Identification of microspore-active promoters that allow targeted manipulation of gene expression at early stages of microgametogenesis in Arabidopsis
- David Honys†1, 2Email author,
- Sung-Aeong Oh†3,
- David Reňák1, 2, 4,
- Maarten Donders3,
- Blanka Šolcová5,
- James Andrew Johnson3,
- Rita Boudová1 and
- David Twell3
© Honys et al; licensee BioMed Central Ltd. 2006
Received: 12 September 2006
Accepted: 21 December 2006
Published: 21 December 2006
The effective functional analysis of male gametophyte development requires new tools enabling the spatially and temporally controlled expression of both marker genes and modified genes of interest. In particular, promoters driving expression at earlier developmental stages including microspores are required.
Transcriptomic datasets covering four progressive stages of male gametophyte development in Arabidopsis were used to select candidate genes showing early expression profiles that were male gametophyte-specific. Promoter-GUS reporter analysis of candidate genes identified three promoters (MSP1, MSP2, and MSP3) that are active in microspores and are otherwise specific to the male gametophyte and tapetum. The MSP1 and MSP2 promoters were used to successfully complement and restore the male transmission of the gametophytic two-in-one (tio) mutant that is cytokinesis-defective at first microspore division.
We demonstrate the effective application of MSP promoters as tools that can be used to elucidate gametophytic gene functions in microspores in a male-specific manner.
The male gametophyte of flowering plants displays a highly reduced structure of two or three cells at maturity and its development provides an excellent system to study many fundamentally important biological processes such as cell polarity, cell division and cell fate determination (reviewed by ). An increasing collection of mutations and genes have been characterized that act gametophytically and have been shown to be important for post-meiotic cell division during pollen development in Arabidopsis. These include MOR1/GEM1  and TIO , whose functions are essential for regular cell polarity and cytokinesis at first microspore division, termed pollen mitosis I. However, mutations in such essential genes cause gametophytic embryo sac defects and sporophytic lethality [2, 3]. This prevents the analysis of homozygous mutations and hinders the functional analysis of their role(s) in specific cell types such as microspores. The native promoters of essential genes such as TIO are not useful tools to examine effects of mis-expression of gene or protein domains during pollen development since their broad activities in other sporophytic tissues can be detrimental to early vegetative development prior to gametophytic development. Moreover the use of well characterized male-gametophyte-specific promoters such as LAT52 enables targeted manipulation of gene expression that is restricted to the vegetative cell during pollen maturation after pollen mitosis I (. Therefore we have faced a practical challenge to identify promoters that allow the targeted manipulation of gene expression in microspores.
A number of male-gametophyte-specific promoters that are active at different developmental stages are known. Most data are available for late pollen promoters. These include petunia chiA , tomato LAT52 and LAT59 [4, 6], rapeseed Bp10 , maize Zm13 [8, 9] and tobacco NTP303 . In Arabidopsis these include the TUA1 , AtPTEN1 , AtSTP6 , AtSTP9  and the late vegetative cell-specific AtVEX1  promoters. Among these, the tomato LAT52 promoter with demonstrated vegetative-cell-specific expression [4, 6] was shown to be highly active in number of plant species and has become widely used as a tool to drive pollen-specific expression [16–20]. More recently, promoters active in generative or sperm cells have been identified from lily  and Arabidopsis [15, 22, 23].
On the other hand, very few promoters have been identified that are active or specifically active at microspore stage. The tobacco NTM19 promoter is the only well characterized promoter that exhibits strict microspore-specific expression with no activity in mature pollen [24, 25]. From rapeseed, Bp4 mRNA was described as microspore-specific , but the Bp4 promoter was later shown to be active only after PMI . However the BnM3.4 promoter was active in tetrads and in free microspores . The potato invGF promoter is initiated in late microspores and is restricted to the male gametophyte . In Arabidopsis available microspore expressed promoters are also limited. The Arabidopsis BCP1 promoter is active in microspores and the tapetum  while the AtSTP2 promoter shows a pattern of activity similar to that of NTM19, but is initiated at tetrad stage .
Transcriptomic analyses based on various microarray experiments including those from isolated microspores and developing pollen now provide genome-wide expression profiles throughout plant development [31–33]. Taking advantage of these public databases, we have asked whether microarray data can be directly exploited in order to identify novel and potentially specific promoters that are first active in Arabidopsis microspores. In this study, we have selected and characterized the activity of three promoters, MSP1, MSP2, and MSP3 that were predicted to be specifically active in microspores and developing pollen. We demonstrate that the MSP1 and MSP2 promoters can drive functional protein expression in microspores in complementation experiments and the utility of MSP promoters as novel male-specific microspore expression tools in Arabidopsis.
Identification of candidate genes
The putative microspore-specific promoter sequences of these genes, At5g59040, At5g46795, At4g26440, were termed MSP1, MSP2 and MSP3 respectively. Selected genes encoded proteins with quite distinct cellular functions. At5g59040 encodes COPT3, a putative pollen-specific member of a copper transporter family . At4g26440 encodes AtWRKY34, a group I WRKY family transcription factor . On the contrary, At5g46795 encodes an expressed protein of unknown function.
Histochemical analysis of promoter specificity
To test the activity of the selected MSP regulatory sequences in vivo, approximately 1 kb upstream genomic fragments were amplified for each of the genes and cloned into the GATEWAY-compatible destination vector, pKGWFS7 . Three MSP-GFP::GUS constructs, MSP1 (At5g59040), MSP2 (At5g46795) and MSP3 (At4g26440), were introduced into wild type Arabidopsis plants and ~40 T1 transformants were analyzed histochemically for GUS activity in inflorescences, bud clusters and flowers. We found that 38/40 MSP1, 36/38 MSP2 and 18/34 MSP3 plants showed GUS expression in flower buds and/or in open flowers. GUS staining in MSP1 plants was clearly detectable in anthers of younger buds than those from MSP2 and MSP3, but gradually became weaker towards anthesis, while GUS expression from MSP2 and MSP3 remained high at later stages including in mature pollen grains.
We also examined GUS expression in seedlings using ~20 lines of T2 generation plants for each construct and found no expression from all three MSP promoters, apart from 5–10% of anomalous lines that showed patchy expression in leaves and roots, or weak expression in stamen vascular tissues. None of the lines examined showed GUS activity in 5 day old seedlings. However interestingly, we consistently observed GUS activity at the distal tip of cotyledons and leaves in 10 day old MSP1-GUS seedlings (Fig. 3V). In summary all 3 MSP promoters were found to be specifically or highly preferentially expressed in microspores, developing pollen and the tapetum (Fig. 3)
Test vectors were built in which MSP1 or MSP2 promoters drive the expression of full length TIO cDNA. pMSP1-TIO and pMSP2-TIO. Both vectors were transformed into tio-3 plants by floral dipping. Double selection for tio-3 (ppt) and pMSP-TIO (kanamycin) constructs led to the isolation of 19 nineteen transformants containing pMSP1-TIO and 10 containing pMSP2-TIO. Plants were screened for the frequency of wild type and mutant spores in mature pollen tetrads after DAPI staining. 18/19 lines from pMSP1-TIO and 4/10 from pMSP2-TIO showed an increase in the frequency of wild type pollen compared to heterozygous tio-3 plants (data not shown). This resulted in the frequent appearance of mature tetrads with three wild type spores and one mutant member, compared with heterozygous tio-3 plants that always showed a 2:2 segregation (Fig. 4).
% mutant pollen
We developed a strategy to identify promoters expressed specifically in Arabidopsis microspores by exploiting in silico analyses and in vivo functional analysis. We tested the specificity and timing of three candidate promoters by promoter-GUS fusion analysis. All three promoters were specifically expressed in anthers with the exception of MSP1 that also showed limited expression in the distal tips of cotyledons and true leaves. Within developing flowers their expression was restricted to microspores, developing pollen and tapetal cells. Furthermore, successful use of the MSP1 and MSP2 promoters for complementation of the tio-3 mutation demonstrated that both promoters directed functional expression in uninucleate microspores before pollen mitosis I. These promoters therefore provide new tools for the functional analysis of genes and proteins expressed during microspore development. Differences in expression profiles were observed between MSP1, that showed earlier expression and a decline in mature pollen, and MSP2 and MSP3, in which expression increased during pollen maturation. These differences in GUS expression profiles were not predicted by the MSP microarray expression profiles that were very similar. This result and the minor expression patterns observed in MSP1 seedlings highlights the need for experimental verification of specificity prior to further practical analyses.
In developing stamens, cell lineages that lead to male gametophytes and tapetal cells can both be traced to archesporial cells derived from the L2 layer of anther primordial . Moreover, there is strong dependence of microspore development on tapetal cell function. In this regard the co-regulation of gene expression in both microspores and tapetum that occurs at early stages of anther development is not surprising. In tobacco, a chalcone-synthase-like gene ( and a chimeric Ca2+calmodulin-dependent protein kinase ) follow this expression pattern. The Brassica campestris Bcp1 gene is also co-expressed in tapetum and microspores . However the corresponding Bgp1 upstream sequences that were active in both tapetum and microspores in B. campestris and Arabidopsis exhibited pollen-specific expression in tobacco. Therefore different cis-acting sequence elements appear to be responsible for coordinated gene expression in tapetum and microspores in Brassicaceae and Solanaceae families . In Arabidopsis, the ABORTED MICROSPOROGENESIS (AMS) gene encodes a MYC-class transcription factor from the basic helix-loop-helix gene family. AMS is coordinately expressed in tapetum and microspores . Interestingly, the MSP3 gene encodes a member of the WRKY transcription factor family that could also have a role in coordinated gene expression in tapetum and microspores.
Taken together, we have characterized three Arabidopsis microspore-expressed promoters MSP1, MSP2 and MSP3 with early expression profiles specific to the male gametophyte and tapetum. The MSP1 and MSP2 promoters were used successfully to complement cytokinesis functions required to complete microspore development. These tools can be applied to manipulate gene expression in microspores and tapetum without detrimental effects that may arise from undesirable gene expression in other sporophytic tissues.
Plant material and spore isolation
Arabidopsis plants were grown in controlled-environment cabinets at 21°C under illumination of 150 mmol m-2 s-1 with a 16-h photoperiod. For spore isolation, Arabidopsis ecotype Landsberg erecta (Ler) plants were used. Isolation of spores and pollen was described . Information on the purity of isolated fractions determined by light microscopy and 4,6-diamino-phenylindole staining and vital staining of isolated spore populations assessed by fluorescein-3,6-diacetate treatment were also described in . Roots were grown from plants in liquid cultures as previously described . Wild-type and transgenic seeds were sterilized according to published procedures [43, 44]. Plants were transformed by floral dipping .
DNA Chip Hybridization and data normalisation and selection of potential target genes
RNA isolation and hybridization of Affymetrix ATH1 genome arrays was described in . Twelve mixed and seventy-five sporophytic datasets used for comparison with the pollen transcriptome were obtained from the NASCArray database  through AffyWatch service . Sporophytic datasets represented seven vegetative tissues (seedlings, leaves, petioles, stems, roots, root hair zone and suspension cell cultures; . Mixed datasets originated from three experiments covering whole inflorescences and flower buds [31, 34] and whole flower development .
All gametophytic and sporophytic datasets were normalized using DNA-Chip Analyzer 1.3 (dChip)  as described previously . All raw and dChip-normalized transcriptomic datasets can be accessed and downloaded through the Arabidopsis Gene Family Profiler (aGFP) database .
PCR primers used
RT-PCR reverse primer
RT-PCR forward primers
Construction of promoter::GUS reporters
Construction of promoter::GUS reporters
To examine the precise gene expression, each tested gene promoter region was fused with GUS to generate the MSP::GUS reporter using GATEWAY cloning system according to manufacturer's instructions (Invitrogen, Carlsbad, CA). Promoter fragments were amplified by two-step PCR from Col-0 genomic DNA isolated from 3-week-old seedlings using the KOD HiFi DNA Polymerase (Novagen, Darmstadt, Germany). For the first step, specific primers with appended adapters complementary to AttB1 and AttB2 sequences were used to generate 1000-bp promoter regions of tested genes (Tab. 2). Purified PCR products were cloned via pDONR201 donor vector (Invitrogen, Carlsbad, CA) into pKGWFS7 destination vector (VIB, Gent, Belgium;  to generate recombinant destination clones pMSP1, pMSP2 and pMSP3. All the recombinant plasmids were transformed into Agrobacterium tumefaciens GV3101 . These strains were used to transform Arabidopsis thaliana ecotype Columbia using the floral dip method . Transgenic progenies were selected either on one-half strength Murashige and Skoog standard medium, supplemented with 50 mg/l kanamycin on 1-week-old seedlings.
Histochemical staining of GUS activity
Histochemical assays for GUS activity in T2 generation of Arabidopsis transgenic plants were performed according to the protocol described previously [44, 51]. Seedlings and inflorescences incubated for 48 h at 37°C in GUS buffer (100 mM Na-phosphate, pH 7.2; 10 mM EDTA, pH 8.0; 0.1% Triton X-100; 2 mM K3Fe [CN]6) supplemented with 1 mM 5-bromo-4-chloro-3-indolyl b-D-glucuronide (X-gluc). GUS stained floral buds were fixed in 70% FAA (Formaldehyde/Acetic acid/Ethanol/water, 5/5/63/27, v/v/v/v) for at least 24 h. After washing with 70% ethanol, samples were dehydrated gradually in the ethanol-butanol series and infiltrated with paraffin. 10 μm thin cross sections were prepared with a microtome (Finesse ME, Shandon). GUS staining patterns were recorded using a Nikon Eclipse E600 microscope (Nikon Instruments, Melville, NY). Images were processed using Adobe Photoshop software (version CS2; Adobe Systems, San Jose, CA).
The full-length TIO cDNA clone, pKS-TIONC19, was modified to insert the ~1 kb MSP1 or MSP2 promoter fragments upstream of the TIO coding sequence using AscI and NotI. MSP promoter-TIO cDNA fusion fragments were subcloned into the binary vector pER10  using AscI and PacI to produce pMSP1-TIO and pMSP2-TIO. Before transformation into Agrobacterium tumefaciens strain GV3101, constructs were verified by restriction enzyme digestion and sequencing. Individual phosphinothricin (ppt) resistant heterozygous tio-3 mutants were transformed by floral dipping . Transformants containing both tio-3 and MSP promoter-TIO T-DNA constructs were selected on 15 cm MS agar plates supplemented with 10 mg/l ppt, 50 mg/l kanamycin, and 200 mg/l cefotaxime. Complementation was analyzed by scoring tio-3 pollen phenotype after 4'-6-Diamidino-2-phenylindole (DAPI) staining and also by scoring the genetic transmission of tio-3 allele through male after test crosses to Col-0 or ms1-1 as described by [2, 53].
DH, DR and RB gratefully acknowledge the financial support from the Grant Agency of the Czech Republic (grant 522/06/0896) and from the Ministry of Education of the Czech Republic (grant LC06004). DT & SAO acknowledge grant support from the Biotechnology and Biological Sciences Research Council.
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