ELONGATA3 is required for shoot meristem cell cycle progression in Arabidopsis thaliana seedlings

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ELONGATA3 is required for shoot meristem cell cycle progression in Arabidopsis thaliana seedlings Anna Skylar, Sean Matsuwaka, Xuelin Wu

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S0012-1606(13)00420-X http://dx.doi.org/10.1016/j.ydbio.2013.08.008 YDBIO6176

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Received date: 27 April 2013 Revised date: 9 August 2013 Accepted date: 12 August 2013 Cite this article as: Anna Skylar, Sean Matsuwaka, Xuelin Wu, ELONGATA3 is required for shoot meristem cell cycle progression in Arabidopsis thaliana seedlings, Developmental Biology, http://dx.doi.org/10.1016/j.ydbio.2013.08.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ELONGATA3 is required for shoot meristem cell cycle progression in

Arabidopsis thaliana seedlings Anna Skylar, Sean Matsuwaka, Xuelin Wu*I Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90089, USA *

Corresponding author. Tel.: +213 740 2165; fax: +213 740 8631. [email protected]

Abstract A key feature of the development of a higher plant is the continuous formation of new organs from the meristems. Originally patterned during embryogenesis, the meristems must activate cell division de novo at the time of germination, in order to initiate post-embryonic development. In a mutagenesis screen aimed at finding new players in early seedling cell division control, we identified ELONGATA3 (ELO3) as a key regulator of meristem cell cycle activation in Arabidopsis. Our results show that plants carrying a hypomorphic allele of ELO3 fail to activate cell division in the meristems following germination, which leads to seedling growth arrest and lethality. Further analyses suggest that this is due to a failure in DNA replication, followed by cell cycle arrest, in the meristematic tissue. Interestingly, the meristem cell cycle arrest in elo3 mutants, but not the later leaf developmental defects that have been linked to the loss of ELO3 activities, can be relieved by the addition of metabolic sugars in the growth medium. This finding points to a new role by which carbohydrate availability promotes meristem growth. Furthermore, growth arrested elo3 mutants suffer a partial loss of shoot meristem identity, which provides further evidence that cell cycle activities can influence the control of tissue identity.

Highlights x

ELO3 is required for cell cycle activation at the time of germination in Arabidopsis.

x

Metabolic sugar rescues meristem cell cycle activity in growth-arrested elo3.

x

ELO3 maintains STM expression in the shoot meristem.

x Keywords Arabidopsis, Shoot meristem, ELO3, Cell division

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Introduction Unlike in higher animals, where postembryonic development is largely limited to the increase in size of the organs that are patterned during embryogenesis, the development of a flowering plant revolves around a recurring theme of organ formation.

Plant

embryonic development achieves two goals: to set up the basic body axes as top (shoot) and bottom (root), and to set aside one group pluripotent cells near each end of the embryonic axis in the primary shoot and root apical meristems. After germination, a series of organs, such as leaves and flowers develop from the shoot apical meristem (SAM) on continuous stem axes, forming the above ground architecture (reviewed in Steeves and Sussex, 1989). Much knowledge has been gained in the past two decades regarding the molecular mechanisms involved in the specification of the SAM. Most of the known regulators can be grouped into two interacting pathways dominated by homeobox transcription factors. On one hand, SHOOTMERISTEMLESS (STM, Barton and Poethig, 1993) is expressed throughout the SAM and is required for preventing its differentiation (Long et al., 1996). On the other, WUSCHEL (WUS, Laux et al., 1996), which is expressed in the organizing center below the stem cell cluster, promotes stem cell proliferation. In addition, WUS activates the receptor kinase pathway encoded by the CLAVATA genes (Clark et al., 1996; Fletcher et al., 1999; Jeong et al., 1999; Mayer et al., 1998), which negatively regulates WUS expression to limit the size of stem cell population (Brand et al., 2002; Schoof et al., 2000). Among them, CLAVATA3 (CLV3) is expressed in the stem cell cluster and has been used as the molecular marker of stem cell activity (Brand et al., 2002). In addition, many other factors have been shown to interact with these two pathways to regulate SAM formation (reviewed in Rieu and Laux, 2009; Veit, 2004).

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After germination, the vegetative SAM must first reactivate cell division from a G1 arrest (Barroco et al., 2005), then increase its size by tissue proliferation while generating new leaves (Medford, 1992). Like other eukaryotic organisms, cell cycle decisions in higher plants rely on cyclin-dependent kinases (CDKs) and their interacting cyclins (CYCs) (Reviewed in De Veylder et al., 2007; Inzé and De Veylder, 2006). In Arabidopsis, all five mitotic CDK’s, CDKA;1, CDKB1;1, CDKB1;1, CDKB2;1, and CDKB2;2, have been shown to be involved in the establishment of a functional shoot meristem in the developing seedlings (Andersen et al., 2008; Barroco et al., 2005; Hemerly et al., 1995; Nowack et al., 2012).

In addition, many pathways regulate

meristem cell division and function by modulating the activities of the mitotic CDK and CYCs (Reviewed in Inagaki and Umeda, 2011; Komaki and Sugimoto, 2012). Compared to the patterning events, the molecular mechanisms that activate the cell cycle in the emerging seedlings are less understood. Previously we identified the Arabidopsis homeobox transcription factor STIMPY (STIP/WOX9, referred to as STIP below, Haecker et al., 2004; Wu et al., 2005) as an essential gene in promoting meristem growth and establishment during early seedling development (Skylar et al., 2010; Wu et al., 2005). Seedlings that lack STIP fail to initiate cell division in the meristematic tissues due to a G2 block, and STIP overexpression results in mis-regulated proliferation at the leaf margin (Wu et al., 2007; Wu et al., 2005). In an attempt to identify additional genes that control early seedling cell division, we carried out a forward genetic screen to isolate genetic suppressors of the STIP over-expression phenotype. Here we report the identification of ELONGATA3 (ELO3), the catalytic subunit of the highly conserved elongator complex in Arabidopsis (Nelissen et al., 2005; Otero et al., 1999; Wittschieben et al., 1999), as another essential gene in promoting cell cycle progression in seedling meristems following germination. We show that the loss of ELO3 results in meristem cell cycle arrest and a partial loss of shoot meristem identity. 3

Materials and Methods Plant materials Plants were grown in long days (16 hours light/8 hours darkness) under about 120 µE m-2 sec-1 light at 22°C. To observe seedling phenotypes, seeds were germinated on 1/2 Murashige Minimal Organics Medium (MS; Phytotechnology Lab) with 0.6% agar after two days of stratification at 4°C. 44 mM of various sugars were added to the media when their effects were assayed.

For testing sucrose’s ability to rescue the growth arrested

small elo3 seedlings, seeds were germinated on 1/2 MS medium.

Twenty growth

arrested small elo3 seedlings were transferred to MS-sucrose medium 3, 7, 10, 14, and 18 days after germination and observed for phenotypic rescue.

Positional cloning of the mutation in 00-11 Map-based cloning was used to identify the gene that was mutated in 00-11. The 00-11 mutant in Col-0 background was crossed to the wild-type Ler-1 plants. F2 and F3 plants displaying the mutant phenotype were tested with polymorphic markers to identify the non-recombining regions of Col-0. The markers were obtained from the Arabidopsis Information Resource (TAIR) sequence collections. Analysis of 1213 F2 and 114 F3 plants located the mutation to a 38.2 kb interval on chromosome 5 between nucleotide position 20453831 and 20492097, which contains 11 genes. Direct sequencing of the coding regions of these 11 genes from three different F3 mutants revealed only one C to T substitution, which resulted in an E to K conversion at amino acid 423 in the ELO3 gene (At5g50320). Based on the existing alleles in the literature, we designated this new allele as elo3-14. For genotyping purpose, the elo3-14 mutation was detected using 5’gtatatagagttcagcgtgatattcctat-3’ and 5’-attgacatcgcgacctgaattccagatt-3’ primers, followed by a HinfI digest of the PCR product.

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Plasmid Construction For ELO3 complementation, 4.5 kb of genomic DNA including the ELO3 coding region, 400 bp upstream of the start codon, and 550 bp downstream of the stop codon, were cloned into the binary vector pMX202 (Wu et al., 2003). 35S::ELO3 was generated by ligating a 4.2 kb genomic fragment, which contains the ELO3 coding region and 550 bp downstream of the stop codon, into the binary vector pCHF3 (Fankhauser and Chory, 1997). To over-express the Radical SAM and HAT domain separately, partial cDNA’s containing amino acid 1-327 or 373-565 was ligated into pCHF3. All constructs were transformed into both Col-0 and elo3-14 and phenotyped in 30-40 independent T1 lines for each construct/background combination. Histological and morphological analysis GUS activity staining was carried out as described (Sessions et al., 1999), using 2 mM potassium ferro and ferri cyanide, at 37qC for 12 to 14 hours. The GUS-stained seedlings were mounted in 30% glycerol for whole-mount analysis or embedded and sectioned to 8 m thickness. Histological staining of the shoot apex sections was carried out according to (Roeder et al., 2003). Whole-mount in situ was performed as described (Friml et al., 2003) except that 10ug/ml of proteinase K (Sigma-Aldrich # P2308) was used for digestion.

in situ

hybridization on tissue sections was performed as previously described (Lie et al., 2012). The Dig-labeled anti-sense probes for ELO3 and STM were generated by in vitro transcription using the respective full-length cDNA as the template. The histone H4 antisense probe was generated against the full-length cDNA of At5g56960, but will also

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hybridize to the remaining histone H4 genes in Arabidopsis due to the high sequence similarities. Samples were photographed on a Zeiss Axio Imager equipped with an AxioCam HRc camera and whole seedlings were imaged using a Leica M165FC stereomicroscope with a DFC295 camera.

Quantitative RT-PCR Total RNA was extracted using the Spectrum Total Plant RNA kit (Sigma), and the firststrand cDNA was obtained using the Enhanced Avian First Strand Synthesis Kit (Sigma). Real-time PCR was carried out with the SYBR-green (Molecular Probes) method on Opticon-2 MJ machines. The relative changes in gene expression levels were determined using the 2-''CT method. Each sample was done in at least two biological replicates, each with three technical replicates, and ACTIN8 was used for normalization. The primers used in this study are: CDKA;1: 5'-aacttctctaccggattataaatctgc-3' and 5'-attaacagcatttt*agaaaggagatcga-3'; CYCB1;1: 5'-acgaaaag*atggagaatatggtgca-3' and 5'-gtagattgcagaagcagctacc-3'; CYCD3;1: 5’-tcaagatttgtcgggtacctcccat-3’ and 5’-agttttcaccttttc*cttggttaagt-3’; CDKB1;2: 5’-ttggatgcatctttg*ccgagat-3’ and 5’-atacctgaaaatatgaagtagttgctgaaa-3’; CDKB1;1: 5’-ccatctcag*caaagatacaaccaac-3’and 5’-gctgatttgggtcttggtcg-3’; CYCA2;1: 5’-tctaaccatccttgg*aaccaaactctac-3’and 5’-ttggtgtgtatagcgataagagtgctt-3’; ACT8: 5’-ttccagcag*atgtggatctcta-3’ and 5’-agaaagaaatgtgatcccgtca-3’ Asterisk (*) indicates exon-intron boundary, and the ACT8 primers were described in (Puthiyaveetil and Allen, 2008).

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Results Identification of 00-11 as a suppressor of stip-D As described in our previous report, ectopic STIP expression in stip-D results in serrated leaf margins (Fig 1A middle, Wu et al., 2005), which indicates mis-regulated cell division patterns. In order to identify additional genes that are required for promoting cell division in young seedlings, we carried out an EMS mutagenesis in stip-D background and identified a mutant line 00-11 as a second-site suppressor of the stip-D leaf phenotype (Fig 1A right). Two classes of phenotype were observed in 00-11 single mutants soon after germination on MS media. 66% of the seedlings (N=1049) failed to initiate growth in both the shoot and the root (Fig 1F) and remained arrested in growth for one to two weeks before death. We refer to this group as the small class. The remaining 34% of 0011 seedlings do initiate post-embryonic development, however very slowly. Eight days after germination, the second pair of true leaves is visible in the wildtype seedlings (Fig 1D), while the large 00-11 mutants only have small first pair of true leaves and their cotyledons are significantly elongated (Fig 1E). We refer to this group as the large class. Although both the elongated leaf phenotype and slow growth persist throughout their life cycle, four weeks after germination, the large 00-11 mutants have generated the same number of leaves as the wildtype plants, and both genotypes have visible inflorescences (Fig 1M, compare to K). This indicates that the slow growth in the large 00-11 is mostly due to delayed leaf enlargement, instead of the rate of primordia initiation. In order to better understand the relationship between STIP and 00-11, we further examined the genetic interactions between STIP and 00-11 using both stip-D and a lossof-function allele of STIP, stip-1. At the seedling stage, stip-D 00-11 plants resemble the 00-11 single mutants except for the epinastic cotyledons (Fig 1C), which is typical of

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stip-D (Fig 1B). Like 00-11, two classes of phenotypes can be found in stip-D 00-11 double mutants: some arrest growth after germination, and the others have retarded development in both the shoot and the root (Fig 1C) and mostly fail to develop beyond the seedling stage. The stip-D 00-11 double mutants that do grow have a phenotype that is intermediate of stip-D and 00-11 (Fig 1P). A similar scenario was found in stip-1 0011 double mutants, where high seedling lethality was observed even after the stip-1 allele was rescued by exogenous sugar. The surviving few have leaves that resemble the 00-11 single mutants but suffer more severe developmental delays than 00-11 (Fig 1R, compare to M). The additive nature of the double mutant phenotypes suggests that STIP and 0011 likely operate in converging pathways in regulating early seedling development.

00-11 mutants have reduced tissue proliferation in the meristems In order to understand the cause of growth disruption in 00-11 mutants, we compared shoot meristem morphology between 8-day-old wildtype and 00-11 mutant seedlings using histological sections. At this developmental stage, the wildtype shoot meristem is already in a dome shape, as detected by the red Safranin O staining (Fig 1G). In comparison, a range of size reductions was found in the shoot meristems of 00-11 mutants. While the growth arrested small class 00-11 seedlings showed little or no meristematic tissue in the shoot apex (Fig 1I, J), the large class 00-11 seedlings have flatter and slightly smaller shoot meristems than those seen in wild type (Fig 1H). A similar change persists in large 00-11 mutants, where the mature inflorescence meristem is slightly flatter with a smaller stem cell cluster, as indicated by the CLV3::GUS reporter (Brand et al., 2002), than the wildtype meristem (Fig 1N, compare to L). Among the known shoot meristem size regulators, the CLV genes are required for negatively regulating the proliferation of the stem cell cluster in the shoot meristem (Clark et al., 1996), and the loss of CLV3 results in a grossly enlarged shoot meristem due to elevated WUS expression (Brand et al., 2002; Schoof et al., 2000). To investigate the 8

relationship between 00-11 and the CLV/WUS pathway, we generated 00-11 clv3-2 double mutants and again observed two classes of phenotypes. Some of the 00-11 clv3-2 appear to be identical to the small class of 00-11 seedlings, which enter growth arrest following germination. The remaining double mutant seedlings resemble the large class of 00-11 during early stages of vegetative development: elongated cotyledons and petioles, as well as extremely slow leaf growth. However, as these seedlings further develop, they evolve into a phenotype that is the combination of clv3-2 and the large 0011 phenotypes, with enlarged shoot meristems and elongated leaves.

This additive

phenotype in the double mutants led us to the conclusion that 00-11 carries a mutation in a gene that acts largely independently of the CLV3/WUS pathway.

00-11 carries a mutation in ELO3 Map-based cloning located the mutation in 00-11 to a 38 kb region on chromosome 5. Direct sequencing of the coding regions of the eleven genes located within this interval identified a C to T change in the 6th exon of ELONGATA3 (ELO3), which encodes a member of the highly conserved elongator complex (Nelissen et al., 2005; Otero et al., 1999; Wittschieben et al., 1999). At the protein level, ELO3 consists of two domains: a Radical SAM domain in the N-terminus and a histone acetyltransferase (HAT) domain in the C-terminus (Nelissen et al., 2005).

The mutation in 00-11 changes a highly

conserved Glu at amino acid position 423 to Lys (Fig 2A), which is expected to disrupt the function of the HAT domain. In line with the meristem phenotype seen in 00-11 seedlings (Fig 1 H-J), in situ hybridization using an anti-sense probe of ELO3 detected ELO3 mRNA in the meristematic regions of both the shoot and the root (Fig 2B-D, Nelissen et al., 2010). In the shoot, it is found in the meristem and the emerging leaves immediately after germination (Fig 2B), and this expression persists and expands into

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young leaves as the seedlings develop (Fig 2C).

In the root, ELO3 expression is

concentrated in the transition zone and the upper meristematic zone (Fig 2D). Complementation using the ELO3 genomic region completely rescued the 00-11 mutants, confirming that the mutation in ELO3 is responsible for generating the 00-11 phenotype. Based on the existing alleles in the literature, we designated 00-11 as elo314. Additionally, ELO3 over-expression under the constitutive CaMV35S promoter was able to fully rescue 00-11 to wild type, and the same transgene caused no phenotypic change in the wildtype plants. As different functions have been attributed to the radical SAM domain and the HAT domain of ELO3 and its homologs in other organisms, we further tested whether a single domain could complement the elo3-14 phenotype. No rescue of the elo3-14 mutant was observed when we over-expressed either domain alone under the CaMV35S promoter, suggesting that a complete ELO3 protein is required, although we cannot exclude the possibility that the truncated proteins were nonfunctional.

Loss of ELO3 results in the failure of meristem growth and maintenance Since ELO3’s role in meristem development has not been described, we focused the rest of this study on the small class of elo3-14 mutants (Fig 1F), in which the primary shoot meristem is very reduced or absent. To investigate whether this defect was caused by changes in meristem patterning, we compared the expression of CLV3 and STM, which represent the two main branches of the shoot meristem patterning pathway, in wildtype and elo3 mutant seedlings. Immediately following germination, no difference could be seen in CLV3 expression between the wildtype (Fig 3A) and elo3 (Fig 3E) seedlings, as detected by the activity of the CLV3::GUS reporter (Brand et al., 2002). One week later, as the wildtype shoot meristem grew into a dome-shaped structure, CLV3::GUS activities increased significantly (Fig 3C). In comparison, the CLV3::GUS domain was present but

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much smaller in elo3-14 of the same age (Fig 3G, compare to C). Surprisingly, we were able to detect CLV3::GUS activity in the mutant shoot meristem at least ten days after germination, indicating that the shoot stem cell population in the growth-arrested elo3 mutants remain active. A different situation was found with STM expression.

Three days after

germination, STM is expressed in the wildtype shoot meristem and is cleared from the emerging leaf primordia (Fig 3B). At this stage, the STM domain encompassed the whole shoot meristem region in the small elo3-14 seedlings (Fig 3F), which is consistent with their failure in leaf initiation. Four days later, while the STM domain expanded in the wildtype shoot meristem (Fig 3D), it was no longer detectable in the growth arrested elo3 mutant seedlings (Fig 3H). This observation showed that although the shoot meristem was correctly patterned in elo3, it is not able to fully maintain its identity after germination.

Small class elo3-14 mutants have replication defects in the meristem The results described above indicate that the observed meristem arrest phenotype in elo314 is caused by defects other than meristem patterning. Because elo3-14 was initially identified as a second-site suppressor of stip-D and other elo3 alleles have been shown to affect DNA synthesis in the growing leaves (Xu et al., 2012), we asked whether the growth arrest phenotype observed in small elo3-14 results from disruptions in meristem DNA replication. The first step in seedling cell cycle activation is DNA replication (Barroco et al., 2005), and new histone molecules must be synthesized during this process. Therefore, the expression levels of the histone genes are a good indication of DNA synthesis activities. Two days after germination, in situ hybridization using an antisense probe against the histone H4 genes detected abundant signals in the shoot apex of the wildtype

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seedlings, both in the meristem and the developing leaf primordia (Fig 4A).

In

comparison, the majority of the elo3-14 seedlings showed very little to no histone H4 RNA signal in the shoot apex (Fig 4C). The remaining samples, which most likely represent the large class of elo3-14, had slightly more histone H4 expression, but remained significantly below the wildtype levels. A very similar result was obtained in the root, where many cells in the wildtype root meristematic zone showed active histone H4 expression (Fig 4B) and nearly no signal could be detected in the roots of the small elo3 seedlings (Fig 4D). These results strongly suggest that DNA replication is not able to proceed in the meristems of the small elo3 seedlings. Because ELO3 is a subunit of the elongator complex, one possible explanation for the lack of histone H4 expression in elo3 mutants is a general failure in transcription activation of the cell cycle genes. We therefore compared the mRNA levels of selected CYC’s and CDK’s between newly emerged wildtype and elo3-14 seedlings using semiquantitative RT-PCR.

Three mitotic CDK’s and three CYC’s that are required for

different stages of cell cycle progression were included in this analysis. Twenty-four hours after radical protrusion, no significant difference could be detected in the mRNA levels of these six genes between the wildtype and the elo3 samples, confirming that the cells were able to activate the transcription of these core cell cycle regulators (Fig 4E). A different scenario emerged a day later, when the small elo3 seedlings began to display signs of growth arrest. Although no difference was found in CDKA;1 expression levels between the two samples, the two CDKB1’s and the three CYC’s all showed approximately fifty percent reduction in their mRNA levels in elo3 mutants (Fig 4F). This implies that, at this stage, the core cell cycle gene expression was being shut off at both the G1 to S and G2 to M transitions in the small elo3 mutants, most likely due to a DNA replication block.

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Exogenous metabolic sugars rescue meristem cell cycle progression in elo3 The phenotypes of small elo3-14 are inconsistent with the previous reports of the elo3 mutant phenotype, which resembled what we observed in the large class of elo3-14 (Nelissen et al., 2005; Xu et al., 2012). Although we cannot exclude the possibility that this is due to allelic differences, a closer look revealed that this discrepancy may be attributed to the different growth conditions used in each study. While it is clear that the small elo3 mutants would be seedling lethal and lost when grown on soil directly (Xu et al., 2012), the elo3 phenotype described by Nelissen and colleagues was based on seedlings grown on sucrose-containing media (Nelissen et al., 2005). This raised the possibility that the addition of sucrose in the growth media could rescue the meristem cell cycle defect observed in the small elo3 mutants. Consistent with this hypothesis, no small class of elo3-14 could be found when we germinated elo3-14 on medium containing 1.5% sucrose (N=498). In addition, glucose and fructose, but not sorbitol, were also able to confer the same rescue effect, suggesting that metabolic sugar is required in this process. As it has been demonstrated that metabolic sugars can stimulate the transcription of a number of core cell cycle genes (Riou-Khamlichi et al., 2000; Skylar et al., 2011), we reasoned that sugar treatment activates cell cycle gene expression in the small elo3 mutants. To test this hypothesis, we compared the expression levels of the six cell cycle regulators in two-day-old elo3 mutants germinated with or without exogenous sucrose. As shown in figure 4F, in wild type, sucrose had little effect in enhancing the transcription of CDKA;1, CYCD3;1, and CYCA2;1, but was able to significantly increase the level of CYCB1;1, CDKB1;1 and CDKB1;2, all of which are specific to the G2 to M phase of the cell cycle. In comparison, all six genes responded to sucrose stimulation in elo3 with two to eight folds of increases over their levels in siblings germinated without

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sucrose. In the cases of CDKA;1, CYCD3;1, and CDKB1;1, their expression levels were raised to approximately twice of the wildtype levels by the presence of sucrose. To determine whether the sucrose rescue effect only occurs at the time of germination, when cell cycle genes were already activated in elo3 mutant, we germinated elo3-14 without supplemented sugar, then transferred the growth arrested small elo3 seedlings to sucrose-containing medium at different time points.

In the small elo3

seedlings that were grown on MS medium for up to seven days, limited root elongation and cotyledon greening could be observed one to two days after the transfer. One week after the transfer, they were able to develop elongated cotyledons and the first pair of true leaves (Fig 5D, compare to B), a phenotype that is identical to that of the large elo3 mutants grown without sucrose (Fig 1E).

To determine how soon the cell cycle

regulators respond to the presence of exogenous sugar, we treated 7-day-old fully arrested small elo3-14 with 1.5% sucrose and compared the expression levels of the six core cell cycle genes before and after the treatment, using qRT-PCR. Compared to the two days it takes to have any visible phenotypic rescue in these seedlings, a moderate increase in the expression levels of all six genes could be detected after four hours of treatment. Twenty-four hours of treatment resulted in four to nine fold of increase in their mRNA levels (Fig 4G).

This led us to the conclusion that metabolic sugar

reactivates cell cycle progression after elo3 enters full growth arrest, and the timing of the initial response suggests that this is one of the early events during the sugar rescue. Further examination revealed that the sugar-rescued elo3 seedlings were able to develop nearly wildtype-sized shoot meristems with a clear increase in histone H4 expression (Fig 5H, compare to F), although it is still below the wildtype levels (Fig 5G). In addition, the stem cell cluster in the rescued elo3 seedlings remains significantly smaller than in the wildtype samples, as measured by the activities of the CLV3::GUS reporter (Fig 5L, compare to I-K). Both phenotypes are consistent with what we observed in the large elo3-14 mutants. 14

Discussion Since its initial identification as an RNA polymerase II-associated protein complex in yeast (Otero et al., 1999), the highly conserved elongator complex has been identified in both animals and plants (Hawkes et al., 2002; Nelissen et al., 2005). In addition to its role in histone acetylation and transcription elongation (Kim et al., 2002; Otero et al., 1999; Winkler et al., 2002), the elongator has been shown to be involved in tRNA modification (Esberg et al., 2006) and mouse zygotic paternal genome demethylation (Okada et al., 2010). In Arabidopsis, a number of processes including leaf development, ABA and drought response, and immune response, are affected by the loss of elongator activity (Chen et al., 2006; DeFraia and Mou, 2011; Nelissen et al., 2005; Zhou et al., 2009).

In this study, we present evidence that ELO3, the catalytic subunit of the

elongator complex in Arabidopsis (Nelissen et al., 2005; Wittschieben et al., 1999), is required for meristem cell cycle activation at the time of germination.

ELO3 plays two roles in seedling cell cycle regulation In a higher plant, the shoot meristem is the source of above-ground development. The activation of meristem cell cycle activities marks the beginning of post-embryonic organ formation and is essential for seedling viability.

Previous studies by Barroco and

colleagues demonstrated that the cells in mature seeds are arrested in G1 phase, and they must proceed to DNA replication and activate the expression of the core cell cycle genes at the time of germination in order to initiate seedling growth (Barroco et al., 2005). The expression analysis of core cell cycle regulators in elo3 mutants (Fig 4) led us to the conclusion that ELO3 is required during DNA replication leading into cell division in the meristems. The incomplete penetrance of the elo3-14 mutant phenotype, as represented by the survival of the large class of mutant seedlings, is most likely due to the

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hypomophic nature of this allele.

Furthermore, this function likely requires the

involvement of the elongator complex, since ELO3 over-expression does not cause any phenotypic change in wildtype plants. Additional studies are needed to explore the molecular mechanisms by which ELO3 and the elongator complex carry out such a diverse range of cellular functions, especially in regard to the involvement of the radical SAM domain in ELO3 during these processes. The first elo3 mutant alleles were isolated based on the changes in their leaf morphology (Nelissen et al., 2005). Further studies showed that the elongated leaf blades were most likely the result of reduced cell division activities in the growing leaf primordia, which has been linked to elevated DNA damage and defects in DNA replication (Xu et al., 2012). Therefore, ELO3 plays two roles in promoting cell division in the developing seedlings: meristem cell cycle activation earlier and tissue proliferation later in leaf primordial, both likely through regulating DNA replication.

A natural

question that arises from these two temporally separated functions of ELO3 is whether they are driven by the same molecular mechanism. We do not believe this is the case based on the following evidence. First, DEFORMED ROOTS AND LEAVES1 (DRL1) encodes a putative regulator of the elongator complex, and mutations in DRL1 result in nearly identical leaf growth defects as observed in the large elo3-14 mutants. However, in contrast to the reduced shoot meristems found in both the large and small elo3-14 mutants (Fig 1 H-J), the drl1 seedlings develop larger and more dome-shaped shoot meristems (Nelissen et al., 2003), suggesting that different mechanisms regulate meristem and leaf cell proliferation.

Moreover, although the exogenously supplied

sugars were able to overcome the cell cycle arrest in the small elo3-14 seedlings (Fig 4F,G), the rescued seedlings do not develop into wildtype plants even when they were maintained on sucrose-containing media (Fig 5D). Instead, they continued to exhibit all the defects that have been linked to the later ELO3 function, such as reduced cell division activities in the shoot apex (Fig 5H), smaller stem cell cluster in the shoot meristem (Fig 16

5L), and extremely slow organ growth (Nelissen et al., 2005; Xu et al., 2012). This partial rescue implies that ELO3 action in the meristem is likely to be different from that in the leaves. It is also worth noting that, unlike what happens during leaf development (Nelissen et al., 2010), the loss of STM expression in the small elo3 is not likely caused by reduced auxin signaling. This is because the STM domain and auxin foci have been shown to be in complementary patterns in the shoot apex (Heisler et al., 2005), which indicates that reduced auxin response should not lead to the loss of STM expression. Although it is unclear how metabolic sugars up-regulate cell cycle gene expression in growth arrested elo3, the timing of CYC and CDK transcriptional response suggests that it is not by direct regulation. Other evidence also showed that exogenous sugar affects the development of seedlings carrying mutations in the elongator complex. For example, other’s (Nelissen et al., 2005) and our own studies observed a severe delay of germination when elo3 seeds were grown with the amount of sucrose that does not affect wild type germination. In addition, a mutation in ELO1, another subunit of the elongator complex, has been reported to have defects in producing carbon assimilates or importing sucrose (Falcone et al., 2007). All these observations point to a complex interplay between the elongator complex and carbon source availability. Cell cycle activities act as a reversible control of tissue identity in the shoot meristem Both tissue identity and the ability to continue to divide are essential for the function of a meristem. In the case of the shoot meristem, the main patterning events take place during early embryogenesis (Long and Barton, 1998; Long et al., 1996; Mayer et al., 1998), and very little cell division occurs in the embryonic meristems (Medford, 1992). At the time of germination, both meristem identity and cell division need to be activated in order to initiate seedling development. It is generally believed that the patterning events, i.e. cell identities, will drive the cell cycle decisions.

However, recent studies raised the

possibility that cell cycle activities can also influence cell identity decisions (Bramsiepe et al., 2010; Gaamouche et al., 2010; Roeder et al., 2012). Previously, we showed that 17

the shoot meristem in a stip mutant arrests cell cycle in G2 and the expression of the stem cell markers, but not STM, were lost as growth arrest sets in (Wu et al., 2005). Here we found that, as a consequence of meristem cell cycle failure, the small elo3-1 seedlings gradually lose STM expression in the shoot meristem (Fig 3F,H).

However, CLV3

expression remains active in the elo3 mutant seedlings of the same age (Fig 3G). This finding, when taken together with the phenotype of stip elo3-14 double mutants and the different types of cell cycle defects caused by the loss of ELO3 or STIP, supports the conclusions that ELO3 and STIP act in two parallel pathways in regulating shoot meristem growth and maintenance post-embryonically and that the activities of the STM and CLV/WUS pathways are coordinated with different phases of the cell cycle. Interestingly, both the cell cycle arrest phenotype and the function of the shoot meristem in stip and elo3 mutants can be relieved by the exogenously provided metabolic sugar. This indicates that, in both cases, the partial loss of meristem identity is not permanent. Instead, the cells in the meristem can regain their appropriate identity when cell cycle is reactivated in the shoot meristem. Conclusions Our results show that, ELO3, the catalytic subunit of the elongator complex in Arabidopsis, is required for meristem cell cycle activation at the time of germination. Similar to its later role in leaf primordial tissue proliferation, ELO3 is likely to act during the DNA replication phase prior to the onset of mitosis in the meristem. In addition, ELO3 maintains STM expression in the shoot meristem, providing another example of how cell cycle activities control cell identity in the shoot meristem.

Acknowledgements We thank Linda Peng for technical assistance; Rüdiger Simon for gifts of materials; Dana Lynn for valuable comments on this manuscript. This work was supported by a grant from NSF (MCB-1122213) to X.W.

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Figures and Figure Legends: Figure 1. 00-11 mutant seedlings have meristem defects. (A) Rosette leaves of 6-week-old Col-0 (left), stip-D (middle), and stip-D 00-11 (right), 00-11 suppresses the leaf margin serration phenotype in stip-D; (B-C) 8-day-old stip-D (B) and stip-D 00-11 double mutant (C) seedlings; (D-F) 8-day-old Col-0 (D), large 00-11 (E), and small 00-11 (F) seedlings grown on MS medium. The development of both classes of 00-11 lags the wild type visibly. (G-J) Histological staining of longitudinal sections through the center of the shoot apices of Col-0 (G), large 00-11 (H), and two samples of the small 00-11 (I, J). The 00-11 mutants show a range of reductions in shoot meristem size and the number of leaves. (K-N) 4-week-old Col-0 (K) and the large class of 00-11 (M), with 00-11 showing elongate petioles and leaves. CLV3::GUS reporter activities (blue) in longitudinal sections through the center of the inflorescence meristems of Col-0 (L) and 00-11 (N) show that the mature shoot meristem in the large 00-11 mutants remain smaller than the wild type. (O-P) phenotype of 6-week-old stip-D (O) and stip-D 00-11 double mutant (P). (Q-R) phenotype of 5-week-old stip-1 (Q) and stip-1 00-11 double mutant (R). The scale bar represents 20 Pm. Figure 2. 00-11 carries a mutation in ELO3. (A) A schematic representation of ELO3 gene structure and the position of the predicted functional domains. The filled boxes represent the coding region, and the unfilled boxes are the untranslated regions. 00-11 is the result of a mis-sense mutation in the 6th exon. (B-C) in situ hybridization using an antisense ELO3 probe detects ELO3 mRNA in the shoot apex of 1-day-old (B) and 7-day-old (C) Col-0 seedlings.

The signal is

concentrated in the meristem and proliferating tissues.

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(D) Whole-mount in situ hybridization detects ELO3 mRNA in the root transition zone and meristematic zone. The scale bar represents 20 Pm. Figure 3. Small elo3-14 seedlings fail to maintain STM expression in the shoot. (A, C, E, G) CLV3::GUS activities in whole-mount Col-0 (A, C) and elo3-14 (E, G) seedlings. Similar levels of GUS activities can be detected in 1-day-old Col-0 (A) and elo3-14 (E) shoots. By 8-day-old, the Col-0 meristem and its CLV3::GUS domain have increased in size (C), while the meristem in elo3-14 remains flat and the CLV3::GUS domain is reduced compared to the earlier time point (G). (B, D, F, H) STM mRNA was detected using an antisense STM probe in Col-0 (B, D) and elo3-14 (F, H). In 3-day-old Col-0, STM is expressed in the shoot meristem but excluded from the emerging leaf primordial (B, arrow head marks the leaf primordia). At the same age, STM is found in a more uniformed pattern in elo3 meristem (F). In 7-day-old seedlings, STM remains active in the shoot meristem of Col-0 (D), but is not detectable in elo3 shoot (H). The scale bar represents 20 Pm. Figure 4. elo3-14 has DNA replication defects in the meristem. (A-D) in situ hybridization using an antisense histone H4 probe detects ample signal in the shoot apex (A) and the root meristem zone (B) in 2-day-old Col-0. Little or no histone H4 expression can be seen in either the shoot (C) or the root (D) of elo3 at the same age. The scale bar represents 20 Pm. (E) Relative mRNA levels of six core cell cycle regulators in 1-day-old Col-0 and elo314 grown on MS medium. The expression levels of each gene are normalized to ACT8. (F) Relative mRNA levels of six core cell cycle regulators in 2-day-old Col-0 and elo314, grown on medium with or without supplemented sucrose. The expression levels of

24

each gene in Col-0 grown without sucrose are artificially assigned the value of 1, and all the measurements are normalized to ACT8. (G) Relative mRNA levels of six core cell cycle regulators in 7-day-old growth arrested elo3-14, before and after 4 hrs and 24hrs of treatment with 1.5% sucrose. The expression levels of each gene before the treatment are artificially assigned the value of 1, and all the measurements are normalized to ACT8.

Figure 5. Exogenous sucrose rescues cell cycle arrest in elo3-14. (A-D) 8-day-old Col-0 (A, C) and elo3 (B, D) seedlings grown on medium with or without supplemental sucrose.

The small growth arrested elo3 (B) can resume

development in the presence of sucrose (D). (E-H) Histone H4 mRNA was detected by in situ hybridization on longitudinal sections through the center of apices of the corresponding seedlings in (A-D). Sucrose reactivates histone H4 expression in elo3 mutants (H, compare to F), however, it is still below the wildtype level (G). (I-L) CLV3::GUS activities in corresponding seedlings in (A-D). Longitudinal sections through the center of the shoot apices show that sucrose stimulates the growth of shoot meristem in elo3, although the CLV3 domain remains significantly smaller than wild type (L, compare to J and K). The scale bar represents 20 Pm.

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5