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 [5
], 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 ). No GFP signal was observed in three transgenic lines of the candidate promoters (Table ), 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
]. 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 [61
]. Those “ubiquitous” promoters, however, are inadequate for meiotic purposes, because they drive gene expression in meiosis at an insufficient level [62
]. For example, fluorescent organelle markers [22
] 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%, [8
]]. 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 [10
]. Furthermore, the AtDMC1
promoter has been used for studies in young seedlings, yielding an efficient expression in a recombination reporter system [11
], 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 [63
]. 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 [5
]. 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 ). 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, [63
]]. 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
], 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
], 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 and Additional file 4, Table S1
) and used the software tool Pscan (Figure 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 [5
] 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 [46
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
], and this finding led to the identification of a MER3 homolog, ROCK-N-ROLLERS
) in Arabidopsis
, supporting that as in budding yeast, both the interference-sensitive and insensitive pathways of recombination crossovers exist in plants [72
]. 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 ). 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 ), 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.