@article{ShahnejatBushehriAlluMehterovetal.2017, author = {Shahnejat-Bushehri, Sara and Allu, Annapurna Devi and Mehterov, Nikolay and Thirumalaikumar, Venkatesh P. and Alseekh, Saleh and Fernie, Alisdair R. and Mueller-Roeber, Bernd and Balazadeh, Salma}, title = {Arabidopsis NAC Transcription Factor JUNGBRUNNEN1 Exerts Conserved Control Over Gibberellin and Brassinosteroid Metabolism and Signaling Genes in Tomato}, series = {Frontiers in plant science}, volume = {8}, journal = {Frontiers in plant science}, publisher = {Frontiers Research Foundation}, address = {Lausanne}, issn = {1664-462X}, doi = {10.3389/fpls.2017.00214}, pages = {13}, year = {2017}, abstract = {The Arabidopsis thaliana NAC transcription factor JUNGBRUNNEN1 (AtJUB1) regulates growth by directly repressing GA3ox1 and DWF4, two key genes involved in gibberellin (GA) and brassinosteroid (BR) biosynthesis, respectively, leading to GA and BR deficiency phenotypes. AtJUB1 also reduces the expression of PIF4, a bHLH transcription factor that positively controls cell elongation, while it stimulates the expression of DELLA genes, which are important repressors of growth. Here, we extend our previous findings by demonstrating that AtJUB1 induces similar GA and BR deficiency phenotypes and changes in gene expression when overexpressed in tomato (Solanum lycopersicum). Importantly, and in accordance with the growth phenotypes observed, AtJUB1 inhibits the expression of growth-supporting genes, namely the tomato orthologs of GA3ox1, DWF4 and PIF4, but activates the expression of DELLA orthologs, by directly binding to their promoters. Overexpression of AtJUB1 in tomato delays fruit ripening, which is accompanied by reduced expression of several ripeningrelated genes, and leads to an increase in the levels of various amino acids (mostly proline, beta-alanine, and phenylalanine), gamma-aminobutyric acid (GABA), and major organic acids including glutamic acid and aspartic acid. The fact that AtJUB1 exerts an inhibitory effect on the GA/BR biosynthesis and PIF4 genes but acts as a direct activator of DELLA genes in both, Arabidopsis and tomato, strongly supports the model that the molecular constituents of the JUNGBRUNNEN1 growth control module are considerably conserved across species.}, language = {en} } @article{WatanabeTohgeBalazadehetal.2018, author = {Watanabe, Mutsumi and Tohge, Takayuki and Balazadeh, Salma and Erban, Alexander and Giavalisco, Patrick and Kopka, Joachim and Mueller-Roeber, Bernd and Fernie, Alisdair R. and Hoefgen, Rainer}, title = {Comprehensive Metabolomics Studies of Plant Developmental Senescence}, series = {Plant Senescence: Methods and Protocols}, volume = {1744}, journal = {Plant Senescence: Methods and Protocols}, publisher = {Humana Press}, address = {Totowa}, isbn = {978-1-4939-7672-0}, issn = {1064-3745}, doi = {10.1007/978-1-4939-7672-0_28}, pages = {339 -- 358}, year = {2018}, abstract = {Leaf senescence is an essential developmental process that involves diverse metabolic changes associated with degradation of macromolecules allowing nutrient recycling and remobilization. In contrast to the significant progress in transcriptomic analysis of leaf senescence, metabolomics analyses have been relatively limited. A broad overview of metabolic changes during leaf senescence including the interactions between various metabolic pathways is required to gain a better understanding of the leaf senescence allowing to link transcriptomics with metabolomics and physiology. In this chapter, we describe how to obtain comprehensive metabolite profiles and how to dissect metabolic shifts during leaf senescence in the model plant Arabidopsis thaliana. Unlike nucleic acid analysis for transcriptomics, a comprehensive metabolite profile can only be achieved by combining a suite of analytic tools. Here, information is provided for measurements of the contents of chlorophyll, soluble proteins, and starch by spectrophotometric methods, ions by ion chromatography, thiols and amino acids by HPLC, primary metabolites by GC/TOF-MS, and secondary metabolites and lipophilic metabolites by LC/ESI-MS. These metabolite profiles provide a rich catalogue of metabolic changes during leaf senescence, which is a helpful database and blueprint to be correlated to future studies such as transcriptome and proteome analyses, forward and reverse genetic studies, or stress-induced senescence studies.}, language = {en} } @article{KamranfarXueTohgeetal.2018, author = {Kamranfar, Iman and Xue, Gang-Ping and Tohge, Takayuki and Sedaghatmehr, Mastoureh and Fernie, Alisdair R. and Balazadeh, Salma and Mueller-Roeber, Bernd}, title = {Transcription factor RD26 is a key regulator of metabolic reprogramming during dark-induced senescence}, series = {New phytologist : international journal of plant science}, volume = {218}, journal = {New phytologist : international journal of plant science}, number = {4}, publisher = {Wiley}, address = {Hoboken}, issn = {0028-646X}, doi = {10.1111/nph.15127}, pages = {1543 -- 1557}, year = {2018}, abstract = {Leaf senescence is a key process in plants that culminates in the degradation of cellular constituents and massive reprogramming of metabolism for the recovery of nutrients from aged leaves for their reuse in newly developing sinks. We used molecular-biological and metabolomics approaches to identify NAC transcription factor (TF) RD26 as an important regulator of metabolic reprogramming in Arabidopsis thaliana. RD26 directly activates CHLOROPLAST VESICULATION (CV), encoding a protein crucial for chloroplast protein degradation, concomitant with an enhanced protein loss in RD26 over-expressors during senescence, but a reduced decline of protein in rd26 knockout mutants. RD26 also directly activates LKR/SDH involved in lysine catabolism, and PES1 important for phytol degradation. Metabolic profiling revealed reduced c-aminobutyric acid (GABA) in RD26 overexpressors, accompanied by the induction of respective catabolic genes. Degradation of lysine, phytol and GABA is instrumental for maintaining mitochondrial respiration in carbon-limiting conditions during senescence. RD26 also supports the degradation of starch and the accumulation of mono-and disaccharides during senescence by directly enhancing the expression of AMY1, SFP1 and SWEET15 involved in carbohydrate metabolism and transport. Collectively, during senescence RD26 acts by controlling the expression of genes across the entire spectrum of the cellular degradation hierarchy.}, language = {en} } @article{OmidbakhshfardFujikuraOlasetal.2018, author = {Omidbakhshfard, Mohammad Amin and Fujikura, Ushio and Olas, Justyna Jadwiga and Xue, Gang-Ping and Balazadeh, Salma and Mueller-Roeber, Bernd}, title = {GROWTH-REGULATING FACTOR 9 negatively regulates arabidopsis leaf growth by controlling ORG3 and restricting cell proliferation in leaf primordia}, series = {PLoS Genetics : a peer-reviewed, open-access journal}, volume = {14}, journal = {PLoS Genetics : a peer-reviewed, open-access journal}, number = {7}, publisher = {PLoS}, address = {San Fransisco}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1007484}, pages = {31}, year = {2018}, abstract = {Leaf growth is a complex process that involves the action of diverse transcription factors (TFs) and their downstream gene regulatory networks. In this study, we focus on the functional characterization of the Arabidopsis thaliana TF GROWTH-REGULATING FACTOR9 (GRF9) and demonstrate that it exerts its negative effect on leaf growth by activating expression of the bZIP TF OBP3-RESPONSIVE GENE 3 (ORG3). While grf9 knockout mutants produce bigger incipient leaf primordia at the shoot apex, rosette leaves and petals than the wild type, the sizes of those organs are reduced in plants overexpressing GRF9 (GRF9ox). Cell measurements demonstrate that changes in leaf size result from alterations in cell numbers rather than cell sizes. Kinematic analysis and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay revealed that GRF9 restricts cell proliferation in the early developing leaf. Performing in vitro binding site selection, we identified the 6-base motif 5'-CTGACA-3' as the core binding site of GRF9. By global transcriptome profiling, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) we identified ORG3 as a direct downstream, and positively regulated target of GRF9. Genetic analysis of grf9 org3 and GRF9ox org3 double mutants reveals that both transcription factors act in a regulatory cascade to control the final leaf dimensions by restricting cell number in the developing leaf.}, language = {en} } @article{DevkarThirumalaikumarXueetal.2019, author = {Devkar, Vikas and Thirumalaikumar, Venkatesh P. and Xue, Gang-Ping and Vallarino, Jose G. and Tureckova, Veronika and Strnad, Miroslav and Fernie, Alisdair R. and Hoefgen, Rainer and Mueller-Roeber, Bernd and Balazadeh, Salma}, title = {Multifaceted regulatory function of tomato SlTAF1 in the response to salinity stress}, series = {New phytologist : international journal of plant science}, volume = {225}, journal = {New phytologist : international journal of plant science}, number = {4}, publisher = {Wiley}, address = {Hoboken}, issn = {0028-646X}, doi = {10.1111/nph.16247}, pages = {1681 -- 1698}, year = {2019}, abstract = {Salinity stress limits plant growth and has a major impact on agricultural productivity. Here, we identify NAC transcription factor SlTAF1 as a regulator of salt tolerance in cultivated tomato (Solanum lycopersicum). While overexpression of SlTAF1 improves salinity tolerance compared with wild-type, lowering SlTAF1 expression causes stronger salinity-induced damage. Under salt stress, shoots of SlTAF1 knockdown plants accumulate more toxic Na+ ions, while SlTAF1 overexpressors accumulate less ions, in accordance with an altered expression of the Na+ transporter genes SlHKT1;1 and SlHKT1;2. Furthermore, stomatal conductance and pore area are increased in SlTAF1 knockdown plants during salinity stress, but decreased in SlTAF1 overexpressors. We identified stress-related transcription factor, abscisic acid metabolism and defence-related genes as potential direct targets of SlTAF1, correlating it with reactive oxygen species scavenging capacity and changes in hormonal response. Salinity-induced changes in tricarboxylic acid cycle intermediates and amino acids are more pronounced in SlTAF1 knockdown than wild-type plants, but less so in SlTAF1 overexpressors. The osmoprotectant proline accumulates more in SlTAF1 overexpressors than knockdown plants. In summary, SlTAF1 controls the tomato's response to salinity stress by combating both osmotic stress and ion toxicity, highlighting this gene as a promising candidate for the future breeding of stress-tolerant crops.}, language = {en} } @article{MaBalazadehMuellerRoeber2019, author = {Ma, Xuemin and Balazadeh, Salma and Mueller-Roeber, Bernd}, title = {Tomato fruit ripening factor NOR controls leaf senescence}, series = {Journal of experimental botany}, volume = {70}, journal = {Journal of experimental botany}, number = {10}, publisher = {Oxford Univ. Press}, address = {Oxford}, issn = {0022-0957}, doi = {10.1093/jxb/erz098}, pages = {2727 -- 2740}, year = {2019}, abstract = {NAC transcription factors (TFs) are important regulators of expressional reprogramming during plant development, stress responses, and leaf senescence. NAC TFs also play important roles in fruit ripening. In tomato (Solanum lycopersicum), one of the best characterized NACs involved in fruit ripening is NON-RIPENING (NOR), and the non-ripening (nor) mutation has been widely used to extend fruit shelf life in elite varieties. Here, we show that NOR additionally controls leaf senescence. Expression of NOR increases with leaf age, and developmental as well as dark-induced senescence are delayed in the nor mutant, while overexpression of NOR promotes leaf senescence. Genes associated with chlorophyll degradation as well as senescence-associated genes (SAGs) show reduced and elevated expression, respectively, in nor mutants and NOR overexpressors. Overexpression of NOR also stimulates leaf senescence in Arabidopsis thaliana. In tomato, NOR supports senescence by directly and positively regulating the expression of several senescence-associated genes including, besides others, SlSAG15 and SlSAG113, SlSGR1, and SlYLS4. Finally, we find that another senescence control NAC TF, namely SlNAP2, acts upstream of NOR to regulate its expression. Our data support a model whereby NAC TFs have often been recruited by higher plants for both the control of leaf senescence and fruit ripening.}, language = {en} } @article{StobieckiSkiryczKerhoasetal.2006, author = {Stobiecki, Maciej and Skirycz, Aleksandra and Kerhoas, L. and Kachlicki, P. and Muth, D. and Einhorn, J. and Mueller-Roeber, Bernd}, title = {Profiling of phenolic glycosidic conjugates in leaves of Arabidopsis thaliana using LC/MS}, series = {Metabolomics : the official journal of the Metabolomics Society}, volume = {2}, journal = {Metabolomics : the official journal of the Metabolomics Society}, publisher = {Springer}, address = {New York}, issn = {1573-3882}, doi = {10.1007/s11306-006-0031-5}, pages = {197 -- 219}, year = {2006}, abstract = {Profiling of plant secondary metabolites is still a very difficult task. Liquid chromatography (LC) or capillary electrophoresis hyphenated with different kinds of detectors are methods of choice for analysis of polar, thermo labile compounds with high molecular masses. We demonstrate the applicability of LC combined with UV diode array or/and mass spectrometric detectors for the unambiguous identification and quantification of flavonoid conjugates isolated from Arahidopsis thaliana leaves of different genotypes and grown in different environmental conditions. During LC/UV/MS/MS analyses we were able to identify tetra-, tri, and di-glycosides of kaempferol, quercetin and isorhamnetin. Based on our results we can conclude that due to the co-elution of different chemical compounds in reversed phase H PLC systems the application of UV detectors does not allow to precisely profile all flavonoid conjugates existing in A. thaliana genotypes. Using MS detection it was possible to unambiguously recognize the glycosylation patterns of the aglycones. However, from the mass spectra we could not conclude neither the anomeric form of the C-1 carbon atoms of sugar moieties in glycosidic bonds between sugars or sugar and aglycone nor the position of the second carbon involved in disaccharides. The applicability of collision induced dissociation techniques (CID MS/MS) for structural analyses of the studied group of plant secondary metabolites with two types of analyzers (triple quadrupole or ion trap) was demonstrated.}, language = {en} } @article{HochreinMachensGremmelsetal.2017, author = {Hochrein, Lena and Machens, Fabian and Gremmels, Juergen and Schulz, Karina and Messerschmidt, Katrin and Mueller-Roeber, Bernd}, title = {AssemblX: a user-friendly toolkit for rapid and reliable multi-gene assemblies}, series = {Nucleic acids research}, volume = {45}, journal = {Nucleic acids research}, publisher = {Oxford Univ. Press}, address = {Oxford}, issn = {0305-1048}, doi = {10.1093/nar/gkx034}, pages = {12}, year = {2017}, abstract = {The assembly of large DNA constructs coding for entire pathways poses a major challenge in the field of synthetic biology. Here, we present AssemblX, a novel, user-friendly and highly efficient multi-gene assembly strategy. The software-assisted AssemblX process allows even unexperienced users to rapidly design, build and test DNA constructs with currently up to 25 functional units, from 75 or more subunits. At the gene level, AssemblX uses scar-free, overlap-based and sequence-independent methods, allowing the unrestricted design of transcriptional units without laborious parts domestication. The assembly into multi-gene modules is enabled via a standardized, highly efficient, polymerase chain reaction-free and virtually sequence-independent scheme, which relies on rare cutting restriction enzymes and optimized adapter sequences. Selection and marker switching strategies render the whole process reliable, rapid and very effective. The assembly product can be easily transferred to any desired expression host, making AssemblX useful for researchers from various fields.}, language = {en} } @article{EbrahimianMotlaghRiboneThirumalaikumaretal.2017, author = {Ebrahimian-Motlagh, Saghar and Ribone, Pamela A. and Thirumalaikumar, Venkatesh P. and Allu, Annapurna Devi and Chan, Raquel L. and Mueller-Roeber, Bernd and Balazadeh, Salma}, title = {JUNGBRUNNEN1 Confers Drought Tolerance Downstream of the HD-Zip I Transcription Factor AtHB13}, series = {Frontiers in plant science}, volume = {8}, journal = {Frontiers in plant science}, publisher = {Frontiers Research Foundation}, address = {Lausanne}, issn = {1664-462X}, doi = {10.3389/fpls.2017.02118}, pages = {12}, year = {2017}, abstract = {Low water availability is the major environmental factor limiting growth and productivity of plants and crops and is therefore considered of high importance for agriculture affected by climate change. Identifying regulatory components controlling the response and tolerance to drought stress is thus of major importance. The NAC transcription factor (TF) JUNGBRUNNEN1 (JUB1) from Arabidopsis thaliana extends leaf longevity under non-stress growth conditions, lowers cellular hydrogen peroxide (H2O2) level, and enhances tolerance against heat stress and salinity. Here, we additionally find that JUB1 strongly increases tolerance to drought stress in Arabidopsis when expressed from both, a constitutive (CaMV 35S) and an abiotic stress-induced (RD29A) promoter. Employing a yeast one-hybrid screen we identified HD-Zip class I TF AtHB13 as an upstream regulator of JUB1. AtHB13 has previously been reported to act as a positive regulator of drought tolerance. AtHB13 and JUB1 thereby establish a joint drought stress control module.}, language = {en} } @article{MesserschmidtHochreinDehmetal.2016, author = {Messerschmidt, Katrin and Hochrein, Lena and Dehm, Daniel and Schulz, Karina and Mueller-Roeber, Bernd}, title = {Characterizing seamless ligation cloning extract for synthetic biological applications}, series = {Analytical biochemistry : methods in the biological sciences}, volume = {509}, journal = {Analytical biochemistry : methods in the biological sciences}, publisher = {Elsevier}, address = {San Diego}, issn = {0003-2697}, doi = {10.1016/j.ab.2016.05.029}, pages = {24 -- 32}, year = {2016}, abstract = {Synthetic biology aims at designing and engineering organisms. The engineering process typically requires the establishment of suitable DNA constructs generated through fusion of multiple protein coding and regulatory sequences. Conventional cloning techniques, including those involving restriction enzymes and ligases, are often of limited scope, in particular when many DNA fragments must be joined or scar-free fusions are mandatory. Overlap-based-cloning methods have the potential to overcome such limitations. One such method uses seamless ligation cloning extract (SLiCE) prepared from Escherichia coli cells for straightforward and efficient in vitro fusion of DNA fragments. Here, we systematically characterized extracts prepared from the unmodified E. coli strain DH10B for SLiCE-mediated cloning and determined DNA sequence-associated parameters that affect cloning efficiency. Our data revealed the virtual absence of length restrictions for vector backbone (up to 13.5 kbp) and insert (90 bp to 1.6 kbp). Furthermore, differences in GC content in homology regions are easily tolerated and the deletion of unwanted vector sequences concomitant with targeted fragment insertion is straightforward. Thus, SLiCE represents a highly versatile DNA fusion method suitable for cloning projects in virtually all molecular. and synthetic biology projects. (C) 2016 Elsevier Inc. All rights reserved.}, language = {en} }