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Keywords:

  • LOS2;
  • AtMBP-1;
  • Alternative translation;
  • abscisic acid signaling;
  • post-translational regulation;
  • ubiquitin ligase

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

The LOS2 gene in Arabidopsis encodes an enolase with 72% amino acid sequence identity with human ENO1. In mammalian cells, the α-enolase (ENO1) gene encodes both a 48 kDa glycolytic enzyme and a 37 kDa transcriptional suppressor protein that are targeted to different cellular compartments. The tumor suppressor c-myc binding protein (MBP-1), which is alternatively translated from the second start codon of ENO1 transcripts, is preferentially localized in nuclei while α-enolase is found in the cytoplasm. We report here that an Arabidopsis MBP-1-like protein (AtMBP-1) is alternatively translated from full-length LOS2 transcripts using a second start codon. Like mammalian MBP-1, this truncated form of LOS2 has little, if any, enolase activity, indicating that an intact N-terminal region of LOS2 is critical for catalysis. AtMBP-1 has a short half-life in vivo and is stabilized by the proteasome inhibitor MG132, indicating that it is degraded via the ubiquitin-dependent proteasome pathway. Arabidopsis plants that over-express AtMBP-1 are hypersensitive to abscisic acid (ABA) during seed germination and show defects in vegetative growth and lateral stem development. AtMBP-1 interacts directly with the E3 ubiquitin ligase AtSAP5 and co-expression of these proteins resulted in destabilization of AtMBP-1 in vivo and abolished the developmental defects associated with AtMBP-1 over-expression. Thus, AtMBP-1 is as a bona fide alternative translation product of LOS2. Accumulation of this factor is limited by ubiquitin-dependent destabilization, apparently mediated by AtSAP5.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

There are many reported examples of truncated protein that have altered cellular functions (Cheng and Cohen, 2007; Kodama and Sano, 2007) and 42% of the proteins in Arabidopsis that lack N-terminal domains due to alternative translational initiation are located in subcellular compartments that differ from those of the full-length isoforms (Chabregas et al., 2003; Kochetov, 2006, 2008). For example, Arabidopsis DNA ligase 1 (AtLIG1) isoforms are translated from a single mRNA through either first or second in-frame AUG codons, and targeted either to mitochondria or nuclei (Sunderland et al., 2004). In addition, plastidic and cytoplasmic/nuclear isoforms of tRNA ligase of Arabidopsis thaliana and Oryza sativa are synthesized by alternative translation initiation through a leaky-scanning mechanism (Christensen et al., 2005). In mammalian systems, the α-enolase (ENO1) gene is a well-studied example of alternative translation initiation. This gene encodes a 48 kDa glycolytic enzyme that catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate, along with a truncated 37 kDa tumor suppressor, known as the c-myc binding protein (MBP-1), which acts as a transcriptional suppressor. These gene products are targeted to different cellular compartments (Ghosh et al., 1999) with the α-enolase found in the cytoplasm and MBP-1 preferentially localized in nuclei (Feo et al., 2000). The ENO1 gene has been intensively studied since the c-myc oncogene, which is often highly expressed in malignant cell types, promotes cell proliferation and directly upregulates the expression of α-enolase, as well as other glycolytic enzymes (Osthus et al., 2000). Expression of MBP-1 in mammalian cells suppresses c-myc expression and results in reduced cell proliferation and apoptosis (Ray, 1995; Ray et al., 1995). MBP-1 binds to the c-myc promoter and negatively regulates its expression as part of a feedback control loop that regulates c-myc expression and glycolytic metabolism. Differential translation of enolase and MBP-1 has been observed under stressful conditions such as limited glucose or low oxygen. Under these conditions, MBP-1 translation is reduced and MBP-1 mediated regulation of c-myc expression is disrupted (Sedoris et al., 2010). MBP-1 is also post-translationally regulated in a proteasome-dependent manner, although the mechanisms that regulate MBP-1 protein turnover remain unclear (Lung et al., 2010).

Previous genetic analysis in Arabidopsis mapped los2, a mutation that affects cold-induced accumulation of stress-responsive transcripts, to an apparent ortholog of ENO1 (Lee et al., 2002). LOS2 encodes an enolase with 72% amino acid sequence identity to human ENO1 and the LOS2 transcript also includes a potential secondary start codon that could be used to translate an MBP-1-like polypeptide. LOS2 was found to bind a c-myc-like element in the promoter of the STZ/ZAT10 gene of Arabidopsis (Lee et al., 2002). Thus, these authors proposed that the effects of LOS2 on stress-responsive gene expression could be mediated through the modulation of STZ/ZAT10 by the AtMBP-1 alternative translation product of LOS2.

In this study, we determined that AtMBP-1 is a specific interacting partner of the E3 ubiquitin ligase AtSAP5 (Kang et al., 2011) by yeast 2-hybrid screening. Our results show that AtMBP-1 is alternatively translated from LOS2 transcripts in plant cells. Over-expression of AtMBP-1 in transgenic Arabidopsis plants leads to increased sensitivity to ABA and stunted vegetative growth. Co-expression of AtSAP5 destabilizes AtMBP-1 in vivo and reverses the growth defects associated with AtMBP-1 expression. Thus, AtMBP-1 acts as a positive regulator of the ABA signaling pathway and its activity appears to be attenuated by ubiquitin-dependent destabilization, mediated by AtSAP5.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

AtMBP-1 interacts with the E3 ubiquitin ligase AtSAP5 in vivo and in vitro

Previously, we identified AtSAP5 as a positive regulator of abiotic stress responses and showed that it acts, in vitro, as an E3 ubiquitin ligase (Kang et al., 2011). In order to identify potential interacting partners of AtSAP5, yeast two-hybrid screening was conducted using AtSAP5 as bait and an Arabidopsis cDNA library as prey. From this screening, 36 cDNA clones were obtained and sequence analysis indicated that six of these cDNAs represented transcripts derived from the LOS2 gene. LOS2 was reported to encode a dual function protein with both α-enolase and DNA binding activities (Lee et al., 2002). This finding raised the possibility that, as in mammalian cells, an Arabidopsis MBP-1-like protein (AtMBP-1) could be produced as an alternative translation product of the LOS2 gene. To determine which of these putative gene products interacts with AtSAP5, specific yeast two-hybrid assays were performed. For this assay, LOS2 and AtMBP-1 coding sequences were cloned into both bait (pGBKT7) and prey (pGADT7) yeast vectors, and each construct was co-transformed into yeast cells with a complementary AtSAP5 construct Figure 1(a). Yeast cells transformed with vectors that co-express AtMBP-1 and AtSAP5 proteins grew well under high stringency conditions, while yeast cells transformed with LOS2/enolase and AtSAP5 did not, indicating strong interaction between AtSAP5 and AtMBP-1 but not between AtSAP5 and LOS2/enolase Figure 1(b). The interaction between AtMBP-1 and AtSAP5 identified through yeast two-hybrid assays was verified with in vitro protein pull-down assays Figure 1(c). His-Trx-AtMBP-1, His-LOS2, and GST-AtSAP5 constructs were transformed into Escherichia. coli and the expressed proteins purified using either His-or GST resin, respectively. His-Trx-AtMBP-1 or His-LOS2 fusion proteins were incubated with the GST-AtSAP5 fusion protein and, after extensive washing, the bound proteins were eluted from the GST resin by reduced glutathione. The eluted proteins were analyzed on immuno-blots probed with anti-His and anti-GST antibodies. Consistent with the results of yeast two- hybrid assay, His-Trx-AtMBP-1 was pulled down by GST-AtSAP5, while His-LOS2 did not bind effectively to GST-AtSAP5 Figure 1(c). To confirm this interaction in vivo, either AtMBP-1–YFP and Flag-AtSAP5, or LOS2–YFP and Flag-AtSAP5 were expressed in Nicotiana benthamiana by agro-infiltration. Protein extracts isolated from the N. benthamiana were subjected to co-immunoprecipitation (Co-IP) assays. Either AtMBP-1–YFP or LOS2–YFP fusion proteins were immunoprecipitated from the plant extracts using the anti-GFP agarose. Flag-AtSAP5 was detected in the AtMBP-1–YFP:Flag-AtSAP5 immunocomplex using the anti-Flag antibody Figure 1(d). By contrast, no signal was detected in reactions using LOS2–YFP and Flag-AtSAP5. To confirm this interaction in plant cells, biomolecular fluorescence complementation (BiFC) analysis was performed by co-infiltrating constructs that encode nYFP-AtSAP5 and AtMBP-1-cYFP into N. benthamiana. Fluorescence signal, indicating interaction between nYFP-AtSAP5 and AtMBP-1-cYFP, was detected in cells of the agro-infiltrated tissue within a nuclear subcompartment, possibly corresponding with the nucleolus Figure 1(e). Together these results indicated that AtMBP-1 and AtSAP5 interact in vivo and co-localize in plant cells.

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Figure 1. The LOS2 truncated form, AtMBP-1 interacts with AtSAP5.

(a) Diagram of LOS2 protein and the truncated LOS2 form (AtMBP-1) used for protein interaction tests with AtSAP5.

(b) AtSAP5 interacts with AtMBP-1, but not with LOS2 in a yeast two-hybrid assay. pGBKT7 vector was used for bait and pGADT7 was used for prey. Each cDNA was cloned to pGBKT7 and pGADT7 vectors. Yeast AH109 cells were co-transformed with a combination of the indicated plasmids. Yeast cells were plated on selective media SD/-L-T or SD/-L-T-H-A+ X-gal and incubated for 5 days. +/+ (pGADT7-Rec/ pGBKT7-53): positive control, −/− (pGADT7-Rec/ pGBKT7-Lam): negative control.

(c) GST pull-down assay of LOS2 or AtMBP-1 with AtSAP5. GST-AtSAP5 recombinant proteins were used to pull-down either His-LOS2 or His-Trx-AtMBP-1 using GST-agarose resin. The bound protein eluted and proteins were detected with anti-His or GST antibodies. NC: negative controls.

(d) Co-immunoprecipitation assay of Flag-AtSAP5:AtMBP-1–YFP or Flag-AtSAP5:LOS2–YFP in vivo. Flag-AtSAP5:AtMBP-1–YFP or Flag-AtSAP5:LOS2–YFP constructs were expressed in Nicotiana benthamiana using agro-infiltration. Cell lysates were immunoprecipitated with anti-GFP agarose. The isolated protein was separated by 10% SDS-PAGE and immunoblotted with anti-GFP or anti-Flag antibodies. * Indicates IgG bands.

(e) Verification of in vivo AtSAP5 and AtMBP-1 interaction in N. benthamiana using Agrobacterium-mediated transient expression system. AtSAP5 was fused to the N-terminal fragment of YFP (nYFP) and AtMBP-1 was fused to the C-terminal fragment of YFP (cYFP). Images were taken under confocal microscopy. Arrows indicate the nuclei. YFP fluorescence, Bright-field, and merged images were captured by fluorescence confocal microscopy.

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AtMBP-1 is translated from LOS2 mRNA

Arabidopsis LOS2 encodes an α-enolase that has 72% amino acid sequence identify with human α-enolase (ENO1) (Lee et al., 2002). The human ENO1 transcript also encodes c-myc binding protein 1 (MBP-1) by translational initiation at an internal start codon located downstream of α-enolase initiation site (Subramanian and Miller, 2000). The LOS2 transcript includes a second AUG translation initiation codon at amino acid position +93 relative to the first in-frame AUG. To address whether LOS2 mRNA can be translated in vivo to produce these two protein isoforms, the functionality of these two in-frame start codons was investigated. Three gene constructs were generated with coding sequences consisting of: (i) full-length LOS2 (ii) a 5′ truncated sequence in which the first AUG codon was deleted, leaving the start codon at position 93 of LOS2 as the first start site; and (iii) a mutated coding sequence in which the sequence at position 93 of LOS2 was changed from a Met codon to a Leu codon, by site-directed mutagenesis Figure 2(a). Vectors that contain these coding sequences, fused at their carboxy-termini to coding sequences for yellow fluorescent protein (YFP) and under transcriptional control of the CaMV 35S promoter, were transformed into Arabidopsis plants. Crude protein samples extracted from transgenic plants that express these gene constructs were analyzed by immunoblot assays probed with anti-GFP. As shown in Figure 2(b), two protein bands, with apparent molecular weights of approximately 78 kDa and 67 kDa, were detected in extracts from plants that express the 35S::LOS2-YFP construct. These molecular masses correspond to the predicted sizes of LOS2–YFP and AtMBP-1–YFP fusion proteins, respectively. As expected, analysis of YFP-tagged proteins in plants that expressed the truncated 35S::AtMBP-1-YFP construct resulted in detection of a single protein band that migrated similarly to the 67 kDa band in extracts from plants that express the 35S::LOS2-YFP construct, while expression of the 35S::mLOS2-YFP construct produced a single band that corresponded to the 78 kDa band from LOS2–YFP expressing plants. This shows that expression of LOS2 mRNA can be differentially translated to produce two protein isoforms in vivo that correspond with full-length LOS2 and truncated AtMBP-1. Expression of AtMBP-1 from the truncated 35S::AtMBP-1 transgene was very weak compared with that from the full-length 35S::LOS2 transgene also see Figure 5(a). This could be due, at least in part, to inefficient translational initiation at the start codon of the truncated transcript compared with initiation at the second AUG of the full-length transcript by a leaky-scanning mechanism.

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Figure 2. Expression of AtMBP-1 and phenotypic analysis of AtMBP-1 over-expressing plants.

(a) Schematic representation of full-length LOS2, N-terminal deletion mutation (AtMBP-1), and site-directed mutation introduced into the LOS2 at position 93 (mLOS2). Black box indicates 35S-CaMV promoter and the yellow box

(b) Protein extracts from eight-day-old transgenic plants that contained each construct were analyzed by immunoblotting with anti-GFP antibody or anti-Flag antibody. Anti-actin antibody was used for loading control.

(c) Phenotype of 10-day-old WT and 35S::AtMBP-1-YFP transgenic seedlings grown on MS medium (upper panel), and seedlings photographed after 3 weeks growth on soil (bottom panel).

(d) Whole plants from 2-month-old matured WT and 35S::AtMBP-1-YFP transgenic plants grown in a growth chamber under 16 h day/8 h night conditions.

(e) Images of the primary inflorescences from plants as in Figure 3(b). Insets show enlarged images of siliques.

(f) Flowering of 4-week-old WT (top in the box) and 35S::AtMBP-1-YFP transgenic plants. Enlarged images of flowers are shown in insets.

(g) Images of seeds after harvest from WT and 35S::AtMBP-1-YFP transgenic plants.

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Analysis of fluorescence using confocal microscopy indicated that recombinant YFP-tagged protein in 35S::LOS2 expressing plants was localized in both cytosolic and nuclear cellular compartments, while the N-terminal truncated AtMBP-1–YFP fusion protein expressed in 35S::AtMBP-1 expressing plants was predominantly localized to nuclei (Figure S1). When the truncated AtMBP-1 isoform was expressed in E. coli, it showed significantly reduced enolase activity compared with full-length LOS2 (Figure S2). This result is consistent with previous reports, which showed that mammalian MBP-1 lacks enolase activity (Bentley and Groudine, 1986) and that an intact N-terminal region is critical for activity in plant enolases (Van der Straeten et al., 1991).

AtMBP-1 over-expression affects plant morphology

Transgenic plants that express the 35S::AtMBP-1 construct exhibited a distinct morphological phenotype that includes retarded vegetative growth and development, when compared with wild-type (WT) Arabidopsis plants. Leaf size, observed at seedling and young plantlet stages, was only slightly reduced in 35S::AtMBP-1 transgenic plants grown on either Murashige and Skoog (MS) medium or soil Figure 2(c); however, as plants grew, significant morphological changes in plant architecture became apparent Figure 2(d, e). As shown in Figure 2(d), plants that constitutively express AtMBP–1 were shorter than WT plants. These plants produced an increased number of flowers and had shorter siliques Figure 2(e). Flowering time was somewhat delayed in AtMBP-1 over-expressing plants Figure 2(f) and slightly increased seed size was observed, compared with WT plants Figure 2(g). Since LOS2 mRNA can also produce AtMBP-1, transgenic plants that express the 35S::LOS2 gene construct showed a phenotype similar to AtMBP-1 over-expressing plants (Figure S3), while transgenic Arabidopsis plants that expressed 35S::mLOS2 grew normally.

AtMBP-1 over-expressing plants are hypersensitive to ABA and abiotic stress

Abscisic acid plays important roles in development and responses to a range of environmental stresses in plants. It has been shown that key regulators involved in ABA-dependent signaling pathway are associated with seed germination and vegetative growth. Arabidopsis plants that ectopically express these factors showed reduced growth and altered responses to ABA (Kang et al., 2002; Sakamoto et al., 2004). To investigate whether AtMBP-1 over-expression can affect ABA sensitivity, seeds from 35S::AtMBP-1 expressing and WT plants were sown on MS plate containing various concentrations of ABA and the rate of germination was determined after stratification and subsequent incubation for 4 days. Germination of seeds from 35S::AtMBP-1 plants was more strongly inhibited by ABA than were seeds from WT plants at all concentrations tested Figure 3(a). At 0.5 μm ABA only 20% of AtMBP-1 over-expressing seeds were able to germinate, while 70% of WT seeds germinated under the same conditions. Growth of AtMBP-1 over-expressing seedlings was almost completely arrested even at 0.25 μm ABA Figure 3(b). Greening and expansion of cotyledon were severely inhibited under these conditions and many 35S::AtMBP-1 seedlings did not produce true leaves. However, there was no obvious difference in root growth between these plants and WT plants in response to ABA (data not shown). These results indicate that ectopic expression of AtMBP-1 leads to ABA hypersensitivity during seed germination and subsequent post-germinative seedling growth. In addition, we tested the ability of AtMBP-1 over-expressing seeds to germinate under salt or osmotic stress. Seeds were sown on medium that contained 150 mm NaCl, 6%, mannitol, or 20% polyethylene glycol (PEG) Figure 3(c–e) and time to germination, in days, was monitored. As shown in Figure 3, stress caused by NaCl, mannitol, or PEG treatment inhibited germination of seeds from AtMBP-1 over-expressing plants to a greater degree than WT seeds, while germination of seed from mLOS2 over-expressing plants on medium that contained 0.5 μm ABA, 150 mm NaCl, or 6% mannitol was similar to WT seeds (Figure S4). These results indicate that, like ABA, AtMBP–1 negatively affects seed germination in response to osmotic stress.

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Figure 3. Transgenic plants that over-express 35S::AtMBP-1 are hypersensitive to ABA.

(a) The percentage of seeds showing root emergence was scored at 4 days after stratification under different ABA concentrations. Bars indicate standard deviation (SD) from three technical replications, n = 30.

(b) Images of WT (Col-0) and 35S::AtMBP-1-YFP transgenic plantlets grown for 6 days on MS medium with or without 0.25 μm ABA.

(c–e) Osmotic stress tolerance of 35::AtMBP-1 plants responding to 150 mm NaCl (c), 6% mannitol (d), 20% polyethylene glycol (PEG) (e). Seeds were germinated and grown for 8 days on MS medium that contained each reagent and germination was counted each day. Bars indicate SD from three technical replications, n = 30.

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AtMBP-1 regulates the expression of genes involved in ABA signaling pathways

The ABA sensitive phenotype seen during germination and seedling growth in 35S::AtMBP-1 transgenic plants prompted us to investigate whether AtMBP-1 expression affects the expression of genes involved in the ABA signaling pathway. Using quantitative RT-PCR analysis, we measured the relative expression of genes involved in ABA signaling from RNA samples from seeding grown for 2 days after stratification on medium supplemented with or without ABA. The expression of ABI1 and ABI2, two members of the PP2C family that act as negative regulators of ABA responses, was not significantly affected in AtMBP-1 expressing plants compared with WT plants under ABA treatment (Figure 4). However, expression of ABI3, ABI4, and ABI5, genes that encode a B3 domain transcription factor, an AP2 transcription factor, and a bZIP transcription factor, respectively, was more responsive to ABA in AtMBP-1 over-expressing plants. In addition, expression of the downstream ABA-responsive genes Em1 and Em6, which are direct targets of ABI3 and ABI5 (Finkelstein et al., 2002), and RAB18 was upregulated more strongly in response to ABA treatments in AtMBP-1 over-expressing plants. Expression of these genes was only weakly affected without ABA treatment, a finding that could indicate that AtMBP-1 affects their expression indirectly by increasing the sensitivity of these plants to ABA. In contrast with ABI5, the response of ABF3 expression to ABA was not affected in 35S::AtMBP-1 plants. Similar expression patterns for these genes were observed using RNA samples from leaves of plants grown for 2 weeks on medium that contained ABA (Figure S5). The expression of genes that encode SNF1-related protein kinase 2 (SnRK2.2 and SnRK2.3) (Fujii et al., 2007) and ABI5 binding proteins (AFP1, 2 and 3) (Lopez-Molina et al., 2003), which act upstream of ABI3 and ABI5 in the ABA signaling pathway, was also tested (Figure S6). The ABA responsiveness of these genes was not affected by AtMBP-1 expression, implying that AtMBP-1 may act downstream of these genes or in a parallel ABA signaling pathway.

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Figure 4. Genes involved in ABA signaling pathway were more strongly up regulated by abscisic acid (ABA) treatments in AtMBP-1 expressing transgenic plants. RNA was extracted from germinated seeds at 2 days after stratification on MS medium that contained 0.25 μm ABA. Quantitative RT-PCR was carried out to monitor gene expression levels. Error bars indicate SD, n = 3.

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Transient cold-responsive expression of STZ/ZAT10 mRNA in los2 mutant plants showed similar induction kinetics to WT plants but expression remained elevated for a longer period (Lee et al., 2002). Conversely, as shown in Figure S6, the increase in STZ/ZAT10 expression at 4°C in 35S::AMBP-1 plants was delayed and partially suppressed, relative to WT plants (Figure S7), a finding that indicated that constitutive expression of AtMBP-1 down-regulates the responsiveness of STZ/ZAT10 to low temperature.

AtMBP-1 instability depends on the ubiquitin-proteasome pathway

Very little AtMBP-1 was observed to accumulate in 35S::AtMBP-1 transgenic plants, despite the fact that AtMBP-1 transcript levels were generally more abundant than those for LOS2 in 35S::LOS2 plants Figure 5(a). To investigate whether this inconsistency reflects post-translational regulation of AtMBP-1, the relative stability of these proteins in plants that constitutively express Flag-LOS2 and AtMBP-1–YFP was examined. To accomplish this effect, levels of AtMBP-1–YFP in 8-day-old transgenic seedlings that were treated with cycloheximide (CHX) in the presence or absence of the ubiquitin-associated proteasome inhibitor, MG132 were monitored at defined time points. As shown in Figure 5(b), AtMBP-1 had an apparent half-life of around 3 h in the absence of new protein synthesis and degradation of AtMBP-1 was largely prevented by MG132 treatment. AtMBP-1 could barely be detected after 6 h treatment with CHX in the absence of MG132, but it was relatively stable for up to 8 h in the presence of MG132, indicating that AtMBP-1 is degraded, in vivo, via the 26S proteasome pathway. In contrast, no significant changes were found in the levels of full-length LOS2 protein after CHX treatment even in the absence of MG132 Figure 5(b). To confirm that the degradation of AtMBP-1 is mediated by a ubiquitin-dependent mechanism, proteins were extracted from 8-day-old 35S::AtMPB-1-YFP transgenic seedlings and WT seedlings in the presence of MG132, and ubiquitinated AtMBP-1 was detected on immuno-blots using GFP- and ubiquitin-specific antibodies Figure 5(c). In these assays, GFP antibodies detected high molecular mass proteins that migrated more slowly than AtMBP-1 and cross-reacted with anti-Ub, a finding that indicated that AtMBP-1 is ubiquitinated in vivo.

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Figure 5. AtMBP-1 degradation via ubiquitin-dependent proteasome pathway.

(a) LOS2 transcript levels from 2 weeks old seedlings of 35S::LOS2-YFP and 35S::AtMBP-1-YFP transgenic lines (upper panel). Western blot analysis of LOS2 and AtMBP-1 proteins extracted from 8-day-old seedling from each transgenic lines (bottom panel).

(b) Eight-day-old 35S::AtMBP-1-YFP seedlings were incubated in MS liquid medium containing 100 μm cycloheximide (CHX), in the presence or absence of 50 μm MG132. Samples were collected at each time point, equal amounts of protein were analyzed by immunoblot using anti-GFP (top) or anti-actin (bottom). 35S::Flag-LOS2 seedlings were treated with cycloheximide (CHX, 100 μm) in MS liquid medium for the indicated time. The protein level was determined by immunoblotting with anti-Flag antibody. Coomassie blue staining showed protein amount loaded in each lane (bottom).

(c) Eight-day-old WT and 35S::AtMBP-1-YFP seedlings were treated with MG132 (50 μm) for 5 h. One milligram of total protein extracts were used to isolate YFP conjugated proteins using anti-GFP agarose beads. GFP- and ubiquitin-specific antibodies were used to detect AtMBP-1–YFP (left) and ubiquitinated AtMBP-1–YFP (right). All experiments were performed in triplicate.

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ABA promotes AtMBP-1 instability in vivo

To test whether ABA could promote AtMBP-1 degradation at the seedling stage of development, 8-day-old 35S::AtMBP-1-YFP transgenic seedlings were treated with CHX for 2 h before addition of ABA. AtMBP-1 protein levels at different time points were detected on immunoblots with GFP-specific antibodies. As shown in Figure 6(a), AtMBP-1 protein levels decreased gradually in seedlings not treated with ABA, which is consistent with results shown in Figure 5(b). However, AtMBP-1 levels declined more rapidly in response to ABA treatment. In seedlings exposed to ABA, the signal from AtMBP-1 at the 3 h time point was weaker than the 6 h time point in untreated samples. This result indicates that ABA increases the rate of AtMBP-1 degradation in vivo by at least two-fold. To determine whether ABA treatments could facilitate the rescue of the reduced vegetative growth phenotype of AtMBP-1 over-expressing plants, ABA was applied at different concentrations to soil-grown 18 day-old WT plants and AtMBP-1 over-expressing plants twice daily for 10 days Figure 6(b). As expected growth of WT plants was inhibited by ABA application; however, AtMBP-1 over-expressing plants showed a dose-dependent increase in growth so that, at higher ABA concentrations, plant size and leaf shape was similar to WT plants under the same ABA treatment conditions. Thus, the growth inhibition mediated by transgenic expression of AtMBP-1 was reversed by ABA treatment.

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Figure 6. ABA facilities AtMBP-1 degradation.

(a) Eight-day-old 35S::AtMBP-1-YFP seedlings were incubated in MS medium containing 500 μm cycloheximide (CHX) for 2 h followed by treatment with or without 50 μm ABA for the indicated time. Equal amounts of protein were loaded on a 10% SDS gel and analyzed by immunoblot using anti-GFP.

(b) Representative plants of wild-type and 35S::AtMBP-1 plants treated with each indicated ABA concentration for 10 days with 18 days old plants.

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AtSAP5 enhances AtMBP-1 protein proteolysis

As shown in Figures 5(b) and 6(a), AtMBP-1 is highly labile with a half-life of approximately 3 h, in vivo. Degradation of AtMBP-1 is substantially blocked by the 26S proteasome inhibitor MG132 and ubiquitinated AtMBP-1 is detectable in CHX-treated plants in vivo, indicating that the instability of this protein is likely to be mediated by ubiquitin-dependent processes. Furthermore, as shown in Figure 2, over-expression of AtMBP-1 resulted in stunted growth, along with other developmental abnormalities. Since AtMBP-1 physically interacts with the E3 ubiquitin ligase AtSAP5 in yeast Figure 1(b), in vitro Figure 1(c) and in plant cells Figure 1(d, e), it seemed likely that the stability of AtMBP-1 could be regulated by AtSAP5-dependent ubiquitination. To investigate whether AtSAP5 expression can affect AtMBP-1 accumulation in vivo, transgenic plants harboring both 35S::AtSAP5 and 35S::AtMBP-1-YFP transgenes were generated by retransformation and the effect of constitutive co-expression AtSAP5/AtMBP-1 on plant development and AtMBP-1–YFP degradation was assayed. As demonstrated by the example shown in Figure 7(a), growth and development of five individual transgenic plants that over-express AtSAP5 along with AtMBP-1 was similar to that of WT plants, indicating that the developmental defects associated with AtMBP-1 over-expression can be rescued by co-expression of AtSAP5.

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Figure 7. AtSAP5 promotes AtMBP-1 protein degradation.

(a) Comparison phenotypes among WT (Col-0), 35S::AtMBP-1, and transgenic plants harboring both 35S::AtSAP5 and 35S::AtMBP-1(top). AtSAP5 and AtMBP-1 transcripts level from WT, 35S::AtMBP-1, and 35S:AtMBP-1/35S::AtSAP5 transgenic plants (bottom).

(b) Eight-day-old 35S::AtMBP-1-YFP and 35S::AtMBP-1-YFP/35S::AtSAP5 transgenic seedlings were treated with 100 μm cycloheximide (CHX) as indicated time. Equal amounts of protein were loaded on the 10% SDS gel and analyzed by immunoblot with anti-GFP.

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The stability of AtMBP-1 was then compared in the crude extracts from 8-day-old 35S::AtMBP-1-YFP and 35S::AtSAP5/35S::AtMBP-1–YFP transgenic seedlings in the presence of CHX. Consistent with the results shown in Figures 5(b) and 6(a), the apparent half-life of AtMPBP-1 in plants that expressed 35S::AtMPB-1-YFP alone was close to 3 h and it remained detectable for up to 6 h Figure 7(b). However, in transgenic plants that express both AtSAP5–YFP and AtMBP-1–YFP, the half-life of AtMBP-1–YFP was reduced to <1 h and AtMBP-1–YFP was nearly undetectable after 3 h of CHX treatment. Thus, it is apparent that AtSAP5 is involved in the destabilization of AtMBP-1 in vivo.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

AtSAP5 includes two conserved zinc-finger motifs, A20-like and An1-like, that play a role in its ubiquitin ligase activity (Kang et al., 2011). Yeast two-hybrid screening showed that AtSAP5 interacts with AtMBP-1, the truncated form of LOS2 (Figure 1). LOS2 was initially identified as a positive regulator of cold stress-responsive gene expression (Lee et al., 2002) and, based on its similarity to the mammalian Eno1 gene, it was proposed that LOS2 is bi-functional, expressing both enolase and transcriptional repressor activity. In the case of Eno1, translational initiation at the 5′ most AUG start codon of the transcript results in expression of α-enolase, while initiation at a second methionine codon, at position 93, results in the translation of AtMBP-1 (Ghosh et al., 1999). As shown in Figure 2, expression of a gene construct that includes the LOS2 cDNA in transgenic Arabidopsis plants results in the production of two translation products. Conversely, expression of a mutated LOS2 transgene, in which the ATG codon at 93 was disrupted, specifically abolished translation of the putative AtMBP-1 polypeptide, providing evidence that LOS2 mRNA can be translated to produce both enolase (which corresponds with Eno1) and AtMBP-1 in plant cells Figure 2(b).

While alternative translation of ENO1 mRNA is clearly one pathway for the production of MBP-1 in mammalian cells (Subramanian and Miller, 2000), a second route for the expression of MBP-1 has also been proposed. Lung et al. (2010) identified a transcript that includes a 5′-UTR sequence derived from the third intron of the ENO1 gene located upstream of the MBP-1 start codon. Sequences within intron III have promoter function and in vitro translation assays indicated that MBP-1 is translated far more efficiently from this transcript than from the ENO1 transcript (Lung et al., 2010). Therefore, alternative transcriptional initiation within this intron could provide an additional route for MBP-1 expression in mammalian cells. The LOS2 gene also contains three introns upstream of the putative AtMBP-1 initiation codon; however, potential transcriptional initiation within them has not been investigated.

While LOS2 is stable, AtMBP-1 is highly labile in vivo. Analysis of its degradation in CHX-treated plant tissues indicated a half-life of approximately 3 h. The stability of AtMBP-1 is substantially increased in tissues treated with the proteasome inhibitor MG132 and ubiquitinated AtMBP-1 products were detected in MG132-treated tissues, a finding that indicated that AtMBP-1 is selectively degraded via ubiquitin-dependent protein processing Figure 5(b, c). The interaction of AtMBP-1 with AtSAP5 suggested to us that AtSAP5 may target AtMBP-1 for ubiquitination and this was confirmed, in vivo, by the more rapid degradation of AtMBP-1 seen in CHX-treated plants that co-express AtSAP5 and AtMBP-1 Figure 7(b). However, despite numerous attempts to optimize the assay, we were unable to detect AtSAP5-dependent ubiquitination of AtMBP-1 in vitro. It is possible that this reaction may require more specific E1 or E2 enzymes or that additional, yet to be unidentified factors are required to enhance polyubiquitin chain assembly. As expression of AtSAP5 is strongly induced by ABA (Kang et al., 2011), the destabilization of AtMBP-1 in plants treated with ABA could result, at least in part, by the ABA-dependent increase in AtSAP5 expression, which provides further evidence for this regulatory relationship. The accumulation of MBP-1 in mammalian cells is also increased in cells treated with MG132, indicating that the stability of this protein is also regulated by ubiquitin-dependent processes (Lung et al., 2010); however, the ubiquitin ligase(s) responsible for targeting MBP-1 have not been identified. It should also be noted that AtSAP5 is encoded by a single member of a family of 14 similar genes in Arabidopsis (Jin et al., 2007). While the individual functions of most of these genes have not been analyzed, our preliminary data showed that AtMBP-1 can interact physically with AtSAP9, another stress-responsive member of the SAP (stress associated protein) gene family, in yeast two-hybrid assays. This result has led us to investigate whether additional members of the Arabidopsis SAP family also interact with AtMBP-1 and regulate its stability.

Seed germination and subsequent seedling development is more sensitive to inhibition by ABA in transgenic lines that constitutively express AtMBP-1 than in WT lines. Furthermore, expression of a subset of ABA-responsive genes, including the positive regulatory factors ABI3, ABI4 and ABI5, along with downstream target genes AtEm1, AtEM6 and RAB18 is induced more strongly in response to ABA treatment in plants that constitutively express AtMBP-1 than in WT plants, while expression of genes that encode negative factors such as ABI1 and ABI2 are not affected. Thus, our results indicate that AtMBP1 is likely to play a positive role in the ABA signaling pathway. ABI3 is a B3-domain-containing transcription factor that acts upstream of ABI5, a basic leucine zipper (bZIP) transcription factor (Finkelstein and Lynch, 2000), to arrest growth during germination (Lopez-Molina et al., 2002). In addition, ABI5 and ABI4, a member of the APETALA2 (AP2) domain family of transcriptional regulators, also function in sugar and salt responses (Bossi et al., 2009). These key regulators are active during limited developmental windows, implying that their expression and accumulation needs to be tightly regulated during plant development (Lopez-Molina et al., 2001).

Regulation of protein stability plays an important role in the post-translational regulation of genes involved in ABA signaling pathways. For example, post-translational regulation of ABI5 depends on interaction with the multidomain E3 ubiquitin ligase KEEP ON GOING (KEG) (Liu and Stone, 2010; Stone et al., 2006) and the RING finger protein AIP2 promotes ABI3 degradation via the 26S proteasome pathway (Zhang et al., 2005). While the phenotype of los2 mutant plants included increased sensitivity to cold in the light, along with decreased response of certain downstream cold-induced genes to low temperature, the responsiveness of these genes to ABA was not compromised (Lee et al., 2002). It is important to note that the los2 mutation, which results in a Gly325 to Ser substitution, is not a null allele. It does not decrease mRNA expression relative to WT plants and the mutant LOS2 protein retains the ability to bind its DNA target site. Thus, the biochemical impact of the los2 lesion on LOS2 function is not known.

MBP-1 is reported to bind to a DNA sequence adjacent to the TATA box of the human c-myc P2 promoter and down-regulate its transcription by interfering with initiation complex formation. LOS2 is also reported to bind, in vitro, to the MBP-1 target in the c-myc promoter and also to a similar site located within the 5′ flanking sequence of the Arabidopsis STZ/ZAT10 gene between nucleotides −774 and −748, relative to the site of transcription initiation (Lee et al., 2002). Compared with WT plants, the cold-responsive expression of STZ/ZAT10 mRNA was extended temporally in los2 mutant plants, possibly indicating compromised attenuation of STZ/ZAT10 expression in response to cold stress. Thus, a LOS2 gene product could be responsible for down-regulation of STZ/ZAT10 transcription following induction by stress exposure. This hypothesis is supported by our finding that the cold-responsive expression of STZ/ZAT10 is significantly reduced in 35S::AtMBP-1 plants (Figure S6). However, while LOS2 was shown to bind upstream of the STZ/ZAT10 gene in vitro, enrichment of LOS2 or AtMBP-1 at this site, or at other loci, in vivo, has not, to our knowledge, been investigated. Thus, it is not clear whether STZ/ZAT10 is the sole, or even the primary target of AtMBP-1 regulation.

The specific role of STZ/ZAT10 in the regulation of stress tolerance in Arabidopsis remains unclear. STZ/ZAT10 binds upstream of stress inducible genes in Arabidopsis, including the master stress-responsive transcriptional regulatory genes DREB1a (CBF3) and AREB2 (ABF4), and suppresses their expression (Sakamoto et al., 2004). However, constitutive expression of this gene in Arabidopsis results in strongly enhanced expression of the oxidative stress-responsive genes ascorbate peroxidase 2 and iron-containing superoxide dismutase. Increased stress tolerance was reported both in plants that constitutively express STZ/ZAT10 and in plants in which expression is suppressed or knocked out (Mittler et al., 2006; Sakamoto et al., 2004). Furthermore, growth of plants that over-express STZ/ZAT10 is stunted while constitutive expression of AtMBP-1, which limits STZ/ZAT10 expression, also resulted in stunted growth. Mittler et al. (2006) proposed that STZ/ZAT10 may play a dual role in the control of plant defenses, causing activation of some stress defense pathways, on the one hand and repressing a different set of defense mechanisms, on the other. Therefore, if the effects of AtMBP-1 and, by extension, AtSAP5 on stress responses are mediated by their regulation of STZ/ZAT10, the ambiguity remains. In addition, both AtMBP-1 and AtSAP5 could have other regulatory targets that are yet to be identified.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

The results presented here show that AtMBP-1 can be expressed in plant cells as an alternative translation product of LOS2. Our results support a model in which accumulation of AtMBP-1 is limited by ubiquitin-dependent destabilization that is regulated by its interaction with the A20/AN1 domain-containing E3 ubiquitin ligase AtSAP5 (Figure 8). While constitutive expression of AtMBP-1 leads to increased sensitivity to ABA and stunted vegetative growth, the growth defect can be reversed by co-expression of AtSAP5, indicating that the capacity of AtSAP5 to attenuate AtMBP-1 may be saturated in 35S::AtMBP-1 plants. It is possible that AtMBP-1 affects stress responses through down-regulation of STZ/ZAT10 gene expression.

image

Figure 8. Hypothetical model of the action of AtSAP5 and AtMBP-1. The LOS2 gene encodes both an enolase involved in glycolysis and a AtMBP-1. AtMBP-1 expression positively affects the responsiveness of plant cell to ABA. This could be mediated by suppression of STZ/ZAT10 gene expression. In response to ABA, AtSAP5 expression is increased and AtSAP5 ubiquitinated and destabilized AtMBP-1, thereby attenuating its positive regulation of ABA response.

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Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

Plasmid construction

Full-length LOS2 and AtMBP-1 were cloned in pENTR/D vector (Invitrogen, http://lifetechnologies.com/) and transferred to pEarlyGate 101 vector (ABRC, CD3-683) via gateway system. LOS2 was used as template to generate site-directed mutagenesis of the AtMB-1 ATG start site. Complementary primers containing ATG to CTA were designed (5′-GACTGCTATTGACAACTTCCTTGTCCATGAACTTGACGGAAC-3′ and 5′-GTTCCGTCAAGTTCATGGACAAGGAAGTTGTCAATAGCAGTC-3′). PCR products were digested with DpnI restriction site to remove non-mutant template DNA. All constructs were verified by sequencing.

Plant material and growth conditions

Seeds of A. thaliana ecotype Columbia 0 (Col-0) were used in this study and all plants were grown at 24°C under 16 h light/8 h dark. Transgenic plants were generated by the floral dip transformation method (Clough and Bent, 1998). Double transgenic plants were obtained by transforming the 35S::AtMBP-1 construct into homozygous T3 35S::AtSAP5 transgenic plants. Seeds were selected on 0.5 × MS medium containing 50 mg/l−1 kanamycin, 6 mg/l−1 Basta, and 100 mg/l−1 cefotaxime. For ABA, sensitivity test, seeds were germinated on MS medium that contained ABA for 4 days and counted for seeds germination. Seedlings with green cotyledon were monitored after 6 days. Seeds were germinated on MS medium containing either 150 mm NaCl, 6% mannitol, or 20% PEG for 8 days and germinated seeds were counted twice every day. All experiments were performed in triplicate. For the phenotype rescue experiment, ABA at different concentrations was applied to WT and 35S::AtMBP-1 over-expressing plants for 10 days, beginning at day 18 after imbibition. For gene expression, either germinated seeds 2 days after stratification or 2-week-old seedlings were harvested. qRT-PCR was performed as described previously (Kang et al., 2011). Triplicate technical replications were performed for each data.

Yeast two- hybrid screening

Yeast two-hybrid screening was carried out using the supplier's instruction (BD Clontech matchmaker 3, http://www.clontech.com/). An Arabidopsis cDNA library prepared from 2-week-old seedling treated with stress (salt, osmotic, cold, ABA) was ligated to the pGADT7 vector and AtSAP5 in pGBKT7 was used as bait. Potential positive clones were identified by DNA sequencing and six of them matched LOS2. To confirm the interaction between AtSAP5 and AtMBP-1, the full-length AtSAP5 and LOS2 cDNAs, or N-terminal truncated AtMBP-1 were inserted into pGADT7 and pGBKT7 and the vectors were transformed into AH109 yeast strain. Transformants were grown on high stringency selective medium (SD/ -His, -Ade, -Leu, -Trp) with 10 mm 3-amino-1,2,4-triazole.

Expression of recombinant proteins and pull-down assay

Recombinant AtSAP5 proteins was expressed and purified as previously described (Kang et al., 2011). Full-length LOS2 and AtMBP-1 cDNAs were recombined with pET105 and pET59 vectors respectively to produce His-tagged fusion proteins (Invitrogen, http://www.lifetechnologies.com/ and Novagen, http://www.emdmillipore.com/life-science-research/novagen/). The plasmids were expressed in Rosetta cells. Bacterial cells were grown in Luria-Bertani (LB) medium containing 50 μg/ml−1 carbenicillin to a density of OD600 = 0.6. Expression of recombinant protein was induced for 6 h at 28°C with 0.2 mm IPTG. Protein was extracted by using CelLytic B cell lysis buffer (Sigma-Aldrich, http://sigmaaldrich.com/). Recombinant fusion proteins were purified using nitrilotriacetate (NTA) resin (Qiagen). For pull-down assays, the recombinant proteins were incubated in binding buffer (150 mm NaCl, 30 mm HEPES pH 7.0, 0.1% NP-40, 10% glycerol, 1 mm DTT) for 1 h. Next, 20 μL GST resin was added, and the mixture incubated for 30 min at room temperature, then washed three times with binding buffer. Pull-down proteins were eluted by sodium dodecyl sulfate (SDS) sample buffer, and were then analyzed by immunoblotting. Experiments were replicated at least three times.

Protein extraction and immunoprecipitation

Here, 8-day-old transgenic seedlings that expressed 35S::AtMBP-1-YFP were treated with indicated concentrations of MG132 (sigma), and/or cycloheximide (sigma), as well as ABA (Sigma). Total protein was extracted from seedlings using CelLytic P Cell lysis reagent (Sigma). For immunoprecipitation, 8-day-old seedlings of 35S::AtMBP-1-YFP plants were extracted in immunoprecipitation (IP) buffer (50 mm Tris pH 7.4, 150 mm NaCl, 1% Triton, 1 mm EDTA, 1 mm MgCl2, and protease inhibitor cocktail). The protein extracts (1 mg) were incubated with monoclonal GFP antibody conjugated with agarose (MLB) at 4°C for 3 h. The precipitated proteins were washed five times with IP buffer and eluted with SDS sample buffer. Eluted samples were subjected to western blot analysis using either anti-GFP antibody (Sigma) or anti-ubiquitin antibody (Boston Biochem, http://www.bostonbiochem.com/). Experiments were replicated at least three times.

In vivo Co-IP and BiFC

Agrobacterium strain GV2260 carrying the Flag-AtSAP5, LOS2–YFP, or Flag-AtSAP5, AtMBP-1–YFP expression vectors was co-infiltrated into N. benthamiana. For in vivo Co-IP, the infiltrated N. benthamiana leaves were harvested 2 days after infiltration and total protein was extracted in IP buffer (50 mm Tris pH 7.4, 150 mm NaCl, 1% Triton, 1 mm EDTA, 1 mm MgCl2, and protease inhibitor cocktail). A 10 μL aliquot of anti-GFP agarose (MBL) was added to the samples, and the mixtures were incubated for 4 h at 4°C with shaking. The immunoprecipitated proteins were washed three times with IP buffer and eluted with SDS sample buffer. Eluted samples were loaded on the protein gels for immunoblot analysis using anti-GFP antibody or anti-Flag antibody (Sigma). For BiFC, AtSAP5 and AtMBP-1 were cloned into the pSITE BiFC nEYFP-C1 or pSITE BiFC-cEYFP-N1 vectors (Marin et al., 2009) using gateway cloning system (Invitrogen). Leaf samples were collected 2 days after agro-infiltration. MG132 (50 μm) was applied 18 h prior to sample harvesting. YFP fluorescence signal was observed with a laser scanning confocal microscope (Leica DM IRE2).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information

This work was funded by Oklahoma Center for Advancement of Science & Technology (OCAST) project number PSB09-002. Support was also provided by the Samuel Roberts Noble Foundation and the Oklahoma Agricultural Experiment Station. The authors thank Dr Million Tadege and Dr Mohamed Fokar for critical reading of the manuscript.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Acknowledgements
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
tpj12312-sup-0001-FigS1-S7.pdfapplication/PDF530K

Figure S1. Confocal images of subcellular localization of LOS2 and AtMBP-1 in 35S::LOS2-YFP and 35S::AtMBP-1-YFP transgenic plants.

Figure S2. Assay of enolase activity of LOS2 and AtMBP-1 expressed in E. coli.

Figure S3. Phenotypes of 5 week old WT, 35S::AtMBP-1-YFP, and 35S::LOS2-YFP overexpressing plants.

Figure S4. Assay of germination for seeds from 35S::mLOS2 transgenic plants.

Figure S5. qRT-PCR was performed to monitor gene expression which is involved in ABA signaling pathway.

Figure S6. The expression of SnRK2s and AFPs in WT and 35S::AtMBP-1 transgenic plant germinated without or with 0.2 µM ABA.

Figure S7. Expression of STZ/ZAT10 in 2-week-old WT and 35S::AtMBP-1 transgenic plants in response to chilling (4°C) treatment.

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