JXB Advance Access originally published online on February 1, 2007
Journal of Experimental Botany 2007 58(5):1083-1098; doi:10.1093/jxb/erl275
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RESEARCH PAPER |
Methyl recycling activities are co-ordinately regulated during plant development
1Department of Biology, University of Waterloo, Waterloo, ON, Canada N2L 3G1
2Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA
3Department of Biology, Trent University, Peterborough, ON, Canada K9J 7B8
To whom correspondence should be addressed. E-mail: moffatt{at}sciborg.uwaterloo.ca
Received 25 August 2006; Revised 12 November 2006 Accepted 15 November 2006
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Abstract |
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A large number of compounds including lignin, phospholipids, pectin, DNA, mRNA, and proteins require methyl groups for their functionality. A detailed study of the expression and activities of two enzymes, adenosine kinase (ADK) and S-adenosylhomocysteine hydrolase (SAHH), which are both required for the maintenance and recycling of S-adenosylmethionine-dependent methylation in plants, was carried out. The abundance and tissue localization of ADK and SAHH transcripts and protein were monitored along with their enzyme activities in leaves, stems, buds, siliques, and roots of Arabidopsis. In all but roots and seed coats, the transcript abundance of ADK and SAHH fluctuated co-ordinately, matching changes in their protein and enzyme activities. To evaluate whether this expression pattern was associated with methyl recycling, the protein content and distribution of S-adenosylmethionine synthetase and phosphoethanolamine N-methyltransferase, a key methyltransferase involved in phospholipid synthesis, were investigated. These were found to accumulate in a pattern similar to ADK and SAHH. ADK and SAHH protein and transcript amounts were shown to fluctuate similarly in tissues accumulating lignin. Additionally, the amounts of ADK and SAHH mRNAs were also found at high levels in inflorescence meristems likely to support their higher rates of cell division. Thus, the results point to a co-ordinated and probably transcriptional regulation of these genes in most organs of Arabidopsis; SAHH abundance is distinctly higher in seeds and roots which suggests it may have a non-methyl-related role in these organs.
Key words: Adenosine kinase, S-adenosylhomocysteine hydrolase, S-adenosylmethionine, Arabidopsis, cytokinin metabolism, methyl recycling, seed development
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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S-Adenosyl-L-methionine (SAM)-dependent transmethylation reactions contribute to a wide array of processes in plants. For instance, methylation is involved in the synthesis of many compounds including phospholipids, pectin, lignin, and choline, as well as the modification of mRNA. Additionally chromatin and histones are continuously subjected to methylation that affects gene expression including gene silencing processes (Moffatt and Weretilnyk, 2001; Rocha et al., 2005). In each case the methyl group of SAM is transferred to a substrate by a specific methyltransferase (MT), and a molecule of S-adenosylhomocysteine (SAH), a strong inhibitor of all known SAM-dependent MTs, is produced (Poulton and Butt, 1976; Poulton, 1981). In eukaryotes and some prokaryotes (Sganga et al., 1992), SAH is rapidly hydrolysed into homocysteine (Hcy) and adenosine (Ado) by S-adenosylhomocysteine hydrolase (SAHH; EC 3.3.1.1 [EC] ) in a reversible reaction whose equilibrium lies towards SAH synthesis. However, the equilibrium is driven towards SAH hydrolysis by the removal of the products of its catabolism, Hcy and Ado. The accumulation of either compound has a strong inhibitory effect on SAHH activity (Poulton, 1981) and, therefore, intracellular levels of either Hcy or Ado must be continually reduced for transmethylation reactions and the regeneration of SAM to proceed (de la Haba and Cantoni, 1959; Poulton and Butt, 1976). While Hcy is used to regenerate SAM or S-methylmethionine in the methyl cycle, three enzymes may contribute to the metabolism of Ado: Ado deaminase (EC 3.5.4.4 [EC] ), Ado nucleosidase (EC 3.2.2.7 [EC] ), and Ado kinase (ADK; EC 2.7.1.20 [EC] ). In plants, ADK mediates the predominant route for Ado recycling (Dancer et al., 1997; Moffatt et al., 2002; Zrenner et al., 2006). This conclusion is based on the finding of very low levels of Ado nucleosidase and Ado deaminase in plants (for a review, Moffatt and Weretilnyk, 2001) and the observation that a deficiency in ADK activity leads to several developmental abnormalities in Arabidopsis plants (Moffatt et al., 2002). Taken together these results indicate that ADK's role in Ado recycling is not compensated by Ado nucleosidase and Ado deaminase activities (Poulton, 1981; Edwards, 1996; Moffatt et al., 2002).
Given that SAHH and ADK activities are essential to sustain methyl recycling, a decrease in either enzyme activity may limit SAM-dependent processes. In addition, since there are hundreds of SAM-dependent MTs, it is not surprising that a deficiency in either ADK or SAHH results in pleiotropic phenotypes in plants (Matsuda et al., 1995; Tanaka et al., 1997; Moffatt et al., 2002). Nicotiana tabacum lines with reduced SAHH levels are stunted, lack apical dominance, and have floral abnormalities (Tanaka et al., 1997). A similar phenotype is observed in ADK-deficient Arabidopsis plants (Moffatt et al., 2002). These changes are associated with hypomethylation of genomic DNA (Tanaka et al., 1997) and, in the case of ADK-deficient lines, SAM-dependent pectin methylation is also reduced (Moffatt et al., 2000). SAHH-deficient Arabidopsis mutants have diminished gene silencing capabilities which are associated with DNA methylation (Rocha et al., 2005).
Despite the great relevance of this cycle to eukaryote cellular metabolism, relatively little is known about how it is regulated or how ADK and SAHH activities accommodate changes in the flux through this cycle. Previous studies of these enzymes have demonstrated that their levels of expression and activities change in response to various environmental stresses and growth conditions (Edwards, 1996; Logemann et al., 2000; Moffatt et al., 2000; Weretilnyk et al., 2001; Suzuki et al., 2003). For instance, in alfalfa, elicitation of the phytoalexin response results in a 6-fold increase of SAM synthetase (SAMS) activity, concomitantly with a transient 2-fold increase of SAHH and Ado nucleosidase (Edwards, 1996). Moreover, salt stress has been associated with higher levels of SAHH and ADK expression and activities in glycine betaine-accumulating species (Weretilnyk et al., 2001). SAHH- encoding transcripts increase 2–3-fold in concert with increases in flavonoid biosynthetic activities, in response to a UV stimulus (Logemann et al., 2000).
Here, the expression patterns of ADK and SAHH in Arabidopsis organs of 5-week-old plants are documented to determine whether changes in their transcript abundance, protein content, and enzyme activities reveal at which level these genes may be regulated. Also evidence that changes in ADK or SAHH expression are linked to methyl-requiring activities was sought. Thus, the transcript abundance and tissue-specific distribution, protein content and localization, as well as the metabolic activities of both enzymes in stems, roots, buds, leaves, flowers, and young and mature siliques were quantified. The results indicate a transcriptional and co-ordinated expression of Arabidopsis ADK and SAHH genes; moreover, their respective protein levels and activities were found to be responsive to the synthesis of SAM-dependent compounds. ADK and SAHH expression in the shoot apical meristem, as well as in the inflorescence meristem, have also been examined by in situ hybridization and immunofluorescence protein localization. Both genes were more highly expressed in the inflorescence meristem. The results substantiate the functional significance of these enzyme activities in methyl recycling, as well as revealing which tissues depend on them most.
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Materials and methods |
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Plant material and growth conditions
Arabidopsis thaliana (accession Columbia) and the det3 mutant [acquired from The Arabidopsis Information Resource (TAIR; www.Arabidopsis.org); CS 6160] seed were suspended in 0.1% (w/v) agar and sown in pots containing a 50:50 v:v mix of ‘Sunshine LC1 Mix’ and ‘Sunshine LG3 Germination Mix’ (JVK). Plants were grown in controlled environment chambers at 20 °C under either long-day (LD) conditions, with 16 h light (150±20 µmol m–2 s–1 photosynthetic photon flux density) and 8 h dark, or short-day (SD) conditions, with 8 h light (150±20 µmol m–2 s–1 photosynthetic photon flux density) and 16 h dark. Arabidopsis wild-type (WT) plants were also grown hydroponically according to the method described in Gibeault et al. (1997).
The expression patterns of ADK and SAHH (including mRNA and protein abundance) enzyme activity were determined in buds, siliques, young and mature leaves, upper and lower regions of the inflorescence stem, and roots of 5-week-old plants. The whole inflorescence of developing buds was designated as one individual organ, simply designated as buds. The other stages of flowers and siliques were sampled based on the classification outlined by Ferrándiz et al. (1999) (see Supplementary Fig. S1 at JXB online).
RT-PCR and gene-specific northerns
The following primers were used for RT-PCR/gene-specific probe synthesis: SAHH1 upper, 5'-AAGCGGTTCAGGTTCGGATCTAC-3'; SAHH1 lower, 5'-GAAGAGTACAACGCTAAACAGAACACAC-3'; SAHH2 upper, 5'-TCGGATTGGGTCTGGTTTGGTCGG-3'; SAHH2 lower, 5'-CAGCCTCTCATAAATCAAAAACAACC-3'; Helicase RH4 upper, 5'-CGGTTCAGATCCGAGTTTGGGAG-3'; RH4 lower, 5'-GACGACGGGTCAAAATCAG-3'.
Enzyme assays
Plant samples were recovered from 5-week-old (LD) and 4-month-old (SD) Arabidopsis plants. Root tip samples were taken from 5-week-old hydroponically grown plants (Gibeault et al., 1997). All subsequent operations were carried out at 4 °C. Fully expanded leaves, buds, stage-12 flowers, stage-17a and -17b siliques, upper inflorescence stem (upper 10 internodes), and bottom inflorescence stem (the lowest internodes) were homogenized in 50 mM HEPES-KOH (pH 7.8), 5 mM dithiothreitol, 1 mM EDTA, 5 mM ascorbic acid, 10 mM boric acid, and 20 mM Na-metabisulphite with the addition of 4% (w/v) polyvinylpyrrolidone (mol. wt. 44 000 Da) (Lorenzin et al., 2001). The amount of buffer used for leaves, stem top, buds, flowers, and 17a siliques was 1:4 (w/v), and for roots, 17b siliques, and stem bottom it was 1:3 (w/v). Protein concentrations were estimated by the method of Bradford (1976) using bovine serum albumin as the standard.
ADK activity was measured in 50 µl reactions as previously described by Moffatt et al. (2000). The SAHH activity in desalted extracts was estimated using a colorimetric reaction (Wolfson et al., 1986). Hcy was used as a reducing reagent for 5, 5' dithio-bis (2-nitrobenzoic acid) (DTNB) to DTNB-thiolate, which results in an increase in the absorbance of the reaction mixture at 412 nm. A 1 ml reaction mixture contained: 50 mM HEPES-KOH (pH 7.8), 1 mM EDTA (pH 8.0), 0.1 mM DTNB (Sigma), 0.1 mM SAH (Sigma), and 0.5 U of Ado deaminase (Roche). For each organ studied the assays were done in three repetitions with six replicates each. The data were statistically analysed with two-way ANOVA and the difference between the means with Student's t-test and 
=0.05.
Antibodies
Anti-SAHH antibodies were raised against native His-tagged recombinant SAHH 1 expressed from pET30a (Novagen) in BL21(DE3)pLysS and affinity-purified by passage over an SAHH affinity column (Gu et al., 1994). Anti-ADK antibodies were described previously by Moffatt et al. (2000). The limits of detection of the purified sera were determined by reacting them with dot blots of serial dilutions of the recombinant proteins. The limits of detection were 0.01 ng for anti-ADK and 0.001 ng for anti-SAHH antibodies, when used at 1:8000 and 1:16 000, respectively. Each antiserum recognizes both isoforms of the target enzyme (e.g. anti-ADK antiserum recognizes both ADK1 and ADK2) and is specific for a polypeptide of the expected molecular mass when used to probe immunoblots of crude extracts of Arabidopsis organs (data not shown).
Immunoblots
The same protein extracts used for the enzyme assays were separated by electrophoresis through 13.5% (v/v) sodium dodecyl sulphate polyacrylamide gels for immunoblotting. Eight micrograms of total protein of each sample were applied to each lane and transferred to a polyvinylidene fluoride membrane. The membranes were probed with affinity-purified polyclonal anti-ADK and anti-SAHH antibodies diluted 1:8000 and 1:16 000, respectively, or polyclonal anti-SAMS antibodies diluted 1:1500 (Schröder et al., 1997).
RNA isolation and electrophoresis
Two methods were used for RNA isolation. The first method, based on the Tripure protocol (Roche), was used for isolating RNA from leaves, buds, flowers, siliques, and roots, following the manufacturer's instructions. The RNAqueous kit (Ambion) in combination with the Plant RNA Isolation Aid (Ambion) was used to recover RNA from stage-17b and -18 siliques which have a high carbohydrate content.
For RNA blotting experiments 10 µg of total RNA extracted from each plant organ were electrophoresed on a 1.5% (w/v) agarose-formaldehyde denaturing gel, and transferred to a nylon membrane. Hybridization was carried out overnight at 60 °C with either SAHH1 or ADK1 32P-labelled coding region probes. Subsequently, the membranes were washed to a final stringency of 1x SSC (300 mM NaCl, 30 mM Na3-citrate, pH 7.0). Bound probe was detected using a Storm 860 phosphorimager following 7 d exposure to the storage phosphor screen.
In situ hybridization
The tissue was prepared as follows. Samples of the inflorescence stems, roots, siliques, and flowers were harvested from Arabidopsis plants and immediately fixed in 4% (v/v) paraformaldehyde in phosphate -buffered saline (PBS) (140 mM NaCl, 2.7 mM KCl, 7.8 mM K2HPO4, 1.5 mM KH2PO4), under vacuum for 1 h at room temperature and overnight at 4 °C. Subsequently, the plant tissue samples were dehydrated, cleared in xylene, and then infiltrated and embedded in Paraplast Plus. Afterwards, the tissue samples were sectioned at a thickness of 10 µm, put on microscope slides, and allowed to dry on a hot plate for 48 h at 45 °C. Sense and antisense digoxigenin (DIG)-UTP (Roche)-labelled RNA probes were synthesized by T3 and T7 RNA polymerases using as templates the same ADK and SAHH cDNAs used in northern hybridizations. Hybridization was done in 50% formamide, 300 mM NaCl, 10 mM TRIS pH 7.5, 1 mM EDTA, 5% dextran sulphate, 1% blocking reagent, and 150 µg ml–1 tRNA at 55 °C. Subsequently, the slides were washed in 2x SSPE (150 mM NaCl, 10 mM NaH2PO4, 1 mM Na-EDTA, pH 7.4) for 15 min at room temperature and 2x SSPE at 40 °C. A final wash was made with 0.1% SSPE at room temperature. Immunological detection of the probes was done using anti-DIG alkaline phosphatase-conjugated antibody (Roche).
Immunofluorescent localization of ADK, SAHH, and phosphoethanolamine N-methyltransferase (PEAMT) proteins and histochemical tests
Tissues samples were fixed and processed as described for in situ hybridization. Sections (10 µm) from stems, buds, and developing siliques were then cut, dewaxed with xylene, and rehydrated in a graded ethanol series.
Samples of stems and siliques were blocked in PBS, 0.5% BSA, 0.1% Triton X-100 for at least 1 h. Afterwards, the slides were rinsed with PBS and then incubated in a wet chamber with the primary antibodies (anti-ADK, 1:100; anti-SAHH, 1:200) in blocking buffer for 1 h. The samples were washed 3x in blocking buffer, and rinsed in PBS 3x for 1 min. Afterwards, the slides were incubated with secondary antibody (anti-rabbit-fluorescein isothiocyanate, 1:100 dilution; Sigma) in a wet chamber in the dark, overnight at room temperature. The slides were kept in PBS, at 4 °C in the dark until observation. Pictures were taken in a Zeiss Axiophot fluorescence microscope, using a digital Nikon Coolpix model 990 camera.
The immunolocalization procedures for seeds were conducted as described in Franklin et al. (1999) with the following modifications: pretreated with 1.4% ß-glucuronidase (w/v) (Sigma) in 7% (w/v) sucrose for 15 min, washed in 1x PBS, and then with 1% Triton X-100, 1 mM EDTA (PBSTE) for 30 min. Sections were blocked as before and incubated overnight with the primary antibodies SAHH and ADK (1:250 dilutions) in blocking buffer. After washing in PBSTE the sections were incubated overnight with 20 µg ml–1 Alexa Fluor488 goat anti-rabbit IgG (Sigma) in blocking buffer. The slides were then washed with PBS, counterstained with propidium iodide (2 µg ml–1, Sigma), and viewed with a Nikon PMC-200 confocal microscope system. The images shown are the sum of the signals captured with the red (propidium iodide) and green (Alexa 488) channels. All images shown were taken at the same magnification and laser power unless noted otherwise.
Hand sections of the basal internode of the inflorescence stems from Arabidopsis WT plants grown for 5 weeks under LD or for 4 months under SD conditions, and det3 mutants grown under LD conditions were tested for the presence of lignin with fluoroglucinol staining solution as described in Johansen (1940).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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There are two genes that encode ADK in the Arabidopsis genome, At3g09820 and At5g03300, designated ADK1 and ADK2, respectively. The steady-state transcript abundance of these genes was investigated previously by RNA blotting and RT-PCR analysis. Both genes are transcribed in all major organs, with ADK1 transcripts being more abundant than those of ADK2 in all cases (Moffatt et al., 2000).
Arabidopsis also possesses two SAHH-encoding sequences, At4g13940 and At3g23810, designated SAHH1 and SAHH2, respectively (Rocha et al., 2005). The transcript abundance analysis of each SAHH gene was determined by hybridization of RNA blots with gene-specific probes and semi-quantitative RT-PCR; both assays indicate that these genes are differentially transcribed (Fig. 1A; data not shown). Whereas SAHH1 transcripts are consistently present in all organs studied, the abundance of SAHH2 transcripts was generally lower, particularly in leaves and roots. A similar analysis of leaves of younger plants indicated a more equal abundance of SAHH1 and 2 (data not shown).
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Plants deficient in both ADK1 and ADK2 activities have numerous morphological abnormalities, whereas mutants lacking SAHH1 expression are embryo lethal and SAHH2 mutants are apparently normal. To begin to address the functional basis for these phentoypes as well as to understand the regulation of the transmethylation cycle in general, it was necessary to try to determine how ADK and SAHH expression is regulated, and identify which organs and cell types expressed them to the greatest extent. Expression of ADK and SAHH was also monitored throughout silique development because this process has well-defined milestones (Ferrándiz et al., 1999); moreover silique dehiscence is abnormal in ADK- and SAHH-deficient plants.
The two Arabidopsis genes coding for each of these enzyme activities share high amino acid identity (92% for ADKs; 95% for SAHHs). The enzyme assays and immunodetection analyses used for this study do not distinguish between the two isoforms in each case. Probes for transcript abundance were also prepared from conserved regions of the gene pairs to evaluate whether total mRNA levels change concurrently with protein abundance.
ADK and SAHH expression in Arabidopsis organs
RNA and immunoblot studies showed that ADK and SAHH transcript and protein levels decreased as plant organs matured. For instance, ADK and SAHH transcript abundance started to drop as siliques expanded (stage 17a), with their respective protein levels falling a stage later (17b) (Fig. 1B). The corresponding enzyme activities displayed similar patterns and reflected the transcript and protein content, with both dropping as the siliques reached maturity (Fig. 1C). A corresponding analysis of ADK and SAHH expression in leaves of different ages showed that younger leaves had higher ADK and SAHH content than older leaves (Fig. 1D).
A similar examination of transcript and protein abundances in different organs revealed a co-ordinate expression pattern for these genes (Fig. 2). Both transcript and protein levels were highest in buds and the upper stem region. Intermediate amounts were observed in the lower stem, while reduced levels were detected in mature leaves and older siliques (Fig. 2A, B). Roots were the only organ that displayed an inconsistency in ADK and SAHH expression. ADK transcripts in roots were detected at intermediate levels compared with other organs, while SAHH mRNAs were abundant (Fig. 2A). Protein and transcript levels for both enzymes were notably higher in the root tips relative to total root tissue (Fig 2B; data not shown).
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ADK and SAHH enzyme activities were also high in root tips, buds, and upper stems, and lower in mature leaves, lower stems, and older siliques (17b), consistent with ADK and SAHH mRNA and protein contents in these organs. The correspondence in transcript abundance and enzyme activities suggests that transcriptional regulation is a key controlling factor in the expression patterns of these genes.
ADK and SAHH protein levels are consistent with those of other enzymes involved in methyl recycling and methyl metabolism
To examine the connection between the activated methyl cycle activities and SAHH and ADK fluctuations, the protein levels of SAMS (EC 2.5.1.6
[EC]
), the enzyme that catalyses the final step of SAM regeneration, and PEAMT (EC 2.1.1.103
[EC]
), a SAM-dependent methyltransferase acting on phosphocholine, the immediate precursor of many phospholipids, were examined (Smith et al., 2000). Immunoblot analysis with specific antibodies for these proteins revealed that the levels of SAMS and PEAMT mirrored the relative ADK and SAHH transcript abundance and activities in most of the organs tested. Higher amounts of SAMS and PEAMT were observed in roots, buds, siliques, and upper stems, consistent with the abundance of ADK and SAHH in these organs (Fig. 2C). Furthermore, the protein abundance of SAMS and PEAMT decreased during leaf development (Fig, 2D). These results indicate a positive correlation between protein levels of SAMS, PEAMT, ADK, and SAHH, reflecting their respective contributions to methyl metabolism.
Tissue-specific expression of ADK and SAHH
ADK and SAHH expression in Arabidopsis meristems: An in situ hybridization study of ADK and SAHH expression in inflorescence and shoot apical meristems revealed that the two tissues have distinct transcript distribution patterns, as shown in Fig. 3. Inflorescence meristems had strikingly high levels of ADK and SAHH transcripts, particularly in the outer layers (Fig. 3A, B); the primordium protuberances, developing ovules, and microspores also bound probes for these genes to a greater extent than surrounding tissues (Fig. 3E, F). Conversely, the signal in the shoot apical meristem was much lower for both genes relative to the signal observed in floral and inflorescence meristems (Fig. 3I, J).
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Recent proteomic and in silico expression-analysis studies of Arabidopsis, Medicago, and barley (Gallardo et al., 2002, 2003; Radchuk et al., 2005) have identified high levels of transcripts encoding methyltransferases, as well as methyl recycling activities, in developing and germinating seeds. Since the present results showed a similar accumulation of ADK and SAHH (data not shown) in situ hybridization was used to localize these transcripts in buds, flowers, and developing siliques of Arabidopsis (Fig. 3C, D, G, H, K, L). In the late floral stages (Fig. 3C, D) ADK and SAHH transcripts were abundant in all tissues of the pistil, with the highest expression associated with the ovules. Both genes were highly transcribed in the vascular tissues of sepals, petals, the gynoecium, and anthers (Fig. 3C, D). Figure 3G and H also shows the presence of ADK and SAHH mRNAs throughout the pericarp in early and intermediate silique stages, with the highest levels in developing embryos and vascular bundles. In most cases, ADK and SAHH transcripts appeared to have similar patterns of distribution. The only exception was observed in the developing seeds, where SAHH transcripts occurred at much higher levels than ADK (compare G and H in Fig. 3).
In developing siliques, transcripts for both ADK and SAHH were observed up to stage 17a, after which both dropped precipitously. Moreover, in all the phases of fruit development analysed here, the mRNA abundance was found to be highest in association with lignifying cells of vascular tissues, particularly in the separation layer of the dehiscence zone or nearby cells; this is particularly evident in the stage-17b sections probed with ADK and stage-17a siliques probed with SAHH (Fig. 3K, L).
To determine whether transcript levels were indicative of the accumulation of ADK and SAHH protein, similar material was examined with affinity-purified antibodies specific for each enzyme. The protein distribution patterns detected by this method reflected the mRNA abundance and provided further evidence for the co-ordinated expression of ADK and SAHH, particularly in lignifying tissues during flower and silique development. Representative images of these results are shown in Fig. 4. In both early and late silique stages, ADK and SAHH accumulated to the highest levels in the vascular bundles and endocarp (Fig. 4A–F).
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Generally, similar expression patterns were observed for ADK and SAHH in flowers, siliques, and meristems. However, this co-ordinated pattern of expression was not observed in the seed coat of Arabidopsis. Late silique stages contained developing seeds with high levels of SAHH in the testa not accompanied by observable ADK, as shown in Fig. 4G–I). This pattern of protein abundance in the seed coat corresponds to the accumulation of ADK and SAHH transcripts detected by in situ hybridization (Fig. 3G, H). To determine whether or not the high levels of SAHH were due to a non-specific association of the protein or SAHH antibody with flavonoids that accumulate in this layer, a similar SAHH/ADK localization experiment was carried out using transparent testa5 (tt5) Arabidopsis mutants whose seeds do not accumulate the brown tannins that characterize the WT (Dong et al., 2001). tt5 seeds accumulated SAHH in the endothelium layer similar to WT seeds (Fig. 4J–L).
ADK and SAHH were also detected in the developing pericarp. It is noteworthy that these enzymes were found associated with endocarp and other lignifying regions of the fruit as shown in Fig. 4A, D. They were particularly prevalent in the ena layer of the endocarp, but disappeared by stage 17b when the ena layer starts to undergo lysis (Fig. 4A, D) (Ferrándiz et al., 1999). Meanwhile, ADK and SAHH levels increased in the enb and the separation layer, reaching a peak at stage 17b (Fig. 4A, B, D, E). The protein distribution patterns for these tissues confirmed the association of ADK and SAHH with lignifying cells of the vascular bundles, dehiscence zone, and enb layer of the silique wall, which also lignifies (Fig. 4A–D). Finally, although ADK and SAHH mRNA levels detected by in situ hybridization and northern analysis were low in stage-17b siliques, ADK and SAHH protein content, as detected by immunofluorescence, was relatively high in these siliques (Fig. 4A, B, D, E).
ADK and SAHH are up-regulated during the lignification process in Arabidopsis inflorescence stems: It is believed that ADK and SAHH are essential to maintain the various SAM-dependent methylations that are required for lignification. However, it is unclear whether the methylated lignin precursors are generated in the lignifying cells or in adjacent cells. According to Hosokawa et al. (2001) the surrounding cells of tracheary elements (TEs) may account for part of their lignification. Therefore, this was investigated by localizing ADK and SAHH mRNA and protein distribution patterns in upper and lower regions of Arabidopsis stems. ADK and SAHH transcripts were observed in cross-sections of all tissues in the upper regions (above the 10th internode) of the inflorescence shoot. In general, higher transcript levels were observed in the vascular tissue and cortex region as shown in Fig. 5. In both upper and lower parts of the stem, transcripts were primarily associated with developing TEs and associated cells (Fig. 5A, B, E, F, I, J, M, N).
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The tissue-specific distribution of ADK and SAHH protein in both upper and lower stems mirrored their mRNA patterns. The proteins were tightly associated with vascular bundles in both regions of the stem. It is interesting to note that parenchyma cells and the developing fibres surrounding the xylem vessels also contained high amounts of both proteins (Fig. 5C, D, G, H, K, L, O, P). Therefore, a strong correlation between both ADK and SAHH transcript and protein levels has been observed in inflorescence of Arabidopsis at these stages stems.
To test the hypothesis that ADK and SAHH transcript and protein levels are increased in these cells due to lignification, their accumulation was examined in stems of Arabidopsis plants grown under an LD photoperiod as well those grown under an SD photoperiod, which leads to increased secondary cell wall growth and lignin accumulation (Chaffey et al., 2002). Also the accumulation of ADK and SAHH in det3 mutants which undergo extensive ectopic lignification of cortical and pith cells (Caño-Delgado et al., 2000) was examined as shown in Fig. 6. Figure 6i–iii depicts the result of a histochemical analysis of lignin accumulation in the lower region of inflorescence stems of WT plants and det3 mutants. The det3 plants accumulated more vascular tissue than did 4-month-old WT plants at a comparable developmental age (Fig. 6i–iii). Moreover, det3 plants underwent the extensive and ectopic lignification of cortical and pith cells verified by Caño-Delgado et al. (2000).
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ADK and SAHH protein levels in the upper part of the stem, as estimated by immunofluorescence detection, were not considerably different among the three samples (see Supplementary Fig. S2 at JXB online). In all cases, ADK and SAHH levels were highest around the vascular bundles. On the other hand, there was a trend of increasing ADK and SAHH accumulation in lower stems of LD WT, SD WT, and det3 plants (see Supplementary Fig. S3 at JXB online). In lower stem regions of WT LD plants, ADK and SAHH were located in the cortex and vascular tissue, whereas in stems of WT SD and det3 plants these proteins were either present in the vascular region (WT SD) or evenly distributed (det3) (see Supplementary Fig. S3 at JXB online).
Assays for ADK activity confirmed these observations. In general, there were no significant differences in ADK activity amongst the three samples in the upper part of the stem, while in the bottom regions there was a statistically significant increase in ADK activity in WT SD plants and the det3 mutant as compared with WT LD plants (Fig. 6iv).
ADK and SAHH transcript and distribution pattern in roots: In contrast to the other organs examined, ADK and SAHH transcript and protein distribution patterns did not exhibit similar expression patterns in roots. In situ hybridization revealed that both genes were highly expressed in the tip region as shown in Fig. 2B. However, in more mature regions, where vascular differentiation is complete, ADK transcript levels dropped dramatically, while SAHH levels remained at detectable levels as shown in Fig. 7. Immunolocalization of these enzymes mirrored the in situ hybridization results, with high signals observed at the tip for both proteins. However, in mature parts of the root only the SAHH signal remained high (Fig. 7). Therefore, it seems that ADK is located primarily at the root tips while SAHH has a more even distribution throughout the root, a pattern clearly different from that observed in other Arabidopsis organs.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Developmental expression of ADK and SAHH
The goal of this work was to investigate the expression patterns of SAHH and ADK in Arabidopsis plants to understand better the contributions of these enzyme activities to plant development. Overall, the results obtained from RNA and protein blots and the enzyme assays showed that the amounts of ADK and SAHH mRNAs, protein, and enzyme activities are higher in developing organs than at maturity (compare Figs 1B, C, 2D). The most logical basis for the commonality in ADK and SAHH expression is their joint involvement in the activated methyl cycle. If so, their increased abundance in younger versus older parts of the plant reflects a higher utilization of SAM for transmethylation in developing organs, the most predominant of which are the metabolic processes involved in cell division and the formation of cell membranes and walls. SAM-dependent methylation events associated with cell division and differentiation include chromatin modification and transcript capping, as well as phospholipid and pectin synthesis for the plasma membranes and walls of the newly dividing and elongating cells.
The association of SAHH activity with mRNA capping is well documented, particularly for the infection cycles of numerous animal viruses. Due to their intensive periods of orchestrated gene expression, viral infections require high levels of SAHH activity to maintain SAM-dependent mRNA capping (Neyts and Clercq, 2003). This is the basis for the sensitivity of viruses such as Ebola (Bray et al., 2000), filovirus (Bray, 2001), and parainfluenza (Clercq, 2001) to SAHH inhibitors. SAHH transcription and its nuclear translocation are correlated with the cell cycle and cell division rate of cultured Xenopus cells (Radomski et al., 1999). Additionally, SAHH up-regulation is also coincident with mRNA transcription rates during embryo development in this species (Radomski et al., 1999), and inhibition of SAHH prevents mRNA capping (Radomski et al., 2002). In plants, SAHH deficiency has been shown to lead to DNA hypomethylation (Tanaka et al., 1997) and a decrease in gene silencing (Rocha et al., 2005). The relatedness of ADK activity to DNA methylation and mRNA capping is less well documented except for the observation that methylation of genomic DNA of ADK-deficient Arabidopsis plants is reduced relative to WT DNA (Perry, 2004).
The decrease in cell division and expansion of mature plant organs, as well as changes in their cellular metabolism would logically lead to a reduction in methyl utilization. The present observations are consistent with this hypothesis. Mature siliques, stems, roots, and leaves have noticeably lower levels of ADK and SAHH expression and enzyme activity.
ADK and SAHH in Arabidopsis meristems: In situ hybridization showed that inflorescence and shoot apical meristems have distinct patterns of ADK and SAHH transcript distribution. Inflorescence meristems, particularly regions with high rates of cell division such as the floral primorida, accumulate high levels of transcripts encoding these enzymes, whereas the vegetative shoot apical meristem did not. These transcriptional differences may reflect rates of cell division in the two meristematic regions. Measurements of mitotic activity in Arabidopsis inflorescence meristems have revealed that the peripheral zone has a higher rate of cell division than the central zone (Laufs et al., 1998); this holds for all meristematic layers (L1, L2, and L3) that comprise the apical meristem. In addition, the mitotic index in developing floral primordia is similar to that in regions of the peripheral zone, denoting that floral primordia have higher rates of cell division (Laufs et al., 1998). On the other hand, the subapical meristematic regions that comprise the shoot meristem-organizing centre and the rib meristem have a mitotic index compatible with that in the central zone, which is indicative of a lower rate of cell division (for reviews, Evans and Barton, 1997; Laufs et al., 1998).
It is interesting to note that cytokinin (CK) distribution patterns in Arabidopsis inflorescence/floral meristems are consistent with ADK and SAHH transcript abundance in the different meristematic regions (Corbesier et al., 2003). Isopentenyladenine and zeatin are found in higher amounts in the inflorescence meristem, and these compounds are conspicuously absent from the subapical pith tissues (Corbesier et al., 2003). CKs in apical regions of Arabidopsis are associated with phenomena such as cell division, floral primordia development, and initiation of vascular tissue differentiation (Corbesier et al., 2003). Moreover there are several reports documenting increases in cell division in the shoot apical meristem during floral transition (Francis, 1992; Bernier, 1997; Laufs et al., 1998). In tobacco, Sinapis alba, and Arabidopsis, the mitotic indices increase during the transition from vegetative to floral growth, which has been associated with the pattern of CK distribution in the meristem (Dewitte et al., 1999; Jacqmard et al., 2003). Thus the differences in ADK and SAHH expression in inflorescence/floral versus vegetative meristems most probably reflect the methylation flux and differential cell division rates of these regions. The observation that ADK and SAHH protein levels fluctuate similarly to those of PEAMT, a key methyltransferase for phospholipid synthesis, supports this hypothesis.
ADK and SAHH expression in several organs
Siliques and stems: At the cellular and tissue levels, in situ hybridization and immunofluorescence detection revealed high levels of ADK and SAHH transcripts and protein in association with lignifying vessels and fibres of stems and siliques (see Figs 3–5
). Consistent with this observation is the indication that the neighbouring cells of TEs play an important role in the differentiation of TEs by intensive chemical exchanges between the TE and its surrounding cells (Fukuda and Komamine, 1980; Ryser and Keller, 1992; Pesquet et al., 2003). Moreover, in Zinnia, TE neighbouring cells reportedly take part in the lignification of the xylem vessels, by providing these cells with lignin monomers (Hosokawa et al., 2001). This has also been demonstrated in other plant groups, such as in conifers, where lignin is supplied from the adjacent parenchyma cells to the secondary walls of differentiating xylem (Savidge, 1989). Even those TEs that have completed programmed cell death may continue to lignify with the help of their surrounding cells (Hosokawa et al., 2001). Therefore, besides being responsible for their own lignification, parenchyma cells may also help with the lignification of the TEs, which may explain why they also have high levels of ADK and SAHH, a feature that is noticeable even in the older region of the xylem (Fig. 5). The association of ADK and SAHH with lignifying cells could also be observed in developing siliques (Fig. 4). The observation that WT plants grown in SD conditions as well as det3 mutants had higher levels of ADK and SAHH protein accumulation than did WT plants grown in LD conditions (see Supplementary Fig. S3 at JXB online) indicates a direct association between lignin synthesis and these methyl-recycling activities. Vasculature differences between stems and roots may contribute to the higher ADK and SAHH expression in stems versus roots. In the primary phase of plant development, the vascular cylinder of Arabidopsis roots is diarch (only two poles of xylem), with vessel walls containing very little secondary wall and lignin. The stem, on the other hand, contains several vascular bundles and may require higher levels of these gene products for differentiation.
EST analysis of transcript abundance from Populus tremula and P. tremuloides, two wood-forming plant species, indicated that genes associated with the phenylalanine ammonium lyase pathway and the synthesis of lignin precursors are strongly up-regulated (Sterky et al., 1998). Interestingly, SAHH and ADK transcript levels increase by similar levels, which is in agreement with the present observations showing that both genes are up-regulated in lignifying cells of Arabidopsis stems. SAMS is also highly abundant in this developing-xylem library (Allona et al., 1998; Sterky et al., 1998). More recently an oligonucleotide array of the majority of Arabidopsis genome ORFs was used to evaluate transcript levels in successive segments of Arabidopsis stems to identify genes associated with lignification. The results of this analysis show that ADK1 is expressed at approximately 5-fold higher levels in the upper region of the stem as compared with the lower region (P=0.01); ADK2 levels are somewhat higher in the elongation region as well, but the difference was not statistically significant; SAHH1 expression was steady from the top of the stem to the bottom, while SAHH2 was not represented on the array (Ehlting et al., 2005). Other microarray experiments accessed via Genvestigator (Zimmermann et al., 2004) reveal that all ADK and SAHH genes are highly expressed in the lower region of the stem. Moreover, transcript levels of SAHH2 are about 3-fold higher than those of SAHH1 in upper stems, while ADK2 levels were slightly higher than those of ADK1, although the difference was much lower than that observed for SAHH genes. Interestingly these data do not follow the trend that has been observed in other organs in which ADK1 and SAHH1 are generally the more highly expressed of the two isoforms. This suggests that ADK2 and SAHH2 may be more responsive to developmental/environmental changes in methyl demand while ADK1 and SAHH1 act to fulfil housekeeping roles; however, this still remains to be demonstrated unequivocally.
Roots and seeds: ADK and SAHH expression profiles changed co-ordinately in all major organs except in roots and developing seeds. While SAHH was found to be evenly distributed throughout the root, ADK was present at higher levels near the tip; seed coats of immature seed accumulated SAHH to high levels while ADK was hardly detectable. The reason for these differences in expression of ADK and SAHH in these organs is not clear. If methyl demand is the primary basis for temporal fluctuations in ADK and SAHH, both should change coincidently. The differential expression of ADK and SAHH in these cases is an intriguing phenomenon that may not be associated with transmethylation activities. Based on microarray analysis, ADK mRNAs are 1.5–2-fold higher in young roots than in older roots (http://jsp.weigelworld.org/expviz/expviz.jsp). Moreover, SAHH expression data given by microarray reveal equal values for young and older roots, which is consistent with the present findings. Although the magnitudes of the differences given by the microarray data for ADK are lower than those predicted by the results presented here, they support the conclusion of discordance in the expression of ADK and SAHH in roots.
A recent detailed study of CK distribution in pea seeds shows that CK levels are high at early embryo stages and decline considerably by the end of embryo development, concomitant with a reduction of isopentenyl nucleotide levels within the pericarp (Quesnelle, 2004). Coincident with the decrease of CKs in other pea seed components, the seed coat becomes the largest CK reservoir in late stages of seed development (Quesnelle, 2004). The reasons for the high CK content in this part of the seed are not yet completely understood, since most of the developmental processes that require CKs are not evident at this point. However, these data present the intriguing observation that CKs are high at the same developmental time and in the same tissue that SAHH accumulation occurred in Arabidopsis seeds.
This observation is particularly interesting because SAHH has been identified as one of two polypeptides in a 130 kDa CK-binding protein of tobacco plants (Mitsui et al., 1993), and SAHH expression in tobacco cell cultures is induced by kinetin (Matsuda et al., 1995; Tanaka et al., 1996). Moreover, tobacco plants with decreased SAHH activity due to antisense SAHH expression have higher CK levels than WT plants, based on a bioassay (Matsuda et al., 1995). Recent analysis of the CK profile of these tobacco SAHH-deficient lines by liquid chromatography–tandem mass spectrometry revealed no difference in total CK levels relative to the WT, although the abundance of specific CK constituents, particularly iP, DZR, and DZRMP was significantly altered (data not shown). Thus, it is clear that, at least in tobacco, reduced SAHH activity is associated with altered CK metabolism.
These results suggest the hypothesis that SAHH contributes to sequestering CKs in the seed coat until later in development when they serve to break dormancy as well as improve the sink strength during germination (Ma et al., 1998; Roitsch and Ehneß, 2000). A change in SAHH abundance may alter CK availability and, thus, CK homeostasis and metabolism. However, this relationship remains to be investigated since numerous soluble proteins have been shown to bind CKs, and a maize CK-binding protein lacks SAHH activity (Romanov and Dietrich, 1995).
These results indicate that, while ADK and SAHH genes are constitutively expressed, they are also transcriptionally regulated in response to methyl demand. The signals mediating this control and whether they act on both copies of each gene have yet to be determined. Since many methylated substrates have essential roles in plant cells, including the modification of chromatin structure, cell wall functionality, and even photosynthesis, understanding the regulation and consequences of methyltransferase activities during development is important. In addition, the basis for the differential expression of ADK and SAHH in roots and developing seeds suggests these enzymes may have non-methyl-related functions as well.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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The following supplementary data are available at JXB online.
Fig. S1. Flower and silique stages as described by Ferrándiz et al. (1999).
Fig. S2. Immunofluorescent localization of ADK and SAHH in upper regions of Arabidopsis inflorescence stem (up to the 10th internode).
Fig. S3. Immunofluorescent localization of ADK and SAHH in lower regions of Arabidopsis inflorescence stem.
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Acknowledgements |
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Anti-SAM synthetase IgG raised against Catharanthus roseus SAMS was the kind gift of Dr J Schröder, Institut für Biologie II, University of Freiburg, Germany. Anti-spinach phosphoethanolamine methyltransferase (PEAMT) antibody was a gift from Dr Elizabeth Weretilnyk, McMaster University, Ontario, Canada. The Arabidopsis Information Resource (TAIR) provided det3 seed. Li-Sen Young and Patrick Masson (University of Wisconsin, Madison) generously provided the image of adenosine kinase immunolocalization in root tips. LAR Pereira was supported by a scholarship from the Brazilian Council of Research (CNPq). This research was funded by Natural Sciences and Engineering Research Council of Canada Discovery Grants to BAM and RJNE.
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* Present address: Department of Botany, University of Brasilia, L2 Norte, Campus Darcy Ribeiro, Brasilia, DF-70910-900, Brazil.
Present address: Department of Experimental Medicine, McGill University, Montreal, QC, Canada H3A 1A3.
Present address: Department of Cell Biology, The University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd, BMSB 781, Oklahoma City, OK 73104, USA.
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