School of Agriculture, Food and Ecosystem Sciences - Research Publications
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ItemProstate apoptosis response-4 mediates TGF-β-induced epithelial-to-mesenchymal transition.(Springer Science and Business Media LLC, 2014-02-06)A growing body of evidence supports that the epithelial-to-mesenchymal transition (EMT), which occurs during cancer development and progression, has a crucial role in metastasis by enhancing the motility of tumor cells. Transforming growth factor-β (TGF-β) is known to induce EMT in a number of cancer cell types; however, the mechanism underlying this transition process is not fully understood. In this study we have demonstrated that TGF-β upregulates the expression of tumor suppressor protein Par-4 (prostate apoptosis response-4) concomitant with the induction of EMT. Mechanistic investigations revealed that exogenous treatment with each TGF-β isoform upregulates Par-4 mRNA and protein levels in parallel levels of phosphorylated Smad2 and IκB-α increase. Disruption of TGF-β signaling by using ALK5 inhibitor, neutralizing TGF-β antibody or phosphoinositide 3-kinase inhibitor reduces endogenous Par-4 levels, suggesting that both Smad and NF-κB pathways are involved in TGF-β-mediated Par-4 upregulation. NF-κB-binding sites in Par-4 promoter have previously been reported; however, using chromatin immunoprecipitation assay we showed that Par-4 promoter region also contains Smad4-binding site. Furthermore, TGF-β promotes nuclear localization of Par-4. Prolonged TGF-β3 treatment disrupts epithelial cell morphology, promotes cell motility and induces upregulation of Snail, vimentin, zinc-finger E-box binding homeobox 1 and N-Cadherin and downregulation of Claudin-1 and E-Cadherin. Forced expression of Par-4, results in the upregulation of vimentin and Snail expression together with increase in cell migration. In contrast, small interfering RNA-mediated silencing of Par-4 expression results in decrease of vimentin and Snail expression and prevents TGF-β-induced EMT. We have also uncovered a role of X-linked inhibitor of apoptosis protein in the regulation of endogenous Par-4 levels through inhibition of caspase-mediated cleavage. In conclusion, our findings suggest that Par-4 is a novel and essential downstream target of TGF-β signaling and acts as an important factor during TGF-β-induced EMT.
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ItemCisplatin-induced caspase activation mediates PTEN cleavage in ovarian cancer cells: a potential mechanism of chemoresistance.(Springer Science and Business Media LLC, 2013-05-10)BACKGROUND: The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor protein is a central negative regulator of the PI3K/AKT signaling cascade and suppresses cell survival as well as cell proliferation. PTEN is found to be either inactivated or mutated in various human malignancies. In the present study, we have investigated the regulation of PTEN during cisplatin induced apoptosis in A2780, A270-CP (cisplatin resistant), OVCAR-3 and SKOV3 ovarian cancer cell lines. METHODS: Cells were treated with 10μM of cisplatin for 24h. Transcript and protein levels were analysed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting, respectively. Immunofluorescence microscopy was used to assess the intracellular localization of PTEN. Proteasome inhibitor and various caspases inhibitors were used to find the mechanism of PTEN degradation. RESULTS: PTEN protein levels were found to be decreased significantly in A2780 cells; however, there was no change in PTEN protein levels in A2780-CP, OVCAR-3 and SKOV3 cells with cisplatin treatment. The decrease in PTEN protein was accompanied with an increase in the levels of AKT phosphorylation (pAKT) in A2780 cells and a decrease of BCL-2. Cisplatin treatment induced the activation/cleavage of caspase-3, -6, -7, -8, -9 in all cell lines tested in this study except the resistant variant A2780-CP cells. In A2780 cells, restoration of PTEN levels was achieved upon pre-treatment with Z-DEVD-FMK (broad range caspases inhibitor) and not with MG132 (proteasome inhibitor) and by overexpression of BCL-2, suggesting that caspases and BCL-2 are involved in the decrease of PTEN protein levels in A2780 cells. CONCLUSION: The decrease in pro-apoptotic PTEN protein levels and increase in survival factor pAKT in A2780 ovarian cancer cells suggest that cisplatin treatment could further exacerbate drug resistance in A2780 ovarian cancer cells.
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ItemMCRiceRepGP: a framework for the identification of genes associated with sexual reproduction in rice(WILEY, 2018-10)Rice is an important cereal crop, being a staple food for over half of the world's population, and sexual reproduction resulting in grain formation underpins global food security. However, despite considerable research efforts, many of the genes, especially long intergenic non-coding RNA (lincRNA) genes, involved in sexual reproduction in rice remain uncharacterized. With an increasing number of public resources becoming available, information from different sources can be combined to perform gene functional annotation. We report the development of MCRiceRepGP, a machine learning framework which integrates heterogeneous evidence and employs multicriteria decision analysis and machine learning to predict coding and lincRNA genes involved in sexual reproduction in rice. The rice genome was reannotated using deep-sequencing transcriptomic data from reproduction-associated tissue/cell types identifying previously unannotated putative protein-coding genes and lincRNAs. MCRiceRepGP was used for genome-wide discovery of sexual reproduction associated coding and lincRNA genes. The protein-coding and lincRNA genes identified have distinct expression profiles, with a large proportion of lincRNAs reaching maximum expression levels in the sperm cells. Some of the genes are potentially linked to male- and female-specific fertility and heat stress tolerance during the reproductive stage. MCRiceRepGP can be used in combination with other genome-wide studies, such as genome-wide association studies, giving greater confidence that the genes identified are associated with the biological process of interest. As more data, especially about mutant plant phenotypes, become available, the power of MCRiceRepGP will grow, providing researchers with a tool to identify candidate genes for future experiments. MCRiceRepGP is available as a web application (http://mcgplannotator.com/MCRiceRepGP/).
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ItemCytochemistry of pollen development in Brachypodium distachyon(SPRINGER WIEN, 2014-08)Brachypodium distachyon is a widely recognized model plant belonging to subfamily Pooideae with a sequenced genome. To gain a better understanding of the male reproductive development in B. distachyon we examined pollen morphology and cytochemical changes of microspore cytoplasm from pollen mother cell stage to mature pollen using light, fluorescent and scanning electron microscopy. Our results show that B. distachyon exhibits a typical monocot-type pollen ontogeny. Meiosis in the pollen mother cells is accomplished by successive cytokinesis generating isobilateral tetrads. Cytochemical examination indicated that microspore cytoplasm contains variable amounts of insoluble carbohydrates and proteins at different developmental stages. Deposition of starch in the cytoplasm of microspores starts at the bicellular stage and continues till the mature pollen stage. The formation of the exine wall progresses by the deposition of sporopollenin from the tapetum layer of the anther. The mature pollen is trinucleate, spheroidal in shape and possesses a single pore with an annulus and operculum. The exine pattern is smooth and of granular type.
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ItemGenomic and molecular analysis of conserved and unique features of soybean PIF4(NATURE PORTFOLIO, 2018-08-22)Phytochrome-interacting factor 4 (PIF4) participates in light signaling by interacting with photoreceptors, phytochromes, and cryptochromes. Although well characterized in Arabidopsis, PIF4's role in crop plants is unknown. Here we performed the first integrated genomics, transcriptomics, and molecular characterization of PIF4 in soybean (Glycine max) plants. Fifteen identified Glycine max PIFs (GmPIFs) grouped into PIF3, PIF4, and PIF8 subfamilies based on their phylogenetic relationships. The GmPIF4 subfamily formed two distinct clades (GmPIF4 I and GmPIF4 II) with different amino acid sequences in the conserved bHLH region. Quantitative transcriptional analysis of soybean plants exposed to different photoperiods and temperatures indicated that all PIF4 I clade GmPIF4s conserved PIF4-like expression. Three out of four GmPIF4 transcripts of the GmPIF4 I clade increased at 35 °C compared to 25 °C under short day conditions. RNA sequencing of soybeans undergoing floral transition showed differential regulation of GmPIF4b, and ectopic GmPIF4b expression in wild type Arabidopsis resulted in an early flowering phenotype. Complementation of GmPIF4b in Arabidopsis pif4-101 mutants partially rescued the mutant phenotype. PIF4 protein levels peaked before dawn, and a GmPIF4b protein variant was observed in soybean plants treated at high temperatures.
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ItemTranscriptome-wide profiling and expression analysis of transcription factor families in a liverwort, Marchantia polymorpha(BMC, 2013-12-23)BACKGROUND: Transcription factors (TFs) are vital elements that regulate transcription and the spatio-temporal expression of genes, thereby ensuring the accurate development and functioning of an organism. The identification of TF-encoding genes in a liverwort, Marchantia polymorpha, offers insights into TF organization in the members of the most basal lineages of land plants (embryophytes). Therefore, a comparison of Marchantia TF genes with other land plants (monocots, dicots, bryophytes) and algae (chlorophytes, rhodophytes) provides the most comprehensive view of the rates of expansion or contraction of TF genes in plant evolution. RESULTS: In this study, we report the identification of TF-encoding transcripts in M. polymorpha for the first time, as evidenced by deep RNA sequencing data. In total, 3,471 putative TF encoding transcripts, distributed in 80 families, were identified, representing 7.4% of the generated Marchantia gametophytic transcriptome dataset. Overall, TF basic functions and distribution across families appear to be conserved when compared to other plant species. However, it is of interest to observe the genesis of novel sequences in 24 TF families and the apparent termination of 2 TF families with the emergence of Marchantia. Out of 24 TF families, 6 are known to be associated with plant reproductive development processes. We also examined the expression pattern of these TF-encoding transcripts in six male and female developmental stages in vegetative and reproductive gametophytic tissues of Marchantia. CONCLUSIONS: The analysis highlighted the importance of Marchantia, a model plant system, in an evolutionary context. The dataset generated here provides a scientific resource for TF gene discovery and other comparative evolutionary studies of land plants.
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ItemSpatial expression of CLAVATA3 in the shoot apical meristem suggests it is not a stem cell marker in soybean(OXFORD UNIV PRESS, 2013-12)CLAVATA3 (CLV3), a stem cell marker in Arabidopsis thaliana, encodes a secreted peptide that maintains the stem cell population within the shoot apical meristem. This work investigated the CLV3 orthologue in a major legume crop, soybean (GmCLV3). Instead of being expressed in the three outermost layers of the meristem as in Arabidopsis, GmCLV3 was expressed deeper in the central zone beneath the fourth layer (L4) of the meristem, overlapping with the expression of soybean WUSCHEL. Subsequent investigation using an alternative stem cell marker (GmLOG1) revealed its expression within layers L2-L4, indicating that GmCLV3 is not a stem cell marker. Overexpression studies of GmCLV3 in Arabidopsis and complementation of clv3-2 mutant suggest similar functional capacity to that of Arabidopsis CLV3. The expression of soybean CLV1, which encodes a receptor for CLV3 in Arabidopsis, was not detectable in the central zone of the meristem via reverse-transcription PCR analysis of amplified RNA from laser-microdissected samples or in situ, implicating a diverged pathway in soybean. This study also reports the novel expression of GmLOG1 in initials of axillary meristem in the boundary region between the SAM and developing leaf primordia, before the expression of GmWUS or GmCLV3, indicating cytokinin as one of the earliest signals in initiating and specifying the stem cell population.
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ItemNovel members of the AGAMOUS LIKE 6 subfamily of MIKCC-type MADS-box genes in soybean(BMC, 2013-07-20)BACKGROUND: The classical (C) MIKC-type MADS-box transcription factors comprise one gene family that plays diverse roles in the flowering process ranging from floral initiation to the development of floral organs. Despite their importance in regulating developmental processes that impact crop yield, they remain largely unexplored in the major legume oilseed crop, soybean. RESULTS: We identified 57 MIKC(c)-type transcription factors from soybean and determined the in silico gene expression profiles of the soybean MIKC(c)-type genes across different tissues. Our study implicates three MIKC(c)-type transcription factors as novel members of the AGAMOUS LIKE 6 (AGL6) subfamily of the MIKC(C)-type MADS-box genes, and we named this sister clade PsMADS3. While similar genes were identified in other legume species, poplar and grape, no such gene is represented in Arabidopsis thaliana or rice. RT-PCR analysis on these three soybean PsMADS3 genes during early floral initiation processes revealed their temporal expression similar to that of APETALA1, a gene known to function as a floral meristem identity gene. However, RNA in situ hybridisation showed that their spatial expression patterns are markedly different from those of APETALA1. CONCLUSION: Legume flower development system differs from that in the model plant, Arabidopsis. There is an overlap in the initiation of different floral whorls in soybean, and inflorescent meristems can revert to leaf production depending on the environmental conditions. MIKC(C)-type MADS-box genes have been shown to play key regulatory roles in different stages of flower development. We identified members of the PsMADS3 sub-clade in legumes that show differential spatial expression during floral initiation, indicating their potential novel roles in the floral initiation process. The results from this study will contribute to a better understanding of legume-specific floral developmental processes.
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ItemThe Dynamics of Soybean Leaf and Shoot Apical Meristem Transcriptome Undergoing Floral Initiation Process(PUBLIC LIBRARY SCIENCE, 2013-06-06)Flowering process governs seed set and thus affects agricultural productivity. Soybean, a major legume crop, requires short-day photoperiod conditions for flowering. While leaf-derived signal(s) are essential for the photoperiod-induced floral initiation process at the shoot apical meristem, molecular events associated with early floral transition stages in either leaves or shoot apical meristems are not well understood. To provide novel insights into the molecular basis of floral initiation, RNA-Seq was used to characterize the soybean transcriptome of leaf and micro-dissected shoot apical meristem at different time points after short-day treatment. Shoot apical meristem expressed a higher number of transcripts in comparison to that of leaf highlighting greater diversity and abundance of transcripts expressed in the shoot apical meristem. A total of 2951 shoot apical meristem and 13,609 leaf sequences with significant profile changes during the time course examined were identified. Most changes in mRNA level occurred after 1short-day treatment. Transcripts involved in mediating responses to stimulus including hormones or in various metabolic processes represent the top enriched GO functional category for the SAM and leaf dataset, respectively. Transcripts associated with protein degradation were also significantly changing in leaf and SAM implicating their involvement in triggering the developmental switch. RNA-Seq analysis of shoot apical meristem and leaf from soybean undergoing floral transition reveal major reprogramming events in leaves and the SAM that point toward hormones gibberellins (GA) and cytokinin as key regulators in the production of systemic flowering signal(s) in leaves. These hormones may form part of the systemic signals in addition to the established florigen, FLOWERING LOCUS T (FT). Further, evidence is emerging that the conversion of shoot apical meristem to inflorescence meristem is linked with the interplay of auxin, cytokinin and GA creating a low cytokinin and high GA environment.