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Several MICRORNA genes belonging to same family or different families are often found in homologous or non-homologous clusters. Among the various classes, head-to-head arranged genes form one of the largest categories of non-canonically organized genes. Such head-to-head arranged, non-canonically organized genes possibly share cis-regulatory region with the intergenic sequence having the potential to function as bi-directional promoter (BDP). The transcriptional regulation of head-to-head arranged genes, especially with bidirectional promoters, remains an enigma. In the past, bidirectional promoters have been characterized for a small set of protein-coding gene pairs in plants; however, to the best of our knowledge, no such study has been carried so far for MICRORNA genes. The present study thus functionally characterizes bidirectional promoters associated with members of MIR395 family, which is evolutionary conserved and is most frequently occurring cluster across plant kingdom. In Arabidopsis thaliana, the MIR395 gene family contains six members with two head-to-head arranged gene pairs- MIR395A-B and MIR395E-F. This organization was found to be conserved at seven loci for MIR395A-B, and eleven loci for MIR395E-F in five Brassica sps. Sequence analysis of the putative bidirectional promoters revealed variation in length, GC content and distribution of strict TATA-box. Comparatively higher level of conservation at both the ends of the bidirectional promoters, corresponding to ca. 250 bp upstream of 5'end of the respective MIRNA precursor, was observed. These conserved regions harbour several abiotic stress (nutrient, salt, drought) and hormone (ABA, ethylene) responsive cis-motifs. Functional characterization of putative bidirectional promoters associated with MIR395A-B and MIR395E-F from Arabidopsis and their respective orthologs from Brassica juncea (Bj_A08 MIR395A-B, Bj_B03 MIR395A-B, Bj_A07.1 MIR395E-F and Bj_A07.2 MIR395E-F) was carried out using a dual-reporter vector with ß-glucuronidase (GUS) and Green Fluorescent Protein (GFP). Analysis of transcriptional regulation of the two reporter genes - GUS and GFP during developmental stages confirmed their bidirectional nature. Orientation-dependent differential reporter activity indicated asymmetric nature of the promoters. Comparison of the reporter activity amongst orthologs, paralogs and homeologs revealed regulatory diversification, an outcome expected in polyploid genomes. Interestingly, reporter gene activities driven by selected bidirectional promoters were also observed in anther and siliques apart vegetative tissues indicating role of miR395 in anther and fruit development. Finally, we evaluated the activity of reporter genes driven under transcriptional regulation of bidirectional promoters under normal and sulfate-deprived conditions which revealed asymmetric inducibility under sulfate-starvation, in agreement with the known role of miR395 in sulfate homeostasis.

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Unravelling genetic networks regulating developmental programs are key to devising and implementing genomics assisted trait modification strategies. It is crucial to understand the role of small RNAs, and the basis of their ability to modify traits. MIR159 has been previously reported to cause defects in anther development in Arabidopsis; however, the complete spectrum and basis of the defects remained unclear. The present study was therefore undertaken to comprehensively investigate the role of miR159 from Brassica juncea in modulating vegetative and reproductive traits. Owing to the polyploid nature of Brassica, paralogous and homeologous copies of MIR159A, MIR159B, and, MIR159C were identified and analysis of the precursor uncovered extensive structural and sequence variation. The MIR159 locus with mature miR159 with perfect target complimentarily with MYB65, was cloned from Brassica juncea var. Varuna for functional characterization by generating constitutively over-expressing lines in Arabidopsis thaliana Col-0. Apart from statistically significant difference in multiple vegetative traits, drastic differences were observed in stamen and pistil. Over-expression of miR159a led to shortening of filament length and loss of tetradynamous condition. Anthers were apiculate, with improper lobe formation, and unsynchronized cellular growth between connective tissue and another lobe development. Analysis revealed arrested meiosis/cytokinesis in microspores, and altered lignin deposition pattern in endothecial walls thus affecting anther dehiscence. In the gynoecium, flaccid, dry stigmatic papillae, and large embryo sac in the female gametophyte was observed. Over-expression of miR159a thus severely affected pollination and seed-set. Analysis of the transcriptome data revealed components of regulatory networks of anther and carpel developmental pathway, and lignin metabolism that are affected. Expression analysis allowed us to position the miR159a-MYB65 module in the genetic network of stamen development, involved in pollen-grain maturation; in GA-mediated regulation of stamen development, and in lignin metabolism. The study, on one hand indicates role of miR159a-MYB65 in regulating multiple aspects of reproductive organ development that can be manipulated for trait modification, but also raises several unaddressed questions such as relationship between miR159a and male-meiosis, miR159a and filament elongation for future investigations. Accession numbers: KC204951-KC204960. Project number PRJNA1035268. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01377-7.

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Myeloblastosis (MYB) family, the largest plant transcription factor family, has been subcategorised based on the number and type of repeats in the MYB domain. In spite of several reports, evolution of MYB genes and repeats remains enigmatic. Brassicaceae members are endowed with complex genomes, including dysploidy because of its unique history with multiple rounds of polyploidisation, genomic fractionations and rearrangements. The present study is an attempt to gain insights into the complexities of MYB family diversity, understand impacts of genome evolution on gene families and develop an evolutionary framework to understand the origin of various subcategories of MYB gene family. We identified and analysed 1129 MYBs that included 1R-, 2R-, 3R- and atypical-MYBs across sixteen species representing protists, fungi, animals and plants and exclude MYB identified from Brassicaceae except Arabidopsis thaliana; in addition, a total of 1137 2R-MYB genes from six Brassicaceae species were also analysed. Comparative analysis revealed predominance of 1R-MYBs in protists, fungi, animals and lower plants. Phylogenetic reconstruction and analysis of selection pressure suggested ancestral nature of R1-type repeat containing 1R-MYBs that might have undergone intragenic duplication to form multi-repeat MYBs. Distinct differences in gene structure between 1R-MYB and 2R-MYBs were observed regarding intron number, the ratio of gene length to coding DNA sequence (CDS) length and the length of exons encoding the MYB domain. Conserved as well as novel and lineage-specific intron phases were identified. Analyses of physicochemical properties revealed drastic differences indicating functional diversification in MYBs. Phylogenetic reconstruction of 1R- and 2R-MYB genes revealed a shared structure-function relationship in clades which was supported when transcriptome data was analysed in silico. Comparative genomics to study distribution pattern and mapping of 2R-MYBs revealed congruency and greater degree of synteny and collinearity among closely related species. Micro-synteny analysis of genomic segments revealed high conservation of genes that are immediately flanking the surrounding tandemly organised 2R-MYBs along with instances of local duplication, reorganisations and genome fractionation. In summary, polyploidy, dysploidy, reshuffling and genome fractionation were found to cause loss or gain of 2R-MYB genes. The findings need to be supported with functional validation to understand gene structure-function relationship along the evolutionary lineage and adaptive strategies based on comparative functional genomics in plants.

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The relationship between evolutionary history, organisation and transcriptional regulation of genes are intrinsically linked. These have been well studied in canonically organised protein-coding genes but not of MIRNAs. In the present study, we investigated the non-canonical arrangement of MIRNAs across taxonomic boundaries from algae to angiosperms employing a combination of genome organization, phylogeny and synteny. We retrieved the complete dataset of MIRNA from twenty-five species to identify and classify based on organisational patterns. The median size of cluster was between 2-5 kb and between 1-20 % of all MIRNAs are organized in head-to-head (with bidirectional promoter), head-to-tail (tandem), and overlapping manner. Although majority of the clusters are composed of MIRNA homologs, 25% of all clusters comprises of non-homologous genes with a potential of generating functional and regulatory complexity. A comparison of phylogeny and organizational patterns revealed that multiple independent events, some of which are species-specific, and some ancient, in different lineages, are responsible for non-canonical organization. Detailed investigation of MIR395 family across the plants revealed a complex origin of non-canonical arrangement through ancient and recent, segmental and local duplications; analysis of MIR399 family revealed major expansion occurred prior to monocot-dicot split, with few lineage-specific events. Evolution of "convergent" organization pattern of non-canonical arrangement originating from independent loci through recurrent event highlights our poor understanding of evolutionary process of MIRNA genes. The present investigation thus paves way for comparative functional genomics to understand the role of non-canonical organization on transcriptional regulation and regulatory diversity in MIRNA gene families.

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Whole-genome and segmental duplications coupled with sequence and functional diversification are responsible for gene family expansion, and morphological and adaptive diversity. Although broad contours of such processes are understood, detailed investigations on regulatory elements, such as miRNA-transcription factor modules, especially in non-model crop plants with complex genomes, are few. The present study was performed to understand evolutionary history of MIR159 family, and changes in the miRNA-binding site (MBS) of the targets MYB33, MYB65, and MYB101 that may affect post-transcriptional gene silencing. We established orthology and paralogy between members of MIR159 family by reconstructing the phylogeny based on 240 precursor sequences sampled across green plants. An unambiguous paralogous relationship between MIR159A and MIR159B was observed only in Brassicaceae which prompted us to analyze the origin of this paralogy. Comparative micro-synteny of ca. 100 kb genomic segments surrounding MIR159A, MIR159B, and MIR159C loci across 15 genomes of Brassicaceae revealed segmental duplication that occurred in the common ancestor of Brassicaceae to be responsible for origin of MIR159A-MIR159B paralogy; extensive gene loss and rearrangements were also encountered. The impact of polyploidy was revealed when the three sub-genomes-least fractionated (LF), moderately fractionated (MF1), and most fractionated (MF2) sub-genomes of Brassica and Camelina sativa-were analyzed. Extensive gene loss was observed among sub-genomes of Brassica, whereas those in Camelina were largely conserved. Analysis of the target MYBs revealed the complete loss of MYB33 homologs in a Brassica lineage-specific manner. Our findings suggest that mature miR159a/b /c are capable of targeting MYB65 across Brassicaceae, MYB33 in all species except Brassica, and MYB101 only in Arabidopsis thaliana. Comparative analysis of the mature miRNA sequence and the miRNA-binding site (MBS) in MYB33, MYB65, and MYB101 showed the complexity of regulatory network that is dependent on strict sequence complementarity potentially leading to regulatory diversity.

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AIM: To study the functional outcome in unstable Hangman s fracture managed with anterior decompression and stabilization with cervical locking plate and tricortical bone graft. MATERIALS AND METHODS: Between 2010 and 2016, 44 patients (range: 19-75 years) with unstable Hangman's fracture underwent anterior decompression and stabilization with cervical locking plate and tricortical bone graft in our institution. RESULT: According to the Levine and Edwards classification, all patients were unstable with Type IA 6 (13.6), Type IIA 35 (79.5%), Type II (0), and Type III (6.8). The mean period of follow-up was 17 months (range: 6-48 months). Neurological recovery was observed in all nine patients. All patients were relieved from axial pain. None of the patients received blood transfusion. All patients showed solid fusion with no complication related to bone graft and plate. CONCLUSION: The anterior C2/C3 discectomy, fusion, and stabilization with cervical locking plate and tricortical bone graft are feasible and safe method in treating HangmanÊs fracture, with the benefit of high primary stability, anatomical reduction, and direct decompression of the spinal cord.