The present work presents an iin silicoi analysis of Upstream Regulatory Modules (URMs) of genes expressed in tapetum specific manner in dicotyledon and monocotyledon plants. In the current analysis, we identified several motifs conserved in these URMs of which ten were observed to be part of known icisi-elements using tools and databases like MEME, PLACE, MAST and TFSEARCH. We also identified that binding sites for two transcription factors, DOF and WRKY71 were found to be present in majority of the URMs.
Tapetum is the innermost layer of the anther wall of plants. It performs the function of a nourishing tissue that remains in continuity with the pollen mother cell through plasmadesmatal connections till the formation of meiocytes occurs in young anther. Tapetum varies from unilayer to multilayer in different plant species and can be uninucleate or multinucleate. Although tapetum cells form a single or at-most a few cell layers in the anther tissue, several studies have been carried out to understand how these cell layers develop and the functions played by them in pollen cell development [
TA29 from Nicotiana tabacum [
The present work is an attempt to identify conserved motifs/cis-elements present in genes expressing in the tapetum tissue of dicotyledon and monocotyledon plants. Further, putative TFs that may bind to these elements have also been predicted. Information generated from this work can be used for experimental validation.
Motif Based Sequence Analysis Suite, MEME suite ver. 4.9.1 [
A literature survey was carried out to identify genes that expressed in a tapetum or anther specific manner. A total of 34 genes, 24 from dicot and 10 from monocot plants were identified and used in the present analysis (
S. No. | Gene name | Accession No. (Acc)/Gene ID (GI) | Plant | Reference | |
---|---|---|---|---|---|
Dicots | 1 | A3 (Ata3) | GI: 3204096 | A. thaliana | DS* |
2 | A6 (Ata6) | GI: 22676 | A. thaliana | [ | |
3 | A9 (Ata9) | GI: 16143 | A. thaliana | [ | |
4 | Ap3 (Atap3) | GI: 940179 | A. thaliana | [ | |
5 | Agp1 (AtAgp1) | Acc: X68211 | A. thaliana | [ | |
6 | Tap2 (AmTap2) | Acc: X55434 | Antirrhinum majus | DS | |
7 | Tapnac | GI: 842404 | A. thaliana | [ | |
8 | Grp18 (At5g07520) | GI: 830645 | A. thaliana | DS | |
9 | Bp4a (BnBp4a) | Acc: X52874 | Brassica napus | [ | |
10 | Bp4c (BnBp4c) | Acc: X52874 | B. napus | [ | |
11 | M1 (Bnm1) | Acc: U86642 | B. napus | DS | |
12 | A9 (Bja9) | AF134410 | B. juncea | DS | |
13 | Bgp1 (BcBgp1) | Acc: X68210 | B. campestris | [ | |
14 | TA29 (NtTA29) | Acc: X52283 | Nicotiana tabacum | [ | |
15 | Ntp303 (NtNtp303) | Acc: X69440 | N. tabacum | [ | |
16 | Ntm19 (NtNtp19) | Acc: X88847 | N. tabacum | [ | |
17 | A37 (Nta37) | Acc: AY090039 | N. tabacum | DS | |
18 | Lat52 (LeLat52) | Acc: 15855 | Lycopersicon esculentum | [ | |
19 | Lat56 (LeLat56) | Acc: X56487 | L. esculentum | [ | |
20 | Lat59 (LeLat59) | Acc: X56488 | L. esculentum | [ | |
21 | TA29 (LeTA29) | GI: AM261325 | L. esculentum | DS | |
22 | Un_char protein/TA29-like | SolyC02g078370 | L. esculentum | DS | |
23 | TA29 (StTA29) | GI: PGSC0003DMG400041062 | Solanum tuberosum | DS | |
24 | Taz1 (PhTaz1) | Acc: AB063169 | Petunia hybrida | [ | |
Monocot | 25 | OsRa8 | Acc: AF042275 | Oryza sativa | [ |
26 | Osg6B | Acc: D21160 | O. sativa | [ | |
27 | Osg4B | Acc: D21159 | O. sativa | [ | |
28 | OsRts2 | Acc: U12171 | O. sativa | DS | |
29 | E1 (Ose1) | Acc: A23333 | O. sativa | Patent: WO 9213956 | |
30 | T42 (Ost42) | Acc: A23332 | O. sativa | Patent: WO 9213956 | |
31 | T72 (Ost72) | Acc: A23331 | O. sativa | Patent: WO 9213956 | |
32 | Af366296 (ZmAf366296) | Acc: AF366296 | Zea mays | DS | |
33 | Af366294 (ZmAf366294) | Acc: AF366294 | Z. mays | DS | |
34 | Af149016 (ZmAf366294) | Acc: AF149016 | Z. mays | [ |
*DS―Direct submission.
The sequences for the respective URMs were downloaded from NCBI website. The sequence files of dicots and monocots URMs thus generated were submitted separately at MEME Tool available online for analysis of conserved motifs. In order to identify the conserved motifs, MEME program was run with different parameters that defined the motif width (5 - 13, 6 - 10 or 6 - 14) and the total number of motifs to be generated was fixed at 10. After identifying the motifs generated using the different widths, it was observed that in most cases, the motif generated with 6 - 14 width encompassed those generated by 5 - 13 or 6 - 10 width. Thus, the 10 motifs generated with 6 - 14 width were taken for further analysis. The position of the motifs in the different URMs as generated by MEME for both datasets and the sequence of the motifs as identified by MAST are presented in
After identifying the conserved motifs in the two datasets of anther/tapetum specific genes, the next step was to analyze if these motifs corresponded to any known cis-elements of plant promoters. This was done by creating strings of the identified motifs and submitting it to the PLACE database. This led to the identification of several known cis-elements. This data was then manually curated and 10 known cis-elements were identified that are enlisted in
We then attempted to see if there was any information about TFs binding to these cis-elements. In order to do so, we first analyzed the presence of transcription factor binding sites using TFSEARCH tool. TFSEARCH searches highly correlated sequence fragments versus a TFMATRIX that is a transcription factor binding site profile database present in “TRANSFAC” database [
Of the 4 identified TFs, it was observed that only DOF and WRKY710S were found to have binding sites in majority of the URMs analyzed. The other 2 were represented in less than 20% of the analyzed URMs.
DOF stands for DNA binding with one finger domain proteins. They are a class of zinc finger transcription factors which are present in algae (Chlamydomonas reinhardtii) and moss (Physcomitrella patens) as single gene spreading
S. No. | Name of cis-element | Putative binding site | Function of the cis-element | TFs | Ref |
---|---|---|---|---|---|
1 | ARR1AT | NGATT; N = A/T/G/C (Motif 5 & 6 of dicot) | ARR1 is a response regulator; found in Arabidopsis; N = G/A/C/T; AGATT is also present in the promoter of rice non-symbiotic haemoglobin-2 (NSHB) gene. | None | [ |
2 | BOX-4 | ATTAAT (Motif 9 of dicot) | Part of a conserved DNA molecule involved in light responsiveness | None | [ |
3 | CAAT-BOX | CAAT (Motif 7 of dicot) | “CAAT promoter consensus sequence” found in legA gene of pea | None | [ |
4 | DOFCOREZM | AAAG (Motif 7 of monocot) | Core dof protein binding site was found in maize; zinc finger, and is unique to plants; Four cDNAs encoding Dof proteins, Dof1, Dof2, Dof3 and PBF, have been isolated maize; PBF is an endosperm specific Dof protein that binds to form prolamin box; and enhances transcription from the promoters of both cytosolic orthophosphate promoter and non-photosynthetic PEPC gene | DOF | [ |
5 | E-BOXNNAPA/ MYCCONSENSUAT | CANNTG; N = A/T/G/C (Motif 7 of dicot) (Motif 5 of monocot) | The site is present in the promoter of rd22 (dehydration-responsive gene) and in many other genes in Arabidopsis; Binding site of ATMYC2 (previously known as rd22BP1); N = A/T/G/C; MYC recognition sequence in CBF3 promoter; Binding site of ICE1 (inducer of CBF expression 1) that regulates the transcription of CBF/DREB1 genes in the cold in Arabidopsis. E-box of napA storage-protein gene of Brassica napus. This sequence is also known as RRE (R response element). | ICE1 | [ |
6 | GTGANTG10 | GTGA (Motif 5 of dicot) | “GTGA motif” found in the promoter of the tobacco late pollen gene g10 which shows homology to pectate lyase and is the putative homologue of the tomato gene lat56; Located between −96 and −93 | None | [ |
7 | POLLENLELAT52 | AGAAA (Motif 1 of monocot) | Regulatory element responsible for pollen specific activation of tomato lat52 gene; Found at −72 to −68 region. Also found in the promoter of tomato endo-beta-mannanase gene. | None | [ |
8 | RAV1AAT | CAACA (Motif 7 of dicot) | RAV1 binding consensus sequence, binds to DNA with bipartite sequence motifs of RAV1-A (CAACA) and RAV1-B (CACCTG); RAV1 protein contains AP2-like and B3-like domains which recognize the CAACA and CACCTG motifs, respectively; The expression level of RAV1 is relatively high in rosette leaves and roots in Arabidopsis | RAV1 | [ |
9 | WRKY710S | TGAC (Motif 3, 4 & 5 of dicot) | A core of TGAC-containing W-box is present in Amy32b promoter; is binding site of rice WRKY71, which acts a transcriptional repressor in the gibberellin signaling pathway; Parsley WRKY proteins bind specifically to TGAC-containing W box elements within the Pathogenesis-Related Class10 (PR-10) genes | WRKY71 | [ |
10 | 10PEHVPSBD | TATTC (Motif 4 of dicot) | “−10 promoter element” present in psbD gene promoter of barley; is required for expression of the psbD (plastid gene) encoding chlorophyll-binding protein of photosystem II reaction center, activated by blue/UV-A light/white light. | None | [ |
across lower and higher plants possessing multiple genes encoding the Dof domain containing protein [
WRKY71 belongs to WRKY family of transcription factors. They are reported to be present across lower eukaryotes (protista) to ferns (pteridophytes) and in plants [
The present analysis has led to the identification of certain elements and TFs that could regulate tapetum specific promoters. However, the role of these needs to be experimentally analysed. This can be done by a “loss-of-function” strategy in which the cis-elements in a given URM are mutated and changes in promoter activity, if any are analysed. In a second strategy, “gain-of function”, a given TF can be ectopically expressed and its influence on the activity of a given URM is recorded.
This work was supported by a grant from University of Delhi, New Delhi. PAS was supported by a research fellowship from Council of Scientific and Industrial Research (CSIR), New Delhi, India.
Sharma, P.A. and Burma, P.K. (2018) An in silico Analysis of Upstream Regulatory Modules (URMs) of Tapetum Specific Genes to Identify Regulatory cis-Elements and Transcription Factors. American Journal of Molecular Biology, 8, 13-25. https://doi.org/10.4236/ajmb.2018.81002