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Originally published In Press as doi:10.1074/jbc.M307787200 on September 5, 2003

J. Biol. Chem., Vol. 278, Issue 46, 45397-45405, November 14, 2003
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S Phase Progression Is Required for Transcriptional Activation of the {beta}-Phaseolin Promoter*

Mahesh B. Chandrasekharan, Guofu Li{ddagger}, Kenneth J. Bishop, and Timothy C. Hall§

From the Institute of Developmental and Molecular Biology and Department of Biology, Texas A&M University, College Station, Texas 77843-3155

Received for publication, July 18, 2003 , and in revised form, September 3, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Elucidating the mechanisms by which the transcription machinery accesses promoters in their chromatin environment is a fundamental aspect of understanding gene regulation. The phas promoter is normally constrained by a rotationally and translationally positioned nucleosome over its TATA region except during embryogenesis when it is potentiated by the presence of Phaseolus vulgaris ABI3-like factor (PvALF), a plant-specific transcription factor, and activated by an abscisic acid (ABA)-induced signal transduction cascade. Ectopic expression of PvALF and the supply of ABA in transgenic tobacco or Arabidopsis leaves can activate expression from phas. We confirmed by [3H]thymidine incorporation that active DNA replication occurred concomitant with the presence of PvALF and ABA. Arrest of DNA synthesis or S phase progression by infiltration of the leaves with replication inhibitors (hydroxyurea, roscovitine, mimosine) strongly inhibited transcriptional activation, especially the ABA-mediated activation step. Similarly, activation of endogenous Arabidopsis MAT and LEA genes in leaf tissue by the presence of ABA and ectopically expressed PvALF was inhibited by DNA replication arrest. No change in transcript levels on the arrest of replication was detected for abi1, abi2, and era1, negative regulators of the ABA signal transduction cascade or for cell cycle components ick1 and aip3. However, a reduction in transcript accumulation for the crucial ABA signaling effector, abi5, occurred upon DNA replication arrest (probably reflected in the decrease in MAT and LEA gene expression). Contrary to the conventional view that ABA inhibits DNA replication, our findings show that ABA acts in concert with S phase progression to activate gene expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The promoter for the {beta}-phaseolin gene (phas, which encodes the major storage protein of Phaseolus vulgaris) is subject to stringent spatial regulation with high levels of expression confined to embryogenesis (1). The presence of a rotationally and translationally constrained nucleosome over the TATA region contributes to transcriptional repression in vegetative tissues (2). During embryogenesis, strong module-specific promoter activity is achieved by a diverse array of DNA-protein interactions (3, 4). An early demonstration of the involvement of ABA1 in phas regulation was the finding of an increase in phaseolin accumulation in excised embryos cultured in the presence of ABA (5). This was corroborated by the enhancement of transcription from the phas promoter upon incubation of transgenic tobacco seeds in the presence of exogenous ABA (6). Further, ABA has been shown to be essential for the activation step of transcription from the phas promoter in transgenic tobacco (3) and, by data reported here, Arabidopsis. The involvement of ABA in gene activation dictates that components of its signal transduction pathway, including enzymes and transcription factors, play direct or indirect roles in this process.

In Arabidopsis, genetic studies have revealed that several loci are involved in responses to ABA (7–9). ABA-insensitive (abi) mutants were isolated using genetic screens to obtain plants with altered sensitivity to germination on media containing inhibitory concentrations of ABA (7). Analyses of the abi3, abi4, and abi5 mutants revealed dramatic changes in seed development with marked effects on transcript accumulation for maturation-specific (MAT) and late embryogenesis-abundant (LEA) genes. Ectopic expression of ABI3 and the supply of exogenous ABA in Arabidopsis leaves have been shown to induce expression of both MAT and LEA genes (10). Similarly, ectopic expression of PvALF (11) in transgenic tobacco leaves, together with exogenous ABA, resulted in phas-driven GUS expression (3).

Fundamental questions are: how does PvALF gain access to the phas promoter, and how does the ABA-stimulated signal transduction cascade activate transcription? An attractive possibility is that PvALF accesses the chromatin-constrained promoter upon displacement of histones during replication (12, 13). This concept is supported by the fact that DNA replication and endoreduplication are characteristic of bean embryogenesis where the phas gene is active (14). A role for DNA replication was further implicated by the observed decrease in de novo activation of the phas promoter by protein synthesis inhibitors in the presence of the replication inhibitors, aphidicolin and hydroxyurea (15). Analyses of Arabidopsis embryos during development revealed the presence of active DNA replication (16) concomitant with the initiation and accumulation of maturation-specific proteins. However, the current view pertaining to the mode of action of ABA is that it blocks cell division by preventing DNA replication and arresting the cell cycle at G1/S (17). To address the role of DNA replication (or S phase progression) in PvALF- and ABA-mediated activation, we employed replication inhibitors and also assessed the effect of S phase arrest on MAT and LEA gene activation. Our results show that S phase progression is required for PvALF- and ABA-mediated promoter activation and is critical for accumulation of the ABA signal effector ABI5.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Construction—The PvAlf coding region (2.3 kb) was obtained by digesting pJIT-PvAlf (11) with SalI and SmaI. The released fragment was ligated to pTA7002 (18) that had been digested with SpeI, end-filled, and then digested with XhoI. The resulting construct, pGVG-PvAlf, yields glucocorticoid-inducible expression of PvALF. The PvAlf coding region was PCR-amplified to incorporate restriction sites for NcoI and SalI and was mobilized into pPCR-Script® (Amp) SK+ (Stratagene). Subsequently, the PvAlf coding region was released by partial digestion with NcoI followed by complete digestion with SalI and ligated to NcoI-SalI-digested vector pET-30(a) (Novagen) to obtain PvAlf/pET. Mobilization of the PvAlf region into pET-30(a) confers a His6 tag and an S-tag to PvALF upon translation (HisSPvALF). The 2.4-kb HisSPvAlf region was PCR-amplified to introduce restriction sites for AscI and PacI, and the PCR product was mobilized into pPCR-Script®. The HisSPvAlf fragment was then released using AscI and PacI and ligated to AscI-PacI-digested vector pER8 (19). The resulting construct, pXVE-HisSPvAlf, yields the estrogen-inducible expression of PvALF in plants.

Plant Transformation—Tobacco leaf discs from line 58.1A, homozygous for –1470phas/uidA (20), were transformed with Agrobacterium strain LBA4404 harboring pGVG-PvAlf (3). Thirty-four hygromycin-resistant (GR1–34) plants were grown to maturity. Following induction of PvALF expression with 90 µM dexamethasone (DEX; Sigma) and activation by 200 µM ABA (Calbiochem), leaves from lines GR3, GR24, and GR30 strongly expressed GUS. Homozygous line GR30 (GVG-PvALF::–1470phas/uidA) was used for the studies reported here.

Arabidopsis plants (ecotype Columbia) were transformed (4, 21) with a mixed culture of Agrobacterium strain GV3101 harboring pXVE-HisSPvAlf or p–1470phas-uidA-3'/HM301K. Twenty-four hygromycin- and kanamycin-resistant T1 plants were obtained. Estradiol- and ABA-mediated induction, followed by histochemical staining, was conducted to evaluate the level and uniformity of phas-driven GUS expression. Genomic DNA blot analysis showed that line Alf5'-2, used in these studies, contained two copies of the two T-DNA cassettes (XVE-HisSPvAlf::Pnos-hptII-nos and –1470phas-uidA-3'::Pnos-nptII-nos).

Hormone and Pharmacological Reagent Treatments—Tobacco leaves from 10–14-day-old seedlings, selected on MS medium containing 50 µg/ml hygromycin, were incubated in liquid basal MS medium with 90 µM DEX (Sigma) and 200 µM ABA (Calbiochem) for 24 h in the dark with gentle agitation at room temperature. The T3 seeds of Arabidopsis line Alf5'-2 were germinated on MS medium containing kanamycin (50 mg/liter) and hygromycin (25 mg/liter) and grown to the onset of rosette leaves. The seedlings were then transferred to MS medium without selection for 10 days. Leaves were subjected to a brief (~2–3 min) vacuum infiltration in liquid basal MS medium containing 10 µM 17{beta}-estradiol (Sigma) and 200 µM ABA and incubated for 10–12 h in the dark with agitation at room temperature. Control treatments of Alf5'-2 leaves with estradiol alone contained the ABA synthesis inhibitor fluridone (100 µM; Crescent Chemical Co.) to prevent endogenous ABA accumulation (22, 23).

For treatments with DNA replication inhibitors, the MS medium containing leaves from lines GR-30 or Alf5'-2 induced with DEX or estradiol, respectively, and ABA as described above, were supplemented with hydroxyurea (HU) (100 or 250 mM; Sigma) or mimosine (1 or 2 mM; Sigma) or roscovitine (100 or 200 µM; Calbiochem).

{beta}-Glucuronidase Assays—Leaves were histochemically stained with X-Gluc, or leaf extracts were used for fluorimetric MUG assays (24) as described in Ref. 4.

DNA Replication and Inhibition—Approximately 100 leaves (10–14 days old) from line Alf5'-2 were incubated in MS medium (100 ml) with or without HU in the presence of [methyl-3H]thymidine (37 MBq/ml, specific activity of 185 GBq/mmol; Amersham Biosciences) at a final concentration of 1 µCi/ml. Two independent experiments were conducted using leaves treated with 100 or 250 mM HU or untreated as a control. Leaves were frozen in liquid nitrogen, and genomic DNA was isolated. MUG assays were conducted on extracts from leaves collected at the fourth interval to measure the effect of inhibitors on phas-driven GUS expression. Incorporation of [3H]dTTP into genomic DNA was determined by scintillation spectrometry (25).

RNase Protection Assay—To generate an antisense riboprobe for PvAlf, a 438-bp fragment corresponding to the acidic activation domain (11) was PCR-amplified and cloned into pPCR-Script®. Riboprobes to the 3' end of the uidA coding region and the 18 S region were as described in Ref. 3. Radiolabeled 32P, antisense uidA, PvAlf, and 18 S riboprobes of 310 nucleotides, 529 nucleotides, or 200 nucleotides, respectively, were synthesized using T3 or T7 RNA polymerase on a HindIII- or EcoRI-linearized plasmid by in vitro transcription (MEGA-scriptTM kit; Ambion). Total RNA was isolated from leaves using RNeasyTM Plant Mini Kit (Qiagen). RNase protection assays were conducted with 10 µg of total RNA using a RPAIII kitTM (Ambion). RNA from line 58.1A was used as a negative control for uidA and PvAlf transcripts, and RNA from line PvAlf-14 was used as a positive control for PvAlf mRNA (3). The protected fragments were analyzed by electrophoresis in a 5% polyacrylamide 8 M urea gel.

RT-PCR Analysis—Total RNA (0.5 µg), isolated from Arabidopsis leaves using an RNeasy Plant Mini Kit and digested with RNase-free DNase (Qiagen), was subjected to RT-PCR analysis employing the Qiagen one-step RT-PCR kit. The RT-PCR reactions included 0.6 µM gene-specific primer and 0.08 µM EF1{alpha}-specific primer (as an internal control). Primer sequences used in assessing transcript abundance and reaction conditions are provided as supplemental material. Following RT-PCR, the products were resolved using agarose (1%) gel electrophoresis and stained with ethidium bromide (1 µg/µl). Images of the stained gels were captured using a digital camera, and relative densitometric intensity (pixels/mm2), normalized relative to EF-1{alpha} (internal control), was obtained using MacBAS v2.5 software (Fuji, Tokyo, Japan). Values shown are for RT-PCR analyses of two entirely independent experiments and were within the linear amplification range.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Involvement of DNA Replication in PvALF- and ABA-mediated Activation of the phas Promoter—Various inhibitors were used to investigate the involvement of DNA replication in PvALF- and ABA-mediated activation of the phas promoter. Mimosine inhibits nascent chain elongation by affecting deoxynucleotide metabolism (26); HU inhibits ribonucleotide diphosphate reductase, thereby depleting cellular dNTP concentration and causing DNA replication arrest (27); roscovitine, a cyclin-dependent kinase inhibitor, blocks the cell cycle at the G1 to S phase transition (28).

DEX was used to induce ectopic expression of PvALF in leaves of the doubly transformed tobacco line GR30 (Fig. 1A). Subsequently, ABA was added to activate phas-driven GUS expression (Fig. 1B, compare GUS activities for DEX alone and DEX+ABA). As shown in Fig. 1B, compared with the DEX+ABA treatment without inhibitor control, 24 h of treatment with mimosine caused 64% (at 1 mM) to 76% (at 2 mM) reduction in GUS activity. Similarly, application of the inhibitor HU gave a 32% (at 100 mM) to 67% (at 250 mM) decrease, and roscovitine, a 79% (at 100 µM) to 94% (at 200 µM) reduction in GUS activity, confirming a role for DNA replication in activation of the phas promoter. Additional support for this conclusion was provided by the increase in GUS activity observed after transferring the leaves to fresh medium without inhibitors and incubation for an additional 24 h (Fig. 1C). Removal of roscovitine resulted in a 7–14-fold increase in GUS activity, whereas the removal of mimosine and HU led to only a 2-fold increase (Fig. 1C). Because the 2-fold increase can be attributed to a doubling of incubation time, only the data for roscovitine represent definitive reversal of inhibition of DNA replication in accordance with previous findings for these inhibitors (29).



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  		FIG. 1.
DNA replication is involved in PvALF- and ABA-mediated activation of the phas promoter. A, experimental design. Leaves from tobacco line GR30 were treated with replication inhibitor (HU, mimosine, or roscovitine). DEX and ABA were added to induce PvALF synthesis and activation of phas/uidA. B, GUS activity (nmol 4-methylumbelliferone/µg protein/h) in the leaves of line GR30 incubated with or without replication inhibitors for 24 h. C, leaves of line GR30 incubated with or without replication inhibitors for 24 h and subsequently transferred to fresh MS medium without inhibitors (R, reversed); GUS activity was determined 24 h later. In B and C, values are shown for three independent experiments; error bars represent standard deviation. D, RNase protection assay of PvAlf, uidA, and 18 S rRNA transcripts in total RNA isolated from GR30 leaves treated with or without replication inhibitors. Total RNA from leaves of line 58.1A (harboring –1470phas/uidA) was used as a negative control for PvAlf and uidA transcripts. Total RNA from leaves of the line PvAlf-14 (58.1A retransformed to contain CaMV35S/PvAlf) was used as a positive control for PvAlf transcripts.

 
The premise that the decrease in GUS activity was a direct outcome of DNA replication arrest rather than a decrease in PvALF production was evaluated by RNase protection assays. In Fig. 1D, data representative of two independent RNase protection assay experiments reveal similar levels of PvAlf transcripts in leaves induced with DEX+ABA in the presence or absence of inhibitors. Furthermore, the decrease in abundance for uidA transcripts as a result of inhibition of replication was assonant to the changes in GUS activities shown in Fig. 1B.

DNA Replication Arrest Affects the ABA-mediated Activation Step—To address if replication arrest specifically affects the ABA-mediated activation step, the regimen shown in Fig. 2A was employed. The rationale for this is that if DNA replication is only required for PvALF to gain access to the promoter (to achieve potentiation), then inhibition of replication following the induction of PvALF should not affect the potentiation step and consequently will not reduce phas-driven GUS expression. Alternatively, observation of a reduction in GUS expression would indicate that inhibition of DNA replication interferes with the ABA-mediated activation step. In fact, the latter situation is indicated by the decrease in GUS activity seen in Fig. 2B for leaves of the tobacco line GR30 induced with DEX, then treated with the inhibitors HU or mimosine, and subsequently treated with ABA.



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  		FIG. 2.
DNA replication plays a role in ABA-mediated activation of the phas promoter. A, experimental design. Leaves from the tobacco line GR30 were treated with DEX, and those from the Arabidopsis line Alf5'-2 were treated with 17{beta}-estradiol to induce PvALF production. The leaves were then treated with replication inhibitors HU (100 mM; 250 mM) or mimosine (1 mM; 2 mM) and ABA. GUS activity (nmol 4-MU/µg protein/h) in GR30 leaves (B) and GUS activity (nmol 4-methylumbelliferone/µg protein/h) in Alf5'-2 leaves (C) incubated with or without replication inhibitors.

 
Diverse components of the ABA signal transduction pathway, such as enzymes and transcription factors, have been discovered in Arabidopsis, but the occurrence of homologous constituents in tobacco is unknown (9). To gain further insight into the concerted mechanism involving DNA replication and components of the ABA pathway, we established an estradiol-inducible system (19) to obtain controlled ectopic expression of PvALF in leaves of Arabidopsis transgenic for –1470phas/uidA. As shown in Fig. 2C, the reduction in GUS activity in inhibitor-treated leaves of the Arabidopsis line recapitulated the results obtained from the tobacco system. RT-PCR analyses were conducted to evaluate changes in the abundance of PvAlf and uidA transcripts. The addition of estradiol and ABA led to high levels of uidA transcripts (Fig. 3A). Application of replication inhibitors resulted in a decrease of uidA transcripts commensurate with the decrease in GUS activity detected using fluorimetric MUG assays (Fig. 2C). Compared with estradiol+ABA-treated leaves, quantitative measurement of the RT-PCR products for uidA (Fig. 3B) revealed a reduction of ~30% for HU-treated (100 mM) and mimosine-treated (1 mM) leaves; a 60% decrease was obtained with 250 mM HU. The level of PvAlf transcripts remained unchanged in both inhibitor-treated and untreated samples (Fig. 3C) showing that the decrease in accumulation of uidA transcripts was not due to a reduction in PvALF production.



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  		FIG. 3.
A, RT-PCR analysis of transcripts for uidA, PvAlf, and histone H4 using total RNA isolated from Alf5'-2 leaves treated or untreated with inhibitors. Panels B–D show transcript levels for the indicated genes normalized relative to that of the internal control, EF-1{alpha}. Error bars denote standard deviation.

 
Evidence for Replication and Its Inhibition in Leaves—Histone H4 synthesis occurs during S phase, and H4 transcripts serve as molecular markers for this stage of the cell cycle (29, 30). High levels of H4 transcripts were detected in leaves treated with estradiol+ABA (Fig. 3, A and D), which are indicative of replication during the induction and synthesis of PvALF. As shown in Fig. 3D, a decrease in accumulation of H4 transcripts upon addition of replication inhibitors was evidenced. The presence of active DNA replication in the tissue used in these experiments was confirmed by a second, direct approach (Fig. 4) in which high levels of [3H]thymidine incorporation into the leaf genomic DNA were obtained under the conditions used for phas promoter potentiation (Fig. 2A); application of HU (100 or 250 mM) virtually eliminated 3H incorporation. Analysis of PvALF+ABA-induced GUS production driven by the phas promoter in the extracts used to determine replication (data not shown) confirmed the production of GUS in the absence of HU and also the reduction of GUS expression in its presence, as found in the experiments depicted in Fig. 2. No decrease in 3H incorporation was observed in the presence of ABA alone or estradiol alone, indicating that these compounds do not inhibit active DNA replication under the experimental conditions employed (data not shown).



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  		FIG. 4.
Hydroxyurea inhibits DNA replication in induced leaves. Expression of PvALF was induced in Alf5'-2 leaves by estradiol for 6 h prior to addition of HU. After 2 h of incubation in the presence of the replication inhibitor, ABA was added to activate transcription from the phas promoter. Over the next 10 h, incorporation of [3H]thymidine was strongly inhibited in the presence of HU.

 
DNA Replication Arrest Affects Activation of MAT and LEA Genes—To determine whether the requirement for DNA replication in PvALF- and ABA-mediated activation is specific to the phas promoter, transcript levels for several Arabidopsis seed-specific genes were analyzed by RT-PCR analyses of total RNA isolated from inhibitor-treated and untreated leaves. The presence of exogenously supplied ABA, in concert with ectopic expression of ABI3 (the Arabidopsis ortholog of bean PvALF), in Arabidopsis leaves stimulates activation of a wide variety of MAT genes (cruciferin C (CRC) and napin (At2S3)) and LEA genes (AtEM1 and M17) (10). Because PvALF and ABI3 are orthologs, it can be predicted that PvALF will activate Arabidopsis MAT and LEA genes in the presence of exogenous ABA. Indeed, although a basal level of At2S3 transcripts was present in both estradiol-treated (+PvALF) and untreated (–PvALF) leaves, induction of PvALF by estradiol in the absence of ABA did not result in the accumulation of transcripts for CRC or AtEM1 (Fig. 5A). As anticipated, sequential addition of estradiol and ABA led to high levels of transcript accumulation for CRC, At2S3, AtEM1, and M17. The addition of DNA replication inhibitors caused a severe decrease in transcript accumulation for CRC (Fig. 5B), At2S3 (Fig. 5C), and M17 (Fig. 5D). Similar to what was observed for phas-driven GUS expression (Fig. 3), a drastic reduction is seen for MAT transcripts. A small decrease was seen for AtEM1 transcripts in inhibitor-treated samples (Fig. 5E). The results provide the first clear demonstration that DNA replication is involved in PvALF- and ABA-mediated regulation of MAT and LEA genes. However, inhibition of replication effectively blocks cell cycle progression from the G1/S phase. Indeed, studies on the establishment of heterochromatin-induced repression of mating loci in yeast have revealed that silencing is DNA replication-independent but is dependent on cell cycle progression (31). Further, studies on the access of yeast heat shock factor to nucleosomal DNA have established that it is not DNA replication per se but the S phase of the cell cycle that is required for activator-nucleosome interaction (32). Thus, the regulation of MAT and LEA genes by PvALF and ABA may be dependent on S phase progression rather than DNA replication.



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  		FIG. 5.
Inhibition of replication causes a severe reduction in CRC, At2S3, and M17 transcripts. A, RT-PCR analyses of transcripts for CRC, At2S3, AtEM1, and M17 using total RNA isolated from Alf5'-2 leaves treated or untreated with inhibitors. Panels B–E show transcript levels for the indicated genes normalized relative to that of the internal control, EF-1{alpha}. Error bars denote standard deviation.

 
DNA Replication Arrest Does Not Augment Negative Regulators of the ABA Signal Cascade—Genes having products shown to possess negative regulatory roles in the ABA signal transduction pathway include protein phosphatases ABI1 and ABI2 (33) and ERA1, a protein farnesyltransferase (34). RT-PCR analyses of total RNA isolated from replication inhibitor-treated and untreated leaves revealed unchanged transcript levels for abi1, abi2, and era1 (Fig. 6), making it unlikely that augmentation of these negative regulators is responsible for the decrease in MAT gene expression.



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  		FIG. 6.
Maturation-specific gene transcript decrease does not result from an increase in negative regulators of the ABA signal transduction pathway. A, RT-PCR analyses of transcripts for abi1, abi2, and era1 using total RNA isolated from Alf5'-2 leaves treated or untreated with inhibitors. Panels B–D show transcript levels for the indicated genes normalized relative to that of the internal control, EF-1{alpha}. Error bars denote standard deviation.

 
Inhibition of DNA Replication Decreases abi5 Transcription—Decreased transcription of MAT and LEA genes could also result from down-regulation of positive regulators of the ABA signal cascade. ick1 encodes an ABA-induced inhibitor of cyclin-dependent kinase that has been implicated in cell cycle arrest (35), and aip3 encodes an ABI3-interacting protein with similarities to human transcription factor C1 that is presumed to play a role in G1/S phase transition (36). However, quantitative analysis of the RT-PCR profiles (Fig. 7A) showed that transcript levels for aip3 (Fig. 7B) and ick1 (Fig. 7C) remained unchanged in inhibitor-treated leaves.



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  		FIG. 7.
Maturation-specific gene transcript decrease correlates with a decrease in abi5 transcripts. A, RT-PCR analyses of transcripts for aip3, ick1, and abi5 using total RNA isolated from Alf5'-2 leaves treated or untreated with inhibitors. Panels B, C, and D show transcript levels for the indicated genes normalized relative to that of the internal control, EF-1{alpha}. Error bars denote standard deviation.

 
ABI5, a b-zip class transcription factor, is a positive regulatory ABA signal effector (37) shown in transient assays to trans-activate phas promoter-driven GUS expression (38). It interacts with abscisic acid-response elements (39) and with ABI3 (40), and its debilitation leads to a severe reduction in LEA proteins (37). As shown in Fig. 7D, a drastic decrease (~50%) in the abundance of abi5 transcripts was observed in total RNA isolated from leaves treated with 250 mM HU. A decrease (~30%) in abi5 transcripts was also evident in total RNA isolated from 100 mM HU-treated and 1 mM mimosine-treated leaves (Fig. 7D). The reduction in abi5 transcripts indicates a role for DNA replication in its regulation and also suggests that the effect of DNA replication on MAT and LEA gene regulation may be achieved indirectly via ABI5.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
PvALF- and ABA-mediated Activation of the phas Promoter Is Cell Cycle-dependent—The observed decrease in phas promoter-driven GUS expression in both tobacco (Figs. 1 and 2B) and Arabidopsis (Fig. 2C) leaves, resulting from inhibition of replication, implicates DNA replication or cell cycle progression in PvALF- and ABA-mediated activation of transcription from the phas promoter. A conclusion to be drawn from the substantial decrease in phas-driven GUS expression in leaves induced to produce PvALF (Fig. 2) and shown to contain active DNA replication (Figs. 3D and 4) prior to replication arrest by inhibitors is that the ABA-mediated activation step is affected by inhibition of replication independent of PvALF-mediated potentiation. Down-regulation of MAT and LEA genes also ensued from the arrest of the cell cycle (Fig. 5) possibly as a consequence of a decrease in ABI5 levels (Fig. 7D).

An alternative possibility for PvALF-mediated activation of the phas promoter is that it is replication-independent. Because our data show that replication, or at least cell cycle progression, is vital for activation, this model cannot be entirely correct. However, the present experiments do not directly address the effect of replication on the potentiation step, and it remains formally possible that this step is affected by replication. Nevertheless, the possibility that PvALF preferentially binds to its target site present as nucleosomal DNA remains attractive. This supposition is strengthened by observations that intact VP1 (41) and PvALF do not bind naked DNA in vitro and that histones can enhance DNA binding by the isolated B3 domain of VP1 and PvALF.2 It may be speculated that, once bound, PvALF can reconfigure the repressive chromatin architecture over the phas promoter by recruiting SWI/SNF or other ATP-dependent chromatin remodeling machinery (42). Alternatively, PvALF may function in an ATP-independent manner similar to that observed for HNF3 (FoxA) and GATA-4 (43).

A Role for Progression through S Phase in ABA-mediated Activation—The dramatic increase in seed ABA content during the maturation phase has been proposed to play a role in cell cycle arrest at the G1/S phase transition (44). ABA has been reported to counter the effect of cytokinins in the shoot meristem by inactivating replication origins, leading to a reduction in DNA synthesis (45). Similarly, in tobacco BY-2 cells, application of ABA prior to G1/S phase transition was found to prevent DNA replication (46). These results are consistent with the concept that the presence of ABA arrests DNA replication. In contrast, in our direct analysis of DNA replication by [3H]thymidine incorporation, there was no decrease in replication upon addition of ABA, whereas replication was dramatically arrested by addition of the DNA synthesis inhibitor HU (Fig. 4). This experiment unequivocally shows that the added ABA was functional because it activated the PvALF-potentiated phas promoter, resulting in GUS synthesis, but it did not cause arrest of DNA replication. Active DNA replication has also been documented during Arabidopsis embryogenesis from the heart to mature embryo stages (16). Because the initiation of transcription from the phas promoter at this stage (47) requires the presence of ABA, it is evident that the production of ABA and continued S phase progression are concurrent. However, a role for ABA in the arrest of cell division (often misinterpreted as an inhibition of DNA synthesis) is likely in late maturation seeds at the S/G2 or G2/M phase boundary.

Ectopic expression of ABI3 (10) or VP1 (48) in the presence of exogenous ABA supports the expression of MAT and LEA genes in vegetative tissues. Similarly, ectopic expression of PvALF and the application of ABA induce robust expression of Arabidopsis MAT (CRC and At2S3) and LEA (AtEM1 and M17) genes in leaves (Fig. 5). This confirms the orthology of PvALF with ABI3 and VP1. Inhibition of DNA replication, and hence arrest of the cell cycle at the S phase, resulted in a decrease in transcription from MAT and LEA genes (Figs. 3 and 5) demonstrating the importance of progression to S phase in PvALF- and ABA-mediated activation.

Requirement of S Phase Progression for ABA-mediated Regulation of ABI5—The observed decrease in ABA-induced MAT and LEA gene expression upon S phase arrest could result from an increase in negative regulators of the ABA signal cascade. However, no increase in transcripts for negative regulators, such as protein phosphatases ABI1 and ABI2 or the farnesyltransferase ERA1, was found (Fig. 6). An alternative possibility is that component(s) involved in both the ABA-signaling pathway and cell cycle phase transitions may be affected on S phase arrest. However, no change in transcript accumulation was evident for ick1, a cyclin-dependent kinase inhibitor that was previously reported to be induced by ABA (35) (Fig. 7C). A yeast two-hybrid screen for interacting partners of VP1 and ABI3 identified AIP3, a protein with sequence similarities to human transcription factor C1 (36, 49) that is thought to have a role in cell cycle control (50). The lack of any substantial decrease for aip3 transcripts in inhibitor-treated samples (Fig. 7B) strongly detracts from it being a major target of DNA replication arrest.

The b-zip class transcription factor ABI5 is an important component of the ABA signal cascade (9, 37). Characterization of an abi5 mutant showed that three LEA genes (AtEm1, AtEm6, and the LeaD34 homolog) were down-regulated, whereas one (M17) was up-regulated. This suggests that ABI5 plays a positive or a negative regulatory role depending on promoter context (37); in our experiments, decreased expression was found for M17 and, to a lesser degree, for AtEM1 (Fig. 4). Recently, it has been shown that ABI5 acts downstream of ABI3 to execute an ABA-dependent post-germination growth arrest (51). It has also been shown by yeast two-hybrid screening that ABI5 interacts with ABI3 (40). Moreover, in transient assays, ABI5 has been demonstrated to trans-activate AtEM6 and phas promoters (38). The decrease in MAT and LEA gene expression (Fig. 5) may be ascribed to down-regulation of abi5 by S phase inhibition. Indeed, as shown in Fig. 7D, a severe decrease is seen for abi5 transcripts in inhibitor-treated samples. This novel finding reveals that S phase progression plays a role in abi5 regulation. Consequently, S phase disruption leads to a decrease in ABI5, thereby causing attenuation of PvALF- (or ABI3) and ABA-mediated activation of MAT and LEA genes.

Studies on post-germination arrest by ABA have shown that its presence stabilizes ABI5 (51) and that ABI5 positively autoregulates abi5 (52). However, this autoregulation is completely lost by the third day of post-germination (51). Two possibilities have been suggested for this loss of ABI5 expression: first, ABA signaling is necessary but not sufficient for abi5 activation; and second, a regulatory mechanism exists in which ABA signaling alters the stability of ABI5 (53). Our data show that S phase progression is required in addition to ABA for abi5 activation and impute S phase progression as the regulatory mechanism that causes altered stability of ABI5. Further, the results reported here suggest that, rather than inhibiting DNA replication (17), ABA acts in concert with S phase progression to activate gene expression in early to mid stages of embryogenesis. In late stages of embryogenesis, termination of S phase may affect ABA-mediated seed programming, perhaps primarily by down-regulating ABI5 levels.


   FOOTNOTES
 
* This work is supported by National Science Foundation Grant MCB-9974706. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains a supplemental table. Back

{ddagger} Present address: Dupont Agriculture and Nutrition, 7300 N. W. 62nd Ave., P. O. Box 1004, Johnston, IA 50131. Back

§ To whom correspondence should be addressed. Tel.: 979-845-7750; Fax: 979-862-4098; E-mail: tim{at}idmb.tamu.edu.

1 The abbreviations used are: ABA, abscisic acid; DEX, dexamethasone; HU, hydroxyurea; LEA, late embryogenesis-abundant genes; MAT, maturation-specific genes; PvALF, Phaseolus vulgaris ABI3-like factor; RT, reverse transcriptase; MS, Murashige and Skoog. Back

2 R. Carranco, M. B. Chandrasekharan, and T. C. Hall, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Nam Hai Chua for providing the vectors pTA7002 and pER8, Elliot Meyerowitz for discussions on cruciferin C gene identity in the TAIR data base, and Robert Goldberg, David Baulcombe, Ann Kirchmaier, Raul Carranco, and Wang Kit Ng for valuable suggestions. James Townsend and Erin Hanover provided excellent technical assistance.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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