Characterization of a set of novel meiotically-active promoters in Arabidopsis
© Li et al.; licensee BioMed Central Ltd. 2012
Received: 9 March 2012
Accepted: 13 June 2012
Published: 9 July 2012
Homologous recombination, together with selection, laid the foundation for traditional plant breeding. The recombination process that takes place during meiotic cell division is crucial for the creation of novel variations of highly desired traits by breeders. Gaining control over this process is important for molecular breeding to achieve more precise, large-scale and quicker plant improvement. As conventional ubiquitous promoters are neither tissue-specific nor efficient in driving gene expression in meiocytes, promoters with high meiotic activities are potential candidates for manipulating the recombination process. So far, only a few meiotically-active promoters have been reported. Recently developed techniques to profile the transcriptome landscape of isolated meiocytes provided the means to discover promoters from genes that are actively expressed in meiosis.
In a screen for meiotically-active promoters, we examined ten promoter sequences that are associated with novel meiotic candidate genes. Each promoter was tested by expressing a GFP reporter gene in Arabidopsis. Characterization of regulatory regions revealed that these meiotically-active promoters possessed conserved motifs and motif arrangement. Some of the promoters unite optimal properties which are invaluable for meiosis-directed studies such as delivering specific gene expression in early meiosis I and/or meiosis II. Furthermore, the examination of homologs of the corresponding genes within green plants points to a great potential of applying the information from Arabidopsis to other species, especially crop plants.
We identified ten novel meiotically-active promoters; which, along with their homologs, are prime candidates to specifically drive gene expression during meiosis in plants and can thus provide important tools for meiosis study and crop breeding.
Meiosis is a key feature in the life cycle of flowering plants during which homologous chromosome pairing, synapsis and recombination are achieved [1–3]. Understanding the mechanisms of the meiotic process is crucial for not only the cell cycle regulation, but also plant breeding, because homologous recombination ensures genetic exchange between homologous chromosomes, generates the genetic variations, and maintains the inheritance of traits. Genome-wide gene expression analyses on isolated meiocytes revealed unique transcriptome-landscapes during male meiosis in the model systems of Arabidopsis and rice [4–7]. Over 1,000 protein coding genes demonstrated preferentially expression in male meiocytes, with a group of 55 genes that have mitochondrial genome origins, and 1,036 transposable element genes were up-regulated in male meiocytes . The observation suggested that there is likely a specific transcription-regulatory mechanism during meiosis. As the first step toward understanding the molecular mechanism, we focus on characterizing the function and regulatory elements in selected candidate meiosis-gene promoters of this study. The objectives are to find common regulatory features in meiotically-active promoters and to explore the potential for applying the promoters in plant meiosis studies and crop breeding.
A prerequisite for meiotical engineering is the availability of effective meiotically-active promoters. However, the widely used CaMV 35 S promoter is not efficient in meiocytes. For example, AtCDC45 encodes a protein required for the normal fertility of the model plant Arabidopsis, and when an AtCDC45-RNAi construct driven by the 35 S promoter was transformed into wild type, only 20 of 59 transformants became sterile (34%), whereas a greater percentage of sterile plants (61%, 45 of 74 transformants) could be obtained by replacing the 35 S promoter with the meiosis-specific DMC1 promoter .
So far, only a limited number of meiotically-active promoters has been reported and investigated. The expression of the meiotic recombination gene AtDMC1 has been reported to be restricted to meiotic cells in anthers and carpels, and a β-glucuronidase (GUS) reporter fused to an AtDMC1 promoter revealed that the reporter gene activity initiated at the stages where meiosis takes place . However, activity of the AtDMC1 promoter is not restricted to meiotic cells [9–11]. MS5 is a gene essential for male meiosis ; in situ hybridization showed that MS5 is localized specifically within anther cells undergoing meiosis .
In yeasts, rodents and human, the expression of genes in meiosis has been well studied [14–18]. The male meiocytes of Arabidopsis are of an extraordinary small size (1% of anther tissues) and are surrounded by somatic anther lobes, making the isolation and analysis of Arabidopsis meiocytes challenging. Recently, the application of effective meiocyte collection methods made it possible to investigate the meiotic transcriptome profile [5, 6], thus allowing the bulk isolation and characterization of meiotically-active promoters. In this study, we experimentally verified the activity of twelve meiotically-active promoters out of fifteen candidate promoters, including ten new promoters.
Transcriptional regulation is critical for many developmental processes, making it important to analyze the transcriptional control to better understand the mechanisms that control spatial and temporal patterning in development . The bulk isolation and characterization of meiotically-active promoters makes the study of important novel cis-regulatory motifs in these sequences feasible.
Comparative transcriptome analysis revealed similarity in meiocyte transcriptomes between organisms, for example, more than 500 single-copy genes are shared by meiotic cells of Arabidopsis, mouse (Mus musculus)  and fission yeast (Schizosaccharomyces pombe) , with a larger number of genes expressed in both mouse and fission yeast, or Arabidopsis and mouse. Therefore, analyzing meiotically-active promoters from Arabidopsis should provide not only clues of meiotic regulatory networks in Arabidopsis, but can also give hints for other species. These meiotically-active promoters or their close relatives could evolve into substantial tools for molecular breeding across species, especially in the plant kingdom where crop improvement is highly desired to cope with global climate changes and the increasing world population.
Identification of meiotically-active promoters
A List of gene IDs and associated primers for the 15 analyzed promoters
(pATDMC1) * pAT3G22880
(pMS5) * pAT4G20900
In control tests, the levels of fluorescence driven by 35 S promoters were determined, including a multicolored set of organelle markers of endoplasmic reticulum, Golgi apparatus, tonoplast, peroxisomes, mitochondria and plastids . The results showed that the 35 S promoter does not lead to the expression of a detectable level of fluorescence in meiotic cell columns, tetrads and pollen. As an example, Additional file 3, Figure S3 shows a signal from an endoplasmic reticulum marker  under the control of a 35 S promoter with dual enhancer elements (d35S) in somatic cells while being undetectable in meiotic cells.
Enriched regulatory elements in meiotically-active promoters
Gene expression is often regulated by the interaction of transcription factors and target cis-regulatory DNA elements in promoters. The identification of potential regulatory elements acting in meiotically-active promoters can be a useful tool for understanding regulatory networks [e.g. [20, 23–26]]. We scanned enriched cis-acting regulatory DNA elements (CREs) in the promoters of our study to obtain clues about possible co-regulation of meiotically-active genes.
Several enriched PLACE motifs are universal or structural CREs that seem also common in meiotically-active promoters, such as TATABOX5 , POLASIG1 and POLASIG3 [28–30]. Interestingly, ROOTMOTIFTAPOX1 , NODCON2GM , RAV1AAT , OSE2ROOTNODULE [34, 35] are all consensus CREs in root and nodule, pointing to a common property of these cells and meiocytes, likely due to their being either in the mitotic or the meiotic process. Many CREs are environment responsive motifs, for example, MYCCONSENSUSAT for cold [36–38], WRKY71OS for gibberellin and pathogenesis [39, 40], ACGTATERD1 and IBOXCORE for light [41, 42], MYBCORE for water stress , GT1GMSCAM4 for pathogen and salt  and WBOXNTERF3 for wounding . Additionally, there is a high similarity to motifs in the promoters of rice sperm-cell-specific genes: the examined meiotically-active promoters share 9 out of 10 common motifs associated with rice sperm cell-specific genes, whereas one detected motif (ROOTMOTIFTAPOX1, ATATT) was only shown in sperm cell-specific genes .
Among these motifs (Figure 4 and Additional file 5, Table S2), MA0044.1 (p = 1.52751e-02) and MA0045.1 (p = 4.32294e-02) are binding sites for the chromatin-associated proteins HMG-1 and HMG-I/Y . The high-mobility group proteins (HMG) are a group of chromosomal proteins that help with transcription, replication, recombination and DNA repair . MA121.1 (p = 9.30809e-02) is a binding site of ARR10, whose multifunctional domain is responsible for both nuclear localization and DNA binding ; MA0096.1 (p = 9.96644e-02) is a binding site of two flower-specific bZIP proteins . These motifs are likely basic elements that confer tissue- or developmental stage-specific activities to their promoters. Additionally, the motifs MA0128.1 (p = 9.03657e-03) and MA0129.1 (p = 4.67192e-02), which were implicated in abscisic acid (ABA)-mediated stress and light signaling , respectively, are found enriched in these promoters ( Additional file 5, Table S2), consistent with the notion that the meiotic process is sensitive to environmental factors and exogenous hormones including light and ABA [e.g. [52–55]].
Furthermore, we used MEME software to search for possible unknown CREs . Three consensus motifs were found present in these promoter sequences ( Additional file 6, Figure S4). These motifs are characterized by enrichment of adenine (or thymine in the reverse complement strands) ( Additional file 6, Figure S4). Similar results were obtained with MClip tool  (data not shown). These adenine-rich motifs could be specific binding sites of transcriptional factors and enhancers . Interestingly, the adenine-rich motifs were also found in promoter regions of 15 selected genes with a documented function in meiosis ( Additional file 7, Table S3 and Additional file 8, Figure S5).
Homologs of the examined meiotically-active genes
The objectives of this study were to identify functional promoters that drive gene expression in meiosis and to find the common cis-regulatory elements that are present in all promoters. Results for meiosis-I, during which homologous recombination occurs, are of special interest to predict homologous recombination-related promoters in crop plants. We have tested 15 promoters that are associated with candidate meiosis genes that were discovered by a previous RNA-Seq experiment on isolated meiocytes , which include 13 promoters of functionally unknown genes and two reported meiotic gene promoters (pDMC1 and pMS5). Among the 13 candidate promoters that have no documented function in meiosis, ten have shown meiotic activity (≈77%) by driving the expression of GFP signal in meiocytes, thus revealing that our preliminary data has a high reliability for isolating meiotically-active promoters (Table 1). No GFP signal was observed in three transgenic lines of the candidate promoters (Table 1), although their respective gene transcripts were up-regulated in meiocytes in the RNA-Seq study. This may be attributed to distinctions in developmental age, time of harvest, sensitivity of the used method, the chosen promoter region or that these promoters function in a chromosome positional dependent manner [59, 60]. Nevertheless, our work provided a significant number of promoters that can drive gene expression in meiosis. These promoters could evolve to be invaluable tools to drive meiotically-active expression in further fundamental meiosis studies as well as in applied molecular breeding.
Until now, most researchers use ubiquitous promoters such as the CaMV 35 S promoter to over-express genes in plants for functional analysis . Those “ubiquitous” promoters, however, are inadequate for meiotic purposes, because they drive gene expression in meiosis at an insufficient level . For example, fluorescent organelle markers  that are driven by a 35 S promoter demonstrated no signal in meiosis stages ( Additional file 3, Figure S3), although there is a strong GFP signal detected in somatic cells ( Additional file 3, Figure S3). In accordance with that, RNAi knock-down of a meiotic mutant is also achieved better by using the meiosis-specific DMC1 promoter than by using the 35 S promoter [61% vs 34%, ]. However, even the established and broadly used DMC1 promoter has its disadvantages in meiosis studies: Doutriaux et al. reported that AtDMC1 is expressed in mitotically active cells from a suspension culture and is even regulated during the mitotic cell cycle, linking it to the processes in proliferating cells . Furthermore, the AtDMC1 promoter has been used for studies in young seedlings, yielding an efficient expression in a recombination reporter system , which is in accordance with the expression data for AtDMC1 obtained with ATH1 microarray chips: The eFP Browser tool displays that DMC1 is also highly expressed in vegetative rosette leaves and especially in the shoot apex and seedling roots . Thus, there is no ultimately optimal meiosis-specific promoter in broad use yet.
The novel candidate meiotically-active promoters from this study should provide more powerful tools for a strict or specified meiotic expression. In our experimental setup, we first chose genes that are highly expressed in meiocytes . We then relied not only on the positive expression that we got with our GFP reporter in meiotic cells but also looked at other tissues, e.g. roots, leaves and stems to validate its nonexistence there. Therefore we defined the promoters here as “meiotically-active” or “homologous recombination-related”, although we cannot completely exclude promoter activity in specific developmental stages or special conditions not covered or detectable by our setup. The decision of which promoter might be best depends on the special application and the preferences of the user, for example if a low or high expression is desired or if the expression should be restricted to a very specific time point in meiosis.
Interestingly, we observed diversified expression patterns in different cell types of meiocytes (cell columns and dissociated meiocytes) resulting from the examined promoters (Table 1). Transgenic lines harboring pAT1G15320:GFP only showed a specific fluorescence signal in dissociated meiocytes but not in meiocyte cell columns ( Additional file 1, Figure S1), which suggested a preferential activity in meiosis-II or after homologous recombination. In contrast, pAT4G40020:GFP plants showed detectable GFP signals only in early meiosis-I meiocytes, pointing to a homologous recombination-specific promoter. In addition to our results that pAT4G40020 drives gene expression at a high level during early meiosis, microarray data of developmental stages indicates that At4G40020 is further expressed only in microspores [eFP Browser, ]. There is also microarray data available for some of our other candidate genes, but not for all of them. Thus, we can confirm their meiosis-specific expression with our experimental setup but cannot completely rule out expression outside meiosis under special conditions or in specific developmental stages. Taken together, we have identified and validated 12 meiotically-active promoters and two of these promoters can be used to specifically address questions regarding roughly meiosis-I (pAT4G40020) or meiosis-II (pAT1G15320). For molecular engineering, expressing genes during prophase-I, the stage of recombination, will be of utmost interest.
Given the complexity and a relatively long duration of meiosis (for example, prophase I lasts 21.3 h) , the temporal specificity of different promoters might be even more confined to individual meiotic stages. In future work, it will be important to test this possibility and investigate the expression even closer to obtain stage-specific promoters which are powerful tools to meet different requirements.
The confirmation of the meiotic activity of the examined promoters also points to a meiotic function of the respective genes. In addition to the already characterized genes AtDMC1 and MS5[9, 12, 65, 66], we have discovered an additional key gene with a role during meiosis by checking the T-DNA insertion mutants for the ten genes without documented function (unpublished data).
The gene transcription in eukaryotes is complex and is largely modulated by transcription factors that bind to regulatory elements within promoters. We scanned the identified promoter set for motifs with binding specificity for known transcription factors from the PLACE collection (Figure 3 and Additional file 4, Table S1) and used the software tool Pscan (Figure 4 and Additional file 5, Table S2). CREs that are common to the meiotically-active promoters from this study may reflect common binding sites for certain transcription factors that are required for meiotic activities (such as the binding sites of HMG-1, HMG-I/Y, ARR10 and bZIP910, Additional file 5, Table S2). It also provides a hint as to know how these promoters are shared by stimulus–response pathways (such as the binding sites of EMBP-1 and TGA1A, Additional file 5, Table S2). We also analyzed the promoters of 15 genes with a documented function in meiosis ( Additional file 7, Table S3) with Pscan. Although they are not all meiosis-specific under the criteria used in  Chen et al. (2010), the identified common elements include not only “basic element” such as HMG-1 binding sites, but also binding sites for proteins involved in gibberellin response and leaf development ( Additional file 9, Table S4). Therefore, it appears that the crosstalk between meiosis and environmental signals, especially hormone signals, are largely through their promoters. These identified CREs can also be further used to design the experimental verification of regulatory elements and the identification of transcriptional factors that regulate meiotically-active gene expression .
Since meiosis is a conserved process in all sexually reproducing eukaryotes, knowledge of gene function from one species could provide useful information transferable to other species. For example, studies in budding yeast (Saccharomyces cerevisiae) have revealed that a MER DNA helicase is required for the interference-sensitive pathway for crossover formation [67–71], and this finding led to the identification of a MER3 homolog, ROCK-N-ROLLERS (RCK) in Arabidopsis, supporting that as in budding yeast, both the interference-sensitive and insensitive pathways of recombination crossovers exist in plants [72, 73]. Analysis of the “family history” of the meiotically-active genes from our study found a wide distribution of homologous sequences in many species in green plants (Viridiplantae), especially in flowering plants (Figure 5). This result suggests a great prospect of transferring the information obtained from Arabidopsis into other plants, including important crops such as soybean, maize, rice and Sorghum. Since low copies of putative homologous genes of AtDMC1, AT4G40020 and AT2G21640 seem to exist (Figure 5), exploring their correspondent promoter sequences in other species should be quite straightforward. For AT2G28090, MS5, AT1G26510, AT1G15320 and AT1G64625, many homologous genes were found in other plants and which might make it more difficult to elucidate “true” homologs and use their promoters; a more appropriate strategy in this case is to try to extend the usage of the promoter sequences from Arabidopsis directly to other plants.
In conclusion, we report here a bulk identification and experimental verification of meiotically-active promoters. The information provides not only invaluable clues about the meiotic regulatory system, but also a potential tool for the application in model and crop plants. In future work, it will be interesting and important to explore the relative activity levels of each promoter.
In conclusion, the ten isolated promoter sequences were confirmed to specifically drive gene expression in meiocytes. The findings can provide important tools for meiosis studies and crop breeding, especially two of these promoters are prime candidates for meiosis directed-studies that desire an expression focused on early meiosis-I and/or meiosis-II.
Plant material and growth conditions
Arabidopsis thaliana (L.) Heynh. Ecotype Columbia (Col-0) was used in this study. Seeds were sown on 50% Sun Gro Professional Growing Mix and 50% Sun Gro SPECIAL BLEND growing medium (Sun Gro Horticulture, USA) and imbibed at 4°C for 3 days in the dark before moving them to long-day conditions (16 h light/8 h dark) at 22°C, 40 to 60% RH and 63 mE`·s-1·m-2 light intensity. The ER marker line ER-gk was obtained from the Arabidopsis Biological Resource Center (ABRC) .
Examination of GFP fluorescence
Young inflorescences were dissected with syringe needles and the anthers were collected using 1xPBS buffer. The meiocytes were squashed out between a microscope slide and a cover glass. The GFP fluorescence was observed under an ERNST LEITZ WETZLAR 307–143.004 microscope (Wetzlar, Germany) and photographed with a SPOT Insight 4 Camera (Diagnostic Instruments, USA).
Cloning, vector construction and plant transformation
To clone the promoters, the genomic sequences upstream of the candidate genes’ start codons were amplified using specific primers as listed in Table 1, in which SalI or PstI restriction sites were introduced for sense primers and NcoI sites for antisense primers. In order to prevent overlap with neighbouring genes or to get the appropriate primer binding sites, the cloned regions ranged from 0.6 Kb to 1.8 Kb, covering the key cis-elements of most promoters [75, 76]. The DMC1 promoter, which has a length of 3.0 Kb, is an exception, since it was cloned in our previous unpublished study. The promoters were cloned into the pCAMBIA1302 expression vector that contains the mgfp5 version of the Aequoria victoria GFP , substituting the CaMV 35 S promoter upstream of the GFP coding sequence. The sequences were confirmed and the plasmids were introduced into Agrobacterium tumefaciens C58. To test the constructs in planta, all plasmids were introduced to the Arabidopsis plants using a floral dip method . Transgenic plants were first screened on medium containing 40 mg/l hygromycin and transferred to soil, and further validated by PCR with the sense primers used in the promoter cloning (Table 1) and an antisense primer near the 5’ end of GFP (GTT GCA TCA CCT TCA CCC TCT).
Analysis of cis-regulatory promoter elements
Known CREs were found by analysis with the PLACE database (http://www.dna.affrc.go.jp/PLACE/) [79–81]. The Pscan program was used to search for significantly overrepresented elements http://184.108.40.206/pscan/, ]. 1000 bp regions with respect to the annotated transcription start site of promoters were analyzed. The frequency matrices and visual logos of the sequences were obtained from the JASPAR CORE database. The p-values were computed by Pscan after z-test. An element was considered to be significantly overrepresented if the p-value was less than 0.1.
We are grateful for the research funds that are provided by the Biotechnology Research and Development Corporation (BRDC) to CC, EFR. CC and EFR are also supported by a NSF-PGRP grant (ISO1025881). The authors would like to thank two anonymous reviewers for their valuable comments and suggestions to improve the quality of the paper, and Ross Peterson, Duane McDowell, Doug Brinkman and Roger Meissner for plant care.
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