein spots are marked in protein maps shown in Fig. 2, and their amino acid sequences are listed in Supplementary Tables 1–6 available at JXB online. Protein identifications and representative graphs for the mean percentage volumes of each of these spots are presented in Table 1 (proteins with similar differential expression patterns in lines 11 and 13), Table 2 (line 13), and Table 3 (line 11). No significant differences in protein spots were observed between wild-type seedlings cultivated with and without DEX.
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Classification of identified proteins
To evaluate the functions of the differentially expressed proteins, the identified proteins were categorized using the criteria described by Bevan et al. (1998). The distribution of the proteins into functional categories is shown in Fig. 3A and Tables 1
–3. Significant fractions of the proteins were involved in protein destination and storage (16%), energy production (14%), and disease/defence (14%). Other highly represented functional categories (11% of identified proteins) were metabolism, signal transduction, and cell structure. Large proportions of proteins for which expression increased following DEX treatment of the transgenic seedlings are involved in photosynthetic processes or chloroplast biogenesis (ATP synthase gamma chain, glyceraldehyde-3-phosphate dehydrogenase A (GAPDHA), F13K23.15 protein, ATP-dependent Clp protease proteolytic subunit, 2-Cys peroxiredoxin BAS1, 50S ribosomal proteins L12-C and L4, and ribosomal protein L21). Proteins with decreased abundance include several associated with amino acid metabolism (cysteine synthase and isovaleryl-CoA dehydrogenase) and defence (myrosinase-binding proteins-like, catalase 3). Sixteen percent of the proteins were ‘unclassified’ and their functions are as yet unidentified.
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The subcellular location of each identified protein was determined according to the TAIR database (www.arabidopsis.org), and the results are summarized in Fig. 3B and Tables 1–3
. The largest group of proteins was localized to the chloroplast (26%), followed by the mitochondria (17%) and nucleus (14%). The finding that a large proportion of differentially expressed proteins in dark-grown seedlings with high CK levels are localized in the chloroplast indicates that CKs play an inductive role in the development of the photosynthetic apparatus in these seedlings.
Comparison of the present data with previous proteomic analyses of light-induced photomorphogenesis
Proteomic changes associated with light-induced photomorphogenesis have been described in Zea mays (Lonosky et al., 2004) and Arabidopsis (Wang et al., 2006). Only one protein (At3g11630) was identified as being differentially expressed in both the present study and that of Wang et al. (2006), and three proteins (At5g45390, At4g04640, and At3g26650) in the present set overlapped with those identified by Lonosky et al. (2004) (Table 4). Interestingly, all four of these proteins are nuclear-encoded chloroplast proteins. No protein common to all three sets was found.
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Comparison of the present proteomic data with published expression profiling data
The limited overlap between the sets of differentially expressed proteins prompted a comparison of the present set with previously observed changes in the transcriptomes of Arabidopsis seedlings during light-induced photomorphogenesis (Ma et al., 2001). To evaluate the effects of the light regime on CK action, the comparison was extended to data obtained from transcriptome profiling of long-day-grown Arabidopsis seedlings treated with exogenous CKs (Brenner et al., 2005) and others with increased levels of endogenous CKs due to ipt gene expression (Hoth et al., 2003). Table 4 summarizes the results of the comparison. Only a few transcripts corresponding to the present protein set were found to be regulated according to the transcriptomic data. Nevertheless, transcripts corresponding to more than half of the nuclear genes encoding chloroplast proteins which were found to be differentially expressed were reportedly up-regulated during light-induced photomorphogenesis. Genes encoding chloroplast proteins represented half of the overlapping set, with transcripts found to be regulated in response to ipt activation (Hoth et al., 2003), while genes encoding mitochondrial proteins prevailed in the set overlapping with those identified by Brenner et al. (2005). Interestingly, opposite effects of CK action were found on the protein and transcript expression levels of a number of genes in dark- and light-grown seedlings.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
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The effect of increased endogenous CK levels on the proteome of dark-grown Arabidopsis seedlings was analysed. Various differentially regulated proteins were detected, a high percentage of which appear to be linked to chloroplast and mitochondrion functions, and seedling architecture. Some of these proteins (or corresponding transcripts) proved to be present in sets of differentially expressed gene products identified in previous proteomic and transcriptomic studies of light-induced photomorphogenesis and transcriptomic analysis of CK-responsive genes, while several others have not been previously associated with these responses.
The pOp/LhGR system as a tool to trigger a partial switch from skotomorphogenesis to photomorphogenesis via regulation of ipt expression
The ability of the pOp/LhGR system to control expression of the ipt gene has been demonstrated by phenotypic evaluations in Arabidopsis and tobacco (Craft et al., 2005; ámalová et al., 2005). In the presented study, the tightness and induction characteristics of the pOp/LhGR system in dark-grown seedlings were found to be comparable with those reported recently for Arabidopsis seedlings cultivated under a long-day light regime (Hradilová et al., 2007; Souek et al., 2007), indicating that the performance of the pOp/LhGR system is not influenced by the light regime. Interestingly, small increases in steady-state ipt transcript levels, resulting in <2-fold increases in CK bases—the biologically active forms of CKs—were sufficient to induce morphological responses, including saturating inhibition of hypocotyl elongation and partial cotyledon opening. These findings indicate that these responses are highly sensitive to the CK dose. If such small increases in CK levels, which moreover were probably restricted to certain tissues or cell types, are involved in the induction of partial light-induced photomorphogenesis, it is very likely that they would not have been noticed in earlier studies (Chory et al., 1991), due to the poorer sensitivity limits of the methods available to quantify CKs at that time. The observed inhibition of hypocotyl elongation is likely to be mediated by the stimulation of ethylene biosynthesis in response to elevated CK levels, as previously shown for CK-treated Arabidopsis and ipt-expressing tobacco seedlings (Cary et al., 1995; Genkov et al., 2003).
Chloroplast proteins represent a major fraction of cytokinin-induced proteins
The differentially expressed proteins detected in the present experiments included a remarkably high percentage of proteins located to chloroplasts—26%, compared with 7.9% predicted for the whole genome (Bevan et al., 1998)—all of which were up-regulated in the CK-overproducing plants. In accordance with the stimulation by CKs of chloroplast biogenesis in etiolated Arabidopsis seedlings (Chory et al., 1991), these up-regulated proteins included ribosomal proteins—the 50S ribosomal protein L12C (spot 1304/2402, Table 1), 50S ribosomal protein L4 (spot 8202/9206, Table 1), and ribosomal protein L21 (spot 9703, Table 2)—and one involved in protein destination, the ATP-dependent Clp protease proteolytic subunit (spot 1105/1105, Table 1). The ribosomal protein L21 has been implicated in the transformation of proplastids to chloroplasts, based on the transcriptional regulation of its gene (Lagrange et al., 1993), and the ATP-dependent Clp protease proteolytic subunit has been found to be essential for chloroplast biogenesis in Arabidopsis (Zheng et al., 2006) and tobacco (Kuroda and Maliga, 2003). In addition, the partial transformation of etioplasts to chloroplasts in the CK-overproducing plants is evidenced by the up-regulation of three proteins involved in photosynthesis; the ATP synthase 
chain (spot 4602/4503, Table 1), chloroplast GAPDH (spot 7702/7702, Table 1), and F13K23.15 protein (GAPDH; spot 7606, Table 3). Furthermore, transcriptional regulation of the AtpC gene encoding the ATP synthase chain by light and CK through the same cis-element has been reported in tobacco (Kusnetsov et al., 1999). Out of nine subunits of the plastid ATP synthase, levels of four (
, β, 
, and ) reportedly increase with increasing duration of exposure to light in etiolated maize seedlings (Lonosky et al., 2004). Induction by light of nuclear genes encoding the chloroplast GAPDH has also been shown at the transcript and protein levels in Arabidopsis (Dewdney et al., 1993) and maize (Lonosky et al., 2004), respectively. The up-regulation of proteins involved directly in photosynthetic reactions was accompanied by increases in the levels of the 2-Cys peroxiredoxin BAS1 protein (spots 2601/2801, Table 1), a stromal enzyme with antioxidant activity implicated in maintaining photosynthesis in a functional state (Baier and Dietz, 1999, and references therein).
Interestingly, the regulatory role of CKs in chloroplast development and function appears to be paralleled by the compartmentation into chloroplasts of significant parts of CK metabolism. Four Arabidopsis isopentenyl transferases are located in the plastids (Takei et al., 2004), and a wide spectrum of CK metabolites have been reportedly found in plastids, together with variations in their relative contents following light and dark treatments (Benková et al., 1999). In addition, the maize β-glucosidase Zm-p60.1, which releases active CKs from corresponding O-glucosides (Brzobohat et al., 1993), has been located to plastids (Kristoffersen et al., 2000), and its accumulation in chloroplasts and plastids of transgenic tobacco leads to perturbation of the zeatin metabolic network (Kiran et al., 2006).
Mitochondrial proteins are down-regulated in response to increased CK levels in dark-grown Arabidopsis seedlings
Mitochondrial proteins represent the second most abundant class of differentially regulated proteins identified in the present experiments (17% of the total, compared with 10% predicted for the whole Arabidopsis genome; Millar et al., 2005). In contrast to chloroplast proteins, identified proteins for which mitochondria were their only predicted destinations were down-regulated. The set of differentially regulated mitochondrial proteins included proteins involved in protein assembly, metabolism, and production of energy, and two proteins with predicted locations in other organelles as well as mitochondria; the peroxisome (catalase3; spot 8501, Table 2) and the chloroplast (zinc metalloprotease; spot 2809, Table 3). None of these proteins has been previously implicated in de-etiolation.
Alterations in cell structure proteins in dark-grown Arabidopsis with increased levels of endogenous CKs
The abundance of several tubulins was found to be regulated by CKs in the dark-grown seedlings (Table 2), in accordance with the reductions in hypocotyl elongation and likely initiation of leaf primordia in seedlings overexpressing ipt [similar to that seen with exogenously applied CKs (Chory et al., 1994)]. For example, down-regulation of -tubulin and β-tubulin transcripts following light treatment of etiolated Arabidopsis seedlings has been reported, and found to correlate with reduced elongation rates (Leu et al., 1995). In addition, the expression level of the -tubulin gene TubA1 has been correlated with cell division activity in pea plants (Stotz and Long, 1999), and the expression of β-tubulin genes has been shown to be promoted by a combination of auxin and CK that induced tracheary element differentiation and cell division in Zinnia mesophyll cells (Yoshimura et al., 1996).
Down-regulation by CKs of cinnamyl-alcohol dehydrogenase (CAD; spots 7709/8001 Table 1) was also observed in dark-grown Arabidopsis seedlings, which may reflect molecular differences between CK-induced partial de-etiolation in darkness and light-induced photomorphogenesis since CAD levels remained constant during lignogenesis in pea shoots induced by illumination in experiments reported by Wilkinson and Butt (1992).
Overlap with previously observed proteomic changes during light-induced de-etiolation
Only two proteins (the RuBisCo -binding subunit 134102/At2g28000 and the 33 kDa subunit of the OEC of PSII 15912247/At5g66570) are present in sets reported to be differentially expressed during light-induced photomorphogenesis in both maize (26 proteins; Lonosky et al., 2004) and Arabidopsis (25 proteins; Wang et al., 2006), neither of which was identified as differentially expressed in the present study. Similarly, little overlap was found between the protein set identified in the present work and either the cited maize set (three common proteins) or the cited Arabidopsis set (one common protein). The lack of substantial overlap between the protein sets may reflect differences in the model plants used, the developmental stage of the starting material, and/or the duration of the treatment, e.g. 7-day-old etiolated seedlings in which CK levels were increased throughout the entire cultivation period were used in this study, while 4-day-old etiolated seedlings subjected to 6–9 h of light treatment were examined by Wang et al. (2006). Some of the differences may also be due to limitations in the proteomic techniques employed, since stronger overlaps were observed with transcriptomic data sets (see below).
Comparison of proteomic and transcriptomic data
To assess similarities between CK- and light-induced photomorphogenesis, the set of differentially expressed proteins identified in the present work was compared with the set of transcripts found to be differentially regulated by light in Arabidopsis seedlings (Ma et al., 2001). Nuclear genes encoding chloroplast proteins form the most abundant group in the 15 genes representing the overlap between the two data sets, and were found to be up-regulated at both transcript and protein levels (Table 4). Interestingly, two of the proteins (the chain of ATP synthase and GAPDH) were also found to be up-regulated during maize greening (Lonosky et al., 2004). Thus, the induction of chloroplast differentiation appears to be the major common effect of CK and light in etiolated seedlings.
To assess similarities between CK action in seedlings grown in darkness and in a standard long-day light regime, the protein set identified in the present work was compared with sets of transcripts that have been previously found to be differentially regulated following ipt induction (Hoth et al., 2003) and CK treatment (Brenner et al., 2005). Interestingly, all five proteins identified in the present study for which the only predicted destination was the mitochondrion were also differentially regulated by CK treatment under standard photoperiods, although two of them display opposite trends (Table 4). Of the eight transcripts identified by Hoth et al. (2003) that are also present in the present proteomics data set, four encode chloroplast proteins. However, under a standard long-day light regime, the four transcripts were found to be down-regulated while in the present study the corresponding proteins were found to be up-regulated in dark-grown seedlings, indicating that CKs may have some opposite effects on plastid development in light and darkness. It is interesting to note that among the proteins detected as being differentially expressed in the present proteomic analysis, the probable annexin (spot 8302, Table 2) is the only one for which the same regulatory trend, i.e. up-regulation, was also found at the transcript level in each of three independent genome-wide transcriptomic analyses of CK action (Hoth et al., 2003; Rashotte et al., 2003; Brenner et al., 2005).
However, drawing conclusions from the comparison requires a great deal of caution, for several reasons. Factors that regulate gene regulation often have differing, or even opposite, effects at the transcript and protein levels. In addition, while virtually all genes can be profiled at the transcript level, the number of genes amenable to analysis at the protein level using state-of-the-art proteomic techniques is still quite limited. Furthermore, the materials and methods used in our proteomic and the cited transcriptomic studies differed in several respects, e.g. the developmental stage of the seedlings and duration of the treatments. Moreover, none of the previous studies included the determination of endogenous CK levels.
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
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In dark-grown Arabidopsis seedlings, even a <2-fold increase in levels of endogenous, biologically active CKs was sufficient to induce a key component of the photomorphogenic developmental programmne—chloroplast biogenesis—as evidenced by the high proportion of chloroplast proteins among the set which were found to be up-regulated, more than half of which are also reportedly up-regulated at the transcript level during light-induced photomorphogenesis (Ma et al., 2001). These observations provide novel insights regarding the involvement of CKs in the multilevel network that regulates photomorphogenesis, in which the activities of auxin, ethylene, and gibberellins are already well established (Nemhauser, 2008, and references therein). Further, in addition to genes that were already known to be involved in CK-induced processes, several novel proteins were identified that appear to be specifically involved in mediating CK action in dark-grown seedlings. Elucidation of the role(s) of these novel CK-regulated proteins in the initiation and/or execution of the photomorphogenetic programme represents a challenge for future work.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataReferences |
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Supplementary data are available at JXB online.
Supplementary Tables 1–6 list the peptide amino acid sequences of the differentially expressed proteins identified by MALDI-MS and LC-MS/MS.
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Acknowledgements |
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We thank Dr Ian Moore for CaMV35S>GR>ipt seeds. This work was supported by grants LC06034, 1M06030, and MSM0021622415 (Ministry of Education of the Czech Republic), IAA600040701 and IAA600040612 (Grant Agency of the Academy of Sciences of the Czech Republic), and AV0Z50040507 and AV0Z50040702 (Academy of Sciences of the Czech Republic).
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