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Keywords:

  • stress response;
  • dedifferentiation;
  • chromatin reorganization;
  • DNA methylation;
  • histone modifications;
  • Arabidopsis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Background: Previous data suggested that senescing cells or cells exposed to acute stress may acquire stem cell properties characterized by open chromatin conformation and by promiscuous expression of transcription factor genes. To further explore the link between stress response and dedifferentiation, we generated transgenic plants in which a reporter AtMBD6-GFP is controlled by a meristem-specific promoter derived from the ANAC2 gene together with the analysis of chromatin conformation. Results: We found that ANAC2 promoter is essentially active in the shoot and the root apical meristems including leaf primordia. ANAC2 was activated in mature leaves following exposure to various stress conditions including protoplasting and dark. This activity was associated with decondensation of pericentric but not of centromeric chromatin. Using epigenetic mutants, ddm1 and kyp/suvh4, we found that compaction at centromeric chromatin persists despite a significant reduction in DNA and histone methylation. Conclusions: Our results suggest that extreme environmental signals trigger plant somatic cells to acquire stem cell properties before assuming a new cell fate. Results also pointed to distinct mechanisms involved in controlling chromatin compaction at chromocenter and that compaction of centromeric chromatin may not be dependent on epigenetic means driven by DDM1 and KYP/SUVH4 chromatin modifier proteins. Developmental Dynamics, 242:1121–1133, 2013. © 2013 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. ACKNOWLEDGMENTS
  8. REFERENCES

It is commonly accepted that in plants, stem cells are localized at distinct regions called meristems. Meristems are of several types (Fahn, 1990) and include the apical meristems located at the apices of the root and the shoot, which are responsible for primary growth leading to elongation of the shoot and the root axes. It is assumed that stem cells are localized within a specific domain of the meristem, such as the central zone of the shoot apical meristem (SAM) that displays reduced mitotic activity (Steeves and Sussex, 1989). Other meristems responsible for secondary growth and thickening of the shoot and root axes are known as lateral meristems and include the vascular cambium that gives rise to secondary growth of the vascular tissues (xylem and phloem) and the phellogen (cork cambium). To date, it is not clear whether stem cells or pluripotent cells also exist in somatic tissues of the plant, as they are in somatic tissues of animals (adult stem cells).

Owing to their sessile nature, many mature plant cells retain totipotency and are capable, under certain conditions (often stress conditions), to dedifferentiate, reenter the cell cycle, proliferate, and regenerate tissues, organs, and entire fertile plants. This capacity is central to a wide range of applications related to in vitro regeneration including micropropagation, germplasm conservation, and formation of genetically modified plants (reviewed in Thorpe, 2007). Dedifferentiation signifies the withdrawal of cells from a given differentiated state into a transient, stem cell–like state that confers pluripotency, a process preceding switch in cell fate such as reentry to the cell cycle and death (Grafi, 2004). One feature characterizing dedifferentiated cells as well as stem cells both in plants and animals is the open chromatin conformation (Grafi, 2004; Meshorer and Misteli, 2006; Gaspar-Maia et al., 2011; Grafi et al., 2011a), which is necessary for maintaining stem cell developmental capacities including self-renewal and differentiation into multiple cell types (Melcer and Meshorer, 2010; Gaspar-Maia et al., 2011).

The transcriptome profile of stem cells has been under intense study in the last decade revealing that in contrast to the idea that stem cells represent a specific entity that is characterized by the expression of specific genes, these cells assume a transient state characterized by open chromatin conformation that confers transcriptional competence and by a promiscuous expression of many marker/transcription factor encoding genes (Zipori, 2004; Meshorer and Misteli, 2006; Grafi et al., 2011c). Likewise, plant cells, acquiring dedifferentiated, stem cell–like state (e.g., protoplasts), are characterized by widespread chromatin decondensation (Zhao et al., 2001; Williams et al., 2003; Tessadori et al., 2007a) associated with overrepresentation of transcription factors of specific families, including ANAC and WRKY (Damri et al., 2009). Recent published data show that senescing cells and dedifferentiating protoplast cells share common features. Both cell types display a large increase in the expression of genes encoding specific transcription factor (TF) families including ANAC, WRKY and b-ZIP (Damri et al., 2009), which are also upregulated in the shoot apical meristem (Yadav et al., 2009) and are often up-regulated under various biotic and abiotic stress conditions (Cheong et al. 2002; Lin and Wu, 2004; Balazadeh et al. 2008; Grafi et al., 2011b).

To further substantiate a link between plant response to stress, dedifferentiation, and SAM stem cells, we generated transgenic plants in which a reporter gene AtMBD6-GFP is controlled by the promoter of the ANAC2 (also known as ATAF1) gene (At1g01720). We selected the promoter of ANAC2 because reported data showed that its activity is upregulated in meristematic cells as well as in dedifferentiating protoplast cells and in cells subjected to various biotic and abiotic stress conditions (Collinge and Boller, 2001; Jensen et al., 2007; Lu et al., 2007; Wu et al., 2009; Damri et al., 2009; Lin and Wu, 2004; Yadav et al., 2009). Thus ATAF1/ANAC2 gene expression may provide a link between stress response and the dedifferentiated, pluripotent state of the cell.

Here we showed that ANAC2 promoter is essentially activated in the apical meristems of the shoot and the root showing almost no expression in mature leaves. However, ANAC2 was activated in leaf cells in response to extreme stress conditions, such as protoplasting, wounding, exposure to dark and heat. We found that activation of ANAC2 was associated with selective reorganization of chromatin, which is independent of DNA and histone methylation.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. ACKNOWLEDGMENTS
  8. REFERENCES

ANAC2 Promoter is Active in Meristems

To further explore a possible link between stress response and the stem cell state, we selected the ANAC2 promoter placed upstream from a reporter gene encoding for a nuclear protein (AtMBD6) fused to GFP, which enables convenient and reliable visualization of positive cells as the reporter protein concentrates in the nucleus, showing peculiar nuclear localization (Zemach et al., 2005). Transient assays showed that introduction of this construct (pUC19-ANAC2Pr:AtMBD6-GFP) into Arabidopsis protoplasts resulted in activation of ANAC2, which directed the transient expression of AtMBD6-GFP, which was restrictively localized to the nucleus (data not shown).

We subcloned the ANAC2Pr:AtMBD6-GFP cassette into the binary vector pGreenII0229, containing resistance to Basta (Glufosinate) as well as the Ti borders required for Agrobacterium-mediated transformation (Hellens et al., 2000). The pGreen-ANAC2Pr:AtMBD6-GFP was transformed into Agrobacterium, which was used to infect Arabidopsis plants by the flower dip method, and to generate transgenic plants (Clough and Bent, 1998). Transgenic plants were primarily selected by their resistance to Basta herbicide and by GFP fluorescence, and several independent homozygote transgenic lines were established and were used for further analysis.

Confocal microscope inspection of transgenic plants showed that ANAC2 promoter is active in the shoot apical meristem at all stages of development tested including auxiliary bud meristem on the inflorescence stem (Fig. 1A), flower meristem (Fig. 1B), as well as in root tips of both the main root and of lateral roots (Fig. 1C). In the shoot apical meristem, ANAC2 promoter was active at all domains including leaf primordia as small as 25 μm in diameter (Fig. 1D); ANAC2 promoter activity persisted in leaf initials of about 200 μm (data not shown).

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Figure 1. ANAC2 promoter is active in meristems in transgenic Arabidopsis plants carrying the ANAC2Pr:AtMBD6-GFP construct. A: Auxiliary bud meristem on inflorescence stem of 21-day-old plants. B: Inflorescence meristem. C: ANAC2 activity in the primary root of a 1-week-old seedling. LI, leaf initial; ABM, auxiliary bud meristem; FM, flower meristem; IFM, inflorescence meristem; RM, root meristem; RC, root cap. D: ANAC2 promoter activity in leaf primordia. Top panels demonstrate ANAC2 promoter activation in four leaf primordia (marked by arrows) in the shoot apical meristem region (marked by a circle) of a young seedling (upper view). Bottom panels show a side view of the same meristem. Chloroplast (AF) is the autofluorescence of chloroplasts. COT, cotyledon; 1st TL, first true leaf; L, leaf.

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Generally, ANAC2 promoter activity in leaves was very low and sporadic, often restricted to fewer cells or cell types. Accordingly, GFP signal was sporadically observed in trichomes of young leaves and at a group of cells located at the tips of both cauline and rosette leaves as well as at the distal region of the cotyledon (data not shown). Rosette leaves often show GFP activity in lobes along the margins of young and mature leaves and in a very stochastic manner also in guard cells (data not shown).

ANAC2 Promoter Is Activated in Dedifferentiating Protoplast Cells

We followed the activation of the ANAC2 promoter in the transgenic plants following protoplast isolation, whereby cells acquire a dedifferentiated, stem cell–like state (Zhao et al., 2001; Grafi, 2004). The majority of fully matured leaf cells were essentially deficient in GFP signal indicating that ANAC2 promoter is not active in these cells. However, protoplasts derived from transgenic leaves displayed nuclear GFP in most (>95%) protoplast cells 24 hr after their isolation (Fig. 2A). Closer observation (Fig. 2B) showed that AtMBD6-GFP was preferentially accumulated at two perinucleolar chromocenters as previously described (Zemach et al., 2005). Detailed analysis revealed that after 8 hr the GFP signal was intense in most protoplasts similarly to the signal obtained after 24 hr. After 2–6 days, the GFP signal intensity was reduced and gradually disappeared from most of the protoplasts as they senesce and die. Some protoplasts, however, were still intact and showed GFP activity even 6 days after isolation.

image

Figure 2. Protoplasting is associated with ANAC2 promoter activation. A: ANAC2 promoter is widely activated in protoplasts 24 hr after isolation. B: Specific nuclear localization to perinucleolar chromocenters of AtMBD6-GFP in an isolated protoplast cell. Nuc, nucleus; Chls, chloroplasts (autofluorescence). Red arrows indicate AtMBD6-GFP signal at perinucleolar chromocenters. Bar = 5 μm. C: Immunoblotting analysis with anti-GFP of AtMBD6-GFP in rosette leaves and protoplasts at the indicated time points after isolation.

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To verify the confocal microscope observations, we analyzed the extent of ANAC2 promoter activation by monitoring the expression of AtMBD6-GFP by immunoblotting using anti-GFP. Total proteins were extracted from protoplasts of transgenic plants at various time points after their isolation and separated on 12% SDS/PAGE and immunoblotted using GFP antibody. While no expression was evident in rosette leaves, the treatment with cell wall–degrading enzymes for isolation of protoplasts resulted in expression of AtMBD6-GFP protein (Fig. 2C). Accordingly, AtMBD6-GFP was evident, though at a low level, 4 hr after isolation and a high amount was accumulated at 8 and 24 hr after protoplast isolation.

ANAC2 Promoter Is Activated in Leaf Cells Following Exposure to Extreme Stress Conditions

We next examined the activation of ANAC2 promoter in leaves following applications of various stress conditions. Previously, we found that tobacco leaf cells induced to senesce by prolonged exposure to dark share common features with dedifferentiating protoplast cells including gene expression profile and chromatin conformation (Damri et al., 2009). We monitored ANAC2 promoter activity in leaves of transgenic Arabidopsis plants following exposure to dark. Transgenic plants exposed to dark for 5 days stopped growing and underwent senescence demonstrated by leaves turning yellow (Fig. 3A, right panel). Yellow leaves showed widespread activation of ANAC2 promoter as the GFP signal was observed in many cells of senescing rosette leaves (Fig 3B). To verify that senescing leaves are not yet committed for death but have acquired a dedifferentiated state, we examined their capacity for proliferation and callus formation following exposure to dark. Indeed, leaves derived from dark-treated plants incubated on callus-inducing medium have an increased success of callus formation than leaf explants derived from untreated plants, and calluses derived from dark treated leaves appeared greener than those derived from untreated leaves (Fig. 3C). Likewise, mechanical wounding of the leaf in the form of a cut resulted in activation of ANAC2 promoter within 24 hr; ANAC2 promoter was activated in leaf cells up to 200–300 μm far from the cutting line (data not shown).

image

Figure 3. ANAC2 promoter is activated in Arabidopsis leaves following exposure to dark. A: Control versus 5-day dark-treated transgenic Arabidopsis plants carrying ANAC2::AtMBD6-GFP. B: Confocal microscope inspection of dark-treated plants showing activation of ANAC2 promoter in many cells of the leaf. Bar = 50 μm. C: Exposure to dark increased survival of callus formation. Intact leaves (20 leaves) derived from control untreated plants or from plants incubated in the dark for the indicated duration (days) were placed on callus inducing medium and inspected for callus formation after 3 weeks.

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Finally, we examined the effect of heat stress on ANAC2 promoter activation by exposing transgenic plants to 37°C for various time periods (18, 20, and 22 hr). ANAC2 promoter activity was inspected immediately after treatment or following 24-hr recovery time. Heat treatment for 20 hr or more resulted in death of plants within a few days after treatment while those subjected to heat stress for 18 hr or less survived (Fig. 4A). Total proteins were extracted from treated leaves and separated on 12% SDS/PAGE followed by immunoblotting using anti-HSP17.6 and anti-GFP. Results showed that exposure to heat for the indicated time periods resulted in an accumulation of a high level of HSP17.6 proteins immediately after treatment, which remained at a high level following 24-hr recovery time (Fig. 4B). Notably, HSP17.6 was not expressed in control untreated plants or in protoplasts (P) 24 hr after their isolation. An abrupt increase in HSP17.6 level was already evident following 2-hr heat treatment (data not shown). Unlike HSP17.6, ANAC2 promoter was almost not active at any time point of exposure to heat when measured immediately after treatment. A notable activation of ANAC2 promoter was evident following 24-hr recovery time by inspection under a confocal microscope and by immunoblotting. Activation was evident in plants subjected to heat stress for 18 and 20 hr (Fig. 4B), but not when plants were heat treated for a short time period (4 hr).

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Figure 4. Heat stress induces ANAC2 promoter activation. A: Survival of Arabidopsis plants following exposure to heat stress (37°C) for various time periods (indicated to the left). Note that after 5 days recovery time, plants subjected to heat treatment for 18 hr but not for 20 and 22 hr have survived. B: Immunoblotting analysis. Total proteins were extracted, separated on 12% SDS/PAGE (coomassie staining), and subjected to immunoblotting using anti-HSP17.6 and anti-GFP. Lane P is a protein extract from protoplasts (P) that was used as a control. Lane M is the protein size markers.

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Stress Induces Changes in Chromatin Structure

To further substantiate the idea that stress might induce cells to acquire a dedifferentiated, stem cell–like state, we sought to examine whether stressed cells display a fundamental feature of pluripotent stem cells, namely, open chromatin conformation (Grafi, 2004; Meshorer and Misteli, 2006; Grafi et al., 2011a). To this end, we analyzed by fluorescence in situ hybridization (FISH) the chromatin conformation of the centromeric and pericentric domains, which normally assume a compact structure. Accordingly, the centromeric 180-bp repeats (CEN180) was used as a probe to monitor the centromeric region while BACs F2H10, T10P12, F2J6, and F28H19 were used to monitor the pericentric region (will be referred to as PC-Chr1). Notably, these BACs contain repetitive DNA sequences of the pericentric region of chromosome 1 and essentially recognize pericentric regions of all chromosomes (Tessadori et al., 2007a). Fixed nuclei prepared from leaf cells and from protoplasts at various time points (0, 24, and 48 hr) after isolation were hybridized with Rhodamin-PC-Chr1 (Fig. 5A) or with Rhodamin-CEN180 (Fig. 5B) and inspected under a confocal microscope. Our results showed (Fig. 5A) that the majority of leaf cell nuclei exhibited pericentric regions as fewer dots adjacent to chromocenters (identified by the intense DAPI staining). In protoplasts, we observed peculiar and dynamic behavior of pericentric chromatin. Immediately after isolation, pericentric chromatin was condensed as in leaf nuclei. However, 24 hr after protoplast isolation pericentric chromatin underwent transient decondensation, which was followed by condensation at 48 hr. Similarly, we found that pericentric chromatin become decondensed following exposure to dark (Damri et al., 2009) or following long exposure to heat stress (data not shown). In contrast with the pericentric region, the centromeric chromatin (CEN180) remains compact 24 hr after protoplast isolation similarly to its compaction in leaf cells (Fig. 5B). Thus, stressing cells by cell wall–degrading enzymes resulted in selective decondensation of pericentric chromatin.

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Figure 5. Protoplasting induces selective chromatin reorganization. FISH analysis was performed to assess the conformation of pericentric chromatin (A) or centromeric chromatin (B). Nuclei prepared from leaves and from protoplasts at the indicated time points after isolation (0, 24, and 48 hr) were subjected to FISH using rhodamine-labeled PC-Chr1 (BACs F2H10, T10P12, F2J6 and F28H19) (A) or rhodamine-labeled CEN180 (B). Note that CEN180 chromatin remains compact in protoplasts. DAPI was used as a counterstain. Bar = 5 μm.

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Maintenance of Centromeric Heterochromatin does not Require Histone and DNA Methylation

In order to assess the importance of DNA and histone methylation for selective chromatin reorganization, we used DNA and histone methylation mutant plants. The ddm1 mutant carries a mutation in the DDM1 (DECREASE IN DNA METHYLATION1) gene encoding for a SWI/SNF chromatin remodeling factor (Jeddeloh et al., 1999) resulting in 70% reduction in cytosine methylation (Vongs et al., 1993) and in dispersion of pericenric chromatin (Soppe et al., 2002). The kyp2 mutant (Ler background) carries a mutation in the KRYPTONITE/SUVH4 gene encoding for histone H3K9 methyltransferase that results in a significant reduction in H3K9 dimethylation as well as in cytosine methylation in the context of CHG (Jackson et al., 2002; Malagnac et al., 2002). Notably, both mutants displayed a significant reduction in H3K9 dimethylation associated with centromeric 180-bp repeats (Vongs et al., 1993; Johnson et al., 2002; Zemach et al., 2005). Since in ddm1 mutant, pericentric chromatin was previously reported to undergo decondensation (Soppe et al., 2002), we examined in ddm1 and kyp2 mutants the chromatin conformation of CEN180 upon protoplasting. Nuclei prepared from leaves and protoplasts of ddm1 and kyp2 mutants were subjected to FISH using rhodamine-CEN180 and inspected under a confocal microscope. Results showed (Fig. 6A, B) that chromatin conformation of CEN180 in both mutants remains compact upon protoplasting as in wild-type Arabidopsis plants despite a significant reduction in DNA and histone methylation associated with CEN180. This suggests that maintenance of centromeric heterochromatin in dedifferentiating protoplasts can occur independently of DNA and histone methylation. Notably, in contrast with ddm1 (Soppe et al., 2002), in suvh4 mutant, pericnteric chromatin retains its compact configuration in leaf cells despite reduction in H3K9 dimethylation (data not shown).

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Figure 6. Maintenance of centromeric heterochromatin in dedifferentiating protoplasts is independent of DNA and histone H3K9 methylation. Nuclei prepared from leaves and protoplasts of the Arabidopsis mutants ddm1 (A) and kyp2 (B) were subjected to FISH using tetramethylrhodamine-5-dUTP-labeled CEN180 and inspected under confocal microscope. DAPI was used as a counterstain. Bar = 5μm.

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We next investigated changes in histone H3 methylation and DNA methylation at specific loci within the pericentric region of chromosome 1 by using chromatin immunoprecipitation (ChIP) assays and methylation sensitive enzymes, respectively. We focused on a pericentric region of chromosome 1 containing several genes including VIP1 previously shown to be upregulated in protoplasts (Avivi et al., 2004), fructose 1,6 biphosphatase (FBP), histidine decarboxylase (HDC) as well as two adjacent genes, namely, At1g43160 (encoding for RAP2.6, a member of the ERF/AP2 transcription factor family) and At1g43560 (encoding for thioredoxin Y2). We examined changes in association of the above-mentioned pericentric genes with modified histones, namely, H3 dimethylated at K4 (H3K4me2), a modification associated with transcriptionally active chromatin, and H3 dimethylated at K9 (H3K9me2), a modification associated with transcriptionally inactive chromatin. Following ChIP, the recovered DNA was subjected to PCR amplification using primers for CEN180 or primers to amplify the indicated pericentric genes. CEN180 was used as a quality control for the ChIP assay, as these repeats are known to be associated mainly with H3K9me2. Indeed, the results showed (Fig. 7) that CEN180 is associated mostly with H3K9me2 both in leaves and in protoplasts. The pericentric genes VIP1, RAP2.6, and At1g43560 were not associated with H3K4me2 or H3K9me2 in leaves, but almost exclusively associated with H3K4me2 in protoplasts. Reverse transcriptase (RT) PCR showed that At1g43160 is not expressed in leaves but is highly upregulated in protoplasts, while At1g43560 is expressed both in leaves and protoplasts (data not shown). FBP was associated with H3K9me2 both in leaves and protoplasts, while HDC that was associated with H3K9me2 in leaves showed also association with H3K4me2 in protoplasts.

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Figure 7. Chromatin immunoprecipitation (ChIP) assay demonstrating changes in association of pericentric genes with modified histones upon protoplasting. ChIP was performed on nuclei from leaves and protoplasts of wild-type Ler ecotype. Chromatin was immunoprecipitated using anti-dimethylated K4 histone H3 (H3K4me2) and anti-dimethylated K9 histone H3 (H3K9me2). Recovered DNAs were subjected to PCR using sets of primers to amplify the centromeric 180-bp repeats (CEN180) or the indicated pericentric genes. Input represents 0.02% of the chromatin subjected to immunoprecipitation.

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Analysis of DNA methylation of pericentric genes (VIP1, HDC, and FBP) as well as of transposable element genes [COPIA4I (At4g16870), IG/LINE (At5g27845) and ATLANTYS2A (At3g60930)] showed (data not shown) that FBP and VIP1 are mostly methylated at the CpG context, the HDC gene as well as the indicated transposable element genes appeared to be methylated in both contexts. However, in all cases, we could not detect significant changes in methylation at CCGG sites upon chromatin decondensation, suggesting that decondensation occurs without notable changes in DNA methylation pattern. Thus, it appears that the ChIP results are not consistent with the DNA methylation data suggesting that multiple mechanisms might be involved in controlling chromatin structure and function at pericentric regions during dedifferentiation.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. ACKNOWLEDGMENTS
  8. REFERENCES

We show here that ANAC2 promoter activity revealed a link between stress response and meristematic cells. Accordingly, ANAC2 promoter is highly active in the root and the shoot apical meristems (RAM and SAM, respectively), the sites of stem cell pool in plants, and activated in leaves subjected to various stress conditions including protoplasting, dark exposure, wounding, and heat treatment. We also showed that dark-induced premature senescence potentiates callus formation, probably via inducing cell dedifferentiating (Damri et al., 2009). This is consistent with the findings that senescing yellowish leaves are not destined for death but can under certain circumstances (e.g., removal of young leaves, application of cytokinins) turn green and regain their photosynthetic capacity (Venkatarayappa et al., 1984; Gan and Amasino, 1995; Buchanan-Wollaston et al., 2003). Most importantly, we showed that stress-induced ANAC2 promoter activity is associated with widespread decondensation of pericentric chromatin, a known feature of stem cells both in plant and animals (Grafi, 2004; Meshorer and Misteli, 2006).

Notably, in the SAM, ANAC2 promoter activity was not restricted to the central zone (CZ) but its expression extended to the periphery of the meristem and to leaf primordia. This suggests that the stem cell state is retained in many cells outside the CZ of the meristem. The meristem is composed of several distinguishable domains that differ in the expression of specific genes. Accordingly, the central zone is characterized by the expression of CLAVATA3 (CLV3), the rib meristem by WUSCHEL (WUS), and the peripheral zone by the FILAMENTOUSFLOWER (FIL). Although stem cells have long been thought to be restricted to the CZ, recent data suggest they are present in all domains within the meristem. This is demonstrated in a recent study showing that cells capable of mediating complete organ regeneration are dispersed in the plant meristem and are not restricted to the “so-called” stem cell niche (CZ); even young leaves/leaf primordia (about 200 μm in length) appeared to contain (stem) cells capable to re-pattern complex tissues (Sena et al., 2009). This is consistent with the proposal that ANAC2 promoter activity may define the stem cell state as our data showed that ANAC2 promoter is active not only within the meristem territory but also in leaf primordia as well as in young leaves (from 50–250 μm in length). Thus, it appears that the stem cell state may persist in plant tissues and organs during early stages of their development and cells within the different domains of the meristem have stem cell properties (Laux, 2003). This is further supported by a recent transcriptome analysis of the SAM domains (Yadav et al., 2009). Scatter plot analysis of the available microarray datasets showed that the gene expression profile of the CZ is highly similar to those of the rib meristem and the peripheral zone (correlation coefficient ∼0.9) suggesting that all SAM domains are essentially alike and cells in all domains retain to some extent stem cell properties.

Open chromatin conformation appears to be an inherent feature characterizing stem cells as well as dedifferentiating cells both in plants and animals (Grafi, 2004; Gaspar-Maia et al., 2011; Grafi et al., 2011a). Indeed, a major attribute of SAM stem cells appears to be a flexible chromatin state demonstrated by the overrepresentation of chromatin modifier genes (CMGs) (Yadav et al., 2009). Remarkably, further analysis of the microarray datasets compiled by Yadav et al. (2009) revealed that among the 445 CMGs, which were represented on the ATH1 array, CZ, RM, and PZ displayed 297, 283, and 282 CMGs, respectively, the expression signal of which is higher than 256 (28). Thus, all domains within the SAM maintain flexible chromatin structure characterized by overrepresentation of CMGs, which is necessary for the establishment of the stem cell state. Recently, it has been shown that dark-induced premature senescence shares common features with dedifferentiating protoplast cells of tobacco including widespread chromatin decondensation and compaction of rRNA gene clusters (Damri et al., 2009). Similarly, decondensation of pericentric heterochromatin was reported during leaf senescence in Arabidopsis (Ay et al., 2009) and following exposure to long (30 hr at 37°C) but not short (3 hr at 37°C) heat stress (Pecinka et al., 2010). Indeed, ANAC2 promoter was activated under various extreme stress conditions including wounding and long exposure to dark. Accumulated data support the hypothesis that plant cells may respond to various environmental cues by undergoing dedifferentiation characterized by chromatin decondensation (Tessadori et al., 2007b; Pecinka et al., 2010; Grafi et al., 2011b). Stress-induced dedifferentiation and/or chromatin relaxation is not unique to plant cells and has also been reported in human cells exposed to oxidative stress (paraquat), UV light, and hydrogen peroxide (Abrahan et al., 2009; Halicka et al., 2009). Indeed, it has recently been proposed that mammalian somatic cells may undergo cell dedifferentiation as an adaptation for extreme stress conditions (Shoshani and Zipori, 2011).

Two features of stress-induced dedifferentiation and chromatin remodeling have been uncovered in the present work. The first is the selective chromatin reorganization, namely, decondensation of preicentric chromatin concomitantly with no change in chromatin conformation of centromeric 180-bp repeats upon protoplasting. The second is the independence of chromatin reorganization on epigenetic marks such as DNA and histone H3K9 dimethylation demonstrated in ddm1 and kyp/suvh4 mutants. Similarly, mutations in Microrchidia (MORC) adenosine triphosphatases (ATPases), namely, ATMORC1 and ATMORC6, were shown to cause pericentric chromatin decondensation and transposable element upregulation without notable changes in DNA and histone methylation (Moissiard et al., 2012). Also, reduction in H3K9 dimethylation in kyp/suvh4 mutant did not result in pericentric chromatin decondensation, further supporting the independence of chromatin compaction on H3K9 dimethylation. However, we cannot exclude the possibility that in the absence of dimethyl H3K9, monomethyl as well as trimethyl H3K9 are involved in maintaining pericentric heterochromatin as has recently been described in animal cells (Pinheiro et al., 2012; Towbin et al., 2012). The gain of dimethyl H3K4 by several pericentric genes is probably related to transcriptional activation of these genes. Accordingly, VIP1, RAP2.6 (At1g43160), and thioredoxin Y2 (At1g43560) are deficient in H3K4me2 in leaf cells but are associated with H3K4me2 in dedifferentiating protoplast cells, concomitantly with their active transcription; in leaves, however, the expression of At1g43560 occurred in the absence of H3K4me2. Taken together, our results suggest that plants have evolved a plethora of mechanisms to control their genome organization and, consequently, gene expression. Chromatin relaxation and/or compaction during stress-induced dedifferentiation appears to occur independently of epigenetic markers driven by the activities of the chromatin modifiers DDM1 and KYP/SUVH4 proteins. Alternatively, maintenance of chromatin compaction at centromeric region may occur via the direct activities of chromatin-remodeling enzymes. These include SWI/SNF chromatin-remodeling factors, single-stranded DNA endonucleases that target weakly hydrogen-bonded regions (i.e., AT-rich) (Grafi and Larkins 1995) within condensed double-stranded DNA leading to chromatin relaxation as well as topoisomerases known to be induced following exposure to stress (Mudgil et al. 2002; Hettiarachchi et al. 2005). Cohesin protein complexes may also be involved in maintaining the compact conformation of centromeric regions (reviewed in Haering and Jessberger, 2012) in somatic cells and dedifferentiating cells independently of DNA and histone methylation.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Construct Preparation and Generation of Transgenic Plants

The ANAC2 promoter (806 bp) derived from the Arabidopsis thaliana At1g01720 gene was amplified using forward: 5′-TCTACTCGAGTCCCTATTGGCTTTACCACTC-3′, and reverse 5′-CACAAGATCTGGAAAGAATGAGAGAAAAGTC-3′ primers flanked with XhoI and BglII restriction enzymes, respectively. The PCR product was restricted with the aforementioned restriction enzymes, and cloned into a pUC19 upstream from the AtMBD6-GFP (Zemach et al., 2005), to generate the pUC19-ANAC2Pr:MBD6-GFP. The ANAC2Pr:MBD6-GFP cassette was excised out by restriction with XhoI and EcoRI and subcloned into the same sites of pGreenII229 binary vector.

Arabidopsis Transformation and Selection

Agrobacterium tumefaciens (GV3101 strain) was transformed with the binary vector pGreen-ANAC2Pr:MBD6-GFP, along with a support vector pSoup. A 5-ml starter culture was grown in LB media for 48 hr, at 28°C, in the presence of 25 μg/ml gentamicin, 50 μg/ml kanamycin, and 5 μg/ml tetracycline. Five hundred milliliters of LB was inoculated and grown until it reached OD600=1. The bacteria was harvested and re-suspended in a solution containing 50 g/L sucrose, and 200 μl/L silwet L-77. Arabidopsis thaliana (Columbia ecotype) plants were infected by the flower dip method, and their seeds were collected. F1 seeds were allowed to germinate and grown for 6–7 days before applying 60 mg/L Basta spray in order to eliminate all non-transgenic seedlings. Few independent transgenic lines were selected, and two lines were used to isolate homozygote plants, by F2 and F3 segregation analysis.

Plant Growth, Tissue Culturing and Stress Application

All Arabidopsis lines (wild type, transgenic, and mutants ddm1, kyp2; kindly provided from E. Richards E. and S. Jacobsen, respectively) were grown in a controlled growth room equipped with fluorescent lights under long day photoperiod (16 hr light and 8 hr dark, light intensity 200 mmol photons m-2 s-1) at 22°C ± 2 and 70% humidity. Plants with 4–5 pairs of leaves were subjected to various stress conditions as indicated in the Results section. Heat treatment was applied by incubating transgenic Arabidopsis plants at 37°C in the dark for various time periods (indicated in the Results section), after which plants were placed in a growth room (mentioned above) for recovery. Leaves were inspected under a confocal microscope for GFP fluorescence or sampled at various time points for protein extraction.

Calluses were generated from Arabidopsis leaves on MS medium supplemented with B5 salts, 3% sucrose, 0.5 mg/L 2,4-D, and 0.5 mg/L benzylaminopurine (BAP).

Protoplasts Preparation and Transient Transformation

Transient expression in protoplasts was performed essentially as described (Sheen, 2002). Arabidopsis fresh leaves were cut into small pieces, incubated in a cell wall–degrading solution containing 1–1.5% cellulase, 0.3–0.5% macerozyme, 0.4M mannitol, 20 mM KCl, 20 mM MES, 10 mM CaCl2, and 0.1% BSA, placed in a vacuum for 20 min, and then shaken for 90–120 min at 50 rpm. The protoplasts were then filtered through a 180-μm mesh, diluted with 1 volume of W5 (150 mM NaCl, 125 mM CaCl2, 5 mM KCl, and 2 mM MES) and pelleted by centrifugation (Room temperature, 2 min at 300g). The protoplasts were re-suspended in W5 solution and incubated for 30 min on ice, before being centrifuged again and re-suspended in 100 μl of MMg solution containing, 0.4 M mannitol and 15 mM MgCl2. Plasmid DNA (5–20 μg) was added to protoplasts and equal volume of 40% PEG solution (in 0.2M mannitol and 0.1 M CaCl2) and the mixture was incubated for 15–30 min. Two volumes of W5 were added to each sample, centrifuged for 2 min, re-suspended in 1 ml of W5, and then incubated at room temperature for 16–24 hr.

Protein Extraction and Immunoblotting

Total proteins were extracted from various organs and tissues of transgenic Arabidopsis plants (ANAC2::AtMBD6-GFP) with NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8, and 0.5% NP-40) supplemented with protease inhibitor cocktail (Sigma, St. Louis, MO) and protein concentration was determined by the Bradford reagent (BioRad, Hercules, CA). Fifteen micrograms of total proteins were resolved by SDS/PAGE and immunoblotted with rabbit polyclonal antibodies to GFP (Abcam, Cambridge, MA) or to HSP17.6 (Agrisera, Vännäs, Sweden). Immuno-detection was performed using secondary antibody of goat anti-rabbit alkaline phosphatase conjugate (Sigma) and BCIP/NBT substrate (Roche, Indianapolis, IN).

Nuclei Preparation and FISH Assay

Nuclei were prepared from leaves and protoplasts as previously described (Zhao et al., 2001). Nuclei were subjected to FISH analysis essentially as described (Avivi et al., 2004). Briefly, fixed nuclei (5–10 μl, kept at −20°C in ethanol:acetic acid/3:1) were spread on a slide, air-dried, and incubated in 100% ethanol for 1 hr at room temperature. Slides were then air-dried and incubated for 6 min in a fixative solution containing freshly prepared 4% paraformaldehyde in 1× SSC. Slides were washed three times, 5 min each, with 2× SSC and subjected to denaturation solution containing 70% formamide in 1× SSC at 60°C for 3 min followed by sequential washes, 3 min each, in 70, 95, and 100% cold ethanol. Slides were air-dried and used for hybridization. BACs (kindly provided by the Arabidopsis Biological Resource Center, ABRC) F2H10, T10P12, F2J6, and F28H19 (Chr 1) were labeled with tetramethylrhodamine-5-dUTP or with fluorescein-12-dUTP (Roche) using the Nick translation reaction essentially as described (Maniatis et al., 1982). Probes were mixed with a hybridization solution (final volume 100 μl) containing 10% (w/v) sodium dextran sulphate, 50% deionized formamide, and 2× SSPE, denatured at 90°C for 5 min, and cooled on ice. Each probe was added to a slide, covered with a cover glass, and incubated at 37°C in the dark for 16–20 hr followed by washings as described (Fransz et al., 1996). Slides were then stained for 10 min with 10 μg/ml diamidinophenylindole (DAPI), washed twice, and mounted in Vectashield (Vector Laboratories, Burlingame, CA).

Chromatin Immunoprecipitation (ChIP)

ChIP was performed on nuclei prepared from leaves and protoplasts of Arabidopsis (Ler ecotype) plants essentially as described (Lawrence et al., 2004) using anti-dimethylated K4 histone H3 (Upstate Biotechnology, East Syracuse, NY), and anti-dimethylated K9 histone H3 (Upstate Biotechnology). Precipitated DNAs were subjected to PCR using the following primers: CEN180-S, 5′-GAGAGGATCCCGTAAGAATTGTATCCTTGTTAG-3′CEN180-AS, 5′-GAGAGAATTCCCTTTAAGATCCGGTTGTGG-3′VIP1-S, 5′-TTACATAATTCCTTCAACTTTT-3′VIP1-AS, 5′-GAGGTTTTCGCAAACCTACC-3′FBP-S, 5′-TTTAACATTATCCCAAACAAA-3′FBP-AS, 5′-TGCTGCGTGATCCATCTTTATC-3′HDC-S, 5′-AACCATCTCTCTTTCTCCTC-3′HDC-AS, 5′-AGTTTGATCAGATTCCAAAGATCAt1g43560-S, 5′-GATAACAAATCCGATTTGTAAG-3′At1g43560-AS, 5′-ATAAAATTGGTTATAACCATTAGC-3′At1g43160-S, 5′- TAAGTCTAGCTAGTTTTGTTACT-3′At1g43160-AS, 5′-GGAGTCACTAGTTAATTCAAC-3′ChIP PCR conditions were 94°C, 5 min; 35 cycles (30 cycles for CEN180) of 94°C, 30 s; 56°C, 30 s; 72°C, 30 s; followed by 72°C, 5 min. PCR products were resolved on 1.2% agarose gel stained with ethidium bromide.

Confocal Laser Scanning Microscope (CLSM) Imaging

Zeiss LSM 510 Meta was used to capture all fluorescence images. A 488-nm laser was used for the excitation of both GFP and chlorophyll, and the received fluorescent light was split using a NFT565 beam splitter, and was detected simultaneously in two channels with BP500-550, and LP650 filters, respectively. Image processing and maximum intensity projections were done with Zeiss LSM Image Browser, Microsoft Powerpoint, and Preview software.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. ACKNOWLEDGMENTS
  8. REFERENCES

We thank The Arabidopsis Biological Resource Center (ABRC), E. Richards and S. Jacobsen S. for providing BAc clones and mutants, G. Granot G. and N. Sikron-Persi for technical assistance, and V. Ransbotyn for helping with bioinformatic analysis. This work was supported by The Israel Science Foundation (ISF) grant No. 476/09 to G. Grafi.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. ACKNOWLEDGMENTS
  8. REFERENCES