scholarly journals Genome-wide survey of the phosphofructokinase family in cassava and functional characterization in response to oxygen-deficient stress

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Haiyan Wang ◽  
Pingjuan Zhao ◽  
Xu Shen ◽  
Zhiqiang Xia ◽  
Xincheng Zhou ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Expression Profiles ◽  
Functional Characterization ◽  
Glycolytic Pathway ◽  
Transcriptional Level ◽  
Subcellular Localisation ◽  
Hypoxic Stress ◽  
Protein Fusion ◽  
Waterlogging Stress ◽  
Cassava Root

Abstract Background Glycolytic pathway is common in all plant organs, especially in oxygen-deficient tissues. Phosphofructokinase (PFK) is a rate-limiting enzyme in the glycolytic pathway and catalyses the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. Cassava (M. esculenta) root is a huge storage organ with low amount of oxygen. However, less is known about the functions of PFK from M. esculenta (MePFK). We conducted a systematic analysis of MePFK genes to explore the function of the MePFK gene family under hypoxic stress. Results We identified 13 MePFK genes and characterised their sequence structure. The phylogenetic tree divided the 13 genes into two groups: nine were MePFKs and four were pyrophosphate-fructose-6-phosphate phosphotransferase (MePFPs). We confirmed by green fluorescent protein fusion protein expression that MePFK03 and MePFPA1 were localised in the chloroplast and cytoplasm, respectively. The expression profiles of the 13 MePFKs detected by quantitative reverse transcription polymerase chain reaction revealed that MePFK02, MePFK03, MePFPA1, MePFPB1 displayed higher expression in leaves, root and flower. The expression of MePFK03, MePFPA1 and MePFPB1 in tuber root increased gradually with plant growth. We confirmed that hypoxia occurred in the cassava root, and the concentration of oxygen was sharply decreasing from the outside to the inside root. The expression of MePFK03, MePFPA1 and MePFPB1 decreased with the decrease in the oxygen concentration in cassava root. Waterlogging stress treatment showed that the transcript level of PPi-dependent MePFP and MeSuSy were up-regulated remarkably and PPi-dependent glycolysis bypass was promoted. Conclusion A systematic survey of phylogenetic relation, molecular characterisation, chromosomal and subcellular localisation and cis-element prediction of MePFKs were performed in cassava. The expression profiles of MePFKs in different development stages, organs and under waterlogging stress showed that MePFPA1 plays an important role during the growth and development of cassava. Combined with the transcriptional level of MeSuSy, we found that pyrophosphate (PPi)-dependent glycolysis bypass was promoted when cassava was under waterlogging stress. The results would provide insights for further studying the function of MePFKs under hypoxic stress.

scholarly journals Genome-wide survey of the phosphofructokinase family in cassava and functional characterization in response to oxygen-deficient stress

2020 ◽  
Author(s):  
Haiyan Wang ◽  
Pingjuan Zhao ◽  
Xu Shen ◽  
Zhiqiang Xia ◽  
Xincheng Zhou ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Expression Profiles ◽  
Functional Characterization ◽  
Glycolytic Pathway ◽  
Transcriptional Level ◽  
Subcellular Localisation ◽  
Hypoxic Stress ◽  
Protein Fusion ◽  
Waterlogging Stress ◽  
Cassava Root

Abstract Background: Glycolytic pathway is common in all plant organs, especially in oxygen-deficient tissues. Phosphofructokinase (PFK) is a rate-limiting enzyme in the glycolytic pathway and catalyses the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. Cassava (Manihot esculenta) root is a huge storage organ with low amount of oxygen. However, less is known about the functions of PFK from M. esculenta (MePFK). We conducted a systematic analysis of MePFK genes to explore the function of the MePFK gene family under hypoxic stress.Results: We identified 13 MePFK genes and characterised their sequence structure. The phylogenetic tree divided the 13 genes into two groups: nine were MePFKs and four were pyrophosphate-fructose-6-phosphate phosphotransferase (MePFPs). We confirmed by green fluorescent protein fusion protein expression that MePFK03 and MePFPA1 were localised in the chloroplast and cytoplasm, respectively. The expression profiles of the 13 MePFKs detected by quantitative reverse transcription polymerase chain reaction revealed that MePFK02, MePFK03, MePFPA1, MePFPB1 and MePFPA2 displayed higher expression in leaves, root and flower. The expression of MePFK03, MePFPA1 and MePFPB1 in tuber root increased gradually with plant growth. We confirmed that hypoxia occurred in the cassava root, and the concentration of oxygen was sharply decreasing from the outside to the inside root. The expression of MePFK03, MePFPA1 and MePFPB1 decreased with the decrease in the oxygen concentration in cassava root. Waterlogging stress treatment showed that the transcript level of PPi-dependent MePFP and MeSuSy were up-regulated remarkably and PPi-dependent glycolysis bypass was promoted. Conclusion: A systematic survey of phylogenetic relation, molecular characterisation, chromosomal and subcellular localisation and cis-element prediction of MePFKs were performed in cassava. The expression profiles of MePFKs in different development stages, organs and under waterlogging stress showed that MePFPA1 plays an important role during the growth and development of cassava. Combined with the transcriptional level of MeSuSy, we found that pyrophosphate (PPi)-dependent glycolysis bypass was promoted when cassava was under waterlogging stress. The results would provide insights for further studying the function of MePFKs under hypoxic stress.

scholarly journals Functional Characterization of a Drought-Responsive Invertase Inhibitor from Maize (Zea mays L.)

2019 ◽  
Vol 20 (17) ◽  
pp. 4081 ◽  
Author(s):  
Lin Chen ◽  
Xiaohong Liu ◽  
Xiaojia Huang ◽  
Wei Luo ◽  
Yuming Long ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Protein Fusion ◽  
Drought Treatment ◽  
Vacuolar Invertase ◽  
Maize Leaves ◽  
Green Fluorescent ◽  
Proteinaceous Inhibitor

Invertases (INVs) play essential roles in plant growth in response to environmental cues. Previous work showed that plant invertases can be post-translationally regulated by small protein inhibitors (INVINHs). Here, this study characterizes a proteinaceous inhibitor of INVs in maize (Zm-INVINH4). A functional analysis of the recombinant Zm-INVINH4 protein revealed that it inhibited both cell wall and vacuolar invertase activities from maize leaves. A Zm-INVINH4::green fluorescent protein fusion experiment indicated that this protein localized in the apoplast. Transcript analysis showed that Zm-INVINH4 is specifically expressed in maize sink tissues, such as the base part of the leaves and young kernels. Moreover, drought stress perturbation significantly induced Zm-INVINH4 expression, which was accompanied with a decrease of cell wall invertase (CWI) activities and an increase of sucrose accumulation in both base parts of the leaves 2 to 7 days after pollinated kernels. In summary, the results support the hypothesis that INV-related sink growth in response to drought treatment is (partially) caused by a silencing of INV activity via drought-induced induction of Zm-INVINH4 protein.

scholarly journals A Mutation in a Novel Yeast Proteasomal Gene,RPN11/MPR1, Produces a Cell Cycle Arrest, Overreplication of Nuclear and Mitochondrial DNA, and an Altered Mitochondrial Morphology

1998 ◽  
Vol 9 (10) ◽  
pp. 2917-2931 ◽  
Author(s):  
Teresa Rinaldi ◽  
Carlo Ricci ◽  
Danilo Porro ◽  
Monique Bolotin-Fukuhara ◽  
Laura Frontali
Keyword(s):  
Mitochondrial Dna ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Mitochondrial Morphology ◽  
Nonpermissive Temperature ◽  
Protein Fusion ◽  
In The Wild ◽  
A Cell ◽  
Green Fluorescent ◽  
Cytoplasmic Structures

We report here the functional characterization of an essentialSaccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of thempr1-1 mutation that causes the following pleiotropic defects. At 24°C growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36°C on either carbon source. Microscopic observation of cells growing on glucose at 24°C shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho°] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.

scholarly journals Profiling of Wheat Class III Peroxidase Genes Derived from Powdery Mildew-Attacked Epidermis Reveals Distinct Sequence-Associated Expression Patterns

2005 ◽  
Vol 18 (7) ◽  
pp. 730-741 ◽  
Author(s):  
Guosheng Liu ◽  
Xiaoyan Sheng ◽  
David L. Greenshields ◽  
Adam Ogieglo ◽  
Susan Kaminskyj ◽  
...  
Keyword(s):  
Powdery Mildew ◽  
Differential Expression ◽  
Fluorescent Protein ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Amino Acid Sequences ◽  
Protein Fusion ◽  
Powdery Mildew Fungus ◽  
Mesophyll Cells ◽  
Signal Molecule

A cDNA library was constructed from leaf epidermis of diploid wheat (Triticum monococcum) infected with the powdery mildew fungus (Blumeria graminis f. sp. tritici) and was screened for genes encoding peroxidases. From 2,500 expressed sequence tags (ESTs), 36 cDNAs representing 10 peroxidase genes (designated TmPRX1 to TmPRX10) were isolated and further characterized. Alignment of the deduced amino acid sequences and phylogenetic clustering with peroxidases from other plant species demonstrated that these peroxidases fall into four distinct groups. Differential expression and tissue-specific localization among the members were observed during the B. graminis f. sp. tritici attack using Northern blots and reverse-transcriptase polymerase chain reaction analyses. Consistent with its abundance in the EST collection, TmPRX1 expression showed the highest induction during pathogen attack and fluctuated in response to the fungal parasitic stages. TmPRX1 to TmPRX6 were expressed predominantly in mesophyll cells, whereas TmPRX7 to TmPRX10, which feature a putative C-terminal propeptide, were detectable mainly in epidermal cells. Using TmPRX8 as a representative, we demonstrated that its C-terminal propeptide was sufficient to target a green fluorescent protein fusion protein to the vacuoles in onion cells. Finally, differential expression profiles of the TmPRXs after abiotic stresses and signal molecule treatments were used to dissect the potential role of these peroxidases in multiple stress and defense pathways.

scholarly journals Repression of hla by rot Is Dependent on sae in Staphylococcus aureus

2008 ◽  
Vol 76 (3) ◽  
pp. 1068-1075 ◽  
Author(s):  
Dongmei Li ◽  
Ambrose Cheung
Keyword(s):  
Staphylococcus Aureus ◽  
Double Mutant ◽  
Northern Blot Analysis ◽  
Fluorescent Protein ◽  
Transcriptional Level ◽  
Protein Fusion ◽  
Two Component System ◽  
Triple Mutant ◽  
Green Fluorescent

ABSTRACT The regulatory locus sae is a two-component system in Staphylococcus aureus that regulates many important virulence factors, including alpha-toxin (encoded by hla) at the transcriptional level. The SarA homologs Rot and SarT were previously shown to be repressors of hla in selected S. aureus backgrounds. To delineate the interaction of rot and sae and the contribution of sarT to hla expression, an assortment of rot and sae isogenic single mutants, a rot sae double mutant, and a rot sae sarT markerless triple mutant were constructed from wild-type strain COL. Using Northern blot analysis and transcriptional reporter gene green fluorescent protein, fusion, and phenotypic assays, we found that the repression of hla by rot is dependent on sae. A rot sae sarT triple mutant was not able to rescue the hla defect of the rot sae double mutant. Among the three sae promoters, the distal sae P3 promoter is the strongest in vitro. Interestingly, the sae P3 promoter activities correlate with hla expression in rot, rot sae, and rot sae sarT mutants of COL. Transcriptional study has also shown that rot repressed sae, especially at the sae P3 promoter. Collectively, our data implicated the importance of sae in the rot-mediated repression of hla in S. aureus.

scholarly journals Relationship of In Vitro Toxicity of Technetium-99m to Subcellular Localisation and Absorbed Dose

2021 ◽  
Vol 22 (24) ◽  
pp. 13466
Author(s):  
Ines M. Costa ◽  
Noor Siksek ◽  
Alessia Volpe ◽  
Francis Man ◽  
Katarzyna M. Osytek ◽  
...  
Keyword(s):  
Dna Damage ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Absorbed Dose ◽  
In Vitro Toxicity ◽  
Subcellular Localisation ◽  
Protein Fusion ◽  
External Beam Irradiation ◽  
Technetium 99M

Auger electron-emitters increasingly attract attention as potential radionuclides for molecular radionuclide therapy in oncology. The radionuclide technetium-99m is widely used for imaging; however, its potential as a therapeutic radionuclide has not yet been fully assessed. We used MDA-MB-231 breast cancer cells engineered to express the human sodium iodide symporter-green fluorescent protein fusion reporter (hNIS-GFP; MDA-MB-231.hNIS-GFP) as a model for controlled cellular radionuclide uptake. Uptake, efflux, and subcellular location of the NIS radiotracer [99mTc]TcO4− were characterised to calculate the nuclear-absorbed dose using Medical Internal Radiation Dose formalism. Radiotoxicity was determined using clonogenic and γ-H2AX assays. The daughter radionuclide technetium-99 or external beam irradiation therapy (EBRT) served as controls. [99mTc]TcO4− in vivo biodistribution in MDA-MB-231.hNIS-GFP tumour-bearing mice was determined by imaging and complemented by ex vivo tissue radioactivity analysis. [99mTc]TcO4− resulted in substantial DNA damage and reduction in the survival fraction (SF) following 24 h incubation in hNIS-expressing cells only. We found that 24,430 decays/cell (30 mBq/cell) were required to achieve SF0.37 (95%-confidence interval = [SF0.31; SF0.43]). Different approaches for determining the subcellular localisation of [99mTc]TcO4− led to SF0.37 nuclear-absorbed doses ranging from 0.33 to 11.7 Gy. In comparison, EBRT of MDA-MB-231.hNIS-GFP cells resulted in an SF0.37 of 2.59 Gy. In vivo retention of [99mTc]TcO4− after 24 h remained high at 28.0% ± 4.5% of the administered activity/gram tissue in MDA-MB-231.hNIS-GFP tumours. [99mTc]TcO4− caused DNA damage and reduced clonogenicity in this model, but only when the radioisotope was taken up into the cells. This data guides the safe use of technetium-99m during imaging and potential future therapeutic applications.

UBE2L6 is Involved in Cisplatin Resistance by Regulating the Transcription of ABCB6

2020 ◽  
Vol 20 (12) ◽  
pp. 1487-1496 ◽  
Author(s):  
Midori Murakami ◽  
Hiroto Izumi ◽  
Tomoko Kurita ◽  
Chiho Koi ◽  
Yasuo Morimoto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Anticancer Agent ◽  
Cisplatin Resistance ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Molecular Target ◽  
Transcriptional Level ◽  
And Control ◽  
Resistant Cells

Background: Cisplatin is an important anticancer agent in cancer chemotherapy, but when resistant cells appear, treatment becomes difficult, and the prognosis is poor. Objective: In this study, we investigated the gene expression profile in cisplatin sensitive and resistant cells, and identified the genes involved in cisplatin resistance. Methods: Comparison of gene expression profiles revealed that UBE2L6 mRNA is highly expressed in resistant cells. To elucidate whether UBE2L6 is involved in the acquisition of cisplatin resistance, UBE2L6- overexpressing cells established from cisplatin-sensitive cells and UBE2L6-silenced cells developed from cisplatin- resistant cells were generated, and the sensitivity of cisplatin was examined. Results: The sensitivity of the UBE2L6-overexpressing cells did not change compared with the control cells, but the UBE2L6-silenced cells were sensitized to cisplatin. To elucidate the mechanism of UBE2L6 in cisplatin resistance, we compared the gene expression profiles of UBE2L6-silenced cells and control cells and found that the level of ABCB6 mRNA involved in cisplatin resistance was decreased. Moreover, ABCB6 promoter activity was partially suppressed in UBE2L6-silenced cells. Conclusion: These results suggest that cisplatin-resistant cells have upregulated UBE2L6 expression and contribute to cisplatin resistance by regulating ABCB6 expression at the transcriptional level. UBE2L6 might be a molecular target that overcomes cisplatin resistance.

scholarly journals Identification of the specific long-noncoding RNAs involved in night-break mediated flowering retardation in Chenopodium quinoa

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Qi Wu ◽  
Yiming Luo ◽  
Xiaoyong Wu ◽  
Xue Bai ◽  
Xueling Ye ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Functional Characterization ◽  
Chenopodium Quinoa ◽  
Noncoding Rnas ◽  
Long Noncoding Rnas ◽  
Rna Seq ◽  
Short Day ◽  
Homologous Genes ◽  
The Relationship ◽  
Encoding Genes

Abstract Background Night-break (NB) has been proven to repress flowering of short-day plants (SDPs). Long-noncoding RNAs (lncRNAs) play key roles in plant flowering. However, investigation of the relationship between lncRNAs and NB responses is still limited, especially in Chenopodium quinoa, an important short-day coarse cereal. Results In this study, we performed strand-specific RNA-seq of leaf samples collected from quinoa seedlings treated by SD and NB. A total of 4914 high-confidence lncRNAs were identified, out of which 91 lncRNAs showed specific responses to SD and NB. Based on the expression profiles, we identified 17 positive- and 7 negative-flowering lncRNAs. Co-expression network analysis indicated that 1653 mRNAs were the common targets of both types of flowering lncRNAs. By mapping these targets to the known flowering pathways in model plants, we found some pivotal flowering homologs, including 2 florigen encoding genes (FT (FLOWERING LOCUS T) and TSF (TWIN SISTER of FT) homologs), 3 circadian clock related genes (EARLY FLOWERING 3 (ELF3), LATE ELONGATED HYPOCOTYL (LHY) and ELONGATED HYPOCOTYL 5 (HY5) homologs), 2 photoreceptor genes (PHYTOCHROME A (PHYA) and CRYPTOCHROME1 (CRY1) homologs), 1 B-BOX type CONSTANS (CO) homolog and 1 RELATED TO ABI3/VP1 (RAV1) homolog, were specifically affected by NB and competed by the positive and negative-flowering lncRNAs. We speculated that these potential flowering lncRNAs may mediate quinoa NB responses by modifying the expression of the floral homologous genes. Conclusions Together, the findings in this study will deepen our understanding of the roles of lncRNAs in NB responses, and provide valuable information for functional characterization in future.

scholarly journals Transcriptome analysis reveals potential function of long non-coding RNAs in 20-hydroxyecdysone regulated autophagy in Bombyx mori

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Huili Qiao ◽  
Jingya Wang ◽  
Yuanzhuo Wang ◽  
Juanjuan Yang ◽  
Bofan Wei ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Target Gene ◽  
Fat Body ◽  
Expression Profiles ◽  
Functional Characterization ◽  
Differentially Expressed ◽  
Post Injection ◽  
Biological Processes ◽  
Potential Target ◽  
Non Coding Rnas

Abstract Background 20-hydroxyecdysone (20E) plays important roles in insect molting and metamorphosis. 20E-induced autophagy has been detected during the larval–pupal transition in different insects. In Bombyx mori, autophagy is induced by 20E in the larval fat body. Long non-coding RNAs (lncRNAs) function in various biological processes in many organisms, including insects. Many lncRNAs have been reported to be potential for autophagy occurrence in mammals, but it has not been investigated in insects. Results RNA libraries from the fat body of B. mori dissected at 2 and 6 h post-injection with 20E were constructed and sequenced, and comprehensive analysis of lncRNAs and mRNAs was performed. A total of 1035 lncRNAs were identified, including 905 lincRNAs and 130 antisense lncRNAs. Compared with mRNAs, lncRNAs had longer transcript length and fewer exons. 132 lncRNAs were found differentially expressed at 2 h post injection, compared with 64 lncRNAs at 6 h post injection. Thirty differentially expressed lncRNAs were common at 2 and 6 h post-injection, and were hypothesized to be associated with the 20E response. Target gene analysis predicted 6493 lncRNA-mRNA cis pairs and 42,797 lncRNA-mRNA trans pairs. The expression profiles of LNC_000560 were highly consistent with its potential target genes, Atg4B, and RNAi of LNC_000560 significantly decreased the expression of LNC_000560 and Atg4B. These results indicated that LNC_000560 was potentially involved in the 20E-induced autophagy of the fat body by regulating Atg4B. Conclusions This study provides the genome-wide identification and functional characterization of lncRNAs associated with 20E-induced autophagy in the fat body of B. mori. LNC_000560 and its potential target gene were identified to be related to 20-regulated autophagy in B. mori. These results will be helpful for further studying the regulatory mechanisms of lncRNAs in autophagy and other biological processes in this insect model.

Sign in / Sign up

Export Citation Format

Share Document